INTRODUCTION:

Diabetes mellitus (DM) is set of chronic metabolic diseases resulting from lack or defects in insulin secretion and insulin action, characterized by increased blood glucose levels, lead to noxious conditions like atherosclerosis, cardiovascular disease and other complications.¹World Health Organization estimated that more than 170 million people are suffering from diabetes globally.² DM is mainly associated with dyslipidaemia and oxidative stress. This condition can cause damage or dysfunction of many of the body’s biological systems like, including nerves ad blood vessels.³ A mortality rate of diabetic patients from cardiovascular complications such as stroke, ischemic heart diseases are high.⁴ DM requires continuous medical care with multifactorial risk-reduction strategies beyond glycaemic.⁵ The maintaining of DM has improved because of advancement in insulin types for many years.⁶

Many studies have been explained that oxidative stress, interferes mainly by hyperglycaemia induced generation of free radicals, which is due to peroxidation of membrane lipids and protein glycation and at the same time, it ceases the antioxidant defence mechanism and leads to cells and tissues damages. Antioxidant are substances which have ability to prevent oxidation process and scavenging free radicals.⁷ Antioxidant are naturally present in foods, a maximum of antioxidants naturally present in foods occur in phenolic structures and especially in flavonoid structure.⁸

Alloxan is a urea derivative where it causes necrosis of beta cells of pancreatic islets. It is well known diabetogenic agent which will induce Type 1 diabetes in experimental animals. The major toxic mechanism of action of alloxan on beta cells of pancreas involves, oxidation of essential sulfhydryl group, inhibition of glucokinase enzyme, generation of free radicals and disturbances in intracellular calcium homeostasis.⁷

Tephrosia genus belongs to fabaceae family with 400 species which is chiefly distributed in Asia, Africa, Australia and America. Tephrosia tinctoria is widely distributed in South India. This genus is well known for its richness in prenylated flavonoids and it possess insect repellent, larvicidal, pesticidal, antimicrobial and anticancer properties.^9,10

MATERIALS AND METHODS:

Collection of plant material:

The whole plant of Tephrosia tinctoria was collected from Kolar district, Karnataka, India. The plant specimen was authenticated by Dr. V. Rama Rao, Department of Botany, Regional Ayurveda Research Institute for Metabolic Disorders, Bengaluru, Karnataka, India, with a Voucher No. 975.

Preparation of extracts:

The whole plant was dried and coarsely powdered. The coarse powder was subjected to successive extraction with petroleum ether, chloroform and ethanol at 50℃ to 60℃ using Soxhlet apparatus. The extraction was continued for 72h. The extracts were filtered and concentrated to a dry mass by using rotary evaporator. The petroleum ether extract was obtained as green residue, chloroform extract as blackish green residue and ethanolic extract as dark brown. All the residues were stored in refrigerator at 2-8℃ for further experimental use.

Experimental animals:

Male Wistar albino rats, weighing 180-200g were used for the experiment. The animals were maintained in the department animal house. Once procured, the animals were acclimatized for ten days under standard husbandry conditions i.e., the animals were housed in polypropylene cages maintained under controlled temperature at 23±2℃, relative humidity 45-55% and with 12:12h day/night cycle. Temperature and humidity were recorded daily using thermometer and hydrometer mounted in animal house. The animals had free access to standard rat pellet along with water ad libitum under strict hygienic conditions. Animals were habituated to laboratory conditions for 48h prior to experimental protocol, to minimize if any non-specific stress. The study protocol was approved by the Institutional Animal Ethical Committee (Reg. No. 18/IAEC/2018-19).

Phytochemical evaluation:

The preliminary phytochemical investigation was carried out with Chloroform and Ethanolic extract of Tephrosia tinctoria (CETT and EETT) for the qualitative identification of phytochemical constituents present in both the extracts by using standard procedure.¹¹

Acute oral toxicity studies:

Acute oral toxicity study was performed by using female albino Wistar rats (150-200g) for CETT and EETT according to the toxic class method 423(2c) as per OECD guidelines. The starting dose level was selected from 300mg/kg b.w. p.o and were given up to 2000 mg/kg b.w. The animals were observed carefully for toxicity signs and mortality for 48h. Thus, the next higher dose (2500 and 5000mg/kg b.w.) were given to observe the signs of mortality. Mortality was not observed in any of the dose ranges after 14 days.

Experimental induction of diabetes in rats:

Wistar albino rats (180-200g) were fasted over-night and injected with alloxan monohydrate in normal saline (0.9% NaCl) at a dose of 120mg/kg b.w intraperitoneally to induce diabetes. After one hour of alloxan administration, the animals were given 5% glucose solution to overcome the early hypoglycaemic condition.^12,13 Two days after alloxan injection, the blood sugar level was measured using glucometer and the rats with blood glucose concentration higher than 200mg/dl were considered as diabetic rats and further used for studies.^14,15

Anti-diabetic activity in diabetic rats:

The diabetic animals were divided into seven groups, each group containing six animals (n=6). Group I served as normal control. Group II served as diabetic control and Group III served as standard control, was treated with Glipizide orally at the dose of 5mg/kg b.w. Group IV and V had received CETT at the dose of 250 and 500mg/kg b.w orally respectively. Group VI and Group VII had received EETT at the dose of 250 and 500mg/kg b.w respectively. All the animals were treated for 14 days. Body weight and blood glucose level were estimated on 0, 7^th and 14^thday of the treatment. At the end of 14^th day, blood samples were again collected from overnight fasted rats by retro orbital plexus for biochemical estimations. All the animals were sacrificed by injecting overdose of ketamine anaesthesia intraperitoneally (150mg/kg b.w) which results in rapid loss of consciousness followed by cardiac or respiratory distress and ultimately leading to the loss of brain function thus minimizing the distress and anxiety experienced by the animals. The pancreas from all the animals were isolated immediately and one animal pancreas from each group was kept in 10% formalin solution for histopathological examination.¹⁶

Assessment of tissue antioxidant enzymes:

The isolated pancreatic tissues were homogenized in 10ml of 100mM KH₂PO₄buffer containing 1mM EDTA (pH 7.0) and the mixture was centrifuged at 12,000rpm for 30 min at 4℃. The supernatant was collected and used for SOD¹⁷and lipid peroxidation assays.¹⁸

Collection of blood and determination of blood parameters:

The blood samples were withdrawn from the individual animals of all the 7 groups by retro-orbital puncture under light ketamine anaesthesia (40mg/kg i.p) in a sterile centrifuge tubes and Centrifuged the samples at 3000rpm for 15min. The blood samples were analysed for the estimations of various biochemical parameters like fasting blood glucose level, insulin level and serum lipid profiles by using respective semi auto-analyser kits.

Statistical analysis:

Data were expressed as mean ± standard error of the mean and the significance of difference between the mean of control and treated groups was considered at p<0.05. One-way analysis of variance (ANOVA) followed by Tukey: compare all pairs of columns was performed in computed treatment.

RESULTS:

Phytochemical screening of Tephrosia tinctoria:

The preliminary phytochemical evaluation of Chloroform and Ethanolic extract of Tephrosia tinctoria (CETT and EETT) revealed the presence of various phytoconstituents like flavonoids, alkaloids, phenolic compounds, saponins, tannins and glycoside.

Acute oral toxicity studies:

Acute oral toxicity of (CETT and EETT) showed no mortality in any of the animals treated with the doses starting from 300 upto and it is found to be safe to use upto 5000mg/kg b.w. Hence 1/10^th of this dose (500mg/kg) was selected as the higher dose and half of this dose (250mg/kg) was selected as lower dose for the further study.

Effect of CETT and EETT on body weight:

Dehydration and loss of body weight is more commonly associated with diabetes mellitus. The effects of CETT and EETT on body weight were measured and represented in the below Table 1. The initial body weights were similar in both normal and diabetic groups, but gradually alloxan-induced diabetic rats showed a significant reduction in body weight when compared to the normal groups. CETT and EETT when given at the dose of 250 and 500mg/kg b.w to diabetic rats caused significant increase in body weight when compared with that to the diabetic group. EETT at the dose of 500mg/kg b.w was showed to be more effective (192.33±1.58) than CETT (183.33±2.27) at the end of the study (14^th day).

Effect of CETT and EETT on fasting blood glucose level:

Effect of extracts on blood glucose levels are shown in Table 2. It was observed that the repeated dose administration of CETT, EETT and Glipizide for 14 days, showed significant decrease in blood glucose levels on day 7 and 14, and the values returned to pre-treatment levels. The pre-treatment fasting blood glucose levels in alloxan induced diabetic rats gradually increased from 223±9.23 to 260.66±9.04 on 14^th day. After continuous administration of the extracts, the hyperglycaemic levels were found to be decreased significantly. EETT at the dose of 500mg/kg b.w was found to be more effective (p<0.001) on 7^th day and14^th day in lowering blood glucose level than CETT on 7^th day (p<0.01) and 14^th day (p<0.001). The Glipizide treated diabetic rats also showed a significant reduction (p<0.001) from on day 7 and 14. Although the hypoglycaemic activity of the extract treated rats were significant when compared to control it was not as effective as the standard Glipizide.

Table 1: Effect of chloroform and ethanolic extracts of Tephrosia tinctoria on body weight of alloxan induced diabetic rats.

Groups

Body weight (g)

0^th day

7^th day

14^th day

Group-1 Normal control

191±4.115

186.83±

5.84

192.33±

4.814

Group-2 Diabetic control (DC)

191.33±

4.55

168.16±

3.32

157.166±4.26

Group-3 Standard

192.16±

3.390

176.5±

6.185^***

182.61±

0.87^***

Group-4 CETT 250mg/kg

192.5±

3.222

169.5±

9.051^**

175.2±

2.017^***

Group-5 CETT 500mg/kg

193.33±

2.728

170.66±

5.63^***

183.33±

2.27^***

Group-6 EETT 250mg/kg

195.16±

2.031

171±

6.148^**

186.16±

3.24^**

Group-7 EETT 500mg/kg

193.33±

3.34

177±

3.307^***

192.33±

1.58^***

Values are expressed as mean±SEM (n=6). One-way ANOVA followed by Tukey: compare all pairs of columns.

Where,

*represents value is significant at p<0.05 vs. DC

** represents value is moderately significant at p<0.01 vs. DC

*** represents value is highly significant at p<0.001 vs. DC

Table 2: Effect of chloroform and ethanolic extracts of Tephrosia tinctoria on blood glucose level in alloxan induced rats.

Groups	Blood glucose level (mg/dl)
Groups	0^th day	7^th day	14^th day
Group-1 Normal control	90.83±8.98	91.66±9.34	92± 9.47
Group-2 Diabetic control (DC)	223±9.23	246.33± 8.015	260.66± 9.04
Group-3 Standard	210.66± 4.56	181±7.169^***	165.5± 6.307^***
Group-4 CETT 250mg/kg	224±6.515	214±4.12^*	208± 5.53^**
Group-5 CETT 500mg/kg	229±9.611	213.83± 8.65^*	190.83± 15.3^***
Group-6 EETT 250mg/kg	213±4.314	203.66± 2.55^**	192± 3.376^***
Group-7 EETT 500mg/kg	218.83± 6.84	196.83± 5.11^***	170.6± 12.96^***

Values are expressed as mean±SEM (n=6). One-way ANOVA followed by Tukey: compare all pairs of columns.

Where,

*represents value is significant at p<0.05 vs. DC

** represents value is moderately significant at p<0.01 vs. DC

*** represents value is highly significant at p<0.001 vs. DC

Effect of CETT and EETT on lipid profile:

The effect of ethanolic and chloroform extracts of Tephrosia tinctoria (CETT and EETT) and glipizide on serum lipid profiles of control and experimental groups were showed in Table 3. The hyperlipidaemic parameters such as serum triglyceride (TG), total cholesterol (TC), and LDL cholesterol (LDL-C) levels were increased, but HDL cholesterol (HDL-C) was decreased in diabetic groups in comparison to the normal control. However, all these parameters except HDL-C were decreased significantly in the diabetic rats treated by 250 and 500mg/kg b.w of CETT, EETT or Glipizide in respective groups. At the end of the study (14^th day), TC, TG and LDL-C levels were decreased significantly (p<0.001) and HDL-C levels were increased significantly (p<0.001) in EETT (500mg/kg) treated and standard treated groups compare to diabetic control group. In, EETT (250mg/kg) treated groups similar effect are shown but moderately significant (p< 0.01). Similarly, in CETT treated group (250mg/kg and 500 mg/kg) moderately decrease (p<0.01) in TC, TG and LDL-C levels and increase in HDL-C level.

Effect of CETT and EETT on insulin level:

When alloxan was injected to rats, it induces pathological lesion in islets of Langerhans of pancreas and resulted in significantly low insulin secretion from beta cells when compared to that of the normal control group. EETT treated groups (250mg/kg and 500mg/kg) showed a highly significant increase (p<0.001) in insulin level and moderately significant increase in insulin levels (250mg/kg and 500mg/kg) in CETT treated groups. EETT was found to be more effective when compared to CETT. The results were expressed in Table 3.

Effect of EETT and CETT on in-vivo antioxidant models:

The SOD activities were significantly decreased in diabetic control than normal control group, but it is significantly increased in extracts (CETT and EETT) treated groups. In Glipizide and EETT (500mg/kg b.w) treated groups showed maximum percentage of inhibition effect of superoxide dismutase (78.5% and 75.65%).

The lipid peroxidation study was showed by percentage of inhibition of TBARS. In diabetic group, there is a significant decrease in the percentage of inhibition of TBARS comparison to normal control group. But in case of Glipizide, CETT and EETT treated groups (Group III to VII) showed a significant increase in the percentage of inhibition of TBARS. The values were expressed in Figure 1.

Figure 1: Effect of chloroform extract and ethanolic extracts of Tephrosia tinctoria on Super dismutase and lipid peroxidation in-vivo antioxidant models.

Histopathological studies:

Histopathological studies of pancreas of normal control group showed normal acini and normal cellular structure with number of islets of Langerhans with normal beta cells. In diabetic control group, maximum damage to the islets of Langerhans and reduced dimensions of islets and least number of beta cells was seen. Regeneration of normal cellular population of islets with a greater number of beta cells was found in Glipizide treated group. Similarly, Partial restoration of normal cellular population of islets with few greater numbers of beta cells was observed in extracts (CETT and EETT) treated groups. EETT (500mg/kg b.w) shown better restoration and regeneration of beta cells compared to CETT (250 and 500mg/kg b.w). The histological slides of all the groups were showed in the Figure 2.

Figure 2: Histopathology studies of pancreas in A) Normal group, B) Diabetic control, C) Standard (Glipizide), D) CETT (250mg/kg), E) CETT (500mg/kg), F) EETT (250mg/kg), G) EETT (500mg/kg)

DISCUSSION:

Diabetes Mellitus (DM) is a major health problem worldwide. Many of the researches have been conducted to overcome the symptoms/complications occurring in DM. The results obtained from this study revealed that CETT and EETT exhibited anti-diabetic and anti-oxidant effect in alloxan induced diabetic rats. Alloxan is widely used as a diabetogenic agent to induce type 1 diabetes in laboratory animals. Alloxan induces DM in experimental animals by inactivating the glucokinase enzyme and the effect is seen by the generation of Reactive Oxygen Species (ROS) which interacts with the DNA of pancreatic islets where fragmentation of DNA takes place in the beta cells.

In the present study, increased blood glucose levels confirmed the occurrence of DM in albino Wistar rats. After the treatment with CETT and EETT blood glucose levels and insulin levels were significantly restored to normal. Treatment with the extracts did not have much impact on the body weight of the animals. However, the body weight did not increase but was very much normal as that of normal control group. The decreased level of insulin secretion in diabetic animals leads to the lack of glucose uptake in the muscles, erythrocytes and central nervous system. In order to balance this deficiency, the tissues take up glucose through gluconeogenesis and at the same time releasing glucose into blood by the liver through glycogenolysis.¹⁹

Herbal plants contain different natural antioxidants, in particular tannins, vitamin C and E which have the ability to maintain β-cells activity and decreasing the intestinal absorption of glucose which maintains blood glucose level.²⁰ Alloxan induced diabetic rats showed, hyperglycemia, increased TC, TG and LDL-C levels, and decreased in HDL-C and insulin levels, due to which there is an increase in the concentration of lipid peroxidation, H₂O₂and nitrate in pancreas and liver. This oxidative stress leads to decrease in antioxidant enzymes.²¹

In this study, the results revealed the stimulatory effect of Tephrosia tinctoria and this could be possibly due to the glucose uptake in peripheral tissues or increase in insulin secretion. The lipid levels are very much higher in diabetic conditions which might lead to the fatal heart diseases.²¹ In the present study, treatment with CETT and EETT significantly decreased the increased levels of triglycerides, total cholesterol and LDL-C levels and also significantly increased the HDL-C and insulin levels in extract treated rats when compared to the diabetic control rats. These effects may be due to the presence of alkaloids and flavonoids in CETT and EETT.

The extract treated animals showed the significant increase in insulin level when compared to the diabetic control similar to that Glipizide treated group, hence the possible mechanism either it might be due to the stimulation of beta cells and release of insulin and activation of the insulin receptors which is major mechanism of action of Glipizide.

The diabetic animals (Group II) showed increased level of free radicals which can causes damage to organs hence leading to oxidative stress. The increased lipid peroxides level in the tissues is due to the failure of antioxidant mechanism.²¹ Exposure to alloxan decreased the activities of the ROS scavenging enzymes viz. SOD.⁷ The present study reveavled that extracts could restore the activity of this antioxidant enzyme and possibly could reduce generation of free radicals and prevent cellular damage. Indeed, extracts supplementation was potentially effective in blunting lipid peroxidation suggesting that extracts possibly have the antioxidant property by reducing membrane lipid peroxidation. Free radicals attack lipids leading to its peroxidation and also covalently bind to microsomal lipids and protein.¹⁰ This results in generation of free radicals which includes super oxide etc., The presence of various phytochemicals in extracts can prevent the free radicals to decrease the oxidative stress caused by diabetic condition. The extracts can increase level of natural antioxidant like SOD, catalase enzymes in tissues, prevent the lipid peroxidation.

The in-vivo antioxidant property of the extracts was supported by previous in-vitro antioxidant studies.¹⁰ Treatment with CETT and EETT ( Group III and Group IV) rats showed reduction in blood glucose levels, which may be due to decreased NO production because, high glucose levels will increases NO production by protein kinase C activation mechanism. This effect was observed due to the Phenolic compounds present in the extract which acts as the natural antioxidants playing an important role in adsorption and neutralization of the nitric oxide-related products, such as nitrites and nitrates.^23-²⁵

The histopathological studies of pancreas, showed that Tephrosia tinctoria have protective effect on pancreas, because there was marked increase in beta cells. Antioxidant properties of extracts have proved to be protective for pancreatic islets and there by helping in the regenaration of β-cells thus showing increase in insulin levels.²⁶

Statistical analysis in the present study, showed both chloroform and ethanolic extracts decreased the blood glucose, triglycerides, cholesterol, LDL-C levels and increased the HDL-C and insulin levels. But ethanol extract (500mg/kg) showed better activity when compared to chloroform extract. The histopathological studies further supported the evidence of exhibiting activity also showed that ethanolic extract has better regeneration of beta cell.

From the results obtained by various observations in CETT and EETT, the flavonoids which are present in CETT and EETT were shown to prevent the increase in blood glucose level in alloxan induced diabetic rats. The present data indicates that Tephrosia tinctoria possesses antidiabetic and antioxidant effect.

CONCLUSION:

The intraperitoneal administration of alloxan produced cardinal symptoms such as hyperglycemia, loss of body weight, impaired lipid and glucose metabolism.

This study concludes that chloroform and ethanolic extracts of Tephrosia tinctoria (CETT and EETT) possess potential antidiabetic activity as it lowers blood glucose level significantly and also lowers the TC, TG and LDL-C, but HDL-C and serum insulin levels significantly increased. The mechanism has point towards the stimulation of beta cells or increasing the glucose utilization in peripheral tissues.

In the present study, both chloroform and ethanolic extracts showed antidiabetic and antioxidant activities but ethanolic extract (500mg/kg b.w) showed better activity when compared to chloroform extract. Hence, this investigation suggests that Tephrosia tinctoria is also produces beneficial effect in complications of the diabetes mellitus and also has protective effect on pancreas which is supported by the histopathological studies.

ACKNOWLEDGEMENT:

We the authors acknowledge the management of College of Pharmaceutical Sciences, Dayananda Sagar University to provide all the research facilities which made this work successful.

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Received on 06.02.2020 Modified on 30.03.2020

Research J. Pharm. and Tech. 2021; 14(7):3727-3732.

DOI: 10.52711/0974-360X.2021.00645

Groups	TC (mg/dl)	TG (mg/dl)	LDL-C (mg/dl)	HDL-C (mg/dl)	Insulin (µIU/ml)
Group-1 Normal control	97.31±7.32	108.96±9.48	73.97±8.87	40.15±1.013	17.34±0.83
Group-2 Diabetic control	189.14±15.82	199.53±7.21	176.51±8.64	28.89±1.28	6.94±0.5001
Group-3 Standard	91.88±8.11^***	141.161±10.02^**	88.175±8.503^***	35.92±1.25^**	14.29±1.190^***
Group-4 CETT 250mg/kg	172.30±9.97^ns	139.655±6.56^**	125.05±3.77^**	36.308±1.00^**	9.41±0.505^**
Group-5 CETT 500mg/kg	127.46±10.75^**	140.36±10.650^**	145.99±6.47^ns	38.325±0.922^***	10.05±0.5047^**
Group-6 EETT 250mg/kg	119.72±10.10^**	138.605±10.58^**	121.91±9.350^***	32.64±1.663^*	11.69±0.32^***
Group-7 EETT 500mg/kg	117.19±9.66^***	112.64±5.195^***	103.40±9.85^***	41.23±1.329^***	13.08±1.008^***