INTRODUCTION:

Medicinal plants are a gift of nature to the management of health in individuals and communities. In case of RA, many of the commonly used drugs are becoming less acceptable due to serious adverse effects. This necessitates the continued search for potent anti-arthritic agents with reduced or no side effects.

Studies based on the ethno-botanical use of plants have often proved to be a more efficient method of drug discovery. According to WHO, still 80% of the world population relies on plant-derived drugs, and WHO has also recommended the evaluation of the effectiveness of plants in conditions where we lack modern safe medicines.¹

Drynaria quercifolia (L.) J. Smith (Polypodiaceae) locally called ‘Marappannakizhangu’ or ‘Attukalkizhangu’. The rhizome is reportedly used by tribal communities in Tamil Nadu and Kerala to treat various diseases. Ethno medicinal information shows that the rhizome of the plant Drynaria quercifolia (L.) J. Smith. was traditionally used for the treatment of body pain, knee pain, joint pain, dyspepsia, diarrhoea, typhoid, cholera, chronic jaundice, fever, and headache and skin diseases.² However, no systematic investigations have been carried out to analyse the anti-arthritic activity of Drynaria quercifolia.

Hence, in the present study an attempt has been made to screen the rhizome of Drynaria quercifolia for its anti-arthritic property in in-vivo model.

MATERIALS AND METHODS:

Materials:

Complete Freund’s adjuvant, Epinephrine, 4,6-diphenyl-1,10-phenanthrolin, Haemoglobin, DTNB, Superoxide dismutase, Acetyl acetone, and Chloramine T were purchased from Sigma Chemicals, U.S.A. Ferric chloride, Thiobarbituric acid (TBA) was purchased from Loba Chemicals, Mumbai. EDTA, p-nitro phenyl phosphate, Sodium acetate tri hydrate, Reduced glutathione, Copper sulphate, Folin-coicalteau reagent, Sodium potassium tartarate, 2,4 dinitro phenyl hydrazine, Ascorbic acid, Pyridine, Glycine, p-nitro phenol, Tris-Hcl buffer, p-dimethyl amino benzaldehyde, Trichloro acetic acid were purchased from Sisco Research Laboratory, Mumbai.

Sample collection and preparation:

Fresh rhizome of Drynaria quercifolia was collected from Kolli hills, Namakkal District, Tamil Nadu (INDIA). The fur portion was removed from the rhizome. The flesh part was sliced and minced. The resulting juice was centrifuged at 4000rpm for 10 minutes. The supernatant was concentrated under reduced pressure and lyophilized to obtain a brown colour residue. The residue was stored in a refrigerator at 4-5ºC until use. The yield (w/w) of the crude extract was found to be 2.7%. Fresh juice and crude extract were dissolved in water to the required concentration and used for the experiments.

Experimental animals:

The Institutional Animal Ethical Committee (IAEC, No: 15/01/AY18) granted permission for the current study, and the procedure complied with CPCSEA's national norms. The 180–200g female albino Wistar rats used in this study were purchased from the King's Institute in Guindy, Chennai, India. They were kept in an institutional animal house with standard environmental conditions (23–1°C, 55–5% humidity, and a 12hour light–dark cycle). They also had unlimited access to water and a standard diet from Hindustan Lever in Bangalore, India.

Anti-arthritic activity:

Anti-arthritic activity was assessed by the Complete Freund’s Adjuvant (CFA) induced arthritic method.^3,4 Arthritis was induced by a single intradermal injection of 0.1ml of Complete Freund’s Adjuvant (CFA) containing 10mg/ml dry heat-killed Mycobacterium tuberculosis in sterile paraffin oil into the left hind paw of rats. Fourteen days were taken for the induction of arthritis after the CFA injection. Rats were divided into five groups of 6 animals each. Group I - normal control rats, received distilled water orally, Group II - arthritic rats received distilled water orally (Negative control), Group III - arthritic rats received standard drug methotrexate (0.75mg/kg) orally (Positive control), Group IV - arthritic rats, received DQ extract (800mg/kg) dissolved in water, Group V - arthritic rats, received DQ fresh juice (30ml/kg) orally. The animals in Groups III, IV, and V were treated with their respective doses of drugs from the 15th day to the 28^th day after induction of arthritis. The change in body weight and swelling (measured using a plethysmometer) in the hind paws were examined periodically. On the 29^th day, blood was collected from the retro-orbital sinus for haematological and biochemical estimation. The animals were sacrificed by using ether anaesthesia. The liver, lungs, kidneys, heart, and hind limbs were removed for biochemical assays and histopathological examination (preserved in 10% formalin).

Measurement of paw swelling:

Changes in the edema of the left hind paw were measured from the ankle using a plethysmometer.

The percentage inhibition of paw volume was determined as:

(V_C-V₀) – (V_t-V₀)

--------------------------- × 100

(V_C-V₀)

Where V_C is the paw volume after induction, V₀ is the paw volume before induction, and V_t is the paw volume after treatment.

Haematological and Biochemical parameters:

The following haematological parameters were analysed by using MDC 4000 vet 3-part haematology analyser: RBC count, Differential Count (Lymphocytes, Neutrophils, Monocytes, and polymorphs), Hb, PCV, platelet count.

The following biochemical parameters were analysed by using Transasia smart batch analyser: Sugar, Cholesterol, TGL, Creatinine, SGPT, SGOT, ALP and Bilirubin.

Bio-chemical assays using tissue homogenates:

Tissue samples collected from all groups of animals were minced into fine pieces and homogenized by using IKAT 25 teflon-homogenizer in 0.1M Tris-HCl buffer with a pH 7.4.

The samples were centrifuged in a centrifuge at 10,000 rpm for 15min. and the supernant was stored at -70ºC for following biochemical assays.

Enzyme Assays:

a) Assays of enzymatic and non-enzymatic antioxidants and lipid peroxide:

The enzymatic antioxidants catalase (CAT), superoxide dismutase (SOD)and glutathione peroxidase (Gpx)were measured in tissue homogenates. The non-enzymatic antioxidants such as reduced glutathione (GSH), vitamin C, vitamin E were determined in tissue homogenates. The extent of lipid peroxidation was measured in tissue homogenates.^5-11

b) Assays of lysosomal enzymes and protein-bound carbohydrates:

The lysosomal enzymes like alkaline phosphatase (ALP), acid phosphatase (ASP) and cathepsin D levels were measured in tissue homogenates. The protein-bound carbohydrates, including hexosamine and uronic acid levels, were determined in homogenates of liver, kidney, and lung tissue.^12-14

c) Assay of hydroxyproline and myeloperoxidase (MPO):

The hydroxyproline level was estimated in liver, lungs, kidneys, and bone homogenates. MPO activity was assayed as an index of neutrophil infiltration into the joints and tissues.^15,16

Histopathological examinations:

At the end of the 28th day, all the animals were sacrificed to collect hind limbs (at the interphalangeal joint). The hind limbs were rinsed in ice-cold 0.9% saline and then fixed in 10% formalin for histopathological examination.

Statistical analysis:

The statistical parameters were analysed by two-way ANOVA (Bonferroni test). The results are expressed as Mean±SD. Significant differences between the groups were identified at p < 0.05.

RESULTS:

Body weight:

Body weight was significantly decreased in the negative control group, i.e, the arthritic control group, when compared with the normal control group. Such a reduction in body weight was not observed in animals treated with standard drug and Drynaria quercifolia (extract and juice). The results are depicted in table 1.

The values are expressed as mean±SD, n=6, a denotes significant at the level of P<0.05 in comparison with 0 day.

Paw volume.

In arthritis induced rats there was an appreciable increase in paw volume in the injected hind legs within 3-5 days. A significant increase (p<0.05) of paw volume was continued up to 20^th day and the increase in volume was stabilized after 20^th day. By this time the tail had become noticeably thickened. A decrease in paw volume was noticed from the 16th day, and a significant reduction in paw volume was observed after the 16^th day in DQ (fresh juice and extract) and standard treated groups. The paw volume changes in the control and perimental groups of rats are shown in Table 2.

Percentage inhibition of paw volume:

The results of percentage inhibition of paw volume depicted in table 3. Percentage inhibition of paw volume showed a gradual increase in positive (III), DQ extract (IV) and DQ fresh juice (V) groups after treatment. Maximum percentage inhibition was observed on day 28 in all the treatment groups. At the end of the study the percentage inhibition of DQ extract group (IV) was like positive group (III).

Table 3. Percentage inhibition of paw volume after treatment

Days	Positive Control	DQ Extract group	DQ Fresh juice group
0	0.000±0.00	0.000±0.00	0.000±0.00
4^th	42.025±17.488	46.787±17.289	42.025±21.731
8^th	54.11±13.419	56.150±19.599	51.869±17.628
12^th	68.01±16.937	68.762±19.061	69.352±12.062
14^th	79.76±14.220	79.919±11.03	70.275±14.530

The values are expressed as mean ± SD, n=6

Haematological analysis:

Table 4 represents the haematological changes associated with arthritic condition after treatment with DQ. Levels of Hb, PCV, platelet and DC were normal in all the groups. Significant (p<0.05) increase of TC and decrease of RBC was observed in arthritic group when compared with normal group.

Biochemical Analysis:

The results of biochemical analysis were shown in table 5. There was a significant increase in ALP and SGPT levels in the negative control group when compared to normal control, this was reversed to normal level by both standard treatment and Drynaria quercifolia treatment. There was a significant increase in SGOT level in the negative group, extract treated group and fresh juice treated group when compared to normal control group.

Biochemical assays using tissue homogenates:

a) Assays of enzymatic and non-enzymatic antioxidants and lipid peroxide:

Fig. 1a, 1b and 1c showed the activities of enzymatic antioxidants CAT, SOD and Gpx in tissue homogenates. In negative control there was a significant decrease catalase level (except heart tissue sample) when compared to normal control. This was significantly (p<0.05) increased by the treatment with standard drug, extract, and fresh juice of DQ. But in liver homogenate there was no significant improvement when compared to control, after treatment with standard drug. In negative control there was a significant decrease SOD and Gpx levels when compared to normal control. This was significantly increased by standard drug, extract, and fresh juice of DQ treatment. By comparing these enzyme levels, the role of DQ in free radical scavenging is well understood.

There was a significant decrease in reduced glutathione, vitamin E and vitamin C levels in the negative control group when compared to normal control group. This was reversed to normal level by the treatment with standard drug (MTx), extract and fresh juice of DQ. Data were represented in fig. 2a, 2b, and 2c.

Table 4. Haematological Parameters

Parameters	Normal Control	Negative Control	Positive Control	DQ Extract group	DQ Fresh juice group
HB (g/dl)	12.84±0.7	9.09±0.5^a	12.06±0.7	12.98±0.2	12.74±0.5
TC (cells/cumm)	6650±0.3	8375.0±4^a	6625±113	6012.5±90	7425.0±76
DC-POLY (%)	21.75±20	22.25±4.3	27.0±5.52	30.50±15	34.63±7.0
DC-LYMPH (%)	75.25±2.8	72.25±4.3	68.25±8.1	66.00±17	60.63±7.7
DC-EOSIN (%)	4.38±2.26	5.50±1.16	5.25±2.12	4.47±1.03	6.75±1.98
RBC (million/cumm)	4.491±0.8	4.887±1.0	4.874±1.0	4.347±0.4	5.374±1.1
PCV (%)	46.25±9.8	37.13±2.4	37.50±2.2	36.50±3.1	37.00±1.6
PC (lakhs/cumm)	2.424±0.2	4.577±1.2	4.545±0.8	4.484±0.1	5.012±0.5

The values are expressed as mean ± SD, n=6, a denotes significant at the level of P < 0.05 in comparison with control.

Table 5. Biochemical Parameters

Parameters	Normal Control	Negative Control	Positive Control	DQ Extract group	DQ Fresh juice group
Glucose (mg/dL)	90.1±3.6	99.25±19.3	90.88±3.35	102.38±9.65	100.75±20.97
Cholesterol (mg/dL)	33.6±3.1	57.63±12.0	53.50±7.5	48.25±9.3	53.38±12.4
Creatinine (mg/dL)	0.3±0.05	0.38±0.08	2.96±0.26	0.49±0.03	0.49±0.07
TGL (mg/dL)	157.8±4.1	128.1±12.4	121.0±15.6	122.5±18.39	154.5±45.11
SGPT (U/L)	100.7±2.4	144.1±10.7^a	108.5±4.2^b	99.6±68.1^b	101.5±66^b
SGOT (U/L)	96.50±1.4	114.5±13.8^a	95.8±2.03	106.6±40.9^a	121.6±48.9^a
ALP (U/L)	128.1±3.5	150.5±55.0^a	122.7±6.2^b	112.7±78.9^b	129.1±68.3^b
Bilirubin (mg/dL) a) T b) C	0.63±0.05 0.11±0.03	0.520±0.108 0.165±0.057	0.468±0.06 0.144±0.01	0.511±0.108 0.150±0.033	0.463±0.12 0.136±0.04

The values are expressed as mean ± SD, n=6, a denotes significant at the level of P< 0.05 comparison with control, b denotes significant

at the level of P < 0.05 comparison with negative control

c) Assay of hydroxyproline and myeloperoxidase (MPO):

In negative control group there was significant increase in hydroxyproline level (in lung and bone samples) was observed when compared to normal control group. This was decreased by the treatment with DQ (extract and fresh juice) and standard drug (MTx). The results were represented in fig. 5a. There was a significant increase in myeloperoxidase level in the negative control group (both in lung and bone samples) when compared to normal control group. This was reversed to normal by the treatment with the DQ (extract) and standard drug but not with DQ fresh juice treatment. The results were shown in fig. 5b.

Histopathological examination of interphalangeal joints

Fig. 6 shows the histopathological examination of bone. Arthritic control group (II) showed the erosion of cartilage and synovial hyperplasia (L), DQ fresh juice treated group showed the mild cartilage erosion (O). There were no abnormalities in normal control, positive control and DQ extract groups.

Fig. 6. Shows the histopathological slides (100X) of bones of Arthritic Wistar albino rats treated with plant extract and standard drug.

K: NC- No abnormality, L: Negative group- Erosion of cartilage and synovial hyperplasia, M: Positive group- No abnormality, N:DQ extract treated group- No abnormality, O:DQ juice treated-Mild cartilage erosion.

DISCUSSION:

Increased paw swelling observed in adjuvant induced arthritic rat was found to be the result of oedema of periarticular tissue. An increase in granulocytes and monocytes has been to be associated with changes in ankle diameter.¹⁷ Changes in body weight are useful index to assess the course of the disease and the response to therapy of anti-inflammatory drugs in question. The loss of body weight observed in arthritic animals may be due to the reduced absorption of glucose and leucin in the rat intestine.¹⁸

The result of present study reveals that the significant increase in body weight during DQ administration is due to restoration of absorption capacity of intestine in arthritic animals. This increase in absorption capacity may be due to the presence of vitamins, minerals, and antioxidant pigments in our plant.

The results also showed that the DQ and standard drug methotrexate significantly suppressed the paw swelling. At the end of the study, there was no significant difference in paw volume of extract and control groups. This shows the effectiveness of our plant in the treatment of inflammation in arthritis.

The haematological parameters did not show any significant difference between the DQ treated groups, methotrexate treated group and normal control. In arthritic control group there was a statistically significant difference (p<0.05) in Hb and TC on comparing with normal control. This shows that the DQ does not affect the haematological parameters.

The biochemical parameters, glucose, cholesterol, creatinine, TGL, bilirubin did not show any statistically significant difference (p<0.05) between the groups. The SGOT, SGPT, and ALP were elevated in arthritic control group compared to normal control. The SGPT and ALP showed a significant difference in DQ treated groups and methotrexate treated groups when compared to normal control, but the levels were within the normal physiological limits.

The enzymatic antioxidants, such as catalase, SOD, and GPx, had reduced activity in the arthritic control group compared to the standard control. But in the DQ-treated group and the methotrexate-treated group, there was no significant decrease compared to the normal control group. The increased production of oxygen free radicals in adjuvant induced arthritic rats leads to decreased activities of both enzymatic and non-enzymatic antioxidants because of their increased consumption during the oxidative stress and cellular lysis.¹⁹

The non-enzymatic antioxidants, such as GSH, vitamin C, and vitamin E, showed increased activity in the DQ-treated groups. But in arthritic control group, there was a significant (p<0.05) decrease. GSH is an important antioxidant that has been shown to destroy ROS and other free radicals through both non-enzymatic and enzymatic mechanisms, as it acts as a substrate for GPx and GST during the breakdown of H₂O₂and lipid peroxides.²⁰ The decreased levels of GSH in adjuvant-induced arthritic rats might also be due to the decreased GR (glutathione reductase) activity.²¹Vitamin C is an effective antioxidant because of its ability to fight against oxygen free radicals and other molecules that trigger rheumatoid inflammation. Also, it serves as a cofactor in collagen synthesis, the main protein in joint tissue and bone. Vitamin E is the major chain-breaking antioxidant. Despite its antioxidant properties, vitamin E also plays a vital role in anti-inflammation by decreasing the expression of pro-inflammatory cytokines, preventing NF-kB activation, and consequently arresting pro-inflammatory gene expression.²² It has been reported that flavonoids are good inhibitors of arachidonic acid peroxidation.²³ They interfere with the radical scavenging system and also increase the function of endogenous antioxidant systems. Administration of DQ decreased the oxidative stress by enhancing the activities of both enzymatic and non-enzymatic antioxidants, which may be due to the presence of flavonoids.

Lipid peroxidation was significantly decreased in DQ, and methotrexate treated groups when compared to arthritic control group. Lipid peroxidation is considered a critical mechanism of the injury that occurs during RA. The malondialdehyde (MDA) concentration in adjuvant-induced arthritic animals suggests a role for free radicals during the phagocytosis of immune complexes in this inflammatory arthropathy.²⁴ This indicates that the administration of DQ inhibited lipid peroxidation.

The lysosomal enzymes like ALP, ACP, and Cathepsin D were significantly elevated in the arthritic group. There was a significant decrease in DQ in the methotrexate-treated groups when compared to the arthritic group. Lysosomal enzymes are the main factors playing a vital role in tissue injury and repair, inflammation, phagocytosis. They participate in pathological processes such as inflammation, degeneration, and rheumatoid arthritis.^25,26 Activities of ALP, ACP and Cathepsin D were significantly increased in arthritic rats, since these are good indices of liver and kidney impairment, which are also considered as the features of adjuvant arthritis.²⁷ Administration of DQ decreases the release of lysosomal enzymes. The reduction of lysosomal enzymes proves the beneficial effect and indirectly confirms the protective role of the plant.

The levels of glycoproteins (hexosamine and uronic acid) were elevated in arthritic group when compared to control group. This elevation indicates the severity of the disease, and this altered glycoprotein metabolism observed in arthritic animals is due to the increased release of acid hydrolases during the arthritic condition. These enzymes are involved in the degradation of structural macromolecules in connective tissues and cartilage proteoglycans.²⁸ In DQ-treated groups, the glycoprotein levels were significantly decreased.

In the arthritic group, the hydroxyproline was significantly increased when compared to normal control group. Hydroxyproline is a biomarker for collagen hydrolysis. The hydroxyproline level was increased in arthritic animals, indicating increased hydrolysis of collagen in tissues and bone cartilage.²⁷ In the DQ treated groups there was a significant decrease in hydroxyproline level compared to arthritic control groups.

Myeloperoxidase activity was significantly increased in the arthritic group when compared to normal control. In the DQ-treated groups and the methotrexate-treated group, there was a significant decrease in myeloperoxidase activity when compared to the arthritic control group. The increased level of MPO in synovial tissues and hind paw of arthritic animals might be due to increased production of hypochlorous acid, which in turn has been reported to have a destructive effect on tissue components, especially those containing unsaturated lipids.^29-31 The result of present shows that DQ has myeloperoxidase inhibition activity.

Histopathological examination of the joint showed mild cartilage erosion in the DQ juice-treated group, but there were no significant changes in the DQ extract-treated group and the methotrexate-treated group. This indicates that DQ extract suppressed the inflammatory changes associated with arthritis which was equivalent to methotrexate.

In conclusion, the present study showed the anti-arthritic potential of DQ. The quantitative enzymatic analysis of tissue homogenates supported the anti-arthritic activity of DQ. DQ extract exhibited better anti-arthritic activity than DQ fresh juice.

CONCLUSION:

In the present study, we investigated the anti-arthritic effect of Drynaria quercifolia rhizhome, in adjuvant induced arthritic rats. Results showed that DQ significantly inhibited the paw swelling, and this inhibition was accompanied by an increase in antioxidant levels, a decrease in the levels of lysosomal enzymes, glycoproteins, hydroxyproline, and myeloperoxidase. Hence, this study validated the antiarthritic potential of fresh juice and extract of Drynaria quercifolia rhizhome.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the respective institutions for their support during the research. RS, UMD and TG are thankful to (Late) Mr. V. Elango, Animal House, CLRI for assistance in the maintenance of experimental animals.

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Received on 18.04.2024 Revised on 16.12.2024

Accepted on 02.06.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5798-5806.

DOI: 10.52711/0974-360X.2025.00836

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Days	Body weight in g
Days	Normal control	Negative control	Positive control	DQ Extract group	DQ Fresh juice group
0	142.00±4.7	142.50±2.6	148.75±3.5	155.63±1.7	156.88±5.3
4^th	141.25±5.0	138.13±2.5	138.13±5.9	145.00±5.3	149.38±4.9
8^th	143.13±4.5	135.00±5.3	138.13±5.9	147.50±3.8	133.13±3.7
12^th	147.13±3.7	133.75±4.4	136.88±6.5	144.38±1.7	132.50±4.6
16^th	148.00±3.7	131.25±4.1	150.00±3.7	160.00±5.3	135.63±4.1
20^th	148.50±4.2	129.38±3.2^a	152.13±4.1	160.65±5.7	135.63±4.1
24^th	150.00±3.6	127.50±2.6^a	155.88±4.3	165.00±4.6	157.88±4.7
28^th	150.88±4.1	127.50±6.5	160.00±7.5	166.88±9.2	168.38±7.9

Days	Paw volume in ml
Days	Normal Control	Negative Control	Positive Control	DQ Extract group	DQ Fresh juice group
0	0.45±0.07	0.55±0.05	0.55±0.05	0.437±0.05	0.50±0.05
4^th	0.45±0.05	1.07±0.08^a	1.21±0.15^a	1.262±0.13^a	1.388±0.20^a
8^th	0.45±0.05	1.037±0.05^a	1.263±0.2^a	1.225±0.14^a	1.188±0.19^a
12^th	0.45±0.07	1.45±0.26^a	1.413±0.2^a	1.525±0.11^a	1.575±0.16^a
16^th	0.43±0.05	1.57±0.10^a	1.02±0.08^ab	1.025±0.19^ab	1.063±0.25^ab
20^th	0.47±0.07	1.66±0.15^a	0.92±0.07^ab	0.937±0.22^ab	1±0.2138^ab
24^th	0.47±0.07	1.66±0.11^a	0.813±0.09^b	0.787±0.19^b	0.838±0.14^ab
28^th	0.47±0.07	1.66±0.11^a	0.713±0.08^b	0.6625±0.10^b	0.825±0.16^ab