European Journal of Medicinal Chemistry 66 (2013) 400e406
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Original article
Antihyperglycemic and antihyperlipidemic activities of 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol in STZ induced diabetic rats Swapna Sirasanagandla a, Ramesh Babu Kasetti a, Abdul Nabi Shaik a, Rajesh Natava a, Venkata Prasad Surtineni a, Suresh Reddy Cirradur b, Apparao Chippada a, * a b
Department of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2012 Received in revised form 12 May 2013 Accepted 13 May 2013 Available online 8 June 2013
Oral administration of 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol (HBPMP) (30 mg/ kg) to Streptozotocin (STZ) rats produced significant antidiabetic activity after 6 h of HBPMP administration. Treatment of the STZ rats with HBPMP (30 mg/kg/day) for 30 days resulted in a significant decrease in their Fasting Blood Glucose (FBG), Serum Total Cholesterol (TC), Low Density LipoproteinCholesterol (LDL-C), Very Low Density Lipoprotein-Cholesterol (VLDL-C) and triglycerides (TG) along with an increase in serum High Density Lipoprotein-Cholesterol (HDL-C) levels. Activities of Serum Aspartate transaminase (AST), Alanine transaminase (ALT) and Alkaline phosphatase (ALP) and levels of blood urea and creatinine were improved to near normal levels in the treated STZ rats indicating the protective role of the HBPMP against liver and kidney damage and its non-toxic property. In conclusion, HBPMP possesses antihyperglycemic and antihyperlipidemic activities. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: HBPMP STZ Hypolipidemic Hypoglycemic activity
1. Introduction Diabetes mellitus (DM) is a metabolic disease as old as mankind and its incidence is considered to be high (4e5%) all over the world [1]. It is also a major cause of disability and hospitalization and it results in significant financial burden [2]. The number of people suffering from the disease worldwide is increasing at an alarming rate with a projected 366 million peoples likely to be diabetic by the year 2030 as against 191 million peoples estimated in 2000 [3]. The management of diabetes mellitus is considered a global problem and successful treatment is yet to be discovered. Besides the classical drug (insulin, sulfonylureas, biguanides and thiazolidinediones) usage for the treatment of diabetes, several species of plants have been described in scientific and popular literature as having hypoglycemic activity [4e6]. Because of their perceived effectiveness, minimal side effects in clinical experience and relatively low costs, herbal drugs are prescribed widely even when their biologically active compounds are unknown [7]. Despite the great interest in the development of novel drugs to revert the
* Corresponding author. Tel.: þ91 877 2289495 (O); fax: þ91 877 2289532. E-mail address:
[email protected] (A. Chippada). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.05.014
burden of complications associated with this disease and the raised interest in the scientific community to evaluate either raw or isolated natural products in experimental studies only a few of them have been tested in humans [8e12]. Although, non-insulin-dependent diabetes mellitus (NIDDM) is more prevalent than insulindependent diabetes mellitus (IDDM), both are chiefly characterized by chronic hyperglycemia with disturbances of carbohydrate, lipid and protein metabolism resulting from defects in insulin secretion, insulin action or both. Hyperglycemia due to insulin deficiency and insulin resistance has been shown to be associated with the pathogenesis of diabetic dyslipidemia, micro- and macro-vascular complications [13,14]. Dyslipidemia is one of the major cardiovascular risk factors. It has been demonstrated that insulin deficiency in DM leads to a variety of disorders in metabolic and regulatory processes, which in turn leads to accumulation of lipids such as TC and TG in diabetic patients. Abnormalities in lipid profile are one of the most common complications in diabetes mellitus found in 40% of diabetic subjects [15]. Diabetes is associated with profound alterations in the plasma lipids, triglycerides and lipoprotein profile and with an increased risk of coronary heart disease. High level of total cholesterol is one of the major risk factors for coronary heart diseases and it is well known that hyperlipidemia leads to atherosclerosis and this is increased during diabetes [16].
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
OH
(1) Fig. 1. 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol.
73.3 70.0 72.5 75 372.5 223.28 102 36.8 206 75.8 72.0 73.16 79.3 373.5 265.16 215.6 54.8 197.6
4h
5.4 3.9 3.5 6.1 17.33 12.8 (17.5%) 20.2** (35.4%) 15.7** (82.3%) 7.8* (21.6%) 76.3 74.16 74.16 73.6 384.33 301.3 248.8 61.4 213.3
3h
3.7 4.2 4.7 1.5 17.5 13.2 (14.5%) 17.9 (34.2%) 21 (68%) 6.9 (12.8%) 76.5 73.16 73.66 71.3 386.66 312.83 252.1 109.5 237
2h
4.3 4.3 4.3 1.52 19.0* 14.0* 21.3 13.2 5 71.26 74.12 74.16 75.7 389.16 357.5 315.2 233.16 243.5
1h
4.8 4.5 4.5 4.7 19.8y 16.5y 19.8y 11.6y 7.5y 0h
Blood glucose at different hours after treatment (mg/dl)
78.1 75.16 74.5 74.6 390.16 365.9 384.4 347.0 272.8 Normal untreated rats Normal rats treated with 20 mg HBPMP/kg Normal rats treated with 30 mg HBPMP/kg Normal rats treated with 60 mg HBPMP/kg STZ untreated rats STZ rats treated with 20 mg HBPMP/kg STZ rats treated with 30 mg HBPMP/kg STZ rats treated with 60 mg HBPMP/kg STZ rats treated with 20 mg glibenclamide/kg
NH
Group
NH
Table 1 Effect of HBPMP on fasting blood glucose levels (mg/dl) of normal and STZ rats.
OH
2.1. Evaluation of antihyperglycemic activity of HBPMP in STZ rats and normal treated rats
yP < 0.0001 compared with the initial (0 h) level of blood glucose of normal rats. **P < 0.0001 compared with the initial (0 h) level of blood glucose in the respective group. *P <0.001 compared with the initial (0 h) level of blood glucose in the respective group. Numbers in parenthesis indicate the percentage of fall in 0 h blood glucose.
2. Results and discussion
The effects of different doses of HBPMP in normal and STZ rats are summarized in Table 1 and shown in Fig. 2. The fasting blood glucose levels of diabetic untreated rats were significantly higher than those of the normal untreated rats. HBPMP did not produce any hypoglycemic activity in normal treated rats. The HBPMP at a dosage of 30 mg/kg produced 73.8% glucose lowering effect in STZ rats after 6 h of treatment. The dose 20 mg/kg has produced a maximum 44% fall in blood glucose after 5 h of treatment. But the dose of 60 mg/kg has produced hypoglycemia in the STZ rats after 3 h of the treatment. Treatment of STZ rats with Glibenclamide (oral hypoglycemic agent) at a dosage of 20 mg/kg resulted in 31% fall of blood glucose after 5 h of treatment. Fig. 3 shows the effect of HBPMP on glucose tolerance in the STZ rats. The administration of HBPMP at a dose of 30 mg/kg along with 2 g/kg glucose load has significantly improved the glucose tolerance in the STZ rats. In the diabetic untreated rats the glucose levels remained higher without much change even after 3 h after glucose load. Whereas in the HBPMP administered STZ rats the glucose
73.8 70.1 72.8 78 372.2 204.8 172.8 49.1 188.1 4.7 3.5 3.3 4.9 15.6 12.4** (39%) 16.6** (44.01%) 10.8 (84.2%) 8.7** (27.5%)
5h
4.5 2.1 3.1 3.6 16.6 11.5** (44.1%) 12.3** (55.2%) 6.8 (85.8%) 5.2** (31%)
6h
4.4 1.8 2.6 5.5 15.6 12.1 (38.9%) 11.7** (73.8%) 9.7 (89.4%) 5.6** (24.4%)
A large number of crude plant extracts and purified substances from plants have been tested in clinical trials for treatment of diabetes. Other than these, many chemically synthesized compounds were also tested for diabetes with decreasing side effects [17]. Previously many synthetic novel compounds were demonstrated for biological activities. Chalcone-based aryl-oxy-propanolamines were tested for antihyperglycemic activity [18] and N-substituted-N-acyl thioureas of 4-substituted piperazines were endowed with local anesthetic, antihyperlipidemic, antiproliferative, antiarrhythmic, analgesic and antiaggregating actions [19]. We have reported earlier that the aminophenol-substituted amino acid ester 3,30 -(1,4-phenylene)-bis-[2-(1-(benzylcarbonyl)-2,3-dihydro-1H-pyrrolidine-2yl)]-2-thiobenzoxaphosphinine has antihyperglycemic and antioxidant activities [20]. The present study was done to demonstrate the antihyperglycemic and antihyperlipidemic activities of its parent compound, HBPMP (Fig. 1) since the aminophenol is more stable than amino acid ester when it is exposed to air.
401
402
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Fig. 2. Effect of HBPMP on fasting blood glucose levels (mg/dl) of normal and STZ rats.
Fig. 3. Effect of HBPMP on glucose tolerance in STZ rats.
levels started falling from the 1st hour after glucose load and there was a consistent decrease in the blood glucose with maximum fall of 56%, (p < 0.0001) by the end of 3 h after glucose load. STZ was selected for induction of diabetes in rats rather than alloxan. STZ is well known for its selective pancreatic b-cell cytotoxicity and has been extensively used to induce diabetes mellitus
in animals [21] and it is less toxic than alloxan and allows a consistent production of diabetes mellitus. STZ is a monofunctional nitrosurea derivative, one of the most commonly used substances to induce diabetes in the experimental animals [22]. The experimental diabetic model used in this study was type 2 since low dose (50 mg/kg) of STZ destroyed half of the population of pancreatic bcells and there were residual b-cells which secreted insufficient insulin causing type 2 diabetes [23]. Earlier, substitute of aminophenol, an amino acid ester was screened for antihyperglycemic and antioxidant activity. The amino acid ester exhibited a significant antihyperglycemic activity (20). Previously many synthetic drugs were screened for treatment for various diseases. Chalcone derivatives from 3,4-methylenedioxybenzaldehyde and substituted acetophenones [24] were synthesized and investigated as antihyperglycemic agents in a glucoseloaded animal model. Influence of chalcone analogues on serum glucose levels in hyperglycemic rats [25], and antidiabetic activity of 2-amino [50 -(4-sulphonyl benzylidine)-2,4-thiazolidnedione]-7chloro-6-flurobenzothiazole in albino rats were evaluated [26]. The present study was carried out to demonstrate the antihyperglycemic and antihyperlipidemic activities of HBPMP. HBPMP decreased the fasting blood glucose levels effectively in STZ rats and also improved the glucose tolerance in glucose-loaded STZ rats. HBPMP did not cause hypoglycemia in normal treated rats. In the dose-dependent study 30 mg of HBPMP/kg produced a
Table 2 Effect of long-term treatment with HBPMP on blood glucose and body weights. Group
Normal untreated rats Normal rats treated with 30 mg HBPMP/kg/day STZ Untreated rats STZ rats treated with 30 mg HBPMP/kg/day STZ rats treated with 20 mg glibenclamide/kg/day F-value Significance
FBG (mg/dl) at different days during the experimental period and change in body weights after treatment with HBPMP. 1st day
15th day
30th day
Change in body weight (g)
89.0 6.4a 90.6 9.8a
82.66 6.05a 80.66 6.05a
94.8 10.4a 98.67 16.3a
þ18 7.5a (þ8.82%) þ16.6 7.8a (þ2.94%)
416.4 24.2c 333 26.5b
404.2 29.15d 247.5 30.14b,c
12.0 25.8d 113.5 11.36b
45 4.5c (30.3%) þ12 4.4d (þ8.57%)
356 16.18b
204 23.9c
188.1 5.2c
9 7.6d (10%)
242.45 0.000
365.57 0.000
369.55 0.000
23.736 0.00
Values are given as mean S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT). Numbers in parenthesis indicate the percentage of gain or loss of body weights.
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maximum (74%) decrease in the fasting blood glucose (from 384.4 19.8 mg/dl to 102 11.7 mg/dl) levels but the dose of 60 mg/ kg caused hypoglycemia in STZ rats while the same dose did not produce any hypoglycemia in normal treated rats. The lack of hypoglycemia in the normal rats after treatment with 60 mg/kg HBPMP could be due to the efficient activity of the counter regulatory hormones to insulin, while it appears to be lacking in the STZ rats. 2.2. Effect of long-term (30 days) treatment with HBPMP on blood glucose and body weights of normal and STZ rats After the long-term treatment with HBPMP (30 mg/kg/day) there was a significant (66%) fall in fasting blood glucose (FBG) levels in the STZ rats, while there was no change in FBG levels in normal treated rats (Table 2). Treatment of STZ rats with glibenclamide for 30 days has resulted in 47% decrease in their blood glucose levels. This shows that the antidiabetic effect of HBPMP is higher than that of the standard antidiabetic drug glibenclamide. At the end of 30 days treatment, the body weights of normal, HBPMP treated normal, HBPMP treated STZ and glibenclamide treated STZ rat groups increased significantly by 18 g, 16.6 g, 12 g and 9 g respectively, whereas the body weights of untreated STZ rat group decreased by 45 g. In the present study, STZ rats have shown marked reduction in their body weights compared to normal rats (Table 2), which could be due to their uncontrolled diabetes. Streptozotocin induced diabetes is characterized by a severe loss in body weight [27]. The decrease in body weight is due to the loss or degradation of structural proteins (since structural proteins are known to contribute to the body weight) to provide amino acids for gluconeogenesis during insulin deficiency resulting in muscle wasting and weight loss [28]. Several workers have also reported a significant decrease in body weights of STZ rats [29]. The weight loss was reverted by the administration of HBPMP/glibenclamide to the STZ rats and this could be due to the improved blood glucose levels in the HBPMP/glibenclamide treated STZ rats. 2.3. Effect of long-term treatment with HBPMP on hyperlipidemia, hepatic and renal function markers Serum TG, TC, LDL- and VLDL-cholesterol levels were significantly higher and HDL-cholesterol levels were significantly lower in
Fig. 4. Effect of long-term treatment with HBPMP on hyperlipidemia in STZ and normal treated groups. Values are given as mean S.D.
403
Table 3 Effect of HBPMP on atherogenic index (AI) and % of protection against atherogenicity. Group
Atherogenic index (AI)
% of protection from atherogenicity
Normal untreated rats Normal rats treated with HBPMP STZ untreated rats STZ rats treated with HBPMP STZ rats treated with glibenclamide
1.32 1.48 4.95 1.64 2.22
73.3 70.1 e 66.8 55.5
the diabetic untreated rats compared to those in normal rats. After the treatment with HBPMP (30 mg/kg/day), a significant reduction in serum TG, TC, LDL- and VLDL-cholesterol and significant increase in the levels of serum HDL-cholesterol was observed (Fig. 4) and treatment with glibenclamide also produced similar changes in STZ rats. Treatment of STZ rats with HBPMP (30 mg/kg/day) showed protection against atherogenicity as evidenced by 66.8% decrease in the atherogenic index of the HBPMP treated STZ rats (Table 3). Abnormalities in lipid profile are one of the most common complications in diabetes mellitus [15]. Diabetes is associated with profound alterations in the plasma lipid, triglycerides and lipoprotein profile and with an increased risk of coronary heart disease [30]. After 30 days treatment with the HBPMP, the levels of HDL-C were significantly increased and the TG, TC, LDL-C and VLDL-C levels were significantly decreased. This marks to be an important advantage in treatment of hypercholesterolemia particularly among Indians where low HDL-cholesterol is the most prevalent lipoprotein abnormality in diabetic subjects [31]. However, it is interesting to find in the present study that treatment with 30 mg HBPMP/kg has not only lowered the TG, TC and LDL-C levels but also enhanced the cardio protective lipid HDLC after 30 days treatment. There is substantial evidence that lowering the TC, particularly the LDL-C level will lead to a reduction in the incidence of coronary heart disease [32], which is still a leading cause of death in diabetic patients [33]. The activities of serum ALT, AST, ALP and the blood levels of urea and creatinine were increased in diabetic untreated rats when compared with normal rats. After treatment with HBPMP at a dosage of 30 mg/kg for 30 days the activities of ALT, AST and ALP (Fig. 5) and levels of creatinine and urea were significantly decreased to near normal levels in the HBPMP treated STZ rats (Table 4). Similar changes were also observed in glibenclamide
Fig. 5. Effect of long-term treatment with HBPMP on serum AST, ALT and ALP activities of normal and STZ rats. Values are given as mean S.D.
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Table 4 Effect of HBPMP on kidney function in normal and STZ rats after 30 days of treatment. Groups
Urea (mg/dl) Before treatment
Normal untreated rats Normal treated with HBPMP STZ untreated rats STZ rats treated with HBPMP STZ rats treated with glibenclamide F-value Significance
a
41.80 0.80 44.7 0.90a 64.20 1.65b 69.50 2.14b 54.61 1.45c 72.138 0.00
Creatinine (mg/dl) After treatment a
42.75 0.85 43.25 1.37a 75.0 1.15b 54.02 1.828c 49.10 4.613c 53.771 0.00
Before treatment 0.294 0.366 0.732 0.508 0.450 42.623 0.00
a
0.082 0.024b 0.044c 0.026d 0.0428d
After treatment 0.308 0.370 0.947 0.462 0.425 429.09 0.000
0.04a 0.021a 0.020b 0.030c 0.047c
Values are given as mean S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT).
treated rats. There were no changes in the hepatic functional markers in normal treated rats (Fig. 5). In the present study a significant increase in the activities of liver function enzymes (AST, ALT and ALP) were observed in diabetic untreated rats compared to normal control rats. Yanardag et al. (2005) [34] and Rajasekaran et al. (2006) [35] reported that in the STZ rats, the levels of ALT and AST activities were significantly increased. Shinde and Goyal (2003) [36], also reported an elevation of serum hepatic enzymes in STZ rats, indicating the hepatotoxic effect of STZ. ALT and AST are directly associated with the conversion of amino acids to ketoacids. Increased protein catabolism accompanying gluconeogenesis and urea formation that are observed in diabetic state might be responsible for the elevation of ALT and AST. Administration of HBPMP lowered the serum AST, ALT and ALP activities in the HBPMP treated STZ rats. Our findings are in agreement with those of Prakasam et al. (2004) [37]. HBPMP has protective effect against liver toxicity caused by STZ. Treatment with HBPMP in normal rats for 30 days did not produce any hepato toxicity. The STZ rats exhibited significantly higher plasma urea, and creatinine levels compared to the normal rats (Table 4). However, supplementation of HBPMP lowered these plasma values to a control range by enhancing the renal function which is generally impaired in STZ rats. 3. Conclusion From all these observations, it is concluded that the HBPMP possess antidiabetic activity and antihyperlipidemic activity. HBPMP effectively reduces the elevated levels of hepatic and renal markers in diabetic treated group, indicating the hepatoprotective and renal protective role of HBPMP. 4. Experimental design The rats were divided into 9 groups for evaluation of antihyperglycemic activity of HBPMP with different doses (20 mg/kg, 30 mg/kg and 60 mg/kg). Among these groups, 2 were control groups (normal control and diabetic control) and the other 7 groups were treated groups with either HBPMP or glibenclamide (standard oral hypoglycemic agent). For the long-term (30 days) study the rats were divided into 5 groups with 2 control groups and 3 treated groups with HBPMP (30 mg/kg/day) or glibenclamide (20 mg/kg/day). 5. Materials and methods 5.1. Induction of diabetes Diabetes was induced in male Wistar albino rats aged 4 months (body weight 180e200 g) by intra peritoneal administration of STZ (single dose of 55 mg/kg) dissolved in freshly prepared 0.01 M
citrate buffer (pH 4.5). After 48 h rats with marked hyperglycemia (fasting blood glucose 250 mg/dl) were selected and used for the study. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages with 12 h light/dark cycle. All the procedures were performed in accordance with the guidelines of institutional Animal Ethics Committee. 5.2. Evaluation of antihyperglycemic activity of HBPMP in normal and STZ rats The animals were divided into 9 groups and each group consisted of 6 rats: Group 1: Normal untreated rats Group 2: Normal treated rats with 20 mg HBPMP/kg Group 3: Normal treated rats with 30 mg HBPMP/kg Group 4: Normal treated rats with 60 mg HBPMP/kg Group 5: STZ untreated rats Group 6: STZ rats treated with 20 mg HBPMP/kg Group 7: STZ rats treated with 30 mg HBPMP/kg Group 8: STZ rats treated with 60 mg HBPMP/kg Group9: STZ rats treated with 20 mg glibenclamide/kg. After an overnight fast the normal treated groups and STZ treated groups received the HBPMP dissolved in distilled water by gastric intubation using a force feeding needle. Normal untreated and diabetic untreated rats were fed with distilled water alone. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 1, 2, 3, 4, 5, & 6 h duration after the administration of HBPMP and blood glucose levels were determined by using dextrostix (glucose oxidase method) with Basic One Touch Accuchec Glucometer. The results were compared with those of the 9th group of rats which were treated with 20 mg glibenclamide/kg. 5.3. Effect of HBPMP on glucose tolerance Two groups of STZ rats each group containing 6 rats were used for this study. 1. STZ untreated rats 2. STZ rats treated with 30 mg HBPMP/kg. The oral glucose tolerance test was (Bonnar-Weir 1998 [38]) performed in overnight fasted STZ rats. Glucose (2 g/kg) was administered orally to both groups of rats using a force feeding needle. The group 2 rats were administered the HBPMP at a dose of 30 mg/kg along with the glucose load. Blood samples were collected from the tail veins of all the animals from 0 h (before glucose administration) to 3 h of glucose administration for
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
estimation of blood glucose using dextrostix with Basic One Touch Accuchec Glucometer (glucose oxidase method). 5.4. Effect of long-term treatment with HBPMP on glycemic control, serum lipid profiles, hepatic and renal function markers in normal and STZ rats The rats were divided into 5 groups and each group consisted of 6 rats. Group 1: Group 2: Group 3: Group 4: Group 5:
Normal untreated rats. Normal rats treated with 30 mg HBPMP/kg/day. STZ untreated rats. STZ rats treated with 30 mg HBPMP/kg/day. STZ rats treated with 20 mg of glibenclamide/kg/day.
The HBPMP or glibenclamide was administered into the animals of the respective groups every day morning for 30 days by gastric intubation with a force feeding needle. All the 5 groups were sacrificed on the 31st day after an overnight fasting by cervical dislocation and then blood, liver, kidney and pancreas were collected and immediately stored at 20 C till further analysis. Body weights of all the animals were recorded prior to the treatment and sacrifice. 5.5. Biochemical parameters Serum total cholesterol (TC), triglycerides (TG), and HDLcholesterol (HDL-C) were estimated according to the methods of Zlatkis et al. [39], Foster and Dunn [40], and Burstein et al. [42] respectively. The serum levels of VLDL- and LDL-cholesterol were calculated using Friedwald et al.’s formula [41]. The atherogenic index (AI) was calculated by using the following formula [43],
Atherogenic indexðAIÞ ¼
Protectionð%Þ ¼
TC HDL C HDL C
AI of control AI of treated group 100: AI of control
Plasma AST and ALT activities were determined by Reitman and Frankel method [44]. Activity of serum alkaline phosphatase was determined by p-nitrophenyl phosphate method [45]. Plasma creatinine and urea were measured by Jaffe’s method [46] and diacetyl monoxime method [47] respectively. 6. Statistical analysis The results were expressed as mean S.D. The statistical analysis of results was carried out using Student’s t-test and one-way analysis (ANOVA) followed by DMRT. References [1] M. Koyuturk, O. Ozsoy-Sacan, S. Bolkent, R. Yanardag, Effect of glurenorm on immunohistochemical changes in pancreatic b cells of rats in experimental diabetes, Indian J. Exp. Biol. 43 (2005) 268e271. [2] A.N. Nagappa, P.A. Thakurdesai, N.V. Rao, J. Singh, Antidiabetic activity of Terminalia catappa Linn. fruits, J. Ethnopharmacol. 88 (2003) 45e50. [3] S.G. Wild, A. Roglic, R. Green, H. King, Global prevalence of diabetes estimated for the year 2000 and projection for 2030, Diabetes Care 27 (2004) 1047e 1054. [4] E.J. Verspohl, Recommended testing in diabetes research, Planta Med. 68 (2002) 581e590. [5] E. de Sousa, L. Zanatta, I. Seifriz, T.B. Creczynski-Pasa, M.G. Pizzolatti, B. Szpoganicz, F.R. Silva, Hypoglycemic effect and antioxidant potential of kaempferol-3,7-O-(a)-dirhamnoside from Bauhinia forficata leaves, J. Nat. Prod. 67 (2004) 829e832.
405
[6] J.R. Colca, Insulin sensitizers may prevent metabolic inflammation, Biochem. Pharmacol. 72 (2006) 125e131. [7] M.S. Valiathan, Healing plants, Curr. Sci. 75 (1998) 1122e1126. [8] J.P. Lieu, M. Zhang, W.Y. Wang, S. Grimsgaard, Chinese herbal medicines, for type-2 diabètes mellitus (3), Cochrane Database Syst. Rev. (2004) 3642. [9] V. Vuksan, J.L. Sievenpiper, Herbal remedies in the management of diabetes: lessons learned from the study of ginseng, Nutr. Metab. Cardiovasc. Dis. 15 (2005) 149e160. [10] L. Johnson, H. Strich, A. Taylor, B. Timmermann, D. Malone, N. Teufel-Shone, R. Drummond, R. Woosley, E. Pereira, A. Martinez, Use of herbal remedies by diabetic Hispanic women in the southwestern United States, Phytother. Res. 20 (2006) 250e255. [11] M. Jung, M. Park, H.C. Lee, Y.H. Kang, E.S. Kang, S.K. Kim, Antidiabetic agents from medicinal plants, Curr. Med. Chem. 13 (2006) 1203e1218. [12] T. Matsui, I.A. Ogunwande, K.J.M. Abesundara, K. Matsumoto, Antihyperglycemic potential of natural products, Mini Rev. Med. Chem. 6 (2006) 109e120. [13] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813e820. [14] T.A. Clark, G.N. Pierce, Cardiovascular complications of non-insulin, dependent diabetes: the JCR: LA-cp rat, J. Pharmacol. Toxicol. Methods 43 (2000) 1e10. [15] K. Ravi, S. Rajasekaran, S. Subramanian, Antihyperglycemic effect of Eugenia jambolana seed kernel on streptozotocin-induced diabetes in rats, Food Chem. Toxicol. 43 (2005) 1433e1439. [16] B.K. Tan, C.H. Tan, P.N. Pushparaj, Anti-diabetic activity of the, semi purified fractions of Averrhoa bilimbi in high fat diet fed-streptozotocin induced STZ rats, Life Sci. 76 (2005) 2827e2839. [17] A.A. Izzo, E. Ernst, Interactions between herbal medicines and prescribed drugs: a systemic review, Drugs 61 (2001) 2163e2175. [18] M. Satyanarayana, P. Tiwari, B.K. Tripathi, A.K. Srivastava, R. Pratap, Synthesis and antihyperglycemic activity of chalcone based aryl oxy propanolamines, Bioorg. Med. Chem. 12 (5) (Mar 1, 2004) 883e889. [19] A. Ranise, O. Spallarossa, S. Schenone, P. Fossa, G. Menozzi, F. Bondavalli, L. Mosti, A. Capuano, F. Mazzeo, G. Falcone, W. Filippelli, Synthesis of Nsubstituted-N-acylthioureas of 4-substituted piperazines endowed with local anaesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions, Farmaco 58 (9) (2003 Sep) 765e780. [20] 2011 S. Swapna, K. Ramesh Babu, S. Abdul nabi, M. Veera Narayana Reddy, C. Suresh Reddy, C. Appa Rao, Antihyperglycemic and antioxidant activities of 3,30 -(1,4-phenylene)-bis [2-(1-(benzyl carbonyl)-2,3-dihydro-1H-pyrrolidine2-yl)-2-thiobenzoxaphosphinine in STZ rats, Int. J. Drug Des. Discov. 3 (1) (JanuaryeMarch 2012) 677e682. [21] K. Raju, R. Balaraman, Antidiabetic mechanisms of saponins of Momordica cymbalaria, Phcog. Mag. 4 (15) (2008). [22] T. Szudelski, The mechanism of alloxan and streptozotocin action in B cells of rat pancreas, Physiol. Res. 50 (2001) 536e546. [23] A. Gomes, J.R. Vedasiromoni, M. Das, R.M. Sharma, D.K. Ganguly, Antihyperglycemic effect of black tea (Camellia sinensis) in rat, J. Ethnopharmacol. 27 (2001) 243e275. [24] A. Ranise, A. Spallarossa, O. Bruno, S. Schenone, P. Fossa, G. Bondavalli, L. MostiL, A. Capuano, F. Mazzeo, G. Falcone, W. Filippelli, Synthesis of Nsubstituted-N-acylthioureas of 4-substituted piperazines endowed with local, anesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions, Farmaco 58 (9) (2003) 765e780. [25] E.H. Alberton, R.G. Damazio, L.H. Cazarolli, L.D. Chiaradia, P.C. Leal, R.J. Nunes, R.A. Yunes, F.R. Silva, Influence of chalcone analogues on serum glucose levels in hyperglycemic rats, Chem. Biol. Interact. 171 (3) (2008) 355e362. [26] P.S. Yadav, Devprakash, G.P. Senthilkumar, Benzothiazole: different methods of synthesis and diverse biological activities, Int. J. Pharmaceut. Sci. Drug Res. 3 (1) (2011) 01e07. [27] L. Al-Shamaorry, S.M. Al-khazrajoi, H.A. Twaij, Hypoglycemic effect of Artemisia herba alba. II. Effect of a valuable extract on some blood parameters in diabetic animals, J. Ethnopharmacol. 43 (1994) 167e171. [28] L. Rajkumar, N. Srinivasan, K. Balasubramanian, P. Govindarajulu, Increased degradation of dermal collagen in diabetic rats, Indian J. Exp. Biol. 29 (1991) 1081e1083. [29] P. Kalaiarasi, K.V. Pugalendi, Antihyperglycemic effect of 18b-glycyrrhetinic acid, aglycone of glycyrrhizin on streptozotocin-diabetic rats, Eur. J. Pharmacol. 606 (2009) 269e273. [30] M. Maghrani, A. Lemhadri, N.A. Zeggwagh, M. Amraoui, M. Haloui, H. Jouad, M. Eddouks, Effects of an aqueous extract of Triticum repens on lipid metabolism in normal and recent-onset STZ rats rats, J. Ethnopharmacol. 90 (2004) 331e337. [31] R. Gupta, H.P. Gupta, N. Kumar, A.K. Joshi, V.P. Gupta, Lipoprotein lipids and prevalence of hyperlipidemia in rural India, J. Cardiovasc. Risk 1 (1994) 179e183. [32] Lipid Research Clinics Programs, The lipid research clinics coronary primary prevention trial results. 11. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering, J. Am. Med. Assoc. 252 (1984) 365e374. [33] J.W. Baynes, Role of oxidative stress in the development of complications in diabetes, Diabetes 40 (1991) 405e.412. [34] R. Yanardag, O. Ozsoy-Sacan, S. Bolkent, H. Orak, O. Karabulut-Bulen, Protective effects of metformin treatment on the liver injury of streptozotocinSTZ rats, Hum. Exp. Toxicol. 24 (2005) 129e135. [35] S. Rajasekaran, K. Ravi, K. Sivagananam, S. Subramanian, Beneficial effects aloevera leaf gel extract on lipid profile status in rats with streptozotocin diabetes, Clin. Exp. Pharmacol. Physiol. 33 (2006) 232e237.
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[36] U.A. Shinde, R.K. Goyal, Effect of chromium picolinate on histopathological alterations in STZ and neonatal STZSTZ rats, J. Cell Mol. Med. 7 (2003) 332e339. [37] A. Prakasam, S. Sethupathy, K.V. Pugalendi, Antiperoxidative and antioxidant effects of Casearia esculenta root extract in streptozotocin-induced rats, J. Biol. Med. 78 (2005) 15e23. [38] S. Bonnar-Weir, Morphological evidence of pancreatic polarity of beta cells within islets of Langerhans, Diabetes 37 (1998) 616e621. [39] A. Zlatkis, B. Zak, A.J. Boyle, D. Mich, A new method for the direct determination of serum cholesterol, J. Lab. Clin. Med. 41 (1953) 486e492. [40] J.B. Foster, R.T. Dunn, Stable reagents for determination of serum triglycerides by colorimetric Hantzsch condensation method, Clin. Chem. 19 (1973) 338e340. [41] W.T. Friedwald, R.I. Levy, D.S. Fredrickson, Estimation of the concentration of LDL-cholesterol in plasma without the use of the preparative utracentrifuge, Clin. Chem. 18 (1972) 499e502.
[42] M. Burstein, H.R. Scholnichk, R. Morin, Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions, J. Lipid Res. 11 (1970) 583e595. [43] T. Suanarunsawat, W.D.N. Ayutthaya, T. Songsak, J. Rattanamahaphoom, Antilipidemic actions of essential oil extracted from Ocimum sanctum L. leaves in rats fed with high cholesterol diet, J. Appl. Biomed. 7 (2009) 45e53. [44] S. Reitman, S. Frankel, Colorimetric method for the determination of serum glutamic oxaloacetic acid and glutamic pyruvic transaminases, Am. J. Clin. Pathol. 28 (1957) 56e63. [45] O.A. Bessey, O.H. Lowry, M.J. Brock, A method for the determination of alkaline phosphatase with five cubic millimeters of serum, J. Biol. Chem. 164 (1946) 321e329. [46] C. Slot, Plasma creatinine determination. A new and specific Jaffe’s reaction method, Scand. J. Clin. Lab. Invest. 17 (1965) 381e387. [47] D.R. Wybenga, J. Di Giorgio, V.J. Pileggi, Manual and automated methods for urea nitrogen measurement in whole serum, Clin. Chem. 17 (1971) 891e895.
Asian Journal of Biochemical and Pharmaceutical Research Issue 3 (Vol. 4) 2014 ISSN: 2231-2560 CODEN (USA): AJBPAD Research Article
Asian Journal of Biochemical and Pharmaceutical Research In -vitro and In-vivo Studies on the Antidiabetic Activity of Stem Bark of Homalium zeylanicum in STZ Induced Diabetic Rats
Natava Rajesh, Srutineni Venkata Prasad, Shaik Abdul Nabi, Sirasanagandla Swapna, Prabhakar Yellanur Konda, Mohammad Subhan Ali and Chippada Appa Rao* Department of Biochemistry, Sri Venkateswara University, Tirupati - 517 502, India.
Received: 12 July 2014; Revised: 02 August 2014; Accepted: 12 August. 2014
Abstract: Homalium zeylanicum (Family: Flacourtiaceae) commonly known as “Liyan or Mukki” has been widely used in traditional system of medicine for diabetes. The crude aqueous suspension, hexane, ethylacetate, methanol and aqueous stem bark extracts of Homalium zeylanicum were examined each at a concentration of 50g/L using an in vitro method and compared to control and standard (Insulin). The methanolic extract showed a significant inhibitory effect on in vitro glucose diffusion and these extracts were also screened at 0.5 g/kg b.w for the in vivo antidiabetic activity on STZ induced diabetic rats using glibenclamide (0.02g/kg b.w) as a reference standard. The methanolic extract showed a significant antidiabetic activity and was more effective in reducing the blood glucose levels compared to that of standard drug glibenclamide, thus validating the traditional claim of the plant. However, more experiments at the clinical levels are required to confirm the utility of this plant by traditional practitioners in the treatment of diabetes mellitus. Keywords: Homalium zeylanicum; Stem bark extracts; Antidiabetic activity; Glucose diffusion; STZ; OGTT.
INTRODUCTION: Diabetes mellitus is wide spread disorder ,which has long been in the history of medicine. This is a chronic and major endocrine disorder caused by inherited and/or acquired deficiency in the production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. Diabetes mellitus is classified as: Insulin-Dependent Diabetes Mellitus (IDDM) and NonInsulin-Dependent Diabetes Mellitus (NIDDM). About 90% of patients are NIDDM with insulin resistance playing a key role in the development of the disease [1]. This is a major threat to global public health that is rapidly getting worse, and the biggest impact is on adults of working age in developing countries. In most developing countries at least one in ten deaths in adults aged 35–64 is attributable to diabetes, and in some the figure is as high as one in five [2]. Diabetes is a common condition and its frequency is dramatically rising all over the world. This disease has reached epidemic proportion and has become one of the most challenging health problems of the 21st century. In 2010, there were 285 million people with diabetes worldwide, and by 2030 this figure is expected to nearly double to reach a total of 439 million [3]. It is becoming the third “killer” of mankind after cancer and cardiovascular diseases, because of its high prevalence, morbidity and mortality [4].
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In modern medicine there is no satisfactory effective therapy to cure diabetes. The management of diabetes without any side effect is still a challenge to the medical system. Numerous therapies designed for the treatment of DM have proven to be fairly effective, but none is ideal due to undesirable side effects and diminution after prolonged use. Currently, the oral hypoglycemic drugs are sulphonylureas and few biguanide groups have been used in the treatment of diabetes mellitus, but they are unable to lower glucose concentration to within normal range and reinstate a normal pattern of glucose homeostasis permanently. Use of these therapies is restricted by their pharmacokinetic properties, secondary failure rates and accomplying side effects [5] Even insulin therapy does not reinstate a permanent normal pattern of glucose homeostasis, and carries an increased risk of atherogenesis and hypoglycemia. Hence the anti diabetic drug discovery has shifted its focus to natural plant sources having minimal side effects [6]. In recent years, finding for novel type of antidiabetic drug from plant materials have been a new direction. World Health Organization (WHO) has also recommended the evaluation of traditional plant treatments for diabetes as they are effective, nontoxic, with less or no side effects and are considered to be excellent candidates for oral therapy. Following the recommendations made by the WHO on the beneficial uses of medicinal plants for the treatment of diabetes mellitus [7], investigations of hypoglycemic agents from medicinal plants have also become more important.Plants have been the major source of drugs in Indian system of medicine and other ancient systems in the world. Earliest description of curative properties of medicinal plants was found in Rig Veda (2500-1800 BC). Charaka Samhita and Sushrutha Samhita give extensive description on various medicinal herbs. Information on medicinal plants in india has been systematically organized [8] Ethanobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes [9], but many plants do not have a scientific scruting. Herbal medicines are used for primary health care, by about 80% of the world population particularly in the developing countries, because of better cultural acceptability, safety, efficacy, potent, inexpensive and lesser side effects [10]. The plant drugs are frequently considered to be less toxic when compared to synthetic drugs [11]. More than 1123plant species have been used to treat diabetes and more than 200 pure compounds have showed, lowering blood glucose activity [12]. There is now a greater interest in the scientific community to evaluate both crude and isolated natural products in experimental studies and traditional medicines have always been proved to be a fruitful source of future drugs to counteract any disease including insulin resistance, consistent with a resurgence of interest in drug discovery from natural products [13, 14, 15]. Plants are rich sources of antidiabetic, antihyperlipidemic and antioxidant agents such as flavonoids, gallotannins, amino acids and other related polyphenols. Flavonoids and polyphenols are being used to treat diabetes [16]. This is based on the fact that, excessive oxidative stress is implicated in the pathology and complications of DM and polyphenols with antioxidant properties exert beneficial antidiabetic effect by correcting the disturbed oxidative milieu in diabetic conditions [17, 18]. Wide array of plantderived active principles were shown to have antidiabetic activity [19]. Homalium zeylanicum commonly known as” Liyan or Mukki” (HZ) is an indigenous plant belonging to the family Flacourtiaceae. The bark and leaf of the plant is having many traditional uses like antidiabetic, rheumatism, wound healing, hepatoprotective and anthelimintic activities[20-23]. The present study 77
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was designed to evaluate the potential antidiabetic activity of the HZ stem bark in streptozotocininduced diabetic rats. MATERIALS AND METHODS: Collection of plant material: Homalium zeylanicum is in and around Tirumala hills, Andhra Pradesh, botanically identified by Dr. Madhava chetti, Dept. of botany, S.V.University, Tirupati. A voucher specimen was deposited in the herbarium, Dept. of botany, S.V.University, Tirupati. The stem bark was shade dried, mechanically ground and the obtained bark powder was used for evaluating its antidiabetic activity. Experimental animals: Male albino wistar rats of age 3-4 months (body weight 180–200 g), were procured from Sri Venkateswara Traders, Bangalore. Animals were housed in clean, dry polypropylene cages and maintained in a well-ventilated animal house with 12 h light- 12 h dark cycle, and provided with free access to standard pellet diet and water ad libitum at uniform laboratory conditions as per the guidelines of Institute Animal Ethics committee. Preparation of solvent extracts of the stem bark of HZ: For the present study, the extracts were prepared by continuous hot percolation method using soxhlet apparatus. 250 gm of bark powder was packed in soxhlet apparatus and extracted using Hexane, Ethyl acetate & Methanol successively. Aqueous extract was prepared by soaking the bark powder in distilled water in a glass jar for two days with occasional stirring and then the solvent was filtered. It was repeated 3 to 4 times until the filtrate give no colouration. All the filtrates were distilled, concentrated under reduced pressure in the Buchi Rotavapour R-200 and the extracts were calculated for their yield and stored for further use. The extracts were designated as HEHZ (Hexane extract), EAEHZ (Ethylacetate extract), MEHZ (Methanol extract) & AEHZ (Aqueous extract) respectively. All the extracts were subjected to phytochemical screening using standard procedures. Phytochemical Tests: The preliminary phytochemical screening of the aqueous crude suspension and different extracts of H. zeylanicum was carried out in order to ascertain the presence of its constituents utilizing standard conventional protocols [24].
Effect of different extracts on glucose diffusion through dialysis membrane (In-vitro antidiabetic activity): A simple model system was used to evaluate the effects of plant extracts on glucose movement in vitro. The model was adapted from a method described by Edwards et al [25] which involved the use of a sealed dialysis tube into which solution of glucose (0.22M) and sodium chloride (0.15M) was introduced and the appearance of glucose in the external solution was measured. The model used in the present experiment consisted of dialysis tubes (6cmX15mm). In each tube 1ml of 50g/liter different extract of the plant in 1% CMC was added to 1ml of 0.15M sodium chloride containing 0.22M D-glucose. 1ml of insulin (40 Units) was used as standard, whereas 1% CMC was used as control. The dialysis tube was sealed at each end placed in a 50ml centrifuge tube containing 45ml of 0.15M sodium chloride. The tubes were placed on an orbital shaker and kept at room
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temperature and the movement of glucose into the external solution was monitored at set time intervals(i.e. , 3,6,12,24 hrs). Evaluation of possible toxicity of methanolic extract of stem bark of HZ: Acute toxicity studies on HZ extract were performed in normal rats. Animals were starved over night and divided into five groups each group consisting of 3 rats. They were fed orally with either the aqueous crude suspension of HZ or MEHZ in increasing doses (0.1, 0.25, 0.5, 0.75 and 1g/kg body weight) and observed for mortality up to 72 h. The animals were observed continuously for 2h following extract administration [26] for the following • Behavioral profile: alertness, restlessness, irritability, and fearfulness. • Neurological profile: spontaneous activity, reactivity, touches response, pain response and gait. • Autonomic profile: defecation and urination. Number of deaths, if any, were recorded after 24 and 72h. Determination of the blood glucose levels: Blood glucose concentrations (mg/dl) were determined by using dextrostix with Basic One Touch Accucheck Glucometer (Glucose oxidase method). Blood samples were collected from the tip of tail at the defined time patterns. Experimental Design: Evaluation of antihyperglycemic activity in H. zeylanicum stem bark aqueous crude suspension: The animals were divided into five g roups and each group consisted of 6 rats. 1. Group I : Diabetic rats treated with 400 mg ACSHZ/kg b.w 2. Group II : Diabetic rats treated with 500 mg ACSHZ/kg b.w 3. Group III : Diabetic rats treated with 600 mg ACSHZ/kg b.w 4. Group IV : Normal control 5. Group V : Diabetic control After an overnight fast, diabetic rats I Group I, II & III (n-6) received the ACSHZ of different concentrations (viz., 400,500 & 600 mg/Kg bw) by gastric intubation using a force feeding needle. Group IV &V served as controls, and they were fed with water alone. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 1, 2, 3, 4, 5, & 6 hours after the administration ACSHZ and blood glucose levels were determined by using dextrostix with Basic One Touch Accucheck Glucometer (Glucose oxidase method).
Evaluation of anti hyperglycemic activity of different extracts of the stem bark of H. zeylanicum: After an overnight fast the diabetic rat groups received the viz hexane extract (HEHZ- dissolved in tween 20), ethyl acetate extract (EAEHZ), methanolic extract (MEHZ) and aqueous extract (AEHZ) (dissolved in water) by gastric intubation using a force feeding needle. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 1, 2, 3, 4, 5, & 6 hours after the administration of extracts and blood glucose levels were determined by using dextrostix with Basic One Touch Accuchec Glucometer (Glucose oxidase method). The animals were divided in to 7 groups and each group consisted of 6 rats. 1. Group I : Diabetic rats treated with 0.5g HEHZ /kg b.w 2. Group II : Diabetic rats treated with 0.5g EAEHZ/kg b.w 79
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3. Group III : Diabetic rats treated with 0.5g MEHZ /kg b.w 4. Group IV : Diabetic rats treated with 0.5g AEHZ/kg b.w 5. Group V : Normal untreated rats 6. Group VI : Diabetic untreated rats 7. Group VII : Diabetic rats treated with 0.02g glibenclamide/kg b.w. 8. Effect of MEHZ on glucose tolerance: The oral glucose tolerance test was [27] performed in overnight fasted diabetic rats. Glucose (2 g/ Kg b.w) was administered orally to both groups of rats using a force feeding needle. The group 2 rats were administered the MEHZ at a dose of 0.5g/k.g b.w along with the glucose load. Blood samples were collected from the tail veins of all the animals from 0hr (before glucose administration) to 3hr of glucose administration for estimation of blood glucose using dextrostix with Basic One Touch Accuchec Glucometer (Glucose oxidase method). Two groups of diabetic rats each group containing 6 rats were used for this study. 1. Group I: Diabetic untreated rats 2. Group II: Diabetic rats treated with 0.5 g MEHZ / k.g b.w Statistical analysis: All the data were statistically evaluated and the results were expressed as mean ± S.D.
RESULTS: The percentage yield of H. zeylanicum stems bark extracts: The percentage yield of hexane, ethylacetate, methanol and aqueous extracts of H. zeylanicum stem bark were 7.8%, 8.6%, 11.9 % and 24 % w/w respectively (Table 1). All the extracts were stored in air tight containers and stored for further use. Preliminary phytochemical screening of the extracts of H. zeylanicum : Preliminary phytochemical screening of the various extracts of H. zeylanicum revealed the presence of various components such as steroids, triterpenes, saponins, alkaloids, carbohydrates, flavonoids, tannins and glycosides among them flavonoids and tannins were the most prominent ones and the results were summarized in Table 2. Effect of different extracts on glucose diffusion through dialysis membrane (In-vitro antidiabetic activity): Among the different extracts the methanolic extract could inhibit the diffusion of glucose more effectively than all other extracts and the glucose concentration in the external solution was 215±6.55 mg/dl whereas in the control the mean glucose concentration in the external solution was 300mg/dl (326±3) at the end of 24 hrs. The invivo inhibitory activity cannot bealways related to in vivo activity. Thus the concept needs to be demonstrated in preclinical animal studies. Accordingly all these extracts were selected for the in vivo study. The effect of various extracts of stem bark of H.zeylanicum on glucose diffusion inhibition was depicted in Fig. 1. Acute toxicity study: Acute toxicity studies revealed the non-toxic nature of stem bark of HZ. The results of acute toxicity studies of the ACSHZ and MEHZ of H.zeylanicum stem bark (0.1, 0.25, 80
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0.5,0.75 and 1g/kg body weight) on the normal rats indicated that the stem bark extracts were non toxic up to the maximum dosage of 1g/kg body weight. There were no lethality or toxic reactions found with any of the selected doses of HZ stem bark suspension or extract until the end of the study period which indicates that the stem bark was non -toxic up to the maximum dosage of 1g/kg body weight. All the animals were alive and active during the observation period. In vivo antidiabetic activity: Evaluation of antihyperglycemic activity in H.zeylanicum stem bark aqueous crude suspension. The effect of different doses of aqueous crude suspension of H.zeylanicum bark on the fasting blood glucose levels of diabetic rats is shown in Table 3. The aqueous crude suspension at a dose of 500 mg/kg b.w produced maximum fall of 61.02% in the blood glucose level of diabetic rats after 6hrs of treatment compared to other doses 400 mg/kg b.w and 600 mg/kg b.w which have produced a 34.39% and 55.07% fall in FBG respectively. All the doses did not produce any hypoglycemic activity. Evaluation of anti hyperglycemic activity of different extracts of the stem bark of HZ: The effect of hexane, ethyl acetate, methanolic and aqueous extracts of H.zeylanicum stem bark on the fasting blood glucose levels of diabetic rats is shown in Table 4. The methanolic extract at a dose of 0.5g/Kg b.w produced the maximum fall of 69.31%, (p<0.0001) in blood glucose levels of the diabetic rats after 6 hrs of treatment. The hexane, ethyl acetate and aqueous extracts each at a dose of 0.5g/Kg also produced antihyperglycemic activity with maximum fall of 12.8%, 22.5% and 48.72% respectively in the blood glucose of the diabetic treated rats. The glibenclamide has produced a maximum fall of 30.9% in blood glucose of the group VII rats after 5hrs of the treatment. The efficacy of methanolic extract (0.5g/Kg b.w) in reducing blood glucose levels in diabetic rats is much higher than that of hexane, ethyl acetate, aqueous extracts (0.5g/kg b.w) and glibenclamide (0.02g/kg b.w). Effect of MEHZ on glucose tolerance: The administration of MEHZ at a dose of 0.5g/k.g b.w along with 2g/k.g b.w glucose load has significantly improved the glucose tolerance in the diabetic rats. In the diabetic untreated rats the glucose levels remained higher without much change even after 3hr after glucose load. Where as in the MEHZ administered diabetic rats the glucose levels started falling from the 1st hr after glucose load and there was a consistent decrease in the blood glucose with a maximum fall of 41.96% (Fig. 2) by the end of 3 hr after glucose load.
DISCUSSION: Diabetes mellitus (DM) is one of the most important health problems worldwide and can be defined as a disease characterized by chronic hyperglycemia, which results from defects in insulin secretion and/or action, giving rise to impaired function in carbohydrate, lipid and protein function [28].Several pathogenic processes are involved in the development of diabetes. These range from autoimmune destruction of the cells of the pancreas with consequent insulin deficiency to abnormalities that result in resistance to insulin action. All forms of DM are characterized by hyperglycemia and the development of diabetes-specific complications. These complications can result in disastrous consequences of economic and social systems, but many synthetic drugs used today failed to complete a long-term glycemic control and alter the course of diabetic complications. Clinically, novel 81
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treatments with fewer side effects are desirable for the control of DM and its complications [29]. Plants play a major role in the discovery of new therapeutic agents and have received much attention as sources of biologically active substances including antioxidants, hypoglycemic and hypolipidemic agents [30]. In pursuit of this goal, several medicinal plants are being investigated for possible hypoglycemic activities based on several approaches including ethanobotanical survey. The hypoglycaemic actions of some of these phytochemical constituents have been evaluated and confirmed in animal models [31,32] suggesting that natural products could serve as a source in the search for effective antidiabetic agents. The traditional system of medicinal plants and practices have been a imperative resource in many countries to control various complications of diabetes mellitus as they are considered to be less toxic and free from side effects than synthetic molecules. Antihyperglycemic activities of most effective plants were in part explained by the ability of the phytoconstituents to increase glucose transport and metabolism in muscle and/or to stimulate insulin secretion [33-39]. The current study was designed to explore the antidiabetic potential of H.zeylanicum stem bark extracts using in vitro assay and the in vivo antidiabetic effect was scrutinized using STZ-induced diabetic model. The methanolic extract showed a significant inhibitory effect on in vitro glucose diffusion. The in vitro inhibitory activity cannot be always related to in vivo activity [40]. Thus the concept needs to be demonstrated in preclinical animal studies. Accordingly all these extracts were selected for the in vivo study. The results of the present study showed that administration of HZACS to STZ induced diabetic rats at a dose of 0.5g/kg b.wt. produced a significant reduction (61%), (p<0.0001) in blood glucose level. Among the different solvent extracts MEHZ at a dose of 0.5g/kg b.wt. produced a significant reduction (69%), (p<0.0001) in blood glucose level in the diabetic rats while the other extracts could produce less antihyperglycemic activity. The administration of MEHZ at a dose of 0.5g/k.g b.w along with 2g/k.g b.w glucose load has significantly improved the glucose tolerance in the diabetic rats with consistent decrease in the blood glucose with a maximum fall of 41.96%. The antidiabetic activity of MEHZ stem bark may be due to its stimulating effect on the remnant beta cells or improvement in insulin action at cellular level or it could also be due to its insulin like effect. However the methanolic extract was found to be more effective than the hexane, ethylacetate and aqueous extract. No dose-dependent effect was observed on increasing the dose further. Such a phenomenon of low hypoglycemic response at higher dose is common with indigenous plants and has been observed earlier with many plants like Aegle marmelos [41], Murraya koenigii [42] and Cinnamomum tamala [43], Eugenia jambolana [44] Terminalia pallida fruit [45], Psacalium decompositum [46]. The decreased activity at a higher dose of the extract could be due to reduced or no effect of components present in the extract at higher doses [47, 48]. It is also likely that the higher doses could not produce the expected higher hypoglycemic effect because of the presence of some other substances, which interfere with the hypoglycemic effect. In this context, the presence of some hyperglycemic compounds also along with hypoglycemic compounds have been reported [49] in three plants, Trigonella foenum graecum (fenugreek) seeds, Ficus bengalensis (banyan tree) bark and Momordica charantia (bitter gourds). So, higher doses of the extract might have higher doses of hyperglycemic compounds. Thus, 0.5g/kg b.wt. was found to be the effective dose of H. zeylanicum on FBG of normal as well as diabetic animals. Further studies are under progress to identify the active antihyperglycemic compound(s) 82
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in methanolic extract. The results of the study strongly suggest that Homalium zeylanicum the treatment of Diabetes mellitus.
is useful in
CONCLUSION: The present investigation is an evidence for the invitro and invivo anti-diabetic activity of Homalium zeylanicum in STZ induced diabetic rats. The provide the evidence that MEHZ could be considered as an excellent candidate for further studies on verifying the mechanisms of hypoglycemic activity, as well as for the isolation and identification of the foremost hypoglycemic phyto-chemical responsible for anti-diabetic activity of the plant. Besides, further comprehensive pharmacological surveys, involving experimental chronic studies, will be of value to assess the possible toxicological effects of this anti-diabetic plant. ACKNOWLEDGEMENTS: The author is grateful to parents for their moral and financial support and Prof. Ch.Apparao for supervising the research work. He also wishes to thank Prof. O.V.S. Reddy, each and every one whoever supported throughout the work.
Fig. 1 Effect of various extracts of stem bark of Homalium zeylanicum (50g/litre) on the movement of glucose out of dialysis tube over 24hr incubation period.
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Fig. 2 Effect of MEHZ on glucose tolerance in STZ induced diabetic rats
Table 1 Percentage yield of different extracts of Homalium zeylanicum Extracts
Percentage of yield
HEHZ
7.8 % w/w
EAEHZ
8.6 % w/w
MEHZ
11.9 % w/w
AEHZ
24 % w/w
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Table 2 Preliminary phytochemical screening of the extracts of Homalium zeylanicum S.No
Components
Aqueous crude
Hexane
suspension
Ethyl
Methanol
Water
acetate
1
Steroids
_
_
_
_
_
2
Triterpenes
+
+
_
+
+
3
Saponins
_
_
_
_
_
4
Alkaloids
+
_
+
+
+
5
Carbohydrates
+
+
+
+
+
6
Flavonoids
++
_
+
++
+
7
Tannins
+
+
+
++
+
8
Glycosides
_
_
_
+
_
“+” indicates presence, “++” indicates rich presence, “_” indicates absence
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Table 3 Effect of ACSHZ on fasting blood glucose levels (mg/dl) in STZ induced diabetic rats S.No
Groups
0h
1h
2h
3h
4h
5h
6h
1
Group-I
347±53*
322±48.5
295±42
268±33.4
239±40.1
199±33.3
182±38.3** (34.39%)
2
Group-II
325±49.4*
317±59.6
289±59.8
259±54.4
226±48.1
185±42.8
146±38.9** (55.07%)
3
Group-III
331±30.6*
313±26.6
269±21.1
236±22.0
199±17.0
163±17.6
129±16.0** (61.02%)
4
Group-IV
77±3.2
86±5
75±9.8
76±3.5
79±3.5
75±4
77±8.1
5
Group-V
385±37.1
354±39
363±30.7
357±16
353±28.4
366±28.5
380±29.5
Group-I, Group-II, & Group-III: Diabetic treated (viz. 400, 500, 600 mg/kg b.w), Group-IV: Normal control, Group-V: Diabetic control. Values are given as mean ± S.D (n = 6). *
P<0.001 compared with the initial level of blood glucose (0hr) of Normal rats.
**
P<0.0001 compared with the initial level of blood glucose (0hr) in the respective group.
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Table 4 Effect of different extracts of HZ stem bark on fasting blood glucose levels (mg/dl) in STZ induced diabetic rats. S.No Groups
0h
1h
2h
3h
4h
5h
6h
1
351±23.3*
343±23.0
336±23.1
330±22.54
320±23.45
313±2402
306±23.4†
Group-I
(12.8%) 2
Group-II
341±33.1*
329±33.4
315±31.7
287±33.7
287±31.8
273±31.6
265±34.1** (22.5%)
3
Group-III
332±53.0*
254±34.2
228±47.7
198±37.8
164±33.0
140±31.9
103±23.4** (69%)
4
Group-IV 345±51.6*
355±53.3
322±54.2
285±56.6
243±51.4
214±53.8
177±46.3** (48.72%)
5
Group-V
6 7
85±5.7
82±1.7
77±7
76±3.7
82±3.5
82±1.7
80±7.5
Group-VI 374±15.8*
366±13.7
363±23.8
370±11.5
342±11
351±25.1
355±12.3
Group-VII 250.5±9**
227.6±14
218.5±13*
208.6±14**
190.3±14**
173±13.6**
190±12
(9.10%)
(12.70%)
(16.7%)
(24%)
(30.9%)
(24.15%)
Group-I, Group-II Group-III, Group-IV: Diabetic treated, Group-V: Normal control, Group-VI: Diabetic control and Group-VII: Diabetic treated with glibenclamide. Values are given as mean ± S.D (n = 6). * P<0.0001 compared with the initial level of blood glucose (0h) of normal rats. †P<0.001 compared with the initial level of blood glucose (0h) in the respective group. ** P<0.0001 compared with the initial level of blood glucose (0h) in the respective group. Numbers in parenthesis indicate the percentage of fall in 0h blood glucose. 87
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REFERENCES: 1. J. H. Fuller, M. J. Shipley, G. J. Rose, R. J. Jarrett and H. Keen., Lancet., 1980, 1, 1373–1376. 2. WHO. Diabetes action now: an initiative of the World Health Organization and the International Diabetic Federation. Geneva, Brussels: WHO, IDF., 2004, 1–20. 3. J. E. Shaw, R. A. Sicree, P. Z. Zimmet., Diabetes Res Clin Pract., 2010, 87, 4–14. 4. W. L. Li, H. C. Zheng, J. Bukuru and N. De Kimpe., J Ethnopharmacol., 2004, 92, 1-21. 5. A. Melinda., Non insulin dependent diabetes mellitus treatment with sulphonylureas in Clinical and Endocrinology and metabolism. Des natures, M. and Hale, P., Balliere- Tindall, London., 1988, 443-453. 6. Y. Nayak, V. P. Veerapur, A. N. Nagappa and M. K. Unnikrishnan, Perspectives inefficacy, safety and clinical evaluation of bioactive natural products. Compendium of Bioactive Natural Products, USA: M/S. Studium Press LLC., 2009, 2,1–30. 7. The WHO Expert Committee on diabetes mellitus. Technical Report Series. World Health Organization., 1980. 8. G.V. Satyavati, A.K. Gupta; Medicinal plants of India eds Indian Council, New Delhi; Vol-II. Shankar, TNB; N.V Shanta, H.P Ramesh, I.A.S. Murthy, V.S. Murthy; Ind. J. Exp. Biol., 1987, 18, 73. 9. P. Pushparaj, C. H. Tan and B. K. H. Tan., J Ethnopharmacol., 2000, 72, 69-76. 10. T. Pullaiah and K. Chandrasekhar Naidu., Antidiabetic plants in india and herbal based antidiabetic research, Regency Publications, New Delhi., 2000, 3-9. 11. L. Pari and J. Uma Maheswari., J Ethanopharmacol., 2000, 14, 1-3. 12. J. K. Grover, S. Yadav and V. Vats., J Ethanopharmacol., 2002, 81, 81-100. 13. F. E. Koehn and G. T. Carter., Nat Rev Drug Discov., 2005, 4, 206–220. 14. M. Bnouham, A. Ziyyat, H. Mekhfi, A. Tahri and A. Legssyer., Int j Diabetes & Metabolism., 2006, 14, 1-25. 15. T. S. Frode and Y. S. Medeiros., J Ethanopharmacol., 2008, 115, 173-183. 16. F. Martinello, S. M. Soares, J. J. Franco, A. C. Santos, A .Sugohara and S. B. Garcia., Food Chem Toxicol., 2006, 44, 810–818. 17. M. A. Abdelmoaty, M. A. Ibrahim, N. S. Ahmed and M. A. Abdelaziz., Indian J. ClinBiochem., 2010, 25, 188–192. 18. A. K. Tiwari and M. J. Rao., Curr Sci., 2002, 83, 30-38. 19. K. R. Prabhakar, V. P. Veerpur, P. Bansal, V. K. Parihar, M. Reddy Kandadi and P. Bhagath Kumar., Chem Biol Interact., 2007, 165, 22-32. 88
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Issue 3 (Vol. 4) 2014 CODEN(USA) : AJBPAD
20. K .Madhavachetty, K .Sivaji and R. K. Tulasi., Flowering of plants of chittoor district, Tirupati., Students offset printers., 2008. 45. 21. S. S. Sandhya, Sai Kumar, K. R. Vinod, David Banji and K. Kumar., Hygeia.J.D.Med., 2011, 3 (1), 11-19. 22. T. Shashank, Ellandala Rajkiran, Nusrath yasmeen, K. Sujatha and K. Vishal Reddy., International Journal of PharmTech Research., 2011, 3(3), 1630-1634 23. K. Gnananath, G. Pavan Kumar, K. Ramakanth Reddy, B. Naveen Kumar and R. Vinod Kumar., International Research Journal of Pharmacy., 2012, 3 (5) 24. J. B. Harborne., Phytochemical Methods. Analysis. A Guide to Modern Techniques of Plant. 3rd ed. Spinger International, India, 2005. 25. C. A. Edwards, N. A. L. Black burn, P. Craigne, J. Daavidson, K. Tomlin, I. T. Sugde, Johnson and N. W. Read., Am J Cli Nutr., 1987, 46, 72-77. 26. M. A. Turner., In: Screening methods in pharmacology. Academic press, New York, 1965; 26. 27. S. Bonner-Weir., Diabetes., 1988, 37, 616-621. 28. Committee Report, Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care., 1997, 20, 1183–1197. 29. S. L. Jeffcoate., Diabetes control and complications: the role of glycated haemoglobin 25 years on. Diabet. Med., 2004, 21, 657–665. 30. R. Marles and R. Farnsworth., Phytomedicine., 1995, 2, 137–189. 31. H. J. Hwang, S. W. Kim, J. M. Lim, J. H. Joo, H. O. Kim and J. W. Yun., Life Sci., 2005, 76, 3069–3080. 32. J. A. Vinson and J. Zhang., J. Agric. Food Chem., 2005, 5,3710– 3713. 33. A. M. Gray and P. R. Flatt., Br J Nutr., 1997, 78, 325–334. 34. A. M. Gray and P. R. Flatt., J Endocrinol., 1998, 157, 259–266. 35. A. M. Gray and P. R. Flatt., Br J Nutr., 1998, 80, 109–114. 36. A. M. Gray and P. R. Flatt., J Nutr., 1998, 128, 2319–2323. 37. A. M. Gray and P. R. Flatt., J Endocrinol., 1999, 160, 409–414. 38. A. M. Gray and P. R. Flatt., Br J Nutr., 1999, 81, 203–209. 39. A. M. Gray, Y. H. A. Abdel-Wahab and P. R. Flatt., J Nutr., 2000, 130, 15–20. 40. M. Rammohan Subramanian, Zaini Asmawi and Amirin Sadikun., Acta Biochimica Polonica., 2008, 55, 391-398.
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Issue 3 (Vol. 4) 2014 CODEN(USA) : AJBPAD
41. S. R. Sharma, S. K. Dwivedi, V. P. Varshney and D. B. Swarup., Phytother Res., 1996, 10, 426–428. 42. A. N. Kesari, R. K. Gupta and G. Wata., J Ethnopharmacol., 2005, 97, 247–251. 43. S. R. Sharma, S. K. Dwivedi and D. Swaroop., Indian J Exp Biol., 1996, 34, 372–374. 44. B. K. Rao, P. R. Sudarshan, M. D .Rajasekhar, N. Nagaraju and Ch. A. Rao., J Ethnopharmacol., 2003, 85, 169–172. 45. F. J. Alarcon-Aguilar, M. Jimenez-Estrada, R. Reyes-Chilpa and R. Roman-Ramos., J Ethnopharmacol., 2000, 72, 21–27. 46. P. S. M. Prince, V. P. Menon and G. Gunasekharan., J Ethnopharmacol., 1999, 64, 53–57. 47. K. B. Rao, M. M. Kesavulu, R. Giri and Ch. A. Rao., J Ethnopharmacol., 2001a, 74, 69–74. 48. K. B. Rao, M. M. Kesavulu, R. Giri and Ch. A. Rao., J Ethnopharmacol., 2001b, 78, 67–71. 49. P. S. Murthy, R. Moorti, S. Pugazhenthi, B. V. Babu, K. M. Prabhu, P. Ratnakar, R. Shukla, D. Puri, G. Dev, U. Rusia and S. Aggarwal, Trends Clin Biochem Lab Med., 2003, 635–639.
*Correspondence Author: Chippada Appa Rao, Department of Biochemistry, Sri Venkateswara University, Tirupati - 517 502, INDIA..
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ISSN 0973 – 9874
J.Pharm.Chem
CODEN: JPCOCM
Journal of Pharmacy and Chemistry (An International Research Journal of Pharmaceutical and Chemical Sciences) Indexed in Chemical Abstract and Index Copernicus www.stfindia.com Volume 6 • Issue 3 • July – September 2012
Antidiabetic and Antioxidant Activities of Piper longum root Aqueous Extract in STZ induced Diabetic Rats 1
1
1
SHAIK ABDUL NABI , MD. SUBAN ALI , RAJESH NATAVA , 1 1 THANDAIAH KRISHNA TILAK , AND CHIPPADA APPA RAO* 1
Department of Biochemistry, Sri Venkateswara University, Tirupati, India-517502
ABSTRACT This study was designed to investigate the antidiabetic and antioxidant activities of the aqueous extract of roots of Piper longum (AQRPL) in streptozotocin (STZ) induced diabetic rats. Oral administration of aqueous extract at a dosage of 200 mg/ kg body weight for 30 days showed significant decrease in fasting blood glucose (FBG), hepatic and renal thiobarbituric acid reactive substances (TBARS) and catalase (CAT) levels. There is a significant improvement in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione - s- transferase (GST) in liver and kidney of STZ induced diabetic rats when compared with untreated diabetic rats. These results clearly indicate that Piper longum (PL) root aqueous extract possess antidiabetic and antioxidant effect in diabetic rats. Keywords: Antidiabetic activity, Piper longum, Streptozotocin, Antioxidant activity and superoxide dismutase.
Introduction Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, glycosurea and negative nitrogen balance and it is mainly due to either lack of insulin secretion from beta cells of pancreas or desensitization of insulin receptors for insulin. It is the most prevalent disease in the world affecting 25% of population and afflicts 150 million people and is set to rise to 300 million by 2025 [1]. It causes number of complications like retinopathy, neuropathy, and peripheral vascular insufficiencies [2].
Oxidative stress is currently suggested as mechanism underlying diabetes and diabetic complications.[3]. During diabetes, persistent hyperglycemia causes increased production of free radicals, especially reactive oxygen species (ROS) for all tissues from glucose auto-oxidation and protein glycosylation. The increase in the level of ROS in diabetes could be due to their increased production and or decreased destruction by non enzymatic and enzymatic antioxidants. The level of these antioxidants critically influences the susceptibility of various tissues to oxidative stress and is associated with the development of complications in diabetes. [4]. *Address for correspondence
[email protected] July - September 2012
30
Oxidants are generated as a result of normal intracellular metabolism in mitochondria and peroxisomes, as well as from a variety of cytosolic enzyme systems. In addition, a number of external agents can trigger ROS production. A sophisticated enzymatic and nonenzymatic antioxidant defence system including catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) counteracts and regulates overall ROS levels to maintain physiological homeostasis. Lowering ROS levels below the homeostatic set point may interrupt the physiological role of oxidants in cellular proliferation and host defence. Similarly, increased ROS may also be detrimental and lead to cell death or to acceleration in ageing and age-related diseases. Traditionally, the impairment caused by increased ROS is thought to result from random damage to proteins, lipids and DNA. In addition to these effects, a rise in ROS levels may also constitute a stress signal that activates specific redox sensitive signalling pathways. Once activated, these diverse signalling pathways may have either damaging or potentially protective functions [5].
In, India Ayurvedic medicine has great importance to treat the diabetes and its complications. Since ancient period, it gains more popularity due to its less toxic effects and more efficacious. Many herbs have been shown to have antidiabetic action in both human and animals [6]. Piper longum under the family of Piperaceae is one such Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
plant used to treat Diabetes . The fruit, commonly known as pippali and its root, called as pippali mula or modi are used for medicinal purpose. It grows all over India, in evergreen forests and is cultivated in Assam, Tamil Nadu and Andhra Pradesh. It is widely used to treat tuberculosis, sleeping problems, respiratory tract infections, chronic gut-related pain, arthritic conditions and diabetes Mellitus. [7].
Materials and Methods Collection of plant material Dry roots of Piper longum, were purchased from the local market and identified by the Botanist, Department of Botany, S.V.University, Tirupati. Voucher specimens (Herbarium Accession Numbers are 713) were deposited in the herbarium, Department of Botany, S.V. University, Tirupati. Preparation of aqueous extract of roots of Piper longum The root powder was soaked in glass jar for 48h at room temperature and solvent was filtered. This was repeated 3–4 times until filtrate gave no coloration. The filtrate was concentrated to dryness under reduced pressure in Buchi Rotavapor R-200 and finally freeze dried. The yield of the extract was 22% (w/w). Induction of diabetes Diabetes was induced in male Wistar albino rats aged 2–3 months (180–200 g body weight) by intraperitoneal administration of STZ (single dose of 50 mg/kg b.w.) dissolved in freshly prepared 0.01M citrate buffer, pH 4.5 . After 72 h rats with marked hyperglycemia (FBG e”250 mg/dl) were selected and used for the study [8]. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages, as per the guidelines of Institute Animal Ethics committee. This study was approved by institutional animal ethics committee (Resolution no: 08/2011- 20129 (i)/a/CPCSEA/ IAEC/SVU/CHA-SAN/dt.25.09.2011). Effect of long term treatment with AQRPL on glycemic control, bodyweights, liver and kidney antioxidant status.
The rats were divided into 5 groups and each group consisted of 6 rats. Group 1 Normal untreated rats. Group 2 Normal rats treated with 200 mg AQRPL /kg b.w/ day. Group 3 Diabetic untreated rats. Group 4 Diabetic rats treated with 200 mg AQRPL /kg b.w/day. Group 5 Diabetic rats treated with 0.02g of glibenclamide/ kg b.w/day. AQRPL or glibenclamide was administered to the rats every day morning for 30 days by gastric intubation using oral gavage. Blood samples were collected from tail July - September 2012
31
th
th
veins before the start of the treatment and on 10 ,20 and th 30 days of the treatment and fasting blood glucose levels were estimated. All the five groups of rats were sacrificed th on the 30 day after an overnight fast, by anesthetizing with anaesthetic ether and further by cervical dislocation. Different tissues including liver and kidney were collected and immediately frozen until the use. Phytochemical analysis was carried out in the aqueous extract by different methods of phytochemical analysis [9]. Analytical procedures Estimation of blood glucose was carried out by glucose oxidase–peroxidase method [10]. The levels of TBARS in tissues were estimated by the method of Fraga et al., 1988.[11] CAT activity was assayed following the method of Sinha .,1972 [12]. SOD activity was assessed according to the method of Kakkar et.al.,1984 [13]. GPx activity was measured as described by Rotruck et.al.,1973 [14]. GST activity was estimated according to the method of Habig et al. 1974[15]. Statistical analysis The results were expressed as mean ± S.D. The statistical analysis of results was carried out using one-way analysis (ANOVA) followed by DMRT.
Results Table.1 Shows effect of long term treatment with the AQRPL on fasting blood glucose and body weights of normal and diabetic rats. The FBG levels were significantly higher in the diabetic groups than those in normals before starting the experiment. However, at the end of 30 days of treatment, there was 68% decrease (P < 0.001) in FBG levels of diabetic rats treated with aqueous extract. The treatment with glibenclamide has produced only 33% decrease in th blood glucose levels in the 5 group of the diabetic rats, while there was a further increase in the blood glucose levels of untreated diabetic rats. There was no change in the FBG levels of normal rats after the treatment with AQRPL. At the end of 30 days treatment, the body weights of normal, normal treated, diabetic treated and standard drug treated group, increased significantly by 25.8g, 31.6g, 24.1g and 21.2 respectively, whereas the body weight of diabetic control group decreased by -33.3g. Table. 2 and table. 3 show the liver and kidney (respectively) levels of TBARS, activities of SOD, CAT, GPx and GST in the normal and experimental groups of rats. There was a significant increase in the levels of TBARS, CAT activity and a significant decrease in the activities of SOD, GPx and GST in both tissues of diabetic rats. The treatment with AQRPL decreased the levels of TBARS, CAT activity and significantly increased activities of SOD, GPx, GST in liver and kidney of diabetic rats.
Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
Table - 1 Effect of long term treatment with the AQRPL on blood glucose and body weights of normal, diabetic and diabetic treated rats Group
Blood glucose(mg/dl) and change in body weights at different days during the experimental period with PLRAE st
1 Day
1
87.1±7.3
2
77±5.3
10 a
a
357±14.98
3
319±30
4
b
th
20 Day
30 Day
93.5±7.5
a
89.3±8.5
a
85.1±6.7
a
+25.8
79.1±7.2
a
79±9.3
a
83.1±6.2
a
+31.6
c
c
d
188±17.3
312.5±28.1
th
Day
424±21
b b
5
th
277±33
433±29
b
106±17.3
c
449±24 a
249.5±17.2
Change in body weights (g)
- 33.3 a
+24.1
208.6±20.9b
+21.2
106.3±21.7 b
F Value
270.185
309.482
419.650
448.880
Significance
0.000
0.000
0.000
0.000
Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT). Table 2: Effect of long term treatment with the AQRPL on TBARS levels and antioxidant enzyme activities in the livers of different experimental animals Group
Lipid Peroxides (nmoles MDA/ml)
1 2 3 4 5 F value Significance
0.128±0.0020 a 0.131±0.0063 c 0.233±0.0081 b 0.163±0.0042 b 0.159±0.0140 164.203 0.000
a
Catalase (U/mg Protein)
Glutathione Peroxidase (U/mgprotein) a
d
17.5±0.492 a 18±0.651 d 48.6±3.18 c 20.6±2.02b c 22.41±2.59 238.980 0.000
0.236±0.02 d 0.218±0.003 a 0.0765±0.004 c 0.159±0.001 b 0.127±0.01 144.296 0.000
Superoxide Dismutase (U/ mg protein) c
15.9±1.14 c 16.5±1.49 a 8.35±0.20 b 13.85±0.52 b 12.5±0.61 76.106 0.000
GlutathioneS- Transferase (U/ mg protein) c
23.7±1.04 c 25.1±1.21 a 10.9±0.62 b 20.25±0.653 b 19.6±1.46 167.820 0.000
Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT).
Discussion The use of plant products in the treatment of diabetes mellitus is becoming advantageous due to the presence of several bioactive compounds with therapeutic potential. In recent years, several researchers have studied the worth of different medicinal plants in controlling DM and delaying the long term effects of DM. Piper longum is one of the herbs mentioned in all ancient scriptures of Ayurveda. Earlier , Shanmugam Manoharan et.al demonstrated that the ethanolic extract of dried fruits of Piper longum July - September 2012
32
has potent antihyperglycemic and antilipidperoxidative activity in alloxan induced diabetic rats [16]. Sheshachala forest (Rayalaseema region, Andhra Pradesh, India), which lie geographically in the South Eastern Ghats are known for the rich heritage of flora. Where the tribes use Piper longum roots to treat Diabetes mellitus [7]. Hence our study was aimed to find out the scientific evidence for the safety and use of the roots of Piper longum to treat DM. In our study STZ was used to induce diabetes mellitus in rats rather than alloxan. At low dose, STZ (50 mg/kg.b.w) partially destruct the beta cell, which secreted insufficient Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
Table - 3 Effect of long term treatment with AQRPL on TBARS levels and antioxidant enzyme activities in the Kidneys of different experimental animals Group
Lipid Peroxides (nmoles MDA/ml)
1 2
0.1376±0.005 a 0.1385±0.010 c 0.2661±0.016 b 0.1731±0.021 b 0.1826±0.019 65.266 0.000
3 4 5 F value Significance
a
Catalase (U/mg protein)
33.4±0.91 a 31±0.70
Glutathione Peroxidase (U/mgprotein)
b
c
0.244±0.007 c 0.253±0.02 a 0.141±0.002 b 0.203±0.005 b 0.191±0.005 63.162 0.000
61.9±1.71c d 39.2±2.25 d 41.7±0.966 484.267 0.000
Superoxide Dismutase (U/ mg protein) d
32.8±0.99 d 33.8±0.70 a 18.2±112 c 28±1.88 b 23.1±1.22 92.459 0.000
Glutathione-STransferase (U/ mg protein) c
25±0.73 c 24±0.79 a 10±1.0 b 21.7±0.88 d 27.8±1.15 320.106 0.000
Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT). insulin causing type 2 diabetes [17]. It is widely accepted animal model and reported to resemble human hyperglycemic non ketotic diabetes mellitus [18]. is often associated with kidney hypertrophy which may contribute to end stage renal damage, hepatotoxicity, oxidative stress and hypercholesterolemia [19,20].
susceptibility to lipid peroxidation [23]. Implication of oxidative stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free radical generation but also due to non-enzymatic protein glycosylation, autooxidation of glucose, impaired antioxidant enzyme, and formation of peroxides [24,25].
The aqueous root extract of the medicinally valued plant Piper longum clearly envisaged hypoglycemic effect by decreasing the fasting blood glucose levels in STZ induced diabetic animals. In the present study, elevated fasting blood glucose levels were observed in diabetic untreated rats compared to normal rats. Administration of PL aqueous extract at a dosage of 200 mg/ kg b.wt for 30 days resulted in significant reduction in fating blood glucose levels in STZ induced diabetic rats. The decreased fasting blood glucose levels may be due to the stimulation effect of PL aqueous extract on the remnant beta cells of islets of Langerhans to release more amount of insulin. Induction of diabetes by STZ leads to loss of body weight due to the increased muscle wasting and loss of tissue proteins [21, 22].
In the present study, we observed a significant increase in lipid peroxide levels (TBARS) in the liver and kidney of diabetic rats compared to normal rats. Administration of AQRPL or glibenclamide potentially abrogated the levels of TBARS in the liver and kidney of diabetic rats. This shows that AQRPL might protect the tissues (liver and kidney) against the cytotoxic action and oxidative stress of streptozotocin. Furthermore, the reduction in lipid peroxidation could be due to the improvement of the glycemic control and increased insulin secretion as insulin decrease the activity of fatty acyl coenzyme A oxidase.
The results obtained with the aqueous extract treatment in chronic diabetic model further clarified the antidiabetic effect of the extract. After 30 days of aqueous extract treatment, gain in body weights were observed in diabetic treated rats and the results were comparable with that of the standard oral hypoglycemic agent glibenclamide. STZ-induced hyperglycemia induces free radical generation which thereby leads to DNA damage, protein degradation, lipid peroxidation and finally culminating into damage to various organs of the body like liver, kidney, brain and eyes. These elevated free radicals and depressed antioxidant defence may lead to cell disruption, oxidative damage to the cell membranes and hence increase the July - September 2012
33
DM is associated with increased formation of free radicals and decrease in antioxidant potential. Due to these events, the balance normally present in cells between radical formation and protection against them is disturbed [26]. An imbalance of oxidant/antioxidant defence systems results in alterations in the activity of antioxidant enzymes, such as SOD, CAT, GR, GPx, and impaired glutathione metabolism [27]. The present data indicates that STZinduced diabetes disrupts actions of liver and kidney antioxidant enzymes. The decreased activities of these enzymes in pancreas may be due to the production of ROS such as superoxide (O 2 ), hydrogen peroxide (H2O2), and hydroxyl radical (OH) that reduce the activity of these enzymes [28,29]. In our study, the activities of SOD, GPx and GST were decreased in diabetic rats compared to normal rats, which could be due to free radical-induced inactivation Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
and glycation of the enzymes in diabetic state [30]. On long-term treatment of diabetic rats AQRPL had reversed the activities of these enzymatic antioxidants, This means that the extracts can reduce the potential glycation of enzymes or they may reduce the production of reactive oxygen free radicals and improve the activities of antioxidant enzymes. In our study the activity of CAT was significantly increased in liver and kidney of diabetic untreated rats. The possible explanation for the increase in catalase activity is that it could be a compensatory mechanism to prevent tissue damage by the increased levels of H2O2 and decreased levels of GPx. In diabetes, it is thought that hypoinsulinemia increases the activity of the enzyme, fatty acyl coenzyme A oxidase, which initiates â-oxidation of fatty acids, resulting in increased levels of H2O2. The CAT activity was restored to near normal in diabetic rats treated with AQRPL, which might be due to decreased LPO levels and increased insulin secretion. Various studies in the past reported conflicting results regarding the status of antioxidant enzymes in diabetes [31,32,33]. Majority of authors reported the decreased enzymatic antioxidant activities (SOD, CAT, GPx and GST) in tissues of diabetic rats [34,35]. In conclusion the present study reveals that the P.longum had antihyperglycemic, and antioxidant agents. The bioactive component(s) responsible for the observed activities are not precisely known but it may be one or more of the phytochemical constituents established to be present in the root aqueous extract. Our phytochemical screening of the extract revealed the presence of alkaloids and glycosides in the P.longum root aqueous extract, which might be the constituents responsible for these activities. Further identification and isolation of these constituents may be fruitful.
References [1]. Vats RK, Kumar V, Kothari A, Mital A, Uma Ramachandran. Emerging targets for diabetes. Curr Sci 2000; 88: 241-247. [2]. Chehade JM, Mooradian AD. A Rational Approach to Drug Therapy of Type 2 Diabetes Mellitus, Disease Management. Drugs 2000 ; 60 (1): 95-113.
Deundorf H. Pharmacokinetics and bioavailability of herbal medicinal products. Phytomed 2002; 9: 1-36. [7]. Madhavachetty, K., Sivaji, K., Tulasirao, K. Flowering plants of chittoor district Andhra Pradesh, India. First edition, published by Students Offset Printers, Tirupati ; 2008. [8]. Gupta S, Kataria M,Gupta PK, Murganandan S, Yashroy RC . Protective role of extracts of neem seeds in diabetes caused by Streptozotocin in rats, J. Ethnopharmacol 2004; 90: 185–189. [9]. Harborne JB 2005; Phytochemical Methods. Analysis. A Guide to Modern Techniques of Plant, third ed. Springer International, India. [10]. Kesari AN, Gupta RK, Singh SK, Diwakar S, Watal G. Hypoglycaemic and antihyperglycaemic activity of Aegle marmelos seed extract in normal and diabetic rats. J. Ethnopharmacol 2006; 107: 374–379. [11]. Fraga, C.G., Leibovitz, B.E., Toppel, A.L. Lipid peroxidation measured as TBARS in tissue slices. Characterisation and comparison with homogenate and microsome. Free RadBio Med 1988; 4, 155-161. [12]. Sinha, K.A. Colorimetric assay of Catalase. Anal Biochem 1972; 47, 389-394. [13]. Kakkar, P., Das, B., Viswanathan, P.N. A modified spectrophotometric assay of Superoxide dismutase. Ind J Biochem Biophys 1984; 21, 130-132. [14]. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B. Selenium: Biochemical role as a component of Glutathione peroxidase. Science 1973; 179, 588-590. [15]. Habig, W.H., Pabst, M.J., Jakoby, W.B. Glutathione transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974 ; 249, 7130-7139. [16]. Shanmugam Manoharan, Simon Silvan, Krishnamoorthi Vasudevan and Subramanian Balakrishnan. Antihyperglycemic and Antilipidperoxidative effects of Piper longum (Linn.) Dried Fruits in Alloxan Induced Diabetic Rats. Journal of Biological sciences 2007; 6 (1):161-168. [17]. Gomes, A., Vedasiromoni, J.R., Das, M., Sharma, R.M., Ganguly, D.K. Antihyperglycemic effect of black tea (Camellia sinensis) in rat. J. Ethnopharmacol. 2001; 27, 243-275.
[3]. SA Moussa. Oxidative stress in Diabetes Mellitus. Romanian J Biophys 2008;18(3):225 236. [4]. Boguslaw Lipinski. Pathophysiology of Oxidative stress in Diabetes mellitus. Journal of Diabetes and its Complications 2001; 15: 203-210.
[18]. Weir GC, Clore ET, Zmachiroski CJ, Bonner-Weir S Islet secretion in a new experiment model for noninsulin dependent diabetes. Diabetes 1981; 5: 30-590.
[5]. Toren Finkel & Nikki J Holbrook. Oxidants, oxidative stress and the biology of ageing. Nature 2000; 408: 239-247.
[19]. Heidland A, Ling H, Vamvakas S, Paczek L (1996) Impaired proteolytic activity as a potential cause of progressive renal disease. Miner Electrolyte Metab 22: 157–61.
[6]. Bhattaram VA, Ceraefe M, Kohlest C, Vest M ,
[20]. Rabkin R, Schechter P, Shi JD, Boner G Protein
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turnover in the hypertrophy in kidney. Miner Electrolyte Metab 1996; 22: 153–6. [21]. Swanston Flat SK, Day C, Bailey CJ, Flatt PR Traditional plant treatment for diabetes: studies in normal and streptozotocin diabetic mice. Diabetologia 1990; 33: 462-464. [22]. Chatterjee MN, Shinde R.Text Book of Medical Biochemistry. Jaypee Brothers Medical Publishers, New Delhi 2002; 317. [23]. Yazdanparast, R., Amin, A., Jamshidi, S. Experimental diabetes treated with Achillea santolina: effect on pancreatic oxidative parameters. J. Ethnopharmacol. 2007 ; 112, 13–18. [24]. Vincent, A.M., Russell, J.W., Low, P., Feldman, E.L. Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 2004; 25, 612–28. [25]. Pari, L., Latha, M. Antidiabetic effect of Scoparia dulcis: effect on lipid peroxidation in streptozotocin diabetes. Gen Physiol Biophys 2005; 24, 13–26. [26]. Nazirogilu, M., Butterworth, P. Protective effects of moderate exercise with dietary vitamin C and E on blood antioxidative defense mechanism in rats with streptozotocin induced diabetes. Can. J. Appl. Physiol. 2005; 30, 172–185. [27]. Maritim, A.C., Sanders, R.A., Watkins, J.B. Diabetes, oxidative stress and antioxidants. A review. J. Biochem. Mol. Toxicol. 2003; 17, 24–38. [28]. Kaleem, M., Asif, M., Ahmed, Q.U., Bano, B. Antidiabetic and antioxidant activity of Annona squamosa extract in streptozotocin-induced diabetic rats. Singapore Med. 2006; J. 47, 670–675.
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35
[29]. Vincent, A.M., Russell, J.W., Low, P., Feldman, E.L. Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 2004; 25, 612–28. [30]. Al-Azzawie, H.F., Alhamdani, M.S. Hypoglycaemic and antioxidant effect of oleuropein in alloxan-induced diabetic rabbits. Life Sci. 2006; 78(12), 1371-1377.
[31]. Selvam, R.., Anuradha, C.V. Lipid perosidation and antioxidative enzyme changes in erythrocytes in diabetes mellitus. Indian J Biochem Biophys. 1988; 25, 268-72. [32]. Kaji, H., Jurasaki, M., Ito, K. Increased lipid peroxide value and glutathione peroxide activity in blood plasma of type II diabetic women. Klin Wochnschr 1985; 63, 765-8. [33]. Matkovics, B., Kotorman, M., Varga, I.S., Hai, D.W., Varga, C. Proantioxidant and rheological studies in the blood of type 2 diabetic patients. Acta physiol. Hung. 1997; 85, 197-112. [34]. Gopalsamy Rajiv Gandhi, Savarimuthu Ignacimuthu, Michael Gabriel Paulraj. Solanum torvum Swartz. fruit containing phenolic compounds shows antidiabetic and antioxidant effects in streptozotocin induced diabetic rats. Food and Chemical Toxicology 49 ; 2725–2733. [35]. Selvan VT, Manikandan L, Senthil Kumar GP, Suresh R, Kakoti BB, Gomathi P, Kumar DA, Saha P, Gupta M, Mazumder UK.Antidiabetic and Antioxidant Effect of Methanol Extract of Artanema sesamoides in Streptatozocin-Induced Diabetic Rats International Journal of Applied Research in Natural Products Vol. 1(1), 2008; pp. 25 33.
Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
ISSN 0973 – 9874
J.Pharm.Chem
CODEN: JPCOCM
Journal of Pharmacy and Chemistry (An International Research Journal of Pharmaceutical and Chemical Sciences) Indexed in Chemical Abstract and Index Copernicus www.stfindia.com
Volume 6 • Issue 3 • July – September 2012
Antihyperglycemic and Antioxidant Effects of Pennisetum glaucum Seed Extracts in STZ Induced Diabetic Rats 1
1
S. VENKATA PRASAD , RAJESH NATAVA , SWAPNA SIRASANAGANDLA1 AND CHIPPADA APPA RAO*1 1
1
Department of Biochemistry, Sri Venkateswara University, Tirupati, India-517502
ABSTRACT Hexane, ethylacetate, methanolic and aqueous extracts of Pennisetum glaucum seeds were prepared and given individually to normal and streptozotocin (STZ) induced diabetic rats after an overnight fast. The blood glucose levels were measured at 0,1,2,3,4,5 and 6 hrs after the treatment. The aqueous extract at a dosage of 250 mg/kg body weight has shown maximal blood glucose lowering effect in diabetic rats. The same dosage did not produce any hypoglycemic activity in normal rats. The antihyperglycemic activity of aqueous extract of Pennisetum glaucum was significantly higher than that of glibenclamide, an oral hypoglycemic agent. All the extracts exhibited dose dependent invitro antioxidant scavenging activites against hydrogen peroxides. Further all the extracts had relatively lower reducing power compared to that of ascorbic acid. In conclusion aqueous extract of seeds of Pennisetum glaucum (AEPG) possess both antihyperglycemic and antioxidant activities. Keywords: Antihyperglycemic, Pennisetum glaucum, Glibenclamide and Antioxidant Activities
Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia arising as a consequence of a relative or absolute deficiency of insulin secretion, resistance to insulin action or both [1]. Type 1 and 2 diabetes are quite different diseases in etiology. While their resulting features have much in common, such as hyperglycemia (high blood sugar), they have many differences and their underlying cause is probably quite different. Hyperglycemic condition causes increased glycosylation leading to biochemical and morphological abnormalities due to altered protein structure which over a period of time develops diabetic complications such as nephropathy, retinopathy, neuropathy and cardiomyopathy [2]. It is a serious endocrine syndrome with poor metabolic control and responsible for increased risk of cardiovascular diseases including atherosclerosis, renal failure, blindness or diabetic cataract worldwide [3,4]. Diabetes mellitus is an increasingly common, potentially evastating, expensive, treatable but incurable life long disease. Globally, the estimated incidence of diabetes mellitus and projection for
Address for Correspondence E-mail:
[email protected] July - September 2012
36
year 2010, as given by International Diabetes Federation is 239 million [5]. The worldwide survey on diabetes reveals that among the entire diabetes cases more than 90% are account to type-II [6]. Many hypoglycemic agents, such as the biguanides and sulfonylureas, are used alone or together with insulin to treat this disease. However, these medications can cause serious side effects [7] motivating a search for safer, more efficacious agents to control diabetes. The synthetic oral hypoglycaemic agents can produce a series of side effects including haematological, gastro-intestinal reactions, hypoglycaemic coma and disturbances of liver and kidney. In addition, they are not suitable for use during pregnancy [8]. World Health Organization (WHO) has recommended the evaluation of traditional plant treatments for diabetes as they are effective, non-toxic, with less or no side effects and are considered to be excellent candidates for oral therapy [9]. Currently, there is growing interest in using herbal remedies for the treatment of DM due to the adverse effects associated with oral hypoglycemic agents and insulin available [10]. Herbal medicines have long been used effectively in treating diseases throughout the world and frequently considered to be less toxic and free from side
Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
effects as compared to synthetic ones [11,12]. Over the years, variety of medicinal plants and their extracts have been reported to be effective in the cure and management of diabetes [13]. Additionally, after the approbations made by WHO on diabetes mellitus [14], exploration on hypoglycemic agents from medicinal plants have become more significant. Antioxidant compounds in the human foods or supplementary diets have been found fruitful against diabetes [15]. Diabetes is usually associated by increased production of the molecules of reactive oxygen species (ROS) and / or impaired antioxidant defense systems, which result oxidative damage leading to ROS mediated diabetic pathogenesis [16]. DM is associated with increased formation of free radicals and decrease in antioxidant potential. Due to these events, the balance normally present in cells between radical formation and protection against them is disturbed [17]. An imbalance of oxidant / antioxidant defense systems results in alterations in the activity of antioxidant enzymes, such as SOD, CAT, GR, GPx, and impaired glutathione metabolism [18]. Pennisetum glaucum belongs to family Poaceae. It is commonly known as pearl millet (sajjalu or gantelu in telugu) in and around the Rayalaseema region, Andhapradesh, India has been found to be appetizer and tonic. Its fruits have been reported as a useful therapy for open facial pimples [19]. P. glaucum, bran, has been considered a good source of dietary fiber [20]. Most of the poor people living in Rayalaseema region use Pearl millet as a source of antidiabetic diet. No work has been reported about the antidiabetic activity of PG on STZ induced diabetic rats. Hence an attempt has been made to investigate the antidiabetic activity of Pennisetum glaucum.
Materials and Methods
To prepare aqueous extract the PG seed powder (1 kg) was soaked in distilled water (3 volumes) in a glass jar for 2 days at room temperature and the solvent was filtered. This was repeated 3 to 4 times until the filtrate gave no coloration. The filtrate was concentrated under reduced pressure in the Buchi rotavapour R-200 and finally freeze dried. The yield of the extract was 29% w/w. Induction of Diabetes Diabetes was induced in healthy male Wistar Albino rats aged 3-4 months, with body weights 180-200g, by single intraperitoneal injection of freshly prepared streptozotocin (50 mg/kg b.w) dissolved in ice cold 0.1M citrate buffer (pH 4.5) after overnight fasting for 12 hours [21]. Since STZ is capable of producing fatal hypoglycemia as a result of massive pancreatic insulin release due to destruction of pancreatic β cells, 6 hrs after STZ administration the rats were kept for next 24 hours on 15% glucose solution to prevent hypoglycemia. Diabetes was assessed by determining the fasting blood glucose after 48 hrs of injection of STZ. The blood glucose levels in STZ rats were increased to markedly higher levels than normal. After a week rats with marked hyperglycemia (blood glucose level ≥ 250 mg/dl) were selected and used for the study.
Experimental design for the evaluation of antihyperglycemic activity of crude suspension of seeds of Pennisetum glaucum in STZ-induced diabetic rats The animals were divided into five groups and each group consisted of six rats: Group 1: Untreated normal rats Group 2: Normal treated rats Group 3: Diabetic untreated rats. Group 4: Diabetic rats treated with 250 mg / kg b.w. of crude suspension
Collection of Plant Material Dry seeds of Pennisetum glaucum were purchased from the local market and identified by the Botanist, Department of Botany, S.V.University, Tirupati. A voucher specimen (Herbarium Accession No.2011) was deposited in the herbarium, Department of Botany, S.V. University, Tirupati. These seeds were dried in shade, powdered. The powder was stored in airtight containers and was used for evaluation of its antidiabetic activity with different solvents extracts. Preparation of extracts
Group 5: Diabetic rats treated with 0.02g glibenclamide / kg b.w.
Hexane, ethyl acetate and methanol extracts Hexane, ethyl acetate and methanol extracts were prepared by successive solvent extraction of PL seed 0 0 powder in soxhlet apparatus at 68 C-70 C. The filtrates obtained were distilled and concentrated under reduced 0 0 pressure at low temperature (40 C to 45 C) in the Buchi rotavapor R-200 and finally freeze dried. The yields of the hexane, ethyl acetate and methanol extracts were 18%, 25% and 28%w/w respectively. All the extracts were stored at 0 0 C in airtight containers until needed for further studies. July - September 2012
Aqueous extract
37
After an overnight fast the group 4 and group 5 animals received the crude suspension of seeds of Pennisetum glaucum and glibenclamide (dissolved in 1ml of distilled water) respectively by gastric intubation using a force feeding needle. Normal and untreated diabetic rats were fed distilled water alone. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 1, 2, 3, 4, 5 and 6 h after the administration of crude suspension and blood glucose levels were determined by glucose oxidase - peroxidase method. Evaluation of antihyperglycemic effect of different extracts of seeds of PG in normal and STZ-induced diabetic rats The animals were divided into Six groups and each group consisted of six rats: Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
Group 1: Untreated normal rats
H2O2 scavenging activity The H202 scavenging ability of the plant extracts was determined according to the method of Ruth et al [24]. A solution of H202 (40 mM) was prepared in phosphate buffer (pH 7.4). 25, 50, 75 & 100 µg/ml concentrations of the plant extracts in 3.4 ml phosphate buffer were added to a H202 solution (0.6mL, 40 mM). The absorbance value of the reaction mixture was recorded at 230 nm. (Butylated hydroxytoluene ) BHT was used as a standard.
Group 2: Untreated diabetic rats Group 3: Diabetic rats treated with 250 mg PG hexane extract /kg b.w. Group 4: Diabetic rats treated with 250 mg PG ethylacetate extract /kg b.w. Group 5: Diabetic rats treated with 250 mg PG methanolic extract/kg b.w. Group 6: Diabetic rats treated with 250 mg PG Aqueous extract/kg b.w.
Reducing power
After an overnight fast the group 3, group 4, group 5, group 6 rats received the hexane (dissolved in 1ml of 5% Tween 80), ethyl acetate, methanol, aqueous extracts (dissolved in 1ml of distilled water) by gastric intubation using a force feeding needle. The untreated normal and diabetic rats were fed distilled water alone. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 1, 2, 3, 4, 5 and 6 h after the administration of PG extracts and blood glucose levels were determined by glucose oxidase–peroxidise method. Effect of AEPG on oral glucose tolerance test in STZinduced diabetic rats (OGTT)
Results and Discussion
Three groups of diabetic rats each group containing six rats were used for this study. Group1: Diabetic untreated rats Group2: Diabetic rats treated with 0.02g glibenclamide/ kg b.w Group 3: Diabetic rats treated with 250 mg/ k.g b.w of AEPG The oral glucose tolerance test was [22]. performed in overnight fasted diabetic rats. Glucose (2 g/ Kg b.w) was administered orally to both groups of rats using a force feeding needle. The group 2 and group 3 rats were administered with AEPG and glibenclamide respectively, along with the glucose load. Blood samples were collected from the tail veins of all the animals from 0 min (before glucose administration) to 120 min of glucose administration for estimation of blood glucose using dextrostix with Basic One Touch Accuchec Glucometer (Glucose oxidase peroxidase method). Phytochemical analysis: Phytochemical analysis was carried out by different methods of phytochemical analysis [23]. Statistical Analysis All values are expressed as Mean ± S.D. The data was statistically analysed by students ‘t’ test.
In spite of the introduction of hypoglycemic agents, diabetes and its related complications continue to be a major medical problem. Since time immemorial patients with non-insulin dependent diabetes mellitus have been treated orally by folklore with a variety of plants extracts in the indigenous Indian system of medicine (Ayurveda). A mention was made on good number of plants for the cure of diabetes or madhumeha and some of them have been experimentally evaluated and the active principles were isolated in India [26,27,28,29]. Such plants and remedies mentioned and used for the treatment of diabetes mellitus date back to the ancient authorities like Bhrigu, charaka, sushruta and vagbatta, the last three called vriddha-trayi of Ayurveda [29]. The effect of crude suspension of Pennisetum glaucum seeds on the fasting blood glucose levels of diabetic rats is given in Table.1. Fasting blood glucose levels of diabetic rats were significantly higher than those in normal rats. But after the treatment with crude suspension of PG a significant decrease in fasting blood glucose levels were observed in diabetic treated groups when compared to those of diabetic untreated group (Group 3).
The effect of hexane, ethyl acetate, methanolic and aqueous extracts on the fasting blood glucose levels of diabetic rats is given in Table.2. The fasting blood glucose levels of diabetic untreated rats were significantly higher than those of normal untreated rats (Group 1). When different extracts of PG were tested for their glucose lowering effects, the ethyl acetate, methanolic and aqueous
Invitro Antioxidant activity The following methods were used to evaluate the invitro anti oxidant activities of the above four extracts. July - September 2012
The reducing power was determined according to the method of Oyaizu [25]. Different concentrations of plant extracts (50, 100,150 and 200 µg/ml) prepared in methanol were mixed with phosphate buffer (2.5 ml, 0.2M, pH 6.6) and potassium ferricyanide [K3Fe (CN)6] (2.5m1,1%). The mixture was incubated at 50°C for 20 min and 2.5m1 of trichloroaceticacid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeC13 (0.5 ml. 0.1%) and the absorbance was measured at 700nm. Increased absorbance of the reaction mixture indicated increased reducing power. BHT was used as a standard.
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Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
Table - 1 Effect of 0.25g/kg. b.w of crude suspension of Pennisetum glaucum seeds on fasting blood glucose levels (mg/dl) in Diabetic rats (Mean + S.D) Groups
Fasting Blood Glucose (mg/dl) levels after treatment with aqueous crude suspension of Pennisetum glaucum 0h
1h
2h
3h
4h
5h
Group 1
96±2.8
92.3±3.9
87.3±9.2
90.3±6.6
93.3±12.6
89±1.4
91.3±06
Group 2
86±2.6
90.3±2.5
89.3±2.9
92.3±8.4
89.6±23.2
90±2.7
93.3±12
Group 3
340.7±31.8†
340.7±31.8
365±28.2
335.3±45.3
381.3±36.7
370.8±32.5
386.3±12.6
Group 4
379.2±23.2 †
165.3±31.8** 158±12.5** -56.40% -58.30%
146±10.3** -61.40%
120.3±10.3** -68.30%
Group 5
283.8±21†
223.5±8.1 ** 214.5±9.7** 209.3±4.8** -21.24% -24.40% -26.35%
199.3±8.8** -29.77%
250.3±37.8** 208±28.6** -33.90% -45% 255.3±16 -10.40%
236.6±11* -16.61%
† P <0.0001 compared with the initial level of blood glucose (0hr) of normal rats. ** P <0.0001 compared with the initial level of blood glucose (0hr) in the respective * P <0.001 compared with the initial level of blood glucose (0hr) in the respective group. Numbers in parenthesis indicate the percentage of fall in 0hr blood glucose.
6h
group.
Table - 2 Assessment of antihyperglycemic effect of different extracts of seeds of PG in STZ-induced diabetic rats GROUPS
Fasting Blood Glucose (mg/dl) levels after treatment with different extracts of Pennisetum glaucum 0h
1h
2h
3h
4h
5h
Group1
96±2.8
92.3±3.9
87.3±9.2
90.3±6.6
93.3±12.6
89±1.4
Group2
340.7±31.8†
340.7±31.8
365±28.2
335.3±45.3
381.3±36.7
370.8±32.5
386.3±12.6
Group3
328.2±21 †
336.4±21
353.8±21
358±21
369.3±01
374.6±23
384.3±7
Group4
284.7±21†
275.3±16 -2.90%
268.6±7 .23 -5.35%
259.3±19 -8.63%
247.8±29 -12.60%
240.6±8.12* -15.57%
236.2±8.13* -16.87%
Group5
364.6±23.2†
320.3±37.8 -12%
294.7±8.2** -19%
270.1±27.4 ** -25.90%
Group6
379.2±23.2†
250.3±37.8** -33.90%
208.3±28.6** -45%
165.3±31.8** -56.40%
251.3±12.3 ** 247.3±37.8** -31.07% -33.07% 158±12.5** -58.30%
146.3±10.3** -61.40%
6h 91.3±06
243.3±37.8** -34% 135.3±10.3** -64%
† P <0.0001 compared with the initial level of blood glucose (0hr) of normal rats. ** P <0.0001 compared with the initial level of blood glucose (0hr) in the respective group. * P <0.001 compared with the initial level of blood glucose (0hr) in the respective group. Numbers in parenthesis indicate the percentage of fall in 0hr blood glucose.
extract at a dosage of 250 mg/kg b.w produced the maximum fall of 16.8%, 34% and 64% respectively, in the FBG levels of diabetic rats after 6 h of treatment. Whereas hexane extract did not show significant antihyperglycemic activity in STZ induced diabetic rats. In the present study 0.25 g/kg b.w. of aqueous extract of PG seed has shown a maximum fall in blood glucose July - September 2012
39
levels in STZ induced diabetic rats, which is significantly higher than the hypoglycemic effect of 20 mg/kg.b.w of glibenclamide in the diabetic treated rats. The onset of st antihyperglycemic action was observed from 1 hr of the treatment and a steady state increase in the action continued up th to 6 hr. The ethyl acetate and methanolic extracts also produced significant but less antihyperglycemic activity in comparison with aqueous extract. They also did not produce Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
Table – 3 Phytochemical constituents of different extracts of PG seeds Sl.No.
Phytochemicals
Hexane
Ethyl acetate
Methanol
Water
1
Steroids
-
-
-
-
2
Triterpenes
+
-
-
-
3
Saponins
+
-
-
-
4
Alkaloids
-
-
+
-
5
Carbohydrates
-
-
+
+
6
Flavonoids
-
-
+
-
7
Tannins
-
-
+
-
8
Glycosides
-
+
+
+
any hypoglycemic activity in normal treated rats. The hexane extract did not show significant antihyperglycemic activity, may be due to the lack of phytochemical constituents like flavonoids and glycosides which are present in ethylacetate, methanolic and aqueous extracts. The results are depicted in table .3
alkaloids, glycosides, saponins, glycolipids, dietary fibres, polysaccharides, peptidoglycans, carbohydrates, amino acids and others obtained from various plant sources have been reported as potent hypoglycemic agents [30].
The phytochemical analysis of PG aqueous extract showed the presence of carbohydrates, proteins, phenols, flavonoids and glycosides. The antidiabetic effect of PG aqueous extract may be due to the presence of more than one antihyperglycemic principle and their synergistic properties. Several phytomolecules including flavonoids,
Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H2O2 can 2+ 2+ probably react with Fe and possibly Cu ions to form hydroxyl radical and this may be the origin of many of its toxic effects [31]. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. Hydroxyl radical is the most reactive among reactive oxygen species (ROS) and it bears the shortest half-life compared with other ROS, induces severe damage to adjacent biomolecules [32]. The scavenging ability of different extracts of seeds of pennisetum glaucum on hydrogen peroxide was shown in Figure 2. Hydrogen peroxide scavenging activity was increased with increasing concentration of the extract (25100 µg/ml). Aqueous extract at a concentration of 100 µg /ml had maximum activity (61%) but which is higher than the standard BHT (57%) at a same concentration.
Fig.1 : Effect of PGAq seed extract on oral glucose tolerance in STZ induced diabetic rats (OGTT)
Fig.2: Scavenging effect of Pennisetum glaucum different extracts and BHT on hydrogen peroxide. Results are mean ± SE of three parallel measurements.
The administration of AEPG at a dose of 250 mg/k.g b.w along with 2g glucose/k.g b.w has significantly improved the glucose tolerance in the diabetic rats. In the diabetic untreated rats the glucose levels remained higher without much change at 120 min after glucose load. Where as in the AEPG and glibenclamide administrated diabetic rats the glucose levels started falling from 30min after glucose load and there was a consistent decrease in the blood glucose levels by the end of 120 min after glucose load. The results are depicted in Fig:1
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Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
[8].
Larner, I. Insulin and oral hypoglycaemic drugs Glucagon. In: Gilman, A.G., Goodman, L.S., Rall, T.W., Murad, F. •Eds.., The Pharmacological Basis of Therapeutics, 7th ed. Macmillan, New York, pp. 1985; 1490_1526.
[9].
Day, C. Traditional plant treatments for diabetes mellitus: pharmaceutical foods. Br. J. Nutr. 1998; 80, 203–208.
[10]. Kameswara, R.B., Giri, R., Kesavulu, M.M., Apparao, C. Herbal medicine:in the management of diabetes mellitus. Manphar Vaidhya Patrika 1997; I,33–35.
Fig.3: The reductive ability of Pennisetum glaucum different extracts and BHT results are mean ± SE of three parallel measurements.
The reducing power has been used as one of the important antioxidant capabilities for medicinal herbs [33,34]. The reducing power of the different extracts of pennisetum glaucum was dose-dependent (figure 3). The aqueous extract at a concentration of 200 µg/ml has produced maximum activity but which is lesser than that of standard BHT at the same concentration. In conclusion the present study showed that the aqueous extract of seeds of pennisetum glaucum not only possess antidiabetic activity but also antioxidant activity. This antioxidant property may reduce oxidative stress in diabetic patients which prevent diabetic complications. Further studies are necessary to isolate the active principle (s) responsible for these activitits.
References [1].
[2].
American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 33 (Suppl. 1): 2010;S62–S69
[11]. Mishra Akansha et al. Antihyperglycemic activity of six edible plants in validated animal models of diabetes mellitus. Indian J SciTechnol 2009;2:80–6. [12]. Prasad SK et al. Antidiabetic activity of some herbal plants in streptozotocin induced diabetic albino rats. Pak J Nutr2009;8:551–7. [13]. Marles RJ, Fransworth NR. Antidiabetic plants and their active constituents. Phytomedicine 1995;2:137–89. [14]. WHO. Expert committee on diabetes mellitus. Technical Report Series 646. Geneva: World Health Organization; 1980. [15]. Arulmozhi,D.K., Veeranjaneyulu, A., Bodhankar, S.L.,. Neonatal streptozotocininduced rat model of Type 2 diabetes mellitus: a glance. Indian J. Pharmacol. 2004; 36,217–221.
[16]. Pitozzi, V., Giovannelli, L., Bardini, G., Rotella, C.M., Dolara, P.,. Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerabiity of polymorphonuclear leukocytes. Mutat. Res. 2003; 529, 129– 133. [17]. Nazirogilu, M., Butterworth, P. Protective effects of moderate exercise with dietary vitamin C and E on blood antioxidative defense mechanism in rats with streptozotocininduced diabetes. Can. J. Appl. Physiol. 2005;30, 172–185. [18]. Maritim, A.C., Sanders, R.A., Watkins, J.B. Diabetes, oxidative stress and antioxidants. A review. J. Biochem. Mol. Toxicol. 2003;17, 24–38.
Arky, R.A. Clinical correlates of metabolic derangements of Diabetes Mellitus.In: Kozak, G.P. (Ed.), Complications of diabetes mellitus. W.B. Saunders, Philadelphia, 1982; pp. 16–20.
[19]. Chopra, R.N., Nayar, S.L., Chopra, I.C. Glossary of IndianMedicinal Plants1956; (CSIR, New Delhi).
[3].
Prasad SK et al. Antidiabetic activity of some herbal plants in streptozotocin induced diabetic albino rats. Pak J Nutr2009; 8:551–7.
[20]. Nandini, Chilkunda D. and Salimath, P. V.Structural features of arabinoxylans from sorghum having good rotimaking quality. Food Chemistry, 2001; 74. pp. 417-422. ISSN 0308-8146.
[4].
Shah Shweta, Bodhankar Subhash, Bhonde Ramesh, Mohan V.Combinative therapeutic approach for better blood sugar level control in alloxan diabetic mice. Int J Diabetes Metab 2006;14:104–5.
[21]. Sekar, N., Kanthasamy, S., William, S., Subramanian, S., Govindasamy, S. Insulinic actions of vanadate in diabetic rats. Pharmacol Res 1990;22, 207-217.
[5].
Gandhi, H.R. Diabetes and coronary artery disease: importance of risk factors. Cardiol. Today. 2001; 1, 31–34.
[22]. Bonnar-Weir, S. Morphological evidence of pancreatic polarity of beta cells within islets of Langerhans. Diabetes 1998;37,616-621.
[6].
National Diabetes Fact sheet, 2005. Centers for Disease Control and Prevention. Available at
.
[23]. Harborne, J.B. Phytochemical Methods. Analysis. A Guide rd to Modern Techniques of Plant. 3 ed. 2005 Spinger International, India.
[7].
Hwang, H.J., Kim, S.W., Lim, J.M., Joo, J.H., Kim, H.O., Kim,K.M., Yun, J.W.,. Hypoglycemic effect of crude exopolysaccharides produced by a medicinal mushroom Phellinus baumii in streptozotocin-induced diabetic rats. Life Sci. 2005;76, 3069–3080.
[24]. Ruth RJ, Cheng Sj, klauning JE. Carcinogenesis 1989;10 : 1003-1008
July - September 2012
41
[25]. Oyaizu M. Jap J Nutr 1986;44: 307-315.
Journal of Pharmacy and Chemistry • Vol.6 • Issue.3
[26]. Chopra RN, Nayer SL, Chopra IC. Glossary of Indian Medicinal Plants, CSIR, V ed. New Delhi, 1956;12: 157. [27]. Al-Awadi, F.M., Gumaa, K.A. Studies on the activities of individual plants of an antidiabetic plant mixture. ActaDiabetologica Latina 1987;24, 37. [28]. Ivorra, M.D., Paya, M., Villar, A. A review of Natural Products and Plants as Potent antidiabetic drugs. J. Ethnopharmacol. 1989; 27(3), 243-275. [29]. Vaidya, A.B., Pandya, S.K. The golden age of Indian Medicine. Bomba Hospital J1971; 3, 95. [30]. Mukherjee, P.K., Maiti, K., Mukherjee, K., Peter, J.
Houghton. Leads from Indian medicinal plants with hypoglycemic potentials. Journal of Ethnopharmacology 2006;106, 1–28. [31]. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, Oxford, Clarendon 1993; 419. [32]. Sakanaka S, Tachibaba Chemistry2005;89:569-575.
Y,
Okada
Y.
Food
[33]. Duh PD, Tu YY, Yen GC. Lebensmittel-Wissenschaft undTechnologie 1999; 32: 269-277. [34]. Duh PD, Yen G. Food Chemistry 1997; 60: 639-645.
European Journal of Medicinal Chemistry 66 (2013) 400e406
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antihyperglycemic and antihyperlipidemic activities of 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol in STZ induced diabetic rats Swapna Sirasanagandla a, Ramesh Babu Kasetti a, Abdul Nabi Shaik a, Rajesh Natava a, Venkata Prasad Surtineni a, Suresh Reddy Cirradur b, Apparao Chippada a, * a b
Department of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2012 Received in revised form 12 May 2013 Accepted 13 May 2013 Available online 8 June 2013
Oral administration of 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol (HBPMP) (30 mg/ kg) to Streptozotocin (STZ) rats produced significant antidiabetic activity after 6 h of HBPMP administration. Treatment of the STZ rats with HBPMP (30 mg/kg/day) for 30 days resulted in a significant decrease in their Fasting Blood Glucose (FBG), Serum Total Cholesterol (TC), Low Density LipoproteinCholesterol (LDL-C), Very Low Density Lipoprotein-Cholesterol (VLDL-C) and triglycerides (TG) along with an increase in serum High Density Lipoprotein-Cholesterol (HDL-C) levels. Activities of Serum Aspartate transaminase (AST), Alanine transaminase (ALT) and Alkaline phosphatase (ALP) and levels of blood urea and creatinine were improved to near normal levels in the treated STZ rats indicating the protective role of the HBPMP against liver and kidney damage and its non-toxic property. In conclusion, HBPMP possesses antihyperglycemic and antihyperlipidemic activities. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: HBPMP STZ Hypolipidemic Hypoglycemic activity
1. Introduction Diabetes mellitus (DM) is a metabolic disease as old as mankind and its incidence is considered to be high (4e5%) all over the world [1]. It is also a major cause of disability and hospitalization and it results in significant financial burden [2]. The number of people suffering from the disease worldwide is increasing at an alarming rate with a projected 366 million peoples likely to be diabetic by the year 2030 as against 191 million peoples estimated in 2000 [3]. The management of diabetes mellitus is considered a global problem and successful treatment is yet to be discovered. Besides the classical drug (insulin, sulfonylureas, biguanides and thiazolidinediones) usage for the treatment of diabetes, several species of plants have been described in scientific and popular literature as having hypoglycemic activity [4e6]. Because of their perceived effectiveness, minimal side effects in clinical experience and relatively low costs, herbal drugs are prescribed widely even when their biologically active compounds are unknown [7]. Despite the great interest in the development of novel drugs to revert the
* Corresponding author. Tel.: þ91 877 2289495 (O); fax: þ91 877 2289532. E-mail address: [email protected] (A. Chippada). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.05.014
burden of complications associated with this disease and the raised interest in the scientific community to evaluate either raw or isolated natural products in experimental studies only a few of them have been tested in humans [8e12]. Although, non-insulin-dependent diabetes mellitus (NIDDM) is more prevalent than insulindependent diabetes mellitus (IDDM), both are chiefly characterized by chronic hyperglycemia with disturbances of carbohydrate, lipid and protein metabolism resulting from defects in insulin secretion, insulin action or both. Hyperglycemia due to insulin deficiency and insulin resistance has been shown to be associated with the pathogenesis of diabetic dyslipidemia, micro- and macro-vascular complications [13,14]. Dyslipidemia is one of the major cardiovascular risk factors. It has been demonstrated that insulin deficiency in DM leads to a variety of disorders in metabolic and regulatory processes, which in turn leads to accumulation of lipids such as TC and TG in diabetic patients. Abnormalities in lipid profile are one of the most common complications in diabetes mellitus found in 40% of diabetic subjects [15]. Diabetes is associated with profound alterations in the plasma lipids, triglycerides and lipoprotein profile and with an increased risk of coronary heart disease. High level of total cholesterol is one of the major risk factors for coronary heart diseases and it is well known that hyperlipidemia leads to atherosclerosis and this is increased during diabetes [16].
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
OH
(1) Fig. 1. 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol.
73.3 70.0 72.5 75 372.5 223.28 102 36.8 206 75.8 72.0 73.16 79.3 373.5 265.16 215.6 54.8 197.6
4h
5.4 3.9 3.5 6.1 17.33 12.8 (17.5%) 20.2** (35.4%) 15.7** (82.3%) 7.8* (21.6%) 76.3 74.16 74.16 73.6 384.33 301.3 248.8 61.4 213.3
3h
3.7 4.2 4.7 1.5 17.5 13.2 (14.5%) 17.9 (34.2%) 21 (68%) 6.9 (12.8%) 76.5 73.16 73.66 71.3 386.66 312.83 252.1 109.5 237
2h
4.3 4.3 4.3 1.52 19.0* 14.0* 21.3 13.2 5 71.26 74.12 74.16 75.7 389.16 357.5 315.2 233.16 243.5
1h
4.8 4.5 4.5 4.7 19.8y 16.5y 19.8y 11.6y 7.5y 0h
Blood glucose at different hours after treatment (mg/dl)
78.1 75.16 74.5 74.6 390.16 365.9 384.4 347.0 272.8 Normal untreated rats Normal rats treated with 20 mg HBPMP/kg Normal rats treated with 30 mg HBPMP/kg Normal rats treated with 60 mg HBPMP/kg STZ untreated rats STZ rats treated with 20 mg HBPMP/kg STZ rats treated with 30 mg HBPMP/kg STZ rats treated with 60 mg HBPMP/kg STZ rats treated with 20 mg glibenclamide/kg
NH
Group
NH
Table 1 Effect of HBPMP on fasting blood glucose levels (mg/dl) of normal and STZ rats.
OH
2.1. Evaluation of antihyperglycemic activity of HBPMP in STZ rats and normal treated rats
yP < 0.0001 compared with the initial (0 h) level of blood glucose of normal rats. **P < 0.0001 compared with the initial (0 h) level of blood glucose in the respective group. *P <0.001 compared with the initial (0 h) level of blood glucose in the respective group. Numbers in parenthesis indicate the percentage of fall in 0 h blood glucose.
2. Results and discussion
The effects of different doses of HBPMP in normal and STZ rats are summarized in Table 1 and shown in Fig. 2. The fasting blood glucose levels of diabetic untreated rats were significantly higher than those of the normal untreated rats. HBPMP did not produce any hypoglycemic activity in normal treated rats. The HBPMP at a dosage of 30 mg/kg produced 73.8% glucose lowering effect in STZ rats after 6 h of treatment. The dose 20 mg/kg has produced a maximum 44% fall in blood glucose after 5 h of treatment. But the dose of 60 mg/kg has produced hypoglycemia in the STZ rats after 3 h of the treatment. Treatment of STZ rats with Glibenclamide (oral hypoglycemic agent) at a dosage of 20 mg/kg resulted in 31% fall of blood glucose after 5 h of treatment. Fig. 3 shows the effect of HBPMP on glucose tolerance in the STZ rats. The administration of HBPMP at a dose of 30 mg/kg along with 2 g/kg glucose load has significantly improved the glucose tolerance in the STZ rats. In the diabetic untreated rats the glucose levels remained higher without much change even after 3 h after glucose load. Whereas in the HBPMP administered STZ rats the glucose
73.8 70.1 72.8 78 372.2 204.8 172.8 49.1 188.1 4.7 3.5 3.3 4.9 15.6 12.4** (39%) 16.6** (44.01%) 10.8 (84.2%) 8.7** (27.5%)
5h
4.5 2.1 3.1 3.6 16.6 11.5** (44.1%) 12.3** (55.2%) 6.8 (85.8%) 5.2** (31%)
6h
4.4 1.8 2.6 5.5 15.6 12.1 (38.9%) 11.7** (73.8%) 9.7 (89.4%) 5.6** (24.4%)
A large number of crude plant extracts and purified substances from plants have been tested in clinical trials for treatment of diabetes. Other than these, many chemically synthesized compounds were also tested for diabetes with decreasing side effects [17]. Previously many synthetic novel compounds were demonstrated for biological activities. Chalcone-based aryl-oxy-propanolamines were tested for antihyperglycemic activity [18] and N-substituted-N-acyl thioureas of 4-substituted piperazines were endowed with local anesthetic, antihyperlipidemic, antiproliferative, antiarrhythmic, analgesic and antiaggregating actions [19]. We have reported earlier that the aminophenol-substituted amino acid ester 3,30 -(1,4-phenylene)-bis-[2-(1-(benzylcarbonyl)-2,3-dihydro-1H-pyrrolidine-2yl)]-2-thiobenzoxaphosphinine has antihyperglycemic and antioxidant activities [20]. The present study was done to demonstrate the antihyperglycemic and antihyperlipidemic activities of its parent compound, HBPMP (Fig. 1) since the aminophenol is more stable than amino acid ester when it is exposed to air.
401
402
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
Fig. 2. Effect of HBPMP on fasting blood glucose levels (mg/dl) of normal and STZ rats.
Fig. 3. Effect of HBPMP on glucose tolerance in STZ rats.
levels started falling from the 1st hour after glucose load and there was a consistent decrease in the blood glucose with maximum fall of 56%, (p < 0.0001) by the end of 3 h after glucose load. STZ was selected for induction of diabetes in rats rather than alloxan. STZ is well known for its selective pancreatic b-cell cytotoxicity and has been extensively used to induce diabetes mellitus
in animals [21] and it is less toxic than alloxan and allows a consistent production of diabetes mellitus. STZ is a monofunctional nitrosurea derivative, one of the most commonly used substances to induce diabetes in the experimental animals [22]. The experimental diabetic model used in this study was type 2 since low dose (50 mg/kg) of STZ destroyed half of the population of pancreatic bcells and there were residual b-cells which secreted insufficient insulin causing type 2 diabetes [23]. Earlier, substitute of aminophenol, an amino acid ester was screened for antihyperglycemic and antioxidant activity. The amino acid ester exhibited a significant antihyperglycemic activity (20). Previously many synthetic drugs were screened for treatment for various diseases. Chalcone derivatives from 3,4-methylenedioxybenzaldehyde and substituted acetophenones [24] were synthesized and investigated as antihyperglycemic agents in a glucoseloaded animal model. Influence of chalcone analogues on serum glucose levels in hyperglycemic rats [25], and antidiabetic activity of 2-amino [50 -(4-sulphonyl benzylidine)-2,4-thiazolidnedione]-7chloro-6-flurobenzothiazole in albino rats were evaluated [26]. The present study was carried out to demonstrate the antihyperglycemic and antihyperlipidemic activities of HBPMP. HBPMP decreased the fasting blood glucose levels effectively in STZ rats and also improved the glucose tolerance in glucose-loaded STZ rats. HBPMP did not cause hypoglycemia in normal treated rats. In the dose-dependent study 30 mg of HBPMP/kg produced a
Table 2 Effect of long-term treatment with HBPMP on blood glucose and body weights. Group
Normal untreated rats Normal rats treated with 30 mg HBPMP/kg/day STZ Untreated rats STZ rats treated with 30 mg HBPMP/kg/day STZ rats treated with 20 mg glibenclamide/kg/day F-value Significance
FBG (mg/dl) at different days during the experimental period and change in body weights after treatment with HBPMP. 1st day
15th day
30th day
Change in body weight (g)
89.0 6.4a 90.6 9.8a
82.66 6.05a 80.66 6.05a
94.8 10.4a 98.67 16.3a
þ18 7.5a (þ8.82%) þ16.6 7.8a (þ2.94%)
416.4 24.2c 333 26.5b
404.2 29.15d 247.5 30.14b,c
12.0 25.8d 113.5 11.36b
45 4.5c (30.3%) þ12 4.4d (þ8.57%)
356 16.18b
204 23.9c
188.1 5.2c
9 7.6d (10%)
242.45 0.000
365.57 0.000
369.55 0.000
23.736 0.00
Values are given as mean S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT). Numbers in parenthesis indicate the percentage of gain or loss of body weights.
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
maximum (74%) decrease in the fasting blood glucose (from 384.4 19.8 mg/dl to 102 11.7 mg/dl) levels but the dose of 60 mg/ kg caused hypoglycemia in STZ rats while the same dose did not produce any hypoglycemia in normal treated rats. The lack of hypoglycemia in the normal rats after treatment with 60 mg/kg HBPMP could be due to the efficient activity of the counter regulatory hormones to insulin, while it appears to be lacking in the STZ rats. 2.2. Effect of long-term (30 days) treatment with HBPMP on blood glucose and body weights of normal and STZ rats After the long-term treatment with HBPMP (30 mg/kg/day) there was a significant (66%) fall in fasting blood glucose (FBG) levels in the STZ rats, while there was no change in FBG levels in normal treated rats (Table 2). Treatment of STZ rats with glibenclamide for 30 days has resulted in 47% decrease in their blood glucose levels. This shows that the antidiabetic effect of HBPMP is higher than that of the standard antidiabetic drug glibenclamide. At the end of 30 days treatment, the body weights of normal, HBPMP treated normal, HBPMP treated STZ and glibenclamide treated STZ rat groups increased significantly by 18 g, 16.6 g, 12 g and 9 g respectively, whereas the body weights of untreated STZ rat group decreased by 45 g. In the present study, STZ rats have shown marked reduction in their body weights compared to normal rats (Table 2), which could be due to their uncontrolled diabetes. Streptozotocin induced diabetes is characterized by a severe loss in body weight [27]. The decrease in body weight is due to the loss or degradation of structural proteins (since structural proteins are known to contribute to the body weight) to provide amino acids for gluconeogenesis during insulin deficiency resulting in muscle wasting and weight loss [28]. Several workers have also reported a significant decrease in body weights of STZ rats [29]. The weight loss was reverted by the administration of HBPMP/glibenclamide to the STZ rats and this could be due to the improved blood glucose levels in the HBPMP/glibenclamide treated STZ rats. 2.3. Effect of long-term treatment with HBPMP on hyperlipidemia, hepatic and renal function markers Serum TG, TC, LDL- and VLDL-cholesterol levels were significantly higher and HDL-cholesterol levels were significantly lower in
Fig. 4. Effect of long-term treatment with HBPMP on hyperlipidemia in STZ and normal treated groups. Values are given as mean S.D.
403
Table 3 Effect of HBPMP on atherogenic index (AI) and % of protection against atherogenicity. Group
Atherogenic index (AI)
% of protection from atherogenicity
Normal untreated rats Normal rats treated with HBPMP STZ untreated rats STZ rats treated with HBPMP STZ rats treated with glibenclamide
1.32 1.48 4.95 1.64 2.22
73.3 70.1 e 66.8 55.5
the diabetic untreated rats compared to those in normal rats. After the treatment with HBPMP (30 mg/kg/day), a significant reduction in serum TG, TC, LDL- and VLDL-cholesterol and significant increase in the levels of serum HDL-cholesterol was observed (Fig. 4) and treatment with glibenclamide also produced similar changes in STZ rats. Treatment of STZ rats with HBPMP (30 mg/kg/day) showed protection against atherogenicity as evidenced by 66.8% decrease in the atherogenic index of the HBPMP treated STZ rats (Table 3). Abnormalities in lipid profile are one of the most common complications in diabetes mellitus [15]. Diabetes is associated with profound alterations in the plasma lipid, triglycerides and lipoprotein profile and with an increased risk of coronary heart disease [30]. After 30 days treatment with the HBPMP, the levels of HDL-C were significantly increased and the TG, TC, LDL-C and VLDL-C levels were significantly decreased. This marks to be an important advantage in treatment of hypercholesterolemia particularly among Indians where low HDL-cholesterol is the most prevalent lipoprotein abnormality in diabetic subjects [31]. However, it is interesting to find in the present study that treatment with 30 mg HBPMP/kg has not only lowered the TG, TC and LDL-C levels but also enhanced the cardio protective lipid HDLC after 30 days treatment. There is substantial evidence that lowering the TC, particularly the LDL-C level will lead to a reduction in the incidence of coronary heart disease [32], which is still a leading cause of death in diabetic patients [33]. The activities of serum ALT, AST, ALP and the blood levels of urea and creatinine were increased in diabetic untreated rats when compared with normal rats. After treatment with HBPMP at a dosage of 30 mg/kg for 30 days the activities of ALT, AST and ALP (Fig. 5) and levels of creatinine and urea were significantly decreased to near normal levels in the HBPMP treated STZ rats (Table 4). Similar changes were also observed in glibenclamide
Fig. 5. Effect of long-term treatment with HBPMP on serum AST, ALT and ALP activities of normal and STZ rats. Values are given as mean S.D.
404
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Table 4 Effect of HBPMP on kidney function in normal and STZ rats after 30 days of treatment. Groups
Urea (mg/dl) Before treatment
Normal untreated rats Normal treated with HBPMP STZ untreated rats STZ rats treated with HBPMP STZ rats treated with glibenclamide F-value Significance
a
41.80 0.80 44.7 0.90a 64.20 1.65b 69.50 2.14b 54.61 1.45c 72.138 0.00
Creatinine (mg/dl) After treatment a
42.75 0.85 43.25 1.37a 75.0 1.15b 54.02 1.828c 49.10 4.613c 53.771 0.00
Before treatment 0.294 0.366 0.732 0.508 0.450 42.623 0.00
a
0.082 0.024b 0.044c 0.026d 0.0428d
After treatment 0.308 0.370 0.947 0.462 0.425 429.09 0.000
0.04a 0.021a 0.020b 0.030c 0.047c
Values are given as mean S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT).
treated rats. There were no changes in the hepatic functional markers in normal treated rats (Fig. 5). In the present study a significant increase in the activities of liver function enzymes (AST, ALT and ALP) were observed in diabetic untreated rats compared to normal control rats. Yanardag et al. (2005) [34] and Rajasekaran et al. (2006) [35] reported that in the STZ rats, the levels of ALT and AST activities were significantly increased. Shinde and Goyal (2003) [36], also reported an elevation of serum hepatic enzymes in STZ rats, indicating the hepatotoxic effect of STZ. ALT and AST are directly associated with the conversion of amino acids to ketoacids. Increased protein catabolism accompanying gluconeogenesis and urea formation that are observed in diabetic state might be responsible for the elevation of ALT and AST. Administration of HBPMP lowered the serum AST, ALT and ALP activities in the HBPMP treated STZ rats. Our findings are in agreement with those of Prakasam et al. (2004) [37]. HBPMP has protective effect against liver toxicity caused by STZ. Treatment with HBPMP in normal rats for 30 days did not produce any hepato toxicity. The STZ rats exhibited significantly higher plasma urea, and creatinine levels compared to the normal rats (Table 4). However, supplementation of HBPMP lowered these plasma values to a control range by enhancing the renal function which is generally impaired in STZ rats. 3. Conclusion From all these observations, it is concluded that the HBPMP possess antidiabetic activity and antihyperlipidemic activity. HBPMP effectively reduces the elevated levels of hepatic and renal markers in diabetic treated group, indicating the hepatoprotective and renal protective role of HBPMP. 4. Experimental design The rats were divided into 9 groups for evaluation of antihyperglycemic activity of HBPMP with different doses (20 mg/kg, 30 mg/kg and 60 mg/kg). Among these groups, 2 were control groups (normal control and diabetic control) and the other 7 groups were treated groups with either HBPMP or glibenclamide (standard oral hypoglycemic agent). For the long-term (30 days) study the rats were divided into 5 groups with 2 control groups and 3 treated groups with HBPMP (30 mg/kg/day) or glibenclamide (20 mg/kg/day). 5. Materials and methods 5.1. Induction of diabetes Diabetes was induced in male Wistar albino rats aged 4 months (body weight 180e200 g) by intra peritoneal administration of STZ (single dose of 55 mg/kg) dissolved in freshly prepared 0.01 M
citrate buffer (pH 4.5). After 48 h rats with marked hyperglycemia (fasting blood glucose 250 mg/dl) were selected and used for the study. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages with 12 h light/dark cycle. All the procedures were performed in accordance with the guidelines of institutional Animal Ethics Committee. 5.2. Evaluation of antihyperglycemic activity of HBPMP in normal and STZ rats The animals were divided into 9 groups and each group consisted of 6 rats: Group 1: Normal untreated rats Group 2: Normal treated rats with 20 mg HBPMP/kg Group 3: Normal treated rats with 30 mg HBPMP/kg Group 4: Normal treated rats with 60 mg HBPMP/kg Group 5: STZ untreated rats Group 6: STZ rats treated with 20 mg HBPMP/kg Group 7: STZ rats treated with 30 mg HBPMP/kg Group 8: STZ rats treated with 60 mg HBPMP/kg Group9: STZ rats treated with 20 mg glibenclamide/kg. After an overnight fast the normal treated groups and STZ treated groups received the HBPMP dissolved in distilled water by gastric intubation using a force feeding needle. Normal untreated and diabetic untreated rats were fed with distilled water alone. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 1, 2, 3, 4, 5, & 6 h duration after the administration of HBPMP and blood glucose levels were determined by using dextrostix (glucose oxidase method) with Basic One Touch Accuchec Glucometer. The results were compared with those of the 9th group of rats which were treated with 20 mg glibenclamide/kg. 5.3. Effect of HBPMP on glucose tolerance Two groups of STZ rats each group containing 6 rats were used for this study. 1. STZ untreated rats 2. STZ rats treated with 30 mg HBPMP/kg. The oral glucose tolerance test was (Bonnar-Weir 1998 [38]) performed in overnight fasted STZ rats. Glucose (2 g/kg) was administered orally to both groups of rats using a force feeding needle. The group 2 rats were administered the HBPMP at a dose of 30 mg/kg along with the glucose load. Blood samples were collected from the tail veins of all the animals from 0 h (before glucose administration) to 3 h of glucose administration for
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
estimation of blood glucose using dextrostix with Basic One Touch Accuchec Glucometer (glucose oxidase method). 5.4. Effect of long-term treatment with HBPMP on glycemic control, serum lipid profiles, hepatic and renal function markers in normal and STZ rats The rats were divided into 5 groups and each group consisted of 6 rats. Group 1: Group 2: Group 3: Group 4: Group 5:
Normal untreated rats. Normal rats treated with 30 mg HBPMP/kg/day. STZ untreated rats. STZ rats treated with 30 mg HBPMP/kg/day. STZ rats treated with 20 mg of glibenclamide/kg/day.
The HBPMP or glibenclamide was administered into the animals of the respective groups every day morning for 30 days by gastric intubation with a force feeding needle. All the 5 groups were sacrificed on the 31st day after an overnight fasting by cervical dislocation and then blood, liver, kidney and pancreas were collected and immediately stored at 20 C till further analysis. Body weights of all the animals were recorded prior to the treatment and sacrifice. 5.5. Biochemical parameters Serum total cholesterol (TC), triglycerides (TG), and HDLcholesterol (HDL-C) were estimated according to the methods of Zlatkis et al. [39], Foster and Dunn [40], and Burstein et al. [42] respectively. The serum levels of VLDL- and LDL-cholesterol were calculated using Friedwald et al.’s formula [41]. The atherogenic index (AI) was calculated by using the following formula [43],
Atherogenic indexðAIÞ ¼
Protectionð%Þ ¼
TC HDL C HDL C
AI of control AI of treated group 100: AI of control
Plasma AST and ALT activities were determined by Reitman and Frankel method [44]. Activity of serum alkaline phosphatase was determined by p-nitrophenyl phosphate method [45]. Plasma creatinine and urea were measured by Jaffe’s method [46] and diacetyl monoxime method [47] respectively. 6. Statistical analysis The results were expressed as mean S.D. The statistical analysis of results was carried out using Student’s t-test and one-way analysis (ANOVA) followed by DMRT. References [1] M. Koyuturk, O. Ozsoy-Sacan, S. Bolkent, R. Yanardag, Effect of glurenorm on immunohistochemical changes in pancreatic b cells of rats in experimental diabetes, Indian J. Exp. Biol. 43 (2005) 268e271. [2] A.N. Nagappa, P.A. Thakurdesai, N.V. Rao, J. Singh, Antidiabetic activity of Terminalia catappa Linn. fruits, J. Ethnopharmacol. 88 (2003) 45e50. [3] S.G. Wild, A. Roglic, R. Green, H. King, Global prevalence of diabetes estimated for the year 2000 and projection for 2030, Diabetes Care 27 (2004) 1047e 1054. [4] E.J. Verspohl, Recommended testing in diabetes research, Planta Med. 68 (2002) 581e590. [5] E. de Sousa, L. Zanatta, I. Seifriz, T.B. Creczynski-Pasa, M.G. Pizzolatti, B. Szpoganicz, F.R. Silva, Hypoglycemic effect and antioxidant potential of kaempferol-3,7-O-(a)-dirhamnoside from Bauhinia forficata leaves, J. Nat. Prod. 67 (2004) 829e832.
405
[6] J.R. Colca, Insulin sensitizers may prevent metabolic inflammation, Biochem. Pharmacol. 72 (2006) 125e131. [7] M.S. Valiathan, Healing plants, Curr. Sci. 75 (1998) 1122e1126. [8] J.P. Lieu, M. Zhang, W.Y. Wang, S. Grimsgaard, Chinese herbal medicines, for type-2 diabètes mellitus (3), Cochrane Database Syst. Rev. (2004) 3642. [9] V. Vuksan, J.L. Sievenpiper, Herbal remedies in the management of diabetes: lessons learned from the study of ginseng, Nutr. Metab. Cardiovasc. Dis. 15 (2005) 149e160. [10] L. Johnson, H. Strich, A. Taylor, B. Timmermann, D. Malone, N. Teufel-Shone, R. Drummond, R. Woosley, E. Pereira, A. Martinez, Use of herbal remedies by diabetic Hispanic women in the southwestern United States, Phytother. Res. 20 (2006) 250e255. [11] M. Jung, M. Park, H.C. Lee, Y.H. Kang, E.S. Kang, S.K. Kim, Antidiabetic agents from medicinal plants, Curr. Med. Chem. 13 (2006) 1203e1218. [12] T. Matsui, I.A. Ogunwande, K.J.M. Abesundara, K. Matsumoto, Antihyperglycemic potential of natural products, Mini Rev. Med. Chem. 6 (2006) 109e120. [13] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813e820. [14] T.A. Clark, G.N. Pierce, Cardiovascular complications of non-insulin, dependent diabetes: the JCR: LA-cp rat, J. Pharmacol. Toxicol. Methods 43 (2000) 1e10. [15] K. Ravi, S. Rajasekaran, S. Subramanian, Antihyperglycemic effect of Eugenia jambolana seed kernel on streptozotocin-induced diabetes in rats, Food Chem. Toxicol. 43 (2005) 1433e1439. [16] B.K. Tan, C.H. Tan, P.N. Pushparaj, Anti-diabetic activity of the, semi purified fractions of Averrhoa bilimbi in high fat diet fed-streptozotocin induced STZ rats, Life Sci. 76 (2005) 2827e2839. [17] A.A. Izzo, E. Ernst, Interactions between herbal medicines and prescribed drugs: a systemic review, Drugs 61 (2001) 2163e2175. [18] M. Satyanarayana, P. Tiwari, B.K. Tripathi, A.K. Srivastava, R. Pratap, Synthesis and antihyperglycemic activity of chalcone based aryl oxy propanolamines, Bioorg. Med. Chem. 12 (5) (Mar 1, 2004) 883e889. [19] A. Ranise, O. Spallarossa, S. Schenone, P. Fossa, G. Menozzi, F. Bondavalli, L. Mosti, A. Capuano, F. Mazzeo, G. Falcone, W. Filippelli, Synthesis of Nsubstituted-N-acylthioureas of 4-substituted piperazines endowed with local anaesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions, Farmaco 58 (9) (2003 Sep) 765e780. [20] 2011 S. Swapna, K. Ramesh Babu, S. Abdul nabi, M. Veera Narayana Reddy, C. Suresh Reddy, C. Appa Rao, Antihyperglycemic and antioxidant activities of 3,30 -(1,4-phenylene)-bis [2-(1-(benzyl carbonyl)-2,3-dihydro-1H-pyrrolidine2-yl)-2-thiobenzoxaphosphinine in STZ rats, Int. J. Drug Des. Discov. 3 (1) (JanuaryeMarch 2012) 677e682. [21] K. Raju, R. Balaraman, Antidiabetic mechanisms of saponins of Momordica cymbalaria, Phcog. Mag. 4 (15) (2008). [22] T. Szudelski, The mechanism of alloxan and streptozotocin action in B cells of rat pancreas, Physiol. Res. 50 (2001) 536e546. [23] A. Gomes, J.R. Vedasiromoni, M. Das, R.M. Sharma, D.K. Ganguly, Antihyperglycemic effect of black tea (Camellia sinensis) in rat, J. Ethnopharmacol. 27 (2001) 243e275. [24] A. Ranise, A. Spallarossa, O. Bruno, S. Schenone, P. Fossa, G. Bondavalli, L. MostiL, A. Capuano, F. Mazzeo, G. Falcone, W. Filippelli, Synthesis of Nsubstituted-N-acylthioureas of 4-substituted piperazines endowed with local, anesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions, Farmaco 58 (9) (2003) 765e780. [25] E.H. Alberton, R.G. Damazio, L.H. Cazarolli, L.D. Chiaradia, P.C. Leal, R.J. Nunes, R.A. Yunes, F.R. Silva, Influence of chalcone analogues on serum glucose levels in hyperglycemic rats, Chem. Biol. Interact. 171 (3) (2008) 355e362. [26] P.S. Yadav, Devprakash, G.P. Senthilkumar, Benzothiazole: different methods of synthesis and diverse biological activities, Int. J. Pharmaceut. Sci. Drug Res. 3 (1) (2011) 01e07. [27] L. Al-Shamaorry, S.M. Al-khazrajoi, H.A. Twaij, Hypoglycemic effect of Artemisia herba alba. II. Effect of a valuable extract on some blood parameters in diabetic animals, J. Ethnopharmacol. 43 (1994) 167e171. [28] L. Rajkumar, N. Srinivasan, K. Balasubramanian, P. Govindarajulu, Increased degradation of dermal collagen in diabetic rats, Indian J. Exp. Biol. 29 (1991) 1081e1083. [29] P. Kalaiarasi, K.V. Pugalendi, Antihyperglycemic effect of 18b-glycyrrhetinic acid, aglycone of glycyrrhizin on streptozotocin-diabetic rats, Eur. J. Pharmacol. 606 (2009) 269e273. [30] M. Maghrani, A. Lemhadri, N.A. Zeggwagh, M. Amraoui, M. Haloui, H. Jouad, M. Eddouks, Effects of an aqueous extract of Triticum repens on lipid metabolism in normal and recent-onset STZ rats rats, J. Ethnopharmacol. 90 (2004) 331e337. [31] R. Gupta, H.P. Gupta, N. Kumar, A.K. Joshi, V.P. Gupta, Lipoprotein lipids and prevalence of hyperlipidemia in rural India, J. Cardiovasc. Risk 1 (1994) 179e183. [32] Lipid Research Clinics Programs, The lipid research clinics coronary primary prevention trial results. 11. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering, J. Am. Med. Assoc. 252 (1984) 365e374. [33] J.W. Baynes, Role of oxidative stress in the development of complications in diabetes, Diabetes 40 (1991) 405e.412. [34] R. Yanardag, O. Ozsoy-Sacan, S. Bolkent, H. Orak, O. Karabulut-Bulen, Protective effects of metformin treatment on the liver injury of streptozotocinSTZ rats, Hum. Exp. Toxicol. 24 (2005) 129e135. [35] S. Rajasekaran, K. Ravi, K. Sivagananam, S. Subramanian, Beneficial effects aloevera leaf gel extract on lipid profile status in rats with streptozotocin diabetes, Clin. Exp. Pharmacol. Physiol. 33 (2006) 232e237.
406
S. Sirasanagandla et al. / European Journal of Medicinal Chemistry 66 (2013) 400e406
[36] U.A. Shinde, R.K. Goyal, Effect of chromium picolinate on histopathological alterations in STZ and neonatal STZSTZ rats, J. Cell Mol. Med. 7 (2003) 332e339. [37] A. Prakasam, S. Sethupathy, K.V. Pugalendi, Antiperoxidative and antioxidant effects of Casearia esculenta root extract in streptozotocin-induced rats, J. Biol. Med. 78 (2005) 15e23. [38] S. Bonnar-Weir, Morphological evidence of pancreatic polarity of beta cells within islets of Langerhans, Diabetes 37 (1998) 616e621. [39] A. Zlatkis, B. Zak, A.J. Boyle, D. Mich, A new method for the direct determination of serum cholesterol, J. Lab. Clin. Med. 41 (1953) 486e492. [40] J.B. Foster, R.T. Dunn, Stable reagents for determination of serum triglycerides by colorimetric Hantzsch condensation method, Clin. Chem. 19 (1973) 338e340. [41] W.T. Friedwald, R.I. Levy, D.S. Fredrickson, Estimation of the concentration of LDL-cholesterol in plasma without the use of the preparative utracentrifuge, Clin. Chem. 18 (1972) 499e502.
[42] M. Burstein, H.R. Scholnichk, R. Morin, Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions, J. Lipid Res. 11 (1970) 583e595. [43] T. Suanarunsawat, W.D.N. Ayutthaya, T. Songsak, J. Rattanamahaphoom, Antilipidemic actions of essential oil extracted from Ocimum sanctum L. leaves in rats fed with high cholesterol diet, J. Appl. Biomed. 7 (2009) 45e53. [44] S. Reitman, S. Frankel, Colorimetric method for the determination of serum glutamic oxaloacetic acid and glutamic pyruvic transaminases, Am. J. Clin. Pathol. 28 (1957) 56e63. [45] O.A. Bessey, O.H. Lowry, M.J. Brock, A method for the determination of alkaline phosphatase with five cubic millimeters of serum, J. Biol. Chem. 164 (1946) 321e329. [46] C. Slot, Plasma creatinine determination. A new and specific Jaffe’s reaction method, Scand. J. Clin. Lab. Invest. 17 (1965) 381e387. [47] D.R. Wybenga, J. Di Giorgio, V.J. Pileggi, Manual and automated methods for urea nitrogen measurement in whole serum, Clin. Chem. 17 (1971) 891e895.
