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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
0378-8741/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.01.017
54
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
55
Table 2
Anti-inflammatory assay of the triterpene
carrageenan-induced mouse paw edema assay
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
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
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
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
0.88
1.58
0.93
1.39
1.39
56
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)
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)
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
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