Effect of a polyherbal formulation in streptozotocin-induced diabetic nephropathy in wistar rats.

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. Copyright:

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.[1] 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.[2] DN associated with morphological and ultrastructural changes within the kidney is determined by the clinical manifestation of the disease.[34]

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.[5] 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.[6] 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].[789] 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

Plant	Phytochemicals	Pharmacological properties
Eugenia jambolona	Jambosine, gallic acid, ellagic acid, corilagin, 3,6-hexahydroxydiphenoylglucose, quercetin, β-sitosterol	AntidiabeticHypolipidemic
Tinospora cordifolia	Berberine, choline, palmatine, magnoflorine, tinosporine, tetrahydropalmatine, isocolumbine, aporphine alkaloids, tinosporides	AntidiabeticAnti-inflammatory
Gymnema sylvestre	Gymnemic acids, gymnemasaponins, flavones, hentriacontane, pentatriacontane	AntidiabeticHypolipidemic anti-inflammatory
Cressa cretica	Quercetin, kampferol-3-O-glucoside, rutin, syringaresinol-β-d-glucoside	Antidiabetic activity
Casearia esculenta	Clerodane diterpenoids, esculentin A, 3-hydroxymethyl xylitol	HypoglycemicAnti-inflammatory
Curcuma longa	Curcumin, desmethoxy curcumin, α and β turmerome, eugenon, campestrol, stigmasterol	AntidiabeticAntihyperlipidemic antioxidant
Swertia chirata	Swertiamarin, mangiferin, swerchirin, sweroside, amaroswerin, amarogentin, chiratin	AntidiabeticAntioxidant
Centratherum anthelminticum	Hexatetracontan-16-ol, 6,9-eicosadiene, butyl 11-hydroxy octadecanoate, hexyl 3-hydroxynonanoate	AntidiabeticAntioxidantAnti-inflammatory
Picrorhiza kurroa	Picroside-I and II, pikuroside, veronicoside, phenol glycosides, a number of cucurbitacin glycosides	AntidiabeticAntihyperlipidemic
Trigonella foenum-graecum	Diosgenin, amino acid 4-hydroxyisoleucine, yamogenin, tigogenin	AntidiabeticAntioxidant
Terminalia chebula	Gallic acid, chebulagic acid, punicalagin, chebulanin, corilagin, neochebulinic acid, ellagic acid	Antidiabetic antioxidant renoprotective, anti-inflammatory
Holarrhena antidysenterica	Holarrifine, kurchamide, kurcholessine, trimethylconkurchine, N-methylholarrhimine	Antidiabetic antioxidant, anti-inflammatory
Pterocarpus marsupium	Pterostilbene, 7-hydroxyflavanone, isoliquiritigenin, liquiritigenin, dihydroxyflavone, marsupsin, pterosupin, p-hydroxybenzaldehyde	Antihyperinsulinemic, anti-hyper triglyceridaemic
Glycyrrhiza glabra	2-beta-glycyrrhizic, glucuronic acid, glycyrrhetinic acid (enoxolone), tannic acid, asparagine, resins	Antidiabetic, antihyperlipidemic anti-inflammatory
Mineral pitch	Benzoic acid, hippuric acid, fatty acid, gums, resins, ellagic acid, triterpenes	AntidiabeticAntioxidantAnti-inflammatory
Tribulus terrestris	Kaempferol, kaempferol3rutinoside, tribuloside, rutin	AntidiabeticHypolipidemicAnti-inflammatory
Withania somnifera	Isopellertierine, anferine, withanolides, withaferins, withanolides	AntihyperglycemicHypolipidemic antioxidant antiinflammatory
Nardostachys jatamansi	Sesquiterpenes, coumarins, jatamansone or valeranone, alpha-patcho-ulense	Antidiabetic antioxidant
Bacopa monnieri	Brahmine, nicotine, herpestine, D-mannitol, hersaponin	Antidiabetic antioxidant

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).[10]

Table 2

Composition of polyherbal formulation

Botanical name	Family	Part used	Weight (g)
Eugenia jambolana	Myrtaceae	Seeds	20
Tinospora cordifolia	Menispermaceae	Roots	10
Gymnema sylvestre	Asclepiadaceae	Leaves	10
Cressa cretica	Convolvulaceae	Leaves	5
Casearia esculenta	Hippocrataceae	Roots	5
Curcuma longa	Zingiberaceae	Rhizome	5
Swertia chirata	Gentianaceae	Leaves	5
Centratherum anthelminticum	Compositae	Seeds	5
Picrorhiza kurroa	Plantaginaceae	Rhizomes	5
Trigonella foenum-graecum	Fabaceae	Seeds	5
Terminalia chebula	Combretaceae	Fruits	5
Holarrhena antidysenterica	Apocynaceae	Bark	2.5
Pterocarpus marsupium	Fabaceae	Leaves	2.5
Glycyrrhiza glabra	Fabaceae	Rhizomes	2.5
Mineral pitch	-	-	2.5
Tribulus terrestris	Zygophyllaceae	Seeds	2.5
Withania somnifera	Solanaceae	Leaves	2.5
Nardostachys jatamansi	Valirenaceae	Rhizomes	2.5
Bacopa monnieri	Plantaginaceae	Leaves	2.5

Experimental animals

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.[1112]

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).[13]

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.[14]

Histopathology of kidney

The kidney tissue was stored in fixing solution (10% buffered formalin) for histopathological examination.

Statistical analysis

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].

Table 3

Effect of polyherbal formulation on lipid profile

Group	TGs (mg/dl)	TC (mg/dl)	HDL (mg/dl)	VLDL (mg/dl)	LDL (mg/dl)
NC	69.4±0.647	87.0±3.06	28.6±1.23	13.8±0.118	38.5±1.92
DNC	175±1.21**	196.0±6.64**	18.4±0.30**	34.8±0.393**	143±4.37**
DN + test 1	132±1.16##	87.3±2.42##	21.2±0.55#	26.3±0.443##	92.46±1.20##
DN + test 2	88.1±1.74##	79.0±1.46##	24.0±0.78##	17.4±0.280##	61.7±2.50##

**P<0.01 compared with NC, ##P<0.01 compared with DNC, #P<0.05 compared with DNC. All values are expressed as mean±SEM. DN rats treated with dose (250 mg/kg/orally) of formulation (DN + test 1), dose (500 mg/kg/orally) of formulation (DN + test 2), respectively. TGs=Triglycerides, TC=Total cholesterol, DN=Diabetic nephropathy, NC=Normal control, DNC=DN control, SEM=Standard error of mean, HDL=High-density lipoprotein, LDL=Low-density lipoprotein, VLDL=Very LDL

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 [Tables 4 and 5].

Table 4

Effect of polyherbal formulation on renal function tests

Group	Urine volume (ml/rat/day)	Urinary urea (mg/dl)	Serum creatinine (mg/dl)	Urine creatinine (mg/dl)
NC	11.3±0.80	5.33±0.98	0.6±0.03	26.6±1.45
DNC	4.0±0.26**	0.01±0.01**	2.40±0.42**	5.8±0.99**
DN + test 1	17.8±1.70##	2.67±1.090#	1.12±0.02##	20.1±1.16##
DN + test 2	13.2±1.05##	3.67±0.33##	0.943±0.02##	23.8±0.70##

**P<0.01 compared with NC, ##P<0.01 compared with DNC, #P<0.05 compared with DNC. All values are expressed as mean±SEM. DN rats treated with dose (250 mg/kg/orally) of formulation (DN + test 1), dose (500 mg/kg/orally) of formulation (DN + test 2), respectively. DN=Diabetic nephropathy, NC=Normal control, DNC=DN control, SEM=Standard error of mean

Table 5

Effect of polyherbal formulation on renal function tests

Group	Protein in urine (mg/day)	UAER (µg/day)	AGES (AU)	Type IV collagen excretion (µg/day)
NC	0.23±0.042	1.8±0.33	197±14.3	16.3±3.48
DNC	24.70±0.422**	16.8±1.51**	455±57.7**	750±76.4**
DN + test 1	0.56±0.098##	6.0±0.57##	252±19.2##	80.0±5.77##
DN + test 2	0.08±0.030##	2.9±0.56##	227±22.0##	68.3±5.63##

**P<0.01 compared with NC, ##P<0.01 compared with DNC. ll values are expressed as mean±SEM. DN rats treated with dose (250 mg/kg/orally) of formulation (DN + test 1), dose (500 mg/kg/orally) of formulation (DN + test 2), respectively. DN=Diabetic nephropathy, NC=Normal control, DNC=DN control, SEM=Standard error of mean, UAER=Urinary albumin excretion rate, AGES=Advanced glycation end products, AU=Arbitrary unit

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].

Table 6

Effect of polyherbal formulation on inflammatory mediators

Group	IL-6 (Pg/ml)	TGF-β1 (%)	TNF-α (Pg/ml)
NC	164±4.80	12.7±2.19	34.2±4.55
DNC	361±11.6**	25.5±2.57**	368±37.1**
DN + test 1	275±5.62##	13.2±1.74##	140±10.6##
DN + test 2	252±8.72##	13.3±1.33##	75.0±7.64##

**P<0.01 compared with NC, ##P<0.01 compared with DNC. All values are expressed as mean±SEM. DN rats treated with dose (250 mg/kg/orally) of formulation (DN + test 1), dose (500 mg/kg/orally) of formulation (DN + test 2), respectively. DN=Diabetic nephropathy, NC=Normal control, DNC=DN control, SEM=Standard error of mean, IL=Interleukin, TGF-β=Transforming growth factor-beta, TNF-α=Tumor necrosis factor-alpha

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)

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.[15] 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.[16] 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.[17] 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.[18] 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.[19] 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.[20] 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).[2122] 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.[23] 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).[24] 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).[2122] 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.

Financial support and sponsorship

This study was financially supported by the Management, RIPER.

Conflicts of interest

There are no conflicts of interest.