α-glucosidase inhibitory and antidiabetic activities of ethanolic extract of pisonia alba span. leaves

α-Glucosidase inhibitory and antidiabetic activity of Pisonia alba International Journal of Integrative Biology
A journal for biology beyond borders
ISSN 0973-8363
α-Glucosidase inhibitory and antidiabetic activities of ethanolic
extract of Pisonia alba Span. leaves
Sunil Christudas1,*, Latha Gopalakrishnan2, Palanisamy Mohanraj 3,
Kalichelvan Kaliyamoorthy1, Paul Agastian 4
1 Department of Pharmacy, Annamalai University, Chidambaram, India 2 Tropical Botanic Garden and Research Institute, Thiruvananthapuram, India 3 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India 4 Entomology Research Institute, Loyola College, Chennai, India Submitted: 12 Feb. 2009; Accepted: 16 Apr. 2009 Abstract
In this study, the ethanolic extract of Pisonia alba has been studied for its α-glucosidase inhibitory and
antidiabetic properties. The extract shows α-glucosidase inhibitory activity in a concentration dependent manner
(IC50 of 416.7 µg/ml). On 15 days administration of the extract (250 and 500 mg/kg) on alloxan induced diabetic
rats resulted in significant (P<0.01) decrease in blood glucose, serum glutamate pyruvate transaminase (SGPT),
serum glutamate oxaloacetate transaminase (SGOT), serum alkaline phosphatase (SALP), cholesterol,
triglycerides levels and increase in HDL levels as compared to pathogenic diabetic rats. These results indicate
that the extract is effective in hyperglycemia and also protects significantly from other metabolic aberrations
caused by alloxan.
Keywords: Pisonia alba, alloxan, α-Glucosidase, antihyperglycemic, glibenclamide.

INTRODUCTION
in the early treatment of DM and in reducing chronic vascular complications (Shim et al., 2003). Diabetes mellitus (DM) is a common metabolic disease characterized by elevated blood glucose levels, One of the therapeutic approaches for reducing resulting from absent or inadequate pancreatic insulin postprandial hyperglycemia in patients with DM is to secretion, with or without current impairment of insulin prevent absorption of carbohydrates after food uptake. action. Currently, diabetes has been estimated to affect Only monosaccharides, such as glucose and fructose, 177 million people world wide and this figure is can be transported out of the intestinal lumen into the projected to increase to 300 million by 2025 (Porter et blood stream. Complex starches, oligosaccharides and al., 2005). Epidemiological studies and clinical trials disaccharides must be broken down into individual strongly support that hyperglycemia is the main cause monosaccharides before being absorbed in the of complications related with coronary artery disease, duodenum and upper jejunum. This digestion is cerebrovascular disease, renal failure, blindness, limb facilitated by enteric enzymes, including pancreatic α- amputation, neurological complications and pre-mature amylase and α-glucosidases that are attached to the death (Lopez 2001). Recent studies suggest that brush border of the intestinal cells. Acarbose and postprandial hyperglycemia could induce the non- miglitol are competitive inhibitors of intestinal α- enzymatic glycosylation of various proteins, resulting glucosidases and reduce the postprandial digestion and in the development of chronic complications. Therefore, absorption of starch and disaccharides (Davis et al., control of postprandial plasma glucose levels is critical 1996). Screening of α-glucosidases inhibitors from plants and synthetic sources is increasing. Inhibitors of these enzymes have been recently developed from *Corresponding author:
natural sources (Shim et al., 2003). Christudas Sunil, Ph.D. Scholar Department of Plant Biology and Biotechnology, School of Life Sciences In herbal medicinal practice many plants are used to Loyola College, Chennai 603203, India treat diabetes mellitus in south India. Most of these Email: [email protected] medicinal plants are not scientifically validated for their International Journal of Integrative Biology
IJIB, 2009, Vol. 6, No. 1, 41 OmicsVista Group, All rights reserved α-Glucosidase inhibitory and antidiabetic activity of Pisonia alba therapeutic efficacy and safety (Babu et al., 2002). animal experiments were carried out according to NIH Though there are numerous traditional medicinal plants guidelines, after getting the approval of the Institute's reported to have hypoglycemic property, many of them Animal Ethics Committee (Reg. No. 176/99/CPCSEA). have proven to be not effective in lowering glucose levels in severe diabetes (Nagarajan et al., 1987). Acute toxicity studies
Furthermore, most of the hypoglycemic agents used in allopathic medicine are reported to have side effects Healthy adult wistar albino rats of either sex, starved including hematological, coma and disturbances of overnight were divided into five groups (n=6) and were liver and kidney. Therefore, there is a need to search orally fed with the PAEE in increasing dose levels of for more effective and safer drugs for diabetes (Pari et 100, 500, 1000, 3000 and 5000 mg/kg body weight al., 1999). continuously for 2 h under behavioral, neurologial, Pisonia alba (Nyctaginaceae), commonly known as autonomic profiles (Turner, 1965). Lettuce Tree, is an evergreen tree 9-12 m high found sparsely wild in the beach forests of Andaman Islands, Inhibition assay for the α-glucosidase
cultivated to a small extent in India and Ceylon. The activity
fresh leaves moistened with Eau-de-Cologne are used to subdue inflammation of a filariosis nature in the legs In order to investigate the inhibitory of PAEE, an in and other parts (Kiritikar et al.,1935). They are used as vitro α-glucosidase inhibition test was performed. diuretic. The root is purgative. A survey of literature Normally α-Glucosidase from yeast is extensively used revealed that Pisonia alba is an untapped candidate for as a screening material for α-glucosidase inhibitors, but antidiabetic activity though it is extensively used in the results do not always agree with those obtained in traditional healing of diabetes in Kerala (Anonymous, mammals. Therefore, we used the mouse small intestine homogenate as an α-glucosidase solution because we speculated that it would better reflect the in vivo state. The inhibitory effect was measured using the method slightly modified from Dahlqvist (Dahlqvist, MATERIALS AND METHODS
1964). After fasting for 20 h, the small intestine between the part immediately below duodenum and the Plant material and extraction procedure
part immediately above the cecum was cut, rinsed with The aerial parts of Pisonia alba were collected from ice cold saline and homogenized with 12 ml of maleate Trivandrum district of Kerala. The plant was buffer (100 mM, pH 6.0). The homogenate was used as the α authenticated by Dr. Mathew Dan, Plant taxonomist of -glucosidase solution. The assay mixture consisted Tropical Botanical Garden and Research Institute. A of 100 mM maleate buffer (pH 6.0), 2% (w/v) each voucher specimen has been deposited at the herbarium sugar substrate solution (100 µl), and the PAEE (50- of TBGRI (TBGT 57019 dated 19/01/07) for future 1000µg/ml). It was pre-incubated for 5 min at 37ºC, and the reaction was initiated by adding the crude α reference. The aerial parts of Pisonia alba were washed thoroughly in tap water, dried under shade and glucosidase solution (50 µl) to it, followed by powdered to coarse particles. The powder (22 gm) was incubation for 10 min at 37ºC. The glucose released in extracted with 3000 ml of ethanol with constant stirring. the reaction mixture was determined with the kit The residue was removed by filtration and the filtrate was evaporated to dryness at 40ºC under reduced decomposition was calculated as percentage ratio to the pressure in a rotary evaporator. The yield of the ethanol amount of glucose obtained when the carbohydrate was extract was found to be 0.165 gm. The extract was completely digested. The rate of prevention was dried in a desiccator and it was referred to as PAEE. It calculated by the following formula: was diluted with 2% Tweeen-80 to desired concentrations and used for the experiments. ( A  A )  B A0 - Amount of glucose produced by the positive control. A1 - Amount of glucose produced by the addition of PAEE Adult male albino rats (250-350 gm), reared in Tropical B - Glucose production value in blank. Botanical Garden and Research Institute's animal house were used for the experiments. They were Effect of PAEE on Oral Glucose Tolerance
grouped and housed in Poly-acrylic cages with six animals per cage and maintained under standard Test (OGTT)
laboratory conditions. (Temperature 24-28ºC, relative The oral glucose tolerance test (Bonner-Weir, 1988) humidity 60-70% and 12 h dark-light cycles). They was performed in overnight fasted (18 h) normal rats. were fed with commercial rat feed (Lipton India Ltd., Rats divided into three groups (n=6) were administered Mumbai, India) and boiled water, ad libitum. All International Journal of Integrative Biology
IJIB, 2009, Vol. 6, No. 1, 42 OmicsVista Group, All rights reserved α-Glucosidase inhibitory and antidiabetic activity of Pisonia alba Blood was collected into heparinized tubes. Table 1: In vitro α-glucosidase inhibition using PAEE.
The serum was analysed for glucose % of inhibition of α-glucosidase
(µg/ml)
Acarbose
50 (µg/ml)
oxaloacetate transaminase, serum alkaline phosphatase, cholesterol, triglycerides and HDL in an autoanalyzer Micro lab 2000 using Ecolin kits. All the values represent (mean ± SE) taken from two experiments each with 3 replicates per dose was tabulated. Statistical analysis

Table 2: Effect of PAEE Oral Glucose Tolerance Test.
The experimental data were expressed as Blood glucose (mg/dl)
mean±SEM. The significance of difference among the various treated groups and control group was analyzed by means of one-way ANOVA followed by Dunnett's multiple comparison test using Graphat Instat Software (San Diego, CA, USA). mg/kg) Values are the mean ± SD, n=6 for all groups significance **P<0.01, compared with vehicle treated control group (One way ANOVA using Dunnett's test). RESULTS AND DISCUSSION
dri nking water, PAEE 250 and 500 mg/kg, respectively. The acute toxicity studies revealed the nontoxic nature Glucose (2 gm/kg) was fed 30 min after the of the PAEE. There was no lethality or any toxic administration of extracts. Blood was withdrawn from reactions found at any of the doses selected until the the retro orbital sinus under ether inhalation at 30, 90 end of the study period. This is not surprising as PAEE and 150 min of glucose administration and glucose is extensively used as vegetable and salad. levels were estimated using the standard glucose reagent kit (Accurex Biomedical Pvt. Ltd. Thane). The PAEE showed a significant inhibitory action of -glucosidase enzyme (Table 1). The results revealed that Effect of PAEE on Alloxan-induced
PAEE showed 5.75% of inhibition at 50 g/ml and for diabetic rats
1000 g/ml inhibition was found to be 83.29%. There Rats were injected with alloxan (60 mg/kg) through tail was a proportionate increase in the percentage of - vein (Babu et al., 2002). Five days later, blood samples glucosidase inhibition in a concentration dependent were drawn and glucose levels were determined to manner (IC50 of 416.7 µg/ml). Acarbose was used as a confirm the development of diabetes. The diabetic rats reference standard for the evaluation of -glucosidase exhibiting glucose levels in the range of 400-450 mg/dl inhibitory action. α-glucosidase is one of a number of were selected to determine the efficacy of PAEE. glucosidases located in the brush-border surface membrane of intestinal cells, and is a key enzyme of Experimental setup
carbohydrate digestion (Caspary, 1978). α-glucosidase inhibitors block the actions of α-glucosidase enzymes The animals were divided into five groups with six in the small intestine, which limits the conversion of animals in each group. oligosaccharides and disaccharides to monosaccharides,  Group I normal control rats administered 2% necessary for gastrointestinal absorption. Postprandial Tween-80 (1 ml), p.o. glucose peaks may be attenuated by delayed glucose  Group II diabetic control rats administered a single absorption. The main benefits attributable to α- daily dose of 2% Tween-80 (1 ml), p.o.  Group III diabetic rats administered PAEE (250 postprandial glycemic levels and in the total range of postprandial glucose levels (Lebovitz, 1997). The  Group IV diabetic rats administered PAEE (500 antidiabetic action of PAEE is attributed to the intestinal α-glucosidase inhibitory activity.  Group V diabetic rats administered reference drug glibenclamide (600 µg/kg b.w/rat/d) in aqueous Oral administration of PAEE at 250 and 500 mg/kg solution orally for 15 days. doses resulted in a significant fall in blood glucose Blood samples were collected in the morning, 1hr after level, 2½ h after a single dose of treatment in glucose drug administration on days 1, 4, 7, 10 and 15 for loaded rats (Table 2). PAEE was effective at both the glucose estimation. After 15 days of treatment, the doses used in depressing the peak value of blood sugar fasted rats were sacrificed by cervical decapitation. at 30 min after glucose loading. At both the dose levels, PAEE completely prevented the elevation of blood International Journal of Integrative Biology
IJIB, 2009, Vol. 6, No. 1, 43 OmicsVista Group, All rights reserved α-Glucosidase inhibitory and antidiabetic activity of Pisonia alba Table 3: Effect of 15 days treatment with PAEE on glucose levels of diabetic rats
Treatments
Blood glucose levels (mg/dl)
Day 1
Diabetic control PAEE (250 mg/kg) PAEE (500 mg/kg) Glibenclamide (600 μg/kg) Values are the mean ± SD, n=6 for all , significance **P<0.01, ***P<0.001 compared with vehicle treated control group (One wa y ANOVA using Dunnett's test).
Table 4: Effect of PAEE on rat serum biochemical parameters after alloxan administration
Cholesterol
Diabetic control 183.45 ± 13.22*** 28.73 ± 1.71*** (500 mg/kg) Diabetic + Glibenclamide (600 µg/kg) Values are the mean ± SD, n=6 for all groups significance **P<0.01, compared with vehicle treated control group (One way ANOVA using Dunnett's test). glucose level caused by oral glucose feeding. This islets or enhanced transport of blood glucose to finding indicates that the PAEE extract might be peripheral (Tulay et al., 2008). Inhibition of proximal producing its hypoglycemic activity by a mechanism tubular reabsorption mechanism for glucose in the independent from the insulin secretion, e.g. by the kidney may also contribute to the blood glucose inhibition of endogenous glucose production (Eddouks lowering effect (Jafri et al., 2000). et al., 2003) or by the inhibition of intestinal glucose absorption (Platel et al., 1997). An increase in the SGPT, SGOT and SALP activities was recorded in diabetic rats in comparison with non In alloxan induced diabetic rats, the blood glucose diabetic rats, indicating an altered liver function in levels were in the range of 400-450 mg/dl which can be diabetic condition. PAEE significantly controlled considered as severe diabetes (Table 3). Administration SGPT, SGOT and SALP values in the alloxan diabetic of PAEE upto 15 days tends to bring the blood glucose rats (Table 4). In diabetic animals a change in the levels towards normal. The PAEE treated groups serum enzymes is directly related to changes in the showed blood glucose levels of 200 mg/dl and 205.95 metabolism in which these enzymes are involved. The mg/dl, when compared with that of diabetic control increased levels of transaminases which are active in groups (203.35 mg/dl). A similar reduction in blood the absence of insulin because of increased availability glucose level was noticed in glibenclamide treated rats of amino acids in diabetes (Bondy et al., 1949; Felig et (203.35 mg/dl). A significant (p<0.01) reduction in al., 1970) are responsible for the increased blood glucose level was noticed among PAEE and gluconeogensis and ketogenesis observed in diabetes. standard drug treated groups when compared with In the present study, the PAEE significantly decreased diabetic control. Alloxan, a β-cytotoxin, induces Aspartate aminotransferase (ASAT) and Alanine chemical diabetes in a wide variety of animal species aminotransferase (ALAT) enzyme activities. Hence, the including rats by damaging the insulin-secreting β-cells improvements noticed in the levels of the enzymes and almost complete destruction of the pancreas. In the studied, namely Glutamate oxaloacetate transaminase present study, PAEE significantly reduced the blood (GOT) and Glutamate pyruvate transaminases (GPT) glucose levels of alloxan diabetic rats indicating the are as a consequence of an improvement in the mechanism possibly by potentiation of pancreatic carbohydrate, fat and protein metabolism. The secretion of insulin from existing residual β-cells of restoration of GOT and GPT after treatment also International Journal of Integrative Biology
IJIB, 2009, Vol. 6, No. 1, 44 OmicsVista Group, All rights reserved α-Glucosidase inhibitory and antidiabetic activity of Pisonia alba indicates a revival of insulin secretion. Elevation of with diabetes ketosis, before and after the administration of insulin. J. Alkaline phosphatase (ALP) has been reported in Clin. Invest., 28: 1126-1133.
diabetic rats (Mishima, 1967) and rabbits (Begum et al., Bonner-Weir S (1988) Morphological evidence of pancreatic polarity 1978). This increase in ALP was significantly reversed of beta cells within islets of langerhans. Diabetes, 37(5): 616-621.
Caspary WF (1978) Sucrose malabsorption in man after ingestion of
alpha-glucosidehydrolase inhibitor. Lancet, 1(8076): 1231-1233.
A decrease in the serum triglycerides and total cholesterol levels and an increase in the HDL Dahlqvist A (1964) Method for assay of intestinal disaccharidases. cholesterol levels were observed (Table 4). A marked Anal. Biochem., 7(1): 18-25.
increase in total cholesterol and decrease in HDL Davis SN and Granner DK (1996) Insulin, oral hypoglycemic agents cholesterol have been observed in untreated diabetic and the pharmacology of endocrine pancreas. The Pharmacological rats. Under normal circumstances insulin activates Basis Of Therapeutics: Pergamon Press, New York, ISBN: enzyme lipoprotein lipase and hydrolyses triglycerides. 10:0071422803, pp: 1487-1517. Insulin deficiency results in failure to activate the Eddouks M, et al. (2003) Inhibition of endogenous glucose enzymes thereby causing hypertriglyceridemia (Annie production accounts for hypoglycemic effect of Spergularia purpurea et al., 2006) The significant control of the levels of in streptozotocin mice. Phytomedicine, 10(6-7): 594-599.
serum lipids in the aqueous extract treated diabetic rats may be directly attributed to improvement in insulin Felig P, et al. (1970) Plasma amino acid levels in diabetic keto
acidosis. Diabetes, 19: 727-729.
levels upon PAEE therapy. Ghosh MN (1984) Fundamentals of Experimental Pharmacology. Scientific Book Agency, Calcutta, ISBN: 81-902965-0-7, pp: 153. investigations at the cellular and molecular level are Jafri MA, et al. (2000) Effect of Punica granatum Linn. (flowers) on underway to elucidate its exact mechanism of action. blood glucose level in normal and alloxan-induced diabetic rats. J.
Ethnopharmacol., 70(3): 309-314.
Kiritikar K and Basu L (1935) Indian Medicinal Plants, Allahabad,
CONCLUSION
India, Vol. III, 2nd ed., pp: 1817-1818. The results obtained from the present study show that Lebovitz HE (1997) Alpha-glucosidase inhibitors. Endocrinol. Metab. the PAEE had beneficial effects on blood glucose Clin. North Am., 26: 539-551.
levels in glucose-fed hyperglycemic and diabetic rats and it also protects significantly from other metabolic Lopez CA (2001) Metabolic syndrome X; a comprehensicve review
of the pathophisiology and recommended therapy. J. Med., 32: 283-
aberrations caused by alloxan, thus scientifically verifying the traditional claim. PAEE appears to be an attractive material for further studies leading to possible Mishimia K (1967) Changes of phosphatase activity in rats with drug development for diabetes. Development of alloxan diabetes. Kyoritsu Yakka Daigaku Kenkyu Nempo, 12: 58-61.
phytomedicines is relatively inexpensive and less time Nagarajan S, et al. (1987) Indigenous plants used in the control of consuming; it is more suited to our economic diabetes. CSIR, New Delhi, ISBN: 81-85038-00-7, pp: 588 conditions than allopathic drug development which is more expensive and spread over several years. Pari L and Uma MJ (1999) Hypoglycaemic effect of Musa sapientum
L. in alloxan-induced diabetic rats. J. Ethnopharmacol., 68: 321-325.
Platel K and Srinivasan K (1997) Plant foods in the management of
diabetes mellitus: vegetables as potential hypoglycaemic agents.
Nahrung, 41(2): 68-74.
The authors thank Mr S Radhakrishna Pillai for technical Porter JR and Barrett TG (2005) Monogenic syndromes of abnormal glucosehomeostasis: understanding of the pathology of insulin resistance and β cell failure.
J. Med. Genet., 42(12): 893-902.
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