|Year : 2019 | Volume
| Issue : 5 | Page : 330-336
Effect of a polyherbal formulation in streptozotocin-induced diabetic nephropathy in wistar rats
Kanala Somasekhar Reddy1, Akkiraju Sudheer1, Bhupalam Pradeepkumar1, Chappidi Suryaprakash Reddy2
1 Department of Pharmacology, Raghavendra Institute of Pharmaceutical Education and Research (RIPER), Anantapuramu, Andhra Pradesh, India
2 Department of Pharmaceutics, Raghavendra Institute of Pharmaceutical Education and Research (RIPER), Anantapuramu, Andhra Pradesh, India
|Date of Submission||23-Apr-2018|
|Date of Decision||11-Oct-2019|
|Date of Acceptance||12-Oct-2019|
|Date of Web Publication||26-Nov-2019|
Dr. Kanala Somasekhar Reddy
Department of Pharmacology, Raghavendra Institute of Pharmaceutical Education and Research, Chiyyedu (Post), Anantapuramu - 515 721, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
OBJECTIVES: Chronic kidney failure among people with diabetes mellitus (DM) is a burgeoning health problem that affects up to 25% of patients with type 2 DM. Current pharmacological treatment for diabetic nephropathy (DN) does not stop the attainment of renal complications. The intention of the current study was to explore the role of a polyherbal formulation (PHF) in diabetic-induced nephropathy in experimental animals.
MATERIALS AND METHODS: Diabetic rats were grouped as follows and underwent the following treatment for about 16 weeks: Group I – normal rats – no treatment, Group II – DN rats – only vehicle (p.o), and Group III and IV – DN rats – PHF orally at 250 and 500 mg/kg, respectively. After the treatment, the animals were sacrificed, and lipid, renal function, and inflammatory markers were estimated. The observed microscopic changes in kidney were analyzed.
RESULTS: Animals administered with PHF exhibited noteworthy decrease in triglycerides, total cholesterol, very low-density lipoprotein (LDL), LDL, serum creatinine, urinary protein, urinary albumin excretion rate, advanced glycation end products, type IV collagen excretion, interleukin-6, transforming growth factor-ß, and tumor necrosis factor-alpha and showed significant increase in high-density lipoprotein, urine volume, urinary urea, and urine creatinine. Histopathological examination established that administration of PHF prohibited kidney damage.
CONCLUSION: Treatment with PHF showed beneficial effect on DN which may be due to the improvement of renal function parameters and hyperlipidemic and inflammatory mediators.
Keywords: Advanced glycation end products, diabetic nephropathy, polyherbal formulation, streptozotocin
|How to cite this article:|
Reddy KS, Sudheer A, Pradeepkumar B, Reddy CS. Effect of a polyherbal formulation in streptozotocin-induced diabetic nephropathy in wistar rats. Indian J Pharmacol 2019;51:330-6
|How to cite this URL:|
Reddy KS, Sudheer A, Pradeepkumar B, Reddy CS. Effect of a polyherbal formulation in streptozotocin-induced diabetic nephropathy in wistar rats. Indian J Pharmacol [serial online] 2019 [cited 2021 Jul 31];51:330-6. Available from: https://www.ijp-online.com/text.asp?2019/51/5/330/271633
| » Introduction|| |
Diabetes mellitus (DM) is associated with hyperglycemia and is due to abnormal carbohydrate metabolism, which relates to relative or absolute impairment in insulin secretion or utilization in the body. Diabetic nephropathy (DN) is a kidney disease or damage of kidney, the exact cause of which is indefinite. However, it is assumed that abandoned elevated blood sugar and elevated blood pressure cause damage to the kidney. DN associated with morphological and ultrastructural changes within the kidney is determined by the clinical manifestation of the disease.,
To prevent diabetic complications, a number of plant extracts have been used as a single agent or else formulations because the safety profile of herbal formulations is better than that of oral hypoglycemic agents. Polyherbal formulation (PHF) is preferred to individual formulation. Still, several herbal formulations such as Cogent db, Hyponidd, Trasina, tincture of punchparna, Diasulin, and Diamed are recommended for diabetes, hyperlipidemia, and oxidative stress.Eugenia jambolana, Tinospora cordifolia, Gymnema sylvestre, Cressa cretica, Casearia esculenta, Curcuma longa, Swertia chirata, Centratherum anthelminticum, Picrorrhiza kurroa, Trigonella foenum-graecum, Terminalia chebula, Holarrhena antidysenterica, Pterocarpus marsupium, Glycyrrhiza glabra, Mineral pitch, Tribulus terrestris, Withania somnifera, Nardostachys jatamansi, and Bacopa monniera have proven antidiabetic, antihyperlipidemic, and antioxidant activities individually [Table 1].,, However, no reports are available regarding their effects synergistically in DN. On the basis of the obtained information, the current investigation has been conducted to assess the outcome of PHF on streptozotocin (STZ)-induced DN in rats.
|Table 1: Phytochemicals present in the plants of polyherbal formulation and their pharmacological properties|
Click here to view
| » Materials and Methods|| |
Collection of medicinal plants
Based on literature, plants which have been reported to possess antidiabetic, antihyperlipidemic, anti-inflammatory, and antioxidant activities are selected. The locally available plants were freshly collected and the remaining herbs were purchased from an Ayurvedic shop in Anantapur and were valid by Professor J. Raveendra Reddy, Division of Pharmacognosy, of our institute.
Polyherbal formulation preparation
The plant materials after air drying were powdered individually in a pulverizer and passed through sieve number 80. Finely powdered 19 herbal ingredients were mixed in the ratio of 8:4 2:2:1, of which eight parts were from single herb, four parts were from two herbs, two parts were from eight herbs, and one part was from the remaining eight herbs (w/w) to obtain a homogenous mixture (order of mixing is mentioned in [Table 2]).
The male and female Wistar rats (180–200 g) were procured from Raghavendra Enterprises, Bengaluru, Karnataka, India. The animals were maintained at standard conditions and protocol permitted by the Institutional Animal Ethics Committee (Protocol No: 878/AC/05/CPCSEA/024/2011).
Acute toxicity study
Female rats were separated into four groups, with three animals in each. Overnight fasted animals from Groups 1–4 received PHF (5, 50, 300, and 2000 mg/kg) orally. During 14 days observational period, no abnormal behavior and death were noticed. Hence, two dose levels were chosen, in which the first dose was one-eighth of the upper dose and the other was twice that of the one-eighth dose (250 and 500 mg/kg) selected for in vivo studies (Organisation for Economic Co-operation and Development guidelines 423).
Administration of polyherbal formulation to rats
First, we took polyherbal churna into a clean motor and grinded it with a pastel and after that, we made the suspension with tween 80. Uniform suspension of churna was given by an oral feeding tube, at the dose of 250 and 500 mg/kg daily between 9.00 am and 10.00 am in order to avoid circadian rhythm.
Induction of diabetes and experimental design
STZ was prepared in citrate buffer on the day of induction and injected intraperitoneally to all the groups except normal control (NC). After 48 h, rats with high glucose (≥250 mg/dl) were segregated into four groups (six/group) and were administered the below-mentioned treatment orally for 16 weeks:
- NC was administered with normal saline
- DN control (DNC) animals were adminstered with vehicle only
- DN rats were administered with PHF 250 mg/kg
- DN rats were administered with PHF 500 mg/kg.
On the last day, serum was separated from blood and stored at 2°C–8°C in a refrigerator until further usage.
Estimation of serum lipid profile
After completion of the treatment schedule, triglyceride (TG) (GPO-PAP method), total cholesterol (TC) (CHOD-PAP method), and high-density lipoprotein (HDL) (precipitating method) were estimated in the collected serum, according to instructions given by commercially available Erba biochemical kits using Erba semi-autoanalyzer (Chem 7, Erba Mannheim, Brentford, London, United Kingdom). Very low-density lipoprotein (VLDL) and LDL were calculated by the Friedewald formula.
Estimation of renal function parameters in serum and urine
Creatinine (Jaffe method) levels were estimated in serum by using Erba Chem-7 commercially available kit. Individual rats of all groups were kept separately in a metabolic cage for 24 h, and the collected urine samples were measured with a measuring cylinder and the urine volume (ml/24 h) was recorded. The collected urine samples were utilized for the estimation of urinary proteins (biuret method), urea (urease L-glutamate dehydrogenase method), and creatinine (Jaffe method) by using Erba Chem-7 commercially available kits. Type IV collagen in urine samples was estimated by assay protocol given by Abcam enzyme-linked immunosorbent assay (ELISA) kits (Cambridge, MA, USA) (ab 6586). Twenty-four hour urinary albumin excretion rate (UAER) was determined by an earlier available formula.,
Estimation of inflammatory cytokines
Inflammatory cytokines in serum: interleukin-6 (IL-6) (ab 100772), transforming growth factor (TGF)-β1 (ab 46780), and tumor necrosis factor-alpha (TNF-α) (ab 9755) levels were estimated according to the procedure by ELISA kits from Abcam (Cambridge, MA, USA).
Determination of advanced glycation end products in kidney homogenate
Advanced glycation end product (AGES) levels in kidney tissue were estimated according to a method described earlier. In brief, homogenized kidney tissue was imbibed overnight with mixture of chloroform and methanol (2:1 v/v). Following decantation, the remaining residue was mixed with 0.1 N NaOH, and the supernatant was collected after centrifugation (5000 rpm/15 min/4°C). Alkali-solubilized AGES were estimated by quantifying the fluorescence of samples in a fluorescence spectrophotometer at 440 nm (emission wavelength) and 370 nm (excitation wavelength) against blank samples of 1 ml of 0.1 N NaOH. One unit of fluorescence is equivalent to 1 mg/ml of bovine serum albumin (as standard). The fluorescence values of sample protein concentration of 1 mg/ml were measured as arbitrary units.
Histopathology of kidney
The kidney tissue was stored in fixing solution (10% buffered formalin) for histopathological examination.
The collected data were evaluated by one-way ANOVA along with Dunnett's multiple comparisons test and noted as mean ± standard error of mean.
| » Results|| |
Polyherbal formulation on serum lipid profiles
Hyperlipidemia was observed in STZ-DN rats with increase in TG, TC, LDL, and VLDL (P < 0.01) and reduction in HDL (P < 0.01). PHF dose dependently (250 and 500 mg/kg) corrected the hyperlipidemia with decrease in serum TG, TC, VLDL, and LDL (P < 0.01) and rise in HDL (P < 0.05, P < 0.01) in correlation with DN rats [Table 3].
Polyherbal formulation on renal function tests
STZ-DN rats displayed a significant decrease in 24-h urine volume, urinary urea, and urine creatinine (P < 0.01) and increase in serum creatinine, protein in urine, UAER, AGES, and type IV collagen excretion (P < 0.01). PHF at 250 and 500 mg/kg body weight (bw) doses demonstrated a significant increase in 24-h urine volume, urine creatinine (P < 0.01), and urinary urea (P < 0.05 and P < 0.01) and a significant decrease in protein in urine, UAER, AGES, and type IV collagen excretion (P < 0.01) in correlation with DN rats [Table 4] and [Table 5].
Polyherbal formulation on inflammatory mediators
STZ-DN rats displayed a considerable rise in IL-6, TGF-β1, and TNF-α (P < 0.01). DN rats with PHF at 250 and 500 mg/kg bw doses demonstrated a significant decrease in IL-6, TGF-β, and TNF-α (P < 0.01) in correlation with DN rats [Table 6].
Histopathology of kidney
Kidney tissues revealed histopathological alteration as follows: (a) normal rats – normal histological architecture; (b and c) DNC rats – accumulation of inflammatory cells, hypotropic glomeruli, and hemorrhagic spots; (d) PHF (250 mg/kg bw) – normal glomerular architecture; (e) PHF (500 mg/kg bw): normal architecture of the tubules and glomeruli [Figure 1].
|Figure 1: Histopathology of rat's kidney (a) Normal rats, (b and c) DNC rats, (d) PHF (250mg/kg b.w), (e) PHF (500mg/kg b.w)|
Click here to view
| » Discussion|| |
DN is the most common complication of DM which results in end-stage renal failure and is observed in 30%–40% of diabetic patients. Among the sufferers of DN, about 50% need either dialysis or kidney replacement, which not only affects their quality of life but also imposes financial burden. Currently available antidiabetic drugs have fewer efficacies toward the prevention and progression of DN and even drugs acting on renin–angiotensin–aldosterone system pathway are clinically approved, but still fail to get considerable results. Although understanding of molecular mechanism underlying DN is not clear, considerable literatures mentioned the importance of hyperglycemia-induced AGES as one of the key factors which further initiates the sequential chain activation of oxidative stress, inflammation, and fibrosis in DN. The pathological complexity of the above motivated many researchers to inculcate the multitargeted therapeutic approach rather than single approach. Moreover, the same thing was practiced in the clinical settings of the alternative system of medicine, such as Ayurveda, Siddha, and Unani in India since ancient times. Hence, we tried to explore the renoprotective effect of an in-house polyherbal churna formulated with herbs which has been proved to possess antidiabetic, antioxidant, anti-inflammatory, and antifibrotic activities. From the literature, it is very clear that lipid/lipoprotein abnormalities are a commonly observed status in type-II DM that are risk factors for renal atherosclerosis formation and further affect the renal blood flow. Pretreatment of DN rats with PHF at doses 250 and 500 mg/kg bw remarkably decreased the TG, TC, VLDL, and LDL and improved the HDL levels dose dependently, and it was clearly witnessed from the existence of known medicinal herbs with antihyperlipidemic active principles of polyherbal churna (Gymnema sylvestre – gymnemic acids, gymnemasaponins). STZ-challenged rats exhibited the characteristic features of DN such as increased amount of serum creatinine, proteins in urine, UAER as well as decrease in urinary urea, urine volume, and urine creatinine, which are the outcomes of hyperglycemia-activated multipathological signaling pathways in DN. PHF pretreatment (250 and 500 mg/kg) corrected the imbalance of pathological characteristics in a dose-dependent manner. Accumulated evidence clear that sustained hyperglycemia enhances the formation of AGES. Interaction of AGES with its RAGE receptor in kidney activated various signaling pathways related to inflammation, fibrosis, and oxidative stress. An increased amount of AGES in kidney tissue was observed in DN rats. Amelioration of AGES levels in kidney tissue by PHF at doses of 250 and 500 mg/kg bw is the best evidence for the nephroprotective mechanism of PHF (Tribulus terrestris – tribuloside, Eugenia jambolana – jambosine)., Type IV collagen is an important component of the extracellular matrix of mesangial and glomerular basement membrane. Excessive deposition of type IV collagen in the extracellular matrix is due to hyperglycemia-induced synthesis and also due to decreased breakdown by the glycation of protein component of collagen, which causes renal fibrosis and excretion of type IV collagen in the urine which is an early hallmark of renal fibrosis. STZ induced DN rats observed with significant increase in urinary excretion of type IV collagen and is efficiently decreased with PHF at doses 250, 500 mg/kg and this suggests that anti-fibrotic mechanism of PHF (Glycyrrhiza glabra – glycyrrhetinic acid, Curcuma longa – curcumin). Accumulation of pro-inflammatory cytokines (IL-6, TNF-α, and TGF-β1) is a well-established event in the progression of DN; to be very specific, TNF-α enhances the production of free radicals and causes apoptosis and necrosis of renal tissue. STZ-induced DN rats showed increased levels of IL-6, TNF-α, and TGF-β1 and treatment with PHF at 250 and 500 mg/kg bw showed reduction of IL-6, TNF-α, and TGF-β1, suggesting the presence of anti-inflammatory constituents (Curcuma longa – curcumin, Tribulus terrestris – tribuloside, and rutin)., Histopathological changes are the most evident indicator of renal damage, and DN causes noticeable glomeruli, tubular damages, and hemorrhagic conditions. PHF efficiently protects the internal structure of kidney. At the end, our results expressed that PHF exhibited better nephroprotective activity by blocking the sequential interaction of oxidative stress, inflammatory pathways, and fibrosis pathways associated with chronic diabetes. Protective effect may be possible due to the presence of known and proven multiple and divergent phytoconstituents of PHF. Hence, PHF can be considered as a therapeutic option in DN. Still, deeper human investigation is necessary to authenticate the useful effects of PHF in DN.
| » Conclusion|| |
At the end, our results expressed that PHF exhibited better nephroprotective activity by blocking the sequential interaction of oxidative stress, inflammatory pathways, and fibrosis pathways associated with chronic diabetes. Protective effect may be possible due to the presence of known and proven multiple and divergent phytoconstituents of PHF. Hence, PHF can be considered as a therapeutic option in DN. Still, deeper human investigation is necessary to authenticate the useful effects of PHF in DN.
The authors convey their heartfelt thanks to the management of Raghavendra Institute of Pharmaceutical Education and Research (RIPER), Anantapuramu, Andhra Pradesh, India, for facilitating chemicals to this research work.
Financial support and sponsorship
This study was financially supported by the Management, RIPER.
Conflicts of interest
There are no conflicts of interest.
| » References|| |
Hasani-Ranjbar S, Larijani B, Abdollahi M. A systematic review of Iranian medicinal plants useful in diabetes mellitus. Arch Med Sci 2008;4:285-92.
Khaki A, Fathiazad F. Diabetic nephropathy-using herbals in diabetic nephropathy prevention and treatment-the role of ginger (Zingiber officinale
) and onion (Allium cepa
) in diabetics' nephropathy. Compend Essays Alternat Ther 2012; p. 206-32.
Toth-Manikowski S, Atta MG. Diabetic kidney disease: Pathophysiology and therapeutic targets. J Diabetes Res 2015; 2015:1-17.
Liu K, Xu H, Lv G, Liu B, Lee MK, Lu C, et al.
Loganin attenuates diabetic nephropathy in C57BL/6J mice with diabetes induced by streptozotocin and fed with diets containing high level of advanced glycation end products. Life Sci 2015;123:78-85.
Makni M, Sefi M, Fetoui H, Garoui el M, Gargouri NK, Boudawara T, et al.
Flax and pumpkin seeds mixture ameliorates diabetic nephropathy in rats. Food Chem Toxicol 2010;48:2407-12.
Patel SS, Shah RS, Goyal RK. Antihyperglycemic, antihyperlipidemic and antioxidant effects of Dihar, a polyherbal ayurvedic formulation in streptozotocin induced diabetic rats. Indian J Exp Biol 2009;47:564-70.
Moradi B, Abbaszadeh S, Shahsavari S, Alizadeh M, Beyranvand F. The most useful medicinal herbs to treat diabetes. Biomed Res Ther 2018;5:2538-51.
Nazarian-Samani Z, Sewell RD, Lorigooini Z, Rafieian-Kopaei M. Medicinal plants with multiple effects on diabetes mellitus and its complications: A systematic review. Curr Diab Rep 2018;18:72.
Choudhury H, Pandey M, Hua CK, Mun CS, Jing JK, Kong L, et al.
An update on natural compounds in the remedy of diabetes mellitus: A systematic review. J Tradit Complement Med 2018;8:361-76.
Pari L, Ramakrishnan R, Venkateswaran S. Antihyperglycaemic effect of Diamed, a herbal formulation, in experimental diabetes in rats. J Pharm Pharmacol 2001;53:1139-43.
Konda VR, Arunachalam R, Eerike M, Rao KR, Radhakrishnan AK, Raghuraman LP, et al.
Nephroprotective effect of ethanolic extract of Azima tetracantha
root in glycerol induced acute renal failure in Wistar albino rats. J Tradit Complement Med 2016;6:347-54.
Zhang S, Xu H, Yu X, Wu Y, Sui D. Metformin ameliorates diabetic nephropathy in a rat model of low-dose streptozotocin-induced diabetes. Exp Ther Med 2017;14:383-90.
Lu HJ, Tzeng TF, Liou SS, Da Lin S, Wu MC, Liu IM. Ruscogenin ameliorates diabetic nephropathy by its anti-inflammatory and anti-fibrotic effects in streptozotocin-induced diabetic rat. BMC Complement Altern Med 2014;14:110.
Perez Gutierrez RM, de Jesus Martinez Ortiz M. Beneficial effect of Azadirachta indica
on advanced glycation end-product in streptozotocin-diabetic rat. Pharm Biol 2014;52:1435-44.
Hsu JD, Wu CC, Hung CN, Wang CJ, Huang HP. Myrciaria cauliflora
extract improves diabetic nephropathy via suppression of oxidative stress and inflammation in streptozotocin-nicotinamide mice. J Food Drug Anal 2016;24:730-7.
Modak M, Dixit P, Londhe J, Ghaskadbi S, Devasagayam TP. Indian herbs and herbal drugs used for the treatment of diabetes. J Clin Biochem Nutr 2007;40:163-73.
Zhang H, Yang Y, Wang Y, Wang B, Li R. Renal-protective effect of thalidomide in streptozotocin-induced diabetic rats through anti-inflammatory pathway. Drug Des Devel Ther 2018;12:89-98.
Thakkar NV, Patel JA. Pharmacological evaluation of “Glyoherb”: A polyherbal formulation on streptozotocin-induced diabetic rats. Int J Diabetes Dev Ctries 2010;30:1-7.
Peng L, Li J, Xu Y, Wang Y, Du H, Liu JS. The protective effect of beraprost sodium on diabetic nephropathy by inhibiting inflammation and p38 MAPK signaling pathway in high-fat diet/streptozotocin-induced diabetic rats. Int J Endocrinol 2016; 2016:1-19.
Saulnier PJ, Wheelock KM, Howell S, Weil EJ, Tanamas SK, Knowler WC, et al.
Advanced glycation end products predict loss of renal function and correlate with lesions of diabetic kidney disease in American Indians with type 2 diabetes. Diabetes 2016;65:3744-53.
Xie R, Zhang H, Wang XZ, Yang XZ, Wu SN, Wang HG, et al.
The protective effect of betulinic acid (BA) diabetic nephropathy on streptozotocin (STZ)-induced diabetic rats. Food Funct 2017;8:299-306.
Zhang J, Zhang Q, Chen X, Liu Y, Xue J, Dahan A, et al.
Revealing synergistic mechanism of multiple components in gandi capsule for diabetic nephropathy therapeutics by network pharmacology. Evid Based Complement Alternat Med 2018;2018:1-11.
Ziyadeh FN, Sharma K, Ericksen M, Wolf G. Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta. J Clin Invest 1994;93:536-42.
Yamamoto Y, Maeshima Y, Kitayama H, Kitamura S, Takazawa Y, Sugiyama H, et al.
Tumstatin peptide, an inhibitor of angiogenesis, prevents glomerular hypertrophy in the early stage of diabetic nephropathy. Diabetes 2004;53:1831-40.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]