全 文 ： 528 Chin J Nat Med Sept. 2013 Vol. 11 No. 5 2013年 9月 第 11卷 第 5期

Chinese Journal of Natural Medicines 2013, 11(5): 05280533
doi: 10.3724/SP.J.1009.2013.00528
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-3-ol (2a) and
24-hydroperoxycycloart-25-en-3-ol (2b) (sample 2) in a 2 : 1 ratio, and another mixture of cycloartenol (3a), lupeol (3b), -amyrin
(3c) and -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, -amyrin fatty acid ester 4c and -amyrin fatty acid ester 4d
(sample 4) in a 3 : 2 : 1 : 1 ratio, linoleic acid, -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 g·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
g·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, -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-3-ol; 24-Hydroperoxycycloart -25-en-3-ol; Cytotox-
icity
[CLC Number] R284.1; R965 [Document code] A [Article ID] 1672-3651(2013)05-0528-06

1 Introduction
Euphorbia hirta L. (Euphorbiaceae) is a diuretic, antidi-
arrhreal, antispasmodic and anti-inflammatory plant. It pro-
motes wound healing, and it is also used for the treatment of
coughs, chronic bronchitis and other pulmonary disorders,

[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: +0632-
5360230, E-mail: consolacion.ragasa@dlsu.edu.ph
These authors have no conflict of interest to declare.
tumors, gonorrhoea, jaundice, dysentery, and boils [1]. The
aerial parts were reported to contain flavonoids: euphorbianin,
leucocyanidol, camphol, quercetin, and quercitol [2-3], poly-
phenols: gallic acid, myricitrin, and 3, 4-di-O-galloylquinic
acid [4-5], tannins: euphorbins A-E [6], triterpenes, and phyto-
sterols [7]. Taraxerone isolated from E. hirta exhibited anti-
microbial activity against fourteen pathogenic bacteria and
six fungi with MIC values between 64-128 g·mL1. In the
brine shrimp lethality assay, taraxerone exhibited an LC50
value of 17.78 g·mL1 [8]. Another study reported that quer-
cetin, myricitrin, 24-methylenecycloartenol, and sitosterol
from E. hirta exhibited dose-dependent anti-inflammatory
activity. Tannins and tannic acid derivatives from E. hirta
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528533
2013年 9月 第 11卷 第 5期 Chin J Nat Med Sept. 2013 Vol. 11 No. 5 529

have antiseptic effects, while taraxerone and 11, 12-oxido-
taraxerol 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-3-ol (2a) and 24-hydroper-
oxycycloart-25-en-3-ol (2b) (sample 1) in a 2 : 1 ratio, and
cycloartenyl (3a), lupeol (3b), -amyrin (3c), and -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), -amyrin fatty acid ester (4c), and -amyrin fatty acid
ester (4d) (sample 4), linoleic acid, -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 re-
ported.
2 Experimental
2.1 General
NMR spectra were recorded on a Varian VNMRS spec-
trometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for
13C NMR spectra. Column chromatography was performed
with silica gel 60 (70-230 mesh), while the TLC was per-
formed 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-3-ol (2a) and 24-hydroperoxy-
cycloart-25-en-3-ol (2b), cycloartenol (3a), lupeol (3b), -amyrin (3c), -amyrin (3d), cycloartenyl fatty acid ester (4a), lupeol
fatty acid ester (4b), -amyrin fatty acid ester (4c), and -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 Qui-
rino 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 fil-
tered. 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
530 Chin J Nat Med Sept. 2013 Vol. 11 No. 5 2013年 9月 第 11卷 第 5期

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 frac-
tion was rechromatographed (4 ×) in 10% EtOAc in petro-
leum 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. Dox-
orubicin, 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-dipheny-
ltetrazolium 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 albi-
cans (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 meas-
ured 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. activ-
ity index (AI) (diameter of clearing zone-diameter of
well)/diameter of well.
3 Structural Identification
Taraxerone (1) 1H NMR (CDCl3) : 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) : 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).
25-Hydroperoxycycloart-23-en-3-ol (2a) 1H NMR
(CDCl3) : 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) : 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-3-ol (2b) 1H NMR
(CDCl3) : 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) : 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) : 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
2013年 9月 第 11卷 第 5期 Chin J Nat Med Sept. 2013 Vol. 11 No. 5 531

(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) : 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).
-Amyrin (3c) colorless solid. 13C NMR (CDCl3): 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).
-Amyrin (3d) colorless solid. 13C NMR (CDCl3) :
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) :
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) : 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).
-Amyrin fatty acid ester (4c) 13C NMR (CDCl3) :
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).
-Amyrin fatty acid ester (4d) 13C NMR (CDCl3) :
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) : 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 ex-
tracts of E. hirta afforded taraxerone 1, a mixture of
25-hydroperoxycycloart-23-en-3-ol (2a) and 24-hydro-
peroxycycloart-25-en-3-ol (2b) (sample 2) in a 2 : 1 ratio,
and a mixture of cycloartenol (3a), lupeol (3b), -amyrin
(3c), and -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, -amyrin fatty acid ester 4c and -amyrin fatty acid
ester 4d (sample 4) in a 3 : 2 : 1 : 1 ratio, linoleic acid,
-sitosterol and squalene from the roots. The 1H NMR spec-
tra 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 reso-
nances at  5.66 (H-23) and 5.50 (H-24) for 2a, and  4.99
and 5.01 (H2-26) for 2b;  4.67 and 4.55 (H2-30) for 3b and
4b;  5.16 (H-12) for 3c and 4c; and  5.11 (H-12) for 3d and
4d; and the cyclopropyl methylene protons at  0.31 and 0.53
(H2-19) for 3a and 4a.
The structures of 1, 2a, and 2b were elucidated by ex-
tensive 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-3-ol (2a) [15], and 24-hydro-
peroxycycloart-25-en-3-ol (2b) [15]. The structures of the
other compounds were identified by comparison of their
13C NMR data (see Experimental) with those reported in the
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528533
532 Chin J Nat Med Sept. 2013 Vol. 11 No. 5 2013年 9月 第 11卷 第 5期

literature for cycloartenol (3a) [16], lupeol (3b) [17], -amyrin
(3c) [17], -amyrin (3d) [17], cycloartenyl fatty acid ester (4a)
[16], lupeol fatty acid ester (4b) [18], -amyrin fatty acid ester
(4c) [19-20], -amyrin fatty acid ester (4d) [18-19, 21], phytol [22],
phytyl fatty acid ester [23], linoleic acid [24], -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 com-
puted. This implied that 1 and sample 3 did not exhibit cyto-
toxic effects against this cell line. Meanwhile, sample 2 (Fig.
2) exhibited good activities, with IC50 values of 4.8 and 4.5
g·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 g·mL1. It is noted that sample 2 is more cyto-
toxic against the normal cell line, AA8 (IC50 3.7 g·mL1)
than the cancer cells, HCT 116 (IC50 4.8 g·mL1) and A549
(IC50 4.5 g·mL1) tested. However, the same trend is ob-
served against the positive control, doxorubicin (IC50 2.1
g·mL1) which is almost as cytotoxic against A549 (IC50 2.2
g·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 accord-
ing to the National Cancer Institute (NCI) guidelines should
be less than 20 and 4 g·mL1, respectively [27]. The IC50
values of sample 2 against the cancer cells, HCT 116 and
A549 are 4.8 g·mL1 and IC50 4.55 g·mL1, respectively.

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

These are close to the doses of pure compounds to be consid-
ered active according to the NCI guidelines.
As part of a continuing search for antimicrobial com-
pounds 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.
Table 1 Antimicrobial test results of 1 and sample 2
Microorganism Compounda (30 g) Clearing Zone (mm)b Activity Index (AI)
1  0
Sample 2 12 0.2

E. coli
Chloramphenicold 30 4.0
1 12 0.2
Sample 2 13 0.3

P. aeruginosa
Chloramphenicold 15 1.5
1 16 0.6
Sample 2 15 0.5

S. aureus
Chloramphenicold 33 4.5
1 13c 0
Sample 2 15c 0

B. subtilis
Chloramphenicold 20 2.3
Sample 2 12 0.2
C.albicans
Canestene, 0.2 g 18 0.8
Sample 2 16 0.6 T. mentagrophytes
Canestene, 0.2 g 55 4.5
A. niger Sample 2 f 0
Canestene, 0.2 g 23 1.3
a Sample–10 mm well diameter; b Average of three trials; c Partial inhibition of growth of test organism; d Chloramphenicol disk-6-mm disc;
eContains 1% clotrimazole; f no clearing zone
IC
50
(μ
g/
m
L
)
Consolacion Y. Ragasa, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 528533
2013年 9月 第 11卷 第 5期 Chin J Nat Med Sept. 2013 Vol. 11 No. 5 533

A previous study reported that 25-hydroperoxy-cyclo
art-23-en-3-ol (2a) showed minimum growth inhibition
concentrations of 2, 2, and 1 g against E. coli, M. luteus and
B. subtilis, respectively[15]. On the other hand,
24-hydroperoxycycloart-25-en-3-ol (2b) exhibited mini-
mum growth inhibition concentrations of 1, 1, and 5 g
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 g·mL1 [8].
Another study reported the antibacterial and antifungal prop-
erties 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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