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MUI Sumsel Yakini Vaksin Meningistis untuk Calhaj Haram

http://republika.co.id/berita/46652/MUI_Sumsel_Yakini_Vaksin_Meningistis_untuk_Calhaj_Haram

MUI Sumsel Yakini Vaksin Meningistis untuk Calhaj Haram

By Republika Newsroom

Senin, 27 April 2009 pukul 11:42:00

PALEMBANG — Majelis Ulama Indonesia Sumatera Selatan (MUI Sumsel) tetap pada sikapnya, meyakini bahwa vaksin meningitis yang digunakan untuk calon jemaah haji atau umrah tersebut haram karena mengandung enzim yang berasal dari binatang babi. Ketua MUI Sumsel KH Sodikun, Senin (27/4) menegaskan, apa yang disampaikan LPPOM MUI Sumsel bersama para pakar sudah melalui diskusi dan pengkajian. “Hasil kajian MUI Sumsel ini sudah kami sampaikan ke MUI pusat melalui forum Rakernas MUI pada November 2008 di Jakarta. Namun apa yang kami sampaikan sampai kini belum ada respon baik dari Menteri Agama dan Menteri Kesehatan,” ujarnya. Menurut Sekretaris MUI Sumsel KH Ayik Farid, “Dalam Rakernas MUI sudah kami sampaikan bahwa proses pembuatan vaksin meningitis tersebut menggunakan enzim porchin dari binatang babi. LPPOM MUI Pusat juga sudah mengakui itu, namun karena sudah ada kontrak pengadaan vaksin tersebut selama lima tahun maka penggunaannya tidak bisa diganti.” Ayik Farid juga mengakui, bahwa temuan MUI Sumsel tersebut sudah melewati forum diskusi dengan para pakar, diantaranya pakar farmakologi Prof Dr T Kamaluddin Ketua Program Pasca Sarjana Universitas Sriwijaya (Unsri), pakar penyakit dalam dan pakar dokter anak. “Jadi apa yang kami sampaikan tentang vaksin meningitis yang mengandung enzim babi bukan tanpa melalui kajian. Kajian ini sudah kami sampaikan ke MUI Pusat melalui forum Rekernas MUI pada Novermber 2008. MUI Sumsel bukan ingin membuat keresahan di tengah masyarakat. MUI Sumsel berharap masalah ini segera menjadi perhatian serius Departemen Agama dan Departemen Kesehatan,” tambah Sodikun. Sementara itu Prof Nasruddin Iljas Ketua LPPOM MUI Sumsel menjelaskan, negara lain seperti Malaysia telah menggunakan vaksin meningitis yang halal dari sapi. “Jadi sudah seharusnya pemerintah pusat, khususnya Departemen Agama segera mencari alternatif pengganti vaksin meningitis yang tidak mengandung binatang babi.” Nasruddin mengatakan, jika produk makanan, obat-obatan serta kosmetik mengandung bahan yang tidak halal maka akan menghambat bahkan menyebabkan ibadah umat Islam sia-sia. “Ini harus menjadi perhatian. Apalagi sekarang marak beredar makanan yang berasal dari daging babi,” tambahnya. Terhadap para jemaah haji yang telah menggunakan vaksin meningitis yang mengandung enzim babi tersebut, Ketua MUI Sumsel menjelaskan, masuknya zat haram ke dalam tubuh para calon jemaah haji itu berakibat menghalangi kemabruran hajinya. Sebab syarat mabrurnya haji, selain bersih secara jiwa, para jamaah haji juga harus bersih secara raga. “Kalau tubuh kita kemasukan zat yang diharamkan maka dapat menghalangi terkabulnya doa. Tapi bagi mereka yang tidak tahu bisa dimaafkan, yang berdosa adalah orang yang mengambil kebijakan dan mengetahui hal itu tapi tetap dilaksanakan,” tegasnya. oed/fif

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Ekstrak Meniran Terbukti Cegah Influenza Jemaah Haji

Ekstrak Meniran Terbukti Cegah Influenza Jemaah Haji
Senin, 30 Maret 2009 | 23:16 WIB

JAKARTA, KOMPAS.com – Pemberian Phyllanthus niruri atau ekstrak meniran bersamaan dengan multivitamin dapat mengurangi risiko Penyakit influenza-like illness (ILI) pada jemaah haji Indonesia selama menjalankan ibadah haji.

Demikian paparan DR.Masdalina Pane, SKM, MKes dan koleganya yang meneliti khasiat meniran untuk mencegah ILI pada jemaah haji Indonesia dari subdirektorat kesehatan haji Depkes RI di Jakarta, Senin (30/03).

Isu kesehatan pada jemaah haji Indonesia selalu menjadi topik hangat tiap tahunnya. Penyakit ILI yang merupakan salah satu Infeksi saluran pernapasan atas (ISPA) yang sering ditemukan pada jemaah haji Indonesia berdasarkan laporan tahunan pelaksanaan haji.

Masdiana menjelaskan influenza-like illness (ILI) secara klinis didefinisikan sebagai ISPA yang disebabkan oleh virus dengan gejala utama batuk kering, demam tinggi sekitar 38,5 derajat celcius, rasa lelah berlebihan, nyeri otot, meriang, demam, sakit kepala, tenggorokan dan hilang nafsu makan.

Ia mengatakan berdasarkan data laporan tahunan penyelenggaran haji, hampir 60% jemaah haji Indonesia tiap tahunnya terkena ISPA, dan 85% nya merupakan ILI dan hanya 15% saja yang benar-benar influenza.

Masdalina mengatakan selama ini jemaah haji Indonesia hanya diberikan vaksin Meningitis meningocous secara gratis sebelum berangkat ke tanah suci. Sementara itu bagi jemaah yang memerlukan vaksin tambahan, biasanya akan diberi vaksin influenza sesuai permintaan jemaah.

Namun menurutnya pemberian vaksin influenza juga tidak dapat mencegah individu terserang ILI, karena strain patogen virus yang menyebabkan ILI berbeda dengan influenzea.

Berangkat dari permasalahan tersebut, maka subdirektorat kesehatan haji Depkes RI bersama dengan Divisi alergi-imunologi klinik departemen penyakit dalam FK UI, RSCM dan didukung oleh PT Dexa Medica mengadakan penelitian dan uji klinis untuk pencegahan ILI pada jemaah haji Indonesia.

dr.Iris Rengganis, SpPD, KAI, salah satu peneliti yang melakukan uji klinis terhadap jemaah haji dari departemen ilmu penyakit dalam FKUI mengatakan penelitian ini dilakukan pada 309 jemaah haji Indonesia tahun 2007/2008(1428 H) dengan kisaran usia 18-65 tahun yang secara klinis dinilai sehat oleh tim kesehatan haji.

“Dalam penelitian ini jemaah dibagi sebara acak menjadi tiga kelompok,” ujar Iris. Kelompok pertama dan kedua hanya diberikan Phyllanthus niruri atau multivitamin saja. Sedangkan kelompok satunya lagi diberikan kombinasi keduanya.

“Produk uji kita berikan dengan dosis dua kali sehari untuk kapsul Phyllanthus niruri 50 mg dan sekali sehari untuk tablet multivitamin selama 40 hari yang meliputi seminggu sebelum berangkat dari tanah air dan selama perjalanan haji,” jelas Iris.

Selama mengikuti penelitian, subyek tidak diperkenankan minum suplemen lainnya. Setelah 40 hari uji coba, selanjutnnya dilakukan evaluasi klinis. Menurut Iris, dari hasil evaluasi klinis didapat data yang menunjukkan pemberian Phyllanthus niruri tunggal kepada kelompok pertama menunjukkan penurunan jumlah jamaah yang terjangkit ILI menjadi 10,5%, kelompok multivitamin tunggal sebesar 12,7 %, dan kelompok kombinasi sebesar hanya 8,1%.

“Hasil penelitian menunjukkan kelompok yang mendapatkan kombinasi keduanya dapat menekan kemungkinan terkena ILI paling kecil diikuti dengan kelompok yang mendapat meniran tunggal. Baru setelah itu kelompok yang hanya mendapat multivitamin,” tutur Iris.

Menurutnya kesimpulan yang didapat adalah pemberian Phyllanthus niruri bersamaan dengan multivitamin dapat mengurangi risiko ILI pada jemaah haji Indonesia selama menjalankan ibadah haji.

Sementara itu peneliti sub bagian patologi Universitas Airlangga, DR.Drs.Suprapto Maat, MS.Apoteker pada kesempatan yang sama menjelaskan bahwa ekstrak tanaman Phyllantus niruri telah dibuktikan secara klinis oleh peneliti yang bernama Thabrew dapat meningkatkan aktivitas sistem komplemen melalui jalur klasik yang pada akhirnya dapat meningkatkan sitotoksisitas sel natural killer (NK) untuk menambah daya tahan tubuh.

Cara Benar Makan Buah

Cara Benar Makan Buah

By Republika Contributor
Selasa, 02 Desember 2008 pukul 12:29:00
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Cara Benar Makan BuahCORBIS.COMTERATUR: Serat pada buah berfungsi efektif membuat tubuh lebih sehat dan segar. Makanlah buah sebelum makan untuk memperoleh manfaat maksimal.

JAKARTA– Di Indonesia, makan buah biasanya dilakukan setelah makan. Umumnya disebut dengan pencuci mulut. Padahal cara makan buah seperti itu, justru tidak akan memperoleh manfaat maksimal.

Sebaliknya, buah seharusnya dimakan saat perut kosong, bukan sebagai makanan penutup yang disantap selesai makan nasi. Jika membiasakan makan buah saat perut kosong, akan membantu pembuangan racun tubuh, sekaligus mensuplai energi yang cukup untuk kegiatan sehari-hari. Terutama bagi Anda yang sedang menjalani diet.

Saat Anda makan potong roti dan seporsi buah, sebenarnya buah akan lebih mudah diserap daripada roti. Di dalam lambung hanya butuh waktu singkat sudah dapat masuk kedalam usus kecil, saat itu roti belum sepenuhnya hancur, akibatnya jalan bagi buah tersebut terhambat.

Makanan dalam lambung akan berubah menjadi asam. Kemudian, asam lambung ditambah lagi dengan buah sehingga mudah menyebabkan buah berubah kualitas. Ujung-ujungnya bisa menimbulkan efek buruk bagi tubuh. Makan buah dengan perut kosong adalah yang salah satu cara terbaik memperoleh zat gizi maksimal.

Pernahkah Anda mendengar orang mengatakan, “setiap kali aku makan semangka suka tersedak” atau “ketika aku makan durian maka lambung terasa mengembang”. Selain itu sering juga orang berkata “selesai makan pisang, aku segera ingin ke toilet”.

Kenyataannya, jika makan buah saat perut kosong, tidak akan timbul masalah tersebut diatas. Buah dan makanan lain dalam lambung akan tercampur hingga membentuk gas tubuh, menyebabkan tersedak atau lambung mengembang. Yang lebih penting lagi, makan buah dengan perut kosong dapat menghindari rambut berubah menjadi putih, botak, syaraf tegang, hitam pada sekeliling mata.

Jangan mengira jeruk, lemon yang rasanya asam dapat membantu asam dalam lambung. Riset menunjukkan, semua buah yang masuk kedalam lambung, setelahnya akan berubah menjadi basa.

Jangan makan buah yang telah dipanaskan, buah yang telah dipanaskan akan hilang semua vitaminnya. Sama dengan tidak memakannya, karena semua vitamin akan hilang melalui pemanasan.

Serat pada buah sangat berfaedah, sebab itu makan buah utuh efeknya jauh lebih bagus daripada hanya minum sari dari buah saja. Saat minum sari buah, minumlah seteguk demi seteguk, sehingga ada waktu yang cukup untuk bercampur dengan air liur.

Tiga hari diet hanya bersantap buah, dapat menjadi cara yang efektif untuk membersihkan lambung dan membuang racun tubuh. Hanya makan buah dan minum air buah perasan, maka hanya butuh tiga hari akan mendapatkan tubuh yang lebih segar.

Memasuki periode “bersantap buah”, Anda akan merasa enak setiap kali makan buah yang berbeda. Sekali-kali juga boleh makan salad buah, dengan demikian selain inovatif juga mendapatkan hasil yang bagus. (berbagai sumber/ri)

Pengaruh Makanan pada Perilaku

Pengaruh Makanan pada Perilaku

By Republika Contributor
Sabtu, 12 Juli 2008 pukul 10:03:00

 

 

Setiap orang beriman diperintahkan Allah SWT untuk senantiasa mengonsumsimakanan yang halal dan baik (mengandung gizi dan vitamin yang cukup). Dua hal tadi–makanan halal danbaik– di samping akan menyebabkan terjaganya kesehatan jasmani, juga akan semakin mendorong meningkatkan kualitas takwa dan syukur kepada Allah SWT.

Hal ini sebagaimana dinyatakan di dalam Alquran surat Al-Baqarah ayat 172,”Hai orang-orang yang beriman, makanlah di antara rezeki yang baik-baik yang Kami berikan kepadamu dan bersyukurlah kepada Allah, jika benar-benar hanya kepada-Nya kamu menyembah.”

Sebaliknya, makanan yang haram, baik substansi maupun cara mendapatkannya,meskipun secara
lahiriyah mengandung gizi dan vitamin yang cukup, akan menumbuhkan perilaku yang buruk dan
merusak, baik bagi dirinya maupun bagi keluarganya di dunia ini maupun di akhirat nanti.

Sabda Rasulullah SAW, ”Setiap daging yang tumbuh dari barang yang haram, maka neraka lebih utama baginya.”

Oleh karena itu yang memerlukan perhatian, pikiran, hati, dan keimanan adalah menentukan
cara mendapatkan makanan atau rezeki yang bersih dan halal itu. Misalnya tidak melalui penipuan, korupsi, membungakan uang, menerima suap, dan cara-cara batil lainnya. Apalagi mengambil harta negara yang seharusnya dijaga dan dimanfaatkan untuk kepentingan dan kesejahteraan masyarakat banyak. Wallahu a’lam.

DPD Surati Menkes Soal Obat Mengandung Babi

DPD Surati Menkes Soal Obat Mengandung Babi
JUMAT, 24 OKTOBER 2008 | 00:36 WIB

MEDAN, KAMIS – Anggota Dewan Perwakilan Daerah (DPD) RI asal Sumatera Utara, Parlindungan Purba, mengatakan, pihaknya akan menyurati Menteri Kesehatan (Menkes) mengenai masalah kandungan unsur lemak babi dalam kapsul obat kapsul yang beredar di Indonesia. 
     
“Kita akan menyurati Menkes agar dalam satu atau dua hari ini agar mengeluarkan pernyataan resmi kalau kapsul obat yang beredar di Indonesia bebas dari kandungan babi,” katanya usai menggelar pertemuan tentang safety food bersama Kepala Balai Besar Pengawasan Obat dan Makanan (BBPOM) Medan dan Dinas Kesehatan Sumut di Medan, Kamis.

Selain itu, ia juga mengimbau kepada seluruh daerah kabupaten/kota untuk wajib melakukan food safety di daerahnya masing-masing, termasuk melakukan pengawasan, obat dan makanan. 

“Ini sesuai dengan prinsip otonomi daerah. Dimana kabupaten/kota sudah seharusnya membuat  laboratorium sendiri untuk melakukan pengawasan obat dan makanan di daerah masing-masing,” katanya.

Kepala BBPOM Medan, Supriyanto Utomo, mengatakan, sampai sejauh ini tidak ditemukan kapsul produk dalam negeri yang mengandung lemak babi. Sementara untuk produk kapsul dari luar negeri, baru bisa masuk ke Indonesia jika ada sertifikasi halal majelis ulama dari negara asal. Bagi negara yang tidak memiliki majelis ulamanya, maka boleh impor ke Indonesia setelah memberikan hasil analisa bahan-bahan  kapsul obatnya melalui lembaga resmi. 

Begitupun, kata dia, pihaknya saat ini sedang melakukan sampling kapsul secara acak. Jika nanti ditemukan satu saja obat kapsul yang terkandung unsur lemak babi, barulah BPOM akan melakukan sampling secara total.
ABD 
Sumber : Antara

Dinkes-MUI Medan Bahas Kapsul Mengandung Lemak Babi

 

Dinkes-MUI Medan Bahas Kapsul Mengandung Lemak Babi

By Republika Contributor
Kamis, 23 Oktober 2008 pukul 21:08:00

MEDAN–Dinas kesehatan Sumut dan MUI Medan diminta duduk bersama dalam satu meja untuk mengkaji hasil penelitian Lembaga Pengkajian dan Penelitian Obat dan Makanan (LPPOM) MUI Medan terkait adanya kandungan lemak babi dalam bahan kapsul. 

“Dinkes dan MUI harus secepatnya mengambil keputusan, apakah benar kapsul yang banyak beredar di pasaran saat ini mengandung lemak babi atau tidak,” kata pengamat sosial Universitas Sumatera Utara (USU) Yos Rizal MSi, di Medan, Kamis. 

Ia mengatakan, jika persoalan ini dibiarkan berlarut-larut, dikhawatirkan masyarakat akan menjadi korban dan semakin bingung, sebab masyarakat muslim akan sangsi untuk mengkonsumsi obat karena ragu atas kehalalannya. 

Sebelumnya, Ketua Majelis Ulama Indonesia (MUI) Medan, Muhammad Hatta, mengatakan, dewasa ini sekitar 80 persen obat-obatan kapsul dari luar negeri yang banyak beredar di masyarakat dicampur dengan lemak babi, seperti misalnya obat insulin. 

“Hal itu sangat mengherankan kenapa pemerintah tidak bisa memberi pengawasan terhadap obat-obatan, termasuk juga makanan, minuman, dan kosmetik yang beredar,” katanya. 

Seharusnya, katanya, pemerintah dapat memberi jaminan keselamatan kepada masyarakat, karena sesuai kategori hukum, apapun yg merusak kesehatan itu dilarang. 

“Untuk itulah, diharapkan RUU tentang sertifikasi halal segera disetujui pemerintah,” katanya. 

Sementara Kasubdin Bina Farmasi dan Makanan Dinkes Sumut, Siskandri, mengatakan hasil penelitian itu sendiri masih belum jelas dari mana asalnya. 

“Kapsul yang beredar di Indonesia itu bebas dari kandungan lemak babi, kecuali obat-obatan impor. Kami telah mengundang MUI Medan untuk membicarakan hal ini (22/10) untuk meminta klarifikasi tentang hasil penelitian mereka, tapi mereka tidak datang,” katanya. 

Menurut dia, Badan POM sendiri sudah melakukan pengawasan secara ketat terhadap obat yang beredar, baik dari luar maupun dalam negeri., bahkan khusus obat impor dimintakan sertifikasi halal dari negara asalnya. 

Untuk kapsul asal dalam negeri hanya diproduksi dua pabrik, yakni, Kapsulindo dan Kapsuljel yang berkedudukan di Jakarta. Kedua pabrik tersebut, produksinya menggunakan bahan dari lemak sapi dan hal itu pun sudah diakui halal oleh MUI pusat. ant/pt

Ekstrak Meniran Bantu Penderita AIDS

Ekstrak Meniran Bantu Penderita AIDS
Kamis, 21 Agustus 2008 | 17:23 WIB

PARA penderita HIV/AIDS kini mendapat sebuah harapan baru dalam meningkatkan kesembuhan. Berdasarkan hasil temuan awal dari Bagian Patologi Klinik Fakultas Kedokteran Universitas Airlangga Surabaya, peluang penderita HIV/AIDS untuk sembuh semakin meningkat dengan mengombinasikan pengobatan antiretroviral dengan terapi adjuvant menggunakan ektrak meniran atau phylanthus.

Seperti diungkap DR.Drs.Suprapto Ma’at, Apt, MS, di Jakarta, Kamis (21/8), ektrak meniran berpotensi meningkatkan harapan kesembuhan para penderita HIV/AIDS karena terbukti dapat meningkatkan kadar salah satu jenis sel pertahanan tubuh Limfosit T – terutama sel T helper (sel Th).

“Ekstrak meniran untuk penderita HIV AIDS bersifat sebagai adjuvant, terutama untuk meningkatkan T-helpernya. Saya akan rencanakan untuk menelitinya lebih lanjut dan sangat yakin hasilnya akan baik,” ungkap  DR. Suprapto dalam diskusi Kolaborasi Jangka Panjang Penelitian dan Industri Farmasi yang digagas PT. Dexa-Medica .

Ektrak menir, jelas Suprapto, pada prinsipnya dapat digunakan sebagai terapi adjuvant pada pengobatan infeksi yang membandel seperti infeksi virus, infeksi jamur, infeksi bakteri, intraseluler dan penyakit infeksi kronis lainnya.

“Adjuvant artinya membantu dalam menanggulangi suatu infeksi. Selain diberikan obat standar,  ditambah dengan stimulan.  Dengan terapi adjuvant, proses penyembuhan penyakit bisa lebih cepat dan yang lebih penting adalah menghilangkan proses kekambuhan,” papar peneliti yang baru mendapat penghargaan BJ Habibie Technology Award 2008 atas riset aplikatifnya tentang tanaman Meniran untuk Stimuno itu.

Kasus unik
Keyakinan DR.Suprapto akan prospek cerah esktrak meniran bagi pengobatan AIDS makin bulat setelah ia menemukan kasus peningkatan sel Th secara signifikan pada seorang pasien di RSUD Dr. Soetomo, Surabaya belum lama ini.

“Beberapa bulan lalu, ada seorang pasien asal Denpasar yang juga anak seorang dokter kandungan. Sakit yang dialami pasien ini awalnya belum diketahui penyebabnya, namun tiga bulan terakhir suhu tubuhnya tak pernah di bawah 39 derajat celcius,” ungkapnya.

Pasien ini, lanjut DR Suprapto, sempat dicurigai menderita enfeksi malaria dan TBC, tetapi upaya pengobatan tak kunjung membuahan hasil. Tim dokter yang terdiri dari beberapa ahli akhirnya menyimpulkan bahwa pasien ini mengalami masalah kekebalan tubuh, sehingga harus diperiksa kadar limfositnya – terutama sel Th (T-helper atau CD4+).

Sel Th ini berfungsi mengaktifkan dan mengatur sel-sel lainnya pada sistem kekebalan (misalnya limfosit B, makrofag dan limfosit T sitotoksik) yang semuanya membantu menghancurkan sel-sel ganas dan organisme asing.

Hasil pemeriksaan T-helper ternyata menunjukkan bahwa kadarnya sangat rendah yakni 52, yang bisa dikategorikan pasien sudah mengidap AIDS stadium lanjut. Dokter lalu memberikan ekstrak manira dengan penambahan dosis secara bertahap setiap bulan dan ternyata jumlah sel Th terus meningkat sebelum akhirnya kembali normal memasuki bulan ketiga.

“Dengan kasus ini, ada rencana untuk melakukan penelitian penggunaan ekstrak meniran di antara pasien HIV/AIDS, terutama AIDS,” ujarnya

DR Suprapto juga telah meminta kepada RSUD Dr Soetomo untuk membantu pasien HIV/AIDS tidak mampu dengan memberikan ekstrak filantus sebagai terapi adjuvant bersama obat atretroviral.
“Saya yakin ekstrak menir nanti akan dapat membantu, bukan mengobati, penyembuhan HIV/AIDS. Atau paling tidak memperbaiki kualitas hidup dan memperpanjang umur penderita,” tegasnya.

Obat Tradisional Pengusir Demam Anak

Obat Tradisional Pengusir Demam Anak
Kamis, 21 Agustus 2008 | 09:30 WIB

Sebagai langkah pertolongan pertama, obat tradisional dapat diandalkan untuk mengatasi demam.

BANYAK orangtua panik bila mendapati suhu tubuh anaknya di atas rata-rata atau sering disebut demam. Sebagai pertolongan pertama, umumnya diberikan obat penurun panas yang berbahan dasar kimia seperti golongan parasetamol, asam salisilat, ibuprofen, dan lain-lain. Jarang sekali orangtua yang langsung teringat  memberikan obat-obatan tradisional.

Padahal, obat-obatan tradisional yang berasal dari tanaman obat ini tak kalah ampuhnya sebagai pengusir demam. Malah, obat-obatan tradisional memiliki kelebihan, yaitu toksisitasnya relatif lebih rendah dibanding obat-obatan kimia. Jadi, relatif lebih aman, bahkan tidak ada efek samping bila penggunaannya benar. Soalnya, kandungan tanaman obat bersifat kompleks dan organis sehingga dapat disetarakan dengan makanan, suatu bahan yang dikonsumsi dengan maksud merekonstruksi organ atau sistem yang rusak. Selain itu, harganya pun lebih murah.

Tiga Jenis Demam

Namun, sebelum mengenal lebih jauh tentang tanaman obat penurun panas, perlu dipahami lebih dulu pengertian demam. Demam pada anak dapat dibedakan menjadi tiga, yaitu:

1. Demam karena infeksi yang suhunya bisa mencapai lebih dari 38°C. Penyebabnya beragam, yakni infeksi virus (seperti flu, cacar, campak, SARS, flu burung, demam berdarah, dan lain-lain) dan bakteri (tifus, radang tenggorokan, dan lain-lain).

2. Demam noninfeksi, seperti kanker, tumor, atau adanya penyakit autoimun seseorang (rematik, lupus, dan lain-lain).

3. Demam fisiologis, seperti kekurangan cairan (dehidrasi), suhu udara yang terlalu panas, dan lain-lain.

Nah, dari ketiganya, hanya demam yang disebabkan oleh infeksi dan noninfeksi sajalah yang memerlukan obat penurun panas. Untuk mempercepat proses penurunan panasnya, selain ramuan tradisional yang diminum, dapat juga diberikan baluran atau kompres untuk membantu.

Akan halnya demam fisiologis, tak diperlukan obat-obatan penurun panas karena umumnya jarang melebihi 380°C. Untuk menurunkan suhu tubuh, cukup diberikan minum yang banyak dan diusahakan berada dalam ruangan berventilasi baik atau berpendingin.

Aneka Obat Tradisional Penurun Panas

Inilah beberapa pilihan obat penurun panas tradisional yang dapat dicoba. Penting diperhatikan, dosis yang tercantum pada ramuan berikut adalah dosis untuk orang dewasa. Bila ingin diberikan kepada anak, bacalah aturan dosis bagi anak dan sesuaikan dengan tingkatan usianya. (Lihat boks: Dosis Aman untuk Anak.)

1. Lempuyang Emprit (Zingiber amaricans)

Memiliki kandungan senyawa minyak atsiri, yaitu sekuiterpenketon yang bermanfaat untuk menurunkan panas. Umumnya yang digunakan adalah rimpangnya; warnanya putih kekuningan dan rasanya pahit.

Caranya: Cuci bersih 10 gram umbi lempuyang emprit. Parut dan tambahkan 1/2 gelas air panas, aduk rata. Setelah dingin, peras, ambil sarinya. Campur dengan 2 sendok makan (sdm) madu bunga kapuk, aduk rata. Berikan 3 kali sehari.

2. Kunyit (Curcuma longa)

Memiliki kandungan minyak atsiri, curcumin, turmeron dan zingiberen yang dapat bermanfaat sebagai antibakteri, antioksidan, dan antiinflamasi (anti-peradangan). Selain sebagai penurun panas, campuran ini juga dapat meningkatkan daya tahan tubuh. Umumnya yang digunakan adalah rimpangnya; warnanya oranye.

Caranya: Cuci bersih 10 gram umbi kunyit. Parut dan tambahkan 1/2 gelas air panas, aduk rata. Setelah dingin, peras, ambil sarinya. Tambahkan dengan perasan 1/2 buah jeruk nipis. Campur dengan 2 sdm madu bunga kapuk, aduk rata. Bagi menjadi 3 bagian campuran madu dan kunyit ini, kemudian berikan 3 kali sehari.

3. Sambiloto (Andrographis paniculata)

Seluruh bagian tanamannya dapat digunakan. Memiliki kandungan andrografolid lactones (zat pahit), diterpene, glucosides dan flavonoid yang dapat menurunkan panas. Bahkan pada tahun 1991 pernah diadakan penelitian di Thailand bahwa 6 g sambiloto per hari sama efektifnya dengan parasetamol.

Caranya: Rebus 10 gram daun sambiloto kering, 25 g umbi kunyit kering (2,5 ibu jari), dan 200 cc air. Rebus hingga mendidih dan airnya tinggal 100 cc, kemudian saring. Setelah hangat, tambahkan 100 cc madu bunga kapuk atau mahoni, aduk rata. Bagi menjadi 3 bagian, berikan 3 kali sehari.

4. Pegagan (Centella asiatica L.)

Tumbuhan yang dikenal pula dengan nama daun kaki kuda ini tumbuh merayap menutupi tanah. Daunnya berwarna hijau dan berbentuk seperti kipas ginjal. Memiliki kandungan triterpenoid, saponin, hydrocotyline, dan vellarine. Bermanfaat untuk menurunkan panas, revitalisasi tubuh dan pembuluh darah serta mampu memperkuat struktur jaringan tubuh. Pegagan juga bersifat menyejukkan atau mendinginkan, menambah tenaga dan menimbulkan selera makan.

Caranya : Rebus 1 genggam pegagan segar dengan 2 gelas air hingga mendidih dan airnya tinggal 1 gelas. Bagi menjadi 3 bagian dan diminum 3 kali sehari.

5. Temulawak (Curcuma xanthorhiza Roxb.)

Penampilan temulawak menyerupai temu putih, hanya warna bunga dan rimpangnya berbeda. Bunga temulawak berwarna putih kuning atau kuning muda, sedangkan temu putih berwarna putih dengan tepi merah. Rimpang temulawak berwarna jingga kecokelatan, sedangkan rimpang bagian dalam temu putih berwarna kuning muda.

Temulawak memiliki zat aktif germacrene, xanthorrhizol, alpha betha curcumena, dan lain-lain. Manfaatnya sebagai antiinflamasi (antiperandangan), antibiotik, serta meningkatkan produksi dan sekresi empedu. Temulawak sejak dahulu banyak digunakan sebagai obat penurun panas, merangsang nafsu makan, mengobati sakit kuning, diare, mag, perut kembung dan pegal-pegal.

Caranya : Cuci bersih 10 gram rimpang temulawak. Parut dan tambahkan 1/2 gelas air panas, aduk rata. Setelah dingin, peras, ambil sarinya. Campur dengan 2 sdm madu bunga kapuk, aduk rata. Bagi menjadi 3 campuran madu dan temulawak, kemudian berikan 3 kali sehari.

6. Bawang merah (Allium cepa L.)

Bawang merah sering digunakan sebagai bumbu dapur. Memiliki kandungan minyak atsiri, sikloaliin, metilaliin, kaemferol, kuersetin, dan floroglusin.

Caranya: Kupas 5 butir bawang merah. Parut kasar dan tambahkan dengan minyak kelapa secukupnya, lalu balurkan ke ubun-ubun dan seluruh tubuh.

7. Daun kembang sepatu (Hibiscus rosa sinensis)

Selain daun kembang sepatu, Anda juga dapat memanfaatkan daun kapuk atau daun sirih. Kembang sepatu mengandung flavonoida, saponin dan polifenol. Daun kapuk mengandung flavonoida, saponin dan tanin. Daun sirih mengandung flavonoida, saponin, polifenol, dan minyak atsiri.

Caranya: Cuci bersih daunnya, keringkan dengan lap bersih, panaskan sebentar di atas api agar lemas. Remas-remas sehingga lemas, olesi dengan minyak kelapa, kompreskan pada perut dan kepala.

8. Meniran (Phyllanthus niruri L.)

Tinggi tanamannya mencapai 1 meter, tumbuh liar, daunnya berbentuk bulat tergolong daun majemuk bersirip genap. Seluruh bagian tanaman ini dapat digunakan. Memiliki kandungan lignan, flavonoid, alkaloid, triterpenoid, tanin, vitamin C, dan lain-lain. Bermanfaat untuk menurunkan panas dan meningkatkan daya tahan tubuh.

Caranya: Rebus 1 genggam meniran segar dengan 2 gelas air hingga mendidih dan airnya tinggal 1 gelas. Bagi menjadi 3 bagian dan diminum 3 kali sehari.

9. Air kelapa muda

Air kelapa muda banyak mengandung mineral, antara lain kalium. Pada saat panas, tubuh akan mengeluarkan banyak keringat untuk menurunkan suhu tubuh. Nah, untuk menggantikan keringat yang keluar, perbanyaklah minum air kelapa.

Dosis Aman untuk Anak

Penggunaan tanaman obat dengan dosis yang tepat tidak akan menimbulkan efek samping dan aman. Berikut dosis yang direkomendasikan untuk anak:

Usia Dosis

Bayi                       1/8 dosis dewasa

2­-5 tahun              1/4 dosis dewasa

6­-9 tahun              1/3 dosis dewasa

10-13 tahun           1/2 dosis dewasa

14-16 tahun          3/4 dosis dewasa

Penulis: Utami Sri Rahayu

Konsultan Ahli: dr Adji Suranto, SpA dari Perhimpunan Dokter Indonesia Pengembang Kesehatan Tradisional Timur (PDPKT DKI Jaya)

45 Mitos dan Fakta Seputar ASI

45 Mitos dan Fakta Seputar ASI
Sabtu, 9 Agustus 2008 | 17:51 WIB

JAKARTA, SABTU – Kekhawatiran bahwa menyusui akan menyebabkan payudara kendur, hanyalah sebuah mitos yang tak terbukti kebenarannya. Presenter Sophie Navita, yang menyusui kedua anaknya mengatakan, mitos tersebut tidak benar. Payudara kendur, kata Sophie, karena kehamilan yang menyebabkan perubahan hormon.

“Payudara memble, itu karena hamil dan perubahan hormon bukan karena menyusui. Kalau nggak mau memble, ya jangan hamil,” ujar Sophie dalam Obrolan Santai “Kembali ke ASI, Sebuah Pilihan Bijak”, di Senayan City, Jakarta Selatan, Sabtu (9/8) sore.

Dalam sebuah buku panduan yang diperbanyak oleh World Vision Indonesia, disebutkan 45 mitos dan fakta tentang ASI. Ingin tahu? Inilah mitos (M) dan fakta (F) tersebut

*M : Menyusui menyebabkan payudara kendur. F : Payudara kendur disebabkan oleh bertambahnya usia dan kehamilan.
*M : Payudara yang berukuran kecil, tidak dapat menghasilkan banyak susu. F : Payudara kecil maupun besar sama-sama dapat menghasilkan banyak susu.
*M : Payudara dengan puting terbenam tidak dapat menyusui. F : Puting terbenam tidak berarti tidak dapat menyusui, karena bayi menyusu pada payudara, bukan pada puting.
*M : ASI pertama (yang berwarna kekuningan) tidak baik bagi bayi. F : ASI pertama (kolostrum) adalah zat terbaik bagi bayi.
*M : Kolostrum / ASI pertama adalah susu basi. F : Kolostrum mengandung zat kekebalan tubuh dan protein yang sangat kaya.
*M : ASI eksklusif berarti tidak boleh memberikan makanan, yang lain boleh. F : ASI ekslusif berarti hanya memberikan ASI saja, yang lain tidak boleh.
*M : ASI eksklusif berarti tidak boleh memberikan susu formula, lainnya boleh. F : ASI eksklusif berarti hanya boleh memberikan ASI saja, yang lain tidak boleh.
*M : ASI eksklusif tidak dapat dilakukan jika ibu bekerja. F : Ibu bekerja tetap dapat memberikan ASI eksklusif.
*M : Hingga usia 6 bulan, ASI saja tidak cukup bagi bayi. F : Semua kebutuhan bayi sampai usia 6 bulan terpenuhi oleh ASI saja.
*M : Pisang dapat menyembuhkan diare pada bayi. F : Makanan padat tidak dapat diolah oleh usus bayi hingga usia 6 bulan.
*M : Pisang dapat membersihkan usus bayi. F : Pisang tidak dapat membersihkan usus bayi melainkan merusak, karena usus bayi belum sanggup mengolah makanan hingga usia 6 bulan.
*M : Susu formula sama baiknya dengan ASI. F : Tidak ada cairan lain apapun yang dapat menggantikan ASI.
*M : Susu formula membuat bayi lebih sehat. F : Hanya jika diberikan ASI eksklusif sampai 6 bulan yang membuat bayi lebih sehat.
*M : Untuk perkembangan otak, susu formula lebih baik daripada ASI. F : ASI mengandung AA/DHA yang sangat penting bagi pertumbuhan otak.
*M : Kombinasi ASI dan formula adalah yang terbaik bagi bayi. F : Yang terbaik bagi bayi hingga usia 6 bulan adalah hanya menerima ASI saja.
*M : Jika ASI belum atau tidak lancar dapat digantikan dengan susu formula. F : Jika ASI belum atau tidak lancar, bayi masih memiliki daya tahan tubuh (tidak akan kelaparan) hingga 2×24 jam sejak lahir, yang dibawa sejak dalam kandungan.
*M : Jika ASI belum keluar, tidak ada gunanya menyusui bayi. F : Jika ASI belum atau tidak lancar, bayi masih memiliki daya tahan tubuh (tidak akan kelaparan) hingga 2×24 jam sejak lahir, yang dibawa sejak dalam kandungan.
*M : Sementara ASI belum keluar, bayi dapat diberikan susu formula atau madu. F : Pemberian makanan lain selain ASI meningkatkan risiko terganggunya usus bayi yang masih belum siap.
*M : Agar bayi tidak kuning dan tidak demam, dapat diberi makanan atau minuman lain sebelum ASI keluar. F : Bayi yang kuning harus banyak menerima sinar matahari pagi dan lebih sering diberi ASI.
*M : Jika bayi terus menangis berati ASI-nya kurang. F : Bayi menangis belum tentu lapar.
*M : Ibu yang banyak minum susu, akan menghasilkan banyak ASI. F : Banyaknya ASI yang dihasilkan tidak dipengaruhi oleh makanan atau minuman yang dikonsumsi ibu. Semakin sering bayi menyusu semakin banyak ASI yang dihasilkan.
*M : Agar menghasilkan banyak ASI, Ibu harus banyak makan sayuran. F : Semakin sering bayi menyusu, semakin banyak ASI yang dihasilkan.
*M : Jika ibu sakit, bayi akan tertular melalui ASI. F : Ketika sakita, tubuh ibu membuat zat kekebalan tubuh yang juga disalurkan kepada bayi melalui ASI sehingga bayi tidak akan sakit.
*M : Ibu yang kurang vitamin tidak dapat menyusui bayinya. F : Ibu yang kurus sekalipun tetap dapat menghasilkan banyak ASI asalkan sering menyusui.
*M : Menyusui tidak boleh dilakukan sambil berbaring. F : Menyusui dapat dilakukan sambil berdiri, duduk ataupun berbaring.
*M : Bayi yang sedang sakit tidak boleh disusui. F : Bayi yang sedang sakit harus lebih sering diberi ASI.
*M : Pemberian air kepada bayi baru lahir hingga usia 6 bulan tidak akan merugikan. F : Pemberian air kepada bayi baru lahir hingga usia 6 bulan hanya akan memenuhi perut bayi sehingga mengurangi ruang untuk ASI yang sangat dibutuhkan bayi.
*M : Bayi baru lahir perlu diberikan air teh agar memiliki tenaga. F : Pemberian air teh kepada bayi baru lahir hingga usia 6 bulan hanya akan memenuhi perut bayi sehingga mengurangi ruang untuk ASI yang sangat dibutuhkan bayi.
*M : Setelah melahirkan, ibu terlalu lelah untuk dapat menyusui bayi. F : Kecuali dalam situasi darurat, ibu yang baru melahirkan mampu menyusui bayinya segera, memeluk dan menyusui bayi adalah penghilang sakit dan rasa lelah ibu.
*M : Bayi baru lahir tidak dapat menyusu sendiri. F : Bayi memiliki naluri kuat untuk mencari puting dalam satu jam pertama setelah lahir.
*M : ASI belum keluar pada hari pertama setelah melahirkan. F : Meskipun tidak terasa, kolostrum (ASI pertama), akan keluar langsung setelah kelahiran. Jumlahnya sedikit, tapi cukup untuk kebutuhan bayi.
*M : Tidak ada gunanya menyusui bayi sejak kelahirannya. F : Kolostrum adalah cairan yang kaya dengan zat kekebalan tubuh dan zat penting lain yang harus dimiliki bayi. Bayi yang menyusui langsung akan merangsang ASI cepat keluar.
*M : Bayi harus dibungkus dan dihangatkan dibawah lampu selama dua jam setelah lahir. F : Bayi bukan anak ayam. Kehangatan terbaik bagi bayi diperoleh melalui kontak kulit bayi ke kulit ibu, karena kehangatan tubuh ibu dapat menyesuaikan dengan kebutuhan bayi. Kontak kulit bayi ke kulit ibu membuat ASI semakin cepat keluar.
*M : ASI pertama/kolostrum sangat sedikit, sehingga bayi lapar dan menangis. F : ASI pertama memang sedikit, tapi cukup untuk memenuhi perut bayi yang hanya dapat diisi sebanyak 4 sendok teh.
*M : Bayi menangis, pasti karena lapar. F : Bayi menangis bisa diakibatkan karena merasa tidak nyaman, merasa tidak aman, merasa sakit, dan sebagainya, belum tentu lapar.
*M : Bayi menangis karena lapar perlu diberi makanan atau minuman lain. F : Jika bayi lapar, beri ASI lagi. Sering-sering diberi ASI tidak akan membuat bayi lapar.
*M : ASI yang penting hanyalah cairan yang berwarna putih. F : Kolostrum/ASI pertama (kekuningan/tidak berwarna) adalah ASI yang paling penting untuk memberikan kekebalan kepada bayi. ASI yang berwarna putih adalah yang paling penting untuk kebutuhan bayi sampai 6 bulan pertama.
*M : Bayi kedinginan sehingga perlu dibedong. F : Bayi baru lahir memang mudah kedinginan, sehingga perlu dipeluk kontak kulit ke kulit, diberi topi, lalu ibu bersama bayi diselimuti. Bedong bayi terlalu ketat akan membuatnya lebih kedinginan.
*M : Kurang tersedia tenaga kesehatan sehingga bayi tidak dapat dibiarkan menyusu sendiri. F : Suami atau anggota keluarga ibu dapat membantu Inisiasi Menyusu Dini.
*M : Kamar bersalin atau kamar operasi sibuk sehingga bayi perlu segera dipisah dari ibunya. F : Sementara sibuk, ibu bisa melakukan Inisiasi Menyusu Dini (IMD).
*M : Ibu harus dijahit sehingga bayi perlu segera dipisah dari ibunya. F : Sementara dijahit, ibu tetap dapat melaksanakan IMD.
*M : Bayi perlu diberikan suntikan vitamin K dan tetes mata segera setelah lahir. F : Benar, tapi dapat ditunda selama 1 jam hingga bayi selesai menyusu awal.
*M : Bayi harus segera dibersihkan setelah lahir. F : Ditunda 1 jam tidak akan mengubah berat dan tinggi bayi.
*M : Tenaga kesehatan belum sependapat tentang pentingnya memberi kesempatan IMD pada bayi yang lahir dengan operasi caesar. F : Mungkin, tapi adalah tugas orangtua untuk membela hak sang bayi. Tenaga kesehatan dapat diberi penjelasan, dan suami atau anggota keluarga dapat membujuk agar bayi dibiarkan untuk IMD.
*M : Ibu belum bisa duduk/duduk miring untuk memberikan ASI. F : Siapa yang mengharuskan duduk? Bayi dapat menyusu pada saat tengkuran di dada ibu.(ING)

ASI Bikin Anak Cerdas, Kok Bisa?

ASI Bikin Anak Cerdas, Kok Bisa?
Senin, 12 Mei 2008 | 17:04 WIB

INGIN memiliki anak Anda yang cerdas? Berikanlah Air Susu Ibu (ASI) sejak lahir kepada bayi Anda karena dalam ASI terkandung sejenis nutrisi penting yang berfungsi untuk belajar dan mengingat lebih baik yang disebut Gangliosida.

Gangliosida merupakan salah satu komponen dari membran sel manusia, terutama membran sel saraf dan otak. Untuk mendapat asupan nutrisi gangliosida optimal, bayi memang sebaiknya mendapat gangliosida dari ASI. Dalam ASI, ada dua jenis gangliosida yaitu, GD3 (disialogangliosides 3) dan GM3 (monosialogangliosides 3).

Gangliosida banyak terdapat pada air susu ibu pada enam minggu pertama masa menyusui. Pada awal menyusui, ASI yang memancar didominasi GD3. Begitu proses menyusui hampir usai, GM3 mendominasi. “Kadar GD3 pada ASI adalah 2-8 mcg/ml. Sedangkan GM3 mencapai 2-14 mcg/ml.

“Karena itu, ibu yang baru melahirkan dianjurkan memberikan ASI kepada bayinya,” ungkap Ines Gulardi MSC dari PT Fonterra Brands Indonesia pada acara media edukasi bertema Peranan Nutrisi Gangliosida (GA) guna mengoptimalkan hubungan antar-sel otak pada bayi dan Balita di Denpasar, Senin.

Didampingi dr I Gusti Ayu Trisna Windiani SPA dari bagian Tumbuh Kembang Anak RSUP Sanglah Denpasar, ia mengatakan, ASI membantu pembentukan hubungan antar-sel otak, membantu kemampuan belajar dan menyimpan memori.

Ia menambahkan, selain GA, tumbuh kembangnya bayi juga dipengaruhi oleh stimulan yang diterima. Oleh sebab itu, orangtua diharapkan dapat menstimulasi anak dengan kasih sayang, berinteraksi dengan lingkungan sekitarnya. Gangliosida yang terdapat dalam ASI itu juga terkandung dalam susu sapi dan sumber makanan lainnya yang bisa dikonsumsi manusia secara aman.

Namun begitu, ASI merupakan makanan yang terbaik bagi bayi. Selain alami, kualitas dan kandungan nutrisinya  tidak tertandingi oleh susu formula terutama bagi pertumbuhan bayi secara maksimal.  Untuk itu, jika seorang ibu tidak dapat menyusui bayinya disarankan untuk berkonsultasi dengan dokter atau ahli kesehatan, ujar Ines Gulardi.

Peminum ASI Kolesterolnya Lebih Baik

Peminum ASI Kolesterolnya Lebih Baik
Selasa, 12 Agustus 2008 | 16:03 WIB

MEMBERIKAN air susu ibu (ASI) sebagai makanan utama adalah keputusan terbaik untuk bayi di awal masa pertumbuhannya. Selain membentuk sistem kekebalan tubuh yang kuat, ASI akan membuat anak tumbuh lebih cerdas dan sehat. Tepatnya, ASI akan menormalkan kolesterol.

Di antara sekian banyak manfaatnya bagi bayi, sebuah penelitian terbaru di AS mengindikasikan bahwa ASI dapat mempengaruhi pembentukan sistem metabolisme lemak dalam darah. Seperti dipublikasikan The American Journal of Clinical Nutrition, pemberian ASI semasa bayi akan membuat seseorang tumbuh lebih sehat saat karena kadar kolesterolnya cenderung lebih baik saat mencapai usia dewasa.

Kesimpulan ini merupakan hasil analsis dan kajian data 17 riset melibatkan 17 ribu partisipan yang memperoleh ASI maupun susu formula. Kajian riset ini, kata peneliti, pada intinya memberi rekomendasi bahwa paparan terhadap ASI secara dini berkaitan dengan rendahnya kadar kolesterol saat menginjak usia dewasa.

“Oleh karena ada bukti substansial bahwa ASI menyediakan manfaat dan proteksi kesehatan  jangka panjang, pemberian ASI sebaiknya diutamakan apabila memungkinkan,” ungkap para peneliti seperti dikutip WebMD.

Dari hasil analisis terungkap,  para partisipan dari tujuh penelitian yang mendapat ASI secara ekslusif tercatat memiliki kadar kolesterol yang sedikit lebih rendah pada masa dewasa dibandingkan mereka yang hanya mendapatkan susu formula.

Dalam tinjauan penelitian sebelumnya, para ahli juga menemukan bahwa kadar kolesterol darah  tercatat lebih tinggi pada bayi-bayi yang diberikan ASI ketimbang bayi yang mendaparkan susu formula. Ketika menginjak usia kanak-kanak,  kadar kolesterol pada kedua kelompok ini tidak menunjukkan perbedaan.

“Penemuan ini membawa kami pada sebuah hipotesa bahwa paparan sejak dini terhadap ASI yang mengandung kolesterol tinggi mungkin dapat mempengaruhi metabolisme kolesterol jangka panjang ,” kata peneliti.

Kajian riset ini didasarkan atas data-data dari 17 studi observasi yang mencatat apakah partisipan mendapatkan ASI atau susu formula dan juga menjalani pengukuran kadar kolesterol saat menginjak usia dewasa. Data diambil dari sekitar  17.000 partisipan — 12.890 di antaranya mendapat ASI dan  4.608 lainnya meminum susus formula. Penelitian juga memperhitungkan faktor lain seperti status sosial ekonomi,  indeks masa tubuh, usia dan kebiasaan merokok.

Alasan mengapa pemberian ASI menyebabkan rendahnya kadar kolesterol saat dewasa, kata para ahli, adalah tingginya kandungan kolesterol dalam ASI  kemungkinan dapat memicu perubahan fisiologis yang mempengaruhi sintesis kolesterol. Namun begitu, peneliti juga menekankan bahwa  faktor lain seperti perbedaan gaya hidup antara orang dewasa yang semasa kecilnya mendapat ASI atau susu formula, mungkin juga berperan.

Mari Dukung Ibu Memberi ASI!

Mari Dukung Ibu Memberi ASI!

Selasa, 5 Agustus 2008 | 19:18 WIB

KANDUNGAN air susu ibu (ASI) ternyata memberi manfaat yang sangat besar bagi bayi bila kita memberikan ASI kepada mereka. Sayangnya, masih banyak yang tidak menyadari bahwa menyusui adalah salah satu hak bayi di awal kehidupannya. Oleh karena itu, sosialisasi tentang pentingnya pemberian ASI eksklusif selama enam bulan perlu ditingkatkan.

“Meski berbagai kalangan meyakini menyusui merupakan naluri, namun kenyataannya menyusui adalah keterampilan yang harus dipelajari dan dipahami para ibu, juga praktisi kesehatan termasuk bidan, dokter spesialis anak, dokter kebidanan dan tenaga medis lain,” kata Ketua Ikatan Dokter Anak Indonesia Badriul Hegar, dalam seminar Bedah ASI, Selasa (5/8), di Jakarta.

Terkait hal itu, IDAI terus mengupayakan sosialisasi ASI kepada anggotanya dan masyarakat dengan berbagai strategi antara lain meningkatkan ilmu pengetahuan tentang ASI, meningkatkan keterampilan dengan memberi pelatihan manajemen laktasi bagi petugas kesehatan dan masyarakat di sejumlah rumah sakit pemerintah dan swasta. Strategi lain adalah menginformasikan tentang ASI melalui media cetak dan elektronik.

Komitmen IDAI adalah terus mengupayakan setiap bayi mendapat haknya di awal kehidupan agar mereka dapat tumbuh dan berkembang secara optimal. Setiap orang tua akan mempunyai perasaan lebih berarti bila mereka dapat memberi yang terbaik untuk anaknya, kata Badriul. Pihaknya berharap, upaya ini akan memberi dukungan para orang tua untuk mendapat emas seperti yang didengungkan pada Pekan ASI Sedunia tahun 2008.

Mendapatkan ASI merupakan hak asasi bayi yang dapat dipenuhi bila para ibu mendapat perlindungan dan bantuan menyeluruh tentang informasi manfaat pemberian ASI. Sejak awal kelahiran, bayi hanya diberi ASI dan disusui sesering mungkin tanpa dibatasi. Bayi bisa mengukur sendiri kemampuan dan kebutuhan cairan yang diperlukan. “Kita hanya perlu meluangkan waktu dan memberi kesempatan padanya untuk mendapat yang terbaik yang dia butuhkan,” ujarnya.

ASI mengandung nilai nutrisi yang secara kuantitas seimbang serta secara kualitas sangat unggul. Komposisi nutrien atau zat gizi yang terkandung di dalam ASI sangat tepat dan ideal untuk tumbuh kembang anak. Di samping itu, komposisi ASI juga menyebabkan bayi dan anak yang mengonsumsi terjaga kesehatannya.

ASI bukan merupakan tempat penularan dari sebagian besar infeksi pada ibu. Oleh karena itu, menyusui merupakan tindakan terbaik bagi ibu dan bayi. Pada sebagian besar kasus ibu menyusui dengan tersangka infeksi, menghentikan menyusui hanya akan mengurangi masukan nutrisi dan manfaat kekebalan dari ASI, kata Badriul menambahkan.

Kecerdasan anak ditentukan oleh interaksi sejumlah faktor. Salah satu faktor penting penentu kecerdasan adalah nutrisi, terutama pemberian ASI sejak lahir untuk waktu cukup lama atau eksklusif enam bulan. ASI juga dapat memenuhi semua kebutuhan dasar anak untuk tumbuh kembang. ASI sebagai makanan bayi paling ideal dan tidak dapat digantikan oleh susu formula sudah tidak dapat disangkal lagi, ujarnya.

Badriul menambahkan, keberhasilan pemberian ASI eksklusif enam bulan dapat diwujudkan dengan motivasi kuat, pengetahuan dasar tentang menyusui, usaha yang terus-menerus, dan dukungan fasilitas persalinan sayang bayi. Pengetahuan dan keterampilan petugas kesehatan harus pula selalu ditingkatkan agar dapat berperan aktif dalam mengatasi kendala yang mungkin timbul selama proses menyusui.

Anak Sapi Huuu…Anak ASI, Yes!

Anak Sapi Huuu…Anak ASI, Yes!
Puluhan anak-anak mengampanyekan Dukung Ibu Menyusui di Senayan City, Jakarta Selatan, Sabtu (9/8).

Sabtu, 9 Agustus 2008 | 15:06 WIB

JAKARTA, SABTU – Sekitar 30 anak berkeliaran di Senayan City, Sabtu (9/8) sore. Ada apa gerangan? Ternyata mereka tengah terlibat aktif mengampanyekan dukungan bagi ibu menyusui. Kegiatan ini merupakan puncak peringatan Pekan ASI se-dunia 1-8 Agustus 2008 yang diadakan World Vision Indonesia (WVI).

Puluhan anak itu berpakaian badut sapi dan memegang sebuah balon berwarna oranye bertuliskan “Dukung Ibu Menyusui”. Tak ketinggalan sebuah kertas bertuliskan “Ayo Dukung Ibu Menyusui” dan “Air Susu Sapi untuk Anak Sapi, Air Susu Ibu untuk Bayi”.

Dari Lantai 6 Senayan City, secara bergerombol mereka menyinggahi setiap lantai dan membagikan puluhan balon kepada pengunjung mal sembari berkata, “Datang ya ke lantai 6.” Di Lantai 6 Senayan City akan diadakan seminar tentang ASI dibawakan oleh pakar ASI dr Utami Lestari dan artis Sophie Navita.

Rombongan anak yang meneriakkan, “Anak Sapi? Huuu…. Anak ASI? Yess!,” sambil mengepalkan tangannya ke udara ini memancing perhatian para pengunjung mal.

Memberi Madu untuk Si Kecil?

Memberi Madu untuk Si Kecil?
Sabtu, 9 Agustus 2008 | 12:17 WIB

Madu memiliki banyak manfaat karena mengandung vitamin dan mineral yang dibutuhkan tubuh. Sebenarnya boleh-boleh saja memberikan madu untuk si kecil, tapi madu untuk bayi dan balita hanya yang refined dan purified with no additive (sudah dibersihkan dan dimurnikan).

Menurut penjelasan dr.Adi Tagor Sp.A dari RS Pondok Indah Jakarta, madu asli dikhawatirkan mengandung bibit penyakit. “Jadi, tak boleh madu asli yang masih ada lilin-lilin atau sarang tawonnya, karena ditakutkan ada bahaya mikrobiologinya, yaitu spora dari clostridium botulinum tadi.” Terlebih buat bayi, “selain harus madu yang steril, juga jangan madu palsu karena bisa menimbulkan berbagai reaksi.”

Madu yang asli, terang dr.Adi Tagor jika menyentuh protein mentah seperti telur, maka telurnya jadi matang, tapi matangnya denaturasi (pematangan secara kimia). “Jadi, kumannya tak mati.”

Itu sebab, Adi Tagor sama sekali tak membenarkan pemberian madu dengan telur mentah untuk bayi dan anak. Dikhawatirkan ada kuman salmonella yang masuk lewat pori-pori telur. “Telur setengah matang itu, kan, kumannya enggak mati. Padahal, kuman ini bisa menyebabkan penyakit tifus atau paratifus. Bayi dan balita paling rentan untuk terkena, apalagi bayi yang belum boleh mendapat imunisasi tifus.”

Jadi, sarannya, paling tidak telurnya harus tiga perempat matang. “Jika untuk orang dewasa telurnya dimasak 5 menit, maka untuk anak harus 9 menit. Namun lebih bagus lagi jika sampai matang karena lebih aman.”

80% Imunitas Balita Ada di Dalam Pencernaan

http://surabaya.detik.com/read/2008/08/01/143338/981377/466/80-imunitas-balita-ada-di-dalam-pencernaan

Jumat, 01/08/2008 14:33 WIB
80% Imunitas Balita Ada di Dalam Pencernaan
Imam Wahyudiyanta – detikSurabaya


detiksurabaya/Imam W

Surabaya – Tidak seperti orang dewasa, seorang balita sangat mudah atau rentan terhadap serangan penyakit. Hal tersebut dikarenakan sistem daya tahan tubuh balita yang memang belum berkembang sepenuhnya seperti sistem daya tahan tubuh orang dewasa.

Komponen pertahanan tubuh atau yang disebut dengan imunitas pada balita sendiri lebih banyak ditentukan oleh faktor dari dalam tubuhnya yakni, di dalam sistem pencernaan. Dan memang 80% komponen imunitas balita berada di dalam sistem pencernaan.

“Saluran cerna yang sehat adalah awal imunitas yang kuat pada balita,” ujar Group Brand Manager Nutrilon, Frinzy Zulkarnain, dalam jumpa pers ‘Protection From the Inside’ di Hotel Sheraton, Jumat (1/8/2008).

Frinzy menambahkan bahwa salah satu solusi untuk meningkatkan imunitas pada balita adalah dengan memberikan nutrisi yang tepat bagi kesehatan saluran cerna. Dan salah satu faktor kunci kesehatan saluran cerna adalah adanya dominasi bakteri baik dibandingkan dengan bakteri patogen.

Pertumbuhan bakteri baik, kata Frinzy, dapat ditingkatkan dengan asupan prebiotik yang merupakan makanan bagi bakteri baik. Prebiotik sendiri salah satunya bisa diperoleh dari susu yang dijual di pasaran.

Frinzy lebih lanjut mengatakan, setidaknya ada 6 manfaat prebiotik bila dikonsumsi oleh balita. Manfaat itu adalah meningkatkan pertumbuhan bakteri baik di dalam saluran cerna, mengurangi pertumbuhan bakteri patogen dalam saluran cerna, menguatkan dinding saluran cerna.

Selain itu prebiotik juga dapat meningkatkan imunitas mukosa saluran cerna, menurunkan insiden infeksi (ISPA dan diare) dan meningkatkan kelembutan BAB sehingga menghindarkan konstipasi.

Namun Frinzy mengingatkan bahwa selain imunitas, hal lain yang diperlukan oleh balita adalah nutrisi penumbuh otak. Namun 2 hal tersebut adalah 2 hal yang berbeda. Dalam arti nutrisi yang digunakan untuk mengembangkan hal itu tidak sama.

“Namun susu yang mempunyai komposisi imunitas pasti juga tidak lupa memberikan komposisi untuk pertumbuhan otak,” tandas Frinzy.(iwd/fat)

Deadline Vaksin Haram

http://hidayatullah.com/index.php?option=com_content&task=view&id=7187&Itemid=1

Deadline Vaksin Haram

Kamis, 10 Juli 2008
Masa berlaku status kedaruratan vaksin berbahan baku haram yang dikeluarkan LPPOM- MUI kepada PT Bio Farma berakhir tahun 2008 ini.  Demikian ungkap LPPOM

Hidayatullah.com–Masa berlaku status kedaruratan vaksin berbahan baku haram yang dikeluarkan LPPOM- MUI kepada PT Bio Farma berakhir tahun 2008 ini.

Namun hingga kini usaha yang dilakukan perusahaan farmasi nasional produsen tunggal vaksin di Indonesia tersebut belum menghasilkan apa-apa.

Demikian diungkap Direktur Eksekutif Lembaga Pengkajian Pangan Obat-obatan dan Kosemetika Majelis Ulama Indonesia (LPPOM MUI), Dr M. Nadratuzzaman Hosen kepada http://www.hidayatullah.com di sela-sela acara Pameran Halal Internasional di Jakarta, pekan lalu.

Menurut Nadratuzzaman, untuk menghasilkan vaksin halal sebenarnya tidak sulit. Yang menjadi masalah, lanjutnya, adalah lemahnya political will pemerintah terhadap masalah halal-haram ini. “Buktinya, Malaysia saja sudah bisa menghasilkan vaksin halal,” tandasnya.

Seperti dilansir Majalah Suara Hidayatullah edisi September 2007, seluruh vaksin yang beredar di dunia saat ini, termasuk vaksin meningitis yang diberikan kepada seluruh jemaah haji,  menggunakan bahan  haram dalam pembuatannya. Di antaranya adalah enzim babi, ginjal kera, ginjal babi, hingga janin bayi hasil aborsi.

Tripsin babi

Salah satu unsur haram yang terdapat dalam vaksin adalah tripsin, enzim yang didapat dari pankreas babi. Menurut penjelasan Ketua Dewan Penasihat LPPOM-MUI, Prof Jurnalis Uddin, tripsin babi sebenarnya bukanlah bahan baku vaksin. Dalam proses pembuatan vaksin, tripsin hanya dipakai sebagai enzim proteolitik (enzim yang digunakan sebagai katalisator pemisah sel/protein).

Pada hasil akhirnya (vaksin), enzim tripsin yang merupakan unsur turunan dari pankreas babi ini tidak terdeteksi lagi. Enzim ini akan mengalami proses pencucian, pemurnian dan penyaringan. ”Hingga jejaknya pun tidak terlihat lagi,” jelas Prof. Jurnalis. Namun karena sudah tersentuh unsur haram dan najis, status kehalalan vaksin jadi bermasalah.

Direktur Pemasaran PT Bio Farma, Sarimuddin Sulaeman mengatakan, Bio Farma sebenarnya telah mengusahakan pengganti tripsin babi sejak tahun 2006.. Penelitian ini memakan waktu tiga tahun. Namun untuk sementara tripsin tersebut masih tetap digunakan.  [surya/www.hidayatullah.com]

Karima Burns: Cintaku kepada Islam tertambat di Istana Al-Hambra

http://hidayatullah.com/index.php?option=com_content&task=view&id=6819&Itemid=1

Karima Burns: Cintaku kepada Islam tertambat di Istana Al-Hambra

Sabtu, 10 Mei 2008

Ketika membuka Al-Quran perasaan yang hadir persis seperti orang yang baru saja menemukan kembali anggota keluarganya yang telah lama hilang

Hidayatullah.com–Karima Kristie Burns, MH, ND nama lengkapnya. Karima (39) dikenal sebagai perempuan dengan banyak bakat. Ya sebagai editor, penulis, guru, dan juga pakar herbalis. Di dunia herbalis dia sangat dikenal lewat konsultasi online di website Herb’n Muslim yang dikelolanya sejak 1994. Sejak masuk Islam, dia membuka usaha Herb’n Muslim yang dikenal dengan teknik penyembuhan alami dan islami. Dia juga telah menulis lebih dari 120 artikel kesehatan yang bisa didownload via websitenya itu. Karima menghabiskan separuh hidupnya di Midwest, Iowa (AS), tempat dia dibesarkan. Dan separuhnya lagi di kawasan Timur Tengah (Mesir dan Arab Saudi).

Karima mulai tertarik dengan metode penyembuhan alami justru ketika berupaya menyembuhkan dirinya sendiri yang mengidap penyakit asma, alergi, mudah panik, depresi, dan beberapa penyakit bagian dalam lainnya. Kala itu dia mencoba dengan terapi alami dan bantuan tumbuh-tumbuhan. Dia berkeliling hingga ke Mesir guna mencari berbagai informasi berkenaan penyembuhan tradisional. Dari kegigihannya itu, dia bahkan berhasil memperoleh gelar formal master of herbalist dan doktor bidang naturopathic tahun 1996 dari Trinity College di Dublin, Irlandia. Naturopathic adalah teknik pengobatan alamiah yang meresepkan herbal untuk para pasiennya. Namun tak banyak yang tahu, ketertarikan Karima kepada Islam justru ketika berkunjung ke Spanyol. Dia mengaku terkagum-kagum dengan tulisan Arab di Istana Al-Hambra di kota Granada. Istana itu sendiri dulunya bekas mesjid hingga bekas kaligrafinya masih ada. Berikut penuturan Karima yang disadur dari beberapa sumber.

***

Kenal Islam di Spanyol

Karima Burns awalnya adalah seorang mahasiswi program sarjana studi kawasan Arab di Universitas Iowa, AS. Karima mengaku Islam hadir di hatinya berawal dari membaca rangkaian tulisan ayat suci Al-Quran dalam rangka penyelesaian tugas kuliahnya. Dan dia tak kuasa menghindar dari bisikan hati itu.

Ceritanya, satu ketika dia dan teman-temannya mengadakan studi tur ke Granada, Spanyol. Granada merupakan salah satu bekas kawasan yang pernah dikuasai Islam selama hampir tujuh abad. Kala itu dia sedang duduk-duduk di Istana Al-Hambra. Istana itu dulunya adalah mesjid. Karima takjub melihat jejeran tulisan di dinding gedung tua itu. Baginya itulah tulisan terindah yang pernah dia lihat.

“Bahasa apa itu?” tanyanya pada salah seorang turis Spanyol. ”Bahasa Arab,” sahut turis lokal itu. Hari berikutnya, tatkala pemandu wisata menanyakan buku panduan dalam bahasa apa yang dia inginkan, Karima menjawab spontan bahasa Arab.

“Apa, bahasa Arab? Anda bisa bahasa Arab?” tanya si pemandu terkejut.

“Tidak, tapi tolong berikan juga yang dalam bahasa Inggris,” sahut Karima.

Di akhir tour tas Karima penuh dengan buku-buku petunjuk wisata dari tiap-tiap kota yang dia singgahi di seluruh Spanyol. Dan semuanya dalam bahasa Arab!

“Tas travel saya sudah terlalu penuh hingga saya bermaksud membuang beberapa potong pakaian dan beberapa barang lainnya agar tasnya bisa muat. Namun, untuk buku-buku bahasa Arab rasanya berat untuk ditinggalkan. Buku-buku itu ibarat emas bagi saya. Saya sering membolak-balik halamannya tiap malam. Kata per kata-nya saya amati dengan seksama. Huruf-hurufnya juga unik, beda dengan huruf latin biasa. Saya membayangkan andainya saja bisa menulis dengan huruf yang demikian indah itu. Waktu itu saya punya pikiran pasti akan sangat berharga jika bisa mengetahui bahasa Arab ini. Saya pun berniat dalam hati untuk belajar bahasa ini. Ya satu saat nanti kala kembali ke kampus di musim gugur,” tukas Karima.

Mencari jawaban

“Ketika itu ada sekitar dua bulan saya meninggalkan keluarga di Iowa untuk mengikuti tour sepanjang kawasan Eropa ini. Sendirian pula. Kala itu usia saya baru 16. Makanya saya kepingin jalan-jalan dulu sembari “melihat dunia”. Itu alasan yang saya katakan pada keluarga dan kawan-kawan. Tapi sebenarnya saya sedang mencari jawaban atas konsep Kristen yang sudah lama saya pendam. Saya meninggalkan gereja (baca: Kristen -red) persis beberapa bulan sebelum berangkat ke Eropa dan belum bisa menentukan pilihan (agama) lain. Saya merasa belum mendapatkan apapun dengan apa yang telah saya pelajari selama ini. Sampai kini pun belum mendapatkan alternatif-alternatif lain,” ungkapnya.

“Tempat dimana saya dibesarkan, yakni Midwest, sebenarnya sangat cocok buat saya. Misalnya hal keyakian, tidak ada yang perlu dipusingkan disana. Mau jadi bagian dari gereja silahkan. Tidak, ya juga ndak masalah. Tapi karena itu pula saya tidak punya gambaran agama lain yang bisa dijadikan alternatif. Makanya ketika ada waktu keliling Eropa saya berharap bisa berjumpa dengan “sesuatu” yang lain itu,” imbuhnya.

“Di gereja tempat kami tinggal, kami hanya boleh melakukan ibadah untuk Yesus dan menyandarkan segala sesuatu padanya agar bisa menyampaikan pesan kepada Tuhan. Secara intuitif saya merasakan bahwa ada sesuatu yang salah dengan dogma itu,” kata dia.

“Saya kala itu dengan patuh pergi ke gereja tiap hari minggu dan sangat serius dengan apa yang saya pelajari tentang kejujuran, murah hati dan saling berkasih sayang. Tapi ada yang bikin saya bingung tatkala melihat jamaag gereja. Sikap mereka tampak begitu beda selama satu hari itu. Apakah Cuma sehari dalam sepekan bersikap jujur, murah hati dan kasih sayang? Apakah mereka cuma bahagia di hari minggu saja? Aku mencari-cari di beberapa buku panduan, namun tak menemukan apa-apa. Ada hal tentang 10 perintah Tuhan yang meliputi hal-hal yang sudah nyata sekali seperti larangan membunuh, mencuri dan berbohong. Uniknya, orang-orang ke gereja seperti tak ada etiket. Misalnya, sejauh yang saya tahu, banyak yang pakai rok mini ke gereja. Ironisnya lagi, ada juga dari mereka pergi ke sekolah minggu hanya karena ada cowok ganteng disana,” tukas Karima.

Kitab Bibel aneka versi

Satu hari Karima berkunjung ke rumah salah seorang dosennya. Disana dia melihat beberapa kitab Bibel tersusun rapi di rak lemari si dosen. “Saya tanya apa itu. Dosennya menjawab bahwa itu kitab Bibel dalam berbagai versi. Saya sebenarnya tak mau mengganggunya dengan pertanyaan seputar Bibel dalam aneka versi itu. Tapi makin dipendam makin sangat mengganggu pikiran. Saya beranikan diri mengamati beberapa dari Bibel itu. Saya terkejut. Memang ada yang benar-benar beda satu versi dengan versi lainnya. Bahkan ada beberapa bab yang tidak sama dengan Bibel kepunyaan saya. Kala itu saya benar-benar bingung. Bahkan mulai timbul perasaan bimbang,” katanya-

Ikut kelas bahasa Arab

Selepas tur Eropa Karima kembali ke kampus dengan perasaan kecewa sebab tak menemukan jawaban yang diharapkannya. Akan tetapi dengan keinginan yang begitu besar akan sebuah bahasa, Karima mengaku tertarik untuk mempelajari bahasa Arab. “Ironis ya, mendapat secercah jawaban yang saya cari-cari justru di dinding istana Al Hambra. Setelah pulang dari Spanyol, butuh dua tahun bagi saya untuk merealisasikan semua itu (masuk Islam-red),” ujarnya.

“Hal pertama sekali yang saya lakukan kala aktif kembali di kampus adalah mendaftar kelas bahasa Arab. Saya amati tampaknya kelas itu tidak begitu diminati. Entah kenapa. Buktinya peserta yang mendaftar cuma tiga. Saya dan dua mahasiswa lainnya. Tapi saya tak ambil pusing,” kata dia. Karima pun langsung tenggelam dengan pelajaran bahasa Arab. Rasa ingin tahunya sangat tinggi, hingga sang dosen takjub melihatnya.

“Saya kerjakan tugas-tugas yang diberikan dengan pulpen khusus untuk menulis huruf kaligrafi. Bahkan seringkali saya pinjam buku-buku dalam bahasa Arab dari dosen hanya untuk melihat huruf-huruf Arab yang ada dalam buku itu. Memasuki tahun kedua di universitas, saya putuskan untuk memilih bidang Studi Timur Tengah. Jadi dengan begitu bisa fokus pada satu kawasan saja. Nah di salah satu mata kuliahnya adalah belajar Al-Quran. Saya gembira bukan main,” aku Karima mengenang.

Kagum dengan Al-Quran

“Satu malam saya buka Al-Quran untuk mengerjakan PR. Heran campur takjub. Makin saya baca makin terasa nikmat. Sulit untuk berhenti membacanya. Persis seperti seseorang baru mendapatkan sebuah novel baru. Ketika itu saya bergumam dalam hati; wow menarik sekali. Inilah yang selama ini saya cari-cari. Semuanya ada dalam Al-Quran. Semua penjelasan betul-betul menarik. Saya sungguh kagum, kitab suci ini menguraikan semua yang juga saya percayai dan saya cari-cari jawabannya selama bertahun-tahun. Sangat jelas disebutkan bahwa hanya ada satu Tuhan yang patut disembah, yakni Allah. Tidak seperti di Kristen, satu dalam tiga,” imbuhnya.

Hari berikutnya Karima kembali ke ruang kelas untuk menanyakan siapa gerangan pengarang kitab itu. Karima melihat ada sebuah nama tertulis di halaman depan Al-Quran itu. “Awalnya saya menyangka itu nama pengarangnya. Misalnya seperti kitab Gospel yang dikarang oleh St. Luke atau kitab-kitab dalam agama lain yang pernah saya pelajari sebelumnya,” kata dia.

Salah seorang dosen Karima yang beragama Kristen memberitahu bahwa itu bukan nama pengarangnya. “Ternyata itu adalah nama penerjemahnya. Masih menurut dosen itu, dia mengutip pernyataan penganut Islam, bahwa tak ada seorang pun yang mampu menulis kitab suci itu. Quran, kata orang Islam, merupakan perkataan Allah dan tidak berubah dari pertama diturunkan hingga saat ini. Al-Quran dibaca dan dihafal banyak orang. Wow…tak perlu saya katakana bagaimana gembiranya hati saya. Makin terpesona dan takjub. Setelah penjelasan itu saya tambah tertarik, bukan hanya mempelajari bahasa Arab, tapi juga mempelajari Islam. Hingga timbul keinginan pergi ke Timur Tengah,” katanya sumringah.

Masuk Islam

Di tahun terakhir kuliah akhirnya Karima mendapat kesempatan mengunjungi Mesir. Salah satu tempat favorit yang ingin dia lihat di sana adalah mesjid. “Saya merasakan seolah-olah sudah jadi bagian dari mereka. Berada di dalam mesjid, keagungan Allah semakin nyata. Dan, seperti biasanya, saya sangat menikmati rangkaian tulisan kaligrafi yang ada di dinding mesjid itu,” kata dia.

Satu hari seorang teman menanyakan kenapa tidak masuk Islam saja kalau memang sudah sangat tertarik. “Tapi saya sudah jadi seorang muslim,” kata Karima. Si teman terkejut mendengar jawaban itu. Tak cuma dia, bahkan Karima sendiri terkejut dengan jawaban spontan yang keluar dari bibirnya. “Tapi kemudian saya sadari hal itu logis dan normal. Islam telah merasuk dalam jiwa saya dan selalu memberikan perasaan lain. Begtupun pernyataan teman saya itu ada benarnya. Kenapa saya tidak masuk Islam saja?” tanya Karima pada dirinya sendiri. Temannya menyarankan agar lebih resmi (masuk Islam) sebaiknya pergi ke mesjid saja dan menyatakan keislaman di hadapan jamaah di sana sebagai saksinya.

“Tanpa menunggu lama saya ikuti sarannya. Ringkas saja, Alhamdulillah, akhirnya saya pun bersyahadat. Pihak mesjid lalu memberikan selembar sertifikat resmi selepas bersyahadat. Tapi sertifikat itu tak penting dan hanya saya simpan dilemari. Sama seperti dokumen-dokumen lain seperti asuransi, ijazah dan lainnya. Tak ada niat menggantung kertas itu di dinding rumah sebagai bukti telah ber-Islam. Bagi saya yang penting sudah jadi seorang muslim,” akunya.

“Kini saya habiskan waktu hanya untuk mempelajari Al-Quran. Ketika membuka Al-Quran perasaan yang hadir persis seperti orang yang baru saja menemukan kembali anggota keluarganya yang telah lama hilang,” ungkap Karima. Di rumahnya Karima tak lupa menggantung foto Istana Al Hambra, tempat dimana dia pertama kali melihat tulisan Arab yang membuat dirinya takjub dan jatuh cinta dengan Al-Quran. Kini, disamping mengelola praktek penyembuhan alaminya dia juga aktif menulis. Ada lebih dari 120 artikel yang telah dia tulis. Umumnya bertema kesehatan. Tulisannya yang terkenal antara lain The “Yoga” of Islamic Prayer, Vetegarian Muslim, dan banyak lainnya lagi. Begitulah. [Zulkarnain Jalil/dari berbagai sumber/www.hidayatullah.com]

Review Buku: Yang Orangtua Harus Tahu tentang Vaksinasi pada Anak

http://www.setiabudi.name/archives/359

Review Buku: Yang Orangtua Harus Tahu tentang Vaksinasi pada Anak
December 18th 2007, on Kesehatan, Ulasan Buku

Buku berjudul Yang Orangtua Harus Tahu tentang Vaksinasi pada Anak ini adalah saduran dari buku berjudul What Your Doctor May Not Tell You About Children’s Vaccinations karangan Stephanie Cave, M.D., F.A.A.F.P bersama Deborah Mitchell.

Diterbitkan dengan ISBN 979-22-349-4 yang diterbitkan pertama kali oleh PT. Gramedia Pustaka Utama cetakan pertamanya pada tahun 2003.

Buku yang sangat memukau saya karena menyajikan banyak informasi mengejutkan tentang vaksinasi yang tidak pernah ditemukan di media informasi apapun.

Selama ini setiap informasi yang kita terima mengenai vaksinasi adalah suatu hal yang harus dilakukan dan memiliki dampak nol persen terhadap kesehatan manusia.

Padahal sebagaimana tertulis dalam lembaran pertama buku ini disebutkan sebagai berikut, “Dalam hal vaksinasi anak, mencegah mungkin tidak lebih baik daripada menyembuhkan”.

Ditutup dengan kalimat berikutnya, “Jangan ambil resiko untuk kesehatan anak Anda! Pelajari lebih lanjut tentang vaksinasi yang ada pada masa kini dengan… ORANG TUA HARUS TENTANG VAKSINASI ANAK”.

Mengapa hal tersebut menjadi penting?

Karena sebagai orang tua, tentunya kita mengharapkan hal terbaik yang dapat kita berikan kepada seluruh anak kita. Hal tersebut hanya dapat diwujudkan jika dan hanya jika kita memiliki informasi yang memadai mengenai apapun yang ingin kita persembahkan kepada mereka.

Fakta-fakta mengejutkan tentang kandungan merkuri yang digunakan dalam sebagian besar vaksin anak saat ini baru salah satu contoh mengerikan tentang vaksin yang harus Anda ketahui.

Berikut ini adalah beberapa hal yang mungkin tidak Anda ketahui tentang vaksin:

1. Beberapa vaksin mengandung racun seperti air raksa (merkuri), almunium dan formalin
2. Di tahun 1998, Pemerintah Perancis menghentikan program vaksinasi berbasis sekolah yang memberikan vaksin Hepatitis B kepada anak-anak usia sekolah karena kasus multiple-sklerosis telah dikaitkan dengan vaksin tersebut dan lebih dari 600 kasus imunitas dan persyarafan telah dilaporkan.
3. Beberapa vaksin dibuat menggunakan bahan yang berasal dari jaringan manusia dari janin yang digugurkan.
4. Kebanyakan negara mewajibkan bahwa saat anak berusia 5 tahun, ia sudah harus menerima 33 dosis dari 10 vaksin.
5. Para dokter hanya melaporkan kurang dari 10 persen kejadian buruk yang berkaitan dengan vaksinasi dan/atau sesudah vaksinasi.

Selain itu salah satu isu keamanan yang menurut buku ini sering diabaikan adalah bahan-bahan tambahan yang terdapat dalam vaksin sebagai berikut:

1. Alumunium

Logam ini ditambahkan ke dalam vaksin dalam bentuk gel atau garam sebagai pendorong terbentuknya antibodi. Alumunium telah dikenal sebagai penyebab kejang, penyakit alzheimer, kerusakan otak dan dimensia (pikun). Logam ini biasanya digunakan pada vaksin-vaksin DPT, DaPT dan Hepatitis B.
2. Benzetonium Khlorida

Benzetonium adalah bahan pengawet dan belum dievaluasi keamanannya untuk dikonsumsi oleh manusia. Biasa digunakan sebagai campuran vaksin anthrax terutama diberikan kepada para personil militer.
3. Etilen Glikol

Biasa digunakan sebagai bahan utama produk antibeku dan digunakan sebagai pengawet vaksin DaPT, polio, Hib dan Hepatitis B.
4. Formaldehid

Bahan kimia yang terkenal sebagai zat karsinogenik (penyebab kanker) yang biasanya digunakan dalam proses pengawetan mayat, fungisida/insektisida, bahan peledak dan pewarna kain.

Selain beracun, menurut Sir Graham S. Wilson pengarang buku The Hazards of Immunization formalin tidak mamadai sebagai pembunuh kuman sehingga maksud penggunaannya sebagai penonaktif kuman dalam vaksin menjadi tidak berfungsi dengan baik.

Akibatnya adalah kuman yang seharusnya dilemahkan dalam vaksin tersebut malah menguat dan menginfeksi penggunanya.
5. Gelatin

Bahan yang dikenal sebagai alergen (bahan pemicu alergi) ini banyak ditemukan dalam vaksin cacar air atau MMR. Bagi kaum Muslim, gelatin menimbulkan isu tambahan karena biasanya bahan dasarnya berasal dari babi.
6. Glutamat

Bahan yang digunakan dalam vaksin sebagai penstabil terhadap panas, cahaya dan kondisi lingkungan lainnya. Bahan ini banyak dikenal sebagai penyebab reaksi buruk kesehatan dan ditemukan pada vaksin varicella.
7. Neomisin

Antibiotik ini digunakan untuk mencegah pertumbuhan kuman di dalam biakan vaksin. Neomisin menyebabkan reaksi alergi pada beberapa orang dan sering ditemukan dalam vaksin MMR dan polio.
8. Fenol

Bahan yang berbahan dasar tar batu bara yang biasanya digunakan dalam produksi bahan pewarna non makanan, pembasmi kuman, plastik, bahan pengawet dan germisida.

Pada dosis tertentu, bahan ini sangat beracun dan lebih bersifat membahayakan daripada merangsang sistem kekebalan tubuh sehingga menjadi berlawanan dengan tujuan utama pembuatan vaksin.

Fenol digunakan untuk pembuatan beberapa vaksin termasuk vaksin tifoid.
9. Streptomisin

Antibiotik ini dikenal menyebabkan reaksi alergi pada beberapa orang dan biasa ditemukan dalam vaksin polio.
10. Timerosal/Merkuri

Bahan yang sangat beracun yang selama beberapa puluh tahun digunakan pada hampir seluruh vaksin yang ada di pasaran. Padahal timerosal/merkuri adalah salah satu bahan kimia yang bertanggung jawab atas tragedi Minamata di Jepang yang menyebabkan lahirnya bayi-bayi yang cacat fisik dan mentalnya.

Berikut ini adalah beberapa kerusakan yang disebabkan keracunan merkuri:

1. Otak bayi masih mengalami perkembangan yang cepat dan merkuri bisa merusak sel otak secara menetap.
2. Sistem kekebalan tubuh bayi masih belum berkembang secara penuh sehingga bayi tidak mempunyai kemampuan melawan serangan benda asing (bakteri, virus dan racun lingkungan) secara benar.
3. Kemampuan tubuh bayi untuk membuang racun dari tubuhnya melalui hati belum berkembang sepenuhnya sehingga zat-zat berbahaya cenderung menetap di dalam tubuhnya seperti merkuri, formalin dan alumunium.
4. Penghambat darah-otak (selaput yang berada di antara darah yang beredar di tubuh dengan otak yang berfungsi bahan-bahan berbahaya mencapai otak) belum mampu menghalangi racun yang bisa merusak otak.
5. Gejala keracunan merkuri yang paling umum antara lain adalah:
* Perubahan suasana hati dan kepribadian, termasuk mudah marah dan malu
* Hilangnya sensasi dan masalah penglihatan serius
* Ketulian dan kecenderungan kesulitan berkomunikasi karenanya
* Kelemahan otot dan tidak adanya koordinasi tubuh yang baik
* Hilangnya/lemahnya ingatan
* Tremor/gemetaran

Belum lagi fakta-fakta yang disajikan dalam buku ini yang mengkaitkan vaksinasi yang berbahaya dengan meningkatnya kasus-kasus autisme saat ini.

Dimana kasus autisme ini ternyata memiliki kemiripan dengan gejala-gejala keracunan merkuri yang banyak digunakan dalam vaksin.

Hal yang menarik lainnya untuk kita di Indonesia yang sedang gencar-gencarnya melakukan vaksinasi polio melalui mulut (oral/dimakan) adalah fakta bahwa sejak tahun 2000 Sentra Pengendalian Penyakit Amerika Serikat sudah menghentikan vaksin oral dan digantikan dengan suntikan.

Mengapa? Karena vaksinasi polio oral terbukti menimbulkan sampai 10 kasus polio per tahun dan dituding menyebabkan gangguan serius pada sistem pencernaan terutama penyumbatan usus!

Lantas mengapa informasi-informasi tersebut cenderung tidak pernah terpublikasikan secara luas?

Alasannya tentu saja sederhana sekali: UANG.

Bisnis produksi dan penjualan vaksin bernilai milyaran dollar Amerika Serikat per tahun! Selain itu banyak sekali bukti-bukti yang kemudian dibungkam menelusuri bahwa penyakit-penyakit saat ini seperti HIV/AIDS, DBD (demam berdarah), flu burung, dsb adalah senjata biologi yang sengaja dikembangkan yang kemudian dilepaskan ke komunitas sehingga mendorong kebutuhan akan obat dan vaksin penyakit-penyakit tersebut.

Saya dan isteri pun akhirnya sepakat untuk tidak memvaksinasi puteri kami. Hal ini kami lakukan setelah berkonsultasi dengan banyak ahli kesehatan (kedokteran, kimia klinis, teknologi kesehatan, dsb).

Apalagi ternyata teman-teman kami yang menjadi atau sedang kuliah menjadi dokter di Eropa secara terang-terangan menyatakan “vaksinasi adalah fiksi seperti cerita manusia mendarat di bulan..”

Nigella sativa – Wikipedia

Nigella sativa

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Nigella sativa
Nigella sativa (left) and Nigella damascena (right)

Nigella sativa (left) and Nigella damascena (right)
Scientific classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Ranunculales
Family: Ranunculaceae
Genus: Nigella
Species: N. sativa
Binomial name
Nigella sativa
L.

Nigella sativa is an annual flowering plant, native to southwest Asia. It grows to 20–30 cm tall, with finely divided, linear (but not thread-like) leaves. The flowers are delicate, and usually coloured pale blue and white, with 5–10 petals. The fruit is a large and inflated capsule composed of 3–7 united follicles, each containing numerous seeds. The seed is used as a spice.

Nigella sativa seed

Nigella sativa seed

In English, Nigella sativa seed is variously called fennel flower, nutmeg flower, Roman coriander, blackseed, black caraway, or black onion seed. Other names used, sometimes misleadingly, are onion seed and black sesame, both of which are similar-looking but unrelated. The seeds are frequently referred to as black cumin (as in Bengali কালো জিরা kalo jira), but this is also used for a different spice, Bunium persicum. The scientific name is a derivative of Latin niger “black”.[1] An older English name gith is now used for the corncockle. In English-speaking countries with large immigrant populations, it is also variously known as kalonji (Hindi कलौंजी kalauṃjī or कलोंजी kaloṃjī), kezah Hebrew קצח), chernushka (Russian), çörek otu (Turkish), habbat albarakah (Arabic حبه البركة ḥabbatu l-barakah “seed of blessing”) or siyah daneh (Persian سیاه‌دانه siyâh dâne).

A commercial pack of kalonji

A commercial pack of kalonji

This potpourri of vernacular names for this plant reflects that its widespread use as a spice is relatively new in the English speaking world[citation needed], and largely associated with immigrants from areas where it is well known. Increasing use is likely to result in one of the names winning out, hopefully one which is unambiguous.

Nigella sativa has a pungent bitter taste and a faint smell of strawberries. It is used primarily in candies and liquors. The variety of naan bread called Peshawari naan is as a rule topped with kalonji seeds. In herbal medicine, Nigella sativa has hypertensive, carminative, and anthelminthic properties[citation needed]. They are eaten by elephants to aid digestion.[citation needed]

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[edit] Historical accounts

According to Zohary and Hopf, archeological evidence about the earliest cultivation of N. sativa “is still scanty”, but they report that N. sativa seeds have been found in several sites from ancient Egypt, including Tutenkhamen‘s tomb.[1] Although its exact role in Egyptian culture is unknown, it is known that items entombed with a pharaoh were carefully selected to assist him in the after life.

The earliest written reference to N. sativa is thought to be in the book of Isaiah in the Old Testament where the reaping of nigella and wheat is contrasted (Isaiah 28: 25, 27). Easton’s Bible dictionary states that the Hebrew word ketsah refers to without doubt to N. sativa (although not all translations are in agreement). According to Zohary and Hopf, N. sativa “was another traditional condiment of the Old World during classical times; and its black seeds were extensively used to flavour food.”[1]

[edit] Use in folk medicine

Nigella sativa has been used for medicinal purposes for centuries, both as a herb and pressed into oil, in Asia, Middle East, and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach and intestinal health, kidney and liver function, circulatory and immune system support, and for general well-being.

In Islam, it is regarded as one of the greatest forms of healing medicine available. Prophet Muhammad once stated that the black seed can heal every disease—except death—as recounted in the following hadith:

Narrated Khalid bin Sa’d:We went out and Ghalib bin Abjar was accompanying us. He fell ill on the way and when we arrived at Medina he was still sick. Ibn Abi ‘Atiq came to visit him andsaid to us, “Treat him with black cumin. Take five or seven seeds and crush them (mix the powder with oil) and drop the resulting mixture into both nostrils, for ‘Aisha has narrated to me that she heard the Prophet saying, ‘This black cumin is healing for all diseases except As-Sam.’ ‘Aisha said, ‘What is As-Sam?’ He said, ‘Death.’ ” (Bukhari)

Ibn Sina, most famous for his volumes called The Canon of Medicine, refers to nigella as the seed that stimulates the body’s energy and helps recovery from fatigue and dispiritedness. It is also included in the list of natural drugs of ‘Tibb-e-Nabavi’, or “Medicine of the Prophet (Muhammad)”, according to the tradition “hold onto the use of the black seeds for in it is healing for all diseases except death” (Sahih Bukhari vol. 7 book 71 # 592).

In the Unani Tibb system of medicine, N. sativa is regarded as a valuable remedy for a number of diseases.

The seeds have been traditionally used in the Middle East and Southeast Asian countries to treat ailments including asthma, bronchitis, rheumatism and related inflammatory diseases, to increase milk production in nursing mothers, to promote digestion and to fight parasitic infections. Its oil has been used to treat skin conditions such as eczema and boils and to treat cold symptoms. Its many uses have earned nigella the Arabic approbation ‘Habbatul barakah’, meaning the seed of blessing.

[edit] Scientific studies

Black cumin oil contains nigellone, which protects guinea pigs from histamine-induced bronchial spasms[citation needed] (perhaps explaining its use to relieve the symptoms of asthma, bronchitis, and coughing).

The presence of an anti-tumor sterol, beta sitosterol, lends credence to its traditional use to treat abscesses and tumors of the abdomen, eyes, and liver.[2]

[edit] As an anti-parasitic

Anticestodal effect of N. sativa seeds was studied in children naturally infected with the respective worm. A single oral administration of 40 mg/kg of N. sativa seeds and equivalent amount of its ethanolic extract were effective in reducing the egg count in the faeces, with a comparable effect to niclosamide. The crude extracts also did not produce any adverse side effects from all the doses tested.[3]

In 1998, Korshom et al. investigated the anti-trematodal activity of N.sativa seeds against a ruminant fluke (Paramphistomum) in sheep.[4] The methanol extract (1 ml/kg) and powder (200 mg/kg) showed high efficacy, comparable to Hapadex (netobimin, 20 mg/kg). (NOTE: methanol is transformed in the body to formaldehyde, and such raw extracts would not be used in a formulated product.)

In 2005, Azza et al. studied the anti-schistosomicidal properties of aqueous extract of N. sativa seeds against Schistosoma mansoni miracidia, cercariae, and the adult worms in vitro. It showed strong biocidal effects against all stages of the parasite and also inhibited egg-laying of adult female worms. [5]

In 2007, Abdulelah and Zainal-Abidin investigated the anti-malarial activities of different extracts of N.sativa seeds against P. berghei. Results indicated strong biocidal effects against the parasite.[6][7]

[edit] See also

[edit] References

  1. ^ a b (2000) Domestication of plants in the Old World, 3, Oxford University Press, p. 206. ISBN 0198503563.
  2. ^ Look for sterols at http://glycoscience.org/glycoscience/linksPage/links.html Click on the 4th listing for the GlycoScience link. (Link is dead of 2008-1-12)
  3. ^ Akhtar, M.S. & Rifaat, S. 1991. Field trial of Saussurea lappa roots against nematodes and Nigella sativa seeds against cestodes in children. Journal of the Pakistan Medical Association 41: 185–187.
  4. ^ Korshom M., Moghney, A.A. & Mandour, A. 1998. Biochemical and parasitological evaluation of Nigella sativa against ruminant fluke (Paramphistomum) in sheep as compared with trematocide “Hapadex”. Assiut. Vaternary Med. J. 39 (78): 238–244.
  5. ^ Azza, M. M., Nadia, M. M. & Sohair, S. M. 2005. Sativa seeds against Schistosoma mansoni different stages. Mem. Inst. Oswaldo. Cruz. Rio de Janeiro 100(2): 205–211.
  6. ^ Abdulelah H.A.A. & Zainal-Abidin B.A.H. 2007. In vivo anti-malarial tests of Nigella sativa (black seed) different extracts. American Journal of Pharmacology and Toxicology 2 (2): 46-50, 2007.
  7. ^ Abdulelah H.A.A. & Zainal-Abidin B.A.H. 2007. Curative and prophylactic anti-malarial activities of Nigella sativa (black seed) in mice. Malaysian Journal of Medical Sciences 14: 209.

[edit] External links

Tanaman Obat untuk penyakit Infeksi

http://www.iptek.net.id/ind/pd_tanobat/search.php

Nama penyakit yang anda cari: infeksi

No Nama Tanaman Nama Latin Nama Lokal/Daerah
1 Daun duduk Desmodium triquetrum [L.] D.C. Genteng cangkeng, ki congcorang, potong kujang,; cen-cen (Sunda), ), daun duduk, sosor bebek, gulu walang,; Gerji,cocor bebek (Jawa). daun duduk (Sumatera); Three-flowered desmodium (Inggris).;
2 Daun Sendok Plantago mayor L. Ki urat, ceuli, c. uncal (Sunda), meloh kiloh, otot-ototan,; Sangkabuah, sangkabuah, sangkuah, sembung otot,; suri pandak (Jawa). daun urat. daun urat-urat, daun sendok,; Ekor angin, kuping menjangan (Sumatera). ; Torongoat (Minahasa). ; Che qian cao (China), ma de, xa tien (Vietnam),; Weegbree (Belanda), plantain, greater plantain, ; Broadleaf plantain, rat’s tail plantain, waybread,; White man’s foot (Inggris).;
3 Kemuning Murraya paniculata [L..] Jack. Kamuning (Sunda), kemuning, kumuning (Jawa).; Kajeni, kemuning, kemoning (Bali), kamoneng (Madura),; Kamuning (Menado, Makasar), kamoni (Bare), palopo (Bugis).; Kamuni (Bima). eseki, tanasa, kamone, kamoni (Maluku).; Jiu li xiang, yueh chu (China), Orange jessamine (Inggris).;
4 Sambiloto Andrographis paniculata Ness. Ki oray, ki peurat, takilo (Sunda). bidara, sadilata, sambilata,; takila (Jawa). pepaitan (Sumatra).; Chuan xin lian, yi jian xi, lan he lian (China), xuyen tam lien,; cong cong (Vietnam). kirata, mahatitka (India/Pakistan).; Creat, green chiretta, halviva, kariyat (Inggris).;
5 Teh Camellia sinensis [L.] Kuntze Enteh (Sunda).; Pu erh cha (China), theler (Perancis), teestrauch (Jerman),; Te (Itali), cha da India (Portugis), tea (Inggris).;
6 Boroco Celosia argentea Linn. Bayam ekor belanda, Bayam kucing, Kuntha, Baya kasubiki; Qing xiang zi (China).;
7 Bunga Matahari Helianthus annuus Linn. bungngong matahuroi, bungka matahari, purbanegara; Bunga panca matoari, bunga teleng matoari, Sungeng; kembang sarengenge, kembhang mataare, bungga ledomata; kembang sangenge, kembhang tampong are; Xiang ri kui (China).;
8 Cakar Ayam Selaginella doederleinii Hieron. Rumput solo, cemara kipas gunung; Shi shang be (China).;
9 Calingcing Oxalis corniculata Linn. Calincing (Indonesia, Jawa), Mala-mala (Maluku); Rempi, semanggen, semanggi gunung, cembicenan (Jawa); Daun asam kecil lela, semanggi (Sumatra); Cu jiang cao (China).;
10 Daun Dewa Gynura segetum (Lour.) Merr. Beluntas cina, daun dewa (Sumatra), Samsit; San qi cao (China).;
11 Jarong Achyranthes aspera Linn. Jarongan, jarong lalaki, daun sangketan, nyarang (jawa).; Sui in sui, sangko hidung (Sulawesi), ; Rai rai, dodinga (Maluku).; Dao kou cao (China).;
12 Jarak Ricinus communis Linn. Jarak, jarak jitun, kaliki (Sunda), Jarak (jawa), Kaleke (Madura),; Gloah, lulang, dulang, jarak, kalikih alang, jarag (Sumatra),; Malasai, kalalei, alale, tangang jara, peleng kaliki jera (Sulawesi); Jarak (Bali), luluk (Roti), paku penuai (Timor), Balacai (Ternate), ; Balacai tamekot (Halmahera), tetanga (Bima), luluk (Roti),; Bi ma (China).;
13 Kembang Pukul Empat Mirabilisjalapa Linn. Kembang pukul empat (Indonesia, Sumatra), ; Kembang pagi sore, bunga waktu kecil (Sumatra); Kederat, segerat, tegerat (Jawa), Kupa oras, cako raha (Maluku); Bunga-bunga paranggi, bunga-bunga parengki (Sulawesi); Pukul ampa, turaga, bodoko sina, bunga tete apa (Sulawesi); Zi Mo li (China).;
14 Kumis Kucing Orthosiphon aristatus (B1) Miq. Kumis kucing, Mamang besar (Indonesia); Kutun, mamam, bunga laba-laba (Jawa); Mao Xu Cao (China).;
15 Pecut Kuda Stachytarpheta jamaicensis (L) Vahl Pecut kuda, Jarongan, Jarong lalaki, ngadi rengga, ; remek getih, jarong, biron, sekar laru, laler mengeng,; rumjarum, ki meurit beureum.; Yu long bian (China).;
16 Pegagan Centella asiatica, (Linn), Urb. Daun kaki kuda (Indonesia), Pegaga (Ujung Pandang); Antanan gede, Antanan rambat (Sunda), Dau tungke (Bugis); Pegagan, Gagan-gagan, Rendeng, Kerok batok (Jawa); Kos tekosan ( Madura), Kori-kori (Halmahera);
17 Rumput Mutiara Hedyotis corymbosa (L.] Lamk. Rumput siku-siku, bunga telor belungkas (Indonesia); Daun mutiara, rumput mutiara (Jakarta); Katepan, urek-urek polo (Jawa), Pengka (Makasar); Shui xian cao (China).;
18 Semanggi Gunung Hydrocotyle sibthorpioides Lam. Pegagan embun, antanan beurit, a. lembut (Sunda).; Andem, katepa’n, rendeng, semanggi (jawa), Salatun; Take cena (Madura), tikim, patikim; Tian hu sui (China).;
19 Sangketan Heliotropium indicum L. Gajahan, langun, uler-uleran, sangketan, cocok bero,; Tlale gajah, tulale gajah (Jawa), Bandotan lombok,; Buntut tikus, ekor anjing, tusuk konde (Sumatera); Da wei yao (China).;
20 Tahi Kotok Tagetes erecta L. Ades, Afrikaantjes; Wan shou ju (China).;

Khasiat Buah Zaitun

http://id.shvoong.com/medicine-and-health/1648264-khasiat-buah-zaitun/

Khasiat Buah Zaitun
Pengarang : Teguh Vedder
Ringkasan oleh : Teguh Vedder
Kunjungan: 230
Diterbitkan di: Agustus 14, 2007

Zaitun sejak lama terkenal dengan khasiatnya untuk kesehatan. Terdiri dari :

Meningkatkan metabolisme
Makan ½ cup buah zaitun setiap hari dapat mencegah kegemukan. Khasiat ini berasal dari lemak tak jenuh tunggal yang mempercepat pembakaran lemak dan mencegah gula diubah menjadi lemak. Selain itu, sebuah studi dalam British Journal of Nutrition menemukan, asam lemak tak jenuh tunggal menstimulir cholecystokinin, sejenis hormone penekan nafsu makan yang mengirim sinyal kenyang ke otak.

Merevitalisasi system imun
Zaitun kaya dengan vitamin E larut lemak, yang melindungi sel-sel dari radikal-radikal bebas yang berbahaya. Antioksidan ini menguatkan system imun, mengurangi penyakit seperti pilek dan flu sampai 30%, begitu menurut para periset di Tufts University di Boston.

Menghaluskan kulit
Makan buah zaitun yang merupakan sumber terkaya oleic acid, membantu mengurangi tampilan garis-garis halus. Asam lemak ini dapat mengenyalkan kulit dan melindungi elastin kulit dari kerusakan.

Meningkatkan sirkulasi
Zaitun adalah sumber istimewa dari polyphenois, senyawa antioksidan yang membantu mencegah penggumpalan darah yang berbahaya. Sebuah studi dalam Journal Of American College of Cardiology mengaitkan senyawa ini dengan peningkatan kadar nitric oxide, molekul jantung sehat yang meningkatkan pelebaran pembuluh darah dan aliran darah.

“VIRGIN COCONUT OIL” (VCO) – Manfaat Ditinjau dari Aspek Kesehatan

http://koranpdhi.com/buletin-edisi7/edisi7-vco.htm

VIRGIN COCONUT OIL” (VCO)
Manfaat Ditinjau dari Aspek Kesehatan

Oleh: Dr. drh. Masdiana Padaga, M.APP.Sc
Fakultas Teknologi Pertanian, Universitas Brawijaya

Minyak kelapa sebenarnya sudah lama dikenal dan digunakan oleh nenek moyang kita, baik untuk keperluan memasak maupun untuk tujuan pengobatan. Akan tetapi dengan adanya mitos bahwa minyak kelapa tidak baik untuk kesehatan maka pemakaian minyak kelapa terpinggirkan dan diganti dengan minyak lainnya. Saat ini minyak kelapa mulai mendapat perhatian dunia karena diketahui mempunyai banyak manfaat. Data empiris menunjukan bahwa penduduk yang mempunyai kebiasaan mengkonsumsi minyak kelapa secara rutin sebagai bagian dari menu makan sehari hari mempunyai system imun yang lebih baik dan bebas dari penyakit kardiovaskuler, kanker dan penyakit-penyakit degeneratif lainnya. Berdasarkan kenyataan tersebut maka penelitian tentang manfaat minyak kelapa ditinjau dari aspek kesehatan semakin meningkat dan terbukti minyak kelapa mengandung asam lemak yang sangat baik untuk kesehatan sehingga Dr. E.V.Carandang dalam paper “Coconut Uses and Issues on Its Health and Nutraceutical Benefits” menyebutkan bahwa minyak kelapa dapat digolongkan sebagai pangan fungsional atau Nutraceutical.

KEUNGGULAN VCO
Minyak kelapa mempunyai keunggulan dibandingkan minyak lainnya karena hampir 50% asam lemak yang terkandung didalamnya adalah asam laurat yang merupakan asam lemak rantai sedang (medium chain fatty acid/ MCFA). Selama ini asam laurat digunakan sebagai bahan baku dalam industri kosmetika, aplikasi dan manfaatnya dalam industri pangan baru diketahui beberapa tahun terakhir. Kandungan asam laurat yang tinggi dapat diperoleh dari minyak kelapa murni yang lebih dikenal dengan nama “VIRGIN COCONUT OIL” atau disingkat VCO. Perbedaan penting antara minyak kelapa biasa dengan VCO adalah pada bahan baku dan proses pembuatan. Minyak kelapa biasa atau dikenal dengan nama RBD coconut oil ( refined, bleached and deodorized) diproses dari bahan baku kopra dengan menggunakan suhu tinggi dan bahan kimia. Sedangkan VCO diproses dari bahan baku kelapa segar dengan suhu rendah dan tanpa menggunakan bahan kimia.

MANFAAT ASAM LEMAK RANTAI SEDANG (MCFA) BAGI KESEHATAN
Menurut Dr. Bruce Fife penulis buku ” The Healing Miracles of coconut oil” , minyak kelapa merupakan sumber utama asam laurat yang tergolong asam lemak rantai sedang atau yang lebih dikenal dengan MCFA (medium chain fatty acid) sehingga minyak kelapa saat ini banyak digunakan dalam formula makanan untuk pasien di rumah sakit dan untuk tambahan susu formula untuk bayi. Tidak seperti asam lemak yang lain, MCFA yang terdapat dalam minak kelapa sangat mudah dicerna dan diserap oleh tubuh untuk selanjutnya digunakan sebagai sumber energi yang paling cepat sehingga sangat membantu proses penyembuhan pasien. Untuk orang-orang yang mempunyai problem pencernaan contohnya dalam kasus Cystic fibrosis, MCFA diperlukan sebagai nutrisi penting yang perlu ditambahkan langsung dalam diet.

Keunggulan MCFA dibandingkan asam lemak rantai panjang (LCFA) adalah pada proses metabolismenya di dalam tubuh. MCFA mempunyai molekul yang lebih kecil sehingga tidak diperlukan energi yang tinggi dan hanya memerlukan sedikit enzim untuk memecah lemak tsb menjadi bentuk yang siap diserap tubuh. Dengan dimikian proses penyerapan dan distribusi dalam tubuh akan berlangsung lebih cepat dan segera digunakan sebagai sumber energi tubuh. Pada saat dikonsumsi MCFA akan segera dipecah oleh enzim-enzim yang terdapat dalam saliva dan cairan lambung sehingga tidak terlalu diperlukan proses pencernaan oleh enzim pemecah lemak yang dihasilkan oleh pancreas sehingga beban kerja pankreas dan sistem pencernaan tidak terlalu berat, Hal ini sangat membantu pasien-pasien yang mempunyai problem metabolisme dan pencernaan terutama malabsorbsi lemak dan vit yang larut dalam lemak. MCFA juga sangat bermanfaat bagi penderita diabetes, obesitas, pankreatitis, gangguan limpa, dan kanker saluran pencernaan.

Pada proses pencernaan lemak lain seperti kolesterol, asam lemak jenuh dan asam lemak tak jenuh rantai pendek dan panjang, sangat diperlukan enzim pankrease untuk memecah lemak menjadi unit yang kecil. Unit-uni ini selanjutnya akan diserap oleh intestin dalam bentuk lipoprotein yang selanjutnya masuk ke hati untuk dirubah menjadi energi dan sebagian disimpan dalam bentuk lemak tubuh. Dalam hal ini proses pencernaan lemak untuk menjadi energi sangat panjang. MCFA tidak dirubah menjadi lipoprotein tapi langsung dipecah menjadi molekul kecil, diserap tubuh masuk ke dalam hati untuk dirubah menjadi energi sehingga kebutuhan energi akan tercukupi dalam waktu singkat dan tidak akan menyebabkan penimbunan lemak tubuh, peningkatan kadar kolesterol dan trigliserida dalam darah.

Di dalam tubuh manusia atau hewan asam laurat akan menjadi monolaurin yaitu monogliserida yang mempunyai efek antibakteri, antiviral dan antiparasit. Dr.Enig dalam artikel “COCONUT : IN SUPPORT OF GOOD HEALTH IN THE 21st CENTURY” menjelaskan beberapa hasil penelitian yang sudah dilakukan sejak tahun 1978 dan terbukti bahwa monolaurin dapat menginaktivkan beberapa bakteri pathogen penting seperti Listeria monocytogenes, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus grup A,F dan G. Minyak kelapa juga mempunyai efek antiaging sehingga dapat digunakan untuk perawatan kulit.

Manfaat minyak kelapa untuk kesehatan telah dibahas secara komprehensif oleh Murray Price dalam buku “COCONUT OIL FOR YOUR HEALTH’ dan sudah diterjemahkan kedalam bahasa Indonesia dengan judul buku TERAPI MINYAK KELAPA. Saat ini penggunaan VCO untuk kesehatan sudah mulai memasyarakat di Indonesia. Namun demikian tetap perlu dicermati bahwa VCO dapat memberikan manfaat bagi kesehatan jika diproduksi dengan proses yang benar dan mengandung asam laurat tidak kurang dari 50%. Mengingat VCO mempunyai manfaat yang cukup banyak bagi kesehatan maka tidak menutup kemungkinan dapat digunakan juga pada bidang kesehatan ternak dan hewan kesayangan.

Penelitian Para Ahli Kedokteran terhadap Habbatus Sauda (Bag 1)

http://www.habbats.com/2007/12/12/penelitian-para-ahli-kedokteran-terhadap-habbatus-sauda-bag-1/
Penelitian Para Ahli Kedokteran terhadap Habbatus Sauda (Bag 1)

* Posted by admin
* December 12, 2007
Habbatus Sauda
Berikut penelitian yang sudah dilakukan oleh para pakar:

“ Habbatus Sauda’ memiliki peranan yang penting pada pencegahan tumor & kanker. Pada penggunaannya dalam waktu yang lama minyak Habbatus sauda’ memperkuat sistem kekebalan tubuh. Selain itu dapat mempercepat pembelahan sel ”. Dr. Stanley Kopok, University of Arizona, USA

“ Habbatus sauda kaya akan beta-sitosterol, yang diketahui memiliki kemampuan mencegah kanker”.
Dr. Michael Tierra L. AC. O.M.D.

” Dengan mengkonsumsi Nigella Sativa dapat melawan kelelahan ”.
Dr. Mohammed Saleh, Kairo-Mesir

” Ekstrak habbatus sauda memiliki khasiat anti-tumor tanpa efek samping seperti apa yang terjadi pada Chemotherapi dan penyinaran”.
Study of Nigella sativa on humans, University of South Carolina. USA

“ Habbatus sauda mempunyai lebih dari 100 bahan aktif dengan berbagai khasiat penyembuhan. Salah satunya adalah impotensi.”
Black Cumin, The Magical Egyptian Herb, Dr. Peter Schleicher, M. D.
Dr.Mohammed Saleh, M.D., Egypt

“ Habbatus sauda’ terbukti memiliki khasiat anti-histamin, anti-oxidant, anti-biotic, anti -mycotic dan penghambat Bronchitis”.
Study of Black Seed Oil on Humans, American Scientists

“ Kombinasi kandungan lipid dan struktur hormon dalam Nigella Sativa dapat meningkatkan air susu bagi ibu menyusui”
University Pochefstroom

” Terapi baru yang efektif, mudah dicerna dan tidak mahal pada penyakit alergi adalah penggunaan asam lemak tidak jenuh dari minyak biji tanaman, terutama minyak Nigella Sativa (Habbatus sauda)”.
Dr. Lutz Bannasch, München – Jerman

” Nigella Sativa (Habbatus sauda mengandungi minyak eter yang dapat membantu pencernaan & mengurangi masalah usus ”.
Prof. Dr. Hildebert Wagner, Institut fur Pharmazeutische Biologie, München – Jerman

” Nigella Sativa mengandung asam lemak tak jenuh, Linoleic & Gammalinolenic yang masuk dalam tubuh. Asam ini memungkinkan untuk pencapaian sintesis yang merupakan sistem kekebalan tubuh yang penting menyebabkan penyakit kronik seperti jerawat dan demam karena alergi, hingga kanser ”.
Dr. Peter Schleicher, M. D., Immunologist, Munich Germany

” Meningkatkan fungsi kekebalan tubuh ”.
US. Patents Section, Antiviral Agents Bulletin # 5, 482, 711

” Habbatus sauda’ merangsang sumsum di tulang & imuniti sel serta pengeluaran terferon, melindungi sel-sel normal melawan virus perusak sel, melawan sel tumor, dan meningkatkan antibodi yang mengeluarkan sel B”.
Cancer Immuno-biology, South Carolina

” Habbatus sauda’ terbukti menyembuhkan 70 % penderita alergi, termasuk di dalamnya alergi serbuk dan debu juga jerawat, serta neurodermitis ( penyakit kulit), asthma dan lemahnya daya kekebalan tubuh. Mengambil habbatus sauda’ secara teratur dapat menahan secara baik penyakit flu ”.
Dr. Peter Schleicher, Immunologie, München-Jerman

Keywords: habbatussauda, jintan hitam, khasiat jintan, jinten hitam, blackseed, nigella sativa

Hembing: Pemanfaatan Herbal Untuk Kesehatan & Pengobatan Penyakit

http://cybermed.cbn.net.id/cbprtl/cybermed/detail.aspx?x=Hembing&y=cybermed%7C0%7C0%7C8%7C98

Pemanfaatan Herbal Untuk Kesehatan & Pengobatan Penyakit
Hembing Fri, 20 Jul 2007 15:45:00 WIB

Kondisi tubuh yang sehat dan bugar merupakan dambaan setiap orang, namun timbulnya penyakit terkadang tidak terduga dan tidak bisa dihindari. Ancaman penyakit bagi tubuh bisa terjadi setiap saat. Adanya perubahan pola hidup telah mengakibatkan perkembangan pola penyakit sehingga timbul berbagai macam penyakit.

Pola kehidupan modern menuntut seseorang untuk bergerak cepat dalam upaya memenuhi berbagai tuntutan kehidupan, sehingga mengakibatkan kelelahan, kurang istirahat, stres, dan faktor-faktor lain yang menyebabkan banyak orang mengalami penurunan daya tahan tubuh. Pola kehidupan modern juga berdampak pada pencemaran lingkungan serta pencemaran makanan oleh zat kimia. Kita menghirup udara kotor yang jenuh dengan asap, partikel-partikel debu, karbon monoksida dan bahan pencemar lainnya.

Kita juga mengkonsumsi makanan dan minuman yang mengandung bakteri, zat karsinogen, dan zat-zat kimia berbahaya lainnya. Kondisi tersebut dapat menekan kerja sistem immun yang menyebabkan mikrobia patogen seperti virus dan bakteri mudah masuk menyerang tubuh sehingga menimbulkan berbagai penyakit.

Kondisi daya tahan tubuh dapat mengalami naik-turun. Hal tersebut dipengaruhi oleh berbagai faktor, antara lain oleh pola hidup seperti pola makan, pola pikir, dan aktivitas sehari-hari. Apabila proporsi salah satu faktor tersebut tidak lagi seimbang pada tubuh, akan mengakibatkan menurunnya daya tahan tubuh.

Gangguan kesehatan yang dapat timbul karena menurunnya daya tahan tubuh sebenarnya dapat diminimalisasi dengan penerapan prinsip-prinsip hidup sehat alami dalam kehidupan sehari-hari, Hidup serasi dengan alam dan penerapan pola hidup alami dengan 3 G, yaitu Gizi seimbang, Gerak badan, dan Gaya hidup sehat dalam kehidupan sehari-hari tentunya dapat meningkatkan kualitas kesehatan.

Pemanfaatan Tanaman Obat untuk Kesehatan Keluarga
Sejak dahulu bangsa Indonesia telah mengenal dan memanfaatkan tumbuhan berkhasiat obat atau herbal sebagai salah satu upaya untuk menanggulangi masalah kesehatan. Alam Indonesia telah menyediakan berbagai solusi dalam menjaga kesehatan, salah satunya melalui terapi tumbuhan berkhasiat obat. Pengetahuan tentang pemanfaatan tumbuhan obat tersebut merupakan warisan budaya bangsa berdasarkan pengetahuan dan pengalaman yang diwariskan secara turun temurun hingga ke generasi sekarang. Sekarang ini beberapa tumbuhan obat telah dikembangkan dan diantaranya telah diteliti untuk menguji efektifitasnya.

Saat ini dengan kembali maraknya gerakan kembali ke alam (back to nature), kecenderungan penggunaan bahan obat alam/ herbal di dunia semakin meningkat. Gerakan tersebut dilatarbelakangi perubahan lingkungan, pola hidup manusia, dan perkembangan pola penyakit. Slogan back to nature yang menunjukan minimnya efek negatif yang ditimbulkan dari penggunaan tumbuhan obat dan juga ekonomis menarik minat masyarakat untuk kembali menggunakan obat-obatan dari bahan alami. Saat ini, semakin banyak industri farmasi baik di negara industri maupun di negara-negara berkembang seperti di Indonesia yang mulai mengembangkan obat-obatan yang bahan bakunya diambil dari alam.

Obat dari bahan alam/herbal diposisikan sebagai antioksidan (menangkal radikal bebas), imuno-modulator (meningkatkan sistem immun) dan mencegah penyakit degeneratif.

Kondisi perekonomian yang terpuruk yang dialami bangsa kita berdampak juga dengan melonjaknya biaya pengobatan dan harga obat-obatan. Di sisi lain, adanya kenyataan bahwa tingkat kebutuhan masyarakat terhadap pengobatan semakin meningkat. Sementara taraf kehidupan sebagian masyarakat kita masih banyak yang kemampuannya pas-pasan. Maka dari itu, pengobatan tradisional yang ekonomis merupakan solusi yang baik untuk menanggulangi masalah tersebut.

Berikut ini beberapa contoh pemanfaatan tanaman obat untuk mengatasi gangguan kesehatan pada keluarga .

PANAS/DEMAM
– 15 gram pegagan + 3 siung bawang merah + 15 gram meniran, dicuci bersih, direbus dengan 500 cc air hingga tersisa 200 cc, tambahkan 2 sendok teh madu, diminum.
(catatan : untuk dewasa diminum sekaligus dan lakukan 2 kali sehari. Untuk anak-anak dibagi menjadi 2-3 kali minum.)

– 4 siung bawang merah dihaluskan + 1 buah air perasan jeruk nipis + minyak kayu putih. Semua bahan dicampur dan diaduk rata, lalu dikompreskan pada ubun-ubun, leher, dada dan punggung anak. (pemakaian luar untuk panas pada anak)

BATUK
5 lembar daun sirih + 25 gram kencur (diris-iris) + 10 gram kulit jeruk mandarin kering + gula batu. Semua bahan dicuci bersih lalu direbus dengan 600 cc air hingga tersisa 300 cc, disaring, airnya diminum.
(untuk dewasa diminum sekaligus dan lakukan 2 kali sehari. Untuk anak-anak 5 tahun ke atas dibagi 3 kali minum.)

SAKIT KEPALA
90 gram daun lidah buaya (dikupas kulitnya) + 15 gram jahe + gula aren, dicuci bersih dan dipotong-potong, lalu direbus dengan 400 cc air hingga tersisa 200 cc, disaring, airnya diminum hangat. Lakukan 2-3 kali sehari.

DIARE
15- 30 gram daun jambu biji segar dan 20 gram kunyit, dicuci bersih, kunyit dipotong-potong, lalu keduanya direbus dengan 600 cc air hingga tersisa 300 cc, disaring, airnya diminum 2-3 kali sehari.

NYERI LAMBUNG/MAAG
30 gram temu lawak + 20 gram kunyit + 25 gram kencur + 80 gram daun lidah buaya (dikupas kulitnya). Semua bahan dicuci bersih dan dipotong-potong,

Pegal Linu, Rematik
30 gram jahe merah + 30 gram temulawak + 30 gram lengkuas + 30 gram kencur + 2 batang sereh + gula aren. Semua bahan dicuci bersih dan dipotong-potong, lalu direbus dengan 600 cc air hingga tersisa 300 cc, disaring, airnya diminum 2 kali sehari.

Infeksi Saluran Kemih, Anyang-Anyangan
30 gram tumbuhan kumis kucing + 60 gram akar alang-alang segar + 60 gram rambut jagung, dicuci bersih lalu direbus dengan 600 cc air hingga tersisa 300 cc, disaring, airnya diminum 2 kali sehari.

Bila mengkonsumsi obat herbal sebaiknya tidak bersamaan dengan obat dokter, beri jarak waktu sekitar 2 jam antar keduanya. Hal tersebut untuk menghindari efek negatif atau adanya interaksi farmakokinetik dengan obat-obat konvensional.

***********

Sumber: hembing

EFEK SPIRULINA (Ganggang Biru Hijau) PADA KEKEBALAN

EFEK SPIRULINA (Ganggang Biru Hijau) PADA KEKEBALAN

http://www.blue-green-algae.org/blue-green-algae.html

Thus, multiple studies on whole blue-green-algae in humans, mice, rats, cats, and chickens have demonstrated an effect on phagocytosis, NK cell function, and inflamma­tion. Some differences exist in the data, including the mild reduction of phagocytic activity in humans after algae con­sumption, in contrast to the increase of phagocytosis among bronchoalveolar macrophages. The cell types and experimental set-ups vary, and further studies are needed to estab­lish the exact biochemical mechanisms involved.

EFFECTS OF BLUE-GREEN ALGAE ON SPECIFIC IMMUNITY
Hayashi et al7 examined the effect of an algae-supple-mented diet on the ability to build a specific immune response

Table 2. Immuno-Modulatory and Anti-Inflammatory Effects of Whole Blue-Green Algae

Algae Species

Introduced as:

Test Species

Effects Reference
Spirulina sp. Food Human Reversal of tobacco-induced oral cancer Mathew et al, 1995
Food Mouse Proportional reduction of IgE, increase of IgA Hayashi et al, 1998
Food Mouse Increased phagocytic activity Increased spleen cell proliferation Increased antibody production Hayashi et al, 1994
Food Chicken Increased phagocytic activity Increased NK cell-mediated anti-tumor activity Increased antibody production Qureshi et al, 1996
Extract

In vitro, cat

Increased phagocytic activity

Qureshi & Ali, 1996

IP injection Rat Inhibition of mast cells Decrease in local allergic reaction Decrease in serum histamine levels Reduced allergy-induced mortality Kim et al, 1998 Yang et al, 1997
Aphanizomenon flos-aquae Food Human Increased transient recruitment of NK cells into tissue Increased mobilization of T and B cells into blood Mild modulation of PMN-mediated phagocytic response Jensen et al, 2000
Food Rat Decreased serum levels of arachidonic acid Kushak et al, 2000
Food Rat Source of linolenic acid (omega-3) Increased serum levels of EPA and DHA Kushak et al, 2000
Extract

In vitro, rat

Activation of macrophages (NF-kappaB, cytokines) Pasco, in press

to sheep red blood cells. After immunizing mice (either once to measure the primary response or twice for the secondary response), they found that mice fed with the algae-supple-mented diet showed increased numbers of splenic IgM anti-body-producing cells when compared to control animals. Interestingly, this finding only held true for the primary immune response, as the IgG antibody production in the sec­ondary immune response was hardly affected. In experiments involving chickens, there were no differences observed in anti-sheep red blood cell antibodies during primary responses, while antibody titers for the secondary response in algae-fed chickens were augmented compared to control animals.8 The differences may reflect the anatomical differences between the rodent and chicken immune systems.

Hayashi et al10 examined other antibody classes such as IgA and IgE in the context of mice orally immunized with a crude shrimp extract. They found that whereby both IgA (intestinal) and IgE (in serum) levels increased with antigen challenge, only IgA levels showed a greater enhancement in secretion with concurrent treatment with Spirulina extract (five-week feeding regimen).10 From this study they con­cluded that bluegreen algae does not seem to induce or enhance food allergic IgE-dependent reactions.  Furthermore, they suggest that when ingested along with or before a poten­tial antigenic threat, bluegreen algae may enhance IgA anti- body levels to protect against food allergies.

Along the same lines, further studies have suggested that blue-green algae may inhibit mast cell-mediated type I aller­gic reactions and even the anaphylactic reaction in rats.11,12 By injecting a blue-green algae extract intraperitoneally (100-1000mg/g body weight) one hour prior to an allergic challenge, mortality induced by the anaphylactic compound 48/80 was decreased, local allergic reaction activated by anti­dinitrophenyl (anti-DNP) IgE was inhibited, and serum hist­amine levels were decreased. In vitro experiments from this group provided similar results.

The effects of blue-green-algae on IgE-production and allergic reactions are encouraging, and warrant further studies in humans.

EFFECTS OF BLUE-GREEN ALGAE ON LEUKOCYTE TRAFFICKING
Much attention with regards to dietary modulation of the immune system has been given to stimulating activity of various immune cell types such as the phagocytic activity of macrophages, or the tumoricidal activity of natural killer cells. However, immune cell trafficking and the recruitment of immune cells from the systemic circulation are of equal importance. A recent study by Jensen et al5 involving

Table 3. Bio-modulatory Effects of Purified Compounds from Blue-Green Algae

Species Compound Effects References
All blue-green algae C-Phycocyanin Anti-inflammatory (reduces leukotriene B4) Free radical scavenger Selective inhibition of COX-2 Reduced tissue damage in acetic acid-induced colitis Hepatoprotective effect Romay 1999 Bhat & Madyastha 2000 Reddy et al, 2000 Gonzalez et al, 1999 Vadiraja et al, 1998
Spirulina Calcium Spirulan (Ca-Sp) Selectively inhibits penetration of virus into host cell (Herpex Simplex, human cytomegalovirus, measles, mumps, Influenza A, HIV-1) Hayashi et al, 1996
Reduces lung metastasis of melanoma cells by inhibition of tumor cell invasion of basal membrane Mishima et al, 1998
Cyanovirin-N Irreversible inactivation of several strains of HIV (inhibited cell-to-cell and virus-to-cell fusion) Boyd, 1997

Extracellular products

Promotion of lactic acid bacteria growth in vitro Parada et al, 1998
Aphanizomenon flos aquae Unknown Polysaccharide Induces apoptosis in some human tumor cell lines Stimulate the macrophage activity Jensen, msp in prep Pasco et al, in press.
Lyngbya lagerheimii Phormidium tenue Sulfolipid Inhibits syncytium formation upon HIV infection Gustafson et al, 1989
Phormidium tenue

Digalactosyl diacylglycerols

Inhibition of chemically induced skin tumors Tokuda et al, 1996

humans demonstrated that the blue-green alga Aphanizomenon flos-aquae was able to trigger within two hours the migra­tion of nearly 40% of the circulating natural killer cells. This effect was significantly more pronounced in long-term consumers than in naïve subjects. In the same study, Aphanizomenon flos-aquae was also shown to stimulate the mobilization of T and B lymphocytes. This effect appeared cell-type specific since no changes were observed on poly­morph nucleated cells.

ANTI-INFLAMMATORY PROPERTIES OF BLUE­GREEN ALGAE
Blue-green algae in general contain a significant amount of carotenoids, namely beta carotene, lycopene, and lutein, providing it with good antioxidant properties. By their quenching action on reactive oxygen species, antioxi­dants carry intrinsic anti-inflammatory properties. However, blue green algae also contains specific anti­inflammatory properties as a result of their high phyco­cyanin content. Phycocyanin is a photoharvesting pigment that provides the intense blue color in blue green algae. It can constitute up to 15% of the dry weight of a blue green algae harvest. C-phycocyanin is a free radical scavenger,26 and has significant hepatoprotective effects.27 Phycocyanin was shown to inhibit inflammation in mouse ears28 and pre-tory effect seemed to be a result of phycocyanin to inhibit the formation of leukotriene B4, an inflammatory metabo­lite of arachidonic acid.28

In a study performed in rats, the blue-green algae Aphanizomenon flos-aquae was also shown to decrease the plasma level of arachidonic acid.30 Aphanizomenon flos­aquae contains significant amounts of the omega-3 alpha-linolenic acid. Omega-3 fatty acids have been shown to inhibit the formation of inflammatory prostaglandins and arachidonate metabolites. Since Spirulina contains signifi­cant amounts of omega-6 gamma-linolenic acid, the anti­inflammatory properties of Spirulina must be due to differ­ent biochemical pathways.

ANTI-VIRAL EFFECTS
As part of its program aimed at discovering new anti­tumor and anti-viral agents in natural sources, the National Cancer Institute isolated extracts of blue-green algae (Lyngbya lagerheimii and Phormidium tenue) that were found to protect human lymphoblastoid T cells from the cytopathic effect of HIV infection.  Upon further investigation, a new class of HIV inhibitory compounds called the sulfonic acid-containing glycolipids were isolated; the pure compounds were found to be strikingly active against the HIV virus in the p24 viral protein and syncytium formation assays.13 Since this discovery, there has been further investigation into other species of blue-green algae for compounds with anti-viral properties. Some compounds worthy of mention include a protein called cyanovirin-N which appears to irreversibly inactivate diverse strains of the HIV virus and to inhibit cell-to-cell and virus-to-cell fusion.14 Other studies using a water-soluble extract of blue-green algae have found a novel sulfat­ed polysaccharide, calcium spirulan (Ca-SP), to be an antivi­ral agent. This compound appears to selectively inhibit the penetration of enveloped viruses (Herpes simplex, human cytomegalovirus, measles virus, mumps virus, influenza A virus, and HIV-1) into host cells, thereby preventing replica-tion.15-17 A review of anti-HIV activity of extracts from blue-green algae has been recently published.18

ANTI-CANCER EFFECTS
An early study on bluegreen algae’s cancer-preventive properties in humans was performed on tobacco-induced oral leukoplakia.19 Mathew et al found that oral supplemen­tation with Spirulina fusiformis resulted in complete regres­sion of 57% of subjects with homogenous leukoplakia. After discontinuation of Spirulina supplementation, almost half of the complete responders developed recurrent lesions.

In other studies, extracts of bluegreen algae have been used to treat cancer in animal models. In one model, inges­tion of an extract of Spirulina and Dunaliella was shown to inhibit chemically-induced carcinogenesis in hamster buc­cal pouches.20,21 Earlier studies often attributed the anti­cancer effect of algae to its content in carotenoids since beta-carotene has been shown to have an effect similar to that of algae extract. A more recent study, however, showed that the sulfated polysaccharide mentioned above, Ca-SP, appears to inhibit tumor invasion and metastasis.22 Both the in vitro and in vivo effects of Ca-SP suggest that the intra­venous administration of Ca-SP reduces the lung metastasis of melanoma cells by inhibiting the tumor invasion of the basement membrane. A water-based extract of Aphanizomenon flos aquae containing high concentrations of phycocyanin inhibited the in vitro growth of one out of four tumor cell lines tested, indicating that at least some tumor cell types may be directly sensitive to killing by phycocyanin (Jensen et al, manuscript in preparation). Another fresh-water blue-green algae, Phormidium tenue, contains several diacyl­glycerol compounds which effectively inhibited chemical-ly-induced skin tumors in mice.23 In addition, Spirulina was shown to have a modulatory effect on hepatic carcinogen metabolizing enzymes.24

Of major interest to ongoing research in inflammation as well as breast cancer is the finding that C-phycocyanin selectively inhibits COX-2, but has no effect on COX-1.25 The COX enzymes are involved in prostaglandin synthesis. Since COX-2 is over-expressed in many breast cancer cells, and inhibition of COX-2 leads to a markedly reduced tumor growth and blocks angiogenesis, the finding that phyco­cyanin specifically interferes with this pathway holds promise.

BLUE-GREEN ALGAE AS A BIOMODULATOR
Besides their effects on the immune system, blue-green algae have also been reported to modulate other systems and improve metabolism. In the past few years increasing attention has been given to the study of the therapeutic effects of blue-green algae. The anecdotal claims for such effects are numer­ous. Although there is limited data from controlled animal or clinical studies, such claims include improvement in condition of Alzheimer’s patients, overall enhancement of immune response, improvement in fibromyalgia, control of hyperten­sion, alleviation of depression and chronic fatigue, increased stamina, healing of internal and external lesions, increased mental acuity, and general improvement in overall well-being. This last section will review the scientific evidence supporting the therapeutic effects of blue-green algae.

EFFECTS ON METABOLISM
Several reports from different labs have shown that cer­tain species of blue-green algae have cholesterol-lowering effects in animal and human models.  In feeding experiments in rats, two studies have reported that the elevation in total cholesterol, LDL, and VLDL cholesterol in serum caused by cholesterol feeding was reduced when the high cholesterol diet was supplemented with 16% and 5% blue-green algae, respectively.31,32 In addition, Kato found that adipohepatosis induced by a high fat and high cholesterol diet was cured rapidly when the diet was supplemented with algae.31 Investigations into the mechanism of this phenomenon led to the finding that the algae-fed group showed a statistically sig­nificant increase in the activity of lipoprotein lipase, a key enzyme in the metabolism of triglyceride-rich lipoproteins.33

The hypocholesterolemic effect of blue green algae was also observed in humans in a study conducted on 30 patients with mild hyperlipidemia and mild hypertension.34 Patients took 4.2 grams of algae or placebo per day, and were observed for two months. At the end of the study, patients taking the algae showed a significant reduction of LDL-cho-lesterol (p<0.05) compared to the control group. LDL cho­lesterol increased back to baseline levels after administration of the algae was discontinued. In addition to lowering LDL cholesterol levels, the atherogenic index (a measure of fat deposition in arteries) declined significantly after four weeks of algae consumption.

In a recent study by Kushak et al, rats were fed the blue-green alga Aphanizomenon flos-aquae and total cholesterol level was monitored. After 43 days, cholesterol levels were significantly decreased when compared to the control group.30 Although Aphanizomenon flos-aquae contains a significant amount of the omega-3 polyunsaturated linolenic

Table 4. Biomodulatory Effects of Whole Blue-Green Algae on Metabolism

Algae Species Introduced as

Test Species

Effects Reference Effects
Aphanizomenon Food Rat Reduction of cholesterol Kushak et al, 2000 Reduction of cholesterol
Food Rat Reduction of blood glucose levels Drapeau et al, 2001 Reduction of blood glucose levels
Food Human Modulation of brain activity (EEG) Walker & Valencia, 1999 Modulation of brain activity (EEG)
Spirulina Food Human Reduced body weight Becker et al, 1986 Reduced body weight
Food Rat Reduction of cholesterol Kato et al, 1984 Reduction of cholesterol
Food Rat Increased activity of lipase Iwata et al, 1990 Increased activity of lipase
Food Rat Reduced glucose levels Takai et al, 1991 Reduced glucose levels
Food Rat Inhibition of maltase and sucrase Kushak et al, 1999 Inhibition of maltase and sucrase
Food Mouse Modulation of carcinogen metabolic enzymes Mittal et al, 1999 Modulation of carcinogen metabolic enzymes
Food Mouse Modulation of lead toxicity Shastri et al, 1999 Modulation of lead toxicity
Food Rat

Increased iron status during pregnancy and lactation

Kapoor & Mehta, 1998

Increased iron status during pregnancy and lactation

Nostoc Food Rat Reduction of cholesterol Hori et al, 1994 Reduction of cholesterol

acid, the effect on cholesterol levels seemed unrelated to the lipid content of the diets. Kushak et al30 proposed that the hypocholesterolemic effect of Aphanizomenon flos-aquae may be due to its chlorophyll content which was shown to stimulate liver function and decrease blood cholesterol.35

In a double-blind crossover study involving human patients, supplementing the diets of obese outpatients with 2.8 grams of bluegreen algae three times daily over a four week period resulted in a statistically significant reduction of body weight.36 In a study measuring the effect of bluegreen algae on glucose levels in diabetic rats, the water-soluble fraction was found to be effective in lowering the serum glucose level at fasting, while the water insoluble fraction suppressed glu­cose levels at glucose loading.37 In another study investigating the effect of the blue-green alga Aphanizomenon flos-aquae on rat intestinal mucosal digestive enzymes, it was observed that this alga specifically inhibited the activity of maltase and sucrase in a dose-dependent manner.38 Furthermore, this decrease in enzymatic activity was accompanied by a dose-dependent decrease in blood glucose.

The overall conclusion is that blue-green algae may have benefits on lipid and sugar metabolism, as well as liver function. Further human studies are needed to address the feasibility of using blue-green algae in conjunction with cholesterol-lowering medication.

OTHER EFFECTS OF BLUE GREEN ALGAE
Other research studies on blue green algae consumption deserve mention. Many reports exist in the literature on its antimicrobial effects.  The secretion of anti-microbial sub­stances is an important part of the competition for ecological niches in the natural environment. However, an interesting caveat exists. In one study, Spirulina was cultured in vitro, and the extracellular medium was shown to stimulate the growth of lactic acid bacteria.39 If the growth-promoting sub-stance(s) exist in sufficient amounts intracellularly, blue green algae may play a role in vivo by supporting friendly gut bacteria. This leads to other facets of health including gut health and nutrient absorption. On that note, consumption of Spirulina was shown to support the iron status and hemoglo­bin of rats during pregnancy and lactation.40 Spirulina fusiformis had a significant protective effect against lead-induced toxicity in rats.41 Finally, a report by Valencia et al has presented evidence that Aphanizomenon flos-aquae accelerates recovery from mild traumatic brain injury.42

CONCLUSION AND SUMMARY
Research results based on the numerous isolated com­pounds from blue green algae warrant the exploration of using whole algae as conjunctive therapy due to the possible synergistic effects of many phytochemicals within the whole algae. The emergence of composite algae supple­ments in contrast to single algae supplements may also yield further anti-inflammatory, immune-boosting, and metabolic benefits. A significant body of data suggests that blue green algae immunoenhancing properties could be useful in the adjunct treatment of various diseases involving 1) sup­pressed or exhausted immune system, and 2) inappropriate immune response including allergies, autoimmune diseases, and chronic inflammatory conditions. The data presented also suggests that blue green algae could be useful as an adjunct in the treatment of cancer and AIDS, and calls for the design of controlled human clinical studies.

REFERENCES

  1. Eisenberg D, Davis R, et al. Trends in alternative medicine use in the United States. 1990-1997.” JAMA. 1998:280(18):1569-1575.
  2. Astin J. Why patients use alternative medicine. JAMA. 1998;279(19):1548-1553.
  3. Sobel D. Rethinking medicine: improving health outcomes with cost-effective psychosocial interventions. Psychosomatic Medicine. 1995;57:234-244.
  4. Furnham A. Forey J. The attitudes, behaviors, and beliefs of patients of conventional vs complementary (alternative) med­icine. J Clini Psych. 1994;50:458-469.
  5. Jensen GS, Ginsberg DI, et al. Consumption of Aphanizomenon flos aquae has rapid effects on the circulation and function of immune cells in humans. JANA. 2000;2(3):50-58.
  6. Qureshi M, Ali R. Spirulina platensis exposure enhances macrophage phagocytic function in cats. Immunopharmacol Immunotoxicol. 1996;18(3):457-463.
  7. Hayashi O, Katoh T, et al. Enhancement of antibody production in mice by dietary Spirulina platensis. J Nutr Sci Vitaminol. 1994;40:431-441.
  8. Qureshi M, Garlich J, et al. Dietary Spirulina platensis enhances humoral and cell-mediated immune function in chickens. Immunopharmacol Immunotoxicol. 1996;18(3):465-476.
  9. Pugh N, Ross SA, ElSohly HN, ElLohly MA, Pasco DS.Isolation of three high molecular weight polysaccharides with potent immunostimulatory activity from Spirulina platensis, Aphanizomenon flos-aquae and Chlorella pyrenoidosa. Planta Medica. In Press.
  10. Hayashi O, Hirahashi T, et al. Class-specific influence of dietary Spirulina platensis on antibody production in mice. J Nutr Sci Vitaminol. 1998;44(6):841-851.
  11. Kim H, Lee E, et al. Inhibitory effect of mast cell-mediated immediate-type allergic reactions in rats by Spirulina. Biochem Pharmacol. 1998;55(7):1071-1076.
  12. Yang H, Lee E, et al. Spirulina platensis inhibits anaphylactic reaction. Life Sci. 1997;61(13):1237-1244.
  13. Gustafson K, Cardellina II J, et al. AIDS-antiviral sulfolipids from cyanobacteria (blue-green algae). J National Cancer Institute.1989;81:1254-1258.
  14. Boyd M. Protein isolated from blue-green algae inactivatesHIV. Antimicrob Agents Chemother. 1997;41:1521-1530.
  15. Hayashi K, Hayashi T, et al. A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti­human immunodeficiency virus activities. AIDS Res Hum Retrovir. 1996;12(15):1463-1471.
  16. Hayashi T, Hayashi K. Calcium spirulan, an inhibitor of enveloped virus replication, from a Blue-Green Alga Spirulina platensis. J Natural Products. 1996;59:83-87.
  17. Ayehunie S, Belay A, et al. Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis. J Aquir Immun Defic Syndr Hum Retrovirol. 1998;18(1):7-12.
  18. Schaeffer DJ, Krylov VS.  Anti-HIV activity of extracts and compounds from algae and cyanobacteria. Ecotoxicology and Environmental Safety. 2000;45:208-227.
  19. Mathew B, Sankaranarayanan R, et al. Evaluation of chemo-prevention of oral cancer with Spirulina fusiformis. Nutr Cancer. 1995;24(2):197-202.
  20. Schwartz J. Shklar G. Regression of experimental hamstercancer by beta carotene and algae extracts. J Oral Maxillofac Surg. 1987;45:510-515.
  21. Schwartz J, Shklar G, et al. Prevention of experimental oralcancer by extracts of Spirulina-Dunaliella algae. Nutr Cancer. 1988;11:127-134.
  22. Mishima T, Murata J, et al. Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated poly­saccharide derived from a blue-green algae, Spirulina platen-sis. Clin Exp Metastasis. 1998;16(6):541-550.
  23. Tokuda H, Nishino H, et al. Inhibition of 12-O-tetrade-canoylphorbol-13-acetate promoted mouse skin papilloma by digalactosyl diacylglycerols from the fresh water cyanobacteri­um Phormidium tenue. Cancer Lett. 1996;104(1):91-95.
  24. Mittal A, Kumar PV, et al.  Modulatory potential of Spirulina fusiformis on carcinogen metabolizing enzymes in Swiss albi­no mice. Phytother Res. 1999;13(2):111-114.
  25. Reddy CM, Bhat VB, et al.              Selective inhibition of cyclooxyge-nase-2 by C-phycocyanin, a biliprotein from Spirulina platen-sis. Biochem Biophys Res Commun. 2000;277(3):599-603.
  26. Bhat VB, Madyastha KM. C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochem Biophys Res Commun. 2000;275(1):20-25.
  27. Vadiraja BB, Gaikwad NW, Madyastha KM. Hepatoprotective effect of C-phycocyanin: protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicity in rats. Biochem Biophys Res Commun. 1998;249(2):428-431.
  28. Romay C, Ledon N, Gonzalez R. Phycocyanin extract reducesleukotriene B4 levels in arachidonic acid-induced mouse-ear inflammation test. J Pharm Pharmacol. 1999;51(5):641-642.
  29. Gonzalez R, Rodriguez S, et al. Anti-inflammatory activity of phycocyanin extract in acetic acid-induced colitis in rats. Pharmacol Res. 1999;39(1):55-59.
  30. Kushak RI, Drapeau C, Van Cott EM.  Favorable effects of blue-green algae Aphanizomenon flos-aquae on rat plasma lipids. JANA. 2000;2(3):59-65.
  31. Kato T, Takemoto K, et al. Effects of Spirulina on dietary hypercholesterolemia in rats. J Jap Soc Nutr Food Science. 1984;37:323-332.
  32. Hori KG, Ishibashi G, et al. Hypocholesterolemic effect of blue-green alga, ishikurage (Nostoc commune) in rats fed atherogenic diet. Plant Foods Hum Nutr. 1994;45:63-70.
  33. Iwata K, Inayama T, et al. Effects of Spirulina platensis on plasma lipoprotein lipase activity in fructose-induced hyper­lipidemic rats. J Nutr Sci and Vitaminology. 1999;36:165-171.
  34. Nakaya N, Honma Y, et al. Cholesterol lowering effect of Spirulina Nutr Rep Int. 1988;37:1329-1337.
  35. Vlad M, Bordas E, Caseanu E, Uza G, Creteanu E, Polinicenco C. Effect of cuprofilin on experimental athero­sclerosis. Biol Trace Elem Res. 1995;48(1):99-109.
  36. Becker E, Jakover B, et al. Clinical and biochemical evalua-tions of the alga Spirulina with regard to its application in the treatment of obesity: a double blind cross-over study. Nutr Rep Int. 1986;33:565-574.
  37. Takai Y, Hosoyamada Y, et al. Effect of water soluble and water insoluble fractions of Spirulina over serum lipids and glucose resistance of rats. J Jap Soc Nutr Food Science. 1991;44:273-277.
  38. Kushak R, VanCott E, Drapeau C, Winter H. Effect of algae Aphanizomenon flos-aquae on digestive enzyme activity and polyunsaturated fatty acids level in blood plasma. Gastroenterol. 1999;116:A559.
  39. Parada JL, Zulpa de Caire G, et al. Lactic acid bacteria growthpromoters from Spirulina platensis. Int J Food Microbiol. 1998;45(3):225-228.
  40. Shastri D, Kumar M, Kumar A. Modulation of lead toxicity by Spirulina fusiformis. Phytother Res. 1999;13(3):258-260.
  41. Kapoor R, Mehta U. Supplementary effect of Spirulina on hematological status of rats during pregnancy and lactation. Plant Foods Hum Nutr. 1998;52(4):315-324.
  42. Valencia A, Walker J.  A multi-axial treatment paradigm for mild traumatic brain injury to achieve reparative functional meta-plasticity. 3rd World Congress on Brain Injury, IBIA, Quebec City, June 1999.
  43. Drapeau C, Kushak RI, Van Cott EM, Winter HH. Blue-green alga Aphanizomenon flos-aquae as a source of dietary polyun­saturated fatty acids and a hypocholesterolemic agent in rats. J Am Chem Soc. In press.

Winter 2001 Vol. 3, No. 4  JANA  28

HAK PATEN: KEGUNAAN NIGELLA SATIVA (HABBATUS SAUDA) UNTUK MENINGKATKAN FUNGSI IMUN (KEKEBALAN TUBUH)

HAK PATEN:
KEGUNAAN NIGELLA SATIVA (HABBATUS  SAUDA) UNTUK MENINGKATKAN FUNGSI IMUN (KEKEBALAN TUBUH)
Use of Nigella sativa to increase immune function

Document Type and Number:
United States Patent 5482711

Abstract:
A pharmaceutical composition containing an extract of the plant Nigella sativa is disclosed for treating cancer, preventing the side effects of anticancer chemotherapy, and for increasing the immune function in humans.

Inventors:
Medenica, Rajko D. (One Ocean Point, Port Royal Plantation, Hilton Head Island, SC, 29928)
Application Number:
08/111631
Filing Date:
08/25/1993
Publication Date:
01/09/1996
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Referenced by:
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Primary Class:
424/776
Other Classes:
514/885, 424/725
International Classes:
A61K39/39; A61P35/00; A61K35/78
Field of Search:
424/195.1, 514/885
US Patent References:
4687761    August, 1987    Liu    514/26    Pharmaceutical composition for increasing immunity and decreasing side effects of anticancer chemotherapy
4945115    July, 1990    Liu    514/731    Process for preparing ferulic acid
Other References:
Elkadi et al. 71st Annual Meeting of the Federation of American Societies for Experimental Biology Washington D.C. Mar. 29-Apr. 2, 1987, 46(4), 1222.
Elkadi et al. Arch AIDS Res 1 (2-3), pp. 232-233, 1987.
Dey, K. L., and Bahadur, R., 1984, Indigenous Drugs of India, International Book Distributors, Dehradun, India.
Kirtikar, K. R., and Basu, B. D., 1987, Indian Medicinal Plants, vol. I, International Book Distributors, Dehradun, India.
Nadkarni, K. M., 1976, “Crocus sativus, Nigella sativa,” Indian Materia Medica, vol. I, K. M. Nadkarni (ed), Bombay Popular Prakashan, Bombay, India.
Agarwal, R.; Kharya, M.D. and Shrivastava, R., 1979, “Antimicrobial Anthelmintic Activites of the Essential Oil of Nigella Sativa Linn.,” Indian J. Exp. Biol. 17:1264-5.
Akhtar, M. S. and Riffat, S., 1991, “Field Trial of Saussurea Lappa Roots Against Nematoides and Nigella Sativa Seeds Against Cestodes in Children,” J.P.M.A. 41(8):185-7.
al-Awadi, F. M.; Khatter, M. A. and Gumaa, K. A., 1985, “On the Mechanism of the Hypoglycaemic Effect of a Plant Extract,” Diabetologia 28:432-4.
al-Awadi, F.; Fatania, H. and Shamte, U., 1991, “The Effect of a Plant’s Mixture Extract on Liver Gluconeogenesis in Streptozotocin Induced Diabetic Rats,” Diabetes Res. (Scotland) 18(4):163-8.
Aruna, K. and Sivaramakrishnan, V. M., 1990, “Plant Products as Protective Agents Against Cancer,” Indian J. Exp. Biol. 28(11):1008-11.
Aruna, K. and Sivaramakrishnan, V. M., 1992, “Anticarcinogenic Effects of Some Indian Plant Products,” Fd. Chem. Toxic. (England) 30(11) :953-6.
Bitterman, W. A.; Farhadian, H.; Abu Samra, C.; Lerner, D.; Amoun, H.; Krapf, D. and Makov, U. E., 1991, “Environmental and Nutritional Factors Significantly Associated with Cancer of the Urinary Tract among Different Ethnic Groups,” Urol. Clin. North Am. 18(3):501-8.
Datta, A. K.; Biswas, A. K. and Ghosh, P. D., 1983, “Chromosomal Variations in Callus Tissues of Two Species of Nigella,” The Nucleus 26(3):173-7.
Elkadi, A. and Kandil, O., 1987, “The Black Seed Nigella Sativa and Immunity: Its Effects on Human Cell Subsets,” Fed.Proc. 45(4):1222.
Finter, N. B., 1969, “Dye Uptake Methods for Assessing Viral Cytopathogenicity and Their Application to Interferon Assays,” J. Gen. Virol. 5:419-427.
Kumar, B. H. and Thakur, S. S., 1989, “Effect of Certain Non-Edible Seed Oils on Growth Regulation in Dysdercus Similis (F),” J. Anim. Morphol. Physiol. 36(2) pp. 209-218.
Medenica, R.; Alonso, K.; Huschart, T. and Tyler, K., 1990, “Tumor Tissue Culture for Determining Efficient Drug for Intra-Arterial, Intra-Hepatic Chemotherapy of Colon Carcinoma Liver Metastasis,” Abstract presented at Conference on Combining BRM with Cytotoxic in the Treatment of Cancer.
Merkel, D. E., Dressler, L. G. and McGuire, W. L., 1987, “Flow Cytometry Cellular DNA Contents and Prognosis in Human Malignancy,” J. Clin. Oncol., 5:1690-1703.
Metcalf, D., 1984, Clonal Culture of Hematopoietic Cells, Elsevier/North American Biomedical Press.
Metcalf, D., 1985, “The Granulocyte-Macrophage Colony-Stimulating Factors,” Science 229:16-22.
Nair, S. C.; Salomi, M. J.; Panikkar, B. and Pannikar, K. R., 1991, “Modulatory Effects of Crocus Sativus and Nigella Sativa Extracts on Cisplatin-Induced Toxicity in Mice,” J. Ethnopharmocol 31(1): 75-83.
Salmon, S. E.; Hamburger, A. W.; Soehnlein, B.; Durie, B. G.; Alberts, D. S. and Moon, T. E., 1978, “Quantitation of Differential Sensitivity of Human-Tumor Stem Cells to Anticancer Drugs,” N. Eng. J. Med. 298:1321-7.
Salomi, N. J.; Nair, S. C.; Jayawardhanan, K. K.; Varghese, C. D. and Panikkar, K. R., 1992, “Anititumour Principles from Nigella Sativa Seeds,” Cancer Lett. 63(1):41-6.
Salomi, M. J.; Nair, S. C. and Panikkar, K. R., 1991, “Inhibitory Effects of Nigella Sativa and Saffron (Crocus Sativus) on Chemical Carcinogenesis in Mice,” Nutr. Cancer 16(1): 67-72.
Sayed, M. D., “Traditional Medicine in Health Care,” 1980, J. Ethnopharmocol. 2(1): 19-22.
Shayeb, N. A. and Mabrouk, S. S., 1984, “Utilization of Some Edible and Medicinal Plants to Inhibit Aflatoxin Formation,” Nutrition Reports International 29(2): 273-289.
Siddiqui, M. B.; Alam, M. M.; Husain, W. and Sharma, G. K., 1988, “Ethno-medical Study of Plants Used for Terminating Pregnancy,” Fitoterapia LIX(3): 250-2.
Srivastava, K. C., 1989, “Extracts from Two Frequently Consumed Spices-Cumin (Cuminum cyminum) and Turmeric (Curcuma Longa)-Inhibit Platelet Aggregation and Alter Eicosanoid Biosynthesis in Human Blood Platelets,” Prostaglandins Leukot Essent Fatty Acids 37(1): 57-64.
Tennekoon, K. H.; Jeevathayaparan, S.; Kurukulasooriya, A. P. and Karunanayake, E. H., 1991, “Possible Hepatotoxicity of Nigella sativa Seeds and Dregea Volubilis Leaves,” J. Ethnopharmocol. 31(3): 283-9.
Vihan, V. S. and Panwar, H. S., 1987, “Galactopoietic Effect of Nigella Sativa (H-Kalonji) in Clinical Cases of Agalactia in Goats.” Indian Vet. J. 64:347-9.
Von Hoff, D. D.; Cowan, J.; Harris, G. and Reisdorf, G., 1981, “Human Tumor Cloning: Feasibility and Clinical Correlations,” Cancer Chemother. Pharmacol. 6:265-271.
Primary Examiner:
Robinson, Douglas W.
Assistant Examiner:
Lee, Howard C.
Attorney, Agent or Firm:
DeWitt Ross & Stevens
Claims:
I claim:

1. A method for activating immune competent cells in humans in order to increase the immune function in humans, the immune competent cells being selected from the group consisting of CD19, HLADR, NKCD3-/CD56+ and CD38, the method comprising administering to humans an effective dose of an extract from Nigella sativa at a concentration which is effective to activate the immune competent cells by reducing the presence of interferon inhibitor factor or lymphokine inhibitor factor.

2. The method of claim 1 wherein the effective dose is between about 20 and about 40 grams of the extract per day.

3. The method of claim 1 wherein the effective dose is about 30 grams of the extract per day.

4. A method for increasing antibody producing B cells in humans, the B cells having minor antigen binding sites, comprising administering to humans an effective dose of an extract from Nigella sativa, at a concentration which is effective to free the tumor antigen binding sites on the B cells thereby increasing the antibody producing B cells.

5. The method of claim 4 wherein the effective dose is between about 20 and about 40 grams of extract per day.

6. The method of claim 4 wherein the effective dose is about 30 grams of extract per day.

Description:

FIELD OF THE INVENTION

The present invention is generally directed to the fields of medicine and pharmacology, and specifically directed to using the plant seed extract of Nigella sativa Linn (N. sativa) in the treatment of cancer, viral diseases, protection from side effects of chemotherapy and growth factor for bone marrow in hematopoiesis.

CITED REFERENCES

A full bibliographic citation of the references cited in this application can be found in the section preceding the claims.

DESCRIPTION OF THE PRIOR ART

A variety of herbal and plant extracts or preparations are available today for treating any number of diseases affecting the human body. Some preparations have been known for literally thousands of years while others are just being discovered to have curative effects. Effective plant extracts are highly desired as a “natural” way to treat a disease. It is believed that natural preparations will not have as much of an adverse effect on the body as synthetic preparations.

One of the primary targets for treatment is cancer. Anticancer remedies are available today which are effective in killing cancer cells. However, many of these medicaments also damage or kill off normal cells or have other serious side effects. It is therefore vitally important to develop an anticancer program which is specific for the cancerous growth in a body, but which is not toxic to the rest of the body system. Ideally, the program will include treatment using natural plant extracts. As used in this disclosure, the term “body” or “patient” can include any warm-blooded mammal, but is specifically intended to refer to the human body.

The medicinal properties of various spices and herbs in general is known (Srivastava, 1989). U.S. Pat. No. 4,986,895 to Grossman et al. is directed to use of water-soluble plant extracts in the treatment of virus skin infections. U.S. Pat. No. 5,178,865 to Ho et al. is directed to the use of Chinese herbal extracts in the treatment of HIV related disease in vitro. A total of 56 herbal extracts were screened for anti-HIV activity using in vitro techniques.

Aruna (1990) also describes the use of spices, leafy vegetables and condiments having diverse medicinal properties. Products of 20 spices or leafy vegetables were screened for anti-carcinogenic activity using induction of glutathione-S-transferase. One of the plants utilized was Cuminum cyminum Linn (C. cyminum), also known as cumin. All of the spices and leafy vegetables were tolerated well and no toxic effects were seen.

Bitterman et al. (1991) disclose a study that was performed on a population of 964 adult patients, of which 28% suffered from malignant diseases of the urinary tract and 72% from a wide spectrum of the nine neurologic diseases. The results conclude that the use of C. cyminum in the diet may contribute to the prevention of diseases mediated by peroxidation of lipids.

Aruna and Sivaramakrishnan (1992) reported on anticarcinogenic properties of some spices. Cumin seeds (C. cyminum Linn) were studied. Cumin seed significantly inhibited some carcinogenesis.

Another plant extract from the plant N. sativa has shown a wide range of medical use. N. sativa is an annual herb belonging to family Ranunculaceae. Other species of Nigella include Nigela arvensis and Nigella damascena. Induction of callus cultures indicates considerable chromosomal variations in callus tissues between the different species of Nigella (Datta, et al. 1983).

N. sativa is characterized by an erect branched stem and alternate finely divided, feathery, grayish-green leaves. The bluish-white, star-shaped flowers are terminal and solitary. Petals are absent. The fruit is a globose capsule with small, black, rough seeds. The plant is cultivated in India, Bangladesh, Turkey, Middle-east and the Mediterranean basin mainly for its seeds or “black cumin” which is almost entirely used for edible and medical purposes, such as spices and for treatment of various diseases.

The ripe seeds of N. sativa, also known as Kalajira or Kalaonji, are known to have a wide range of medicinal uses (Kirtikar et al. 1982, and Chopra et al. 1982). The constituents of the seeds include saponin, an essential oil, a bitter compound (nigellone) and tanners. These substances have been shown to have diuretic (Nadkarni 1976), cholagogic and antispasmodic (Tennekoon, et al. 1991), carminative (Shayeb and Mabrouk, 1984), galactogogic (Vihan 1987), antibacterial (Hassan, et al. 1989), antifungal (Agarwal, et al 1979), anthelminthic (Akhtar 1991) and emmenagogic (Siddiqui et al. 1988) properties. al-Awadi, et al. (1985) have demonstrated an antidiabetic effect of N. sativa plant extract.

N. sativa has been reported to be used in Egyptian folk medicine as a diuretic and carminative (Sayed 1980). The oil is used in the treatment of asthma, respiratory oppression and coughs. The active principle, nigellone, has been isolated from the volatile oil fraction and is reported to be useful in the treatment of bronchial asthma.

The petroleum ether extract of the seeds at 1000-62.5 parts per million (ppm) concentrations was found to have the same activities as growth regulating juvenile hormone when tested against the fifth instar larvae of Dysdercus similis (Kumar et al. 1987).

al-Awadi et al. (1981) is directed to the study of the effect of a plant’s mixture extract containing N. sativa on liver gluconeogenesis. The researchers report that non-insulin dependent diabetes mellitus is treated in Kuwait by a plant mixture extract, which contains N. sativa. In this study, a powdered mixture of equal portions of N. sativa, Linn, Commiphora myrrh, Eng, Ferula Asafoetida, Linn, Aloe vera, Linn, and olibanum was boiled in distilled water for 10 minutes. Diabetic animals were given a daily dose by gastric intubation. The results indicate that the anti-diabetic action of the plant’s extract is at least partly mediated through decreased liver gluconeogenesis.

Elkadi and Kandil (1987) are directed to N. sativa and its effect on human T-cells. N. sativa was tested in volunteers with a low helper T-cell to suppressor T-cell ratio. The results indicated an increase in the helper T-cell population in the experimental group. Further, the helper T-cell to suppressor T-cell ratio increased while the ratio within the control groups remain the same.

Nair et al. (1991) investigated the effects of N. sativa as potential protective agents against cisplatin-induced toxicity in mice. Some protective effects were shown by the use of N. sativa extracts.

Salomi, N. J., et al. (1992) studied N. sativa seeds containing certain fatty acids for antitumor activities against Ehrlich ascites carcinoma (EAC), Dalton’s lymphonia ascites (DLA) and Sarcoma-180 (S-180) cells. The paper presents the results of in vitro and in-vivo antitumor experiments. The active antitumor principle was isolated. It was found that the active principle was cytotoxic for EAC cells, KB cells and lymphocytes.

Salomi et al. (1991) reported the effect of the active principle isolated from N. sativa in inhibiting chemically induced skin carcinogenesis. Intraperitoneal administration of N. sativa extract was shown to prevent the incidents of soft tissues sarcomas and reduced tumor diameters in treated groups.

Metcalf (1984, 1985) reported the inhibitory effects of N. sativa on chemical carcinogenesis in mice. However, there was no evidence in these papers of the destruction of human tumor cells.

SUMMARY OF THE INVENTION

The present invention provides an anticancer remedy and treatment which has, as its active ingredient, the extract of the plant N. sativa. When used properly, the medicament of the present invention is useful in treating cancer, preventing toxicity of anticancer drugs in human body and in increasing immune function.

The present invention is specifically directed to a pharmaceutical composition for the treatment of cancer comprising a pharmaceutical preparation consisting essentially of an extract from Nigella sativa, at a concentration which is effective to destroy cancer cells in a patient.

Additionally, the present invention is directed to a pharmaceutical composition for the treatment of the side effects of anticancer therapy consisting essentially of an extract from Nigella sativa, at a concentration of the extract which is effective to reduce the side effects of anticancer therapy.

The present invention is also directed to a method for treating humans suffering from the side effects of anticancer chemotherapy using the extract of Nigella sativa, comprising administering to humans effective doses of the composition described above.

Further, the present invention is directed to a method for increasing the immune function in humans comprising administering to humans effective doses of an extract from Nigella sativa, at a concentration of the extract which is effective to increase the immune function.

The present invention is also directed to a method for protecting the normal cells from cytopathic effects of virus, comprising administering to humans effective doses of an extract from Nigella sativa, at a concentration of the extract which is effective to protect against the cytopathic effects of the virus.

The present invention is still further directed to a method for increasing antibody producing B cells, comprising administering to humans an extract from Nigella sativa, at a concentration which is effective to increase the antibody-producing B cells.

The present invention is also directed to a process for inhibiting tumor cells without affecting normal or nontumor cells in a patient comprising administering to a patient an extract from Nigella sativa, at a concentration which is effective to inhibit the tumor cells without affecting nontumor cells.

The present invention is also directed to a method for stimulating bone marrow formation in humans comprising administering to humans effective doses of an extract from Nigella sativa, at a concentration of the extract which is effective to stimulate bone marrow formation.

In general, N. sativa extract helps stimulate bone marrow cells, protects the normal cells from cytopathic effects of virus, destroys tumor cells and increases antibody producing B cells. It protects the bone marrow against chemotherapy and at the same time, can act as an anticancer agent. All these factors makes N. sativa seed extract an ideal candidate to be used as vaccine for cancer prevention and cure.

N. sativa plant extract is more effective than standard chemotherapeutic anti-cancer drugs. N. sativa extract stimulates bone marrow cells, protects the normal cells from cytopathic effects of virus, destroys tumor cells and increases antibody producing B cells. Further, it protects the bone marrow against chemotherapy and can act as an anti-cancer agent.

N. sativa plant extract also has been found to help restore immune competent cells in immunosuppressed cancer patients and to overstimulate bone marrow formation in normal individuals.

N. sativa extract helps free tumor antigen binding sites on B cells. The administration of the N. sativa extract, rather than the increase of the immune competent cell number, has been found to help free tumor antigen binding sites on B cells, thereby elevating the CD19 and associated cell population. When the antigen binding site on the immunoglobulin molecule on the surface of B cells is free because of N. sativa treatment, it binds to the tumor associated antigen thereby generating an immune response against the antigen.

Protection of Human Amniotic “WISH” cells from cytopathic effects of vesicular stomatitis virus (VSV) was also observed upon administration of N. sativa plant extract. Additionally, the serum interferon level is found to increase; and, hence, the plant extract of N. sativa has interferon-like antiviral activity. This is an example of interferon level increasing in the circulation, preventing viral diseases and, in addition, possibly curing viral diseases.

N. sativa promotes anti-tumor activity. Data from pharmacosensitivity screening indicates anti-tumor activity of N. sativa plant extract mainly against melanoma and colon cancer types. N. sativa plant extract destroys tumor cells and leaves normal cells alone, possibly because of its ability to bind to cell surface asialofeutin (lectin) in diseased cells, which causes aggregation and clumping of tumor cells. It also blocks enzymes and inappropriate gene products involved in nucleic acid synthesis and metabolism.

Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the elevation in CD19, HLADR, NK CD3-/CD56+ and CD38 populations of peripheral blood cells from cancer patients upon incubation with N. sativa extract over a 18 hour period at 37° C. in 5% CO 2 incubator as shown in Experiment 2.

FIG. 2 is a graph illustrating the percent protection of 3.5×10 4 WISH cells from the cytopathic effects (CPE) of vesicular stomatitis virus (VSV) by serial dilutions of N. sativa extract as described in Experiment 3.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The following definitions will be used for the present application:

B cells: B cells or B lymphocytes secrete proteins/antibodies that protect the human body against infections.

CD3: Cluster Differentiation 3. These are the antibodies which indicate Activated T lymphocytes which are used by the body for its protection against foreign harmful germs.

CD19: Cluster Differentiation 19. These are antibodies which help detect B lymphocytes. Elevation in CD19 indicates an elevation in B lymphocytes and vice versa.

CD56: Cluster Differentiation 56 are antibodies which inhibit Natural Killer target cell interactions in certain systems.

G-CSF: Granulocyte-Colony Stimulating Factor

GM-CSF: Granulocyte Macrophage-Colony Stimulating Factor.

HLADR: Human Leukocyte antigen DR. DR designates a genetic locus or the antigen of the major histocompatibility complex corresponding to the locus. An increase in HLADR indicates an elevation in immunological parameters against the disease.

NK: Natural Killer cells. NK is an indication to detect the Natural Killer cells.

NKCD3-/CD56+: Natural Killer Cluster Differentiation 3-/Cluster Differentiation 56+.

Preparation of N. sativa Extract:

The process for preparing the N. sativa extract comprises grinding the seeds and separating the extract of the N. sativa with appropriate solvents such as alcohol or water, removing lipids by extraction with ether or petroleum ether, crystallization or chromatographic fractionation and then mixing its components in the desired proportion. The N. sativa extract can then be prepared in 3 forms–oil, fluid and crystal–by processes known to the art. The preferred process for the preparation of N. sativa extract is described in Experiment 1.

Administration:

The extract of N. sativa may be administered by itself or in admixture with an appropriate excipient or carrier. The preparation may be administered to the patient by enteral, such as oral or rectal, and parenteral, such as intraperitoneal, intramuscular, intravenous or subcutaneous route. The preparation may also be administered in combination with supplements, such as antiviral agents, immune modulators, antibodies, other chemotherapeutic agents, or combinations thereof. The preparation may additionally be administered in dosage form, such as by capsules, tablets, suppositories or the like.

Dosages:

N. sativa has been found to be most effective when administered at a dosage of 30 g per day, with an effective range of 20-40 g per day.

N. sativa extract is also known to confer protection of human amniotic “WISH” cells against cytopathic effects of vesicular stomatitis virus (VSV). The plant extract at a dilution of 1:1000 when incubated with 3.5×10 4 WISH cells for 18 to 24 hours at 37° centigrade gave maximum protection.

Theoretically, a patient weighing 70 kg has about 7×10 13 cells in its body and N. sativa will be useful at a dosage between about 20 and 40 g per day and preferably about 30 g per day to protect against viral attack in virus endemic areas.

The invention is further illustrated by the following experiments and tests but not limited by the following experiments:

EXPERIMENTS

To evaluate the usefulness of N. sativa for cancer treatment, the activity of the N. sativa extract was measured on bone marrow and peripheral white blood cells. In vitro antiviral antitumor and growth factor like activity were also measured.

N. sativa extract was incubated with bone marrow cells to determine the growth of the cells. The results were compared with that of bone marrow growth factors and biological response modifiers (GM-CSF, G-CSF, erythropoietin, interferon, IL-2, and STS). Mouse connective cells and human amnion or “WISH” cells were also assayed.

The following calculations were used in the examples:

Calculation of Percent Elevation: ##EQU1## in which R T is the average cell count of the experimental well with extract/growth factors, R C is the average background cell count with control, R MAX is the average maximum elevation without extract/growth factor.

Calculation of Percent Inhibition: ##EQU2## in which R T is the average cell count of the experimental well with extract/Abrin control, R C is the average background cell count with control, R MAX is the average maximum inhibition without extract/Abrin control.

Following are descriptions of some procedures used in the experiments. Universal safety precautions, known to the art, must be observed when handling all biological materials.

Procedure 1

Flow Cytometry Direct Immunofluorescence Staining

Automated Flow Cytometry (FC) provides an efficient, sensitive and quantitative method to analyze cell populations, sub-populations, and their components in suspension. Cells express and shed surface antigens throughout their life indicating their classification, stage of maturation, activation state and disease state. Monoclonal antibodies (mABs) have been developed that specifically bind these surface antigens. Established clinical applications are in leukemia and lymphoma diagnosis, T-cell subset analysis, monitoring transplant rejection, monitoring the effects of chemotherapeutic agents on different cell types and measuring cellular activation.

Whole blood, bone marrow or cellular suspension is first treated with a red blood cell lysing solution, washed, and then mixed with a labeled mAB against a specific membrane antigen. The direct staining procedure is then followed by analysis with the FACSCAN Immunocytometry Systems flow cytometer (FACSCAN Users’ Guide, Becton Dickinson).

All specimens are generally labeled with the following information: 1) Patient name; 2) Date drawn; 3) Time drawn; 4) Phlebotomist’s initials; and 5) Panel number. Patient specimens are identified by the nurse or phlebotomist generally by checking the wristband to verify the name of the patient. A unique tracking number is assigned to each individual specimen to assure proper specimen tracking.

Peripheral Blood is aseptically collected in an appropriate container such as a lavender top (EDTA anti-coagulant) vacutainer tube and delivered at room temperature preferably within 48 hours. The minimum amount required is approximately 2 ml whole blood per panel ordered. The optimum amount required is approximately 3 ml whole blood per panel ordered

The bone marrow (BM) specimen is aseptically collected in a container such as a lavender top (EDTA) vacutainer tube and transported at room temperature within 48 hours. The minimum amount required is approximately 2 ml of BM specimen per panel ordered. The optimum amount is approximately 3 ml of BM specimen per panel ordered.

Solid tumors include those from the breast, lymph node, colon, ovarian, lung, and skin. Reference is made to the HTCA Pharmacosensitivity Procedure (Procedure 5, infra.) for a description of the procedures. The tumor should be delivered at room temperature within 48 hours of extraction. The minimum amount required is approximately 2 ml of cells at a concentration of 1×10 6 /ml per panel ordered. The optimum amount is approximately 3 ml of cells at a concentration of 1×10 6 /ml per panel ordered.

Materials:

The materials required are as follows:

EDTA Vacutainer Blood Collection Tubes

12×75 mm plastic test tubes

Serofuge

FACSCAN Flow Cytometer

Micropipettes (20 ul and 100 ul)

Micropipet Tips

Repeater Pipet

Small Beakers

Gauze Squares

Monoclonal Antibodies

Lysing Solution

PBS

0.5% p-Formaldehyde

Transport Media

50 ml Conical Tubes

Repeater Pipet Tips

Safety Shield

Sorvall TR6000 centrifuge

Coulter Cytotrol control cells

Reagents:

The reagents are prepared as follows:

A. Monoclonal antibodies–see package inserts for proper reconstitution and storage of various mABs. For Coulter mABs, reconstitute according to package insert and then make a 1:2 dilution in sterile water and place in an appropriate container.

B. Lysing solution–(Becton-Dickinson order #92-0002). To prepare 1× working solution, add 10 ml 10× lysing stock solution to 90 ml distilled water. Mix well. Store at room temperature. Discard after one week.

C. Phosphate Buffered Saline (PBS) without Ca++ and Mg++ (Gibco order #310-4190AJ)

D. 0.5% Paraformaldehyde. Dissolve 2.5 g paraformaldehyde (Baker S-898-7) in 500 ml of 1× PBS without Ca++ or Mg++.

E. Coulter Cytotrol control cells. Reconstitute Cytotrol control cells according to manufacturers’ package insert. These cells must be prepared fresh daily.

Quality Control:

A leukogate and a negative control tube must be run for each patient. Tubes for Cytotrol control cells should be included for each mAB used that day.

Procedure:

Label test tubes with patient name and appropriate mAB. Using a micropipet, to each tube add 20 ul of the appropriate antibody. Using a micropipet, to each tube add 100 ul of the patient whole blood. Vortex each tube. Incubate the tubes at room temperature for 15 minutes. Using the repeater pipet (set on 4) add 2 ml of 1× lysing solution to each tube. Incubate the tubes at room temperature for 10 minutes. Centrifuge tubes in serofuge set on high for 3 minutes or in Sorvall TR6000 for 5 minutes at 3000 RPM. Pour off supernatant in waste beaker, being careful not to splash. Blot top of tube on gauze square. Vortex each tube to break pellet (use safety shield). Using repeater pipet (set on 4) add 1 ml of 1× PBS to each tube and vortex each tube (use safety shield). Centrifuge tubes in a serofuge set on high for 3 minutes .or in Sorvall TR6000 for 5 minutes at 3000 RPM. Pour off supernatant in waste beaker being careful not to splash. Blot top of tube on gauze square. Vortex tubes to break pellet (use safety shield). Using repeater pipet (set on 2) add 0.5 ml of 0.5% paraformaldehyde. Vortex. Keep tubes covered with parafilm and in the refrigerator until ready for flow cytometric analysis.

Specimen Analysis:

Analyze each tube on the FACSCAN flow cytometer using Simulset software for surface marker evaluation. (Refer to FACSCAN Users’ Guide and Simulset Software Manual.) Use the complete blood count (CBC) information (be sure CBC was drawn same date and time as FC sample) to enter the total white blood count and % lymphocytes.

Procedure 2

Colony Forming Cells

In order to be able to calculate the potency of the bone marrow, the stem cell assay is performed. Information on the status of Colony Forming Units (CFU) before therapy is performed is obtained. This will allow one to precisely define the medication according to the possible toxicities developed. Also, this will allow the prediction of the toxicity of the medication. The character of CFU when stem cells are treated with different medications will help determine predominance of development of cell lines. It has been demonstrated that interferon prolongs myelopoietic differentiation; therefore, the number of CFU will increase. This will demonstrate efficiency of interferon on bone marrow recovery and confirm the protective activity of interferon to the bone marrow.

With different hemopoietic growth factors, CFU development for the different cell lines can be directed.

Specimen Requirements, Collection and Handling:

Specimen Requirements: Bone marrow sufficient to yield a minimum of 4.0 ml and an optimum of 5.0 ml mononuclear cells at a concentration of 1.0×10 6 cells/ml.

Bone Marrow collection procedure: Prepare 4-6 50 ml Falcon tubes containing 20 ml of tissue transport media. Transport the media in the cooler with frozen ice chips to the procedure. A mask, gloves and gown must be worn during the procedure. Inject 2500 units of preservative-free heparin into the media tubes using sterile technique. This must be done no earlier then 15 minutes before obtaining the bone marrow specimen. Mix well. When bone marrow is handed to the technologist in a syringe, carefully inject into the media tubes, rinsing with media twice. Discard syringe in the Sharps container. Cap tube and mix well. Label the specimen with the following: 1) Patient’s full name; 2) Date specimen was received; 3) Time specimen was received; and 4) Initials of technologist who obtained the specimen. Place in cooler with ice chips for transportation.

Materials:

The following materials are necessary:

Ficoll-Hypaque

Capped Test Tubes [Sterile] 15 ml

Falcon Tubes-50 ml

Sterile Laminar Flow Hood with UV Light

Centrifuge

Sterile Disposable Pipettes

1.0 ml

5.0 ml

10.0 ml

25.0 ml

RPMI 1640 [1×]

Iscove’s Modified Dulbecco’s Medium [1×]

Fetal Bovine Serum

Penicillin/Streptomycin

L-Glutamine [100×]

Trace Elements [100×]

2-Mercaptoethanol

Insulin Transferrin-Sodium Selenite Media Supplement (ITS)

Glass Test Tubes 12×75 ml

Trypan Blue Stain (0.4%)

Hemocytometer With Cover Slips

Microscopes (Light, Inverted)

Tissue Culture Flasks-25 CM 2

Somatostatin-50 μg/ml (Sandoz-Sandostatin)

Interferon-3×10 6 U/ml (Roferon A-Hoffman LaRoche)

Interleukin-2 (Boehringer Mannheim GMBH Cat #799068)

GM-CSF (Amgen Cat #13050)

G-CSF (Amgen Cat #10050)

Epogen (Amgen Cat #06050)

PIXY 321 (Immunex)

CO 2 Incubator

Hand Counter

Ice Chips

Media and Reagent Preparation:

After preparation of each media, the final product is labeled with the following: 1) Name of Product; 2) Preparation Date; 3) Expiration Date; 4) Storage Recommendations; and 5) Technologist’s Initials. 10% RPMI 1640 (1×) G,G Fortified:
______________________________________
AMOUNT COMPONENT
______________________________________

500.0 ml RPMI 1640 (1X) with L-Glutamine
50.0 ml Fetal Bovine Serum (Qualified, Heat
Inactivated)
5.0 ml L-Glutamine (100X), 200 mM
1.5 ml Penicillin/Streptomycin
2.0 ml Trace Element Mix (100X),
Lyophilized
0.5 ml 2-Mercaptoethanol
5.0 ml ITS Media Supplement, Lophilized,
Gamma-Irradiated
______________________________________

All components are combined under a sterile laminar flow hood, and sterilized by filtration using a 0.22 micron membrane filter with a 60 micron prefilter. The prepared media is labeled and stored at 2°-10° C.

IMDM/RMPI 1640 [1×] (Tissue Transport Media):
______________________________________
AMOUNT COMPONENT
______________________________________

500.0 ml RPMI 1640 [1X]
500.0 ml IMDM [1X]
100.0 ml FBS
5.0 ml Penicillin/Streptomycin
10.0 ml L-Glutamine (100X)
5.0 ml Trace Element Mix (100X)
5.0 ml Nonessential Amino Acids
(100X)
0.5 ml MEM Vitamins
0.5 ml 2-Mercaptoethanol
5.0 ml ITS
______________________________________

All components are combined under a sterile laminar flow hood, and sterilized by filtration using a 0.22 micron membrane filter with a 60 micron prefilter. The prepared media is labeled and stored at 2°-10° C.

Interferon:

Use 1 vial of Roferon A Interferon available from Hoffmann-LaRoche Inc. (3,000,000 U). Take 1 ml Interferon and add 39 ml of RPMI(1×) media to yield a concentration of 75,000 U/ml. Make 40 1 ml aliquots of this dilution and freeze. For the Colony Forming Assay use 1 ml of the 75,000 U/ml Interferon for each 25 cm 2 flask containing 4.0 ml of media and 0.5 ml of cells.

Interleukin-2:

Thaw interleukin-2 and take 5 ml of interleukin-2 containing 200 units per ml. Immediately freeze remaining interleukin-2 into 5 ml aliquots. Add 5 ml of RPMI(1×) media to 5 ml of interleukin-2 containing 200 units per ml to obtain a concentration of 100 units/ml. To 10 tubes containing 9 ml of the media, add 1 ml of interleukin-2 at a concentration of 100 units/ml to obtain a concentration of 10 units/ml. These aliquots are stable for 30 days at 4°-8° C. For colony forming assay, use 0.5 ml (or 5 units) for each 25 cm 2 flask containing 4.5 ml of medium and 0.5 ml of cells.

Somatostatin:

Somatostatin is available in single vials containing 50 μg/ml and 100 μg/ml from Sandoz Ltd. Store at 4°-8° C. For the Colony Forming Assay, use 1.0 ml of STS containing 50 μ/ml for each 25 cm 2 flask containing 4.0 ml of media and 0.5 ml of cells. This procedure is written for 50 μg/ml vials. If 100 μg/ml vials are used, use 0.5 ml of Somatostatin and 4.5 ml of media for a 25 cm 2 flask.

GM-CSF:

Dilute 250 ul of GM-CSF containing 50,000 units in 50 ml of media to obtain 1000 units/ml. Prepare aliquots of 5 ml each containing 5,000 units in sterile tubes with caps. Label each aliquot: GM-CSF–5,000 units (1,000 units/ml). Close nine of these aliquots with sterile adhesive and store at 4° C. Aliquot #10, containing 1,000 units/ml (total 5,000 units), will be used as follows: Pipet one ml into each of 5 sterile tubes. Add 19 ml of media to obtain 50 units/ml. (Each tube will contain a total of 1,000 units). Label each of these aliquots: GM-CSF–50 units/ml (total 1,000 units). Close 4 of these aliquots with sterile adhesive and store at 4° C. Aliquot #5 will be marked “IN USE” and stored at 4° C. For colony forming assay, use 50 ul (or 2.5 units) for 25 cm 2 flask containing 5.0 ml of medium and 0.5 ml of cells.

G-CSF:

Dilute 125 ul of G-CSF, containing 60,000 units in 50 ml of media to obtain 1200 units/ml. Prepare aliquots of 5 ml each containing 6,000 units in sterile tubes with caps. Label each aliquot: G-CSF–6,000 units (1,200 units/ml). Add the date. Close 9 of these aliquots with sterile adhesive and store at 4° C. Aliquot #10 containing 5 ml of 1,200 units/ml (total 6,000 units), will be used as follows: Pipet one ml into each of 5 sterile tubes. Add 19 ml of media to obtain 60 units/ml (each tube will contain a total of 1,200 units). Label each of these aliquots: G-CSF–60 units/ml (total 1,200 units). Add the date. Close 4 of these aliquots with sterile adhesive and store at 4° C. Aliquot #5 will be marked “IN USE” and stored at 4° C. For colony forming assay, use 50 μl (or 3 units) for each 25 cm 2 flask containing 5.0 ml of media and 0.5 ml of cells.

Epogen:

To one ml of epogen containing 2,000 units, add 19 ml of media to yield 20 ml of 2,000 units or 100 units/ml. Aliquot 2 ml into each of 10 cryovials. Label as follows: Epogen–100 units/ml and date. Store at 4°-8° C. For colony forming assay, use 50 μl (or 5 units) for each 25 cm 2 flask containing 5.0 ml of medium and 0.5 ml of cells.

PIXY 321-GM-CSF/IL-3:

Using 15 ml sterile capped tubes, dilute 1 ml of Pixy 321 (be sure to quantitatively transfer by rinsing vials) with 9 ml of 10% BSA and mix well. This will yield a neutralizing effect of 1.0×10 6 units/ml. Aliquot 1.0 ml of this solution of 1×10 6 units/ml into each of 9 cryovials. Label each vial with: name, concentration and expiration date. Store at -70° C. To the remaining 1 ml of 1.0×10 6 units/ml, add 9 ml of 10% BSA and mix well. This will yield a neutralizing effect of 1.0×10 5 units/ml. Aliquot 1.0 ml of this solution of 1×10 5 units/ml into each of 9 cryovials. Label each vial with: name, concentration and expiration date. Store at -70° C. To the remaining 1 ml of 1.0×10 5 units/ml, add 9 ml of 10% BSA and mix well. This will yield a neutralizing effect of 1.0×10 4 units/ml. Aliquot 1.0 ml of this solution of 1×10 4 units/ml into each of 9 cryovials. Label each with: name, concentration, and expiration date. Store at 70° C. To the remaining 1 ml of 1.0×10 4 units/ml, add 9 ml of 10% BSA and mfx well. This will yield a neutralizing effect of 1.0×10 3 units/ml. Aliquot 1.0 ml of this solution into each of 10 cryovials. Label each with: name, “Working Pixy,” concentration, and expiration date. Store at -70° C. One vial may be thawed and stored at 4° C. for use in the assay. Take 1 ml aliquot of 1.0×10 3 units/ml out of the -70° C. freezer and thaw. This will be marked “in use” and stored at 4° C. For Colony Forming Assay, use 50 μL (or 2.5 units) for 25 cm 2 flask.

PROCEDURE

Sample Processing:

Using a sterile pipet, dispense 20-25 ml of histopaque into the appropriate number of capped sterile 50 ml conical tubes. Using another sterile pipet, gently layer the bone marrow at a 45° C. angle onto the histopaque using a 1:1 ratio of histopaque to specimen. Centrifuge at 1600 RPM for 20 minutes in refrigerated centrifuge at 4° C. (brake on 2). Remove and discard the supernatant. Remove the cell interface and place in RPMI(1×) in a pre-labeled tube. Wash cells twice with RPMI(1×) media and centrifuge at 1600 RPM for 5 minutes with a brake of 2. Remove and discard the supernatant. Resuspend the cell pellet in 5 ml of RPMI(1×) medium.

Perform cell count:

In a 12×75 ml glass tube, combine:

0.1 ml of well-mixed cell suspension

0.2 ml of 0.4% Trypan Blue stain

0.7 ml of media

This is a 1:10 dilution.

Mix well and charge one chamber of the hemocytometer with the cell suspension. Under the light microscope, count the viable cells (those which have not absorbed the Trypan Blue) in the four corner 1 mm 2 squares. Count the total number of cells (stained and unstained) in the four corner 1 mm 2 squares.

Calculation for Cell Concentration:

1. Perform the viable cell count using the following equation: Average number of viable cells per square×dilution factor×hemacytometer factor =cells/ml

Sample calculation: 20 Cells (average # of viable cells per square)×10 4 ×10 (dilution factor)=2×10 6 cells/ml.

2. Determine the % viability using the following formula: Viable cell count×100=% viability total cell count

3. Adjust volume to amount needed for the appropriate cell concentration for the assay (this assay requires 1×10 6 cells/ml) using the following formula: (V 1 C 1 =V 2 C 2 )

For example:

V 1 =10 ml V 2 =?

C 1 =2×10 6 cells/mlC 2 =1×10 6 cells/ml ##EQU3## 10 ml×2×10 6 cells/ml=V 2 1×10 6 cells/ml

20 ml=V 2

20 ml-10 ml=10 ml

Ten ml of media must be added to have a volume of 1×10 6 cells/ml.

4. Prepare the flasks: label eight 25 cm 2 liquid tissue culture flasks with the following information: 1) Patient name; 2) Tracking number; 3) Date; 4) Growth Factor or BRM.

5. To each of the 25 cm 2 flasks, add bone marrow cells (BMC), growth factor or biological response modifier (BRM), and media according to Table 1:
TABLE 1
______________________________________
Growth Factor BMC BRM Media
______________________________________

BMC + M 0.5 ml — 5.0 ml
(Control)
BMC + IFN + M 0.5 ml 1.0 ml 4.0 ml
BMC + IL2 + M 0.5 ml 0.5 ml 4.5 ml
BMC + STS + M 0.5 ml 1.0 ml 4.0 ml
BMC + GM-CSF + M
0.5 ml 50 μl
5.0 ml
BMC + G-CSF + M 0.5 ml 50 μl
5.0 ml
BMC + EPO + M 0.5 ml 50 μl
5.0 ml
BMC + PIXY + M 0.5 ml 50 μl
5.0 ml
______________________________________

NOTE: The final volume in all flasks will be 5.5 ml.

NOTE: The final volume in all flasks will be 5.5 ml.

6. Incubate the flasks in the 37° C. and 5% CO 2 incubator. The flasks are to be read and fed as needed, taking care to treat each flask in the assay the same.

Reading and Reporting Results:

At days 7, 14 and 21, scan each of the flasks of the colony forming assay for colonies under the inverted microscope. A colony is defined as 40 or more cells adhering to the bottom of the flask. Floating colonies are not to be counted. Scan the entire flask and report the total number of colonies counted for each flask on the colony forming assay report form.

If no growth is seen at the end of 21 days, report as “No Growth” and hold the flasks for an additional 7 days. Flasks may be discarded after the final report is reviewed by the Medical Director.

Procedure 3

Immunomodulatory Testing

The prediction of clinical or in vitro response to cancer therapy and the corresponding determination of optimum patient treatment through the use of in vitro assays has been the goal of many investigators. The immunomodulatory assay is a procedure to determine how the patient will react to certain biological response modifiers in vitro. It is a necessary addition to other forms of testing in that it can determine if a patient’s serum naturally contains the factors which are able to suppress or activate the growth of tumor cells.

In this procedure, the patient’s serum, and/or WBC’s, is combined with various biological response modifiers (BRM) and co-cultured with a tumor cell line. The percentage of stimulation or percentage of inhibition of the tumor cell growth is determined. This information enables the oncologist to determine appropriate and customized treatment for each patient.

SPECIMEN REQUIREMENTS AND COLLECTION

Label all specimens with patient name, date drawn, time drawn, and phlebotomist’s initial. A unique tracking number will be assigned to each individual specimen to assure proper specimen tracking.

Requirements:

1. Whole blood (WB)–10 ml

2. Serum–3 ml

3. Bone Marrow (BM)–one BM aspiration in 25 ml of transport media. The optimum amount is 3 ml at 1×10 6 cells per ml.

4. Plasmapheresis specimen–3 ml

Both a whole blood specimen and a serum specimen are required for complete immunomodulatory evaluation. However, in the event that only one of the two specimens (WB or serum) is available, partial immunomodulatory testing may be performed.

Collection, Processing, and Handling:

The patient will be properly identified by the nurse or phlebotomist (i.e., check wristband, verify patient name).

Specimens will be aseptically collected by the nurse or a phlebotomist. Gloves and lab coat must be worn while drawing or handling the specimen and “universal precautions” must be observed.

1. Whole Blood

a. Collection–Add 2500 units of preservative free heparin (Calciparine) to a 10 ml red top vacutainer tube. Perform venipuncture and draw the patient’s blood into tube. Label all tubes with patient’s name, date and time collected. Invert several times to mix anticoagulant. Refrigerate tube until ready for separation of the mononuclear layer by the density gradient procedure (Reference is made to Procedure 4, infra., at part B-2).

Minimum amount whole blood=7 ml

Optimum amount whole blood=10 ml

b. Unacceptable Specimens–Clotted specimens, grossly hemolyzed specimens, or frozen specimens are unacceptable.

c. Rejection of Specimens–When any criteria are not met, the unacceptable specimens may be tested if necessary.

2. Serum

a. Collection–Use a red top vacutainer tube to perform venipuncture and draw 10 ml of the patient’s blood into the tube. Centrifuge the serum specimen at 2000 RPM for 10 minutes at 25° C. Refrigerate tube until testing. Aseptically draw off serum and place in a separate tube.

Minimum amount serum=3 ml

Optimum amount serum=5 ml

b. Unacceptable Specimens–Specimens with excessive fibrin clotting are unacceptable.

c. Rejection of Specimens–When any criteria are not met, the unacceptable specimens may be tested if necessary.

3. Plasmapheresis

a. Collection–Add 2500 units of preservative free heparin (Calciparine) to a 10 ml red top vacutainer tube. Draw from the plasma collection container at the beginning of the plasmapheresis procedure. Label tube #1. Refrigerate until testing. Minimum amount of plasma=3 ml

Optimum amount of plasma=5 ml

b. Unacceptable Specimens–Specimens with excessive fibrin clotting are unacceptable.

c. Rejection of Specimens–When any criteria are not met, the unacceptable specimens may be tested if necessary.

4. Bone Marrow

a. Collection–Not more than 15 minutes prior to the BM procedure, add 2500 units of Calciparine per 25 ml of tissue transport media using aseptic techniques. Using aseptic techniques, quickly add the BM specimens to the transport tube and mix well. Refrigerate the tube until ready for separation of the mononuclear layer by the density gradient procedure (see part IV, section B-2).

b. Unacceptable Specimens–Grossly clotted specimens, grossly hemolyzed specimens, or frozen specimens are unacceptable.

c. Rejection of Specimens–When any criteria are not met, the unacceptable specimens may be tested if necessary.

MATERIALS:

The following materials are required for this procedure:

Laminar Flow Hood

Bacto-Agar

Balance

Weighing Paper

Weighing Tools

Capped Bottles (Sterile) (-250 ml, -500 ml)

Distilled Water (Sterile)

Microwave Oven

Autoclave

RPMI 1640 Media

Gentamicin

Disposable Pipettes (Sterile) (-1 ml, -2 ml, -5 ml, -10 ml, -25 ml)

Glutamine

Trace Elements

2-Mercaptoethanol with Dulbecco’s PBS

ITS Solution

Fetal Bovine Serum

Test Tubes (Capped) (-5 ml, -10 ml)

Petri Dishes (Sterile) (Regular 100×15 mm,

Gridded 35 mm)

Falcon Tubes

Conical Tube (Sterile-Plastic)

Trypan Blue Stain (0.4.about.)

Hemocytometer with Cover Slips

Microscopes (Tissue Culture Inverted Microscope, Light Microscope)

pH Paper (Narrow Spectrum)

Graduated Cylinders (50 ml, 100 ml)

Histopaque-1077

Water Bath

Suction Pipetter

Refrigerated Centrifuge

CO 2 Incubator

Biological Response Modifiers (Recombinant Interferon Alpha 2a, Recombinant Interleukin-2, mAB to Interferon Alpha)

Vortex Mixer

Refrigerator (2-8 C.)

Hand Counter

Sharps Container

Pasteur Pipettes

Dropper Bulbs

-70° C. Freezer

Precise Surface Disinfectant

Micro Glassware Disinfectant/Cleanser

Beaker (250 ml)

Betadine Disinfecting Solution

Syringes (1 cc) (Sterile)

Needles (Various Gauges)

Vacutainer Tubes (Red Tops and “Tiger Tops”)

MEDIA AND REAGENT PREPARATION

Prepare Agar:

Lower layer (2.5% working solution): Weigh out 2.50 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix.

Upper layer (1.5% working solution): Weigh out 1.50 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix. Place both bottles in the microwave with caps loosened. Microwave for 60 seconds at full power, mixing every 20 seconds. Avoid boiling. The agar will appear clear when ready. If solution boils over, do not use it. Re-make the agar solution. Loosely tape cap with autoclave indicator tape and autoclave both solutions for 20 minutes at 22 psi and 250°-270° F. Place both bottles in the water bath under the hood. This bath must be kept at 45°-50° C. at all times. The agar should cool down to 50° C. before use. Swirl agar to mix well before using.

Prepare Media:

After preparation of each media, label the final product with the following: 1) Name of Product; 2) Preparation Date; 3) Expiration Date; 4) Storage Recommendations; 5) Technologist’s Initials

10% RPMI 1640 (1×) G,G fortified:
______________________________________
AMOUNT COMPONENT
______________________________________

500.0 ml RPMI 1640 (1X) with L-Glutamine
50.0 ml Fetal Bovine Serum, Qualified, Heat
Inactivated
5.0 ml L-Glutamine (100X) 200 mM
2.5 ml Penicillin/Streptomycin
2.0 ml Trace Element Mix (100X), Lyophilized
0.5 ml 2-Mercaptoethanol
5.0 ml Insulin-Transferrin-Sodium Selenite, Media
Supplement, Lyophilized, Gamma-Irradiated
______________________________________

Combine all components under a sterile laminar flow hood. Sterilize by filtration using a 0.22 micron cellulose acetate membrane filter with a 60 micron prefilter. Store the prepared media at 2°-10° C.

10% RPMI 1640 (2×) G,G Fortified:
______________________________________
AMOUNT COMPONENT
______________________________________

500.0 ml RPMI 1640 (2X) Powdered Cell Culture Medium
with L-Glutamine (Reconstitute one powder
pack with a total of 500.0 ml of sterile
distilled water)
50.0 ml Fetal Bovine Serum, Qualified, Heat
Inactivated
5.0 ml L-Glutamine (100X) 200 mM
2.5 ml Penicillin/Streptomycin
2.0 ml Trace Element Mix (100X), Lyophilized
0.5 ml 2-Mercaptoethanol
5.0 ml Insulin-Transferrin-Sodium Selenite, Media
Supplement, Lyophilized, Gamma-Irradiated
______________________________________

Under a sterile laminar flow hood, measure out 450 ml of sterile distilled water and place in a sterile 500 ml reagent bottle. Add the powdered medium to the water with gentle stirring. Rinse out the inside of the package to remove all traces of powder. Add 1.0 g of NaHCO 3 per 500 ml of medium. Dilute with sterile distilled water to 500 ml. Adjust the pH of the preparation to 0.2-0.3 below the desired final working pH (pH units will usually rise 0.1-0.3 upon filtration); use of 1N NaOH or 1N HCl is recommended. After the pH has been adjusted, keep the container closed until the medium is filtered. Sterilize immediately by membrane filtration using a 0.22 micron cellulose acetate membrane filter with a 60 micron prefilter. Store the prepared media at 2°-10° C.

Prepare Lower Layer Stock Media:
______________________________________
125 ml Fetal Bovine Serum 125 ml 2 × G, G Fortified Solution 250 ml 1 × G, G Fortified Solution
______________________________________

Mix, label, and store at 2-10° C.

Prepare Upper Layer Stock Media:
______________________________________
150 ml Fetal Bovine Serum 150 ml 2 × G, G Fortified Solution 150 ml 1 × G, G Fortified Solution
______________________________________

Preparation of Drugs:

Prepare a working concentration for the drugs to be tested. All drug dilutions are made with RPMI 1640 1× G,G Fortified.

To prepare IFN Alpha-2a in a 10 ml capped tube, mix 0.1 ml Roferon (3,000,000 units/ml) with 9.9 ml 1× G,G, Media. Label tube with name of drug, preparation date, expiration date (1 week from the day of preparation), concentration (30,000 u/ml) and technician’s initials.

To prepare IL-2 in 10 ml capped tube: Remove working stock aliquot of IL-2 (10 units/ml) from refrigerator. If the aliquot is not available in refrigerator, follow the IL-2 aliquot procedure found in the aliquot procedure book. The stock tubes are kept in the -48° C. freezer. The dosage for testing will be 0.1 ml, which will equal 1 unit of interleukin-2. Therefore, 1 unit of interleukin-2 will be tested in the immunomodulatory procedure. Interleukin-2, in this media, can be maintained for 30 days at 2°-8° C.

To prepare LI-8-ABIFN (mAB to IFN alpha 2A) in a 10 ml capped tube, remove working aliquot (neutralizing effect of 1000 units/ml from freezer). The dosage will be 0.1 ml in the assay to achieve a concentration of 100 units of neutralizing ABIFN.

PROCEDURE:

Lower Layer (2.5% agar):

Use lower layer stock media and dispense 8 ml of stock media into the appropriate number of sterile 15 ml tubes with caps. (Approximately 8 plates can be made from 1 tube.) Keep capped at room temperature until needed to prepare lower layer plates or store capped overnight at 2°-8° C. for use the following day. Warm to room temperature before use. When ready to prepare the lower layer plates work quickly to prevent premature solidification of the agar. Using a sterile 10 ml pipet draw up 2.0 ml of the 2.5% agar mixture. Dispense the 2.0 ml into one of the 15.0 ml plastic tubes containing 8 ml of lower layer media, and mix twice by drawing up and dispensing into the tube. Draw up 9.0 ml of the mixture and dispense 1.0 ml into each 35 mm gridded plate. Swirl to cover the bottom of the plate. Take care to prevent bubbles. Prepare only 8 plates at a time so that the agar does not solidify before it is plated. Allow plates to sit undisturbed on a flat surface until the agar has solidified. Discard any plates with uneven agar distribution. Store plates at room temperature for same day use or overnight in a 37° C. CO 2 incubator for use the following day.

Preparing the Cells Lines:

Generally, L929 cells are used in the assay, but colon, prostate, breast or ovarian cells may also be used.

For L929 cells, microscopically examine the liquid culture flasks of the desired cell line and determine by confluency which flasks to use. Remove supernatant with pipet and discard. Add 5 ml Trypsin. Expose to cell layer and remove 2 ml. Incubate flasks with Trypsin for minute. Hit flask with the palm of your hand to knock cells off. Add 7 ml of L929 cell media to stop reaction. Transfer cells to a sterile 50 ml conical tube, add more media (to bring to 20 ml). Centrifuge tube 1600 RPM for 5 minutes with brake on 2. Remove supernatant and resuspend pellet in L929 cell media. Centrifuge cells at 1600 RPM for 5 minutes with brake on 2. Remove supernatant, resuspend pellet in appropriate media.

Preparing the Patient Cells (from whole blood with Calciparine, or BM Specimen with Calciparine):

Into an appropriate size (depends on sample volume) capped plastic tubes labeled with each patient’s name, dispense the appropriate volume of histopague (use equal amounts of histopaque to equal amounts of whole blood, i.e., if a whole blood tube containing 8 ml of blood is received, dispense 4 ml of histopaque and 4 ml of whole blood into each of two 15 ml capped tubes. If 75 ml of BM is received, dispense approximately 18.75 of histopaque and 18.75 of BM into four 50 ml conical tubes. Layer in the whole blood very slowly at a 45° C. angle on the histopaque. Centrifuge at 1600 RPM for 20 minutes in centrifuge at 4° C., brake on 2. Remove and discard the supernatant. Remove the cell interface and place in a separate tube containing 10.about. RPMI 1× G,G media. Dilute the cells with media quickly because the histopaque is very toxic to the cells if left on them for extended periods of time. Wash cells twice with 10.about. RPMI 1× G,G media. Centrifuge at 1600 RPM for 5 minutes with the brake on 2. Remove and discard the supernatant. Resuspend the cell pellet in 5 ml RPMI 1× G,G media.

PERFORM CELL COUNT:

In a 12×75 ml glass tube, combine: 0.7 Media; 0.2; Trypan Blue; and 0.1 Cell Suspension. This is a 1:10 Dilution. Mix well. Charge one chamber of the hemocytometer with the cell suspension. Under the light microscope, count the live cells (those which have not absorbed Trypan Blue) in the 4 corner 1 mm 2 squares. The concentration of cell lines and patient cells for the IM assay will be as follows (cells per ml):

L-Cells5×10 5

Colon1×10 5

Prostate5×10 4

Breast5×10 4

Ovarian2×10 4

Patient1×10 6

Count the total number of cells (stained and unstained) in the 4 corner 1 mm 2 squares.

CALCULATION FOR CELL COUNT:

Use the following cell count formula: Cells per ml=average # cells×dilution factor×hemocytometer factor.

EXAMPLE CALCULATION:

Patient cells are brought up in 10 ml of media. 0.1 ml of cells are added to 0.2 ml of Trypan Blue and 0.7 ml of media. 4-1 mm 2 squares were counted and cell count=80 cells. 20 cells (80 cells 4-1 mm 2 squares=average # of cells)×10 (dilution factor)×10 4 =2×10 6 cells/ml.

Calculate % viability using the following formula: % of viable cells×100=% viability total # of cells

Adjust volume to amount needed for the appropriate cell concentration for the assay: (V 1 C 1 =V 2 C 2 )

V 1 =10 ml V 2 =?

C 1 =2×10 6 cells/ml C 2 =1×10 6 cells/ml ##EQU4## 20 ml=V 2 20 ml-10 ml=10 ml

Ten ml of media must be added to have a volume of 1×10 6 cells/ml.

Label the appropriate number of plates with the following information: 1) Specimen number; 2) Patient name; 3) Name of BRM; 4) Setup date.

Preparing Upper Layer:

Label and arrange 15 ml sterile capped tubes for each control, patient serum, patient whole blood sample, that you need in a test tube rack according to Table 2 (infra.). Be sure to include a cell control, and a control for each BRM to be tested. The cell control should contain everything the others contain except for BRM. A separate set of control tubes must be set up for each cell line assayed (colon, L cell, prostate). Dispense the upper layer stock media into each tube, then add appropriate amounts of BRM, ABIFN, cell line and serum or patient whole blood. See Table 2 as follows:
TABLE 2
________________________________________________________ __________________
FOLLOW THIS TABLE FOR UPPER LAYER VOLUMES (ALL IN ml): PTWBC UPPER OR L AGAR TOTAL LAYER BRM ABIFN SERUM CELLS 1.5% VOLUME
________________________________________________________ __________________

CELL 2.1 — — — 0.3 0.6 3.0 ml
CONTROL
IFN 2.0 0.1 — — 0.3 0.6 3.0 ml
CONTROL
IL2 2.0 0.1 — — 0.3 0.6 3.0 ml
CONTROL
ABIFN
CONTROL 2.0 — 0.1 — 0.3 0.6 3.0 ml
PT. SERUM
1.9 — — 0.2 0.3 0.6 3.0 ml
PT. SERUM/
1.8 — 0.1 0.2 0.3 0.6 3.0 ml
ABIFN
PT. SERUM/
1.8 0.1 — 0.2 0.3 0.6 3.0 ml
IFN
PT. SERUM/
1.8 0.1 — 0.2 0.3 0.6 3.0 ml
IL2
PT. WBC 1.9 — — 0.2 0.3 0.6 3.0 ml
PT. WBC/
1.7 — — 0.2 0.3 0.6 3.0 ml
SERUM 0.2
PT. WBC/
1.6 — 0.1 0.2 0.3 0.6 3.0 ml
SERUM/ABIF 0.2
PT. WBC/
1.8 0.1 — 0.2 0.3 0.6 3.0 ml
IFN
PT. WBC/
1.6 0.1 — 0.2 0.3 0.6 3.0 ml
IFN/SERUM 0.2
PT. WBC/
1.8 0.1 — 0.2 0.3 0.6 3.0 ml
IL-2
PT. WBC/
1.6 0.1 — 0.2 0.3 0.6 3.0 ml
IL-2/SERUM 0.2
________________________________________________________ __________________

Add all components except agar to the appropriate tube. For the following, one tube should be finished before moving on to the next. With a 2.0 ml pipet, draw up 0.6 ml of 1.5% agar. Dispense the agar into the tubes one at a time, starting with tube #1. Mix by aspirating and dispensing twice. Draw up 2.2 ml of solution and dispense 1.0 ml into each gridded petri plate (on top of the other layer) taking care not to produce bubbles in the agar. Gently swirl plate to allow for even distribution. Allow agar to solidify, and then add distilled water (approximately 0.5 ml) to humidity plate and place in large labeled petri plate. Repeat steps 5-7 for each plate to be set up. Incubate the cultures at 37° C. in a humid, 5% CO 2 -enriched atmosphere.

REPORTING RESULTS:

Reading semi-solid culture plates:

Read the plates after 7 days as follows: Scan the plate for colonies and aggregates. If they are present, count all the squares to obtain the number of aggregates and colonies in the plate (aggregates=4-20 cells per cluster) (colony is >20 cells per cluster). If no colonies or aggregates are present, count all individual cells (cell line and patient’s WBC’s) in 4 squares and take the average. Multiply the average by 15×13.36 (15=# of square in the center row) (13.36=area of the plate). Plates may be read at 5, 6 or 8 days. Record all data in scientific notation on the report form.

Procedure 4

Human Interferon Assay

Interferons are cytokines that have the ability to inhibit the growth of viruses and to protect infected cells against viral cytopathic effects. The immunoregulatory functions of interferons such as the increase in natural killer (NK) lymphocyte activity, the increase in histocompatibility antigens, the activation of monocyte/macrophages, and B-cell function have also proven to be of clinical importance. The first natural interferon was discovered by Isaac and Linderman in 1975, and recombinant interferon alpha 2 was registered by the FDA in 1986 ushering in a new phase of biotherapy. The goal of this assay is to determine the level of interferon in international units/ml in patient’s serum. Potency of the human interferon of serum samples and controls will be determined using WISH cells challenged with Vesicular Stomatitis Virus measuring the cytopathic effect.

SPECIMEN REQUIREMENTS, COLLECTION AND HANDLING

Specimens are labeled with the following information: 1) Patient name; 2) Date drawn; 3) Time drawn; 4) Phlebotomist’s initials; 5) Test name.

Serum-Amount required: Optimum=3.0 ml Minimum=1.0 ml

The specimen must be aseptically collected in a “tiger” top vacutainer tube, allowed to clot, then centrifuged at room temperature for 10 minutes at 2000 rpm. The serum should be poured over into a sterile plastic tube, capped, appropriately labeled, and stored frozen until the assay is performed.

Plasmapheresis–Amount required: Optimum=3.0 ml Minimum=1.0 ml

The specimen must be collected using sterile technique into a 10 ml red top vacutainer tube from the plasma bag obtained during the plasmapheresis procedure. Add 2500 units of calciparine to the 10 ml red top vacutainer tube containing the plasmapheresis sample and mix well. In addition to the information required above, the specimen should be labeled with the bag number from which it was obtained, i.e. #1, #2, etc. The specimen must be poured over into a sterile plastic tube, capped, appropriately labeled, and stored frozen until the assay is performed.

EQUIPMENT:

The following equipment is needed for this procedure:

Sterile laminar flow hood with ultra-violet light

Freezer -70° C.

Refrigerator 2°-8° C.

Autoclave

Analytical balance

Refrigerated centrifuge

CO 2 incubator

Spectrophotometer (Bio-Rad 96-well plate reader with 540 nm filter)

96-well microtiter plates–sterile flat bottom with covers Falcon 3872

Pipet aid

Micropipettes

Costar octapettes–50 ul, 100 ul, & 200 ul

Wheaton multi-channel pipet–50-200 ul range

MLA-40 ul

Sterile disposable:

Pipettes (1 ml, 5 ml, 10 ml, and 25 ml)

Pipet tips Wheaton 851247

Plastic troughs

Gauze

Cryovials

50 ml conical tubes with caps

Test tubes

Culture flasks (75 cm and 150 cm)

Kimtex wipers

Biohazardous waste can and bags

Stainless steel pan with cover

MATERIALS:

The following materials are needed for this procedure:

Basal Medium Eagle 1× (BME), 500 ml Gibco Cat. No. 320-1010AJ

Fetal Bovine Serum, 100 ml Hyclone Cat. No.

A-1111-D

L-Glutamine, Cat. No. 320-5030AG

Hepes Buffer Solution (1M), 100 ml Gibco Cat. No. 380-5630AG

Penicillin/Streptomycin

WISH cells (Human Amnion) ATCC 25-CCL

Vesicular Stomatitis Virus ATCC

Human Interferon (alpha) Reference NIAID Cat. No. Ga23-902-530

Gamma Interferon control

Neutral red dye Sigma No. N-2880

Phosphate buffered saline (PBS)

PBS without Ca++ and Mg++, Ph 7.4 Gibco

Cat. No. 310-4190AJ

PBS with Ca++ and Mg++, Ph 6.8 Gibco

Cat. No. 310-4040AJ

Glacial acetic acid Sigma Cat. No. A-6283

Distilled water

Ethyl Alcohol Aldrich Cat. No. 18,738-0

Patient sera

One-Stroke Environ Calgon Vestal No. 539708

Trypsin-EDTA 1×, 100 ml Gibco Cat. No. 610-520OAG

Trypan Blue Gibco 6305250AG

REAGENT PREPARATION:

All media must be filtered for sterility, labeled with name, preparation and expiration dates, storage information, and technician’s initials. Prepared media is stable for three months at 2°-8° C.

BME with 15% FBS:

Add 75 ml of FBS, 5.0 ml Pen/Strep 10 ml glutamine, and 5 ml hepes to a 500 ml bottle of BME. (For feeding WISH cells.)

BME with 10% FBS:

Add 50 ml of fetal bovine serum, 2.5 ml Pen/Strep, 10 ml glutamine, and 5 ml hepes to a 500 ml bottle of BME.

BME with 2% FBS:

Add 10 ml of FBS, 2.5 ml Pen/Strep, 10 ml glutamine, and 5 ml hepes to a 500 ml bottle of BME.

Vesicular Stomatitis Virus (VSV):

Prepare two 150 cm 2 culture flasks of WISH cells. Discard the media when the cells become confluent. Wash once with fresh BME with 2% FBS. Dilute a VSV stock preparation 1000 times with BME with 2% FBS. Inoculate 8-10 ml per flask of the 1:1000 VSV into each WISH cell flask such that it covers the entire cell layer. Incubate the flasks for 1 hour at 37° C. in a 5% CO2 incubator. Add 15 ml of the BME with 2% FBS to each flask and incubate at 37° C. in a 5% CO2 incubator for 1-3 days or until cell layers show nearly complete viral cytopathic effect (CPE). Harvest the culture fluid and clarify by centrifugation at 3000 RPM for 20 minutes at 4° C. Aliquot the virus containing supernatant into 10 ml tubes and store at -70° C. This virus stock usually contains 1×10 9 plaque-forming units/ml when its infectivity is quantitated in WISH cells. Dilute one 10 ml stock VSV tube 1:10 with BME with 2% FBS to yield a working VSV of 1×10 8 plaque forming units/ml. Aliquot 1 ml of working VSV into cryovials and store at -70° C. Dilutions of 1:100, 1:200, and 1:300 of working VSV should be made and inoculated onto confluent WISH cells in a 96-well plate to determine the dilution that yields nearly 100% CPE in 24-48 hours.

Human Alpha Interferon (National Institute of Health: catalog #Ga23-902-530):

Reconstitute with 1.0 ml of sterile distilled water being careful to avoid any loss of material in the neck of the ampule. Dilute to a concentration of 105 IU/ml of interferon with BME with 2% FBS (no Pen/Strep and no hepes). Aliquot 200 ul into cryovials and store in -70° C. freezer. The aliquots are stable for two years at this temperature.

Neutral Red Dye:

Prepare 0.1% stock solution of Neutral Red Dye by adding 500 mg of red dye powder to 500 ml of distilled water. Mix with magnetic stir bar at room temperature for 1 hour. Autoclave for 10 minutes at 15 psi. Cool. Label and store stock Neutral Red Dye in a brown bottle and refrigerate. Staining Solution–prepare a 15% staining solution by adding 85 ml PBS with Ca + + and MG + + (Ph 6.85) to 15 ml stock Neutral Red Dye.

Eluting Solution:

To 1 ml glacial acetic acid add 49 ml distilled water and 50 ml 100% ethanol. Label and store in glass capped bottle at room temperature.

Gamma Interferon Control:

Obtain Actimmune (3.0×10 6 units/ml). Dilute 0.05 ml (50 ul) of Actimmune with 49.95 ml of BME with 2% FBS and mix well to obtain a concentration of 3000 units/ml. Aliquot 10 ml into each of 5 sterile capped tubes and label with “stock IFN gamma,” 3000 units/ml, preparation and expiration dates. (Stable for 2 years at -70° C.). Dilute 1 ml of stock IFN gamma with 9.0 ml of BME with 2% BME for a working concentration of 300 units/ml. Aliquot 200 ul of this concentration into cryovials, label, and store at -40° C. (Stable for 2 years.). Run in parallel with current control to obtain range prior to putting into use.

PROCEDURE:

Day One:

Generate a worksheet from the interferon data base for 24 samples. Number the worksheet with #1 as the reference, patient samples #2-#25, the gamma control #26, and the alpha control #27. Retrieve the patient samples, interferon reference and controls from the freezer. Verify samples with the worksheet. Label nine sterile 96-well microtiter plates with sample numbers (run in duplicate), plate number and date. Two 150 cm 2 flasks of confluent WISH cells are harvested and washed by the liquid culture technician. The tube of cells is labeled with the name of the cells, the passage number, date, and technician’s initials. Note: The passage of WISH cells should be less than P250. Before this passage is reached, cells of younger passage should be retrieved from liquid nitrogen and started in liquid culture.

Perform the cell count in the following manner: in a 12×75 ml tube, combine 0.5 ml of WISH cells and 0.5 ml of 0.4% Trypan Blue (1:2 dilution). Mix well and charge the hemacytometer chamber. Under the light microscope, count the viable WISH cells in the four corner 1 mm 2 squares and divide by 4 to obtain the average for 1 square. Count the total number of cells in the four corner 1 mm 2 squares and divide by 4 to obtain the average for 1 square. Determine the % viability by dividing the viable cell count by the total cell count and multiplying by 100. Record the % viability on the cell count worksheet.

Determine the number of cells/ml by using the following equation: Avg. # of viable cells/square×dilution factor×hemacytometer factor=cells/ml

Dilute the WISH cells to 3.5×10 5 cells/ml according to the following formula: V 1 C 1 =V 2 C 2

For example:

Volume 1 =40 ml Concentration 1 =2.0×10 6

Volume 2 =? Concentration 2 =3.5×10 5 ##EQU5## V 2 =229 ml

Add 189 ml of BME with 10% FBS to the original volume of 40 ml to obtain 3.5×10 5 cells/ml. Set aside diluted WISH cells until ready to be added to the microtiter plates. Using sterile techniques, add 100 ul of 10% BME to each well (including blanks) of the 96-well microtiter plates. To the wells in column #1, B-G, add an additional 60 ul of 10% BME. To well positions B1 and C1 of plate #1 (each sample will be run in duplicate) add 40 ul of the reference. To well positions D1 and E1 add 40 ul of patient serum. To each successive pair of wells, add 40 ul of the appropriate patient serum. In addition to the patient samples, set up a gamma interferon control and an alpha interferon control (reference) on the last plate of the assay. Make serial dilutions with the 100 ul octapet from column 1 to column 10 and rows B-G by mixing up and down several times, then carrying 100 ul to the next column through the 10th column. Discard the remaining 100 ul from the 10th column. Add 100 ul of the 3.5×10 5 cells/ml WISH cell suspension to all wells except blank wells in rows A and H. Designate column 11 as cell control and column 12 as viral control on each plate. Incubate plates 18-24 hours at 37° C. at 5% CO 2 . Lay out the stainless steel pan containing OneStroke Environ, diapers and gauze squares in hood with white light overnight.

Day Two:

Check plates under the inverted microscope for confluency. Check for toxicity of patient sera (black edges). Using sterile techniques, dump the media from the plates into the stainless steel pan containing OneStroke Environ, blot on gauze. Wash the cells by adding 100 ul of BME with 2% FBS to all the wells except the blank wells, dump, and blot. Add 50 ul of 2% BME to column 11. Add 100 ul of 2% BME to all wells (including blanks). Add 50 ul VSV (in a concentration that yields 90% CPE in the viral control in 24-48 hours) to all the sample wells in columns 1-10 and column 12. Incubate 24-48 hours at 37° C. with 5% CO 2 .

Day Three:

Check plates under the microscope for 90-100% CPE in the viral control. Look for 50% CPE in the reference and samples. The 50% CPE should fall between columns 4 and 6 for the reference. Incubate plates until this is achieved. Place the stainless steel pan containing One-Stroke Environ along with Kimtex wipers sufficient for blotting the plates under the hood. Dump plates and blot. Add 100 ul of 15% neutral red dye to all the sample wells. Incubate at 37° C. with 5% CO 2 for 45 minutes. Dump plates and blot. Rinse with 200 ul PBS at pH 6.85. Dump and blot. Plates may be left to dry inverted on Kimtex wipers on a tray for several days before eluting. Elute by adding 100 ul of the eluting solution to each sample well.

Read on the microplate reader with a 540 nm filter. Select Microman from the menu to access the Microplate Manager program. Under Analysis, select Multiple Readings.

Select:

Single Wavelength

540 nm

Mixing duration–5 seconds

Reading per plate–1

Number of plates–9

Filename–date (i.e., 0722)

Remove cover of microtiter plate #1 and place in the reader. Select Start. After all the plates have been read, print the raw data reports as follows: Under file, select Open and Lotus Results. Arrow down to filename, or type in the file name and plate number. Select Open. If message appears “Save current results . . . ,” say NO. When Raw Data Report appears on the screen, under File, select Print. Select Raw Data Report and OK. Under file select Close. Repeat for each plate to be printed.

RESULTS:

A. Average the optical density (O.D.) values for the cell controls (column 11) and viral controls (column 12).

B. Average the O.D. values for the duplicates of the patients and reference samples–i.e. average B & C for each column 1-10, average D & E for each column 1-10, etc.

C. To determine the O.D. at 50%: ##EQU6##

D. To enter the O.D. values and their corresponding IFN dilutions:

1. Enter the 1st average O.D., press X-Y

2. Enter corresponding 1st IFN dilution, press LOG, then Ε+

3. Enter the 2nd average O.D., press X-Y

4. Enter corresponding 2nd IFN dilution, press LOG, then Ε+

5. Enter the 3rd average O.D., press X-Y

6. Enter corresponding 3rd IFN dilution, press LOG, then Ε+

E. To determine the correlation coefficient (r):

1.Press 2nd, then CORR (÷)

2. The correlation coefficient (r) should be -1.0±0.1.

3. Enter O.D. 50%, press 2nd, then y’ (x)

F. To convert the titer (log 10) to units/ml, press INV, then LOG

G. To clear data, press 2nd, then STO, then CE/C

H. To convert IFN units/ml to International Reference Units/ml:

1. IFN units/ml of Reference=Factor Int’l Ref. Units/ml of Reference

2. Sample IFN units/ml=Sample titer (IRU/ml) Factor

Procedure 5

Human Tumor Colony-Forming Chemosensitivity Assay

(Pharmacosensitivity)

The Human Tumor Colony Assay (HTCA: clonogenic or tumor stem cell assay) is an in vitro culture system employing semi-solid medium support originally described by Salmon and Hamburger, et al. Using HTCA, the growth and chemosensitivity of clonogenic tumor cells present in fresh biopsy specimens of human tumors can be investigated. Since this technique was first described, there has been a marked increase in the direct study of human tumors in vitro. Excellent evidence has been obtained which establishes that colonies grown in HTCA are comprised of tumor cells and that clonogenic cells within tumor colonies have the property of self-renewal (the defining property of a tumor stem cell). Chemosensitivity testing with specific agents in HTCA has documented striking degrees of heterogeneity in drug sensitivity from patient to patient, even for tumors of the same histopathology. Clinical correlations have been made between in vitro chemosensitivity and the response of patients with metastatic cancer to chemotherapy. In a series of trials, HTCA has had a 71% true-positive rate and a 91.about. true-positive rate for predicting the drug sensitivity and resistance, respectively, of cancer patients to specific chemotherapeutic agents. The assay thus appears to be a prognostic factor which identifies chemosensitive patients and which may allow some individualization of chemotherapy.

Tumor specimens, either from solid tumor masses, malignant ascites, or bone marrow are mechanically disaggregated into a suspension which is as close to a “single-cell suspension.about.as possible. These cells along with chemotherapeutic agents, biological response modifiers, and hormones are suspended in an agar-containing culture medium and then layered or “plated” onto a semi-solid underlayer. The underlayer prevents normal human fibroblasts, a major tumor stromal component, from adhering to the culture dish bottom and forming colonies which might be confused with colonies of tumor-cell origin. After 7 days, 14 days, and 21 days of incubation in a humid, CO2-enriched atmosphere at 37° C., colonies (greater than 20 cells) and aggregates (4-20 cells) are counted. A negative control and a positive control are set up for each tumor. From this data the effect of each specific drug on the tumor can be determined.

SPECIMEN REQUIREMENTS, COLLECTION AND HANDLING:

This assay may be performed on the following specimen types:

Solid tumors obtained during surgery or biopsy

Bone marrow aspirates and biopsies

Ascites

Pleural fluids

Thoracentesis fluids

The sample should be sufficient to yield an optimum of 20 ml of 1.0×10 6 viable cells/ml. A minimum of 12 ml of 1.0×10 6 viable cells/ml is acceptable. All specimens should be labeled with patient name, collection date, tumor type, and initials of technician.

MATERIALS:

The following materials are needed for this procedure:

Laminar Flow Hood

Bacto-Agar

Analytical Balance

Weighing Paper

Weighing Tools

Sterile Capped Bottles (100 ml, 250 ml, 500 ml; and 1,000

Distilled Water

Microwave Oven

Autoclave

RPMI 1640 Media (1× and 2×)

HCL (1N)

Sodium Bicarbonate

Penicillin/Streptomycin

Disposable Pipettes (sterile) (1 ml; 2 ml; 5 ml; 10 ml, 25 ml)

Select-A-Pette Pipetter (1 ml)

Select-A-Pette Tips (sterile)

Eppendorf Pipettes

Eppendorf Combitips (sterile) (2.5 ml; 5.0 ml; 12.5 ml)

Glutamine

Trace Elements

2-Mercaptoethanol With Dulbecco’s PBS

ITS Solution

Fetal Bovine Serum

Test Tubes (12×75 Glass-disposable; 15 ml Capped-sterile and disposable)

Petri Dishes (sterile) (Routine-100×15 mm; Gridded Plates-35×10 mm)

Sterile Capped 50 ml Conical Tubes (Falcon)

Homogenizer

Trypan Blue Stain (0.4.about.)

Hemocytometer With Cover Slips

Microscopes (Inverted; Light)

Hand Tally Counter

Ph Paper (narrow spectrum)

Graduated Cylinders (50 ml and 100 ml)

Histopaque-1077

Water Bath

Pipet Suction Aide

Refrigerated Centrifuge

Liquid Culture Flasks (25 cm 2 )

CO 2 Incubator

Chemotherapeutic Drugs

Biological Response Modifiers

Hormones

Vortex Mixer

Refrigerator (2°-10° C.)

Scalpel (disposable and sterile)

Sharps Container Pasteur Pipettes 53/4″ (disposable)

Dropper Bulbs

-70° C. Freezer

Precise Surface Disinfectant

70% Alcohol In Squeeze Bottle

Contrad Glassware Disinfectant/Cleanser

Thermometer (-20° C.-120° C.)

Beaker (500 ml)

Betadine Disinfecting Solution

Syringes (disposable and sterile) (1 cc; 10 cc)

Needles (various gauges)

MEDIA AND REAGENT PREPARATION:

Agar:

Lower layer=2.5% working solution: Measure out 2.5 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix.

Upper layer=1.5% working solution: Measure out 1.50 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix.

Place both bottles in the microwave with caps loosened. Microwave for 60 seconds at full power, mixing every 20 seconds. Avoid boiling. The agar will appear clear when ready. If the solutions boil over, do not use, remake the agar. Place a piece of autoclave tape on each of the loosely capped bottles and autoclave the solutions for 20 minutes at 22 psi. Place both bottles in the water bath under the hood. THIS BATH MUST BE KEPT AT 45°-50° C. AT ALL TIMES. The agar should cool down to 50° C. before use. Swirl agar to mix well before using. After preparation of each media, label the final product according to procedures previously described.

MEDIA:

10% RPMI 1640 (1×):
______________________________________
AMOUNT COMPONENT
______________________________________

500.0 ml RPMI 1640 (1X) with L-Glutamine
50.0 ml Fetal Bovine Serum, Qualified, Heat
Inactivated
5.0 ml L-Glutamine (100X) 200 mM
1.5 ml Penicillin/Streptomycin
2.0 ml Trace Element Mix (100X) Lyophilized
0.5 ml 2-Mercaptoethanol
5.0 ml Insulin-Transferrin-Sodium Selenite
Media Supplement, Lophilized, Gamma-
Irradiated
______________________________________

Combine all components under a sterile laminar flow hood. Sterilize by filtration using a 0.22 micron membrane filter with a 60 micron prefilter. Label and store the prepared media at 2°-10° C.

10% RMPI 1640 (2×) G,G Fortified:
______________________________________
AMOUNT COMPONENT
______________________________________

500.0 ml RPMI 1640 (2X) Powdered Cell Culture
Medium With L-Glutamine (Reconstitute
one powder pack with a total of 500.0 ml
of sterile DISTILLED WATER)
50.0 ml Fetal Bovine Serum, Qualified, Heat
Inactivated
5.0 ml L-Glutamine (100X) 200 mM
2.5 ml Penicillin/Streptomycin
2.0 ml Trace Element Mix (100X) Lyophilized
0.5 ml 2-Mercaptoethanol
5.0 ml Insulin-Transferrin-Sodium Selenite
Media Supplement; Lophilized; Gamma-
Irradiated
______________________________________

Under a sterile laminar flow hood, measure out 450 ml of sterile distilled water and place in a sterile 500 ml reagent bottle. Add the powdered medium to the water with gentle stirring. Rinse out the inside of the package to remove all traces of powder. Add 1.0 G OF NaHCO 3 per. 500 ml of medium. Dilute with sterile DH 2 O to 500 ml volume. Adjust the pH of the preparation to 0.2-0.3 below the desired final working pH of 7.2 (pH units will usually rise 0.1-0.3 upon filtration); use of 1N NaOH or 1N HCl is recommended. After the Ph has been adjusted, keep the container closed until the medium is filtered. Sterilize immediately by membrane filtration using a 0.22 micron membrane filter with a 60 micron prefilter (positive pressure is recommended). Label and store the prepared media at 2°-10° C.

Lower Layer Stock Media:

125 mlFetal Bovine Serum

125 ml2×G,G Fortified Solution

250 ml1×G,G Fortified Solution

Mix, label, and store at 2°-10° C.

Upper Layer Stock Media:

150 mlFetal Bovine Serum

150 ml2×G,G Fortified Solution

150 ml1×G,G Fortified Solution

Mix, label, and store at 2°-10° C.

Preparation of Drugs:

Working aliquots of each drug, hormone, and biological response modifier are prepared every two weeks according to the aliquot procedure. The working aliquots are stored at 4° C. and are ready for use.

PROCEDURE:

Prepare the Lower Layer Tubes:

Pipet 8 ml of lower layer stock media into 15 ml sterile capped tubes. (NOTE: Determine the number of plates needed by multiplying the number of drugs on the testing panel ×2. One lower layer tube yields approximately 8 lower layer plates.) Keep capped at room temperature until needed to prepare lower layer plates or store capped overnight at 28° C. for use the following day. Be sure to warm to room temperature before use.

Plate the lower layer plates as follows (WORK QUICKLY TO PREVENT PREMATURE SOLIDIFICATION OF THE AGAR): With a sterile 10 ml pipet, draw up 2.0 ml of the 2.5% agar mixture. Dispense the 2.0 ml of agar into one of the 15.0 ml plastic tubes containing 8 ml of lower layer media and mix twice by drawing up and dispensing into the tube. Draw up 9.0 ml of the mixture and dispense 1.0 ml into each of eight sterile 35 mm gridded plates. Swirl to cover the bottom of the plate. Take care to prevent bubbles. Allow plates to sit undisturbed on a flat surface until the agar has solidified. Discard any plates with uneven agar distribution. Store plates at room temperature for same day use or store overnight ln a 37° C. CO2 incubator for use the following day.

Prepare the Cell Suspension:

Solid tumor: In a sterile petri dish, using sterile scalpel and forceps, cut the tumor specimen into pea-size portions. Save the original transport media for centrifugation. Place the specimen in a glass conical tube that has a matched loose-fitting homogenizer. Add 5.0 ml of transport media. Gently homogenize the tumor pieces a small portion at a time and transfer the cell suspension to a sterile 50 ml conical tube. When all of the tumor has been homogenized, mix well and then allow cell suspension to settle, undisturbed, for one minute (this will allow clumps of tissue to settle out). Pipet the supernatant into a sterile tube, dilute with RPMI 1× with 10% FBS and centrifuge at 1600 RPM for 5 minutes. Remove the supernatant, resuspend the cells in RPMI 1× with 10% FBS, and perform cell count and viability.

Liquid tumor (ascites, bone marrow, etc.): Using a sterile pipet, dispense 20-25 ml of histopaque into the appropriate number of capped sterile 50 ml conical tubes. Using another sterile pipet, gently layer the ascites, bone marrow or cell suspension at a 45° C. angle onto the histopaque using a 1:1 ratio of histopaque to specimen. Centrifuge at 1600 RPM for 20 minutes in the refrigerated centrifuge at 4° C. (brake on 2). Remove and discard the supernatant. Remove the cell interface and place in RPMI (1×) GG fortified in a pre-labeled tube. DILUTE THE CELLS WITH THE MEDIA QUICKLY BECAUSE THE HISTOPAQUE IS VERY TOXIC TO THE CELLS IF LEFT ON THEM FOR EXTENDED PERIODS OF TIME. Wash cells twice with RPMI 1640 (1×) media and centrifuge at 1600 RPM for 5 minutes with a brake of 2. Remove and discard the supernatant. Resuspend the cell pellet in 5 ml of RPMI 1× medium.

Perform Cell Count:

In a 12×75 ml glass tube, combine:

0.1 ml of well-mixed cell suspension

0.2 ml of 0.4% Trypan Blue stain

0.7 ml of media

This is a 1:10 Dilution.

Mix well and charge one chamber of the hemocytometer with the cell suspension. Under the light microscope, count the viable tumor cells (those which have not absorbed the Trypan Blue) in the four corner 1 mm 2 squares. Count the total number of tumor cells (stained and unstained) in the four corner 1 mm 2 squares.

Calculation for Cell Concentration:

Perform the viable cell count using the following equation: Average # of viable cells per square×dilution factor×hemacytometer factor

Sample calculation: 20 Cells (average # of viable cells per square)×10 4 ×10 (dilution factor)=2×10 6 cells/ml.

Determine the % viability using the following formula: ##EQU7##

Adjust volume to amount needed for the appropriate cell concentration for the assay (this assay requires 1×10 6 cells/ml) using the following formula: (V 1 C 1 =V 2 C 2 )

For example:

V 1 =10 ml V 2 =?

C 1 =2×10 6 cells/ml C 2 =1×10 6 cells/ml ##EQU8## 20ml=V 2 20 ml-10 ml=10 ml

Ten ml of media are needed to have a volume of 1×10 6 cells/ml.

Prepare Upper Layer Tubes:

Pull and arrange the prepared drugs that are to be tested against the tumor cells. Label the appropriate number of 15 ml capped tubes with the names of the drugs. Arrange the tubes in a test tube rack. Use the Table 3 as follows for upper layer volumes (all in ml):
TABLE 1
______________________________________
UPPER LAYER AGAR MEDIA CELLS DRUGS 1.5%
______________________________________

CONTROL 2.1 0.3 — 0.6
ABRIN 1.8 0.3 0.3 0.6
DRUG #1 1.8 0.3 0.3 0.6
DRUG #2 1.8 0.3 0.3 0.6
DRUG #3 1.8 0.3 0.3 0.6
______________________________________

Add all components except agar to the appropriate tubes. For the following steps, one tube should be finished before moving on to the next. With a 2.0 ml pipet, draw up 0.6 ml of 1.5% agar. Dispense the agar into the tube, one at a time, starting with tube #1. Mix by aspirating and dispensing twice. Draw up 2.2 ml of the solution and dispense 1.0 ml into each of two gridded plates (on top of the other layer) taking care not to produce bubbles in the agar. Gently swirl plate to allow even distribution. Allow agar to solidify undisturbed, and then add approximately 0.5 ml of distilled water to a third “half plate” in the large petri dish labeled with the corresponding drug. Repeat steps 5-7 for each drug to be tested. Incubate the cultures at 37° C. in a humid, 5% CO 2 enriched atmosphere.

Liquid Culture:

With the remaining cell suspension liquid cultures as follows: Label two 25 cm 2 liquid culture flasks. Add RPMI 1640 1× Fortified plus FBS and cell suspension to the flasks as follows:
______________________________________
MEDIA CELLS
______________________________________

25 cm 2 4.0 ml 0.5-1.0 ml
______________________________________

Incubate the cultures at 37° C. in a humid, 5% CO 2 enriched atmosphere. Leave the caps loose for CO 2 exchange.

DNA Analysis:

If requested by the physician, save at least 1 ml for DNA. (NOTE: The cells do not need to be alive for DNA testing.) Label the tube with the patient name, date, specimen type, and tracking number and place in the DNA beaker in the refrigerator.

Liquid Nitrogen:

Prepare a minimum of 2 cryovials for long-term storage in liquid nitrogen according to the freezing protocol.

Immunomodulation:

If requested by the physician, save at least 2.0 ml for the assay and store refrigerated until the IM assay is performed.

Reporting Results:

Within 24 hours after setting up the pharmacosensitivity, read the cell control and abrin plates under the inverted scope. Scan the plates in entirety. Count any clumps of cells and record as a background count on the worksheet. Always scan the entire plate for colonies and aggregates using a hand tally counter to record the number of each. If no colonies or aggregates are present, count the number of cells in four squares. Divide by 4 to obtain the average number of cells in one square, multiply by 15, then by 13.36 to obtain the number of cells in the entire 35 mm plate. Use scientific notation to report the colonies, aggregates and/or cells for any number greater than 3 digits. Representative colonies, aggregates, and/or cells must be photomicrographed and logged in the log book prior to discarding plates. Verify that controls fall within expected range. If not, document in the comment section and notify the Supervisor immediately. Do not discard the assay; it may be accepted at the discretion of the Medical Director.

Experiment 1

Activity of N. sativa on bone marrow cells

Preparation of N. sativa:

1. Dried seeds are finely ground, and 5 grams (5 g) of the powder is extracted with 95% ethanol 3 times, by soxhlet extraction. A Wheaton soxhlet extraction apparatus consists of 3 parts: the bottom part is the flask in which the 95% ethanol is heated; the middle portion is the extractor; and the top the Allihn condenser, in which there is an inlet and outlet for running cold water through the condenser. Dried N. sativa seed powder is packed in filter paper and placed in the extractor.

2. The pooled extracts are evaporated under reduced pressure to a known volume of 10 ml and loaded onto a silica gel (Keiselgel 60: Fluka) column and eluted with 95% methanol/water (9:1). In the column, silica gel suspended in methanol/water is layered on sand. N. sativa extract is added on top of the silica gel, followed by the eluting solvent.

3. The active fraction (as indicated by brown color) is collected and separated by preparing thin layer chromatography (TLC). On commercially available chromatography plates, a band of N. sativa active compound is spotted and run in a TLC jar using chloroform as the solvent system.

4. The active spot at the solvent front is removed, eluted with methanol (overnight), and evaporated under reduced pressure to render the product alcohol free.

5. The extract is then available as a soluble solution and is then stored at 2° C.

The approximate content of the active compound in the seed in 2.2% (w/w).

Calculation of active compound in N. sativa extract proceeded as follows: the active compound present in seed extract is 2.2% w/w. One gram of seeds was added to 10 ml assay media. Therefore, active compound added was 22 mg/ml media. Finally 40 μl from this was taken for assay. Hence, the active compound actually added was 88 μg. This was further diluted 1:10, 1:100 and 1:1000 to have a working concentration of 8.8 μg, 880 ng and 88 ng respectively.

Colony Forming Unit Assay

Specimen: Bone marrow sufficient to yield an optimum of 5.0 ml mononuclear cells at a concentration of 1.0×10 6 cells/ml was collected.

Assay: The assay was performed according to a modified process Metcalf (1984, 1985), which is incorporated herein by reference. The process is described in detail above in Procedure 2. To each 25 cm 2 flask, 1×10 6 bone marrow cells are added, and incubated at 37° C. with 5% CO 2 and 100 μl of N. sativa plant extract.

Immunofluorescence Staining of Cells: The peripheral blood cells were collected aseptically. For every panel of analysis 3.0 ml whole blood was needed. The assay was conducted according to processes disclosed in and partially modified from Karen (1989) and Merkel et al. (1987), which are incorporated herein by reference. The process is described in detail above in Procedure 1.

The N. sativa plant extract was incubated with peripheral blood cells from cancer patients over an 18 hour period at 37° C. in a 5% CO 2 incubator. Gates for the antibodies CD3, CD19, HLADR, LAK CD3+/CD56+, NK CD3-/CD56+, CD38, CD37, and CD33 surface markers were set on a Becton Dickinson FacScan serial No. 81326. These antibodies are designed to assist in the characteristics of the types of cells. The cluster designation (CD) is used to group two or more mABs that have statistically similar expression on normal and neoplastic cells and cell lines, as well as similar recognition of the same antigen or binding site (epitope). While mABs in a CD group usually define different epitopes on the same antigen, some CD groups, primarily non-lymphoid clusters, contain mABs that do not bind to the same antigen.

The results obtained from Experiment 1 were compared with that of bone marrow growth factors and biological response modifiers namely GM-CSF, G-CSF, erythropoietin, interferon, IL-2 and STS.

At days 7, 14 and 21, each flask was scanned under a Reichart Jung Biostar inverted microscope. A colony is defined as 40 or more cells adhering to the bottom of the flask.

The colony forming cell unit (CFU) assay on 1×10 6 bone marrow cells obtained from 2 cancer patients was performed with and without incubation with 100 μl of N. sativa plant extract.

Results: The colony forming cell unit assay on 1×10 6 bone marrow cells post 15 days incubation with 100 μl of plant extract showed a 600% elevation. This elevation was 125% after 21 days of incubation. Among the different growth factors/biological response modifiers, IL-2 and GM-CSF gave the maximum elevation of 120 and 115% after incubation with bone marrow cells for 15 days. There was not much detection of CFU elevation post 21-day incubation with growth factor/biological response modifiers. Interferon showed marginal elevation after a 15-day incubation, but negligible detectable response after 21 days.

N. sativa plant extract helps restore the immune competent cells in immunosuppressed cancer patients. The colony forming cell unit assay indicates increase in CFU count when bone marrow cells from cancer patients were incubated with N. sativa plant extract as well as with different growth factors indicating that N. sativa plant extracts mimic the potential application of these several growth factors. Even though the plant extract was used without dissolving, it had no toxic effects against human bone marrow cells. Hence, N. sativa plant extract helps restore the immune competent cells in immunosupressed cancer patients and over-stimulates bone marrow in normal individuals.

Experiment 2

Correlation Between Elevation in Immune Competent Cells/Hematopoiesis and
N. sativa Extract

Immunomodulatory Testing

Immunomodulatory testing evaluates the activity of patients’ sera and white blood cells in relation to interferon, interleukin and lymphokine demonstrating the presence of interferon inhibitor factor and lymphokine inhibitor factor in the experimental procedure. A detailed description of immunomodulatory testing is found above in Procedure 3.

To test for possible correlation between elevation in immune competent cells/hematopoiesis and N. sativa extract, the various surface markers of immune competent cells were analyzed by automated flow cytometry as described above in Procedure 3.

The results indicate an elevation in CD19, HLADR, NK CD3-/CD56+ and CD38 population. There was not much alteration in CD3, CD37 and CD33 surface marker index. A proliferation of connective tissue `L` cells was observed upon incubation with cancer patient serum and N. sativa extract at 37° centigrade in 5% CO 2 incubator for 7 days.

Cell Line: The mouse connective tissue L929 cells were obtained from the ATCC and grown in MEM medium (GIBCO) with 20% fetal equine serum (BioWhittaker) as described above.

Assay: The assay was conducted according to the process described in Medenica, et al. (1990), which is incorporated herein by reference. Proliferation of 5×10 5 `L` cells/ml was checked on a double layer agar procedure in which lower 1.5% agar layer media contained 125 ml FBS, 125 ml 2× G.G. fortified solution and 250 ml 1× G.G. fortified solution. The upper layer stock media contained 150 ml FBS, 150 ml 2× G.G. fortified solution and 150 ml 1× G.G. fortified solution. To 2.0 ml of upper layer, 0.1 ml of N. sativa extract, 0.3 ml of `L` cells and 0.6 ml of 1.5% agar was added to make a total volume 3.0 ml. Growth of `L` cells were analyzed under an inverted microscope type Reichert Jung biostar post incubation at 37° centigrade and 5% CO 2 after 7 days.

The results are illustrated in Table 4 as follows:
TABLE 4
______________________________________
CELL % INCUBATION MIXTURE NUMBER ELEVATION
______________________________________

`L` CELLS & MEDIA 2.0 × 10 4
0
`L` CELLS & IFN & MEDIA
2.7 × 10 4
35
`L` CELLS & IL-2 & MEDIA
2.3 × 10 4
15
`L` CELLS & SERUM & PLANT
4.7 × 10 4
135
EXTRACT
`L` CELLS & WBC & PLANT
7.4 × 10 4
270
EXTRACT
______________________________________

Connective tissue `L` cells at a concentration of 5×10 5 were incubated with IFN, IL-2 or plant extract at 37° C. in a humid 5% CO 2 incubator for 7 days. The SD of the data was <10%.

Results: Peripheral blood cells from cancer patients upon incubation with N. sativa extract over an 18-hour period at 37° C. in 5% CO 2 incubator showed elevation in CD19, HLADR, NK CD3-/CD56+ and CD38 populations as shown in FIG. 1. CD19 surface marker index showed an elevation of 90% as compared to control, while HLADR, NKCD3-/CD56+ and CD38 population showed elevations of 44, 38 and 5% respectively. There was not much alteration in the CD3 population.

N. sativa extract, rather than the increase in immune competent cell number, helped free tumor antigen binding sites on B cells, thereby elevating the CD19 and associated cell population. Data from flow cytometric analysis indicates elevation in CD19, HLADR, NK CD3-/CD56+ and CD38 population. There was not much alteration in CD3, CD37 and CD33 surface marker index. This data clearly indicates proliferation in immune competent cells, mainly the humoral response against tumor antigens. Thus, the extract, rather than the increase in immune competent cell number, helps free tumor antigen binding sites on B cells, thereby elevating the CD19 and associated cell population.

Connective tissue `L` cells at a concentration of 5×10 5 upon incubation with N. sativa plant extract and patient serum/white blood cells showed elevation in cell number. `L` cells when incubated with media alone was 2.0×10 4 , which increased to 4.7×10 4 and to 7.4×10 4 upon incubation with cancer patient serum and plant extract and cancer patient white blood cells and plant extract, respectively. This indicated an elevation of 135 and 270% respectively as shown in Table 4 (supra.) On the other hand, interferon and interleukin-2 gave marginal elevations of 35 and 15% only upon incubation with 5×10 5 `L` cells in an ideally same environment as that of N. sativa plant extract.

Immunomodulatory evaluation indicates enhanced proliferation of the connective tissue `L` cells in presence of N. sativa extract than in its absence, thereby indicating positive immunomodulatory effect of the extract. Similar results were also obtained with patient white blood cells.

Data indicates presence of an interferon inhibitor factor (IIF) in cancer patient serum due to which there is no proliferation of the `L` cells when they were incubated with patient serum alone. IIF and lymphokine inhibitor factor (LIF) in patient serum get suppressed post treatment with N. sativa plant extract, thereby exhibiting enhanced proliferation of `L` cells. The IIF cannot be identified with antibodies against interferon.

IIF is a factor which does not allow the production of autologous (self) interferon in the human body. When the inducer initiates the production of interferon, the human body produces another protein which neutralizes the possibility for production of interferon. LIF includes all factors that suppress cytokine activity, including interferons.

The increased stimulation of connective cells by the reduction of IIF and LIF allows the human body to produce its own interferon, which then interferes with controlling connective tissue cell production. For this reason, N. sativa has potential use in connective tissue diseases such as rheumatoid arthritis, lupus and autoimmune diseases.

Experiment 3

Cytopathic Effects of Vesicular Stomatitis Virus

Cell Line: The human amnion or “WISH” cells were obtained from the American Type Cell Culture facility (ATCC) and grown in Basal Eagle Medium (BEM) (GIBCO) with 15% fetal bovine serum.

Virus: Vesicular stomatitis virus (VSV) was obtained from the ATCC. The VSV stock solution was diluted 1000× with BEM containing 2% fetal bovine serum (FBS).

Assay: The assay was conducted as described above in Procedure 4. For determining the amount of protection conferred to the “WISH” cells by N. sativa plant extract, complete viral cytopathic effect (CPE) was calculated by inoculating 8-10 ml per flask of the 1:1000 VSV into each WISH cell flask (containing 3.5×10 5 cells/ml) such that it covered the entire cell layer. The flasks were incubated for 1 hour at 37° C. in a 5% CO 2 incubator. Fifteen ml of the BMEM with 2% FBS were added to each flask and incubated at 37° C. in a 5% CO 2 incubator for 1-3 days or until cell layer showed complete viral cytopathic effect (CPE). N. sativa plant extract was serially diluted in a 96-well polystyrene plate. To 40 μl of the extract was added 100 μl of the 3.5×10 5 cells/ml “WISH” cell suspension and the plates were incubated for approximately 18-24 hours at 37° C. at 5% CO 2 . In the control wells, no extract was added. Next day (after 18-24 hours) the plates were observed under Nikon TMS microscope for confluency. Fifty μl VSV were added (in a concentration to yield 100% CPE), and Incubated 24-48 hours at 37° C. with 5% CO 2 .

The next day, the plates were observed under a Nikon TMS microscope for 100% CPE in the viral control wells. Once satisfied, the plates were dumped and blotted. One hundred μl of 15% neutral red dye were added to all wells. The wells were incubated at 37° C. in 5% CO 2 incubator for 45 minutes. The plates were dumped, blotted and rinsed with 200 μl PBS at pH 6.85. The plates were again dumped and blotted. The cells were eluted by adding 100 μl of the eluting solution to each well. The plates were read on Bio-Rad Model 3550 microplate reader.

The concentration of vesicular stomatitis virus (VSV) producing 100% cytopathic effect (CPE) of human amniotic “WISH” cells was first determined, which was 1×10 5 plaque forming units. Optimum protection to WISH cells conferred by N. sativa extract was then calculated by serially diluting N. sativa extract and adding in between WISH cells and VSV.

Results: The results depicted in FIG. 2 indicate protection of “WISH” cells in the order of 25 and 65% upon incubation with 1:10 and 1:100 diluted N. sativa plant extract. There was no noticeable cytopathic effect when N. sativa was added at a dilution of 1:1000. Approximate active compound concentrations were calculated according to materials and methods. Serum interferon levels were measured after plant extract administration according a modified procedure of Finter (1969).

Protection of Human Amniotic “WISH” cells from cytopathic effects of vesicular stomatitis virus (VSV) was observed upon administration of 1:1000 diluted N. sativa plant extract. This had an active compound concentration of 88 ng. At a lower dilution, there was some effect. Theoretically, a patient weighing 70 kg has about 7×10 13 cells in its body and N. sativa will be useful in the dosage of 20-40 g to protect against viral attack in virus endemic areas. Upon administration of the extract, the serum interferon level is found to increase and hence N. sativa has interferon-like antiviral activity. The extract possibly blocks all receptor sites on “WISH” cells which are essential for attachment of the virus to cell membrane.

Experiment 4

Pharmacosensitivity Assay

The tumor specimens can be of 2 types: solid tumor and liquid tumor.

Solid tumor: 2 grams of tumor specimen were excised, cut into pea size portions and gently homogenized in a Potter-Elvehjam homogenizer with 10% FBS, centrifuged at 1600 rpm for 20 minutes in a Mistral 3000i refrigerated centrifuge at 4° C. Cells were finally suspended in media RPMI 1640 with 10% FBS into a fine suspension.

Liquid tumor: The specimen was collected from the ascitic fluid of cancer patients. The ascitic fluid was gently layered on a histopaque using a 1:1 ratio of the histopaque to specimen, and centrifuged at 1600 rpm for 20 minutes in a Mistral 3000i refrigerated centrifuge at 4° C. Cells were finally suspended in RPMI with 10% FBS.

Assay: The assay was performed according to the modified processes of Von Hoff, et al. (1981) and Salmon, et al. (1978), which are incorporated herein by reference, as described above in Procedure 5. Tumoricidal activity of N. sativa was checked on a double agar layer. The lower layer of warm 2.5% Bacto-agar working solution was poured into 35 mm grid petri dishes. The plates were then allowed to sit undisturbed on a flat surface until the agar solidified. To 2.1 ml of the upper layer media containing 150 ml FBS, 150 ml 2× G.G. fortified solution and 150 ml 1× G.G. fortified solution, 0.3 ml of the tumor cells and 0.6 ml of the 1.5% agar were added.

The upper layer was carefully layered on top of the lower layer in the 35 mm grid petri dish. Tumor specimens grown without the extract and with `Abrin` were used as controls. Tumor cell growth in the presence of the plant extract was monitored 7, 14 and 21 days post incubation at 37° C. and 5% CO 2 , at which time the cells were counted under a Reichert-Jung biostar inverted microscope. Cells from tumor specimens [8.2×10 4 ] were grown with media/Abrin plant extract on a double agar layer and incubated for 7, 14 and 21 days at 37° C. in a humid 5% CO 2 incubator. The results are shown on Table 5 as follows:
TABLE 5
______________________________________
INCU- BATION INCUBATION TIME MIX- % % % TURE 7 days Inh 14 days Inh 21 days Inh
______________________________________

TUMOR 8.2 × 10 4
0 10.0 × 10 4
0 10.5 × 10 4
0
CELL &
MEDIA
TUMOR 8.1 × 10 4
1.2 9.8 × 10 4
2.0 10.3 × 10 4
1.9
CELL &
ABRIN
TUMOR 5.3 × 10 4
35.0 5.4 × 10 4
46.0 5.5 × 10 4
48.0
CELL &
EX-
TRACT
______________________________________

The SD of the data was <10%. Percentage of inhibition was calculated as described in Materials & Methods.

The results are analyzed on the basis of pharmacosensitivity assay in which tumor cell growth was monitored. Seven, 14 and 21 days post incubation with plant extract indicated tumoricidal activity. Hence, N. sativa extract possessed a multifaceted toxic-free cure for cancer.

Results: Tumor specimens (2 grams) either from solid tumor masses or malignant ascites were mechanically homogenized into a suspension containing 8.2×10 4 cells. Incubation of tumor cells at 37° C. in a humid 5% CO 2 incubator for 7, 14 and 21 days with N. sativa plant extract indicated a percent inhibition of 35, 46 and 48, respectively. Incubation with Abrin in an ideally similar condition did not exhibit any noticeable inhibition.

N. sativa promotes anti-tumor activity. Data from pharmacosensitivity screening indicates anti-tumor activity of N. sativa plant extract, as cell growth in culture was inhibited anywhere between 25-90%. In vitro tumor study report, using rapid screen for early pharmacosensitivity screening, in which cells were incubated with N. sativa plant extract for 2 hours, gave similar results. Hence, the extract indicates anti-tumor function mainly against melanoma and colon cancer types. N. sativa plant extract destroys tumor cells and leaves normal cells alone, possibly because of its ability to bind to cell surface asialofeutin (lectin) in diseased cells, which causes aggregation and clumping of tumor cells. It also blocks enzymes and inappropriate gene products involved in nucleic acid synthesis and metabolism.

It is understood that the invention is not confined to the particular construction and arrangement herein described, but embraces such modified forms thereof as come within the scope of the claims following the bibliographic citations.

BIBLIOGRAPHY

Agarwal, R., Kharya, M. D., and Shrivastava, R., 1979, “Antimicrobial anthelmintic activities of the essential oil of Nigella sativa Linn,” Indian J. Exp. Biol. 17, 1264.

Akhtar, M. S.; Riffat, S., 1991, “Field Trial of Saussurea Lappa Roots Against Nematodes and Nigella Sativa Seeds Against Cestodes in Children,” JPMA J Pak Med Assoc 41(8)185-7.

al-Awadi, F. M., Khattar, M. A. and Gumaa, K. A., 1985, “On the Mechanism of the Hypoglycemic Effect of a Plant Extract,” Diabetologia 28, 432-434.

al-Awadi, F.; Fatania, H.; Shamte, U., 1991, “The Effect of a Plant’s Mixture Extract on Liver Gluconeogenesis in Streptozotocin Induced Diabetic Rats,” Diabetes Res (Scotland) 18(4):163-8.

Aruna, K.; Sivaramakrishnan, V. M., 1990, “Plant Products as Protective Agents Against Cancer,” Indian J Exp Biol 28(11):1008-11.

Aruna, K.; Sivaramakrishnan, V. M., 1992, “Anticarcinogenic Effects of Some Indian Plant Products,” Food Chem Toxicol (England) 30(11)953-6.

Bitterman, W. A.; Farhadian, H.; Abu Samra, C.; Lerner, D.; Amoun, H.; Krapf, D;. Makov, U. E., 1991, “Environmental and Nutritional Factors Significantly Associated with Cancer of the Urinary Tract among Different Ethnic Groups,” Urol Clin North Am 18(3)501-8.

Chopra, R. N., Chopra, I. C., Handa, K. L., and Kapur, L. D., 1982, Indigenous Drugs of India, Academic Publishers; Calcutta, India.

Datta, A. K., Biswas, A. K. and Ghosh, P. D., 1983, “Chromosomal variations in callus tissues of two species of Nigella sativa, L.,” The nucleus 26(3) 173-177.

Elkadi, A ; Kandil, O., 1987, “The Black Seed Nigella sativa and Immunity Its Effects on Human Cell Subsets,” Fed Proc 45(4)1222.

Finter, N. B., 1969, “Dye Uptake Methods for Assessing Viral Cytopathogenicity and Their Application to Interferon Assays, ” J. Gen. Virol. 5, 419-427.

Karen, D. F., 1989, “Flow Cytometry in Clinical Diagnosis,” American Society of Clinical Pathology (press).

Kirtikar, K. R. and Basu, B. D., 1982, Indian Medicinal Plants, Vol. I, Bishen Singh and Mahendra Pal Singh, eds., Dehra Dun, India.

Kumar, B. H. and Thakur, S. S., 1989, “Effect of Certain Non-edible Seed Oils on Growth Regulation in Disdercus Similis (F), J. Anim. Morphol. Physiol. 36(2) pp. 209-218.

Medenica, R., Alonso, K., Huschart, T. and Tyler, K., 1990, “Tumor Tissue Culture for Determining Efficient Drug for Intra-Arterial, Intra-Hepatic Chemotherapy and Interferon of Colon Carcinoma Liver Metastasis,” Abstract presented at Conference on Combining BRM with Cytotoxic in the Treatment of Cancer.

Merkel, D. E., Dressier, L. G. and McGuire, W. L., 1987, “Flow Cytometry Cellular DNA Contents and Prognosis in Human Malignancy,” J. of Clinical Oncology, 5, 1690-1703.

Metcalf, D., 1984, Clonal Culture of Hematopoietic Cells, Elsevier/North American Biomedical Press.

Metcalf, D., 1985, “The Granulocyte Macrophage Colony Stimulating Factors,” Science 229, 16-22.

Nadkarni, K. M., 1976, “Crocus sativus, Nigella sativa,” Indian Materia Medica, K. M. Nadkarni (ed) Bombay, India; Popular Prakashan, Vol 1, pg. 386-411.

Nair, S. C.; Salomi, M. J.; Panikkar, B.; Pannikar, K. R., 1991, “Modulatory Effects of Crocus sativus and Nigella sativa Extracts on Cisplatin-Induced Toxicity in Mice,” J Ethnopharmocol 31(1): 75-83.

Salmon, S. E., Hamburger, A. W., Soehnlein, B., 1978, “Quantitation of Differential Sensitivity of Human Tumor Stem Cells to Anticancer Drugs, , ” N. Eng. J. Med. 298, 1321-1327.

Salomi, N. J.; Nair, S. C.; Jayawardhanan, K. K.; Varghese, C. D.; Panikkar, K. R., 1992, “Anititumour Principles from Nigella sativa Seeds,” Cancer Lett 63(1):41-6.

Salomi, M. J.; Nair, S. C.; Panikkar, K. R., 1991, “Inhibitory Effects of Nigella sativa and Saffron (Crocus Sativus) on Chemical Carcinogenesis in Mice,” Nutr Cancer 16(1):67-72.

Sayed, M. D., “Traditional Medicine in Health Care,” 1980, J Ethnopharmocol 2(1):19-22.

Shayeb, N. A. and Mabrouk, S. S., 1984, “Utilization of Some Edible and Medicinal Plants to Inhibit Aflatoxin Formation,” Nutrition Reports International 29(2).

Siddiqui, M. B., Alam, M. M., Husain, W. and Sharma, G. K., 1988, “Ethno-medical Study of Plants used for Terminating Pregnancy,” Fitoterapia V. LIX, no. 3, 250.

Salmon, S. E.; Hamburger, A. W.; Soehnlein, B.; et al., 1978, “Quantitation of differential sensitivity of human tumor stem cells to anticancer drugs,” N Engl J Med 298:1321-1327.

Srivastava, K. C., 1989, “Extracts from Two Frequently Consumed Spices–Cumin (Cuminum cyminum) and Turmeric (Curcuma Longa)–Inhibit Platelet Aggregation and Alter Eicosanoid Biosynthesis in Human Blood Platelets,” Prostaglandins Leukot Essent Fatty Acids 37(1)57-64.

Tennekoon, K. H.; Jeevathayaparan, S.; Kurukulasooriya, A. P.; Karunayake, E. H., 1991, “Possible Hepatotoxicity of Nigella sativa Seeds and Dregea Volubilis Leaves,” J Ethnopharmocol 31(3):283-9.

Vihan, V. S. and Panwar, H. S., 1987, “Galactopoietic Effect of Nigella sativa (H-Kalonji) in Clinical Cases of Agalactia in Goats, ” Indian Vet. J. 64, 347-349.

Von Hoff, D. D., Cowan, J., Harris, J. and Reisdorf, G., 1981, “Human Tumor Cloning: Feasibility and Clinical Correlations,” Cancer Chemother. Pharmacol. 6, 265-271.