Salah satu alasan kenapa masih rendahnya jumlah

PAKET INFORMASI TERSELEKSI

Tanaman Obat Seri: Sambung Nyawa

S

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DAUN SAMBUNG NYAWA, Gynura procumbens (Lour.) Merr, PENGANTAR RENCANA PENELITIAN KHASIAT OBAT TRADISIONAL

EFEK HEPATOPROTEKTIF EKSTRAK ETANOL DAUN DEWA (Gynura procumbens (Lour) Merr) PADA TIKUS TERINDUKSI PARASETAMOL

Soekrijanto

Jason Merari Peranginangin

Bina Widya : majalah ilmiah, No. 28, 1997:24-33

Biomedika. Vol. 3, No. 1, 2010

Abstrak: -

Abstract:

EFEK ANTIPROLIFERASI EKSTRAK ETANOLIK DAUNGynura procumbens (Lour.)Merr.PADA SEL PARU TIKUS JANTAN YANG DIINDUKSI 7,12 DIMETILBENZ[A]ANTRASEN Wasito, Hendri; Murwantib, Retno; Meiyanto, Edy STATISTIKA: Forum Teori dan Aplikasi Statistika, Vol. 7, No. 1, 2007:13-20 Abstrak: Telah dilakukan penelitian untuk mengetahui efek ekstrak etanolik daun G. Procumbens terhadap aktivitas proliferasi sel paru tikus jantan galur Sprague Dawley yang diinduksi oleh 7,12- dimetilbenz[a]antrasen (DMBA). Hasil pengamatan preparat histopatologi organ paru tikus dengan pengecatan H&E dan AgNOR pada minggu ke-16 serta analisis statistik dengan metode non parametrik Kruskal-wallis Test dan diteruskan dengan Mann-Whitney Test menunjukkan bahwa pemberian DMBA 20 mg/kg BB dua kali seminggu selama 3 minggu meningkatkan aktivitas proliferasi sel paru tikus jantan, namun belum dapat menunjukkan insidensi kanker paru serta pemberian ekstrak G. procumbens 300 mg/kg BB dan 750 mg/Kg BB belum dapat menghambat proliferasi sel paru tikus jantan yang diinduksi DMBA 20 mg/kg BB.

Hepatoprotective effect of ethanol extract of Dewa leaf (Gynura procumbens (Lour) Merr) at mouse induceed parasetamol has been investigated, as a mean to obtains data and scientific evidence of hepatoprotective effect of ethanol extract of dewa leaf, as result of treatment of hepatotoksin parasetamol. This study applies 30 white Wistar mouse divided to 6 group. Group I is given by parasetamol dose 200 mg/200 g BB, group II as negative control is given by condensation CMC 1 %, group III as positive control is given by asetilsistein dose 3,6 mg/200 g BB, group IV, V, VI as a group treatment is given by dewa leaf extract successively 1,32; 2,75; and 5,50 mg/200 g BB. Blood taken away from vein lateralis tail to determine activity GPT and GOT-serum activity. Result of study indicates that dewa leaf extract given oral can reduce GPT-serum activity 57 U/L; 46,6 U/l; 39 U/l to control parasetamol, GOTserum activity downwards 51,2 U/L; 38,6 U/L; 30,2 U/L to control parasetamol. Based on GPT and GOT-serum activity, inferential that ethanol extract of dewa leaf (Gynura procumbens (Lour) Merr) measures up to hepatoprotektif with effective dose 5,50 mg/200 gBB.

DAFTAR ISI EKSPRESI CYP1A1 DAN GSTU HEPATOSIT TERINDUKSI 7,12-DIMENTILBENZ(A)ANTRASENA DAN PENGARUH PEMBERIAN EKSTRAK ETANOLIKGynura procumbens Iwan Sahrial Hamid;Sugiyanto;Edy Melyanto;Sitarina Widyarini Majalah Farmasi Indonesia, Vol. 20, No. 4, 2009:198-206 Abstrak: Sitokrom P450 (CYP) dan glutathione S-transferase merupakan system enzim yang mempunyai pengaruh biologik pada karsinogen.Demikian pula, enzim ini diduga berkaitan dengan perkembangan kanker payudara, sehingga menjadi target potensial untuk senyawa kemopreventif.Tujuan dari penelltian ini adalah untuk membandingkan ekspresi CYP1A1 dan GSTµ di antara kelompok perlakuan.Ekspresi CYP1A1 dan GSTµ ditentukan secara kuantitatif dari jaringan hepar tikus galur Sprague dawley betina, umur 40 hari sejumlah 18 ekor.Dilakukan pengelompokan secara acak ke dalam enam kelompok perlakuan. Kelompok base line (tanpa pemberian DMBA dan ekstrak etanolik), kelompok induksi kanker dengan DMBA, dua kelompok perlakuan diberi DMBA setelah pemberian ekstrak etanolik Gynura procumbens dengan dua dosis, yaitu dosis 300 dan 750 mg/ kg BB. Dua kelompok perlakuan terakhir hanya diberi perlakuan terdiri dari dua dosis ekstrak etanolik tanpa inisiasi DMBA.Pemberian ekstrak dilakukan selama tiga minggu dan inisiasi DMBA dilakukan pada minggu ke dua dan ke tiga dari awal pemberian ekstrak.Pengamatan ekspresi CYP1A1 dan GSTµ dilakukan dengan metode imunohistokimia. Ekspresi CYP1A1 pada kelompok perlakuan DMBA menunjukkan jumlah yang tertingi secara signifikan (p<0,05) dibanding kelompok perlakuan yang lain. Sebaliknya pada ekspresi GSTµ menunjukkan jumlah yang paling rendah (p<0,05). Hasil penelitian menunjukkan bahwa pemberian ekstrak etanolik daun Gynura

procumbens 300 mg/kg BB dapat menghambat ekspresi CYP1A1 lebih kuat disbanding kelompok yang lain dan induksi level GSTµ. Kesimpulan yang diambil adalah bahwa ekstrak etanolik daun Gynura procumbens mempunyai kemampuan berperan sebagai blocking agent dalam mencegah inisiasi tahap karsinogenesis, sehingga dapat dipakai sebagai agen kemopreventif pada karsinogenesis mamae.

EKSPRESI PROTEIN p53 PADA KELENJAR MAMMAE TIKUS GALUR SPRAGUE DAWLEY SETELAH INISIASI DIMETHYLBENZ(A) ANTRASEN (DMBA) DAN PEMBERIAN KEMOPREVENTIF Gynura procumbenz Iwan Sahrial Hamid; Sugiyanto; Eddy Meiyanto; Sitarina Widyarini Media Kedokteran Hewan, Vol. 24, No. 3, 2008:171-176 Abstract: Gynura procumbens (Lour.) Merr is used in Indonesian folk medicine to treat cancer. Previous researches have shown that ethanolic extract from G. procumbens-leaves could inhibit the growth of lung tumor of rats, have an antiangiogenic, antimutagenic and cytotoxic effect to myeloma and vero cells. They also have an antiinflammation effect. The aim of this experiment is to evaluate the inhibitory effect of ethanolic extract leaves of Gynura procumbens (Lour) Merr administrated at pre initiation period on 7,12dimethylbenz(a)anthlacene (DMBA) induced rat mammary carcinoma as Chemoprevention agent from natural substance. Eightheen female Sprague Dawley rats with the age of 49-60 day were randomly divided into 6 group of three. Group I, positive control animals, rats aged 54 day were given DMBA 20 mg/kg BB (intragasbic) twice a week for 5 weeks. Group II, treatment group

DAFTAR ISI animals, rats were given ethanolic extract leaves of G. procumbens, rats were given DMBA (doses, frequency, and treatment same as 1st group) and these extracts with dose 300 mg/kg BB were given 2 weeks prior to initiation with DMBA and at the same time with the administration of the initiator. Group III, same like 2nd group, but animals were given these extracts with dose 750 mg/kg BB. Group IV, negative control animals, rats aged 40 day were given ethanolic extract leaves of G. procumbens with dose 300 mg/kg BB, everyday during 7 weeks. Group V, same as 5th group, these extracts with dose 750 mg/kg BB and Group VI as base line. Initiations with DMBA in all groups were started at 3 weeks of age. Necropsy was performed at 12 weeks after last DMBA treatment has been terminated for microscopic examination using Immunohistochemistry staining to analyze the expression of p53 protein. Result of this study demonstrated that etanohc extract leaves of G. procumbens 300 and 750 mg/kg BB could decrease mutant type p53, therefore it should be taken into account for chemopreventing agent in mammary tumor model.

GYNURA PROCUMBENS ETHANOLIC EXTRACT PROMOTES LYMPHOCYTE ACTIVATION AND REGULATORY T CELL GENERATION IN VITRO Dwijayanti, Dinia Rizqi Journal of Tropical Life Science, Vol. 5, No. 1, 2015:14-19 Abstract: invaded pathogen. Cellular and humoral immune system mediated by immunocompetent cells such as CD4+ T cells, CD8+ T cells, CD4+CD25+ T cells, and B220 cells play important role for maintaining immunological surveillance. The purpose of this study was to determine the effect of ethanolic extract of G. Procumbens leaves (EEGL) on the profile of CD4+ T cells, CD4+CD25+ T cells, and

B220+ cells. Splenic cells were isolated from BALB/c mice and cultured in RPMI1640 medium in the presence of EEGL. After 4 days of incubation, cells were harvested, stained with antibodies and analyzed by flow cytometer. The data were analyzed by one-way ANOVA with α= 0.05 and Tukey test using SPSS 16.0 for windows. The results showed that the extract of G. Procumbens could increase proliferation of CD4+CD62L- T cell, CD4+CD25+ T cells, and B220+ cells compared to the control. Here, we showed the biological effect of G. procumbens as medicinal herb with immunomodulatory activity and the dose of 0.1 µg/ml and 1.0 µg/ml could promote T cell activation compared to the highest dose of 10 µg/ml. Interestingly, the dose of 10 µg/ml rather promote than inhibit B cell proliferation.

Gynura procumbens PREVENTS CHEMORESISTANCE THROUGH INHIBITION MDR1 EXPRESSION ON MCF-7 BREAST CANCER CELL LINE AND SENSITIZES THE CELLS TO DOXORUBICIN Nunuk Aries Nurulita; Edy Meiyanto; Sugiyanto; Eishou Matsuda; Masashi Kawaichi Indonesian Journal of Biotechnology , Vol. 17, No. 1, 2012:51-60 Abstract: The long-term exposure of doxorubicin (Dox) causes enhancement in MDR1 expression that leads to breast cancer cell resistance. This protein become a serious problem in cancer treatment and also well-known as negative prognostic factor in breast cancer malignancies. The new approach using natural chemopreventive substance was developed to inhibit this resistance progress. This study was aimed to investigate whether ethyl acetate fraction of Gynura procumnens (FEG) can prevent chemoresistance through suppressing the MDR1 protein expression. MCF-7 cell was used as

DAFTAR ISI chemoresistance cell model. The MCF-7 cells were maintained with 100 nM Dox-contained medium for fi ve weeks. The chemoprevention effect of FEG was investigated by treated MCF-7/Dox with sub-toxic concentration of FEG. The cytotoxic properties of MCF-7 cells were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide) assay. Immunofl uorescence and western blotting analysis was performed to detect the MDR1 expression. MCF-7/Dox cells need higher concentration for inhibiting cell growth, were compared with MCF7, shown by IC50 value. The MDR1 protein level elevated after Dox exposure in time dependent manner. The FEG treatment decreased MDR-1 protein level with dose dependent manner. FEG in combination with DOX potentiates the DOX effect on breast cancer cell growth inhibition. The FEG prevents the chemoresistance development in breast cancer cell line, MCF-7 induced by Dox through inhibiting MDR1 expression. The additional of FEG enhances Dox effect on cell death induction. Thus, FEG could be developed as co-chemotherapy agent for reverse multidrug resistance.

IMMUNOMODULATOR TESTING ON ETHANOL EXTRACT OF Gynura Procumbens LEAVES TO MUS MUSCULUS ADAPTIVE IMMUNE SYSTEM: IN VITRO STUDY

variations of G. procumbens extract on biological aspect of CD4+CD62L-, CD4+CD62L+, CD8+CD62Land CD8+CD62L+ T cells. G. procumbens extract concentrations that used in this experiment were 0 μg /ml, 0.1 μg/ml, 1 μg/ml, and 10 μg/ml. Spleen cells were cultured for 4 days in 5% CO2 incubator at a temperature of 37˚C. Cultured cells were harvested and analyzed by flowcytometry to asses cell surface molecule expression. The resulting data were tabulated and analyzed using ANOVA analysis with a significance of 0.05% on SPSS version 16. Results showed that the extract of G. procumbens can increase the proliferation of CD4+CD62L-, CD4+CD62L+, CD8+CD62L- and CD8+CD62L+ T cells compared to the control. Dose of 1 μg/ml showed the highest effect to promote cell activation compared with the dose of 0.1 μg/ml and 10 μg/ml. Dose of 10 μg/ml could suppress CD4+CD62L-, CD4+CD62L+, CD8+CD62Land CD8+CD62L+ T cells development. This study suggests that the ethanol extract of G. procumbens has benefit as an immunomodulator and involved in the immune system.

ISOLASI DAN UJI ANTIBAKTERI BATANG SAMBUNG NYAWA (Gynura Procumbens Lour) UMUR PANEN 1, 4 DAN 7 BULAN Aryanti; Harsojo; Syafria, Yefni; Ermayanti, Tri Muji

Dwijayanti, Dinia Rizqi

Jurnal Bahan Alam Indonesia, Vol. 6, No. 2, 2007: 43-45

The Journal of Experimental Life Science, Vol. 4, No. 1, 2014:10-14

Abstract:

Abstract: Immunomodulator is a substance that has an ability to modulate the activity and function of immune system. Gynura procumbens supposed to has benefit as an immunomodulator because of it afficacy to cure many diseases. The aim of this study is to determine the effect and dose

The research of isolation of sambung nyawa (Gynura procumbens Lour) stem and its anti bacteria activities on Escherichia coli, Staphylococcus aureus and Samonella typhimurium have been conducted on their 1, 4 and 7 months harvesting. Sambung nyawa stem dried and extracted by ethanol to obtain the ethanol extract, and then tested to three

DAFTAR ISI kind of bacteria with the concentration of 100 and 50 mg/l, and the compound group testing also have been done. Ethanol extract separated by column chromatography using chloroform, ethyl acetate and methanol as solvent to obtain the active compound. The fraction of column chromatography tested again on the bacteria. The result showed that sambung nyawa stem contain of antibacterial compound was very active on S. aureus compared two others with the clear zone was 13,5 mm same as size of positive control (amoxilin antibiotic). The harvesting age of 7 month more active as antibacterial and the compound group testing positive than age 1 and 4 months.

ISOLASI MIKROBA ENDOFITIK DARI TANAMAN OBAT SAMBUNG NYAWA (Gynura procombens) DAN ANALISIS POTENSINYA SEBAGAI ANTIMIKROBA Rumella Simarmata; Harmastini Sukiman

Sylvia

Lekatompessy;

Berkala Penelitian Hayati, Vol. 13, No. 1, 2007:8590 Abstract: Sambung nyawa (Gynura procumbens) has many beneficial effects to human health. suIch as decreasing blood pressure, maintaining blood sugar level (hypoglycaemic), decreasing cholesterol, a remedy for kidney trouble, antibacterial and lessen the inflamation (antiinflamation). This research was undertaken to discover the potency of endophytic microbes from sambung nyawa as antimicrobial agents. The purpose of this research was to screen the endophytic bacteria and the endophytic fimgi having antimicrobial activity, which were isolated from stems, leaves, roots and fruits of medical plants, sambung nyawa. The antimicrobial activity was determined by measuring the growth inhibition of pathogenic

microbes i.e Candida albicafls, Escherichia coli, Pseudomonas sp. and Bacillus subtilis. A total of 38 isolates of bacteria and 15 isolates of fungi were obtained from sambung nyawa. Analysis demonstrated that, 45% isolates of bacteria and 20% isolates of fungi exhibited inhibitory activity. Antimicrobial activity was found in 21% of the isolates that inhibited the growth of e. albicans, E. coli, Pseudomonas sp., and B. subtilis, whereas 24% of isolates had activity only against B. subtilis. Isolate of endophytic bacteria USN 1.1 and USN 2.3 showed the most Significant of inhibition zone. The Inhibition zone of the isolate USN 1.1 to C. albicans, E. coli, Pseudomonas sp, and B. subtilis were 2.318 cm2, 0.969 cm2, 0.796 cm2, and 0.381 cm2, respectively. The Inhibition zone of the isolate USN 2,3 to C. albicans, E. coli, Pseudomonas sp., and B. subtilis were 3.01 cm2, 0.519 cm2, 0.588 cm2 and 0.83 cm2, respectively. These results indicated that endophytic bacteria and endophyticfimgi could be a promising source for antimicrobial agents.

KO-KEMOTERAPI EKSTRAK ETANOLIK DAUN SAMBUNG NYAWA (Gynura procumbens (Lour.) Merr.) DAN DOXORUBICIN PADA SEL KANKER PAYUDARA Jenie, Riris Istighfari Majalah Farmasi Indonesia, Vol. 18, No. 2, 2007: 81-87 Abstrak: Penggunaan kombinasi kemoterapi, yaitu senyawa kemoprevensi yang bersifat non-toksik atau lebih tidak toksik dikombinasikan dengan agen kemoterapi, diketahui mampu meningkatkan sensitifitas sel kanker serta efikasi kemoterapi dengan penurunan toksisitas terhadap jaringan normal.Penelitian ini bertujuan untuk mengetahui efek sinergisme aplikasi kokemoterapi ekstrak etanolik daun Sambung Nyawa (ESN) atau Gynura

DAFTAR ISI procumbens (Lour.)Merr.terhadap sel kanker payudara. Uji MTT digunakan untuk mengukur besarnya efek penghambatan pertumbuhan sel T47D oleh adanya perlakuan kombinasi ESN-Dox, kemudian ditentukan index kombinasinya (IK) untuk menetapkan apakah efeknya sinergis, aditif atau antagonis. Perlakuan dengan ESN (25-500 µg/ mL) selama 48 jam menghasilkan penghambatan pertumbuhan sel sebesar 12,97 persen, dengan IC50 90 µg/mL sedangkan perlakuan dengan Dox (1,8-90 nM) sebesar 16-83persen dengan IC50 50 nM. Kombinasi ESN-Dox memberikan efek sinergis (IK<1).Hasil penelitian ini menunjukkan bahwa ESN berpotensi meningkatkan efikasi agen kemoterapi dalam terapi pengobatan kanker dengan menurunkan toksisitas agen kemoterapi tersebut pada jaringan normal.

MOTODE ADAPTASI TANAMAN SAMBUNG NYAWA TERHADAP CAHAYA-UV UNTUK MENINGKATKAN PRODUKSI FLAVONOID Widodo, Winarso D.; Kurniawati, Ani; P, Edi Djauhari Jurnal Ilmu Pertanian Indonesia, Vol. 13, No. 3, 2008:164-172 Abstrak: -

PENGAMBILAN MINYAK ATSIRI DARI DAUN SAMBUNG NYAWA DENGAN METODA EKSTRAKSI Rosdiana Moeksin; Anna Fetriana; A. Maharanti Jurnal Rekayasa Sriwijaya, Vol. 17, No. 3, 2008:4245 Abstrak: Secara tradisional daun sambung nyawa dipergunakan masyarakat sebagai obat.Oleh

karena itu, dilakukan pengambilan minyak atsirinya dengan metode ekstraksi menggunakan pelarut ethanol.Hal ini ditunjukkan dari hasil yang didapat bahwa daun yang dikeringkan relatif lebih baik dan minyak atsirinya lebih banyak.

PRODUCING THE JELLY MADE OF SAMBUNG NYAWA AND STEVIA LEAVESTO DECREASE THE GLUCOSE LEVEL IN THE BLOOD Tinting Hastuti, Wari; Irfantiningtyas Sari, Hasna; Wirastiti, Athika; Ratnasari; Trihantoro, Saptaka Pelit: Jurnal Penelitian Mahasiswa UNY,Vol. 8, No. 1, 2013:83-91 Abstract: This research was aimed to prove whether the jelly of Sambung Nyawa leaves and stevia could lower the blood glucose level as an alternative option for people with diabetes.The method used in this research was a method of diabetes test glucose monohydrate. In this method glucose monohydrate was induced. The subject in this research was the jelly of Sambung Nyawa made of Sambung Nyawa leaves as the basic material added with a natural sweetener made of stevia leaves. The objects of this research were Wistar mice. The products of Sambung Nyawa jelly and the steviawere given to Wistar mice that had been induced by glucose monohydrate. The rats were divided into three groups. The first served as a control group, the second group was given the same dosage variation, and the third group was a different dosage variation. Based on the results of this research, it was found out that there was a decrease in the blood sugar levels in the mice that had been induced by glucose monohydrate after being given the product of Sambung Nyawa jelly and stevia. It can be seen from the data obtained in the second group. In the group three mice were induced with glucose monohydrate dilution of 1.67 grams with 3 ml of blood sugar levels with

DAFTAR ISI an average of 82.67 mg / dl blood glucose levels decreased to 90 mg / dl. In the third group the data could not be retrieved because the mice died before the sugar level was checked after being induced with glucose monohydrate. Based on the results of experimental data it can be concluded that the products of Sambung Nyawa jelly and stevia could lower the blood glucose levels. The blood glucose levels were measured by using a glucometer.

had lower effect on T47D cell. Quercetin did not seem as the main active compound of FEG. At this study, FEG caused less inhibition on the growth of NIH3T3 cells than that of on all cell lines. Selectivity index (SI) of FEG on WiDr, MCF-7 and T47D were 4.97, 2.77 and 7.79 respectively. According to the datas obtained, FEG possesses moderate to high cytotoxicity properties on WiDr, MCF-7 and T47D cells. FEG demonstrates selective effect against cancer cells and reveals prospective properties as cancer chemoprevention agent.

SELECTIVITY OF ETHYL ACETATE FRACTION OF Gynura Procumbens ON COLON CANCER AND BREAST CANCER

SENYAWA ALKOLOID DARI EKSTRAK ASETON DAUN SAMBUNGAN NYAWA Gynura procumbens L.(GpL)

Nurulita, Nunuk Aries; Meiyanto, Edy; Sugiyanto

Darminto;Muharram

Indonesian Journal of Cancer Chemoprevention, Vol. 2, No. 3, 2011:274-280

Bionature: Jurnal Kajian, Penelitian, Dan Pengajaran Biologi, Vol. 10, No. 1, 2009:20-23

Abstract:

Abstract:

Gynura procumbens is widely used as traditional remedy in South-East Asia. Gynura procumbens exhibites anti inflammatory, antioxidant, and reduced blood pressure activity. The aim of this study was to determine chromatographic profile of ethyl acetate fraction of Gynura procumbens (FEG) and to investigate its cytotoxic properties and selectivity to colon cancer and breast cancer cancer cells. The chromatographic profile of FEG was determined using HPTLC densitometric and HPLC. MTT (3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide) assay was performed to determine the growth inhibitory effect of FEG on the growth of WiDr, MCF-7, and T47D cells. NIH3T3, a normal cells was used to determine the selectivity of FEG, which contained small amount of quercetin as identified from chromatographic profile both HPTLC and HPLC. FEG inhibited cell growth of WiDr, of MCF-7 and of T47D cells in time dependent manner. Quercetin affected cell growth inhibition approximately two fold higher at WiDr and MCF-7, whereas FEG

These research aims to isolate and identify alkaloid compound in acetone extracts from sambung nyawa leafs Gynura procumbens L. This compound obtained trough keeping the plant and isolation. The isolation consists of extraction, fractionation, purification, and identification. The identification consists of melting point test, three phase system test, and Dragendorf test. The results of the research are: 90 mg of white crystal with the range of its melting point is 134-136° C. The crystals indicate positively with Dragendorf test.These results indicate that the compound can be classified in the alkaloid compound.

DAFTAR ISI THE NUMBER OF MACROPHAGES AND HETEROPHILS ON CHICK EMBRYO CHORIOALLANTOIC MEMBRANE AFTER Gynura procumbens (Lour) Merr EXTRACT TREATMENT AND BFGF INDUCTION Hamid, Iwan Sahrial; P, Yuseni Kusuma; Bijanti, Retno; Aksono, E Bimo Indonesian Journal of Cancer Chemoprevention, Vol. 3, No. 2, 2012:399-404 Abstract: Antiangiogenesis (inhibition of new blood vessels formation) has become a strategy to inhibit cancer development. The aim of this experiment was to investigate antiangiogenic effect of Gynura procumbens (Lour) Merr focusing on the decreasing of the number of macrophages and heterophils on chick embryo chorioallantoic membrane. Nine-days-aged-eggs were divided into six groups (eight eggs each group). Group I (positive control) eggs were induced with bFGF+Tris HCl. Group II (negative control) eggs were treated with DMSO+Tris HCl. Group III (treatment I) eggs were induced with 60 ng bFGF and treated with ethanolic extract of G. procumbens leaves with the dose of 60 µg. The following treatment groups, i.e. group IV (treatment II), group V (treatment III), and group VI (treatment IV) were treated with increasing dose of extract, starting from 75 µg, 90 µg, and the last was 110 µg. Eggs were incubated until they reach the age of twelve days to observe macrophages, while to observe heterophils, eggs were incubated until the age of seventeen days. Based on haematoxylin-eosin staining, macrophages in the treatment groups were less than the control positive group (bFGF+Tris HCl), but based on giemsa staining, the effect of Gynura procumbens in decreasing the number of heterophils could not be observed because some blood smears. These analysis suggest that the ethanolic extract of Gynura procumbens leaves can perform as antiangiogenic agent decreasing the number of macrophages.

UJI EFEK ANTIINFLAMASI DARI EKSTRAK DAUN DEWA (Gynura procumbens (lour) Merr) PADA TIKUS PUTIH JANTAN DENGAN PARAMETER SERUM CRP Sindhu Winata;Irwan Setiabudi;Liliek S. Hermanu Jurnal Obat Bahan Alam, Vol. 7, No. 2, 2008:152159 Abstrak: -

Statistika, Vol. 7 No. 1, 13 – 20 Mei 2007

Efek Antiproliferasi Ekstrak Etanolik Daun Gynura procumbens (Lour.) Merr. pada Sel Paru Tikus Jantan yang Diinduksi 7,12 Dimetilbenz[a]antrasen Hendri Wasitoa), Retno Murwantib), Edy Meiyantoc) A)

Jurusan Farmasi FMIPA Universitas Islam Bandung B,C) Fakultas Farmasi Universitas Gadjah Mada

Abstrak Telah dilakukan penelitian untuk mengetahui efek ekstrak etanolik daun G. procumbens terhadap aktivitas proliferasi sel paru tikus jantan galur Sprague Dawley yang diinduksi oleh 7,12dimetilbenz[a]antrasen (DMBA). Hasil pengamatan preparat histopatologi organ paru tikus dengan pengecatan H&E dan AgNOR pada minggu ke-16 serta analisis statistik dengan metode non parametrik Kruskal-wallis Test dan diteruskan dengan Mann-Whitney Test menunjukkan bahwa pemberian DMBA 20 mg/kg BB dua kali seminggu selama 3 minggu meningkatkan aktivitas proliferasi sel paru tikus jantan, namun belum dapat menunjukkan insidensi kanker paru serta pemberian ekstrak G. procumbens 300 mg/kg BB dan 750 mg/Kg BB belum dapat menghambat proliferasi sel paru tikus jantan yang diinduksi DMBA 20 mg/kg BB. Kata kunci : G. procumbens, proliferasi, sel paru, DMBA

1. Pendahuluan Penyakit kanker masih menjadi masalah kesehatan di dunia, angka kematian karena penyakit ini setiap tahun terus bertambah. Kanker paru merupakan penyebab utama kematian akibat kanker di dunia. Setiap tahun diperkirakan 178.100 kasus baru kanker paru terjadi di Amerika, dan 160.400 orang akan mengalami kematian akibat penyakit mematikan ini meskipun telah dilakukan terapi yang baik (Forgacs et al., 2001). Di Indonesia diperkirakan sedikitnya akan terdapat 170 – 190 kasus kanker per 100.000 orang (Tjindarbumi dan Mangunkusumo, 2002). Penemuan tanaman obat yang menunjukkan efek farmakologis terhadap penyakit kanker terutama yang telah mengalami uji secara ilmiah telah memberikan alternatif dalam mengatasi dan mengobati penyakit kanker (Novalina, 2003). Secara ilmiah telah dibuktikan bahwa tanaman G. procumbens yang memiliki kandungan kimia antara lain flavonoid, saponin, alkaloid, tanin dan minyak atsiri (Sudarto, 1985) mempunyai aktivitas sebagai antikanker baik secara in vitro (Arianti, 1998) maupun in vivo (Sugiyanto et al., 1993 ) serta bersifat antiangiogenik (Jenie, 2003). Berdasarkan hal-hal tersebut di atas, penelitian dan pengembangan G. procumbens sebagai obat kanker menjadi sangat penting untuk terus dilakukan. Pada penelitian ini dilakukan penelitian mengenai efek antiproliferasi dari ekstrak etanolik G. procumbens terhadap sel paru tikus jantan dengan 7,12-dimetilbenz[a]antrasen (DMBA) sebagai agen penginduksi kanker.

2. Metodelogi Bahan dan Alat Bahan simplisia yang digunakan adalah daun G. procumbens yang diambil dari daerah Ngaglik, Sleman, Yogyakarta pada bulan Juli 2004. Tanaman G. procumbens yang digunakan dideterminasi di Laboratorium Farmakognosi, Bagian Biologi Farmasi, Fakultas Farmasi UGM.

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14 Hendri Wasito, Retno Murwanti, Edy Meiyanto

Simplisia dibersihkan, dikeringkan, diserbuk menggunakan blender dan diekstrak dengan alat sokhlet memakai etanol 96 %. Bahan kimia yang digunakan berupa etanol 96 %, alkohol 70 %, eter anasthetikum, akuades, 7,12-dimetilbenz[a]antrasen (DMBA) (Sigma Chem., Steinherm) sebagai agen penginduksi kanker paru, CMC-Na sebagai pensuspensi ekstrak, minyak jagung (corn oil ) sebagai pelarut DMBA, buffer formalin 10 %, paraffin, minyak imersi, pewarna sediaan histologik (Hematoksilin dan Eosin), serta perak nitrat (AgNO3) sebagai pewarna pengecatan AgNOR. Subyek uji yang digunakan berupa tikus (Rattus norvegicus) jantan galur Sprague Dawley umur 40 hari yang diperoleh dari Unit Penyediaan Hewan Percobaan (UPHP) UGM. Pakan mencit berupa pellet dan minum dari air ledeng yang masing-masing diberikan secara ad libitum. Alat-alat yang digunakan berupa alat bedah, kandang tikus, alat pengecatan histologik H&E dan AgNOR, sarung tangan, masker, cassette untuk memproses jaringan, bedding, kertas saring, kapas, staining jars, spuit injeksi oral 1 ml dan 3 ml, blender, labu sokhlet, waterbath, cawan porselin, kipas angin, vortex, mortir dan stamper, labu takar, pipet tetes, timbangan analitik, neraca elektrik, mikroskop binokuler, kamera digital, dan alat-alat gelas yang lazim.

Jalannya Penelitian Tikus jantan Sprague Dawley umur 40 hari diadaptasikan di kandang percobaan selama satu minggu sebelum diberikan perlakuan. Tikus dibagi menjadi 4 kelompok, tiap-tiap kelompok sebanyak 10 ekor. Kelompok I sebagai kontrol pelarut diberi CMC-Na 0,5 % dengan volume disesuaikan dengan bobot badan tikus. Pemberian dilakukan setiap hari secara peroral dari minggu ke-1 sampai minggu ke-5 percobaan. Kelompok II sebagai kontrol DMBA diberikan larutan DMBA dalam minyak jagung (corn oil) secara peroral dengan dosis 20 mg/kg BB sebanyak dua hari sekali selama 3 minggu pada minggu ke-2 sampai minggu ke-4. Kelompok III dan IV sebagai dosis ekstrak 300 mg/kg BB dan dosis ekstrak 750 mg/kg BB diberikan ekstrak dalam pelarut CMC Na 0,5 % secara peroral setiap hari selama 5 minggu, larutan DMBA diberikan setelah minggu pertama pemberian ekstrak dengan dosis 20 mg/kg BB dan diberikan satu jam setelah pemberian ekstrak uji. Frekuensi pemberian larutan DMBA disesuaikan dengan perlakuan kelompok II. Pada minggu ke-6 sampai ke-16 tidak dilakukan perlakuan, hanya diberi makan pelet dan minum air. Kemudian pada minggu ke-16 dilakukan pengorbanan hewan uji dengan dekapitasi dan dilakukan nekropsi untuk diambil organ paru untuk diamati secara makroskopik dan mikroskopik histopatologinya dengan pengecatan H&E dan AgNOR. Data pengamatan mAgNOR dianalisis secara statistik dengan metode nonparametrik Kruskal Wallis yang diteruskan dengan Uji Mann-Whitney (SPSS ver.12).

Gambar 1. Protokol penelitian efek antiproliferasi ekstrak etanolik daun G. procumbens pada selparu tikus jantan yang diinduksi DMBA

Statistika, Vol. 7, No. 1, Mei 2007

Efek Antiproliferasi Ekstrak Etanolik Daun Gynura procumbens (Lour.) Merr. pada 15 Sel Paru Tikus Jantan yang Diinduksi 7,12 Dimetilbenz[a]antrasen 3. Hasil dan Pembahasan Pengamatan terhadap uji antiproliferasi dilakukan pada minggu ke-16 secara makroskopis dan mikroskopis terhadap kelompok kontrol dan kelompok perlakuan dengan ekstrak etanolik daun G. procumbens. Pemeriksaan mikroskopis meliputi analisis histopatologi sel paru dengan pewarnaan H&E dan aktivitas proliferasi dengan pewarnaan AgNOR. Dari hasil nekropsi pada akhir minggu ke-16, secara makroskopis tidak ditemukan perubahan yang signifikan berupa insidensi kanker pada paru tikus kelompok kontrol DMBA 20 mg/kg BB maupun kelompok kontrol perlakuan yang ditunjukkan oleh gambaran morfologi organ paru, serta tidak ditemukan adanya nodul tumor pada organ paru yang diamati.

A

C

B

D

Gambar 2. Gambaran makroskopis paru tikus (A) tikus kontol DMBA 20 mg/kg BB, (B) tikus kontrol pelarut CMC-Na 0,5 %, (C) tikus dosis ekstrak 300 mg/kg BB, (D) tikus dosis ekstrak 750 mg/kg BB Perubahan yang terjadi meliputi reaksi radang pada paru, perubahan warna paru dari merah menjadi merah agak pudar, permukaan paru yang berubah lebih kasar terutama pada kelompok kontrol DMBA 20 mg/kg BB, serta timbulnya timbunan lemak pada kelompok kontrol DMBA 20 mg/kg BB dan perlakuan dosis ekstrak 300 mg/kg BB dan 750 mg/kg BB. Pengamatan histopatologi dengan pengecatan H&E yang dilakukan terhadap sel paru tikus normal (kontrol pelarut CMC-Na 0,5 %), tikus perlakuan dosis ekstrak 300 mg/kg BB dan 750 mg/kg BB maupun tikus kelompok kontrol DMBA 20 mg/kg BB tidak menunjukkan terjadinya perubahan spesifik yang mengindikasikan terjadinya kanker pada sel paru akibat induksi DMBA. Tidak adanya insidensi kanker pada sel paru baik pada tikus kelompok kontrol DMBA 20 mg/kg BB, kelompok perlakuan dosis ekstrak 300 mg/kg BB dan 750 mg/kg BB, mengakibatkan pengaruh ekstrak etanolik daun G. procumbens terhadap penghambatan kanker dan aktivitas proliferasi pada sel paru tikus jantan yang diinduksi DMBA 20 mg/kg BB dengan metode pengecatan Hematoksilin dan Eosin (H&E) tidak bisa teramati dengan jelas.



Gambar 3. Gambaran histopatologi H&E paru tikus (A) tikus kontol DMBA 20 mg/kg BB, (B) tikus kontrol Pelarut CMC-Na 0,5 %, (C) tikus dosis ekstrak 300 mg/kg BB, (D) tikus dosis ekstrak 750 mg/kg BB (perbesaran 20x10) Keterangan : a. bronkus, b. bronkeolus, c. septa alveoli, d. lumen, e. sel radang limfosit Gambaran histopatologi jaringan paru pada kelompok kontrol pelarut CMC-Na 0,5 % tidak ditemukan adanya perubahan spesifik, sel-sel paru tampak normal, sel-sel epitel bronkus masih tersusun selapis, meskipun terdapat pertumbuhan epitel bronkeolus dan bronkus ke arah lumen disertai adanya reaksi radang dengan infiltrasi limfosit pada daerah sekitar bronkeolus. Pada kelompok tikus kontrol DMBA 20 mg/kg BB ditemukan pertumbuhan epitel bronkeolus dan bronkus ke arah lumen, selain itu juga ditemukan penebalan septa interveoli dengan infiltrasi sel radang limfosit disekitar bronkus dan bronkeolus. Pada kelompok dosis ekstrak 300 mg/kg BB sebagian besar tidak ditemukan perubahan spesifik pada sel paru, hanya sebagian kecil tikus mengalami kongesti dan ditemukan infiltrasi sel radang limfosit pada jaringan intertitial serta pada septa alveoli. Sedangkan hasil pemeriksaan histopatologi pada kelompok perlakuan dosis ekstrak 750 mg/kg BB menunjukkan sebagian besar paru tikus tidak mengalami perubahan yang spesifik, pada beberapa paru ditemukan proliferasi sel sub mukosa dari bronkus kearah lumen. Adanya beberapa perubahan pada jaringan paru yang diamati bukanlah karena hasil dari perlakuan, kemungkinan disebabkan oleh proses iritasi kronis seperti proses radang yang disebabkan oleh agen infeksi atau kondisi lingkungan kandang yang kurang memadai. Dengan pewarnaan silver, protein AgNOR akan tampak sebagai titik hitam (black dot) yang kemudian dapat dihitung. Aktivitas proliferasi disini diukur dengan nilai mAgNOR, yaitu perbandingan antara jumlah titik hitam dalam sel (black dot) dengan jumlah sel. Semakin tinggi tingkat proliferasi maka semakin banyak titik hitam yang teramati (Rizali dan Auerkari, 2003).


Efek Antiproliferasi Ekstrak Etanolik Daun Gynura procumbens (Lour.) Merr. pada 17 Sel Paru Tikus Jantan yang Diinduksi 7,12 Dimetilbenz[a]antrasen

Gambar 4. Gambaran histopatologi hasil pewarnaan AgNOR dari sel paru tikus: (A) tikus kontol DMBA 20 mg/kg BB, (B) tikus kontrol pelarut CMC-Na 0,5 %, (C) tikus dosis ekstrak 300 mg/kg BB, (D) tikus dosis ekstrak 750 mg/kg BB (perbesaran 100x10), Ketetangan : : NOR yang tercat silver Nilai mAgNOR (rata-rata ± SD) untuk kelompok kontrol pelarut CMC-Na 0,5 % adalah 1,31 ± 0,066; kelompok kontrol DMBA 20 mg/kg BB sebesar 1,69 ± 0,156; kelompok perlakuan dosis ekstrak 300 mg/kg BB adalah 1,65 ± 0,182; sedangkan kelompok perlakuan dosis ekstrak 750 mg/kg BB adalah 1,66 ± 0,094. Kelompok kontrol DMBA 20 mg/kg BB memiliki nilai mAgNOR terbesar dan kelompok kontrol pelarut CMC-Na 0,5 % memiliki nilai mAgNOR paling kecil dibandingkan kelompok yang lain. Hal ini menunjukkan bahwa tingkat proliferasi sel-sel paru pada tikus kelompok kontrol DMBA paling tinggi dan tingkat proliferasi untuk tikus kelompok kontrol pelarut CMCNa 0,5 % paling rendah. Tingkat proliferasi pada kelompok kontrol DMBA 20 mg/kg BB belum menunjukkan terbentuknya kanker, pada keadaan jaringan normal rata-rata jumlah titik hitam (mAgNOR) sebanyak 1,2 ± 0,1 dan pada jaringan yang menunjukkan terbentuknya kanker memiliki nilai mAgNOR sebanyak 3,8 ± 0,6 atau bisa lebih besar lagi yakni sebanyak 4,8 ± 1,1 (Rizali dan Auerkari, 2003).

Gambar 5. Efek ekstrak etanol daun G. procumbens terhadap aktivitas proliferasi sel paru tikus jantan yang diinduksi DMBA



Analisis statistik dengan menggunakan uji nonparametrik Kruskal Wallis dilanjutkan dengan uji Mann Whitney menunjukkan terjadinya perbedaan signifikan antara nilai mAgNOR kelompok kontrol pelarut CMC-Na 0,5 % dengan kelompok kontrol DMBA 20 mg/kg BB serta kelompok perlakuan dosis ekstrak 300 mg/kg BB dan 750 mg/kg BB. Penurunan aktivitas proliferasi akibat pemberian DMBA tidak signifikan pada pemberian dosis ekstrak 300 mg/kg BB dan 750 mg/kg BB. Karena penurunan aktivitas proliferasi sel-sel paru oleh ekstrak etanolik daun G. procumbens tidak menunjukkan adanya perbedaan yang bermakna (p<0,05), sehingga dapat dikatakan bahwa pemberian ekstrak etanolik G. procumbens dengan dosis 300 mg/kg BB dan 750 mg/kg BB belum dapat menghambat proliferasi sel paru tikus jantan yang diinduksi DMBA 20 mg/kg BB dua hari sekali selama 3 minggu. Hal ini terjadi mungkin karena adanya kemampuan DMBA untuk menginduksi enzim Glutation S-Transferase (GST). Enzim Glutation-S-Transferase (GST) merupakan enzim pemetabolisme fase II yang mampu mendetoksifikasi senyawa asing, xenobiotika atau karsinogen. Kusharyanti (2004) melaporkan bahwa pemberian DMBA dapat menginduksi aktivitas maupun ekspresi enzim GST di hati, selain itu juga pemberian ekstrak etanolik daun G. procumbens pada tikus jantan yang diinduksi DMBA dapat meningkatkan ekspresi GST. Peningkatan aktivitas enzim GST akan meningkatkan kecepatan detoksifikasi metabolit DMBA. Metabolit reaktifnya tidak akan membentuk adduct dengan DNA karena ekskresi metabolit tersebut ditingkatkan sehingga menurunkan aktivitas karsinogennya. Senyawa metabolit DMBA berupa 3,4-epoksida DMBA dapat berinteraksi dengan glutation S-transferase menghasilkan konjugat glutation yang dapat segera dimetabolisir membentuk asam merkapturat sehingga akan meningkatkan kecepatan eliminasi dari DMBA. Tidak adanya insidensi kanker juga dapat dipengaruhi oleh kurang lamanya waktu pengamatan mengingat kanker merupakan penyakit yang berbanding linear dengan waktu. Karsinogenesis membutuhkan akumulasi perubahan seluler yang membutuhkan waktu untuk memperolehnya. Semakin lama waktu pengamatan, probabilitas insidensi kanker semakin besar. Kanker merupakan hasil proliferasi klonal sel tunggal memerlukan waktu yang lama dari sel tunggal yang terubah (transformed cell) untuk tumbuh menjadi nodul yang cukup besar yang memberikan tanda dan gejala klinis. Pada penelitian ini, subyek uji dikorbankan 12 minggu setelah pemberian DMBA terakhir, namun belum mampu menimbulkan kanker pada paru.

4. Kesimpulan dan Saran Kesimpulan Pemberian DMBA dosis 20 mg/kg BB dua hari sekali dalam waktu 3 minggu pada tikus jantan meningkatkan aktivitas proliferasi sel paru tikus jantan, namun belum dapat menunjukkan insidensi kanker paru, dan pemberian ekstrak etanolik daun G. procumbens 300 mg/kg BB dan 750 mg/kg BB belum dapat menghambat proliferasi sel paru tikus jantan yang diinduksi DMBA.

Saran Perlu dilakukan penelitian lebih lanjut untuk menghasilkan insidensi kanker pada sel paru tikus jantan dengan DMBA maupun oleh senyawa penginduksi kanker lainnya.

Ucapan Terima Kasih Terima kasih kepada Cancer Research Group Fakultas Farmasi UGM yang telah memberikan bantuan dan dukungan selama proses penelitian.

5. Daftar Pustaka Arianti, S., 1998, Aktivitas Biologis Ekstrak Etanol Daun Gynura procumbens (Lour.) Merr. Terhadap Kultur Sel Vero dan Kultur Sel Mieloma, Skripsi, Fakultas Farmasi Universitas Gadjah Mada, Yogyakarta.


Efek Antiproliferasi Ekstrak Etanolik Daun Gynura procumbens (Lour.) Merr. pada 19 Sel Paru Tikus Jantan yang Diinduksi 7,12 Dimetilbenz[a]antrasen Forgacs, E., Zochbauer-Muller, S., Olah, E., dan Minna, J.D., 2001, Molecular Genetic Abnormalities in the Pathogenesis of Human Lung Cancer, Pathology Oncology Research, Vol 7, No 1. Jenie, R.I., 2003, Efek Antiangiogenik Ekstrak Etanolik Daun Dewa (Gynura procumbens (Lour.) Merr.) pada Membran Korio Alantois (CAM) Embrio Ayam Terinduksi b-FGF, Skripsi, Fakultas Farmasi Universitas Gadjah Mada, Yogyakarta. Novalina, 2003, Penggunaan Tanaman Obat Sebagai Upaya Alternatif Dalam Terapi Kanker, Makalah Pribadi Pengantar Ke Falsafah Sains, Program Pasca Sarjana IPB , Bogor. Kusharyanti, I., 2004, Efek Ekstrak Etanol Daun Dewa (Gynura procumbens (Lour) Merr) Terhadap Aktivitas Enzim Glutation S-Transferase Pada Hepar Tikus Jantan Terinduksi 7,12Dimetilbenz(α)antrasen, Skripsi, Fakultas Farmasi, Universitas Gadjah Mada, Yogyakarta Rizali, E., dan Auerkari, E. I., 2003, Teknik Pewarnaan Silver (AgNOR) Sebagai Salah Satu Cara Menentukan Aktivitas Proliferasi sel Tumor dan Apoptosis, Jurnal Kedokteran Gigi Indonesia, 10(3): 41-45 Sudarto, B., dan Pramono, S., 1985, Skrining Fitokimia Daun Dewa (Gynura procumbens), Lour. Merr. yang Diduga Berkhasiat Sebagai Anti-kanker, PPPT-UGM, Lembaga Penelitian UGM, Yogyakarta. Sugiyanto, Sudarto, B., dan Meiyanto, E., 1993, Efek Penghambatan Karsinogenisitas Benzo(a)piren Oleh Preparat Tradisional Tanaman Gynura sp. Dan identifikasi Awal Senyawa yang Berkhasiat, Laporan Penelitian P4M DitJen DikTi, Fak. Farmasi UGM, Yogyakarta. Sugiyanto, Sudarto, B., Meiyanto, E., Nugroho, A.E., dan Jenie, U.A., 2003, Aktivitas Antikarsinogenik Senyawa yang Berasal dari Tumbuhan, Majalah Farmasi Indonesia, 14 (4), 216-225. Tjindarbumi, D., dan Mangunkusumo, R., 2002, cancer in Indonesia, Present and future, Jpn J Clin Oncol : 32 (Suplement 1), S17-S21.




EFEK HEPATOPROTEKTIF EKSTRAK ETANOL DAUN DEWA (Gynura procumbens (Lour) Merr) PADA TIKUS TERINDUKSI PARASETAMOL HEPATOPROTECTIVE EFFECT OF ETHANOL EXTRACT OF DEWA LEAF (Gynura procumbens (Lour) Merr) AT MOUSE INDUCEED BY PARASETAMOL Jason Merari Peranginangin Fakultas Farmasi Universitas Setia Budi Jl. Letjen Sutoyo, Mojosongo, Surakarta 57127 ABSTRAK Telah dilakukan penelitian efek hepatoprotektif ekstrak etanol daun dewa (Gynura procumbens (Lour) Merr) pada tikus terinduksi parasetamol dengan tujuan untuk memperoleh data dan bukti ilmiah efek hepatoprotektif dari ekstrak etanol daun dewa akibat perlakuan hepatotoksin parasetamol. Penelitian ini menggunakan 30 ekor tikus putih jantan galur Wistar yang dibagi menjadi 6 kelompok. Kelompok I diberikan parasetamol dosis 200 mg/200 g BB, kelompok II sebagai kontrol negatif diberikan larutan CMC 1 %, kelompok III sebagai kontrol positif diberikan asetilsistein dosis 3,6 mg/200 g BB, kelompok IV, V, VI sebagai kelompok perlakuan diberi ekstrak daun dewa berturut-turut 1,32; 2,75; dan 5,50 mg/200 g BB. Darah diambil dari vena lateralis ekor untuk ditetapkan aktivitas GPT dan GOT-serumnya. Hasil penelitian menunjukkan bahwa ekstrak daun dewa yang diberikan secara oral mampu menurunkan aktivitas GPT-serum sebesar 57 U/L; 46,6 U/L; 39 U/L terhadap kontrol parasetamol, aktivitas GOT-serum turun sebesar 51,2 U/L; 38,6 U/L; 30,2 U/L terhadap kontrol parasetamol. Berdasarkan data aktivitas GPT dan GOT-serum yang ditemukan serta tatacara analisis hasil yang diterapkan, dapat disimpulkan bahwa ekstrak etanol daun dewa (Gynura procumbens (Lour) Merr) memiliki sifat hepatoprotektif dengan dosis efektif 5,50 mg/200 gBB Kata kunci: efek hepatoprotektif, daun dewa (Gynura procumbens (Lour) Merr), aktivitas GPT dan GOT, parasetamol. ABSTRACT Hepatoprotective effect of ethanol extract of Dewa leaf (Gynura procumbens (Lour) Merr) at mouse induceed parasetamol has been investigated, as a mean to obtains data and scientific evidence of hepatoprotective effect of ethanol extract of dewa leaf, as result of treatment of hepatotoksin parasetamol. This study applies 30 white Wistar mouse divided to 6 group. Group I is given by parasetamol dose 200 mg/200 g BB, group II as negative control is given by condensation CMC 1 %, group III as positive control is given by asetilsistein dose 3,6 mg/200 g BB, group IV, V, VI as a group treatment is given by dewa leaf extract successively 1,32; 2,75; and 5,50 mg/200 g BB. Blood taken away from vein lateralis tail to determine activity GPT and GOT-serum activity. Result of study indicates that dewa leaf extract given oral can reduce GPT-serum activity 57 U/l; 46,6 U/l; 39 U/l to control parasetamol, GOT-serum activity downwards 51,2 U/l; 38,6 U/l; 30,2 U/l to control parasetamol. Based on GPT and GOT-serum activity, inferential that ethanol extract of dewa leaf (Gynura procumbens (Lour) Merr) measures up to hepatoprotektif with effective dose 5,50 mg/200 gBB. Key words : hepatoprotective effect, daun dewa (Gynura procumbens (Lour) Merr), GPT dan GOT activity, parasetamol.

PENDAHULUAN Penggunaan bahan alam untuk pengobatan merupakan hal yang umum di Indonesia, terlihat dari banyaknya produk ramuan tradisional baik yang telah diolah dengan teknologi modern maupun secara sederhana yang beredar di masyarakat. Dari alam telah diperoleh berbagai macam obat-obatan seperti atropin, berbagai macam antibiotik, kina, reserpin dan masih banyak lagi. Mengingat hal tersebut perlu adanya pengujian untuk membuktikan khasiat suatu bahan alam karena masih banyak yang didasarkan pada pengalaman saja (Raflizar dkk. 2006). Hati memegang peranan yang sangat penting dalam fungsi fisiologis tubuh. Hati merupakan tempat pembentukan lipid, albumin, dan beberapa protein plasma. Selain itu juga merupakan organ penting dalam proses biotransformasi senyawa endogen maupun senyawa eksogen, misalnya amonia, hormon steroid, dan obat. Metabolisme karbohidrat, protein, dan lipid juga terjadi di hati. Demikian pula proses detoksifikasi atau inaktivasi obat atau senyawa beracun lainnya dilakukan oleh hati, sehingga dapat dikatakan hati mempunyai fungsi pertahanan dan pelindung bagi tubuh (Wyngaarden,1982). Hepatotoksin didefinisikan sebagai senyawa kimia yang memiliki efek toksik pada sel hati. Dengan dosis berlebihan (dosis toksik) atau pemejanan dalam jangka waktu yang lama senyawa bersangkutan dapat menimbulkan kerusakan hati akut, subakut maupun kronis. Terdapat dua macam senyawa hepatotoksin yaitu hepatotoksin hakiki (hepatotoksin terramalkan) dan hepatotoksin tak terramalkan. Hepatotoksin hakiki adalah golongan senyawa yang memiliki sifat dasar toksik terhadap hati, misal : karbon tetraklorida (CCl 4), kloroform, etionin dan parasetamol. Senyawa-senyawa tersebut dapat menyebabkan kerusakan hati pada semua individu. Hepatotoksin tak terramalkan adalah golongan senyawa yang bersifat toksik terhadap hati, tetapi hanya dapat mengakibatkan hepatitis pada individu yang hipersensitif terhadap senyawa ini, misal : isoniazida (INH), halotan dan sulfanamida (Zimmerman, 1978). Dalam penelitian ini, yang akan digunakan sebagai model hepatotoksin adalah parasetamol. Salah satu tanaman yang menarik diteliti sebagai hepatoprotektor adalah daun dewa, karena mengandung senyawa flavonoid, di mana senyawa ini mempunyai aktivitas sebagai antioksidan, oleh adanya gugus hidroksi fenolik dalam struktur molekulnya. Parasetamol merupakan devirat para amino fenol yang berkhasiat sebagai analgesik-antipiretik. Sebagian besar parasetamol mengalami metabolisme di hati dan hanya 2-5% dari dosis terapi dieskresi dalam bentuk tidak berubah dalam urine. Meskipun demikian perubahan hayati parasetamol lebih dominan dalam hati, namun dapat juga terjadi dalam usus dan ginjal (Dollery, 1991). Test fungsi hati yang paling sensitif adalah dengan memonitor peningkatan aktivitas enzim Glutamin Oksaloasetat Transaminase (GOT) dan aktivitas enzim Glutamin Purivat Transaminase (GPT) (Rhodes et al. 1995). Perubahan aktivitas enzim GOT dan GPT merupakan petunjuk hilangnya integritas sel hepatosit dan sebagai indikator yang akurat dari hepatitis akut (Sullivan et al. 1992), akan tetapi perlu diperhatikan bahwa aktivitas enzim GPT merupakan indikator kerusakan hati yang lebih baik dari GOT (Hinton et al. 1995). METODE PENELITIAN Bahan

Bahan yang digunakan adalah daun dewa (Gynura procumbens (Lour) Merr) yang diperoleh dari Tawangmangu, Karanganyar, Jawa Tengah. Hewan uji yang digunakan adalah tikus putih Jantan galur Wistar umur 2,5-3,5 bulan dengan berat badan 180-200 gram, yang diperoleh dari unit Pengembangan Hewan Percobaan UGM, Yogyakarta. Bahan untuk model hepatotoksin parasetamol diperoleh dari Apotek Kimia Farma Surakarta, Parasetamol disuspensikan dalam CMC 1 %. Kontrol hepatoprotektor yang digunakan yaitu asetilsistein, diperoleh dari Apotek Kimia Farma, Surakarta. Kit GPT (ALAT) untuk mengukur aktivitas GPT-serum dan kit GOT (ASAT) untuk mengukur aktivitas GOT-serum, etanol 70%.

Alat Penangas air, neraca analitik, fotometer 4010, sentrifuge, spuit injeksi (Nipro) 1 ml dan jarum oral, pipa kapiler, tabung reaksi, klinipet, yellow tip, gelas piala, pengaduk, botol penampung, corong gelas, dan kain flanel. Pembuatan ekstrak etanol daun dewa Serbuk daun dewa sebanyak 50 gram dimasukkan botol, ditambah etanol 70% sebanyak 375 ml dan digojok, kemudian didiamkan selama 5 hari, penggojokan dilakukan 3 kali sehari. Setelah 5 hari maserat disaring dengan kain flanel, ampas hasil penyaringan ditambah etanol 70% secukupnya, dilakukan penggojokan dan dilakukan penyaringan kembali sampai diperoleh 500 ml maserat. Hasil maserasi diuapkan di atas penangas sampai diperoleh ekstrak yang kental. Penentuan dosis sediaan uji Dosis sediaan dihitung dari dosis empiris yang kemudian dikonversikan ke dalam dosis ekstrak. Peringkat dosis dihitung dari perkalian dosis empiris yang didapat, yaitu setengah kali dosis empiris (0,5 DE), satu kali dosis empiris (DE), dan dua kali dosis empiris (2 DE). Di mana 0,5 DE = 1,32 g/200 gBB; DE = 2,75 g/200 gBB dan 2 DE = 5,50 g/200 gBB. Pembuatan suspensi dan penentuan dosis parasetamol Suspensi parasetamol dalam CMC 1% dibuat dengan cara melarutkan sejumlah gram parasetamol yang telah ditimbang ke dalam CMC 1% hingga konsentrasi yang telah ditetapkan sebelumnya, yaitu dosis hepatotoksik. Dosis parasetamol yang diberikan secara peroral pada tikus jantan sebesar 0,5 g-1,0 g/kgBB (Anonim, 1993). Dosis parasetamol yang digunakan adalah 1,0 g/kgBB. Jadi dosis untuk tikus adalah 200 mg/200 g BB. Penentuan dosis asetilsistein Dosis yang digunakan sebagai hepatoprotektor untuk manusia adalah 200 mg/KgBB (Tan, H.T. 2002). Faktor konversi dari manusia dengan berat badan 70 kg ke tikus putih dengan berat badan 200 g adalah 0,018. Jadi dosis untuk tikus = 200 x 0,018 = 3,6 mg/200 gBB. Pengelompokan dan perlakuan hewan uji Tiga puluh lima ekor tikus dibagi dalam 6 kelompok. Tikus kelompok I diberi parasetamol dosis hepatotoksik 200 mg/200 g BB. Kelompok II diberi larutan CMC 1% sebagai kontrol negatif. Kelompok III diberi asetilsistein dosis 3,6 g/200 g BB sebagai kontrol positif. Kelompok IV, V dan VI masing-masing diberikan ekstrak etanol daun dewa dengan dosis berturut-turut 1,32 mg/200 gBB; 2,75 mg/200 gBB; dan 5,50 mg/200 gBB. Kelompok I, II dan III pada hari ke dua diambil cuplikan darahnya melalui vena mata menggunakan pipa kapiler untuk diambil serumnya, dan diukur aktivitas GPT dan GOTserumnya secara spektrofotometri. Pemberian ekstrak etanol daun dewa sesuai peringkat dosis, dilakukan satu kali sehari selama 7 hari berturut-turut. Pada hari ke-8, kelompok tikus IV, V dan VI diberi parasetamol dosis hepatotoksik. Sesudah 48 jam (hari ke sepuluh), semua tikus pada kelompok IV, V dan VI diambil cuplikan darahnya melalui vena mata menggunakan pipa kapiler, ditampung dalam tabung reaksi dan didiamkan selama 15 menit kemudian disentrifuge dengan kecepatan 3500 rpm selama 15 menit, dan diukur aktivitas GPT dan GOT-serumnya.

Penetapan aktivitas enzim GPT dan GOT Darah tikus yang telah diambil ditampung dalam tabung sentrifuge, kemudian disentrifuge agar sel-sel darah mengendap dan terpisah dari plasmanya. Pada analisis fotometri aktivitas GPT dan GOT ini dilakukan sejumlah reaksi pada tabel 1 Tabel 1. Penetapan aktivitas enzim GPT dan GOT Serum atau plasma

50 µl

Larutan reagen

500 µl

Dicampur dan ditambahkan dengan dengan pipet, setelah 1 menit Start reagen

50 µl

Campur dan baca penurunan resapan dalam 1 menit Reagen yang digunakan adalah sebagai berikut: KH 2PO4, K2HPO4 L-alanin, 2-Oxoglutarat, ,

NADH, LDH, NaOH, Gliserol, NaHCO3. Aktivitas enzim dibaca pada panjang gelombang 340 nm, suhu 370C, dengan faktor 1745. Aktivitas GPT dinyatakan dalam U/l. HASIL DAN PEMBAHASAN Aktivitas enzim GPT dan GOT dapat dilihat pada tabel 2 dan 3 . Kehepatotoksikan parasetamol pada tikus dikaji dari nilai aktivitas enzim GPT dan GOT setelah pemberian parasetamol dosis 200 mg/200 gBB dibandingkan dengan kelompok tanpa perlakuan. Rata-rata nilai aktivitas enzim GPT untuk kelompok kontrol parasetamol adalah 128,2 U/l, kelompok kontrol negatif (CMC 1%) adalah 127,4 U/l, kelompok kontrol positif adalah 31 U/l, kelompok IV adalah 57 U/l, kelompok V adalah 46,6 U/l, dan kelompok VI adalah 39 U/l. Rata-rata nilai aktivitas enzim GOT untuk kelompok kontrol parasetamol adalah 109,8 U/l, kelompok kontrol negatif (CMC 1%) adalah 107,6 U/l, kelompok kontrol positif adalah 22 U/l, kelompok IV adalah 51,2 U/l, kelompok V adalah 38,6 U/l, dan kelompok VI adalah 30,2 U/l. Penggunaan dosis berlebih dari parasetamol dapat merusak organ hati. Hal ini disebabkan oleh karena terbentuknya metabolit toksik didalam hati, yang diikat oleh glutathion, tetapi pada dosis yang lebih tinggi persediaan glutathion telah terpakai seluruhnya dan terjadilah pengikatan pada molekul makro lainnya dari sel-sel hati hingga mengakibatkan kerusakan di dalam hati. Pemberian asetilsistein merupakan suatu cara untuk mencegah kerusakan hati bila dosis parasetamol berlebih, karena asetilsistein merupakan prekusor langsung glutathion. Tabel 2. Aktivitas GPT-serum akibat praperlakuan ekstrak etanol daun dewa

Kel. I II III IV V VI

Perlakuan Kontrol parasetamol Kontrol negatif Kontrol positif Ekstrak daun dewa 1,32 mg/200 g BB Ekstrak daun dewa 2,75 mg/200 g BB Ekstrak daun dewa 5,50 mg/200 g BB

Aktivitas GPT Purata ± SD (U/l)

I

II

III

5

128,2 ± 5,8

-

(+) 0,6

(+) 313,5(b)

5 5 5

127,4 ± 3,2 31,0 ± 2,7 57,0 ± 6,2

(-) 0,6 (-) 75,8(b) (-) 55,5(b)

(-) 75,7(b) (-) 55,3(b)

(+) 310,9(b) (+) 83,8(b)

5

46,6 ± 3,4

(-) 63,7(b)

(-) 63,4(b)

(+) 50,3(b)

5

39,0 ± 1,6

(-) 69,6(b)

(-) 69,4(b)

(+) 25,8

n

% perbedaan terhadap

Tabel 3. Aktivitas GOT-serum akibat praperlakuan ekstrak etanol daun dewa

Kel.

Perlakuan

n

Aktivitas GOT Purata ± SD (U/l)

% perbedaan terhadap I

II

III

I

Kontrol parasetamol

5

109,8 ± 7,2

-

(+) 2,0

(+) 399,1(b)

II III

Kontrol negatif Kontrol positif

5 5

107,6 ± 8,7 22 ± 1,6

(-) 79,6(b)

(+) 389,1 -

IV

Ekstrak daun dewa 1,32 mg/200 g BB Ekstrak daun dewa 2,75 mg/200 g BB Ekstrak daun dewa 5,50 mg/200 g BB

5

51,2 ± 3,9

(-) 2,0 (-) 80,0(b) (-) 53,4

(-) 52,4

(b)

(+) 132,7

5

38,6 ± 2,4

(-) 64,1(b)

(+) 75,5(b)

5

30,2 ± 2,6

(-) (b) 64,8 (-) 72,5(b)

(-) 71,9 (b)

(+) 37,3

V VI

(b)

(b)

Keterangan : (-) = mengalami penurunan ; (+) = mengalami peningkatan ; I = dibandingkan kontrol parasetamol; II = dibandingkan kontrol negatif larutan CMC 1%; III = dibandingkan kontrol positif (b) asetilsistein; = perbedaan bermakna (p<0,05)

Kontrol parasetamol dipakai untuk menilai apakah dengan pemberian parasetamol dapat menaikkan aktivitas GPT dan GOT, sehingga dapat dilihat pada hasil penelitian bahwa pemberian parasetamol dengan dosis beriebihan dapat merusak hati ditunjukkan dengan kenaikan aktivitas GPT sebesar 128,2 ± 5,8 U/l dan GOT sebesar 109,8 ± 7,2 U/l. Hal ini sama dengan kontrol negatif (CMC 1%) yang digunakan untuk melihat apakah pemberian CMC 1% dapat mempengaruhi hasil penelitian. Ternyata pemberian CMC 1 % tidak mempengaruhi karena tidak dapat menurunkan atau meningkatkan aktivitas GPT dan GOT. Pada tabel 2 aktivitas GPT tikus yang diberi praperlakuan ekstrak daun dewa dosis 1,32; 2,75; dan 5,50 mg/200 gBB berturut-turut ditemukan sebesar 57,0; 46,6; 39,0 U/l. Bila dibandingkan dengan tikus yang diberi parasetamol saja (kelompok I) aktivitas GPT tersebut secara bermakna (p<0,05) 55,5%; 63,7%; 69,6% lebih rendah. Temuan ini menunjukkan bahwa ekstrak daun dewa dapat menghambat atau mencegah peningkatan aktivitas GPTserum oleh parasetamol. Berarti kemungkinan besar ekstrak daun dewa mampu mencegah kerusakan sel hati karena parasetamol. Semakin tinggi dosis ekstrak daun dewa semakin besar daya hambat kerusakan sel hati. Sedangkan bila dibandingkan dengan tikus yang diberikan kontrol positif asetilsistein (kelompok III) aktivitas GPT dosis 1,32 dan 2,75 mg/200 gBB secara bermakna (p<0,05) 83,8% dan 50,3% lebih tinggi, dosis 5,50 mg/200 gBB secara tidak bermakna 25,8% lebih tinggi. Hal ini berarti bahwa ekstrak daun dewa dosis 5,50 mg/200 gBB mempunyai potensi daya hepatoprotektif yang setara dengan asetilsistein. Pada tabel 3 aktivitas GOT tikus yang diberi praperlakuan ekstrak daun dewa dosis 1,32; 2,75; dan 5,50 mg/200 gBB berturut-turut ditemukan sebesar 51,2; 38,6; 30,2 U/l. Bila dibandingkan dengan tikus yang diberi parasetamol saja (kelompok I) aktivitas GPT tersebut secara bermakna (p<0,05) 53,4%; 64,8%; 72,5% lebih rendah. Temuan ini menunjukkan bahwa ekstrak daun dewa dapat menghambat atau mencegah peningkatan aktivitas GOTserum oleh parasetamol. Berarti kemungkinan besar ekstrak daun dewa mampu mencegah kerusakan sel hati karena parasetamol. Semakin tinggi dosis ekstrak daun dewa semakin besar daya hambat kerusakan sel hati. Sedangkan bila dibandingkan dengan tikus yang diberikan kontrol positif asetilsistein (kelompok III) aktivitas GOT dosis 1,32 dan 2,75 mg/200 gBB secara bermakna (p<0,05) 132,7% dan 75,5% lebih tinggi, dosis 5,50 mg/200 gBB secara tidak bermakna 37,3% lebih tinggi. Hal ini berarti bahwa ekstrak daun dewa dosis 5,50 mg/200 gBB mempunyai potensi daya hepatoprotektif yang setara dengan asetilsistein.

Dengan demikian dapat dinyatakan bahwa ekstrak etanol daun dewa terbukti memiliki sifat hepatoprotektif terhadap kehepatotoksikan parasetamol. Ditemukan bahwa semakin besar dosis ekstrak menunjukkan efek hepatoprotektif yang semakin besar pula, tetapi ekstrak dosis 1,32 dan 2,25 mg/200 gBB memiliki kemampuan menurunkan aktivitas GPT dan GOT-serum yang lebih rendah secara nyata dibandingkan asetilsistein, sedangkan ekstrak dosis 5,50 mg/200 gBB memiliki kemampuan menurunkan aktivitas GPT dan GOT-serum yang tidak berbeda secara nyata dengan asetilsistein. Sehingga dapat dikatakan ekstrak daun dewa dosis 5,50 mg/200 gBB memiliki efek hepatoprotektif yang lebih baik daripada dosis 1,32 dan 2,75 mg/200 gBB. Kandungan kimia daun dewa yang diduga memiliki efek hepatoprotektif adalah senyawa flavonoid, di mana senyawa ini mempunyai aktivitas sebagai antioksidan, oleh adanya gugus hidroksi fenolik dalam struktur molekulnya. Efek flavonoid terhadap macam-macam organisme sangat banyak macamnya antara lain, karena flavonoid sering merupakan senyawa pereduksi yang baik, banyak menghambat reaksi oksidasi. Flavonoid bertindak sebagai penampung yang baik bagi radikal hidroksi dan superoksida serta dengan demikian melindungi lipid membran terhadap reaksi yang merusak. Aktivitas antioksidannya mungkin dapat menjelaskan mengapa flavonoid tertentu merupakan komponen aktif tumbuhan yang digunakan secara tradisional untuk mengobati gangguan fungsi hati (Robinson, 1995). KESIMPULAN Berdasarkan data aktivitas GPT dan GOT-serum tikus yang ditemukan serta tatacara analisis hasil yang diterapkan, dapat disimpulkan bahwa ekstrak etanol daun dewa (Gynura procumbens (Lour) Merr) memiliki sifat hepatoprotektif dengan dosis efektif 5,50 mg/200 gBB. DAFTAR PUSTAKA Anonim. 1993. Penapisan Farmakologi. Departemen Kesehatan RI. 69-71. Dollery S.C. 1991. Therapeutic Drugs. Churchill Living Stone. New York. 13-15. Hinton, Richard H. and Grasso. 1995. Hepatotoxicity in Animals Models in Toxicology. Shayne Coxgad and Christopher P. Chengelist (Sds), New York. Marcel Dekker Inc. 555-591. Raflizar dkk. 2006. Cermin Dunia Kedokteran No. 150: 10-14. Rhodes C, Thomas M, Athis, and Janet, 1995. Prinsiples of Testing for Acute Toxic Effects dalam General and Applied Toxicology. Abridged Editions. London and Basingstoke. Macmillan Press Ltd. 39-57. Robinson,T. 1995. Kandungan Organik Tumbuhan Tinggi. Institut Teknologi Bandung, Bandung. Sullivan, John B. Kriager. Gary R. 1992. Hazardous Materials Toxicology Clinical Prinsiples of Enviromental Health. Baltimore Maryland. Williams and Willkins. 109118. Tan H.T. dan Rahardja K. 2002. Obat-Obat Penting. Edisi kelima. PT Elex Media Komputindo. Jakarta. Voigt R. 1995. Buku Pelajaran Teknologi Farmasi. Universitas Indonesia. Jakarta. Wyngaarden J.B. 1982. The Textbook of Medicine. Vol I, 169, W.B. Saunders Co. Philadelphia. Zimmerman H.J. 1978. Hepatotoxicity. Appleton Century Crofts. New York.

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Gynura procumbens Ethanolic Extract Promotes Lymphocyte Activation and Regulatory T Cell Generation In Vitro Dinia Rizqi Dwijayanti1, Muhaimin Rifa'i1* 1

Department of Biology, Facullty of Mathematic and Natural Sciences, University of Brawijaya, Malang, Indonesia ABSTRACT

Immune system is a system of biological structures and processes within organism directed to protect against invaded pathogen. Cellular and humoral immune system mediated by immunocompetent cells such as CD4+ T cells, CD8+ T cells, CD4+CD25+ T cells, and B220 cells play important role for maintaining immunological surveillance. The purpose of this study was to determine the effect of ethanolic extract of G. procumbens leaves (EEGL) on the profile of CD4+ T cells, CD4+CD25+ T cells, and B220+ cells. Splenic cells were isolated from BALB/c mice and cultured in RPMI1640 medium in the presence of EEGL. After 4 days of incubation, cells were harvested, stained with antibodies and analyzed by flow cytometer. The data were analyzed by one-way ANOVA with α= 0.05 and Tukey test using SPSS 16.0 for windows. The results showed that the extract of G. procumbens could increase proliferation of CD4+CD62L- T cell, CD4+CD25+ T cells, and B220+ cells compared to the control. Here, we showed the biological effect of G. procumbens as medicinal herb with immunomodulatory activity and the dose of 0.1 µg/ml and 1.0 µg/ml could promote T cell activation compared to the highest dose of 10 µg/ml. Interestingly, the dose of 10 µg/ml rather promote than inhibit B cell proliferation. Keywords: B220+, CD4+, CD4+CD25+, Gynura procumbens, proliferation known that the extract of G. procumbens containing anti-hyperglycemic cures diabetes mellitus by decreasing blood glucose level. G. procumbens has a hypoglycemic effect and its leaves extract has an anti-hyperlipidemia property [4-7]. Another benefit of its leaves was reported to cure inflammation of eyes, toothache, rheumatic, cysts, and tumors. Anticancer effect of G. procumbens plants is associated with high antioxidant content. In addition, some studies indicated that the ethanol extract of G. procumbens leaves is able to inhibit the process of angiogenesis [8-10]. G. procumbens is also known for its anti-angiogenesis agent with an ability to inhibit the formation of new blood vessels so that the distribution of food to cancer cells is inhibited. However, tumor progression is not only mediated by angiogenesis but also the over expression of mortalin molecules [11-15]. A qualitative analysis of G. procumbens by thinlayer chromatography had detected the presence of sterols, triterpenes, phenolic compounds, polyphenols, and essential oils in that medicinal plant. G. procum*Corresponding author: bens contains sterols, sterol glycosides, quercetin, Muhaimin Rifa’i, Ph.D. kaempferol-3-O-neohesperidosida, kaempferol-3-glucoDepartment of Biology, Faculty of Mathematics and Natural side, quercetin-3-ramnosil, galactoside, and quercetinSciences, University of Brawijaya, Malang 65145, Indonesia Phone/Fax: 62-341-575841/62-341-575841, e-mail: [email protected] 3-O-ramnosil glucoside [1-6,15,16]. The results of

INTRODUCTION

Immune system is a defense system which protects the body from pathogen. It consists of cellular and humoral immune system. Cellular immune system is the immune system mediated by T cells either T helper cells (CD4+) or cytotoxic T cells (CD8+). Besides, the cellular immune system cells also contain the regulatory T cells CD4+CD25+ cells which have a function to control the performance of CD4+ and CD8+ T lymphocytes. Humoral immune system is the immune system involving activation of B cells (B220+) and antibody production. Immune system can be activated by the presence of foreign antigens as well as by the induction of immunomodulatory compounds [1,2]. G. procumbens is native plant from China, Myanmar and some Asian countries such as Indonesia, Malaysia, and Thailand. In Indonesia it is widely grown in Java, Sumatra, and Bali [3]. Based on the results of previous studies, G. procumbens leaves have benefit to cure various kinds of diseases. It has been

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Gynura procumbens Ethanolic Extract Promotes Lymphocyte Activation

study showed that the polar fraction of ethanol G. procumbens leaves has three classes of flavonoids: flavones, auron, and flavonols.16 Flavonoids contained in the ethanol extract of G. procumbens are immunomodulatory compound. An immunomodulatory compound has worked to increase or suppress the body's defense mechanisms, both specific and non-specific, as well as cellular and humoral defense mechanisms. Most immunomodulatory compounds enhance the immune system by increasing the proliferation of immunocompetent cells [17].

molecule staining.

Flow cytometry Analysis Antibodies used in the study were FITC-conjugated rat anti-mouse CD4, PE-conjugated anti-mouse CD25 (clone PC61.5), PE/Cy5-conjugated anti-mouse CD62L (clone MEL-14), and PE/Cy5-conjugated anti-mouse B220. Samples were incubated in the ice box for 30 minutes. Each sample was added with 500 µl PBS and transferred to the flow cytometry cuvettes. Samples were ready for running with flow cytometry. Data Analysis Flow cytometry results were visualized by BD CellQuest PRO™ software, tabulated, and analyzed by ANOVA analysis with a significance of 0.05% in SPSS version 16 for windows.

MATERIALS AND METHODS

Culture Preparation In this experiment we used RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS), 1% of antibiotics penicillin and streptomycin, 50 µM of 2-mercaptoethanol, and 1.0% α-CD3 (supernatant). This medium was filtered with cell strainer (Millipore membrane). G. procumbens extract powder was 0.2 g and dissolved in 200 ml of sterile water as a stock solution with 1 mg/ml concentration. 100 µl of stock solution was diluted in 9900 µl medium as a dose 1 medium with 10 µg/ml concentration. Dose 1 medium was also filtered with cell strainer and transferred to a new propylene tube. 500 µl of dose 1 medium was diluted in 4500 µl medium, to obtain dose 2 medium with 1 µg/ml concentration. 500 µl of dose 2 medium was diluted in of 4500 µl medium, and it would be dose 3 medium with 0.1 µg/ml concentration.

Data analysis Data analysis was performed using SPSS 16 with Kruskal-Wallis test, Independent T-test, and Pearson correlation test with α<0.05. RESULTS AND DISCUSSION

G. procumbens extract had an ability to activate CD4 T cells indicated by the loss of CD62L molecule. G. procumbens leaves with 10 µg/ml concentration was able to increase a relative number of activated T cells significantly (p<0.05). At these concentrations, the relative number of CD4+CD62L+ T cells was 61.90% and the relative number of CD4+CD62L- T cells was 38.10%. G. procumbens leaves ethanol extract in cell culture showed a significant increase (p>0.05) of CD4+CD25+ regulatory T cells compared with controls (Figure 2). The relative number of CD4+CD25+ T cells in control was 14.92%, while relative number of cells in the dose 1 reached 18.66%. G. procumbens leaves ethanol extract was able to increase the relative number of B220+ cells from 10.96% in the control to be 26.97% in the dose 1 extract (Figure 3). Based on statistical tests with one-way ANOVA, the results differ significantly with p<0.05. However, another case with CD4 + T cells both CD4+CD62L- and CD4+CD25+ T cells, extract with dose 2 and dose 3 actually gave effect to the decrease of relative B220+ cell when compared to a control namely 8.27% and 7.79%. The relative number of cells in the control was significantly different (p<0.05%) with dose 2 and 3 treatments, while between dose 2 and 3 did not differ significantly. The decrease in the relative number of B220+ cells at dose 2 and 3 compared to the control indicated that G.

Cell Isolation and Calculation Spleen was isolated and washed in petri dish containing PBS, transferred to another petri dish which also contained PBS and then crushed. The suspension was transferred into a polypropylene tube and then centrifuged with a speed of 1500 rpm for 5 minutes at 10˚C. Pellet was resuspended in 1 ml of medium. Nucleated cells were calculated by taking 10 µl of this suspension and added with 90 µl Evans blue then the number of cells was counted with haemocytometer. Cell culture Splenic cells (3x106) were cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin, 2-mercaptoethanol 50 µM, and 1% α-CD3 supernatant in 48 well plates. Cell cultures were incubated in 5% of CO2 at 37˚C for 4 days. After 4 days, cells were harvested then centrifuged with a speed of 1500 rpm for 5 minutes at 4˚C. Pellets were then subjected to cell surface JTLS | J. Trop. Life. Science

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Figure 1. T cell stimulation using ethanol extract of G. procumbens for four days increased the number of activated T cells. Spleen cells were cultured in RPMI medium for four days. The up left panel is control without G. procumbens ethanol extract addition. In the up right panel, cell culture was added with 10 µg/ml ethanol extract of G. procumbens. In low left panel, cell culture was added with 1 µg/ml ethanol extract of G. procumbens. Meanwhile, in low right panel cell culture was added with 0.1 µg/ml ethanol extract of G. procumbens. On day 4, cell culture was harvested and analyzed by flow cytometry. Naïve (CD4+CD62L+) and activated (CD4+CD62L-) T cells were presented in relative number. Data were mean ± SD values of five mice in each group.

procumbens has role as immunostimulant, besides it may also act as an immunosuppressant. The immunosuppressant has opposite works with immunostimulant. Immunosuppressant tends to inhibit the transcription process of cytokines so that cytokines play role as the little cell activation levels [1]. CD62L are molecules mediating naive T cell migration to the peripheral lymphoid organs which are the site of initiation of the immune response [1]. In the control treatment, the relative number of naive CD4 T cells (CD4+CD62L+) was 70.88% and the relative number of CD4 T cells that had been activated (CD4+CD62L-) was 29.12% (Figure 1). It showed that in the control treatment, CD4 T cells had not been activated. The administration of extract with 1 µg/ml and

0.1 µg/ml concentrations showed higher cell activation significantly than that of control and treatment with a dose of 10 µg/ml (p<0.05). The relative number of CD4+CD62L+ T cells at concentration of 1 µg/ml and 0.1 µg/ml extract were 51.81% and 50.91% respectively, whereas CD4+D62L- T cells were 48.19% and 49.09% respectively. The extract dose of 1 µg/ml and 0.1 µg/ml to activate CD4 T cells showed no significant difference (p>0.05). This data showed that the ethanol extract of G. procumbens leaves was able to increase the relative number of activated CD4 T cells. This increase provided evidence that G. procumbens has an ability as immunostimulant. Immunostimulatory compound is able to inhibit the activity of mitogen-Activated Protein Kinase (MAPK) [18]. MAPK is

Figure 2. T cell stimulation using ethanol extract of G. procumbens for four days showed the increase of CD4+CD25+ T cells. Spleen cells were cultured in RPMI medium for four days. The up left panel is control without addition of ethanol extract of G. procumbens. In the up right panel, cell culture was added with 10 µg/ml ethanol extract of G. procumbens, in low left panel cell culture was added with 1 µg/ml ethanol extract of G. procumbens, and in low right panel cell culture was added with 0.1 µg/ml ethanol extract of G. procumbens. On day 4, cell culture was harvested and analyzed by flow cytometry. CD4+CD25+ T cells were presented in relative number. Data were mean ± SD values of five mice in each group. JTLS | J. Trop. Life. Science

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Figure 2. Culture cell stimulation using ethanol extract of G. procumbens for four days showed the increase of B220 + cells. Spleen cells were cultured in RPMI medium for four days. In the in up left panel, cell culture was without ethanol extract of G. procumbens. In up right panel cell culture was added with 10 µg/ml ethanol extract of G. procumbens, in low left panel cell culture was added with 1 µg/ml ethanol extract of G. procumbens, and in low right panel cell culture was added with 0.1 µg/ml ethanol extract of G. procumbens. On day 4, cell culture was harvested and analyzed by using flow cytometry. B220+ cells were presented in relative number. Data were mean ± SD values of five mice in each group.

CD4+CD25+ T cells have important role in the immune system to create tolerant and maintain normal homeostasis. CD4+CD25+ T cells are able to control effector cell functions that have been activated [23-24]. This ability is necessary to avoid the presence of autoreactivity effector cells. The activation of B lymphocytes (B220+) and antibody production include in humoral immune response to protect the extracellular area [1]. Activation of B220 + cell is also one of the most important parts in the body defense mechanism. The increase in relative number of B220+ cells after administration of G. procumbens extract was closely associated with the increase of MAPK and IL-2 production. Craxton et al. further explained that MAPK also has a function to activate Nuclear Factor Kappa b (NF-kb). NF-kb is a transcription factor which stimulates B220+ cells to proliferate and differentiate [25]. Under these conditions, the G. procumbens leave’s ethanolic extract may become an immunostimulator as well as immunosuppresant. According to Schroeter et al., the action of compound is very complex, sometimes synergistic and antagonistic; in the other time it depends on the specific components used, cell type, concentration, and experimental design. The statement proved that the difference in cell types respond differently to the same exposure of compound with the same concentration. In particular, this is the case in dose 2 and 3 whose effect is the increase in the relative number of T cells CD4 +CD62L- T cells and CD4+CD25+, but it gives the effect of decrease in the relative number of B220+ cells [26].

also known to play role in the activation of immunocompetent cells because it can induce the increase of cytokines IL-2. Exposure to IL-2 on the cell will cause cyclin D2 and cyclin E concentration increase, and serves to activate cyclin-dependent kinases (cdk) [19]. IL-2 is also capable to reduce the concentration of p27 protein that plays role in the inhibition of cdk activity. If the active cdk and p27 are inhibited then the cells will be induced to resume cell cycle from G1 to S phase and the cells will proliferate [20]. Regulatory T cells are CD4+ T cell population which express CD25 molecule, CD4+CD25+. CD25 molecule is the alpha chain of the IL-2 receptor (IL2Ra) [21-22]. The treatments of dose 2 and dose 3 resulted in increasing the relative number of cells namely 25.11% and 25.14%, respectively. These numbers had significant differences (p<0.05) compared to control and dose 1 namely 14.92% and 18.66%, respectively. The changes of CD4+CD25- T cells into CD4+CD25+ could occur when CD4 T cells were activated by stimuli such as immunomodulator substances. An increase in the number of CD4+CD25+ T cells related to the increase of IL-2 production resulted by the G. procumbens stimulation. In addition to the direct stimulus of G. procumbens extract, CD4+ T cells which have been activated will also secrete IL-2 as a growth factor for both itself and other cells, one of which is a CD4+CD25+ T cell.1 The increasing number of CD4+CD25+ T cells is also related to the role of CD4+CD25+ T cells in the regulatory mechanism to balance the number of CD4+CD62L- T cells, CD4+ T cells that have been activated. According to Rifa’i et al., JTLS | J. Trop. Life. Science

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CONCLUSIONS

Based on the results and discussion, it can be concluded that the extract of G. procumbens may increase proliferation of CD4+CD62L- T cell, T CD4+CD25+, and B220+ compared to the control. 1 µg/ml and 0.1 µg/ml doses showed the highest ability of T cell activation compared to 10 µg/ml dose, but it gives a suppressant effect on B cells. 10 µg/ml dose has the highest ability for B cell activation compared to the 1 µg/ml and 0.1 µg/ml doses.

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12. ACKNOWLEDGMENT

The authors would like to thank University of Brawijaya Malang, Indonesia for providing study necessities, and Indonesian Ministry of Higher Education for supporting the financial under the Competitive Research Grant Scheme. Authors also want to thank Indriya R, S.Si., Dewi S., S.Si., M.Si., Ahmad Sony, S.Si., M.Si., and Bambang P., S.Si., for their support in conducting this experiment.

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REFERENCES

1. 2. 3. 4.

5.

6.

7.

8. 9.

Rifa’i M (2013) CD4+CD25+ Regulatory T Cells Prevent Detrimental Autoimmune Reactions. Int J. The Open Autoimmunity Journal. Bentham Open Publisher. 5: 1-5. Murphy K, Travers P, Walport M (2008) Immunobiology. 7th edition. Garland Science, Taylor & Francis Group, LLC. USA. Backer CA, Brink VD (1965) Flora of Java (Spermatophytes Only). Noordhoff-Groningen. Netherlands. Akowuah GA, Amirin S, Mariam, Aminah A (2001) Blood sugar lowering activity of Gynura procumbens leaves extracts. Journal of Tropical Medicinal Plants. 2: 5– 10. Akowuah GA, Sadikun A, Mariam A (2002) Flavonoid identification and hypo-glycaemic studies of butanol fraction from Gynura procumbens. Pharmaceutical Biology. 40: 405–410. Hassan Z, Yam MF, Ahmad M, Yusof APM (2010) Antidiabetic Properties and Mechanism of Action of Gynura procumbens Water Extract in Streptozotocin-Induced Diabetic Rats. Molecules. 15: 9008-9023. Zhang XF and Tan BK (2000) Effects of an Ethanolic Extract of Gynura procumbens Serum Glucose, Cholesterol and Triglyceride Levels in Normal and StreptozotocinInduced Diabetic Rats. Singapore Medical Journal. 41: 9– 13. Dalimartha S (2001) Indonesian Medicinal Plants. 1st edition. Trubus Agriwidia. Jakarta. Poh TF, Ng HK, Hoe SZ, Lam SK (2013) Gynura procumbens Causes Vasodilation by Inhibiting An-


15.

16.

17.

18.

19.

20.

21.

18

giotensin II and Enhancing Bradykinin Actions. J Cardiovasc Pharmacol. doi:10.1097/FJC.0b013e31828685b3. Hew C Sen, Khoo BY, Gam LH (2013) The Anti-Cancer Property of Proteins Extracted from Gynura procumbens (Lour.) Merr. PLoS One. 8:doi:10.1371/journal.pone. 0068524. Seow LJ, Beh HK, Majid AMSA, Murugaiyah V, Ismail N, Asmawi MZ (2011) Anti-angiogenic activity of Gynura segetum leaf extracts and its fractions. J Ethnopharmacol. 134:221–7. doi:10.1016/j.jep.2010.12.007. Wang H, Zhou JW, Fu DH, Zhou Y, Cheng WZ, Liu ZL (2013) Gynura procumbens ethanolic extract suppresses osteosarcoma cell proliferation and metastasis in vitro. Oncol Lett. 6:113–7. doi:10.3892/ol.2013.1315. Puangpronpitag D, Chaichanadee S, Naowaratwattana W, Sittiwet C, Thammasarn K, et al. (2010) Evaluation of nutritional value and antioxidative properties of the medicinal plant Gynura procumbens extract. Asian Journal of Plant Sciences. 9: 146–151. Jenie RI and Meiyanto E (2008) Combination of Sambung Nyawa (Gynura procumbens) Leaves Ethyl Acetate Fraction-Doxorubicin Induces Apoptosis in Human Breast Cancer T47D cells. Gajah Mada University, Yogyakarta. 15. Utomo DH, Widodo N, Rifa’i M (2012) Identifications small molecules inhibitor of p53-mortalin complex for cancer drug using virtual screening. Bioinformation. 8(9):426-429 Hew CS, Gam LH (2011) Proteome analysis of abundant proteins extracted from the leaf of Gynura procumbens (Lour.) Merr. Applied Biochemistry and Biotechnology. 165: 1577–1586. Rachmut IH, Samuels N, Melnick SJ, Ramachandran C, Sharabi Y, Pavlovsky A, et al. (2013) Immunomodulatory effects of the botanical compound LCS101: implications for cancer treatment. Onco Targets Ther. 6: 437–45. doi: 10.2147/OTT.S42038. Middleton E, Kandaswami C, and Theoharides TC (2000) The Effects of Plant Flavonoids on Mammalian Cells: Implications for Inflammation, Heart Disease, and Cancer. Journal Pharmacol. 52(4): 673-751. Nworu CS, Akah PA, Okoye FBC, Proksch P, and Esimone CO (2010) The Effects of Phyllanthus niruri Aqueus Ectract on the Activation of Murine Lymphocytes and Bone Marrow-Derived Macrophages. Immunological Investigations Informa Healthcare. 39: 245-267. Fathir A, Rifa’i M, Widodo (2014) Activity Of Aqueous Leaf Extract Of Horseradish Tree On Helper T- Cell And Cytotoxic T- Cell In Mice Infected With Salmonella Thypi. Veteriner J. 15 (1): 144-122. Rifai’i M (2010) Andrographolide ameliorate rheumatoid arthritis by promoting the development of regulatory T Volume 5 | Number 1 | January | 2015


latory T cells controlling hyperglycemia. SpringerPlus. 3 (1): 1-8 25. Craxton A, Shu G, Graves JD, Saklatvala J, Krebs EG, and Clark EA (1998) P38 MAPK is Required for CD40-Induced Gene Wxpression and Proliferatio in B Lymphocytes. Journal of Immunology.161: 3225-3236. 26. Schroeter H, Boyd C, Spencer JPE, Williams RJ, Cadenas E, and Rice-Evans C (2002) MAPK Signaling in Neuro Degeneration: Influences of Flavonoids and of Nitric Oxide. Neurobiology of Aging. 23: 861–880.

cells. Journal of Tropical Life Science. 1(1): 5-8. 22. Lee YH and Rifa'i M (2011) CD4 +CD25+FOXP3+ Regulatory T Cells In Allogeneic Hematopoietic Cell Transplantation. Journal of Tropical Life Science. 1(2): 69-75. 23. Rifa'i M, Shi Z, Zhang SY, Lee YH, Shiku H, Isobe K, Suzuki H (2008) CD8+CD122+ regulatory T cells recognize activated T cells via conventional MHC class I– αβTCR interaction and become IL-10-producing active regulatory cells. Int Immunology. 20(7): 937-947. 24. Rifa’i M, Widodo N (2014) Significance of propolis administration for homeostasis of CD4 +CD25+ immunoregu-


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Vol. 17, No.I.J. 1, pp.51-60 Biotech.

Gynura procumbens Prevents Chemoresistance through Inhibition MDR1 Expression on MCF-7 Breast Cancer Cell Line and Sensitizes the Cells to Doxorubicin Nunuk Aries Nurulita1,2*, Edy Meiyanto3, Sugiyanto3, Eishou Matsuda4, and Masashi Kawaichi4 Faculty of Pharmacy Universitas Muhammadiyah Purwokerto, Purwokerto, Indonesia Graduate Program of Pharmaceutical Science, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia 3 Cancer Chemoprevention Research Centre, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia 4 Laboratorium of Gene Function of Animal, Nara Institute of Science and Technology, Nara, Japan 1 2

Abstract

The long-term exposure of doxorubicin (Dox) causes enhancement in MDR1 expression that leads to breast cancer cell resistance. This protein become a serious problem in cancer treatment and also well-known as negative prognostic factor in breast cancer malignancies. The new approach using natural chemopreventive substance was developed to inhibit this resistance progress. This study was aimed to investigate whether ethyl acetate fraction of Gynura procumnens (FEG) can prevent chemoresistance through suppressing the MDR1 protein expression. MCF-7 cell was used as chemoresistance cell model. The MCF-7 cells were maintained with 100 nM Dox-contained medium for ve weeks. The chemoprevention effect of FEG was investigated by treated MCF-7/Dox with sub-toxic concentration of FEG. The cytotoxic properties of MCF-7 cells were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Immunouorescence and western blotting analysis was performed to detect the MDR1 expression. MCF-7/Dox cells need higher concentration for inhibiting cell growth, were compared with MCF-7, shown by IC50 value. The MDR1 protein level elevated after Dox exposure in time dependent manner. The FEG treatment decreased MDR-1 protein level with dose dependent manner. FEG in combination with DOX potentiates the DOX effect on breast cancer cell growth inhibition. The FEG prevents the chemoresistance development in breast cancer cell line, MCF-7 induced by Dox through inhibiting MDR1 expression. The additional of FEG enhances Dox effect on cell death induction. Thus, FEG could be developed as co-chemotherapy agent for reverse multidrug resistance. Keywords : Gynura procumbens, Chemoresistance, MDR1, MCF-7, MCF-7/Dox

drug inactivation, drug efux, drug target mutation, and the failure of apoptosis inisitian (Davis et al., 2003; Notarbartolo et al., 2005). Over expression of drug transporter such as P-gp plays a major role in causing multi drug resistance. P-gp is an adenosine triphosphatase (ATPase), a member of ATPbinding cassette (ABC) transporter encoded by MDR1 gene. In normal cells, P-gp licalized at ephitel cell and has a functioan to eliminate xenobiotic and metabolit endogen. However, in some human cancers P-gp overexpression has high corelation with the decreasing

Introduction Drug resistance becomes a major obstacle to the successful of cancer chemotherapy. There are several mechanisms mediate the drug resistance mechanism including *Corresponding author : Nunuk Aries Nurulita, Faculty of Pharmacy Universitas Muhammadiyah Purwakerto, Jl. Raya Dukuhwaluh, Kembaran, Purwokerto, Jawa Tengah Indonesia Telp. : +62-281-636751, E-mail: [email protected]

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survival and become poor prognosis on cancer development (Leonessa and Clarke, 2003). There is positive corelation between overall MDR1 expression and grade of breast cancer tumor (Surowiak et al., 2005). This protein become a serious problem in cancer treatment and also well-known as negative prognostic factor in breast cancer malignancies. P-gp substrates include substances in variable structure, chemical properties and drug mechanism. Several cancer chemotherapy drugs such as anthracyclines, taxanes, and epipodhophylotoxins already well-known as the substrate of this protein. Long time exposure of these agents on cancer cells triggers the transcriptional activity of MDR1 lead the increasing of P-gp (Germann, 1996; Nielsen et al., 1996). The elevation of P-gp protein leads to chemotherapy failure due to cell resistance at several type of cancer. Doxorubicin which belongs to anthracyclin group could induce breast cancer multi drug resistance after long time exposure. DOX treatment increase P-gp expression level through Ras/Raf1 activation (Davis et al., 2003). P-gp-mediated drug efux decreases intracellular Dox uptake and decrease its efcacy. The development of P-gp inhibitors led to the third generation. Unfortunately, most of P-gp inhibitors that have been developed giving disappointing results. New approach using natural substances with moderate or low cytotoxic properties become a promising hope for reversing multi drug resistance due to MDR1 over expression. Natural avonoids have antitumor properties and others pharmacological activities related with its antitumor such as anti-inammatory and antioxidant. Flavonoid, especially flavones and flavonol show modulation effect on cancer cell growth through cell cycle arrest and apoptosis induction (Limtrakul et al., 2007). Quercetin and kaempferol down regulate multidrug resistance gene on human erythroleukemic K562/A cells. It causes decreasing of cells sensitivity to adriamycin

(Yanqiu et al., 2011). Quercetin sensitizes cell resistance to daunorubicin through suppression on MDR1 expression and P-gp activity at human pancreatic carcinoma (Borska et al., 2010). Gynura procumbens is commonly used as herbal medicines in South-East Asia, especially Indonesia, Malaysia, and Thailand. The extract and fraction of this plant have high anti-inammatory and antioxidant properties (Iskander et al., 2002; Rosidah et al., 2008). The ethyl acetate fraction of G. procumbens (FEG) may contain quercetin and kaempferol. FEG sensitizes MCF-7 breast cancer cells to Doxorubicin treatment led to cell death. FEG modulates the microtubule integrity followed by cell cycle arrest and cell growth inhibition (Nurulita et al., 2012). In this study, we subject FEG to doxorubicin-resistance cell. We use MCF-7 cells treated by 100 nM doxorubicin for ve week as a model of cell resistance. We investigate the modulation on their sensitivity to doxorubicin, P-gp level, and cell growth. Materials and Methods Plant material The leaves of G. Procumbens were obtained from Balai Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional (BP2TO2T) Indonesia. Dried powdered leaves were rst extracted with ethanol 96%, and concentrated by evaporation under reduced pressure and the temperature was kept below 40oC. The extract was diluted in hot water and then fractionated with n-hexane. The aqueous fraction then was fractionated again with ethyl acetate. The ethyl acetate fraction was concentrated by evaporation under reduced pressure and the temperature was kept below 40oC. The extract was stored at 4oC until used. Cells and chemicals MCF-7 cells were obtained from Laboratorium of Gene Function in Animal, Nara Institute of Science and Technology (NAIST), Japan. MCF-7/DOX cells were

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developed from MCF-7 cell that induced with 100 nM DOX for ve weeks. The resistance development was checked by P-gp levels. The MCF-7 cells were routinely growed in DMEM (Nacalay Tesque) supplemented with 10% FBS (PAA Laboratories), 1% v/v Penicillinstreptomycin (Sigma), and 1 mM L-glutamine (Nacalay Tesque) at 37 oC in 5% CO2. MCF-7/ DOX cells were maintained in 25 nM DOXcontained medium. Doxorubicin (DOX) and 5-Fluorouracil (5-FU) (Ebewe) was purchased from PT. Ferron Par Pharmaceutical Cikarang, Indonesia, Dimethyl sulfoxide (DMSO) (Sigma, Aldrich, Germany), penicillin and streptomycin (Gibco), Tripsin (Sigma), 3-(4,5dimethyltiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT) (Sigma), triton X-100, 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen) are at analytical degree.

Gel electrophoresis and immunoblotting Cells were recovered, washed in PBS, and lysed for 30 min on ice using lysis buffer (20 mM Tris-HCl, pH 8, 0.5 mM EDTA, 1% NP40, 25 mM NaCl, and complete inhibitors of protease. Cells extract were centrifuged at 15.000 rpm for 20 min at 4oC to separate insoluble material. The protein concentration was determined using Bradford assay. Equal amount of each sample were mixed with SDS loading buffer, boiled for 3 min and subjected to SDS-PAGE at 120 V followed by electro blotting to Polyvinylidene uoride (PVDF) membranes for 2 h at 100 V. Membranes were blocked using 5% skim milk in PBS at room temperature for 1 hand subsequently probed with the primary antibody of interest. Blots were exposed by Chemilumi-oneSuper (Nacalay Tesque).

Antibodies Primary antibodies that used in western blotting and immunostaining were anti P-Glycoprotein (JSB-1) (GeneTex, Inc.), anti α-Tubulin (Santa Cruz), and secondary antibodies were diluted in PBS containing 5% skim milk, 0,05% Tween (blocking buffer). For P-gp detection, the anti-P-gp and its secondary antibody were diluted in Can Get Signal (CGS) solution (Cosmo Bio).

Results Long treatment of sub toxic concentration of DOX induced resistance cells development. Breast cancer cells become less sensitive to chemotherapy agen during the cells resistance development. After five weeks induced by DOX, MCF-7 cells decreased its sensitivity to DOX treatment, as shown by IC50 value (Table 1). Table 1. IC50 value of DOX and FEG on MCF-7 and MCF-7/DOX cells

Cells growth assay Cells (104 cells/well) were seeded in 96-well plate (100 μl/well). After 24 h of incubation, the medium was replaced with DOX, FEG, and its combination-containing medium. The medium was discarded and replaced with MTT-containing medium (0.5 mg/ml) after 24 h incubation and incubated again for further 4 h at 37oC, 5% CO2. The reaction was stopped using 10%SDS in 0.1 N HCl solution and was incubated for overnight in light protected chamber, to dissolve formasan salt. The absorbance of each well was measured by ELISA reader at 595 nm. The ratio between treated and control cells absorbance referred to percentage (%) of cells viability.

Sample DOX FEG

Cell Line MCF-7 MCF-7/Dox MCF-7 MCF-7/Dox

IC50 value * 410 ± 44 nM 921 ± 40 nM 259 ± 29 μg/ml 161 ± 40 μg/ml

*average from two independent experiments

DOX resulted cell growth inhibition more than 2-fold higher on MCF-7/DOX sub line than that of MCF-7 cell line (Figure 1A). FEG treatment showed the opposite results. FEG caused cell growth inhibition stronger on MCF-7/DOX sub line compared with MCF-7 cell (Figure 1B).

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A CellViability(%)

120

MCFͲ7

100

MCFͲ7/Dox

80 60 40 20 0 0

200

400

600

800

1000

1200

Dox(nM)

B CellViability(%)

120

MCFͲ7 MCFͲ7/Dox

100 80 60 40 20 0 0

50

100

150 FEG(μg/mL)

200

250

300

Figure 1. MCF-7/DOX cells are less sensitive to DOX (A) but more sensitive to FEG (B). Cells (104 cells/well) were cultured at 96-well plate, then treated with indicated concentration of DOX or FEG for 24 h incubation. Cells Viability was determined with MTT assay. This result represents from 3 independent experiments. The data was statistically analized by two tailedT-test analysis with p < 0,05.

DOX induced the elevation of P-gp level DOX-induced MCF-7 cells showed the elevation of P-gp expression level in time dependent manner (Figure 2A). expression level of this protein was correlated with less sensitivity of MCF-7/DOX cells to DOX. FEG treatment decreased P-gp level in dose dependent pattern (Figure 2B). To make confirmation of this phenomenon, both immunobloting and immunofluorescence were conducted. FEG decreased the green uorescence intensity that refer P-gp protein decreasing (Figure 3). DOX induces breast cancer cell resistance due to increasing of

P-gp expression level. The P-gp protein has 170 kDa molecule size. This protein is hyperglycosilated as such appears as a diffuse band on Western blot results (Patel et al., 2002). We also found the additional band below P-gp protein size (Figure 2). The lower molecules weight bands was detected using the JSB-1 antibody for P-gp represent degraded product of P-gp protein. JSB-1 antibody recognizes a cytoplasmic epitope of P-gp and does not cross-react with other protein such as MDR3 or muscle myosin, as does the C219 antibody (Leonessa and Clarke, 2003).

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A

T47D

MCF-7

M MCF7//Dox1

T47D

MCF-7

MCFM 7/Dox

FMCF 7/Doox2

MCF7/Dox3

MCF7/Dox4

MC CF7/Dox FEG G 25

MCF7/Dox FEG 50

MCF7/Dox FEG 100

P-gp

Į--Tubulin

B P-gp

Į--Tubulin

Figure 2. DOX induced P-gp expression on pada MCF-7 cells (A) and FEG prevents the elevation of this protein expression (B). Cells (106 cells/well) were seeded at 6-well plate, then treated with DOX, FEG or its combination for 24 h. Cell lysate then analyzed with Western blotting. Anti-α-tubulin was used for loading control. Dox1, Dox2, Dox3, and Dox4 refer to induction time for 2, 3, 4, and 5 weeks, respectively.

anthrasiclin, epipodhophylotoxins, and rhodamine. The exposure of These substrate to cancer cells could trigger transcriptional activity of MDR-1 (Germann, 1996; Lampidis et al., 1989; Nielsen et al., 1996). Previous study have found the long time exposure of DOX induced cancer cells resistance through the increasing level of P-gp (Abolhoda et al., 1999). In this study MCF-7 cells were used as resistance model of breast cancer. This cells were developed by long term sub toxic concentration of DOX induction. MCF-7 cells are cell line that express MDR1 and Breast cancer resistance protein (BCRP) that will be triggered its expression after several chemotherapy agent induction (Faneyte et al., 2002). MDR-1 expression elevates at patient after long time chemotherapy agent administration and had strong correlation with cancer chemotherapy

FEG reversed cell resistance and increased the sensitivity of MCF-7/DOX cells to DOX treatment To investigate whether FEG could reverse the cell resistance development on MCF-7/DOX, MCF-7 cells were treated with DOX, FEG, or its combination and observed FEG reversal effect and its modulation on cell growth. Combination treatment of DOX+FEG suppressed cell growth o both of MCF-7 and MCF-7/DOX cells. The inhibition effect of combination treatment on MCF-7/ DOX cells more signicance compared to MCF-7 cells (Figure 4). Discussion P-glycoprotein (P-gp), a protein encoded by MDR-1 gene, has important function as ATP-dependent pump of drug efux. P-gp has specic substrate, such as vinca alkaloids,

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Figure 3. DOX induced P-gp expression on pada MCF-7 cells and FEG prevents the elevation of this protein expression in concentration dependent manner. Cells (5.104 cells/well) were seeded on cover slip at 24-well plate. After treated with or without DOX+FEG for 24 h, then stained with anti-α-Tubulin (green uorescence) and counterstained with DAPI (blue uorescence). Cells were observed under uorescence microscope with 400x magnication.

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Cell Viability (%)

100

MCF-7/DOX

80 60 40 20 0

Figure 4. FEG increased the sensitivity of MCF-7/DOX cells to DOX. Cells (104 cells/well) were cultured at 96-well plate, then treated with indicated concentration of DOX, FEG and its combination for 24 hours incubation. Cells Viability was determined with MTT assay. This result represents from 2 independent experiments with similar results. The data was statistically analized by two tailed-Ttest analysis. *p = 0,1 and **p = 0,028.

*

**

Figure 4. FEG increased the sensitivity of MCF-7/DOX cells to DOX. Cells (104 cells/well) were cultured at 96-well plate, then treated with indicated concentration of DOX, FEG and its combination for 24 h incubation. Cells Viability was determined with MTT assay. This result represents from 2 independent experiments with similar results. The data was statistically analized by two tailed-T-test analysis. *p = 0,1 and **p = 0,028.

failure (Leonessa and Clarke, 2003). This protein has identied as negative prognostic in cancer diseases development. Sub toxic concentration of DOX for ve weeks decreased MCF-7 cells sensitivity to this agent, shown by IC50 value become more than 2 fold higher compared with MCF-7 cells. DOX exposure to MCF-7 cells lead to increasing expression of P-gp in time dependent manner. MCF-7 cells after ve weeks DOX induction (MCF-7/DOX) shown the significance increasing of this protein level compared with MCF-7 cells. The P-gp level has strong correlation with the low sensitivity of MCF-7/DOX to DOX exposure. The attempts to overcome drug resistance mediated by MDR-1 activation and elevation of P-gp level were developed the P-gp inhibitors. The agent that could suppress P-gp expression will increase uptake of chemotherapy agent and may influence its clinical efficacy. Flavonoids

could modulate the expression and activation of P-gp. There are several mechanisms mediate their effect: changing membrane cells permeability, suppressing P-gp expression, inhibition ATPase activity, and occupation substrate binding site at P-gp, then changes its conformation (Arora et al., 2000; Boumendjel et al., 2002; Drori et al., 1995). Natural compounds such as avonoids and polyphenol have signicant biological properties related to multidrug resistance development on cancer cells. Biochanin A and sylimarin inhibit drug efux from the cells by P-gp pump, and produce synergistic effect when combined with DOX at MCF-7 and MDA435/LCC6. Both compound decreased IC50 value of daunomycin significantly at MCF-7/Adr cells (Zhang and Morris, 2003; Chung et al., 2005). (-)-Epigallocatechin (EGC), (-)-epicatechin gallate (ECG), dan (-)-epigallocatechin gallate (EGCG) increase daunomycin accumulation at P-gp overexpressing cells, KB-C2 (Kitagawa et al.,

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2004). Flavonoid and polyphenol have a promising properties to be developed as P-gp inhibitor to prevent cell resistance. FEG treatment enhances DOX cytotoxicity signicantly at MCF-7/DOX cells, higher than the effect on MCF-7 cells. The inhibition of cells growth exhibited synergistic effect of combination treatment DOX+FEG in dose dependent manner. FEG treatment decreases P-gp expression level at MCF-7/DOX cells linear to the given concentration. The ethyl acetate fraction of G. procumbens (FEG) was suggested have avon and or avonol with hydroxyl group modied to be metoksi at 3 and 4’, and asetoksi at 7 posisition (Sugiyanto et al., 2003). The ethyl acetate fraction of G. procumbens (FEG) may contain quercetin and kaempferol (Nurulita et al., 2012). FEG may modulate MDR-1 expression through the same mechanism with quercetin and kaempferol. Quercetin inhibits the members of MDR1 family, such as : P-gp, MRP, and BCRP, competitively (Cooray et al., 2004; Scambia et al., 1994; van Zanden et al., 2005). Kaempferol and quercetin decrease P-gp expression and its activity at MDR KB-V1 cells (Limtrakul et al., 2005). Quercetin and kaempferol prevent cell resistance development through down regulate multidrug resistance gene on human erythroleukemic K562/A cells. It reverses cells sensitivity to adriamycin (Yanqiu et al., 2011). Quercetin suppresses MDR1 expression and P-gp activity at human pancreatic carcinoma. The net influx of daunorubicin increases when given together with quercetin (Borska et al., 2010). One study on structure activity relationship concludes avonoid with –OH group at 5 dan 7, with maximal have three total hydroxyl groups, and have strong inhibition on P-gp activity. Double bond at C2 and 3 as shown at avon generates its planarity, lead to intercalate with hydrophobic amino acids residue of P-gp (Sheu et al., 2010). Quercetin and kaempferol modulate both of MDR-1 gene and P-gp protein level and also its activity that reverse multidrug resistance on many type of cancer cell lines.

This study found that FEG decrease P-gp expression level at MCF-7/DOX cells. This results were suggested to the decreasing P-gp expression that may affect on the increasing of DOX accumulation at cancer cells. In the next study, monitoring the net uptake of DOX during co-treatment of FEG should be considered. This effect may precede through several possibility, such as suppression on transcriptional activity of MDR-1 gene, translation, or post translation. As the conclusion, FEG prevents MCF-7 cells from developing resistance through down-regulating P-gp expression. FEG in combination with DOX potentiates the DOX effect on breast cancer cell growth inhibition. Thus, FEG could be developed as co-chemotherapy agent for reversing multidrug resistance. Acknowledgement This work was supported by Sandwichlike program 2010/2011, managing by Directorate General of Higher Education Republic of Indonesia, 2010 References Abolhoda, A., Wilson, A.E., Ross, H. Danenberg, P.V. Burt, M., and Scotto, K.W. 1999, Rapid activation of MDR1 gene expression in human metastatic sarcoma after in vivo exposure to doxorubicin, Clin. Cancer Res., 5, 3352-3356 Arora, A. Byrem, T.M., Nair, M.G., and Strasburg, G.M. 2000, Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids, Arch Biochem. Biophys., 373, 102-109. Borska, S., Sopel, M., Chmielewska, M., Zabel, M., and Dziegiel, P. 2010, Quercetin as a potential modulator of P-glycoprotein expression and function in cells of human pancreatic carcinoma line resistant to daunorubicin, Molecules, 15, 857-870. Boumendjel, A., Di Pietro, A., Dumontet, C., and Barron, D. 2002, Recent advances in the discovery of flavonoids and

58

Nurulita et al.

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analogues with high afnity binding to P-glycoprotein responsible for cancer cell multidrug resistance, Med. Res. Rev., 22, 512-529. Chung, S.Y., Sung, M.K., Kim, N.H., Jang, J.O., Go, E.J., and Lee, H.J. 2005, Inhibition of P-glycoprotein by natural product in human breast cancer cells, Arch. Pharm. Res., 28(7), 823-828 Cooray, H.C., Janvilisri, T., van Veen, H.W., Hladky, S.B., and Barrand, M.A. 2004, Interaction of the breast cancer resistance protein with plant polyphenols, Biochem. Biophys. Res. Comm., 317, 269–275 Davis, J.M., Navolanic, P.M., WeinsteinOppenheimer, C.R., Steelman, L.S., Wei H., Konopleva, M., Blagosklonny, M.V., and McCubrey, J.A. 2003, Raf-1 and Bcl-2 induce distinct and common pathways that contribute to breast cancer drug resistance, Clin. Cancer Res., 9, 1161-1170. Drori, S., Eytan, G.D., and Assaraf, Y.G. 1995, Potentiation of anticancer-drug Cytotoxicity by multidrug-resistance chemosensitizers involves alterations in membrane uidity leading to increased membrane permeability, Eur. J. Biochem., 228, 1020-1029. Faneyte, I.F. Kristel, P.M.P. Maliepaard, M. Scheffer, G.L.Scheper,R.J. Schellens, J.H.M. and van de Vijver, 2002, Expression of the breast cancer resistance protein in breast cancer, Clin. Cancer Res., 8,1068-1074. Germann, U.A. 1996, P-glycoprotein – mediator of multidrug resistance in tumor cells, Eur. J. Cancer. 32A, 927-944 Iskander, M.N., Song, Y., Coupar, I.M., and Jiratchariyakul,W. 2002, Antiinamatory screening of medicinal plant G. Procumbens, Plant Foods Hum. Nutr., 57, 233-244. Kitagawa, S., Nabekura, T., and Kamiyama, S. 2004, Inhibition of P-glycoprotein function by tea catechins in KB-C2 cells. J. Pharm. Pharmacol., 56, 1001-1005.

Leonessa, F. and Clarke, R. 2003, ATP binding cassette transporters and drug resistance in breast cancer, Endocr. Relat. Cancer, 10, 43-73. Lampidis, T.J., Castello, C.,del Giglio, A., Pressman, B.C.,Viallet, P., Trevorrow, K.W. ,Valet, G.K., Tapiero, H., and Savaraj, N. 1989, Relevance of the chemical charge of rhodamine dyes to multiple drug resistance, Biochem. Pharmacol., 38, 4267-4271. Limtrakul, P., Khantamat, O., and Pintha, K. 2007, Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. J. Chemother., 17, 86-95. Nielsen, D., Maare, C. and Skovsgaard,T. 1996, Cellular resistance to anthracyclins, Gen. Pharmacol., 27, 251-255. Notarbartolo, M., Poma, P., Perri, D., Dusonchet, L., Cervello, M., and Alessandro, N. 2005, Antitumor effects of curcumin, alone or in combination with cisplatin or doxorubicin, on human hepatic cancer cells. Analysis of their possible relationship to changes in NFкB activation levels and in IAP gene expression, Cancer Lett., 224, 53-65. Nurulita, N.A., Meiyanto, E., Sugiyanto, Matsuda, E., and Kawaichi, M., 2012, Gynura procumbens modulates the microtubules integrity and enhances distinct mechanism on doxorubicin and 5-urouracil-induced breast cancer cell death, Orient. Pharm. Exp. Med., DOI 10.1007/s13596-012-0063-5. Nurulita, N.A., Meiyanto, E., and Sugiyanto, 2012, Selectivity of ethyl acetate fraction of Gynura procumbens on colon cancer and breast cancer, Indo. J. Cancer Chemoprev., 3(4) Pattel, V.A., Dunn, M.J., and Sorokin, A. 2002. Regulation of MDR-1 (P-glycoprotein) by cyclogenase 2. J. Biol. Chem., 277(41), 38915-38920. Rosidah, Yam, M.F., Sadikun, A. and Asmawi, M.Z. 2008, Antioxidant potential of Gynura procumbens, Pharmaceutic. Biol., 46, 616-625.

59

Nurulita et al.

I.J. Biotech.

Scambia, G., Ranelletti, F.O., Panici, P.B., De Vincenzo, R., Bonanno, G., Ferrandina,G., Piantelli, M., Bussa, S., Rumi, C., and Cianfriglia, M. 1994, Quercetin potentiates the effect of adriamycin in a multidrugresistant MCF-7 human breastcancer cell line: P-glycoprotein as a possible target, Cancer Chemother. Pharmacol., 34, 459-464. Sheu, M.T., Liou, Y.B., Kao, Y.H., Lin, Y.K., and Ho,H.O. 2010, A quantitative structure –activity relationship for the modulation effects of flavonoids on P-glycoprotein-mediated transport, Chem. Pharm. Bull., 58(9), 1187-1194. Surowiak, P., Materna, V., Matkowski, R., Szczuraszek, K., Kornafel, J., Wojnar, A., Pudelko, M., Dietel, M., Denkert, C., Zabel, M., and Lage, H. 2005. Relationship between the expression of cyclooxigenase 2 and MDR1/Pglycoprotein in invasive breast cancers and their prognostic signicance, Breast Cancer Res., 7, R862-R870. Sugiyanto, B., Sudarto, Meiyanto, E., Nugroho, A.E., and Jenie, U.A. 2003, The anticarcinogenic activity of plants compounds, Indo. J. Pharm., 14(4), 216225. Van Zanden, J. J., Wortelboer, H. M., Bijlsma, S., Punt, A., Usta, M., Bladeren, P.J., Rietjens, I.M., and Cnubben, N.H. 2005, Quantitative structure activity relationship studies on the avonoid mediated inhibition of multidrug resistance proteins 1 and 2. Biochem. Pharmacol., 69, 699–708, 2005. Yanqiu, H., Linjuan, C., Jin, W., Hongjun, H., Yongjin, S., Guobin, X., and Hanyun, R. 2011. The effect of quercetin and kaempferol on multidrug resistance and the expression of related genes in human erythroleukimia K562/A cells, African J. Biotech, 10(62), 13399-13406. Zhang, S. and Morris, M.E. 2003, Effects of the flavonoids biochanin A, morin, phloretin, and silymarin on P-glycoprotein-mediated transport, J.Pharmacol. Exp. Ther., 304, 1258-1267.

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Immunomodulator of ethanol extract of G. procumbens leaves to M. musculus immune (Dwijayanti and Rifa’i)

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Immunomodulator Testing on Ethanol Extract of Gynura procumbens Leaves to Mus musculus Adaptive Immune System: in Vitro Study Dinia Rizqi Dwijayanti and Muhaimin Rifa’i* Department of Biology, Faculty of Mathemathic and Natural Sciences, University of Brawijaya, Malang, Indonesia Abstract Immunomodulator is a substance that has an ability to modulate the activity and function of immune system. Gynura procumbens supposed to has benefit as an immunomodulator because of it afficacy to cure many diseases. The aim of + this study is to determine the effect and dose variations of G. procumbens extract on biological aspect of CD4 CD62L , + + + + + CD4 CD62L , CD8 CD62L and CD8 CD62L T cells. G. procumbens extract concentrations that used in this experiment were 0 µg /ml, 0.1 µg/ml, 1 µg/ml, and 10 µg/ml. Spleen cells were cultured for 4 days in 5% CO2 incubator at a temperature of 37˚C. Cultured cells were harvested and analyzed by flowcytometry to asses cell surface molecule expression. The resulting data were tabulated and analyzed using ANOVA analysis with a significance of 0.05% on SPSS + version 16. Results showed that the extract of G. procumbens can increase the proliferation of CD4 CD62L , + + + + + CD4 CD62L , CD8 CD62L and CD8 CD62L T cells compared to the control. Dose of 1 µg/ml showed the highest effect to promote cell activation compared with the dose of 0.1 µg/ml and 10 µg/ml. Dose of 10 µg/ml could suppress + + + + + + CD4 CD62L , CD4 CD62L , CD8 CD62L and CD8 CD62L T cells development. This study suggests that the ethanol extract of G. procumbens has benefit as an immunomodulator and involved in the immune system. Keywords: Gynura procumbens, immunity, immunomodulator, in vitro, lymphocytes.

INTRODUCTION Immunomodulator is a substance that has an ability to modulate the activity and function of the immune system. Immunomodulator has ability to influence the number of cells that play + role in the adaptive immune system such as CD4 + and CD8 T cells. Research on immunomodulator that has been done used the recombinant cytokines. Recombinant cytokines that were used were interferon-γ (IFN-γ) in combination with vaccines [1,2]. The weakness of this substance are unstable, easily degraded, and have negative effects such as neutrophilia or defective neutrophil function, lymphophenia and monocytophenia [3]. Based on that case, we need another alternative such as active compound in a plant that has immunomodulator activity. One of them is Gynura procumbens or Sambung Nyawa. G. procumbens has been long used by people in Java as a traditional medicine. Based on recent studies, leaves of G. procumbens have a lot functions such as anti-cancer, anti-inflammation of the kidneys and as anti-diabetic [4]. The 

Correspondence address: Muhaimin Rifa’i Email : [email protected] Address : Laboratory of Animal Physiology, University of Brawijaya, Jl. Veteran, Malang, 65145

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immunological side G. procumbens such as immunomodulator activity has not been studied yet. In fact, with so many functions of G. procumbens, it is possible that these plants have immunomodulator capabilities that can affect the activity of immunocompetent cells such as + + + + CD4 CD62L , CD4 CD62L , CD8 CD62L and + + CD8 CD62L T cells. This study aimed to determine the effect of G. procumbens and dose variations of G. procumbens extract on the + + + + number of CD4 CD62L , CD4 CD62L , CD8 CD62L + + and CD8 CD62L T cells. MATERIALS AND METHODS Medium Preparation Culture medium that was used in this study was RPMI medium with antibiotics penicillin and streptomycin. The medium were put in 2 propylene tubes, each as much as 5 ml and used as control medium. G. procumbens extract powder weighed 0.2 gr and diluted with 200 ml of sterile water and then homogenized. Then, a stock solution with a concentration of 1mg/ml. 100 µl was diluted with 9900 µl control medium to obtain a dose of 3 medium with a concentration 10 µg/ml. 500 µl dose in 3 medium was diluted with of 4500 µl control medium to obtain a dose 2 medium with a concentration 1 µg/ml. 500 µl dose 2 medium was diluted with of 4500 µl control medium to obtain a dose 1

ISSN. 2087-2852 E-ISSN. 2338-1655

Immunomodulator of ethanol extract of G. procumbens leaves to M. musculus immune (Dwijayanti and Rifa’i) medium with a concentration 0.1 µg/ml. Dose 1, 2, and 3 medium filtered with cell strainer (Millipore membrane) and transferred into a new propylene tube. After filtered, 4 types of medium were added with 1 µl of 2-mercaptoethanol, 10% Fetal Bovine Serum (FBS), and 1% α-CD3. Cell Isolation and Counting Spleen organ was isolated from mice. Spleen was washed in petri dish that contained PBS. The spleen was transferred to another petri dish which also contained PBS, and then crushed. The suspension was filtered and transferred into propylene tube, then added with PBS until 12 ml suspension. Suspension of cell was centrifuged with a speed of 2500 rpm for 5 minutes at 4˚C. Pellet resuspended in 1 ml of control medium. This suspension taken 20 µl and added with 80 µl evans blue and then homogenized by pipetting. The number of cells was counted with haemocytometer. Cell culture Each medium, i.e. control, dose 10 µg/ml, 1 µg/ml, and 0.1 µg/ml were added with + 122 µl of cell suspension and mixed gentle. Each of these medium which contain cell was inserted into the well in plate. The cells were incubated in a 5% CO2 incubator at 37˚C for 4 days. After 4 days, the cells were harvested and then centrifuge with a speed of 2500 rpm for 5 min at 4˚C. Pellet was resuspended in 1 ml PBS. Each sample was taken 20 µl and added with 80 µl evans blue after that was homogenized by pipetting to count the number of cells. Each sample was also taken 300 µl and transferred into micro tube containing 500 µl PBS. A sample in 3 micro tubes was centrifuged with a speed of 2500 rpm for 5 minutes at 4˚C. Pellet stained with antibodies with extracellular staining. Flowcytometry Test Pellet in micro tube was added with 50 µl antibody. Antibodies that were used were FITCconjugated rat anti-mouse CD4, PE-conjugated rat anti-mouse CD8, and PE/Cy5-conjugated rat anti-mouse CD62L. After that, the samples were homogenized by pipetting and incubated in the ice box for 20 minutes. Each sample in a micro tube was transferred to the flowcytometry cuvettes and added with 300 µl PBS. Next, the samples were ready for running with flowcytometer.


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Experimental Design and Data Analysis The design of this study was experimental research with a completely randomized design consist of 4 treatments (control, dose 10 µg/ml, 1 µg/ml, and 0.1 µg/ml). Flowcytometry results were visualized using BD CellQuest PRO™ software then the resulting data were tabulated and analyzed using ANOVA analysis with a significance of 0.05% on SPSS version 16. RESULT AND DISCUSSION + + + Population of CD4 CD62L and CD4 CD62L T Cells Extract of G. procumbens with 4 different doses gave significant different results on the + activation of CD4 T lymphocytes (Fig. 1). Control treatment 0 µg/ml indicated the relative + numbers of CD4 CD62L T cells were 23.91%, and 3 the absolute numbers of cells were 5376x10 cells. Dose of 0.1 µg/ml in cell culture showed greater cell numbers compared with control with 3 relative number 29.54% as many 17015.1x10 cells. Dose of 1 µg/ml showed the highest absolute number of 4 treatments given with the 3 absolute number 30272.5x10 cells (39.41%). The absolute numbers of cells in a 10 µg/ml dose 3 were 9301x10 cells (37.67%). This number was decreased compared to dose 0.1 µg/ml and 1 µg/ml, but still higher than the control.

Figure 1. Population of CD4+CD62L- and CD4+CD62L+ T Cells: A. Relative number; B. Absolute number.

ISSN. 2087-2852 E-ISSN. 2338-1655

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Immunomodulator of ethanol extract of G. procumbens leaves to M. musculus immune (Dwijayanti and Rifa’i) Ethanol extract of G. procumbens was also + + affected the proliferation of CD4 CD62L T cells. The relative numbers of the cells in the control treatment were 52.29% and the absolute 3 numbers were 6551.2x10 cells. Relative + + numbers of CD4 CD62L T cells in the 0.1 µg/ml dose of the extract were 37.31% and the 3 absolute numbers were 8822.5x10 cells. The relative numbers of cells in the 1 µg/ml dose treatment were 33.12% with the absolute 3 numbers 15164.6x10 cells. Treatment dose of 10 µg/ml showed the relative number of 33.53% 3 with the absolute number 8733.7x10 cells. + The pattern of CD4 CD62L T cell proliferation showed the same trend with the proliferation of + + CD4 CD62L T cells. The number of cells increased at treatment doses 0.1 µg/ml, 1 µg/ml and 10 µg/ml compared to the control treatment. Dose 1 µg/ml showed the highest cell number compared to other treatments. The number of cells decreased in a dose of 10 µg/ml when compared to dose 0.1 µg/ml and 1 µg/ml. + Comparison of the number of CD4 CD62L T cells + + and CD4 CD62L T cell can be seen clearly that in + + the control treatment CD4 CD62L T cell count + more than CD4 CD62L T cells, while cells after treatment with the G. procumbens extract given, + + the number of CD4 CD62L T cells tend to + decrease compared to CD4 CD62L T cells. CD62L was a molecule that is owned by the naive cells [5]. It showed that extracts of G. procumbens has + + + the ability to activate CD4 T cells so CD4 CD62L + T cells lose CD62L molecule into CD4 CD62L T cells. + The increase of activated CD4 T cells number after the administration of extracts G. procumbens supposedly because the content of flavonoids and saponin in G. procumbens as an immunostimulant. According to Middleton et al. [6], flavonoids have the ability to trigger the activity of MAP Kinase. Mitogen can stimulate the increase of IL-2. IL-2 is a proliferation and differentiation factor of immunocompetent cells. Concentration of cyclin D2 and cyclin E would have increased when the cells were exposed to IL-2. IL-2 was also served to inhibit the p27 concentration. Under these conditions, IL-2 was able to induce cell cycle continuation of the G1 phase to the S phase of the cell cycle so it proliferated actively [7]. Saponin has the ability to increase cytokine IFN [8]. Lee et al. [9] and Shi et al. [10] mentioned that IFN can stimulate the


up-regulation of MHC-II expression so that more + T cells differentiate into CD4 T cells. The decrease of the number of cells in the dose 3 treatment showed that G. procumbens at a dose of 10 µg/ml have immunosuppressive + effects on CD4 T cells. This immunosuppressive effect was also obtained from the ethanol extract of leaf flavonoids in G. procumbens. According to Schroeter et al. [11], the actions of flavonoids were very complex, sometimes synergistic and antagonistic at times depend on the specific components, cell type, concentration, and experimental design. Based on this statement, it can be seen that the concentration of flavonoids in the given doses influenced the cells. Immunosuppressant tends to inhibit the transcription of cytokines so the numbers of cytokines such as IL-2 and IFN that play role in cell activation were decreasing [5]. +

-

+

+

Population of CD8 CD62L and CD8 CD62L T Cells Extract of G. procumbens in cell cultures + influence the activation of CD8 T lymphocytes + (Fig. 2). The relative numbers of CD8 CD62L T cells in a control treatment were 4.04% and 3 absolute number of cells 2421.1x10 cells. Treatment with the G. procumbens ethanol extract showed the higher proliferation compared with control treatment. The numbers 3 of cells in dose 1 treatment were 4584.6x10 cells 3 (4.70%), dose 2 were 7767.5x10 cells (3.38%), 3 and dose 3 were 3518.2x10 cells (2.93%). Same + with CD4 T cells, a dose 2 also showed the + highest proliferation ability of the CD8 T cells. Extract of G. procumbens cell culture also + + affect proliferation of CD8 CD62L T cell. The + + relative number of CD8 CD62L T cells in the control treatment were 2.07% and the absolute 3 number were 404x10 cells. The dose 0.1 µg/ml treatment showed the relative number of cells 3 3.31% and the absolute number were 615.5x10 cells. At a dose 1 µg/ml, the relative number of 3 cells 2.63% was as many 1029.1x10 cells. While at a dose 10 µg/ml, the relative number of cells 3 1.95% was as many 292.8x10 cells. These results + + indicated that proliferation of CD8 CD62L T cell was higher in the treatment with the G. procumbens extract dose 0.1 µg/ml and dose 1 µg/ml, while the dose 10 µg/ml cell number decreased compared to the control. + The increase in CD8 T cells was affected by the increase in cytokines IL-2 and IFN that

ISSN. 2087-2852 E-ISSN. 2338-1655

Immunomodulator of ethanol extract of G. procumbens leaves to M. musculus immune (Dwijayanti and Rifa’i) induced flavonoid and saponin in G. procumbens extracts as described previously. The increase of + + CD8 T cells were also influenced by CD4 T cells + that activated because CD4 T cells that were activated would be differentiated into Th1 that producing IL 2 and IFN- [12]. Dose 10 µg/ml + provides suppressive effect on CD8 T cells because this dose also has suppressive effects on + + CD4 T cells. IL-2 that produced by CD4 T cells as addition used for the up-regulation itself and + itwas also used by CD8 T cells as a stimulant for + proliferation. It is explained further that the CD8 T cells have a higher affinity than the affinity of + CD4 T cells in the use of IL-2 [12].

Figure 2.

Population of CD8+CD62L- and CD8+CD62L+ T Cells: A. Relative number; B. Absolute number.

CONCLUSION We concluded that the extract of G. procumbens can increase the proliferation of + + + + CD4 CD62L , CD4 CD62L , CD8 CD62L , and + + CD8 CD62L T cells. Dose of 1 µg/ml showed the highest cell activation capability compared to the dose of 0.1 µg/ml and 10µg/ml. Dose of 10 µg/ml + was given suppressants effect to CD4 CD62L , + + + + + CD4 CD62L , CD8 CD62L , and CD8 CD62L T cells because at that dose the number of cells was decreasing.


13

ACKNOWLEDGEMENT The first author thanks to Muhaimin Rifa`i, S.Si., PhD.Med.Sc., as the supervisor of this research. We also thank Widodo, S.Si., M.Si, PhD.Med.Sc., DR. Ir. Sasmito Djati, MS and Drs. Aris Soewondo, M.Si. as the reviewers of this paper. Thank is also delivered to Indriya R., Dewi S., S.Si., M.Si., Ahmad Sony, S.Si., M.Si., Bambang P., S.Si., and Uwais A.Q., S.Si. for supporting me in this research. REFERENCES [1] Anderson, K.P., E.H. Fennie, and T. Yilmo. 1989. Enhancement of a secondary antibody response to vesicular stomatitis virus G protein by IFN- γ treatment at primary immunization. Immun. 140. 35993604. [2] Cao, M., O. Sasaki, A. Yamada, and J. Imanishi. 1992. Enhancement of the protective effect of inactivated influenza virus vaccine by Cytokines. Vaccine. 10. 238 – 242. [3] Lowenthal, J.W., B. Lambrecht, T.P. van Den Berg, M.E. Andrew, A.D.G. Strom, and A.G. D. Bean. 2000. Avian Cytokines-the natural approach to therapeutics. Developmental and Comparative Immunology. 24. 355-365. [4] Nugroho, Y. A., B. Wahjoedi, and A. Chozin. 1997. Information of Pharmacology and Phytochemical research of Gynura procumbens (Lour.) Merr. Proceeding of National Seminar of Indonesian Herbal XII. [5] Rifa’i, M. 2011. Imunology and Bioregulator. UB Press. Malang. [6] Middleton, E., C. Kandaswami, and T.C. Theoharides. 2000. The effects of plant Flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. 52. 673751. [7] Fathir, A. 2013. Immunity modulation of Mus musculus infected with Salmonella thypi after the induction of Moringa oleifera Lam. leaves extract. Master Thesis. Department of Biology, University of Brawijaya. Malang. [8] Cheeke, P.R. 2000. Actual and potential aplication of Yucca schidigere and Quillaja saponaria Saponin in human and animal nutrition. Anim Sci. 77. 1-10. [9] Lee, Y.H., Y.Ishida, M. Rifa’i, Z. Shi, K. Isobe, and H. Suzuki. 2008. Essential role of CD8+CD122+ regulatory T cells in the ISSN. 2087-2852 E-ISSN. 2338-1655

Immunomodulator of ethanol extract of G. procumbens leaves to M. musculus immune (Dwijayanti and Rifa’i)

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recovery from experimental autoimmune encephalomyelitis. Immunology. 180. 825832. [10] Shi, Z., M. Rifa’i, Y.H. Lee, H. Shiku, K. Isobe, and H. Suzuki. 2008. Importance of CD80/CD86–CD28 interactions in the recognition of target cells by CD8+CD122+ regulatory T cells. Immunology. 124. 121128. [11] Schroeter H., C. Boyd, J.P.E. Spencer, R.J. Williams, E. Cadenas, C. Rice-Evans. 2002. MAPK Signaling in neuro degeneration: influences of Flavonoids and of nitric oxide. Neurobiology of Aging. 23. 861–880. [12] Rifa’i, M., Z. Shi, S.Y. Zhang, Y.H. Lee, H. Shiku, K. Isobe, and H. Suzuki. 2008. CD8+CD12+ regulatory T cells recognize activated T cells via conventional MHC class I–αβTCR interaction and become IL-10producing active regulatory cells. International immunology. 20. 937-947.


ISSN. 2087-2852 E-ISSN. 2338-1655

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ISOLASI DAN UJI ANTIBAKTERI BATANG SAMBUNG NYAWA (Gynura procumbens Lour) UMUR PANEN 1, 4 DAN 7 BULAN (Isolation And Antibacteria Testing of Sambung Nyawa (Gynura procumbens Lour) Stick Age 1, 4 And 7 Months Harvesting)

1,2 3 4

Aryanti1, Harsojo2, Yefni Syafria3 Tri Muji Ermayanti4 Pusat Aplikasi Teknologi Isotop dan Radiasi, BATAN Institut Sains dan Teknologi Nasional Pusat Penelitian Bioteknologi, LIPI Abstract

The research of isolation of sambung nyawa (Gynura procumbens Lour) stick and its anti bacteria activities on Escherichia coli, Staphylococcus aureus and Samonella typhimurium have been conducted on their 1, 4 and 7 months harvesting. Sambung nyawa stick dried and extracted by ethanol to obtain the ethanol extract, and then tested to three kind of bacteria with the concentration of 100 and 50 mg/l, and the compound group testing also have been done. Ethanol extract separated by column chromatography using chloroform, ethyl acetate and methanol as solvent to obtain the active compound. The fraction of column chromatography tested again on the bacteria. The result showed that sambung nyawa stick contain of antibacterial compound was very active on S.aureus compared two others with the clear zone was 13.5 mm same as size of positive control (amoxilin antibiotic). The harvesting age of 7 month more active as antibacterial and the compound group testing positive than age 1 and 4 months. Keywords: Isolation, Antibacteria, Gynura procumbens Lour Naskah diterima tanggal Mei 2006, disetujui dimuat tanggal Juli 2006 Alamat koresponden: Gedung BPPT Lantai 2, Jl. M.H. Thamrin No. 8 Jakarta 10430 PENDAHULUAN Penyakit infeksi merupakan penyakit yang banyak diderita masyarakat Indonesia sejaka dulu, infeksi akibat bakteri S.aureus, E.coli, S.typhii, Vibrio cholera menimbulkan infeksi pada usus, sedang pada kulit seperti S.aureus dan Pseudomonas auruginosa. Berbagai laporan menyatakan bahwa pengobatan penyakit infeksi pada masyarakat Indonesia banyak yang mengandalkan tanaman sebagai obat. Rimpang kunyit sangat sering digunakan untuk mengobati penyakit infeksi ini (1) demikian juga tanaman sambung nyawa Tanaman sambung nyawa (Gynura procumbens Lour) adalah tanaman semak semusim merupakan terna menahun termasuk famili Compositae. Tanaman ini sering rancu dengan tanaman daun dewa (Gynura pseudochina DC), namun kedua tanaman ini dapat dibedakan dari umbi yang hanya terdapat pada tanaman daun dewa. Sambung nyawa telah dimanfaatkan penduduk Indonesia sebagai obat alami untuk penyembuhan penyakit limpa, ginjal, kulit, menurunkan gula darah, menurunkan tekanan darah antikarsinogenik dan antibiotic. Para dokter Klinik Herbal Karyasari telah merekomendasikan penggunaan daun sambung nyawa untuk pengobatan hipertensi, kolesterol, diabetes dan gangguan lambung. Menurut Winarto fraksi butanol daun sambung nyawa dapat menurunkan kadar gula darah tikus dengan sangat signifikan, demikian juga ekstrak etanol dapat menurunkan hingga 23 % sel kanker pada mencit dan pada konsentrasi 82,8 ìg/ml dapat menghambat

pertumbuhan sel vero dan mieloma. Menurut Sudarto (3), minyak atsiri sambung nyawa aktif membunuh bakteri Staphylococcus aureus. Bakteri S.aureus merupakan bakteri pathogen yang dapat menyebabkan infeksi impetigo dan furuncle yang cukup serius. Aktifnya tanaman sambung nyawa menghambat pertumbuhan bakteri maupun mengobati berbagai penyakit menandakan bahwa tanaman ini mengandung berbagai senyawa metabolit. Senyawa metabolit yang terkandung pada bagian daun sambung nyawa berupa flavonoid, glikosida kuersetin, saponin, steroid dan minyak atsiri. Senyawa flavonoid dan tannin merupakan senyawa yang banyak bersifat sebagai antibakteri. Beberapa senyawa flavonoid pada tanaman daun sendok (Plantago major) telah diketahui aktif sebagai antibakteri. Flavonoid yang terdapat pada ekstrak etanol tanaman leunca (Solanum ningrum) aktif terhadap bakteri E.coli dan S.aureus, demikian juga flavonoid scutellin dari tanaman Scutellin baicalensis berkhasiat sebagai antibakteri E.coli, S.aureus dan B. subtilis (4). Kandungan senyawa pada bagian batang dan akar belum banyak dilakukan penelitian. Kandungan senyawa obat yang terdapat pada suatu tanaman sangat dipengaruhi oleh lingkungan seperti struktur tanah, pengaruh cuaca, cara bercocok tanam dan waktu panen tanaman. Tanaman sambung nyawa umumnya dapat dipanen setelah umur 4 bulan, kemudian dilakukan peremejaan hingga dapat dipanen selama 4 tahun. Penelitian ini bertujuan untuk mendapatkan fraksi

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Tabel I. Zona hambat (mm) berbagai bakteri oleh ekstrak etanol batang sambung nyawa beberapa umur panen pada konsentrasi 100 dan 50 mg/ml Bakteri uji E.coli S.aureus S.typhimurium

Umur 1 bulan 100 mg/ml 50 mg/ml 7,5 7 8,5 8,5 3 0

Umur 4 bulan 100 mg/ml 50 mg/ml 8,5 8,5 11 9 8,5 5,5

Umur 7 bulan 100 mg/ml 50 mg/ml 9 8,5 12 10,5 9,5 8

Tabel II. Uji golongan senyawa kimia paa batang sambung nyawa umur 1,4 dan 7 bulan Hasil uji pada umur panen ( bulan ) 1 4 1. Alkaloid + ++ 2. Flavonoid + ++ 3. Sapoin + ++ 4. Steroid/terpen ++ Keterangan : + = warna agak jelas, ++ = warna jelas, +++ = warna sangat jelas No.

Golongan senyawa

aktif antibakteri dari batang sambung nyawa yang dipanen umur 1, 4 dan 7 bulan serta mendapatkan umur panen terbaik pada tanaman sambung nyawa yang di tanam di lapang. METODE Bahan. Penelitian ini menggunakan bagian batang tanaman sambung nyawa (Gynura procumbens Lour.) umur panen 1, 4 dan 7 bulan diperoleh dari kebun percobaan Bioteknologi – LIPI, Cibinong. Bakteri yang diuji adalah Escherichia coli, Staphylococcus aureus dan Salmonella typhimurium dari PATIR – BATAN. Bahan kimia yang digunakan berkualitas pro analitik dari Merck Darmstad Jerman. Ekstraksi dan Isolasi Senyawa Bioaktif. Batang sambung nyawa yang telah kering dihaluskan dan diekstrak dengan dengan etanol. Ekstrak etanol kemudian dipisahkan menggunakan kromatografi kolom dengan pelarut klroform/etil asetat, etil asetat, etil asetat/methanol dan metanol sebagai fasa gerak, sedang silikagel digunakan sebagai fasa diam. Hasil pemisahan kolom ditampung dalam botol vial dan dilakukan uji kromatografi lapis tipis untuk mendapatkan fraksi yang mempunyai spot yang sama digabung menjadi satu fraksi dengan menggunakan uap amnoia untuk uji adanya flavonoid. Uji Aktivitas Antibakteri Bakteri E.coli, S. aureus dan S.typhimurium dengan konsentrasi 10 6 dibiakkan pada media Muller Hinton Agar ( MHA ). Kemudian kertas cakram steril ditetesi larutan uji konsentrasi 100 dan 50 mg/ml dan dibiarkan sisa pelarut menguap hingga kering, lalu kertas cakram diletakkan di atas bakteri yang telah berada dalam cawan petri. Cawan selanjutnya diinkubasi pada suhu 370C selama 24 jam dan keesokan harinya diamati lebar zona hambat yang diakibatkan oleh ekstrak etanol maupun fraksi hasil kromatografi kolom. Untuk control negative

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7 +++ +++ +++ +++

digunakan pelarut methanol, sedang control positif yaitu antibiotic amoxilin dengan konsentrasi 2,083 mg/ml. Uji Golongan Senyawa Kimia Golongan senyawa alkaloid dilakukan dengan menggunakan pereaksi Dragendroff dan Mayer yang memberikan endapan merah bata dan endapan putih yang menunjukkan adanya alkaloid. Adanya senyawa flavonoid terbentuknya warna merah jingga oleh pereaksi deteksi flavonoid, sedang adanya saponin menimbulkan busa yang stabil. Deteksi adanya steroid dan terpen ditunjukkan dengan terbentuknya warna merah-hijau-violet-biru dengan pereaksi eter dan asetat anhidrat HASIL DAN PEMBAHASAN Hasil penelitian isolasi dan uji daya antibakteri dari ekstrak batang sambung nyawa umur panen 1, 4 dan 7 bulan dapat dilihat pada Tabel 1. Dari tabel terlihat bahwa ekstrak etanol umur panen 1 bulan tidak memberikan zona hambat yang cukup nyata terhadap bakteri S.typhimurium dan setelah batang berumur 7 bulan zona hambat yang terbentuk cukup baik. Bila dibandingkan diantara ketiga bakteri, S.aureus sedikit sensitive dengan ekstrak batang sambung nyawa meskipun perbedaanya tidak terlalu signifikan. Umur panen 4 dan 7 memperlihatkan sifat antibakteri yang hampir sama. Uji golongan senyawa kimia yang terdapat pada batang sambung nyawa dapat dilihat pada Tabel II.Data menujukkan bahwa makin tua umur panen, makin kuat warna identifikasi golongan senyawa yang terdapat pada bagian batang sambung nyawa. Hal ini dapat diasumsikan bahwa makin tua umur tanaman makin terakumulasi senyawa bioaktif yang terdapat pada suatu tanaman. Menurut Paniego and Giuletti (5) senyawa artemisinin yang terdapat pada tanaman Artemisia annua makin meningkat kandungannya pada waktu tanaman akan dan sedang berbunga. Tanaman sambung nyawa umumnya mulai dipanen atau dipetik daunnya untuk obat herba semenjak tanaman berumur 4 bulan. Dari data diatas dapat

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Tabel III. Uji daya antibakteri fraksi 3 batang sambung nyawa umur panen 1, 4 dan 7 bulan terhadap tiga jenis bakteri

Bakteri E .coli

S.aureus

S.typhimurium

Umur panen ( bulan ) 1 4 7 1 4 7 1 4 7

Zona hambat ( mm ) pada konsentrasi 100 mg/ml 7,5 8,5 9,5 9 10,5 13,5 7 7 8

dikatan bahwa tanaman umur 4 bulan sudah dapat dimanfaatkan karena kandungan senyawa metabolit sekunder yang umumnya aktif terhadap berbagai penyakit dan keaktivan batang terhadap tiga jenis bakteri juga tidak terlalu berbeda dengan umur 7 bulan, maka tanaman umur 4 bulan ini sudah dapat dipanen untuk pengobatan. Hasil pemisahan ekstrak etanol dengan kromatogarafi menghasilkan 5 fraksi, fraksi 2 dan 3 memberikan spot adanya senyawa flavonoid, yang merupakan senyawa dominant sebagai antibakteri, nilai fraksi retensi spot dengan pelarut kloroform/etil asetat yakni 0,53 dan 0,67 bercak berwarna hijau kekuningan dengan uap ammonia. Uji fraksi 3 terhadap ketiga jenis bakteri E.coli, S.aureus dan S. typhimurium dapat dilihat pada Tabel III. Dari tabel menujukkan bahwa fraksi 3 dari batang sambung nyawa umur panen 7 bulan konsentrasi 100 mg/ ml memberikan efek yang sama dengan antibiotic amoksilin konsentrasi 2,083 mg/ml terhadap S.aureus. Antibiotik amoksilin merupakan antibiotic dengan daya kerja broad spectrum yang aktif terhadap bakteri gram bakteri maupun gram positif. Zona hambat yang terbetuk di sekeliling kertas cakram sangat jernih sehingga zona hambat akibat fraksi batang sambung nyawa ini memberikan efek zona hambat total. Hasil uji aktivitas antibakteri pada fraksi ini dapat dikatakan sebagai antibakteri kuat karena menurut Davis Stout ketentuan kekuatan suatu zat uji terhadap bakteri bila ukuran zona hambat 20 mm atau lebih disebut sebagai sangat kuat, bila 10 mm – 20 mm kuat dan 5 – 10 mm dikatakan sebagai zat uji bersifat sedang dan dibawah 5 mm bersifat lemah. Menurut Tomimori et.al ( 6 ) senyawa flavonoid scutellin dari tanaman Scutellin baicalensis aktif terhadap bakteri E.coli, S.aureus dan B.subtilis, demikian juga flavonoid pada ekstrak etanol leunca ( Solanum ningrum ) berkhasiat sebagai antibakteri E. coli dan S.aureus. Data fraksi 3 menujukkan bahwa mempunyai aktivitas kuat terhadap bakteri S. aureus, sedang dua bakteri lainnya bersifat sedang. Adanya warna hijau kekuningan diperkirakan flavonoid jenis flavon dan flavonol, senyawa

zona hambat ( mm ) pada konsentrasi 50 mg/ml 7,5 6 8 7,5 8 10,5 7 6 6

Zona hambat ( mm ) pada kontrol positif ( amoksilin ) 18

13,5

15,5

ini mampu membentuk kompleks dengan protein pada dinding sel, sehingga aktivitas sel terganggu dan menimbulkan kematian pada bakteri. Menurut Tan et.al dan Zheng ( 7, 8 ) senyawa terpen dan flavonoid yang terdapat pada tanaman A.annua aktif sebagai antibakteri KESIMPULAN Dari hasil penelitian ini dapat disimpulkan bahwa tanaman sambung nyawa aktif sebagai anti bakteri, umur panen 4 bulan tanaman sudah dapat dimanfaatkan sebagai antibakteri. Tanaman ini lebih aktif terhadap bakteri S.aureus daripada E.coli dan S.typhimurium. DAFTAR RUJUKAN 1. Dzulkarnaian, B., Sundari D dan Chozin A. 1996. Tanaman obat bersifat antibakteri di Indonesia. Cermin Dunia Kedokteran No. 110 : 35 48 2. Winarto, W.P dan Tim Karyasari. 2003. Sambung Nyawa Budidaya dan Pemanfaatan untuk Obat. Penerbit Swadaya, Jakarta. 3. Sudarto, B. 1990. Studi farmakognosi tumbuhan Gynura procumbens Lour. Tesis, UGM Jogyakarta. p. 7 - 10 4. DePadua, L.S., N. Bunyapraphatsara and R.H.M.J. Lemmens. 1999. Plants Resources of South-East Asia, Bogor, Indonesia 5. Paniego, N.B., and A.M. Giuletti 1994. Artemisia annua : dedifferentiated and differentiated cultures. Plant Cell Tissue & Organ Culture. 36 : 163 – 168. 6. Tomimori, T., Miyaichi Y, Imoto Y, Kiu H, and Namba T 1988. Sudies on the falvonoid constituents of the aerial part of Scutellaria discolor. Chemical and Pharmaceutical Buletin 36 (9) : 3654 – 3658. 7. Zheng, G.Q. 1994. Cytotoxic of terpenoid and flavonoids from Artemisia annua. Planta Medica. 60 : 54 – 57. 8. Tan, R.X., Zheng W.F, and Tang H.Q. 1998. Biologically active substances from the genus Artemisia. Planta Medica 64 : 295 – 302.

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PRODUCING THE JELLY MADE OF SAMBUNG NYAWA AND STEVIA LEAVES TO DECREASE THE GLUCOSE LEVEL IN THE BLOOD Wari Tinting Hastuti, Hasna Irfantiningtyas Sari, Athika Wirastiti, Ratnasari, dan Saptaka Trihantoro Mahasiswa FMIPA Universitas Negeri Yogyakarta Abstract This research was aimed to prove whether the jelly of “Sambung Nyawa” leaves and stevia could lower the blood glucose level as an alternative option for people with diabetes. The method used in this research was a method of diabetes test glucose monohydrate. In this method glucose monohydrate was induced. The subject in this research was the jelly of “Sambung Nyawa” made of “Sambung Nyawa” leaves as the basic material added with a natural sweetener made of stevia leaves. The objects of this research were Wistar mice. The products of “Sambung Nyawa” jelly and the stevia were given to Wistar mice that had been induced by glucose monohydrate. The rats were divided into three groups. The first served as a control group, the second group was given the same dosage variation, and the third group was a different dosage variation. Based on the results of this research, it was found out that there was a decrease in the blood sugar levels in the mice that had been induced by glucose monohydrate after being given the product of “Sambung Nyawa” jelly and stevia. It can be seen from the data obtained in the second group. In the group three mice were induced with glucose monohydrate dilution of 1.67 grams with 3 ml of blood sugar levels with an average of 82.67 mg / dl blood glucose levels decreased to 90 mg / dl. In the third group the data could not be retrieved because the mice died before the sugar level was checked after being induced with glucose monohydrate. Based on the results of experimental data it can be concluded that the products of “Sambung Nyawa” jelly and stevia could lower the blood glucose levels. The blood glucose levels were measured by using a glucometer. Keywords: “Sambung Nyawa” leaf, stevia leaf, Wistar mice, blood glucose levels

PENDAHULUAN Saat ini, penyakit diabetes masih menjadi permasalahan karena setiap tahunnya penderita diabetes semakin me-

83

ngalami peningkatan. Berdasarkan data yang diperoleh dari Organisasi Kesehatan Dunia (World Health Organitation/ WHO), diperkirakan jumlah penyandang

84

diabetes di Indonesia akan melonjak drastis. Pada tahun 2000 jumlah penderita diabetes sekitar 8,4 juta dan diprediksi akan meningkat menjadi 21,3 juta di tahun 2030. Indonesia menduduki urutan ke-4 setelah Amerika Serikat, Tiongkok dan India (Perkumpulan Endokrinologi Indonesia, 2006). Penyakit diabetes memiliki beberapa tipe salah satunya adalah diabetes mellitus. Diabetes mellitus (DM) adalah penyakit metabolik pankreas yang ditandai dengan keadaan hiperglikemia atau meningkatnya kadar gula darah (Powers, Alvin C, 2005). Penderita diabetes tidak bisa memproduksi insulin dalam jumlah yang cukup, sehingga terjadi kelebihan gula di dalam tubuh. Kelebihan gula yang kronis di dalam darah (hiperglikemia) tersebut akan menjadi racun bagi tubuh. Cara mengobati diabetes salah satunya dengan mengembalikan kerja pankreas seperti sedia kala. Hal ini membutuhkan biaya yang cukup mahal. Untuk itu perlu langkah awal mencegah penyakit diabetes di Indonesia semakin bertambah. Sebagai Negara yang kaya akan floranya, Indonesia memiliki banyak tanaman herbal yang dapat dimanfaatkan sebagai obat herbal seperti daun sambung nyawa (Gynuraprocumbens back) yang memiliki senyawa flavonoid. Dengan karya yang inofatif, daun sambung nyawa dapat diolah menjadi produk pangan berupa Jelly. Produk maPELITA, Volume VIII, Nomor 1, April 2013

Universitas Negeri Yogyakarta

kanan jelly merupakan makanan yang dapat dikonsumsi oleh semua kalangan umur. Selain itu, jelly sambung nyawa diduga dapat menurunkan kadar gula darah, dilihat dari adanya kandungan flavonoid. Seperti pembuatan jelly pada umumnya, jelly berbahan dasar sambung nyawa ini mudah dilakukan dengan cara yang sederhana. Tujuan dalam penelitian ini dapat membuktikan asumsi bahwa daun sambung nyawa dapat menurunkan kadar gula darah dan mengetahui cara pembuatan jelly daun sambung nyawa. Untuk mengurangi rasa ketir dari daun sambung nyawa, maka perlu ditambahkan daun stevia sebagai pemanis alami. Selain itu, untuk membuktikan jelly sambung nyawa dapat menurunkan kadar gula darah, maka dilakukan eksperimen pada tikus putih wistar jantan yang diinduksi dengan glukosa monohidrat. Pemilihan tikus wistar sebagai objek penelitian karena memiliki susunan organ yang hampir mirip dengan manusia dan biasa digunakan dalam objek penelitian.

KAJIAN PUSTAKA Diabetes Mellitus Diabetes Mellitus merupakan gangguan metabolik klinis yang tidak dapat disembuhkan, tetapi dapat dikontrol, yang dikarakteristikkan dengan hiperglikemia karena defisiensi insulin atau ketidakadekuatan insulin. Diabetes melli-


tus dengan hiperglikemia menyebabkan meningkatnya stres oksidatif dan pertahanan antioksidan disturbance (Barbara Engram, 1999).

Sambung Nyawa Di Indonesia, daun sambung nyawa memiliki beberapa nama daerah seperti; daun dewa (Melayu) (Heyne, 1987; Wijayakusuma et al., 1992), sambung nyawa dan ngokilo (Jawa) (Thomas, 1989). Tanaman Gynuraprocumbens berbentuk perdu tegak bila masih muda dan dapat merambat setelah cukup tua. Bila daunnya diremas menghasilkan bau aromatis. Batangnya segi empat beruas-ruas, panjang ruas dari pangkal sampai ke ujung semakin pendek, ruas berwarna hijau dengan bercak ungu. Daun tunggal bentuk elips memanjang atau bulat telur terbalik tersebar, tepi daun bertoreh dan berambut halus. Tangkai daun panjang ½-3 ½ cm, helaian daun panjang 3 ½-12 ½ cm, dan lebar 1- 5 ½ cm. Helaian daun bagian atas berwarna hijau sedangkan bagian bawah berwarna hijau muda dan mengkilat. Kedua permukaan daun berambut pendek. Tulang daun menyirip dan menonjol pada permukaan daun bagian bawah. Pada tiap pangkal ruas terdapat tunas kecil berwarna hijau kekuningan. Tumbuhan ini mempunyai bunga bongkol, di dalam bongkol terdapat bunga tabung berwarna kuning oranye coklat kemerahan panjang 1-1 ½ cm,

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berbau tidak enak. Tiap tangkai daun dan helai daunnya mempunyai banyak sel kelenjar minyak (Perry, 1980; Van Steenis, 1975; Backer and Van den Brink, 1965; Sodoadisewoyo, 1953). Daun tanaman Gynuraprocumbens mengandung senyawa flavonoid, sterol tak jenuh, triterpen, polifenol dan minyak atsiri (Pramono dan Sudarto, 1985). Hasil penelitian lain melaporkan bahwa tumbuhan ini mengandung senyawa flavonoid, tanin, saponin, steroid, triterpenoid, asam klorogenat, asam kafeat, asam vanilat, asam para kumarat, asam p-hidroksi benzoat (Suganda et al., 1988), asparaginase (Mulyadi, 1989). Sedangkan hasil analisis kualitatif dengan metode kromatografi lapis tipis yang dilakukan Sudarsono et al. (2002) mendeteksi adanya sterol, triterpen, senyawa fenolik, polifenol, dan minyak atsiri. Sugiyanto et al. (2003) juga menyatakan berdasarkan penelitian yang dilakukan bahwa dalam fraksi polar etanol daun tanaman Gynuraprocumbens terdapat tiga flavonoid golongan flavon dan flavonol. Gynuraprocumbens mengandung sterols, glikosida sterol, quercetin, kaempferol-3-O-neohesperidosida, kaempferol-3-glukosida, quercetin-3-rhamnosyl(16)galaktosida, quercetin-3-O-rhamnosyl(1-6)glukosida. Stevia

Stevia termasuk dalam jenis keluarga bunga matahari atau Asteraceae.

Producing the Jelly Made of Sambung Nyawa and Stevia Leaves to Decrease the Glucose Level in the Blood

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Banyak tumbuh di daerah subtropis dan tropis di wilayah barat Amerika Utara dan Amerika Selatan. Spesies yang satu ini umumnya dikenal sebagai sweetleaf, daun manis, sugarleaf atau stevia. Saat ini stevia telah dibudidayakan dan digunakan dalam makanan di Asia Timur termasuk Cina, Korea, Taiwan, Thailand dan Malaysia. Bentuk daunnya seperti kemangi, berukuran kecil dan berwarna hujau. Daunnya bertumpuk-tumpuk dalam satu batang, berbiji dan bertunas. Rasa daun ini 300 kali dari gula, karena itu daun ini dapat dijadikan pemanis pengganti gula. Untuk mengolah daun ini biasanya melalui teknik kristalisasi menggunakan etanol atau metanol sebagai pelarutnya. Pada penelitian yang dilakukan tahun 2009, ditemukan bahwa Stevia memiliki senyawa anti-hiperglikemia, anti-hipertensi, anti-tumor, anti-diare, diuretik dan imunomodulator (www.food.detik.com).

Tikus Wistar Tikus Wistar merupakan tikus albino spesies Rattus norvegicus. Jenis galur ini dikembangkan di Institut Wistar pada tahun 1906 untuk digunakan dalam biologi dan penelitian medis. Jenis tikus ini galur tikus pertama yang dikembangkan sebagai model organisme. Tikus Wistar saat ini menjadi salah satu yang strain tikus paling populer yang digunakan untuk penelitian labora-

PELITA, Volume VIII, Nomor 1, April 2013


torium. Ciri tikus ini adalah mempunyai kepala lebar, telinga panjang, dan memiliki ekor panjang (tidak melebihi panjang tubuhnya). Galur tikus Sprague Dawley dan Long-Evans dikembangkan dari tikus galus Wistar. Tikus Wistar lebih aktif daripada jenis lain seperti tikus Sprague dawley (www.dokterternak.wordpress.com). METODE PENELITIAN Penelitian tentang produk jelly sambung nyawa dan stevia sebagai penurun kadar gula darah dilaksanakan mulai bulan Juni sampai Juli di rumah peneliti dan Laboratorium Chem-Mix Pratama Kretek Jambidan Banguntapan Bantul Yogyakarta. Jenis penelitian yang digunakan adalah penelitian eksperimen dengan memanfaatkan tanaman herbal daun sambung nyawa (Gyanura procumbens) dan stevia. Dengan menggunakan metode uji diabetes induksi glukosa monohidrat serta uji laboraturium secara kuantitatif. Dalam metode ini, subjek dalam penelitian adalah jelly sambung nyawa dengan pemanis alami daun stevia. Objek dalam penelitian ini adalah tikus wistar putih jantan. HASIL DAN PEMBAHASAN Penelitian ini dilaksanakan melalui empat tahapan yaitu: (1) tahap pembuatan jelly, (2) tahap penginduksian tikus, (3) tahap pemberian jelly sambung


nyawa pada tikus, (4) tahap penelitian sampel jelly sambung nyawa. Metode ini menggunakan uji diabetes induksi glukosa monohidrat menggunakan tikus putih jantan wistar yang berusia dua bulan dengan rata-rata berat badan 150 gram. Metode kedua dengan uji kuantitatif pada sampel jelly sambung nyawa.

Tahap Pembuatan Jelly Pembuatan jelly sambung nyawa dan stevia menggunakan bahan dasar berupa daun sambung nyawa sebanyak 12 lembar dan daun stevia kering satu sendok teh yang disedu dengan air hangat. Daun sambung nyawa dihaluskan kemudian diambil sarinya dengan diperas menggunakan air sebanyak 80 ml, mencampurkan 60 ml seduhan daun stevia dan memasak campuran tersebut selama 10 menit. Menambahkan sedikit nutria jell untuk memadatkan makanan. Pembuatan jelly sambung nyawa ini cukup sederhana dan mudah dilakukan.

Penginduksian pada Tikus Tikus putih jantan wistar sebelum digunakan untuk percobaan dikondisikan dulu dengan lingkungannya selama satu minggu.Kemudian dicek gula darahnya dengan glukometer (ONETOUCH Select SimpleTM) selanjutnya tikus diinduksi dengan glukosa monohidrat.Sebanyak sepuluh tikus diuji dengan perlakuan yang

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berbeda menghasilkan data sebagai berikut. Kelompok I : terdiri dari empat tikus tanpa glukosa monohidrat. Kelompok II : terdiri dari tiga tikus dengan dosis glukosa monohidrat 1,67 gram yang dilarutkan dengan 3 ml air. Kelompok III : tikus diinduksi dengan tiga variasi glukosa monohidrat.  Tikus variasi pertama dengan dosis glukosa monohidrat 5,68 gram dilarutkan dalam 7 ml air.  Tikus variasi dua dengan dosis glukosa monohidrat 2,46 gram dilarutkan dalam 3 ml air.  Tikus variasi tiga dengan dosis glikosa monohidrat 2,28 gram dilarutkan dalam 4 ml air.

Kadar Gula Darah Sebelum Penyuntikan (Diinduksi Glukosa Monohidrat) Kelompok I

Kadar Gula Darah (mg/dl) 83

83

82

83

Rerata

82.75


88

Kelompok II

Kadar Gula Darah (mg/dl) 83

82

III


Rerata

83

82.666 66667

83

83

83

83

Sebagian besar tikus memiliki gula darah yang rendah sebelum diinduksi glukosa monohidrat.Satu minggu setelah penginduksian tikus dicek kembali gula darahnya untuk memastikan bahwa tikus memiliki gula darah yang tinggi. Hasil percobaannya sebagai berikut. Kadar Gula Darah Satu Minggu Setelah Penyuntikan Kelompok I

II III

Kadar Gula Darah Tikus (mg/dl)

Rerata

Tidak disuntik

Tidak disuntik

Tidak disuntik

0

196

132.6666667

Mati

0

109

Mati

Mati

Tahap Pemberian Jelly Daun Sambung Nyawa Pada tahap ini tikus putih diberi makan jelly sambung nyawa selama tiga kali. Kadar Gula Darah Setelah Pemberian Produk Jelly

Tidak disuntik 93

Satu minggu setelah diinduksi glukosa monohidrat pada tikus kelompok I tidak terjadi perubahan kadar gula darah karena tidak diinduksi glukosa monohidrat dan kadar gula darah tikus tidak diuji kembali. Tikus kelompok II terjadi perubahan gula darah untuk tikus pertama naik menjadi 93 mg/dl, tikus kedua naik menjadi 109 mg/dl, dan tikus ketiga naik menjadi 196 mg/dl. Sedangkan untuk tikus kelompok III tidak terjadi perubahan karena beberapa faktor tikus ini mati, hal ini diduga kuat karena variansi glukosa monohidrat yang diberikan terlalu banyak, sehingga daya tahan tubuh tikus tidak kuat dan mengalami diabetes. Selain itu, dapat dilihat tikus yang mati mengalami pembusukan pada bagian belakang tubuhnya.

PELITA, Volume VIII, Nomor 1, April 2013

Kelompok I

Kadar gula darah Tikus (mg/dl)

Rerata

Tidak disuntik Tidak disuntik

0

Tidak disuntik

Tidak disuntik


Kelompok II

III

Kadar gula darah Tikus (mg/dl)

Rerata

83

90

Mati

0

91

96

Mati

Mati

89

Pada kelompok pertama, tidak terjadi perubahan kadar gula karena tidak diberi perlakuan. Pada kelompok kedua terjadi penurunan kadar gula darah. Tikus pertama yang kadar gula darah setelah diinduksi sebesar 93 mg/dl turun menjadi 91 mg/dl setelah diberi jelly sambung nyawa. Tikus kedua yang kadar gula darah setelah diinduksi sebesar 109 mg/dl turun menjadi 96 mg/dl setelah diberi jelly sambung nyawa. Tikus ketiga yang kadar gula darah setelah dinduksi sebesar 196 mg/dl turun menjadi 90 setelah diberi jelly sambung nyawa. Penurunan yang sangat signifikan ini diduga disebabkan karena kondisi tikus saat akan dicek kadar gula darahnya konNo 1

Kode Sample

Satu Sample Jelly Sambung Nyawa

disinya tegang sehingga dapat mempengaruhi produksi hormon insulin. Dalam percobaan ini, dilakukan uji ulang pada sampel gula darah 91 mg/dl setelah diberikan produk jelly sambung nyawa kadar gula darahnya turun menjadi 82 ml/dl, sehingga mengalami penurunan sekitar 9 mg/dl. Hal tersebut membuktikan bahwa jelly sambung nyawa dapat menurunkan kadar gula dalam darah, kira-kira penurunnya sebesar 1-10 mg/dl. Pemberian jelly dari daun sambung nyawa dan stevia dilakukan selama satu minggu. Jelly sambung nyawa diberikan tiga kali dalam sehari dengan volume kira-kira 140 ml.

Penelitian Sampel Uji Jelly Daun Sambung Nyawa Uji makanan secara kuantitatif dilakukan di Laboratorium Chem-Mix Pratama (Kretek Jambidan Banguntapan Bantul Yogyakarta). Uji ini digunakan untuk mengetahui kadar gula total dan flavonoid dalam jelly sambung nyawa. Hasil penelitian yang didapatkan sebagai berikut.

Analisis

Gula Total Flavonoid

Sample jelly sambung nyawa yaitu 120 ml dan didapatkan kadar gula darah dalam sample ini rata-rata adalah

Ulangan 1 0,4807 %

69,8224 ppm

Ulangan 2 0,4884 %

71,1915 ppm

0,48455% artinya 0,48455 gram dalam 100 gram jelly memiliki massa 99,5116 (100-0,48455=99,515). Kandungan sen-


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yawa flavonoid dalam sample ini adalah 70.50695 ppm (mg/ml) sehingga produk ini memiliki kadar glukosa yang rendah dan senyawa flavonoid yang cukup banyak.

PENUTUP Simpulan  Berdasarkan hasil data percobaan dapat disimpulkan bahwa produk jelly sambung nyawa dan stevia dapat dikatakan sebagai antidiabet karena terjadi penurunan gula darah pada tikus yang telah diinduksi glukosa monohidrat setelah diberi produk jelly sambung nyawa dan stevia.  Cara pembuatan jelly sambung nyawa dan stevia dapat dilakukan seperti yang terlampir prosedur penelitian di atas.

Saran

Penelitian tentang produk jelly sambung nyawa dan stevia sebagai penurun kadar gula darah harus memperhatikan beberapa hal sebagai berikut.  Sebaiknya percobaan dilakukan di laboratorium hewan.  Alangkah baiknya perlu dilakukan penenlitian lebih lanjut DAFTAR PUSTAKA Abacker, C.A. dan Van Den Brink, R.C.B. 1965. Flora of Java (Spermatophytes Only), Vol II, N.V.P, 363-364, PELITA, Volume VIII, Nomor 1, April 2013


424-425. Noordhoff-Groningen, The Netherlands.

Maryani, Herti dan Suharmiati. 2004. Khasiat dan Manfaat Daun Sambung Nyawa dan Daun Dewa. Agromedia Pustaka.

Masdar, Huriatul , dkk. 16 April 2013. Diabetes Melitus. Retrivedfrom: http://repository.unri.ac.id:80/ha ndle/123456789/2920. 27 Juli 2013.

Meiyanto, E., Sugiyanto, dan Sudarto, B. 1997. Uji Antikarsinogenik dan Antimutagenik Preparat Tradisional Daun Gynura procumbens (Lour.) Merr. Fakultas Farmasi UGM, Prosiding Seminar Nasional Tumbuhan Obat Indonesia XII, 32.

Nindya Helvy Pramita. (n.d). 2013. Khasiat daun sambung nyawa (Gynura procumbens (Lour Merr). Retrived From:http://jamu.biologi.ub.ac.id/?page_id=784.

Perkumpulan Endokrinologi Indonesia. 2006. Konsensus Pengelolaan dan Pencegahan Diabetes Melitus Tipe 2 di Indonesia 2006. Jakarta.

Perry, L.M. 1980. The Medical Plants of East and Southeast Asia: Attributed


Properties and Uses, 94-95. London: The MIT Press.

Powers, Alvin C. “Diabetes Mellitus”. Harrison’s: Principles of Internal Medicine. 2005; 324 (16): 21522179.

Sudarsono, dkk. 2002. Tumbuhan Obat II, Hasil Penelitian, Sifat-sifat dan Penggunaan. 96-100. Pusat Studi Obat Tradisional. Universitas Gadjah Mada, Yogyakarta.

Suganda, A., Sudiro, I., dan Ganthina. 1988. Skrining Fitokimia dan Asam Fenolat Daun Dewa (Gynura procumbens (Luor) Merr), Simposium Penelitian Tumbuhan Obat III, Universitas Indonesia, Jakarta.

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Sugiyanto, Sudarto, dkk. 2003. Aktivitas Antikarsinogenik Senyawa yang Berasal dari Tumbuhan, Majalah Farmasi Indonesia, 14 (4), 216225.

Thomas, A.N.S. 1989. Tanaman Obat Tradisional. 120-121. Yogyakarta: Penerbit Kanisius.

Winarto dan Hakim, Luqman.18 April 2013, Diabetes Melitus, Retrived From :ttp://repository.unri.ac.id:80/handle/123456789/2920, 27 Juli 2013.

Sugiyanto, Sudarto, B., dan Meiyanto, E. 1993. Efek Penghambatan Karsinogenisitas Benzo(a)piren Oleh Preparat Tradisional Tanaman Gynura sp. dan identifikasi Awal Senyawa yang Berkhasiat. Laporan Penelitian P4M Ditjen Dikti, Fak. Farmasi UGM, Yogyakarta.


Indonesian Journal of Cancer Chemoprevention, 2011, 2(3): 274-280 ISSN: 2088 - 197

Selectivity of Ethyl Acetate Fraction of Gynura Procumbens on Colon Cancer and Breast Cancer Nunuk Aries Nurulita1),2) *, Edy Meiyanto3), Sugiyanto3),4) 1) Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Jawa Tengah, Graduate Program of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 3) Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 4) Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara,Yogyakarta

2)

Abstract Gynura procumbens is widely used as traditional remedy in South-East Asia. Gynura procumbens exhibites anti inflammatory, antioxidant, and reduced blood pressure activity. The aim of this study was to determine chromatographic profile of ethyl acetate fraction of Gynura procumbens (FEG) and to investigate its cytotoxic properties and selectivity to colon cancerand breast cancer cancer cells. The chromatographic profile of FEG was determined using HPTLC densitometric and HPLC. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the growth inhibitory effect of FEG on the growth of WiDr, MCF-7, and T47D cells. NIH3T3, a normal cells was used to determine the selectivity of FEG, which contained small amount of quercetin as identified from chromatographic profile both HPTLC and HPLC. FEG inhibited cell growth of WiDr, of MCF-7 and of T47D cells in time dependent manner. Quercetin affected cell growth inhibition approximately two fold higher at WiDr and MCF-7, whereas FEG had lower effect on T47D cell. Quercetin did not seem as the main active compound of FEG. At this study, FEG caused less inhibition on the growth of NIH3T3 cells than that of on all cell lines. Selectivity index (SI) of FEG on WiDr, MCF-7 and T47D were 4.97, 2.77 and 7.79 respectively. According to the datas obtained, FEG possesses moderate to high cytotoxicity properties on WiDr, MCF-7 and T47D cells. FEG demonstrates selective effect against cancer cells and reveals prospective properties as cancer chemoprevention agent. Keywords: Gynura procumbens, colon cancer, breast cancer, cytotoxicity, selectivity

INTRODUCTION Breast cancer and colon cancer are the two of the most five deadly cancer diseases together with lung cancer, stomach cancer and hepatocarcinoma. Approximately 1,2 million cases of colorectal cancer are expected to occur in 2007. While 1,3 million new cases of invasive breast cancer are expected in 2007 (Garcia et al., 2007). Breast cancer caused 519.000 death per year, while colon cancer causes 639.000 patient death per year (WHO, 2006). In Indonesia, breast cancer prevalence is the second range after cervix cancer and become the major caused on women mortality. Combination therapy using surgery, chemotherapy and radotherapy resulted dissatisfied achievement. Many cancer chemotherapeutic drugs present relatively poor selectivity for neoplastic cells. The failure of cancer chemotherapy partly is resulted from the low selectivity of anticancer drugs and the

unclear of molecular target on cancer. Drugs with low selectivity affect several side effect on patient. Research efforts have been carried on to overcome these problems by developing more effective and selective anticancer drugs through consideration of the molecular target and mechanisms. Plant bioactive compounds are normally cheaper and produce lower side effects respected to chemotherapy. Thus, the searching for active compounds extracted from plant is an important path of research in the discovery of novel anticancer candidates. Gynura procumbens (G. procumbens) is a medicinal plant with several biological activity related with anticancer. G. procumbens extract and its fractions exhibit high antioxidant properties shown by xantine oxidase inhibition and radical scavenging activity.

*Corresponding author email:[email protected]

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Nurulita et al. ISSN: 2088-01972

The uncontrolled production of radical oxygen species (ROS) and unbalance antioxidant protective system have an important part on cancer initiation and development (Rosidah et al., 2008). These natural antioxidants may become a beneficial agent to be developed as chemoprevention substance for cancer disease. G. procumbens inhibited carcinogenesis process of lung cancer (Sugiyanto et al., 2003) and breast cancer (Meiyanto et al., 2007a; Meiyanto et al., 2007b) and has antimutagenic properties on benzo(a)pyrene-induced mice lung cancer (Meiyanto, 1996). The phenolic compounds of G. procumbens suppressed cells proliferation and induced apoptosis at HeLa (Meiyanto dan Septisetyani, 2005), and T74D cells (Maryati, 2006), and also has antiangiogenic properties (Jenie et al., 2007). Phenolic compounds of G. procumbens decreased Cox-2 expression (in vivo) and elevated p53 and Bax expression (in vitro) (Maryati et al., 2006). To our knowledge, no specific study had been reported addressing the selective cytotoxic effects of FEG on colon cancer WiDr and breast cancer MCF-7 and T47D and normal NIH3T3 cell lines. Thus, in this present study, we have evaluated the in vitro cytoxicity of FEG on those cells. Results from both cancerous and normal cell were then compared to determine the selective activity.

METHODS Chemicals and Reagents Material were used in this study: DMEM (Nacalay, Japan), Fetal Bovine Serum (FBS) (PAA), Dimethyl sulfoxide (DMSO) (Sigma, Aldrich, Germany), penicillin and streptomycin (Gibco), tripsin (Sigma), 3-(4,5dimethyltiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulphate (SDS). Tris–HCl, ethanol, n-hexane, and ethyl acetate and fraction of ethyl acetate G. procumbens (FEG). The fractions was dissolved in DMSO with 0,1 % as maximum concentration. All of the chemicals were on the higher degree of quality. Human cell lines Human colon cancer cell WiDr and breast cancer cell MCF-7 and T47D was provided from Cancer Chemoprevention Research Centre (CCRC) collection. NIH3T3 cell was kindly provided from Prof. Masashi Kawaichi, Laboratorium of Gene Function in Animal, Nara Institute of Science and

Technology. WiDr cells were cultured in Rosewell Park Memorial Institute (RPMI) containing 10% heat-inactivated fetal bovine serum (FBS) 1 % Penicillin-streptomycin (v/v), and L-glutamine (1 mM) at 37 oC and 5% CO2. MCF-7 and T47D cells were routinely grown in Dulbecco’s modified eagle’s medium (DMEM) and added the same supplement as that to RPMI medium. NIH3T3 cells were routinely grown in DMEM supplemented with 10% heatinactivated fetal calf serum (FCS) (PAA Labortories), 1 % Penicillin-streptomycin (v/v), and L-glutamine (1 mM) at 37 oC and 5% CO2. Plant material The leaves of G. Procumbens were obtained from the collection of Balai Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional (BP2TO2T) Indonesia, and was determined at Laboratorium of Pharmacognocy, Faculty of Pharmacy Gadjah Mada University, Indonesia. Dry powder of Gynura procumbent was re-macerated with 96 % ethanol for three time with three days maceration for each process. All filtrate then evaporated using rotary evaporator until a thick liquid extract was obtained. The ethanolic extract was dissolved in hot aquadest then fractionated using n-hexane. The water fraction then continued to fractionate with ethyl acetate. The ethyl acetate fraction was evaporated using rotary evaporator. The ethyl acetate extract was concentrated by evaporation under reduced pressure and the temperature was kept not more than 40oC. The extract was stored at 4oC prior to use. Determination of chromatographic profile of FEG Chromatography profile of FEG was determined using high performance thin layer chromatography (HPTLC) dan high performance liquid chromatography (HPLC). Mobile phase that used for HPTLC was toluene:chloroform:aceton:acetic acid (4:4:4:1) and using silica gel 60 F254 (Merck) as stationary phase. ultraviolet (UV) detection at 254 nm in wave length was used for spot visualization. The all spots were scanned using TLC Scanner (CAMAG), deuterium and wolfram (D2 & W) lamp, at 380 nm. Coloumn silica Symmetry C18 (lenght x internal diameter: 150 mm x 4.6 mm, particle size 15 mm) was used as stationery phase for HPLC. The mobile phase was acetonitrile : formic acid (25:75). The eluent was detected by ultraviolet-visible (UVVIS) detector at 360 visualization of the spot. 275


Quercetin (QUER) (Sigma-Aldrich, 98% HPLC, CAS No. 117-39-5)) and kaempferol (KAEMP) (Sigma-Aldrich, 90% HPLC, CAS No. 520-18-3) were used as marker. Cytotoxicity assessments Cells (104 cells/well) were cultured at 96-well plate. After 24 hours growth, cells culture medium was replaced with 5-FU, CISP, and/or FEG-contained medium. After incubated for 24 hours, the medium was replaced with MTT-containing medium (0,5 mg/mL) and incubated for 4 hours at 37oC, 5 % CO2. The reaction was stopped with 10% SDS in 0,1 N HCl solution and was incubated for overnight in light protected chamber, to dissolve formasan salt. The absorbance of each well was measured with ELISA reader at 595 nm. The ratio between treated and control cells absorbance refer to percentage (%) of viable cells. Selectivity index (SI) In the present study, the degree of selectivity of FEG refered to the previous report (Badisa et al., 2006), was calculated using formula: SI=LC50 in normal cells /LC50 in cancer cell lines, where LC50 is the concentration required to kill 50% of the cell population.

RESULTS Chromatography profile of ethyl acetate fraction of Gynura procumbens Ethyl acetate fraction of Gynura procumbens was fractionated twice separately, produced two different batch of ethyl acetate fraction (FEG). This FEG was obtained from liquid-liquid fractionation of ethanolic extract using n-heksane-hot water and continued with ethyl acetate-water. From the two fractionations were obtained percentage of FEG 1,6 % and 1,7 %, respectively. FEG was analized its chromatographic profile using High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography (HPLC) as shown on Fig. 1 and 2 respectively. Quercetin (QUER) and

kaempferol (KAEMP) were used as marker compound. Both FEG from different batch had similar chromatographic profile as result of HPTLC and HPLC. Most of chromatogram refered to flavonoid compound. From the scanning of maksimum wave lenght we concluded FEG may contained quercetin, but not kaempferol. However the amount of quercetin was not dominated the active compound of FEG. Inhibitory effect of FEG on WiDr, MCF-7 and T47D cells growth To determine the potency of FEG as anticancer agent, first the cytotoxicity properties were examined in WiDr colon cancer cell, MCF7 and T47D breast cancer cells. Cell viability was examined using MTT assay method after 24 hours incubation. FEG caused cell growth inhibition on WiDr, MCF-7 and T47D cells in dose dependent manner with IC50 value 119 μg/mL, 214 μg/mL and 76 μg/mL, respectively (Table I). FEG had moderate to high potency as cytotoxic agent. The highest potency was shown on T47D cell. Quercetin also caused cell growth inhibition on three cells in dose dependent manner with IC50 value 56 μg/mL, 169 μg/mL, 127 μg/mL, respectively. These results demonstrated that quercetin was not the main active compound of FEG. FEG shows lower cell growth inhibition in normal cells than that of cancer cells Cytotoxicity results showed moderate to high potency of FEG to be developed as anticancer agent. In order to determine the selectivity of FEG, we performed cytotoxic properties of FEG on normal cells, NIH3T3 (Fig.3). FEG gave no significant effect up to 400 μg/mL on NIH3T3 cell growth after 24 hours incubation. While after 72 hours, FEG caused cell growth inhibition at minimal concentration 600 μg/mL. IC50 value of FEG was measured after 24 and 72 hours of FEG treatment, resulted 592 μg/mL and 538 μg/mL, respectively. FEG shows lower cell growth inhibition in NIH3T3 than that in WiDr, MCF-7 and T47D cells. FEG shows lower inhibition on the growth of normal cells than that of cancer cell.

276


Figure I. Chromatogram profile of FEG was compared with Quercetin (QUER) and Kaempferol (KAEMP) as marker using HPTLC. FEG, QUER and KAEMP wre analized using HPTLC with silica gel 60 F254 as stationary phase and toluene:kloroform:aseton:asam asetat (4:4:4:1) as mobile phase (Lalla et al., 2003). Silica plate was visualized under UV light 254 nm then scanned using densitometer, 380 nm.

Figure 2. Chromatogram profile of FEG-contained compounds using HPLC. Chromatogram profile of FEG1 (A), FEG2 (B), and QUER (C). FEG and QUER were diluted in ethyl acetate with the indicated concentrations as described at method. FEG and quercetin solution was analyzed using HPC with Asetonitril : asam formiat (25:75) as mobile phase and silica coloumn symmetry C18, detector UV-VIS.

277


Figure 3. The effect of FEG on NIH3T3 cells growth. FEG treatment up to 400 μg/mL caused no significant cell growth inhibition. Cells (104 cells/well) were seeded at 96-well plate. After 24 hours growed, cells were treated with FEG (0-1000 μg/mL) for 24 and 72 hours. Cells viability were determined using MTT assay. T Test analysis, with 95% confidency was used to conclude the significance of cells growth inhibition. *FEG inhibit cell growt significantly (p>0,05). Table I. IC50 Value of FEG and QUER on WiDr, MCF-7 and T47D cells and The selectivity index (SI) which represents IC50 of FEG for normal cell line/IC50 for cancerous cell line after 24 hours incubation treatment.

Cells

Sample

IC50 Value*

WiDr

FEG QUER

119 μg/mL 56 μg/mL

MCF-7

FEG QUER


2.77

T47D

FEG QUER


7.79

Selectivity Index (SI) 4.97

*represented from al least 2 (two) independent experiments.

DISCUSSION Many researchers are now interested in examining the use of herbal medicines as a health care method. Herbal medicines continue to be accepted forms of treatment in the Orient, and the plant derived drugs based on traditional practices represent a huge proportion of the pharmaceutical production in modern Western countries (Newall et al., 1996 Schulz et al., 1998). Development of biologically targeted agents that exploit differences between cancerous and normal cells and permit greater specificity for cancer cells with less damage to

normal cells is still the ultimate goal in the field of antineoplastic drug discovery (Adam, 2001). Until now, no ideal cytotoxicity assay has been developed; hence, it is always advisable to support results with more assays where possible. Besides, it is important that of compare the cytotoxicity of a novel compound against several cell lines and even with other commercial cytotoxic agents. In this study, we showed the profile of bioactive compounds of ethyl acetate fraction of G. procumbens (FEG). We also confirmed the cytotoxicity properties of FEG and its selectivity directed human colon cancer and breast cancer cells.

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HPTLC analysis of FEG shown three main spots, similar with previous results from study of Sugiyanto et al., (2003). Previous study showed 3 (three) main spots that had been identified as flavon/flavonol. The first flavonoid was identified as flavonol derivate with 5 –OH and 3,7 –OCH3. He second flavonoid was flavonol derivate with 5,7 –OH and 3-Oglucosil. The third flavonoid was similar with the second flavonoid. Flavonol derivative that has 7 alkoksi, 3’ and 4’ di-OH, and 3,5 di-OH (Sugiyanto et al., 2003). The third flavonoid was quercetin derivative with alkoksi substitution on 7 –OH. Previous results also found quercetin, kaempferol and its derivate in G. procumbens (Akowuah et al., 2002). Methanolic extract may contain kaempferol-3-O-rutinosida dan astragalin (Rosidah et al., 2009), and its ethanolic extract has been identified as kaempferol-3-O-rutinosida, quercetin-3-Orutinosida, dan isobioquercetin compounds (Kim et al., 2011). HPLC results of FEG showed chromatogram peak with retention time (tR) value of 41.7 similar to the tR value of quercetin 41.6. Therefore FEG may contain quercetin. However there are others compound also contained in the chromatogram peak. FEG shows moderate to high cytotoxicity properties on colon cancer and breast cancer as indicated on the IC50 value at WiDr, MCF-7 and T47D cells. While quercetin presented higher potency (almost 3 fold) on cytotoxicity compared to FEG on WiDr and MCF-7 cells. Conversely quercetin demonstrated lower potency on cytotoxic properties on T47D cells. This phenomenon may cause by different cells characteristic or different target of action. The results of cytotoxicity assay showed that quercetin does not seem as the main active compound of FEG. There are other compounds contained in FEG that have pivotal portion on its cytotoxicity properties.

Since FEG had high potency of cytotoxicity, its impotant to reveal the selectivity of this fraction. FEG produced no significance effect on cell growth inhibition up to 400 μg/mL concentration at NIH3T3 cells. Selectivity of FEG on WiDr and T47D cells higher than that on T47D. FEG exhibited very high selectivity on WiDr and MCF-7 cells as shown by selectivity index (SI), that is > 3. Even as on MCF-7, FEG demonstated moderate selectivity properties.

CONCLUSION The present in vitro studies FEG possesses moderate to high cytotoxicity properties on WiDr, MCF-7 and T47D cells. FEG shows selective effect against cancer cells. Further FEG reveals prospective properties to be developed as cancer chemoprevention agent.

AUTHOR’S CONTRIBUTION Nunuk Aris Nurulita produced the concept and design of this study, acquisition of data, analysis and interpretation and statistical of data, and drafted the manuscript. Prof. Dr. Edy Meiyanto and Prof. Dr. Sugiyanto interpreted data and reviewed the manuscript. All authors have already read and approved the final manuscript.

ACKNOWLEDGEMENTS This work was supported in part by Research Grants of Hibah Doktor and Hibah Bersaing, managed by the Directorate General of Higher Education Republic of Indonesia, 2010/2011.

REFERENCES Adams, J., 2001, Proteasome inhibition in cancer: Development of PS-341. Sem. Oncol., 28, 613-619. Akowuah, G.A., Sadikun, A., and Mariam, A., 2002. Flavonoid identification and hypoglycaemic studies of butanol fraction from Gynura procumbens, Pharmaceut. Biol., 40, 405–410. Badisa, R.B., Lambert, A.T., 2006, Ikediobi CO and Walker EH: Selective anticancer

activity of pure licamichauxiioic-B acid in culture cell lines. Pharmaceut Biol., 44(2), 141-145 Garcia, M., Jemal, A., Ward, E.M., Center, M.M., Hao, Y., Siegel, R.L., and Thun, M.J., 2007, Global Cancer Facts & Figures 2007, Atlanta, GA: American Cancer Society. Jenie, R.I., Meiyanto, E., Murwanti, R., 2006, Antiangiogenic effect of sambung nyawa leaves (Gynura procumbens (Lour.) Merr.) etanolic extract on chick embryo 279


chorioallantoic membrane (CAM). Indonesian Journal of Pharmacy, 17(1), 5055. Kim, J., Lee, C.W., Kim, E.K., Lee, S.J., Park, N.H., Kim, H.S., Kim, H.K., Char, K., Jang, Y.P., and Kim, J.W., 2011, Inhibition effect of Gynura procumbens extract on UV-Binduced matrix-metalloproteinase expression in human dermal fibroblast, J. Ethnopharmacol., 137(1), 427-433. Maryati, Meiyanto, E., and Riyanto, S., 2005, Uji Sitotoksik dan isolasi flavonoid dari fraksi etil asetat daun Gynura procumbens (Lour) Merr, Pharmacon., 6(2), 46-50. Meiyanto, E. and Septisetyani, E.P., 2005, Antiproliferative and apoptotic effect of fenolic fraction of ethanolic extract of Gynura procumbens (Lour.) Merr. against HeLa Cells. Artocarpus., 5(2), 74-80. Meiyanto, E., Susilowati, S., Tasminatun, S., Murwanti, R., and Sugiyanto, 2007, Chemopreventive effect of ethanolic extract of Gynura procumbens (Lour), Merr on the carcinogenesis of Rat breast cancer development, Indonesian Journal of Pharmacy, 18(3), 154-161. Meiyanto, E., 1996, Efek Antimutagenik Beberapa Fraksi Ekstrak Alkohol Daun Gynura procumbens (Lour)Merr, Laporan

Penelitian, Fakultas Farmasi UGM, Yogyakarta. Meiyanto, E., Tasminatun, S., Susilowati, S., Murwanti, R., and Sugiyanto, 2007b, Penghambatan Karsinogenesis Kanker Payudara Tikus terinduksi DMBA pada fase Post Inisiasi oleh ekstrak etanolik Gynura procumbens (Lour) Merr., Majalah Farmasi Indonesia., 18(4), 154-161. Newall, C.A., Anderson, L.A., and Phillipson, J.D., Herbal Medicines. A Guide for Healthcare Professionals; Pharmaceutical Press: London, UK, 1996 Rosidah, Yam. M.F., Sadikun, A, and Asmawi M.Z., 2008, Antioxidant potential of Gynura procumbens, Pharmaceutical Biol., 46, 616-625. Schulz, V., Rudolf, H., and Tyler, V.E., 1998, Rational Phytotherapy. A Physician’s Guide to Herbal Medicine, 3rd ed., Springer, Berlin, Germany. Sugiyanto, Sudarto, Meiyanto, E., Nugroho, A.E., and Jenie, U.A., 2003, The anticarcinogenic activity of plants compounds. Indonesian Journal of Pharmacy, 14(4), 216-225. WHO, 2006, Cancer Control, Knowlendge into Action, WHO Guide for Effective Programes, www.who.int/cancer.

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Indonesian Journal of Cancer Chemoprevention, 2012, 3(2): 399-404 ISSN: 2088 - 0197

The Number of Macrophages and Heterophils on Chick Embryo Chorioallantoic Membrane After Gynura procumbens (Lour) Merr Extract Treatment and bFgF Induction Iwan Sahrial Hamid1)*, Yuseni Kusuma P.2), Retno Bijanti1), E. Bimo Aksono1) 1)

Division of Basic Veteriner, Faculty of Veterinary, Universitas Airlangga, Jl. Mulyorejo, Surabaya, 60115, Indonesia, Tel. +62-031-5992785 2) Faculty of Veterinary, Universitas Airlangga, Surabaya, Jl. Mulyorejo, Surabaya, 60115 Indonesia, Tel. +62-031-5992785

Abstract Antiangiogenesis (inhibition of new blood vessels formation) has become a strategy to inhibit cancer development. The aim of this experiment was to investigate antiangiogenic effect of Gynura procumbens (Lour) Merr focusing on the decreasing of the number of macrophages and heterophils on chick embryo chorioallantoic membrane. Nine-days-agedeggs were divided into six groups (eight eggs each group). Group I (positive control) eggs were induced with bFGF+Tris HCl. Group II (negative control) eggs were treated with DMSO+Tris HCl. Group III (treatment I) eggs were induced with 60 ng bFGF and treated with ethanolic extract of G. procumbens leaves with the dose of 60 µg. The following treatment groups, i.e. group IV (treatment II), group V (treatment III), and group VI (treatment IV) were treated with increasing dose of extract, starting from 75 µg, 90 µg, and the last was 110 µg. Eggs were incubated until they reach the age of twelve days to observe macrophages, while to observe heterophils, eggs were incubated until the age of seventeen days. Based on haematoxylin-eosin staining, macrophages in the treatment groups were less than the control positive group (bFGF+Tris HCl), but based on giemsa staining, the effect of Gynura procumbens in decreasing the number of heterophils could not be observed because some blood smears. These analysis suggest that the ethanolic extract of Gynura procumbens leaves can perform as antiangiogenic agent decreasing the number of macrophages. Keywords: antiangiogenic, macrophages, heterophils, Gynura procumbens

INTRODUCTION Angiogenesis is new blood formation as part of normal process in the body, playing and important role in growth and development. Angiogenesis occurs during recovery, such as during the formation of new tissue after damage. Unfortunately, angiogenesis is also involved in carcinogenesis, that turns cancer into its uncontrolled malignant state (Folkman, 1998). Angiogenesis involves cells engaged with inflammation, including macrophages and heterophils, facilitated by matrix metalloproteinase (MMP) (Zijlstra et al., 2004; Zijlstra et al., 2005). According to the study conducted by Deryugina and Quigley (2006) discussed in Ardi et al. (2007), the role of MMP in the development of tumour and

metastasis is complex. MMP acts as the main mediator in growth factor activation, e.g. bFGF, receptor bioavailability and signaling, cell adhesion and motility, apoptosis and survival mechanism, angiogenesis and immune response, and also immune surveillance. To date, more than 20 proteases that belongs to MMP family have been discovered (Rundhaug, 2005). MMP-9 secreted by heterophils and MMP-13 secreted by macrophages as part of MMP family involved in angiogenesis, as proangiogenic proteinase (Zijlstra et al., 2004; Zijlstra et al., 2005).Plenty strategies have been applied in cancer treatment. However, existing methods faced various clinical problems. Corresponding author email: : [email protected]

399

Hamid et al. ISSN: 2088 - 0197

Hence, novel strategies are being developed continuously. One strategy of suppressing carcinogenesis is by inhibiting angiogenesis. Jenie et al. (2006) reported that G.procumbens leaves possess antiangiogenic activity. G. procumbens also showed pharmacological effect to cancer, since its extract contains flavonoid, quercetin, polyphenol, alkaloid, and also other substances that are able to inhibit carcinogenesis (Hamid et al., 2009). This study was conducted to observe G. procumbens leave ethanolic extract’s ability to suppress cancer growth by inhibiting angiogenesis. Antiangiogenic effect will be observed by evaluating its ability to decrease the number of macrophages and heterophils.

MATERIALS AND METHODS Materials Another chemicals used were ethanol 70%, sterile aquabidest, formaline buffer 10% for TAB chorioallantoic membrane preservation, haematoxyllin-eosin for macrophage-histology and counting staining, and methanol-giemsa for heterophils counting staining (Thompson and Samuel, 1966). Antiangiogenic Activity Observation Eggshell were marked for air space boundary, embryo location, and the area above the embryo that later would be perforated with a 1x1 cm square window. The embryo location was detected by candling on the eggs. The pole of the eggshells containing air space and the eggshell part above the embryo was washed with ethanol 70%. Then, those two areas were perforated with a minidrill. The air inside the air space was aspirated by using a rubber ball until the chorioallantoic that adhered to the egg membrane detached. Egg position was mantained horizontal during the process conducted in a dark room by candling, so that the chorioallantoic membrane and the artificial air space formed above the embryo could be seen. Next, eggs were washed with ethanol 70% again and kept horizontally in laminar air flow hood. Artificial air space were positioned at the upper part. Eggshell above the embryo were perforated with a minidrill to make a 1 cm2

hole. Test solution was implanted into formed chorioallantoic membrane through this window. There were 6 groups, each containing 8 eggs, half were for machrophages observation, while another four were for heterophils observation. Group 1 was implanted with paper disc containing 60 ng bFGF and tris-HCl as positive control. The 2nd group was implanted with paper disc containing tris-HCl and DMSO 2% as negative control. Group 3, as the first treatment group, was implanted with paper disc containing 60 ng bFGF and 60 µg Gynura procumbens leave ethanolic extract (GLE). Another treatment groups were group 4, 5, and 6, implanted with paper disc containing 60 ng bFGF and 75 µg, 90 µg, and 110 µg GLE, respectively. After treatment, eggs were incubated at 39ºC and 60% RH for 72 hours (Ribatti et al., 1997). On the 13th day, four embryonic eggs from each group were kept in the freezer for 24 hours to kill the embryo. Following it, TAB was opened by cutting the shell into two, starting from the area that was close to the air space. Egg content was slowly extracted to prevent chorioallantoic membrane detachment. Chorioallantoic membrane around the paperdisc were then cut and kept in formaline buffer 10% for preservation. The membrane then were used to made the histopathology preparation stained with haematoxyllin-eosin for macrophages number observation. Preparations were conducted in Laboratorium Patologi Fakultas Kedokteran Universitas Gadjah Mada. The other four eggs remaining from each group were used for heterophils observation. Heterophils could be isolated from 17 days-aged TAB (Zijlstra et al., 2005). Embryonic chicken eggs were opened by cutting the shell. Blood was extracted from the blood vessel of the egg to be made into blood preparations with Romanowsky method. The preparations were then used for heterophils counting. Observation was conducted by using Olympus ® CX-21 microscope with 1000x magnification on six different window of view. Data acquired was analyzed statistically by using One-way Analysis of Variance (ANOVA), continued with Duncan post-hoc test (p<0.05).

400


RESULTS AND DISCUSSION Effect of GLE on Number of Macrophages The result of hematoxylin-eosinstained embryonic chicken egg

chorioallantoic membrane macrophage counting is shown on Table I. Histology observation is shown in Fig. 1.

Table 1. Effect of GLE on mean of macrophages number on each group. Differently superscritpted mean shows significant different compared to each other (p < 0.05).

1

Number of Macrophages (x ± SD)

2

21.50d + 2.65

3

76.50b + 6.56

4

50.25c + 7.57

5

40.75c + 2.87

6

24.00d + 6.83

Group

104.50a + 16.46

Figure 1. Histology observation on (A) Group 1 as positive control, (B) Group 2 as negative control, (C) Group 3, (D) Group 4, (E) Group 5, and (F) Group 6 as treatment groups. Preparations were stained with haematoxylin-eosin, 1000x magnification. Black arrows shown macrophages.

The mean of the number of macrophages of all GLE-treated groups (Group 3 to 6) with different doses, that are 60 μg, 75 μg, 90 μg, and 110 μg, respectively, shown a significant decrease compared to positive control group (Group 1) (p < 0.05). Moreover, Group 6, 110 μg GLE-treated group, had shown a decrease of the number of macrophage that is similar to its normal state, shown by Group 2 as the negative control group.

The number of macrophage in each GLE-treated group showed significant difference among each other (p < 0.05). These data showed that GLE could decrease the number of macrophages. It is possible that GLE contains certain substance(s) that possess angiogenesis inhibitory activity by decreasing macrophages infiltration. A component in G. procumbens having antiangiogenic activity is flavonoid. The study support the result reported by Jenie

401


et al. (2006), proving that flavonoid contained in G. procumbens could inhibit angiogenesis of TAB chorioallantoic membrane. According to the study on Citrus reticulata conducted by Chrisnanto et al. (2008), treatment of Citrus reticulata peels extract containing a huge amount of tangeretin (a polymethoxy flavonoid) and nobiletin yielded less number of macrophages compared to bFGF-onlytreated group in microscopic observation of hematoxylin-eosin-stained TAB chorioallantoic membrane histopathological preparations . Macrophages are known to secrete angiogenic factors, such as VEGF, bFGF, and interleukin-8 (IL-8) that are able to induce angiogenesis and Tumor Necrosis Factor-α (TNF-α) that later would increase the number of VEGF, bFGF, and IL-8 receptors (Lee et al., 2006). The decrease of the number of macrophages could possibly occured by cell cycle arrest induced by tangeretin (Chrisnanto et al., 2008). It has been reported that several flavonoids, such as apigenin, genistein, and quercetin were able to induce premitotic Growth phase-2/Mitotic phase (G2/M) arrest in several cell lines, while some others were also able to induce presynthetic Growth phase-1 (G1) arrest (Pan et al., 2002). Pan et al. (2002) also reported that tangeretin found in Citrus reticulata peels could induce G1 arrest by increasing Cyclin-dependent Kinase Inhibitors (CKIs), such as p27 and p21 in colon cancer cell lines. The possible mechanism of cell cycle arrest by the extract might be the same as the mechanism of cell cycle arrest by flavonoids contained in GLE. GLE could possibly increase CKIs, such as p21, p27, and/or p57, inhibiting Cyclin-dependent Kinases (CDKs), that later would suppress cyclins expression or increase p53 level, resulting in cell cycle arrest (Meiyanto et al., 2007) and the decrease of the number of macrophages. p53 a regulatory protein that could induce p21 expression, hence causing G1 arrest (Meiyanto et al., 2007). G1 phase is the phase when cells are getting prepared for DNA synthesis together with RNA and protein biosynthesis (Hartono, 2009). Flavonoid contained in G. procumbens extract could also suppress topoisomarase I and II expression that play a role in DNA supercoil conversion. Topoisomerase inhibitor may stabilize topoisomerase complex, causing the formation of DNA nick

hence undergoes a damage. DNA damage will further increse the expression of proapoptotic proteins, such as Bax and Bak, and decrease antiapoptotic proteins Bcl-2 and Bcl-XL (Ren et al., 2003). Here, we may see that flavonoids play a role in inducing apoptosis. According to the discussion above, it is very likely that flavonoids in GLE could decrese the number of macrophages by similar mechanism. Effect of GLE on the Number of Heterophils The effect of GLE on heterophils could not be observed yet, since the preparations did not allow a valid observation, resulting in insufficient replication. The problem was caused by the lysed nucleus, causing it impossible to differ heterophils and the other leukocytes, worsen by the precitipated staining agent interfering cell observation. Nucleus lysis may occur because of insufficient duration of fixation. The minimum duration of fixation is 5 minutes. It could be modified, depending on the quality of staining agent used (Bijanti et al., 2010). The precipitation of staining agent was caused by the solution that was kept uncovered, the solution that was not filtered properly prior to usage, and dirty object glass (WHO, 2006). The preparations also shown too intense blue color of the cells and blue background. Too intense blue color of the cells may caused by the blood smear that is too thick, insufficient time for the buffer to dissolve, too high buffer pH, too long staining duration, too high staining agent concentration and viscosity, impurities in staining agent stock, and light exposure on staining agent stock solution.The blue background yielded may be caused by the too quick fixation process and too long duration time prior to fixation (WHO, 2006). This study concluded that GLE treatment could decrease the number of macrophages of bFGF-induced TAB chorioallantoic membrane histopahological preparations. Unfortunately, the effect of GLE on the number of heterophils could not be observed because of the improper preparation of the blood smears that did not allow a valid observation, resulting in insufficient replication.

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REFERENCES Ardi, V.C., T.A. Kupriyanova, E.I. Deryugina, and J.P. Quigley. 2007. Human Neutrophils Uniquely Release TIMP-Free MMP-9 to Provide a Potent Catalytic Stimulator of Angiogenesis. J. PubMed., 104 (51), 20262–20267. Bijanti, R., R.B. Utomo, R.S. Wahyuni, S. Budhy, and M.G.A. Yuliani. 2010. Penuntun Pratika Patologi Klinik Veteriner Cetakan Keempat. Laboratorium Patologi Klinik Veteriner Fakultas Kedokteran Hewan Universitas Airlangga. Chrisnanto, E., Adelina, R., Putri, D. D. P., Sahid, M.N.A., Setyaningtias, D., Jenie, R.I., and Meiyanto, E., 2008, Antiangiogenic Effect of Ethanol Extract of Citrus reticulata Peel in the Chorio Allantoic Membrane (CAM) Induced by bFGF. Proceeding The International Symposium on Molecular Targeted Therapy. ISBN: 978-979-95107-6-1, Fakultas Farmasi Universitas Gadjah Mada, 57-66. Folkman, J., 1998, Angiogenic Therapy of the Human Heart Circulation, 97, 628–9. Hamid, I.S., Sugiyanto, E. Meiyanto, and S. Widyarini. 2009. Ekspresi Protein p53 pada Kelenjar Mammae Tikus Galur Sprague Dawley Setelah Inisiasi Dimethylbenz(a)antrasen (DMBA) dan Pemberian Kemopreventif Gynura procumbens. Media Kedokteran Hewan, 24 (3), 171-176. Hartono, N.W.B., 2009, Pengaruh Alpinia galanga (Lengkuas) terhadap Aktivitas Proliferasi Sel dan Indeks Apoptosis pada Adenokarsinoma Mamma Mencit C3H [Tesis]. Universitas Diponegoro. Jenie, R.I. and E. Meiyanto. 2006. Efek Antiangiogenik Ekstrak Etanolik Daun Sambung Nyawa (Gynura procumbens (Lour.) Merr.) pada Membran Korio Alantois (CAM) Embrio Ayam. Indonesian Journal of Pharmacy, 17(1), 50 – 55.

Lee, C., K.J. Liu and T.S. Huang. 2006. Tumor Associated Macrophage : Its Role in Tumor Angiogenesis. Journal of Cancer Molecules, 2(4),135-140. Meiyanto E., Susilowati S., Tasminatun S, Murwanti R., and Sugiyanto, 2007, Penghambatan karsinogenesis kanker payudara tikus terinduksi DMBA pada fase post inisiasi oleh ekstrak etanolik daun Gynura procumbens (Lour), Merr , Indonesian Journal of Pharmacy, 18(4), 169-175. Pan, M.H., W.J. Chen, S. L., Ho, C.H., and Lin, J. K., 2002, Tangeretin Induces Cell Cycle Through Inhibiting Cyclin Dependent Kinase 2 and 4 Activities As Well As Elevating Cdk Inhibitor p21 in Human Colorectal Carcinoma Cells, Carsinogenesis, 23, 1677-1684. Ren, W., Qiao Z., Wang H., Zhu L., and Zhang, L., 2003, Flavonoids: Promising Anticancer Agents, Medicinal Research Review, 23(4), 519-534. Ribatti, D., A. Gulandris, M. Bastaki, A. Vacca, Iurlaro, M., Roncali, L., and Presta, M., 1997, New Model for the Study of Angiogenesis and Antiangiogenesis in the Chick Embryo Chorioallantoic Membrane: The Gelatin Sponge/Chorioallantoic Membrane Assay. Journal of Vascular Research, 34, 455-463. Rundhaug, J.E. 2005. Matrix Metalloproteinase and Angiogenesis. J. Cell. Mol. Med., 9(2), 267-285. Thompson and W. Samuel, 1966, Selected Histochemical and Histopathological Methods, Charles C. Thomas, Springfield, IL. World Health Organization. 2006. Preparation and Staining of Blood Films. Guidelines on Standard Operating Procedures for Haematology. Blood Safety and Clinical Technology. Zijlstra, A., Aimes RT, and Zhu D. 2004. Collagenolysis-Dependent

403


Angiogenesis Mediated by Matrix Metalloproteinase-13 (Collagenase3). J Biol Chem., 279, 27633-27645. Zijlstra, A., Seandel, M., Kupriyanova, T.A., Patridge, J.J., Madsen, M. A., HahnDantona, E.A., Quigley, J.P., and Deryugina, E.I., 2005, Proangiogenic Role of Neutrophil-like Inflammatory Heterophils during Neovascularization Induced by Growth Factors and Human Tumor Cell. DOI 10.1182/blood2005-04-1458.

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