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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new triterpenoid from Sapium baccatum (Euphorbiaceae) a
ac
L.M. Ramadhan Al Muqarrabun , Norizan Ahmat , S. Ruzaina S. a
a
a
Aris , Norhazana Norizan , Nurdiana Shamsulrijal , Farida Z.M. a
a
b
c
Yusof , M. Nazip Suratman , M. Izwan M. Yusof & Fatimah Salim a
Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia b
Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia c
Atta-ur-Rahman Institute for Natural Product Discovery (AuRins), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia Published online: 04 Apr 2014.
To cite this article: L.M. Ramadhan Al Muqarrabun, Norizan Ahmat, S. Ruzaina S. Aris, Norhazana Norizan, Nurdiana Shamsulrijal, Farida Z.M. Yusof, M. Nazip Suratman, M. Izwan M. Yusof & Fatimah Salim (2014) A new triterpenoid from Sapium baccatum (Euphorbiaceae), Natural Product Research: Formerly Natural Product Letters, 28:13, 1003-1009, DOI: 10.1080/14786419.2014.903396 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903396
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 13, 1003–1009, http://dx.doi.org/10.1080/14786419.2014.903396
A new triterpenoid from Sapium baccatum (Euphorbiaceae) L.M. Ramadhan Al Muqarrabuna, Norizan Ahmatac*, S. Ruzaina S. Arisa, Norhazana Norizana, Nurdiana Shamsulrijala, Farida Z.M. Yusofa, M. Nazip Suratmana, M. Izwan M. Yusofb and Fatimah Salimc a
Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia; Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia; cAtta-ur-Rahman Institute for Natural Product Discovery (AuRins), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia
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b
(Received 6 January 2014; final version received 8 March 2014) A new triterpene, malaytaraxerate (1), and four known compounds, taraxerol (2), taraxerone (3), docosyl isoferulate (4) and docosanoic acid 20 ,30 -dihydroxypropyl ester (5), were isolated from the acetone extract of Sapium baccatum stem bark. The structures of the isolated compounds were determined using several spectroscopic methods, including UV – Vis, FT-IR, 1D and 2D NMR, and mass spectrometry. Major isolated compounds were assayed for cytotoxicity. The chemotaxonomic significance of this plant was also studied. Keywords: Euphorbiaceae; Sapium baccatum; triterpenoid; malaytaraxerate; cytotoxicity
1. Introduction Sapium baccatum (Euphorbiaceae), also known as ludai (Malay), is a medicinal plant which belongs to the family Euphorbiaceae. This species commonly grows in Malaysia, Sumatera and Borneo (Kalimantan) (Esser 1999). It is widely used as timber tree and as wayside plant. The fruits are edible, mealy and sweet (Heyne 1950) and have been used to treat ulcer by the folk in Pahang, Malaysia (Eswani et al. 2010). Panthong et al. (1998) reported antiinflammatory activity displayed by the major alkaloid isolated from this species, bukittinggine (Arbain et al. 1990). Besides bukittinggine, there are only a few number of compounds that have been isolated from the leaves of this species; which include 3-acetoxy aleuritolic acid, 1hexacosanol, b-sitosterol, 3,30 -di-O-methyl ellagic acid (Khastigir et al. 1969; Ray et al. 1975), baccatin (Saha et al. 1977), taraxerol (2), taraxerone (3), lupeol, betulin and stigmasterol (Ahmed et al. 2010). However, up until now, the chemistry of the stem bark of this plant has not been reported. Thus, conducting further study is important in order to enrich the knowledge about the chemistry of the species. In this study, a new triterpene, malaytaraxerate (1), and four known compounds, taraxerol (2), taraxerone (3), docosyl isoferulate (4) and docosanoic acid 20 ,30 -dihydroxypropyl ester (5), were isolated from the stem bark of S. baccatum. The cytotoxicity of the isolated compounds against two cancer cell lines, human colorectal cancer (HT-29) and mammary breast cancer (MDA-MB), and normal cell line 3T3, were also studied.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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L.M.R. Al Muqarrabun et al.
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2. Results and discussion 2.1. Structure determination of malaytaraxerate The methanolic extract of the stem bark of S. baccatum was fractionated using vacuum liquid chromatography (VLC) and purified using radial chromatography (RC) and recrystallisation yielding five compounds (1 –5) (Figure 1), including a new triterpene named malaytaraxerate (1). The structural elucidation of the compounds was determined using several spectroscopic methods including mass spectrometry, UV – Vis, FT-IR, NMR 1D and 2D (HMQC, HMBC, COSY and NOESY), and by comparing with reported data. Compound 1 was isolated as a white crystal. The molecular formula C31H48O4 was generated using HR-ESI-MS [M þ H]þ ion at m/z 485.2916 (calc. for C31H49O4 485.71836). The UV spectrum indicated that there was no conjugated double bond in the molecule; this was deducted by the presence of only one absorbance band at 205.54 nm. The FT-IR spectral data revealed a broad absorbance band at 3430 cm21 representing the stretching vibration of OZH bond. The presence of carbonyl group was indicated by a strong stretching band of CvO at 1734 cm21 and the vibration band at 1034 cm21 indicated the CZO bending representing an ester/carboxylic acid group. The CZC(vO)ZO asymmetrical stretching vibration was indicated by absorbance band at 1245 cm21. The Csp3ZH stretching band appeared at 2935 cm21, while absorption bands at 1457 and 1382 cm21 indicated the asymmetrical and symmetrical bending vibrations of Csp3ZH of the methyl groups, respectively. An olefinic group was detected in this compound which was indicated by an absorbance peak of symmetrical CvC stretching at 1690 cm21 and Csp2ZH stretching band at 2858 cm21. Compound 1 contained 31 carbons observed using APT experiment of 13C NMR. The compound consisted of eight methyls, nine methylenes, five methines and nine quarternary carbons. The DBE value of eight indicated that this compound was a pentacyclic triterpene containing three double bonds. A carbon at dC 80.9 (C-3) indicated the presence of an oxygenattaching carbon, while the carbon –carbon double bond in the molecule was revealed by a pair of highly deshielded carbon signals at dC 160.5 (C-14) and 116.8 (C-15). A highly deshielded quarternary carbon at dC 183.4 (C-27) represented a carbonyl carbon of carboxylic acid, while signal at dC 171.0 (C-10 ) indicated a carbonyl carbon which is typical to ester group. One methyl signal at dC 21.3 (C-20 ) indicated the presence of a methyl adjacent to a carbonyl carbon, forming an acetate group.
Figure 1. Compounds isolated from the stem bark of S. baccatum.
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According to 1H NMR, there was a proton at dH 4.46 (dd, J ¼ 9.0 and 6.6, H-3) integrated for one proton, indicating a proton attached to an oxygen-attaching carbon, which in this case is C-3. There was only one highly deshielded proton at dH 5.52 (H-15) indicating the olefinic proton that attaches to a double-bond carbon (C-15). This explains that the sole carbon –carbon double bond in the molecule was constructed by a quarternary carbon (C-14) and a methine (C-15). Eight singlet methyls were detected in this compound suggesting that all the methyl groups were attached to quarternary carbons. This kind of skeletal structure is characteristic to the oleananetype triterpene. Seven methyls were located in the ring system while the other one was adjacent to a carbonyl carbon. A singlet methylene proton signal appeared at dH 1.25 indicating that the methylene (C-21) was located between two quarternary carbons (C-17 and C-20). The structure of 1 was confirmed using 2D NMR including HMBC, COSY and NOESY. The HMBC spectrum revealed a strong correlation between acetate methyl (C-20 ) and the carbonyl carbon (C-10 ) as well as between C-20 and the oxymethine (C-3). This indicated that the acetate group was bound to oxymethine (C-3). The correlations between H-15 and C-8 as well as between H-16 and C-14 confirmed the position of the double bond in the molecule. The position of the carboxylic acid group was confirmed by the correlations between H-17 and H-21. The COSY spectrum displayed the correlations between the oxymethine proton (H-3) and H-2, while the olefinic proton (H-15) shows a correlation with H-16 as described in Figure 2(a). NOESY spectrum revealed correlation between H-15 and H-16 confirming the position of the double bond in the molecule (Figure 2(b)). Judging from the structure, 1 was proposed to be synthesised via a pathway similar to that of taraxerol (2) and taraxerone (3). Lupanyl cation underwent a series of rearrangement reactions to form a pentacyclic triterpene with four hexagonal rings and one pentagonal ring due to the loss of one methylene from the molecule. The methyl group (C-26) was attached to C-13, while a double bond was formed on C-14 and C-15 by deprotonation of C-15. The methyl group at C-27 was hydroxylated, and converted into carboxylic acid. The reactions did not stop here. The hydroxyl group attached to oxymethine C-3 underwent an esterification reaction with acetic acid to form compound 1 (Figure 3).
2.2. Chemotaxonomy significance According to the author’s literature search, only six species of genus Sapium have been phytochemically studied so far, including Sapium insigne, Sapium haematospermum, Sapium sebiferum, Sapium indicum, Sapium rigidifolium and S. baccatum. This genus has been reported to contain various types of compounds including flavonoids, terpenoids and a few numbers of other classes (Srivastava & Agnihotri 1985; Brooks et al. 1987; Siems et al. 1993; Chumkaew
Figure 2. Malaytaraxerate 2D correlation NMR: (a) HMBC (
), COSY(
) and (b) NOESY.
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Figure 3. Plausible biogenetic pathway of malaytaraxerate.
et al. 2003; Woldemichael et al. 2004; Huang et al. 2007; Devkota et al. 2009; Liu et al. 2012). Diterpenoids are the most dominant compounds found in the studied species. However, no such compounds have been reported to occur in S. baccatum. Up until now, only 11 compounds have been reported from S. baccatum including an alkaloid, bukittinggine (Arbain et al. 1990), six triterpenes, lupeol, betulin (Ahmed et al. 2010), aleuritolic acid, 3-acetoxy aleutoric acid (Khastigir et al. 1969), taraxerol (2) and taraxerone (3), two sterols, b-sitosterol and stigmasterol (Ahmed et al. 2010), 3,30 -di-O-methyl ellagic acid (Khastigir et al. 1969; Ray et al. 1975) and baccatin (Saha et al. 1977). Of the six Sapium species, S. haematospermum was the only one reported to biologically synthesise triterpenes, displaying its close taxonomical relationship with S. baccatum in the genus. 2.3. Biological activity In this study, three compounds isolated from this plant (3, 4 and 5) were assayed for anticancer activity against HT-29 and MDA-MB cell lines. Their toxicity towards normal cell was also tested against 3T3 cell line. Compounds 1 and 2 could not be tested due to insufficient amount. The activity was expressed as the percentage of cell viability after being treated with the tested compounds for 72 h. Doxorubicin was used as positive control. The results indicated that all the tested compounds exhibited weak anticancer property against HT-29 and MDA-MB cancer cell lines. Amongst the tested compounds, 5 demonstrated the strongest cytotoxicity against HT-29 cells with an IC50 of 64.49 mg/mL, while 3 exhibited the strongest activity against MDA-MB cells with IC50 of 42.16 mg/mL. None of the tested compounds displayed significant toxicity towards the normal cell line 3T3, indicated by IC50 of . 100 mg/mL. The cytotoxicity of the tested compounds were significantly weaker compared
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with that of doxorubicin (IC50 ¼ 0.02 mg/mL against HT-29, IC50 ¼ 0.05 mg/mL against MDA-MB).
3. Experimental
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3.1. General experimental procedures The spectrophotometer instruments used in this research were Perkin – Elmer Lambda 35 UV – Vis (Waltham, USA), Perkin – Elmer FT-IR (Waltham, USA), Bruker NMR (Billerica, USA) 300 MHz for 1H and 75 MHz for 13C, and Agilent Technologies GC – MS (Santa Clara, USA). The chromatographic separation and purification were conducted using the following adsorbents: Silica Gel 60 PF254 (Merck, Darmstadt, Germany) catalogue number: 1.07747.2500) for VLC, Silica Gel 60 PF254 containing gypsum (Merck catalogue number: 1.07749.1000) for RC, Silica Gel 60 (0.040 – 0.063 mm) (Merck catalogue number: 1.09385.1000) for column chromatography, and Silica Gel 60 (0.2 –0.5 mm) (Merck catalogue number: 1.07733.1000) for sample. Thin-layer chromatography (TLC) Silica Gel 60 PF254 (aluminium sheets) (Merck catalogue number: 1.05554.0001) were used for TLC analysis.
3.2. Plant materials The sample used in this research was the stem bark of S. baccatum which was collected from the Reserved Forest UiTM Jengka, Pahang, Malaysia. The plant sample was identified by the botanist at the Forest Research Institute of Malaysia (FRIM), and a voucher specimen has been deposited at the Herbarium of FRIM with the voucher number FRI 52032.
3.3. Isolation and purification The ground, air-dried stem bark of S. baccatum (800 g) was extracted with 5 L of acetone at room temperature. The acetone extract was concentrated at 508C with rotary evaporator under reduced pressure to yield 156 g of crude extract. The tannins were separated by dissolving the crude extract in MeOH and then fractionating it with diethyl ether. The ether fraction was then concentrated again to yield95 g of crude extract with less tannin. The crude extract was subjected to VLC with column diameter of 17 cm and eluted with the mixtures of n-hexane and EtOAc with increasing polarity (starting with n-hexane/EtOAc, 9:1) to yield five fractions (fraction 1 – 5). A white solid was formed during the VLC. The solid was washed with acetone to yield 3 (100 mg). Fraction 3 (20 g) was subjected to VLC starting with CHCl3/MeOH (9:1) to yield four fractions (fraction 3.1 –3.4). Fraction 3.1 (31 mg) was subjected to RC with n-hexane/EtOAc, 9.5:0.5 to yield 2 (2.6 mg). Fraction 3.2 (256 mg) was subjected to RC with n-hexane/EtOAc (9:1) to yield 1 (12 mg). Fraction 3.3 (0.9 g) was subjected to RC with the same solvent systems to yield 4 (32.7 mg) and 5 (44.7 mg) Malaytaraxerate (1). White crystal. Melting point: 2358C. HR-ESI-MS [M þ H]þ at m/z: 485.2916 (calc. for C31H49O4 485.71836). UV – Vis lmax: 205.54 nm. IR y max (KBr disc) cm21: 3430, 2935, 2858, 1734, 1690, 1457, 1382, 1245, 1208, 1171, 1034, 938, 828. 1H NMR (CDCl3, 300 MHz): dH 4.46 (1H, dd, J ¼ 9.0, 6.6, H-3), 5.52 (1H, dd, J ¼ 7.8, 3.6, H-15), 0.84 (3H, s, H-22), 0.87 (3H, s, H-23), 0.94 (3H, s, H-24), 0.92 (3H, s, H-25), 0.95 (3H, s, H-26), 0.93 (3H, s, H-28), 0.91 (3H, s, H-29), 2.03 (3H, s, H-20 ). 13C NMR (75 MHz): dC 37.3 (C-1), 23.4 (C-2), 80.9 (C-3), 37.6 (C-4), 55.5 (C-5), 18.7 (C-6), 40.7 (C-7), 38.9 (C-8), 41.4 (C-9), 37.9 (C-10), 17.3 (C-11), 30.7 (C-12), 29.3 (C-13), 160.5 (C-14), 116.8 (C-15), 31.3 (C-16), 51.4 (C-17), 49.0 (C-18), 33.6 (C-19), 33.3 (C-20), 29.7 (C-21), 27.9 (C-22), 16.6 (C-23), 15.6 (C-24), 22.4 (C-25), 26.2 (C-26), 183.4 (C-27), 31.9 (C-28), 28.7 (C-29), 171.0 (C-10 ), 21.3 (C-20 ).
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3.4. Cytotoxicity 3.4.1. Cell lines and cell culture condition
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Cancer cell lines, HT-29 (colon colorectal adenocarcinoma, ATCC HTB-38) and MDAMB (mammary gland/breast, ATCC HTB-26), were maintained in RPMI 1640 (Sigma, St. Louis, MO, USA), supplemented with 10% of foetal bovine serum (FBS, PAA Laboratories, Pasching, Austria) and 1% of penicillin/streptomycin. The normal cell line, 3T3 (Mus musculus embryo, ATCC CRL-1658), was maintained in DMEM (PAA Laboratories), supplemented with 10% of FBS (PAA Laboratories) and 1% of penicillin/streptomycin. Cell numbers were counted by a haematocytometer, and the viability of cells was determined using trypan blue reagent. Cells of 80 –85% confluence were harvested and plated onto 96 flat bottom well plates for experimental use. In all experiments, the cells were incubated in a CO2 incubator at 378C with 5% CO2 overnight prior to treatment. 3.4.2. Cell viability assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay Stock solutions of 10 mg/mL were prepared in dimethyl sulfoxide (DMSO) and serial dilutions were made in culture media prior to use. The final concentrations of DMSO did not exceed 0.1% (v/v), a concentration which is non-toxic to the cells (Li et al. 2007). The cells treated with pure compounds (0.01 – 100 mg/mL) were incubated for 72 h after which the MTT assay was carried out as described by Mosmann (1983), but with slight modifications. After 72 h, supernatants were discarded and 50 mL of MTT stock solution (5 mg/mL) was added to each well and the plates were further incubated for 4 h. DMSO (100 mL) was added to each well to solubilise the water-insoluble purple formazan crystals. The amount of MTT – formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 570 nm using microplate reader (Tecan). The percentage of cytotoxic activity compared with the untreated cells was determined using the following equation: Cellviability ¼
OD of treated cell £ 100%: OD of control cell
ð1Þ
Data generated were used to plot a dose – response curve. Cytotoxic activity was expressed as the mean concentration of test sample required to kill 50% of the cell population. Doxorubicin was used as the positive reference in this study. 4. Conclusions The phytochemical investigation of the stem bark of Sapium baccatum resulted in the isolation of a new triterpenoid named malaytaraxerate (1) along with four known compounds including taraxerol (2), taraxerone (3), docosyl isoferulate (4), and docosanoic acid 20 ,30 -dihydroxypropyl ester (5). Compounds 3, 4 and 5 exhibited weak cytotoxicity towards HT-29 and MDA-MB cell lines. Supplementary material Supplementary material relating to this article is available online, alongside Tables S1 and S2 and Figures S1– S3. Acknowledgements The authors would like to thank the Faculty of Applied Sciences, Universiti Teknologi MARA for financing this research project.
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References Ahmed Y, Sohrab MH, Al-Reza SM, Tareq FS, Hasan CM, Sattar MA. 2010. Antimicrobial and cytotoxic constituents from leaves of Sapium baccatum. Food Chem. 48:549–552. Arbain D, Byrne LT, Cannon JR, Patrick VA, White AH. 1990. (2)-Bukittinggine, the major alkaloid of Sapium baccatum: crystal structure and absolute configuration of bukitttinggine hydrobromide. Aust J Chem. 43:185–190. Brooks G, Morrice NA, Ellis C, Aitken A, Evans AT, Evans FJ. 1987. Toxic phorbol esters from Chinese tallow stimulate protein kinase C. Toxicon. 25(11):1229–1233. Chumkaew P, Karalai C, Ponglimanont C, Chantrapromma K. 2003. Antimycobacterial activity of phorbol esters from the fruits of Sapium indicum. J Nat Prod. 66:540–543. Devkota HP, Basnet P, Yahara S. 2009. Diterpene esters and phenolic compounds from Sapium insigne (Royle) Benth. ex Hook fil. Chem Pharm Bull. 57(11):1289–1291. Esser HJA. 1999. Partial revision of the Hippomaneae (Euphorbiaceae) in Malaysia. Blumea. 44:149–215. Eswani N, Kudus KA, Nazre M, Noor AGA. 2010. Medicinal plant diversity and vegetation analysis of logged over hill forest of Tekai Tembeling Forest Reserve, Jerantut, Pahang. J Agric Sci. 2(3):189–210. Heyne K. 1950. De Nuttige Planten van Indonesie. 3rd ed. Bandung: N.V. Uitgeverijw van Hoeve-‘s-Gravenhage. Huang S, Fujioka T, Yoshida M, Ishimaru K. 2007. A new chalcone glycoside from Sapium sebiferum. J Nat Med. 61:339–341. Khastigir HN, Pradhan BP, Misra DR. 1969. Terpenoids and related compounds: part VIII. Chemical investigation of Sapium baccatum Roxb. J Indian Chem Soc. 46:663–664. Li YL, Gan GP, Zhang HZ, Li CL, Huang YP, Liu YW, Liu JW. 2007. A flavonoid glycoside isolated from Smilax china L. rhizomes in vitro anticancer effects on human cancer cell lines. J Ethnopharmacol. 113:115–124. Liu HB, Zhang H, Yu JH, Xu CH, Ding J, Yue JM. 2012. Cytotoxic diterpenoids from Sapium insigne. J Nat Prod. 75:722–725. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55–63. Panthong A, Kanjanapothi D, Thitiponpunt Y, Taesotikul T, Arbain D. 1998. Anti-inflammatory activity of the alkaloid bukittinggine from Sapium baccatum. Plant Med. 64:530–535. Ray TK, Misra DR, Khastgir HN. 1975. Phytosterols in Euphorbiaceae and Rutaceae. Phytochemistry. 14:1876–1877. Saha B, Naskar DB, Misra DR, Pradhan BP, Khastigir HN. 1977. Baccatin, a novel nor-triterpene peroxide isolated from Sapium baccatum Roxb. Tetrahedron Lett. 35:3095–3098. Siems K, Jakupovic J, Castro V, Poveda L. 1993. Rigidol, an unusual diterpene from Sapium rifidifolium. Phytochemistry. 33(6):1465– 1468. Srivastava SK, Agnihotri VK. 1985. 3-O-acetylcycloart-23-en-25-ol from the roots of Sapium insigne. J Nat Prod. 48:496–497. Woldemichael GM, Lugo MTG, Franzblau SG, Wang Y, Suarez E, Timmermann BN. 2004. Mycobacterium tuberculosis growth inhibition by constituents of Sapium haematospermum. J Nat Prod. 67:598–603.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new triterpenoid from Sapium baccatum (Euphorbiaceae) a
ac
L.M. Ramadhan Al Muqarrabun , Norizan Ahmat , S. Ruzaina S. a
a
a
Aris , Norhazana Norizan , Nurdiana Shamsulrijal , Farida Z.M. a
a
b
c
Yusof , M. Nazip Suratman , M. Izwan M. Yusof & Fatimah Salim a
Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia b
Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia c
Atta-ur-Rahman Institute for Natural Product Discovery (AuRins), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia Published online: 04 Apr 2014.
To cite this article: L.M. Ramadhan Al Muqarrabun, Norizan Ahmat, S. Ruzaina S. Aris, Norhazana Norizan, Nurdiana Shamsulrijal, Farida Z.M. Yusof, M. Nazip Suratman, M. Izwan M. Yusof & Fatimah Salim (2014) A new triterpenoid from Sapium baccatum (Euphorbiaceae), Natural Product Research: Formerly Natural Product Letters, 28:13, 1003-1009, DOI: 10.1080/14786419.2014.903396 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903396
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 13, 1003–1009, http://dx.doi.org/10.1080/14786419.2014.903396
A new triterpenoid from Sapium baccatum (Euphorbiaceae) L.M. Ramadhan Al Muqarrabuna, Norizan Ahmatac*, S. Ruzaina S. Arisa, Norhazana Norizana, Nurdiana Shamsulrijala, Farida Z.M. Yusofa, M. Nazip Suratmana, M. Izwan M. Yusofb and Fatimah Salimc a
Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia; Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia; cAtta-ur-Rahman Institute for Natural Product Discovery (AuRins), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Kuala Selangor, Malaysia
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b
(Received 6 January 2014; final version received 8 March 2014) A new triterpene, malaytaraxerate (1), and four known compounds, taraxerol (2), taraxerone (3), docosyl isoferulate (4) and docosanoic acid 20 ,30 -dihydroxypropyl ester (5), were isolated from the acetone extract of Sapium baccatum stem bark. The structures of the isolated compounds were determined using several spectroscopic methods, including UV – Vis, FT-IR, 1D and 2D NMR, and mass spectrometry. Major isolated compounds were assayed for cytotoxicity. The chemotaxonomic significance of this plant was also studied. Keywords: Euphorbiaceae; Sapium baccatum; triterpenoid; malaytaraxerate; cytotoxicity
1. Introduction Sapium baccatum (Euphorbiaceae), also known as ludai (Malay), is a medicinal plant which belongs to the family Euphorbiaceae. This species commonly grows in Malaysia, Sumatera and Borneo (Kalimantan) (Esser 1999). It is widely used as timber tree and as wayside plant. The fruits are edible, mealy and sweet (Heyne 1950) and have been used to treat ulcer by the folk in Pahang, Malaysia (Eswani et al. 2010). Panthong et al. (1998) reported antiinflammatory activity displayed by the major alkaloid isolated from this species, bukittinggine (Arbain et al. 1990). Besides bukittinggine, there are only a few number of compounds that have been isolated from the leaves of this species; which include 3-acetoxy aleuritolic acid, 1hexacosanol, b-sitosterol, 3,30 -di-O-methyl ellagic acid (Khastigir et al. 1969; Ray et al. 1975), baccatin (Saha et al. 1977), taraxerol (2), taraxerone (3), lupeol, betulin and stigmasterol (Ahmed et al. 2010). However, up until now, the chemistry of the stem bark of this plant has not been reported. Thus, conducting further study is important in order to enrich the knowledge about the chemistry of the species. In this study, a new triterpene, malaytaraxerate (1), and four known compounds, taraxerol (2), taraxerone (3), docosyl isoferulate (4) and docosanoic acid 20 ,30 -dihydroxypropyl ester (5), were isolated from the stem bark of S. baccatum. The cytotoxicity of the isolated compounds against two cancer cell lines, human colorectal cancer (HT-29) and mammary breast cancer (MDA-MB), and normal cell line 3T3, were also studied.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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2. Results and discussion 2.1. Structure determination of malaytaraxerate The methanolic extract of the stem bark of S. baccatum was fractionated using vacuum liquid chromatography (VLC) and purified using radial chromatography (RC) and recrystallisation yielding five compounds (1 –5) (Figure 1), including a new triterpene named malaytaraxerate (1). The structural elucidation of the compounds was determined using several spectroscopic methods including mass spectrometry, UV – Vis, FT-IR, NMR 1D and 2D (HMQC, HMBC, COSY and NOESY), and by comparing with reported data. Compound 1 was isolated as a white crystal. The molecular formula C31H48O4 was generated using HR-ESI-MS [M þ H]þ ion at m/z 485.2916 (calc. for C31H49O4 485.71836). The UV spectrum indicated that there was no conjugated double bond in the molecule; this was deducted by the presence of only one absorbance band at 205.54 nm. The FT-IR spectral data revealed a broad absorbance band at 3430 cm21 representing the stretching vibration of OZH bond. The presence of carbonyl group was indicated by a strong stretching band of CvO at 1734 cm21 and the vibration band at 1034 cm21 indicated the CZO bending representing an ester/carboxylic acid group. The CZC(vO)ZO asymmetrical stretching vibration was indicated by absorbance band at 1245 cm21. The Csp3ZH stretching band appeared at 2935 cm21, while absorption bands at 1457 and 1382 cm21 indicated the asymmetrical and symmetrical bending vibrations of Csp3ZH of the methyl groups, respectively. An olefinic group was detected in this compound which was indicated by an absorbance peak of symmetrical CvC stretching at 1690 cm21 and Csp2ZH stretching band at 2858 cm21. Compound 1 contained 31 carbons observed using APT experiment of 13C NMR. The compound consisted of eight methyls, nine methylenes, five methines and nine quarternary carbons. The DBE value of eight indicated that this compound was a pentacyclic triterpene containing three double bonds. A carbon at dC 80.9 (C-3) indicated the presence of an oxygenattaching carbon, while the carbon –carbon double bond in the molecule was revealed by a pair of highly deshielded carbon signals at dC 160.5 (C-14) and 116.8 (C-15). A highly deshielded quarternary carbon at dC 183.4 (C-27) represented a carbonyl carbon of carboxylic acid, while signal at dC 171.0 (C-10 ) indicated a carbonyl carbon which is typical to ester group. One methyl signal at dC 21.3 (C-20 ) indicated the presence of a methyl adjacent to a carbonyl carbon, forming an acetate group.
Figure 1. Compounds isolated from the stem bark of S. baccatum.
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According to 1H NMR, there was a proton at dH 4.46 (dd, J ¼ 9.0 and 6.6, H-3) integrated for one proton, indicating a proton attached to an oxygen-attaching carbon, which in this case is C-3. There was only one highly deshielded proton at dH 5.52 (H-15) indicating the olefinic proton that attaches to a double-bond carbon (C-15). This explains that the sole carbon –carbon double bond in the molecule was constructed by a quarternary carbon (C-14) and a methine (C-15). Eight singlet methyls were detected in this compound suggesting that all the methyl groups were attached to quarternary carbons. This kind of skeletal structure is characteristic to the oleananetype triterpene. Seven methyls were located in the ring system while the other one was adjacent to a carbonyl carbon. A singlet methylene proton signal appeared at dH 1.25 indicating that the methylene (C-21) was located between two quarternary carbons (C-17 and C-20). The structure of 1 was confirmed using 2D NMR including HMBC, COSY and NOESY. The HMBC spectrum revealed a strong correlation between acetate methyl (C-20 ) and the carbonyl carbon (C-10 ) as well as between C-20 and the oxymethine (C-3). This indicated that the acetate group was bound to oxymethine (C-3). The correlations between H-15 and C-8 as well as between H-16 and C-14 confirmed the position of the double bond in the molecule. The position of the carboxylic acid group was confirmed by the correlations between H-17 and H-21. The COSY spectrum displayed the correlations between the oxymethine proton (H-3) and H-2, while the olefinic proton (H-15) shows a correlation with H-16 as described in Figure 2(a). NOESY spectrum revealed correlation between H-15 and H-16 confirming the position of the double bond in the molecule (Figure 2(b)). Judging from the structure, 1 was proposed to be synthesised via a pathway similar to that of taraxerol (2) and taraxerone (3). Lupanyl cation underwent a series of rearrangement reactions to form a pentacyclic triterpene with four hexagonal rings and one pentagonal ring due to the loss of one methylene from the molecule. The methyl group (C-26) was attached to C-13, while a double bond was formed on C-14 and C-15 by deprotonation of C-15. The methyl group at C-27 was hydroxylated, and converted into carboxylic acid. The reactions did not stop here. The hydroxyl group attached to oxymethine C-3 underwent an esterification reaction with acetic acid to form compound 1 (Figure 3).
2.2. Chemotaxonomy significance According to the author’s literature search, only six species of genus Sapium have been phytochemically studied so far, including Sapium insigne, Sapium haematospermum, Sapium sebiferum, Sapium indicum, Sapium rigidifolium and S. baccatum. This genus has been reported to contain various types of compounds including flavonoids, terpenoids and a few numbers of other classes (Srivastava & Agnihotri 1985; Brooks et al. 1987; Siems et al. 1993; Chumkaew
Figure 2. Malaytaraxerate 2D correlation NMR: (a) HMBC (
), COSY(
) and (b) NOESY.
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Figure 3. Plausible biogenetic pathway of malaytaraxerate.
et al. 2003; Woldemichael et al. 2004; Huang et al. 2007; Devkota et al. 2009; Liu et al. 2012). Diterpenoids are the most dominant compounds found in the studied species. However, no such compounds have been reported to occur in S. baccatum. Up until now, only 11 compounds have been reported from S. baccatum including an alkaloid, bukittinggine (Arbain et al. 1990), six triterpenes, lupeol, betulin (Ahmed et al. 2010), aleuritolic acid, 3-acetoxy aleutoric acid (Khastigir et al. 1969), taraxerol (2) and taraxerone (3), two sterols, b-sitosterol and stigmasterol (Ahmed et al. 2010), 3,30 -di-O-methyl ellagic acid (Khastigir et al. 1969; Ray et al. 1975) and baccatin (Saha et al. 1977). Of the six Sapium species, S. haematospermum was the only one reported to biologically synthesise triterpenes, displaying its close taxonomical relationship with S. baccatum in the genus. 2.3. Biological activity In this study, three compounds isolated from this plant (3, 4 and 5) were assayed for anticancer activity against HT-29 and MDA-MB cell lines. Their toxicity towards normal cell was also tested against 3T3 cell line. Compounds 1 and 2 could not be tested due to insufficient amount. The activity was expressed as the percentage of cell viability after being treated with the tested compounds for 72 h. Doxorubicin was used as positive control. The results indicated that all the tested compounds exhibited weak anticancer property against HT-29 and MDA-MB cancer cell lines. Amongst the tested compounds, 5 demonstrated the strongest cytotoxicity against HT-29 cells with an IC50 of 64.49 mg/mL, while 3 exhibited the strongest activity against MDA-MB cells with IC50 of 42.16 mg/mL. None of the tested compounds displayed significant toxicity towards the normal cell line 3T3, indicated by IC50 of . 100 mg/mL. The cytotoxicity of the tested compounds were significantly weaker compared
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with that of doxorubicin (IC50 ¼ 0.02 mg/mL against HT-29, IC50 ¼ 0.05 mg/mL against MDA-MB).
3. Experimental
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3.1. General experimental procedures The spectrophotometer instruments used in this research were Perkin – Elmer Lambda 35 UV – Vis (Waltham, USA), Perkin – Elmer FT-IR (Waltham, USA), Bruker NMR (Billerica, USA) 300 MHz for 1H and 75 MHz for 13C, and Agilent Technologies GC – MS (Santa Clara, USA). The chromatographic separation and purification were conducted using the following adsorbents: Silica Gel 60 PF254 (Merck, Darmstadt, Germany) catalogue number: 1.07747.2500) for VLC, Silica Gel 60 PF254 containing gypsum (Merck catalogue number: 1.07749.1000) for RC, Silica Gel 60 (0.040 – 0.063 mm) (Merck catalogue number: 1.09385.1000) for column chromatography, and Silica Gel 60 (0.2 –0.5 mm) (Merck catalogue number: 1.07733.1000) for sample. Thin-layer chromatography (TLC) Silica Gel 60 PF254 (aluminium sheets) (Merck catalogue number: 1.05554.0001) were used for TLC analysis.
3.2. Plant materials The sample used in this research was the stem bark of S. baccatum which was collected from the Reserved Forest UiTM Jengka, Pahang, Malaysia. The plant sample was identified by the botanist at the Forest Research Institute of Malaysia (FRIM), and a voucher specimen has been deposited at the Herbarium of FRIM with the voucher number FRI 52032.
3.3. Isolation and purification The ground, air-dried stem bark of S. baccatum (800 g) was extracted with 5 L of acetone at room temperature. The acetone extract was concentrated at 508C with rotary evaporator under reduced pressure to yield 156 g of crude extract. The tannins were separated by dissolving the crude extract in MeOH and then fractionating it with diethyl ether. The ether fraction was then concentrated again to yield95 g of crude extract with less tannin. The crude extract was subjected to VLC with column diameter of 17 cm and eluted with the mixtures of n-hexane and EtOAc with increasing polarity (starting with n-hexane/EtOAc, 9:1) to yield five fractions (fraction 1 – 5). A white solid was formed during the VLC. The solid was washed with acetone to yield 3 (100 mg). Fraction 3 (20 g) was subjected to VLC starting with CHCl3/MeOH (9:1) to yield four fractions (fraction 3.1 –3.4). Fraction 3.1 (31 mg) was subjected to RC with n-hexane/EtOAc, 9.5:0.5 to yield 2 (2.6 mg). Fraction 3.2 (256 mg) was subjected to RC with n-hexane/EtOAc (9:1) to yield 1 (12 mg). Fraction 3.3 (0.9 g) was subjected to RC with the same solvent systems to yield 4 (32.7 mg) and 5 (44.7 mg) Malaytaraxerate (1). White crystal. Melting point: 2358C. HR-ESI-MS [M þ H]þ at m/z: 485.2916 (calc. for C31H49O4 485.71836). UV – Vis lmax: 205.54 nm. IR y max (KBr disc) cm21: 3430, 2935, 2858, 1734, 1690, 1457, 1382, 1245, 1208, 1171, 1034, 938, 828. 1H NMR (CDCl3, 300 MHz): dH 4.46 (1H, dd, J ¼ 9.0, 6.6, H-3), 5.52 (1H, dd, J ¼ 7.8, 3.6, H-15), 0.84 (3H, s, H-22), 0.87 (3H, s, H-23), 0.94 (3H, s, H-24), 0.92 (3H, s, H-25), 0.95 (3H, s, H-26), 0.93 (3H, s, H-28), 0.91 (3H, s, H-29), 2.03 (3H, s, H-20 ). 13C NMR (75 MHz): dC 37.3 (C-1), 23.4 (C-2), 80.9 (C-3), 37.6 (C-4), 55.5 (C-5), 18.7 (C-6), 40.7 (C-7), 38.9 (C-8), 41.4 (C-9), 37.9 (C-10), 17.3 (C-11), 30.7 (C-12), 29.3 (C-13), 160.5 (C-14), 116.8 (C-15), 31.3 (C-16), 51.4 (C-17), 49.0 (C-18), 33.6 (C-19), 33.3 (C-20), 29.7 (C-21), 27.9 (C-22), 16.6 (C-23), 15.6 (C-24), 22.4 (C-25), 26.2 (C-26), 183.4 (C-27), 31.9 (C-28), 28.7 (C-29), 171.0 (C-10 ), 21.3 (C-20 ).
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3.4. Cytotoxicity 3.4.1. Cell lines and cell culture condition
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Cancer cell lines, HT-29 (colon colorectal adenocarcinoma, ATCC HTB-38) and MDAMB (mammary gland/breast, ATCC HTB-26), were maintained in RPMI 1640 (Sigma, St. Louis, MO, USA), supplemented with 10% of foetal bovine serum (FBS, PAA Laboratories, Pasching, Austria) and 1% of penicillin/streptomycin. The normal cell line, 3T3 (Mus musculus embryo, ATCC CRL-1658), was maintained in DMEM (PAA Laboratories), supplemented with 10% of FBS (PAA Laboratories) and 1% of penicillin/streptomycin. Cell numbers were counted by a haematocytometer, and the viability of cells was determined using trypan blue reagent. Cells of 80 –85% confluence were harvested and plated onto 96 flat bottom well plates for experimental use. In all experiments, the cells were incubated in a CO2 incubator at 378C with 5% CO2 overnight prior to treatment. 3.4.2. Cell viability assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay Stock solutions of 10 mg/mL were prepared in dimethyl sulfoxide (DMSO) and serial dilutions were made in culture media prior to use. The final concentrations of DMSO did not exceed 0.1% (v/v), a concentration which is non-toxic to the cells (Li et al. 2007). The cells treated with pure compounds (0.01 – 100 mg/mL) were incubated for 72 h after which the MTT assay was carried out as described by Mosmann (1983), but with slight modifications. After 72 h, supernatants were discarded and 50 mL of MTT stock solution (5 mg/mL) was added to each well and the plates were further incubated for 4 h. DMSO (100 mL) was added to each well to solubilise the water-insoluble purple formazan crystals. The amount of MTT – formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 570 nm using microplate reader (Tecan). The percentage of cytotoxic activity compared with the untreated cells was determined using the following equation: Cellviability ¼
OD of treated cell £ 100%: OD of control cell
ð1Þ
Data generated were used to plot a dose – response curve. Cytotoxic activity was expressed as the mean concentration of test sample required to kill 50% of the cell population. Doxorubicin was used as the positive reference in this study. 4. Conclusions The phytochemical investigation of the stem bark of Sapium baccatum resulted in the isolation of a new triterpenoid named malaytaraxerate (1) along with four known compounds including taraxerol (2), taraxerone (3), docosyl isoferulate (4), and docosanoic acid 20 ,30 -dihydroxypropyl ester (5). Compounds 3, 4 and 5 exhibited weak cytotoxicity towards HT-29 and MDA-MB cell lines. Supplementary material Supplementary material relating to this article is available online, alongside Tables S1 and S2 and Figures S1– S3. Acknowledgements The authors would like to thank the Faculty of Applied Sciences, Universiti Teknologi MARA for financing this research project.
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