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Journal of Ethnopharmacology 92 (2004) 5356

Evaluation of the bioactivity of triterpene mixture isolated


from Carmona retusa (Vahl.) Masam leaves
Irene M. Villaseor , Arlyn P. Canlas, Karen M. Faustino, Katherine G. Plana
Institute of Chemistry, University of the Philippines, Diliman, 1101 Quezon City, Philippines
Received 2 February 2003; received in revised form 13 January 2004; accepted 26 January 2004

Abstract
The major constituent of Carmona retusa (Vahl.) Masam. leaves is an intractable mixture of triterpenes, namely -amyrin (43.7%), -amyrin
(24.9%), and baurenol (31.4%). At a dosage of 100 mg/kg mouse, the triterpene mixture exhibited 51% analgesic activity but only showed 20%
anti-inflammatory activity. KruskalWallis one-way analysis of variance by ranks showed that the triterpene mixture is as active as mefenamic
acid, a commercially available analgesic, at = 0.01. The charcoal tracing test showed a 29% anti-diarrheal activity for the triterpene mixture,
which increased to 55% at a dosage of 250 mg/kg mouse. At the higher dosage, the triterpene mixture differed significantly from its solvent
control at = 0.01. Results of the micronucleus test showed that the triterpene mixture did not exhibit mutagenic nor anti-mutagenic activity
at = 0.001. There was no significant decrease in blood glucose levels (bgl) in alloxan-induced diabetic mice after administration of the
triterpene mixture. The triterpene mixture was inactive against Escherichia coli and possessed moderate activities against Staphylococcus
aureus, Candida albicans, and Trichophyton mentagrophytes.
2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Analgesic; Anti-diarrheal; -Amyrin; -Amyrin; Baurenol; Carmona retusa (Vahl.); Masam

1. Introduction
The Department of Health of the Philippines included in
its primary health care the use of medicinal plant preparations, targeted symptomatics rather than curatives, and came
up with a list of 10 priority plants for dosage formulation
and clinical trials. Carmona retusa (Vahl.) Masam. (Boraginaceae) is one of these priority plants under the Department
of Science and TechnologyPhilippine Council for Health
Research and DevelopmentNational Integrated Program on
Medicinal Plants. The dried leaves are available as 250 mg
tablets and are recommended as anti-colic and anti-diarrheal.
The tablets have undergone clinical trial phases 1 and 2 as
well (Cortes-Maramba et al., 1991). The leaves are used as
a stomachic, for cough, fever, and secondary and constitutional syphilis (de Padua et al., 1982; Quisumbing, 1978).
During the processing for the isolation of an anti-mutagenic
constituent (Villaseor and Edu, 1993; Villaseor et al.,
1993), white solids crystallized out in huge quantities.
GCMS analysis showed an intractable mixture of triterpenes consisting of structural isomers: -amyrin, -amyrin,
and baurenol (Villaseor et al., 1992).
Corresponding

author. Fax: +63-2-9205427.


E-mail address: ivillase@chem.upd.edu.ph (I.M. Villaseor).

Our present research is on the determination of the


anti-diarrheal constituent from the leaves of Carmona retusa. The triterpene mixture was again isolated as the major
constituent and they turned out to be anti-diarrheal. Apprehensive that we might again miss their other bioactivities,
we investigated this triterpene mixture for its possible
analgesic, anti-inflammatory, mutagenic, anti-mutagenic,
anti-diabetic, and anti-microbial activities.

2. Materials and methods


2.1. Plant material
Carmona retusa leaves were bought from a herbal store.
A voucher specimen was deposited at the Dr. Jose Vera
Santos Herbarium at the Institute of Biology, University of
the Philippines, Diliman with accession number 08903 and
authenticated by L.L. Co.
2.2. Isolation and identification of active compounds
Air-dried Carmona retusa leaves were homogenized
in methanol, filtered and concentrated in vacuo. The
MeOH extract was then partitioned between water and

0378-8741/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved.
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54

I.M. Villaseor et al. / Journal of Ethnopharmacology 92 (2004) 5356

hexane. The hexane layer was collected and concentrated in


vacuo.
The hexane extract (21.7 g) was subjected to vacuum liquid chromatography using a 9.5 cm column filled to 7 cm
height of silica gel 60G (Merck) as stationary phase. The
column was eluted with 500 ml hexane, 10% gradient ratios
of EtOAc in hexane, and EtOAc. The column fractions were
pooled according to analytical thin layer chromatography
results.
The fraction eluted out with 20% EtOAc in hexane was
rechromatographed using silica gel and eluted with hexane
and 2% gradient ratios of EtOAc in hexane. Yellowish crystals, precipitated out in 4% EtOAc in hexane, were collected
and washed with acetone until white crystals were obtained.
The white crystals were analyzed by GCMS at the
National Pesticide Analytical Laboratory of the Bureau
of Plant Industry using an HP 6890 spectrophotometer:
J&W 125-0212 column (30.0 m length, 320.0 m diameter, 0.25 m film thickness), helium was used as carrier
gas, 1.0 psi, 1 l sample splitless injection at an isothermal
temperature of 240 C, constant flow of 1.3 ml/min.
2.3. Animals
Swiss Webster albino mice were purchased from the Bureau of Animal Industry and were acclimatized for at least
a week before the start of the experiments. Five mice were
used for each test sample.
2.4. Anti-diarrheal assay: charcoal tracing method
(PCHRD, 1989)
The charcoal meal, which was used as an indicator, was
prepared as follows: 2 g of activated charcoal was mixed
with 20 ml of castor oil to form a uniform suspension. This
suspension was then mixed with 10 ml of coconut oil.
After 12 h fasting, the triterpene mixture was administered orally to mice at dosages of 100 and 250 mg/kg BW
of mouse. After 1 h, the mice were fed with the charcoal
meal. Twenty minutes later, they were sacrificed by cervical dislocations, their abdomens were opened, and their
small intestines were excised from the pylorus to the cecum. The total length of the intestine and the length that
the charcoal meal has traveled were measured. Loperamide
(Immodium ) was used as reference.

control, was dissolved in water (3.5 mg/kg mouse) and was


administered orally.
2.6. Mutagenicity/antimutagenicity bioassay: micronucleus
test
Swiss Webster albino mice, 712 weeks old, were used.
The required weight of the triterpene mixture (100 mg/kg
BW of mouse) was dissolved in corn oil while the required
weight (55 mg/kg BW of mouse) of tetracycline (Upjohn)
was dissolved in distilled water. Tetracycline, the positive
control, was administered orally using a gavage while the
test sample was injected intraperitoneally to the test animals.
The protocol used was described by Schmid (1975).
2.7. Anti-diabetic assay: alloxan-induced (Esmerino et al.,
1998)
Male mice were fasted for 24 h with water ad libitum and
their blood glucose levels (bgl) were then determined using
one touch basic blood glucose meter and test strips. Diabetes was induced by intraperitoneal injection of alloxan at
a dosage of 150 mg/kg mouse. Their bgl were again determined after 2 days. Animals with bgl over 13.8 mmol/l were
considered diabetic. The test samples were then administered orally at t = 0. The bgl were monitored at 30-min intervals. The triterpene mixture (100 mg/kg BW of mouse) was
dissolved in dimethylsulfoxide while glipizide (0.77 mg/kg
BW of mouse), the positive control, was dissolved in
water.
2.8. Anti-inflammatory bioassay: carrageenan-induced
mouse paw edema test (Winter et al., 1962)
The initial volume of the mouse hind paw was measured
using a plethysmometer. One hour after intraperitoneal injection of the triterpene mixture at a dosage of 100 mg/kg
BW of mouse, 0.01 ml carrageenan was injected intradermally into the left hind paw. After 3 h, the volume of the
mouse hind paw was measured. The difference in the initial
and final volumes was then computed and compared with
those of the solvent control. Indomethacin, the positive control, was administered orally at a dosage of 1.4 mg/kg BW
of mouse.
2.9. Antimicrobial assay: agar cup method

2.5. Analgesic bioassay: acetic acid-induced writhing test


(Villaseor et al., 2002)
Approximately 30 min after administration of the test
samples, 0.7% acetic acid was injected intraperitoneally
(0.2 ml/20 g mouse). The number of writhings for each
mouse was then counted for 15 min starting from 5 min
after acetic acid injection. The triterpene mixture was dissolved in corn oil and was injected intraperitoneally at a
dosage of 100 mg/kg mouse. Mefenamic acid, the positive

The anti-microbial assay was done by the Microbiological Services Laboratory of the Natural Sciences Research
Institute using the agar cup method. The test organisms used
were Escherichia coli UPCC 1195, Staphylococcus aureus
UPCC 1143, Candida albicans UPCC 2168, and Trichophyton mentagrophytes UPCC 4193. Aliquots of the cell suspensions of the bacteria, yeast and molds were transferred
into pre-poured nutrient agar, glucose yeast peptone agar and
potato dextrose agar plates, respectively. The plates were

I.M. Villaseor et al. / Journal of Ethnopharmacology 92 (2004) 5356

55

incubated at room temperature for 24, 48, and 72 h, respectively.

Table 2
Anti-inflammatory assay of the triterpene
carrageenan-induced mouse paw edema assay

2.10. Statistical analysis

Test samples

Dosage
(mg/kg)

Average change in
volume (ml) S.D.

Percentage
inhibition

Triterpene mixture

100
250

0.016 0.016
0.030 0.024

20
50

Data were analyzed using the KruskalWallis one-way


analysis of variance by ranks. This is an extremely useful test
for deciding whether k independent samples are from different populations. For the anti-diabetic assay, t-test: paired
two sample for means was used.

Corn oil

10 ml

0.020 0.012

1.4

0.011 0.012

10 ml

0.036 0.022

Indomethacin
Water

mixture

using

the

69

Table 3
Anti-diarrheal assay of the triterpene mixture using the charcoal tracing
method

3. Results and discussion


The gas chromatogram of the triterpene mixture (1.33%
yield) showed the presence of three compounds with retention times of 19.84, 21.00, and 23.23 min with relative
amounts of 24.9, 43.7, and 31.4%, respectively. Their mass
spectra showed that they are structural isomers with a molecular ion peak at m/e 426 and the following prominent peaks
with their relative abundances: Rt = 19.84 min426 (8%),
218 (100%), 205 (42%), 189 (10%); Rt = 21.00 min426
(12%), 218 (100%), 205 (20%), 189 (16%); Rt = 426
(32%), 411 (28%), 393 (8%), 247 (100%), 229 (62%), 205
(14%), 187 (8%). Reverse fit data and comparison with literature (Villaseor et al., 1992) showed that Rt = 19.84 min
was -amyrin, Rt = 21.00 min was -amyrin, and Rt =
23.23 min was baurenol.
At a dosage of 100 mg/kg BW of mouse, the triterpene
mixture exhibited analgesic activity, as shown by a 51% reduction in the number of writhes induced by acetic acid
(Table 1). The computed Kruskal Wallis statistic (KW =
14.46) showed that there were differences in the responses
of the mice to the different treatments at 0.01 level of significance. Comparisons of the treatments versus solvent controls showed that mefenamic acid differed from water at
= 0.01 while the triterpene mixture differed from corn
oil at = 0.10. Comparison between treatments showed
that the triterpene mixture is as active as mefenamic acid, a
commercially available analgesic, at = 0.01.
The triterpene mixture did not exhibit anti-inflammatory
activity (Table 2). KW: 4.89 indicated differences in responses only at = 0.20. At this level of significance, indomethacin differed from water while the triterpene mixture
is statistically similar to corn oil.
Table 1
Analgesic assay of the triterpene mixture using the acetic acid-induced
writhing test
Test samples

Dosage
(mg/kg)

Average number
of writhes S.D.

Triterpene mixture
Corn oil
Mefenamic acid
Water

100
10 ml
3.5
10 ml

26
53
13
54

12
12
9
6

Percentage
inhibition
51
76

Test samples

Dosage
(mg/kg)

Average percentage
distance (mm)
traveleda S.D.

Percentage
inhibition

Triterpene mixture

100
250

43.8 5.7
28.1 26.3

29
55

Corn oil

10 ml

61.9 13.8

Loperamide

10

36.5 7.7

Water

10 ml

65.9 7.2

45

Percentage of distance traveled = (distance traveled by the charcoal/


length of the small intestine) 100.
a

The triterpene mixture exhibited a 29 and 55% antidiarrheal activity at dosages of 100 and 250 mg/kg BW of
mouse, respectively (Table 3). KW: 13.79 denoted differences in responses at = 0.01. At this level of significance,
the variances of loperamide and the triterpene mixture at
250 mg/kg BW of mouse differed significantly from their
solvent controls, while that of the triterpene mixture at
100 mg/kg BW of mouse was not significantly different
from that of corn oil.
Results of the micronucleus test (Table 4) showed that the
triterpene mixture is not mutagenic as it approximates the
number of MN-PCE induced by corn oil, the solvent control.
It is not anti-mutagenic because it was not able to decrease
the number of MN-PCE induced by tetracycline, the positive
control. KW: 24.66 implied that there were differences in
responses at = 0.001. At this level of significance, the
triterpene mixture is neither mutagenic nor anti-mutagenic
Table 4
Mutagenicity and anti-mutagenicity assay of the triterpene mixture using
the micronucleus test
Test samples

Dosage
(mg/kg)

(Number of MN-PCEa /
1000 PCEb ) S.D.

Triterpene mixture
Tetracycline
Corn oil
Triterpenes + tetracycline
Corn oil + tetracycline

100
55
10 ml
100/55
10 ml/55

1.44
5.66
1.11
5.22
5.22

a
b

Micronucleated polychromatic erythrocytes.


Polychromatic erythrocytes.

0.88
1.58
0.93
1.39
1.39

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I.M. Villaseor et al. / Journal of Ethnopharmacology 92 (2004) 5356

Table 5
Anti-diabetic assay of the triterpene mixture using alloxan-induced diabetic mice
Test samples

Normal bgl
(mmol/l)

Alloxan-induced bgl
t = 0 (mmol/l)

Blood glucose levels (bgl) at 30-min intervals (percentage decrease in bgla )


t = 30 min

t = 60 min

t = 90 min

t = 120 min

t = 150 min

Triterpene mixture
DMSO
Glipizide
Water

2.9
3.0
2.7
3.3

20.0
18.7
23.5
25.9

15.5
18.0
19.5
25.4

16.1
17.9
19.0
25.6

16.3
17.9
19.8
24.6

15.9
18.0
19.5
24.1

15.8
18.0
18.5
23.9

(22.5)
(3.7)
(17.0)
(1.9)

(19.5)
(4.3)
(19.1)
(1.2)

(18.5)
(4.3)
(15.7)
(5.0)

(20.5)
(3.7)
(17.0)
(7.7)

(21.0)
(3.7)
(21.2)
(7.7)

Percentage of decrease in bgl = (alloxan-induced bgl sample bgl)/alloxan-induced bgl 100.

Table 6
Anti-microbial assay of the triterpene mixture using the agar cup method
Organism

Test samplesa

Average
clearing
zone (mm)

Antimicrobial
indexb

Escherichia coli

Triterpene mixture
Tetracycline

0
25

0
1.5

Staphylococcus
aureus

Triterpene mixture
Chloramphenicol

13
25

0.3
1.5

Candida albicans

Triterpene mixture
Clotrimazole

18
25

0.8
1.5

Trichophyton
mentagrophytes

Triterpene mixture
Clotrimazole

16
40

0.6
3.0

a 30 l of 1 g/l solution for the positive controls; 200 l of


10,000 ppm for the triterpene mixture.
b Antimicrobial index = (diameter of clearing zone diameter of
well)/diameter of well.

also available as 250 mg tablets. They are used in the symptomatic relief of diarrhea. Our results showed that the major
constituent of Carmona retusa, an intractable triterpene mixture, may relieve diarrhea and the stomach ache associated
with it. The triterpene mixture exhibited potent analgesic and
anti-diarrheal activities. It did not exhibit mutagenic activity
and hence, it is safe to use as a herbal medicine. Hence, results of this study validated the popular and safe use of the
plant as an anti-diarrheal.

Acknowledgements
The authors would like to thank the National Research
Council of the Philippines (NRCP) for the funding support.

References
because its variance is statistically similar to that of the
solvent control.
Diabetes was induced by intraperitoneal injection of alloxan using a dosage of 150 mg/kg BW of mouse. The
triterpene mixture was tested for its anti-diabetic activity
at a dosage of 100 mg/kg BW of mouse. It proved to be a
more effective anti-diabetic than the known and commercially available glipizide (Table 5) in terms of percentage
decrease in bgl. However, statistical analysis using t-test:
paired two sample for means showed that there was a significant decrease for glipizide during the first 30 min at =
0.10 (tstat : 2.14 > tcrit : 1.64) but there was none for the
triterpene mixture (tstat : 0.56 < tcrit : 1.64).
The triterpene mixture showed a weak to moderate
anti-candidal activity against Candida albicans and antidermatophytic against Trichophyton mentagrophytes, which
causes 90% of chronic dermatophyte infections, but is inactive against Escherichia coli (Table 6). KW: 11.0 showed
differences in treatments at = 0.10. At this level of significance, the triterpene mixture did not show the same activity
as the positive controls, chloramphenicol and clotrimazole.

4. Conclusion
Carmona retusa is used as tea in the Philippines. A decoction is usually prepared from the dried leaves. They are

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