Correlation Between Cytotoxic Activity on MCF-7 Cell and Total Steroid Level on Trigonella Foenum-Graecum L

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Correlation between Cytotoxic Activity on MCF-7 Cell and Total Steroid Level on Trigonella foenum-graecum L. Korelasi aktivitas sitotoksik pada sel MCF-7 dan kadar steroid total Trigonella foenum-graecum L.

Kurnia Agustini*, Wahono Sumaryono*, Azri Fitria** *Center for Pharmaceuticals and Medical Technology Laboratory for Development of Industrial Agro and Biomedical Technology Agency for the Assessment and Application of Technology ( BPPT), Jakarta **Departement of Pharmacy, Faculty of Medicine and Health Science State Islamic University Syarif Hidayatullah Jakarta e-mail: [email protected]

ABSTRAK

Trigonella foenum-graecum L. (TFG) adalah salah satu tumbuhan obat yang mengandung beberapa steroid sapogenin seperti diosgenin, yamogenin, gitogenin, tigogenin, dan trigoneosid, serta alkaloid trigonellin. TFG memiliki aktivitas antidiabetik, estrogenik, dan antikanker. Penelitian ini bertujuan untuk menguji korelasi aktivitas sitotoksik TGF pada sel kanker payudara MCF-7 serta kadar steroid total. Ekstrak metanol TGF difraksi- sitotoksik pada sel MCF-7 diuji dengan uji MTT [3-(4,5-dimetiltiazol-2-il)2,5-difeniltetrazolium]. Kadar ste- roidnasi dengantotal ditetapkan etil asetat, dengan n-heksan, metode dan spektrofotometri.n-butanol. Tiap fraksi Hasil dipisahkan penelitian denganmenunjukkan kromatografi tidak terdapat kolom. Aktivitas korelasi antara aktivitas sitotoksik dengan kadar steroid total. Sehingga dapat disimpulkan bahwa efek sitotoksik TFG bukan merupakan kontribusi dari komponen steroid.

Kata kunci: Trigonella foenum-graecum L., MTT, MCF-7, kadar steroid total

ABSTRACT Trigonella foenum-graecum (TFG) is one of medicinal plants containing some steroidal sapogenins such as diosgenin, yamogenin, gitogenin, tigogenin and trigoneoside, also alkaloid trigonellin, which has many activity as antidiabetic, estrogenic and also as anti cancer. The aim of this research is to investigate the correlation of its cytotoxic activity on breast cancer cell line, MCF-7, with its total steroid level. Samples was prepared by fractioned the methanolic TFG with ethylacetate, n-hexane and n-buthanol. Each fraction then was separated by chromatographic coloumn. Cytotoxic activity was investigated using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium) assay on MCF-7 cell, while total steroid level was done using spectrophotometric methods. Results showed that there was no correlation between cytotoxic activity and total steroid level. Thus we can conclude that the cytotoxic effect from TFG is not caused by its steroid compounds.

Key words: Trigonella foenum graecum L., MTT, MCF-7, total steroid level.

26 Volume 4, No. 1, Agustus 2011 CORRELATION BETWEEN CYTOTOXIC ACTIVITY ON MCF-7 CELL AND TOTAL STEROID LEVEL ON Trigonella foenum-graecum L. Korelasi aktivitas sitotoksik pada sel MCF-7 dan kadar steroid total Trigonella foenum-graecum L.

INTRODUCTION Fenugreek seed or Foenigraeci semen is dried seed from Trigonella foenum-graecum L. (TFG), Leguminosae, (MMI, 1979). Geographical distribution of TFG is indigenous to the Mediter- ranean region, China, India and Indonesia (WHO, 2007). TFG contains some sapogenin steroid ingredients, e.g. diosgenin, precursor for sexual hormone (Evans, 2002), its isomer Yamogenin (Dewick, 1997), gitogenin, tigogenin, and trigoneoside (saponine like estrogen) which have effect as phytoestrogen for menopause symptoms therapy. Sapogenins are the aglycones or non- Protodioscin saccharide of saponins, one of phytochemical compound family. Sapogenins contain steroid or other triterpene frameworks as their key organic feature. Some steroidal sapogenins can serve as a practical starting point for semisynthesis of par- ticular steroid hormones. Tigogenin TFG contains diosgenin in base free form 0.8-2.2 % (Wiryowidagdo, 2000). TFG also con- Figure 1. Some sapogenine steroids of Trigonella tains alkaloids (trigonelline, an alkaloid pyridine, foenum-graecum L. - coside or ester form, isovitexin, orientin, vicenin, Empirically TFG was use as aphrodisiac, gentianin and karpain), flavonoids (vitexin in gly quercetin and luteolin), fatty oil 20-30%, essen- carminative, diuretic, emmenagogue, emollient, tial oil, saponine, nicotinamide, choline, bitter galactogogue and tonic (WHO, 2007). Many re- compound and mucilage (Evans, 2002). searches, preclinical and clinical, showed that TFG have activity for diabetic disease (Annida, 2004). TFG also predicted as Phytoestrogen cause have

and immature female rat (Agustini, 2007). It can estrogenic effect on ovariectomized female rat induce uterine contraction, so it should be avoid- ed during pregnancy. Phytoestrogen is used as alternative for Hormone Replacement Therapy (HRT) to help reducing menopause symptoms. It can be used for long term until the body can make

Volume 4, No. 1, Agustus 2011 27 Kurnia Agustini*, Wahono Sumaryono*, Azri Fitria**

adaptation on the new level hormone (Badziad, MATERIALS AND METHODS 2003). Samples Preparation Extracts of TFG seeds and some of their sa- TFG seeds were obtained from Tawang- ponine constituents also have been found to have mangu, Central Java, Indonesia. Seeds were dried and grind, then were extracted with methanol. has been evaluated in the Ehrlich ascites carci- The methanolic extract was fractioned with n- anticarcinogenic activity in different setting. It hexane, ethylacetic (EtOAc) and n-buthanol. Fu- 70% inhibition of tumor cell growth compared ther, each fraction then separated by vacuum liq- noma model in BALB/c mice, where it affected with controls (Sur, 2001). Hibasami et al., (2003) uid chromatography using various eluens. Every suggest that growth inhibition of human leuke- extract and fraction was dried with vacuum ro- mia HL-60 cells by protodioscin, isolated from tary evaporator. TFG seeds, results from the induction of apopto- Total Steroid Analysis (Chapagin et al., 2005) sis. Moalic et al., (2001) reported that diosgenin 1 mg dried extract/ fraction diluted in 2 inhibits cell proliferation in the human osteosar- mL ethylacetate in a tube, then 1 ml reagent A coma 1547 cell line by induction of apoptosis and (contains p-anysaldehyde and ethylacetate (0.5 G1 phase cell cycle arrest. : 99.5)) and 1 ml reagent B (contains sulfuric Refer to data that showed TFG having es- acid glacial and ethyl acetate (1:1)) were added. trogenic activity as phytoestrogen, inspiring to Tube was put in water bath 600C for 10 minutes study its possibility as Selective Estrogen Recep- till color was occurred and then cooled in an- tor Modulators (SERMs) candidate. SERMs such other water bath 250C for 10 minutes. Color was as tamoxifen, which are used clinically for the measured by Spectrophotometer UV Vis 423nm, treatment of breast cancer, act as estrogen ago- against ethyl acetate solution as reagent blank. nists in certain tissues but exhibit antiestrogenic Results were compared with curve standard of Diosgenin (Sigma). several researches about SERMs candidates for Cell Culture effects in others (Rosenbaum, 2000). There are post tumor surgery therapy from phytoestrogen The cell lines MCF-7 (Human Breast Ad- pure compounds, such as dammarane, a sapoge- enocarcinoma) was obtained from Laboratory nine steroid from ginseng (Oh, 1999), Genistein for Development of Industrial Agro and Biomedi- cal Technology (LAPTIAB-BPPT) Indonesia. Cells stilbene from grape (Baht, 2001). were routinely maintained and grown in 75 cm2 (an isoflavone from soybean) and resveratrol, a 0 This research was done to investigate the C, 5% CO2 correlation between TFG’s cytotoxicity on hu- atmosphere. The growth medium was prepared flasks at 37 and in a 95% humidified man breast cancer cell line MCF7 and its total as following: RPMI 1640, Gibco life Technolo- steroids level in methanolic extract and its frac- gies with phenol red and 2 mM glutamine, 100 tioned (hexane, buthanol and ethylacetate). U/ml penicillin, 0.1 mg/ml Streptomycin, 1 mM sodium pyruvate and supplemented with 10%

28 Volume 4, No. 1, Agustus 2011 CORRELATION BETWEEN CYTOTOXIC ACTIVITY ON MCF-7 CELL AND TOTAL STEROID LEVEL ON Trigonella foenum-graecum L. Korelasi aktivitas sitotoksik pada sel MCF-7 dan kadar steroid total Trigonella foenum-graecum L.

Foetal Bovine Serum (FBS, Gibco Life Technolo- RESULTS AND DISCUSSION gies) which already heat inactivated at 560C for TFG was extracted with methanol to 30 min. Passaging of cells was carried out using obtain more chemical phyto compound in it. 4 ml of trypsin-EDTA at room temperature for 75 After that, methanolic extract was fractioned cm2 by vacuum liquid chromatography and divided ml media with 10% FBS were used to reduce the into three fractions based on its polarity, they flask, respectively for 3 min. After that, 10 action of trypsin on cells. After centrifugation, are non-polar fraction (n-hexane fraction), semi- the obtained cells were platted. polar fraction (ethyl acetic fraction) and polar Cytotoxicity test with MTT method fraction (Buthanolic fraction). Methanolic extract Cells were platted into 96-well plates and other three fractions then were analyzed for (10,000 cells/well) in medium RPMI with phenol cytotoxicity activity on human breast cancer cell- red containing 10%Fetal Bovine Serum (FBS), line, MCF-7, and its total steroidal level. Result of 100U/ml penicillin, 0.1 mg/ml streptomycin these analyze are shown by Figure 2. Ethylacetic and 1 mM sodium pyruvate, then incubated for fraction gives the lowest IC50 (41.48 ppm) and 0 24 hours at 37 C, 5% CO2 and in a 95% humidi- the highest level of total steroid (20.033ppm) was given by n-buthanolic fraction, while the changed with samples (extracts and phases of lowest total steroid level was given by n-hexane fied atmosphere. After 24 hours, medium was - fraction (3.693ppm) and less cytotoxicity activity tion and incubated for another 24 hours at 370C, was given by methanolic extract (186.007 ppm). TFG) in growth medium in different concentra 5% CO2 From this data we can see that three fraction are Assays were done in wide range concentration, more active than its methanolic extract. We can and in a 95% humidified atmosphere. from 10 ppm until 500 ppm, divide into six varia- predict that the characteristic of active compound tion concentration. on MCF-7 cell is semi-polar. After 24 hours treatment, the cells were

Then the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- washed with Phosphate Buffer Saline (PBS). Diphenyltetrazolium) solution in medium, was added followed by incubation for 4 hours at 370C,

5% CO2 The crystal of formazan blue will be formed. Af- and in a 95% humidified atmosphere. ter that, reaction was stopped by added Sodium Dodecyl Sulphate (SDS) into every well. Leave plate in dark place for 12 hours (overnight). The intensity of the color formed was measured by ELISA reader at 570nm.

Volume 4, No. 1, Agustus 2011 29 Kurnia Agustini*, Wahono Sumaryono*, Azri Fitria**

Figure 3. Comparative of cytotoxicity activity on Figure 2. Comparative of cytotoxicity activity on MCF7 (IC50) and its total steroid level from column MCF7 (IC50) and its total steroid level chromatographic fractions of Ethylacetic fraction.

fractionation. from methanolic extract and its first Screening then continued by separated the ethylacetic fraction using vacuum column chromatographic with gradient elucidation using mixed dichloromethane and ethanol. All fractions were grouped based on their spots in thin layer chromatography. Results of cytotoxic activity and its steroid level analyze were showed in Figure 3.

The most active fraction is EA2 with IC50= 3.812 ppm and the less active fraction is EA4 with

IC50 = 2662 ppm, while the highest level of total steroid was given by EA3 fraction (27.533 ppm) and the lowest level of total steroid was given by EA1 fraction (13.417 ppm). Also in these results of ethyl acetate fraction, the same pattern of total steroid level and its cytotoxic activity are not found.

30 Volume 4, No. 1, Agustus 2011 CORRELATION BETWEEN CYTOTOXIC ACTIVITY ON MCF-7 CELL AND TOTAL STEROID LEVEL ON Trigonella foenum-graecum L. Korelasi aktivitas sitotoksik pada sel MCF-7 dan kadar steroid total Trigonella foenum-graecum L.

= 396.19ppm, while the highest level of total steroid was given by HX4 fraction (25.055 ppm) and the lowest level of total steroid was given by HX2 fraction (3.92 ppm). This research showed that there was no correlation between total steroid level and its cytotoxicity activity on breast cancer cell line, MCF-7. As we know, the mayor sapogenin steroid from TFG, diosgenin, has been investigated in many researches for its cytotoxicity activity. Moalic et al. (2001) reported that diosgenin inhibits cell proliferation in the human osteosarcoma 1547 cell line by induction of apoptosis and G1 phase cell cycle arrest. Raju et al. (2004) also reported that diosgenin, a steroid saponin of Trigonella foenum-graecum (fenugreek), inhibits azoxymethane-induced aberrant crypt foci formation in F344 rats and induces apoptosis in HT-29 colon cancer cells. Yoshihiro et al. (2001) studied about cytotoxicity activities on human promyelocytic leukemia cells, HL-6 and structure-cytotoxic relationships of steroidal saponins. They found that the activities Figure 4. Comparative of cytotoxicity activity on MCF7 of some sapogenin steroids were sensitive to the (IC50) and its total steroid level from column chromatographic fractions of n-Hexane monosaccharides constituting the sugar moieties fraction. and their sequences, as well as to the structures of the aglycons. They also concluded that structure– To investigate more about the correlation of activity relationships of (25R)-Spirost-5-en-3b-ol these two parameters, the screening to n-Hexane (Diosgenin) glycoside derivatives Diosgenin b-D- fraction also was done. After separated by column glucoside showed no cytotoxic activity against chromatography using also gradient elucidation HL-60 cells (IC50>20ppm) of mixed dichloromethane and ethanol, each fraction then screened by MTT assay and their CONCLUSIONS level of Steroids. Results are showed in Figure 4. The most active TFG fraction on MCF-7 cell- The most active fraction is HX4 with IC = 35.57 50 line is ethylacetic fraction, with IC50 41.48 ppm and ppm and the less active fraction is HX3 with IC 50 its fraction EA2 (IC50= 3.812). The highest level

Volume 4, No. 1, Agustus 2011 31 Kurnia Agustini*, Wahono Sumaryono*, Azri Fitria**

Badziad, Ali. 2003. Endokrinologi Ginekologi. fraction (25.31 ppm). While from coloumn Media Aesculapius. Fakultas Kedokteran of total steroid in first fractination is buthanolic chromatographic results of ethylacetic fraction, Universitas Indonesia, Jakarta: xxiv + 167p. the highest total steroid level given by EA3 Jakarta (27.533 ppm). The cytotoxic activity on MCF-7 of Bhat KPL., Lantvit D., Christov K., Mehta RG., Moon TFG maybe caused by another group compound RC., and Pezzuto JM. 2001. Estrogenic and except its sapogenin steroid. The isolation of Antiestrogenic Properties of Resveratrol in active compound should be continue. So we can Mammary Tumor Models. Cancer Research conclude that there was no correlation between 61(20):7456-7463. cytotoxic activity on MCF-7 cell and total steroid Chapagain, Bishnu, Zeev Wiesman. 2005. Variation in diosgenin level in seed kernels is not caused by its steroid compounds. among different provenances of Balanites level. The cytotoxic effect on MCF-7 cell from TFG aegyptiaca Del (Zygophyllaceae) and its ACKNOWLEDGEMENT correlation with oil content. Departement of We would like to thank Rahma Micho Widy- biotechnology Engineering, The Institute for anto from cell culture laboratory of LAPTIAB who Applied Research, Ben-Gurion University of has done the maintenance of MCF-7 cells. We the Negev, Israel. Hal 1211. also would like to thank Siti Aisyah and Purnama Dewick, P.M. 1997. Medicinal Natural Products. A Dwi Tistianto, bachelor students of Departement Biosynthetic Approach. John Wiley & Sons, of Pharmacy, Faculty of Medicine and Health Sci- New York: x + 466p. ence, State Islamic University Syarif Hidayatullah Evans, C.W. 2002. Pharmacognosy. 15th edition. Jakarta, who have helped in extraction and frac- W.B. Saunders, London: xi + 585p. tination work in laboratorium of phytochemistry, Hibasami H., Moteki H., Ishikawa K., Katsuzaki H., LAPTIAB, Serpong. Imai K., Yoshioka K., Ishii Y., and Komiya T. 2003. Protodioscin isolated from fenugreek REFERENCES (Trigonella foenum-graecum L.) induces cell Agustini, Kurnia, Sumali W., Dadang K. 2007. death and morphological change indicative Estrogenic Effect of Fenugreek (Trigonella of apoptosis in leukemic cell line H-60, but foenum-graecum L.) on White Female Rats. not in gastric cancer cell line KATO III. Int. Conference Proceedings “Women’s Health J. Mol. Med., 11(1): 23-6 and Traditional Medicine”, International Moalic S., Liagre B., Corbiere C., Bianchi A., Dauca Medicine and Medicinal Plants, Surabaya. M., Bordji K., and Beneytout JL. 2001. A Annida B., Prince SMP. 2004. Supplementation plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in streptozotocin-induced diabetic rats. J. Med. osteosarcoma cells. FEBS Lett; 506(3):225- of fenugreek leaves lower lipid profile in Food., 7(2): 153-156 30.

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on MCF-7 human breast cancer cells. Intl. Antiproliferating effects of ginsenoside Rh2 Journal of Oncology., 14(5):869-875. Raju J., Patlolla JM., Swamy MV., Rao CV. 2004. Diosgenin, a steroid saponin of Trigonella foenum-graecum (fenugreek), inhibits azoxymethane-induced aberrant crypt foci formation in F344 rats and induces apoptosis in HT-29 colon cancer cells. Cancer Epidemiology Biomarkers Prevention. 13(8): 1392-8. Smith RSM., and Osborne MP. 2000. Breast cancer chemoprevention. Am. J. Surg. 180: 249- 251. Sur P., Das M., Gomes A., Vedasiromoni JR., Sahu NP., Banarjee S., Sharma RM., Ganguly DK. 2001. Trigonella foenum-graecum (fenugreek) seed extract as an antineoplastic agent. Phytother Res., 15 (3):257-9. Mimaki Y., Yokosuka A., Kuroda M., Sashida Y. 2001. Cytotoxic Activities and Structure-Cytotoxic Relationship of Steroidal Saponins. Biol. Pharm. Bull., 24(11): 1286-9. Wiryowidagdo, Sumali. 2001. Kimia dan Farmakologi Bahan Alam. Universitas Indonesia, Jakarta: viii + 339p.

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