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: kurnia_atini@yahoo.com
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 ak-tivitas sitotoksik TGF pada sel kanker payudara MCF-7 serta kadar steroid total. Ekstrak metanol TGF
difraksi-nasi dengan etil asetat, n-heksan, dan n-butanol. Tiap fraksi dipisahkan dengan kromatografi kolom. Aktivitas
sitotoksik pada sel MCF-7 diuji dengan uji MTT [3-(4,5-dimetiltiazol-2-il)2,5-difeniltetrazolium]. Kadar ste-roid total ditetapkan dengan metode spektrofotometri. Hasil penelitian menunjukkan tidak terdapat 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.
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 trigo-neoside (saponine like estrogen) which have ef-fect as phytoestrogen for menopause symptoms therapy. Sapogenins are the aglycones or non-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.
TFG contains diosgenin in base free form 0.8-2.2 % (Wiryowidagdo, 2000). TFG also con-tains alkaloids (trigonelline, an alkaloid pyridine,
gentianin and karpain), flavonoids (vitexin in gly -coside or ester form, isovitexin, orientin, vicenin, quercetin and luteolin), fatty oil 20-30%, essen-tial oil, saponine, nicotinamide, choline, bitter compound and mucilage (Evans, 2002).
Protodioscin
Tigogenin
Figure 1. Some sapogenine steroids of Trigonella foenum-graecum L.
Empirically TFG was use as aphrodisiac, carminative, diuretic, emmenagogue, emollient, galactogogue and tonic (WHO, 2007). Many re-searches, preclinical and clinical, showed that TFG have activity for diabetic disease (Annida, 2004). TFG also predicted as Phytoestrogen cause have
estrogenic effect on ovariectomized female rat
adaptation on the new level hormone (Badziad, 2003).
Extracts of TFG seeds and some of their sa-ponine constituents also have been found to have
anticarcinogenic activity in different setting. It
has been evaluated in the Ehrlich ascites
carci-noma model in BALB/c mice, where it affected
70% inhibition of tumor cell growth compared with controls (Sur, 2001). Hibasami et al., (2003) suggest that growth inhibition of human leuke-mia HL-60 cells by protodioscin, isolated from TFG seeds, results from the induction of apopto-sis. Moalic et al., (2001) reported that diosgenin inhibits cell proliferation in the human osteosar-coma 1547 cell line by induction of apoptosis and G1 phase cell cycle arrest.
Refer to data that showed TFG having es-trogenic activity as phytoestrogen, inspiring to study its possibility as Selective Estrogen Recep-tor ModulaRecep-tors (SERMs) candidate. SERMs such as tamoxifen, which are used clinically for the treatment of breast cancer, act as estrogen ago-nists in certain tissues but exhibit antiestrogenic
effects in others (Rosenbaum, 2000). There are
several researches about SERMs candidates for post tumor surgery therapy from phytoestrogen pure compounds, such as dammarane, a sapoge-nine steroid from ginseng (Oh, 1999), Genistein
(an isoflavone from soybean) and resveratrol, a
stilbene from grape (Baht, 2001).
This research was done to investigate the correlation between TFG’s cytotoxicity on hu-man breast cancer cell line MCF7 and its total steroids level in methanolic extract and its frac-tioned (hexane, buthanol and ethylacetate).
MATERIALS AND METHODS Samples Preparation
TFG seeds were obtained from Tawang-mangu, Central Java, Indonesia. Seeds were dried and grind, then were extracted with methanol. The methanolic extract was fractioned with n-hexane, ethylacetic (EtOAc) and n-buthanol. Fu-ther, each fraction then separated by vacuum liq-uid chromatography using various eluens. Every extract and fraction was dried with vacuum ro-tary evaporator.
Total Steroid Analysis (Chapagin et al., 2005)
1 mg dried extract/ fraction diluted in 2 mL ethylacetate in a tube, then 1 ml reagent A (contains p-anysaldehyde and ethylacetate (0.5 : 99.5)) and 1 ml reagent B (contains sulfuric acid glacial and ethyl acetate (1:1)) were added. Tube was put in water bath 600C for 10 minutes
till color was occurred and then cooled in an-other water bath 250C for 10 minutes. Color was
measured by Spectrophotometer UV Vis 423nm, against ethyl acetate solution as reagent blank. Results were compared with curve standard of Diosgenin (Sigma).
Cell Culture
The cell lines MCF-7 (Human Breast Ad-enocarcinoma) was obtained from Laboratory for Development of Industrial Agro and Biomedi-cal Technology (LAPTIAB-BPPT) Indonesia. Cells were routinely maintained and grown in 75 cm2
flasks at 370C, 5% CO
2 and in a 95% humidified
Foetal Bovine Serum (FBS, Gibco Life Technolo-gies) which already heat inactivated at 560C for
30 min. Passaging of cells was carried out using 4 ml of trypsin-EDTA at room temperature for 75 cm2 flask, respectively for 3 min. After that, 10
ml media with 10% FBS were used to reduce the action of trypsin on cells. After centrifugation, the obtained cells were platted.
Cytotoxicity test with MTT method
Cells were platted into 96-well plates (10,000 cells/well) in medium RPMI with phenol red containing 10%Fetal Bovine Serum (FBS), 100U/ml penicillin, 0.1 mg/ml streptomycin and 1 mM sodium pyruvate, then incubated for 24 hours at 370C, 5% CO
2 and in a 95%
humidi-fied atmosphere. After 24 hours, medium was
changed with samples (extracts and phases of
TFG) in growth medium in different concentra -tion and incubated for another 24 hours at 370C,
5% CO2 and in a 95% humidified atmosphere. Assays were done in wide range concentration, from 10 ppm until 500 ppm, divide into six varia-tion concentravaria-tion.
After 24 hours treatment, the cells were
washed with Phosphate Buffer Saline (PBS).
Then the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium) solution in medium, was added followed by incubation for 4 hours at 370C,
5% CO2 and in a 95% humidified atmosphere. The crystal of formazan blue will be formed. Af-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.
RESULTS AND DISCUSSION
Figure 2. Comparative of cytotoxicity activity on MCF7 (IC50) and its total steroid level
from methanolic extract and its first
fractionation.
Figure 3. Comparative of cytotoxicity activity on MCF7 (IC50) and its total steroid level from column
chromatographic fractions of Ethylacetic fraction.
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.
Figure 4. Comparative of cytotoxicity activity on MCF7 (IC50) and its total steroid level from column chromatographic fractions of n-Hexane fraction.
To investigate more about the correlation of these two parameters, the screening to n-Hexane fraction also was done. After separated by column chromatography using also gradient elucidation of mixed dichloromethane and ethanol, each fraction then screened by MTT assay and their level of Steroids. Results are showed in Figure 4. The most active fraction is HX4 with IC50 = 35.57 ppm and the less active fraction is HX3 with IC50
= 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 of some sapogenin steroids were sensitive to the monosaccharides constituting the sugar moieties and their sequences, as well as to the structures of the aglycons. They also concluded that structure– activity relationships of (25R)-Spirost-5-en-3b-ol (Diosgenin) glycoside derivatives Diosgenin b-D-glucoside showed no cytotoxic activity against HL-60 cells (IC50>20ppm)
CONCLUSIONS
of total steroid in first fractination is buthanolic
fraction (25.31 ppm). While from coloumn chromatographic results of ethylacetic fraction, the highest total steroid level given by EA3 (27.533 ppm). The cytotoxic activity on MCF-7 of TFG maybe caused by another group compound except its sapogenin steroid. The isolation of active compound should be continue. So we can conclude that there was no correlation between cytotoxic activity on MCF-7 cell and total steroid
level. The cytotoxic effect on MCF-7 cell from TFG
is not caused by its steroid compounds.
ACKNOWLEDGEMENT
We would like to thank Rahma Micho Widy-anto from cell culture laboratory of LAPTIAB who has done the maintenance of MCF-7 cells. We also would like to thank Siti Aisyah and Purnama Dwi Tistianto, bachelor students of Departement of Pharmacy, Faculty of Medicine and Health Sci-ence, State Islamic University Syarif Hidayatullah Jakarta, who have helped in extraction and frac-tination work in laboratorium of phytochemistry, LAPTIAB, Serpong.
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