Hypoglycemic Effect of Bumelia sartorum Polyphenolic Rich Extracts

Halliny S. Ruelaa,*, Katia C. C. Sabinob, Ivana C. R. Lealc, Ana M. LandeiraFernandezd, Michelle R. A. de Almeidae, Talita S. M. Rochad, Ricardo M. Kustera,e,*
a

Biotecnologia Vegetal, Centro de Cincias da Sade, Cidade Universitria,

Universidade Federal do Rio de Janeiro, 21921-590, Rio de Janeiro, RJ, Brazil.

b

Instituto de Biologia Roberto Alcntara Gomes, na Universidade do Estado do

Rio de Janeiro, 20551-030, Rio de Janeiro, RJ, Brazil.

c

Faculdade de Farmcia, Cidade Universitria Campus Maca, Universidade

Federal do Rio de Janeiro, 27930-560, Maca, RJ, Brazil.

d

Instituto de Bioqumica Mdica, Centro de Cincias da Sade, Cidade

Universitria, Universidade Federal do Rio de Janeiro, 21921-590, Rio de Janeiro, RJ, Brazil.
e

Ncleo de Pesquisas de Produtos Naturais, Centro de Cincias da Sade,

Cidade Universitria, Universidade Federal do Rio de Janeiro, 21921-590, Rio de Janeiro, RJ, Brazil

*Corresponding authors: fone: 55 21 25626795; fax: 55 21 25626512 E-mail address: hallinyruela@ufrj.br; kuster@nppn.ufrj.br

ABSTRACT

Diabetes mellitus is a chronic metabolic disorder characterized by a high blood glucose concentration. Bumelia sartorum Mart. (Sapotaceae) is used ethnomedicinally for the treatment of several diseases, including Diabetes mellitus. Assessment of B. sartorum hypoglycemic activity was performed from the blood glucose level in normoglycemic mice after samples administration by oral gavage. The hypothesis that sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition could prolong the increase in cytoplasmic Ca2+ concentration and thus leading to an increase of insulin released was evaluated. The enzyme inhibition was measured by ATP hydrolysis using SERCA1 isolated from rabbit skeletal muscle. The total phenolics were determined by the Folin-Ciocalteau method. The EtOAc partition and F5 fraction obtained thereof, both of them rich in polyphenolic compounds, like procyanidins B and phenolic acids, were shown to have significant hypoglycemic effect on normoglycemic mice, comparable to glibenclamide, that was used as a positive control. Both samples promoted a significantly inhibition of the SERCA enzyme.

Keywords: Bumelia sartorum; Hypoglycemic activity; Phenolic compounds; Sapotaceae; SERCA.

1. Introduction

Diabetes Mellitus is defined as a group of metabolic diseases characterized by blood glucose high levels, polydipsia, polyuria and polyphagia, muscle weakness and weight loss. It can progress to visual complications, poor kidney function and neurological disorders. Diabetic patients also suffer increasing risk of myocardial infarction, stroking and lower limb amputations, being the cardiovascular complications the leading causes of death (ADA, 2008). The type 2 diabetes is more common than type 1, accounting for 80-90% of the total cases, affecting mainly adults over 40 years of age (McGill & Felton, 2007). Despite the controllable character, diabetes is emerging as an epidemic of large proportions. Its prevalence is increasing due to the growth and the aging population, as well as by changes in life style such as fatty feeding and physical inactivity. According to data from the World Health Organization (WHO), the expectance by the year 2030 is that the world will have more than three hundred and sixty million diabetics (Wild et al., 2004). Therefore this work justify the search for new effective drugs for Diabetes Mellitus treatment, using regional medicinal plants, providing a more accessible service for the population (Rocha et al., 2006). In northeastern Brazil, Bumelia sartorum Mart. has long been used in Brazilian folklore for the treatment of several diseases, including Diabetes mellitus (Almeida et al., 1985). The plant is commonly known as quixaba, quixabeira and rompe-gibo. It is a large and tall (10-15 m) tree from the Sapotaceae family and distributed from the North of Minas Gerais to Piau

(Silva et al., 2004). Phytochemical studies showed the presence of triterpenoids and steroids such as the (2,3,4)-2,3,23-trihydroxyoleana-5,12-dien-28-oic acid, or basic acid, that was identified as a hydrolysis product from the ethanol extract (Almeida et al., 1985; Naik et al., 1991). In a previous study, we described the abundant presence of polyphenolic compounds in B. sartorum extracts, such as catechin and epicatechin as well as type-B procyanidins (Ruela et al., 2011). During the stimulation by glucose and other secretagogues, the exocytosis of insulin-containing granules from pancreatic -cells is trigger by an increase in intracellular Ca2+ concentrations ([Ca2+]i). The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump is responsible for transporting Ca2+ from the cytosol back into the lumen of the endoplasmic reticulum (ER) at the expense of ATP hydrolysis, and thus decreasing the insulin secretion (Worley et al., 1994). In mouse pancreatic islets, inhibition of the SERCA pump increased the amplitude of the glucose-triggered [Ca2+]i oscillations and abolished the slow release of Ca2+ from the ER (Arredouani et al., 2002). In this work we measured the effect of the B. sartorum polyphenolic enriched samples in the rate of ATP hydrolisys catalyzed by the SERCA1 from rabbit skeletal muscle. We hypostatized that the medicinal hypoglycemic effect of the B. sartorum might be by the regulation of this enzyme. In addition this work also shows the total phenolic content and the ferrous ion-chelating ability of the B. sartorum extracts.

2. Experimental

2.1. Plant material

Bumelia sartorum was collected in 2006, in Cabrob city (0830' south; 3918' west), Pernambuco State, during Brazilian summer. An exsiccate was deposited in herbarium of Institute of Biology (RFA-34154), at Federal University of Rio de Janeiro. According to our previous work (Ruela et al., 2011) methanol crude extract was obtained from B. sartorum ground barks by maceration for seven days at room temperature. From this, hexane, dichloromethane, ethyl acetate and butanol partitions were obtained by liquid-liquid partition. The ethyl acetate partition was chromatographed on a Diaion HP-20 column with stepwise gradient with H2O:MeOH (9:10:1) resulting, among others, the F5 fraction tested here. In accordance with the popular use of this plant, the aqueous extract was also prepared beyond decoction, cooking 20 g of grounded barks in 1 liter of boiling water for 15 minutes. This material was then frozen and lyophilized in lyophilizer Labconco.

2.2. Determination of glycemia in normoglycemic animals

Assessment of B. sartorum hypoglycemic activity was performed determining the blood glucose level in normoglycemic mice according to Menezes et al. (2007) with minor modifications.

All the samples were prepared using 0.9% saline solution as vehicle. Animals (Swiss webster, female, 25-30 g) were left 12 h fasting, receiving only water, before the onset and during the whole experiment. After fasting, the samples were administered to the animals (n = 6) by oral gavage at 250 mg/kg, defined from our previous toxicological work (Ruela et al., 2011). Blood collection was performed through an incision in the animal tail, and blood glucose level was determined by using the Glucose Monitoring System AccuChek Active (Roche). The positive control group received glibenclamide (Geolab) at 10 mg/kg, which is in accordance with Adisa et al., 2010. The negative control group received only 0.9% saline solution. The glucose in each group was observed at the start time (zero time) and 1, 2, 3 and 5 h after the beginning of the experiment. This test was conducted after agreement by the Ethics Committee of Federal University of Rio de Janeiro, under registration IMPPG 014.

2.3. Sarcoplasmic Reticulum Vesicles Isolation

Vesicles derived from sarcoplamic reticulum were isolated from rabbit skeletal muscle as previously described by Eletr & Inesi (1972).

2.4. Ca2+-ATPase activity

ATP hydrolysis was measured using the colorimetric method of Fiske & Subbarow (1925), adapted by Landeira-Fernandez et al. (2004), using two

types of reaction medium: one containing 50 mM Mops-Tris pH 7,0, 2 mM MgCl2, 10 M CaCl2, 1 mM ATP and water, and the second medium containing all the reagent plus 0.1 M KCl. The EtOAc and F5 fractions (at 1, 10, 20, 40, 60, 80 and 100 g/ml) were selected for this test once they have shown a lowering effect on blood glucose levels of normoglycemic animals. The reaction was started by the addition of the enzyme (20 g/ml) diluted in a solution containing 0.2 mM Mops-Tris (pH 7.0), 10 M CaCl2, 0.1 M KCl and water, achieving a final reaction volume of 600 l. After incubation by 15 min at 35 C, the reaction was stopped by adding 400 l of trichloroacetic acid (TCA) 50%. The control was prepared without the vegetal samples. Two different blank tubes were also prepared: one in the absence of the fractions and another with the highest concentration of the fractions, both tubes were stopped by TCA immediately after adding the enzyme (time zero). The assays were put on ice then bring back to ambient temperature and then were added 400 l of ammonium molybdate and 100 l of reductive solution (consisting of 0.25% 1-amino-2naphthol-4sulphonic acid, 0.5% Na2SO3 and 15% NaHSO3). The color intensity was measured after 20 min at 660 nm. The activity of the SERCA was calculated as a percent of the control activity (100%) considering no vegetal samples added, after deducting the blank values.

2.5. Total phenolics content

Total phenolics were determined by the Folin-Ciocalteau method according to Victrio et al. (2010) with minor modifications. The samples

(methanol and aqueous extracts, hexane, dichloromethane, ethyl acetate and butanol partitions and the F5 fraction) were dissolved in ethanol at 500 g/ml. After 5 min, 50 l of the diluted sample and 100 l of 10% Folin-Ciocalteau reagent (Spectrum) were added to 100 l of 7.5% sodium carbonate (Vetec ), and the contents were mixed. The mixture was homogenized and incubated at room temperature for 2 h. Absorbance was measured at 740 nm in a microplate reader (Biot-Tek Instruments Inc., modelo Quant) using gallic acid (SigmaAldrich) as standard (5, 10, 25, 50, 125 and 250 g/ml). Two controls were employed: (1) Folin-Ciocalteau + sodium carbonate and (2) sample solution. Quantification of phenolic compounds was determined for regression equation of calibration curves: y = 0.0047x + 0.3666 (R2 = 0.911) and expressed as mg of gallic acid equivalents (GAE) per 1 g of dried extracts. All measurements were performed in triplicate.

2.7. Statistical analysis

The results represent the mean standard deviation of three or more independent experiments. The data comparison was performed by Students t test or ANOVA followed by Tukey test, using the GraphPad Prism software, considering the differences statistically significants when p<0.05.

3. Results

3.1. Determination of glycemia in normoglycemic animals

The B. sartorum hypoglycemic activity can be seen in Table 1. The hypoglycemic action was more pronounced in animals that received the EtOAc partition and F5 fraction when compared to other groups. After the first hour of experiment, the group treated with F5 fraction already showed a statistically significant reduction in blood glucose compared to control group. The hypoglycemia induced by EtOAc partition was observed in the second hour of experiment. After five hours, both samples showed reductions of blood glucose level comparable to the glibenclamide group.

3.2. Ca2+-ATPase activity

Ca2+ is involved in the regulation of a variety physiological process such as muscle contraction, platelet activation, release of neurotransmitter, cell migration, and metabolism. Also, Ca2+ is involved in the regulation of insulin release from pancreatic -cells. Thus, we tested the effect of the B. sartorum EtOAc partition and F5 fraction in the rate of ATP hydrolysis catalyzed by the Ca2+-ATPase. These samples were selected due to the promising results found with the physiological glycemia experiments (Table 1). As shown in Fig 1. both EtOAc (Fig. 1A) and F5 (Fig. 1B) were able to completely inhibit the activity of the SERCA enzyme. The calculation of the IC50 values for these samples suggests that the F5 fraction is significantly more active, with IC50 of 15.13 g/ml, than the EtOAc partition with the IC50 of 22.47 g/ml. The addition of KCl to the reaction medium, did not significantly change the control activity of the

SERCA pump and also KCl was not able to reverse the inhibition promoted by the B. sartorum extracts. This result differs from previous data showing that the SERCA1 can be inhibited by sulfated polysaccharides and KCl is able to revert this inhibition (Da Costa & Landeira-Fernandez, 2009) and shows that the inhibition promoted by the B. sartorum samples can be physiological. Taken together these results contribute to the hypothesis that the physiological hypoglycemic effect of B. sartorum might be due to sustained high cytoplasmic Ca2+ concentrations. In the presence of the phenolic compounds the rate of ATP catalyzed by the SERCA pump is inhibited and consequently not able to transport Ca2+ from the cytosol to the lumen of the ER, promoting greater release of insulin.

3.3. Total phenolics content

The spectrometric quantification of phenolic compounds using the FolinCiocalteu method is one of the most widely used techniques. The reagent is a mixture of fosfomolibdic and fosfotungstic acids, in which molybdenum and tungsten are in the 6 + oxidation state but in the presence of certain reducing agents, such as phenolic compounds, they form the blue molybdenum and blue tungsten in which the average oxidation state of metals is between 5 and 6 and whose color allows the determination of the reducing substances concentration (Sousa et al., 2007). All samples (Figure 2) showed high levels of phenolic compounds, when compared to data reported for other species known to be rich

in phenolic substances (Sousa et al. 2007; Victrio et al., 2010). Only the butanol fraction showed no phenolic content measured by this method.

4. Discussion

In a previous study, the chemical constituents profile of B. sartorum ethyl acetate partition was evaluated and the presence of polyphenolic compounds, such as (+)-catechin and (-)-epicatechin and type-B procyanidins were observed (Ruela et al., 2011). The abundant presence of polyphenols in B. sartorum extracts was confirmed by the determination of total phenols content by the Folin-Ciocalteu method. Except the butanol extract and especially the EtOAc partition, as well as the F5 fraction (515 and 555 mg GAE/g, respectively), showed high levels of phenolic compounds, when compared with data from other vegetal species described in the literature (Sousa et al. 2007; Victrio et al., 2010). Because there is large amount of phenolic substances in this species, traces of these appear even in the more apolar samples (hexane and dichloromethane partitions). The formation of free radicals occurs as a natural consequence of cellular metabolism. But it is known that oxidative stress, characterized by an intense overload of reactive oxygen species, can be extremely damaging to cellular structures. The polyphenols classes are particularly useful in the prevention of human pathologies in which free radical production plays a key role, as atherosclerosis, ischemic injury, degenerative neurological diseases (Parkinson and Alzheimer), inflammation and cancer (Sakano et al., 2005).

The pathogenesis of diabetes also includes oxidative stress, such as causing -cell destruction in type I diabetes or in the inflammatory process involved in the development of type 2 diabetes (Adisa et al., 2010). Besides oxidative stress, hyperglycemia frame is almost always accompanied by lipids and proteins dysfunctions and these metabolic changes may lead to the development of secondary diseases such as cataract, microangiopathy, nephropathy, atherosclerosis, infections and neurological changes (Rocha et al. 2006). The hypoglycemic activity displayed by the EtOAc partition and F5 fraction was very significant. Such samples caused a reduction in mice blood glucose in a higher rate than glibenclamide, reaching a similar result as that after 5 hours of treatment. The hypoglycemic effect of the crude ethanol extract of B. sartorum had already been reported by Almeida and colleagues (1985). The authors found a reduction of blood glucose in mice, rats and rabbits after 8 hours of extract administration, and they further suggested that this effect is due to an increased release of insulin and glucose uptake by skeletal muscle. Naik and colleagues (1991) stated to the bassic acid, triterpene isolated after acid hydrolysis of the ethanol extract, the hypoglycemic agent of this plant. But the predominant presence of phenolic compounds in extracts of this species and the results presented here for the EtOAc and F5 samples suggest that these substances are the responsible for the glucose-lowering effect. The

hypoglycemic potential of (+)-epicatechin, (-)-catechin and type B-procyanidins are widely described in literature. As examples of source of polyphenol substances used in diabetes treatment, we can cite the crude extract of

Pterocarpus marsupium (Lamba et al., 2000) and green tea (Camellia sinensis) (Sharangi, 2009). Several studies on the role of natural products in the various mechanisms of diabetes pathophysiology have been made in recent decades. Plant phenolic derivatives such as (+)-catechin, (-)-epicatechin,

epigallocatechin, tannic acid and isoflavones exert inhibitory activity on the amylase and -glucosidase enzymes, contributing to the suppressive activity of postprandial hyperglycemia in some vegetable products (Da Silva & CechineloFilho, 2002). Cao and colleagues (2007) found that the aqueous extract of cinnamon, a source of type-A procyanidins, has the potential to increase the amount of proteins involved in signaling for insulin release, glucose transport and anti-inflammatory response. In this work, it was evaluated the hypothesis that SERCA inhibition could prolong the increase of cytoplasmic Ca2+, thereby leading to an increased release of insulin. The Ca2+ ion is essential in the metabolism, functioning as second messenger in several cellular events. Two enzymes are mainly responsible for reducing their cytoplasmic concentration: a Ca2+-ATPase of sarcoplasmic/endoplasmic reticulum (SERCA) and Ca2+-ATPase of plasmatic membrane (PMCA). The SERCA actively removes Ca+2 from the cytoplasm by transporting it into the grid, using for that the energy released by the ATP molecules breakdown for this purpose. The PMCA removes Ca+2 from the cytoplasm also actively transporting it into the extracellular environment (Rocha et al., 2006). Although we did not used the SERCA3 isoform that is mainly expressed in the pancreatic -cells, this enzyme is highly conserved among the

different isoforms (Prasad et al., 2004). We decided to use the SERCA1 isoform because of its abundance in the rabbit white skeletal muscle, being easy to measure the activity. The stimulation of insulin secretion by Langerhans islets is initiated by plasma membrane depolarization and a consequent increase in the concentration of free Ca2+ intracellular, through a combination of flow caused by depolarization and activation of voltage-dependent Ca2+ channels and also by the release to the cytoplasm of Ca2+ stored in endoplasmic reticulum (Roe et al., 1994). Some studies also showed that Ca2+ depletion of endoplasmic reticulum results in activation of an internal current, inducing depolarization, which facilitates ion influx through voltage-dependent Ca2+ channels (Worley et al., 1994). The EtOAc and F5 samples were able to inhibit the SERCA activity quite significantly, since no such inhibitory effect was reversed in the presence of KCl, as had been described for some substances (Da Costa & LandeiraFernandez, 2009). The physiological relevance of this observation is the possibility of using SERCA inhibitors as novel hypoglycemic agents.

5. Conclusion

This work confirms the popular use of B. sartorum as a hypoglycemic medicinal plant. Polyphenolic rich samples were able to reduce blood glucose in normoglycemic mice and to inhibit SERCA activity. However, further research is required to evaluate the practical values of a therapeutic application.

Acknowledgement

This study was supported by grants from CNPq, FINEP, CAPES, FAPERJ (Brazil) and DAAD (Germany).

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Fig. 1. Percentage of SERCA1 activity inhibition by EtOAc partition (A) and F5 fraction (B) in the presence () or absence () of 0.1 M KCl. The 100% enzyme activity was 2.8 0.4 mol Pi/mg/15min for control in absence of 0.1 M KCl and 4.3 0.8 mol Pi/mg/15min in presence of 0.1 M KCl. Values are means S.D. (n = 3).

Fig. 2. Total phenolic content in the B. sartorum samples. Aq: crude aqueous extract; Me: crude methanolic extract; Hex: n-hexane partition; Di:

dichloromethane partition; Ac: ethyl acetate partition; Bu: butanolic partition; F5: F5 fraction, obtained from ethyl acetate partition. Values are means S.D. (n = 3).

Table 1. Determination of fasting blood glucose (mg/dl) in normoglycemic animals. Values are means S.D. (n = 6). * p<0.05, vs 0.9% saline.

Figure 1.

Figure 2.
555 485.7 515.4

600 500

mg GAE/g

400 300 200 100

354.5 236.3 208.2

ND
0
Aq Me Hex Di Ac Bu F5

Table 1. Treatment 0.9% Saline Glibenclamide Aqueous Extract MeOH Extract AcOEt Partition F5 Fraction Time: 0 119 6.0 115 2.5 121 5.9 116 5.8 103 7.0 106 6.0 1h 117 4.6 105 11.8 96 9.5 125 10.0 112 3.0 *98 6.9 2h 93 3.4 93 3.7 96 8.5 98 1.5 *71 6.6 *79 1.1 3h 81 3.8 82 5.0 78 11.1 94 5.0 *55 5.8 *65 3.7 5h 74 1.8 *51 4.9 64 6.2 83 6.1 *32 7.9 *56 6.5