Pengaruh ion Tembaga dan Kobalt terhadap kandungan Kumarin dan profil kromatografi lapis tipis metabolit sekunder kultur sel pacar air (Impatiens balsamina L.)

ZAINAB, Dr. C.J. Soegihardjo, Apt

2007 | Tesis | S2 Ilmu Farmasi

Senyawa golongan kumarin mempunyai aktivitas biologi yang sangat beragam diantaranya sebagai antikoagulan, estrogenik, fotosensitifitas pada kulit, antimikroba, vasodilator, moluskasidal, antelmintik, antimalaria, antitumor, anti- HIV, antivirus, Ca antagonis, sitostatik, inhibitor 5-lipooksigenase, inhibitor monoamin yang sangat potensial digunakan sebagai obat. Pembentukan kultur sel Impatiens balsamina L. sudah pernah diteliti dan mampu memproduksi senyawasenyawa derivat kumarin, skopoletin dan isofraksidin. Penelitian ini dilakukan dengan tujuan mengetahui pengaruh penambahan ion tembaga dan kobalt terhadap kandungan kumarin dan profil kromatogram lapis tipis metabolit sekunder kultur suspensi sel I. balsamina. Upaya peningkatan kadar kumarin dalam kultur suspensi sel I. balsamina dilakukan dengan penambahan elisitor ion Cu dan ion Co dalam media cair MS dengan penambahan kinetin 1 mg/l dan 2,4 D 0,1 mg/l (1K; 0,1D) yang diinisiasi dari kalus yang ditumbuhkan pada media padat MS (1K; 0,1D). Konsentrasi ion Cu dan Co yang ditambahkan dalam media cair MS adalah 10, 20 dan 30 kali konsentrasi Cu dan Co pada MS normal atau setara dengan 250, 500 dan 750 μg/l. Kultur suspensi sel pada media perlakuan diinkubasi pada suhu 22-280C dengan kondisi gelap terus dan di shaker. Kultur suspensi sel dipanen setelah diinkubasi selama 30 hari kemudian dianalisis berat basah dan berat keringnya. Analisis kualitatif metabolit sekunder kultur dilakukan terhadap kandungan naftokinon, flavonoid, kumarin dan saponin dengan metode kromatografi lapis tipis. Analisis kuantitatif kandungan kumarin dalam kultur suspensi sel dilakukan dengan metode TLC Scanner. Hasil analisis berat basah dan berat kering, penambahan ion Cu (250-750 μg/l) menyebabkan penurunan berat basah 77,87-95,29% dan penurunan berat kering 62,38-76,79%, sedangkan pada ion Co (250-750 μg/l) terjadi penurunan berat basah 83,72-99,99% dan berat kering 85,81-98,22% pada suspensi sel setelah diinkubasi selama 30 hari kecuali pada Co 750 μg/l terjadi peningkatan berat basah sampai 118,35%. Penambahan ion Cu dan ion Co pada kultur suspensi sel secara kualitatif tidak mempengaruhi profil kromatogram metabolit sekunder dibandingkan dengan kontrol suspensi sel, tetapi hanya terdeteksi senyawa kumarin dan saponin triterpenoid saja sedangkan pada tanaman pacar air terdeteksi senyawa naftokinon, flavonoid, kumarin dan saponin triterpenoid. Penambahan ion Cu dan ion Co mempengaruhi kadar senyawa golongan kumarin secara kualitatif dan kuantitatif. Secara kualitatif penambahan ion Cu hanya dihasilkan kumarin dengan Rf 0,72 saja dengan kadar tertinggi 147,53 mg%. Penambahan ion Co mampu menghasilkan senyawa kumarin lengkap (Rf 0,23, 0,32 dan 0,72) dengan kadar tertinggi 259,60 mg% (ion Co 750 μg/l).

Coumarins displays various bioactivities in vitro such as anticoagulant, estrogenic, dermal photosensitising, antimicrobial, vasodilator, molluscicidal, antihelmintic, sedative and hipnotic, analgesic, antimalarial, antitumor, anti-HIV, antiviral, Ca antagonistic, cytostatic, inhibition of 5-lipoxygenase, inhibition of monoamine oxydase which is very potencial for medicine. The study on cell cultures of Impatiens balsamina L. have been done before and the cell cultures were capable of producing coumarins derivate, scopoletin and isofraxidin. So the aim of these research was to study the effect of addition copper and cobalt metal ions into cell culture of I. balsamina on the yield of coumarins and thin layer chromatogram profile of secondary metabolite in cell cultures. Increasing the yield of coumarins in cell cultures of I. balsamina, was achieved by addition of elicitor Cu and Co metal ions in the liquid MS media supplemented with 1,0 mg/l kinetin and 0,1 mg/l 2,4-dichlorophenoxyacetic acid (1K; 0,1D) which initiated from calli grown on solid MS media (1K; 0,1D). Concentration of Cu and Co metal ions which used in this research were 10, 20, and 30 fold of normal MS media (250, 500, 750 μg/l). The cell culture on treatment media were incubated at 22-28 0C with continuous dark periode on rotary shaker. The 30 days old cell cultures were harvested and analyzed of fresh and dry weight. Qualitative analysis of secondary metabolite in cell cultures were done on naphthoquinones, flavonoids, coumarins, and saponins content with TLC methode. Quantitative analysis the yield of coumarins in cell cultures were measured by TLC Scanner. The addition of various concentration of Cu metal ion (250-750 μg/l) decreased the fresh weight to 77,87-95,29% and dry weight to 62,38-76,79% of cell cultures; while the various concentration of Co metal ion (250-750 μg/l) decreased the fresh weight to 83,72-99,99% and dry weight to 85,81-98,22% after incubation under continuous dark condition for 30 days. However, at the concentration of Co (750 μg/l) caused increasing fresh weight to 118,35%. The addition of both Cu and Co metal ions had no influence on the chromatogram profile of secondary metabolite compared to cell cultures (control) according to TLC result, but only coumarins and triterpenoid saponins were detected. While naphthoquinones, flavonoids, coumarins and triterpenoid saponins were detected in the mother plant. The addition of Cu and Co metal ions had influence both qualytative and quantitative of coumarins content. The addition of Cu metal ion could only produce coumarin (Rf 0,72) and the highest coumarin production detected was 147,53 mg%. While the addition of metal ion Co could produce complete coumarins as mother plant (Rf 0,23, 0,32 and 0,72) with highest coumarin production was 259,60 mg% (ion Co 750 μg/l).

Kata Kunci : Impatiens balsamina L., kultur suspensi sel, elisitor, ion Cu dan Co, kumarin, cell suspension cultures, elicitor, Cu and Co ions, coumarins.