Abstract
Phytochemical investigation of the ethyl acetate-soluble fraction of stem bark extract of an African medicinal plant Terminalia brownii led to the isolation of a new oleanane-type triterpenoid, along with seven known triterpenoids, seven ellagic acid derivatives, and 3-O-β-D-glucopyranosyl-β-sitosterol. The new compound was identified using spectroscopic methods, notably 1D- and 2D NMR, as 3β,24-O-ethylidenyl-2α,19α-dihydroxyolean-12-en-28-oic acid. The isolated compounds were evaluated for their antimicrobial and antiplasmodial activities. Two compounds with a galloyl group (4 and 6) were found to be active against chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum, whereas three ellagic acid derivatives (5–7) were found active against three species of fungi and one species of bacteria.
Keywords: Terminalia brownii; Antimicrobial; Antiplasmodial; 3β, 24-O-Ethylidenyl-2α,19α-dihydroxyolean-12-en-28-oic acid
Terminalia (Combretaceae) species, distributed in the tropics and sub-tropic regions, are famous for their usefulness in traditional medicine [1]. The main compounds reported from this genus are tannins, flavonoids, pentacyclic triterpenoids and their glycosidic derivatives. Some of these compounds have been reported to have biological properties such as antimalarial, antifungal, antibacterial, antidiabetic, antioxidant, immunoregulatory and cytotoxic activities [1–7]. T. brownii Fresen. occurs in parts of Eastern and Central Africa [8], where it is used traditionally as a remedy for malaria, yellow fever, diarrhea, ulcers, cough, hepatitis, stomach ache and sexually transmitted diseases. Previous studies on the plant reported the antimicrobial activity of its extracts and fractions [8, 9]. Studies carried out in Kenya reported three triterpenoids [9], and another study reported one chromone derivative from plant material collected in Tanzania [10]. However, the reported data on characterization and quantification of compounds from T. brownii are rather inadequate.
Owing to the extensive use of T. brownii in traditional medicine and the observed activity of its extracts, we have investigated it for antiparasitic and antimicrobial constituents. Therefore, this communication reports the isolation of oleanane-type triterpenoids and ellagic acid derivatives from the plant, and evaluation of their antiplasmodial and antimicrobial activities. We also report the first isolation of 3β,24-O-ethylidenyl-2α,19α-dihydroxyolean-12-en-28-oic acid (1) from either a natural or synthetic source.
Antiplasmodial evaluation of the fractions obtained from liquid-liquid partitioning of the EtOH extract from the stem bark of T. brownii indicated that activity was concentrated in the ethyl acetate fraction. Chromatographic procedures on this fraction, as described in the Experimental Section, yielded fifteen known compounds and one new oleanane-type triterpenoid (1). The known compounds isolated were identified using NMR and MS data, and by comparison with published data for arjunic acid (2) [11], sericic acid (3) [12], 23-galloylarjunic acid (4) [13], tomentosic acid, [14], arjungenin [15], sericoside [16], arjunglucoside I [15] and 3-O-β-D-glucopyranosyl-β-sitosterol [17]. Other compounds are 3-O-methylellagic acid (5), 3,3′-di-O-methylellagic acid, 3,3′,4-tri-O-methylellagic acid [18], 4-(α-L-rhamnopyranosyl)ellagic acid [19], 3-O-methyl-4-(α-L-rhamnopyranosyl)ellagic acid [18], 4-O-(3″,4″-di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (6) [20] and diellagic lactone (7) [21] (Figure 1).
Figure 1.
Structures of compounds 1–7.
Compound 1 was isolated as white amorphous powder with the molecular formula C32H50O6, as determined by HRESIMS (negative mode, m/z 529.3651 [M-H]−), with eight degrees of unsaturation. The IR absorption bands at 3442 (br), 1686 and 1644 indicated the presence of hydroxyl, carboxyl and olefinic functionalities. Comprehensive 1H- and 13C-NMR data are shown in Table 1. The 1H NMR spectrum displayed, among other signals, six tertiary methyls (δH 0.74, 0.93, 0.96, 1.05, 1.29, 1.35) and one secondary methyl (δH 1.25, d, J=5.2 Hz). The 13C NMR and DEPT indicated the presence of 32 carbons. The low field region had a carboxyl carbon (δC 180.9), two olefinic carbons (δC 123.1 d, 143.3 s), one acetal carbon (δC 92.4 d), three oxymethine carbons (δC 85.7, 80.9, 61.7) and one oxymethylene carbon (δC 69.6). The high field region had seven methyl, eight methylene, three methine and six quaternary carbons.
Table 1.
1H and 13C NMR spectroscopic data for compound 1
| Position | δCa | δHb |
|---|---|---|
| 1 | 45.2, t | 0.89, m; 2.00, m |
| 2 | 61.7, d | 4.40, dd (10.4, 4.0) |
| 3 | 85.7, d | 3.13, dd (10.0) |
| 4 | 37.2, s | |
| 5 | 55.4, d | 1.00, m |
| 6 | 17.4, t | |
| 7 | 32.6, t | |
| 8 | 39.3, s | |
| 9 | 47.4, d | 1.78, m |
| 10 | 37.6, s | |
| 11 | 27.9, t | |
| 12 | 123.1, d | 5.31, br s |
| 13 | 143.3, s | |
| 14 | 41.2, s | |
| 15 | 28.0, t | |
| 16 | 23.6, t | |
| 17 | 45.2, s | |
| 18 | 43.7, d | 3.04, br s |
| 19 | 80.9, d | 3.24, d (2.4) |
| 20 | 34.6, s | |
| 21 | 27.1, t | 1.75, m |
| 22 | 32.6, t | |
| 23 | 25.2, q | 1.35, s |
| 24 | 69.6, t | 3.43, d (12.4); 4.02, d (12.0) |
| 25 | 15.9, q | 1.05, s |
| 26 | 16.5, q | 0.74, s |
| 27 | 23.7, q | 1.29, s |
| 28 | 180.9, s | |
| 29 | 27.3, q | 0.92, s |
| 30 | 23.7, q | 0.96, s |
| 1′ | 92.4, d | 5.02, dd (5.2, 1.2) |
| 2′ | 19.9, q | 1.25, d (5.2) |
Recorded at 100 MHz in CD3OD.
Recorded at 400 MHz in CD3OD.
The carbon signals were indicative of an olean-12-en skeleton and correlated closely to those of sericic acid 3 [12], except for the additional acetal methine carbon (δC 92.4) and a secondary methyl carbon (δC 19.9) of 1. Proton signals for the additional groups (δH 5.02 d, J=5.2 Hz and δH 1.25 d, J=5.2 Hz) showed COSY correlation, suggesting an ethylidene group of an acetal moiety. The ethylidene was assigned as connected to the oxygenated carbon atoms of C-3 and C-24 forming a six membered 1,3-dioxane moiety [4], as confirmed by HMBC correlations made by H-3 (δH 3.13) and H-24 (δH 3.43) with the acetal carbon (δC 92.4), as well as the downfield shift of C-3 and C-24 compared with those of sericic acid 3. Figure 2 shows some important HMBC and NOESY correlations that were used to confirm the structure.
Figure 2.
Selected HMBC and NOESY correlations of 1.
In vitro antimicrobial and antiplasmodial activities were evaluated for all the compounds, except 1, which was isolated in small quantities as a minor compound. Antimicrobial activity was evaluated against the fungi Candida albicans, C. glabrata, C. krusei, Cryptococcus neoformans, and Aspergillus fumigatus, and the bacteria Staphylococcus aureus, methicillin-resistant S. aureus (MRS), Escherichia coli, Pseudomonas aeruginosa, and Mycobaterium intracellulare (see Table 2). Activity was observed for ellagic acid derivatives 5, 6 and 7, as well as for the triterpene arjunic acid (2), and was stronger towards C. glabrata compared with other Candida species. The most potent activity observed was for diellagic lactone 7 with an IC50 of 0.32 μg/mL. The antiplasmodial activity was evaluated against chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum. Results are summarized in Table 3. Activity was observed for 4 and 6, both of which have galloyl group(s). All compounds tested were not cytotoxic to VERO cells, a mammalian cell line.
Table 2.
Antimicrobial activity of isolated compounds as IC50 in μg/mL
| C. albicans | C. glabrata | C. crusei | C. neoformans | P. aeruginosa | |
|---|---|---|---|---|---|
| 2 | - | - | - | 14.5 | - |
| 5 | - | 2.7 | - | 18.0 | - |
| 6 | 15.0 | 0.6 | 4.3 | 4.7 | 8.8 |
| 7 | 19.4 | 0.3 | 1.7 | 7.6 | 8.4 |
| Amphotericin B | 0.2 | 0.2 | 0.5 | 0.2 | NT |
| Ciprofloxacin | NT | NT | NT | NT | 0.06 |
NT = not tested; - = not active.
Table 3.
Antiplasmodial activity of isolated compounds
| P. falciparum (D6) | P. falciparum (W2) | VERO | |||
|---|---|---|---|---|---|
|
|
|||||
| IC50 (μg/mL) | SI | IC50 (μg/mL) | SI | IC50 | |
| n-Hexane extract | - | - | - | - | NC |
| Ethyl acetate extract | <5.3 | >9 | <5.3 | >9 | NC |
| Aqueous extract | 47.1 | >1 | 27.4 | >1.7 | NC |
| 4 | 4.5 | >1 | 2.8 | >1.7 | NC |
| 6 | 4.7 | >1 | 4.7 | >1 | NC |
| Artemisinin | <0.03 | >9 | <0.03 | >9 | NC |
| Chloroquine | <0.03 | >9 | 0.15 | >1.4 | NC |
SI = selectivity index (ratio of IC50 vs. VERO cells to P. falciparum strain); NC = not cytotoxic at the highest test concentration; NT = not tested; - = not active.
Experimental
Plant material
The stem bark of Terminalia brownii was collected from Machakos County, Kenya, in November 2011, and identified at the University Herbarium, School of Biological Sciences, University of Nairobi, where a voucher specimen (JM2011/502) was deposited. The stem bark was air dried in shade and pulverized.
General experimental procedures
1H- and 13C-NMR spectra were recorded on a Varian-Mercury 400 spectrometer. Optical rotations were measured in MeOH using an AUTOPOL IV® instrument at ambient temperature. HRMS were obtained by direct injection using a Bruker Bioapex-FTMS with electrospray ionization (ESI). IR spectra were taken as films on a Bruker Tensor 27 FTIR instrument. UV spectra were obtained in MeOH using a Hewlett-Packard 8453 spectrophotometer. Column chromatography used either silica gel 60 (Merck, 0.063–0.2 mm) or silica gel RP C-18 (EMD, 0.04-0.063 mm) or Sephadex LH-20 (Fluka) as stationary phases. Analytical TLC was carried out using factory prepared aluminum plates (0.25 mm) coated with silica gel (60 F254, Merck) and compounds were visualized by observing under UV light at 254 or 365 nm, followed by spraying with 1% vanillin-H2SO4 spray reagent and heating.
Extraction and isolation
Dried and pulverized stem bark (0.6 Kg) was extracted by cold percolation at room temperature using 1:1 CH2Cl2/MeOH (3×2 L, each 24 h), followed by 100% methanol (1×2 L, 24 h). The filtrates were concentrated in a rotory evaporator and combined to give 5 g of black-brown gummy extract, which was suspended in methanol/water (2:8, 100 mL) and partitioned successively with n-hexane and EtOAc (2×200 mL each) to give 0.2 g of n-hexane extract, 2.1 g of ethyl acetate extract and 2.2 g of residual water extract. Antiplasmodial assays showed that the activity was concentrated in the ethyl acetate fraction, which was chosen for isolation of compounds.
The ethyl acetate extract (2.0 g) was chromatographed over RP C-18 silica gel (100 g, 3×30 cm) eluted with H2O/MeCN 8:2 (800 mL), 7:3, (1200 mL), 6:4 (600 mL), 1:1 (1000 mL), 6:4 (400 mL), 8:2 (300 mL) and 100% MeCN (1000 mL). 260 fractions of 20 mL each were collected and combined according to similarities in their TLC patterns. Repeated CC and crystallization afforded the compounds in the following order: 4-(α-L-rhamnopyranosyl)ellagic acid (14.6 mg, afforded by crystallization of fractions 22–28 in MeOH/CHCl3), 3-O-methyl-4-(α-L-rhamnopyranosyl)ellagic acid (47 mg, crystallized from fractions 44–51, H2O/MeCN; 7:3), arjunglucoside I (5.7 mg, afforded by cleaning fractions 52–53 by Sephadex LH-20 CC, using MeOH/CHCl3; 7:3), diellagic lactone 7 (6.5 mg, precipitated from MeOH solution of fractions 54–56), sericoside (15.2 mg, obtained by Sephadex LH-20 CC of fractions 61–65 with MeOH/CHCl3; 7:3), 3-O-methylellagic acid 5 (4.0 mg, obtained by Sephadex LH-20 CC of fractions 61–65 with MeOH/CHCl3; 7:3), 4-O-(3″,4″-di-O-galloyl-α-L-rhamnopyranosyl) ellagic acid 6 (21.1 mg, afforded by Sephadex LH-20 CC of fractions 77–81 with MeOH/CHCl3; 7:3), 3,3′-di-O-methylellagic acid (13 mg, crystallized in fractions 95–100, H2O/MeCN; 7:3), 3,3′,4-tri-O-methylellagic acid (5.2 mg, obtained by silica gel CC of fractions 113–118 using CHCl3/MeOH; 9:1), arjugenin (14.6 mg, afforded by silica gel CC of fractions 126–130, using CHCl3/MeOH; 92:8), tomentosic acid (16.0 mg, afforded by silica gel CC of fractions 132–139, using CHCl3/MeOH; 92:8), 23-galloylarjunic acid 4 (6.5 mg, afforded by silica gel CC of fractions 126–130, using CHCl3/MeOH; 92:8), sericic acid 3 (168 mg, crystallized from fractions 144–161, H2O/MeCN; 1:1), arjunic acid 2 (6.8 mg, afforded by silica gel CC of fractions 177–194, using CHCl3/MeOH; 94:6), 3β,24-O-ethylidenyl-2α,19α-dihydroxyolean-12-en-28-oic acid 1 (2.8 mg, afforded by silica gel CC of fractions 177–194, using CHCl3/MeOH; 94:6) and 3-O-β-D-glucopyranosyl-β-sitosterol (4.0 mg, afforded by silica gel CC of fractions 255–258, using CHCl3/MeOH; 93:7).
Identification of the compounds
The known compounds (vide infra) were identified using NMR spectroscopic methods (1H NMR, 13C NMR, DEPT, COSY, HSQC), and MS, as well as by direct comparison with published spectroscopic data and structures [12–21].
3β,24-O-Ethylidenyl-2α,19α-dihydroxyolean-12-en-28-oic acid (1)
White amorphous powder.
Rf : 0.45 (CHCl3/MeOH 93:7).
[α]D 25: −49.99 (c 0.2 mg/mL).
UV: 270 nm, 229 nm.
IR: 3442 cm−1 (OH), 1686 cm−1 (carboxyl), 1644 cm−1 (olefinic).
1H and 13C-NMR: see Table 1; also Supporting Information.
HR-ESIMS m/z: 529.3651 [M-H]−, 575.3688 [M+HCOO]−, 1059.7018 [2M-H]−, 548.3846 [M+H2O]+.
Antimicrobial assay
Susceptibility testing was performed at the National Center for Natural Products Research, University of Mississippi, using a modified version of the CLSI methods, as described by Samoylenko et al [22]. The organisms were obtained from the American Type Culture Collection (Manassas, VA) and include the fungi Candida albicans ATCC 90028, C. glabrata ATCC 90030, C. krusei ATCC 6258, Cryptococcus neoformans ATCC 90113 and Aspergillus fumigatus ATCC 204305, as well as the bacteria Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus ATCC 33591 (MRS), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Mycobacterium intracellulare ATCC 23068. Drug controls were included in each assay, ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and amphotericin B (ICN Biomedicals, Ohio) for fungi.
Antiplasmodial assay
The in vitro antiplasmodial activity was measured using the assay protocol based on a colorimetric method that determines the parasitic lactate dehydrogenase (pLDH) activity [22,23]. The assay was performed in a 96-well microplate and included two P. falciparum strains [Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine-resistant)]. DMSO, artemisinin and chloroquine were included in each assay as vehicle and drug controls, respectively.
Supplementary Material
Acknowledgments
The authors thank Mr John Trott and Ms Marsha Wright for assistance in biological work, and Dr Bharathi Avula for HRMS and Mr Frank Wiggers for running the NMR spectra. This work is supported by US Department of Defense CDMRP, grant No. W81XWH-09, the NIH, NIAID, Division of AIDS, grant No. AI 27094, and the USDA ARS Specific Cooperative Agreement No. 58-6408-1-603. JOM would like to acknowledge International Science Program, Uppsala University, for research grant (KEN-02) for performing part of this work.
References
- 1.Liu M, Katerere DR, Gray AI, Seidel V. Phytochemical and antifungal studies on Terminalia mollis and Terminalia brachystemma. Fitoterapia. 2009;80:369–373. doi: 10.1016/j.fitote.2009.05.006. [DOI] [PubMed] [Google Scholar]
- 2.Cao S, Brodie PJ, Miller JS, Randrianaivo R, Ratovoson F, Birkinshaw C, Andriantsiferana R, Rasamison VE, Kingston DGI. Antiproliferative xanthones of Terminalia calcicola from the Madagascar rain forest. Journal of Natural Products. 2007;70:679–681. doi: 10.1021/np060627g. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cao S, Brodie PJ, Callmander M, Randrianaivo R, Rakotobe E, Rasamison VE, Kingston DGI. Saponins and a lignan derivative of Terminalia tropophylla from the Madagascar dry forest. Phytochemistry. 2010;71:95–99. doi: 10.1016/j.phytochem.2009.09.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ponou BK, Teponno RB, Ricciutelli M, Quassinti L, Bramucci M, Lupidi G, Barboni L, Tapondjou LA. Dimeric antioxidant and cytotoxic triterpenoid saponins from Terminalia ivorensis A. Chev. Phytochemistry. 2010;71:2108–2115. doi: 10.1016/j.phytochem.2010.08.020. [DOI] [PubMed] [Google Scholar]
- 5.Saxena M, Yadav S, Bawankule DU, Srivastava SK, Pal A, Mishra R, Gupta MM, Darokar MP, Priyanka, Khanuja SPS. An immunomodulator from Terminalia arjuna and biological evaluation of its derivatives. Natural Product Communications. 2008;3:891–894. [Google Scholar]
- 6.Nkobole N, Houghton PJ, Hussein A, Lall N. Antidiabetic activity of Terminalia sericea constituents. Natural Product Communications. 2011;6:1585–1588. [PubMed] [Google Scholar]
- 7.Pham AT, Malterud KE, Paulsen BS, Diallo D, Wangensteen H. DPPH radical scavenging and xanthine oxidase inhibitory activity of Terminalia macroptera leaves. Natural Product Communications. 2011;6:1125–1128. [PubMed] [Google Scholar]
- 8.Mbwambo ZH, Moshi MJ, Masimba MJ, Kapingu MC, Nondo RSO. Antimicrobial activity and brine shrimp toxicity of extracts of Terminalia brownii roots and stem. BMC Complementary and Alternative Medicine. 2007;7:9–11. doi: 10.1186/1472-6882-7-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Opiyo SA, Manguro LOA, Owuor PO, Ochieng CO, Ateka EM, Lemmen P. Antimicrobial compounds from Terminalia brownii against sweet potato pathogens. Open Natural Product Journal. 2011;1:116–120. [Google Scholar]
- 10.Negishi H, Maoka T, Njelekela M, Yasui N, Juman S, Mtabaji J, Miki T, Nara Y, Yamori Y, Ikeda K. New chromone derivative terminalianone from African plant Terminalia brownii Fresen (Combretaceae) in Tanzania. Journal of Asian Natural Products Research. 2011;13:281–283. doi: 10.1080/10286020.2011.552431. [DOI] [PubMed] [Google Scholar]
- 11.Row LR, Murty PS, Subbarao GSR, Sastry CSP, Rao KVJ. Chemical examination of Terminalia species: Part XII—Isolation and structure determination of arjunic acid, a new trihydroxytriterpene carboxylic acid from the Terminalia arjuna bark. Indian Journal of Chemistry. 1970;8:716–721. [Google Scholar]
- 12.Yeo H, Park S-Y, Kim J. A-ring contracted triterpenoid from Rosa multiflora. Phytochemistry. 1998;48:1399–1401. [Google Scholar]
- 13.Vogler B, Conrad J, Hiller W, Klaiber I, Roos G, Sandor P. In: Schreier P, editor. Can LC-NMR serve as a tool for natural products elucidation?; Natural product analysis: Chromatography, biological testing [symposium]; Wuerzburg, Germany. Sept 1997; 1998. pp. 143–146. [Google Scholar]
- 14.Schneider WP, Caron EL, Hinman JW. Occurrence of tomentosic acid in extracts of Bixa orellana. Journal of Organic Chemistry. 1965;30:2856–2857. doi: 10.1021/jo01019a519. [DOI] [PubMed] [Google Scholar]
- 15.Reddy BM, Rao NK, Ramesh M, Rao AVNA, Lin L-J, Lin L-Z, Cordell GA. Chemical investigation of the fruits of Terminalia chebula. International Journal of Pharmacognosy. 1994;32:352–356. [Google Scholar]
- 16.Romussi G, Parodi B, Falsone G. Minor triterpenoids from Quercus ilex L.: seric acid and sericoside. Part 7: Constituents of Cupuliferae. Pharmazie. 1983;38:787–788. [Google Scholar]
- 17.Lee D-Y, Lee S-J, Kwak H-Y, Jung L, Heo J, Hong S, Kim G-W, Nam-In Baek N-I. Sterols isolated from Nuruk (Rhizopus oryzae KSD-815) inhibit the migration of cancer cells. Journal of Microbiology and Biotechnology. 2009;19:1328–1332. doi: 10.4014/jmb.0902.072. [DOI] [PubMed] [Google Scholar]
- 18.Hillis WE, Yazaki Y. Properties of some methylellagic acids and their glycosides. Phytochemistry. 1973;12:2963–2968. [Google Scholar]
- 19.Yang S-W, Zhou B-N, Wisse JH, Evans R, Van-der-Werff H, Miller JS, Kingston DGI. Three new ellagic acid derivatives from the bark of Eschweilera coriacea from the Suriname rain forest. Journal of Natural Products. 1998;61:901–906. doi: 10.1021/np980046u. [DOI] [PubMed] [Google Scholar]
- 20.Pfundstein B, El Desouky SK, Hull WE, Haubner R, Erben G, Owen RW. Polyphenolic compounds in the fruits of Egyptian medicinal plants (Terminalia bellerica, Terminalia chebula and Terminalia horrida): Characterization, quantitation and determination of antioxidant capacities. Phytochemistry. 2010;71:1132–1148. doi: 10.1016/j.phytochem.2010.03.018. [DOI] [PubMed] [Google Scholar]
- 21.Yukata K, Masazumi W. Diellagic lactone and anti-inflamatoryagent. 05310745 A Patent JP. 1993
- 22.Samoylenko V, Jacob MR, Khan SI, Zhao J, Tekwani BL, Midiwo JO, Walker LA, Muhammad I. Antimicrobial, antiparasitic and cytotoxic spermine alkaloids from Albizia schimperiana. Natural Product Communications. 2009;4:791–796. [PMC free article] [PubMed] [Google Scholar]
- 23.Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, Hinriches DJ. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. The American Journal of Tropical Medicine and Hygiene. 1993;48:739–741. doi: 10.4269/ajtmh.1993.48.739. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.