Antidiabetic Effect of Memecylon Talbotianum Leaf Methanol Active Fraction in Male Wistar Rats

Bharathi, Jameel, Ghassam, Bajpe, and Prakash: Antidiabetic Effect of Memecylon Talbotianum Leaf Methanol Active Fraction in Male Wistar Rats

Authors

INTRODUCTION

Medicinal plants play an appreciable role in the development of modern herbal medicines where many diseases such as cancer, liver diseases and arthritis find no complete cure in allopathy. The bioactive compounds of medicinal plants are used as antidiabetic, chemotherapeutic, anti-inflammatory and anti-arthritic agents where no satisfactory cure is available in modern medicines. Although different classes of drugs are available to control diabetes, still it is a challenging task to develop a better molecule which is devoid of undesirable adverse effects compared to existing drugs. Therefore, the search for more effective and safer hypoglycemic agents is important area of active research and the efficacy of antidiabetic medicinal plants has been increasingly documented.1

Memecylon (Melastomataceae) belongs to the genus of shrubs and trees which consists of about 150 species, mainly in the old world tropics, used in Ayurveda and Siddha systems of medicine for the treatment of leucorrhoea, polyuria, diabetes, bacterial infections, and viral diseases including herpes and chickenpox.2. The phytoconstituents of the aerial plant parts including β-amyrin, sitosterol, oleanolic acid, ursolic acid and umbelactone were shown to have various pharmacological activities.3-5

Memecylon talbotianum Brandis, is one of the endemic plants of Western Ghats of India which is distributed in moist deciduous to evergreen forests of Karnataka, Tamil Nadu, Sri Lanka and Malaya and this plant is used as a folk medicine, although in recent years, the anti-inflammatory, antidiabetic, antioxidant and antimicrobial activities of other species of the genus Memecylon have been reported.6-7 So far no scientific report is available to support the antidiabetic activity of M. talbotianum.

The present study investigates in vitro and in vivo antidiabetic activity of methanol active fractions of M. talbotianum leaves against streptozotocin-induced diabetes mellitus in male Wistar rats. The phytoconstituents present in M. talbotianum was characterized by high-performance liquid chromatography (HPLC) analysis. The possible mechanisms involved in the antidiabetic activity of M. talbotianum were also investigated.

MATERIALS AND METHODS

Chemicals

Streptozotocin, metformin 500 mg tablets, thiobarbituric acid, glutathione oxidase, glutathione reductase, pyrogallol, Triton-X 100, bovine serum albumin, propidium iodide, α- amylase, α- glucosidase, acarbose and HPLC standards were obtained from Sigma Aldrich. All other solvents and chemicals were purchased from Merck Biosciences. Rabbit polyclonal IgG, Insulin (H-86), rabbit anti-mouse insulin antibody, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody were obtained from Santa Curz Biotechnology Co. Limited.

Plant Materials

Fresh aerial parts of M. talbotianum collected from the Western Ghats of India, during June 2015 were authenticated by a taxonomist, Prof Sampath Kumara K.K., at University of Mysore (UOM). The voucher specimen (No. # IOE LP0002) were deposited at the Department of Biotechnology, UOM. The leaves of the plant were washed under running tap water to remove adhering contaminants, air dried in shade and pulverized in a mechanical grinder to obtain coarse powder.

Preparation of extracts and partial purification

Ground leaf powder (250 g) was extracted sequentially using 500 mL of non-polar and polar solvents in increasing polarity hexane < ethyl acetate < methanol <water using Soxhlet apparatus by continuous hot percolation (boiling point, 52°C to 62°C) until the solvent became colorless. The resultant solvent extracts were concentrated in a SpeedVac (Savant SPD 2010, Thermo Scientific, Germany) under reduced pressure. The yield from these solvents were - hexane (5.37 g); ethyl acetate (6.2 g); methanol (8.23 g) and water (2.32 g) from 250 g dry weight of leaf. Only methanol extract was selected for further purification as it exhibited highest antidiabetic activity. Partial purification was carried out using silica gel column chromatography 6 g of methanol extract were mixed with 3 g of silica gel as stationary phase loaded on to the column. A glass column (50 cm×2 cm dia.) was equilibrated systematically by passing ethyl acetate repetitively. The methanol extract was loaded on to the packed glass column. Flow rate was set to 10 mL/min. The column was eluted with ethyl acetate-methanol mobile phase with the following increasing polarity: 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100. Six fractions were collected from the methanol extract to obtain active fractions. The effluents were collected in a flask and allowed to dry. The yields of six fractions were 0.126, 0.236, 0.322, 0.233, 0.323, and 0.422 g. In vitro (bioassays) α-amylase and α-glucosidase inhibitory activities were carried out using all fractions and the active fraction was identified and named as M. talbotianum leaf methanol active fraction (MTLMAF).

Analysis of phytoconstituents using High Performance Liquid Chromatography (HPLC)

HPLC separation of MTLMAF was carried out as described Ruangchakpet and Sajjaanantakul 8. A thermo hypersil gold (4.6 mm × 150 cm, 5 μm) column/PDA maintained at 35°C with different detection times was used in the study. The mobile phase A used was water containing 2.5% formic acid and mobile phase B used was acetonitrile; water and acetic acid in the ratio of 45:54:1; flow rate was 1mL/min; injection volume 10 μL. The retention time for each eluted component in the analyzed sample was compared with the following standards: rutin, protocatechuic acid, quercetin, synapic acid, cinnamic acid, chlorogenic acid and synergic acid.

In vitro antidiabetic studies
Inhibition of α-amylase and α-glucosidase

Screening of crude extracts and eluted methanol fractions for pancreatic α-amylase (PPA) and α-glucosidase inhibition was carried out according to previous methods 9-10.

In vivo studies
Experimental Animals

The study was carried out on healthy adult male Wistar rats in the weight range of (180-200 g), selected from an inbred group housed in designed cages and maintained under standard conditions of temperature (23 ± 1°C) and humidity (55-60%) with a 12–-h light/dark cycles for a minimum of one week before use. Rats were put into 6 groups each group with 6 rats on the basis of initial weight and kept in separate cages; all rats consumed rodent diet and tap water was provided ad libitum. Clinical monitoring of the animals was performed weekly to evaluate body weight and blood glucose and insulin levels. All animals were cared for according to the guiding principle in the care and use of animals in Central Animal House Facility, DOS in Zoology, UOM. The animal care and experimental procedures performed were in compliance with the Regulations for Animal Research and Animal Ethical Committee of the UOM (Animal Order No: UOM/IAEC/07/2013).

Experimental induction of diabetes and treatment

Diabetes was induced in male Wistar rats by intraperitoneal administration of STZ (single dose of 60 mg/kg body weight) 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 animals were cared as per the guidelines of Institute Animal Ethics committee 11. For animal treatments, extracts were reconstituted in water and animals were force fed orally by using 25-27 gauge feeding needles.

Selection of dose of the extract

LD50 was determined as per the Organisation for Economic Co-operation and Development (OECD) guidelines for fixing the dose for biological evaluation. The LD50 of the extracts falls under category 5 values with no death and no signs of acute toxicity at dose of 2000 mg/kg. The biological evaluation of the extracts was carried out at dose levels of 100 and 200 mg/kg body weight.

Experimental design

The rats were divided into six groups, each having six animals; each group was given the following treatment:

Group I : Normal control rats

Group II : STZ-induced diabetic control rats.

Group III : Diabetic rats with MTLMAF (100 mg/kg)

Group IV : Diabetic rats with MTLMAF (200 mg/kg)

Group V : Control rats with only MTLMAF (1000 mg/kg)

Group VI : Diabetic rats with Standard Metformin (100 mg/kg)

Body weight and Blood glucose estimation

Blood samples were collected from tip of rat tail and blood glucose levels were estimated on 1, 7, 14, 21 and 28 days of treatment using GlucocardTM 01-mini Blood Glucose Meter. Body weight was measured initially and during treatment period. Glucose tolerance test was performed as described by Matteucci and Giampietro 12.

Analysis of biochemical parameters and serum enzymes

On completion of the treatment period, rats were anesthetized by inhalation and blood was drawn by tail vein puncture and cardiac puncture. The whole blood was centrifuged at 2000 × g at 4°C for 10 min. Serum samples were collected and stored at -20°C for further use. The levels of triglycerides, total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) in the serum samples were measured using commercial assay kits as obtained from Agappe Diagnostics as per the manufacturer’s protocol using a semi autoanalyser (Mispa Excel Chemical Analyser). Results are expressed as units/dL.

Analysis of antioxidant enzymes

The liver was excised, weighed and homogenized (10% w/v) in ice-cold 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 4000 × g at 4°C for 10 min and the supernatant obtained was stored at -20°C. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were measured from the supernatant. SOD activity was measured using pyrogallol (2 mM) auto-oxidation in Tris buffer 13. CAT activity was assayed by using H2O2 as the substrate in phosphate buffer 14 and GSH was estimated using Ellman’s reagent 15.

Estimation of protein

Protein was determined by the method of Lowry 16 using bovine serum albumin as standard, at 660 nm.

Histopathology of liver and pancreatic tissues

For the histopathological studies, pancreas and liver tissue samples were collected and fixed in 10% buffered formalin, embeded in paraffin wax. Paraffin embeded tissue sections (4 μm each) were deparaffinized, rehydrated, and subjected to hematoxylin and eosin staining and examined microscopically at 1×400 magnification 17.

Immunostaining studies of pancreatic tissues

Immunohistochemistry was performed on formalin fixed, paraffin-embedded hepatic and pancreatic tissues. Pancreatic samples were immunostained with rabbit polyclonal IgG Insulin (H-86) according to manufacturer’s instructions. For immunostaining studies, the pancreatic tissues were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h at 4°C, immersed in 0.1 M phosphate buffered saline (PBS) at room temperature, washed with PBS, allowed to permeablize with 0.1% Triton-X 100 and again washed with PBS. The primary antibody rabbit anti-mouse insulin antibody (1:50) in PBS + 30 mg/mL BSA+ 0.1% Triton X-100 were added, incubated for 90 min and washed 3 times consequently with 0.1% Triton X-100 in PBS. Secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:200 was added at proper dilutions in PBS + BSA) washed 3 times with 0.2% Triton X-100. The tissue sections were incubated for 60 min at room temperature, and nuclei were stained with propidium iodide (PI-1:1000) for one min and washed with PBS, observed and analysed using confocal laser scanning imaging facility (LSM 710, Carl Zeiss, Germany) equipped with an excitation filter 515-560 and magnification at 20x 18.

Statistical analysis

The experiment was carried out with three replications. Data are expressed as mean ± standard deviation (SD) (n = 6). Differences between treatment means were compared for significance using Kruskal-Wallis analysis of variance at P≤0.05.

RESULTS

In vitro antidiabetic studies
Inhibition of α-amylase and α-glucosidase

The IC50 values for α-amylase of crude methanol, ethyl acetate and water extracts including standard acarbose were found to be 160, 130, 122, 120 μg at 240 μg/reaction respectively (Fig 1A), and the IC50 values for α-glucosidase were found to be 9.1, 7.9 and 7.0 μg for methanol and ethyl acetate extracts, including standard acarbose at 15 μg/reaction (Fig 1B). Further methanol extract which has shown the lowest IC50 value was partially purified using silica gel column chromatography to obtain active fraction and fractions were further eluted. Among these eluted fractions, the fraction ethyl acetate-methanol ratio of 20:80 showed the highest α-amylase and α-glucosidase inhibition with IC50 values of 180 and 10 μg (Fig 1A & 1B) respectively. Hence this fraction was selected as active fraction named as M. talbotianum leaf methanol active fraction (MTLMAF) and used in all in vivo studies.

Analysis of phytoconstituents using high performance
liquid chromatography (HPLC)

The phytochemical analysis of MTLMAF by HPLC revealed the presence of several phenolic constituents and flavonoids. The constituents included protocatechuic acid, synergic acid, ferulic acid, sinapic acid, rutin, chromoic acid, quercetin and cinnamic acid (Fig 2).

In vivo studies
Body weight and Blood glucose estimation

The initial and final (28th day) body weights of rats are summarized in Fig 3A & 3B. During the course of experiment, the body weight of normal control animals significantly increased (3.2% over their initial readings) after 28 days of treatment, whereas a significant (P<0.05) decrease (5.3%) was observed in diabetic control rats. Administration of MTLMAF or the standard metformin to diabetic rats reversed this weight loss. Significant (P<0.05) increases in the final body weight of these animals was observed. As shown in Fig 3A, the level of glucose increased by three folds on injection of STZ. On oral administration of MTLMAF at the concentration of 100 and 200 mg/kg/day to diabetic rats, the glucose level was reduced by 50% in the first week of treatment, when compared to diabetic control animals. At the end of the treatment, this reduction reached to 78%.

The glucose tolerance test performed with 100 mg of MTLMAF treatment showed improved glycemic control after 30 min of treatment. There was about 72% reduction in glucose level after 150 min of treatment when compared to diabetic rats (data not shown).

Figure 1

In vitro antidiabetic property represented through inhibition of α-amylase and α-glucosidase enzyme by different solvent extracts of M. talbotianum.

Figure 1: Inhibition of α-amylase (concentration per total reaction mixture 250 μL) and α-glucosidase (concentration per total reaction mixture 100 μL) of various solvent extracts from Memecylon talbotianum. Acarbose was used as a positive control. Results are expressed as percentage of the control. Each value is expressed as Mean ± SD, n=3, p<0.05 compared to positive control.

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Figure 2

Analysis of phytoconstituents of MTLMAF based on their retention time comparison with standard phytoconstituents

Figure 2: HPLC profile of MTLMAF. Thermo hypersil gold (4.6 mm × 150 cm, 5μm,) column maintained at 35°C was used in the study. The binary mobile phase consisted of 2.5% formic acid (A) and acetonitrile; water and acetic acid (45:54:1) (B). The gradient elution was as follows: 18% B for 9 min, 18-50% B over 20 min, 50% B for 16 min. A 10 μL aliquot of sample solution was injected into the column. The flow rate was maintained at 1 mL / min. Chromatographic data were recorded by PDA at different detection times. The presence of bioactives was detected based on their retention time.

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Figure 3

Effect of MTLMAF on glucose level in Streptozotocin-induced diabetic male wistar rats.

Figure 3A & 3B: Values are expressed as Mean ± SD (n=6). Gp 1- Normal rats, Gp II –STZ-induced diabetic control rats, Gp III - Diabetic rats with MTLMAF (100 mg/kg), Gp IV - Diabetic rats with MTLMAF (200 mg/kg), Gp V - Control rats with only MTLMAF (1000 mg/kg), Gp VI - Diabetic rats with Standard Metformin 100 mg/kg.

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Table 1

Effect of MTLMAF on biochemical parameters in Streptozotocin-induced male Wistar diabetic rats

ProfilesControlDiabetic controlDiabetic + 100mg MTLMAFDiabetic + 200mg MTLMAFNon-diabetic + only MTLMAF 1000mgDiabetic +Metformin 100mg
Serum cholesterolb68.00 ± 7.1184.33 ± 8.60#94.21 ± 5.50*86.04 ± 10.01*79.01 ± 10.04*77.66 ± 13.08*
Serum triglyceridesb71.25 ± 24.33155.01 ± 63.55#100.33 ± 9.71*100.66 ± 13.31*77.33 ± 14.01*91.01 ± 36.03*
HDL cholesterolb38.25 ± 1.2517.66 ± 1.15#28.66 ± 1.15*24.01 ± 8.01*41.66 ± 3.05*26.33 ± 6.65*
LDL cholesterolb42.01 ± 2.1686.06 ± 13.37#80.86 ± 1.22*71.06 ± 4.71*49.86 ± 5.31*77.13 ± 14.25*
VLDL cholesterolb14.65 ± 4.4929.01 ± 12.71#20.06 ± 1.94*21.13 ± 2.66*18.08 ± 3.03*24.02 ± 7.27*
CHOL:HDLCHOLb2.75 ± 0.203.63 ± 0.46#2.08 ± 0.31*3.43 ± 0.57*2.53 ± 0.37*2.02 ± 3.98*
LDL:HDL ratiob1.10 ± 0.124.90 ± 0.92#2.49 ± 0.23*3.36 ± 0.47*1.19 ± 0.26*2.92 ± 0.68*
Blood Ureab15.2 5± 4.3419.01 ± 9.01#18.01 ± 1.03*18.08 ± 2.02*16.07 ± 1.08*19.01 ± 6.08*
Creatinineb0.62 ± 0.060.68 ± 0.08#0.64 ± 0.15*0.69 ± 0.06*0.60 ± 0.06*0.68 ± 0.08*
SGOT(AST)a567.05 ± 65.01602.33 ± 151.01#576.33 ± 40.09*549.33 ± 70.43*547.66 ± 21.36*497.66 ± 109.02*
SGPT(ALP)a187.00 ± 189.05208.03 ± 17.01#79.01 ± 15.71*106.33 ± 33.85*176.33 ± 12.66*75.33 ± 15.63*
APa321.25 ± 42.04503.02 ± 106.78#430.33 ± 37.44*440.66 ± 17.07*440.01 ± 46.93*477.66 ± 190.43*
Total protein7.00 ± 0.458.43 ± 0.63#7.22 ± 0.24*7.05 ± 0.12*6.80 ± 0.21*7.11 ± 0.36*

Values are expressed as Mean ± SD (n=6 for each group)

# denotes P<0.05 when compared Normal control group;

* denotes P<0.05 when compared to diabetic control group. MTLMAF – M. talbotianum leaf methanol active fraction.

a – values expressed as units of activity /mg protein.

b – values are expressed as units /dL

Table 2

Effect of MTLMAF on enzymatic and non-enzymatic antioxidants in the liver tissues of Streptozotocin-induced diabetic male Wistar rats

GroupsCatalase aSODaGSHb
Control32.7 ± 1.424.7 ± 1.240.1 ± 1.9
Diabetic control22.4 ± 1.8#17.8 ± 0.9#18.2 ± 0.9#
Diabetic +100mg MTLMAF26.8 ± 1.6*21.7 ± 1.7*32.1 ± 0.9*
Diabetic + 200mg MTLMAF29.7 ± 0.8*23.8 ± 1.2*38.9 ± 0.6*
Non diabetic +1000mg MTLMAF30.7 ± 1.4*24.0 ± 0.9*42.1± 0.9*
Diabetic +Metformin 100mg36.4 ± 1.8*25.8 ± 0.2*42.2 ± 2.3*

Values are expressed as Mean ± SD (n=6 for each group).

# denotes P<0.05 when compared Normal control group

* denotes P<0.05 when compared to diabetic control group. MTLMAF – M. talbotianum leaf methanol active fraction.

a – values expressed as units of activity /mg protein.

b – values are expressed as units /dL

Analysis of biochemical parameters and serum enzymes

The effect of MTLMAF on these lipids were estimated their concentrations in all our experimental groups. Administration of MTLMAF, significantly decreased the glucose level after four weeks (312 ± 14 mg/dL in diabetic animals to 134 ± 13 mg/dL in MTLMAF treated animals) and controlled the levels of TG (155 ± 63.55 units/dL in diabetic and 100.66 ± 13.31 units/dL in treated animals), LDL (86.06 ± 13.37 units/dL in diabetic and 71.06 ± 4.71 units/dL in treated animals), HDL (17.66 ± 1.15units/dL in diabetic and 24.01 ± 8.01units/dL in treated animals), VLDL (29.01 ± 12.71units/dL in diabetic and 20.06 ± 1.94 units/dL in treated animals). In diabetic control group the concentrations of TC, LDL, VLDL, TG were found elevated whereas the concentration of HDL was decreased. Treatment with MTLMAF and metformin showed marked reversal of changes in the serum lipid parameters as compared to diabetic rats. The activities of SGOT, SGPT and ALP enzymes were found elevated in Group II as compared to Group I treated rats. In Group III and Group IV animals which received with MTLMAF showed a marked decrease in the enzyme activities, whereas protein concentration was found enhanced (Table 1).

Analysis of antioxidant enzymes

The treatment of diabetic animals with MTLMAF (100-200 mg/kg) increased the activities of catalase (19-35%), SOD (21-33%) and glutathione (50-56%) when compared to untreated diabetic rats. Hence, MTLMAF effectively controlled the oxidative stress under disease conditions by increasing the activities of these antioxidants. The levels of enzymatic and non-enzymatic antioxidants in the liver tissues of various groups of experimental animals are presented in Table 2.

Histopathology of pancreatic and liver tissues

Histopathological studies of the pancreatic and liver tissues were performed and results are shown in Fig 4A & 4B. Group I rats showed normal architecture of the pancreas with dark, granulated beta cells in the islet of Langerhans. Induction of diabetes using streptozotocin resulted in damaged, shrunken islets. There is evidence of atrophic changes, vacoulation, infiltration of lymphocytes and damage in connective tissue parenchyma of islets in these diabetic rats. The abnormal histopathology of pancreas due to streptozotocin-induced diabetes was reversed in the MTLMAF and metformin treated diabetic animals. Treatment with MTLMAF in Group III animals resulted in appearance of less damaged islets cells when compared to untreated diabetic rats (Group II). The recovery in metformin-treated group was evident by the near normal size islets and granulated beta cells (Fig 4A). The histopathological studies of the liver tissues were also performed (Fig 4B). In Group I animals the portal tracts were composed of portal vein radical, and bile duct radical. The central veins appeared normal. In diabetic Group II rats the hepatocytes showed global microvesicular steatosis, dilated bile duct with inflammatory cell infiltration in the portal area. In the nondiabetic rats treated with MTLMAF, the hepatocytes, portal tracts and central veins appeared normal. In Group III animals, the hepatocytes showed mild inflammation, central veins appeared normal and no steatosis was observed. However, diabetic metformin treated group showed maximum connective tissue restoration (Fig 4B).

Figure 4

Histopathology of Streptozotocin-induced diabetic male wistar rat pancreatic and liver tissues

Figure 4A & 4B: Histopathology of rat pancreatic and liver tissue Gp 1- Normal rats, Gp II –STZ-induced diabetic control rats, Gp III - Diabetic rats with MTLMAF (100 mg/kg), Gp IV - Diabetic rats with MTLMAF (200 mg/kg), Gp V - Control rats with only MTLMAF (1000 mg/kg), Gp VI - Diabetic rats with Standard Metformin 100 mg/kg as visualized by fluroscence microscopy.

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Figure 5

Pathological and Immunohistochemical staining of Streptozotocin-induced diabetic male wistar rat pancreatic tissues

Figure 5: Pathological and Immunohistochemical analysis of rat pancreas streptozotocin induced diabetic male Wistar rats were treated with MTLMAF the pancreas were isolated for anti-insulin immunostaining against insulin antibodies as visualized by the confocal microscopy equipped with an excitation filter 515-560 and magnification at 20x, scale bar: 10 μm. Gp 1- Normal rats, Gp II STZ-induced diabetic control rats, Gp III - Diabetic rats with MTLMAF (100 mg/kg), Gp IV - Diabetic rats with MTLMAF (200 mg/kg), Gp V - Control rats with only MTLMAF (1000 mg/kg), Gp VI - Diabetic rats with Standard Metformin 100 mg/kg. An Arrow mark indicates co-localization.

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Immunostaining studies of pancreatic tissues

Weak insulin immunoreactivity was detected in the pancreas of STZ treated rats compared to control group. Whereas in the tissues of MTLMAF treated group, significant increased immunoreactivity of insulin was observed as evidenced by co-localization (Fig 5).

DISCUSSION

Streptozotocin induces necrosis of beta cells resulting in diabetes mellitus 19 and used to induce experimental diabetes in animals. The long term effects of diabetes include damage, dysfunction and failure of various organs 20. According to WHO reports, there are 347 million people worldwide suffering from diabetes and deaths may double between 2005 and 2030 21. The management of diabetes without any side effects is still a challenge to the medical profession. Further, after the recommendations made by WHO on diabetes mellitus 22, investigation on hypoglycemic agents from medicinal plants has received greater attention. More than 800 plants are reported to be in use as traditional remedies for diabetes 23.

The study was aimed to investigate the therapeutic potential of M. talbotianum with respect to diabetes by employing in vitro and in vivo studies. Carbohydrate hydrolyzing enzymes α-amylase and α-glucosidase are responsible for glucose generation from diet. α-Amylase hydrolyzes α-1,4-glycocidic bonds and splits up starch components into smaller oligosaccharides and disaccharides, such as maltose. The α-glucosidases hydrolyze disaccharides to monosaccharides. Inhibition of these two enzymes is important for management of diabetes. Extracts of green tea have α-amylase inhibitory activity 24 and a number of plant extracts have been shown to exert antidiabetic property through α-glucosidase inhibition. The methanol extract of M. talbotianum showing the highest IC50 value was partially purified and named as M. talbotianum leaf methanol active fraction (MTLMAF) and used in all in vivo studies in this study.

The HPLC analysis of MTLMAF revealed the presence of phenolic and flavonoid constituents. These constituents are proven to have antioxidant and antidiabetic properties 25. Among these protocatechuic acid, quercetin and rutin are the major compounds. Antidiabetic properties of these compounds have been shown by several reports 26-28. Hence, it can be predicted that these molecules have prominent roles in controlling diabetes and its related complications. But, other molecules present may also get involved, especially in the protection of pancreatic damage and in controlling associated biochemical parameters.

Diabetic animals tend to lose their weight due to altered metabolism 3. During the course of experiment, the body weight of normal control animals significantly increased after 28 days of treatment, whereas a significant decrease was observed in diabetic control rats. STZ is known to increase glucose levels in experimental animals by decreasing the production of insulin causing damage to pancreatic cells. The observed values are comparable to metformin treated group rats 29.

Decreases in the concentration of HDL and high levels of cholesterol, lipoprotein and triglycerides along with the altered cholesterol/HDL ratio are the predictors of coronary heart disease including atherosclerosis 30. Diabetes is also associated with profound alterations in plasma lipid, triglycerides and lipoprotein profile. Consequently these may lead to heart ailments, which are the major causes of morbidity and deaths in diabetic subjects 31. Therefore, to evaluate the effect of MTLMAF on these lipids, we estimated their concentrations in all our experimental groups. Salacia L. (Celastraceae) species have been widely used as antidiabetic medicine in ayurveda from several years. Chronic oral administration of the S. oblonga Wall. root extract reduced the cardiac TG and other lipid contents as demonstrated by 32. The results which obtained with MTLMAF were compared with those of salacia species33, 34. The liver function is assessed by SGOT, SGPT and ALP. Any alteration in the activity of these enzymes is the indication for liver malfunctioning 23. Ohaeri 24 found that liver was necrotized and the activities of these enzymes were increased in STZ-induced diabetic rat serum. This may be mainly due to leakage of these enzymes from liver cytosol into blood stream. To further validate the change in the activities of these enzymes, we performed the histopathological studies of liver. These results indicated the liver protective effect of MTLMAF in diabetic animals. Increased levels of urea and creatinine in the serum are considered as significant markers of renal dysfunction, which are generally evident in chronic diabetes 30. In this study no significant changes in these were observed and hence studies on kidney damage was not considered.

Several in vitro and in vivo studies have demonstrated that superoxide generation is increased in diabetes which is associated with β-cell dysfunction along with the decrease in the activities of endogenous antioxidants 35. The main players of endogenous antioxidant defense of the islets are SOD, catalase, glutathione and GPx enzymes and glutathione molecule 36. Therefore, induction of these endogenous antioxidants may strengthen islets against detrimental effect of ROS. Several compounds such as flavonoids isolated from plants have antioxidant properties. The plant extracts which are used in treatment of diabetic complications have shown to ameliorate the activities of these antioxidants 1. In the present study we found that treatment of diabetic animals with MTLMAF increased the activities of catalase, SOD and glutathione when compared to untreated diabetic rats. Hence, MTLMAF effectively controlled the oxidative stress under disease conditions by increasing the activities of these antioxidants.

Histopathological and immunostaining studies of the pancreatic and liver tissues indicated the protective effect of MTLMAF in diabetic animals, which is in accordance with previous reports 30. This supports our other performed experiments. Since diabetes is a chronic metabolic disease which requires long term treatment, determining the safety of the drug is crucial. Hence, we evaluated the toxicity of MTLMAF up to the dose level of 1000 mg/kg. No significant changes either in the body weight or behavior of treated animals were observed. Several biochemical parameters were investigated and histopathological observations were made. But no noticeable change in any of the tested parameters during the entire treatment phase was observed. Thus it can be concluded that MTLMAF does not produce any adverse toxic effect on treated animals.

CONCLUSION

MTLMAF from Memecylon talbotianum plant helps to control the STZ induced diabetes. Improves lipid profile, reduce the risk of oxidative stress and, ameliorate liver and pancreatic damage. However, detailed mechanistic studies are needed for validation of the active compound(s), before developing a therapeutically potential antidiabetic molecule.

PICTORIAL ABSTRACT

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SUMMARY

  • The Memecylon talbotianum is an indigenous medicinal plant used in ethno medicine including ayurveda and siddha mainly used in the treatment of skin diseases.

  • Diabetes was induced in male Wistar rats by using STZ (2-deoxy-2- (3- (methyl-3-nitrosoureido) - D-glucopyranose).

  • in vitro and in vivo antidiabetic properties of MTLMAF including protection against tissue damage was evaluated.

  • Our study shows that MTLMAF of Memecylon talbotianum helps in controlling glucose level by its protective effect on pancreatic damage in STZ induced diabetic rats along with modulating other physiological parameters.

ACKNOWLEDGEMENT

The authors acknowledge the support form Ministry of Human Resource Development and University Grant Commission under Institution of Excellence scheme awarded to University of Mysore (F. No. 8-3/2008-U.I) and UGC fellowship scheme (Or. No. DV9/192/NON-NETFS/2013-14 dated: 11-11-2013).

ABOUT AUTHORS

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Dr. Bharathi, T. R. Is a Scientist (EU- alba), Department of studies in Biotechnology, University of Mysore, Mysuru. Her research is focused on DNA barcoding, metabolite profiling, characterization and assaying bioactive potential of Medicinal plants including seed technology.

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Dr. Prakash, H. S. Professor, Department of studies in Biotechnology, University of Mysore, Mysuru has research interests in Plant and microbial biotechnology, molecular diagnostics, molecular markers and metabolite diversity, metagenomics, bioprospecting of medicinal plants and endophytes. He has several publications and patents to his credit. At present he is handling a three crore European Union coordinated project on “Novel strategies to increase the yield of sorghum and millets”, along with partners from Denmark, Burkina Paso and Tanzania. He has visited USA, Japan, Italy, Denmark, Germany, China, Tanzania, Bangladesh, Nepal and Vietnam on academic assignments. He has demonstrated the role of peroxidise, lipoxygenase, proton-ATPase, Superoxide dismutase, their isoforms and transcriptomes in biotic and abiotic stress. He has shown the diversity of bark endophytes in medicinal trees, their bioactive potential and characterized novel metabolites. He has identified promising disease-tolerant lines for various diseases. The role of PGPR and RNA interference in virus disease management was demonstrated. He has developed serological and molecular diagnostic tools for the identification of plant.

Notes

[11] Conflicts of interest CONFLICT OF INTEREST

None declared.

ABBREVIATION USED

STZ

2-deoxy-2- (3- (methyl-3-nitrosoureido) - D-glucopyranose

MTLMAF

Memecylon talbotianum leaf methanol active fraction

OECD

Organisation for Economic Co-operation and Development

ALP

alkaline phosphatase

AST

aspartate transaminase

ALT

alanine transaminase

CAT

catalase

GSH

glutathione

SOD

superoxide dismutase

HE

hematoxylin-eosin

PBS

phosphate buffered saline

HRP

Horseradish peroxidase

PI

Propidium iodide

Notes

[12] Supported by Source(s) of support: UGC fellowship scheme

Acknowledgement: The authors acknowledge the support from UGC fellowship scheme (Or. No. DV9/192/NON-NETFS/2013–14 dated: 11-11-2013) and Ministry of Human Resource Development and University Grant Commission, Government of India, under the Institution of Excellence scheme awarded to the University of Mysore, Mysore, India (F. No. 8-3/2008-U. I).

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