INTRODUCTION:

Diabetes mellitus is defined as a metabolic disorder manifested by augmentation in blood glucose level or hyperglycaemia. It is caused by complete or virtual deficiency of insulin, frequently resistance of insulin release¹. Persistent hyperglycaemia causes abiding deterioration, damage, resistance dysregulation in physiological function and ultimately failure of organs, mainly the kidney, nerves, eyes and cardiovascular system². It was reported that in 2017 there are 451 million people with diabetes worldwide. These data forecast to increases by 2045 to 693 million^3,4. In most of Western countries, persistent hyperglycaemia leads to nephropathy and renal damage, about 20-30% of all diabetes case was suggested to develop evidence of diabetic nephropathy apparent from microalbuminuria to macroalbuminuria and cause renal failure⁵. Therefore, uncontrolled diabetes mellitus is the prime factor in induction and propagation of nephropathy. In diabetic nephropathy disrupted pathophysiological mechanism in renal alteration including podocyte loss, thickening of Glomerular basement membrane, interstitial fibrosis, mesangial cell expansion, glomerulosclerosis and tubular atrophy^6-11.

During diabetic nephropathy these renal change result in albuminuria, raise blood urea nitrogen and serum creatinine levels and diminish in GFR^12-16. Thus, interruption of the process of diabetic nephropathy would manage and improve the health of many Diabetes Mellitus patients. Several research studies have shown that superior complication control is helpful in slowing the development of nephropathy in diabetes,^17,18. Although, a great extent of research work shows that, kidney is damage quickly in diabetic case. Patients with diabetes kidney failure undergo either kidney transplantation or painful dialysis both of them are harmful as well as economically worst ^19.

At present, an assortment of drugs/medicine accessible for management of diabetic nephropathy but mostly are allopathic medicine and multiple drug therapy having adverse effects. Instead of allopathic medicine, herbal medicine having less adverse effects. Recently a lot of research on herbal based drugs, Calotropis gigantea exhibit many beneficial medical properties including anti-diabetic activity. Calotropis gigantea flowers were massively used by Vaids/Ayurvedic practitioners for management of diseases listed as bronchial asthma, diabetes mellitus, nervous disorders and arthritis^20,21. Calotropis gigantea chloroform extract of flowers and leaves halts insulin induced resistance in high fructose diet²². Calotropis gigantea has rich in reported Phytoconstituents including proteases, 3'-methylbutanoates of amyrin²³, flavanol glycosides²⁴, calotropins²⁵, stigmasterol and sitosterols²⁶, cardenolides²⁷, and pregnanone²⁸. Roots of Calotropis gigantea extremely used in treatment of eczema, ulceration, syphilis, leprosy, antidiarrhoeal, and respiratory disorders in the Indian Traditional system of medicine²⁹. Antipyretic and analgesic activities ³⁰ of the alcoholic extract of root peels of Calotropis gigantea were identified and reported in mice^31,32. The C. gigantea fresh milky juice reported as a gastrointestinal irritant, violent purgative and also being used for inducing abortion ³³. The latex extract showed proteolytic activity having ability to being hydrolyze casein, in a dose dependent mien³⁴. It has also been reported for Antiamoebic³⁵, wound healing^36,37, hepatoprotective³⁸, and antioxidant^39,40Anti-inflammatory⁴¹, Anticoagulant⁴², Antifertility⁴³. Therefore, plan of the recent study was map out to investigate possible nephroprotective effect of Calotropis gigantea using stem bark extract of C. gigantea in streptozotocin-induced diabetic nephropathy in rats. The images of the plant parts are shown in Figure A.

Figure A: Calotropis gigantea

MATERIAL AND METHODS:

Chemicals and Reagents:

Streptozotocin (STZ) (MP Biomedicals, India), ethanol, sulphanilamide, butanol, citric acid, petroleum ether, ethylene diamine tetra acetic (EDTA), Ellman’s reagent, ferric chloride, glacial acetic acid, sodium, acarbose (SRL, research laboratories Pvt. Ltd. New Delhi, India), Metformin (hydrochloride), NADPH, napthylethylene -diamine, p-nitrophenyl-α-D glucopyranoside pyridine (pNGP), potassium dihydrogen phosphate, potassium ferricyanide, sodium nitroprusside, sodium hydroxide, sodium lauryl sulphate, soluble starch and iodine (HI Media, Laboratories Pvt. Ltd, Mumbai, India) and all other chemicals used are analytical grade.

Collection and authentication of plant:

The whole plant was collected from the local area of Hisar, Haryana during April, 2019 and a voucher specimen was deposited and authenticated by Dr S.S. Yadav, Assistant Professor, Department of botany, Maharshi Dayanand University Rohtak, Haryana.

Preparation of extract:

Fresh stem bark of Calotropis gigantea were collected and washed properly with tap water followed by distilled water, then shade dried at room temperature. Bark was powdered using a mechanical grinder. The bark powder was extracted in ethanol using Soxhlet assembly.

Experimental animals:

Healthy Wistar albino rats (150-200g) of either sex were acquired from diseases free small animal house, Lala Lajpat Rai University of Veterinary and Animal science Hisar (Haryana) for evaluation of anti-hyperglycemic and associated diabetic nephropathy activity of stem bark extract of Calotropis gigantea. Experimental protocol was approved by the Institutional Animals Ethics Committee in its meeting was held on Oct, 2017 (CPCSEA Registration No.436/GO/ReBi/S/2001).

Development of Diabetes Mellitus (DM) Type II in rats: Induction of experimental DM in overnight fasted rats by administration of single dose Streptozotocin (60 mg/kg, i.p.) added in 0.1 M cold citrate buffer having 4.5 pH. After 6 h of STZ administration rats received 10% dextrose solution for next 24 h. The blood glucose level was checked after 72 hours of Streptozotocin administration by using automatic glucose analyzer (Gluco Dr. Auto Glucometer) and serum glucose level more than 250 mg/dl were selected for study and considered as diabetic rats.

Assessment of diabetic nephropathy:

The Nephropathic complication develops, after 4 weeks the single administration (60mg/kg) of Streptozotocin, was evaluated in rats by measuring serum creatinine, uric acid and urea level by using commercially accessible kits.

Experimental groups of animal study:

Group for Diabetes Mellitus Type 2 (Streptozotocin induced Diabetes)

Group 1: Normal control rats.

Group 2: STZ+NA (Diabetic control).

Group 3: STZ+NA+ Metformin (50mg/kg) were administered to the diabetic rats (Standard).

Group 4: Extracts of Calotropis gigantea (200 mg/kg, p.o).

Group 5: Extracts of Calotropis gigantea (500 mg/kg, p.o).

Sample collection:

Blood sample: After 56^th days the blood (2 ml) was collected from retro orbital method and serum separated by blood, centrifuge for 10 min at 3000 rpm. This obtained serum was used for the estimation of bio-chemical parameters such as glucose, lipid profile, protein, creatinine, urea and uric acid.

Organs collection and storage:

The animals were sacrificed under mild anesthesia after collection of blood from intra-ocular vein by cervical dislocation technique. The kidney sample was washed in normal saline and stored in 10% formalin for further use.

Estimation of parameters:

I. Glucose estimation: The blood glucose level was checked after 72 hours of Streptozotocin administration. Blood sample was collected from pricking of tail by hypodermic sterile needle.

II. Serum creatinine level estimation:

Estimation of serum creatinine level by using available kit by using Alkaline picrate Method (Angstrom Biotech, Pvt. Ltd., Gujrat India) (Table 1).

III. Blood urea nitrogen level:

Blood urea nitrogen level calculated by marketable accessible kit using Mod. Berthelot (Reckon Diagnostics, Pvt. Ltd., Gujrat, India) (Table 1).

IV. Urine protein estimation:

Urine protein was estimated by available diagnostic kit Crest biosystems, Goa, India.

Method: Pyrogallol red (Table 1). ^{3,5,29,30,44,64}

Table 1: Indicates the various parameters with method

S. No.	Parameter	Methods	Colored Complex	Absorbance
I	Serum Creatinine	By Alkaline picrate	+	510
II	Blood Urea nitrogen	By Berthelot	+	578
III	Urine Protein	By Pyrogallol red method	+	600

Present = +

Lipid Profile Estimation:

Estimation of lipid profile by calculated the concentration of low density lipoproteins (LDL), High density lipoproteins (HDL), Total cholesterol (TC) level in the serum. The concentration was estimated by accessible diagnostic kits (Erba Diagnostic, Baddi, H.P).

Renal Oxidative Stress:

Renal oxidative stress was estimating by thio-barbituric acid reactive method (TBAR), reduced glutathione method (GSH), superoxide dismutase (SOD) and Catalase (Table 2).

Table 2: Indicates the various oxidative stress estimating parameters with method

S. No.	Parameter	Method	Absorbance
1	Renal oxidative stress	Renal TBARS	532
2		GSH	412
3		Catalase	620
4		SOD	560

Statistical analysis:

All values were expressed as mean ± S.D. The result obtained from different group of animals were statistically analyze by using one way ANOVA, followed by Turkey’s multiple comparison test. The ‘p’ value of less than 0.05 was considered statistically significant and the ‘p’ values were of two tailed.

RESULTS:

In the current research study administration of CG (500 mg/kg) and CG (200 mg/kg) daily p.o., after 4 weeks produce prominent effect on various experimental parameters performed in STZ induced diabetes nephropathy in rats as compared to normal group rats. The rats having blood glucose level 250 mg/dL or more than after 72 hrs are considered as diabetic rats and admitted in the study. Treatment with CG (200 mg/kg/daily p.o) and CG (500 mg/kg/daily p.o) were started after 4 weeks of STZ administration and the treatments were continued for 8 weeks. All the experimental parameters were performed at the completion of 8th weeks of study design.

Effect of Calotropis gigantea stem bark extract on serum glucose level:

Administration of CG 500 mg/kg to diabetic rats significantly (p<0.0001) lower the blood glucose level as compared to normal control. But as compared to diabetic control group, CG 500 mg/kg significantly (p<0.0001) decreased the blood glucose level. CG 200 mg/kg did not significantly lower the blood glucose level as in comparison to diabetes control rats (Table 3).

Effect of Calotropis gigantea stem bark extract on serum creatinine level and blood urea nitrogen level: In comparison to normal rats the level of serum creatinine and blood urea nitrogen were found to be higher in diabetic group rats. Treatment with (CG 200 mg/kg) and (CG 500 mg/kg) decreased the diabetes induced serum creatinine, blood urea nitrogen level as compared to normal group rats. CG 500 mg/kg shows significantly (p<0.0001) results as compared to CG 200 mg/kg. (Table 3)

Effect of Calotropis gigantea extract on serum protein:

An enhanced level of micoproteinuria was found in diabetic rats group as compared to normal rats group. CG 500 mg/kg and metformin significantly decreased the diabetes induced micorpoteinuria. But CG 200 mg did not significantly reduced the diabetes induced microproteinuria in diabetic rats (Table 3). Moreover, in diabetic rats, generation of various renal pathological disorientation including microvascular condensation, Glomerular wall distortion, mesangial, tubular damage, cell expansion.

Effect of Calotropis gigantea stem bark extract on Lipid profile:

Table 3 shown that significant (p<0.0001) increased level of serum triglyceride (TG), total cholesterol and low density lipoprotein (LDL) further subsequent decline in HDL cholesterol level were found in diabetic rats as compared to lipid profile of normal rats. Treatment with CG 500 mg/kg, CG 200 mg/kg decreased the diabetes-induced elevated serum triglyceride, total cholesterol and low density lipoprotein significantly. As compared to 200 mg/kg, 500 mg/kg showed significant result (p<0.0001) in enhance the HDL level in the diabetic rats.

Antioxidant activity of Calotropis gigantea stem bark extract on renal TBARS, GSH and Catalase level in STZ induced diabetic nephropathy: The current study indicates that renoprotective effect of Calotropis gigantea (CG) in STZ induced diabetes nephropathy. An elevated level of renal TBARS and decrease level of GSH, catalase and SOD was noted in diabetic rats in comparison to normal group rats. CG 200 mg/kg, CG 500 mg/kg and metformin significantly (p<0.0001) decreased the renal TBARS level, elevated the GSH, catalse and SOD level as compared to the normal rats as show in Figures 1-4 respectively. This indicated that’s CG shows antioxidants activity.

Fig. 1: Effect of different treatment on renal TBARS in STZ induced diabetic Nephropathy

Fig. 2: Effect of different treatment on reduced glutathione level in STZ induced diabetic nephropathy

Fig. 3: Effect of different treatment on Catalase level in STZ induced diabetic nephropathy

Fig. 4: Effect of different treatment on Superoxide dismutase level in STZ induced diabetic nephropathy

Table 3: Effect of different treatment on various parameters in STZ-induced diabetic nephropathy.

Parameters	Different Experimental Groups
Parameters	Normal Control	Diabetic Control	Metformin	CG 200	CG 500
Blood glucose (mg/dl)	102.7± 3.98	287.8± 6.14^a	118.7± 4.32^***###	248.2± 6.43^***	206± 15.42^***###
Serum creatinine (mg/dl)	0.75± 0.02	1.96± 0.05^***	1.02± 0.02^###	1.55± 0.07^***	1.35± 0.04^***###
Protein in urine (mg/dl)	5.03± 0.302	34.95± 0.76^***	7.42± 0.72^###	25.24± 0.95^***	21.91± 0.68^***###
Blood urea nitrogen (mg/dl)	10.51± 0.56	35.93± 0.38^***	17.79± 0.34^***###	29.98± 0.75^***###	25.99± 0.78^***###
TG (mg/dl)	147.7± 1.46	308.0± 1.16^***	181.5± 0.861^*###	262.1± 5.19^***###	230.3± 2.25^***###
TC (mg/dl)	52.42± 1.35	105.8± 1.234^***	54.23± 1.199^###	90.09± 0.50^***####	79.49± 1.32^***###
LDL	59.39± 2.93	156.2± 2.58^***	76.22± 1.08^***###	124.4± 0.69^***###	101.8± 3.17^***###
HDL (mg/dl)	49.57± 0.88	32.54± 0.90^***	43.77± 0.50^**###	35.87± 0.40^***##	39.53± 0.58^***###

Data is expressed as Mean ± SEM and analysed by using One Way ANOVA followed by Turkey’s test. *** P<0.0001, **P<0.001 and *P<0.05 as compared to normal control. ^###P<0.0001,^##P<0.001 and ^#P<0.05 as compared to diabetic rats. NC- Normal control, DC- Diabetic control, CG- Calotropis gigantea, TG-Triglyceride, TC- Total cholesterol, LDL- low density lipoprotein, HDL- High density lipoprotein.

DISCUSSION:

Current research study shows the nephroprotective effect of stem bark extract of Calotropis gigantea in experimentally induced type II diabetes in rats amid the outset of nephropathy. We found out that treatment with either Calotropis gigantea 500 mg/kg shows prominent effect in prevention and progression of experimentally induced diabetic nephropathy in the diabetic rat. The increased levels of blood urea nitrogen and serum creatinine are considered as prime marker of kidney dysfunction^45,46,47. Other parameter including proteinuria that may associate with the development of tubular structural disorientation and glomerulosclerosis⁴⁸. In the study, as compared to normal rats a discernible augmentation in blood urea nitrogen and serum creatinine was found in diabetic rats after STZ administration. It is the preferable sign of the progression of renal functional and physiological abnormalities. The obtained results strongly recommended the incidence and progression of diabetic nephropathy. In current research study, Calotropis gigantea at different doses barred the increased levels of blood urea nitrogen, serum creatinine and it may recommended the protective effect of this herbal drug extract in preventing the foregoing renal structural abnormalities and physiological functions in rats having diabetic nephropathy. Treatment with Calotropis gigantea affects the elevated level of serum glucose in diabetic rats. It is signifies that defensive effect of Calotropis gigantea in halting the incidence of diabetic nephropathy is regarded to their direct nephroprotective action in diabetic rats.

The diabetes mellitus-associated increased lipid level is also an individualistic risk factor in the pathogenesis of diabetic nephropathy^49-52. The abnormal regulation of lipid absorption leads to increases the value of LDL, TG, TC and decreases the HDL value in the systemic circulation. The preferment of free lipids circulation might lead to pathogenesis of renal disease^{8, 53, 54}. In this study marked decrease in HDL level while low density lipoprotein, triglyceride level and serum total cholesterol, and was noted to be markedly high in diabetic rats after STZ administration. These consequences further considerable that diabetes associated lipid profile alteration impart into the pathogenesis of renal disease. Treatment with Calotropis gigantea 500 mg/kg significantly impaired the diabetes mellitus induced augmentation in low density lipoprotein, serum total cholesterol and triglyceride level in diabetic rats. Thus it might be certainly recommended that prominent protective result of Calotropis gigantea 500 mg/kg against the commencement of nephropathy complication in diabetic rats might be due to efficiently decreases the level of circulating lipids. Numerous clinical trial studies indicated that the nephroprotective effect of various drugs in diabetic patients, but the therapies showed various complications^55-59.

The present research study demonstrated that treatment with Calotropis gigantea 500 mg/kg effective in prevention the diabetes mellitus-induced nephropathy. Persistent high glucose level leads to oxidative stress further cause deterioration of the structural and physiological functions of the kidney⁶⁰. The augmented level in TBARS and decreased in GSH, catalase and SOD are considered as major index of the development of oxidative stress^61-64. Current research design shows that diabetic rat after STZ administration increases in the renal TBARS and a marked reduces in renal GSH, SOD and catalase. The obtained results firmly suggested that renal oxidative stress could contribute pivotal role in renal structural and functional abnormalities in diabetic rats. Treatment with Calotropis gigantea 500 mg/kg prominently decreases the diabetes-induced oxidative stress. However, Calotropis gigantea 200 mg/kg did not develop any effective and significant role in lowering the oxidative stress of kidney of diabetic rats. From present study, it may be accomplished that the diabetes-induced renal lipid alteration and oxidative stress, along with hyperglycemia, play a crucial role in the generation of nephropathy complication in diabetic rats. Herbal medicine put a valuable effect in management and treatment of diabetic complications. Sole therapy of modern allopathic and herbal drugs are in the management of diabetes is not up to mark.

CONCLUSION:

The obtained result showed that extracts at different doses Calotropis gigantea shows nephroprotective competence. The combination of modern medicine with Ayurvedic herbal medicine put an ultimate role in prevention the diabetes complications and other ailments. Herbal formulation and preparation shows tremendous pharmacological activities. Ayurvedic herbs/herbal drugs have no side effect, complicated medical occurrences and shows healthy outcomes. The herbal drugs also having economical affordable. Most of the herbal drugs are easily available, so more exploration and advanced research is needed to make a platform to find more active constituents helpful in preventing and treatment of various diseases and complications. Modern era of research is towards the development of herbal based medicine system for the betterment of health and wealth of human life.

List of abbreviations

DM TBARS	: :	Diabetes mellitus Thiobarbituric acid reactive substances
STZ	:	Streptozotocin
CG	:	Calotropis gigantea
SOD	:	Superoxidase
GSH	:	Reduced Glutathione
CPCSEA	:	Committee for the Purpose of Control and Supervision of Experiments on Animals

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE:

CPCSEA Registration No.436/GO/ReBi/S/2001 dated on Oct, 2017.

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Received on 03.02.2021 Modified on 27.03.2021

Research J. Pharm. and Tech. 2022; 15(3):1090-1096.

DOI: 10.52711/0974-360X.2022.00182