Pharmacological and Biochemical Evaluation of Anti-arthritic Activity of Punica Granatum Extracts in FCA Induced Arthritis in Wistar Rats

The present studies were executed on Freund’s complete adjuvant (0.1 ml) induced arthritic Wistar rats to explore the folkloric use of the seeds. This study describes the effect of Punica granatum Linn’s Ethanolic (PGSE) and Chloroform (PGSC) extract within the FCA-induced arthritis rat paw oedema, vagaries in behaviour, haematological and alterations in biochemical parameters in the developed and progression of arthritis phases. There was a sig-ni(cid:976)icant rise in the paw swelling (volume) of rats and reduction in (BW) body weight, with FCA-induced arthritic rats. In contrast, PGSE and PGSC with the dose of 200mg/kg and 400mg/kg and Diclofenac (20mg/kg) treated group showed a substantial decrease in paw volume and the normal improvement in body weight to the positive control group. The altered level of haematological parameters, including, Hb, RBC, WBC, and ESR, in arthritic rats, have been substantially regained to normal by PGSE and PGSC treatment at the dosage of 200 mg/kg/p.o and 400 mg/kg/p.o in both the developed and progression of arthritis phases. In this study, Anti-in(cid:976)lammatory action of the PGSE and PGSC with carrageenan-induced paw oedema has also been investigated. Thus, percentage (%) inhibition of PGSE and PGSC were found to be 91.8 % and 86.71, respectively, concerning Diclofenac sodium (93.14%), this gave the evidence of dose-dependent action potential of Punica granatum as anti-in(cid:976)lammatory activity.

Punica granatum, Freund's complete adjuvant (FCA), Diclofenac sodium, Carrageenan, Arthritis A The present studies were executed on Freund's complete adjuvant (0.1 ml) induced arthritic Wistar rats to explore the folkloric use of the seeds. This study describes the effect of Punica granatum Linn's Ethanolic (PGSE) and Chloroform (PGSC) extract within the FCA-induced arthritis rat paw oedema, vagaries in behaviour, haematological and alterations in biochemical parameters in the developed and progression of arthritis phases. There was a signi icant rise in the paw swelling (volume) of rats and reduction in (BW) body weight, with FCA-induced arthritic rats. In contrast, PGSE and PGSC with the dose of 200mg/kg and 400mg/kg and Diclofenac (20mg/kg) treated group showed a substantial decrease in paw volume and the normal improvement in body weight to the positive control group. The altered level of haematological parameters, including, Hb, RBC, WBC, and ESR, in arthritic rats, have been substantially regained to normal by PGSE and PGSC treatment at the dosage of 200 mg/kg/p.o and 400 mg/kg/p.o in both the developed and progression of arthritis phases. In this study, Anti-in lammatory action of the PGSE and PGSC with carrageenan-induced paw oedema has also been investigated. Thus, percentage (%) inhibition of PGSE and PGSC were found to be 91.8 % and 86.71, respectively, concerning Diclofenac sodium (93.14%), this gave the evidence of dose-dependent action potential of Punica granatum as anti-in lammatory activity.

INTRODUCTION
Plants have always been used for its medicinal tenacities since the primaeval period, and the medicinal plants and their parts play an essential role in developing countries for potent therapeutic agents. Rheumatoid arthritis is a known autoimmune, musculoskeletal, infectious ailment, in lammatory, metabolic, affecting all the joints protected by synovium contributing to destructive polyarthritis and other body organs and in lammation is the human/body's immune response to injury, wound or other damage to start healing processes and eliminate harmful stimuli (Guo et al., 2018).
The root cause of the autoimmune disease (RA) remains unclear, but it has stimulated the production of new medications and revolutionized care. Rheumatoid arthritis induction of the immune response involves speci ic CD4 + T cells, most perspective as a response to an unfamiliar exogenous or endogenous antigen and the recruited monocytes, macrophages, and ibroblasts, therefore, contain cytokines, including, tumour necrosis factora (TNF-a) and synovial cavity interleukin-1 and these cytokines are crucial to the destructive cascade, which ultimately triggers the development of, MMP, matrix metalloproteinases and osteoclasts, resulting in bone and soft tissue irreversible damage (Olsen and Stein, 2004).
The pomegranate is the contemplate "a pharmacy for itself" in Ayurvedic medicine, the roots as well as bark thought to possess an anthelmintic and vermifuge property. Still, the peels are potent astringent, remedy for diarrhoea and oral aphthae (Ali and Sharma, 2006). The dried and extracted lowers are commonly used in hematuria, haemorrhoids, dysentery and hemoptysis, but the powdered buds were used to treat bronchitis (Ross et al., 2001).
Punica granatum L. (Anar or Pomegranate) is an identi ied deciduous shrub or, can say, small tree, growth of 1.8-4.6m tall, belonging to the family Punicaceae. The fruits are globose, shiny berry types, and 5-7.6 cm in diameter, but when get matured, the colour is reddish or yellowish-green. The seeds are crunchy having acidic pulp enclosed in a membranous skin (Qnais et al., 2007).
There have even been much preliminary toxicity studies and approved data in mice/rats (rodents) to report Punica granatum as non-toxic at all concentrations/doses, especially at a high dose (Braga et al., 2005), and there are various compounds present in P.granatum fruits; among these compounds, the most therapeutic phytochemicals are polyphenolics, lavonoids, alkaloids, ellagic, as well as gallic acid both (Anibal et al., 2013).
Consequently, this study was intended to explore and estimate the pharmacological and biochemical parameters of the Punica granatum ethanolic and chloroform seed extract in FCA-induced arthritis in Wistar rats. These current studies were also done to determine/evaluate an anti-in lammatory potential of the pomegranate seed extracts (ethanolic & chloroform) in Carrageenan-induced paw oedema in Wistar rats.

Experimental Animal
Wistar rats of either sex weighing 150-240g were acquired from College Animal House Facility, KIET School of Pharmacy (KSOP), Ghaziabad. In KSOP, the animal house temperature was maintained at 24-25 ± 2o C, and the animals were kept under the standard laboratory condition, i.e. 12 hours light and 12 hours dark cycle, in polypropylene cages. The animals had free access to good diet pellets (Pranave Agro Industries Ltd, New Delhi) and water ad libitum. The IAEC, Institutional Animal Ethics Committee, approved the protocol of KIET School of Pharmacy, Ghaziabad, with the Registration number (1099/PO/Re/S/07/CPCSEA).

Plant Material
The seeds of Punica granatum were purchased from Chawla & Co, Chandini Chowk, Delhi, and they were authenticated from the CSIR -National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India and the authentication number or Ref. No. for the plant is NSCAIR/RHMD/Consult/2019/3540-41.

Preparation of Punica granatum seeds extracts
The P. granatum seeds were obtained from an authorized vendor, dried mainly in the shade or dark, and then pulverized with an (electric) blender (Wang et al., 2019).
A portion of the powder (250 g) was thoroughly extracted for 16-18 hours in a Soxhlet apparatus with 400 ml of absolute ethanol and chloroform. The extract iltrates were concentrated to dryness using the distillation process and stored at the appropriate temperature .

Morphological and Standardization of Plant
The Plant material was evaluated for its morphological characteristics, and all the standardization parameters such as foreign matters, moisture content, extractive values as well as ash value. These parameters were necessary for the standard objectives of the plant to determine its safety, ef icacy, quality, quantity, and stability.
The existence of these active chemical components in plant extracts is determined by preliminary qualitative phytochemical analysis according to the procedure described in (Lee et al., 2010;Yadav et al., 2014).

Evaluation of In-vitro anti-arthritic activity
Two in-vitro models were selected for the study, and these are:

Inhibition of Protein Denaturation Model
A reaction mixture (50ml) was prepared with a 28ml phosphate buffer (pH 6.4, PBS), 2 ml fresh egg albumin, and 20 ml of varying extract and Diclofenac sodium concentrations as a reference so that the inal concentrations were 50, 100, 200, 400, 800, 1000, 2000µg/ml. The double-distilled water with parallel concentrations was used in control, and the above reaction mixture was mainly incubated (BOD incubator) at 36-37 ºC (±2) for 15 minutes.
After that, it was heated for 5 minutes at 70 ºC 9,13 . Keep it aside for cooling, and their absorbance was considered at 660nm using UV visible spectrophotometer. All of the above solutions go for viscosity test using Ostwald viscometer, and percentage inhibition was determined using the formula (Mittal et al., 2013;Bensaad et al., 2017).
× 100 where, Vt = absorbance of the test samples; Vc = absorbance of control.

Preparation of reagents
Alsevers solutions were prepared with the addition of sodium citrate (0.8%), 0.42% (NaCl) sodium chloride, 2% dextrose and citric acid (0.05%), in distilled water to mark up the volume up to 100ml with some distilled water and sterilized. 0.85 gm of sodium chloride (NaCl) was dissolved with 100ml distilled water, and this above solution was used as an isotonic saline solution. The hypotonic solution was prepared with adding 0.36 gm NaCl (sodium chloride) to 100ml of distilled water. Phosphate buffer containing 0.19gm potassium dihydrogen phosphate, 8gm of sodium chloride, 2.38 gm disodium hydrogen phosphate and pH at 7.4.

Assay of membrane stabilizing activity
The HRBC method was done to assess or evaluate the in-vitro anti-in lammatory action potential in the samples. A person who had not taken NSAIDs 14 days before the experiment was preferred for blood collection and collected blood was mixed with Alsevers solution in equal volume, and then centrifugation was done for 12-15 minutes at 3000rpm. After centrifugation, decanted the supernatant liquid using a micropipette and washed the remaining packed cells with isosaline solution 3-4 times, then made a 10% suspension solution from it. The hydroalcoholic doses of extracts were prepared (100,200, 400, 800 and 1600µg/mL) respectively with distilled water. A solution was prepared to have a concentration of 1ml phosphate buffer, 0.5ml of HRBC suspension, 2ml hyposaline and 0.5 ml of drug extract. It was placed in an incubator on 37 • C (±2) for 25-30 minutes and then centrifuged at 3000rpm for 20 min, and then the supernatant liquid was again decanted. Its haemoglobin content estimated using spectrophotometrically at 560 nm (Mittal et al., 2013). Diclofenac sodium having (50, 100,200, 400, 800 and 1600µg/mL) concentration were taken for standard drugs and a control part was done by excluding all extracts, and distilled water in place of hyposaline. The percentage protection or HRBC membrane stabilization percentage and % hemolysis was calculated using the below formula (Kota et al., 2018;Gautam et al., 2013).

Carrageenan-induced paw oedema in rats
The carrageenan-induced right hind paw oedema approach has already been carried out for determining the anti-in lammatory action. In short, acute in lammation was caused through a sub plantar injection of 0.1 ml of 1 per cent carrageenan suspension in the normal saline, in rats' right hind paw, 1h after an oral administration of test material (Sarker et al., 2012;Kavitha et al., 2011). Water Plethysmometer or Vernier calliper was used to determine oedema volume before and 1, 3 and 5 hours after the carrageenan injection. An hour earlier, the carrageenan injection, sample or test substance was administered, and the control group with the vehicle only (Bhandary et al., 2014;Kamble and Nazia, 2017). Diclofenac was taken as reference anti-in lammatory agent, with a dosage of 20 mg/kg bodyweight (Ratheesh and Helen, 2007;Sarker et al., 2012). The control group (vehicle) was used to compare the inhibitory effect on the oedema formation to calculate the percentage inhibition. The formula that is used for the calculation of the above mention parameter is as (Lin et al., 1999;Kavitha et al., 2011) and the protocol followed for the same study were taken as Table 1.
where, Ec = Edema rate of the control group; Et = Edema rate of the treated group.

Freund's Complete Adjuvant (FCA) induced arthritis in rats
Arthritis was induced with a single 0.1 mL intradermal injection of Freund's Complete Adjuvant (FCA) containing 1 mg.mL-1 Mycobacterium tuberculosis H37Ra suspension in a sterile paraf in oil into the footpad of Wistar rats' left hind paw for all animal groups, except for normal control group rats. The rats were carefully anaesthetized with the ether inhalation before and during the adjuvant injection because the adjuvant's very viscid nature exerts dif iculty while being injected (Petchi et al., 2015). Treatment with Punica granatum extracts, Diclofenac and normal control (normal saline) was    started on the 14th day after induction of arthritis and continued till 28 days (Wang et al., 2019;Kothari et al., 2011). After Freund's complete adjuvant injection, the paw volume for all taken animal group was measured by plethysmograph or Vernier calliper at 0, 7,14, 17, 21 and 28 days (Mittal et al., 2013;Das et al., 2012). On the 28th day (last day), blood was withdrawn using the retro-orbital process to the evaluation of the haematological param-eters, i.e. RBCs, WBCs, Hb, and ESR (Kumar et al., 2018;Umar et al., 2012) and the protocol followed for the same were as Table 2.

Statistical Analysis
All data were depicted as mean ± standard value deviation error, (SEM), and its statistical signi icances were achieved using common one-way variance analysis (ANOVA), also followed by Dunnett's   Multiple Comparison Test, where P<0.001 was considered statistically relevant using Graph Pad Prism version 8 (Hasan et al., 2015).

Morphological Evaluation of Punica granatum seeds
It was done to compared, weighed, counted and listed to determine differences or similarities in plant taxa by using these characters to de ine, classify and characterize plants. The results of all possible morphological parameters are observed and noted (Table 3).

Standardization Parameters of Punica granatum seeds
The standardization study was conducted to ensure consistency and the purity, protection and ef icacy of medicinal plants and the observed results were

Figure 1: Effects of standard (Diclofenac sodium), PGSE and PGSC on change in
Carrageenan-induced paw oedema (mm), and compared with the in lammatory control group at aP < 0.05. Data was taken in mean ± SEM, (n = 6).

Phytochemical Screening of Punica granatum
The phytochemical study was performed using aqueous, ethanolic and chloroform seeds extracts of Punica granatum, and it has given the preliminary con irmation of all compounds present in the extracts (Table 5).

Protein Denaturation Study
Anti-in lammatory activity of Diclofenac and seeds' extracts of Punica granatum on protein denaturation inhibition test was recorded. It has shown the concentration-dependent potential for antiin lammatory activities when compared against standard Diclofenac drug (Table 6, Table 7).

Human red blood cell (HRBC) membrane stabilization method
The anti-in lammatory action of Diclofenac and extracts of Punica granatum on human red blood cell (HRBC) membrane stabilization method were recorded. It has shown the concentrationdependent potential for anti-in lammatory activity when compared against Diclofenac sodium drug (Table 8, Table 9).

Carrageenan-induced Paw oedema
Carrageenan (0.1%) administration caused in lammation just within 1 hr, and after injection of this irritant, the peak effects were reached around 4-5 hours. The effects of the ethanolic, as well as     chloroform seeds, extract of Punica granatum seeds with the dosage of 200mg/kg on the carrageenaninduced paw oedema was then recorded. It has shown the time-dependent action against the control group and shown similar effects as evaluated against standard, Diclofenac sodium (20mg/kg), drug ( Figure 1). The data was statistical analysis using one-way ANOVA test that is followed by the Dunnett' test.

Bodyweight
The body weight was therefore measured at each of these days (0, 7, 14, 21, 28 days) and PGSE and PGSC (200 and 400 mg/kg), produced substantial increases (P< 0.05) and dose-dependent increases from day 14th till day 28th compared with the arthritic rats. Thus, results were shown in Figure 2.

Paw Volume
The decrease in paw swelling or volume is a constraint used to examine an anti-arthritic property of many drugs. PGSEL and PGSCH, produced signi icant dosage-dependent decreases from day 14th to day 28th, when compared to the standard and arthritic rats (Figure 3).

Joint Diameter
PGSE and PGSC (200 and 400mg/ kg) treatment showed signi icant with dose-dependent decreases (P < 0.05) from day 14 th to day 28 th compared to control and arthritis group. Hence, the results were summarized in Figure 4.

Biochemical Parameter
In this study, the arthritic control groups' biochemical parameters showed a slight rise in both the SGOT and SGPT levels and standard drug treatment group, it was increased signi icantly, but PGSE and PGSC treatment (PGSEL and PGSCH), produced signi icant decreases in the both SGOT and SGPT levels. The results were presented in Figure 5.

Haematological Parameters
The haematological parameters of arthritic control group rats showed lessening in the both RBC count and haemoglobin (Hb) levels and with an increase in the WBC and ESR levels. PGSE and PGSC treatment (PGSEL & PGSEH) produced signi icant increases in RBC and Hb, with substantial decreases in WBC and ESR ( Figure 6).

DISCUSSION
In the present investigation, the pharmacological and biochemical evaluation of Punica granatum seeds extracts (ethanolic and chloroform) in FCA-induced arthritis and Carrageenan-induced paw oedema in Wistar rats were studied. As found in some studies, several biologically vigorous and therapeutic active phytocompounds, namely, terpenoids, lavonoids, steroids, alkaloids, tannins, glycosides, and phenolic compounds, mainly are accountable for important anti-arthritic as well as anti-in lammatory activity inside numerous plant extracts. Increases in test sample absorbances against control indicate protein stabilization. Viscosities of protein solution has been stated to increase on denaturation. Thus, in the followed present study, both the seeds extract of Punica granatum has shown concentration-dependent percentage inhibition of tissue protein denaturation with % protection of membranes, suggesting its therapeutic anti-in lammatory activity to diclofenac sodium and control group. The results for the invitro study were con irmed by a practical evaluation of PGSE and PGSC in the in-vivo model. This activity was due to the availability of active phytochemicals and their ability to reduce in lammatory cytokine concentrations.

CONCLUSION
PGSE and PGSC have shown the highest concentration-dependent action in protein denaturation and HRBC membrane stabilization at the dosage of 2000µg/ml and 1000µg/ml, respectively. Punica granatum at the given 200 mg/kg dose level and 400mg/kg, p.o was shown to decrease the volume of rats' paw oedema and could normalize behavioural, haematological as well as biochemical irregularities in adjuvant-induced arthritic rats in the both developed and developing of FCAinduced arthritis, indicating momentous recovery in rheumatoid arthritis.

Future Aspects
The exact mechanism of action of Punica granatum on adjuvant-induced arthritis is not evident with these studies. The activity of Punica granatum on proin lammatory mediators including TNFan Interleukins and other related mediators will be carried out in future to study its mechanisms.

ACKNOWLEDGEMENT
Authors are thankful to KIET School of Pharmacy, KIET Group of Institutions, Delhi-NCR, Ghaziabad to provide the lab facility and all the required infrastructure to accomplish the research work.