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Short communication
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Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoides on human ovarian cancer cell lines
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Okiemute Rosa Johnson-Ajinwo, Alan Richardson, Wen-Wu Li ∗ Guy Hilton Research Centre, Institute for Science and Technology in Medicine, Keele University, Thornburrow Drive, ST4 7QB Stoke-on-Trent, United Kingdom
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Article history: Received 9 July 2014 Received in revised form 11 September 2014 Accepted 29 September 2014
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Keywords: Ovarian cancer Margaritaria discoidea GC–MS Gallic acid Securinine
Margaritaria discoidea (Baill.) G. L. Webster (Euphorbiaceae) is a well-known medicinal plant in Africa used for the treatment of various diseases. So far, no cytotoxic effects of the plant extracts on the cancer cell lines have been reported. Aim of the study: To evaluate the cytotoxicity against human ovarian cancer cells of the extracts of M. discoidea and characterize the major bioactive compounds. Methods: Both organic and aqueous extracts of this plant were obtained by maceration. The sulforhodamine B cell proliferation assay was used for evaluation of their cytotoxic activities and the potential bioactive compounds were characterized by gas chromatography–mass spectrometry. Results: The organic extract of M. discoidea showed stronger cytotoxicity than the aqueous extract with IC50 values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 g/ml on OVCAR-8, A2780 and cisplatin-resistant A2780cis ovarian cancer cells, respectively. The organic extract was further subjected to bioassay-guided fractionation by partitioning with n-hexane, ethyl acetate, and n-butanol in water. The ethyl acetate fraction was the most potent on the three ovarian cancer cell lines. A GC–MS analysis of trimethylsilyl derivatives of this fraction indicated the presence of phenolic compounds such as gallic acid and the alkaloid securinine. The IC50 values of these two compounds were determined to be in the range of 5–16 M, which indicated that they could contribute to the cytotoxic activity of the extract of M. discoidea. Conclusions: This study has evaluated the cytotoxicity of stem bark extracts of M. discoidea against ovarian cancer cells and provided a basis of further development of this plant for the treatment of ovarian cancer. © 2014 Published by Elsevier GmbH.
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Introduction Ovarian cancer is the second most common gynaecologic cancer among women and the ninth most prevalent cancer in the US (Siegel et al., 2013). The standard treatment for ovarian cancer is the use of paclitaxel and platinum-based therapy after aggressive surgical reduction and this has improved survival. However, the reemergence of cancer in a drug-resistant form frequently results in a poor overall survival rate. Natural products have played an important role in the development of new medicines. Currently, more
Abbreviations: BSTFA, N,O-bis(trimethylsily)trifluoroacetamide; EI, electron ionization; FBS, foetal bovine serum; GC–MS, gas chromatography–mass spectrometry; NCI, National Cancer Institute; SRB, sulforhodamine B; TCA, trichloroacetic acid; TMCS, chlorotrimethylsilane; TMSi, trimethylsilyl. ∗ Corresponding author. Tel.: +44 01782 674382; fax: +44 01782 747319. E-mail address: [email protected] (W.-W. Li).
than 50% of all approved drugs (not limited to cancer) are natural products and their derivatives (Cragg et al., 2009). Margaritaria discoidea (Baill.) G. L. Webster [synonym Phyllanthus discoideus (Baill.) Müll. Arg.)] (Euphorbiaceae) is a tree native to Africa and has been traditionally used for the treatment of onchocerciasis, wounds and skin infections (Mensah et al., 1990), and oedema (Obiri et al., 2014). Pharmacological studies have showed that it has uterontonic, anti-malarial (Weenen et al., 1990), antibacterial (Mensah et al., 1990), anti-inflammatory and analgesic, filaricidal, free-radical scavenging (Ekuadzi et al., 2013), anti-allergic, and anti-arthritic activities (Obiri et al., 2014). Many Phyllanthus species which are closely related to the genus Margaritaria (both in the Euphorbiaceae family) have been shown to inhibit the growth of human breast, lung, gynecologic, colon and ovarian (Jia et al., 2013) cancer cells. The Phyllanthus species have been shown to be a source of alkaloids, phenolics and terpenoids (Patel et al., 2011). Previously, alkaloids such as viroallosecurinine and securinine (Mensah et al., 1990; Weenen et al., 1990), and
http://dx.doi.org/10.1016/j.phymed.2014.09.008 0944-7113/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Johnson-Ajinwo, O.R., et al., Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoides on human ovarian cancer cell lines. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.09.008
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Fig. 1. Mean dose-response curves of organic extract (A) of M. discoidea and gallic acid (B) on the A2780 ovarian cancer cell line. The mean values of their IC50 are expressed as mean ± SEM (n = 3).
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flavonoids have been isolated from M. discoidea (Ekuadzi et al., 2013). However, to date, no cytotoxic effects of the M. discoidea extracts on the cancer cells have been reported. Here we evaluated cytotoxicity of the extracts of M. discoidea on three ovarian cancer cell lines and characterized their chemical composition.
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Materials and methods
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Plant material and reagents The stem bark of M. discoidea was obtained from a bio-reserve in Nsukka, Enugu state, Nigeria in February 2013. The plant was authenticated by Mr. A.O. Ozioko and a voucher specimen (INTERCEDD/1579) was deposited in the herbarium at the International Centre for Ethnomedicine and Drug Development (InterCEDD), Nsukka, Nigeria. Trichloroacetic acid (TCA) was purchased from Fisher Scientific, U.K. Trypsin-EDTA solution, Trizma base, sulforhodamine B (SRB) sodium salt, glacial acetic acid, paclitaxel, 3,4dihydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, gallic acid, trans-catechin, trans-gallocatechin, N,O-bis(trimethylsily) trifluoroacetamide (BSTFA) with 1% chlorotrimethylsilane (TMCS), and pyridine were purchased from Sigma–Aldrich. Securinine was purchased from Abcam Biochemicals, U.K. RMPI Media, foetal bovine serum (FBS), and penicillin-streptomycin was purchased from Lonza, Switzerland. Extraction of plant materials The plant materials were extracted according to the method of extraction used by National Cancer Institute, USA (NCI) (McCloud, 2010). A pulverized plant material (80 g) was macerated in a mixture of dichloromethane and methanol (1:1). Methanol was further added to the residue and allowed to stand to yield the methanol extract. The two extract solutions were combined and evaporated to dryness on a rotary evaporator under 40 ◦ C to yield the organic extract (4.0 g). Furthermore, deionised water was added to the plant residue to obtain the aqueous extract (1.6 g). The organic extract of M. discoidea was further partitioned in water with organic solvents of increasing polarity to obtain the n-hexane fraction (0.5 g), the ethyl acetate fraction (0.8 g), the nbutanol fraction (1.5 g) and the aqueous fraction (0.9 g). Cell culture The human ovarian cancer cell lines OVCAR-8, A2780 and cisplatin-resistant A2780cis were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin-streptomycin (50 U/ml) and glutamine (2 mM).
Cell growth assay The SRB assay (Vichai and Kirtikara, 2006) was used to evaluate the effects of the plant extracts and pure compounds on the growth of ovarian cancer cell lines (Supporting information).
Analysis of the M. discoidea bioactive fraction by gas chromatography–mass spectrometry (GC–MS) The bioactive ethyl acetate fraction of M. discoidea (1.0 mg) was added to 10 l of pyridine and 50 l of BSTFA (with 1% TMCS). The obtained solution was incubated in the oven at a temperature of 37 ◦ C for 2 h to prepare the trimethylsilyl (TMSi) derivatives for subsequent analyses using GC–MS (Li and Barz, 2005, 2006). The GC–MS system consisted of an Agilent 7890 A gas chromatography system with split injection (250 ◦ C; 1:10), coupled to an Agilent MS model 5975 C MSD with triple axis detector (Agilent Technologies, USA). The instrument’s column is a 30 m HP5-MS column [(5% phenyl)-methylpolysiloxane, 0.25 mm i.d., df = 0.25 m] (Johnson-Ajinwo and Li, 2014). The gas chromatography began with an oven temperature of 60 ◦ C for 2 min, which increased to 300 ◦ C at the rate of 10 ◦ C/min, and was then held at 300 ◦ C for 4 min under a constant helium pressure (10 psi). The mass spectrometry utilized an electron ionization (EI) source at 70 eV with an ion source temperature of 230 ◦ C. The compounds were identified by matching their EI-MS spectra with those in the NIST/EPA/NIH 2011 Mass Spectral Library using MSD ChemStation (Agilent Technologies, USA).
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Results and discussion
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In vitro anti-ovarian cancer activity of the M. discoidea extracts
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The organic and aqueous extracts of M. discoidea were tested for their effects on the proliferation of OVCAR-8, A2780 and A2780cis ovarian cancer cell lines using a cell growth assay (Fig. 1 and Table 1). The organic extract of M. discoidea inhibited growth of the cultures with IC50 values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 g/ml on OVCAR-8, A2780 (Fig. 1) and A2780cis ovarian cancer cells, respectively. Bioassay-guided fractionation of the organic extract of M. discoidea was further carried out to identify the possible anti-cancer compounds. Solvent fractionation of the crude organic extract of M. discoidea yielded n-hexane, ethyl acetate, nbutanol and aqueous fractions. The cytotoxicity of these fractions was assayed and the results are also presented in Table 1. The ethyl acetate fraction showed the most potent cytotoxic effects on the OVCAR-8, A2780 and A2780cis ovarian cancer cell lines.
Please cite this article in press as: Johnson-Ajinwo, O.R., et al., Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoides on human ovarian cancer cell lines. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.09.008
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Fractions and pure compounds
OVCAR-8 (g/ml)
Organic extract Aqueous extract n-Hexane fraction Ethyl acetate fraction n-Butanol fraction Water fraction Securinine (16) Gallic acid (17) Paclitaxel
14.4 66.2 33.7 11.6 95.4 63.9 5.2 16.2 10.0
± ± ± ± ± ± ± ± ±
3.0 1.3 2.2 1.5 15.2 7.2 0.1 M 0.5 M 4.0 nM
A2780 (g/ml)
14.2 ± 1.2 37.9 ± 5.1 27.0 ± 1.3 7.3 ± 0.7 >100 91.2 ± 0.9 2.7 ± 0.7 M 6.2 ± 0.3 M 9.8 ± 1.3 nM
A2780cis (g/ml)
34.7 ± 0.5 >75 >75 24.8 ± 0.5 71.6 ± 1.2 >75 5.4 ± 0.3 M 6.5 ± 0.4 M Not determined
Chemical characterization of the bioactive fraction of M. discoidea In order to identify the compounds that were responsible for the growth inhibitory properties of the M. discoidea extracts, the TMSi derivatives of the ethyl acetate fraction were made and subjected to analysis by GC–MS. The gas chromatogram of the ethyl acetate fraction of M. discoidea (Fig. 2) demonstrated a large number of compounds, most of which were identified by comparison of their mass spectra with those in the NIST library and 29 compounds (1–29) were identified and listed (Supporting information). Among them, the major compounds were cis-gallocatechin (28, 21.6%), trans-gallocatechin (29, 43.0%), sucrose (25, 7.16%), (Z,Z)-9, 12-octadecadienoic acid (22, 2.05%), and the alkaloid securinine (16, 2.06%) (Supporting information). Some minor compounds such as vanillic acid (9, 0.24%), gallic acid (17, 0.20%), cis-catechin (26, 1.23%) and trans-catechin (27, 0.89%) were also identified. The assignment of compounds 9, 16, 17, 27 and 29 was further confirmed by comparison with their standard samples by their identical retention times with the reference compounds. Securinine (16) was previously isolated from P. discoideus (Mensah et al., 1990) and P. amarus (Patel et al., 2011) and showed antibacterial activity. Gallic acid (17) and ellgagic acid were isolated from P. urinaria (Huang et al., 2010) and P. amarus (Patel et al., 2011). After literature search of the possible biological activities of the identified compounds (1–29), we suspected that securinine and gallic acid could contribute to the cytotoxicity of the extract of
Fig. 2. GC–MS of TMSi derivatives of the M. discoidea ethyl acetate fraction with the strongest cytotoxicity against ovarian cancer cells.
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M. discoidea. Therefore, we determined IC50 values of these two pure compounds. They both showed potent in vitro anti-ovarian cancer activities with IC50 s ranging between 5 and 16 M on all three ovarian cell lines (Fig. 1 and Table 1). The anticancer properties of securinine against colon cancer SW480 cell (Xia et al., 2011) and myeloid leukaemia cell lines (Gupta et al., 2011) have been previously reported. Gallic acid has been previously reported to inhibit in vitro cell proliferation on a range of cancer cell lines including ovarian cell lines and carcinogenesis in animal models (Verma et al., 2013). Gallic acid also induced apoptosis in esophageal, prostate carcinoma, colon adenocarcinoma and cervical cancer cells but not in non-cancerous cells (Verma et al., 2013). In conclusion, the cytotoxic effects of stem bark extracts of M. discoidea on three ovarian cancer cell lines have been evaluated. Bioassay-guided fractionation of the M. discoidea extracts, chemical analysis by GC–MS and cell proliferation assay showed that securinine and gallic acid could be the cytotoxic compounds in the M. discoidea. This study has provided a basis for the future development of M. discoidea for the potential treatment of ovarian cancer. Conflict of interest All authors have no conflict of interest to disclose. Acknowledgements This work was supported by Nigerian ETF (a PhD studentship to Q2 Okiemute R. Johnson-Ajinwo) and Keele University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.phymed.2014.09.008. References Cragg, G.M., Grothaus, P.G., Newman, D.J., 2009. Impact of natural products on developing new anti-cancer agents. Chem. Rev. 109, 3012–3043. Ekuadzi, E., Dickson, R., Fleischer, T., Annan, K., Pistorius, D., Oberer, L., Gibbons, S., 2013. Flavonoid glycosides from the stem bark of Margaritaria discoidea Q3 demonstrate antibacterial and free radical scavenging activities. Phytother. Res.: PTR. Gupta, K., Chakrabarti, A., Rana, S., Ramdeo, R., Roth, B.L., Agarwal, M.L., Tse, W., Agarwal, M.K., Wald, D.N., 2011. Securinine, a myeloid differentiation agent with therapeutic potential for AML. PLoS ONE 6, e21203. Huang, S.T., Pang, J.H., Yang, R.C., 2010. Anti-cancer effects of Phyllanthus urinaria and relevant mechanisms. Chang Gung Med. J. 33, 477–487. Jia, L., Jin, H., Zhou, J., Chen, L., Lu, Y., Ming, Y., Yu, Y., 2013. A potential antitumor herbal medicine, Corilagin, inhibits ovarian cancer cell growth through blocking the TGF-beta signaling pathways. BMC Complement. Altern. Med. 13, 33. Johnson-Ajinwo, O.R., Li, W.W., 2014. Stable isotope dilution gas chromatography–mass spectrometry for quantification of thymoquinone in black cumin seed oil. J. Agric. Food Chem. 62, 5466–5471. Li, W.W., Barz, W., 2005. Biotechnological production of 8,4 -oxynorneolignans by elicitation of Echinacea purpurea cell cultures. Tetrahedron Lett. 46, 2973–2977. Li, W.W., Barz, W., 2006. Structure and accumulation of phenolics in elicited Echinacea purpurea cell cultures. Planta Med. 72, 248–254. McCloud, T.G., 2010. High throughput extraction of plant, marine and fungal specimens for preservation of biologically active molecules. Molecules 15, 4526–4563. Mensah, J.L., Lagarde, I., Ceschin, C., Michel, G., Gleye, J., Fouraste, I., 1990. Antibacterial activity of the leaves of Phyllanthus discoideus. J. Ethnopharmacol. 28, 129–133. Obiri, D.D., Osafo, N., Oppong-Sarfo, J., Prah, J.K., 2014. Margaritaria discoidea (Euphorbiaceae) stem bark extract attenuates allergy and Freund’s adjuvantinduced arthritis in rodents. Pharmacogn. Res. 6, 163–171. Patel, J.R., Tripathi, P., Sharma, V., Chauhan, N.S., Dixit, V.K., 2011. Phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review. J. Ethnopharmacol. 138, 286–313. Siegel, R., Naishadham, D., Jemal, A., 2013. Cancer statistics, 2013. CA: Cancer J. Clin. 63, 11–30.
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Verma, S., Singh, A., Mishra, A., 2013. Gallic acid: molecular rival of cancer. Environ. Toxicol. Pharmacol. 35, 473–485. Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116. Weenen, H., Nkunya, M.H., Bray, D.H., Mwasumbi, L.B., Kinabo, L.S., Kilimali, V.A., Wijnberg, J.B., 1990. Antimalarial compounds containing an alpha,
beta-unsaturated carbonyl moiety from Tanzanian medicinal plants. Planta Med. 56, 371–373. Xia, Y.H., Cheng, C.R., Yao, S.Y., Zhang, Q., Wang, Y., Ji, Z.N., 2011. l-Securinine induced the human colon cancer SW480 cell autophagy and its molecular mechanism. Fitoterapia 82, 1258–1264.
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PHYMED 51733 1–4
Phytomedicine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Short communication
1
Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoides on human ovarian cancer cell lines
2
3
4 5 6
Q1
Okiemute Rosa Johnson-Ajinwo, Alan Richardson, Wen-Wu Li ∗ Guy Hilton Research Centre, Institute for Science and Technology in Medicine, Keele University, Thornburrow Drive, ST4 7QB Stoke-on-Trent, United Kingdom
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a r t i c l e
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a b s t r a c t
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Article history: Received 9 July 2014 Received in revised form 11 September 2014 Accepted 29 September 2014
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Keywords: Ovarian cancer Margaritaria discoidea GC–MS Gallic acid Securinine
Margaritaria discoidea (Baill.) G. L. Webster (Euphorbiaceae) is a well-known medicinal plant in Africa used for the treatment of various diseases. So far, no cytotoxic effects of the plant extracts on the cancer cell lines have been reported. Aim of the study: To evaluate the cytotoxicity against human ovarian cancer cells of the extracts of M. discoidea and characterize the major bioactive compounds. Methods: Both organic and aqueous extracts of this plant were obtained by maceration. The sulforhodamine B cell proliferation assay was used for evaluation of their cytotoxic activities and the potential bioactive compounds were characterized by gas chromatography–mass spectrometry. Results: The organic extract of M. discoidea showed stronger cytotoxicity than the aqueous extract with IC50 values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 g/ml on OVCAR-8, A2780 and cisplatin-resistant A2780cis ovarian cancer cells, respectively. The organic extract was further subjected to bioassay-guided fractionation by partitioning with n-hexane, ethyl acetate, and n-butanol in water. The ethyl acetate fraction was the most potent on the three ovarian cancer cell lines. A GC–MS analysis of trimethylsilyl derivatives of this fraction indicated the presence of phenolic compounds such as gallic acid and the alkaloid securinine. The IC50 values of these two compounds were determined to be in the range of 5–16 M, which indicated that they could contribute to the cytotoxic activity of the extract of M. discoidea. Conclusions: This study has evaluated the cytotoxicity of stem bark extracts of M. discoidea against ovarian cancer cells and provided a basis of further development of this plant for the treatment of ovarian cancer. © 2014 Published by Elsevier GmbH.
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Introduction Ovarian cancer is the second most common gynaecologic cancer among women and the ninth most prevalent cancer in the US (Siegel et al., 2013). The standard treatment for ovarian cancer is the use of paclitaxel and platinum-based therapy after aggressive surgical reduction and this has improved survival. However, the reemergence of cancer in a drug-resistant form frequently results in a poor overall survival rate. Natural products have played an important role in the development of new medicines. Currently, more
Abbreviations: BSTFA, N,O-bis(trimethylsily)trifluoroacetamide; EI, electron ionization; FBS, foetal bovine serum; GC–MS, gas chromatography–mass spectrometry; NCI, National Cancer Institute; SRB, sulforhodamine B; TCA, trichloroacetic acid; TMCS, chlorotrimethylsilane; TMSi, trimethylsilyl. ∗ Corresponding author. Tel.: +44 01782 674382; fax: +44 01782 747319. E-mail address: [email protected] (W.-W. Li).
than 50% of all approved drugs (not limited to cancer) are natural products and their derivatives (Cragg et al., 2009). Margaritaria discoidea (Baill.) G. L. Webster [synonym Phyllanthus discoideus (Baill.) Müll. Arg.)] (Euphorbiaceae) is a tree native to Africa and has been traditionally used for the treatment of onchocerciasis, wounds and skin infections (Mensah et al., 1990), and oedema (Obiri et al., 2014). Pharmacological studies have showed that it has uterontonic, anti-malarial (Weenen et al., 1990), antibacterial (Mensah et al., 1990), anti-inflammatory and analgesic, filaricidal, free-radical scavenging (Ekuadzi et al., 2013), anti-allergic, and anti-arthritic activities (Obiri et al., 2014). Many Phyllanthus species which are closely related to the genus Margaritaria (both in the Euphorbiaceae family) have been shown to inhibit the growth of human breast, lung, gynecologic, colon and ovarian (Jia et al., 2013) cancer cells. The Phyllanthus species have been shown to be a source of alkaloids, phenolics and terpenoids (Patel et al., 2011). Previously, alkaloids such as viroallosecurinine and securinine (Mensah et al., 1990; Weenen et al., 1990), and
http://dx.doi.org/10.1016/j.phymed.2014.09.008 0944-7113/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Johnson-Ajinwo, O.R., et al., Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoides on human ovarian cancer cell lines. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.09.008
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Fig. 1. Mean dose-response curves of organic extract (A) of M. discoidea and gallic acid (B) on the A2780 ovarian cancer cell line. The mean values of their IC50 are expressed as mean ± SEM (n = 3).
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flavonoids have been isolated from M. discoidea (Ekuadzi et al., 2013). However, to date, no cytotoxic effects of the M. discoidea extracts on the cancer cells have been reported. Here we evaluated cytotoxicity of the extracts of M. discoidea on three ovarian cancer cell lines and characterized their chemical composition.
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Materials and methods
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Plant material and reagents The stem bark of M. discoidea was obtained from a bio-reserve in Nsukka, Enugu state, Nigeria in February 2013. The plant was authenticated by Mr. A.O. Ozioko and a voucher specimen (INTERCEDD/1579) was deposited in the herbarium at the International Centre for Ethnomedicine and Drug Development (InterCEDD), Nsukka, Nigeria. Trichloroacetic acid (TCA) was purchased from Fisher Scientific, U.K. Trypsin-EDTA solution, Trizma base, sulforhodamine B (SRB) sodium salt, glacial acetic acid, paclitaxel, 3,4dihydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, gallic acid, trans-catechin, trans-gallocatechin, N,O-bis(trimethylsily) trifluoroacetamide (BSTFA) with 1% chlorotrimethylsilane (TMCS), and pyridine were purchased from Sigma–Aldrich. Securinine was purchased from Abcam Biochemicals, U.K. RMPI Media, foetal bovine serum (FBS), and penicillin-streptomycin was purchased from Lonza, Switzerland. Extraction of plant materials The plant materials were extracted according to the method of extraction used by National Cancer Institute, USA (NCI) (McCloud, 2010). A pulverized plant material (80 g) was macerated in a mixture of dichloromethane and methanol (1:1). Methanol was further added to the residue and allowed to stand to yield the methanol extract. The two extract solutions were combined and evaporated to dryness on a rotary evaporator under 40 ◦ C to yield the organic extract (4.0 g). Furthermore, deionised water was added to the plant residue to obtain the aqueous extract (1.6 g). The organic extract of M. discoidea was further partitioned in water with organic solvents of increasing polarity to obtain the n-hexane fraction (0.5 g), the ethyl acetate fraction (0.8 g), the nbutanol fraction (1.5 g) and the aqueous fraction (0.9 g). Cell culture The human ovarian cancer cell lines OVCAR-8, A2780 and cisplatin-resistant A2780cis were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin-streptomycin (50 U/ml) and glutamine (2 mM).
Cell growth assay The SRB assay (Vichai and Kirtikara, 2006) was used to evaluate the effects of the plant extracts and pure compounds on the growth of ovarian cancer cell lines (Supporting information).
Analysis of the M. discoidea bioactive fraction by gas chromatography–mass spectrometry (GC–MS) The bioactive ethyl acetate fraction of M. discoidea (1.0 mg) was added to 10 l of pyridine and 50 l of BSTFA (with 1% TMCS). The obtained solution was incubated in the oven at a temperature of 37 ◦ C for 2 h to prepare the trimethylsilyl (TMSi) derivatives for subsequent analyses using GC–MS (Li and Barz, 2005, 2006). The GC–MS system consisted of an Agilent 7890 A gas chromatography system with split injection (250 ◦ C; 1:10), coupled to an Agilent MS model 5975 C MSD with triple axis detector (Agilent Technologies, USA). The instrument’s column is a 30 m HP5-MS column [(5% phenyl)-methylpolysiloxane, 0.25 mm i.d., df = 0.25 m] (Johnson-Ajinwo and Li, 2014). The gas chromatography began with an oven temperature of 60 ◦ C for 2 min, which increased to 300 ◦ C at the rate of 10 ◦ C/min, and was then held at 300 ◦ C for 4 min under a constant helium pressure (10 psi). The mass spectrometry utilized an electron ionization (EI) source at 70 eV with an ion source temperature of 230 ◦ C. The compounds were identified by matching their EI-MS spectra with those in the NIST/EPA/NIH 2011 Mass Spectral Library using MSD ChemStation (Agilent Technologies, USA).
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In vitro anti-ovarian cancer activity of the M. discoidea extracts
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The organic and aqueous extracts of M. discoidea were tested for their effects on the proliferation of OVCAR-8, A2780 and A2780cis ovarian cancer cell lines using a cell growth assay (Fig. 1 and Table 1). The organic extract of M. discoidea inhibited growth of the cultures with IC50 values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 g/ml on OVCAR-8, A2780 (Fig. 1) and A2780cis ovarian cancer cells, respectively. Bioassay-guided fractionation of the organic extract of M. discoidea was further carried out to identify the possible anti-cancer compounds. Solvent fractionation of the crude organic extract of M. discoidea yielded n-hexane, ethyl acetate, nbutanol and aqueous fractions. The cytotoxicity of these fractions was assayed and the results are also presented in Table 1. The ethyl acetate fraction showed the most potent cytotoxic effects on the OVCAR-8, A2780 and A2780cis ovarian cancer cell lines.
Please cite this article in press as: Johnson-Ajinwo, O.R., et al., Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoides on human ovarian cancer cell lines. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.09.008
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Fractions and pure compounds
OVCAR-8 (g/ml)
Organic extract Aqueous extract n-Hexane fraction Ethyl acetate fraction n-Butanol fraction Water fraction Securinine (16) Gallic acid (17) Paclitaxel
14.4 66.2 33.7 11.6 95.4 63.9 5.2 16.2 10.0
± ± ± ± ± ± ± ± ±
3.0 1.3 2.2 1.5 15.2 7.2 0.1 M 0.5 M 4.0 nM
A2780 (g/ml)
14.2 ± 1.2 37.9 ± 5.1 27.0 ± 1.3 7.3 ± 0.7 >100 91.2 ± 0.9 2.7 ± 0.7 M 6.2 ± 0.3 M 9.8 ± 1.3 nM
A2780cis (g/ml)
34.7 ± 0.5 >75 >75 24.8 ± 0.5 71.6 ± 1.2 >75 5.4 ± 0.3 M 6.5 ± 0.4 M Not determined
Chemical characterization of the bioactive fraction of M. discoidea In order to identify the compounds that were responsible for the growth inhibitory properties of the M. discoidea extracts, the TMSi derivatives of the ethyl acetate fraction were made and subjected to analysis by GC–MS. The gas chromatogram of the ethyl acetate fraction of M. discoidea (Fig. 2) demonstrated a large number of compounds, most of which were identified by comparison of their mass spectra with those in the NIST library and 29 compounds (1–29) were identified and listed (Supporting information). Among them, the major compounds were cis-gallocatechin (28, 21.6%), trans-gallocatechin (29, 43.0%), sucrose (25, 7.16%), (Z,Z)-9, 12-octadecadienoic acid (22, 2.05%), and the alkaloid securinine (16, 2.06%) (Supporting information). Some minor compounds such as vanillic acid (9, 0.24%), gallic acid (17, 0.20%), cis-catechin (26, 1.23%) and trans-catechin (27, 0.89%) were also identified. The assignment of compounds 9, 16, 17, 27 and 29 was further confirmed by comparison with their standard samples by their identical retention times with the reference compounds. Securinine (16) was previously isolated from P. discoideus (Mensah et al., 1990) and P. amarus (Patel et al., 2011) and showed antibacterial activity. Gallic acid (17) and ellgagic acid were isolated from P. urinaria (Huang et al., 2010) and P. amarus (Patel et al., 2011). After literature search of the possible biological activities of the identified compounds (1–29), we suspected that securinine and gallic acid could contribute to the cytotoxicity of the extract of
Fig. 2. GC–MS of TMSi derivatives of the M. discoidea ethyl acetate fraction with the strongest cytotoxicity against ovarian cancer cells.
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M. discoidea. Therefore, we determined IC50 values of these two pure compounds. They both showed potent in vitro anti-ovarian cancer activities with IC50 s ranging between 5 and 16 M on all three ovarian cell lines (Fig. 1 and Table 1). The anticancer properties of securinine against colon cancer SW480 cell (Xia et al., 2011) and myeloid leukaemia cell lines (Gupta et al., 2011) have been previously reported. Gallic acid has been previously reported to inhibit in vitro cell proliferation on a range of cancer cell lines including ovarian cell lines and carcinogenesis in animal models (Verma et al., 2013). Gallic acid also induced apoptosis in esophageal, prostate carcinoma, colon adenocarcinoma and cervical cancer cells but not in non-cancerous cells (Verma et al., 2013). In conclusion, the cytotoxic effects of stem bark extracts of M. discoidea on three ovarian cancer cell lines have been evaluated. Bioassay-guided fractionation of the M. discoidea extracts, chemical analysis by GC–MS and cell proliferation assay showed that securinine and gallic acid could be the cytotoxic compounds in the M. discoidea. This study has provided a basis for the future development of M. discoidea for the potential treatment of ovarian cancer. Conflict of interest All authors have no conflict of interest to disclose. Acknowledgements This work was supported by Nigerian ETF (a PhD studentship to Q2 Okiemute R. Johnson-Ajinwo) and Keele University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.phymed.2014.09.008. References Cragg, G.M., Grothaus, P.G., Newman, D.J., 2009. Impact of natural products on developing new anti-cancer agents. Chem. Rev. 109, 3012–3043. Ekuadzi, E., Dickson, R., Fleischer, T., Annan, K., Pistorius, D., Oberer, L., Gibbons, S., 2013. Flavonoid glycosides from the stem bark of Margaritaria discoidea Q3 demonstrate antibacterial and free radical scavenging activities. Phytother. Res.: PTR. Gupta, K., Chakrabarti, A., Rana, S., Ramdeo, R., Roth, B.L., Agarwal, M.L., Tse, W., Agarwal, M.K., Wald, D.N., 2011. Securinine, a myeloid differentiation agent with therapeutic potential for AML. PLoS ONE 6, e21203. Huang, S.T., Pang, J.H., Yang, R.C., 2010. Anti-cancer effects of Phyllanthus urinaria and relevant mechanisms. Chang Gung Med. J. 33, 477–487. Jia, L., Jin, H., Zhou, J., Chen, L., Lu, Y., Ming, Y., Yu, Y., 2013. A potential antitumor herbal medicine, Corilagin, inhibits ovarian cancer cell growth through blocking the TGF-beta signaling pathways. BMC Complement. Altern. Med. 13, 33. Johnson-Ajinwo, O.R., Li, W.W., 2014. Stable isotope dilution gas chromatography–mass spectrometry for quantification of thymoquinone in black cumin seed oil. J. Agric. Food Chem. 62, 5466–5471. Li, W.W., Barz, W., 2005. Biotechnological production of 8,4 -oxynorneolignans by elicitation of Echinacea purpurea cell cultures. Tetrahedron Lett. 46, 2973–2977. Li, W.W., Barz, W., 2006. Structure and accumulation of phenolics in elicited Echinacea purpurea cell cultures. Planta Med. 72, 248–254. McCloud, T.G., 2010. High throughput extraction of plant, marine and fungal specimens for preservation of biologically active molecules. Molecules 15, 4526–4563. Mensah, J.L., Lagarde, I., Ceschin, C., Michel, G., Gleye, J., Fouraste, I., 1990. Antibacterial activity of the leaves of Phyllanthus discoideus. J. Ethnopharmacol. 28, 129–133. Obiri, D.D., Osafo, N., Oppong-Sarfo, J., Prah, J.K., 2014. Margaritaria discoidea (Euphorbiaceae) stem bark extract attenuates allergy and Freund’s adjuvantinduced arthritis in rodents. Pharmacogn. Res. 6, 163–171. Patel, J.R., Tripathi, P., Sharma, V., Chauhan, N.S., Dixit, V.K., 2011. Phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review. J. Ethnopharmacol. 138, 286–313. Siegel, R., Naishadham, D., Jemal, A., 2013. Cancer statistics, 2013. CA: Cancer J. Clin. 63, 11–30.
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beta-unsaturated carbonyl moiety from Tanzanian medicinal plants. Planta Med. 56, 371–373. Xia, Y.H., Cheng, C.R., Yao, S.Y., Zhang, Q., Wang, Y., Ji, Z.N., 2011. l-Securinine induced the human colon cancer SW480 cell autophagy and its molecular mechanism. Fitoterapia 82, 1258–1264.
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