Preparation of hydroethanolic extract of Tephrosia purpurea
The plant was identified as Tephrosia purpurea (Linn.) pers. and a Voucher no: GGV/BOT/H/FAB/DKS/210 was obtained at Botany Department, Guru Ghasidas University, Chhattisgarh, India. Whole plant of Tephrosia purpurea was collected form Guru Ghasidas University campus and air dried in shade for 3 weeks and pulverized. 15 grams of pulverized sample was extracted by Soxhlet apparatus (worm method) for 30 minutes using 70% (v/v) ethanol in water. The hydroethanolic extract was concentrated in a rotary evaporator and lyophilized. The yield of the extract (2.9g, 19.33%) was obtained preserved for further use.
Reverse phase-HPLC instrumentation and chromatographic conditions
The isocratic reverse phase HPLC analysis was carried out in a Shimadzu HPLC system (LC-20AD) consisted with double pumps (LC 20-AD), column oven (CTO-20AC), sample injector system (SIL-20ACHT) and a spectrophotometric detector (SPD-20A) with a computer assisted analysis software (Shimadzu LC solution 1.25sp1). The stationary phase used for this analysis was a reverse-phase C18 column (150mm × 4.6 mm, 5 𝜇m) and the mobile phase consisted of aqueous acid solution (0.5 M phosphoric acid) : methanol (50 : 50 v/v) mixture with a stable flow rate of 0.9 mL/min. The mobile phase was filtered through 0.45 μm membrane filter and degassed by ultrasonic bath prior to use. The temperature of the column was maintained at room temperature (25 ± 2ºC). 20 𝜇L of each sample were filtered through a 0.22 μm nylon membrane before injecting into the HPLC system for analysis. The presence of bioactive phytochemicals including tannic acid, ascorbic acid, catechin, gallic acid, caffeic acid, rutin, vanillic acid, morin, malic acid, quercetin and ellagic acid were investigated. Presence of these compounds were confirmed by comparing chromatogram peaks of plant extract with reference standards by comparing their retention time. Stock solutions of 40 ppm for all the standard references and 2000 ppm for plant extract were prepared in mobile phase. The detection wavelength was 280 nm for tannic acid, 265 nm for ascorbic acid, 276 nm for catechin, 220 nm for gallic acid, 324 nm for caffeic acid, 260 nm rutin, 220 nm for vanillic acid, 370 nm for morin, 230 nm for malic acid, 256 nm for quercetin and 368 nm for ellagic acid. The detection wavelength for Tephrosia purpurea extract was 269 nm. The absorption spectra were determined by UV-VIS spectroscopy.
UV–Vis spectra analysis
Hydroethanolic (70% ethanol in water) extract of Tephrosia purpurea at 1000 ppm concentration was observed by UV–Vis spectroscopy within the ranges between 200 nm and 800 nm to identify phytoconstituents containing σ-bonds, π-bonds and lone pair of electrons, chromophores and aromatic rings present in it.
Particle size analysis (PSA)
The crude plant extract, having various particle size ranges were determined using particle size analyzer (Shimadzu’s SALD- 2300). Sample was dissolved in 20 ml 70% ethanol followed by sonication for 10 minutes. Particle sizes were measured based on the time dependent scattering of laser light by the particles with a pump speed of 5.0 cm3/min.
Thermogravimetry analysis (TGA)
The changes in mass of Tephrosia purpurea extract over the time with changes in temperature were assessed. Dynamic thermogravimetric curves of extract were obtained by thermogravimetric analysis with Shimadzu’s TGA -50, with 10°C/min heating rate ranging from 25°C to a temperature of 800°C under nitrogen atmosphere with a flow of 100 mL/min. The plant extract was packed in a platinum crucible and subjected to analysis. Calcium oxalate monohydrate was used for calibration of the instrument to characterize weight loss steps.
Differential scanning colorimetry (DSC)
The DSC curves of Tephrosia purpurea were obtained using a Shimadzu calorimeter, model Shimadzu’s DSC-60 Plus. The instrument was coupled to a photovisual system and an oven with a temperature range of 20-300°C. The whole analysis was done under nitrogen atmosphere with constant flow of 100 ml/min and a heating rate of 5°C/min. The plant extract was packed in alumina crucible for analysis.
X-ray diffraction (XRD)
The structural analysis of Tephrosia purpurea extract was carried out by using X ray diffraction techniques on Rigaku’s Mini Flex 600 diffractometer. The Tephrosia purpurea extract was subjected to Cu-Kα radiations (λ=0.154056 nm, 2θ= 2-90°) operating at a voltage of 40 kV and a current of 15 mA, at 10°C/min speed at a drive axis of 2θ. The average size of the crystallite (D in nm) was also calculated from the XRD peak using Debye Scherrer’s relation [17, 18].
Where, k= 0.9 is the Scherrer’s constant, λ= 1.540562Å is wavelength of X-rays and β (in radian) is the full width of half maxima (FWHM) of the diffraction peak at an angle θ.
Fourier transform infrared spectroscopy (FT-IR)
Functional groups are responsible for medicinal properties of plants. The whole plant extract of Tephrosia purpurea was subject to FT-IR analysis on a Perkin Elmer’s Spectrum Two using KBr stressed disks within a spectral range of 4000–450 cm−1 to determine presence of possible functional groups.
Experimental animals
Experimental animals (Wistar rats, 150±10g) were purchased from Defense Research and Development Establishment, Gwalior, and kept in departmental animal house for 15 days to get acclimatized. Animals were exposed to a 12 h periodic light and dark cycle with a constant temperature about 25°±2°C. Rats were provided with palleted rat diet and water ad libitum.
Animal right
Animal care and experimental procedures were carried out by sincerely following the guidelines set by the committee for the purpose of control and supervision of experiments on animal, India. The experimental procedure was approved by institutional animal ethics committee (994/Ere/Go/06/CPCSEA) on 01/08/2016 (Ref:162/IAEC/Pharmacy/2016).
Chemicals
Pure and analytical grade chemicals were procured from Sisco Research Laboratories Pvt. Ltd (SRL) and Himedia Laboratories Pvt. Ltd., India in this study. LPS (Lot # 114M4009V) was procured from Sigma Aldrich Co Ltd, USA whereas D-GalN (Batch # 3647503) was procured from SRL, India.
D-GalN and LPS model of acute multiorgan injury
The D-GalN and LPS was prepared in saline (0.9% NaCl in water). Acute organ injury was induced by D-GalN (300 mg/2ml/kg) and LPS (50µg/2ml/kg) administered intraperitonially as per previous scientific literature [19].
Preparation of different doses of Tephrosia purpurea extract
Freeze dried plant extract was dissolved in distilled water using 1% (w/v) gum acacia as the suspending agent [19]. Three different doses of Tephrosia purpurea (50 mg/kg, 100 mg/kg and 200 mg/kg) were taken for evaluation where the highest dose (200 mg/kg) corresponds to 1/10th of the LD50.
Experimental design
Well acclimatized thirty-six animals were divided equally into six groups. Group I: designated as control, received 1% gum acacia as vehicle. Group II: designated as TP per se, received 200 mg/kg dose of Tephrosia purpurea extract. Group III: designated as D-GalN+LPS (experimental control), received 300 mg/kg dose of D-GalN followed by 50 µg/kg dose of LPS. Group IV-VI: designated as TP50, TP100 and TP200 respectively, received 50, 100 and 200 mg/kg dose of Tephrosia purpurea extract respectively along with D-GalN and LPS. Vehicle and different doses of Tephrosia purpurea were given orally for 6 days straight to their respective groups. The D-GalN followed by LPS (only after 1 hour of D-GalN) were given intraperitonially to group III to VI on the 6th day evening. All the animals were fasted overnight and sacrificed 16 hours after D-GalN and LPS treatment using diethyl ether. To generate an individual data point in each of the independent experiments, samples were used in triplicates.
Hematological study
Blood was collected by puncturing retro-orbital venous sinus into heparinized tubes and kept at 4°C for hematological studies. Analysis of blood for determining white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), hemoglobulin (Hb), hematocrit (HCT), lymphocyte and monocyte count was done by semi-automatic blood analyzer (Analytica HEMA 2062+) [20].
Serological study
The blood was also collected in heparin free tubes and allowed to clot at room temperature for 30 minutes. The blood was centrifuged to obtain serum for determination of serological parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, urea, uric acid, creatinine, glucose, triglyceride and cholesterol with the help of respective diagnostic kits following direction of use given on kit manual (The ERBA Chem 5 v 3 Germany).
Oxidative stress assessment in hepatic and renal tissue samples
For determination of lipid peroxidation (LPO), 10% homogenates of hepatic and renal tissues were prepared in KCl solution. The LPO in liver, kidney and microsomes were determined from quantified TBARS [21].
Antioxidant status assessment in hepatic and renal tissue samples
Sucrose solution (1%) was used to prepare homogenates to assess reduced glutathione (GSH) [22]. NaCl (0.9%) was used to prepare tissue homogenates to determine superoxide dismutase [23] and catalase activities [24].
Lipid status in hepato-renal tissue samples
To determine triglycerides [25] and cholesterol [26] in tissue, homogenates of liver and kidney were prepared in chilled hypotonic solution.
Microsomal CYP2E1 activity, protein and lipid peroxidation
Microsomes were prepared by CaCl2 precipitation method [27]. The CYP2E1 activity was determined in terms of aniline hydroxylase activity using aniline as a substrate [28]. Microsomal protein was assessed using Folin and Ciocalteu phenol reagent [29].
Histopathological preparation of hepatic and renal tissue samples
Small pieces of liver and kidney were immediately fixed in Bouin’s fixative. Fixed tissues were further processed and embedded in paraffin wax before cutting 05 mm thick sections. Sections were placed on a glass slide and stained with hematoxylin and eosin (H&E) stain [30]. Approximately 10-15 observations for each slide were taken with light microscope.
From the above-mentioned results, it can be clearly observed that, 200 mg/kg dose of Tephrosia purpurea perform better than the other two lower doses. Thus, only 200 mg/kg dose of Tephrosia purpurea treated groups was further considered for determination of antioxidant enzymes of GSH cycle, proinflammatory cytokines, electron microscopy, genotoxicity and vital parameters of RBC degradation cycle.
Enzymes of GSH cycle in hepato-renal tissue samples
Glutathione reductase [31], glutathione peroxidase [32], glucose-6-phosphate dehydrogenase [33] and glutathione-S-transferase [34] were determined from tissue homogenate freshly prepared in 1.15% KCl solution.
Assessment of proinflammatory cytokines in serum
Proinflammatory cytokines including tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6) were assessed in serum using ELISA kits (Ray Biotech, Inc; Norcross, GA 30092 USA) to assess inflammation.
RBC degradation cycle in hepatic tissue sample
Hepatic hemoglobin [35], heme content [36], ferritin [37], hemosiderin [38], iron [39], bilirubin [40], biliverdin [41], biliverdin reductase [42] and hemoxygenase-1 (HO-1) enzyme [43], were determined in freshly prepared liver homogenate.
Processing of hepatic and renal tissue samples for ultra-structural observation
Liver and kidney tissue of about 1 mm3 in size were fixed immediately in Karnovsky’s fixative and then in 1% osmium tetroxide [44]. Fixed tissues were eventually embedded in resin and were cut into 70-90 nm thick sections and placed on a copper grid. The grid along with tissue sections were stained with positive staining (Uranyl acetate and lead citrate) prior to the observation with transmission electron microscope [45, 46].
Genotoxicity study in hepato-renal tissue samples
Single cell gel electrophoresis study was performed to observe genotoxicity [47]. Liver tissue homogenate (10%) was taken on a thin film of agarose spread glass slide. Slides were placed in electrophoresis buffer to start electrophoresis. Slides were immediately washed with HCl buffer and stained with ethidium bromide to observe with a microscope. Images were analyzed by ImageJ software.
Statistical analysis
Results are expressed as mean ± SE (n=6). Data were analyzed for statistical significance using one-way analysis of variance (P≤0.05) followed by Tukey’s post hoc honestly significant difference test (post hoc HSD test) to draw a comparison among different treatment groups (P≤0.05) [48] using Microsoft Excel worksheet-2019. A p value ≤0.05 was considered as significant.