|Year : 2011 | Volume
| Issue : 3 | Page : 189-193
Cucurbitacins-type triterpene with potent activity on mouse embryonic fibroblast from Cucumis prophetarum, cucurbitaceae
Seif-Eldin N Ayyad1, Ahmed Abdel-Lateff2, Salim A Basaif1, Thomas Shier3
1 Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Saud Arabia
2 Department of Health Information Technology, Jeddah Community College, King AbdulAziz University, P.O. Box 80283, Jeddah 21589, Saudi Arabia; Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt
3 Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
|Date of Submission||11-May-2011|
|Date of Decision||25-Jun-2011|
|Date of Web Publication||16-Sep-2011|
Seif-Eldin N Ayyad
Department of Chemistry, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Kingdom of Saud Arabia
Source of Support: SABIC, the Saudi Arabian Company for Basic Industries,
for the fi nancial support of this work (MS/8/68), through the collaboration with
the Deanship of Scientifi c Research (DSR) at King Abdul-Aziz University, Conflict of Interest: None
| Abstract|| |
Background: Higher plants are considered as a well-known source of the potent anticancer metabolites with diversity of chemical structures. For instance, taxol is an amazing diterpene alkaloid had been lunched since 1990. Objective: To isolate the major compounds from the fruit extract of Cucumis prophetarum, Cucurbitaceae, which are mainly responsible for the bioactivities as anticancer. Materials and Methods: Plant material was shady air dried, extracted with equal volume of chloroform/methanol, and fractionated with different adsorbents. The structures of obtained pure compounds were elucidated with different spectroscopic techniques employing 1D ( 1 H and 13 C) and 2D (COSY, HMQC and HMBC) NMR (Nuclear Magnetic Resonance Spectrometry) and ESI-MS (Eelectrospray Ionization Mass Spectrometry) spectroscopy. The pure isolates were tested towards human cancer cell lines, mouse embryonic fibroblast (NIH3T3) and virally transformed form (KA3IT). Results: Two cucurbitacins derivatives, dihydocucurbitacin B (1) and cucurbitacin B (2), had been obtained. Compounds 1 and 2 showed potent inhibitory activities toward NIH3T3 and KA31T with IC 50 0.2, 0.15, 2.5 and 2.0 μg/ml, respectively. Conclusion: The naturally cucurbitacin derivatives (dihydocucurbitacin B and cucurbitacin B) showed potent activities towards NIH3T3 and KA31T, could be considered as a lead of discovering a new anticancer natural drug.
Keywords: Cucumis prophetarum, cucurbitacin B, dihydrocucurbitacin B, mouse embryonic fibroblast and virally transformed form
|How to cite this article:|
Ayyad SEN, Abdel-Lateff A, Basaif SA, Shier T. Cucurbitacins-type triterpene with potent activity on mouse embryonic fibroblast from Cucumis prophetarum, cucurbitaceae. Phcog Res 2011;3:189-93
|How to cite this URL:|
Ayyad SEN, Abdel-Lateff A, Basaif SA, Shier T. Cucurbitacins-type triterpene with potent activity on mouse embryonic fibroblast from Cucumis prophetarum, cucurbitaceae. Phcog Res [serial online] 2011 [cited 2020 May 29];3:189-93. Available from: http://www.phcogres.com/text.asp?2011/3/3/189/85006
| Introduction|| |
Generally, there are two different approaches used for discovering of antitumor compounds; bio-chemical approach and target-based approach. The first approach has gained a significant attention in the last decades.  This resulted in discovery of antitumor agents. For instance, Food and Drug Administration (FDA) approved imatinib mesilate as a first-line treatment for chronic myelogenous leukemia.  The use of high-throughput screening is aimed at discovery of anticancer agents employing mouse embryonic fibroblast (NIH3T3) and virally transformed form (KA3IT) cells. The selection of these cells based on that they are adherent, easily manipulated, and well characterized. ,
The Cucurbitaceae are mostly known prostrate or climbing herbaceous annuals plants comprising about 125 genera and 960 species, includes the melons and gourd crops like cucumbers. The family is predominantly distributed around the tropics, where edible fruits are grown. The diversity of the cucurbitacins' activities, especially cytotoxicity and antifeedants, is a good evidence for further investigations. ,, Recently, they were exploited for their antitumor properties, differential cytotoxicity toward renal, brain tumor, and melanoma cell lines,  inhibition of cell adhesion,  and finally, antifungal effects.  A computer survey includes science finder data base, indicated that a number of cucurbitacins were isolated from genus Cucumis. For example, cucurbitacins were isolated from Cucumis prophetarum: cucurbitacin (B, E, I, O, P, and Q1); dihydocucurbitacin (D and E), isocucurbitacin (B, D, and E) and dihydroisocucurbitacin (D and E). ,,,,
In continuation of our research program which interested in the isolation of the bioactive secondary metabolites from marine macro organisms or higher plants, collected from Saudi Arabia. ,, The fruits of Cucumis prophetarum L., belongs to family Cucurbitaceae, wild plant growing in the desert of Makah, 80 km from Jeddah Saudi Arabia. The total extract (Chloroform: Methanol [1: 1]) had been fractionated using different chromatographic techniques, led to purification of two cucurbitacins derivatives; dihydocucurbitacin B ( 1 ) and cucurbitacin B ( 2 ). The compounds 1 and 2 [Figure 1] showed potent inhibitory activities toward mouse embryonic fi broblast (NIH3T3) and virally transformed form (KA3IT) cells with IC 50 0.2, 0.15, 2.5, and 2.0 μg/ml, respectively.
| Materials and Methods|| |
Chromatographic material: silica gel type 60-120 mesh.was used form Column Chromatography (CC). Silica gel GF 254 was used for Thin Layer Chromatography (TLC). Finally, silica gel 60 F 254 was used for Preparative Thin Layer Chromatography (PTLC). Electron impact mass spectra were determined at 70 ev on a Kratos EIMS-25 instrument. All NMR spectra were recorded on (1D and 2D) NMR spectra were recorded on a Varian VI-500 MHz spectrometer. LC-CIMS was performed using an API 2000, LC MS/MS from Applied Biosystems/MDS Sciex. Bruker Avance 300 DPX and 500 DRX spectrometers in CDCl 3 . Chemical shifts are given δ (ppm) relative to TMS as internal standard. The spray reagent used is: Anisaldehyde-sulphuric acid. A freshly prepared solution was made by adding concentrated sulphuric acid (1 ml) to a solution of anisaldehyde (0.5 ml) in acetic acid (50 ml).
Cucumis prophetarum L. was collected from wild plants growing from the desert of Makah, Saudi Arabia. The fresh fruits were separated, air-dried, and powdered. A voucher sample was deposited at the Chemistry Department, Faculty of Science King Abdulaziz University, Jeddah, Saudi Arabia.
Extraction and purification
The fruits of C. prophetarum (500 gm) were extracted twice by chloroform: methanol (1:1) at room temperature. The extract was concentrated under reduced pressure and led to yellowish brown residue (25 gm). This material was chromatographed on a column of silica gel. The total extract was fractionated by NP silica (500 gm, 80 cm Χ 2.5 cm) employing pet. Ether/CHCl 3 /MeOH (50 ml each fraction). The fractionation was followed by TLC using anisaldehyde-sulphuric acid as spraying reagent. The fraction eluted by chloroform: methanol (9: 1) was collected and purified by Sephadex LH 20 using methanol: chloroform (3:1) followed by preparative TLC silica gel and chloroform: Methanol (9+1), led to 1 (200 mg) and 2 (100 mg)
Cytotoxic assays , were performed using two proliferating mouse cell lines, a normal fibroblast line NIH3T3 and a virally transformed form KA3IT. Samples of extract or pure compound (5 mg) were dissolved in 62.2 μl of Dimethyl sulfoxide (DMSO), and working solutions made by diluting 20 μl of the DMSO solution into 2 ml of sterile medium (Dulbecco's modified Eagle's medium, Sigma Chemical Co. St. Louis, MO, USA). Two-fold or 2.5-fold dilutions of the extracts of pure compounds from 200 μg/ml to 0.5 μg/ml were prepared in triplicate in the wells of 96-well culture trays (Falcon Micro Test III, # 3072, Becton Dickinson Labware, Lincoln Park, NJ, USA) in 200 μl of medium containing 5% (v/v) calf serum (Hyclone Laboratories, Logon, Utah, USA). Inoculums of 2 \ 103 cells were added to each well in a 100 μl aliquot of 10% calf serum in medium. The 96-well trays of cells were cultured under standard conditions until uninhibited cultures (control) became confluent. The contents of the wells were decanted, and each cell layer washed with a small amount of the medium. The wells were filled with formal saline (3.7% w/v formaldehyde in 0.15 ml NaCl), and allowed to stand at room temperature for at least 30 minutes. The trays was washed with tap water, and attached cells stained by adding two drops of 0.5% (w/v) crystal violet solution in 20% (v/v) aqueous methanol added to each well. The trays was washed with tap water, and the IC 50 estimated visually as the approximate concentration that causes 50% reduction in the number of stained cells adhering to the bottom of the wells.
| Results and Discussion|| |
The chromatographic purification of the total extract obtained from fruits of C. prophetarum resulted in the isolation of two compounds belong to cucurbitacins-type triterpens; dihydocucurbitacin B ( 1 ) and cucurbitacin B ( 2 ). They identified by employing 1D ( 1 H and 13 C) and 2D (COSY, HMQC and HMBC) NMR, and ESI-MS spectroscopy. The pure isolates were tested towards human cancer cell lines, mouse embryonic fibroblast (NIH3T3) and virally transformed form (KA3IT). The compounds 1 and 2 showed cytotoxic activities toward NIH3T3 and KA31T with IC 50 0.2, 0.15, 2.5, and 2.0 μg/ml, respectively.
The structure elucidation of 1, commenced when the molecular formula of C 32 H 48 O 8 was established by LC-ESI-MS at 583 [M + + Na]. This result was validated by HRESIMS, m/z 583.3245 [M+ Na] + . The 13 C NMR spectra ( 1 H decoupled and DEPT) of 1 showed 32 resonances attributable to 9 X CH 3 , 7 X CH 2 , 6 X CH, and 10 X C [Table 1]. Three of the eight elements of unsaturation, as indicated by the molecular formula of 1, deduced to be a carbonyl group appeared at δc 213.9, 212.9, and 212.0, for C-3, C-11, and C-22, respectively, and an olefinic proton at δc 5.79 (J= 5.5) assigned for H-6 of the double bond at ∆ (5)6 . The molecule, thus, has four rings. As the 1 H and 13 C NMR data enabled all but three of the hydrogen atoms within 1 to be accounted for, it was evident that the remaining three protons were present as part of a hydroxyl functions. After association of all the protons with directly bonded carbons via 2D NMR (HMQC) spectral measurements, it was possible to deduce the structure of 1 by interpretation of the 1 H- 1 H COSY and 1 H - 13 C HMBC spectra. From the 1 H- 1 H COSY spectrum of 1, a 1 H- 1 H spin system between H-2 and H 2 -1 and between H 2 -1 and H-10 was observed. Long-range C-H correlations observed between the resonances of H-8 and those of, C-6, C-7, C-9, C-10 and C-19; between H 2 -1 and C-2, C-3, C-5, C-8, C-9, and C-19; between H-2 and C-1, C-3, C-4, and C-10; between H-6 and C-4, C-5, C-7, C-8, and C-10; between H 3 -29 and C-3, C-4, and C-5 and between H 3 -30 and C-3, C-4, and C-5 established ring A and B, which are fused together. HMBC correlations, this time observed between H 3 -28 and C-8, C-14, and C-15, as well as correlations between H-16 and C-13, C-14, C-15, and C-17, and also correlations between H 3 -18 and C-12, C-13, C-14, C-17 were observed. Long-range C-H correlations observed between the resonances of H-12 and C-11, C-13, C-17, were observed. Ring C was established by deduction through the previous data. From the 1 H- 1 H COSY spectrum of 1, a 1 H- 1 H spin system between H 2 -15 and H-14 and H 2 -16 and between H 2 -16 and H-17 were observed indicated the C-C bond between C-14 to C-15 and C-15 to C-16 and C-16 to C-17. Ring D was established based on 1 H- 1 H COSY and 1 H - 13 C HMBC spectra. The main skeleton of 1 was established as steroidal derivative. From 1 H- 1 H COSY, correlations were observed between H 2 -23 and H 2 -24. Extensive investigation of the HMBC correlation between H 3 -21 and C-20 and C-22 (C=O) was observed. Long range correlations between C-22 and H 2 -23 and H 2 -24 led to establishing the side chain as iso-heptane derivative. This side chain is attached to the steroidal nucleus the connection between C-17 and C-20 based on HMBC correlations, between H 3 -21 and C-17 and C-20. The positions of the three hydroxyl groups were assigned by examining the correlation obtained from the 1 H and 13 C NMR chemical shifts and supported by the 1 H- 1 H COSY, 1 H - 13 C HMBC spectral data. The remaining from the structure of 1, is the acetate moiety, which connected to C-25 based on the 13 C chemical shift. The spectral data of 1, is well fitted with published data with dihydocucurbitacin B, which was isolated from Bryonia cretica. It is isolated from the first time from C. prophetarum of Saudi source.
|Table 1: 1H (CDCl3, 500 MHz) and 133, 125 MHz) spectral data of compound 1 and 2a|
Click here to view
The structure of 2 was constructed based on the molecular formula of C 32 H 46 O 8 Na, which abstracted from the ESI-MS m/z 581 [M + +Na] and HRESI MS m/z 581.3114[M + + Na]. After extensive studying of the 1 H and 13 C NMR spectral data indicated that the doublet at 4.42 (J =13.0, 6.0) and quartet at 4.35 (J = 8.0) were assigned for H-2 and H-16, respectively. A normal H 2 -1 shift at 2.30 (1H, ddd, J = 13.0, 6.0, 3.0 Hz), 1.24 (1H, q, I = 13.0 Hz). An acetate signal was clear from 1 H and 13 C shifts at 2.08 and 170.2 ppm, respectively. The spectra also exhibited two double bond one at 5.79 (d, J =5.5) and the other at 7.05(1 H, d, J = 16.0 Hz), 6.48 (1 H, d, J = 16.0 Hz), which were assigned for C-6 and C 23 =C 24 , respectively. The structures also have three carbonyl groups by 13 C NMR at 213.0, 212.2, and 202.4, for C-3, C-11 and C-22, respectively. The structure also has eight methyls at δ 1.57, 1.55, 1.44, 1.36, 1.34, 1.29, 1.08, and 0.98 ppm. It was clear from the spectral data of 2, that it is cucurbitacin B, which was published before. ,
The potent activities of compounds 1 and 2 toward NIH3T3 and KA31 will open the gate for new era of discovering anticancer drug especially for the steroidal compounds, which lead to discovering of new anticancer natural drugs.
| Conclusion|| |
This manuscript investigates the fractionation of Cucumis prophetarum, Cucurbitaceae aiming at finding or discovering a bioactive metabolites. This study afforded dihydocucurbitacin B ( 1 ) and cucurbitacin B ( 2 ) [Figure 1]. The structures of the two compounds were elucidated by spectroscopic analyses including: 1D ( 1 H and 13 C) and 2D (COSY, HMQC and HMBC) NMR, and ESI-MS spectroscopy. The cytotoxicity of 1 and 2 , towards human cancer cell lines, mouse embryonic fibroblast (NIH3T3) and virally transformed form (KA3IT) cells, has been estimated. The compound 1 and 2 had potent inhibitory activities toward NIH3T3 and KA31T with IC 50 0.2 and 0.15, 2.5, and 2.0 μg/ml, respectively.
| Acknowledgment|| |
The authors would like to acknowledge SABIC, the Saudi Arabian Company for Basic Industries, for the financial support of this work (MS/8/68), through the collaboration with the Deanship of Scientific Research (DSR) at King Abdul-Aziz University.
| References|| |
|1.||Yip KW, Mao X, Au PY, Hedley DW, Chow S, Dalili S, et al. Benzethonium chloride: A novel anticancer agent identified by using a cell-based small-molecule screen. Clin Cancer Res 2006;12:5557-69. |
|2.||Stockwell BR. Chemical genetics: Ligand-based discovery of gene function. Nat Rev Genet 2000;1:116-25. |
|3.||Druker BJ, Lydon NB. Lessons learned from the development of tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:3-7. |
|4.||Lavie D, Glotter E. The cucurbitacins, a group of tetracyclic triterpenes. Fortschr Chem Org Naturst 1971;29:307-62. |
|5.||Halaweish FT. Cucurbitacins in tissue cultures of Bryonia dioica Jacq., PhD thesis. University of Wales: Cardiff, UK; 1987. |
|6.||Miro M. Cucurbitacins and their pharmacological effects. Phytother Res 1995;9:159-68. |
|7.||Fuller RW, Cardellina JH, Cragg GM, Body MR. Cucurbitacin: Differential cytotoxicity, dereplication and first isolation from Gonystylus keithii. J Nat Prod 1994;57:1442-5. |
|8.||Musza LL, Speight P, Mcelhiney S, Brown CT, Gillum AM, Cooper R, et al. Cucurbitacins: Cell adhesion inhibitor from Conobea scoparioides. J Nat Prod 1994;57:1498-502. |
|9.||Bar-nun N, Mayer AM. Cucurbitacins-repressor of induction of laccase formation. Phytochemistry 1989;28:1369-71. |
|10.||Al-Rawi A. Wild plants of Iraq with their distribution. Baghdad: Government Press; 1964. |
|11.||Afifi MS, Ross SA, Elsohly MA, Naeem ZE, Halawelshi FT. Cucurbitacins of Cucumis prophetarum and Cucumis prophetarum. J Chem Ecol 1999;25:847-59. |
|12.||Atta-Ur-Rahman A, Ahmed VU, Khan MA, Zehra F. Isolation and structure of cucurbitacin Q1. Phytochemistry 1973;12:2741-3. |
|13.||Gitter S, Gallily R, Shohat B, Lavie D. Studies on antitumor on the antitumor effects of cucurbitacins. Cancer Res 1961;21:516-21. |
|14.||Gallily R, Shohat B, Kalish J, Gitter S, Lavie D. Further studies on the antitumor effect of cucurbitacins. Cancer Res 1962;22:1038-45. |
|15.||Matsuda H, Nakashima S, Abdel-Halim OB, Morikawa T, Yoshikawa M. Cucurbitane-type triterpenes with anti-proliferative effects on U937 cells from an Egyptian natural medicine, Bryonia cretica: Structures of new triterpene glycosides, bryoniaosides A and B. Chem Pharm Bull 2010;58:747-51. |
|16.||Alarif WM, Ayyad SE, Al-lihaibi SS. Acyclic Diterpenoid from the Red Alga Gracilaria Foliifera. Rev Latinoam Quím 2010;38:52-8. |
|17.||Alarif, WM, Abou-Elnaga ZS, Ayyad SE, Al-Lihaibi SS. Insecticidal metabolites from the green alga Caulerpa racemosa. Clean-Soil, Air, Water 2010;38:548-57. |
|18.||Ayyad SE, Makki MS, Al-Kayal NS, Basaif SA, El-Foty KO, Asiri AM, et al. Cytotoxic and Protective DNA damage of three new Diterpenoids from the brown alga Dictoyota dichotoma. Eur J Med Chem 2011;46:175-82. |
|19.||Abbas HK, Mirocha CJ, Shier WT, Gunther R. Procedures for bioassay, extraction and purification of wortmannin, the hemorrhagic factor produced by Fusarium oxysporum N17B grown on rice. J Assoc Off Anal Chem 1992;75:474. |
|20.||Shier WT. An undergraduate experiment to demonstrate the use of cytotoxic drugs in cancer chemotherapy. Am J Pharm Educ 1983;47:216. |
|This article has been cited by|
||Penicillium verruculosum SG: a source of polyketide and bioactive compounds with varying cytotoxic activities against normal and cancer lines
| ||Salma Gul Shah,W. Thomas Shier,W. Thomas Jamaluddin,Nawaz Tahir,Abdul Hameed,Safia Ahmad,Naeem Ali |
| ||Archives of Microbiology. 2014; 196(4): 267 |
|[Pubmed] | [DOI]|
||Selective cytotoxic effects on human breast carcinoma of new methoxylated flavonoids from Euryops arabicus grown in Saudi Arabia
| ||Walied M. Alarif,Ahmed Abdel-Lateff,Ahmed M. Al-Abd,Salim A. Basaif,Farid A. Badria,Maher Shams,Seif-Eldin N. Ayyad |
| ||European Journal of Medicinal Chemistry. 2013; 66: 204 |
|[Pubmed] | [DOI]|