2.1. Chemicals & reagentsGallic acid (GA), pyrogallol (PG), (+)-catechin hydrate (CH), vanillic acid (VA), caffeic acid (CA), syringic acid (SA), (−)-epicatechin(EC), vanillin (VL), p-coumaric acid (PCA), trans-ferulic acid (FA), ellagic acid (EA), rutin hydrate (RH), rosmarinic acid (RA), myricetin(MC), kaempferol (KF), and quercetin hydrate (QU) used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile(HPLC), acetic acid (HPLC), methanol (HPLC), and ethanol were obtained from Merck (Darmstadt, Germany).
2.2. Plant collection and extract preparation: Fresh litchi leaves were collected from a local market of Dhaka Bangladesh. The plant specimen was authenticated from NationalHerbarium situated in Mirpur, Dhaka and a voucher specimen was also deposited there for reference purpose in future (DACB 47694). The leaves were shade-dried and ground into fine powder. The crude ethanol extract was prepared by maceration of the powder with ethanol (100%)at room temperature (27 °C) and the solvent (ethanol) was evaporated in a rotary evaporator (at 40 °C). The sticky dark-greenish crude extract was obtained after the evaporation of the solvents. Finally, this extract was used for the phytochemical analysis and treatment of the experimental rats.
2.3. HPLC analysis detection and quantification of selected phenolic compounds in the litchi leaf extract were performed by HPLC-DAD analysis as demon-strated by Hossain et al. along with some alterations (Hossain et al.,2016). The process was carried out on a Dionex UltiMate 3000 system equipped with a quaternary separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). The separation was performed using Acclaim®C18(5 μm) Dionex column (4.6 × 250 mm) with a flow rate of one ml per minute at 30 °C and a volume of 20 μL for injection. Acetonitrile (solvent A), acetic acid (solvent B, pH 3.0), and methanol(solvent C) composed the mobile phase that followed a gradient elution of the following program: 5%A/95%B (0–10 min), 10%A/90%B(11–15 min), 15%A/70%B/15%C (16–25 min), 20%A/60%B/20%C(26–30 min), 30%A/40%B/30%C (31–35 min), 40%A/50%B/10%C(36–40 min), and 5%A/95%B (41–45 min).
2.4. Experimental animals: Animal protocols were reviewed and approved by the institutional animal Care and Use Committee of North South University (AEC-005–2017), Dhaka, Bangladesh. Thirty-five male Long Evans rats aging from 12 to 14 weeks (180–200 g of weight) were procured from the animal House of the Department of Pharmaceutical Sciences of the aforementioned university to conduct this experiment. All the rats were caged in a controlled room environment (temperature 22 ± 2 °C; 55%humidity; 12 h light/dark cycles) and they had free access to standard chow diet and drinking water ad libitum.
2.5. Surgery and post-surgery treatment of rats: The left renal artery was surgically clipped to establish 2K1C rat model following procedures described previously (Alam et al., 2015). Animals were allowed to recover post-surgery and 2K1C model was confirmed by the sustained elevation of blood pressure by about20 mmHg in the rats having clipped left renal artery (Alam et al., 2015). One group of animals spared from clipping served as the control (GroupI) whereas Group II to Group V animals had a surgical clipping of the left renal artery. To evaluate the in-vivo antioxidant, anti-inflammatory, and nephroprotective effects of litchi leaf, animals were then treated for a period of four weeks as follows: Group I and Group II animals received normal food and water; Group III, Group IV, and Group V animals received ethanol extract of litchi leaf at the dose of 50, 100 and 200 mg/kg, respectively. At the end of the treatment period, animals were euthanized with an intraperitoneal injection of ketamine followed by their blood withdrawal for biochemical analysis. Tissues from the heart, kidney, spleen, and liver were surgically removed as well for biochemical and microscopic evaluations.
2.5. Surgery and post-surgery treatment of rats: The left renal artery was surgically clipped to establish 2K1C rat model following procedures described previously (Alam et al., 2015). Animals were allowed to recover post-surgery and 2K1C model was confirmed by the sustained elevation of blood pressure by about20 mmHg in the rats having clipped left renal artery (Alam et al., 2015). One group of animals spared from clipping served as the control (GroupI) whereas Group II to Group V animals had surgical clipping of the left renal artery. To evaluate in-vivo antioxidant, anti-inflammatory, and nephroprotective effects of litchi leaf, animals were then treated for a period of four weeks as follows: Group I and Group II animals received normal food and water; Group III, Group IV, and Group V animals received ethanol extract of litchi leaf at the dose of 50, 100 and 200 mg/kg, respectively. At the end of the treatment period, animals were euthanized with an intraperitoneal injection of ketamine followed by their blood withdrawal for biochemical analysis. Tissues from the heart, kidney, spleen, and liver were surgically removed as well for biochemical and microscopic evaluations. kidney function markers including creatinine and uric acid; and cardiac function markers such as creatine kinase-Muscle/Brain (CK-MB) were measured according to manufacturer’s instructions (DCI Diagnostics, Hungary).
2.11. Histopathological examination for the histopathological evaluation, isolated kidney and heart tissues of the experimental rats were initially fixed in 10% NeutralBuffered Formalin (NBF) followed by their treatment with graded ethanol and xylene. Following careful embedment of these fixed, pro-cessed tissues into paraffin blocks to avoid air entry, the blocks were efficiently cut with a rotary microtome machine into thin, delicate slices of about 5 μm which were then taken on fresh slides and finally colored with hematoxylin/eosin (H & E) to evaluate any inflammatory cell infiltration in the tissues. The tissue sections were also stained withPicrosirius Red to notice and analyze any fibrotic changes. After completion of the routine staining, all the slides with stained tissue slices were snapped and evaluated meticulously using a light microscope at40X magnification (Zeiss Axioscope).
2.12. Statistical analysis: Graph Pad Prism (version 6.01) was used for statistical analyses. Values were expressed as the mean ± SEM (standard error of mean). The student’s t-test was used for comparison of paired and unpaired data, and ANOVA along with Newman-Keuls post-doc test was used for multiple comparisons.p < 0.05was considered to be statistically significant.