Anuj Kumar², Akhilesh Kumar Rana², Amit Singh² and Alok Singh¹*

¹Department of Pharmacology, All India Institute of Medical Sciences Raipur Chhattisgarh, India.

²Institute of Medical Sciences, Banaras Hindu University, Varanasi Uttar Pradesh India.

Corresponding Author E-mail: draloksingh@aiimsraipur.edu.in

DOI : https://dx.doi.org/10.13005/bpj/1613

Abstract

To study the effect of methanolic extract of Phyllanthus niruri on animal model of Diabetes Mellitus. Diabetes Mellitus was induced in rats by injecting Streptozotocin (60mg/kg) intraperitonealy. Blood glucose was measured on day 3 by GOD-POD method. Rats having fasting blood glucose >250 mg/dl were further selected for study. Four groups were created i.e. Control, Control+Streptozotocin, Streptozotocin+ Metformin(75mg/kg) and Streptozotocin+ extract of P. niruri (250mg/kg). Each group was consisting of 6 rats of either sex. Metformin and experimentalextract were administered for 21 days. Blood Glucose was measured on day 7 and 21. Triglyceride, Cholesterol and Leptin level were also measure by commercially available kit. Anti-oxidant potential was assessed by estimating extent of Lipid peroxidation (LPO) by Malondialdehyde (MDA), Nitric oxide (NO), Superoxide dismutase (SOD) and Glutathione (GSH) in four different tissues i.e. Liver, Kidney, Pancreas, Muscle on day 21. Unpaired and paired student’s t-test were applied for statistical analysis using SPSS Software. The extract of P. niruri showed significant decrease in blood glucose level on day 21 (p-0.04). The treatment didn’t show significant lowering of Leptin and Cholesterol level however Triglyceride level was significantly reduced (p-0.05). The treatment group showed improvement in oxidative stress by increasing SOD and GSH and decreasing LPO and NO activity. The study showed anti-hyperglycemic and anti-oxidative properties of methanolic extract of P.niruri.

Keywords

Anti-hyperglycemic; Anti-oxidant; Phyllanthus niruri

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Copy the following to cite this article: Kumar A, Rana A. K, Singh A, Singh A. Effect of Methanolic Extract of Phyllanthus niruri on Leptin Level in Animal Model of Diabetes Mellitus. Biomed Pharmacol J 2019;12(1).

Copy the following to cite this URL: Kumar A, Rana A. K, Singh A, Singh A. Effect of Methanolic Extract of Phyllanthus niruri on Leptin Level in Animal Model of Diabetes Mellitus. Biomed Pharmacol J 2019;12(1). Available from: https://bit.ly/2V0CN1R

Introduction

Diabetes Mellitus (DM) is a major metabolic disorder involving glucose and lipid metabolism and is characterized by hyperglycemia. Diabetes Mellitus is among commonest non-communicable diseases and most of countries are facing its havoc. Latest data suggests that the worldwide prevalence of diabetes is on rise affecting 382 million people in 2013 and likely to affect 592 million people by year 2035¹ and India is also facing similar situation and over 62 million people living with diabetes.² Diabetic patients may have absolute or relative deficiency of insulin (Type1 DM) or tissue resistance to insulin (Type 2 DM). Type 2 DM accounts for the maximum number of cases (90%)³ and monogenic forms are responsible for 5% cases of DM.⁴ Diabetes causes secondary pathophysiologic changes in different organ systems leading to micro-vascular and macro vascular complications and increasing co-morbidities. There are seven different class of drugs which are currently being used in management of Diabetes Mellitus but effective management of complications remains a problem.⁵ Uncontrolled DM along with overproduction of reactive oxygen species (ROS), and decreased antioxidant enzymatic pathways are contributory to various complications of DM.⁶ Important ROS are superoxide, hydrogen peroxide, hydroxyl radicals, and hypochlorous acid. These are generated through several metabolic processes and scavenged by efficient anti-oxidant system so that accumulation of ROS doesn’t occur. Important antioxidant enzymes are Superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and reduced glutathione (GSH). Any mismatch in the action of these enzymes normally leads to faulty disposal of free radicals and its accumulation. The ROS are responsible for the oxidation of tissues leading to lipid peroxidation and damage to cell. Role of free radicals and oxidative stress in diabetes mellitus is well documented.⁷ Free radicals damage cellular membranes by releasing intracellular components like lysosomal enzymes, which lead to further tissue damage. These radicals promote lipid peroxidation and membrane damage by cross-linking proteins, lipids and nucleic acids.⁸

Currently the drugs available to manage DM are hypoglycemic agents which primarily reduce blood glucose level. They offer very less advantage to correct other pathological aspects of DM. Plants can be source of agents which not only can reduce blood glucose level but also show benefit in correction of pathological aspects of DM. Numerous plants have shown anti-hyperglycemic potential in animal model of DM.⁹ Phyllanthus niruri, commonly known as ‘stone breaker’, belongs to family Euphorbiaceae is an indigenous plant. The plant is of medicinal importance for numerous ailments like renal stones, dyspepsia, hepatotoxicity, antihyperlglycemic.¹⁰ Systemic studies for anti-hyperglycemic and antioxidant potential are less, in the present study medicinal plant Phyllanthus nirurui is selected for hypoglycemic and anti-oxidative property.

Streptozotocin (STZ) can be used to induce both Type 1 and Type 2 DM.¹¹ It is taken up by beta cell of pancreas by GLUT2.¹² Damage to beta cell is mediated by alkylation of DNA. STZ is also an NO donor which also contribute to its cytotoxic action.¹³ Free radicals generated by STZ is supposed to be ultimate cause of beta cell damage.

Leptin is primarily produced in adipose tissue and its level reflects energy status and it has got significant role in energy homeostasis, glucose and lipid metabolism.¹⁴ Leptin is considered to act on central pathways and affect peripheral insulin sensitivity which are independent of its effects on food intake and weight.¹⁵ Effect of plant extracts on leptin level will also be assessed in present work.

Material and Methods

Plant Material and Extract Preparation

The dried aerial part of Phyllanthus niruri was procured from the botanical garden of Banaras Hindu University (BHU), Varanasi and identified with the department of Dravyaguna, Institute of Medical Sciences, BHU. The dried aerial part of Phyllanthus niruri of 500 grams was crushed into small pieces and prepared by adding sufficient amount of methanol in a glass jar for 72 hours and then filtered off. The methanolic extract of Phyllanthus niruri was vacuum dried and stored at -20°C until further use.

Animals

Inbred Charles-Foster (CF) albino rats (150-200 g) of either sex, were obtained from the central animal house of Institute of Medical Sciences, Banaras Hindu University,Varanasi. They were kept in the departmental animal house at 26 ± 2°C and relative humidity 44-56%, light and dark cycles of 10 and 14 h respectively for 1 week before and during the experiments. Animals were provided with standard rodent pellet diet and the food was withdrawn 18-24 h before the experiment though water was allowed ad libitum. Approval from the Institutional Animal Ethical Committee was taken prior to the experimental work.

Drug Treatment

A total of 32 rats were obtained from Central Animal House and 24 rats were chosen for study divided into 6 rats per group. Diabetes was induced in adult rats weighing between 150-200 g by a single intra-peritoneal injection of streptozocin (STZ, 60 mg/kg) dissolved in citrate buffer (pH 4.5) and fed normally thereafter.¹⁶ On day 3 the fasting plasma glucose was assessed to validate the model. Details of groups are as under:

Group A- Control: The control rats received citrate buffer only as per their body weight.

Group B- Streptozotocin with no Treatment: In this group Streptozotocin was given and followed for 21 days without any other treatment.

Group C- Standard: This group received Metformin (75 mg/kg) suspended in 1% carboxymethyl cellulose (CMC) in distilled water.

Group D- Treatment Group: This group received extract of Phyllanthus niruri (250 mg/kg) orally once daily for 21 days.

Glycemic Study

Fasting plasma glucose levels were estimated on day 7 and day 21 after extract administration. Blood samples were collected from the retro-orbital plexus. Rats showing fasting plasma glucose over 250 mg/dL under fasting conditions were used for the study. Plasma glucose was estimated by GOD-POD method.

Estimation of Triglycerides and Cholesterol

Blood samples were collected from the retro-orbital plexus of the rat and serum was separated by centrifugation at 3000 rpm for 3 minutes. Triglycerides level was estimated in the unhemolysed serum by (GPO-PAP Method) using Triglycerides Test Kit (Coral Clinical System.) Total cholesterol levels were estimated in the unhemolysed serum by (CHOD-PAP Method) using Cholesterol Kit (Span Diagnostics Pvt. Ltd. India).

Estimation of free radicals and anti-oxidants

Test and standard drugs were given orally daily for 21 days and on the day of the experiment the animals were then sacrificed and various parameters was calculated in tissue and serum as described earlier. Liver, pancreas, kidney and muscle tissue were homogenized (5%) in ice cold 0.9 % saline with a Potter – Elvehjem glass homogenizer for 30 sec. The homogenate was then centrifuged at 800xg for 10 min followed by centrifugation of the supernatant at 12,000-x g for 15 min and the obtained supernatant was used for the following estimations.¹⁷

Antioxidants viz. superoxide dismutase SOD (nmol/g wet tissue)¹⁸ and reduced glutathione GSH (nmol/g wet tissue)¹⁹ and free radicals viz. extent of lipid peroxidation LPO by estimating MDA (nmol/g wet tissue)¹⁸ and nitric oxide NO (mmol/g wet tissue)²⁰ were estimated in homogenate.

Estimation of Leptin

Leptin was estimated in serum by commercially available ELISA kit. The kit was obtained from Kinesis Dx catalog no. K11-0562.

Statistical Analysis

The statistical analysis was carried out using paired and unpaired t test. The values are represented as Mean ± SD. P <0.05 was considered significant.

Results

The study was conducted in department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University. The results are as under:

In Group A all animals completed study. In Group B, 3 animals died during the study and rest completed the study. In Group C, 2 animals didn’t develop required blood glucose level and hence excluded from study. In Group D, one animal died and one didn’t develop required blood glucose so excluded from study.

After Streptozotocin administration, on day 3 the blood glucose level was markedly increased (274±11.9 mg/dl) as compare to control (99.7±17.7 mg/dl). Group D i.e. treatment group showed a decrease in blood glucose level by 2.9% at day 7 and 49.7% at day 21. Group C i.e. Standard showed a decrease in blood glucose level by 50.27% at day 21. (Table 1). The extract of Phyllanthus niruri showed statically significant decrease in blood glucose level at day 21 (p – 0.041). At day 7 plant was showing slight decrease in blood glucose level which was not statistically significant.

 

Table 1: Effect on Blood Glucose Level (mg/dl).

Day	Group A	Group B	Group C	Group D
0	96.5±18.97	104.66±12.83	93.33±16.23	89.16±13.76
3rd	99.66±17.66	274±11.93	277.83±9.08	276.33±113.62
7th	97.66±6.74	279.83±12.62	202.5±3.78	268.5±113.21
21th	97.16±6.73	285.66±11.20	138.16±6.31	139±10.37

 

On day 21, blood Cholesterol and Triglycerides level were assessed. In Group B blood cholesterol and triglyceride level were higher than Group A which was control group indicating deranged lipid profile There was significant difference in blood triglyceride level between Group B and Group D. In Group D, the level was significantly less as compared to Group B (p value 0.05). The result shows Group D was having better Triglyceride Profile than Group B. Group B and Group D were having similar cholesterol level (p value 0.36). It seems that extract of P. niruri didn’t offer any change in Cholesterol level (Table 2).

 

Table 2: Effect on Plasma Triglycerides and Cholesterols Level and Serum Leptin on Day 21.

Parameters	Group A	Group B	Group C	Group D
Leptin (ng/ml)	4.5±0.14	4.6±0.11	4.4 ±0.15	4.5±0.15
Triglycerides(mg/dl)	74.33±1.87	97.5±4.92	81.83 ± 5.7	81.66±8.26
Cholesterols (mg/dl)	108± 6.72	142.6±10.63	117.5 ± 8.96	147.3±8.73

 

On day 21, blood Leptin level was assessed. In Group B blood leptin level was higher than Group A (Table 2). Group D showed decrease in leptin level, as compared to Group B which was not statistically significant (p value 0.18).

On day 21, markers of oxidative stress i.e. extent of Lipid Peroxidation (LPO) and Nitric Oxide (NO) level were assessed in four different tissues homogenate i.e Liver, Kidney, Pancreas, Muscle. Both are indicators of oxidative stress. In Group B, the level of both were increased on day 21 compared to Group A in all tissue samples showing increase in oxidative stress.

The extent of LPO level significantly decreased in Group D as compared to Group B in tissue sample of Kidney, Pancreas and Muscle having p value 0.026, 0.017 and 0.046 respectively. (Table 3)

The NO level significantly decreased in Group D as compared to Group B in tissue sample of Kidney, Pancreas and muscle having p value 0.004, 0.017 and 0.002 respectively. (Table 3).

 

Table 3: Effect on Lipid Peroxidation (nmol/g wet tissue) and Nitric Oxide ( mmol/g wet tissue) on Day 21.

Groups & Tissue Homogenate Lipid Peroxidation	Group A	Group B	Group C	Group D	P – value
Liver	217.5± 27.02	196 ±20.89	218.66 ±28.73	211.5±20.44	0.285
Kidney	196 ± 11.11	158.66 ± 26.25	187.5 ± 12.38	192±13.99	0.026*
Pancreas	216.5 ± 16.63	179.16 ± 16.63	220.16 ± 19.27	195.5±12.83	0.017*
Muscle	187.33 ± 9.77	132.33 ± 21.33	190 ± 12.21	173.33±10.50	0.046*
Groups & Tissue Homogenate Nitric Oxide
Liver	232±6.75	197.5±7.23	216.5±4.08	204.5± 5.61	0.092
Kidney	202.83±5.84	167.5±7.44	184.66±9.09	183.16 ± 7.08	0.004*
Pancreas	199.33±7.04	157.33±14.06	190.16±9.19	184.16 ± 11.7	0.017*
Muscle	198.33±8.21	164±8.69	187.5±9.37	184± 8.24	0.002*

 

On day 21, anti-oxidants i.e. Glutathione (GSH) and Super-oxide Dismutase (SOD) level were assessed in four different tissues homogenate i.e Liver, Kidney, Pancreas, Muscle. Both are part of inherent defense mechanism against oxidative stress. In Group B, the level of both these anti-oxidants were reduced on day 21 compared to Group A in all tissue samples showing decreased defense against oxidative stress.

The SOD level significantly increased in Group D as compared to Group B in tissue sample of Kidney and Pancreas having p value 0.012 and 0.032respectively. (Table 4).

The GSH level significantly increased in Group D as compared to Group B in tissue sample of Liver, Kidney and Pancreas having p value 0.02, 0.024 and 0.01 respectively. (Table 4).

Table 4: Effect on Superoxide Dismutase ( units/ g wet tissue) and Glutathione (mmol/g wet tissue)  level on Day 21.

Groups & Tissue Homogenate Superoxide Dismutase (units/ g wet tissue)	Group A	Group B	Group C	Group D	P – value
Liver	35.61±4.3	43.08±10.63	39.66±3.2	44.16±3.3	0.332
Kidney	54.7±1.31	58.08±1.31	56.78±2.79	55.38±1.4	0.012*
Pancreas	49.41±4.5	54.91±3.05	50.33±3.1	50.41±3.1	0.032*
Muscle	45.33±2.9	49.66±3.5	46.91±2.9	52.16±3.9	0.295
Groups & Tissue Homogenate Glutathione (mmol/g wet tissue)
Liver	5.7±0.91	7.5±0.53	6.4±0.73	6.28±0.49	0.020*
Kidney	7±0.50	10.4±0.72	8.33±0.56	9.23±0.82	0.024*
Pancreas	7.15±0.70	8.71±.94	7.5±0.89	7.4±0.70	0.010*
Muscle	7.61±.66	9.65±1.02	8.41±1.22	9.33±1.17	.631

 

Discussion

In the present study, diabetes in rats was induced by Streptozotocin and we selected the Phyllanthus niruri commonly known as stone breaker. The study includes the status of various diabetic parameters in streptozotocin-induced diabetic rats. The present study was therefore intended to examine the effect of methanolic dried aerial part of P. niruri  streptozocin-induced diabetic rats, using metformin as a reference drug.

niruri, belongs to family Euphorbiaceae, an indigenous antidiabetic plant popularly used in South India for diabetes mellitus. Hypoglycemic effect P. niruri is documented.²¹ Our present result with P. niruri also showed significant antihyperglycemic effect in diabetic rats after 21 days of treatment, after seven days of treatment the blood glucose was reduced but the effect was not statistically significant. Significant decrease in fasting plasma glucose after 21 days may indicate that P.niruri can be beneficial on prolonged use, however the decrease in fasting plasma glucose started from after 7 days itself. This may have insulinomimetic activity or may affect insulin resistance also.²²

Group B rats exhibited increases in plasma Cholesterol and Triglycerides level. Diabetes is known to associated with increased plasma and cholesterol and triglyceride levels.²³ Currently, P.niruri extract administration led to significant decline in triglyceride level. The significant decreases in triglycerides is similar to the result reported earlier in hyper lipemic rats treated with Phyllanthus niruri extract.²⁴ We did not find significant reduction in plasma cholesterol level which is similar to previous study.²² This finding suggests that the plant extract may have activity like fibrates class of drug which act via PPAR-g and indicated for treatment of hypertriglycridemia.²⁵

Sustained hyperglycaemia leads to increased lipid peroxidation, production of superoxide, and resulting in oxidative stress.^26,27 GSH and SOD are important endogenous anti-oxidative defence against oxidative stress.⁸ SOD is a free radical scavenger which protects cells from oxidative damage.²⁸ Group B animals were found to be have lower level of GSH and SOD on day 21 in all the tissue homogenate i.e Liver, Kidney, Pancreas, Muscle compared to Group A. The treatment group i.e. Group D was found to have statistically significant higher level of GSH and SOD in all tissue homogenate of kidney, pancreas and showing anti-oxidant effects. These finding are similar to results reported by authors.^29,21 All tissue homogenate of Group B was having higher concentration than Group A. Kidney, Pancreas and muscle homogenate showed significant reduction in level indicating less oxidative damage. These finding are similar to results reported by authors.^29,21 Anti-oxidant activity in Kidney and liver supports its activity as stone-breaker and prevention of hepatotoxicity.^30,31 Group D showed decrease in Serum Leptin level, although the result was not statistically significant but it was following similar trend reported earlier in study.²² Decrease in Leptin level can be corelated with decrease in insulin resistance as both are positively corelated. On long term use the extract can be effective in reversing insulin resistance.

Conclusion

In light of the present results, our work indicates that methanolic extracts of P.niruri have good anti-diabetic activity. Our study showed significant improvement in anti-oxidant parameters i.e. increase in GSH & SOD while decrease in NO, LPO in various tissue homogenate (Muscle, kidney, Liver and Pancreas) after 21 days of administration of extract of P.niruri indicating its anti-oxidant potential. Downward trend in Leptin level may be correlated with decrease in insulin resistance.

Acknowledgements

The author(s) received no specific funding for this work.

Conflict of Interest

There is no conflict of interest.

Funding Source

Intramural grant of Banaras Hindu University.

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