Scholarly article on topic 'Anti-hyperglycemic activity of leaves extract of Hyptis suaveolens L. Poit in streptozotocin induced diabetic rats'

Anti-hyperglycemic activity of leaves extract of Hyptis suaveolens L. Poit in streptozotocin induced diabetic rats Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Shanti Bhushan Mishra, Amita Verma, Alok Mukerjee, M Vijayakumar

Abstract Objective To evaluate the antihyperglycemic activity of leaves of Hyptis suaveolens using streptozotocin model. Methods Hyptis suaveolens extract (HSE) 250 and 500 mg/kg body weight was administered orally to streptozotocin induced diabetes, once daily for 21 days. Results A significant reduction in blood glucose was observed in diabetic animals treated with HSE at different doses when compared with diabetic rats. Levels of triglyceride, total cholesterol, low density lipoprotein, very low density lipoprotein were decreased while administering HSE at different doses, compared with their control values in diabetic animals. Conclusions Our results show that HSE possesses significant antihyperglycemic activity which might be attributed to stimulating effects on glucose utilization and antioxidant enzyme.

Academic research paper on topic "Anti-hyperglycemic activity of leaves extract of Hyptis suaveolens L. Poit in streptozotocin induced diabetic rats"

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Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

Document heading doi:

Anti-hyperglycemic activity of leaves extract of Hyptis suaveolens L. Poit in streptozotocin induced diabetic rats

Shanti Bhushan Mishra1*, Amita Verma2, Alok Mukerjee1, Vijayakumar M3

'United Institute of Pharmacy, UCER, Allahabad, India

Faculty of Health, Medical Sciences, Indigenous & Alternative Systems of Medicine, Shiats, Allahabad, India 3Ethnopharmacology Division, National Botanical Research Institute, Lucknow, India

ARTICLE INFO

ABSTRACT

Article history:

Received 29 April 2011

Received in revised form 25 May 2011

Accepted 15 July 2011

Available online 20 September 2011

Keywords: Hyptis suaveolens Antidiabetic Ethanolic extract Glibenclamide Streptozotocin

Objective: To evaluate the antihyperglycemic activity of leaves of Hyptis suaveolens using streptozotocin model. Methods: Hyptis suaveolens extract (HSE) 250 and 500 mg/kg body weight was administered orally to streptozotocin induced diabetes, once daily for 21 days. Results: A significant reduction in blood glucose was observed in diabetic animals treated with HSE at different doses when compared with diabetic rats. Levels of triglyceride, total cholesterol, low density lipoprotein, very low density lipoprotein were decreased while administering HSE at different doses, compared with their control values in diabetic animals. Conclusions: Our results show that HSE possesses significant antihyperglycemic activity which might be attributed to stimulating effects on glucose utilization and antioxidant enzyme.

1. Introduction

The concept of developing drugs from plants in indigenous medical system is much older, while in some cases direct links between a local and biomedical use exists, in other cases the relationship is much more complexHL Hyptis is a genus of Lamiaceae with about 400 species. One of the species from genus, Hyptis suaveolens Poit (referred as Hyptis hereajter) is naturalized in India[2]. Its distribution is tropical and subtropical and it is not commonly found over 500 m. A plenty of traditional herbal medicinal practices have been used in the diagnosis, prevention and treatment of diabetes. The plant, Hyptis suaveolens (L) Poit commonly known as Wilayati tulsi is an ethnobotanically important medicinal plant. The plant has been considered as an obnoxious weed, distributed throughout the tropics and subtropics. Almost all parts of this plant are being used in traditional medicine to treat various diseases. Leaves of Hyptis suaveolens have been utilized as a stimulant,

Corresponding author: Shanti Bhushan Mishra, United Institute of Pharmacy, UCER, Allahabad, India.

Tel: +918004279457

Fax: 91(0532) 2687142

E-mail: shantipharma15@gmail.com

carminative, sudorific, galactogogue and as a cure for parasitic cutaneous diseases[3]. Crude leaf extract is also used as a relief to colic and stomachache. Leaves and twigs are considered to be antispasmodic and used in antirheumatic and antisuporific baths, antiinflammatory, antifertility agents[4] and also applied as an antiseptic in burns, wounds, and various skin complaints. The decoction of roots is highly valued as appetizer and is reported to contain urosolic acid, a natural HIV-integrase inhibitorl5]. Though there is no scientific evidence to support the antidiabetic effect of Hyptis suaveolens, tribal men continue to use the plant in the treatment of diabetes. The objective of this investigation was to ascertain the scientific basis for the use of this plant in the treatment of diabetes using streptozotocin model.

2. Materials and methods

2.1. Plant extract preparation

Dried leaves of Hyptis suaveolens (Lamiaceae) were procured from in and around Shevaroy hills, Yercaud in the month of April 2009 and were identified and authenticated by taxonomist of the Botanical Survey of India, Yercaud,

Tamilnadu, India. The coarsely powdered material was exhaustively extracted thrice with 50% aqueous ethanol, and the yield of Hyptis suaveolens extract (HSE) was 9.5% (w/w)

2.2. Animals

Sprague-Dawley rats (150-200 g) and Swiss albino mice (20-25 g) of either sex and of approximately the same age were procured from the animal house of the Central Drug Research Institute, Lucknow. They were kept in the departmental animal house at (26±2)°C and relative humidity 44%-56% in polypropylene cages. Animals were exposed to alternate 12 hrs of darkness and light each. Animals were provided with standard rodent pellet diet (Dayal, India) and the food was withdrawn18-24 h before the experiment though water was allowed ad libitum. The experimental protocol has been approved by the Institutional Animal Ethics Committee and by the regulatory body of the government.

2.3. Chemicals

Streptozotocin was purchased from Sigma Aldrich Chemicals Pvt, Ltd, Bangalore. All other chemicals and reagents used were of analytical grade.

2.4. Experimental induction of diabetes

All animals were allowed to adapt to cages for 3 days, after which they were fasted overnight. Diabetes was induced in rats by intra peritoneal injection of streptozotocin at a dose of 50 mg/kg, dissolved in normal saline[6]. After streptozotocin treatment, all animals were given free access to food and water. Blood glucose levels were measured 2 days after streptozotocin injection and used as parameters to match pairs of diabetic rats with similar level of severity. Only rats with fasting blood glucose levels higher than 200 mg/dL were considered to be diabetic and were used in the experiment. The mean blood concentration of glucose in normoglycemic rats was 95 mg/dL. Animals were randomly assigned to four different groups ie. group II to V. Group

I served as normal control containing 6 normal rats. All treatments started 2 days after Streptozotocin injection. Vehicles and the drugs were administered orally using oral gavage tube daily for 3 weeks. Blood samples were collected for the measurement of blood glucose level from the tail vein on 0 day, 7th, 14th and 21st day. The blood glucose level was determined by glucometer (one touch). Values of sample treated were compared with that of the standard group which was treated with glibenclamide. Then animals were sacrificed by cervical dislocation. The liver, kidney and pancreas were exposed and perfused with cold saline phosphate buffer of pH 7.4 for histopathological examination. Blood free liver and kidney were taken out and homogenized in a glass Teflon homogenizer separately (10% w/v). Incubation was done at 37 C under controlled conditions for biochemical estimation. The collected blood samples were immediately centrifuged at 2 500 rpm for 15 min. The serum separated was collected in fresh serum tubes and stored in refrigerator (2-4 C) after tightly capped[7].

2.5. Antioxidant assay

2.5.1. DPPH radical scavenging activity

The free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical, DPPH[8]. A 0.1 mM solution of DPPH in methanol was prepared and 1 mL of this solution was added to 3 mL of control ie. standard butylated hydroxyl toluene (BHT) at different concentration (25-100 t g/mL) and test solutions at different concentrations (5-100 tg/mL) in different test tubes. Thirty minutes later, the absorbance were measured at 517 nm.

2.5.2. Nitric oxide scavenging activity

Nitric oxide scavenging activity was measured by the spectrophotometry method[9]. Sodium nitroprusside (5 mM) in phosphate-buffer saline was mixed with a control without the test compound, but with an equivalent amount of methanol. Test solutions at different concentrations (5-100 t g/mL) were dissolved in methanol and incubated at 25 C for 30 min. After 30 min, to 1.5 mL of the incubated solution was diluted with 1.5 mL of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% naphthyl ethylenediamine dichloride). The absorbance of the chromophore formed during the diazotization of the nitrile with sulphanilamide and the subsequent coupling with naphthyethylene diamine dihydrochloride was measured at 546 nm.

2.5.3. Superoxide scavenging activity

Superoxide scavenging was carried out using the alkaline dimethyl sulfoxide (DMSO) method[9]. Solid potassium superoxide was allowed to stand in contact with dry DMSO for at least 24 h and the solution was filtered immediately before use. The filtrate (200 t L) was added to 2.8 mL of an aquous solution containing nitroblue tetrazolium (56 t M), ethylenediaminetetraacetic acid (EDTA) (10 t M) and potassium phosphate buffer (10 t M, pH 7.4). Test solutions at different concentrations (5-100 t g/mL) were added and absorbances were recorded at 560 nm against the control.

2.5.4. Hydroxyl radical scavenging activity

The scavenging capacity for hydroxyl radical was determined according to the modified method[10]. The assay was performed by adding 0.1 mL of EDTA, 0.01 mL of ferric chloride, 0.1 mL of hydrogen peroxide, 0.36 mL of deoxyribose, 1.0 mL of test solutions (5-100 t g/mL) in distilled water, 0.33 mL of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid were dissolved in sequence. The mixture was then incubated at 37 C for 1 hr and 1.0 mL portion of the incubated mixture was mixed with 10% TCA and 1.0 mL of 0.5% TBA to develop the pink chromogen and measured at 532 nm.

2.6. Statistical analysis

Data were statistically calculated by utilizing one way ANOVA and expressed as mean±SEM followed by Donnett's t-test using computerized graph pad in stat version 3.05 graph pad software USA. Differences of P<0.05 were considered statistically significant.

3. Results

3.1. Anti-hyperglycemic activity of Hyptis suaveolens

The effect of 50% ethanolic extract of leaves of Hyptis suaveolens on streptozotocin-induced animals is shown in Table 1 and Table 2. Table 1 showed the effect of oral administration of 250 mg/kg/day and 500 mg/kg/day of HSE on blood glucose level. After 21 days, at the end of treatment, reduction in blood glucose level of treated rats with dose 250 mg/kg was 116.50-219.50 mg/dL (P< 0.01). After oral administration of 500 mg/kg of HSE, blood glucose level in 21 days was 98.83-237.00 mg/dL(P< 0.01). While in case of glibenclamide 600 f g/kg, it was 94.50-232.33 mg/dL (P< 0.01). Table 2 showed that the serum triglyceride (TG), total cholesterol, low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels were significantly higher in diabetic rats compared to those in normal and drug treated rats, while high density lipoprotein (HDL) levels were significantly decreased in the model rats compared to those in normal rats. After the treatment with 50% ethanolic extract of Hyptis suaveolens in the diabetic rats, a significant reduction in serum cholesterol, TG, LDL and VLDL were observed. In antioxidant assay, potential decrease in the concentration of DPPH radial due to scavenging property of methanolic extract of Hyptis suaveolens (MEHS) and BHT showed significant free radical scavenging activity viz. 86.44% and 91.86%, respectively at 100 fg/mL. The scavenging of nitric oxide by MEHS and BHT was concentration dependent. There was a moderate inhibition of nitric oxide formation with the maximum inhibition being 74.12% and 82.14%, respectively at 100 f g/mL MEHS and BHT. The MEHS and BHT showed a moderate inhibition of the superoxide radical 75.50% and 81.87%, respectively at 100 f g/mL.

The effect of MEHS and BHT on hydroxyl radical and iron (Il)-dependent deoxyribose damage was protected significantly at all concentrations. The percentage of inhibition of hydroxyl radical being 68.84% and 73.56% respectively at 100 f g/mL(Table 3).

3.2. Histopathological studies

3.2.1. Pancreas

Microscopically examined pancreas section of control group (1A) showed normal architecture with acini of serous epithelial cells along with nest of endocrine cells separated by fibrocollagenous stroma into lobules. No fibrosis or any inflammation was seen.

Microscopically examined pancreas section of diabetic control group (1B) showed architecture with acini of serous epithelial cells along with nest of endocrine cells separated by fibrocollagenous stroma into lobules. Focal lymphocytic infiltrate was present in stroma.

Microscopically examined pancreas section of 50% ethanolic extract of leaves of Hyptis suaveolens treated group (1C and 1D) showed normal architecture with acini of serous epithelial cells along with nest of endocrine cells separated by fibrocollagenous stroma into lobules. No fibrosis or any inflammation was seen.

The normal architecture was restored to the same as that of the standard drug (Glibenclamide) treated pancreas (1E).

Figure 1. Histopathology of pancreas.

A: Control group; B: Diabetic control group; C: 50% ethanolic extract of leaves of Hyptis suaveolens treated group at 250 mg/kg; D: 50% ethanolic extract of leaves of Hyptis suaveolens treated group at 500 mg/kg; E: Glibenclamide treated group.

3.2.2. Kidney

Microscopically examined kidney section of control group (2A) showed the glomerulli appear normal. The tubules stroma appeared normal.

Microscopically examined kidney section of diabetic control group (2B) showed the moderate increase in mesengial cellularity and matrix. Tubules appeared normal and lined by single layer of cuboidal cells. The stroma revealed focal lymphoplasmacytic infiltrates.

Microscopically examined kidney section of 50% ethanolic extract of leaves of Hyptis suaveolens treated group (2C) showed the moderate increase in mesengial cellularity and matrix. Tubules appeared normal. The stroma showed mild infiltration by lymphocytes, plasma cells and glomeruli and mild increase in mesengial cellularity. The tubules appeared normal. The stroma was infiltrated by lymphoplasmacytic cells (2D).

The normal architecture was restored to the same as that of the standard drug (Glibenclamide) treated kidney (2E).

3.2.3. Liver

Microscopically examined liver section of control group (3A) showed normal architecture with central veins, sinusoids and portal triads appeared normal. Hepatocytes showed moderate cytoplasm and round to oval nuclei.

Microscopically examined liver section of diabetic control group (3B) showed architecture was distorted. The central veins showed congestion. Hepatocytes showed feathery degeneration. Portal triads showed peri-portal inflammation

composed of lymphocytes.

chronic venous congestion and patchy necrosis. The portal triads showed hepatocytes had feathery degeneration and mild chronic inflammation composed of lymphocytes. Portal triads were normal. Hepatocytes appeared normal and showed moderate cytoplasm and round to oval nuclei. There was no feathery degeneration of the hepatocytes (3D).

Figure 2. Histopathology of kidney.

A: Control group; B: Diabetic control group; C: 50% ethanolic extract of leaves of Hyptis suaveolens treated group at 250 mg/kg; D: 50% ethanolic extract of leaves of Hyptis suaveolens treated group at 500 mg/kg; E: Glibenclamide treated group.

Microscopically examined liver section of 50% ethanolic extract of leaves of Hyptis suaveolens treated group (3C) showed the architecture was partially effaced. There was

Table 1

Figure 3. Histopathology of liver.

A: Control group; B: Diabetic control group; C: 50% ethanolic extract of leaves of Hyptis suaveolens treated group at 250 mg/kg; D: 50% ethanolic extract of leaves of Hyptis suaveolens treated group at 500 mg/kg; E: Glibenclamide treated group.

Effects of 50% ethanolic extract of leaves of Hyptis suaveolens on blood glucose levels in streptozotocin induced diabetic rats(Mean ± SEM)(mg/ dL).

Treatment 0 day 7 days 14 days 21 days

Normal control 76.16±5.36 75.66 ± 3.94 75.00±4.96 75.00±4.61

Diabetic control 220.83±19.00 214.50± 10.60** 211.33±20.30** 208.16±17.38 **

HSE(250 mg/kg) 219.50±13.25 191.00±15.35 164.50±19.45 116.83±19.16 AA

HSE( 500 mg/kg) 237.00±15.00 190.16±16.14 132.66±11.01 A 98.83±10.55 AA

Glibenclamide(600 ^ g/kg) 232.33±13.90 184.83±12.80 129.83±19.20A 94.50±5.46 AA

** P<0.01 vs. normal control; AP<0.05,AAP<0.01 vs. diabetic control.

Table 2

Effects of 50% ethanolic extract of leaves of Hyptis suaveolens on the total cholesterol and triglyceride level in blood serum after 21 days(Mean : SEM)(mg/dL).

Treatment TG Total cholesterol HDL LDL VLDL

Normal control 67.25 ± 4.54 74.36 ± 5.46 23.80 ± 2.10 35.30 ± 3.30 13.30 ± 1.70

Diabetic control 112.62 ± 6.36 152.86 ± 7.54** 13.30 ± 3.10** 71.70 ± 8.30** 30.30 ± 1.10**

HSE (250 mg/kg) 71.54 ± 5.25 78.36 ± 5.38A 18.20 ± 0.90aa 47.20 ± 6.70A 20.70 ± 2.10A

HSE (500 mg/kg) 68.32 ± 4.37 72.52 ± 6.56AA 18.90 ± 0.90aa 45.20 ± 5.30A 19.00 ± 2.00A

Glibenclamide(600 ^ g/kg) 82.46 ± 4.23 89.52 ± 6.46AA 19.20 ± 1.00AA 40.20 ±4.50AA 17.80 ± 2.00AA

P<0.01 vs. normal control; AP<0.05,AAP<0.01 vs. diabetic control.

Table 3

Free radical scavenging activity of MEHS (Mean± SEM)(%).

Drug Concentration ( j g/mL) DPPH radical inhibition Nitric oxide Superoxide inhibition Hydroxyl radical inhibition

MEHS 5 11.240±0.517 10.590±0.112 11.870±0.016 11.040±0.135

10 22.360±0.453 22.720±0.451 26.690±0.592 24.580±0.876

25 51.440±0.004 51.140±0.009 68.460±0.158 54.630±0.745

50 72.640±0.005* 72.920±0.001* 70.430±0.364* 61.930±0.984*

100 86.440±0.002** 74.120±0.001** 75.500±0.654** 68.840±0.647**

BHT 25 83.120±0.141 76.690±0.054 75.480±0.784 68.650±0.386

50 86.940±0.125* 80.120±1.215* 76.020±0.887* 71.880±0.423*

100 91.860±0.156** 82.140±0.512** 81.870±1.246** 73.560±0.368**

*P<0.05, ** P<0.01 as compared to standard.

The normal architecture was restored to the same as that of the standard drug (Glibenclamide) treated liver (3E). It was evident that 50% ethanolic extract of leaves of Hyptis suaveolens possessed good antidiabetic activity.

Conflict of interest statement

We declare that we have no conflict of interest.

4. Discussion

Streptozotocin as an antibiotic and anticancer agent has been widely used for inducing type I diabetes in a variety of animals by affecting degeneration and necrosis of pancreatic P -cells[n]. It has been demonstrated that insulin deficiency in diabetes mellitus leads to a variety of derangements in metabolic and regulatory process, which in turns leads to accumulation of lipids such as cholesterol and triglyceride in diabetic patients. The abnormal high concentration of serum lipids in the diabetic subject is mainly due to increase in the mobilization of free fatty acids from the peripheral fat depots. In oxidative stress, where large quantities of reactive oxygen species (ROS) like hydrogen peroxide, superoxide, hydrogen radical, singlet oxygen and nitrogen species are generated, one of the earliest responses to stress. These ROS have a role in disease and aging in animals. The antioxidative system protects the organism against ROS-induced oxidative damage. There are restrictions on the use of synthetic antioxidants such as BHT, as they are suspected to be carcinogenic. Natural antioxidants therefore have gained importance^]. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of DPPH radical is determined by the decrease in absorbance at 517 nm induced by antioxidants^]. The significant decrease in the concentration of DPPH radical is due to the scavenging ability of MEHS. Nitric oxide is a potent pleiotropic mediator of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity. MEHS was shown significant scavenging activity. Hydroxyl radicals are the major active oxygen species that cause lipid oxidation and enormous biological damage[14]. The MEHS has potent antioxidant and free radical scavenging effects in different in-vitro systems. The present studies demonstrated that the 50% ethanolic extract of Hyptis suaveolens had an antihyperglycemic effect in the streptozotocin induced diabetic rats when administered orally along with antioxidant as well as decrease the cholesterol and triglyceride levels in the significant manner. The current study provides some useful insight into the antihyperglycemic potency of Hyptis suaveolens leaves in streptozotocin induced diabetes. However, we suggest that further work should be carried out at molecular level to find out the absolute mechanism of action of plant Hyptis suaveolens in experimental diabetes.

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