^*1Department of Biochemistry, Patna University,

²Department of Biochemistry RMRIMS, (ICMR), Patna

³Post Graduate Department of Botany, Patna University, Patna

Corresponding author email: kumud_rnj@rediffmail.com

Article Publishing History

INTRODUCTION

Type 2 Diabetes is a chronic metabolic disorder with marked hyperglycemia, reduced insulin sensitivity, increased oxidative stress and altered carbohydrate, fat, and protein metabolism. There are currently 425 million people with diabetes worldwide, and this number is expected to reach 629 million by 2045, with type 2 diabetes (T2DM) being the most expressive form of the disease Current evidence has suggested that oxidative stress due to reactive oxygen species (ROS) and nitrogen species plays an important role in the pathogenesis of chronic diseases such as type 2 Diabetes mellitus (Ahmad et al., 2017). Prolonged hyperglycemia accompanied with higher reactive oxygen species (ROS) may develop into macro and microvascular diseases which are the main causes of morbidity and mortality worldwide associated with T2DM (Goycheva et al., 2017). ROS in association with hyperglycemia enhances four major molecular pathways pertaining to tissue damage. The four major pathways are increased hexosamine flux, activation of Protein Kinase C pathways (PKC), enhanced polyol pathways and increased advanced glycation end product (AGEs) (Ahmad et al., 2017, IDF, 2017 and ADA, 2018 ).

Monosaccharide such as fructose, glucose, glyceraldehyde etc, reacts non-enzymatically with an amino group of protein, lipid, and nucleic acid to form nascent macromolecule which is termed as advanced glycation end product (AGEs), which is hardly metabolized or expelled from the body. Formation of AGE results in interaction with receptor of AGEs namely Receptor of advanced glycation end product (RAGE) which mediates activation of cytosolic intermediates for the formation of ROS (Ahmad et al., 2017). To neutralize the reactive oxygen species animal body is equipped with various types of Enzymatic (Catalase, Superoxide dismutase, GPX, GST, etc) and non-enzymatic (Ascorbate, Tocopherol, lycopene, beta carotene, glutathione, etc) antioxidant molecules. The antioxidant molecule scavenges the various types of radicals into molecular oxygen and other essential components which don’t harm to tissues (York-Duran et al., 2019).

Oxidative stress has a significant role in further complications of type 2 Diabetes Mellitus. An increase in ROS level leads to elevated or dysregulated production of antioxidants like catalase, superoxide dismutase, glutathione peroxidase, etc. The variation in the availability of the mentioned parameters pronounces the tissue susceptibility to the oxidative stress pertaining to the appearance of diabetes and its associated complications (He et al., 2017). This enhanced oxidative stress causes severe tissue damage in the liver, kidney, brain, heart, Pancreas etc, because of a lack of counterbalance between increased ROS and antioxidant. This imbalance could be nullified by an exogenous supply of anti-oxidant and hypoglycemic botanicals (Jha et al., 2016; Wilson et al., 2018 Seo et al., 2019).

The present research is aims to reveal the potential of the Gymnema sylvestre herbal extract to nullify the complication of the Diabetes. The belief for the use of plant-based product is still a primary option in Indian subcontinent. In such case phytotherapy becomes the relevant topic to probe a natural diabetic treatment naturally because it contains both active ingredient as well as anti-oxidant to cater the need to reduce glucose use to combat AGEs and its related complications (Kajal and Singh, 2018).

The desired plant, Gymnema sylvestre is a large woody climber running over the tops of high trees and belongs to the family Asclepiadaceae. The vernacular names of G. sylvestre are in English-Periploca of the woods, Hindi-Gurmar, Telugu-Podapatri, and Sanskrit- AjaboUi. The plant is an herb native to the tropical forests of southern and central India and Sri Lanka (Pham et al., 2018). Wistar norvegicus male rats were included in the experiment and diabetogenic material was Alloxan because it is widely used to create an experimental diabetic model in rats due to its selective destruction of pancreatic β cell because of its free radical production upon decomposition.

It exhibits multiphasic changes in blood glucose, insulin concentration accompanied by histo-architectural changes pertaining to necrotic cell death (Radenković et al., 2016). There is a lack of literature availability in the organ tissue oxidative stress caused due to diabetes and its amelioration by Gymnema sylvestre. The present study examines the efficacy and potential of the desired plant in reduction of oxidative stress in different organs and its hypoglycemic properties in diabetic subjects (Pham et al., 2018).

MATERIAL AND METHODS

For the present research work healthy male wistar rats (Rattus norvegicus) of weight ranging 180-200 gram were selected and provided ambient physical and physiological condition as per the standard protocol and all the experimental protocol was carried based on the guideline adopted by Mahavir Cancer Sansthan ethical comittee, Phulwari sariff Patna.

Induction of diabetes: Diabetes was induced by repeated dose of Alloxan monohydrate 100 mg/kg b.wt in cold citrate buffer bearing pH 4.5.

Plant materials: Leaves of Gymnema sylvestre: Preparation of herbal extract: Freshly harvested plant samples were washed under running tap water blotted with filter paper and was dried in the shade at room temperature. The dried plant materials i.e., leaf of Gymnema sylvestre was subjected to converted into fine powder which was then soaked with absolute ethanol and kept in dark for 48 hours. After that the entire extract of plant was filtered using Whatman filter paper till the clear material appeared. The solvent containing secondary metabolite of the plant was mounted on the vacuum rotary evaporator at 40⁰C. The extract was kept on the vacuum rotary till the thick paste appeared devoid of any solvent material. The thick paste colloidal was lyophillysed in lyophyliser (Labconco, USA). The lyophilized plant extract was stored in deep freezer at ^_80⁰ C until further test.

Sample collection: After the treatment of the extract for 10, 20, and 30 days respectively the tissues (Liver, Kidney, and heart) collected were for anti-oxidant quantification. For anti-oxidant analysis the tissue sample were subjected to preparation of post mitochondrial supernatant (PMS).

Chemicals and reagents: All the reagents were prepared in the laboratory using high grade chemical. Glucose estimation was done by GOD POD method and the estimation of the antioxidant parameters were carried out by the published standard literature like Estimation Of Catalase was done by (Sinha AK, 1972), estimation of total reduced Glutathione (GSH) (Boyne and Ellman, 1972), estimation of Ascorbic acid (Newman et al., 2000; Omaye et al., 1979), estimation of Glutathione Peroxidase (Rostruck et al., 1979), estimation of Glutathione-s-transferase by (Habig et al., 1974), and quantification of Superoxide dismutase (SOD) was done by (Marklund S and Marklund G, 1974).

RESULTS AND DISCUSSION

Recent reports of chemotherapeutics resistant in treatment of diabetes mellitus have forced the research community to look towards nature for the better remedy and hence the herbal drugs are emerging as a future hope in control of diabetes mellitus.

Table 1. Estimation of fasting plasma glucose in Gymnema sylvestre treated Diabetic rats

Fasting Plasma Glucose (mg/dl)
Control	Alloxan treated	Diabetic 10 Days GSE Treated	Diabetic 20 Days GSE Treated	Diabetic 30 Days GSE Treated
90.00±7.90*	368.0±14.40*	264.0±20.74*	196.0±11.40*	122.0±7.82*

Table represents fluctuation in the glucose concentration in the classified groups. The diabetic rats showed significant recovery after treatment with TCE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range tests compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *p<0.001.

Table 2. Estimation of Glutathione Content and Enzyme Activity of Glutathione Peroxidase (GSH-Px), Glutathione-S-transferase (GST), a Catalase in Liver PMS of Gymnema sylvestre treated diabetic wistar rat.

Enzyme activity
	Glutathione content	GSH-Px	GST	Catalase
	(μg/mL)	(nmol/NADPH oxidized/min)	(units/mg protein/min)	(mU/mg protein)
Control	218.8±12.09*	2.33±0.17*	182.0±10.37*	325.5±7.19*
Alloxan treated	77.95±5.10*	0.11±0.00*	74.10±10.39*	102.2±8.65*
Diabetic 10 Days GSE Treated	170.2±14.59 ns	0.32±0.00 ns	87.60±7.09***	140.0±11.18 ns
Diabetic 20 Days GSE Treated	86.79±9.60*	1.2±0.12*	113.6±7.40*	188.2±7.49*
Diabetic 30 Days GSE Treated	136.4±6.54*	2.11±0.17*	135.2±7.59*	221.0±49.15*

Table represents fluctuation in the mentioned parameters in the classified groups. The diabetic rats showed significant recovery after treatment with GSE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range test compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *** p<0.05, **p<0.01, *p<0.0001, Ns Non significant, >0.05.

Table 3. Estimation of Glutathione Content and Enzyme Activity of Glutathione Peroxidase (GSH-Px), Glutathione-S-transferase (GST), a Catalase in Kidney PMS of Gymnema sylvestre treated diabetic wistar rat

Enzyme activity
	Glutathione content	GSH-Px	GST	Catalase
	(μg/mL)	(nmol/NADPH oxidized/min)	(units/mg protein/min)	(mU/mg protein)
Control	140.4±7.62*	3.05±0.27*	85.88±6.42*	243.4±5.76*
Alloxan treated	72.68±10.32*	0.61±0.11*	39.84±3.70*	116.5±11.73*
Diabetic 10 Days GSE Treated	83.56±4.358***	1.30±0.21*	52.46±5.30**	141.0±8.21**
Diabetic 20 Days GSE Treated	98.30±5.03*	2.30±0.4*	61.60±6.42*	180.0±7.90*
Diabetic 30 Days GSE Treated	120.3±7.91*	2.89±0.10*	70.40±3.57*	207.0±10.37*

Table represents fluctuation in the mentioned parameters in the classified groups. The diabetic rats showed significant recovery after treatment with GSE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range test compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *** p<0.05, **p<0.01, *p<0.0001, Ns Non significant, >0.05.

Table 4.  Estimation of Glutathione Content and Enzyme Activity of Glutathione Peroxidase (GSH-Px), Glutathione-S-transferase (GST), a Catalase in Heart PMS of Gymnema sylvestre treated diabetic wistar rat

Enzyme activity
	Glutathione content	GSH-Px	GST	Catalase
	(μg/mL)	(nmol/NADPH oxidized/min)	(units/mg protein/min)	(mU/mg protein)
Control	125.9±7.590*	3.05±0.27*	85.88±6.42*	243.4±5.76*
Alloxan treated	67.45±6.028*	0.61±0.11*	39.84±3.70*	116.5±11.73*
Diabetic 10 Days GSE Treated	77.73±5.760***	1.30±0.21**	52.46±5.30***	141.0±8.21 ns
Diabetic 20 Days GSE Treated	87.21±7.900*	2.30±0.4*	61.60±6.42*	180.0±7.90***
Diabetic 30 Days GSE Treated	100.9±7.872*	2.89±0.10*	70.40±3.57*	207.0±10.37*

Table represents fluctuation in the mentioned parameters in the classified groups. The diabetic rats showed significant recovery after treatment with GSE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range test compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *** p<0.05, **p<0.01, *p<0.0001, Ns Non significant, >0.05

Table 5. Estimation of Glutathione Content and Enzyme Activity of Ascorbic Acid, Total Thiol (TSH), superoxide dismutase (SOD) in Liver PMS of Gymnema sylvestre treated diabetic wistar rat.

Enzyme activity
Ascorbic Acid		Total thiol (TSH)			SOD
(mg/dl)		(nmol/NADPHoxidized/min)			(units/mg protein/min)
Control	3.139±0.09*		11.19±0.82*	5.42±0.60*
Alloxan Treated	1.084±0.18*		2.136±0.25*	16.05±0.75*
Diabetes 10 days GSE Treated	1.646±0.16*		4.240±0.39**	13.36±0.40
Diabetes 20 days GSE Treated	2.420±0.30*		6.034±0.37*	10.84±0.59*
Diabetes 30 days GSE Treated	2.99±0.08*		8.482±1.19*	8.27±0.73*

significant recovery after treatment with GSE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range test compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *** p<0.05, **p<0.01, *p<0.0001, Ns Non significant, >0.05

Table 6. Estimation of Ascorbic Acid, Total Thiol (TSH), superoxide dismutase (SOD) in Kidney PMS of Gymnema sylvestre treated diabetic wistar rat.

Enzyme activity
Ascorbic Acid		Total thiol (TSH)		SOD
(mg/dl)		(nmol/NADPHoxidized/min)		(units/mg protein/min)
Control	3.202±0.21*		5.640±0.53*		3.66±0.52*
Alloxan Treated	0.7240±0.12*		2.438±0.22*		9.0±0.79*
Diabetes 10 days GSE Treated	1.818±0.26*		3.416±0.33*		7.23±0.49*
Diabetes 20 days GSE Treated	2.418±0.22*		3.976±0.31*		5.92±0.55*
Diabetes 30 days GSE Treated	2.864±0.13*		5.106±0.30*		4.68±0.42***

Table represents fluctuation in the mentioned parameters in the classified groups. The diabetic rats showed significant recovery after treatment with GSE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range test compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *** p<0.05, **p<0.01, *p<0.0001, Ns Non significant, >0.05

Table 7. Estimation of Ascorbic Acid, Total Thiol (TSH), superoxide dismutase (SOD) in Heart PMS of Gymnema sylvestre treated diabetic wistar rat

Enzyme activity
Ascorbic Acid		Total thiol (TSH)			SOD
(mg/dl)		(nmol/NADPHoxidized/min)			(units/mg protein/min)
Control	2.742±0.14*		5.150±0.80*	3.48±0.39*
Alloxan Treated	1.192±0.06*		0.724±0.09*	6.22±0.53*
Diabetes 10 days GSE Treated	1.872±0.09*		1.842±0.20*	5.33±0.39*
Diabetes 20 days GSE Treated	2.192±0.07*		2.280±0.21*	4.80±0.26*
Diabetes 30 days GSE Treated	2.424±0.08*		3.090±0.31*	4.17±0.32***

Table represents fluctuation in the mentioned parameters in the classified groups. The diabetic rats showed significant recovery after treatment with GSE extract for the subsequent days. Values are mean ± SD (n=5). Significant level was calculated by Tukey multiple range test compared from the entire column after the ANOVA. The level of significance represented here for diabetic VS treated group having p value *** p<0.05, **p<0.01, *p<0.0001, Ns Non significant, >0.05

Effect of Gymnema sylvestre ethanolic extract on Glutathione peroxidase (GPx), and catalase (CAT) in the Liver, Kidney and Heart in the groups: For the assessment of peroxide radical level, glutathione peroxidase and catalase quantification were performed. Lowered glutathione peroxidase and catalase level signifies high peroxide stress in Diabetic subjects due to less availability of the related enzymes (Kaskoos et al., 2015). Gymnema sylvestre significantly restored the activity of glutathione peroxidase towards normal range (liver 90%, kidney 94%, and heart 86 %.)  (Table II-IV) which was accompanied by significant recovery of catalase in the tissues after treatment of the animal with the extract of Gymnema sylvestre (liver 67.89%, kidney 85.18%, and heart 88.02%) (Table II-IV). The result obtained was as par with the findings (Porkodi et al., 2020).

Superoxide dismutase (SOD) catalyses the dismutation of superoxide anion (O^2-) into H₂O₂ and molecular oxygen (Wang et al., 2018). Diabetes rats treated with Gymnema sylvestre extract showed significant recovery (liver 65.53%, kidney 78.20%, and heart 83.45%) (Table V-VII) and this study was in accordance with the findings of (Priya et al., 2017).

Vitamin C plays a vital role against oxidative stress and helps to overcome it (Spoelstra et al., 2018). Phyto-extract administration has led to increase in tissue Ascorbate. Treatment with ethanolic extract of Gymnema sylvestre showed significant recovery in organs under investigation (liver liver 95.5%, kidney 89.37%, and heart 88.32%) (Table V-VII). Increase in vitamin C after drug administration reduced prolonged hyperglycemic induced oxidative stress and the study found itself as par with the findings of (Madani et al., 2015).

CONCLUSION

Diabetes is a multifactorial disorder which affects the individual in many ways. Nephropathy, cardiovascular complication, intestinal disorder, hepatic damage, retinopathy, and neuropathy are some of the glimpse of the Diabetes complications. And behind each disorder macromolecule glycosylation and free radical induced tissue damages are the key ingredients. The present study reveals that the Gymnema sylvestre ethanolic extract as an important herbal drug in alleviation of the fasting plasma glucose and the oxidative stress significantly. Reduction in the oxidative stress in the tissues of the liver, kidney, and heart and hypoglycemic activities pronounces the effectiveness of the herb.  With the increase in diabetes population at an alarming rate demands an urgent need for more effective research and assessment to find out the active phytochemical ingredients with exact active anti-diabetic mechanism. In this regard, a long and continuous research accompanied with large sample size, and translational study is required.

ACKNOWLEDGEMENTS

This work was carried with the help of Department of Science Technology (DST) New Delhi in the form of DST Inspire Fellowship.

Conflict of interest:The authors declare there is no any conflict of interest.

Ethical Clearance Statement: The Current Research Work Was Ethically Approved by the Institutional Review Board (IRB) of Biochemistry, of Patna University, Patna Bihar India.

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