Scholarly article on topic 'Searching for antihyperglycemic phytomolecules through bioassay-guided solvent fractionation and subfractionation from hydro-methanolic (2:3) extract of Tamarindus indica Linn. seeds in streptozotocin-induced diabetic rat'

Searching for antihyperglycemic phytomolecules through bioassay-guided solvent fractionation and subfractionation from hydro-methanolic (2:3) extract of Tamarindus indica Linn. seeds in streptozotocin-induced diabetic rat Academic research paper on "Biological sciences"

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{antidiabetic / "high performance liquid chromatography" / "high performance thin layer chromatography" / "serum insulin" / " Tamarindus indica Linn"}

Abstract of research paper on Biological sciences, author of scientific article — Debasis De, Kausik Chatterjee, Kishalay Jana, Kazi Monjur Ali, Tushar Kanti Bera, et al.

Abstract The study identified the most effective fraction and subfraction of hydro-methanolic extract (2:3) of the seed of Tamarindus indica Linn. (T. indica) having antidiabetic activity in rats with diabetes induced by streptozotocin (STZ). The effective fraction and subfraction of T. indica were subjected to an antidiabetic study in STZ-induced diabetic rats at two dose levels, 100 mg/kg and 25 mg/kg body weight twice a day. Serum insulin, glycosylated hemoglobin, carbohydrate metabolic enzymes, and transaminases were assessed and the histopathology of the pancreas was examined after 8 weeks of treatment and compared to the vehicle control. Treatment of n-hexane fraction at a dose of 100 mg/kg twice a day for 56 days in STZ-induced diabetic rat resulted in a significant reduction in fasting blood glucose and glycosylated hemoglobin levels along with a rise in serum insulin and glycogen contents in hepatic and skeletal muscle in comparison to chloroform, ethyl acetate, or n-butanol fraction treated groups as well as the untreated diabetic group. The most antidiabetic activity of n-hexane fraction had been highlighted by the activities of hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, and lactate dehydrogenase in the liver, kidney, cardiac, and skeletal muscle in respect with groups treated with other fractions. Two subfractions, A and B, were obtained from the n-hexane fraction using petroleum ether, of which subfraction B was more bioactive considering the above biosensors and was comparable with glibenclamide, a standard antihyperglycemic drug. Chromatographic study by high performance thin layer chromatography focused on two components of subfraction B, P1 and P2 where P1 is predominant, conformed by high performance liquid chromatography. The dose of subfraction B was 25 mg/kg twice a day i.e., 1/4 dose of the n-hexane fraction. The n-hexane fraction and subfraction B of T. indica are free from hepatic and renotoxicity according to the study of serum transaminase.

Academic research paper on topic "Searching for antihyperglycemic phytomolecules through bioassay-guided solvent fractionation and subfractionation from hydro-methanolic (2:3) extract of Tamarindus indica Linn. seeds in streptozotocin-induced diabetic rat"

Biomarkers and Genomic Medicine (2013) 5, 164-174

Available online at www.sciencedirect.com

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ORIGINAL ARTICLE

Searching for antihyperglycemic phytomolecules through bioassay-guided solvent fractionation and subfractionation from hydro-methanolic (2:3) extract of Tamarindus indica Linn. seeds in streptozotocin-induced diabetic rat

CrossMark

Debasis Dea, Kausik Chatterjeea, Kishalay Jana a,

Kazi Monjur Alia, Tushar Kanti Beraa, Ramapati Samanta b,

Debidas Ghosh a *

a Department of Bio-Medical Laboratory Science and Management (UGC Innovative Programme Funded Department), Vidyasagar University, Midnapore, West Bengal, India

b Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India

Received 21 May 2013; received in revised form 10 August 2013; accepted 12 September 2013 Available online 27 November 2013

KEYWORDS

antidiabetic; high performance liquid

chromatography; high performance thin layer chromatography; serum insulin; Tamarindus indica Linn

Abstract The study identified the most effective fraction and subfraction of hydro-methano-lic extract (2:3) of the seed of Tamarindus indica Linn. (T. indica) having antidiabetic activity in rats with diabetes induced by streptozotocin (STZ). The effective fraction and subfraction of T. indica were subjected to an antidiabetic study in STZ-induced diabetic rats at two dose levels, 100 mg/kg and 25 mg/kg body weight twice a day. Serum insulin, glycosylated hemoglobin, carbohydrate metabolic enzymes, and transaminases were assessed and the histopathol-ogy of the pancreas was examined after 8 weeks of treatment and compared to the vehicle control. Treatment of n-hexane fraction at a dose of 100 mg/kg twice a day for 56 days in STZ-induced diabetic rat resulted in a significant reduction in fasting blood glucose and glyco-sylated hemoglobin levels along with a rise in serum insulin and glycogen contents in hepatic and skeletal muscle in comparison to chloroform, ethyl acetate, or n-butanol fraction treated groups as well as the untreated diabetic group. The most antidiabetic activity of n-hexane

* Corresponding author. Department of Bio-Medical Laboratory Science and Management, Vidyasagar University, Midnapore 721102, West Bengal, India.

E-mail address: debidas_ghosh@yahoo.co.in (D. Ghosh).

2214-0247/$36 Copyright © 2013, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.10167j.bgm.2013.09.001

fraction had been highlighted by the activities of hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, and lactate dehydrogenase in the liver, kidney, cardiac, and skeletal muscle in respect with groups treated with other fractions. Two subfractions, A and B, were obtained from the n-hexane fraction using petroleum ether, of which subfraction B was more bioactive considering the above biosensors and was comparable with glibenclamide, a standard antihyperglycemic drug. Chromatographic study by high performance thin layer chromatography focused on two components of subfraction B, P1 and P2 where P1 is predominant, conformed by high performance liquid chromatography. The dose of subfraction B was 25 mg/kg twice a day i.e., 1/4 dose of the n-hexane fraction. The n-hexane fraction and subfraction B of T. indica are free from hepatic and renotoxicity according to the study of serum transaminase.

Copyright © 2013, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.

Introduction

Diabetes mellitus is a chronic and major endocrine disorder caused by a deficiency in the production of insulin by the pancreas or by the ineffectiveness of the insulin produced.1 Currently there are over 150 million diabetics worldwide and this is likely to increase to 300 million or more by 2025. Statistical projection in India suggest that the number of diabetics will rise from 15 million in 1995 to 57 million in 2025 making India the country with the highest number of diabetics in the world.2,3 Recent studies on geographical and ethnic influences have shown that people of Indian origin are highly prone to diabetes.4 A considerable amount of money is spent on antidiabetic drugs, so the introduction of indigenous herbal compounds in the management of diabetes mellitus will greatly simplify the management and make it less expensive. Compared with synthetic drugs, plant drugs are frequently considered to be less toxic with fewer side effects.5

Tamarindus indica Linn. is a large and tall tree belonging to the family Caesalpiniaceae that is found all over India. These fruits are found mainly in summer; the seed coat is brownish black in color and the kernel is white. Pharmacological studies of the plant have revealed that T. indica possess anti-snake venom, antibacterial, antifungal, anti-inflammatory, antimalarial, antioxidant, and hepator-egenerative activities.6-9 In our previous work, aqueous extract of seed of T. indica has also studied as an antidia-betic agent.10 The antidiabetic efficacy of the extract was compared with that of glibenclamide, a standard oral antidiabetic drug.

The present study was designed to investigate the most active fraction and subfraction of the hydromethanolic extract of the seed of T. indica against hyperglycemia in experimental diabetes along with the isolation of active plant ingredients through high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) mediated purification.

Materials and methods Collection of plant material

The plant material i.e. seeds of T. indica were collected during the summer season (May-June) from the local

market and agricultural fields of Midnapore and authenticated by the Department of Botany, Vidyasagar University, West Bengal, India. The specimens were preserved in the Department of Bio-Medical Laboratory Science and Management as BMLSM-No.-03. The seeds required for the study were first separated from the pulp. The collected seeds were cleaned properly with tap water and then with deionized water. The cleaned seeds of the plant were dried completely at 40 °C in the drying oven. The dried seeds were pulverized separately in an electrical grinder and were kept in self-sealing polythene bags to avoid any contamination and for future experimental studies.

Preparation of hydro-methanolic extracts

Pulverized seeds (5000 g) of T. indica were placed in a 20 L percolator and maceration was carried out with 10 L hydro-methanolic solution (H2O:MeOH 40:60) at 25°C to avoid any degradation or deactivation of the active compound (s). The slurry was stirred intermittently for1 hourand left overnight. The extract was collected on the Day 2 after 24 hours of extraction process and then freshly prepared 5 L hydro-methanolic solution was added to the extraction chamber and the slurry was stirred again with a glass rod. The same procedure was repeated again on Day 3 with another 5 L of solvent mixture and the extract was collected on Day 4. The extract was filtered first by cotton filter and then by Whatman filters paper (No.1). The filtrate was evaporated under reduced pressure by Rotavapour (BUCHI-R124; Buchi, Flawil, Switzerland) at 40°C for the complete removal of methanol. Finally plain aqueous filtrate (9.5 L free from methanol) was lyophilized on VirTis bench top K lyophilizer (S.P Scientific, Warminster, PA, USA). The lyophilized extract (920 g) was collected and put into the amber colored glass containers, which were stored in the refrigerator under vacuum for subsequent fractionation and experimental studies. The lyophilized extract was a mixture of dark brownish sticky layer and light brownish solid powder (slightly hygroscopic in nature).

Bioassay guided fractionation

In a 5 L separating flask, 600 g of lyophilized extract of T. indica were dissolved with 2 L of hydromethanolic solution

Table 1 Comparative study of different fractions and subtraction of the seed of Tamarindus indica Linn. on body weight in male albino rat: duration-dependent response.

Groups

Duration of treatment and body weight (g)

Day 1 Day 28 Day 56

Control 145.58 ± 4.63a 172.24 ± 3.42a 198.62 ± 2.14'

Untreated diabetic 147.31 ± 3.82a 135.53 ± 3.14b 124.82 ± 3.751

Diabetic - h n-hexane fraction 147.74 ± 4.64a 168.65 ± 2.67a 193.45 ± 2.56'

Diabetic - chloroform fraction 145.56 ± 3.14a 161.35 ± 3.18c 182.54 ± 3.75'

Diabetic - h ethyl acetate fraction 147.54 ± 3.65a 156.72 ± 2.25d 168.24 ± 3.21'

Diabetic - h n-butanol fraction 145.80 ± 3.95a 154.63 ± 2.87d 161.76 ± 3.31'

Diabetic - h subfraction A fraction 147.47 ± 4.46a 165.45 ± 3.14a 190.24 ± 4.61

Diabetic - h subfraction B fraction 148.79 ± 5.14a 170.62 ± 4.13a 196.45 ± 4.62'

Diabetic - glibenclamide 146.36 ± 3.27a 171.26 ± 2.84a 194.42 ± 4.71

Data are expressed as mean ± standard error of the mean, n = 6, analysis of variance followed by multiple comparisons two-tail t test. Values with different superscripts (a-d) differ from each other significantly (p < 0.05).

and solvent fractionation was carried out using solvents with increasing polarity (n-hexane, chloroform, ethyl acetate, and n-butanol). Thin layer chromatography was carried out to monitor the progress in fractionation. All fractionates were collected separately and dried under reduced pressure (20-200 mbar) using a rotavapour instrument at 40°C. From 600 g of lyophilized extract of T. indica, 2.5 g of n-hexane fraction, 19.4 g of chloroform fraction, 61.8 g of ethyl acetate fraction, and 138.5 g of n-butanol fraction were obtained. All the doses were administered orally through gavage twice a day.

Subfraction of n-hexane fraction by petroleum ether

A 1 g sample of n-hexane fraction was taken into an eppendorf tube (T-2795, Sigma Aldrich (P-10), Taratalla Road, 2nd floor, Kol-700 088) (2 mL) and 1 mL of petroleum ether was added to it. The solution was then vortexed for 2 minutes for proper mixing. Finally the solution was centri-fuged at 2800g for 5 minutes. After centrifugation, it was observed that the solution had separated into two distinct layers. The upper portion was liquid and oily in nature with greenish color (subfraction A) whereas the lower portion was a white colored solid compound (subfraction B). From 1 g of n-hexane fraction, 672 mg of subfraction A and 328 mg of subfraction B were obtained. Subfraction A and B were administered orally through gavage.

Chemicals and reagents

Streptozotocin (STZ; Sigma-Aldrich Diagnostic Ltd., St Louis, MO, USA) and glibenclamide (Medicare Pharmacy, Allahabad, India) were purchased. Kits for the estimation of glucose, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) were from Span Diagnostics, Surat, India. All other chemicals and reagents used for the study were of analytical grade obtained from E. Merck (Mumbai, India) or HIMEDIA (Mumbai, India).

Induction of diabetes mellitus

Diabetes was induced in male Wistar albino rats aged 3 months, body weight 145 ± 5 g by single intramuscular injection of STZ at the dose of 4 mg/0.1 mL of citrate buffer/ 100 g body weight. After48 h rats with marked hyperglycemia (fasting blood glucose > 250 mg/dL and <400 mg/dL) were selected and used for the study. Animals were housed at an ambient temperature of 25 ± 1 °C with a 12-hour light/dark cycle and fed with rat pellets diet (Hindustan Lever Limited, Bangalore, India) and water ad libitum in plastic cages, as per the guidelines of the Vidyasagar University, Midnapore, West Bengal, India Animal Ethics committee. The diabetic condition was tested for 7 successive days to monitor the stabilization of diabetes.

Experimental design

Screening for antihyperglycemic activity of the different fractions and subfractions of n-hexane fraction of seed of T. indica.

The rats were divided into nine groups and each group consisted of six rats: Group 1, control rats; Group 2, diabetic untreated rats; Group 3, diabetic rats treated with 100 mg/kg n-hexane fraction twice a day; Group 4, diabetic rats treated with 100 mg/kg chloroform fraction twice a day; Group 5, diabetic rats treated with 100 mg/kg ethyl acetate fraction twice a day; Group 6, diabetic rats treated with 100 mg/kg n-butanol fraction twice a day; Group 7, diabetic rats treated with 25 mg subfraction A/kg twice a day; Group 8, diabetic rats treated with 25 mg subfraction B/kg twice a day; and Group 9, diabetic rats treated with 20 mg/kg glibenclamide twice a day.

Every day, the first oral dose of the said fractions and subfractions was given 1 hour prior to the supply of animal feeds in the morning (at 08:00) and the second oral dose was administered after 2 hours of the cleaning of feed box at 17:00. Feeds were supplied again to the animals after 1 hour of the second oral administration of the said fractions and subfractions. Animals of Group 1 and Group 2 were

Table 2 Corrective effect of 56 days of treatment with various fractions and subfraction of Tamarindus indica Linn. from hydromethanolic extract on blood glucose level in STZ-induced diabetic rats.

Groups Fasting blood glucose level (mg/dL)

Day 1 (day of STZ injection) Day 7 (day of fraction treatment) Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56

Control 74.03 ± 2.42 a 73.45 ± 2.35a 75.21 ± 2.54 a 77.86 ± 1.67 a 75.25 ± 2.31 a 78.75 ± 3.36a 81.45 ± 2.82a 79.47 ± 3.04a 77.37 ± 3.24a

Untreated diabetic 75.58 ± 3.12a 337.32 ± 2.84b 335.54 ± 2.53b 338.63 ± 2.75 b 341.52 ± 2.14 b 339.36 ± 1.95b 345.53 ± 1.83b 342.52 ± 2.02b 337.54 ± 2.03b

Diabetic + n-hexane 75.87 ± 2.80a 348.62 ± 3.34b 211.37 ± 3.49c 158.57 ± 2.15c 128.92 ± 2.15c 115.74 ± 2.67c 82.54 ± 2.84a 80.54 ± 3.24a 82.35 ± 2.62a

fraction

Diabetic + chloroform 76.56 ± 1.98a 344.78 ± 2.03b 264.43 ± 2.51d 231.43 ± 2.56d 188.32 ± 1.76d 167.43 ± 2.41d 140.64 ± 3.18c 134.36 ± 2.72c 139.53 ± 2.27c

fraction

Diabetic + ethyl acetate 74.63 ± 2.41a 341.26 ± 2.63b 268.54 ± 4.24d 235.42 ± 3.83d 205.82 ± 4.52e 195.52 ± 2.52e 121.53 ± 2.63d 119.52 ± 2.17d 117.53 ± 2.62d

fraction

Diabetic + n-butanol 76.57 ± 2.64a 347.63 ± 4.23b 296.45 ± 3.54e 249.34 ± 4.23e 203.34 ± 3.65e 195.45 ± 3.04f 212.35 ± 2.52e 226.54 ± 2.63e 228.64 ± 3.05e

fraction

Diabetic + subfraction A 75.72 ± 2.53a 349.47 ± 5.21b 302.65 ± 4.65f 278.75 ± 5.54f 247.54 ± 4.43f 179.54 ± 3.65f 148.76 ± 3.54f 124.65 ± 4.63f 122.54 ± 3.66d

fraction

Diabetic + subfraction B 76.48 ± 3.54a 350.61 ± 5.13b 212.73 ± 4.56c 162.65 ± 3.87c 129.56 ± 3.56c 115.63 ± 2.65c 81.65 ± 2.85a 81.48 ± 3.16a 79.42 ± 2.87a

fraction

Diabetic + glibenclamide 77.36 ± 3.15a 346.31 ± 2.42b 219.67 ± 3.12c 167.61 ± 2.82c 148.35 ± 2.42c 119.54 ± 2.05c 92.52 ± 2.15g 94.43 ± 3.02g 93.73 ± 2.53f

Data are expressed as mean ± standard error of the mean, n = 6, analysis of variance followed by multiple comparisons two-tail t test. Values with different superscripts (a-g) differ from each other significantly (p < 0.05).

b 1 4

Serum insulin

□ Control □ Diabetic + n-butanol fraction

□ Untreated diabetic □ Diabetic + subfraction A

□ Diabetic + n-hexane fraction □ Diabetic + subfraction B

□ Diabetic + chloroform fraction □ Diabetic + glibenclamide

□ Diabetic + ethyl acetate fraction

Figure 1 Protective effect of n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A, and subfraction B on serum insulin level in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a-e) differ from each other significantly at the level of p < 0.05.

Skeletal muscle

□ Control

□ Untreated diabetic

□ Diabetic + n-hexane fraction

□ Diabetic + chloroform fraction

□ Diabetic + ethyl acetate fraction

□ Diabetic + n-butanol fraction

□ Diabetic + subfraction A

□ Diabetic + subfraction B

□ Diabetic + glibenclamide

Figure 3 Comparative analysis of n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A, and subfraction B on glycogen level in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a-e) differ from each other significantly at the level of p < 0.05.

subjected to gavage of 0.5 mL water/100 g body weight/ rat/day for 49 days at the time of fractions, subfractions, and glibenclamide administration to the animals of Groups 3—9, to keep all the animals under the same experimental condition and stress imposition, if any, due to the administration of fractions and animal handling. Starting from Day 1 of fractions and subfractions administration to the diabetic rats, fasting blood glucose levels (12 hours after feed delivery) were measured in all groups on every week. On Day 56 of the experiment peripheral blood was collected by retro orbital puncture under light anesthesia at the 12-

Glycosylated hemoglobin

□ Control □ Diabetic + n-butanol fraction

□ Untreated diabetic □ Diabetic + subfraction A

□ Diabetic + n-hexane fraction □ Diabetic + subfraction B

□ Diabetic + chloroform fraction □ Diabetic + glibenclamide

□ Diabetic + ethyl acetate fraction

Figure 2 Effect of n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A, and subfraction B on glycosylated hemoglobin level in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a-d) differ from each other significantly at the level of p < 0.05.

hour fasting state and the fasting glucose level was monitored. All the animals were sacrificed at the fasting state by light ether anesthesia followed by decapitation after recording the final body weight. Blood was collected from the dorsal aorta by a syringe and the serum was separated by centrifugation at 2800 g for 5 minutes for the estimation of serum insulin and serum toxicity study. The liver, kidney, skeletal muscle, and cardiac muscle were dissected out and stored at -20°C for the quantification of hepatic and skeletal muscle glycogen, assessment of the activities of the carbohydrate metabolic enzymes—hexokinase, glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphatase (G6P), and lactate dehydrogenase (LDH) in the liver, kidney, skeletal muscle, and cardiac muscle. Blood was used for the quantification of glycated hemoglobin.

Measurement of fasting blood glucose level

At the time of grouping of the animals, fasting blood glucose level was measured. After every 6 days (on every 7th day) of treatment, fasting blood glucose was further recorded from all the animals of all groups. The blood samples were withdrawn from the animals by retro-orbital puncture under light anesthesia. The plasma glucose estimation was done by the glucose oxidase/peroxidase11 method using a standard kit obtained from Span Diagnostics.

Biochemical measurement

Serum insulin levels were measured by following the methods of Burgi et al (1988)12 using rat insulin enzyme linked immunosorbent assay kit (Millipore Corporation, Billerica, MA, USA). Glycosylated hemoglobin level was measured according to Chandalia et al (1980).13 The hepatic glycogen level was measured according to the standard methods of

Hexokinase

Liver Kidney Skeletal muscle Cardiac muscle

□ Control □ Diabetic + n-butanol fraction

□ Untreated diabetic □ Diabetic + subfraction A

□ Diabetic + n-hexane fraction □ Diabetic + subfraction B

□ Diabetic + chloroform fraction □ Diabetic + glibenclamide

□ Diabetic + ethyl acetate fraction

Figure 4 Protective effect of n-hexane, chloroform, ethyl acetate, n-butanol, subtraction A, and subtraction B on hexokinase in liver, kidney, skeletal muscle, and cardiac muscle in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a—e) differ from each other significantly, p < 0.05.

Kemp and Van Hejnigen.14The activities of the carbohydrate metabolic were assayed by previously described meth-ods.15—18 SGOT and SGPT activities were measured by the methods of Henry et al19 using a semiautoanalyzer kit obtained from Merck Limited.

Histological parameters

The midpart of the pancreas was collected from all the groups to avoid error due to the variation in size of islets in different parts of the pancreas. The pancreas was fixed in a neutral buffered formalin solution (10%) and dehydrated in a graded series (50%, 70%, 80%, 90%, 95%, and 100%) of ethanol. The tissue was embedded in Paraplast, sectioned into approximately 4 mm thicknesses and stained with hematoxylin and eosin. The tissues were observed using a light microscope. Diameters of the pancreatic islets were measured by computerized microphotography using software (For microphotography: AverPac Version 2.5, AverMedia Technologies Inc. Fremont, CA, USA. For

measurement: DeWinter Caliper Pro-Software, Version 3.0, DeWinter Optical Inc. West Patel Nagar, Delhi, India).20

Statistical analysis

Data were evaluated statistically using one-way analysis of variance (ANOVA) followed by multiple comparison two-tail t test by using Origin Lab (Version 6.0) software (Origin Lab Corporation. North Antton, One Round House Plaza, MA 01060, USA). A p value < 0.05 was considered to indicate statistical significance. Data are presented as mean ± standard error of the mean.

Results Body weight

Table 1 shows that the final body weight of the untreated control group increased significantly compared to the beginning

Glucose-6-phosphatase

□ Control □ Diabetic + n-butanol fraction

□ Untreated diabetic □ Diabetic + subfraction A

□ Diabetic + n-hexane fraction □ Diabetic + subfraction B

□ Diabetic + chloroform fraction □ Diabetic + glibenclamide

□ Diabetic + ethyl acetate fraction

Figure 5 Correction of glucose-6-phosphatase activities in liver, kidney, skeletal muscle, and cardiac muscle with the treatment of n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A and subfraction B in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a—d) differ from each other significantly at the level of p < 0.05.

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Glucose-6-phosphate dehydrogenase

£ 60 -

Kidney

Skeletal muscle

Cardiac muscle

□ Control

□ Untreated diabetic

□ Diabetic + n-hexane fraction

□ Diabetic + chloroform fraction

□ Diabetic + ethyl acetate fraction

□ Diabetic + n-butanol fraction

□ Diabetic + subfraction A

□ Diabetic + subfraction B

□ Diabetic + glibenclamide

Figure 6 Comparative analysis of the correction in glucose-6-phosphate dehydrogenase in liver, kidney, skeletal muscle, and cardiac muscle in streptozotocin-induced diabetic rat by n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A, and subfraction B treatment. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a—d) differ from each other significantly, p < 0.05.

of the experiment, whereas a significant decrease in body weight was observed in the diabetic group in respect to the control. Administration of n-hexane, chloroform, ethyl acetate, and n-butanol fractions and subfractions A and B of T. indica resulted a significant increase in body weight compared to the diabetic group. The recovery in body weight was significant with the n-hexane fraction and subfraction B in comparison to other fractions and subfraction A treated groups and this change was comparable to the glibenclamide treated group.

Fasting blood glucose level

Table 2 shows that there was a significant decrease in fasting blood glucose levels in diabetic treated rats after the administration of n-hexane, chloroform, ethyl acetate, and n-butanol fractions at the dose of 100 mg/kg twice a day. Administration of n-hexane fraction of T. indica to

diabetic animals resulting in a more significant corrective effect than other fractions on the blood glucose level. After the treatment of subfraction A or B from the n-hexane fraction there was a significant reduction of blood glucose level in the diabetic group but subfraction B was more effective in this concern. The n-hexane fraction and subfraction B also had more potent action than the glibencla-mide in this concern.

Serum insulin

Serum levels of insulin were decreased significantly in untreated diabetic test animals as compared with nondiabetic control animals. Administration of n-hexane fraction of T. indica to diabetic animals resulted in a significant corrective effect than other fractions on the levels of insulin. Administration of subfraction A, subfraction B, or glibenclamide to

.!£ 50

Lactate dehydrogenase

Kidney

Skeletal muscle

Cardiac muscle

□ Control

□ Untreated diabetic

□ Diabetic + n-hexane fraction

□ Diabetic + chloroform fraction

□ Diabetic + ethyl acetate fraction

□ Diabetic + n-butanol fraction

□ Diabetic + subfraction A

□ Diabetic + subfraction B

□ Diabetic + glibenclamide

Figure 7 Comparative analysis of n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A, and subfraction B on lactate dehydrogenase activities in liver, kidney, skeletal muscle, and cardiac muscle in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a—e) differ from each other significantly at the level of p < 0.05.

=> 1.5 1

Figure 8 Resettlement on the activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) after administered of n-hexane and subfraction B in streptozotocin-induced diabetic rat. Each bar represents mean ± standard error of the mean, n = 6 for each group. Analysis of variance followed by multiple comparison two-tail t test. Values of bar diagrams with different superscripts (a—c) differ from each other significantly at the level of p < 0.05.

diabetic animals resulted in a significant corrective effect and again, subfraction B was more effective (Fig. 1 ).

Glycosylated hemoglobin

Glycosylated hemoglobin level is one of the important indicators of a diabetic condition, and the level of the parameter was increased significantly in the diabetic group in comparison with the control group. After administration of n-hexane, chloroform, ethyl acetate, and n-butanol extracts of T. indica in a STZ-induced diabetic rat, the level of this parameter was recovered towards the control level. After the treatment of n-hexane subfractions there was a significant reduction of glycosylated hemoglobin level in the diabetic group. Subfraction B was more effective in this concern than subfraction A or glibenclamide (Fig. 2).

Hepatic and skeletal muscle glycogen

The levels of hepatic and skeletal muscle glycogen were decreased in the untreated diabetic group in comparison with the nondiabetic control group. The administration of n-hexane, chloroform, ethyl acetate, or n-butanol fraction to diabetic animals resulted in a significant recovery in this parameter, but the administration of n-hexane produced the most significant recovery in the test animals in comparison with those treated with any other individual fraction. The administration of subfraction A or B from n-hexane fraction or glibenclamide to the diabetic animal resulted in a significant recovery in the level of liver glycogen, but the administration of subfraction B was more effective in comparison with those animals treated with subfraction A or glibenclamide (Fig. 3).

Hexokinase, G6P, G6PDH, and LDH in hepatic, kidney, skeletal muscle, and cardiac muscle

Figs. 4—7 show that the activities of hexokinase and G6PDH in hepatic, kidney, skeletal muscle, and cardiac

muscle were decreased, but the activities of G6P and lactate dehydrogenase in these tissues were increased in the untreated diabetic group in comparison with the nondiabetic control group. The administration of the n-hexane, chloroform, ethyl acetate, and n-butanol extracts to diabetic animals resulted in a significant recovery in these metabolic reactions but the administration of n-hexane produced a more significant recovery to normal in comparison with those animals treated with any of the individual fractions. The administration of n-hexane subfractions to a diabetic animal resulted in a significant recovery in the metabolic reactions. Subfraction B resulted in a more significant recovery in the levels of these parameters towards normal in the test animals in comparison with those animals treated with subfraction A or glibenclamide.

SGOT and SGPT

Activities of SGOT and SGPT were increased in the diabetic group compared to the control group. After administration of the n-hexane fraction and subfraction B, there was a significant recovery in the levels of these parameters. Subfraction B was more effective than glibenclamide (Fig. 8).

Histological study

The diameter of pancreatic islets as well as the count of islet cells was significantly decreased in the STZ-induced diabetic group in respect to the control group. The values of these parameters were significantly recovered after the treatment of n-hexane, chloroform, ethyl acetate, n-butanol, subfraction A, or subfraction B in diabetic rats but the most promising result was obtained after the treatment with n-hexane fraction and subfraction B (Fig. 9).

HPTLC fingerprinting of the most effective fraction and subfraction

To identify the number of compounds present in the active n-hexane fraction of T. indica, HPTLC fingerprinting was performed using chloroform:methanol:ethyl acetate 90:5:5 v/v as a suitable solvent system deduced from a pilot study. From this study it was observed that the fraction was a mixture of six different compounds. HPTLC finger printing was also carried out to examine the number of compounds present in subfraction B. After the HPTLC study it was observed that subfraction B contained two different compounds in which one was very prominent (Pi) on chromatographic plate and the other was very faint (P2; Fig. 10).

HPLC analysis of the most effective subfraction

Analytical RP-HPLC (Waters Corporation, 34 Maple Street, Milford, MA 01757, USA) was performed to find out the number of compounds present in the solid subfraction of petroleum ether (subfraction B) of the n-hexane fraction of seed of T. indica (Fig. 11). This study was also carried out to establish the purity percentage of the isolated compound as well the 1max values. HPLC analysis was performed on the Water Alliance

Figure 9 (A) Representative sample of pancreatic tissue of vehicle control rat focusing on the normal islet diameter. (B) Diminution in the diameter of islet in the representative pancreatic tissue sample of streptozotocin (STZ)-induced diabetic rat. (C) Representative pancreatic tissue sample showing recovery in islet cell diameter after n-hexane fraction of hydro-methanolic extract treatment in a streptozotocin-induced diabetic rat. (D) Pancreatic tissue sample showing recovery in islet cell diameter after treatment of chloroform fraction of hydro-methanol extract of seed of Tamarindus indica Linn. in a STZ-induced diabetic rat. (E) Pancreatic tissue sample showing recovery in islet cell diameter after treatment of ethyl acetate fraction of hydromethanol extract in a STZ-induced diabetic rat. (F) Pancreatic tissue sample showing recovery in islet cell diameter after treatment of n-butanol fraction of hydro-methanol extract in a STZ-induced diabetic rat. (G) Pancreatic tissue sample showing recovery in islet cell diameter after treatment of subfraction A in a STZ-induced diabetic rat. (H) Pancreatic tissue sample showing recovery in islet cell diameter after treatment of subfraction B in a STZ-induced diabetic rat. (I) Representative pancreatic tissue sample showing recovery in islet cell diameter after glibenclamide treatment.

HPLC system equipped with 2695 separation module 2996 Photodiode Array Detector, performance plus four-channel inline Degasserauto injector, quaternary pump, and column oven. The HPLC analysis was carried out on the Thermo Hypersil BDS C18 (4.6 mm x 250 mm, 5 mm) column using a premixed solvent system as a mobile phase with a flow rate of 1 mL/minute and Isocratic Illusion Technique. The column temperature was maintained at 30oC and detection was performed at 254 nm. For the HPLC study, the white solid layer was separately dissolved with a mobile phase (ethyl aceta-te:hexane:isopropyl alcohol 60:30:10) The solution was sonicated for 15 minutes and then filtered through MilliporeMillex Syringe filter unit (0.45 mm). The sample solution of 10 mL

injected through the autoinjector fitted with a 200-mL syringe and 100-mL sample loop. The separated compounds scanned across the entire UV range on three-dimensional spectral mode to record the photodiode array detector spectra. HPLC chromatograms of isolated compounds were recorded at 254 nm. Data from the Water Alliance HPLC system were acquired and processed using Water Millennium32 software.

Discussion

The present study was conducted to identify the possible active principle(s) having antihyperglycemic activity

Figure 10 High performance thin layer chromatogram of subfraction B, Scan at 254 nm of different concentration (3 dimensional), five tracks of different volume (according to behind to in front 5 mL, 10 mL, 15 mL, 20 mL, 25 ml ) of the same sample are shown here.

present in the n-hexane fraction followed by subfractions of T. indica seed. The n-hexane fraction showed a more significant reduction than other fractions in blood glucose levels in STZ-induced diabetic rats. The n-hexane fraction produced a maximum antidiabetic activity, as has been established from the comparative study with glibenclamide in the diabetic rats.

The levels of blood glucose, serum insulin, and glycosy-lated hemoglobin were noted in normal and experimental groups of rats. There was a significant elevation in blood glucose and glycosylated hemoglobin, whereas the level of serum insulin decreased in the diabetic group when compared with the control group.21 Administration of n-hexane fraction and subfraction B of T. indica resulted in a

Figure 11 Representative high performance liquid chro-matogram of petroleum ether subfraction B from the n-hexane fraction.

significant recovery to near normal values as that of the standard drug glibenclamide treatment.

Restoration in the glycogen level with fraction treatment may be explained by the recovery of insulin, as insulin controls the activity of glycogen synthetase.22 Such recovery of insulin has been indicated by a serum insulin level assessment as well as from histological evaluation of islets.

For the assessment of the antidiabetic potency of the plant extract, we measured the activity of hepatic G6P, an important enzyme for glycogenolysis.23 Similarly G6PDH and hexokinase are two enzymes that are under the positive control of insulin.24 In the diabetic state, the activities of these three enzymes are altered as reported previ-ously.25'26 The n-hexane fraction and subfraction B are able to recover the activities of these enzymatic biosensors significantly and are comparable to glibenclamide, which may be due to the recovery of insulin.

LDH is a bifunctional enzyme involved in the glycolytic pathway. The LDH system reflects the nicotinamide adenine dinucleotide (NAD+)/NADH ratio indicated by the lactate/pyruvate ratio of hepatocyte cytosol. In different fractions of T. indica or glibenclamide treated groups of rats, the reduction in the LDH activity is probably due to the regulation of NAD+/ NADH ratio by the oxidation of NADH.

There was no toxicity of this extract as proved by SGOT and SGPT activities, as these enzymes are important sensors for toxicity assessment.27 In respect to LD50 values and maximum nonfatal doses studies revealed the nontoxic nature of the n-hexane fraction and subfraction B of this plant. There was no lethality or any toxic reactions found at any of the doses selected up to the end of the study period. According to a toxicity classification,28 the n-hexane fraction of T. indica was nontoxic.

To find the number of active biomolecules for the management of STZ-induced a diabetic subfraction study using petroleum ether was conducted. Subfraction B was more effective than subfraction A at a dose that was 1/4 of the dose of n-hexane fraction. From HPLC analysis it has been found that there are two components named as P1 and P2, where P1 was more prevalent in the petroleum ether subfraction B. Therefore, it may be concluded that the single phytomolecule P1 may be the causative molecule for the management of diabetes mellitus from the seed of T. indica. Further work is necessary for characterization of the biologically active ingredients present in the seed of T. indica.

Acknowledgments

The authors are thankful to the Indian Council of Medical Research (ICMR), New Delhi, India, for its financial support.

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