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Topical anti-inflammatory activity of Bauhinia tarapotensis leaves
Phytomedicine 9: 646–653, 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves S. Sosa1, A. Braca2, G. Altinier1, R. Della Loggia1, I. Morelli2, and A. Tubaro1 1 2
Dipartimento di Economia e Merceologia, Università di Trieste, Italia Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Italia
Summary The topical anti-inflammatory properties of Bauhinia tarapotensis Benth. (Leguminosae) leaves have been studied by the inhibition of the croton oil-induced ear edema in mice. A bioassay-guided fractionation showed an interesting anti-inflammatory activity of the chloroform extract, that justifies the activity of the whole herbal drug. The main anti-inflammatory principles of B. tarapotensis leaves are triterpenic acids of ursane and oleanane series. The antiphlogistic activity of mixtures constituted of two ursane and oleanane isomers with different hydroxylation pattern, in the ratio 2:1, is comparable to that of indomethacin (ID50 ranging from 95 to 147 µg/cm2 and 93 µg/cm2, respectively). Key words: Bauhinia tarapotensis, anti-inflammatory activity, triterpenes, croton oil ear test
Introduction Bauhinia tarapotensis Benth. (Leguminosae) is a small tree growing in Ecuador (South America), where it is commonly known as “pata de vaca”. The plant leaves are traditionally used for their anti-inflammatory and decongestant properties (Cordero, 1950), whereas the bark is employed as antidiarrhoeal remedy (Kohn, 1992). Previous study on the methanol extract of B. tarapotensis leaves revealed antioxidant and radical scavenger properties, due to the presence of different antioxidant principles, such as cyclohexenone, lignans, and phenylethanoids derivatives (Braca et al., 2001). It is well known that free radicals are oxidizing agents produced in large amounts by phagocytic leukocytes during inflammatory conditions. They are also involved in tissue injury associated with inflammations (Conner and Grisham, 1996). Actually, some antioxidants possess antiphlogistic properties and attenuate the tissue injuries occurring in some inflammatory disorders (Conner and Grisham, 1996; Cuzzocrea et al., 0944-7113/02/09/07-646 $ 15.00/0
2001). Consequently, we hypothesised that the antioxidant and radical scavenger principles of B. tarapotensis leaves could possess also an anti-inflammatory activity, that could justify the use of the plant material in the traditional medicine. Therefore, the present study was undertaken to evaluate the anti-inflammatory properties of B. tarapotensis leaves and to identify the compounds responsible for this effect. To this aim, the plant material was submitted to a bioassay-oriented fractionation, evaluating the anti-inflammatory activity as inhibition of the croton oil-induced ear edema in mice, after topical application (Tubaro et al., 1985). This approach showed that the anti-inflammatory activity of B. tarapotensis leaves was concentrated in their chloroform extract, which accounted for the total activity of the plant material. Moreover, phytochemical and pharmacological investigation of the chloroform extract led to the identification of different triterpenic acid derivatives with anti-inflammatory activity.
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves
Materials and Methods Plant material
The leaves of B. tarapotensis were collected from the Pastaza region (Ecuador) in July 1995. The plant material was identified by Dr. Medardo Tapia, Escuela Superior Politecnico de Chimborazo (Ecuador), where a voucher specimen was deposited. Instruments and chemicals
NMR spectra were recorded on a AC-200 (200 MHz) instrument, using CD3OD and CDCl3 as solvents; the chemical shifts were expressed in d (ppm) referring to solvent peaks: δH 3.31 and δH 7.24 and δC 49.0 and δC 77.0, respectively. EIMS were obtained on a VG ZAB spectrometer (70 eV). TLC was performed on precoated Kieselgel 60 F254 plates (Merck, Frankfurt, Germany) with n-hexane-chloroform (9:1), chloroformmethanol (95:5) and chloroform-methanol (9:1) as eluents. Compounds were detected by spraying with Ce(SO4)2/H2SO4 (Sigma-Aldrich, Milano, Italy) solution followed by heating. Column chromatography was obtained over silica gel (40–63 µm, Merck, Frankfurt, Germany). Croton oil and indomethacin were Sigma products (St Louis, USA). Ursolic acid and oleanolic acid were supplied by Indena S.p.A. (Milano, Italy) and Roth (Karlsruhe, Germany). Ketamine hydrochloride was purchased from Virbac S.r.l. (Milano, Italy). The other reagents and solvents, of analytical grade, were purchased from Carlo Erba (Milano, Italy). Extraction and fractionation procedure
The leaves of B. tarapotensis (400 g) were air dried at 40 °C and the dried material was then powdered and submitted to sequential extractions with n-hexane, chloroform, chloroform-methanol (9:1), methanol, and water, by exhaustive maceration (3 times × 2 l). The extracts were filtered and concentrated in vacuum to give the corresponding residues (Figure 1). Part of the chloroform extract (0.5 g) was partitioned by consecutive extractions between diethyl ether and water containing 1% NaOH (100:250 ml, for several times). The organic layer was dried in vacuum giving fraction I, whereas the combined alkaline aqueous layers were acidified with acetic acid to pH 5 and then re-extracted with diethyl ether several times. The combined diethyl ether layers were dried in vacuum to give fraction II (Fig. 1). Isolation and identification procedure
Part of the chloroform extract (8.0 g) was submitted to a column chromatographic separation over silica gel employing gradient elution from chloroform to methanol. Ursolic acid (1) and oleanolic acid (2) were obtained from the chloroform fraction, 2α-hydroxyursolic acid (3), 2α-hydroxyoleanolic or maslinic acid (4), 2α,3αdihydroxy-urs-12-en-28-oic acid (5), and 2α,3α-dihydroxy-olean-12-en-28-oic acid (6) were purified from
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the chloroform-MeOH 99:1 fraction, and 2α,3α,23-trihydroxy-urs-12-en-28-oic acid (7) and 2α,α3,23-trihydroxy-olean-12-en-28-oic acid (8) were isolated from the chloroform-MeOH 97:3 fraction. Pure 3β-O-palmitoyl-lupeol (9) was purified from the n-hexane extract by silica gel column chromatography eluting with nhexane-chloroform 9:1. The structures of all compounds were determined spectroscopically by 1H and 13 C NMR and chromatographically by comparison with references. The data obtained agreed with those reported in the literature (Mahato and Kundo, 1994; Kojima and Ogura, 1989; Lin and Tome, 1988). Anti-inflammatory activity assay
The topical anti-inflammatory activity was evaluated as inhibition of the croton oil-induced ear edema in mice (Tubaro et al., 1985). The animal experiments complied with the Italian D.L. n. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC). Male CD-1 mice (28–32 g; Harlan-Italy, Udine, Italy) were anaesthetised with ketamine hydrochloride (145 mg/kg, intraperitoneally). Cutaneous inflammation was induced on the inner surface of the right ear (surface: about 1 cm2) of anaesthetised mice by application of 80 µg of croton oil dissolved in an appropriate vehicle, as reported below. Control animals received only the irritant solution, while the other animals received both the irritant and the substances under testing, dissolved in the same vehicle used for the relevant controls. The vehicles used were acetone or, for the aqueous extract and its controls, 42% aqueous ethanol (v/v). At the maximum of the edematous response, six hours later, mice were sacrificed and a plug (6 mm ∅) was removed from both the treated (right) and the untreated (left) ears. The edematous response was measured as the weight difference between the two plugs. The anti-inflammatory activity was expressed as percentage of the edema reduction in treated mice compared to the relevant control mice. The non steroidal anti-inflammatory drug (NSAID) indomethacin was used as reference compound. Statistical analysis
Pharmacological data were analysed by the Student’s ttest, and a probability level lower than 0.05 was considered as statistically significant. ID50 values (dose giving 50% edema inhibition) were calculated by graphic interpolation of the dose-effect curves.
Results Bioassay-oriented fractionation and screening of the anti-inflammatory activity
The subsequent extractions of B. tarapotensis leaves with solvents of increasing polarity gave n-hexane,
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chloroform, chloroform-methanol (9:1), methanol, and water extracts in the amounts of 2.4, 14.0, 8.5, 19.0, and 8.0 g (corresponding to 0.6, 3.5, 2.1, 4.7, and 2.0% of the dry plant material), respectively. Each extract was evaluated for its anti-inflammatory activity at the dose of 300 µg/cm 2. The results on the anti-edema activity of the extracts are reported in Table 1. All the extracts were able to reduce the edematous response to a certain extent, being the chloroform one the most active. Therefore, the chloroform extract, which induced 89% edema inhibition at the dose of 300 µg/cm 2, was further investigated in order to evaluate its contribution to the global activity of the starting plant material. To this aim, its effect was compared to that of a virtual total extract, prepared by pooling n-hexane (4.6%), chloroform (27.0%), chloroform-methanol (16.4%), methanol (36.6%), and aqueous (15.4%) ex-
tracts, on the basis of their extraction yields. The total extract (300 µg/cm 2) or the equivalent dose of the chloroform extract (81 µg/cm 2) induced similar edema reduction (81 and 76%, respectively), indicating that the chloroform extract gives the highest contribution to the activity of B. tarapotensis leaves (Table 2). Phytochemical analysis of the chloroform extract and isolation of triterpenic acids
Phytochemical analysis of the chloroform extract by silica gel column chromatography revealed triterpenic acids as its main constituents. Therefore, a triterpenic acid enriched fraction was separated from the chloroform extract. In particular, by repeated partitions between diethyl ether and water containing 1% NaOH, the extract was separated in two main fractions: frac-
Fig. 1. Fractionation procedure for B. tarapotensis leaves.
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves
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Table 1. Anti-inflammatory activity of B. tarapotensis extracts after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
Control n-Hexane extract Chloroform extract Chloroform-methanol extract Methanol extract Control Aqueous extract
— 300 300 300 300 — 300
10 11 10 10 10 10 11
8.3 ± 0.2 3.4 ± 0.4* 0.9 ± 0.2* 4.5 ± 0.4* 4.9 ± 0.4* 7.0 ± 0.4 4.1 ± 0.5*
— 59 89 46 41 — 41
Edema values are expressed as mean ± S.E.; *p < 0.001, using Student’s t-test, as compared with the respective controls.
Fig. 2. Structure of compounds 1–9.
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Table 2. Anti-inflammatory activity of the total and the chloroform extracts of B. tarapotensis after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
Control Total extract Chloroform extract
— 300 81
10 10 10
7.3 ± 0.2 1.4 ± 0.2* 1.8 ± 0.2*
— 81 76
Edema values are expressed as mean ± S.E.; *p < 0.001, using Student’s t-test, as compared with the respective controls.
Table 3. Anti-inflammatory activity of fractions I and II from the chloroform extract of B. tarapotensis after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
Control Chloroform extract Fraction I Fraction II
— 300 63a 210a
10 10 10 10
7.1 ± 0.3 0.6 ± 0.1* 5.3 ± 0.3* 0.7 ± 0.2*
— 92 25 90
Edema values are expressed as mean ± S.E.; *p < 0.05, using Student’s t-test, as compared with the respective controls; adose of the fraction equivalent to 300 µg of the chloroform extract.
Table 4. Anti-inflammatory activity of some triterpenic acids from B. tarapotensis after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
ID50 (µg/cm 2)
Control 3β-hydroxy-urs-12-en-28-oic acid (1)/ 3β-hydroxy-olean-12-en-28-oic acid (2) (2:1)
— 37.5 75 150
30 10 10 10
7.8 ± 0.3 6.8 ± 0.4* 5.1 ± 0.6* 2.2 ± 0.3*
— 13 35 72
— 95
2α,3β-dihydroxy-urs-12-en-28-oic acid (3)/ 2α,3β-dihydroxy-olean-12-en-28-oic acid (4) (2:1)
37.5 75 150
10 10 10
5.9 ± 0.4* 5.0 ± 0.3* 3.9 ± 0.4*
24 36 50
146
2α,3α-dihydroxy-urs-12-en-28-oic acid (5)/ 2α,3α-dihydroxy-olean-12-en-28-oic acid (6) (2:1)
37.5 75 150
10 10 10
6.1 ± 0.5* 5.5 ± 0.4* 2.7 ± 0.3*
22 29 65
107
2α,3α,23-trihydroxy-urs-12-en-28-oic acid (7)/ 2α,3α,23-trihydroxy-olean-12-en-28-oic acid (8) (2:1)
37.5 75 150
10 10 10
7.9 ± 0.4 6.5 ± 0.3* 3.6 ± 0.3*
–1 17 54
147
2α,3β-dihydroxy-urs-12-en-28-oic-acid (3)
25 50 100
10 10 11
8.1 ± 0.3 6.8 ± 0.5 6.2 ± 0.4*
–4 13 21
n.e.
3β-O-palmitoyl-lupeol (9)
50 100 200
11 10 10
6.9 ± 0.3* 5.3 ± 0.5* 4.0 ± 0.3*
12 32 49
204
3β-hydroxy-urs-12-en-28-oic acid (1) 3β-hydroxy-olean-12-en-28-oic acid (2) Indomethacin
100 100 90
10 10 10
1.9 ± 0.2* 4.3 ± 0.5* 4.1 ± 0.3*
76 45 47
Edema values are expressed as mean ± S.E.; *p<0.05, using Student’s t-test, as compared with the respective controls; n.e. = not evaluable.
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves tions I (106.0 mg) and II (352.1 mg), representing 21 and 70% of the parent extract, respectively (Fig. 1). Comparison by TLC analysis confirmed that fraction II contained the same triterpenic acids identified in the chloroform extract, while fraction I contained pigments together with a small amount of ursolic acid (1). In particular, the following triterpenic acids of ursane and oleanane series were identified in fraction II: 3β-hydroxy-urs-12-en-28-oic acid (ursolic acid; 1), 3β-hydroxy-olean-12-en-28-oic acid (oleanolic acid; 2), 2α,3β-dihydroxy-urs-12-en-28-oic acid (2α-hydroxyursolic acid; 3), 2,3-dihydroxy-olean-12-en-28-oic acid (2α-hydroxyoleanolic or maslinic acid; 4), 2α,3α-dihydroxy-urs-12-en-28-oic acid (5), 2α,3α-dihydroxyolean-12-en-28-oic acid (6), 2α,3α,23-trihydroxy-urs12-en-28-oic acid (7), and 2α,3α,23-trihydroxy-olean12-en-28-oic acid (8) (Fig. 2). Separation of the chloroform extract by column chromatography over silica gel allowed to isolate four mixtures of these triterpenes, each of them constituted of two structural isomers 1–2, 3–4, 5–6, and 7–8, in the ratio ursane:oleanane derivatives 2:1. Only a small amount of 2α,3β-dihydroxyurs-12-en-28-oic acid (3) was separated as pure compound. Anti-inflammatory activity of the chloroform extract fractions
Fractions I and II were evaluated for their anti-inflammatory activity at the respective doses of 63 and 210 µg/cm 2 that, on the basis of their fractionation yield, correspond to 300 µg of the parent chloroform extract. While fraction I provoked only 25% edema reduction, fraction II showed the same activity of the chloroform extract (90 and 92% inhibition, respectively) (Table 3). Therefore, fraction II, rich in triterpenic acids, accounts for all the activity of the parent chloroform extract. Anti-inflammatory activity of the triterpenic acids
The four mixtures of triterpenic acids isomers and the compound 3 were submitted to the anti-inflammatory activity assay. In addition, also the activity of 3β-Opalmitoyl-lupeol (9) (Figure 2), isolated from the nhexane extract, was studied. The pure triterpenes ursolic acid (1) and oleanolic acid (2), as well as the NSAID indomethacin, were used as reference compounds. As shown in Table 4, all the couples of isomers induced a dose-dependent edema reduction and ursolic and oleanolic acid mixture (compounds 1 and 2) was the most active. The ID50 values (dose giving 50% edema inhibition) of the isomer couples ranged between 95 and 147 µg/cm 2, showing an activity higher
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than that of the pure compound 3, which induced only 21% edema reduction at the highest dose (100 µg/cm 2). As reference, 100 µg/cm 2 of ursolic acid (1) or oleanolic acid (2) induced 76 and 45% edema inhibition, respectively, while 90 µg/cm 2 of indomethacin, a dose level proximal to its ID50 value (ID50 = 93 µg/cm 2; Ismaili et al., 2001), reduced the edematous response by 47%. Also the pure compound 9 showed a dose-dependent edema inhibition, but lower than that of the reference compounds ursolic and oleanolic acids as well as of the triterpenic acids mixtures (ID50 = 204 µg/cm 2). Therefore, the mixtures of ursane and oleanane derivatives (2:1) showed the highest activity, comparable to that of the NSAID.
Discussion The present results demonstrate that the leaves of B. tarapotensis possess anti-inflammatory properties, supporting their popular use for the treatment of inflammatory-based disorders. In particular, five extracts of the leaves significantly inhibited the croton oil-induced ear edema in mice, among which the chloroform one was the most active. This extract justifies also the anti-inflammatory activity of the whole herbal drug of B. tarapotensis. While the antioxidant components of B. tarapotensis leaves were previously found in their methanol extract and identified as various compounds of different structures (Braca et al., 2001), the anti-inflammatory principles are concentrated in the chloroform extract. Therefore, the in vitro antioxidant effect of the methanol extract and of some of its constituents does not seem to significantly contribute to the anti-inflammatory activity of the plant leaves. In fact, the bioassay-guided fractionation led to identify some triterpenic acids as the anti-inflammatory principles of B. tarapotensis leaves. Four mixtures of two structural isomers of ursane and oleanane derivatives, in the ratio 2:1, were isolated for the anti-inflammatory activity assay. The components of these mixtures were identified as ursolic acid (1), oleanolic acid (2), 2α-hydroxyursolic acid (3), 2αhydroxyoleanolic or maslinic acid (4), 2α,3α-dihydroxy-urs-12-en-28-oic acid (5), 2α,3α-dihydroxyolean-12-en-28-oic acid (6), 2α,3α,23-trihydroxy-urs12-en-28-oic acid (7), and 2α,3α,23-trihydroxy-olean12-en-28-oic acid (8). The four mixtures of these compounds, as couples of isomers 1–2, 3–4, 5–6, and 7–8, exhibited an interesting anti-inflammatory effect (ID50 ranging from 95 to 147 µg/cm 2), comparable to that of the NSAID indomethacin (ID50 = 93 µg/cm 2). It was observed that ursolic acid (1) and oleanolic acid (2) mixture (2:1) exhibited the strongest antiphlogistic effect (ID50 = 95 µg/cm 2), showing a potency
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similar to that of indomethacin (ID50 = 93 µg/cm 2; Ismaili et al., 2001). On the contrary, their hydroxylation at 2α position (compounds 3 and 4) involves a loss of activity (ID50 = 146 µg/cm 2). Similarly, a loss of activity was observed also after the 2α-hydroxylation of the pure compound ursolic acid (1): while 100 µg/cm 2 of ursolic acid (1) induced 76% edema inhibition, its 2αhydroxylated derivative (3) reduced the edematous response only by 21%. On the contrary, when the hydroxylation is accompanied by a single conformational change of the hydroxy group from 3β to 3α position, like in the mixture of compounds 5 and 6, the activity was almost similar to that of ursolic and oleanolic acid mixture (ID50 = 107 and 95 µg/cm 2, respectively). However, further addition of a hydroxy group at 23 position (compounds 7 and 8) involves a decrease of the anti-inflammatory effect (ID50 = 147 µg/cm 2). The anti-inflammatory activity of triterpenic acids and, in particular, that of ursolic and oleanolic acid, was previously observed both after topical application (Huang et al., 1994; Recio et al., 1995a, b; Máñez et al., 1997; Ismaili et al., 2001) and after systemic administration (Kosuge et al., 1985; Singh et al., 1992; Kapil and Sharma, 1995; Recio et al., 1995a, b). Among the other triterpenic acids isolated from B. tarapotensis leaves, an in vivo anti-inflammatory activity was only reported for 2α-hydroxyoleanolic acid (Shimizu et al., 1986), while 2α-hydroxyursolic and 2α,3α-dihydroxyurs-12-en-28-oic acids were shown to inhibit in vitro mast cells degranulation and/or nitric oxide production, which could be related to an in vivo antiphlogistic effect (Ryu et al., 2000). Moreover, ursolic and oleanolic acids and their derivatives were identified as the main anti-inflammatory constituents of other plants belonging to different families and, particularly, to some species of the Lamiaceae family, such as Prunella vulgaris (Ryu et al., 2000), Salvia officinalis (Baricevic et al., 2001), and Thymus willdenowii (Ismaili et al., 2001). Their anti-inflammatory effects have been attributed to the inhibition of different events of an inflammatory reaction, such as histamine release, cyclooxygenase-2, and 5-lipooxygenase pathways of arachidonic acid metabolism, elastase activity, complement activity and nitric oxide production (Kapil and Sharma, 1995; Liu, 1995; Ringbom et al., 1998; Suh et al., 1998; Díaz et al., 2000; Ryu et al., 2000). Consequently, also the preparations of Bauhinia tarapotensis leaves, containing ursane and oleanane derivatives, could exert their antiphlogistic effect acting on multiple targets of the inflammatory reaction. In conclusion, the obtained results confirm the validity of B. tarapotensis leaves use in the traditional medicine for the treatment of inflammatory conditions, due to the presence of triterpenic acids belonging to the ursane and oleanane series.
Acknowledgements
This work was supported by a grant of the Italian Ministry for University and Scientific Research (Project “Piante medicinali ed alimentari di Paesi in via di sviluppo: studio chimico e biologico”).
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Address A. Tubaro, Dipartimento di Economia e Merceologia, Via A. Valerio, 6 - 34127 Trieste, Italia Tel.: ++39-040-558 7910; Fax: ++39-040-558 3215; e-mail: [email protected]
Phytomedicine
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves S. Sosa1, A. Braca2, G. Altinier1, R. Della Loggia1, I. Morelli2, and A. Tubaro1 1 2
Dipartimento di Economia e Merceologia, Università di Trieste, Italia Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Italia
Summary The topical anti-inflammatory properties of Bauhinia tarapotensis Benth. (Leguminosae) leaves have been studied by the inhibition of the croton oil-induced ear edema in mice. A bioassay-guided fractionation showed an interesting anti-inflammatory activity of the chloroform extract, that justifies the activity of the whole herbal drug. The main anti-inflammatory principles of B. tarapotensis leaves are triterpenic acids of ursane and oleanane series. The antiphlogistic activity of mixtures constituted of two ursane and oleanane isomers with different hydroxylation pattern, in the ratio 2:1, is comparable to that of indomethacin (ID50 ranging from 95 to 147 µg/cm2 and 93 µg/cm2, respectively). Key words: Bauhinia tarapotensis, anti-inflammatory activity, triterpenes, croton oil ear test
Introduction Bauhinia tarapotensis Benth. (Leguminosae) is a small tree growing in Ecuador (South America), where it is commonly known as “pata de vaca”. The plant leaves are traditionally used for their anti-inflammatory and decongestant properties (Cordero, 1950), whereas the bark is employed as antidiarrhoeal remedy (Kohn, 1992). Previous study on the methanol extract of B. tarapotensis leaves revealed antioxidant and radical scavenger properties, due to the presence of different antioxidant principles, such as cyclohexenone, lignans, and phenylethanoids derivatives (Braca et al., 2001). It is well known that free radicals are oxidizing agents produced in large amounts by phagocytic leukocytes during inflammatory conditions. They are also involved in tissue injury associated with inflammations (Conner and Grisham, 1996). Actually, some antioxidants possess antiphlogistic properties and attenuate the tissue injuries occurring in some inflammatory disorders (Conner and Grisham, 1996; Cuzzocrea et al., 0944-7113/02/09/07-646 $ 15.00/0
2001). Consequently, we hypothesised that the antioxidant and radical scavenger principles of B. tarapotensis leaves could possess also an anti-inflammatory activity, that could justify the use of the plant material in the traditional medicine. Therefore, the present study was undertaken to evaluate the anti-inflammatory properties of B. tarapotensis leaves and to identify the compounds responsible for this effect. To this aim, the plant material was submitted to a bioassay-oriented fractionation, evaluating the anti-inflammatory activity as inhibition of the croton oil-induced ear edema in mice, after topical application (Tubaro et al., 1985). This approach showed that the anti-inflammatory activity of B. tarapotensis leaves was concentrated in their chloroform extract, which accounted for the total activity of the plant material. Moreover, phytochemical and pharmacological investigation of the chloroform extract led to the identification of different triterpenic acid derivatives with anti-inflammatory activity.
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves
Materials and Methods Plant material
The leaves of B. tarapotensis were collected from the Pastaza region (Ecuador) in July 1995. The plant material was identified by Dr. Medardo Tapia, Escuela Superior Politecnico de Chimborazo (Ecuador), where a voucher specimen was deposited. Instruments and chemicals
NMR spectra were recorded on a AC-200 (200 MHz) instrument, using CD3OD and CDCl3 as solvents; the chemical shifts were expressed in d (ppm) referring to solvent peaks: δH 3.31 and δH 7.24 and δC 49.0 and δC 77.0, respectively. EIMS were obtained on a VG ZAB spectrometer (70 eV). TLC was performed on precoated Kieselgel 60 F254 plates (Merck, Frankfurt, Germany) with n-hexane-chloroform (9:1), chloroformmethanol (95:5) and chloroform-methanol (9:1) as eluents. Compounds were detected by spraying with Ce(SO4)2/H2SO4 (Sigma-Aldrich, Milano, Italy) solution followed by heating. Column chromatography was obtained over silica gel (40–63 µm, Merck, Frankfurt, Germany). Croton oil and indomethacin were Sigma products (St Louis, USA). Ursolic acid and oleanolic acid were supplied by Indena S.p.A. (Milano, Italy) and Roth (Karlsruhe, Germany). Ketamine hydrochloride was purchased from Virbac S.r.l. (Milano, Italy). The other reagents and solvents, of analytical grade, were purchased from Carlo Erba (Milano, Italy). Extraction and fractionation procedure
The leaves of B. tarapotensis (400 g) were air dried at 40 °C and the dried material was then powdered and submitted to sequential extractions with n-hexane, chloroform, chloroform-methanol (9:1), methanol, and water, by exhaustive maceration (3 times × 2 l). The extracts were filtered and concentrated in vacuum to give the corresponding residues (Figure 1). Part of the chloroform extract (0.5 g) was partitioned by consecutive extractions between diethyl ether and water containing 1% NaOH (100:250 ml, for several times). The organic layer was dried in vacuum giving fraction I, whereas the combined alkaline aqueous layers were acidified with acetic acid to pH 5 and then re-extracted with diethyl ether several times. The combined diethyl ether layers were dried in vacuum to give fraction II (Fig. 1). Isolation and identification procedure
Part of the chloroform extract (8.0 g) was submitted to a column chromatographic separation over silica gel employing gradient elution from chloroform to methanol. Ursolic acid (1) and oleanolic acid (2) were obtained from the chloroform fraction, 2α-hydroxyursolic acid (3), 2α-hydroxyoleanolic or maslinic acid (4), 2α,3αdihydroxy-urs-12-en-28-oic acid (5), and 2α,3α-dihydroxy-olean-12-en-28-oic acid (6) were purified from
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the chloroform-MeOH 99:1 fraction, and 2α,3α,23-trihydroxy-urs-12-en-28-oic acid (7) and 2α,α3,23-trihydroxy-olean-12-en-28-oic acid (8) were isolated from the chloroform-MeOH 97:3 fraction. Pure 3β-O-palmitoyl-lupeol (9) was purified from the n-hexane extract by silica gel column chromatography eluting with nhexane-chloroform 9:1. The structures of all compounds were determined spectroscopically by 1H and 13 C NMR and chromatographically by comparison with references. The data obtained agreed with those reported in the literature (Mahato and Kundo, 1994; Kojima and Ogura, 1989; Lin and Tome, 1988). Anti-inflammatory activity assay
The topical anti-inflammatory activity was evaluated as inhibition of the croton oil-induced ear edema in mice (Tubaro et al., 1985). The animal experiments complied with the Italian D.L. n. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC). Male CD-1 mice (28–32 g; Harlan-Italy, Udine, Italy) were anaesthetised with ketamine hydrochloride (145 mg/kg, intraperitoneally). Cutaneous inflammation was induced on the inner surface of the right ear (surface: about 1 cm2) of anaesthetised mice by application of 80 µg of croton oil dissolved in an appropriate vehicle, as reported below. Control animals received only the irritant solution, while the other animals received both the irritant and the substances under testing, dissolved in the same vehicle used for the relevant controls. The vehicles used were acetone or, for the aqueous extract and its controls, 42% aqueous ethanol (v/v). At the maximum of the edematous response, six hours later, mice were sacrificed and a plug (6 mm ∅) was removed from both the treated (right) and the untreated (left) ears. The edematous response was measured as the weight difference between the two plugs. The anti-inflammatory activity was expressed as percentage of the edema reduction in treated mice compared to the relevant control mice. The non steroidal anti-inflammatory drug (NSAID) indomethacin was used as reference compound. Statistical analysis
Pharmacological data were analysed by the Student’s ttest, and a probability level lower than 0.05 was considered as statistically significant. ID50 values (dose giving 50% edema inhibition) were calculated by graphic interpolation of the dose-effect curves.
Results Bioassay-oriented fractionation and screening of the anti-inflammatory activity
The subsequent extractions of B. tarapotensis leaves with solvents of increasing polarity gave n-hexane,
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chloroform, chloroform-methanol (9:1), methanol, and water extracts in the amounts of 2.4, 14.0, 8.5, 19.0, and 8.0 g (corresponding to 0.6, 3.5, 2.1, 4.7, and 2.0% of the dry plant material), respectively. Each extract was evaluated for its anti-inflammatory activity at the dose of 300 µg/cm 2. The results on the anti-edema activity of the extracts are reported in Table 1. All the extracts were able to reduce the edematous response to a certain extent, being the chloroform one the most active. Therefore, the chloroform extract, which induced 89% edema inhibition at the dose of 300 µg/cm 2, was further investigated in order to evaluate its contribution to the global activity of the starting plant material. To this aim, its effect was compared to that of a virtual total extract, prepared by pooling n-hexane (4.6%), chloroform (27.0%), chloroform-methanol (16.4%), methanol (36.6%), and aqueous (15.4%) ex-
tracts, on the basis of their extraction yields. The total extract (300 µg/cm 2) or the equivalent dose of the chloroform extract (81 µg/cm 2) induced similar edema reduction (81 and 76%, respectively), indicating that the chloroform extract gives the highest contribution to the activity of B. tarapotensis leaves (Table 2). Phytochemical analysis of the chloroform extract and isolation of triterpenic acids
Phytochemical analysis of the chloroform extract by silica gel column chromatography revealed triterpenic acids as its main constituents. Therefore, a triterpenic acid enriched fraction was separated from the chloroform extract. In particular, by repeated partitions between diethyl ether and water containing 1% NaOH, the extract was separated in two main fractions: frac-
Fig. 1. Fractionation procedure for B. tarapotensis leaves.
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves
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Table 1. Anti-inflammatory activity of B. tarapotensis extracts after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
Control n-Hexane extract Chloroform extract Chloroform-methanol extract Methanol extract Control Aqueous extract
— 300 300 300 300 — 300
10 11 10 10 10 10 11
8.3 ± 0.2 3.4 ± 0.4* 0.9 ± 0.2* 4.5 ± 0.4* 4.9 ± 0.4* 7.0 ± 0.4 4.1 ± 0.5*
— 59 89 46 41 — 41
Edema values are expressed as mean ± S.E.; *p < 0.001, using Student’s t-test, as compared with the respective controls.
Fig. 2. Structure of compounds 1–9.
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Table 2. Anti-inflammatory activity of the total and the chloroform extracts of B. tarapotensis after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
Control Total extract Chloroform extract
— 300 81
10 10 10
7.3 ± 0.2 1.4 ± 0.2* 1.8 ± 0.2*
— 81 76
Edema values are expressed as mean ± S.E.; *p < 0.001, using Student’s t-test, as compared with the respective controls.
Table 3. Anti-inflammatory activity of fractions I and II from the chloroform extract of B. tarapotensis after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
Control Chloroform extract Fraction I Fraction II
— 300 63a 210a
10 10 10 10
7.1 ± 0.3 0.6 ± 0.1* 5.3 ± 0.3* 0.7 ± 0.2*
— 92 25 90
Edema values are expressed as mean ± S.E.; *p < 0.05, using Student’s t-test, as compared with the respective controls; adose of the fraction equivalent to 300 µg of the chloroform extract.
Table 4. Anti-inflammatory activity of some triterpenic acids from B. tarapotensis after 6 h induction of the croton oil ear inflammation in mice. Substance
Dose (µg/cm 2)
No. of mice
Edema (mg)
Edema reduction (%)
ID50 (µg/cm 2)
Control 3β-hydroxy-urs-12-en-28-oic acid (1)/ 3β-hydroxy-olean-12-en-28-oic acid (2) (2:1)
— 37.5 75 150
30 10 10 10
7.8 ± 0.3 6.8 ± 0.4* 5.1 ± 0.6* 2.2 ± 0.3*
— 13 35 72
— 95
2α,3β-dihydroxy-urs-12-en-28-oic acid (3)/ 2α,3β-dihydroxy-olean-12-en-28-oic acid (4) (2:1)
37.5 75 150
10 10 10
5.9 ± 0.4* 5.0 ± 0.3* 3.9 ± 0.4*
24 36 50
146
2α,3α-dihydroxy-urs-12-en-28-oic acid (5)/ 2α,3α-dihydroxy-olean-12-en-28-oic acid (6) (2:1)
37.5 75 150
10 10 10
6.1 ± 0.5* 5.5 ± 0.4* 2.7 ± 0.3*
22 29 65
107
2α,3α,23-trihydroxy-urs-12-en-28-oic acid (7)/ 2α,3α,23-trihydroxy-olean-12-en-28-oic acid (8) (2:1)
37.5 75 150
10 10 10
7.9 ± 0.4 6.5 ± 0.3* 3.6 ± 0.3*
–1 17 54
147
2α,3β-dihydroxy-urs-12-en-28-oic-acid (3)
25 50 100
10 10 11
8.1 ± 0.3 6.8 ± 0.5 6.2 ± 0.4*
–4 13 21
n.e.
3β-O-palmitoyl-lupeol (9)
50 100 200
11 10 10
6.9 ± 0.3* 5.3 ± 0.5* 4.0 ± 0.3*
12 32 49
204
3β-hydroxy-urs-12-en-28-oic acid (1) 3β-hydroxy-olean-12-en-28-oic acid (2) Indomethacin
100 100 90
10 10 10
1.9 ± 0.2* 4.3 ± 0.5* 4.1 ± 0.3*
76 45 47
Edema values are expressed as mean ± S.E.; *p<0.05, using Student’s t-test, as compared with the respective controls; n.e. = not evaluable.
Topical anti-inflammatory activity of Bauhinia tarapotensis leaves tions I (106.0 mg) and II (352.1 mg), representing 21 and 70% of the parent extract, respectively (Fig. 1). Comparison by TLC analysis confirmed that fraction II contained the same triterpenic acids identified in the chloroform extract, while fraction I contained pigments together with a small amount of ursolic acid (1). In particular, the following triterpenic acids of ursane and oleanane series were identified in fraction II: 3β-hydroxy-urs-12-en-28-oic acid (ursolic acid; 1), 3β-hydroxy-olean-12-en-28-oic acid (oleanolic acid; 2), 2α,3β-dihydroxy-urs-12-en-28-oic acid (2α-hydroxyursolic acid; 3), 2,3-dihydroxy-olean-12-en-28-oic acid (2α-hydroxyoleanolic or maslinic acid; 4), 2α,3α-dihydroxy-urs-12-en-28-oic acid (5), 2α,3α-dihydroxyolean-12-en-28-oic acid (6), 2α,3α,23-trihydroxy-urs12-en-28-oic acid (7), and 2α,3α,23-trihydroxy-olean12-en-28-oic acid (8) (Fig. 2). Separation of the chloroform extract by column chromatography over silica gel allowed to isolate four mixtures of these triterpenes, each of them constituted of two structural isomers 1–2, 3–4, 5–6, and 7–8, in the ratio ursane:oleanane derivatives 2:1. Only a small amount of 2α,3β-dihydroxyurs-12-en-28-oic acid (3) was separated as pure compound. Anti-inflammatory activity of the chloroform extract fractions
Fractions I and II were evaluated for their anti-inflammatory activity at the respective doses of 63 and 210 µg/cm 2 that, on the basis of their fractionation yield, correspond to 300 µg of the parent chloroform extract. While fraction I provoked only 25% edema reduction, fraction II showed the same activity of the chloroform extract (90 and 92% inhibition, respectively) (Table 3). Therefore, fraction II, rich in triterpenic acids, accounts for all the activity of the parent chloroform extract. Anti-inflammatory activity of the triterpenic acids
The four mixtures of triterpenic acids isomers and the compound 3 were submitted to the anti-inflammatory activity assay. In addition, also the activity of 3β-Opalmitoyl-lupeol (9) (Figure 2), isolated from the nhexane extract, was studied. The pure triterpenes ursolic acid (1) and oleanolic acid (2), as well as the NSAID indomethacin, were used as reference compounds. As shown in Table 4, all the couples of isomers induced a dose-dependent edema reduction and ursolic and oleanolic acid mixture (compounds 1 and 2) was the most active. The ID50 values (dose giving 50% edema inhibition) of the isomer couples ranged between 95 and 147 µg/cm 2, showing an activity higher
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than that of the pure compound 3, which induced only 21% edema reduction at the highest dose (100 µg/cm 2). As reference, 100 µg/cm 2 of ursolic acid (1) or oleanolic acid (2) induced 76 and 45% edema inhibition, respectively, while 90 µg/cm 2 of indomethacin, a dose level proximal to its ID50 value (ID50 = 93 µg/cm 2; Ismaili et al., 2001), reduced the edematous response by 47%. Also the pure compound 9 showed a dose-dependent edema inhibition, but lower than that of the reference compounds ursolic and oleanolic acids as well as of the triterpenic acids mixtures (ID50 = 204 µg/cm 2). Therefore, the mixtures of ursane and oleanane derivatives (2:1) showed the highest activity, comparable to that of the NSAID.
Discussion The present results demonstrate that the leaves of B. tarapotensis possess anti-inflammatory properties, supporting their popular use for the treatment of inflammatory-based disorders. In particular, five extracts of the leaves significantly inhibited the croton oil-induced ear edema in mice, among which the chloroform one was the most active. This extract justifies also the anti-inflammatory activity of the whole herbal drug of B. tarapotensis. While the antioxidant components of B. tarapotensis leaves were previously found in their methanol extract and identified as various compounds of different structures (Braca et al., 2001), the anti-inflammatory principles are concentrated in the chloroform extract. Therefore, the in vitro antioxidant effect of the methanol extract and of some of its constituents does not seem to significantly contribute to the anti-inflammatory activity of the plant leaves. In fact, the bioassay-guided fractionation led to identify some triterpenic acids as the anti-inflammatory principles of B. tarapotensis leaves. Four mixtures of two structural isomers of ursane and oleanane derivatives, in the ratio 2:1, were isolated for the anti-inflammatory activity assay. The components of these mixtures were identified as ursolic acid (1), oleanolic acid (2), 2α-hydroxyursolic acid (3), 2αhydroxyoleanolic or maslinic acid (4), 2α,3α-dihydroxy-urs-12-en-28-oic acid (5), 2α,3α-dihydroxyolean-12-en-28-oic acid (6), 2α,3α,23-trihydroxy-urs12-en-28-oic acid (7), and 2α,3α,23-trihydroxy-olean12-en-28-oic acid (8). The four mixtures of these compounds, as couples of isomers 1–2, 3–4, 5–6, and 7–8, exhibited an interesting anti-inflammatory effect (ID50 ranging from 95 to 147 µg/cm 2), comparable to that of the NSAID indomethacin (ID50 = 93 µg/cm 2). It was observed that ursolic acid (1) and oleanolic acid (2) mixture (2:1) exhibited the strongest antiphlogistic effect (ID50 = 95 µg/cm 2), showing a potency
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similar to that of indomethacin (ID50 = 93 µg/cm 2; Ismaili et al., 2001). On the contrary, their hydroxylation at 2α position (compounds 3 and 4) involves a loss of activity (ID50 = 146 µg/cm 2). Similarly, a loss of activity was observed also after the 2α-hydroxylation of the pure compound ursolic acid (1): while 100 µg/cm 2 of ursolic acid (1) induced 76% edema inhibition, its 2αhydroxylated derivative (3) reduced the edematous response only by 21%. On the contrary, when the hydroxylation is accompanied by a single conformational change of the hydroxy group from 3β to 3α position, like in the mixture of compounds 5 and 6, the activity was almost similar to that of ursolic and oleanolic acid mixture (ID50 = 107 and 95 µg/cm 2, respectively). However, further addition of a hydroxy group at 23 position (compounds 7 and 8) involves a decrease of the anti-inflammatory effect (ID50 = 147 µg/cm 2). The anti-inflammatory activity of triterpenic acids and, in particular, that of ursolic and oleanolic acid, was previously observed both after topical application (Huang et al., 1994; Recio et al., 1995a, b; Máñez et al., 1997; Ismaili et al., 2001) and after systemic administration (Kosuge et al., 1985; Singh et al., 1992; Kapil and Sharma, 1995; Recio et al., 1995a, b). Among the other triterpenic acids isolated from B. tarapotensis leaves, an in vivo anti-inflammatory activity was only reported for 2α-hydroxyoleanolic acid (Shimizu et al., 1986), while 2α-hydroxyursolic and 2α,3α-dihydroxyurs-12-en-28-oic acids were shown to inhibit in vitro mast cells degranulation and/or nitric oxide production, which could be related to an in vivo antiphlogistic effect (Ryu et al., 2000). Moreover, ursolic and oleanolic acids and their derivatives were identified as the main anti-inflammatory constituents of other plants belonging to different families and, particularly, to some species of the Lamiaceae family, such as Prunella vulgaris (Ryu et al., 2000), Salvia officinalis (Baricevic et al., 2001), and Thymus willdenowii (Ismaili et al., 2001). Their anti-inflammatory effects have been attributed to the inhibition of different events of an inflammatory reaction, such as histamine release, cyclooxygenase-2, and 5-lipooxygenase pathways of arachidonic acid metabolism, elastase activity, complement activity and nitric oxide production (Kapil and Sharma, 1995; Liu, 1995; Ringbom et al., 1998; Suh et al., 1998; Díaz et al., 2000; Ryu et al., 2000). Consequently, also the preparations of Bauhinia tarapotensis leaves, containing ursane and oleanane derivatives, could exert their antiphlogistic effect acting on multiple targets of the inflammatory reaction. In conclusion, the obtained results confirm the validity of B. tarapotensis leaves use in the traditional medicine for the treatment of inflammatory conditions, due to the presence of triterpenic acids belonging to the ursane and oleanane series.
Acknowledgements
This work was supported by a grant of the Italian Ministry for University and Scientific Research (Project “Piante medicinali ed alimentari di Paesi in via di sviluppo: studio chimico e biologico”).
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