Kadali S. L .D. V. R. M, Das M. C, Vijayaraghavan R, Kumar M. V. Evaluation of Antidiabetic Activity of Aqueous and Ethanolic Extracts of Leaves of Chloroxylon Swietenia in Streptozotocin (Stz) Induced Diabetes in Albino Rats. Biomed Pharmacol J 2017;10(3).

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S. L .D. V. Ramana Murty Kadali¹, M. C. Das², R. Vijayaraghavan³ and M. Vijay Kumar⁴

¹Department of Research, Saveetha University, Chennai.

²Department of Pharmacology, NRI Medical College, Guntur, AP.

³Department of Research, Saveetha University, Chennai.

⁴Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Science, Deralakatte, Mangalore.

Corresponding Author E-mail: murtypharma@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1239

Abstract

Toevaluate antidiabetic activity of Chloroxylon swietenia in STZ induced diabetes in albino rats  Forty two albino rats were randomly divided into seven groups (n=6). Diabetes was induced by intraperitoneal injection of streptozotocin (60mg/Kg). Distilled water, Tween 80, glibenclamide, Chloroxylon swieteniaaqueous extract(CSAE), ethanolic extracts (CSEE) of 200 and 400mg/kg were given orally for 14 days to the normal control, diabetic control, standard group and test groups respectively. Glucose, TC (total cholesterol), TG (triglyceride), HDL (High density lipoprotein), AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase), creatinine, direct bilirubin and indirect bilirubinlevels were estimated. ANOVA followed by Student-Newman-Keuls test was used to analyze the data. CSAE of 200mg/kg showed a significantreduction in glucose, ALT, TB and ALP levels in diabetic rats.CSAE of 400mg/kg showed a significant decrease in glucose, AST, ALT, TB, DB and ALP levels in diabetic rats.CSEE of 200mg/kg showed a significant decrease in glucose, ALT, TB,ALPandcreatininelevels in diabetic rats.CSEE of 400mg/kg showed a significant decrease in glucose, AST, ALT, TB, DB and ALP levels in diabetic rats. Both extracts show antidiabetic activity in STZ induced diabetes.

Keywords

Antidiabetic activity; AST; ALT; ALP;Chloroxylon swietenia; creatinine and Bilirubin streptozotocin;

Introduction

Diabetes mellitus is an endocrine, metabolic disorders caused by relative or an absolute lack of insulin.¹According to International Diabetes Federation (IDF), worldwide 382 million people were affected by diabetesin 2013 and it is expected to raise to 592 million by 2035. IDF estimates 65 million diabetic patientsin India in 2013and it is expected to cross 109 million by 2030.²In India diabetic patients are increasing day by daymay be because of the change in food pattern, i.e. fast food diet intake and change in lifestyle.³Management of diabetes is a tough task. The medicines used in diabetic treatment are either too costlier or have adverseeffectslike hypoglycemic coma, insulin resistance, hypersensitivity and metallic taste etc.⁴ Hence, in the recent years, herbal compoundsare gaining popularity in both developed and developing countries because of their natural origin, low adverse effects.⁵Ethnobotanical information indicates that around 800 medicinal plants having hypoglycemic or antidiabetic potential.⁶ Herbal plants are abundant in India.Hence the search for safer and effective antidiabetic agents has become the current research area.⁵

An ethnobotanicalstudy was carried on the medicinal plants often used for the management of diabetes in Warangal district, Andhra Pradesh by traditional healers.Chloroxylon swieteniais the one of the plant used by the traditional healers for diabetes.^7,8Even though medicinal plants are widely used, the effective treatment of the disease has not been verified with scientific standards. Only a few plants usedfor diabetesin traditional medicineare scientifically audited in vivo.⁹Chloroxylon swietenia belongs to the Rutaceae family. Common name – satinwood, Telugu name – billu, bildu,billedu, Tamil name- porasu orvaaimaram.Chloroxylon swietenia has been reported to have anti-inflammatory activity,¹⁰ mosquitocidal activity,^11-13 antioxidant activity,¹⁴ analgesic activity,¹⁴ anthelmintic activity¹⁵,antimicrobial activity.^15-17Antidiabetic activity was reported with this plant, but with different parts of stem, bark and whole plant.^18,19Invitro antidiabetic activity was reported with the leaf extract of Chloroxylon swietenia in our previous report.²⁰Based on the claims and available evidence, it was thought worthwhile to investigate Chloroxylon swietenia for diabetes in animal models.

Materials and Methods

Plant Material and Extract Preparation

The leaves of Chloroxylon swietenia were collected locally and authenticated by Dr. Shiva Kumari, Department of Botany, Andhra Loyola College.After shade-dried (Temp<40°C.), plant material was grounded into a moderately coarse powder. The aqueous extract was made by maceration and the ethanolic extract was made by using soxhlet apparatus. The extract was allowed to dry. The dried extract was weighed.  The % yield of each plant extract was calculated. The % of yield obtained was 8.96 and 9.16% for alcoholic and aqueous extracts respectively. Both the extracts were preserved in the refrigerator till further use.

Experimental Design

Both sexes of albino rats weighing 250-300g were used. Rats werefedwith a standard pellet diet and water ad libitum. Animals were kept in a controlled environment (12 h/12 h light/night) and temperature (27±2°C).Before starting the experiment, rats were allowed to acclimatize to the laboratory conditions. All the animal experiments were approved by the institutional animal ethics committee(36/IAEC/NRIMC/2013-14) in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimentation on Animals.

Induction of Diabetes

STZ was freshly prepared by dissolving in citrate buffer (0.01M, PH-4.5) and kept on ice prior to practice. The overnight fasted rats were made diabetes with a single intraperitoneal injection of STZ (60 mg/kg). After 4hrs STZ administration 5% glucose was administered orally in drinking water for a day to overcome the early hypoglycemic phase.Rats were allowed to stabilize for three days.On the third day (72hrs)blood samples were drawn to estimate the blood glucose concentration to confirm the development of diabetes.Rats with plasma glucose above 250 mg/dL were considered as diabetic and used in the study.Both the test extracts and standard drug treatment were given orally for 14days. Blood was collected by the retro-orbitalpuncture under light ether anesthesia on 1, 7 & 14^th day of treatment schedule for biochemical estimations.

Rats were randomly allocated into 7groups (n=6) (Table-1).

Table 1: Grouping of animals

CSAE ₌ Chloroxylon swietenia aqueous extract, CSEE ₌ Chloroxylon swietenia Ethanolic extract

Biochemical Estimations

Serum was used to estimate the biochemical parameters like ALP,AST, ALT, TB, DB, creatinine, TG, HDL and TC using commercially available kits.LDL and VLDL values were calculated by using Friedewald’s formula²¹ as mentioned below

VLDL= TG/5

LDL= TC-HDL-VLDL

Atherogenic index (AI) values were calculated by using formula as given below²²

AI= (TC-HDL) /HDL

Statistical Analysis

The data were expressed as mean ± standard error (SE). The Significance of differences among the groups were assessed by using ANOVA, followed by Student-Newman-Keuls test. p<0.05 (5%) were considered as significant.

Results

Effect on Blood Glucose

The effect of extracts on glucose level is illustrated in Graph-1. Statistical analysis at ‘0’ (zero) dayby One-way ANOVA revealed that there was no significant (P>0.05) difference among the groups. Further, statistical analysis on the 7^thday of medication showed a significant (P<0.05) difference among the groups. There was a significant elevation of blood glucose level in diabetic control as compared to normal control rats.Student-Newman-Keulstest revealed that glibenclamide, CSAE₁, CSAE₂, CSEE₁ and CSEE₂treated groups shows asignificant reduction in blood glucose level as compared to the diabetic control. Similarly, statistical analysis at 14^thday showed that there was significant (P<0.05) difference among the groups. Student-Newman-Keuls test revealed that glibenclamide, CSAE₁, CSAE₂, CSEE₁ and CSEE₂treated groups shows asignificant reduction in blood glucose level as compared to the diabetic control. Further analysis by ANOVA followed by Student-Newman-Keuls test revealed that glibenclamide, CSAE₁, CSEE₁ and CSEE₂treated groups shows no significant difference between the groups when test groups are compared with standard (glibenclamide) control. This result indicates that test groups produced the effect almost equal to the standard group.

Graph 1: The effect of glibenclamide, CSAE and CSEE on plasma glucose (FBS) in diabetic rats.Mean + SE (n = 6). a Statistically significant from control group. b Statistically significant from STZ group.   Click here to View graph

Effect on AST and ALT

The changes in the AST and ALT levels of all the groups are illustrated in Graph-4. Statistical analysis by One-way ANOVA revealed that there was asignificant difference among the groups [p<0.05].There was a significant elevation of AST and ALT level in diabetic control as compared to normal control rats. Glibenclamide, CSAE₁, CSAE₂, CSEE₁ and CSEE₂treated groups show asignificant reduction in ALT level as compared to the diabetic control. CSAE₁ and CSEE₁reduced the AST level when compared to the diabetic control, butthe reduction is not statistically significant (p>0.05). Both the extracts of 400mg/kg dose shows significant(p<0.05) reduction in the AST levels.

Graph 2: The effect of CSAE and CAEEon TC, TG, HDL, LDL and VLDL in diabetic rats.Mean + SE (n = 6).a Statistically significant from control group. b Statistically significant from STZ group.   Click here to View graph

Effect on Total Bilirubin and Direct Bilirubin

The changes in the total bilirubin and direct bilirubin levels of all the groups are illustrated in Graph-5.There was a significant elevation of total bilirubin and direct bilirubin levels in diabetic control as compared to normal control rats.CSAE₁, CSAE₂, CSEE₁ and CSEE₂ treated groups shows asignificant reduction in the total bilirubin level as compared to the diabetic control. CSEE of 400mg/kg dose shows the significant reduction inthe direct bilirubin level when compared to the diabetic control.

Graph 3: The effect of glibenclamide, CSAE and CSEE on AI, CRI and % protection in diabetic rats.Mean + SE (n = 6). a Statistically significant from control group. b Statistically significant from STZ group.*Statistically significant from glibenclamide group.   Click here to View graph

Effect on ALP and Creatinine

ALP and creatinine levels are significantly elevated in diabetic controls as compared to the normal control (Graph-4 and 5). Both extracts of all the doses significantly reduced the elevated ALP levels. CSEE of 200mg/kg showed asignificant decrease in the creatinine levels.

Graph 4: The effect of glibenclamide, CSAE and CSEE on AST, ALT and ALP in diabetic rats.Mean + SE (n = 6). a Statistically significant from control group. b Statistically significant from STZ group.   Click here to View graph

Graph 5: The effect of glibenclamide, CASE and CSEE on total bilirubin (TB), direct bilirubin (DB) and creatinine (Cr) in diabetic rats.Mean + SE (n = 6). a Statistically significant from control group.b Statistically significant from STZ group.   Click here to View graph

Effect on Lipid Profile

There was a significant rise in TC, TG, VLDL and LDL levels in the diabetic control in comparison to the normal rats(Graphs-6). Both doses of CSAE showed asignificant reduction in TC level. CSEE of 200mg/kg showed a significant reduction in the TG levels. CSAE (400mg/kg), CSEE(200mg/kg) and CSEE(400mg/kg) showed a significant elevation of HDL levels in comparison to the normal and diabetic control. Both extracts of all the doses significantly reduced the LDL levels in comparison to diabetic control.

AI and CRI values are significantly higher in diabetic control compared to the normal rats (Graphs-2).  Standard drug and test extracts significantly reduce the AI and CRI values. Glibenclamide, CSAE₁, CSAE₂, CSEE₁ and CSEE₂ showed 86.5, 64.6, 89.3,94 and 94.5 % protection respectively (Graphs-3).

Discussion

The development of safer medicines for diabetes is still a challenge for researchers working in this area.²³The experimental data on herbal medication can offer new functional leads to reduce toxicity, time and money are the three main hurdles in drug development. It is correctly stated that ‘laboratories to clinics’ becomes ’clinics to laboratories’ is a true reverse pharmacology approach. ²⁴The development of modern treatment methods requires animal models that mimic the range of pathophysiological changes visualized in diabetic humans. ²³

Streptozotocin is commonly using chemical to induce diabetes in rodents than the other chemical inducing agents like alloxan, gold thioglucose etc because of its less toxicity and specificity.²⁵Streptozotocin (2-deoxy-2-(3-(methyl-3- nitrosoureido)-D-glucopyranose) is obtained from Streptomycetes achromogenesand is used to induce both type-1 and type-2 diabetes.STZ is taken up by pancreatic β-cells via glucose transporter GLUT2. STZ alkylates the DNA leads to the β-cell death. STZ is a nitric oxide (NO) donor and this NO destroys the pancreatic islet cells.²⁶

Both doses of CSAE and CSEE significantly decreased the glucose level as compared to the diabetic control rats. This effect may be due the decrease in glucose absorption from the intestines or induction of glycogenic process along with decrease in glyconeogenesis and glycogenolysis.²⁷

Dyslipidemia is a most common complication observed in chemical induced diabetes and presents a serious risk of vascular disease.⁶  Inthe present study, raise in TC and TG levels were observed in diabetic control rats.In diabetes the abnormal high levels of lipids are due to, an increase in the mobilization of free fatty acids from fat deposits due to the less utilization of glucose.³ Hypertriglyceridemia is amost common abnormality in diabetes.²⁷

The serum lipid levels are generally high in diabetes; mapping a major risk factor for coronary heart disease.⁶ Excess levels of TC and LDL are major coronary risk factors. The C. swietenia leaf extractreduced the TC, TG and LDL levels, where as it increased the cardioprotective lipid HDL levels significantly. It has been proved that raise in HDL levels is associated with a reduction in coronary risk.²⁸In the present study, it has been observed that the C. swietenia leaf extract mitigated the raised TC and LDL levels in diabetic rats. Further, it has been indicated that TG itself is independently linked to coronary heart disease²⁹and in the present study, the plant extracts lowered TG levels in diabetic rats. The atherogenic index and the coronary risk index were very high in the diabetic rats.²⁸Standard drug and plant extracts significantly reduced the AI, CRI as to the normalrats. % protection was increased with the dose, CSEE has shown more protection than the CSAE.

Diabetes is one of the common causes for a liver disease which includes abnormal liver enzymes, cirrhosis, hepatocellular carcinoma and acute liver failure.The AST, ALT, ALP , TB, and DB levels were raised in lever injury.²⁷ These enzymes are considered as asensitive indicator of liver injury.Chloroxylonswietenia leaf extracts reduced the AST level, it shows the protective effect on theliver. The rise in ALP levels,indicates bone disease, bile tract obstruction or liver disease. C. swieteniaextracts lowered the ALP levels, suggesting its protective effect on liver function.

Diabetes affects the kidney, result in the development of diabetic nephropathy. Serum creatinine levels reveal the kidney function.³⁰CSEE (200mg/kg) significantly reduced the creatinine levels.

Conclusion

It is evident that C. swietenia leafextracts contain antihyperglycemic agents capable of reducing the blood glucose level.

Acknowledgement

The authors are thankful to the colleagues and management for facilitating to accomplish this project.

Conflicts of Interest

None.

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