Chemicals
Collagen, Complete Freund’s Adjuvant, Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich. Dexamethasone was procured from Qualigens Fine Chemicals, India. C-RP kit (ERBA diagnostics kit), IL-17A ELISA kit (KRISHGEN Biosystem), Rat-specific MMP-1 and MMP-9 ELISA kits (Elabscience) Mouse-specific (TNF-α, IL-6) and rat-specific (TNF-α, IL-6) ELISA Kit was procured from BD Biosciences, USA. NFkB-p65 primary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA), N-(1-Naphthyl) ethylene-diamine dihydrochloride (NED) SD fine chem limited, Secondary antibody (Jackson immune).
Plant Material
G. glabra roots were obtained from Dabur India and authenticated by Dr. S. C. Singh, taxonomist (superannuated) of our institute.
Extract Preparation
The 2 Kg of G. glabra roots were cut into small parts, dried under a shaded environment, and powdered with a mechanical grinder. The powder roots were extracted with 4 litres of the ethanol-water mixture (1:1) at room temperature for 6 hours. This process was repeated for three times (4 litres X 3 times). After filtration, the combined hydroalcoholic solution was evaporated on the rota evaporator, giving 376 g of hydroalcoholic extract. This extract was subjected to acid hydrolysis in the presence of 5% HCl solution, and the resulting reaction mixture was extracted with ethylacetate which gave 81.6 g of ethyl acetate extract on concentration. The ethyl acetate extract was fractionated on silica gel (60–120 mesh size) chromatography using a mixture of ethyl acetate and chloroform solvent system as an eluent. The solvent system changes as a gradient mode from 100% CHCl3 to 25% Ethyl acetate: CHCl3. First, elution of column from CHCl3 and 5% ethyl acetate: CHCl3 solvent systems provided fractions containing an oily mixture. After that, other non-polar compounds came out at around 8–10% ethyl acetate: CHCl3 solvent system. Then isoliquiritigenin followed by liquiritigenin came out around 12–15% ethyl acetate: CHCl3 solvent system. Other polar compounds were eluted from 20–25% ethyl acetate: CHCl3 solvent system. Discarding the first oily fraction and last polar compounds containing fractions, all the middle fractions eluted between 8–15% ethyl acetate:CHCl3 solvent systems were combined and concentrated on a rota evaporator to provide 29.1 g of ISL and LTG enrich final extract, which was coded as IVT-21. The schematic representation of enrich extract preparation is given in Fig. 1.
Chemical characterization of IVT-21
Authentic Standard Compounds (ISL and LTG) were procured from Sigma-Aldrich. The HPLC analyses were carried out on Waters Alliance 2695 separation module equipped with a 2998 photodiode array detector (Waters, Milford, MA, USA). Separation was carried out using a Phenomenex C18 Luna® column (5 um; 250 x 4.6 mm). The TLC analyses were performed on silica gel TLC plates of Merck, India, with the fluorescent indicator (F254), and TLC plates were visualized by ultraviolet light, and spots were developed by vanillin–sulphuric acid spraying reagent. Column Chromatography was performed on 60–120 mesh size silica gel (Merck, India).
In-vitro study
Primary Peritoneal Macrophages Isolation From Experimental Mice
Swiss albino mice (24 ± 2) were used for the primary macrophage cells isolation Institutional Animal Ethics Committee (IAEC) approved protocols (CIMAP/IAEC/2020-23/10). 1.0 mL peptone (1%) in saline was injected in peritoneal cavity of animals. Cells were isolated by phosphate buffer saline (PBS), pH-7.4 from mice in intraperitoneal region. Cell viability was checked by trypan blue exclusion and the viable macrophages at the concentration of 1.0× 106 live cells/mL were used for the experimentation.
Anti-inflammatory profile of IVT-21 and ISL, LTG present in IVT-21
For the analysis of anti-inflammatory profile macrophages were seeded in 48 cell culture plate (0.5 million cells /well) in DMEM in 5% CO2 incubator at 37°C. Cultured primary macrophages were treated with IVT-21, LTG and ISL at dose of 3, 10 and 30 µg/mL and dexamethasone (used as a standard drug) at a dose of 1 µg/ml. After 30 minutes of treatment, primary macrophages were stimulated with LPS at dose of 0.1µg/mL. After six hours LPS stimulation, supernatants were collected and kept at -80ºC for the quantification of pro-inflammatory markers like TNF-α and IL-6 through ELISA (BD Biosciences, USA). Percentage (%) inhibition of pro-inflammatory markers was calculated as given equation:
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Cell Toxicity
Mice peritoneal primary macrophages were seeded in 96 cell culture plate (0.2 million cells /well) in DMEM (Dulbecco modified Eagle medium, Sigma) supplemented with 10% fetal bovine serum with 1× stabilized antibiotic–antimycotic solution (Sigma) in 5% CO2 incubator at 37°C. Cell viability assay of IVT-21 was studied by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium) test. Briefly, after culture of peritoneal macrophages, cells were treated with IVT-21 (10, 30 & 100 µg/mL) for 24 hours. Macrophages were incubated with MTT dye for four hours. After that the media was discarded and add 100µL DMSO and kept for 10 minutes at room temperature and measurement of absorbance taken at 550 nm (Spectramax; Molecular Devices, USA). Percentage (%) of survival = (average experimental absorbance / average control absorbance) Х 100.
In-vivo study
Wister’s rats (180–200 gram) were used in this study. All rats were obtained from institute animal house and acclimatized to experimental room for one week before experiment. All animals were ad libitum drinking water and feed with pellet animal feed and maintained under standard environmental temperature (22–24°C) and a 12 hours light/12 hours dark cycle circumstance. Animal experiments were carried out as per the appropriate procedure by the institutional animal ethics committee (IAEC) approved protocols (CIMAP/IAEC/2020-23/08) followed by the committee for the purpose of control and supervision of experimental animals (CPCSEA), Government of India (Registration no: 400/01/AB/CPCSEA).
Induction Of Rheumatoid Arthritis In Rats
Healthy and acclimatized animals were separated into 6 groups [n = 6 (male = 3 and female = 3)]. One of the groups served as normal control whereas, the other 5 groups were subjected to CIA induction. CIA was induced by the subcutaneous injection of 0.2 mL mixer of collagen and Freund’s complete adjuvant (1:1 ratio) at the base of tail (Except normal group) on days 1. Collagen was dissolved in 0.05M acetic acid (4 mg/mL) and emulsified with Freund’s complete adjuvant in equal volume. Booster dose was inserted in to the base of the tail (subcutaneous) avoiding the primary injection sites after 7 days of the primary immunization. The development of arthritis symptoms was detected after the 7–10 days of second immunization, paw and knee joint inflammation is the main visible sign of arthritis. After 3 weeks of the primary immunization animals were treated with test extract and standard drug for 21 days. Group- 1 (Normal control), group- 2 (CIA + Vehicle control) treated with 1 mL 0.7% CMC, group 3–5 orally treated with IVT-21 at dose of 30,100,300 mg /kg respectably and group 6 received dexamethasone (0.1 mg/kg) used as a standard drug.
Behavioral Assessment Of Arthritis Index And Arthritis Score
Thickness of the joint and paw was measured by screw gauge on day 0,21,28,35 and 42 and arthritis index (AI) was calculated by given equation: AI (%) = [(Thickness of paw and joints on day x - Thickness of paw and joints on day 0)/ Thickness of paw and joints on day 0] Х100. Visual monitoring of the arthritis score were performed to measure the severity of arthritis in all animals. The Arthritis scores ranged from 0 to 4 per paw, and were noted once a week for 21 days after the treatment. Scores were added and highest arthritis score was 16 per rat, with the highest arthritis score of 4 for each paw. Score 0 = no sign of arthritis, 1 = a low inflammation and/or redness, 2 = moderate inflammation and redness in joint and paw, 3 = extraordinary edema with limited joint activity, and 4 = excessive edema with difficulty in walking.
Mechanical Pain Thresholds Measurement
PAM instrument (pressure application measurement) (ugobasile, Italy; Model No.-38500) was used for the measurement of knee joint withdrawal threshold in gram force. It has a force transducer, fitted on thumb and placed on rat’s knee joint. Mechanical force (300 gram force/second) was gradually increased and applied by the operator. To avoid the tissue damage mechanical force was applied only for 5 seconds. An attempt to withdraw the joint from operator’s thumb and forefinger was taken an indication of the test end point. The limb withdrawal threshold (LWT) measurement was repeated for 3 times and mean LWT was calculated (Barton et al., 2007).
Quantification Of Inflammatory Markers
Six weeks after the first immunization, blood was collected by orbital plexus of experimental animals, and separated the serum for quantifying the inflammatory markers TNF-α, IL-6, IL-17A, MMP-1, MMP-9, C-RP, NO as per manufacture's instruction.
Histopathology And Immunohistochemistry (Ihc)
On day 42 (end of experiment day) animals were anesthetized and sacrificed, the knee joints were cut carefully, removed the skin and fixed in 10% formalin buffer. After the fixation and decalcification, the samples were processed and sectioned into 4 µm and stained with hematoxylin and eosin (H and E) staining (Suvarna et al., 2018).
Immunohistochemistry was used to measure NFкB-p65 in the knee joint tissue of experimental rats. Knee joint tissue section of five mm thickness were cut and taken on the poly- l- lysine coated slides. Tissue section were dewaxed and dehydrated then antigen retrieval and quenching step by EDTA and PBS + H2O2 (4:1) respectably. After that section were blocked with 5% BSA for 10 min and then incubated with NFкB-p65 primary antibodies (Santa Cruz, CA, USA) for 1.5 hours at room temperature. After primary antibody incubation tissue section were washed with PBS and HRP-conjugated secondary antibody (Jackson immune) was overlaid and incubated for 30 minutes at room temperature. Tissue section washed with PBS and overlaid with DAB (3´3 - diaminobenzidine) to detect antibody binding. Sections were counterstained using hematoxylin solution. The stained sections were dehydrated, mounted and microscopic analysis were carried out (Sereika et al., 2018). Staining intensity was assessed on a semi quantitative five-point scale (0 = no expression, 1 = very low expression, 2 = low expression, 3 = high expression, and 4 = very high expression) by percentage of stained cells and synovial membrane damaged area (Choy et al., 2002). Immunohistochemical/ NFkB-p65 protein expression assessment was evaluated in a blinded manner by Olympus CX21iLED light microscope.
Rna Isolation And Rt-pcr Analysis
RNA isolation and RT-PCR analysis
After anesthetized and survival dislocation hind paw of experimental rats were cut, claws and skin were dissected, then paw tissues were directly frozen in liquid nitrogen then stored at -80˚C. Paw tissues were used for quantitative RT- PCR analysis. Approximately 45-50mg tissue was cut from frozen paw and homogenized in 1mL triazole regent by tissue homogenizer and isolate the total RNA with TRI-reagent (Sigma). Purity of RNA was checked by ration of A 260 /A 280 nm using Nano Drop (Thermo Scientific). The RNA sample showing A 260 /A 280 ratio 1.95–2.05 and A 260 /A 230 ratio > 1.5 were used for synthesis of cDNA using commercially available cDNA synthesis kit (Applied Biosystems) according to manufacturer’s protocol. The Real time PCR reaction was performed with 12.5 µL of Power SYBR Green PCR master mix (Applied Biosystems) 0.8 µL of forward and reverse primers and 1.0 µL of sample cDNA. PCR reaction efficiency was optimized, and the final concentration of each primer was 0.5 µM. Real time PCR was carried out in Applied Biosystems QuantStudio™ 5 System using PCR master mix (SYBER green) the relative expression of TNFα, IL-6, IL-1 β, MMP-9, IL-17A mRNA was normalized to the amount of GADPH in the same cDNA using the relative quantification 2 − ΔΔCt method. The fold change in target gene cDNA relative to the GADPH internal control was determined using the following formula: Fold change = 2−ΔΔCt, where [ΔΔ Ct = (Ct,target gene – Ct,GAPDH) treatment − (Ct,target gene – Ct,GAPDH) Control]. Primer sequences for analysis of TNFα, IL-6, IL-1 β, COX-2, MMP-9, IL-17A and GAPDH are described in Table 1.
Acute Toxicity Study In Swiss Albino Mice
Safety evaluation of IVT-21 was done as per reported method (Babu et al., 2021) at single acute oral doses at 2000 mg/kg.
Statistical Analysis
Results are expressed as the means ± standard error of mean (SEM) and compared the statistical significance of vehicle verses treatment groups by ANOVA. Differences with a P value < 0.05 were considered significant.