THE ACTIVITY OF EURYCOMANONE DERIVATIVES ON CANCER CELL LINESHTML Full Text
THE ACTIVITY OF EURYCOMANONE DERIVATIVES ON CANCER CELL LINES
H. Yusuf * 1, D. Satria 2 and Zulkarnain 3
Department of Pharmacology 1, Department of Pathology Anatomy 2, Department of Physiology 3, Faculty of Medicine Universitas Syiah Kuala, 23111 Banda Aceh, Indonesia.
ABSTRACT: Eurycomanone from the roots of Eurycoma longifolia Jack (ELJ) has been reported to exhibit anticancer activity. Four of ester eurycomanone derivatives: eurycomanone dibutyrate, eurycomanone monovalerate, eurycomanone dimethoxybenzene, and eurycomanone disuccinate were synthesized for knowing their activities on cancer cell lines T47D, MCF-7, Hela, WIDR and Vero cells. The cancer activity was evaluated by the MTT colorimetric assay method. The results showed that eurycomanone has anticancer activity on T47D, MCF-7, Hela, WIDR cancer cells with IC50 values (1.17 ± 0.09; 3.96 ± 0.02; 2.95 ± 0.08; 1.45 ± 0.01 µg/mL), and no toxic to Vero cells (609.89 ± 29.77 µg/mL). Its derivatives eurycomanone dibutyrate have anticancer activity on T47D, MCF-7, Hela, WIDR cancer cells with IC50 values (25.16 ± 2.25; 21.56 ± 4.55; 29.32 ± 1.25; 149.42 ± 12.50µg/mL), eurycomanone monovalerate (25.59 ± 1.31; 22.48 ± 1.25; 30.14 ± 1.89; 91.88 ± 8.90 µg/mL), eurycomanone dimethoxybenzene ( 102.77 ± 2.56; 38.83 ± 2.55; 66.65 ± 1.90; 51.61 ± 2.37µg/mL), eurycomanone disuccinate (218.94 ± 9.30; 198.87 ± 5.50; 166.67 ± 12.34; 145.39 ± 6.67 µg/mL) respectively. The conclusion of this study showed eurycomanone dibutyrate, eurycomanone monovalerate have low activity as anticancer and safe to Vero cells. Eurycomanone dimethoxybenzoate are safe to Vero cells, besides that eurycomanone disuccinate toxic to Vero cells.
Anticancer, Eurycomanone, Eurycoma longifolia Jack, Synthesis
INTRODUCTION: Natural compounds from plants have been proven as a source of lead compounds for developing new drugs 1, 2. Eurycomanone is a natural pentacyclic quassinoid obtained from the roots of Eurycoma longifolia Jack in the family of Simaroubaceae 3. This popular plant uses as an aphrodisiac and the roots usually taken for preparing pharmaceutical products 4.
Various natural compounds have been isolated and characterized, mostly from the extract of the ELJ roots. The compounds have been reported to have a wide pharmacological activities such as anti-malarial, anticancer and antipyretic and anti-ulcer 5. Eurycomanone is one of the major natural quassinoids isolated from the roots and has exhibited cytotoxic activities against selected cancer cell line 3, 4.
Its pharmacological potency has been proven in numerous in-vitro and in-vivo experimental laboratory. But until now very limited efforts to develop this compound for obtaining its derivatives. Therefore, this study was conducted for knowing the anticancer activity of synthesized eurycomanone derivatives on selected cancer cells lines (T47D, MCF-7, Hela, WIDR) and Vero cells by MTT Colorimetric Assay Methods 6. The synthesis of eurycomanone derivatives was done by using eurycomanone natural compound and esterified without using a protecting agent. Esterifying of eurycomanone by using pharmacophore agents such as butirylchloride, valeroylchloride, para methoxy benzoyl chloride, and succinate anhydride. This study was approved by The Institutional Ethics Commission, Faculty of Medicine Universitas Syiah Kuala with number: 46/KE/FK/2017).
MATERIALS AND METHODS:
Materials: The plant and roots of ELJ were taken after 4 years of cultivation and identified by a specialist. The certificate of identification with number BF/123/Ident/Det/III/2010. The used of pharmacophore agent in this study (butirylchloride, valeroylchloride, para methoxy benzoyl chloride, and succinate anhydride) were pro analytic grade.
Methods: The roots of ELJ (10kg) were cleaned with tap water and then dried in the oven at 40 ºC. After cutting in small pieces, the dried roots were ground into a crude powder and stored in the desiccators. Then the crude powder was soaked in 30L methanol at room temperature and stirred regularly. The liquid extract was filtered and concentrated in a rotary evaporator at 40 ºC to produce the methanolic extract.
Isolation of Eurycomanone: Before isolation the methanolic extract of ElJ was subjected to vacum liquid chromatography (VLC) by using stationary phase silica gel and the mixed mobile phase chloroform: methanol: water in the ratio (5:5:1; 3:7:1: 1:9:1). Fractions with similar Rf values on thin layer chromatography (TLC) which were monitored at UV lamp at 254 nm, then pooled and used for isolation of eurycomanone as starting material for synthesizing its derivatives.
Isolation of eurycomanone was done by preparative thin layer chromatography (PTLC) using silica gel PF254 as stationary phase and the mixed mobile phase ethyl acetate: methanol: water in ratio 80: 20: 1. Repeated isolation, purification, and crystallization were done to get the pure compound. The structure and its purity were confirmed by comparison with detailed spectroscopic data in published reports (UV, IR, NMR, LCMS-ESI positive ion, DEPT, COSY, HMQC, and HMBC).
Synthesis of Eurycomanone Derivatives: Synthesis of eurycomanone derivatives can be performed by simple esterification without using a protecting agent. Eurycomanone which is isolated from the extract of ELJ roots (50 mg, 0,1225 mmol) was dissolved in cold pyridine (1 mL) and pharmacophore agent (butiryl chloride, valeroyl chloride, para methoxy benzoyl chloride, and succinate anhydride 0.49 mmol, respectively) dissolved in cold chloroform. The solution of pharmacophore agent was added slowly to the eurycomanone solution at 0 ºC, and the reaction mixture was stirred for 1 hour in an ice bath. After that, the reaction mixture was refluxed and stirred using magnetic heat stirrer for 6-8 h, and every 2 h checked the product by TLC.
After the esterification process ended, the mixture was extracted three times with 10 ml of cold ethyl acetate. The ethyl acetate layer is washed three times with 10 mL cold water and then dried with sodium sulfate anhydrate. After filtration and drying, the cooled precipitate is poured in methanol and preparing for detailed spectroscopic analysis and used for anticancer activity testing.
Testing of Anticancer Activity: The anticancer activity test of eurycomanone and its derivatives on Vero cells and cancer cell lines (T47D, MCF-7, Hela, WIDR) were carried out by MTT Colorimetric Assay Methods. The tested compounds were used at concentration 25; 12,5; 6,25; 3,125; 1,57625, 0,78125 µg/mL and prepared from the substock solutions by serial dilution of media to give a volume of 100 µL in each microtitre plate well. The concentration of tested compounds was prepared in triplicate. As standard drug used doxorubicine and 5- fluorouracil in the same concentration. Then each well was added with 100 µL of 104 mL of cells in complete growth media, respectively. As controls were used the cells and media that were placed into 96 well microplates then incubated for 24 h at 37 ºC, 5% CO2 and 90% humidity.
After incubation, the media was removed and 100 µL of the new medium and 10 µL MTT was added. Then, it was incubated again for 4 h and next to the media was aspirated and 100 µL SDS 10% in 0.001N HCl added. The microplate was re-incubated for 24 h in room temperature and its absorbance was read at λ 405 nm (Vero cells) and 595 nm (cancer cell lines) by ELISA reader. The IC50 value on Vero cells and cancer cell lines were determined by probit regression analysis.
RESULTS AND DISCUSSION: Firstly, after maceration, the solid extract of ELJ roots is yielded ± 6%, then after fractionation yielded ± 2.5% of eurycomanone fraction. Isolation of eurycomanone from this fraction was performed by preparative thin layer chromatography (TLC) and yielded ± 0.04%.
Eurycomanone is the potential quassinoid anticancer was structurally esterified by using acyl chloride and carboxylic anhydride to influence their activity and cytotoxicity to cancer cell lines and normal cells. Synthesis of its derivatives by esterification is attempted to increase activity, decreasing toxicity, or improving other pharmacological profiles. In finding new anticancer with better activity than the previous compound, it was esterificated OH group in eurycomanone structure by butiryl chloride, valeryl chloride, para-methoxybenzoyl chloride, and succinic anhydride. The result of esterification yielded eurycomanone dibutyrate (60.35%), eurycomanone monovalerate (55.10%), eurycomanone dimethoxybenzoate (60.10%) and eurycomanone disuccinate (65.25%). The chemical structure of all tested compounds had been analyzed by spectroscopic analysis.
Eurycomanone as starting material has formula C20H24O9 (MW 408.02; m.p 254º-257 ºC) and its derivatives eurycomanone dibutyrate C28H36O11 (MW 548,94; m.p 241-243 ºC), eurycomanone monovalerate C25H32O10 (MW 492,8; m.p 235-237 ºC), eurycomanone dimethoxybenzoate C36H36O13 (MW 676.13; m.p 225-228 ºC) and eurycomanone disuccinate C28H30O15 (MW 606,86, m.p 251-254 ºC). The evaluation of tested compounds on Vero cell is aimed for knowing the safety of these compounds on normal cells. In addition, these compounds are also used for examining their activities growth inhibition of cancer cell lines.
The IC50 and selectivity index values of these compounds on cancer cell lines and Vero cells are showed in Table 1 and 2.
TABLE 1: THE IC50 OF EURYCOMANONE AND ITS DERIVATIVES ON SELECTED CANCER CELL LINES AND VERO CELLS
|Tested compounds||IC50 Values (µg/mL)|
|Eurycomanone||609.8 ± 29.7||1.1 ± 0.1||3.9 ± 0.0||2.9 ± 0.0||1.4 ± 0.0|
|E. dibutyrate||219.2 ± 11.3||25.1 ± 2.2||21.5 ± 4.5||29.3 ± 1.2||149.4 ± 12.5|
|E. monovalerate||92.4 ± 7.5||25.5 ± 1.3||22.4 ± 1.2||30.1 ± 1.8||91.8 ± 8.9|
|E dimethoxy benzoate||13.2 ± 6.9||102.7 ± 2.5||38.8 ± 2.5||66.6 ± 1.9||51.6 ± 2.3|
|E. disuccinate||12.7 ± 2.8||218.9 ± 9.3||198.8 ± 5.5||166.6 ± 12.3||145.3 ± 6.6|
|Doxorubicine||3.5 ± 0.6||1.9 ± 0.1||4.6 ± 0.1||3.5 ± 0.0||41.8 ± 2.2|
TABLE 2: THE SELECTIVITY INDEX OF EURYCOMANONE AND ITS DERIVETIVES ON SELECTED CANCER CELL LINES
|Tested compounds||Selectivity Indexes|
|E dimethoxy benzoate||128.7||340.6||198.4||256.2|
In-vitro screening of anticancer activity of eurycomanone and its derivatives is based on the ability of the compounds to inhibit the growth of cancer cell lines in medium culture. Previous studies showed the anticancer activity of eurycomanone on various cancer cell lines has IC50 value on MCF-7 is 4.40 ± 0.42 µg/mL 7; 3.63 ± 0.11 µg/mL 8; and less than 2.5 µg/mL 9; 1.1 µg/mL 10. The anticancer activity of eurycomanone on Hela cells has IC50 value 2.13 ± 0.09 µg/mL 7.
The result of the test toward four synthesized compounds showed that eurycomanone more potent than monoacetylated and diacetylated eurycomanone to selected cancer cell lines above. Some structural requirements, like an α, β-unsaturated ketone in the A ring, an oxymethylene bridge in the C ring and an ester function in C-15 in the D ring are considered very important for the anticancer activity which is presented by quassinoids 11, 12.
CONCLUSION: The data suggest that eurycomanone has anticancer activity more potential than its derivatives on selected cancer cell lines (T47D, MCF-7, Hela and WIDR) and safe to normal Vero cells. Eurycomanone dibutyrate, eurycomanone monovalerate and eurycomanone dimethoxybenzoate have a low activity as anticancer and safe to Vero cells with selectivity index (IS) more than 3, besides that eurycomanone disuccinate toxic to Vero cells.
ACKNOWLEDGEMENT: This study was supported by the Ministry of Education and Culture of Indonesia. The authors would like to thank Universitas Syiah Kuala for this grant. The authors are grateful to the Indonesian Institute of Sciences (LIPI) for analyzing our synthesis compounds.
CONFLICT OF INTEREST: There is no conflict of interest in this study
- Orlikova B, Legrand N, Panning J, Dicato M and Diederich M: Anti-inflammatory and anticancer drugs from nature. Cancer Treat. Res 2014; 159: 123-43.
- Hajjouli S, Chateauvieux S, Teiten MH, Orlikova B, Schumacher M, Dicato M, Choo CY and Diederich M: Eurycomanone and Eurycomanol from Eurycoma longifolia Jack as Regulators of Signaling Pathways Involved in Proliferation, Cell Death and Inflammation. Molecules 2014; 19: 14649-66.
- Hnin ET, Zaid H, Mohamed IN and Shuid AN: Eurycoma longifolia, a potential phytomedicine for the treatment of cancer: Evidence of p53-mediated apoptosis in cancerous cells. Current Drug Targets 2018; 19(10): 1109-26.
- Li CH, Liao JW, Liao PL, Huang WK, Tse LS, Lin CH, Kang JJ and Cheng YW: Evaluation of Acute13-week subchronic toxicity and genotoxicity of the powdered root of Tongkat Ali (Eurycoma longifolia Jack). Evidence-Based Complementary and Alternative Med 2013; 1-11.
- Nurani LH: Isolation and free radicals scavenging activity of isolate-1, ethyl acetate fraction, and ethanolic extract of Pasak Bumi (Eurycoma longifolia Jack) root. Faculty of Pharmacy Ahmad Dahlan University, Yogyakarta 2013.
- Mosman T: Rapid colorimetric assay for celluler growth and survival application to proliferation and cytotoxicity assays. Journal of Immunology Method 1983; 65: 55-63.
- Tee TT and Hawariah LP: Induces of Apoptosiss by longifolia Jack extract. Anticancer Res 2005; 25: 2205 -14.
- Nurhasanah M and Hawariah LP: Eurycomanone induces apoptosis through the Up regulation of p53 in human cervical carcinoma cells. Paper Research International Conference on Chemical Sciences (ICCS-2007), Yogyakarta, Indonesia 2007.
- Kuo PC, Damu AG, Lee KK and Wu TS: Cytotoxic and antimalarial constituent from the roots of Eurycoma longifolia. Journal of Bioorganic Medicinal Chemistry 2004; 12: 537-44.
- Kardono LBS, Angerhofer CK, Tsauri S, Padmawinata K, Pezzuto JM and Kinghorn AD: Cytotoxic and antimalarial constituents of the roots of Eurycoma longifolia. Journal of Natural Product 1991; 54: 1360-67.
- Kupchan SM, Britton RW, Lacadie JA, Ziegler MF and Siegel CWJ: The Isolation and Structural Elucidation of Bruceantin and Bruceantinol, New Potent Antileukemic Quassinoids from Bruceae antidysenterica. Journal Organic Chemistry 1975; 40: 648-54.
- Kupchan SM, Fessle DC, Eakin MA and Giacobbe TJ: Reaction of alpha methylen lactone tumor promoter inhibitors with model biological nucleophiles. Science 1970; 168: 376-77.
How to cite this article:
Yusuf H, Satria D and Zulkarnain: The activity of eurycomanone derivatives on cancer cell lines. Int J Pharm Sci & Res 2019; 10(6): 2947-50. doi: 10.13040/IJPSR.0975-8232.10(6).2947-50.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
H. Yusuf *, D. Satria and Zulkarnain
Department of Pharmacology, Faculty of Medicine Universitas Syiah Kuala, Banda Aceh, Indonesia.
02 October 2018
18 January 2019
14 February 2019
01 June 2019