Efek Non Genomik Dan Genomik Ekstrak Daun Ceplukan (Physalis Minima L) Pada Kultur Sel Endotel Manusia (Huvecs) Nur Permatasari, Nurdiana, Setyawati Karyono Fakultas Kedokteran, Universitas Brawijaya Abstrak Daun ceplukan secara empiris digunakan untuk menurunkan tekanan darah. Komponen yang terkandung di daun ceplukan antara lain physalin, senyawa yang mempunyai gugus steroid. Pada penelitian ini akan dibuktikan efek non genomik dan genomik ekstrak daun ceplukan pada sel endotel. Disamping itu akan dibuktikan juga peran physalin pada efek estrogenlike tersebut. Penelitian dilakukan pada HUVECs. Daun ceplukan dari Materia Medika Batu diekstrak menggunakan alkohol. Physalin didapatkan dari hasil TLC (Indochimie,USA). Efek non genomik diamati dengan mengevaluasi kadar Ca2+ sitosolik, menggunakan teknik Fura-2AM., pada HUVECs yang diinkubasi ekstrak selama 10 menit. Efek genomik diamati dengan menghitung jumlah sel yang mengekspresikan inducible nitric oside synthase(iNOS) menggunakan teknik immuno staining pada HUVECs yang diinkubasi ekstrak selama 120 menit. Pada semua kelompok, diukur nitric oxide (NO), dengan mengukur nitrat dan nitrit menggunakan spektrofotometer. Sedangkan peran physalin dilakukan dengan menginkubasikan physalin selama 10 dan 120 menit untuk dievaluasi sel yang mengekspresikan eNOS. Hasil penelitian menunjukkan ekstrak daun ceplukan konsentrasi 0.0025%,0.005% dan 0.05% meningkatkan kadar Ca2+ sitosolik HUVECs (arbitrary unit) secara signifikan (p<0.001) dibandingkan HUVECs tanpa perlakuan. Pada evaluasi iNOS, ekstrak dengan konsentrasi 0.0025% dan 0.005 % pada HUVECs dengan masa inkubasi 120 menit, jumlah sel yang mengekpresikan iNOs meningkat signifikan (p<0.001) dibandingkan masa inkubasi 10 menit. Jumlah NO pada semua kelompok perlakuan meningkat. Disamping itu hasil evaluasi eNOS pada kultur sel yang diinkubasi physalin (2.10-3 %, 8.10-4 % dan 4.10-5 %) pada HUVECs selama 10 dan 120 menit terlihat memicu ekspresi eNOS. Disamping itu terlihat juga adanya kecenderungan bahwa semakin lama inkubasi physalin, semakin banyak jumlah sel dan kepadatan gambaran eNOS. Pada penelitian ini dapat disimpulkan ekstrak daun ceplukan meningkatkan pelepasan NO dari sel endotel, diduga terkait dengan mekanisme non genomik dan mekanisme genomik. Diduga physalin yang memberikan efek yang menyerupai estrogen tersebut. Kata kunci: daun ceplukan – Ca2+ sitosolik – NO - iNOS
Non genomic and genomic effects of the leaves of ceplukan (Physalis minima L) in cultured human endothelial cells (HUVECs) Abstract Non genomic and genomic effects of the leaves of ceplukan (physalis minima l) in cultured human endothelial cells (huvecs).The leaves of ceplukan was empirically used for decreasing blood pressure. The experiments were study the non genomic and genomic effects of the extract of the leaves of ceplukan in endothelial cells. In addition, the experiments were also study the role of physalin on that estrogenlike effects. The experiments was done in HUVECs. The leaves of ceplukan were carried from Materia Medika Batu and were extract with alcohol. Physalin was carried from Indochimie (USA). The Non genomic effect was approved by measuring cytosolic calcium concentration in HUVECs exposed to the extract for 10 minutes. The calcium cytosolic was measured using probe fura-2AM and observed by optical method. Genomic effect was determined by evaluating the expression of inducible nitric oxide synthase (iNOS) in HUVECs exposed to the extract for 120 minutes.The iNOs was evaluated using immuno staining technique All of the groups of experiment were measured the production of nitric oxide (NO) using indirectly
measurement (nitrate and nitrite) by spectrophotometer. To study the effects of physalin, the huvecs was incubated with physalin at dose of 2.10-3 % ,8.10-4 % and 4.10-5 % for 10 and 120 minutes , respectively. The results showed that the extract of ceplukan in the concentration of 0.0025%,0.005% and 0.05% increased cytosolic calcium concentration (arbitrary unit) significantly (p<0.001) from the normal group. The evaluation of iNOS showed that the incubation of extract in the concentration of 0.0025% and 0.005% for 120 minutes increased the amount of cells that expressed iNOS significantly (p<0.001) than the incubation of the extract for 10 minutes. All of the groups of experiment increased the production of NO. This experiments had also showed that the incubation of physalin at dose of .10-3 % ,8.10-4 % and 4.10-5 % for 10 and 120 minutes could increase the expresion of eNOS, respectively. In addition, the results had also showed that the amounts of cell that expressed eNOS tend to be increased related to the duration of physalin exposure. The conclusion of the experiments, the extract of the leaves of ceplukan can induce NO production and stimulate the calcium signal transduction and NOS expression. The effects maybe represents of a non genomic and genomic effects. In addition, we also suggest that physalin is the active compound of that “estrogen like effects” Keywords: the leaves of ceplukan – Ca2+ cytosolic – NO – iNOS
Daftar Pustaka
Boulanger and Vanhoutte. 1994. The endothelium: a pivotal role in health and cardiovascular disease. Servier International Carvalho AP., Meireles LA., Malcata FX.1998. Rapid spectrophotometric determination of nitrates and nitrites in marine aqueous culture media. Analysis 26:347-351 Chambliss KL, Sghaul PW. 2002. Rapid activaton of endothelial NO synthase by estrogen: evidence for a steroid receptor fast-action complex in caveolae. Steroids 67:413-419. Efendi M, 1998. Pengaruh Alkaloid Daun Ciplukan (Physalis minima L) terhadap Kontraktilitas Sediaan Aorta Terpisah Kelinci dengan Stimulasi Noradrenalin Eksogen. Skripsi S1. Malang: Fakultas MIPA Universitas Brawijaya. Gupta A and Gupta R. A survey of Plants for Presence of Cholinesterase Activity. Phytochemistry. 1997; 46 (5): 827-831. Heyne K, 1987. Tumbuhan Berguna Indonesia. Jilid 2. Diterjemahkan oleh Badan Litbang Kehutanan. Jakarta: Yayasan Sarana Warna Jaya. Kirson I, Zaretskii Z and Glotter E, 1976. in: Sen G and Pathak HD. Physalin L, A 13, 14–seco16, 24 cyclosteroid from Physalis minima. Phytochemistry. 1995; 39 (35): 12451246. Khoiriyah F, Permatasari N.2004. Efek ekstrak daun ceplukan (Physalis minima L) pada pembuluh darah terisolasi tikus. Tugas akhir. Matsuura T, Kawai M, Nakashima R and Butsugan, 1970. In: Sen G and Pathak HD. Physalin L, A 13, 14– seco-16, 24 cyclosteroid from Physalis minima. Phytochemistry. 1995 ; 39 (35): 1245-1246. Mershan JL, Baker RS, Clark KE.2002. Estrogen increases iNOS expression in ovine coronary artery. Am.J.Physiol. Heart Circ Physiol, 283:1169-1180 Mohana K, Uma R and Purushottaman KK, 1979. in: Sen G and Pathak HD. Physalin L, A 13, 14–seco-16, 24 cyclosteroid from Physalis minima. Phytochemistry. 1995; 39 (35): 1245-1246. Morgan D. 1996.Isolaation and culture of human umbilical vein endothelial cells. In (Jones GE, eds), Human cell culture protocols. New Jersey: Humana Press, pp101-110 Munzel T, Just H, Harrison D. 1997. The physiology and pathophysiology of nitric oxide/superoxide system. In (Born and Swartz, eds), Vascular Endothelium, Physiology, Pathology and Therapeutics Opportunities. Stuttgart: Schattaeur, pp 205-220. Mulchandani NB, Iyer SS ang Badheka LP, 1979. in: Sen G and Pathak HD. Physalin L, A 13 , 14–seco16, 24 cyclosteroid from Physalis minima. Phytochemistry. 1995; 39 (35): 1245-1246. Reed D, 2002. Ground Cherry and Physalis, (on line), (http//www.2ttnThewildcom wildflowers_ southwesternUS, diakses 20 Oktober 2004). Ritcher RK, 1998. Reporting Biological Assay Result on Tropical Medical Plant to Host Country Collaborators, (on line), (http// www.rain tree. com/mullaca.htm, diakses 20 November 2002) Row LR, Sarma NS, Matsuura T and Nakashima I, 1978, in: Sen G and Pathak HD. Physalin L, A 13, 14– seco-16, 24 cyclosteroid from Physalis minima. Phytochemistry. 1995 ; 39 (35): 1245-1246. Sen G and Pathak HD. Physalin L, A 13, 14–seco16, 24 cyclosteroid from Physalis minima. Phytochemistry. 1995; 39(35): 1245-1246
Setiawan, 1982. Pemeriksaan Senyawa Cecendet (Physalis minima Linn). Skripsi. Bandung: Farmasi Fakultas MIPA Institut Teknologi Bandung. Singh AP, 2002. Withanolides-Much Talked Natural Product, (on line), (http://www.Healthstores.com/ayurveda/mar 2002_newsletter.htm, diakses 20 Oktober 2004). Stennis VCGGJ, 2003. Flora. Jakarta: PT Pradnya Paramita. hlm. 363-364. Wakhidatin N, 1996. Pengaruh Ekstrak Alkohol dari Daun Physalis minima L (ciplukan) terhadap Kontraktilitas Sediaan Arteri Terpisah Tikus (Rattus rattus Strain Wistar). Skripsi. Malang: Fakultas MIPA Universitas Brawijaya. Tjitrosoepomo G, 1989. Taksonomi Tumbuhan (Spermatophyta). Yogyakarta: Gadjah Mada University Press. hlm. 32,355. Volk T, Hensel M, Kox W. 1997. Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species in role hydroxyperoxide. Moll Cell Biochem 173:11-21
