Chinese Journal of Natural Medicines 2013, 11(5): 0528−0533
Chinese Journal of Natural Medicines
Triterpenes from Euphorbia hirta and their cytotoxicity Consolacion Y. Ragasa*, Kimberly B. Cornelio Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, Manila 1004, Philippines Available online 20 Sept. 2013
[ABSTRACT] AIM: To investigate the chemical constituents of the stems, leaves and roots of Euphorbia hirta, and to test for the cytotoxic and antimicrobial potentials of the major constituents of the plant. METHODS: The compounds were isolated by silica gel chromatography and their structures were elucidated by NMR spectroscopy. The cytotoxicity tests were conducted using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, while the antimicrobial tests employed the agar well method. RESULTS: The air-dried stems of E. hirta afforded taraxerone 1, a mixture of 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b) (sample 2) in a 2 : 1 ratio, and another mixture of cycloartenol (3a), lupeol (3b), α-amyrin (3c) and β-amyrin (3d) (sample 3) in a 0.5 : 4 : 1 : 1 ratio. The air-dried leaves of E. hirta yielded sample 2 in a 3 : 2 ratio, sample 3 in a 2 : 3 : 1 : 1 ratio, phytol and phytyl fatty acid ester, while the roots afforded sample 2 in a 2 : 1 ratio, sample 3 in a 2 : 1 : 1 : 1 ratio, a mixture of cycloartenyl fatty acid ester 4a, lupeol fatty acid ester 4b, α-amyrin fatty acid ester 4c and β-amyrin fatty acid ester 4d (sample 4) in a 3 : 2 : 1 : 1 ratio, linoleic acid, β-sitosterol and squalene. Compound 1 from the stems, sample 2 from the leaves, and sample 3 from the stems were assessed for cytotoxicity against a human cancer cell line, colon carcinoma (HCT 116). Sample 2 showed good activity with an IC50 value of 4.8 μg·mL−1, while 1 and sample 3 were inactive against HCT 116. Sample 2 was further tested for cytotoxicity against non-small cell lung adenocarcinoma (A549). It showed good activity against this cell line with an IC50 value of 4.5 μg·mL−1. Antimicrobial assays were conducted on 1 and sample 2. Results of the study indicated that 1 was active against the bacteria: Pseudomonas aeruginosa and Staphylococcus aureus, but was inactive against Escherichia coli and Bacillus subtilis. Sample 2 was active against the bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli and fungi: Candida albicans and Trichophyton mentagrophytes. It was inactive against Bacillus subtilis and Aspergillus niger. CONCLUSIONS: The triterpenes: 2a, 2b, 3a, 3b, 3c and 3d were obtained from the stems, roots and leaves of E. hirta. Taraxerol (1) was only isolated from the stems, the leaves yielded phytol and phytyl fatty acid esters, while the roots afforded 4a-4d, linoleic acid, β-sitosterol, and squalene. Triterpene 1 and sample 2 were found to exhibit antimicrobial activities. Thus, these compounds are some of the active principles of E. hirta which is used in wound healing and the treatment of boils. The cytotoxic properties of sample 2 imply that triterpenes 2a and 2b contribute to the anticancer activity of E. hirta. [KEY WORDS] Euphorbia hirta; Asteraceae; 25-Hydroperoxycycloart-23-en-3β-ol; 24-Hydroperoxycycloart -25-en-3β-ol; Cytotoxicity [CLC Number] R284.1; R965
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[Document code] A
[Article ID] 1672-3651(2013)05-0528-06
Introduction
Euphorbia hirta L. (Euphorbiaceae) is a diuretic, antidiarrhreal, antispasmodic and anti-inflammatory plant. It promotes wound healing, and it is also used for the treatment of
[Received on] 20-Jun.-2012 [Research funding] This project was supported by the grant from the De La Salle University Science Foundation. [*Corresponding author] Consolacion Y. Ragasa: Tel/Fax: +06325360230, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
coughs, chronic bronchitis and other pulmonary disorders, tumors, gonorrhoea, jaundice, dysentery, and boils [1]. The aerial parts were reported to contain flavonoids: euphorbianin, leucocyanidol, camphol, quercetin, and quercitol [2-3], polyphenols: gallic acid, myricitrin, and 3, 4-di-O-galloylquinic acid [4-5], tannins: euphorbins A-E [6], triterpenes, and phytosterols [7]. Taraxerone isolated from E. hirta exhibited antimicrobial activity against fourteen pathogenic bacteria and six fungi with MIC values between 64-128 μg·mL−1. In the brine shrimp lethality assay, taraxerone exhibited an LC50 value of 17.78 μg·mL−1 [8]. Another study reported that quercetin, myricitrin, 24-methylenecycloartenol, and sitosterol from E. hirta exhibited dose-dependent anti-inflammatory
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activity. Tannins and tannic acid derivatives from E. hirta have antiseptic effects, while taraxerone and 11α, 12α-oxidotaraxerol exhibited antibacterial and antifungal properties. The effectiveness of E. hirta in treating asthma may be due to the synergistic relationships between flavonoids, sterols, and triterpenoids [9]. This study reports the isolation of taraxerone (1), 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b) (sample 1) in a 2 : 1 ratio, and cycloartenyl (3a), lupeol (3b), α-amyrin (3c), and β-amyrin (3d) (sample 2) from the stem; sample 2, sample 3, phytol and phytyl fatty acid ester from the leaves; sample 2, sample 3, cycloartenol fatty acid ester (4a), lupeol fatty acid ester (4b), α-amyrin fatty acid ester (4c), and β-amyrin fatty acid ester (4d)
(sample 4), linoleic acid, β-sitosterol, and squalene from the roots of E. hirta (Fig. 1). This is the first report of the isolation of 2a and 2b from E. hirta. The cytotoxicity test results on 1, sample 2 and sample 3 using the MTT assay and the antimicrobial activities of 1 and sample 2 are also reported.
2
Experimental
2.1 General NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for 13 C NMR spectra. Column chromatography was performed with silica gel 60 (70-230 mesh), while the TLC was performed with plastic-backed plates coated with silica gel F254. The plates were visualized with vanillin-H2SO4 and warming.
Fig. 1 Triterpenes from Euphorbia hirta taraxerone (1), 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b), cycloartenol (3a), lupeol (3b), α-amyrin (3c), β-amyrin (3d), cycloartenyl fatty acid ester (4a), lupeol fatty acid ester (4b), α-amyrin fatty acid ester (4c), and β-amyrin fatty acid ester (4d)
2.2
Plant material Euphorbia hirta L. samples were collected from Sinait, Ilocos Sur, Philippines in September. The specimens of the plant were authenticated by T. E. Guevara of the Varietal Improvement Section at the Bureau of Plant Industry in Quirino Avenue, Manila, Philippines. 2.3 Extraction and isolation Air-dried Euphorbia hirta stems, leaves, and roots weighing 357, 1 432, and 75 g, respectively were ground in an Osterizer, soaked in CH2Cl2 for three days, and then filtered. The filtrates were evaporated in vacuo to afford crude extracts weighing 8.50 g for the stems, 46.70 g for the leaves, and 1.30 g for the roots. 2.3.1 Isolation of constituents from the stems of E. hirta The crude extract (8.50 g) was fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% increment) as eluents. The 10% acetone in CH2Cl2 fraction was rechromatographed (3 ×) with 5% ethyl acetate in petroleum ether to afford 1 (10 mg). The 20% ace-
tone in CH2Cl2 fraction was rechromatographed (5 ×) in 10% EtOAc in petroleum ether to afford 3a-3b (sample 3, 12 mg). The 30% acetone in CH2Cl2 fraction was rechromatographed with 12.5% EtOAc in petroleum ether. Purification involved further rechromatography in 20% EtOAc in petroleum ether to afford 2a-2b (sample 2, 4 mg). 2.3.2 Isolation of constituents from the leaves of E. hirta The crude extract (46.70 g) was fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% increment) as eluents. The 50% acetone in CH2Cl2 fraction was rechromatographed in 5% EtOAc in petroleum ether. The less polar fractions were combined and rechromatographed in 7.5% EtOAc in petroleum ether to afford 3a-3b (sample 3, 7 mg). The more polar fractions were combined and rechromatographed in 12.5% EtOAc in petroleum ether to afford 2a-2b (sample 2, 6 mg). 2.3.3 Isolation of constituents from the roots of E. hirta The crude extract (1.30 g) was fractionated by silica gel chromatography using increasing proportions of acetone in
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
CH2Cl2 (10% increment) as eluents. The CH2Cl2 fraction was rechromatographed with 1% EtOAc in petroleum ether. The less polar fractions afforded squalene (10 mg), while the more polar fractions yielded 4a-4d (sample 4, 6 mg). The 30% acetone in CH2Cl2 fraction was rechromatographed with 10% EtOAc in petroleum ether. Purification involved further rechromatography in 12.5% EtOAc in petroleum ether to afford sitosterol (15 mg). The 40% acetone in CH2Cl2 fraction was rechromatographed (4 ×) in 10% EtOAc in petroleum ether and then washed with petroleum ether to give 3a-3d (sample 3, 4 mg). The 50% acetone in CH2Cl2 fraction was rechromatographed (5 ×) in 20% EtOAc in petroleum ether to afford sample 2a-2b (sample 2, 2 mg). 2.4 Bioassays 2.4.1 Cytotoxicity test Four milligrams each of 1, sample 2 and sample 3 were dissolved in dimethyl sulfoxide (DMSO) (1 mL) to make 4 mg·mL−1 solutions. Compound 1, sample 2 and sample 3 were tested for cytotoxic activity against a human cancer cell line, colon carcinoma (HCT116) at the Institute of Biology, University of the Philippines, Diliman, Quezon City. Doxorubicin, an anticancer drug and DMSO were used as the positive and negative controls, respectively. Those that gave positive results with the HCT116 cell line were further tested against a human lung non-small cell adenocarcinoma (A549) cell line and the non-cancer cell line Chinese hamster ovary cells (AA8). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay reported in the literature was employed [10-12]. 2.4.2 Antimicrobial assays The microorganisms used in these tests were obtained from the University of the Philippines Culture Collection (UPCC). These are Pseudomonas aeruginosa (UPCC 1244), Bacillus subtilis (UPCC 1149), Escherichia coli (UPCC 1195), Staphylococcus aureus (UPCC 1143), Candida albicans (UPCC 2168), Trichophyton mentagrophytes (UPCC 4193) and Aspergillus niger (UPCC 3701). Compound 1 and sample 2 were tested for antimicrobial activity against these microorganisms. The test compound (30 mg) was dissolved in 95% ethanol. The positive control for the bacteria is a chloramphenicol sample (HiMedia Laboratories, Ltd.) which contains 30 mg chloramphenicol in a 6 mm disc. The positive control for the fungi is Canesten (Bayer) which contains 1% chlotrimazole. The antimicrobial assay procedure reported in the literature [13] was employed. The clearing zone was measured in millimeters, and the average diameter of the clearing zones was calculated. The diameter of the well for the test compounds was 10 mm. The activity index was computed by subtracting the diameter of the well from the diameter of the clearing zones divided by the diameter of the well, e.g. activity index (AI) (diameter of clearing zone-diameter of well)/diameter of well.
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Structural Identification
Taraxerone (1) 1H NMR (CDCl3) δ: 1.87, 1.36 (H2-1), 2.31, 2.55 (H2-2), 1.32 (H-5), 1.55, 1.60 (H2-6), 1.01, 1.38 (H2-7), 1.50 (H-9), 1.54, 1.64 (H2-11), 0.96, 1.32 (H2-12), 5.54 (H-15), 1.63, 1.90 (H2-16), 0.96 (H-18), 1.37, 2.05 (H2-19), 1.55, 1.62 (H2-21), 1.24, 1.35 (H2-22), 1.07 (s, H3-23), 1.06 (s, H3-24), 1.08 (H3-25), 0.91 (s, H3-26), 1.13 (s, H3-27), 0.82 (s, H3-28), 0.95 (s, H3-29), 0.89 (s, H3-30); 13C NMR (CDCl3) δ: 38.3 (C-1), 34.1 (C-2), 217.6 (C-3), 47.6 (C-4), 55.8 (C-5), 19.9 (C-6), 35.1 (C-7), 38.6 (C-8), 48.7 (C-9), 35.8 (C-10), 17.4 (C-11), 37.7 (C-12), 37.7 (C-13), 157.6 (C-14), 117.2 (C-15), 36.7 (C-16), 37.5 (C-17), 48.8 (C-18), 40.6 (C-19), 28.8 (C-20), 33.5 (C-21), 33.1 (C-22), 26.1 (C-23), 21.3 (C-24), 14.8 (C-25), 29.8 (C-26), 25.6 (C-27), 29.9 (C-28), 33.3 (C-29), 21.5 (C-30). 1 25-Hydroperoxycycloart-23-en-3β-ol (2a) H NMR (CDCl3) δ: 1.22, 1.53 (H2-1), 1.55, 1.73 (H2-2), 3.25 (H-3), 1.26 (H-5), 0.78, 1.58 (H2-6), 1.05, 1.30 (H2-7), 1.48 (H-8), 1.08, 1.96 (H2-11), 1.58, 1.58 (H2-12), 1.27, 1.29 (H2-15), 1.28, 1.88 (H2-16), 1.56 (H-17), 0.94 (H3-18), 0.31, 0.53 (H2-19), 1.46 (H-20), 0.84 (s, H3-21), 1.76, 2.20 (H2-22), 5.66 (ddd, J = 6.6, 9.0, 15.6 Hz, H-23), 5.50 (d, J = 15.6 Hz, H-24), 1.32 (s, H3-26), 1.32 (s, H3-27), 0.79 (s, H3-28), 0.93 (s, H3-29), 0.86 (s, H3-30); 13C NMR (CDCl3) δ: 31.9 (C-1), 30.3 (C-2), 78.8 (C-3), 40.5 (C-4), 47.1 (C-5), 21.1 (C-6), 26.0 (C-7), 47.9 (C-8), 19.9 (C-9), 26.1 (C-10), 26.4 (C-11), 32.8 (C-12), 45.3 (C-13), 48.8 (C-14), 35.5 (C-15), 28.1 (C-16), 52.0 (C-17), 18.1 (C-18), 29.9 (C-19), 36.3 (C-20), 18.3 (C-21), 39.4 (C-22), 130.7 (C-23), 134.4 (C-24), 82.3 (C-25), 24.32 (C-26), 24.39 (C-27), 25.4 (C-28), 14.0 (C-29), 19.3 (C-30). 24-Hydroperoxycycloart-25-en-3β-ol (2b) 1H NMR (CDCl3) δ: 1.22, 1.53 (H2-1), 1.55, 1.73 (H2-2), 3.25 (H-3), 1.26 (H-5), 0.78, 1.58 (H2-6), 1.05, 1.30 (H2-7), 1.48 (H-8), 1.08, 1.96 (H2-11), 1.58, 1.58 (H2-12), 1.27, 1.29 (H2-15), 1.28, 1.88 (H2-16), 1.56 (H-17), 0.94 (H3-18), 0.31, 0.53 (H2-19), 1.46 (H-20), 0.84 (s, H3-21), 1.56, 1.57 (H2-22), 1.35, 1.64 (H2-23), 4.22 (H-24), 4.99, 5.01 (H2-26), 1.72 (s, H3-27), 0.79 (s, H3-28), 0.93 (s, H3-29), 0.86 (s, H3-30); 13C NMR (CDCl3) δ: 31.9 (C-1), 30.3 (C-2), 78.8 (C-3), 40.5 (C-4), 47.1 (C-5), 21.1 (C-6), 26.0 (C-7), 49.9 (C-8), 19.9 (C-9), 26.1 (C-10), 26.4 (C-11), 32.8 (C-12), 45.3 (C-13), 48.8 (C-14), 35.5 (C-15), 28.1 (C-16), 52.0 (C-17), 18.1 (C-18), 29.9 (C-19), 36.3 (C-20), 18.3 (C-21), 32.8 (C-22), 27.5 (C-23), 90.1 (C-24), 144.0 (C-25), 114.3 (C-26), 17.0 (C-27), 25.4 (C-28), 14.0 (C-29), 19.3 (C-30). Cycloartenol (3a) 13C NMR (CDCl3) δ: 31.3 (C-1), 26.6 (C-2), 78.8 (C-3), 39.6 (C-4), 47.2 (C-5), 20.9 (C-6), 28.1 (C-7), 47.7 (C-8), 19.7 (C-9), 26.0 (C-10), 25.7 (C-11), 35.6 (C-12), 45.3 (C-13), 48.3 (C-14), 32.8 (C-15), 26.6 (C-16), 52.2 (C-17), 18.0 (C-18), 29.9 (C-19), 35.6 (C-20), 18.3 (C-21), 36.9 (C-22), 25.1 (C-23), 125.3 (C-24), 130.7
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(C-25), 17.5 (C-26), 25.7 (C-27), 19.3 (C-28), 15.4 (C-29), 25.4 (C-30). Lupeol (3b) 13C NMR (CDCl3) δ: 38.7 (C-1), 27.4 (C-2), 79.0 (C-3), 38.9 (C-4), 55.3 (C-5), 18.3 (C-6), 34.3 (C-7), 40.8 (C-8), 50.4 (C-9), 37.2 (C-10), 20.9 (C-11), 25.1 (C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.6 (C-16), 43.0 (C-17), 48.0 (C-18), 48.3 (C-19), 151.0 (C-20), 29.8 (C-21), 40.0 (C-22), 28.0 (C-23), 15.4 (C-24), 16.1 (C-25), 16.0 (C-26), 14.5 (C-27), 18.0 (C-28), 18.3 (C-29), 109.3 (C-30). α-Amyrin (3c) colorless solid. 13C NMR (CDCl3)δ: 38.8 (C-1), 27.3 (C-2), 79.1 (C-3), 38.8 (C-4), 55.2 (C-5), 18.3 (C-6), 32.9 (C-7), 40.0 (C-8), 47.7 (C-9), 36.9 (C-10), 23.3 (C-11), 124.4 (C-12), 139.6 (C-13), 42.1 (C-14), 28.7 (C-15), 26.6 (C-16), 33.7 (C-17), 59.1 (C-18), 39.6 (C-19), 39.7 (C-20), 31.2 (C-21), 41.5 (C-22), 28.1 (C-23), 15.7 (C-24), 15.7 (C-25), 16.9 (C-26), 23.4 (C-27), 28.1 (C-28), 17.5 (C-29), 21.4 (C-30). β-Amyrin (3d) colorless solid. 13C NMR (CDCl3) δ: 38.7 (C-1), 27.3 (C-2), 79.1 (C-3), 38.8 (C-4), 55.2 (C-5), 18.4 (C-6), 32.6 (C-7), 38.8 (C-8), 47.7 (C-9), 37.2 (C-10), 23.5 (C-11), 121.7 (C-12), 145.2 (C-13), 41.5 (C-14), 26.1 (C-15), 27.3 (C-16), 32.5 (C-17), 47.7 (C-18), 46.8 (C-19), 31.2 (C-20), 34.7 (C-21), 37.2 (C-22), 28.1 (C-23), 15.6 (C-24), 15.6 (C-25), 16.9 (C-26), 26.0 (C-27), 28.4 (C-28), 33.3 (C-29), 23.7 (C-30). Cycloartenyl fatty acid ester (4a) 13C NMR (CDCl3) δ: 32.1 (C-1), 30.6 (C-2), 80.6 (C-3), 40.8 (C-4), 47.2 (C-5), 20.9 (C-6), 26.0 (C-7), 48.0 (C-8), 20.1 (C-9), 26.0 (C-10), 26.5 (C-11), 32.8 (C-12), 45.3 (C-13), 48.3 (C-14), 35.6 (C-15), 28.1 (C-16), 52.2 (C-17), 18.0 (C-18), 29.8 (C-19), 36.0 (C-20), 18.3 (C-21), 36.3 (C-22), 25.1 (C-23), 125.2 (C-24), 130.9 (C-25), 22.7 (C-26, C-27), 25.4 (C-28), 14.5 (C-29), 19.3 (C-30), 173.7 (C-1′), 34 9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2 – 29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). Lupeol fatty acid ester (4b) 13C NMR (CDCl3) δ: 38.9 (C-1), 27.4 (C-2), 80.6 (C-3), 38.9 (C-4), 55.4 (C-5), 18.3 (C-6), 34.2 (C-7), 40.8 (C-8), 50.3 (C-9), 37.1 (C-10), 20.9 (C-11), 25.1 (C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.6 (C-16), 43.0 (C-17), 48.0 (C-18), 48.3 (C-19), 151.0 (C-20), 29.8 (C-21), 40.0 (C-22), 28.0 (C-23), 15.5 (C-24), 16.2 (C-25), 16.1 (C-26), 14.5 (C-27), 18.0 (C-28), 18.3 (C-29), 109.3 (C-30), 173.7 (C-1′), 34.9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2-29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). α-Amyrin fatty acid ester (4c) 13C NMR (CDCl3) δ: 38.9 (C-1), 27.2 (C-2), 80.6 (C-3), 38.9 (C-4), 55.2 (C-5), 18.3 (C-6), 32.9 (C-7), 40.0 (C-8), 47.6 (C-9), 37.0 (C-10), 23.2 (C-11), 124.3 (C-12), 139.6 (C-13), 42.1 (C-14), 28.7 (C-15), 26.5 (C-16), 33.7 (C-17), 59.0 (C-18), 39.6 (C-19), 39.6 (C-20), 31.2 (C-21), 41.5 (C-22), 28.1 (C-23), 15.7 (C-24), 15.7 (C-25), 16.9 (C-26), 23.4 (C-27), 28.1 (C-28),
17.5 (C-29), 21.4 (C-30), 173.7 (C-1′), 34 9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2-29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). β-Amyrin fatty acid ester (4d) 13C NMR (CDCl3) δ: 38.9 (C-1), 27.2 (C-2), 80.6 (C-3), 38.9 (C-4), 55.2 (C-5), 18.5 (C-6), 32.6 (C-7), 38.9 (C-8), 47.6 (C-9), 37.1 (C-10), 23.7 (C-11), 121.6 (C-12), 145.2 (C-13), 41.5 (C-14), 26.1 (C-15), 27.2 (C-16), 32.4 (C-17), 47.8 (C-18), 46.8 (C-19), 31.2 (C-20), 34.7 (C-21), 37.1 (C-22), 28.1 (C-23), 15.7 (C-24), 15.7 (C-25), 16.9 (C-26), 26.0 (C-27), 28.2 (C-28), 33.3 (C-29), 23.7 (C-30), 173.7 (C-1′), 34 9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2-29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). β-Sitosterol 13C NMR (CDCl3) δ: 37.2 (C-1), 31.7 (C-2), 71.8 (C-3), 42.3 (C-4), 140.7 (C-5), 121.7 (C-6), 31.9 (C-7), 31.9 (C-8), 50.1 (C-9), 36.1 (C-10), 21.1 (C-11), 39.8 (C-12), 42.3 (C-13), 56.8 (C-14), 24.3 (C-15), 28.2 (C-16), 56.0 (C-17), 11.9 (C-18), 19.4 (C-19), 36.2 (C-20), 19.0 (C-21), 33.9 (C-22), 29.1 (C-23), 45.8 (C-24), 26.0 (C-25), 18.8 (C-26), 19.8 (C-27), 23.1 (C-28), 11.9 (C-29).
4
Results and Discussion
Silica gel chromatography of the dichloromethane extracts of E. hirta afforded taraxerone 1, a mixture of 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b) (sample 2) in a 2 : 1 ratio, and a mixture of cycloartenol (3a), lupeol (3b), α-amyrin (3c), and β-amyrin (3d) (sample 3) in a 0.5 : 4 : 1 : 1 ratio from the stems; sample 2 in a 3 : 2 ratio, sample 3 in a 2 : 2 : 1 : 1 ratio, phytol and phytyl fatty acid ester from the leaves; and sample 2 in a 2 : 1 ratio, sample 3 in a 2 : 1 : 1 : 1 ratio, a mixture of cycloartenyl fatty acid ester 4a, lupeol fatty acid ester 4b, α-amyrin fatty acid ester 4c and β-amyrin fatty acid ester 4d (sample 4) in a 3 : 2 : 1 : 1 ratio, linoleic acid, β-sitosterol and squalene from the roots. The 1H NMR spectra of samples 2-4 indicated resonances for mixtures of compounds as deduced from the integrals and disparity in single hydrogen peaks. The ratios of the triterpenes were determined from the integrations of the olefinic proton resonances at δ 5.66 (H-23) and 5.50 (H-24) for 2a, and δ 4.99 and 5.01 (H2-26) for 2b; δ 4.67 and 4.55 (H2-30) for 3b and 4b; δ 5.16 (H-12) for 3c and 4c; and δ 5.11 (H-12) for 3d and 4d; and the cyclopropyl methylene protons at δ 0.31 and 0.53 (H2-19) for 3a and 4a. The structures of 1, 2a, and 2b were elucidated by extensive 1D and 2D NMR spectroscopy, and confirmed by comparison of their 13C NMR data (see Experimental) with those reported in the literature for taraxerone (1) [14] , 25-hydroperoxycycloart-23-en-3β-ol (2a) [15], and 24-hydroperoxycycloart-25-en-3β-ol (2b) [15]. The structures of the other compounds were identified by comparison of their 13 C NMR data (see Experimental) with those reported in the
literature for cycloartenol (3a) [16], lupeol (3b) [17], α-amyrin (3c) [17], β-amyrin (3d) [17], cycloartenyl fatty acid ester (4a) [16] , lupeol fatty acid ester (4b) [18], α-amyrin fatty acid ester (4c) [19-20], β-amyrin fatty acid ester (4d) [18-19, 21], phytol [22], phytyl fatty acid ester [23], linoleic acid [24], β-sitosterol [25], and squalene [26]. The cytotoxicity tests on 1 and sample 3 did not give linear interpolation with the human cancer cell line colon carcinoma (HCT 116), thus the IC50 value could not be computed. This implied that 1 and sample 3 did not exhibit cytotoxic effects against this cell line. Meanwhile, sample 2 (Fig. 2) exhibited good activities, with IC50 values of 4.8 and 4.5 μg·mL−1 against the human cancer cell line colon carcinoma (HCT 116) and the non-small cell lung adenocarcinoma (A549), respectively. Sample 2 was also cytotoxic against the non-cancer Chinese hamster ovary AA8 cell line with an IC50 value of 3.7 μg·mL−1. It is noted that sample 2 is more cytotoxic against the normal cell line, AA8 (IC50 3.7 μg·mL−1) than the cancer cells, HCT 116 (IC50 4.8 μg·mL−1) and A549 (IC50 4.5 μg·mL−1) tested. However, the same trend is observed against the positive control, doxorubicin (IC50 2.1 μg·mL−1) which is almost as cytotoxic against A549 (IC50 2.2 μg·mL−1) (Fig. 2). Moreover, Doxorubicin is more cytotoxic than sample 2. The suggested effective doses for IC50 values for plant extracts and pure compounds to be considered active according to the National Cancer Institute (NCI) guidelines should be less than 20 and 4 μg·mL−1, respectively [27]. The IC50 values of sample 2 against the cancer cells, HCT 116 and A549 are 4.8 μg·mL−1 and IC50 4.55 μg·mL−1, respectively. Table 1
E. coli
P. aeruginosa
S. aureus
B. subtilis C.albicans T. mentagrophytes
These are close to the doses of pure compounds to be considered active according to the NCI guidelines. As part of a continuing search for antimicrobial compounds from Philippine medicinal plants, 1 and sample 2 were tested for possible antimicrobial activities by the agar well method. Results of the study (Table 1) indicated that 1 was active against the bacteria: P. aeruginosa and S. aureus with activity index (AI) of 0.2 and 0.6, respectively, but was inactive against E. coli and B. subtilis. Sample 2 was active against the bacteria: P. aeruginosa (AI = 0.3), S. aureus (AI = 0.5) and E. coli (AI = 0.2) and fungi: C. albicans (AI = 0.2) and T. mentagrophytes (AI = 0.6). It was inactive against B. subtilis and A. niger.
Compounda (30 μg)
Clearing Zone (mm)b
1
−
0
Sample 2
12
0.2
Chloramphenicold
30
4.0
1
12
0.2
A. niger
Activity Index (AI)
Sample 2
13
0.3
Chloramphenicold
15
1.5
1
16
0.6
Sample 2
15
0.5
Chloramphenicold
33
4.5
c
1
13
Sample 2
15c
0
Chloramphenicold
20
2.3
0
Sample 2
12
0.2
Canestene, 0.2 g
18
0.8
Sample 2
16
0.6
Canesten , 0.2 g
55
4.5
Sample 2
−f
0
Canestene, 0.2 g
23
1.3
e
e
Fig. 2 Inhibitory concentrations at 50% (IC50) of sample 2 from the dichloromethane extract of the leaves of E. hirta tested against human cancer cell lines: colon carcinoma HCT 116 and lung adenocarcinoma A549, and the non-cancer Chinese hamster ovary AA8 using the MTT assay. Each value is the mean of four trials with three replicates per trial and with SD indicated by bars
Antimicrobial test results of 1 and sample 2 Microorganism
a
IC50 (μg/mL)
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
Sample–10 mm well diameter; b Average of three trials; c Partial inhibition of growth of test organism; d Chloramphenicol disk-6-mm disc; Contains 1% clotrimazole; f no clearing zone
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
A previous study reported that 25-hydroperoxy-cyclo art-23-en-3β-ol (2a) showed minimum growth inhibition concentrations of 2, 2, and 1 μg against E. coli, M. luteus and B. subtilis, respectively[15]. On the other hand, 24-hydroperoxycycloart-25-en-3β-ol (2b) exhibited minimum growth inhibition concentrations of 1, 1, and 5 μg against E. coli, M. luteus and B. subtilis, respectively [15]. Taraxerone isolated from E. hirta was also reported to exhibit antimicrobial activities against fourteen pathogenic bacteria and six fungi with MIC values between 64-128 μg·mL−1 [8]. Another study reported the antibacterial and antifungal properties of taraxerone [9].
Acknowledgments The MTT assay was conducted at the Institute of Biology, University of the Philippines, Diliman, Quezon City. The antimicrobial tests were conducted at the University of the Philippines-Natural Sciences Research Institute.
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Chinese Journal of Natural Medicines
Triterpenes from Euphorbia hirta and their cytotoxicity Consolacion Y. Ragasa*, Kimberly B. Cornelio Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, Manila 1004, Philippines Available online 20 Sept. 2013
[ABSTRACT] AIM: To investigate the chemical constituents of the stems, leaves and roots of Euphorbia hirta, and to test for the cytotoxic and antimicrobial potentials of the major constituents of the plant. METHODS: The compounds were isolated by silica gel chromatography and their structures were elucidated by NMR spectroscopy. The cytotoxicity tests were conducted using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, while the antimicrobial tests employed the agar well method. RESULTS: The air-dried stems of E. hirta afforded taraxerone 1, a mixture of 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b) (sample 2) in a 2 : 1 ratio, and another mixture of cycloartenol (3a), lupeol (3b), α-amyrin (3c) and β-amyrin (3d) (sample 3) in a 0.5 : 4 : 1 : 1 ratio. The air-dried leaves of E. hirta yielded sample 2 in a 3 : 2 ratio, sample 3 in a 2 : 3 : 1 : 1 ratio, phytol and phytyl fatty acid ester, while the roots afforded sample 2 in a 2 : 1 ratio, sample 3 in a 2 : 1 : 1 : 1 ratio, a mixture of cycloartenyl fatty acid ester 4a, lupeol fatty acid ester 4b, α-amyrin fatty acid ester 4c and β-amyrin fatty acid ester 4d (sample 4) in a 3 : 2 : 1 : 1 ratio, linoleic acid, β-sitosterol and squalene. Compound 1 from the stems, sample 2 from the leaves, and sample 3 from the stems were assessed for cytotoxicity against a human cancer cell line, colon carcinoma (HCT 116). Sample 2 showed good activity with an IC50 value of 4.8 μg·mL−1, while 1 and sample 3 were inactive against HCT 116. Sample 2 was further tested for cytotoxicity against non-small cell lung adenocarcinoma (A549). It showed good activity against this cell line with an IC50 value of 4.5 μg·mL−1. Antimicrobial assays were conducted on 1 and sample 2. Results of the study indicated that 1 was active against the bacteria: Pseudomonas aeruginosa and Staphylococcus aureus, but was inactive against Escherichia coli and Bacillus subtilis. Sample 2 was active against the bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli and fungi: Candida albicans and Trichophyton mentagrophytes. It was inactive against Bacillus subtilis and Aspergillus niger. CONCLUSIONS: The triterpenes: 2a, 2b, 3a, 3b, 3c and 3d were obtained from the stems, roots and leaves of E. hirta. Taraxerol (1) was only isolated from the stems, the leaves yielded phytol and phytyl fatty acid esters, while the roots afforded 4a-4d, linoleic acid, β-sitosterol, and squalene. Triterpene 1 and sample 2 were found to exhibit antimicrobial activities. Thus, these compounds are some of the active principles of E. hirta which is used in wound healing and the treatment of boils. The cytotoxic properties of sample 2 imply that triterpenes 2a and 2b contribute to the anticancer activity of E. hirta. [KEY WORDS] Euphorbia hirta; Asteraceae; 25-Hydroperoxycycloart-23-en-3β-ol; 24-Hydroperoxycycloart -25-en-3β-ol; Cytotoxicity [CLC Number] R284.1; R965
1
[Document code] A
[Article ID] 1672-3651(2013)05-0528-06
Introduction
Euphorbia hirta L. (Euphorbiaceae) is a diuretic, antidiarrhreal, antispasmodic and anti-inflammatory plant. It promotes wound healing, and it is also used for the treatment of
[Received on] 20-Jun.-2012 [Research funding] This project was supported by the grant from the De La Salle University Science Foundation. [*Corresponding author] Consolacion Y. Ragasa: Tel/Fax: +06325360230, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
coughs, chronic bronchitis and other pulmonary disorders, tumors, gonorrhoea, jaundice, dysentery, and boils [1]. The aerial parts were reported to contain flavonoids: euphorbianin, leucocyanidol, camphol, quercetin, and quercitol [2-3], polyphenols: gallic acid, myricitrin, and 3, 4-di-O-galloylquinic acid [4-5], tannins: euphorbins A-E [6], triterpenes, and phytosterols [7]. Taraxerone isolated from E. hirta exhibited antimicrobial activity against fourteen pathogenic bacteria and six fungi with MIC values between 64-128 μg·mL−1. In the brine shrimp lethality assay, taraxerone exhibited an LC50 value of 17.78 μg·mL−1 [8]. Another study reported that quercetin, myricitrin, 24-methylenecycloartenol, and sitosterol from E. hirta exhibited dose-dependent anti-inflammatory
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
activity. Tannins and tannic acid derivatives from E. hirta have antiseptic effects, while taraxerone and 11α, 12α-oxidotaraxerol exhibited antibacterial and antifungal properties. The effectiveness of E. hirta in treating asthma may be due to the synergistic relationships between flavonoids, sterols, and triterpenoids [9]. This study reports the isolation of taraxerone (1), 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b) (sample 1) in a 2 : 1 ratio, and cycloartenyl (3a), lupeol (3b), α-amyrin (3c), and β-amyrin (3d) (sample 2) from the stem; sample 2, sample 3, phytol and phytyl fatty acid ester from the leaves; sample 2, sample 3, cycloartenol fatty acid ester (4a), lupeol fatty acid ester (4b), α-amyrin fatty acid ester (4c), and β-amyrin fatty acid ester (4d)
(sample 4), linoleic acid, β-sitosterol, and squalene from the roots of E. hirta (Fig. 1). This is the first report of the isolation of 2a and 2b from E. hirta. The cytotoxicity test results on 1, sample 2 and sample 3 using the MTT assay and the antimicrobial activities of 1 and sample 2 are also reported.
2
Experimental
2.1 General NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for 13 C NMR spectra. Column chromatography was performed with silica gel 60 (70-230 mesh), while the TLC was performed with plastic-backed plates coated with silica gel F254. The plates were visualized with vanillin-H2SO4 and warming.
Fig. 1 Triterpenes from Euphorbia hirta taraxerone (1), 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b), cycloartenol (3a), lupeol (3b), α-amyrin (3c), β-amyrin (3d), cycloartenyl fatty acid ester (4a), lupeol fatty acid ester (4b), α-amyrin fatty acid ester (4c), and β-amyrin fatty acid ester (4d)
2.2
Plant material Euphorbia hirta L. samples were collected from Sinait, Ilocos Sur, Philippines in September. The specimens of the plant were authenticated by T. E. Guevara of the Varietal Improvement Section at the Bureau of Plant Industry in Quirino Avenue, Manila, Philippines. 2.3 Extraction and isolation Air-dried Euphorbia hirta stems, leaves, and roots weighing 357, 1 432, and 75 g, respectively were ground in an Osterizer, soaked in CH2Cl2 for three days, and then filtered. The filtrates were evaporated in vacuo to afford crude extracts weighing 8.50 g for the stems, 46.70 g for the leaves, and 1.30 g for the roots. 2.3.1 Isolation of constituents from the stems of E. hirta The crude extract (8.50 g) was fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% increment) as eluents. The 10% acetone in CH2Cl2 fraction was rechromatographed (3 ×) with 5% ethyl acetate in petroleum ether to afford 1 (10 mg). The 20% ace-
tone in CH2Cl2 fraction was rechromatographed (5 ×) in 10% EtOAc in petroleum ether to afford 3a-3b (sample 3, 12 mg). The 30% acetone in CH2Cl2 fraction was rechromatographed with 12.5% EtOAc in petroleum ether. Purification involved further rechromatography in 20% EtOAc in petroleum ether to afford 2a-2b (sample 2, 4 mg). 2.3.2 Isolation of constituents from the leaves of E. hirta The crude extract (46.70 g) was fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% increment) as eluents. The 50% acetone in CH2Cl2 fraction was rechromatographed in 5% EtOAc in petroleum ether. The less polar fractions were combined and rechromatographed in 7.5% EtOAc in petroleum ether to afford 3a-3b (sample 3, 7 mg). The more polar fractions were combined and rechromatographed in 12.5% EtOAc in petroleum ether to afford 2a-2b (sample 2, 6 mg). 2.3.3 Isolation of constituents from the roots of E. hirta The crude extract (1.30 g) was fractionated by silica gel chromatography using increasing proportions of acetone in
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
CH2Cl2 (10% increment) as eluents. The CH2Cl2 fraction was rechromatographed with 1% EtOAc in petroleum ether. The less polar fractions afforded squalene (10 mg), while the more polar fractions yielded 4a-4d (sample 4, 6 mg). The 30% acetone in CH2Cl2 fraction was rechromatographed with 10% EtOAc in petroleum ether. Purification involved further rechromatography in 12.5% EtOAc in petroleum ether to afford sitosterol (15 mg). The 40% acetone in CH2Cl2 fraction was rechromatographed (4 ×) in 10% EtOAc in petroleum ether and then washed with petroleum ether to give 3a-3d (sample 3, 4 mg). The 50% acetone in CH2Cl2 fraction was rechromatographed (5 ×) in 20% EtOAc in petroleum ether to afford sample 2a-2b (sample 2, 2 mg). 2.4 Bioassays 2.4.1 Cytotoxicity test Four milligrams each of 1, sample 2 and sample 3 were dissolved in dimethyl sulfoxide (DMSO) (1 mL) to make 4 mg·mL−1 solutions. Compound 1, sample 2 and sample 3 were tested for cytotoxic activity against a human cancer cell line, colon carcinoma (HCT116) at the Institute of Biology, University of the Philippines, Diliman, Quezon City. Doxorubicin, an anticancer drug and DMSO were used as the positive and negative controls, respectively. Those that gave positive results with the HCT116 cell line were further tested against a human lung non-small cell adenocarcinoma (A549) cell line and the non-cancer cell line Chinese hamster ovary cells (AA8). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay reported in the literature was employed [10-12]. 2.4.2 Antimicrobial assays The microorganisms used in these tests were obtained from the University of the Philippines Culture Collection (UPCC). These are Pseudomonas aeruginosa (UPCC 1244), Bacillus subtilis (UPCC 1149), Escherichia coli (UPCC 1195), Staphylococcus aureus (UPCC 1143), Candida albicans (UPCC 2168), Trichophyton mentagrophytes (UPCC 4193) and Aspergillus niger (UPCC 3701). Compound 1 and sample 2 were tested for antimicrobial activity against these microorganisms. The test compound (30 mg) was dissolved in 95% ethanol. The positive control for the bacteria is a chloramphenicol sample (HiMedia Laboratories, Ltd.) which contains 30 mg chloramphenicol in a 6 mm disc. The positive control for the fungi is Canesten (Bayer) which contains 1% chlotrimazole. The antimicrobial assay procedure reported in the literature [13] was employed. The clearing zone was measured in millimeters, and the average diameter of the clearing zones was calculated. The diameter of the well for the test compounds was 10 mm. The activity index was computed by subtracting the diameter of the well from the diameter of the clearing zones divided by the diameter of the well, e.g. activity index (AI) (diameter of clearing zone-diameter of well)/diameter of well.
3
Structural Identification
Taraxerone (1) 1H NMR (CDCl3) δ: 1.87, 1.36 (H2-1), 2.31, 2.55 (H2-2), 1.32 (H-5), 1.55, 1.60 (H2-6), 1.01, 1.38 (H2-7), 1.50 (H-9), 1.54, 1.64 (H2-11), 0.96, 1.32 (H2-12), 5.54 (H-15), 1.63, 1.90 (H2-16), 0.96 (H-18), 1.37, 2.05 (H2-19), 1.55, 1.62 (H2-21), 1.24, 1.35 (H2-22), 1.07 (s, H3-23), 1.06 (s, H3-24), 1.08 (H3-25), 0.91 (s, H3-26), 1.13 (s, H3-27), 0.82 (s, H3-28), 0.95 (s, H3-29), 0.89 (s, H3-30); 13C NMR (CDCl3) δ: 38.3 (C-1), 34.1 (C-2), 217.6 (C-3), 47.6 (C-4), 55.8 (C-5), 19.9 (C-6), 35.1 (C-7), 38.6 (C-8), 48.7 (C-9), 35.8 (C-10), 17.4 (C-11), 37.7 (C-12), 37.7 (C-13), 157.6 (C-14), 117.2 (C-15), 36.7 (C-16), 37.5 (C-17), 48.8 (C-18), 40.6 (C-19), 28.8 (C-20), 33.5 (C-21), 33.1 (C-22), 26.1 (C-23), 21.3 (C-24), 14.8 (C-25), 29.8 (C-26), 25.6 (C-27), 29.9 (C-28), 33.3 (C-29), 21.5 (C-30). 1 25-Hydroperoxycycloart-23-en-3β-ol (2a) H NMR (CDCl3) δ: 1.22, 1.53 (H2-1), 1.55, 1.73 (H2-2), 3.25 (H-3), 1.26 (H-5), 0.78, 1.58 (H2-6), 1.05, 1.30 (H2-7), 1.48 (H-8), 1.08, 1.96 (H2-11), 1.58, 1.58 (H2-12), 1.27, 1.29 (H2-15), 1.28, 1.88 (H2-16), 1.56 (H-17), 0.94 (H3-18), 0.31, 0.53 (H2-19), 1.46 (H-20), 0.84 (s, H3-21), 1.76, 2.20 (H2-22), 5.66 (ddd, J = 6.6, 9.0, 15.6 Hz, H-23), 5.50 (d, J = 15.6 Hz, H-24), 1.32 (s, H3-26), 1.32 (s, H3-27), 0.79 (s, H3-28), 0.93 (s, H3-29), 0.86 (s, H3-30); 13C NMR (CDCl3) δ: 31.9 (C-1), 30.3 (C-2), 78.8 (C-3), 40.5 (C-4), 47.1 (C-5), 21.1 (C-6), 26.0 (C-7), 47.9 (C-8), 19.9 (C-9), 26.1 (C-10), 26.4 (C-11), 32.8 (C-12), 45.3 (C-13), 48.8 (C-14), 35.5 (C-15), 28.1 (C-16), 52.0 (C-17), 18.1 (C-18), 29.9 (C-19), 36.3 (C-20), 18.3 (C-21), 39.4 (C-22), 130.7 (C-23), 134.4 (C-24), 82.3 (C-25), 24.32 (C-26), 24.39 (C-27), 25.4 (C-28), 14.0 (C-29), 19.3 (C-30). 24-Hydroperoxycycloart-25-en-3β-ol (2b) 1H NMR (CDCl3) δ: 1.22, 1.53 (H2-1), 1.55, 1.73 (H2-2), 3.25 (H-3), 1.26 (H-5), 0.78, 1.58 (H2-6), 1.05, 1.30 (H2-7), 1.48 (H-8), 1.08, 1.96 (H2-11), 1.58, 1.58 (H2-12), 1.27, 1.29 (H2-15), 1.28, 1.88 (H2-16), 1.56 (H-17), 0.94 (H3-18), 0.31, 0.53 (H2-19), 1.46 (H-20), 0.84 (s, H3-21), 1.56, 1.57 (H2-22), 1.35, 1.64 (H2-23), 4.22 (H-24), 4.99, 5.01 (H2-26), 1.72 (s, H3-27), 0.79 (s, H3-28), 0.93 (s, H3-29), 0.86 (s, H3-30); 13C NMR (CDCl3) δ: 31.9 (C-1), 30.3 (C-2), 78.8 (C-3), 40.5 (C-4), 47.1 (C-5), 21.1 (C-6), 26.0 (C-7), 49.9 (C-8), 19.9 (C-9), 26.1 (C-10), 26.4 (C-11), 32.8 (C-12), 45.3 (C-13), 48.8 (C-14), 35.5 (C-15), 28.1 (C-16), 52.0 (C-17), 18.1 (C-18), 29.9 (C-19), 36.3 (C-20), 18.3 (C-21), 32.8 (C-22), 27.5 (C-23), 90.1 (C-24), 144.0 (C-25), 114.3 (C-26), 17.0 (C-27), 25.4 (C-28), 14.0 (C-29), 19.3 (C-30). Cycloartenol (3a) 13C NMR (CDCl3) δ: 31.3 (C-1), 26.6 (C-2), 78.8 (C-3), 39.6 (C-4), 47.2 (C-5), 20.9 (C-6), 28.1 (C-7), 47.7 (C-8), 19.7 (C-9), 26.0 (C-10), 25.7 (C-11), 35.6 (C-12), 45.3 (C-13), 48.3 (C-14), 32.8 (C-15), 26.6 (C-16), 52.2 (C-17), 18.0 (C-18), 29.9 (C-19), 35.6 (C-20), 18.3 (C-21), 36.9 (C-22), 25.1 (C-23), 125.3 (C-24), 130.7
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
(C-25), 17.5 (C-26), 25.7 (C-27), 19.3 (C-28), 15.4 (C-29), 25.4 (C-30). Lupeol (3b) 13C NMR (CDCl3) δ: 38.7 (C-1), 27.4 (C-2), 79.0 (C-3), 38.9 (C-4), 55.3 (C-5), 18.3 (C-6), 34.3 (C-7), 40.8 (C-8), 50.4 (C-9), 37.2 (C-10), 20.9 (C-11), 25.1 (C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.6 (C-16), 43.0 (C-17), 48.0 (C-18), 48.3 (C-19), 151.0 (C-20), 29.8 (C-21), 40.0 (C-22), 28.0 (C-23), 15.4 (C-24), 16.1 (C-25), 16.0 (C-26), 14.5 (C-27), 18.0 (C-28), 18.3 (C-29), 109.3 (C-30). α-Amyrin (3c) colorless solid. 13C NMR (CDCl3)δ: 38.8 (C-1), 27.3 (C-2), 79.1 (C-3), 38.8 (C-4), 55.2 (C-5), 18.3 (C-6), 32.9 (C-7), 40.0 (C-8), 47.7 (C-9), 36.9 (C-10), 23.3 (C-11), 124.4 (C-12), 139.6 (C-13), 42.1 (C-14), 28.7 (C-15), 26.6 (C-16), 33.7 (C-17), 59.1 (C-18), 39.6 (C-19), 39.7 (C-20), 31.2 (C-21), 41.5 (C-22), 28.1 (C-23), 15.7 (C-24), 15.7 (C-25), 16.9 (C-26), 23.4 (C-27), 28.1 (C-28), 17.5 (C-29), 21.4 (C-30). β-Amyrin (3d) colorless solid. 13C NMR (CDCl3) δ: 38.7 (C-1), 27.3 (C-2), 79.1 (C-3), 38.8 (C-4), 55.2 (C-5), 18.4 (C-6), 32.6 (C-7), 38.8 (C-8), 47.7 (C-9), 37.2 (C-10), 23.5 (C-11), 121.7 (C-12), 145.2 (C-13), 41.5 (C-14), 26.1 (C-15), 27.3 (C-16), 32.5 (C-17), 47.7 (C-18), 46.8 (C-19), 31.2 (C-20), 34.7 (C-21), 37.2 (C-22), 28.1 (C-23), 15.6 (C-24), 15.6 (C-25), 16.9 (C-26), 26.0 (C-27), 28.4 (C-28), 33.3 (C-29), 23.7 (C-30). Cycloartenyl fatty acid ester (4a) 13C NMR (CDCl3) δ: 32.1 (C-1), 30.6 (C-2), 80.6 (C-3), 40.8 (C-4), 47.2 (C-5), 20.9 (C-6), 26.0 (C-7), 48.0 (C-8), 20.1 (C-9), 26.0 (C-10), 26.5 (C-11), 32.8 (C-12), 45.3 (C-13), 48.3 (C-14), 35.6 (C-15), 28.1 (C-16), 52.2 (C-17), 18.0 (C-18), 29.8 (C-19), 36.0 (C-20), 18.3 (C-21), 36.3 (C-22), 25.1 (C-23), 125.2 (C-24), 130.9 (C-25), 22.7 (C-26, C-27), 25.4 (C-28), 14.5 (C-29), 19.3 (C-30), 173.7 (C-1′), 34 9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2 – 29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). Lupeol fatty acid ester (4b) 13C NMR (CDCl3) δ: 38.9 (C-1), 27.4 (C-2), 80.6 (C-3), 38.9 (C-4), 55.4 (C-5), 18.3 (C-6), 34.2 (C-7), 40.8 (C-8), 50.3 (C-9), 37.1 (C-10), 20.9 (C-11), 25.1 (C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.6 (C-16), 43.0 (C-17), 48.0 (C-18), 48.3 (C-19), 151.0 (C-20), 29.8 (C-21), 40.0 (C-22), 28.0 (C-23), 15.5 (C-24), 16.2 (C-25), 16.1 (C-26), 14.5 (C-27), 18.0 (C-28), 18.3 (C-29), 109.3 (C-30), 173.7 (C-1′), 34.9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2-29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). α-Amyrin fatty acid ester (4c) 13C NMR (CDCl3) δ: 38.9 (C-1), 27.2 (C-2), 80.6 (C-3), 38.9 (C-4), 55.2 (C-5), 18.3 (C-6), 32.9 (C-7), 40.0 (C-8), 47.6 (C-9), 37.0 (C-10), 23.2 (C-11), 124.3 (C-12), 139.6 (C-13), 42.1 (C-14), 28.7 (C-15), 26.5 (C-16), 33.7 (C-17), 59.0 (C-18), 39.6 (C-19), 39.6 (C-20), 31.2 (C-21), 41.5 (C-22), 28.1 (C-23), 15.7 (C-24), 15.7 (C-25), 16.9 (C-26), 23.4 (C-27), 28.1 (C-28),
17.5 (C-29), 21.4 (C-30), 173.7 (C-1′), 34 9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2-29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). β-Amyrin fatty acid ester (4d) 13C NMR (CDCl3) δ: 38.9 (C-1), 27.2 (C-2), 80.6 (C-3), 38.9 (C-4), 55.2 (C-5), 18.5 (C-6), 32.6 (C-7), 38.9 (C-8), 47.6 (C-9), 37.1 (C-10), 23.7 (C-11), 121.6 (C-12), 145.2 (C-13), 41.5 (C-14), 26.1 (C-15), 27.2 (C-16), 32.4 (C-17), 47.8 (C-18), 46.8 (C-19), 31.2 (C-20), 34.7 (C-21), 37.1 (C-22), 28.1 (C-23), 15.7 (C-24), 15.7 (C-25), 16.9 (C-26), 26.0 (C-27), 28.2 (C-28), 33.3 (C-29), 23.7 (C-30), 173.7 (C-1′), 34 9 (C-2′), 31.9 (C-3′), 22.7, 25.2, 29.2-29.8 (CH2)n, 130.2, 130.0, 129.9, 129.8 (CH=CH), 14.1 (CH3). β-Sitosterol 13C NMR (CDCl3) δ: 37.2 (C-1), 31.7 (C-2), 71.8 (C-3), 42.3 (C-4), 140.7 (C-5), 121.7 (C-6), 31.9 (C-7), 31.9 (C-8), 50.1 (C-9), 36.1 (C-10), 21.1 (C-11), 39.8 (C-12), 42.3 (C-13), 56.8 (C-14), 24.3 (C-15), 28.2 (C-16), 56.0 (C-17), 11.9 (C-18), 19.4 (C-19), 36.2 (C-20), 19.0 (C-21), 33.9 (C-22), 29.1 (C-23), 45.8 (C-24), 26.0 (C-25), 18.8 (C-26), 19.8 (C-27), 23.1 (C-28), 11.9 (C-29).
4
Results and Discussion
Silica gel chromatography of the dichloromethane extracts of E. hirta afforded taraxerone 1, a mixture of 25-hydroperoxycycloart-23-en-3β-ol (2a) and 24-hydroperoxycycloart-25-en-3β-ol (2b) (sample 2) in a 2 : 1 ratio, and a mixture of cycloartenol (3a), lupeol (3b), α-amyrin (3c), and β-amyrin (3d) (sample 3) in a 0.5 : 4 : 1 : 1 ratio from the stems; sample 2 in a 3 : 2 ratio, sample 3 in a 2 : 2 : 1 : 1 ratio, phytol and phytyl fatty acid ester from the leaves; and sample 2 in a 2 : 1 ratio, sample 3 in a 2 : 1 : 1 : 1 ratio, a mixture of cycloartenyl fatty acid ester 4a, lupeol fatty acid ester 4b, α-amyrin fatty acid ester 4c and β-amyrin fatty acid ester 4d (sample 4) in a 3 : 2 : 1 : 1 ratio, linoleic acid, β-sitosterol and squalene from the roots. The 1H NMR spectra of samples 2-4 indicated resonances for mixtures of compounds as deduced from the integrals and disparity in single hydrogen peaks. The ratios of the triterpenes were determined from the integrations of the olefinic proton resonances at δ 5.66 (H-23) and 5.50 (H-24) for 2a, and δ 4.99 and 5.01 (H2-26) for 2b; δ 4.67 and 4.55 (H2-30) for 3b and 4b; δ 5.16 (H-12) for 3c and 4c; and δ 5.11 (H-12) for 3d and 4d; and the cyclopropyl methylene protons at δ 0.31 and 0.53 (H2-19) for 3a and 4a. The structures of 1, 2a, and 2b were elucidated by extensive 1D and 2D NMR spectroscopy, and confirmed by comparison of their 13C NMR data (see Experimental) with those reported in the literature for taraxerone (1) [14] , 25-hydroperoxycycloart-23-en-3β-ol (2a) [15], and 24-hydroperoxycycloart-25-en-3β-ol (2b) [15]. The structures of the other compounds were identified by comparison of their 13 C NMR data (see Experimental) with those reported in the
literature for cycloartenol (3a) [16], lupeol (3b) [17], α-amyrin (3c) [17], β-amyrin (3d) [17], cycloartenyl fatty acid ester (4a) [16] , lupeol fatty acid ester (4b) [18], α-amyrin fatty acid ester (4c) [19-20], β-amyrin fatty acid ester (4d) [18-19, 21], phytol [22], phytyl fatty acid ester [23], linoleic acid [24], β-sitosterol [25], and squalene [26]. The cytotoxicity tests on 1 and sample 3 did not give linear interpolation with the human cancer cell line colon carcinoma (HCT 116), thus the IC50 value could not be computed. This implied that 1 and sample 3 did not exhibit cytotoxic effects against this cell line. Meanwhile, sample 2 (Fig. 2) exhibited good activities, with IC50 values of 4.8 and 4.5 μg·mL−1 against the human cancer cell line colon carcinoma (HCT 116) and the non-small cell lung adenocarcinoma (A549), respectively. Sample 2 was also cytotoxic against the non-cancer Chinese hamster ovary AA8 cell line with an IC50 value of 3.7 μg·mL−1. It is noted that sample 2 is more cytotoxic against the normal cell line, AA8 (IC50 3.7 μg·mL−1) than the cancer cells, HCT 116 (IC50 4.8 μg·mL−1) and A549 (IC50 4.5 μg·mL−1) tested. However, the same trend is observed against the positive control, doxorubicin (IC50 2.1 μg·mL−1) which is almost as cytotoxic against A549 (IC50 2.2 μg·mL−1) (Fig. 2). Moreover, Doxorubicin is more cytotoxic than sample 2. The suggested effective doses for IC50 values for plant extracts and pure compounds to be considered active according to the National Cancer Institute (NCI) guidelines should be less than 20 and 4 μg·mL−1, respectively [27]. The IC50 values of sample 2 against the cancer cells, HCT 116 and A549 are 4.8 μg·mL−1 and IC50 4.55 μg·mL−1, respectively. Table 1
E. coli
P. aeruginosa
S. aureus
B. subtilis C.albicans T. mentagrophytes
These are close to the doses of pure compounds to be considered active according to the NCI guidelines. As part of a continuing search for antimicrobial compounds from Philippine medicinal plants, 1 and sample 2 were tested for possible antimicrobial activities by the agar well method. Results of the study (Table 1) indicated that 1 was active against the bacteria: P. aeruginosa and S. aureus with activity index (AI) of 0.2 and 0.6, respectively, but was inactive against E. coli and B. subtilis. Sample 2 was active against the bacteria: P. aeruginosa (AI = 0.3), S. aureus (AI = 0.5) and E. coli (AI = 0.2) and fungi: C. albicans (AI = 0.2) and T. mentagrophytes (AI = 0.6). It was inactive against B. subtilis and A. niger.
Compounda (30 μg)
Clearing Zone (mm)b
1
−
0
Sample 2
12
0.2
Chloramphenicold
30
4.0
1
12
0.2
A. niger
Activity Index (AI)
Sample 2
13
0.3
Chloramphenicold
15
1.5
1
16
0.6
Sample 2
15
0.5
Chloramphenicold
33
4.5
c
1
13
Sample 2
15c
0
Chloramphenicold
20
2.3
0
Sample 2
12
0.2
Canestene, 0.2 g
18
0.8
Sample 2
16
0.6
Canesten , 0.2 g
55
4.5
Sample 2
−f
0
Canestene, 0.2 g
23
1.3
e
e
Fig. 2 Inhibitory concentrations at 50% (IC50) of sample 2 from the dichloromethane extract of the leaves of E. hirta tested against human cancer cell lines: colon carcinoma HCT 116 and lung adenocarcinoma A549, and the non-cancer Chinese hamster ovary AA8 using the MTT assay. Each value is the mean of four trials with three replicates per trial and with SD indicated by bars
Antimicrobial test results of 1 and sample 2 Microorganism
a
IC50 (μg/mL)
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
Sample–10 mm well diameter; b Average of three trials; c Partial inhibition of growth of test organism; d Chloramphenicol disk-6-mm disc; Contains 1% clotrimazole; f no clearing zone
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528−533
A previous study reported that 25-hydroperoxy-cyclo art-23-en-3β-ol (2a) showed minimum growth inhibition concentrations of 2, 2, and 1 μg against E. coli, M. luteus and B. subtilis, respectively[15]. On the other hand, 24-hydroperoxycycloart-25-en-3β-ol (2b) exhibited minimum growth inhibition concentrations of 1, 1, and 5 μg against E. coli, M. luteus and B. subtilis, respectively [15]. Taraxerone isolated from E. hirta was also reported to exhibit antimicrobial activities against fourteen pathogenic bacteria and six fungi with MIC values between 64-128 μg·mL−1 [8]. Another study reported the antibacterial and antifungal properties of taraxerone [9].
Acknowledgments The MTT assay was conducted at the Institute of Biology, University of the Philippines, Diliman, Quezon City. The antimicrobial tests were conducted at the University of the Philippines-Natural Sciences Research Institute.
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