Journal of Ethnopharmacology 97 (2005) 1–6
Flavonoids and antiulcerogenic activity from Byrsonima crassa leaves extracts M. Sannomiyaa , Vitor B. Fonsecab , M.A. da Silvaa , L.R.M. Rochab , L.C. dos Santosa , C.A. Hiruma-Limab , A.R.M. Souza Britoc , W. Vilegasa,∗ a
Departamento de Qu´ımica Orgˆanica, Instituto de Qu´ımica, Rua Francisco Degni s/n, Bairro Quitandinha, c.p. 355, CEP 14800-900, UNESP, Araraquara, SP, Brazil b Departamento de Fisiologia, Instituto de Biociˆ encias, c.p. 510, CEP 18618-000 UNESP, Botucatu, SP, Brazil c Departamento de Fisiologia e Biof´ısica, Instituto de Biologia, c.p. 6109, CEP 13083-970, UNICAMP, Campinas, SP, Brazil Received 23 October 2003; received in revised form 5 May 2004; accepted 11 September 2004 Available online 13 December 2004
Abstract Byrsonima crassa Niedenzu (IK) (Malpighiaceae) is used in Brazilian folk medicine for the treatment of diseases related mainly to gastric ulcers. In this study, we evaluated the potential antiulcerogenic effect of three different extracts obtained from the leaves of Byrsonima crassa namely hydromethanolic (80% MeOH), methanolic (MeOH) and chloroformic extracts (CHCl3 ). The oral administration (250, 500 and 1000 mg/kg) of all the extracts reduced the formation of lesions associated with HCl/ethanol administration in mice. The 80% MeOH extract significantly reduced the incidence of gastric lesions by 74, 78 and 92% at doses of 250, 500 and 1000 mg/kg, respectively (P < 0.01). The MeOH extract reduced the ulceration by 93 and 99% only at the doses of 500 and 1000 mg/kg (P < 0.01). The lower gastroprotective action (69%) was observed when animals were treated with CHCl3 extract at the dose of 1000 mg/kg (P < 0.01). Phytochemical investigation of Byrsonima crassa afforded five known substances: quercetin-3-O--d-galactopyranoside, quercetin-3-O-␣-l-arabinopyranoside, the biflavonoid amentoflavone, (+)-catechin and (−)-epicatechin. The presence of these phenolic compounds may probably explain the antiulcerogenic effect of the extracts of Byrsonima crassa leaves. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Byrsonima crassa; Antiulcer activity; Flavonoids; Malpighiaceae
1. Introduction Although there are many products used for the treatment of gastric ulcers, most of these drugs produce several adverse reactions (Ariyphisi et al., 1986). Plant extracts are among the most attractive sources for developing new drugs and have been shown to produce promising results in the treatment of gastric ulcers (Hiruma-Lima et al., 2000a, 2000b, 2001). In traditional medicine for example, several plants have been ∗
Corresponding author. Tel.: +55 16 2016668; fax: +55 16 2227932. E-mail address:
[email protected] (W. Vilegas).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.09.053
used to treat gastrointestinal disorders, including gastric ulcers (Toma et al., 2002). The Central Brazil is one of highest biogeographic regions of the world and also the most threatened (Klink, 1996). Recent lists of the Central Brazil flora register some 6253 native vascular plants species included in 150–160 families (Mendonc¸a et al., 1998). Many of these plants are used as medicine by people living in the Cerrado area (Almeida et al., 1998). An ethnopharmacological survey carried out in the Cerrado of Central Brazil showed a high number of plants used to treat gastric pain and gastritis (Silva et al., 2000).
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M. Sannomiya et al. / Journal of Ethnopharmacology 97 (2005) 1–6
In Brazil, members of the genus Byrsonima (Malpighiaceae) are employed not only in folk medicine but also as food to make juice, jellies and liquor in Northeast and Northeastern Brazil. Byrsonima crassa Niedenzu (IK) is a native species from the Cerrado formation. It is popularly known as murici-cascudo or murici-vermelho and its fruits are used as food (Silva et al., 2001). There are several properties attributed to the bark and leaves of Byrsonima crassa including antiemetic, diuretic, febrifuge, to treat ulcer, gastritis and diarrhea (Silva et al., 2001). The antimicrobial activity of the organic extracts from roots and stems of Byrsonima crassifolia has been described by Martinez-Vasquez et al. (1999) whereas Berger et al. (1998) reported the trypanocidal activity of this species. Heinrich (2003) also proved the antispasmodic activity from this plant in a fraction rich in flavonoids. Another species, Byrsonima verbascifolia was shown to possess antiviral activity (Lopez et al., 2001). Phytochemical investigations from Byrsonima crassifolia, Byrsonima microphylla and Byrsonima verbascifolia revealed the occurrence of sulphonoglycolipids, steroids, triterpenes, aromatic esters, amino acids and proanthocyanidins (Gottlieb et al., 1975; Amarquaye et al., 1994; Bejar et al., 1995; Geiss et al., 1995; Rastrelli et al., 1997; Mendes et al., 1999). We have previously reported the isolation of amentoflavone, quercetin-3-O--d-galactopyranoside and quercetin-3-O-␣-l-arabinopyranoside from the extracts of Byrsonima crassa leaves (Sannomiya et al., 2004). Despite the popular use of Byrsonima crassa as a medicinal plant, there are no data on the pharmacological effect of leaves extracts on the gastrointestinal system. Thus, interest in this plant is justifiable because of its potential medicinal value. Considering that aqueous infusions of Byrsonima crassa leaves are commonly used as phytomedicine, the present study has been conducted to assess the gastroprotective effect of the polar (80% MeOH and MeOH) and apolar (CHCl3 ) extracts by the HCl/ethanol-induced ulcer model in mice. Some of the plant constituents were isolated and identified.
by Eduardo Ribeiro dos Santos. A voucher specimen (no. 3377) is deposited at the Herbarium of the Tocantins University. 2.3. Extract preparation and isolation The air-dried and powdered leaves (2.0 kg) of Byrsonima crassa were extracted exhaustively with chloroform, methanol and 80% methanol, successively at room temperature (48 h for each solvent). Solvents were evaporated at 60 ◦ C under reduced pressure to afford the CHCl3 (53.8 g), MeOH (158.3 g) and 80% MeOH extract (95.7 g). The yields (w/w) for the CHCl3 , MeOH and 80% MeOH extracts from the dried powders of Byrsonima crassa leaves were 2.69, 7.91 and 4.79%, respectively. Some 4.0 g of the MeOH extract of Byrsonima crassa was permeated on a Sephadex LH-20 (Pharmacia) column (100 cm × 5 cm), eluted with MeOH. Fractions (8 ml) were collected and checked by TLC on Si gel eluted with CHCl3 /MeOH (80:20) and revealed either with NP/PEG (diphenylaminoborate/polyethyleneglycol) reagent or anisaldehyde/sulfuric acid solution (Wagner et al., 1984). Fractions 129–141 (95.0 mg) were purified by repeated CC on microcrystalline cellulose (Merck) eluted with CHCl3 /MeOH (80:20) yielding compound 5 (6.0 mg). Fractions 88–95 (69.0 mg) were further purified by HPLC using MeOH/H2 O (1:1) as eluent to afford 3 (15.0 mg). Fractions 82–87 (122.0 mg) were purified by silica CC using EtOAc/n-PrOH/H2 O 140:8:80 (upper phase) as eluent affording 4 (14.0 mg) and a mixture of compounds 1 + 2 (30.0 mg). The antioxidant activity of the extracts and pure compounds isolated from Byrsonima crassa were evaluated by TLC revealed with 1,1-diphenyl-2-picrylhydrazyl (DPPH) reagent (Fauconneau et al., 1997). The NMR spectra in DMSO-d6 were obtained using a Varian INOVA 500 spectrometer, operating at 500 MHz for 1 H and 125 MHz for 13 C and 2D NMR (1 H-1 H COSY, HMQC, TOCSY and HMBC). Chemical shifts are given as δ (ppm) using TMS as an internal standard. 2.4. Analyses of the compounds by TLC and HPLC
2. Materials and methods 2.1. Drugs and chemicals Ethanol, hydrochloric acid, lansoprazole (Medley, Brazil) were used in the present study. Lansoprazole, 80% MeOH and MeOH extracts were dissolved in 0.9% NaCl solution and CHCl3 extract was suspended in 8% Tween 80® just before use. 2.2. Plant material Leaves of Byrsonima crassa Niedenzu (IK) were collected at Porto Nacional, Tocantins State, Brazil and authenticated
An aliquot of the ethyl acetate fraction and the collected fractions were analyzed using silica gel TLC plates on glass (20 cm × 20 cm, Aldrich) developed with a solvent mixture composed of CHCl3 /MeOH (85:15, v/v). The spots on the TLC plates were observed under UV lamp (254 nm). Fractions of similar retention factors (RF ) were combined, weighed and further analysed using a Varian, ProStar HPLC system equipped with a RP-18 column (250 mm × 4.60 mm i.d., 5 m, Phenomenex Luna). The mobile phase was water (A) and acetonitrile (B) starting with linear gradient elution of 27–41% of B in 50 min and 41–100% in 70 min eluted at a flow-rate of 1.0 ml/min, and the effluent was monitored using a ProStar 330 photodiode-array ultraviolet detection (PDA) system at 254 nm.
M. Sannomiya et al. / Journal of Ethnopharmacology 97 (2005) 1–6
2.5. Animals Male Swiss albino mice (25–35 g) from the Central Animal House of the Universidade Estadual Julio de Mesquita Filho (UNESP/Botucatu) were used. The animals were fed with a certified Nuvilab CR-a® (Nuvital) diet and free access to tap water under standard conditions of 12 h dark–12 h light and temperature (21 ± 1%). Fasting (24 h) was used prior to all assays because standard drugs and crude extracts were always administered orally by gavage. Moreover, the animals were kept in cages with raised floors of wide wire mesh to prevent coprophagy. The protocols were approved by the UNESP Institutional Animal Care and Use Committee, following the recommendations of the Canadian Council on Animal Care (Olfert et al., 1993). 2.6. Antiulcerogenic activity 2.6.1. HCl/ethanol-induced ulcer The antiulcerogenic activity of MeOH, 80% MeOH and CHCl3 extracts from Byrsonima crassa leaves was studied in HCl/ethanol-induced gastric ulcer. The experiment was made as described by Mizui and Doteuchi (1983) with modification. Mice were allotted into groups of 5–8 animals, fasted for 24 h prior to receiving an oral dose of the vehicle, 8% Tween 80® or saline (10 ml/kg), lansoprazole (30 mg/kg) and MeOH, 80% MeOH and CHCl3 extracts (250, 500 and 1000 mg/kg). After 50 min all groups were orally treated with 0.2 ml of a 0.3 M HCl/60% ethanol solution (HCl/ethanol) for gastric-ulcer induction. Animals were killed 1 h after the administration of HCl/ethanol, and the stomachs excised and inflated by saline injection (2 ml).
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The extent of the lesions was measured, and the lesion index expressed as the sum of all lesions as described by Szelenyi and Thiemer (1978). The pH of gastric juice was recorded with a digital pH meter (PA 200, Marconi S.A, Brazil). 2.7. Statistical analysis Results were expressed as the mean ± S.E.M. Statistical significance was determined by one-way analysis of variance followed by Dunnett’s test, with the level of significance set at P < 0.05.
3. Results and discussion The effect of the three preparations from Byrsonima crassa (MeOH, 80% MeOH and CHCl3 extracts) on gastric ulcers induced by a damaging agent (HCl/ethanol solution) were first investigated in mice and the results are shown in Table 1. Oral administration of HCl/ethanol solution to the control group clearly produced the expected characteristic zonal necrotizing mucosal lesions. HCl/ethanol solution induced both long ulcers and petechial lesions within a relatively short time. Pretreatment with CHCl3 extract, given orally at doses of 250, 500 and 1000 mg/kg and lansoprazole (positive control) administered at a dose of 30 mg/kg, induced a significant protective effect in variable degrees. The lesion index for the control group (vehicle) of the HCl/ethanolinduced gastric ulcers was 58.9 ± 2.73 mm. The HCl/ethanol induced acute ulcer model evaluates the capacity of the drug to protect the gastric mucosa. Ethanol treatment induces solubilization of mucus constituents in the stomach with a con-
Table 1 Effects of lansoprazole and different doses of MeOH, 80% MeOH and CHCl3 extracts of Byrsonima crassa leaves in gastric lesions induced by HCl/ethanol solution in mice Treatments (p.o.) Control Lansoprazole MeOH
Control Lansoprazole 80% MeOH
Control Lansoprazole CHCl3
Dose (mg/kg)
N
pH (unit)
Ulcer index (mm)
Inhibition (%)
– 30
8 8
4.62 ± 0.37 6.62 ± 0.18**
43.2 ± 5.11 12.50 ± 3.56**
– 71
250 500 1000
8 8 7
3.25 ± 0.25** 2.28 ± 0.18** 2.00 ± 0.26**
34.10 ± 6.86 3.00 ± 1.05** 0.50 ± 0.34**
21 93 99
– 30
13 13
2.85 ± 0.29 6.61 ± 0.29**
54.00 ± 3.08 20.60 ± 3.16**
– 62
250 500 1000
11 11 11
2.36 ± 0.15 2.27 ± 0.19 2.36 ± 0.20
13.90 ± 3.04** 12.10 ± 1.20** 4.54 ± 1.03**
74 78 92
– 30
13 13
3.15 ± 0.19 5.61 ± 0.14**
58.90 ± 2.73 19.60 ± 2.22**
– 67
250 500 1000
10 10 10
3.20 ± 0.29 3.20 ± 0.25 3.40 ± 0.22
24.10 ± 2.69** 25.40 ± 3.59** 18.50 ± 1.98**
59 57 69
Results are presented as the mean ± S.E.M. MeOH extract: ANOVA F(4,32) = 51.3 (P < 0.05) by pH and 17.6 (P < 0.05) by ulcerative index. Dunnett’s test: ** P < 0.01. 80% MeOH extract: ANOVA F ** P < 0.01.CHCl extract: (4,54) = 62.4 (P < 0.01) by pH and 57.6 (P < 0.01) by ulcerative index. Dunnett’s test: 3 ANOVA F(4,51) = 27.6 (P < 0.01) by pH and 40.77 (P < 0.01) by ulcerative index. Dunnett’s test: ** P < 0.01.
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Fig. 1. HPLC profile of the 80% MeOH and MeOH extracts of Byrsonima crassa: 1: (+)-catechin; 2: (−)-epicatechin; 3: quercetin-3-O--d-galactoside; 4: quercetin-3-O-␣-l-arabinoside; 5: amentoflavone. Chromatographic conditions: see text.
comitant fall in the transmucosa potential difference, and increase Na+ and K+ flow into the lumen, pepsin secretion, the loss of H+ ions and the histamine content in the lumen. The ethanol also depresses tissue levels of DNA, RNA and proteins, leading to flow stasis and injured areas (Szabo, 1987). The presence of HCl solution in this lesion only accelerates the process of gastric ulcerogenesis (Sun et al., 1991). The antiulcer drug lansoprazole (30 mg/kg) and the CHCl3 extract (250, 500 and 1000 mg/kg) significantly inhibited (P < 0.01) ulcer formation by 59, 57 and 69%, respectively. The MeOH extract (500 and 1000 mg/kg) significantly inhibited ulcer formation (P < 0.01) by 93 and 99%, respectively, whereas treatment with the 80% MeOH extract (250, 500 and 1000 mg/kg body weight) significantly inhibited ulcer formation by 74, 78 and 92%, respectively, and significantly decreased (P < 0.01) the hemorrhagic lesions and provided dose-response characterization. Therefore, our results showed that polar extracts provided better antiulcer activity than the apolar extract. Several mechanisms have been suggested for the effect of antiulcerogenic compounds, including increasing the gastric hexosamine level and enhancing the strength of the gastric barrier either physically or by blocking the H+ , K+ –ATPase pump (Repetto and Llesuy, 2002). Probably, the compounds present in the tested plant might exert their activities by one or more of these proposed mechanisms. Recently, studies have implicated the generation of oxygen derived free radicals and lipid peroxidation as one of the most important mechanisms involved in the pathogenesis of gastric ulcer. Antioxidants are known to inhibit lipid peroxidation and scavenge free radicals (Di Carlo et al., 1999). Chromatographic evaluation of the CHCl3 , MeOH and 80% MeOH extracts by TLC revealed with DPPH solution showed the presence of yellowish spots, indicating the antioxidant activity of the substances present only in the MeOH and 80% MeOH extracts. The HPLC analyses showed the similarity between the chemical profiles of these polar extracts (Fig. 1). Plates revealed with NP/PEG reagent produced intense orange and yellow spots characteristic of flavonoids and plates revealed with anisaldehyde/sulfuric acid solution also
produced reddish spots, suggesting the presence of catechin derivatives. Since the MeOH extract presented the best antiulcer response, we performed its phytochemical investigation. Fractionation of an aliquot of the crude extract by GPC followed by purification by several chromatographic procedures led to the isolation of the biflavonoid amentoflavone 5, quercetin-3-O-␣-d-arabinopyranoside 4, quercetin-3-O-d-galactopyranoside 3, (−)-epicatechin 2 and (+)-catechin 1, identified by comparison of their spectroscopic data with those reported in literature (Agrawal, 1989; Harborne, 1996). TLC evaluation of the pure substances isolated from the MeOH extract of Byrsonima crassa with the DPPH reagent produced the typical yellowish spots for all tested compounds, thus indicating their free radical scavenging effect. Flavonoids and catechins are secondary metabolites present in plants and have attracted the attention of many researchers because the wide range of their biological activities (Harborne, 1996). There are many studies related to the antiulcerogenic properties of flavonoids (Gracioso et al., 2002; Gonzalez and Di Stasi, 2002). Especially interesting are those compounds that have a catechol nucleus that has been related with the antioxidant activity (Azuma et al., 2000). Galati et al. (2003) also describe that there is a correlation between the antioxidant and the antiulcer activity of the flavonoids. The presence of (−)-epicatechin 2 and (+)-catechin 1 in the MeOH and 80% MeOH extracts may also contribute for the gastroprotective activity observed in Byrsonima crassa since these compounds were reported to inhibit lipid peroxidation as well as to possess a very potent antioxidant activity (Iwai et al., 2001). Moreover, quercetin derivatives also possess a catechol nucleus at the B-ring and well-known antioxidant activity (Makris and Rossiter, 2001). Literature also reports that amentoflavone 5 displays both antioxidant and antiulcer activities (Goel et al., 1988; Cholbi et al., 1991). Therefore, our results obtained by TLC analysis with the DPPH reagent and recently with high-speed countercurrent chromatography (Sannomiya et al., 2004) confirm the presence and identity of at least some antioxidant compounds in the polar extracts.
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4. Conclusions This study showed that the polar extracts of Byrsonima crassa exhibited significant antiulcerogenic effect. Phytochemical evaluation carried out on the MeOH extract afforded amentoflavone, quercetin derivatives and catechins. Since catechins and flavonoids were the secondary metabolites found in the active extracts of Byrsonima crassa, we pose the question if these substances are related to the observed protection against gastric ulcers. They also may be involved in the scavenging of the reactive oxygen species on the surface of gastric mucosa, thus protecting cells from gastric injury. Additional experiments are in progress to further evaluate the isolated compounds in order to investigate the mechanisms of action and will be published in the future.
Acknowledgements We thank the Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP) for a grant to M.S and for fundings from Biota-Fapesp Program and to Conselho Nacional de Desenvolvimento Cient´ıfico Tecnol´ogico (CNPq) for a grant to W.V. and to A.R.M.S.B. We also thank CAPES for a fellowship to M.A.S.
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