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Bauhinia Extract Clinical Studies
 
The leaves of green plants as well as a cyanobacterium, a red alga, and fungi contain insulin-like antigens.
Silva LB, Santos SS, Azevedo CR, Cruz MA, Venâncio TM, Cavalcante CP, Uchôa AF, Astolfi Filho S, Oliveira AE, Fernandes KV, Xavier-Filho J.
 
Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brasil.
 
We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution.

PMID: 11887207 [PubMed - indexed for MEDLINE]


Bauhinia candicans stimulation of glucose uptake in isolated gastric glands of normal and diabetic rabbits.
Fuentes O, Alarcón J.
 
Laboratorio de Fisiología, Facultad de Ciencias, Universidad del Bío-Bío, Campus Chillán, Avenida Andrés Bello s/n, P.O. Box 447, Chillán, Chile. ofuentes@ubiobio.cl
 
A butanol extract of dried leaves of Bahuinia candicans showed, in vitro, a stimulatory effect of glucose uptake in isolated gastric glands of normal and alloxan-diabetic rabbits. The extract (0.001-0.07 mg/mg protein) produced a dose-dependent effect and was similar to the effect of insulin (1-100 nM). These findings suggest that the extract contains compound(s) that efficiently increase glucose transport at the basolateral side of isolated gastric glands.

PMID: 16690222 [PubMed - indexed for MEDLINE]

Acute effect of Bauhinia forficata on serum glucose levels in normal and alloxan-induced diabetic rats.
Silva FR, Szpoganicz B, Pizzolatti MG, Willrich MA, de Sousa E.

UFSC Departamento de Bioquímica, Centro de Ciências Biológicas, Campus Universitário, Bairro Trindade, Cx Postal 5069, CEP-88040-970, SC, Florianópolis, Brazil. mena@mboxl.ufsc.br
 
Experimental diabetes was used to study the acute effect of the n-butanol fraction of Bauhinia forficata Link (Leguminosae) (BF) leaves on the serum glucose levels of rats. Body weight was measured on the day of diabetes induction and on the day of the experiment. Levels of glucose were determined at different doses and times following treatment with BF or with vehicle in normal, diabetic and hyperglycemic normal rats. Oral administration of n-BuOH fraction led to a significant blood glucose-lowering effect in normal and diabetic rats. However, in glucose-fed hyperglycemic normal rats, the maximum dose of this fraction failed to decrease blood glucose levels. The hypoglycemic effect was observed at doses of 500 and 600 mg/kg after 1 and 2 h treatment respectively, in normal rats. The maximum effect of BF was detected at 1 h with 800 mg/kg in diabetic animals and this profile was maintained for the next 3 h. Treatment of normal and alloxan-induced diabetic rats with BF decreased glucose levels, while this fraction was devoid of hypoglycemic effect in glucose-fed hyperglycemic normal rats.
PMID: 12413705 [PubMed - indexed for MEDLINE]

Hypoglycemic effect and antioxidant potential of kaempferol-3,7-O-(alpha)-dirhamnoside from Bauhinia forficata leaves.
de Sousa E, Zanatta L, Seifriz I, Creczynski-Pasa TB, Pizzolatti MG, Szpoganicz B, Silva FR.

Departamento de Química, Centro de Ciências Físicas e Matemáticas, Campus Universitário, Bairro Trindade. Cx. Postal 5069, CEP 88040-970, Florianópolis, SC, Brazil.
 
In vivo and in vitro treatments were carried out to investigate the effects of kaempferol-3,7-O-(alpha)-dirhamnoside (kaempferitrin), a major flavonoid compound of the n-butanol fraction from Bauhiniaforficata leaves, on serum glucose levels, as well as its antioxidant potential. Oral administration of kaempferitrin led to a significant hypoglycemic effect in normal and in alloxan-induced diabetic rats. In normal rats, blood glucose lowering was observed only with the higher dose of kaempferitrin (200 mg/kg) at 1 h after treatment. However, the hypoglycemic effect of kaempferitrin in diabetic rats was evident at all doses tested (50, 100, and 200 mg/kg), and this profile was maintained throughout the period studied for both higher doses. Additionally, in glucose-fed hyperglycemic normal rats, the kaempferitrin failed to decrease blood glucose levels. In vitro antioxidant properties or action against reactive oxygen species of this compound was also evaluated. The compound showed high reactivity with 1,1-diphenyl-2-picryl hydrazyl (DPPH), IC(50) of 84.0 +/- 7.8 microM, inhibited myeloperoxidase activity with K(0.5) = 86 +/- 9.9 microM, and decreased lipid peroxidation, induced by ascorbyl radical either in microsomes or in asolectin and phosphatidylcholine liposomes, with IC(50)'s of 320 +/- 14.1, 223 +/- 8.3, and 112 +/- 8.8 microM, respectively.

PMID: 15165145 [PubMed - indexed for MEDLINE]

 
Advances in research of chemical constituents and pharmacological activites of Bauhinia]
Shang XY, Liu W, Zhao CW.
Beijing Union University, Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing 100083, China. shangxiaoya@ygi.edu.cn
The research advances based on the related references were summarized in the last thirty years. Bauhinia contained many kinds of chemical constituents, primarily including flavanoids, steroids, terpenoid and so on, some of them were firstly obtained from the nature. Many plants of the Bauhinia are used in traditional medicine for their interesting biological activities such as antidiabetic, antiinflammatory, antimicrobial, analgesic, astringent and diuretic effects. This paper gives an overview of phytochemical and pharmacological research in Bauhinia, and it has been classified accordding to the chemical structure characteristics. To provide more material to draw on for further development and utilization resources of Bauhinia.

PMID: 18590204 [PubMed - in process]

 
Anti-diabetic activity of Bauhinia forficata decoction in streptozotocin-diabetic rats.
Pepato MT, Keller EH, Baviera AM, Kettelhut IC, Vendramini RC, Brunetti IL.

Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, UNESP, R Expedicionários do Brasil 1621, SP, Araraquara, Brazil. pepatomt@fcfar.unesp.br
 
The effects of using Bauhinia forficata leaf decoction (150 g leaf/l water; 35.2+/-7.8 ml/100 g body weight mean daily dose) as a drinking-water substitute for about 1 month on streptozotocin-diabetes (STZ-diabetes) in male Wistar rats were investigated. The physico-metabolic parameters measured were: body weight, food and liquid intake, urinary volume, hepatic glycogen, serum triglycerides and cholesterol, plasma glucose, urinary glucose and urea, and the weight of epididymal and retroperitoneal adipose tissue and soleus and extensor digitorum longus muscles. The STZ-diabetic rats treated with decoction showed a significant reduction in serum and urinary glucose and urinary urea as compared to the STZ-diabetic control, no difference being seen between decoction-treated and -untreated non-diabetic rats. The other physico-metabolic factors showed no changes in treated STZ-diabetic rats. The improvement in carbohydrate metabolism seen in the rats treated with Bauhinia forficata decoction does not appear to be linked to the inhibition of glycogenolysis or the stimulation of glycogenesis nor does it appear to act in a way similar to insulin or the sulfonylureas, although it may act by the inhibition of neoglycogenesis in a manner similar to that of the biguanides.
PMID: 12065150 [PubMed - indexed for MEDLINE]

 
Clinical trial of Myrcia uniflora and Bauhinia forficata leaf extracts in normal and diabetic patients.
Russo EM, Reichelt AA, De-Sá JR, Furlanetto RP, Moisés RC, Kasamatsu TS, Chacra AR.
 
Disciplina de Endocrinologia, Escola Paulista de Medicina, São Paulo, Brasil.
 
1. Myrcia uniflora and Bauhinia forficata were compared with placebo for their hypoglycemic effect in randomized cross-over double-blind studies in 2 groups of normal subjects (10 subjects each) and 2 groups of Type II diabetic patients (18 in the M. uniflora group and 16 in the B. forficata group). The protocol with each plant lasted 56 days. 2. After the ingestion of infusions of 3 g leaves/day of M. uniflora and B. forficata leaves, no acute or chronic effects on plasma glucose levels or glycated hemoglobin were found in either group. However, plasma insulin levels in the diabetic group were lower after M. uniflora than after placebo. 3. Among other clinical parameters tested, a statistically significant difference was found only in the alkaline phosphatase level after placebo compared with that after M. uniflora in the normal group. 4. There were no differences in any clinical parameters after the use of placebo or of B. forficata. 5. We conclude that infusions prepared from the leaves of M. uniflora or B. forficata have no hypoglycemic effect on normal subjects or Type II diabetic patients.

PMID: 2201413 [PubMed - indexed for MEDLINE]

Effects of Bauhinia megalandra aqueous leaf extract on intestinal glucose absorption and uptake by enterocyte brush border membrane vesicles.
Gonzalez-Mujica F, Motta N, Márquez AH, Capote-Zulueta J.
 
Sección de Bioquímica Médica, Instituto de Medicina Experimental, Facultad de Medicina, Universidad Central de Venezuela, Apartado Postal 50587, Sabana Grande, Caracas, Venezuela. gonzalef@latinmail.com
 
Aqueous extract of Bauhinia megalandra leaves was able to inhibit the intestinal glucose absorption in a concentration-dependent way and additive to phlorizine. Moreover, B. megalandra leaf extract drastically reduced the 14C-glucose uptake by enterocyte brush border membrane vesicles. The B. megalandra leaf extract administrated orally, simultaneously with glucose improved the glucose tolerance with a significant reduction of the 30-min peak. The extract did not have an effect on the glucose tolerance when glucose was administrated subcutaneously.

PMID: 12628399 [PubMed - indexed for MEDLINE

Effect of Bauhinia forficata aqueous extract on the maternal-fetal outcome and oxidative stress biomarkers of streptozotocin-induced diabetic rats.

Volpato GT, Damasceno DC, Rudge MV, Padovani CR, Calderon IM.

Laboratório de Pesquisa Experimental de Ginecologia e Obstetrícia, Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina de Botucatu, UNESP, Distrito de Rubião Jr s/n, 18610-000 Botucatu, SP, Brazil. gtvolpato@yahoo.com
 
ETHNOPHARMACOLOGICAL RELEVANCE: Bauhinia forficata Link, commonly known as "paw-of-cow", is widely used in Brazilian folk medicine for the treatment of diabetes. AIM OF THIS STUDY: To evaluate the effect of Bauhinia forficata treatment on maternal-fetal outcome and antioxidant systems of streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Virgin female Wistar rats were injected with 40 mg/kg streptozotocin before mating. Oral administration of an aqueous extract of Bauhinia forficata leaves was given to non-diabetic and diabetic pregnant rats at increasing doses: 500 mg/kg from 0 to 4th day of pregnancy, 600 mg/kg from 5th to 14th day and 1000 mg/kg from 15th to 20th day. At day 21 of pregnancy the rats were anaesthetized with ether and a maternal blood sample was collected for the determination superoxide dismutase (SOD) and reduced glutathione (GSH). The gravid uterus was weighed with its contents and fetuses were analyzed. RESULTS AND CONCLUSION: The data showed that the diabetic dams presented an increased glycemic level, resorption, placental weight, placental index, and fetal anomalies, and reduced GSH and SOD determinations, live fetuses, maternal weight gain, gravid uterine weight, and fetal weight. It was also verified that Bauhinia forficata treatment had no hypoglycemic effect, did not improve maternal outcomes in diabetic rats, but it contributed to maintain GSH concentration similarly to non-diabetic groups, suggesting relation with the decreased incidence of visceral anomalies.

PMID: 18155864 [PubMed - indexed for MEDLINE]

 
Antioxidant activity of leaf extracts from Bauhinia monandra.
Argolo AC, Sant'Ana AE, Pletsch M, Coelho LC.

Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, S/N Cidade Universitária, Pernambuco, Brazil.
 
Bauhinia monandra Kurz. is used in Brazil for the treatment of diabetes. Since this activity may be correlated with the presence of antioxidant compounds, leaf extracts of B. monandra were evaluated for their radical scavenging capacity (RSC). An ethanolic extract was taken up in aqueous methanol and partitioned with hexane, chloroform, ethyl acetate to yield three organic extracts together with remaining aqueous extract. The RSC was determined spectrophotometrically using 1,1-diphenylpicrylhydrazyl free radical (DPPH). The chloroform and ethyl acetate extracts were the most appropriate as sources of antioxidant compounds as shown by their inhibition concentration (IC50) and inhibition percentage (IP) values. The antioxidant activity of such extracts was attributed to the presence of three compounds of different polarities (flavonoids and steroids). The chloroform and ethyl acetate extracts exhibited an IC50 of approximately 2 mg/g DPPH and IP values in the range of 60-65%. The results indicate that the extracts of B. monandra have a very potent antioxidant activity, compared with the pure catechins used as positive controls and with other plant extracts.

PMID: 15246449 [PubMed - indexed for MEDLINE]

Anti-inflammatory activity of a novel flavonol glycoside from the Bauhinia variegata Linn.
Yadava RN, Reddy VM.

Natural Products Laboratory, Department of Chemistry, Dr. H.S. Gour University, Sagar (M.P.) 470003, India. rnyadava@rediffmail.com

Bauhinia variegata Linn. (Leguminosae) is commonly known as 'Kachnar' in Hindi. It is distributed almost throught India. Its powdered bark is traditionally used for tonic, astrain, ulcers. It is also useful in skin diseases. The roots are used as antidote to snake poison. The present article deals with the isolation and structural elucidation of a novel flavonol glycoside 5,7,3',4'-tetrahydroxy-3-methoxy-7-O-alpha-L-rhamnopyranosyl(1-->3)-O-beta-galactopyranoside (1) from the roots of Bauhinia Variegata and its structure was identified by spectral analysis and chemical degradations. The novel compound (1) showed anti-inflammatory activity.

PMID: 12737399 [PubMed - indexed for MEDLINE]

Antinociceptive, anti-inflammatory and antipyretic properties of the aqueous extract of Bauhinia purpurea leaves in experimental animals.
Zakaria ZA, Wen LY, Abdul Rahman NI, Abdul Ayub AH, Sulaiman MR, Gopalan HK.

Faculty of Pharmacy, Universiti Teknologi Mara, Shah Alam, Selangor, Malaysia. shaza8174@yahoo.com
OBJECTIVE: The present study was carried out to determine the antinociceptive, anti-inflammatory and antipyretic activities of the aqueous extract of Bauhinia purpurea leaves using animal models. MATERIALS AND METHODS: The air-dried, powdered leaves (approximately 20 g) were soaked in distilled water (1:20; w/v) at room temperature for 72 h and the supernatant obtained was freeze-dried. The crude dried extract (approximately 2.4 g) was prepared in doses of 6.0, 30.0 and 60.0 mg/kg, and subjected to the respective antinociceptive (abdominal constriction, hot plate and formalin tests), anti-inflammatory (carrageenan-induced paw edema test) and antipyretic (brewer's yeast-induced pyrexia test) assays. RESULTS:The results obtained indicate that the extract possessed significant (p < 0.05) antinociceptive, anti-inflammatory and antipyretic activities, which were not dependent on the doses of extract used. The highest concentration of extract was less effective as an anti-inflammatory and an antipyretic agent. CONCLUSION: This study showed that the aqueous extract of B. purpurea leaves possesses potential pharmacological activities that require further investigation and, thus, confirms the folklore use of the plant in the treatment of ailments associated with pain and inflammation. Copyright 2007 S. Karger AG, Basel.

PMID: 17917444 [PubMed - indexed for MEDLINE]


Antiinflammatory activities of flavonoids and a triterpene caffeate isolated from Bauhinia variegata.
Rao YK, Fang SH, Tzeng YM.

Institute of Biotechnology, Chaoyang University of Technology, Wufeng, Taiwan, ROC.
In the continuing search for novel antiinflammatory agents, six flavonoids, namely kaempferol (1), ombuin (2), kaempferol 7,4'-dimethyl ether 3-O-beta-D-glucopyranoside (3), kaempferol 3-O-beta-D-glucopyranoside (4), isorhamnetin 3-O-beta-D-glucopyranoside (5) and hesperidin (6), together with one triterpene caffeate, 3beta-trans-(3,4-dihydroxycinnamoyloxy)olean-12-en-28-oic acid (7) were isolated from the non-woody aerial parts of Bauhinia variegata. Compounds 1-7 were evaluated as inhibitors of some macrophage functions involved in the inflammatory process. These seven compounds significantly and dose dependently inhibited lipopolysaccharide (LPS) and interferon (IFN)-gamma induced nitric oxide (NO), and cytokines [tumor necrosis factor (TNF)-alpha and interleukin (IL)-12]. The concentration causing a 50% inhibition (IC50) of NO, TNF-alpha and IL-12 production by compounds 1, 2 and 7 was approximately 30, 50 and 10 microM, respectively, while at 50, 200 and 40 microM compounds 3, 4, and 5, 6 showed 15-30% inhibition, respectively. On the other hand, compounds 3 and 7 showed no inhibitory effect, while compounds 1, 4-6 reduced by around 10-30% the synthesis of NO by macrophages, when inducible NO synthase was already expressed with LPS/IFN-gamma for 24 h. These experimental findings lend pharmacological support to the suggested folkloric uses of the plant B. variegata in the management of inflammatory conditions.

PMID: 18384188 [PubMed - indexed for MEDLINE]

 
Hypoglycemic activity of two Brazilian Bauhinia species: Bauhinia forficata L. and Bauhinia monandra Kurz.
Atividade hipoglicemiante de duas espécies de Bauhinia brasileira: Bauhinia forficata L. and Bauhinia monandra Kurz.
Fábio de Sousa MenezesI, II, *; Andréa Barreto Mattos MintoI; Halliny Siqueira RuelaIII; Ricardo Machado KusterIII; Helen SheridanII; Neil FrankishII
IDepartamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Centro de Ciências da Saúde, Cidade Universitária, 21941-590, Rio de Janeiro, RJ, Brazil
IISchool of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Universtity of Dublin, 23 Westland Row, Dublin 2, Ireland
IIINúcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Bloco H, Cidade Universitária, 21941-590, Rio de Janeiro, RJ, Brazil
ABSTRACT
The hypoglycemic activity of aqueous extracts from Bauhinia forficata L. and Bauhinia monandra Kurz leaves (10% w/v) was evaluated in normoglycemic mice. Both extracts have shown hypoglycemic activity using this methodology. It was also possible to isolate two flavonoids from B. forficata L., 3,7-di-O-a-rhamnopyranosylquercetin and 3,7-di-O-a-rhamnopyranosylkaempferol (kaempferitrin), whose structures were elucidated by usual NMR techniques. Only the quercetin derivative was identified in B. monandra aqueous extract by HPLC.

Keywords: Bauhinia forficata, Bauhinia monandra, Leguminosae, hypoglycemic activity, flavonoids, HPLC.
RESUMO
Extratos aquosos das folhas de Bauhinia forficata L. e Bauhinia monandra Kurz (10% p/v) foram testados em camundongos normoglicêmicos, objetivando averiguar a sua atividade hipoglicemiante. Ambos os extratos mostraram atividade hipoglicemiante na metodologia empregada. Ainda, foi possível isolar de B. forficata L. dois flavonóides, quercetina-3,7-O-dirhamnosido e kaempferol-3,7-O-dirhamnosido, sendo as estruturas estabelecidas por técnicas clássicas de RMN. Apenas o derivado da quercetina foi identificado no extrato aquoso de Bauhinia monandra por CLAE.
 
Unitermos: Bauhinia forficata, Bauhinia monandra, Leguminosae, atividade hipoglicemiante, flavonoides, CLAE.
INTRODUCTION
Plants belonging to the genus Bauhinia are known in Brazil as "Pata-de-vaca" or "Unha-de-boi". Their species belong to the Leguminosae family and encompass circa 300 plants cultivated in tropical areas worldwide (Miyake et al., 1986). Aqueous extracts from Bauhinia species leaves, stems and roots, mainly B. cheilantha, B. forficata, B. glabra, B. rufescens, B. splendens and B. ungulata, are widely used in Brazil and in other countries in the treatment of several diseases, specially infections, pain processes and diabetes (Achenbach et al., 1988; Ritter et al., 2002; Pereira et al., 2004; Macedo; Ferreira, 2004; Morais et al., 2005; Silva et al., 2006). Several Bauhinia species are utilized as folk medicines worldwide, including Africa, Asia, South America and Central America (Ayensu, 1978).

Phytochemical and pharmacological studies with species of this genus are ongoing and, consequently a wide range of compounds have been isolated and identified, including lactones, flavonoids, terpenoids, glicolipids, glucosyl steroids, steroids, tannins and quinines (Cechinel-Filho et al., 1996, Silva et al., 2000; Mendes et al., 2006). One early study, by Bhartiya and Gupta (1981), identified 3,4-dihydroxychalcone-4-O-b-L-arabinopyranosyl-O-b-D-galactopyranoside from B. purpurea seeds. Roots and stems of this plant are popularly used in the treatment of diarrhea in India. During the same period studies with the methanolic extract (2 g/kg) of B. racemosa flowers in gastric ulcer model induced by acetylsalicylic acid in rats have shown that this extract significantly reduced the ulcer index (Akhtar et al., 1981). A glucosyl steroid has been isolated from B. candicans aerial parts and identified as sitosterol-3-O-a-D-riburonofuranoside (Irribarren; Pomilio, 1985). Achenbach et al. (1988), in a phytochemical study of B. manca stems have isolated 3-O-galloylepicatequine as its main constituent. They also identified gallic acid, cinnamic acid, b-sitosterol and b-D-glucosyl-sitosterol in significant concentrations. Alkaloids were not detected.
All the extracts from B. splendens leaves, stems, barks and roots, except the hydroalcoholic extract from its roots, have shown significant analgesic effect (10 mg/kg) in a pain model induced by acetic acid 0.6% in mice, being more efficient than salicylic acid and paracetamol. In the same study the chemical constituents from leaves, stems, barks and roots extracts were evaluated by thin layer and column chromatography. Identified compounds include ethyl gallate, rutin and quercetin (Cechinel-Filho et al., 1996).

In a more recent study, Taylor et al. (1996), on Bauhinia species used in folk medicine in Nepal, the methanolic extract from B. vahlii roots was found to be one of the most active in the treatment of virus-induced diseases with specific activity toward Herpes simplex. A later chemical study of the methanolic extract obtained from B. purpurea wood led to the isolation and identification of 6-butyl-3-hydroxyflavanone (Kuo et al., 1998). More recently the ethanolic extract from B. guianensis stem barks has shown a very good activity against Plasmodium vinckei (50 mg/kg) (Muñoz et al., 2000) in an evaluation of one hundred and fifty six different species belonging to different families indicated by "Chacabos" (native community of Amazon Region) for the treatment of several diseases such as digestive complaints, ulcer, diarrhea, malaria, etc. Yadava and Tripathi (2000) have isolated 5,6-dihydroxy-7-methoxy-flavone-6-O-b-D-xilopyranoside from the chloroform fraction of the total ethanolic extract from B. purpurea barks. Extracts from the bark of B. guianensis stems have been shown to possess antinociceptive effects in the acetic acid writhing model in mice, as well as anti-inflammatory activity in the carrageenan, dextran and histamine paw edema model in rats (Silva; Cechinel-Filho, 2002; Falcão et al., 2005).
 
Based on the study of Cechinel-Filho et al. (1996) with B. splendens, Silva and Cechinel-Filho (2000) have studied B. forficata, used by the Brazilian Pharmacopoeia for the physical-chemical and botany parameters for the quality control of this species. The phytochemical study of this species showed the presence of a kaempferol-3,7-dirhamnoside (kaempferitrine, lespedin) in the methanolic extracts of leaves and b-sitosterol from the hexane fraction. A comparative study carried out with all the vegetative organs of this species (leaves, roots and barks) has proved that steroids and terpenes are present in all of them, being more concentrated in the leaves. Lespedin was detected only in B. forficata leaves and because of that, this compound can be considered the chemical marker of this species for quality control purposes, present in leaves and absent in other parts.
The documented use and potential of Bauhinia species to act as hypoglycemic agents have begun to receive much attention. Bauhinia divaricata, known in Mexico as "pezunã de vaca", has been studied regarding its hypoglycemic activity and its aqueous extract (obtained from leaves) showed a relatively significant hypoglycemic effect in normal mice (Gupta et al., 1980). It has been established that the aqueous extract from B. megalandra inhibits the glucose intestinal absorption (Gonzales-Mujica et al., 2003) in rats. However, the hypoglycemic activity of Bauhinia species has been most extensively researched in B. forficata, which is considered by the rural population in Brazil as the "true pata-de-vaca". B. forticata Link. had its hypoglycemic activity confirmed by Juliane (1929, 1931). This species leaves are currently used in Brazil, in the form of aqueous extract (Modesto-Filho, 1988; Barbosa-Filho et al., 2005). However, a later study by Coimbra-Teixeira et al. (1992) has shown that the alcoholic leaves extract do not make the glucose blood levels lower in diabetic rats induced by streptozotocin.
 
Due to conflicting scientific results and the fact that Bauhinia species are widely used in Brazil as hypoglycemic plants, without any control, using popular knowledge that correlates the color of the flowers with the suggested activity power, we have decided to investigate the hypoglycemic activity of the aqueous extract of two Bauhinia species used in the State of Rio de Janeiro, Brazil. The selected species are Bauhinia forficata L. and Bauhinia monandra Kurz. A second objective is to try and isolate one or more markers for these species that could be responsible, at least in part, for their activity in normoglycemic mice, and could find potential use for quality control purposes.
MATERIAL AND METHODS

Plant material

Bauhinia forficata L. and B. monandra Kurz were collected in different parts of Rio de Janeiro State and prepared for the study. These species were identified by Professor Ângela Studart da Fonseca Vaz from the Botanical Garden of Rio de Janeiro, specialist in Leguminosae family. Two voucher samples are deposited in the Botanical Garden of Rio de Janeiro Herbarium under the numbers 3456 and 3765 respectively.
 
Extracts preparation and animals

The aqueous extracts from the two mentioned species were prepared in the concentration of 10% (w/v) using dried leaves (2 g). Further their preparation they were administered to Swiss mice (male, medium weight between 20 and 25 g). Animals were maintained in room temperature with free access to water and food.
Animal care and research protocols were in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Biomedical Science Institute/UFRJ-Ethical Committee for animal research.
Animal blood collection and glucose level determination

All blood samples were collected by orbital puncture, obtained from the ophthalmic plexus, in animals under anesthesia with ethylic ether. Blood was collected in heparin capillaries for micro hematocrit with capacity of 70 mL and centrifuged at 11000 rpm for 5 minutes in order to separate plasma. After its separation, an aliquot of 20 mL was collected and glucose-oxidase (color reagent) was added in a glass tube, named sample tube (S). Standard solution (20 mL) + 2 mL of a glucose-oxidase solution was used as positive control (C) and 2 mL of glucose-oxidase solution was used as blank (B). The plasmatic glycemia was achieved by the glucose/oxidase methodology. In this method, hydrogen peroxide formed by the catalytic action of the enzyme glucose-oxidase in D-glucose was measured by the oxidation of o-dianisidine in the presence of peroxidase.

Determination of glycemia in normoglycemic animals

Mice (n = 8) were used to verify the hypoglycemic activity from the two studied species of Bauhinia which the aqueous extract was administered by oral route to male mice in a concentration of 10% (w/v).

Animals were left fasting receiving water since 12 hours before the onset and during all the experiment. The basal glycemia (time = zero) was determined and further each group received its appropriate treatment. Each control group received distillated water 0.1 mL/10g of weight, p.o. and each treated group received aqueous extract (10% w/v) of one of the two tested Bauhinia species 0.1 mL/10g of weight, p.o.

The glycemia of each group was observed in 2, 4, 6 and 8 hours after the onset of the experiment with or without the administration of aqueous extract.
Separation, isolation and identification of the suggestive active compounds
The high performance liquid chromatography was made in a Shimadzu CBM 10ھ apparatus equipped with a LC-10AD pump and SPD-M10A DAD detector, manual injection valve Rheodyne with 20 mL of capacity, column Lichrosorb (Merk N. 738342) RP-18 (250 x 4 mm, internal diameter 10 mm). The monitoring was in fix wavelength (254 nm). The mobile phase was formed by 1% phosphoric acid solution in water (Solvent A) and 1% phosphoric acid solution in methanol Solvent B) flowing at 1 mL/mim. The gradient beginning with 30% of B growing to 40% of B in ten minutes. This composition remaining isocratic up to 20 minutes when another gradient was started going to 100% of B in 60 minutes, maximum time of the chromatographic process. Using this methodology, it could be possible to isolate from Bauhinia forficata aqueous extract, two glucosyl flavonoids: 3,7-di-O-a-rhamnopyranosylkaempferol, kaempferitrin, (RT = 24 minutes) and 3,7-di-O-a-rhamnopyranosyl quercetin (RT = 32 minutes).
 
These compounds were identified by 1H NMR (200 MHz) and 13C NMR (50 MHz) spectra registered in a Varian Star-200 apparatus, with deuterated solvents and TMS as internal standard. The 400 MHz 1H NMR was registered in a Bruker DRX apparatus using the same conditions. After elucidated each structure, data were compared with literature previously published one and by his way, it was possible to confirm the identity of each compound. The analysis of B. monandra aqueous extract and co-injection with the isolated molecules from B. forficata made possible to visualise or not the same compounds.
3,7-Di-O-a-rhamnopyranosylkaempferol..1H NMR (200 MHz, DMSO-d6, TMS): d (ppm) 0.8-1.1 (methyl groups of rhamnose, 2 d), 3.1-4.0 (signals concerning the H of the sugar moiety of this molecule) 5.3 (sl, anomeric H from rhamnose in the position 3), 5.55 (sl, H anomeric H from rhamnose in the position 7), 6.45 (sl, H-8), 6.75 (sl, H-6), 6.9 (d, J 8.43 Hz, 2H, H-3 and H-5), 7.75(d, J 8.42 Hz, 2H, H-2and H-6). 1H NMR (200 MHz, CD3OD, TMS): d (ppm) 0.9-1.3 (methyl groups of rhamnose, 2 d), 3.3-3.9 (signals concerning the H of the sugar moiety of this molecule), 5.4 (d, J 1.6 Hz, H anomeric H from rhamnose in the position 3), 5.6 (d, J 1.16, H anomeric H from rhamnose in the position 7), 6.44 (d, J 2.1 Hz, H-8), 6.7 (d, J 2.0 Hz, H-6), 6.94 (d, J 8.80 Hz, 2H, H-3 and H-5), 7.78 (d, J 8.80 Hz, 2H, H-2and H-6). 13C NMR (50 MHz, CD3OD, TMS): d (ppm) 17.7 (C-1); 18.1 (C-6-Rha); 71.2 (C-5-Rha); 71.6 (C-5-Rha); 71.8 (C-2-Rha); 72.1 (C-3-Rha, C-2-Rha, C-3-Rha); 73.1 (C-4-Rha); 73.5 (C-4-Rha); 95.5 (C-8); 99.8 (C-6); 100.5 (C-1-Rha); 103.5 (C-1-Rha); 107.5 (C-8a); 116.6 (C-3); 122.3 (C-1); 131.9 (C-2); 136.4 (C-3); 157.9 (C-4a); 159.7 (C-2); 161.7 (C-4); 162.9 (C-5); 163.5 (C-7); 179.7 (C-4).
3,7-Di-O-a-rhamnopyranosylquercetin. 1H NMR (400 MHz, CD3OD, TMS): d (ppm) 0.95-1.25 (methyl groups of rhamnose, 2 d), 3.34-4.25 (signals concerning the H of the sugar moiety of this molecule), 5.37 (d, J 1.37 Hz, H anomeric H from rhamnose in the position 3), 5.56 (sl, H anomeric H from rhamnose in the position 7), 6.47 (d, J 2.05 Hz, H-8), 6.73 (d, J 2.05 Hz, H-6), 6.92 (d, J 8.21 Hz, H-3), 7.33 (dd, J 2.05 Hz and J 8,21 Hz, H-2), 7.37 (d, J 2.05 Hz, H-6).
Statistical evaluation
Data related to the animals glycemia were analyzed by an experimental design completely casual, applying variance analysis (ANOVA) in order to verify significant differences between the groups, with 5% of significance level (p < 0.05). To compare control and experimental groups, t-Student test was applied. Non-pared t-Student test was applied to compare the glycemia average in each measured time. The results were expressed as an arithmetic average of glycemia in each measured time, with standard deviation. Each aqueous extract was evaluated in triplicate.
RESULTS AND DISCUSSION
Evaluation of the chromatograms of the aqueous extract of Bauhinia forficata and Bauhinia monandra by UV led to the visualization of signals which we believed to be glucosyl flavonoids (or flavonoid glycosides). In the case of Bauhinia forficata it was possible to isolate and identify two flavonoids with the following retention times: 3,7-di-O-a-rhamnopyranosylkaempferol, kaempferitrin, (24 minutes) and 3,7-di-O-a-rhamnopyranosylquercetin (32 minutes) (Figure 1). These compounds structures were established by NMR techniques. 3,7-di-O-a-rhamnopyranosylquercetin was also identified in the aqueous extract of Bauhinia monandra by comparison of both UV and retention data and by co-injection using HPLC. Table 1 shows the relative amount of both flavonoids in the two extracts. The quantitative relation between kaempferitrin and 3,7-di-O-a-rhamnopyranosylquercetin observed in B. forficata aqueous extract used in this work is in accordance with those showed to another sample of B. forficata aqueous extract from Itajaí (South part of Brazil), which was also seen when they evaluated B. forficata fluid extract (Pinheiro et al., 2006). Flavonoids were also identified in the aqueous extract of B. monandra, although their structures have not been elucidated (Argolo et al., 2004).
 
The hypoglycemic activity was established for both species using the described methodology as it can be seen in Graphic 1. The results found in this work match some results previously published, at least for B. forficata (Jorge et al., 2004). In the same study, the isolated molecule (kampferitrin) was also evaluated on 14C-glucose uptake in rat soleus muscle and has shown insulinomimetic effects. The insulin-like effects of B. forficata aqueous extract were also evaluated in mice after scorpion envenoming. Although no insulinomimetic effect has been shown, this extract was able to enhance the venom lethality (Vasconcelos et al., 2004).
Based on these evidences, the hypoglycemic activity shown in this paper can be related to the presence of glucosyl flavonoids which have different qualitative and quantitative profiles in both extracts. Both species had a very pronounced effect in the in vitro method used to establish activity, showing them to be very promising hypoglycemic agents. It is interesting to note that no relationship was observed between the color of the flowers and the strength of the hypoglycemic activity. Also, hypoglycemic activity for other Bauhinia species has been shown using different methodologies (Fuentes et al., 2004; Almeida et al., 2006; Fuentes; Alarcón, 2006).Further research is needed to permit us to completely understand the way the aqueous extracts make the glucose blood level lower. It is now our intention to test the pure isolated molecules.
 
Further studies also include the evaluation of these extracts and the extracts belonging to other Brazilian Bauhinia species, as well as their isolated compounds in beta-cell line culture (Asfari et al., 1992), to evaluate the efficacy of each extract and isolated compound in the elicitating the insulin production and release.
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Received 11/23/06. Accepted 02/23/07

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