Pentahydroxy avonoid isolated from Madhuca indica ameliorated adjuvant-induced arthritis: Possible role of TNF-α, IL’s, NF-kβ, Ikβα, COX-2, P2X7

Background Rheumatoid arthritis (RA) is a chronic autoimmune disorder associated with progressive joint disability. Madhuca indica J. F. Gmel (family Sapotaceae) is an Indian medicinal plant reported to have an array of pharmacological properties. Objective To evaluate the anti-arthritic activity of isolated phytoconstituent from methanolic extract of Madhuca indica Leaves (MI-ALC) and its possible mechanism of action in FCA induced experimental arthritis. HPTLC, FTIR, and LC-MS spectral analysis of phytoconstituent isolated from MI-ALC conrmed the structure as 3,5,7,3′,4′- Pentahydroxy avone (i.e., QTN). Treatment with QTN (10 and 20 mg/kg) showed signicant inhibition (p < 0.05) in FCA-induced increased paw volume, joint diameter, paw withdrawal threshold, and latency. The elevated synovial oxido-nitrosative stress and protein levels of TNF-α, IL-1β, and IL-6 were signicantly reduced (p < 0.05)by QTN. Western blot analysis revealed QNT signicantly ameliorated (p < 0.05) up-regulated NF-kβ, Ikβα, COX-2, and P2X7 protein expressions. group, *p < 0.05 as compared to AIA control and $ p < 0.05 as compared to each other. AIA: Adjuvant-induced arthritis, LF: Leunomide, QTN: 3,5,7,3′,4′-Pentahydroxy avone, RBC: Red blood cells, WBC: White blood cells, ESR: Erythrocyte Sedimentation Rate. SOD: Superoxide dismutase, GSH: Glutathione Peroxidase, MDA: Malondialdehyde, NO: Nitric Oxide, TNF-α: Tumor necrosis factor-alpha, IL’s: Interleukins.


Introduction
Rheumatoid arthritis (RA) is a chronic, autoimmune, and in ammatory disease mainly characterized by in ammation in the joint and synovial membrane, deformity, destruction of cartilage, and pain, leading to loss of joint function restricted limbs motions 1, 2 . RA shows symmetrical progression affecting metacarpophalangeal and metatarsophalangeal joints, proximal interphalangeal, ankle, and wrist 3 . This immune-in ammatory arthritis affects almost 1-2%, i.e., 20 million of the general population worldwide, and the incidence is three times higher in women between the age of 40 and 50 years than men 4,5 . It is associated with signi cantly higher lifetime costs along with a poor quality of life.
Researchers documented that the in ux of immune-in ammatory cells and the release of pro-nociceptive and pro-in ammatory mediators into joints leads to synovial in ammation 2,4 . The release of in ammatory mediators such as prostaglandins and leukotrienes, also activates primary nociceptive bers, which results in joint hyperalgesia and nociception 2 . Moreover, cyclooxygenases (COX) enzymes play a vital role in activating immune cells and the induction of in ammatory cytokines 6, 7 . Furthermore, elevated generation of free radicals, including hydroxyl radicals, reactive oxygen species (ROS), and reactive nitrogen species (RNS), have an ability to modulate the structure of membrane lipids, DNA, proteins, and cartilage 2 . Thus, the generation of free radicals is suggested as one of the important mechanisms during the pathogenesis of RA 2 . Based on such underlying mechanisms, signi cant effort has been taken during the last few decades to develop new therapeutic moieties for RA management.
The use of therapies such as Non-steroidal anti-in ammatory drugs (NSAIDs) (such as aceclofenac, ibuprofen), disease-modifying anti-rheumatic drugs (DMARDs) (such as Cyclosporin A, methotrexate), anti-CD20 and anti-tumor necrosis factor (anti-TNFα) (such as adalimumab, in iximab) are currently available remedies for the management of RA 2 . These treatments halt the progression of disease via suppression of the immunological response. However, these therapies provide symptomatic relief in the fraction of patients, as well as are associated with several side effects such as gastrointestinal discomfort, an increase in cardiovascular risk, and nephrotoxicity 2 . Thus, there is a need for conventional and alternative therapy with signi cant safety and e cacy.
Natural products, especially those derived from herbs, have contributed to the development of modern therapeutic drugs since ancient times. Herbal medicinal products target speci cally de ned mediators of in ammation and pain. The major bene t of using herbs and other natural products lies in their limited or no undesirable side effects 8 . Therefore, the interdisciplinary efforts of researchers are aimed at identifying new herbal products, and de ning their mechanisms of action has been reinforced. This has facilitated the discovery and development of safe and effective natural products to treat chronic pain 1 .
Various animal models of Adjuvant induced arthritis (AIA) have been utilized extensively to analyze the potential of such herbal moieties. Freund's complete adjuvant (FCA)-induced arthritis is one of the most employed AIA animal models of arthritis that possesses many common features with human rheumatoid arthritis 1,2,9,10 .

Animals
Female Wistar rats (150-180 g) were obtained from the animal house of Xi'an Central Hospital. The rats were housed in polypropylene cages at 24 ± 1°C with a 12h:12h dark-light cycle, free access to standard pellet feed, and ltered water. All experiments were carried out between 08:00 h and 17:00 h in a quiet laboratory. The research protocol (no. XCH202013771) was approved by the Institutional animal ethics committee (IAEC) of Xi'an Central Hospital.

Preparation, isolation, and characterization of an isolated molecule from methanolic extract of Madhuca indica
It was carried out according to a previously reported method 17 . Brie y, weighed quantity (500 g) of airdried powder (Mesh size-16) of the leaves of Madhuca indica J. F. Gmel. was macerated with distilled methanol at room temperature by soaking it with eventual stirring for 7 days and ltered. The ltrate was dried in a tray dryer maintained at 60°C. Semisolid methanolic extract of Madhuca indica J. F. Gmel. (MI-ALC) was obtained. The preliminary phytochemical screening of MI-ALC was carried out according to the earlier reported methods 17 . Phytochemical analysis of MI-ALC was performed for the identi cation of phytochemicals like alkaloids, avonoids, steroid & phenols, etc.
MI-ALC further fractionates with chloroform as per the previously reported method 17 . Brie y, the parent methanolic extract (MI-ALC) was fractionated with chloroform. A weighed quantity (20 g) of the chloroform fraction was mixed with 70 mL of acetone and subjected to column chromatography (height, 60 cm; diameter, 3 cm) eluting with a mobile phase containing acetone: n-hexane (0.5:9.5). Elution was carried at the ow rate of 2 mL/min. Their polarity order increased the polarity of the mobile phase, and fractions (50 mL each) were collected in the amber-colored bottle. The remaining loaded material in the column that cannot be eluted with the mobile phase was eluted with methanol and collected as methanol fraction and completed column chromatography. A standard stock solution of quercetin was prepared by dissolving 10 mg of the drug in 10 ml of methanol to get a concentration of 1000 µg/ml. With the help of microsyringe (100 µL capacity), 5 µL of the solution was diluted to 100 µL to get a nal concentration of 50 µg/mL (50 ng/µL). Different mobile phases containing various ratios of Benzene, Methanol, Acetone, Toluene, Formic Acid, and Ethyl acetate were examined. Finally, the mobile phase containing Toluene: Ethyl Acetate: Formic Acid (6: 3.5: 0.5 v/v/v) was selected as optimal for obtaining well-de ned and resolved peaks. Other chromatographic conditions like chamber saturation time, run length, sample application rate, and volume, the distance between tracks, detection wavelength were optimized to give reproducible Rf value for the drug. The standard stock solution was scanned over 200-400 nm, and the spectra were obtained. It was observed that the drug showed considerable absorbance at 370 nm, as shown in Supplementary File 1.
A powdered extract (20 mg) was weighed and transferred to a 10 ml volumetric ask containing about 5 ml of methanol, ultrasonicated for 5 min. The solution was ltered using Whatman paper No. 41 and further diluted to 10 ml with methanol. 1 µl volume of sample solution was applied on the HPTLC plate. After chromatographic development, the peak area of the QTN spot in the sample chromatograph was measured, and the concentration of QTN in the sample was estimated from the calibration curve. The standard stock solution of quercetin (50 ng/µl) was applied on the HPTLC plate in a range of 1-10 µl with the help of CAMAG 100 µL sample syringe using Linomat 5 sample applicator to obtain a nal concentration 50-500 ng/band for quercetin. The plate was developed and scanned under the above established chromatographic conditions. The calibration curve of quercetin was plotted of peak area vs. concentration.
The nal drying of the test sample was carried out on a Rotavac under reduced pressure. Out of all the fractions, fraction D was further processed for isolation of active compound(s) by preparative TLC. From this fraction D, 5 compounds were isolated, labeled as D1, D2, D3, D4, and D5, respectively. It was further subjected to preparative HPTLC for the nal puri cation of the compound. Isolation of active constituent was carried out by preparative HPTLC. The dried component was further analyzed by HPTLC for the determination of Rf and λmax. The chemical structure of the isolated compound was elucidated by FT-IR and LC-MS spectroscopy.

Adjuvant-induced polyarthritis (AIA)
On day 0 of the study, AIA was induced in female rats (150-200 g) (5 groups, i.e., group II to VI, n = 18) by a single intradermal injection of 0.1 ml FCA (Sigma Aldrich, St. Louis, USA) into the tail of the rats 19 . FCA contains 0.6 mg heat-inactivated Mycobacterium tuberculosis H37Ra emulsi ed in a sterile mixture of para n oil, saline, and Tween 80. The 32 days were allowed to develop arthritis. A separate group of rats (group I, n = 18) was maintained as normal and did not receive FCA. After the development of AIA (after 32 days), animals were received either distilled water (10 ml/kg, p.o. i.e., group I and II) or le unomide (10 mg/Kg, as a standard, i.e., group III) or QTN (5, 10 and 20 mg/kg, i.e., group IV to VI) for next 28 days 17 .
Paw volume was determined by using a plethysmometer (UGO Basile Italy) 20

Blood withdrawal and biochemical analysis
On the last day of study (on day 60), rats were sacri ced by cervical dislocation, and blood was withdrawn for Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP) 20 and hematological measurements (Red blood cell (RBC), hemoglobin (Hb), White blood cell (WBC), and platelets (PLT)). The serum was separated by centrifugation using an Eppendorf cryocentrifuge and used for serum turbidity measurement 23 . The levels of serum albumin, alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), and total cholesterol (TC) were measured by a spectrophotometer (UV-visible spectrophotometer, Jasco V-530, Tokyo, Japan) using a commercially available reagent kits according to the procedure provided by the manufacturer (Accurex Biomedical Pvt. Ltd., Mumbai, India).

Tissue preparation and biochemical analysis
All animals were sacri ced at the end of the study; the synovial tissues were immediately isolated. Tissue homogenates were prepared with 0.1 M Tris-HCl buffer (pH 7.4), and supernatant of homogenates was employed to estimate superoxide dismutase (SOD), reduced glutathione (GSH), lipid peroxidation (MDA), and nitric oxide (NO) as described previously 24 . The levels of TNF-α, IL-1β, and IL-6 were determined by Enzyme-linked immunosorbent assay (ELISA) using commercial kits (Thermo Fisher Scienti c, USA) as per manufacturers' instructions.
2.7. Determination of NF-kβ, Ikβα, COX-2, and P2X7 protein expressions Synovial tissue was sonicated in Tissue Protein Extraction Reagent (Thermo Fisher Scienti c, Inc.). The lysates were centrifuged at 10,000 X g 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 sulfatepolyacrylamide gel electrophoresis) and transferred onto polyvinylidene di uoride 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 NF-kβ, Ikβα, COX-2, P2X7, and GAPDH. Then they were incubated with secondary antibody at 37°C for 2 h. Protein bands were visualized using the Chemiluminescent kit (Bio-Rad Laboratories, Inc.), GAPDH served as the loading control.

Histopathology of tibiotarsal joint
Ankle joints from the 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, depara nated, and stained using hematoxylin and eosin (H and E) stain. The specimens were mounted on slides by the use of 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 histopathology features specimen and in ltration of cells in epithelium and sub-epithelium. The intensity of histological aberrations in the tibiotarsal joint was graded as Grade 0 (not present or very slight)l; Grade 1 (mild); Grade 2 (moderate); and Grade 3 (severe) as described in the literature.

Statistical analysis
Data were expressed as Means ± standard error of the mean (SEM). All statistical tests were performed using Prism 5.0 (Graph Pad, San Diego CA, USA) statistical software. The data were considered signi cant at p < 0.05. 3.2. Effects of QTN on FCA-induced alteration in body weight, joint diameter, paw volume, paw withdrawal latency, and paw withdrawal threshold in arthritic rats There was a signi cant decrease (p < 0.05) in body weight, whereas a signi cant increased (p < 0.05) in joint diameter, paw volume, paw withdrawal latency, and paw withdrawal threshold in AIA control rats as compared to normal rats. Administration of Le unomide (10 mg/kg) signi cantly inhibited (p < 0.05) FCAinduced alterations in body weight, joint diameter, paw volume, paw withdrawal latency, and paw withdrawal threshold as compared to AIA control rats. Whereas administration of QTN (10 and 20 mg/kg) also signi cantly increase (p < 0.05) body weight and signi cantly decreased (p < 0.05) joint diameter, paw volume, paw withdrawal latency, and paw withdrawal threshold as compared to AIA control rats. (Table 1   3.3. Effects of QTN on FCA-induced alteration in AST, ALT, ALP, albumin, CRP, and rheumatoid factor in arthritic rats The levels of serum AST, ALT, ALP, and CRP were signi cantly (p < 0.05) increased, whereas serum albumin level signi cantly (p < 0.05) decreased in the AIA control rats as compared to normal rats.
Le unomide (10 mg/kg) treatment signi cantly (p < 0.05) increased serum albumin level whereas it signi cantly (p < 0.05) decreased serum AST, ALT, ALP and CRP levels as compared to AIA control rats. The FCA-induced alterations in serum AST, ALT, ALP, albumin, and CRP levels were signi cantly (p < 0.05) attenuated by QTN (10 and 20 mg/kg) as compared to AIA control. These alterations in levels of serum AST, ALT, ALP, albumin, and CRP were more signi cantly (p < 0.05) attenuated by le unomide (10 mg/kg) as compared to QTN treatment. (Table 2)

Effects of QTN on FCA-induced alteration in synovial oxido-nitrosative stress in arthritic rats
The levels of synovial SOD and GSH were signi cantly decreased (p < 0.05), whereas levels of synovial MDA and NO was increased signi cantly (p < 0.05) in AIA control rats as compared to normal rats. Le unomide (10 mg/kg) treatment signi cantly increased (p < 0.05) synovial SOD and GSH levels, whereas signi cant decreased (p < 0.05) synovial MDA and NO levels as compared to AIA control rats. Similarly, administration of QTN (10 and 20 mg/kg) signi cantly attenuated (p < 0.05) FCA-induced increased synovial oxido-nitrosative stress as compared to AIA control rats. However, elevated MDA and decreased GSH levels were more signi cantly (p < 0.05) attenuated by QTN (20 mg/kg) treatment as compared to Le unomide (10 mg/kg). (Table 4)

Histopathology of the tibiotarsal joint in FCA-induced AIA in arthritic rats
Intraperitoneal administration of FCA resulted in signi cant histological aberrations in the tibiotarsal joint of AIA control rats re ected by signi cant increase (p < 0.05) in in ammatory in ltration, synovial proliferation, cartilage erosion, and pannus formation (Fig. 4B) as compared to normal rats. Whereas, tibiotarsal joint of normal rats showed the presence of minimal in ammatory in ltration, synovial proliferation, and pannus formation (Fig. 4A). Le unomide (10 mg/kg) treatment signi cantly inhibited (p < 0.05) FCA-induced histological aberrations in tibiotarsal joint (Fig. 4C) as compared to AIA control rats. However, when compared with AIA control rats, QTN (5 mg/kg) treatment failed to produce any signi cant protection against FCA-induced histological aberrations in the tibiotarsal joint (Fig. 4D).

Discussion
Rheumatoid arthritis (RA) is a chronic autoimmune illness mainly associated with progressive joint disability and cartilage damage due to the release of multiple in ammatory mediators. It has been well reported that females between the age of 40 and 50 have a signi cant risk for the development of RA than males 5 . Current treatment therapies such as NSAIDs, DMARDs, etc., have their limitations of wellknown side effects, variations in e cacy, and high cost 2 . Thus, the various researcher has explored the safety and effectiveness of various therapeutic moieties from plant origin for RA management. Madhuca indica is a traditional medicine rich in various phytoconstituents dominant with the presence of avonoids. It has been widely used to manage various in ammatory disorders due to its inhibitory potential against histamine, serotonin, prostaglandin, and COX-2 25 . Thus, in the present investigation, we have evaluated the anti-arthritic potential of isolated phytoconstituent (3,5,7,3′,4′-Pentahydroxy avone, i.e., QTN) from methanolic extract of Madhuca indica Leaves in female Wistar rats after subplantar administration of FCA. QTN exerts its antiarthritic potential via inhibition of oxido-nitrosative stress, proin ammatory cytokines (TNF-α, IL-1β, and IL-6), and NF-kβ, Ikβα, COX-2, and P2X7 expressions.
It has been reported that cachexia, i.e., marked decrease in body weight, is a characteristic feature of many chronic diseases, including cancer, heart or renal failure, diabetes, and Crohn's disease 26-31 .
Clinically it has been shown that rheumatoid arthritis (RA) exhibited hypermetabolism and accelerated protein breakdown, which is a major reason for increased morbidity and premature mortality in those patients 32 . In the present study, there was a signi cant reduction in the body weight recorded in AIA control rats even before the manifestation of external signs of the illness, such as destruction of joint integrity and function disability. The result of the present study is in line with the ndings of the previous researcher 33 . It has been reported that decreased body weight affected by immune in ammation and elevated levels of pro-in ammatory cytokines (such as TNF-α and IL's) are thought to play a vital role in the regulation of leptin activity 34 . In the present study, rats administered with FCA showed a signi cant reduction of body weight, which might be via a decrease in leptin levels, whereas administration of QTN showed the signi cant attenuation in FCA induced decreased body weight which might be due to inhibition of the release of in ammatory mediators.
During the in ammatory insult, the release of pro-in ammatory mediators (such as prostaglandin E2) and pro-in ammatory cytokines are responsible for the initiation of pain promotes nociceptor sensitization resulted in a decrease threshold 35,36 . Most anti-in ammatory agents possess analgesic, i.e., reduction of allodynia and hyperalgesia as an essential ancillary property, which is widely utilized to increase pain threshold in various animal models [37][38][39] . It has been well established that AIA-induced arthritis is associated with altered allodynia and hyperalgesia 2, 10, 40 , and in the present investigation also intradermal administration of FCA resulted in a signi cant reduction of allodynia and hyperalgesia evaluated by an array of behavioral assessment using Randall-Selitto, von Frey hair, and Hargreaves test. However, the administration of QTN showed signi cant attenuation in FCA induced alteration of allodynia and hyperalgesia by virtue of its anti-in ammatory potential.
The researcher showed that elevated paw thickness is evidence of arthritis induction 2, 10, 40, 41 . Determination of paw thickness using a plethysmometer is the well-established and standardized method during AIA-induced arthritis 2, 10, 40 . In the present study, the in amed 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 signi cantly decreased paw thickness, which might be due to inhibition of the release of in ammatory mediators, indicating its anti-in ammatory potential in FCA-induced arthritis. This potential of cytokine blockage in pain nervous bers by QTN might be responsible for increased pain threshold to exert its basis of analgesic effect. The presence of avonoid moiety in the methanolic extract of Madhuca indica could be responsible for its anti-in ammatory, analgesic, and anti-nociceptive activities. The present investigation results corroborate with the ndings of the previous investigator where phytoconstituents isolated from methanolic extract of Madhuca indica showed anti-in ammatory potential via inhibition of TNF-α and IL-1β 16 .
It has been reported that AIA is associated with the diminished Hb, and RBC levels represent the anemic condition of arthritic rats, which is a common diagnostic feature in patients with chronic arthritis 20,42 .
This decreased Hb, and RBC levels in AIA rats may be due to either sequestering or insu cient iron in the reticuloendothelial system and synovial or failure of bone marrow response erythropoietin along with the destruction of premature red blood cells 42  Thus, the reduction in the ESR and improvement in RBC and Hb count brought about by QTN treatment indicate the signi cant recovery from the anemic condition and further support its anti-arthritic effect.
In the present investigation, a battery of serum chemistry tests was assessed to determine the functionality of the vital organs like the liver after administration of FCA. There were signi cant alterations in liver functions after chronic oral administration of FCA, re ected by an increase in albumin, ALT, AST, and ALP levels. It has been reported that albumin corresponds to 50% of the total protein 47 . An elevated level of serum albumin after the FCA administration corroborated with an increased level of complete protein in synovial tissue. AST and ALT are the two cytoplasmic enzymes present in abundance in the liver 48 , representing the liver function, and alteration in their levels re ects hepatic toxicity 49 .
Administration of FCA caused a signi cant elevation in the AST and ALT level, thus produces hepatotoxicity. A recent study has documented that arthritis patients are associated with primary liver disease clinically 50 . Findings of the present investigation also suggest that FCA induced arthritis is associated with hepatotoxicity, which was re ected by elevated AST and ALT levels. However, the administration of QTN signi cantly attenuated these elevated levels of hepatotoxicity markers suggesting its hepatoprotective role, which might contribute to its antiarthritic potential.
Oxidative stress plays a central role in the induction and maintenance of painful arthritis 50 . It has been documented that increased production of reactive oxygen species such as hydrogen peroxide, hydroxyl, and superoxide radicals contributes to elevated oxidative stress 4,10,20 . This elevated oxidative stress further depletion of protective antioxidant moieties (SOD and GSH) that resulted in the elevation of lipid peroxidation (MDA), causing damage to the macromolecules in vital biomembrane 22,37,51 . In the present study, the synovial SOD and GSH were signi cantly decreased, whereas the MDA level increased signi cantly after the FCA administration. However, treatment with QTN signi cantly attenuated FCAinduced decreased SOD and GSH in the synovial tissue suggesting its antioxidant potential that might support its antiarthritic mechanism. The result of the present investigation is in line with the ndings of the previous researcher, where QTN isolated from Madhuca indica exerts its potential via inhibition of elevated oxidative stress 17 .
The researcher has well-identi ed CRP as a vital marker during various in ammatory diseases. Moreover, rheumatoid arthritis patients also exhibit increased serum CRP levels associated with in ammation and tissue destruction 40 . Furthermore, a couple of inducible in ammatory enzymes, including nitric oxide (NO) and COX-2, play a key role in the activation of an in ammatory network of mediators 43 . Numerous nings documented the linkage between elevated nitric oxide and the release of pro-in ammatory cytokines (TNF-α and IL-1β) in local synovial uid 6, 43, 52 . Furthermore, COX-2, an isoenzyme, is abundantly present in activated macrophages responsible for the synthesis of prostaglandins that mediate various in ammatory reactions 53 . Thus, dual inhibition of these inducible in ammatory enzymes (NO and COX-2) would be important in terms of symptomatic relief from pain and in ammation. In the present investigation, the activity of NO and COX-2 signi cantly decreased after administration of QTN, which might, in turn, in ammation and exerts its anti-nociceptive potential in FCA induced rats to modulate the paw withdrawal latency.
Cytokines such as TNF-α and IL's play a vital role in RA's pathogenesis 2 . The release of these proin ammatory cytokines in response to antigen-stimulated immune response cause recruitment, activation, and deposition of polymorphonuclear neutrophils (PMNs) into the joint space 9, 54 . Further, these PMN's caused the elevated response of ROS, which damages cartilage and joint 55 . The researcher reported that differentiation and proliferation of T and B cells as well as their resorption into bone inducted by TNF-α and IL-6 whereas IL-1β responsible for modulation of immune response via production of nitric oxide (NO) and prostaglandin 7,56 . Recent evidence demonstrated an elevated response of proin ammatory cytokines in RA patients 57 . Thus, measures have been oriented towards the administration of anti-TNF-α antibodies to manage RA 2 . In the present investigation, the FCA administration resulted in an elevated response of these pro-in ammatory cytokines in the synovial uid. In contrast, treatment with QTN ameliorated this in ux of cytokines. The results of the present investigation are in accordance with the ndings of the previous researcher. In contrast, the administration of Madhuca indica signi cantly inhibited the elevated response of TNF-α and IL-1β 17 , thus exerts its anti-in ammatory potential to modulate the pathogenesis of disease.
Numerous researchers suggested that the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) plays a central dogma role in the induction and maintenance of immune-in ammatory disease modulation of various biomolecules such as COX-2 and pro-in ammatory cytokines 10,53 . During the resting state, the NF-kB remains unstimulated and retain in 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 58 . This cascade leads to NF-kB translocation from cytoplasm to nucleus, where it modulates the expression of various genes, including pro-in ammatory cytokines 58 . In the present investigation, activated expression of IκBα and NF-kB signi cantly up-regulated in synovial of AIA control rats after administration of FCA whereas QTN treatment signi cantly down-regulated these expressions of pro-in ammatory cytokines via inhibition of phosphorylation of IκBα and thus inactivation of NF-kB.
Purinergic Receptor-X 7 (P2X7), a protein-coding gene from the purinoceptors family, has been implicated in the induction and maintenance of various diseases associated with bone and cartilage, including rheumatoid arthritis 59 . The researcher documented its vital role in bone remodeling via the release of various pro-in ammatory factors such as prostaglandins and IL-1β into the synovial uid 59 . It has been suggested that activation of ectonucleotidases degrade extracellular ATP, which results in the formation of active molecules such as adenosine or pyrophosphates 60 . These active molecules further promote the activation of alternative macrophages and thus initiate the release of pro-in ammatory signaling 60 .
Therefore, extracellular metabolism of ATP by P2X7 modulate the sequence of in ammatory in ux and thus initiate the pathogenesis of RA 59 . In this view, inhibition of P2X7 receptor activation would be bene cial for the management of RA. In the present study, treatment with QTN signi cantly inhibits the activation of P2X7, which might reduce the bone and cartilage damage in RA.
Recently an array of isolated phytoconstituents from herbal origin has been implicated in the management of arthritis clinically. Studies 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 61 . Furthermore, a researcher suggested that the moiety bearing carbonyl group at C-4 and a hydroxyl group at C-3 or C-5 in their structure have a chelation ability with metal ions to exert its antioxidant potential 24 . In the present investigation, isolated moiety from methanolic extract of leaves of Madhuca indica, i.e., QTN (3,5,7,3′,4′-Pentahydroxy avone) also possesses such hydroxyl and carbonyl groups in its structure, holding promising antioxidant potential.
Thus, QTN can be considered as a potential therapeutic moiety for the management of RA clinically.

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