Appraisal of Anti-Arthritic Potential of Quercus Leucotrichophora Methanolic Extract in Complete Freund’s Adjuvant Induced Arthritic Rats; a Mechanistic Study

This research work was conducted to validate the folkloric use and therapeutic potential of Quercus leucotrichophora (QL) leaf methanolic and aqueous extracts against inammation and arthritis and to determine the chemical composition by HPLC. The in-vitro anti-oxidant and anti-inammatory activities were carried out along with in-vivo assays such as carrageenan induced paw edema, xylene induced ear edema and Complete Freund’s Adjuvant induced arthritis in Wistar rats. The CFA (0.1 ml) was inoculated to the left hind paw at day 1 to induce arthritis and oral dosing with QLME at 150, 300 and 600 mg/kg was begun at 8 th day till the 28 th day in all groups while methotrexate was given as standard treatment. There was a noteworthy (p<0.05-0.0001) restoration in body weight, paw edema, arthritic index, altered blood parameters and oxidative stress biomarkers in treated rats as compared to diseased group. Moreover, QLME considerably (p<0.0001) downregulated TNF-α, IL-6, IL-1β, COX-2, and NF-κB, while signicantly (p<0.0001) upregulated IL-10, I-κB, IL-4 in relation to diseased group. The QLME exhibited no mortality in acute toxicity study. It was concluded that QLME possessed substantial anti-inammatory and anti-arthritic potential at all dosage levels, mainly at 600 mg/kg might be due to presence of quercetin, sinapic acid and ferulic acid.

Qualitative and quantitative analysis The QL extracts were evaluated for phytochemicals like alkaloids, tannins, phenols, saponins, avonoids, steroids, terpenoids, proteins and glycosides by standard procedures (Saleem et al. 2020a).
For the determination of total phenolic contents (TPC), extract (0.5 ml) was mixed with 2.2 ml of distilled water (DW) and 0.15 ml of 5% NaNO 2 solution. Then after 6 min, 0.3 ml of 10% AlCl 3 .6H 2 O was mixed and left for 5 min. Then 1 ml of 1N NaOH solution was added and vortexed, later the absorbance was taken at 510 nm. Gallic acid was used as standard (Saleem et al. 2020b).
For total avonoid contents (TFC), the stock sample (10 mg/ml) of 100 µl was added to 2 ml of 2% Na 2 CO 3 and left for 2 min at room temperature. Afterwards, 100 µl of 50% Folin-Ciocalteu's reagent was added. Catechin was used as reference. After 30 min incubation at room temperature, absorbance was determined at 765 nm (Cheruth et al. 2016).

High performance liquid chromatography (HPLC) analysis
The HPLC analysis of QLME and QLAE was performed by the previous procedure (Asif et al. 2020). The sample preparation was done by adding 10 mg of extract in 5 ml DW and then ethanol 12 ml was mixed. After 5 min, 6 ml DW was added and paused for 5 min along with the addition of 10 ml of 15 M HCl and then placed in the oven for 2 h at 90°C. Syringe lter was used for injection of the sample into HPLC. The separation of compounds was done by using Shim-Pack Column (Shimadzu, Japan) CLC-ODS. The mobile phase included methanol and acetonitrile in 30:70 as solvent A, while double DW with glacial acetic acid (0.5%) as solvent B. The UV-visible detector (SPD-10AV) was used at 280 nm wavelength. The retention time of standards was used to identify and quantify detected compounds.
In-vitro evaluation 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay For this, 2 ml DPPH solution (0.04/100 mg methanol) was mixed in 1 ml solution of plant extract and 1 ml of methanol. The twofold dilution method was used to prepare the sample solution of extracts in methanol. Absorbance was taken after 30 min at 517 nm using ascorbic acid as a reference (Saleem et al. 2020a). The % DPPH radical scavenging of the mean was calculated.

Inhibition of protein denaturation assay
In egg albumin (EA) denaturation assay, a 5 ml reaction mixture contained 0.2 ml of EA (from fresh egg of hen), phosphate buffered saline 2.8 ml (PBS) of pH 6.4 and 2 ml of plant extracts (50, 100, 200, 400, 800 & 1600 µg/ml), while same volumes of piroxicam solution and DW were used as standard and control solutions respectively in place of extract solutions. Afterwards, these mixtures were incubated for 15 min at 37±2 o C. Later, warmed for 5 min at 70 o C. The absorbance of mixture was taken at 660 nm (Akhtar 2020).
For the BSA denaturation assay, an earlier procedure was followed (Saleem et al. 2019). Brie y, test control (0.5 ml) contained 0.45 ml BSA (5% w/v) and 0.05 ml of extract dilutions. The product control solution contained DW (0.45 ml) in place of BSA solution. Standard solution contained piroxicam instead of extract solution. The pH was adjusted to 6.3 and incubated for 20 min at 37 C afterward, heated at 57°C for 3 min. The absorbance was taken at 660 nm.

Human red blood cells (HRBC) membrane stabilization assay
This assay was performed according to a previous procedure (Saleem et al. 2019). In short, 3 ml blood from healthy volunteer was mixed with Alsever's solution and RBCs suspension (10% v/v) was prepared using isosaline solution after centrifugation at 3000 rpm for 15 min.
Test solution contained phosphate buffer (1 ml), hypotonic saline (2 ml), 0.5 ml extract and 10% human red blood cells (0.5 ml). Test control solution contained DW while the standard solution contained piroxicam instead of extract. The solutions were incubated for 30 min at 37°C after that centrifugation at 3000 rpm for 15 min. The sample absorbance was taken at 560 nm and % protection was calculated.

Study design for in-vivo evaluation
The Wistar rats (120-170 g) were indiscriminately allocated into six groups (n=6), Group I served as normal control (NC) and was provided with DW. Group II was disease control (DC) and provided with DW.
Group III was standard control (SC) and given piroxicam (10 mg/kg). Group IV, V and VI received with QLME and QLAQ at 150, 300 and 600 mg/kg via oral route respectively.

Xylene induced ear edema
An earlier procedure was adopted for xylene induced ear edema (Shabbir et al. 2018). Brie y, a drop of xylene was applied to right ear's inner surface of each animal except NC, 30 min post-administration of the above treatments. Afterwards, the anesthetized rats were slaughtered after 15 min to remove and weigh both ears. The percentage inhibition was calculated.
Increase in weight = right ear weight -left ear weight % inhibition = (C-T)/ (C) ×100 C: control; T: treatment Carrageenan induced paw edema The rats in all groups were treated as aforementioned. One hour later, 0.1 ml carrageenan (1% w/v) solution was administered via sub-cutaneous injection in left hind paw (sub-plantar region) of all rats except NC. Paw diameter (mm) was measured at 0, 1, 2, 3, 4 till 8th hour using a digital Vernier caliper (Shabbir et al. 2018).

Complete Freund's Adjuvant (CFA) induced arthritis
The results of in-vitro anti-oxidant and anti-in ammatory studies along with acute in-vivo antiin ammatory studies revealed that QLME effectively reduced free radicals and showed profound antiin ammatory activity than QLAQ. Therefore, QLME was further evaluated by using CFA induced arthritic model.
At day 1, 0.1 ml CFA (Sigma Aldrich®, UK) was injected in sub-plantar region of the left hind paw in all except normal control rats (Han et al. 2016). The study design was same as above mentioned except SC was treated with methotrexate (MTX) (1 mg/kg/week) by intraperitoneal route. Treatment groups received QLME at 150, 300 and 600 mg/kg/day via oral gavage from 8 to till 28th day (21 days).

Arthritis evaluation
The body weight and diameter of the left hind paw was determined before rst immunization and then at 7, 12, 16, 20, 24, and 28th day post-CFA administration. The percentage inhibition of paw edema was measured. The severity of arthritis was assessed by an arthritic scoring system that ranged from 0 to 4 scales. In arthritic scoring, 0 and 1 and 2 meant no swelling, swelling of toe joints and swelling of both toes and toe joints respectively. Moreover, 3 and 4 meant ankle joint swelling and entire paw swelling leading to immobility respectively (Han et al. 2016).

Hematological and biochemical evaluation
After 28th day, the blood was collected by cardiac puncture in plain and EDTA containing tubes from anesthetized rats. The commercially available kit (Antec Diagnostic Products®, UK) was utilized to determine RF, alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), while automated chemistry analyzer (Microlab 300®, Germany) was used for urea and creatinine determination. The automated hemocytometer (Sysmex, Roche®, Germany) was used to count complete blood count (CBC).

Immune organ and histopathological evaluation
The blood collected by cardiac puncture in EDTA tube was processed for RNA extraction. The abdomen was dissected to remove the spleen and thymus, which were then washed with DW and weighed (Saleem et al. 2020c). For histopathological evaluation, left hind limb ankle joints were removed, rinsed with DW and placed for 24 h in 10% v/v neutral buffered formalin. The decalci cation of joints was done by decalcifying solution (10% w/v EDTA). After mounting on slides, the tissues were stained and slides were observed for histopathological changes under light microscope at 40X (Shabbir et al. 2018).
Quanti cation by real time (RT)-PCR For the estimation of IL-4, IL-10, IL-6, IL-1β, NF-κB, TNF-α, I-κB and COX-2 in blood of rats, RT-PCR was used. The RNA was removed from the collected blood by the TRIzol method by using kit procedure (Invitrogen®Pure Link RNA). The complementary DNA synthesis was done using protocol of kit manufacturer (K1622: Thermo Scienti c®, Germany). For quanti cation and ampli cation, the kit of SYBR Select Master Mix (Applied Biosystems Thermo Scienti c®, Germany) was used on qRT-PCR (Applied Biosystems Thermo Scienti c®,). The primers were selected from previous study using GAPDH as housekeeping gene (Saleem et al. 2020d).

Assessment of oxidative stress biomarkers
After 28th day, rats were sacri ced and liver was taken out for estimating superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) level. The 10% liver homogenate (LH) was prepared (Akhtar et al. 2016). The Lowry's method was used for valuation of protein content in LH (Saleem et al. 2021). The SOD and CAT, and MDA in the LH were estimated by xanthine oxidase, hydrogen peroxide and Thiobarbituric acid (TBA) assays respectively (Bhangale & Acharya 2016).

Acute toxicity study
It was performed by following OECD guidelines 425 Up and down procedure with slight changes (Saleem et al. 2020b). The female rats (120-170 g) were distributed in to two groups (n=5). Brie y, 2000 mg/kg dose of QLME was given and observed for change in behavior, gait, movements, respiration and mortality at 0.5, 1, 2, and 4hr till 48th hour. While in NC, only 1ml DW was given. The rats were observed for clinical signs of toxicity like mortality, body temperature, respiratory rate and motor movements during this period. The bodyweight was also measured on 1, 7 and 14th day.

Statistical analysis
The results were described as mean ± standard deviation (S.D) and were inspected by One-way and Twoway analysis of variance (ANOVA) followed by multiple comparison test using GraphPad Prism® software version 7.0. The signi cance level was considered at p< 0.05.

Results
The percentage yield of QLME and QLAQ was 6.1% and 4.05% respectively.

Phytochemicals analysis
It was observed that tannins, phenols, glycosides, terpenoids and avonoids were present in QLME and QLAQ while alkaloids and carbohydrates were present only in QLAQ. Saponins, proteins and steroids were absent in both extracts. The QLME contained higher amount of phenolic acid and avonoids (TPC: 201.2 ± 0.72 mg GAE/g and TFC: 30.30 ± 0.66 mg CE/g) than QLAQ (TPC:188.07 ± 0.50 mg GAE/g and TFC: 23.37 ± 0.85 mg CE/g).

Quantitative analysis
The HPLC analysis of QLME revealed that it contained the highest amount of quercetin (265.53 ppm) followed by sinapic acid (73.86 ppm). The QLAQ had the highest amount of p-coumaric acid (255.34 ppm) followed by catechin (236.9 ppm). Phenolic acid and avonoids detected in the plant extracts are presented in Table 1. In-vitro antioxidant activity The DPPH assay revealed that both extracts exhibited profound dose dependent antioxidant activity. The highest % scavenging was presented by 1600 µg/ml solution. The antioxidant activity of QLME (67.64 ± 0.43%) and QLAQ (63.09 ± 0.65%) at 1600 µg/ml was signi cantly (p<0.0001) different compared to ascorbic acid (72.75 ± 0.21%) as shown in Figure 1 Inhibition of protein denaturation The EA and BSA denaturation inhibition assays revealed that both extracts inhibited protein denaturation at all concentrations. In egg albumin assay, the maximum % inhibition was shown by QLME (85.42 ± 0.42%) and QLAQ (79.44 ± 0.52%) at 1600 µg/ml that was signi cantly different as compared to piroxicam (75.13 ± 0.81%). In BSA assay, the maximum per inhibition exerted by QLME (87.54 ± 0.73%) and QLAQ (91.74 ± 0.44%) at 1600 µg/ml that was considerably different from piroxicam (84.05 ± 0.54%) as displayed in Figure 1.

Effect on xylene induced ear edema
There was a considerable increase in ear weight of DC in comparison to NC. The QLME (91.37 ± 3.24%) and QLAQ (86.42 ±2.88%) signi cantly (p<0.001) reduced ear edema at 600 mg/kg compared to piroxicam (54.94 ± 2.31%) as presented in Figure 2.

Effect on carrageenan induced paw edema
The carrageenan administration produced a considerable increase in paw diameter in DC rats as compared to NC. All treated groups showed signi cant (p<0.05) reduction in paw edema in comparison with DC at respective time interval ( Table 2). The maximum reduction in paw edema exhibited by QLME (3.59 ± 0.08 mm) at 600 mg/kg was notably (p<0.0001) different as compared to piroxicam (3.72 ± 0.49 mm). The maximum % inhibition was exhibited by QLME (29.03%) at 600 mg/kg as compared to other groups as given in Table 2. Results as presented mean ± S.D. (n=6); Two-way ANOVA; Tukey's test; a", a"', a: p<0.01, 0.001 and 0.0001; 'b' in contrast to NC and c', c'', c"', c: p< 0.05, 0.01, 0.001 and 0.0001 to Standard control.

Effect on paw diameter of arthritic rats
The anti-arthritic effect of QLME was evaluated against CFA induced arthritis in rats. On 8th day, there was a considerable rise in paw diameter in all arthritic rats in contrast to NC. A substantial increase in paw diameter in DC rats was noted until the 28th day compared to NC. The QLME 600 mg/kg (4.05 ± 0.07 mm) considerably (p<0.05) reduced paw edema along with MTX treated rats (3.92 ± 0.16 mm) from 16 to 28th day. The maximum % inhibition was exhibited by MTX (50.02%) followed by QLME 600 mg/kg/day treated rats (48.34%) at 28th day. The inhibitory effect of QLME 600 mg/kg on paw edema was insigni cantly varied from MTX on 16 to 28th (Table 3). Results as mean ± S.D. (n=6); Two-way ANOVA, Tukey's test; 'a' (p<0.0001) in contrast to DC and c", c"' and c: p< 0.01, 0.001, 0.0001 to SC.

Effect on body weight
There was a signi cant decline in body weight after CFA inoculation in all groups compared to NC at 8th day. The fall in weight was continuous in DC from the 8th day as compared to NC till the end of the study. However, treatment with QLME (150-600 mg/kg) and MTX caused a signi cant (p<0.05) restoration of body weight from 16 to 28th day compared to DC. The MTX (178.4 ± 3.65 g) and QLME 600 mg/kg/day (175.3 ± 5.19 g) treated rats exhibited considerable (p<0.05) restoration in body weight on the 28th day, in contrast to DC while the effect of QLME 600 mg/kg/day on body weight was insigni cantly varied from MTX on respective day as given in Table 4.

Effect on arthritic index
During whole study, the DC showed continuous increase in arthritic index. The treatment groups exhibited substantial (p<0.05) restoration of the arthritic index from day 16 till the end of the study. The maximum arthritic index was observed in DC at 28th day (4.25 ± 0.96). The plant extract exerted the most pronounced effect on the 28th (Figure 3).

Effect on blood parameters
There was a remarkable (p<0.0001) decrease in the level of hemoglobin (Hb) and RBC's in DC as compared to NC (Figure 4 and 5). However, there was a signi cant (p<0.05) rise in platelets, c-reactive protein (CRP), erythrocyte sedimentation rate (ESR), RF and total leukocyte counts (TLC) in DC in comparison to NC. All the treatment groups at 150-600 mg/kg restored the hematological parameters in arthritic rats as shown in Figure 4 and 5.
There were non-signi cant changes by induction of arthritis on urea and creatinine in arthritic rats. The levels of ALT, ALP and AST were considerably (p<0.05) higher in DC as compared to NC, and all the treatment groups signi cantly reduced their level in arthritic rats ( Figure 5).

Effect on weight of spleen and thymus
The weight of the spleen and thymus increased in DC than NC. The weight of spleen and thymus was signi cantly (p <0.05) restored by QLME (spleen: 0.45 ± 0.03 g; thymus: 0.23 ± 0.02 g) at 600 mg/kg and MTX (spleen: 0.42 ± 0.01 g; thymus: 0.20 ± 0.02 g) treated groups in contrast to DC as shown in Figure 6.

Effect on oxidative stress biomarker
The oxidative stress was developed after CFA immunization in all rats that was restored with extract at all tested dosage and MTX treated rats (Figure 8). The activities of CAT and SOD were signi cantly (p<0.05) lowered in DC than NC. There was also an increased level of MDA in LH of DC as compared to NC. All the treatment signi cantly (p<0.05) improved the activities of CAT and SOD as well as reduced the level of MDA in arthritic rats as compared to DC. The QLME 600 mg/kg  Figure 8.

Effect on joint histopathology
At the end of 28th day, the histopathology of ankle joints showed the formation of pannus, in ammation, bone erosion and mononuclear cell in ltration in DC ( Figure 10). Furthermore, NC rats were devoid of bone erosion and in ammation ( Figure 10). The treatment with QLME (150, 300, 600 mg/kg) and MTX profoundly reduced in ammation, pannus formation, bone erosion and cellular in ltration in arthritic groups in comparison to DC as exhibited in Figure 10c, d, e and f.

Acute toxicity study
After QLME 2000mg/kg administration, the marked changes in behavioral and physiological parameters were not observed. The QLME LD 50 was more than 2000 mg/kg as no mortality was noticed within 14 days of extracts administration. There were non-signi cant (p>0.05) changes in body weight of the treated group (140.3±1.53 g) as compared to NC (139.3 ± 2.52 g) at 14th day.

Discussion
The purpose of the current study was to assess the anti-in ammatory and anti-arthritic potential of QL.
The QLME extract showing notable anti-oxidant and anti-in ammatory activities were further evaluated in animals by using CFA induced arthritis.
The excessive production of free radicals causes oxidative stress, a hallmark of numerous diseases like arthritis. The medicinal plants containing anti-oxidants reduce the intensity of disease associated with oxidative stress in human. The QLME and QLAQ exhibited prominent anti-oxidant activity probably due to occurrence of high amounts of TFC and TPC. The avonoids and phenols having high amount of hydroxyl group in plant reduced free radicals and halted oxidative stress as previously described (Arulselvan et al. 2016). The QLME was safe in acute toxicity study as it did not cause mortality and LD 50 was more than 2000 mg/kg.
The phytochemicals like quercetin, sinapic acid, p-coumaric acid etc. protected lysosomal degradation via free radical scavenging activity (Oyeleke et al. 2018). The denaturation of protein leads to RA by the production of autoantibodies at the target site. It occurred in the presence of strong stimulus like heat, resulting in loss of the structure of protein (Akhtar 2020). Therefore, it can be assumed that the QLME showed the maximum inhibition of protein denaturation and HRBC membrane stabilization than QLAQ due to the presence of higher amount of avonoids and phenols as detected by HPLC analysis. It has been reported in previous studies that plants containing phenolic compounds like ferulic, p-coumaric, The xylene and carrageenan are considered as acute models of in ammation. The xylene is partially involved in Substance-P release, causing neurogenic in ammation with swelling leading to release of kinins and histamine. The QLME reduced ear in ammation more than QLAQ and its activity was comparable to piroxicam. The similar nding was reported previously by other Quercus species (Shabbir et al. 2018). Carrageenan induced in ammation by its actions on complement system or via mediators of in ammation like histamine, prostaglandins (PG's) and 5-HT. In current ndings, carrageenan signi cantly reduced paw edema by the inhibition of release of in ammatory mediators like PG's, serotonin and histamine by the activated immune cells. The comparison with DC group revealed that QLME had shown profound anti-in ammatory activity followed by QLAQ (Peerzada et al. 2020).
The sinapic acid is derived from cinnamic acid that showed its anti-oxidant, anti-in ammatory, anxiolytic and anti-cancer activities ( The expression of pro-in ammatory cytokines (IL-6, IL-1β, NF-κB, COX-2 and TNF-α) were reduced with treatment at all dosage levels as evidenced from the improvement in blood parameters of arthritic rats also.
IL-4 is involved in Th2 cell production. The IL-4 and IL-10 halt pro-in ammatory cytokine release from monocytes as well as synovial broblasts and their level was reduced in RA patients as also obvious in DC animals in the current study (Mateen et al. 2016). The levels of anti-in ammatory cytokines were increased in treated rats as co-evidenced from the reduction of paw in ammation.
As reported in previous studies, quercetin caused an inhibition of late phase in ammation by the inhibition of TNF-α, lipoxygenase, COX-2 and phospholipase A2 (Yin et al. 2019). The QLME at 600 mg/kg/day showed profound anti-arthritic activity comparable to MTX treated group. The presence of quercetin, sinapic acid, ferulic acid, and other phenolic and avonoids in QLME might be responsible for its anti-arthritic potential by downregulating pro-in ammatory and upregulating anti-in ammatory cytokines in treated rats (Foyet et al. 2015).
The systemic biomarkers of arthritis like RF, CRP, ESR, HB, RBCs, WBCs and platelets were notably altered in arthritic rats that were restored by treatment (Shabbir et al. 2018). Enhanced ROS generation occurred because of increased discharge of in ammatory cytokines from activated immune cells like neutrophils and macrophages ultimately headed to synovium cellular in ltration (Ishibashi 2013). In the current study, oxidative stress was reduced in treated arthritic rats in contrast to DC. Numerous other species of Quercus genus such as Q. dilatata, Q. incana, Q. sideroxyla, Q. durifolia, and Q. eduardii and Q.

Conclusion
It was concluded that QL exhibited in-vitro anti-oxidant and anti-in ammatory activity along with in-vivo anti-arthritic activity in Wistar rats might be due to the presence of Ferulic acid, sinapic acid, catechin and quercetin as detected with HPLC. QLME exhibited anti-arthritic potential by restoring paw edema, body and immune organ weight. Additionally, QLME reinstated altered blood parameters, pro-, antiin ammatory and oxidative stress biomarkers in treated rats. Current ndings suggested the QL leaves for the treatment of in ammation and arthritis. There is an immense need for activity guided fractionation of QLME to isolate anti-arthritic component. Moreover, there should be detailed toxicity study of QLME to assure its safety for human use.
Declarations Figure 3 The effect of Quercus leucotrichophora on the arthritic index Results as mean ± S.D. (n=6); Two-way ANOVA; Tukey's test; a', a", a"' and a: p< 0.05, 0.01, 0.001, 0.0001 to Disease control. Treatment groups were insigni cant in comparison to standard control.

Figure 4
The effects of Quercus leucotrichophora extract on blood parameters in CFA induced arthritis Data as mean ± S.D (n=6); One-way ANOVA; Tukey's test; 'a': p<0.05 to Disease control, 'b': p<0.0001varied from Normal control and 'c' from Standard control.

Figure 5
The effects of Quercus leucotrichophora extract on various blood parameters in CFA induced arthritis Data as mean ± S.D (n=6). One-way ANOVA; Tukey's test; a': p<0.05 to Disease control, 'b': p<0.0001varied from Normal control and 'c' from Standard control.  Effects of Quercus leucotrichophora extract on in ammatory biomarkers Results as mean ± S.D. One-way ANOVA; Tukey's test; 'a': p <0.0001 to Disease control and 'b': p<0.0001 from Normal control. c', c", c"', c: p<0.05, 0.01, 0.001, 0.0001 to Standard control.