O E r P H a b P c a A R A A K G A N N I u ( i n l e t s 0 c Revista Brasileira de Farmacognosia 29 (2019) 234–240 ww w . elsev ier .com/ locate /b jp riginal article ffect of Withania somnifera on gentamicin induced renal lesions in ats rem Kumar Govindappa a,∗, Vidhi Gautam b, Syamantak Mani Tripathi b, Yash Pal Sahnib, allur Lakshmana Shetty Raghavendra c Department of Biomedical Engineering, Schools of Medicine and Engineering, University of Alabama at Birmingham, Birmingham, United States Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Jabalpur, Madhya radesh, India Departamento de Ginecologia e Obstetrícia, Câmpus de Botucatu, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP, Brazil r t i c l e i n f o rticle history: eceived 4 November 2018 ccepted 17 December 2018 vailable online 23 January 2019 eywords: entamicin ntioxidant ephrotoxicity ephroprotective and nephrocurative a b s t r a c t Gentamicin induced renal complications are well known in humans and animals. Medicinal properties of Withania somnifera (L.) Dunal, Solanaceae, are recognized to improve renal functions. However, the phar- macological function of W. somnifera is not completely understood. We sought to unravel medicinal therapeutic function of W. somnifera on gentamicin-induced nephrotoxicity in wistar rats. Twenty- four adult male wistar rats evenly divided into four groups to evaluate in vivo nephroprotective and nephrocurative function of W. somnifera in gentamicin induced nephrotoxic rats. Experimental design as follows: Group I, saline control for 21 days; Group II, gentamicin nephrotoxic control for eight days; Group III, alcoholic extract of W. somnifera for 13 days + simultaneous administration of gentamicin and W. somnifera, from day 14 to 21 (nephroprotective) and Group IV, gentamicin for 8 days + alcoholic extract of W. somnifera from day 9 to 21 (nephrocurative). End of experiment, respective serum and kidney tissue samples used to analyze renal function. Withania somnifera as a nephroprotective and nephrocurative molecule significantly restore the renal function on gentamicin-induced nephrotoxi- city. This phenomenon is accompanied with significantly reduced blood urea nitrogen, creatine, alkaline phosphatase, gamma-glutamyl transferase, albumin, total protein, calcium, potassium and kidney malon- dialdehyde concentrations. Additionally, W. somnifera significantly increased antioxidant activities of glutathione and superoxide dismutase to protect renal tissue damage from gentamicin in wistar rats. Over all, W. somnifera treated nephroprotective animal shows improved recovery compared to nephrocuartive. The nephroprotective or nephrocurative effect of W. somnifera could be due to inherent antioxidant and free-radical-scavenging principle(s). In the near future, biologically active compounds of W. somnifera (withanolides) could appear as a novel therapeutic molecule for renal disorders. rasil he CC © 2019 Sociedade B access article under t ntroduction Gentamicin (GM) is an important aminoglycoside antibiotic sed reliably to treat serious and life threatening infections Mycobacterium infection, septicemia, complicated urinary tract nfection, endocarditis, peritonitis, etc.) mainly caused by gram- egative bacteria in human and animals. However, its clinical use is imited due to incidence of nephrotoxicity and ototoxicity (Krause t al., 2016; Jiang et al., 2017). Nephrotoxicity induced by gen- amicin manifests clinically as nonoliguric renal failure, with a low rise in creatinine, blood urea nitrogen, also accompanied with ∗ Corresponding author. E-mail: pgovindappa@pennstatehealth.psu.edu (P.K. Govindappa). https://doi.org/10.1016/j.bjp.2018.12.005 102-695X/© 2019 Sociedade Brasileira de Farmacognosia. Published by Elsevier Edit reativecommons.org/licenses/by-nc-nd/4.0/). eira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). proteinuria, enzymuria, glycosuria and electrolyte impairments (Mingeot-Leclercq and Tulkens, 1999). Although the exact pathophysiology of gentamicin induced nep- hrotoxicity is still unclear and it seems to be linked with generation of destructive reactive oxygen species (ROS) in renal cells. Even- tually, overproduction of ROS potentiates oxidative damage of biomolecules (lipids, proteins, DNA) by altering malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione per- oxidase (GPx) in prime renal failure (Khan et al., 2011). Herbal products have a special place in the world of pharma- ceuticals with their safety, efficacy and cost effectiveness. The World Health Organization reported that 80% of the world’s popu- lation depends on medicinal plants for their healthcare needs and more than 30% of the pharmaceutical preparations based on plants ora Ltda. This is an open access article under the CC BY-NC-ND license (http:// https://doi.org/10.1016/j.bjp.2018.12.005 www.elsevier.com/locate/bjp http://crossmark.crossref.org/dialog/?doi=10.1016/j.bjp.2018.12.005&domain=pdf https://orcid.org/0000-0001-8069-234X https://orcid.org/0000-0001-5004-1837 https://orcid.org/0000-0002-5992-3980 https://orcid.org/0000-0003-4300-109X http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:pgovindappa@pennstatehealth.psu.edu https://doi.org/10.1016/j.bjp.2018.12.005 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ eira de ( b I m a a w t a a p A m g M P m A J b S m D o s d b t c 3 Q l fl 1 m A A C ( s o 0 w a t w 2 o s s t T c P.K. Govindappa et al. / Revista Brasil Jeyanthi and Subramanian, 2009). Withania somnifera (L.) Dunal elongs to Solanceae family, commonly known as Ashwagandha, ndian ginseng and Winter cherry used in ayurvedic and indigenous edicine for over 3000 years. The biologically active chemical constituents of W. somnifera re alkaloids (isopelletierine, anferine), steroidal lactones (with- nolides, withaferins), saponins (sitoindoside VII and VIII) and ithanolides (sitoindoside XI and X); used widely for therapeu- ic purpose of immunomodulatory, antitumor, anti-inflammatory, ntioxidant, anti-ageing, antidepressant, anxiolytic, hematopoietic nd aphrodisiac . To our knowledge, as of today there is no profound nephro- rotective or nephrocurative evidence on traditional medicine. ccordingly, the present study is design to understand phar- acological renal function of alcoholic extract W. somnifera on entamicin-induced nephrotoxicity in Wistar rats. aterial and methods lant materials and alcoholic extraction Withania somnifera (L.) Dunal, Solanaceae, roots obtained com- ercially from Department of Aromatic and Medicinal Plants, griculture College, Jawaharlal Nehru Krishi Vishwa Vidyalaya, abalpur (M.P.), India. The roots were identified and authenticated y Prof. Kappali S.A., Botanist, Department of Botany, Basaveshwar cience College, Bagalkot, Karnataka, India. The voucher speci- en was prepared and deposited (Bsc/Pharmacy/81/2010) in the epartment of Pharmacology, Hangal Shri Kumareshwar College f Pharmacy, Bagalkot, Karnataka, India. Withania somnifera roots, ubject to shade dry for fine powder preparations. These pow- ers utilized to extract pharmacologically active phytochemicals y alcoholic extract method using Soxhlet apparatus. After extrac- ion, excess alcoholic solvent evaporated at 60 ◦C until the extracts oncentrated to a paste and dried by vacuum drying method at 5 ◦C. uantification of bioactive compound using high performance iquid chromatography (HPLC) Withania somnifera extract (500 mg) taken in a clean and dried ask, 50 ml of HPLC grade methanol was added and refluxed for 0 min, cooled and sonicated for 6 min. The final volume of 100 ml ade up with methanol. HPLC analysis performed on a Waters lliance 2690 HPLC system, equipped with a 2998 Photodiode rray Detector (Waters, Milford, MA, USA). For all separations, a 18 column (250 × 4.60 mm, 5 �m particle size) from Phenomenex Torrance, CA, USA) was used. The mobile phase consisted of olvent A; dissolved 0.14 g of anhydrous potassium dihydrogen rthophosphate (KH2PO4) in 900 ml of HPLC grade water, added .5 ml of orthophosphoric acid and volume was made to 1000 ml ith water. The mixture was filtered through 0.45 �m membrane nd sonicated for 3 min. The sonicated content was quantitatively ransferred to HPLC column with solvent B (acetonitrile) which ere applied in the following gradient elution: 95A/5B, 55A/45B, 0A/80B, 20A/80B, 55A/45B, 95A/5B, 95A/5B from respective time f 0.01, 18, 25, 28, 35, 40 and 45 min. The separation flow rate and ample volume were set to 1.5 �l and 20 �l, respectively and all eparations were monitored at 227 nm. Then injected three times he standard preparation and calculated the mean area and the RSD. hen after injected 20 �l of sample preparation and recorded the hromatogram at 227 nm. Farmacognosia 29 (2019) 234–240 235 Animal procurement and care Healthy male Wistar rats (8–10 weeks, 180–200 g) procured from laboratory animal breeding centre, college of Veterinary Sci- ence and Animal Husbandry, Jabalpur (M.P.), India. All animals were maintained as per the protocol outlined in the publication of the Committee for the Purpose of Control and Supervision of Experi- ments on Animals (CPCSEA). Institutional Animal Ethics Committee (IAEC) bearing approval No. 268/IAEC/MPPCVV/2010 approved the experimental procedures. Acute oral toxicity study of Withania somnifera The acute oral toxicity study was conducted according to the OECD guideline 423 (Acute toxic class method). The limit test dose of W. somnifera (2000 mg kg−1), administered by gavage using a stomach tube. Animals observed individually at least once during the first 30 min, with special attention for first 4 h and daily there- after for a total of 14 days. The extract was found to be devoid of mortality at 2000 mg kg−1. Hence, 2500 mg kg−1 was considered as LD50 cutoff value and 1/5th (500 mg kg−1, p.o.) of the dose selected for the screening of nephroprotective and nephrocurative activities. Experimental protocol Gentamicin sulphate (40 mg ml−1 injection, Wockhardt Ltd., Mumbai, India) and all other chemicals used in this experiment meets analytical grade. Twenty-four experimental rats randomly divided into four different groups (n = 6): Group I, normal saline (1 ml kg−1, once a day for 21 days, i.p.); Group II, GM (80 mg kg−1, once a day for 8 days, i.p.); Group III, W. somnifera (500 mg kg−1, once a day for 13 days, p.o.) pre-treatment + simultaneous admin- istration of gentamicin and W. somnifera (i.p., 80 mg kg−1 and p.o., 500 mg kg−1, once a day from day 14 to day 21) is called as nephro- protective/NP group; Group IV, GM (80 mg kg−1, once a day for 8 days, i.p.) pre-exposure + W. somnifera (500 mg kg−1, once a day from day 9 to day 21, p.o.) is called as nephrocurative/NC group. Forced motor activity Experimental rats were habituated and completed six training sessions (three sessions daily at 15 min resting interval) on rotarod at 5–20 rpm for 5 min (Rotarod EM-35, Microteknik, India). On the test day of experiment, trained rats were placed on the rotating rod at 10 rpm for 5 min and the latency to fall (the time it took for the rat to fall from the rod) was measured. The variations in animals were used to identify and compare alterations in motor coordination (Hernández-López et al., 2017). Relative kidney weight Rats were fasted overnight on day 21 of experiment and the live body weight (g) was recorded on day 22 before euthanizing. Kidney was isolated and weighed (g) (absolute organ weight) to calculate relative organ weight of each animal. Relative kidney weight (g) = Absolute kidney weight (g) Body weight of rat on sacrifice day (g) × 100 Serum and tissue sampling Experimental rats were fasted overnight and anesthetized using isoflurane to collect blood samples (via retro-orbital plexus) for serum isolation. The harvested serum stored at −80 ◦C until analysis of biochemical parameters using Erba Chem 5 Plus V2 Biochem- istry Analyzer. Before harvesting kidneys, all animals were perfused 236 P.K. Govindappa et al. / Revista Brasileira de Farmacognosia 29 (2019) 234–240 mV 200 150 100 50 0 0 5 10 15 20 25 30 35 40 45 1Det.A ch1 W ith an os id e V W ith an os id e IV W ith af er in A W ith as tr am on ol id e W ith an ol id e A W ith an ol id e B tract ( o s A b S o P A a f s g T g l s t t p m T w d t w a o a t b c t D m 1 t t ence of withanolides; withanolide A, withanolide B, withanoside IV, withanoside V, 12-deoxy withastramonolide and withaferin in the root extract of W. somnifera and their relative percentage are mentioned in Table 1. Table 1 Percentage of withanolides in the root extract of Withania somnifera by HPLC. Peak Number retention time Active ingredient Area Area% 1 16.753 Withanoside IV 703 633 14.042 2 20.606 Withanoside V 715 829 14.285 3 21.140 Withaferin A 2 414 057 48.176 4 21.960 Withastramonolide 472 909 9.438 Fig. 1. Chromatogram of Withania somnifera root ex 0.9% sodium chloride) and then stored at −80 ◦C (for estimation of xidative stress markers) and 10% neutral buffered formaldehyde olution (for histopathological studies). ssessment of electrolytes Serum samples subject to analyze electrolytes, Na+ and K+ y using Flame Photometer (Flame Photometer 128, Systronic). odium and potassium standards were prepared for the analysis f samples by plotting a straight-line graph as guided by Hawk’s hysiological Chemistry. ssessment of oxidative stress Kidneys homogenized in ice cold 0.9% sodium chloride using homogenizer. The homogenates were centrifuged at 800 × g or 5 min at 4 ◦C to separate the nuclear debris. The supernatant o obtained was centrifuged at 5000 × g for 15 min at 4 ◦C to et the postmitochondrial supernatant (Karadeniz et al., 2008). hese samples were used to analyze lipid peroxidation (MDA), lutathione (GSH) and superoxide dismutase (SOD) activity by fol- owing in-house laboratory protocol using Helios double beam pectrophotometer. The brief protocol of MDA, GSH and SOD quan- ification is as follows. Determination of MDA: One ml of 33% (dilution in PBS) of kidney issue supernatant was incubated at 37 ± 0.5 ◦C for 2 h. To each sam- le 1 ml of 10% (w/v) trichloroacetic acid was added. After thorough ixing, the reaction mixture was centrifuged at 100 × g for 10 min. o 1 ml of supernatant 1 ml of 0.67% (w/v) of thiobarbituric acid as added and kept in boiling water bath for 10 min, cooled and iluted with 1 ml of distilled water. Blank was made by adding all he reagents except the kidney tissue supernatant. The absorbance as read at 535 nm (Shafiq-Ur-Rehman, 1984). Determination of GSH: Kidney tissue supernatant (0.2 ml) was dded to 4 ml of 0.08 N H2SO4 and mixed carefully. After 10 min f standing at room temperature, 0.5 ml of tungstate solution was dded to clear the brown haemolysate. The tube was stoppered and he mixture was shaken vigorously for 5 min in order to avoid bub- le formation on the top of supernatant. The suspension was then entrifuged for 15 min at 100 × g at room temperature. After cen- rifugation, 2 ml of supernatant, 2.5 ml of TRIS buffer and 0.2 ml of TNB [5,5-dithiobis(2-nitrobenzoic acid)] reagent was added and ixed well. Absorbance was measured at 412 nm (Beutler et al., 963). Determination of SOD: In a set of test tubes 650 �l of PBS was aken. To this, 30 �l MTT [3-(4,5-dimethyl thiazol-2-4) 2,5-diphelyl etrazolium bromide] was added. Then, 75 �l of pyrogallol was min by using high performance liquid chromatography. added to both the tubes. This mixture was incubated for 5 min at room temperature. To stop this reaction, 750 �l of dimethyl sulf- oxide was added to both the tubes and finally 10 �l of sample was added to the second tube to find out the percentage of reduction of MTT formation. The colour development was read at 570 nm (Madesh and Balasubramanian, 1998). Histopathology Kidneys fixed in 10% neutral buffered formaldehyde solution (pH 7), dehydrated in graded anhydrous ethanol and embedded in paraffin. Fine sections of 5 �m of thickness were obtained, mounted on glass slides and counter-stained with haematoxylin and eosin (H and E) for light microscopic analyses (Cardiff et al., 2014). Vet- erinary pathologist who was unaware of the treatments carried out interpretation of histopathological changes. Statistical analysis The data were analyzed using Student’s t-test or one-way analy- sis of variance (ANOVA). All the values are presented as mean ± SE. The probability (p) values of ≤0.05 were considered statistically significant. Results Quantification of withanolides by HPLC A HPLC analysis was performed to estimate withanolides in the root extract of W. somnifera The chromatogram (Fig. 1) depicts pres- 5 22.150 N/A 131 542 2.625 6 22.786 Withanolide A 472 719 9.434 7 25.965 Withanolide B 100 238 2.000 Total 5 010 927 100.000 P.K. Govindappa et al. / Revista Brasileira de Farmacognosia 29 (2019) 234–240 237 180.00 b b b b b b 0.70 a a a 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0.60 0.50 0.40 0.30 0.20 0.10 0.00 175.00 170.00 165.00 160.00 155.00 150.00 145.00 140.00 Control GM NP NC Control GM NP NC Control GM NP NC B od y w ei gh t ( g) R el at iv e ki dn ey to b od y w ei gh t ( g) F or ce d m ot or a ct iv ity ( m in ) A B C F ) Body weight (g); (B) relative kidney weight (g); (C) forced motor activity (min) shows s ed to gentamicin. E w ( o N T ( I n t E m m ( a E c n p f a r b E t ( c K t a a C Table 3 Effect of Withania somnifera on gentamicin induced serum electrolyte changes. Results of serum electrolyte parameters (potassium and calcium) significantly (ap ≤ 0.05) decreased in gentamicin treatment rats. Withania somnifera treatment groups, nephroprotective and nephrocurative significantly (bp ≤ 0.05) increased the altered serum electrolytes in gentamicin induction (n = 6). Group Sodium (mEq l−1) Potassium (mEq l−1) Calcium (mg dl−1) Control 138.53 ± 1.74 6.20 ± 0.74 7.91 ± 0.10 GM 142.52 ± 2.68 4.55 ± 1.01a 5.14 ± 0.07a T E r ig. 2. Effect of Withania somnifera on gentamicin induced nephrotoxic changes. (A ignificant (bp ≤ 0.05, n = 6) improvement in W. somnifera treatment rats as compar ffect of Withania somnifera on body weight and relative kidney eight Animals of gentamicin (GM) treatment shows significant ap ≤ 0.05) reduction in body weight as compare to normal control n day 22. However, as compare to GM treatment, animals of NP and C shows significant (bp ≤ 0.05) increase in body weight (Fig. 2A). he relative kidney weight (g) of GM treatment alone significantly ap ≤ 0.05) increased as compare to control, NP and NC treatment. nterestingly, ameliorative effect of W. somnifera (NP and NC) sig- ificantly (bp ≤ 0.05) retained relative kidney weight (g) against GM reatment (Fig. 2B). ffect of Withania somnifera on forced motor activity Gentamicin treatment significantly (ap ≤ 0.05) reduced forced otor activity (min) as compare to control, NP and NC treat- ent. Intriguingly, the forced motor activity fortified significantly bp ≤ 0.05) in NP and NC treatment as compare to GM treatment lone (Fig. 2C). ffect of Withania somnifera on renal biochemical parameters GM treatment significantly (ap ≤ 0.05) increased serum con- entrations of biochemical parameters: creatinine, blood urea itrogen, total protein, albumin, �-glutamyltransferase, alkaline hosphatase, alanine aminotransferase and aspartate aminotrans- erase as compared to control. However, interestingly per oral dministration of W. somnifera (NP and NC) significantly (bp ≤ 0.05) educed elevated serum concentrations of biochemical parameters y GM (Table 2). ffect of Withania somnifera on renal electrolytes Restorative effect of W. somnifera on GM induced serum elec- rolytes (Na+, K+ and Ca2+) changes depicted in Table 3. W. somnifera NP and NC) and GM treated group did not show any appreciable hanges in Na+ concentrations as compared to control. However, + and Ca2+ levels were significantly (ap ≤ 0.05) reduced in GM reatment as compared to control. Intriguingly, administration of lcoholic extract of W. somnifera significantly (bp ≤ 0.05) moder- ted effects of GM by protecting serum concentrations of K+ and a2+. able 2 ffect of Withania somnifera on GM induced serum biochemical changes. Results of renal b ats. Withania somnifera treatment groups, nephroprotective and nephrocurative significa Group Cr (mg dl−1) BUN (mg dl−1) Total Prot. (g dl−1) ALB (g dl−1 Control 0.32 ± 0.01 34.09 ± 1.09 7.12 ± 0.08 3.05 ± 0.0 GM 0.90 ± 0.04a 78.17 ± 2.45a 9.91 ± 0.59a 3.74 ± 0.0 NP 0.37 ± 0.01b 37.42 ± 0.35b 7.46 ± 0.05b 3.14 ± 0.0 NC 0.40 ± 0.02b 39.92 ± 0.81b 7.85 ± 0.11b 3.16 ± 0.0 NP 140.50 ± 1.48 6.33 ± 0.78b 7.21 ± 0.10b NC 140.75 ± 1.84 5.68 ± 0.87b 7.16 ± 0.20b Effect of Withania somnifera on renal tissue enzymes The effect of W. somnifera on GM-induced changes in lipid per- oxides (LPO), superoxide dismutase (SOD) and glutathione (GSH) kidney tissue is depicted in Table 4. Lipid peroxide concentration significantly (ap ≤ 0.05) increased in GM treatment, whereas SOD and GSH levels significantly (ap ≤ 0.05) reduced as compared to control. Administration of alcoholic extract of W. somnifera signifi- cantly (bp ≤ 0.05) subverted nephrotoxic effects of GM by reducing LPO and increasing SOD and GSH concentrations. Effect of Withania somnifera on renal histopathological changes Gross examinations of kidneys in GM treated animals were evi- dently pale and swollen in character (Fig. 3A). Inquisitively, W. somnifera with its antioxidant and rejuvenate function effectively reduce the renal lesions of GM in nephroprotective and nephrocu- rative treatment (Fig. 3B and C). Haematoxylin and eosin (H and E) staining of kidney in GM treatment showed an extensive and marked congestion, interstitial haemorrhages and tubular necrosis (Fig. 3A). On the other hand, tubular injuries were also markedly reduced in nephroprotective treatment (Fig. 3B). However, histo- logical lesions in nephrocurative treatment showed minimal to mild interstitial nephritis with slightly detached (flattened) but otherwise normal tubular cells (Fig. 3C). Discussion Gentamicin is a bactericidal broad-spectrum antibiotic, com- monly used in veterinary practice to treat acute serious infections. Despite of therapeutic application, its usage is limited due to its iochemical parameters significantly (ap ≤ 0.05) increased in gentamicin treatment ntly (bp ≤ 0.05) reduced the biochemical changes in gentamicin induction (n = 6). ) GGT (IU l−1) ALP (IU l−1) ALT (IU l−1) AST (IU l−1) 6 2.52 ± 0.10 140.2 ± 2.11 46.29 ± 1.30 56.59 ± 1.60 9a 5.40 ± 0.23a 256.51 ± 5.21a 75.84 ± 3.70a 83.01 ± 1.47a 5b 3.09 ± 0.03b 141.32 ± 1.45b 47.61 ± 1.32b 58.77 ± 1.32b 3b 3.23 ± 0.10b 143.54 ± 1.30b 50.87 ± 1.00b 59.76 ± 0.94b 238 P.K. Govindappa et al. / Revista Brasileira de Farmacognosia 29 (2019) 234–240 Table 4 Effect of Withania somnifera on gentamicin induced renal tissues enzyme changes. Results of LPO and GSH and SOD significantly (ap ≤ 0.05) altered in renal tissues of gentamicin treatment. Withania somnifera treatment groups, nephroprotective and nephrocurative significantly (bp ≤ 0.05) attenuated altered renal enzymes (n = 6). Group LPO (nM MDA g−1 of tissue) GSH (�M GSH g−1 of tissue) SOD (U g−1 of tissue) Control 71.85 ± 1.08 71.74 ± 3.73 876.83 ± 0.32 GM 126.71 ± 1.45a 37.38 ± 4.06a 574.87 ± 0.36a NP 73.83 ± 1.56b 60.77 ± 3.66b 843.34 ± 0.68b NC 80.59 ± 0.51b 58.79 ± 4.67b 806.98 ± 0.71b A B C Gentamicin Nephroprotective Nephrocurative G ro ss le si on s M ic ro sc op ic le si on s Fig. 3. Effect of Withania somnifera on renal gross and histopathological changes in gentamicin treatment. Gross lesions of kidney shows diffused pale and swollen condition in gentamicin treated animals (A), which are effectively ameliorated in nephroprotective (B) and nephrocurative (C) treatment of W. somnifera. (A) Haematoxylin and eosin ( rrhag ( roprot s s h i e v m f 2 i f g a m 2 p h t h ( a c h t ( l a a t r b H and E) staining of kidney sections shows marked congestion, interstitial haemo ×200). These lesions were effectively ameliorated in W. somnifera treatment, neph lightly flattened tubular cells observed (n = 6). evere acute nephrotoxicity (Ahn et al., 2012). Several studies ave reported that oxygen-free radicals are prime offenders of GM nduced acute renal failure (Karahan et al., 2005). However, the xact mechanism by which GM induces renal damage is elusive. Among therapeutic antioxidants, deferoxamine, methimazole, itamin E, vitamin C, diethyl dithiocarbamate, l-histidinol and thy- oquinone are extensively studied to protect renal physiological unction (Karahan et al., 2005; Yaman and Balikci, 2010; Tavafi et al., 012). Uncertainly, none of these compounds has proved to be clin- cally efficient in patients. Recently, with this concern attention is ocused on natural antioxidant sources that are able to ameliorate entamicin-induced nephrotoxicities. These natural antioxidants ct as an alternative medicine to synthetic antioxidants, which ay cause serious or deleterious adverse effects (Sodimbaku et al., 016). W. somnifera is such a plant containing rich antioxidant princi- les to treat renal disorders. In support to our findings, investigators ave shown that hydromethanolic extract of W. somnifera exerts herapeutic efficiency in regulation of lead nitrate induced nep- rotoxicity in Swiss albino mice and that could be attributed to i) presence of natural antioxidants (ii) its free radical scavenging nd antioxidant properties and (iii) excess removal of urea related ompounds (Davis and Kuttan, 2000; Jayaprakasam et al., 2003). It as also been reported that the root extract of W. somnifera tends o reverse the changes in lipid peroxidation and damage to cells Dhuley, 2000). With these scientific backgrounds, we have iso- ated and quantified biologically active compounds of W. somnifera s withanolides (withanolide A, withanolide B, withanoside IV-V nd 12-deoxy withastramonolide and withaferin A) by HPLC. After hat, we focused to understand renal protective and renal curative ole of W. somnifera in Wistar rats. In our findings GM treatment significantly decrease the live ody weight of rats and similar findings were reported in rats es, desquamated epithelial cells and tubular necrosis in gentamicin treated group ective group (×100) (B), as compared to nephroprotective (×200) (C) group, where administered with GM (80 mg kg−1 IM) for six consecutive days (Ali et al., 1992). This could be due to cumulative accumulation of GM in renal tissues, which leads to decrease in food intake and body weight due to consequence of renal injuries. This impair- ment causes subsequent loss of tubular cells to reabsorb water that leads to dehydration and loss of body weight. Unfortunately, urine volume of rats was not measured in this experiment. However, interestingly there were no significant alterations in kidney and body weight ratios of rats treated with W. somnifera in NP and NC group. The alleviation of GM-induced live body weight reduction denotes the palliative effect of W. somnifera on nephrotoxicity. Serum creatinine and BUN are well-known biomarkers to detect early phase of renal damage and in chronic renal damage, the ele- vated levels of total protein, ALP, AST and ALT are remarkable (Lopez-Giacoman and Madero, 2015; Abuelezz et al., 2016; Teo and Endre, 2017). In our study, GM (80 mg kg−1 b.wt., i.p.) signif- icantly increased the levels of creatine, blood urea nitrogen, total protein, ALP, AST and ALT levels in serum. Additionally, GGT lev- els also increased in serum due to damage in renal brush border of epithelial cells in the proximal tubule and it is a sensitive indi- cator of GM toxicity (McMahon and Waikar, 2013). Intriguingly, the concentration of serum biochemical parameters significantly reduced in W. somnifera treatment. This could be due to antioxidant properties of withanolides viz., withanolide A-B, withanoside IV- V, 12-deoxy withastramonolide and withaferin. These alterations in biochemical parameters were well correlated with the renal histopathological lesions (Bhattacharya et al., 1997; Jeyanthi and Subramanian, 2009). Gentamicin, independently of cell injury, inhibits variety of cell membrane transporters of both the brush border and the baso- lateral membranes leading to electrolyte abnormalities. Transport inhibition not only affects tubular reabsorption but also compro- mises cell viability, which ultimately results in necrosis or apoptosis eira de ( t n t r c m o n o b a c r T s w p a 2 i t l 2 m i A p ( i a r e l g k H r G c w w o S n m n C t i t a A t P.K. Govindappa et al. / Revista Brasil Rai et al., 2003; Raghavan and Weisz, 2016). The significant reduc- ion in calcium and potassium concentration and simultaneous on-significant slight increase in sodium ions observed in GM reatment. GM probably by antagonizing calcium ions reduces the elease of acetylcholine from the motor nerve endings and produce urare-like effects (Lee et al., 2013; Rhee et al., 2015). This could itigate forced motor activities in rats. Nephroprotective effects f W. somnifera shows improved ameliorative action on the kid- eys as compared to their nephrocurative effects. The observations f Jeyanthi and Subramanian (2009); also supports our findings y reporting the ameliorative effect of W. somnifera on electrolyte bnormalities in GM intoxicated rats. Several investigators have reported that, aminoglycoside as a lassic antibiotic capable of causing nephrotoxicity by inducing eactive oxygen species (Randjelovic et al., 2017; Perazella, 2018). he elevated level of MDA (a marker of lipid peroxidation in tis- ues) results in the reduction of polyunsaturated fatty acid content, hich serves as substrate of free radicals particularly, hydrogen eroxide and superoxide. These aggravated free radicals affect ntioxidant functions of SOD, catalase, GSH and GPx (Niki et al., 008). Thus, there is an imbalance between oxidant and antiox- dant status leading to cellular damage. Other report also shows hat, cationic interaction of aminoglycosides with anionic phospho- ipid (kidneys) induces nephrotoxicity (Wiland and Szechciński, 003). By following ionic interaction process, GM as a catalyst ight form chelation complex with iron (iron-GM complex), to nduce oxidative free radicals in renal tissues (Yanagida et al., 2004). ccordingly, our result also shows an increase in lipid peroxidation roducts (MDA) and decrease in nonenzymic (GSH) and enzymic SOD) antioxidants in renal tissues of GM treatment. However, nterestingly W. somnifera retains MDA, GSH and SOD levels in NP nd NC groups and is probably due to (i) detoxification of potential eactive oxygen species and (ii) restoration of non-enzymatic and nzymatic antioxidant functions. These oxidative changes well correlated with the renal histo- ogical changes, by revealing interstitial haemorrhages, epithelial ranular degeneration and marked tubular necrosis in GM treated idney (Sayed-Ahmed and Nagi, 2007; Yaman and Balikci, 2010). owever, co-administration of W. somnifera effectively reverse enal damage with marked reduction in tubular damage induced by M. Overall, our result shows that renal injury induced by gentami- in is a manifest of elevated MDA, total protein, BUN and Cr levels ith decreased electrolytes and antioxidant enzymes. W. somnifera ith its antioxidant function significantly alleviates adverse effects f GM. tudy limitation We showed the ameliorative effect of W. somnifera extracts on ephrotoxicity. However, more studies are required to understand echanistic pathways of specific bioactive compounds of W. som- ifera on gentamicin induced nephrotoxicity. onclusion The present study specifies nephroprotective and nephrocura- ive medicinal values of W. somnifera root extract. Hence, validates ts traditional use as an ethnomedicine to cure renal ailments. In he near future, bioactive compounds of W. somnifera could appear s a novel biomolecule to protect oxidative stress. uthor’s contribution All authors contributed significantly to the work presented in his manuscript. YPS and VG supervised the whole experiments. Farmacognosia 29 (2019) 234–240 239 PKG and VG conceptualized the project and performed the experi- ments. PKG, VG, SMT and RHL organized, analyzed and interpreted the results. PKG and SMT wrote the manuscript draft. RHL and SMT read the manuscript and provided critical assessment and concep- tual insights. Conflicts of interest The authors declare no conflicts of interest. Ethical disclosures Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regula- tions of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Dec- laration of Helsinki). Confidentiality of data. The authors declare that no patient data appear in this article. Right to privacy and informed consent. The authors declare that no patient data appear in this article. 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http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30633-1/sbref0175 Effect of Withania somnifera on gentamicin induced renal lesions in rats Introduction Material and methods Plant materials and alcoholic extraction Quantification of bioactive compound using high performance liquid chromatography (HPLC) Animal procurement and care Acute oral toxicity study of Withania somnifera Experimental protocol Forced motor activity Relative kidney weight Serum and tissue sampling Assessment of electrolytes Assessment of oxidative stress Histopathology Statistical analysis Results Quantification of withanolides by HPLC Effect of Withania somnifera on body weight and relative kidney weight Effect of Withania somnifera on forced motor activity Effect of Withania somnifera on renal biochemical parameters Effect of Withania somnifera on renal electrolytes Effect of Withania somnifera on renal tissue enzymes Effect of Withania somnifera on renal histopathological changes Discussion Study limitation Conclusion Author's contribution Conflicts of interest Ethical disclosures Protection of human and animal subjects Confidentiality of data Right to privacy and informed consent Acknowledgements References