Pentahydroxy flavonoid isolated from Madhuca indica ameliorated adjuvant-induced arthritis via modulation of inflammatory pathways

Rheumatoid arthritis (RA) is an autoimmune disease associated with advanced joint dysfunction. Madhuca indica J. F. Gmel, from the family Sapotaceae, is an Indian medicinal plant reported to have an array of pharmacological properties. The aim of present investigation was to determine the anti-arthritic potential of an isolated phytoconstituent from methanolic leaf extract of Madhuca indica (MI-ALC) against FCA-induced experimental arthritis. Polyarthritis was induced in female rats (strain: Wistar) via an intradermal injection of FCA (0.1 mL) into the tail. Polyarthritis developed after 32 days of FCA administration. Then rats were treated orally with an isolated phytoconstituent from MI-ALC at doses of 5, 10, and 20 mg/kg. Findings suggested that High-Performance Thin-Layer Chromatography, Fourier-Transform Infrared Spectroscopy, and Liquid Chromatography-Mass Spectrometry spectral analyses of the phytoconstituent isolated from MI-ALC confirmed the structure as 3,5,7,3′,4′-Pentahydroxy flavone (i.e., QTN). Treatment with QTN (10 and 20 mg/kg) showed significant (p < 0.05) inhibition of increased joint diameter, paw volume, paw withdrawal threshold, and latency. The elevated synovial oxidative stress (Superoxide dismutase, reduced glutathione, and malondialdehyde) and protein levels of Tumor necrosis factor-α (TNF-α) and Interleukin (ILs) were markedly (p < 0.05) reduced by QTN. It also effectively (p < 0.05) ameliorated cyclooxygenase-2 (COX-2), Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kβ) and its inhibitor-α (Ikβα), and ATP-activated P2 purinergic receptors (P2X7) protein expressions as determined by western blot analysis. In conclusion, QTN ameliorates FCA-induced hyperalgesia through modulation of elevated inflammatory release (NF-kβ, Ikβα, P2X7, and COX-2), oxido-nitrosative stress, and pro-inflammatory cytokines (ILs and TNF-α) in experimental rats.

www.nature.com/scientificreports/ tion phase (acetone: n-hexane (0.5:9.5)) at a flow rate of 2 mL/min. HPTLC analysis of various fractions (A-H) was performed with the following reagents: • A stock solution of standard (quercetin) at 1000 µg/mL with a final concentration of 50 ng/µL • Mobile phase of Toluene: Ethyl Acetate: Formic Acid (6: 3.5: 0.5 v/v/v) The drug was observed to show a considerable absorbance at 370 nm. Among all the fractions, active compound isolation was carried out in fraction D using preparative HPTLC. Furthermore, FT-IR and LC-MS spectroscopy were used to elucidate the chemical structure of the isolated compound.
Adjuvant-induced polyarthritis (AIA). FCA (0.1 mL, intradermal) was injected into the tail of the rats to induce AIA in female rats (150-180 g; 5 groups, i.e., group II to VI, n = 18 each group) 19 . Following the injection, 32 days were allowed for development of arthritis. A separate group of rats (group I, n = 18) was maintained as normal and did not receive FCA. After the development of AIA (32 days), animals received either dimethyl sulfoxide (DMSO) (0.5%, 10 mg/kg, p.o. in group I and II) or leflunomide (10 mg/kg in 0.5% DMSO, as a standard in group III) or QTN (5, 10 and 20 mg/kg in 0.5% DMSO in group IV to VI) for the next 28 days 14 . A solution of leflunomide or QTN was freshly prepared daily in 0.5% DMSO and administered to rats by oral gavage (1 mL) according to their dose based on body weight of rats.
Plethysmometer (UGO Basile Italy) was used to determine paw volume 20 . Pain latency against mechanical hyperalgesia (paw withdrawal threshold) was determined by using Randall-Selitto, i.e., paw pressure test (Ugo Basile Model 7200) as well as von Frey hair application 20 . Hargreaves apparatus (UGO Basile, SRL Biological Research Apparatus, Italy) was used to determine paw withdrawal latency 21,22 . Biochemical estimation. On the last day of study (day 60), rats were anesthetized with ether and retroorbital plexus was used to withdraw blood. After blood withdrawal, rats were sacrificed by cervical dislocation. Immediately synovial tissues were isolated and stored at − 70 °C. Levels of ESR (Erythrocyte Sedimentation Rate), CRP (C-reactive protein), WBC (White blood cell), Hb (hemoglobin), RBC (Red blood cell), and PLT (platelets) were estimated in blood 20 , whereas turbidity, albumin, AST (aspartate transaminase), ALP (alkaline phosphatase), ALT (alanine transaminase), and TC (total cholesterol) were measured in serum using reagent kits (Accurex Biomedical Pvt. Ltd., Mumbai, India) 23 .
Determination of synovial pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) levels. Pro-inflammatory cytokine levels, i.e., TNF-α, IL-1β, and IL-6 of synovial tissue (n = 5-6) were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's protocol (Bethyl Laboratories Inc., Montgomery, TX, USA). TNF-α, IL-1β, and IL-6 were determined from a standard curve for the combination of these cytokines. The concentrations were expressed as pg/mg of protein. Briefly, the quantifications of TNF-α, IL-1β, and IL-6 were done in accordance to the protocol provided with Bethyl Laboratories Inc Rat TNF-α, IL-1β, and IL-6 immunoassay kit. Rat TNF-α, IL-1β, and IL-6 immunoassay was a 4.5 h solid-phase ELISA designed to measure rat TNF-α, IL-1β, and IL-6 levels. The assay employed a sandwich enzyme immunoassay principle. A monoclonal antibody specific to rat TNF-α, IL-1β, and IL-6 was pre-coated on the microplates. Standards, control, and samples were pipetted into the wells, and any rat TNF-α, IL-1β, and IL-6 present in the sample was thus bound by the immobilized antibody. After washing away the unbound substance, an enzymelinked polyclonal antibody specific to rat TNF-α, IL-1β, and IL-6 was pipetted into the microtitre wells. Any unbound antibody was washed off, and then a substrate solution was added to the wells. The enzymatic reaction produced a blue product that turned yellow when the stop solution was added. The intensity of the color generated was measured, which was proportional to the amount of rat TNF-α, IL-1β, and IL-6 bound in the initial steps. A standard curve was run on each assay plate using recombinants of the TNF-α, IL-1β, and IL-6 in serial dilutions. The sample values were then read, and calculations were made according to the standard curve. Values were expressed as means ± SEM. The levels of TNF-α, IL-1β, and IL-6 were expressed as units per mL.
Determination of synovial protein expressions of COX-2, Ikβα, NF-kβ, and P2X7. The protein expressions of COX-2, Ikβα, NF-kβ, and P2X7 in synovial tissue (n = 4) were determined using Western blot analysis according to a method reported elsewhere 24 . Briefly, synovial tissue was sonicated in tissue protein extraction reagent (Thermo Fisher Scientific, Inc.). The lysates were centrifuged at 10,000 xg for 10 min at 4 °C. Protein concentration was determined using a Bicinchoninic Acid (BCA) assay kit (Beyotime Shanghai, China) on ice for 30 min. Equal amounts of extracted protein samples (50 μg) were separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk at 37 °C for 1 h and incubated overnight at 4 °C with the primary antibodies that recognized COX-2, Ikβα, NF-kβ, P2X7 and GAPDH. Anti-rabbit horseradish-linked IgG was used as the secondary antibody, which was incubated at 37 °C for 2 h. Protein bands were visualized using Chemiluminescent kit (Bio-Rad Laboratories, Inc.) and GAPDH served as the loading control. www.nature.com/scientificreports/ Histopathology of tibiotarsal joint. Ankle joints from three rats of each group were separated, cleaned, washed in cold physiological saline, and preserved in 10% formaldehyde solution until histopathological studies. At the time of staining, sections of tibiotarsal joints were cut (5 μm thickness) with the help of microtome, deparaffinated, and stained using hematoxylin and eosin (H and E) stain. The specimens were mounted on slides using Distrene Phthalate Xylene (DPX). Sections were examined under a light microscope (Olympus DP71, DP-BSW Ver.03.03, Olympus Medical Systems India Private Limited, India) to obtain a general impression of the histopathological features of the specimen and infiltration of cells in epithelium and sub-epithelium. The intensity of histological aberrations in the tibiotarsal joint was graded as Grade 0 (not present)l; Grade 1 (slight/minimal); Grade 2 (mild); Grade 3 (moderate) or Grade 4 (severe) as described in the literature 25 .
Statistical analysis. GraphPad Prism 5.0 software (GraphPad, San Diego, CA) was used to perform data analysis. Data are expressed as mean ± standard error mean (SEM) and analyzed using One-Way ANOVA followed by Tukey's multiple range post hoc analysis (for parametric tests including body weight, joint diameter, paw volume, paw withdrawal latency, paw withdrawal threshold, serum AST, ALT, ALP, albumin, CRP, rheumatoid factor, hematological parameters, ESR, antioxidant parameters, cytokines levels and protein expressions (COX-2, Ikβα, NF-kβ, and P2X7) or Kruskal-Wallis test for post hoc analysis (non-parametric tests, i.e., histopathology score). A value of p < 0.05 was considered to be statistically significant. Body weight, paw volume, joint diameter, paw withdrawal threshold, and latency. Body weight markedly decreased (p < 0.05), whereas joint diameter, paw volume, paw withdrawal threshold, and latency increased significantly (p < 0.05) in AIA control rats compared to normal rats. Treatment with leflunomide (10 mg/kg) effectively inhibited (p < 0.05) FCA-induced alterations in body weight, paw volume, joint diameter, paw withdrawal threshold, and latency compared to AIA control rats. QTN (10 and 20 mg/kg) treatment predominately increased (p < 0.05) body weight and significantly decreased (p < 0.05) paw volume, joint diameter, paw withdrawal threshold, and latency when compared with AIA control rats. (Table 1 and Fig. 2).
AST, ALT, ALP, albumin, CRP, and rheumatoid factor. When compared to normal rats, serum ALT, AST, ALP, and CRP markedly (p < 0.05) increased, whereas serum albumin level significantly (p < 0.05) decreased in the AIA control rats. Leflunomide (10 mg/kg) treatment effectively (p < 0.05) increased albumin level in serum whereas it markedly (p < 0.05) reduced levels of ALT, AST, ALP and CRP in serum compared to AIA control rats. The FCA-induced alterations in serum ALT, AST, ALP, CRP, and albumin levels were significantly (p < 0.05) attenuated by QTN (10 and 20 mg/kg) when compared with AIA control. These alterations in levels of serum AST, ALT, ALP, albumin, and CRP were more effectively (p < 0.05) attenuated by leflunomide (10 mg/kg) when compared to QTN treatment. ( Table 2).
The subplantar administration of FCA resulted in a marked increase (p < 0.05) in the rheumatoid factor in AIA control rats compared to normal rats. As compared to AIA control rats, leflunomide (10 mg/kg) and QTN (10 and 20 mg/kg) treatment significant decreased (p < 0.05) rheumatoid factor. (Table 2).
Hematological parameters and ESR levels. A significant decrease (p < 0.05) in RBCs and Hb levels, whereas a marked increase (p < 0.05) in WBCs, platelet, and ESR levels were observed in AIA control rats when compared with normal rats. Treatment with Leflunomide (10 mg/kg) effectively ameliorated (p < 0.05) hematological and ESR variations when compared with AIA control rats. QTN (10 and 20 mg/kg) significantly increased (p < 0.05) levels of RBCs and Hb whereas effectively reduced (p < 0.05) WBCs, platelet and ESR levels when compared with AIA control rats. However, leflunomide (10 mg/kg) more significantly (p < 0.05) attenuated FCA-induced hematological and ESR variations as compared to QTN treatment. (Table 3).

Discussion
Madhuca indica is a traditional medicine rich in various phytoconstituents dominant with the presence of flavonoids. It has been widely used to manage various inflammatory disorders due to its inhibitory potential against histamine, serotonin, prostaglandin, and COX-2 16 . In the present study, we have assessed the anti-arthritic effect of an isolated phytoconstituent (3,5,7,3′,4′-Pentahydroxy flavone, i.e., QTN) from Madhuca indica Leaves on female Wistar rats after subplantar administration of FCA. The findings of the present investigation suggested that methanolic extract from Madhuca indica leaves exhibit anti-arthritic activity and its phytoconstituent 3,5,7,3′,4′-Pentahydroxy flavone (i.e., QTN) is responsible for its anti-arthritic potential. Furthermore, QTN exerts its anti-arthritic potential via attenuation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), oxidonitrosative stress, and NF-kβ, Ikβα, COX-2, and P2X7 expressions. Results of present investigation are in line with the findings of previous investigators where administration of Pentahydroxy flavone, i.e., quercetin ameliorated collagen-induced arthritis 26 and adjuvant arthritis 27 in experimental animal models. www.nature.com/scientificreports/ Previous studies showed that elevated paw thickness is an evidence of arthritis induction 2,7,28,29 . Determination of paw thickness using a plethysmometer is a well-established and standardized method during AIA-induced arthritis 2,7,29 . In the present study, the inflamed rat's edematous hind paw was estimated using a plethysmometer. It was further subjected to a constant force to assess the pain threshold that was examined by the Randall-Selitto assay method. Treatment with QTN significantly decreased paw thickness, which might be due to inflammatory mediator inhibition suggesting its anti-inflammatory property against FCA-induced arthritis. This potential of cytokine blockage in pain nervous fibers by QTN might be responsible for increased pain threshold testifying its analgesic effect. The presence of flavonoid moiety in the methanolic extract of Madhuca indica could be responsible for its anti-inflammatory, analgesic, and anti-nociceptive activities. The findings of the current study corroborate the results of earlier investigators where Madhuca indica phytoconstituents showed anti-inflammatory potential via reduction of TNF-α and IL-1β 13 .
Oxidative stress plays a central role in the activation and maintenance of painful arthritis 30 . It has been reported that increased production of ROS (reactive oxygen species) such as hydroxyl, hydrogen peroxide, and superoxide radicals contribute to elevated oxidative stress 4,7,20 . This elevated oxidative stress further decreased protective antioxidant moieties (GSH and SOD) that caused elevation of MDA (lipid peroxidation), thus damaging biomembrane macromolecules 22,31,32 . The FCA administration caused a marked reduction in synovial GSH and SOD, whereas the MDA level increased effectively. However, treatment with QTN significantly attenuated FCA-induced decreased GSH and SOD in the synovial tissue suggesting its antioxidant potential that might support its anti-arthritic mechanism. These findings are in accordance with the results of an earlier study where QTN isolated from Madhuca indica was shown to exert its potential via attenuation of oxidative stress 14 .
Numerous researchers have suggested that the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) plays a vital role in the induction and maintenance of immune-inflammatory disease modulation of various biomolecules such as COX-2 and pro-inflammatory cytokines 7,33 . During the resting state, the NF-kB remains unstimulated and retains an inactive state in the cytoplasm. Whereas, IκB kinase, which is an enzyme complex, plays an essential role in the upstream NF-κB signal transduction pathway, and its phosphorylated activation leads to subsequent ubiquitination and degradation of 26S proteasome 34 . This cascade leads to NF-kB translocation to the nucleus from the cytoplasm, where it modulates the expression of various genes, including pro-inflammatory cytokines 34 . In the present investigation, activated expressions of IκBα and NF-kB were found to be significantly up-regulated in synovial of AIA control rats after administration of FCA whereas QTN treatment significantly down-regulated these expressions of pro-inflammatory cytokines via attenuation of IκBα phosphorylation and thus inactivation of NF-kB.
Purinergic Receptor-X 7 (P2X7), a protein-coding gene from the purinoceptors family, has been suggested to play a central role in the induction and maintenance of an array of diseases associated with bone cartilage such as RA 35 . It is known to play a vital role in bone remodeling via activation of various mediators, including IL-1β and prostaglandins, in the synovial fluid 35 . It has been suggested that activation of ectonucleotidases degrade extracellular ATP, which results in the formation of active molecules such as adenosine or pyrophosphates 36 . These active molecules further promote the activation of alternative macrophages and thus initiate the release of pro-inflammatory signaling 36 . Therefore, extracellular metabolism of ATP by P2X7 modulates the sequence of inflammatory influx and thus initiates the pathogenesis of RA 35 . In this view, inhibition of P2X7 receptor activation would be beneficial for the management of RA. In the current investigation, treatment with QTN significantly inhibits P2X7 activation, which might reduce the bone and cartilage damage in RA. www.nature.com/scientificreports/ Recently an array of isolated phytoconstituents from herbal origin have been implicated in the management of arthritis clinically. Studies have investigated the potential of various moieties such as Pycnogenol® from Pinus pinaster Aiton, Curcuminoids from Curcuma longa, Bromelain from Ananas comosus, etc. for the symptomatic relief of RA 37 . Furthermore, a researcher suggested that a moiety bearing carbonyl group at C-4 and a hydroxyl group at C-3 or C-5 in their structure have chelation ability with metal ions, helpful in exerting its antioxidant property 24 . In the current study, an isolated moiety from methanolic extract of leaves of Madhuca indica, i.e., QTN (3,5,7,3′,4′-Pentahydroxy flavone) also bears such hydroxyl and carbonyl groups in its structure, indicating promising antioxidant potential. Thus, QTN can be considered as a potential therapeutic moiety for the management of RA clinically.

Data availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request.