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Evaluation of the bioactivity of triterpene mixture isolated from Carmona retusa (Vahl.) Masam leaves
Journal of Ethnopharmacology 92 (2004) 53–56
Evaluation of the bioactivity of triterpene mixture isolated from Carmona retusa (Vahl.) Masam leaves Irene M. Villaseñor∗ , 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. Kruskal–Wallis one-way analysis of variance by ranks showed that the triterpene mixture is as active as mefenamic acid, a commercially available analgesic, at α = 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 α = 0.01. Results of the micronucleus test showed that the triterpene mixture did not exhibit mutagenic nor anti-mutagenic activity at α = 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 Technology–Philippine Council for Health Research and Development–National 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 (Villaseñor and Edu, 1993; Villaseñor et al., 1993), white solids crystallized out in huge quantities. GC–MS analysis showed an intractable mixture of triterpenes consisting of structural isomers: ␣-amyrin, -amyrin, and baurenol (Villaseñor et al., 1992). ∗ Corresponding
author. Fax: +63-2-9205427. E-mail address: [email protected] (I.M. Villaseñor).
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. doi:10.1016/j.jep.2004.01.017
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I.M. Villaseñor et al. / Journal of Ethnopharmacology 92 (2004) 53–56
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 GC–MS 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, 7–12 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 (Villaseñor 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. Villaseñor et al. / Journal of Ethnopharmacology 92 (2004) 53–56
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 Kruskal–Wallis 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 min—426 (8%), 218 (100%), 205 (42%), 189 (10%); Rt = 21.00 min—426 (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 (Villaseñor 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 α = 0.01 while the triterpene mixture differed from corn oil at α = 0.10. Comparison between treatments showed that the triterpene mixture is as active as mefenamic acid, a commercially available analgesic, at α = 0.01. The triterpene mixture did not exhibit anti-inflammatory activity (Table 2). KW: 4.89 indicated differences in responses only at α = 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 α = 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 α = 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. Villaseñor et al. / Journal of Ethnopharmacology 92 (2004) 53–56
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)
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
a
(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 α = 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 α = 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
Cortes-Maramba, N.P., Dayrit, F.M., de Castro, N.F., Estrada, H.R., Lim-Sylianco, C.Y., Lingao, A.L., Quijano, R.F., Quintana, E.G., 1991. Selection and scientific validation of medicinal plants for primary health care. Technical Report Series No. 12. Philippine Council for Health Research and Development, Department of Science and Technology. de Padua, L., Lugod, G., Pancho, J., 1982. Handbook on Philippine Medicinal Plants, vol. 1. Documentation and Information Section, Office of the Director of Research, UPLB, Philippines, p. 21. Esmerino, L.A., Ranali, J., Rodrigues Jr., A.L., 1998. Blood glucose determination in normal and alloxan-diabetic rats after administration of local anesthetics containing vasoconstrictors. Brazilian Dental Journal 9, 33–37. PCHRD (Philippine Council for Health Research and Development), 1989. Recommended Bioassay Procedures for Medicinal Plants Products. Department of Science and Technology, p. 4. Quisumbing, E., 1978. Medicinal Plants of the Philippines. Katha Publishing Co., Inc. Philippines, p. 773. Schmid, W., 1975. The micronucleus test. Mutation Research 31, 9–15. Villaseñor, I.M., Edu, D.A., Bremner, J.B., 1993. Structure of an antimutagen from Carmona retusa leaves. Carcinogenesis 14, 123–125. Villaseñor, I.M., Edu, D.A., 1993. Antimutagen from the leaves of Carmona retusa (Vahl.) Masam. Mutation Research 298, 215–218. Villaseñor, I.M., Edu, D.A., Bremner, J.B., 1992. Triterpenoids of Carmona retusa (Vahl.) Masam. Philippine Journal of Science 121, 413– 416. Villaseñor, I.M., Canlas, A.P., Pascua, M.P.I., Sabando, M.N., Soliven, L.A.P., 2002. Bioactivity studies on Cassia alata Linn. leaf extracts. Phytotherapy Research 16, 93–96. Winter, C., Risley, E., Nuss, G., 1962. Carrageenan-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. In: Proceedings of the Society for Experimental Biology and Medicine III, p. 544.
Evaluation of the bioactivity of triterpene mixture isolated from Carmona retusa (Vahl.) Masam leaves Irene M. Villaseñor∗ , 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. Kruskal–Wallis one-way analysis of variance by ranks showed that the triterpene mixture is as active as mefenamic acid, a commercially available analgesic, at α = 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 α = 0.01. Results of the micronucleus test showed that the triterpene mixture did not exhibit mutagenic nor anti-mutagenic activity at α = 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 Technology–Philippine Council for Health Research and Development–National 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 (Villaseñor and Edu, 1993; Villaseñor et al., 1993), white solids crystallized out in huge quantities. GC–MS analysis showed an intractable mixture of triterpenes consisting of structural isomers: ␣-amyrin, -amyrin, and baurenol (Villaseñor et al., 1992). ∗ Corresponding
author. Fax: +63-2-9205427. E-mail address: [email protected] (I.M. Villaseñor).
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. doi:10.1016/j.jep.2004.01.017
54
I.M. Villaseñor et al. / Journal of Ethnopharmacology 92 (2004) 53–56
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 GC–MS 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, 7–12 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 (Villaseñor 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. Villaseñor et al. / Journal of Ethnopharmacology 92 (2004) 53–56
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 Kruskal–Wallis 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 min—426 (8%), 218 (100%), 205 (42%), 189 (10%); Rt = 21.00 min—426 (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 (Villaseñor 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 α = 0.01 while the triterpene mixture differed from corn oil at α = 0.10. Comparison between treatments showed that the triterpene mixture is as active as mefenamic acid, a commercially available analgesic, at α = 0.01. The triterpene mixture did not exhibit anti-inflammatory activity (Table 2). KW: 4.89 indicated differences in responses only at α = 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 α = 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 α = 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
56
I.M. Villaseñor et al. / Journal of Ethnopharmacology 92 (2004) 53–56
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)
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
a
(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 α = 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 α = 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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