Evaluation of possible mechanisms of three plants for blood glucose control in diabetes

This study was conducted to provide the evidence for the mechanism of antidiabetic activity of Cocculus orbiculatus, Leea indica and Ventilago maderaspatana. This was accomplished by employing methods like uptake of glucose, glycogen synthesis and inhibition of α-glucosidase. For uptake of glucose, diaphragms were dissected out in Tyrode solution with 2% glucose and assayed for glucose content. In glycogen synthesis methodology liver, skeletal muscle and cardiac muscles were isolated, homogenized and glycogen content was analyzed. In α-glucosidase enzyme inhibition procedure involved estimation of α-glucosidase enzyme inhibition. All the three plant extracts exhibited significant (p<0.05 p<0.01) anti-diabetic activity by increasing glucose uptake, glycogen synthesis and inhibiting α-glucosidase enzyme. Among the three plants, V. maderaspatana (500 mg/kg) exhibited higher glucose uptake, glycogen content and α-glucosidase inhibition activity (IC50 145 μg/mL). The present experimental results evidenced the anti-diabetic activity of three plants by all the three mechanisms. Article Info Received: 8 September 2015 Accepted: 8 November 2015 Available Online: 24 January 2016 DOI: 10.3329/bjp.v11i1.24932 Cite this article: Dalu D, Dhulipala S. Evaluation of possible mechanisms of three plants for blood glucose control in diabetes. Bangladesh J Pharmacol. 2016; 11: 224 -30. Evaluation of possible mechanisms of three plants for blood glucose control in diabetes Damayanthi Dalu1 and Satyavati Dhulipala2 Department of Pharmacology, Netaji Institute of Pharmaceutical Science, Toopranpet, Choutuppal, Nalgonda 508 252, Telangana, India; Satyavati Dhulipala, Department of Pharmacology, Brilliant College of Pharmacy, Abdullapurmet, Hayatnagar, Ranga Reddy, Hyderabad 501 505, Telangana, India. This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor plants Cocculus orbiculatus, Leea indica and Ventilago maderaspatana in the treatment of diabetes (Damayanthi and Satyavati, 2015); Damayanthi et al., 2014; Damayanthi and Satyavati, 2015). But, till date there are no scientific evaluation reports available to support the mechanisms responsible for anti-diabetic activity. Therefore, the present investigation was aimed to ascertain in vivo anti-diabetic activity by methods such as glucose uptake activity using isolated rat diaphragm and glycogen synthesis in liver, skeletal muscle and cardiac muscle and in vitro anti-diabetic activity by inhibiting α-glucosidase enzyme. Materials and Methods


Introduction
Diabetes mellitus is characterized by impaired production of insulin and/or diminished stimulation of insulin sensitive peripheral tissues associated with a marked decrease in glucose uptake and metabolism in response to insulin. The defective glucose transport system plays a significant role in the pathogenesis of peripheral insulin resistance. Glucose uptake in target tissues is a crucial step in maintaining glucose homeostasis and in lowering the postprandial glucose load (Shulman, 2000). Direct stimulation of glucose transport and metabolism in muscle and fat cells lead to enhanced glucose utilization. To attenuate the glucose uptake by peripheral cells, biguanides are employed. Cellular assays are utilized to investigate the mechanism of action of natural compounds using isolated rat diaphragms. It is highly preferred to explore modern anti-diabetic agents from natural sources that stimulate glucose uptake/ disposal by peripheral tissues such as adipose tissue or muscle cells.
The pivotal enzyme for carbohydrate digestion is αglucosidase. This is a therapeutic target for the modulation of postprandial hyperglycemia, the earliest abnormality that occurs in NIDDM . Dietary carbohydrates are the major source for blood glucose. These carbohydrates are hydrolyzed by α-glucosidase, so as to be absorbed by small intestine. Therefore, the most effective treatment is to inhibit the activity of αglucosidase (Krentz and Bailey, 2005). α-Glucosidase inhibitors such as acarbose, miglitol and voglibose reduce postprandial hyperglycemia by inhibiting the activity of carbohydrate digesting enzymes and delaying glucose absorption.
Previously, we have reported anti-diabetic, anti-hyperlipidemic and anti-oxidant activity of three medicinal plants Cocculus orbiculatus, Leea indica and Ventilago maderaspatana in the treatment of diabetes ; Damayanthi et al., 2014;. But, till date there are no scientific evaluation reports available to support the mechanisms responsible for anti-diabetic activity. Therefore, the present investigation was aimed to ascertain in vivo anti-diabetic activity by methods such as glucose uptake activity using isolated rat diaphragm and glycogen synthesis in liver, skeletal muscle and cardiac muscle and in vitro anti-diabetic activity by inhibiting α-glucosidase enzyme.

Plant materials
Aerial parts of C. orbiculatus were collected from Tirumala forest area, Tirupathi. L. indica leaves were procured from Karthikavanam forest area, Dhulapally, Hyderabad. V. maderaspatana roots were obtained from Tirumala forest area, Tirupathi. The plants were authenticated by Prof. Madhava Chetty, Department of Botany, Sri Venkateshwara University, Tirupathi, India.

Preparation of plant extract
Aerial parts of C. orbiculatus were finely powedered, packed in soxhlet apparatus and then extracted with hydroalcohol (60:40). Leaves of L. indica were air dried at room temperature, coarsely powdered extracted by maceration with hydroalcohol (3:1). V. maderaspatana roots were finely powered and extracted by using soxhlet apparatus with hydroalcohol solvent (60:40). Percentage yield of the plants C. orbiculatus, L. indica, V. maderaspatana was found to be 16.7, 25.6 and 15.8% respectively.

Preliminary phytochemical analysis
All the three plant extracts were subjected to prelimnary phytochemical analysis to determine the phytoconstituents employing standard tests (Harbone, 1998).

α-glucosidase inhibitory assay
This assay was assigned to investigate the in vitro inhibitory activity of three plant extracts on α-glucosidase enzyme. α-Glucosidase (100 µL of 1 U/mL) was mixed with phosphate buffer (100 µL, pH 7.0) containing 100 µL of three plant extracts (25-1600 µL) or standard drug acarbose (0.1-3.2 µg/mL). This mixture is incubated at 37°C for 60 min in maltose solution. Later the mixture is kept in boiling water for 2 min and cooled. The boiling stops the α-glucosidase action on maltose. Glucose reagent (2 mL) was added and absorbance is measured at 540 nm to estimate the amount of liberated glucose by the action of α-glucosidase (Kuppusamy et al., 2011)

Glucose uptake in normal and streptozotocin-induced diabetic rats
The glucose uptake using rat hemidiaphragm was estimated according to the method reported elsewhere (Walass and Walass, 1952;Chattopadhyay et al., 1992), but with some modifications. After 18 hours fasting; rats were killed by decapitation. Diaphragms were dissected out quickly with minimal trauma and divided into two halves. Hemidiaphragms were then rinsed in cold Tyrode solution (without glucose) to remove blood clots. Then these were weighed and placed in test tubes. The volumes in all test tubes were made equal by adding distilled water. The test tubes were incubated for 30 min at 37°C in an atmosphere of 100% oxygen and were shaken at 140 cycles/min. Hemidiaphragms were taken out. Glucose content of the incubated medium before and after incubation was measured. This was carried out by employing Erba Diagnostic Mannheim kit using GOD-POD method (Barham and Trinder, 1972) and Erba Mannheim Chem-7 semiautoanalyzer. Glucose uptake was calculated as the difference between initial and final glucose content. Glucose uptake was expressed as mg/g of tissue per 30 min of incubation. Rats were divided into 12 groups; six in normal group and six in diabetic control group of five rats each. In normal group: Group I (normal rats; received 2 mL Tyrode solution with 2% glucose); Group II (received Tyrode solution and 0.6 mL of 0.4 IU/mL insulin); Group III (administered Tyrode solution and standard drug, metformin-2 mL of 0.1%); Group IV (received Tyrode solution and 300 mg/kg of C. orbiculatus); Group V (received Tyrode solution-400 mg/kg of L. indica); Group VI (administered Tyrode solution and 500 mg/kg V. maderaspatana).

Glycogen estimation in normal and streptozotocininduced diabetic rats
The glycogen content in liver, skeletal muscle and cardiac muscle was estimated by Carroll et al., (1956). Rats were divided into ten groups: Five in normal group and five in diabetic control group of five rats each.
Normal group: Group I (normal rats; received 1% sodium carboxymethyl cellulose); Group II (received standard drug, glibenclamide 10 mg/kg); Group III (administered 300 mg/kg of C. orbiculatus); Group IV (administered 400 mg/kg of L. indica) Group V (administered 500 mg/kg V. maderaspatana) Diabetic control group: Group VI (diabetic control group, untreated); Group VII (diabetic control received standard drug, glibenclamide 10 mg/ kg); Group III (diabetic control administered 300 mg/ kg of C. orbiculatus); Group IV (diabetic control administered 400 mg/kg of L. indica); Group V (diabetic control administered 500 mg/kg of V. maderaspatana) After 18 hours of fasting, plant extracts were administered to different groups. Two hours later they were sacrificed by decapitation. The liver, skeletal muscle and cardiac muscle were isolated, weighed and homogenized using 10 mL of 4% trichloroacetic acid and centrifuged for 10 min. Supernatant was decanted and precipitate is discarded. To 2 mL of supernatant 4 mL of anthrone reagent was added. Later test tubes were allowed to cool for 30 min. Absorbance was measured at 620 nm using spectrophotometer. Glycogen content was expressed as milligram for 100 g of tissue.
Glycogen content = DU x 0.2 x volume of the extract x 1000 DS x weight of the tissue DU= Absorbance of the sample; DS= Absorbance of the standard

Statistical analysis
The experimental results were presented as mean ± standard error mean (SEM). Statistical analysis was performed by graphpad instat version 3.2. Probability value of analysis p<0.01 and p<0.05 was considered to be statistically significant.

α-Glucosidase inhibitory activity
C. orbiculatus, L. india and V. maderaspatana hydroalcoholic extracts exhibited 8.3, 10.3 and 15.1% inhibition of α-glucosidase activity at 25 µg/mL and 85.7, 90.3 and 95.8% inhibition at 1600 µg/mL respectively (Table II). The IC50 values were 325, 265 and 145 µg/mL. IC50 value of acarbose was found to be 0.2 µg/mL. Table III show glucose uptake in an isolated rat hemidiaphragm muscle of normal and diabetic animals. Addition of C. orbiculatus, L. india and V. maderaspatana hydroalcoholic extracts elicited significant increase in glucose uptake by the rat hemidiaphragm in normal animals. V. maderaspatana seemed to be more effective in enhancing peripheral glucose uptake than L. indica, C. orbiculatus and metformin. The glucose uptake by rat hemidiaphragm was significantly higher in all the groups when compared to control group. Diabetic control animals exhibited significant increase in glucose uptake of C. orbiculatus, L. india and V. maderaspatana (37.7, 40.8 and 48.0% respectively). Glucose uptake of V. maderaspatana was significantly higher when compared to L. india and C. orbiculatus

Effect on glycogen content in liver, skeletal muscle and cardiac muscle in normal animals
Hydroalcoholic extracts of C. orbiculatus, L. india and V. maderaspatana showed significantly increased glycogen content in liver (Table IV). Increase in glycogen content was more for V. maderaspatana than L. india, C. orbiculatus and glibenclamide. Skeletal muscle glycogen content was increased more for all the three plant extracts. Increase was greater for V. maderaspatana than L. india, C. orbiculatus and glibenclamide. Significant increase in cardiac muscle glycogen content was observed for V. maderaspatana than L. india and C. orbiculatus. V. maderaspatana exhibited increased cardiac muscle glycogen content than L. india, C. orbiculatus and glibenclamide.

Effect on glycogen content in liver, skeletal muscle and cardiac muscle in diabetic animals
Three plants have elicited significantly increased liver glycogen content. V. maderaspatana manifested higher liver glycogen content than L. indica and C. orbiculatus. Significantly increased skeletal muscle glycogen content was observed for V. maderaspatana L. indica and C. orbiculatus. Glycogen content was more in V. maderaspatana than L. indica and C. orbiculatus . Cardiac muscle glycogen content increase was observed for all the three plants. Among these V. maderaspatana exhibited increased glycogen content than L. indica and C. orbiculatus.

Discussion
In current study, we investigated the mechanism for anti-diabetic activity of three plants (C. orbiculatus, L. and V. maderaspatana) by employing glucose uptake, glycogen synthesis and α-glucosidase enzyme inhibition methods. Three plants showed significant uptake of glucose and are more or less effective than insulin. V. maderaspatana has evidenced greater uptake of glucose than L. india, C. orbiculatus, metformin and comparatively equal to insulin. Glibenclamide showed increased glycogen content in muscle cells. Therapy with the three plants evidenced increased glucose uptake and thereby glycogen storage in liver, skeletal muscle and cardiac muscle. V. maderaspatana increased glycogen content greater than L. india and C. orbiculatus. The three plants also evidenced α-glucosidase inhibitory activity among which V. maderaspatana exhibited greater activity.
The impaired glucose uptake is linked with decrease in the translocation of glut 4 and is the major cause of insulin resistance. Metformin and insulin stimulate glucose uptake in muscle cells by increasing GLUT 4 (Klip and Leiter, 1990). Berberine the reported active constituent of C. orbiculatus has been investigated to activate AMPK in skeletal muscle and adipose tissue that lead to 162.5 ± 2.9 b 40 ± 0.9 b 41.0 ± 0.7 b 139 ± 1.0 b 32.7 ± 1.0 b 36.7 ± 1.3 b increased glucose uptake (Yin et al., 2008;Cheng et al., 2006;Kim et al., 2007;Lee et al., 2006;Zhou et al., 2007;Wang et al., 2004;Ko et al., 2005). Similarly gallic acid the constituent of L. india has been implicated to mediate insulin stimulated glucose transport in muscle and adipocytes (Vishnu Prasad et al., 2010). Finally VMHAE possess emodin that mediated Glut 1 and Glut 4 expression to enhance glucose uptake in skeletal muscles and adipocytes (Ying Yang et al., 2007). Glibenclamide increased glycogen content in muscle but adverse effects like hypoglycemia and weight gain limits the use. Hence drugs that attenuate glycogen content without adverse effects were desirable for long term anti-diabetic therapy. The three plants evidenced enhanced glucose uptake and glycogen storage in liver, skeletal muscle and cardiac muscle. The increased glycogen levels by the three plants might be due to the berberine of C. orbiculatus that evidenced glucose utilization in Hep G2 cells and 3T3-L1 adipocytes increasing glycogen content (Yin J et al., 2002;. Gallic acid, the constituent of L. india by insulin secretagogue action produced increased glycogen levels (Punithavathi et al., 2011). Emodin of V. maderaspatana, the natural PPARγ activator, up-regulated PPARγ and increased glycogen content (Yonemitsu et al., 2001). Acarbose, the α-glucosidase inhibitor has common side effects (flatulence and abdominal bloating). Herbal drugs devoid of side effects are desired to improve compliance for diabetic patients. The constituents like berberine, β-sitosterol and emodin of the plants have reported α-glucosidase inhibitory activity (Pan et al., 2003;Sunil Kumar et al., 2013;Yang et al., 2014). Thus active constituents of the experimental study might be responsible for the elicited α-glucosidase inhibitory activity of C. orbiculatus, L. indica and V. maderaspatana .

Conclusion
The experimental reports evidenced C. orbiculatus, L. indica and V. maderaspatana plants act through multiple mechanisms like increased glucose uptake, glycogen synthesis in muscle cells and α-glucosidase inhibition to control blood glucose in diabetes. V. maderaspatana elicited higher antidiabetic activity compared to L. indica and C. orbiculatus.

Financial Support
Self-funded

Ethical Issue
Experiments were conducted in accordance with the guidelines of CPCSEA REGD No. 878/ac/05/CPCSEA/21/2015. The study protocol was approved by Institutional Animal ethics committee.

Conflict of Interest
Authors declare no competing interest