Evaluation Of Anti-Rheumatoid Arthritis Activity of Curly Kale (Brassica Oleracea Var. Sabellica) Leaves in Freund’s Complete Adjuvant Induced Arthritis in Wistar Albino Rats

Mayukh Sarkar; Anjali Yadav; Arpita Jena; Surabhi K. S.; Vinayak Pawar; Yamini Durga

doi:10.5281/zenodo.15738477

Research Paper | Open Access
Volume 03 | Issue 06 | Article Id IJPS/250306255

Evaluation Of Anti-Rheumatoid Arthritis Activity of Curly Kale (Brassica Oleracea Var. Sabellica) Leaves in Freund’s Complete Adjuvant Induced Arthritis in Wistar Albino Rats
Mayukh Sarkar* Anjali Yadav Arpita Jena Surabhi K. S. Vinayak Pawar Yamini Durga
Department of Pharmacology, Krupanidhi College of Pharmacy, Bangalore.

Abstract

Objective: To evaluate the anti-rheumatoid arthiritic activity of curly kale (Brassica oleracea var. Sabellica) leaves in Freund’s complete adjuvant induced arthritis in Wistar albino rats. Materials and Methods: The extract of curly kale leaves was administered orally to the rats in 250mg and 500mg after inducing inflammation with Complete Freund's Adjuvant (CFA). Physical parameters (body weight, paw volume, grip strength) and haematological measures (RBC, Hb, ESR, WBC count, platelets) were assessed on days 0, 7, 13, 17, and 22. Radiological comparisons with a standard and histopathological studies were also conducted. Results: Curly kale leaf extract, at doses of 250mg/kg and 500mg/kg, shows a marked decrease in paw volume in rats with induced inflammation. The extract also elevated RBC and Hb levels toward normal, while effectively mitigating increased WBC count and ESR. The treated rats exhibited protection from bone destruction, reduced soft tissue swelling, and preserved joint integrity, especially at the higher dose of 500mg/kg. Conclusion: This study reveals that the aqueous extract of Curly kale leaves showed an powerful anti-rheumatoid activity.

Keywords

Curly Kale. Complete Freund's Adjuvant. Indomethacin. Inflammation, NSAID'S. Rheumatoid Arthritis

Introduction

Rheumatoid arthritis (RA) is an autoimmune disorder that causes chronic, symmetrical inflammation, and systemic manifestations and it affects approximately 0.3 to 1% of the global population. Mostly this disease occurs in between 50 and 60 years of age and women being more frequently affected than men. In individuals with RA, joint deformities are seen, along with a progressive loss of function, cartilage degradation, and bone damage. NSAIDs and DMARDs are used to manage pain and inflammation in rheumatoid arthritis (RA). Treatment aims to minimize pain and prevent bone deformity, with ibuprofen and naproxen being common NSAIDs. Despite recent progress, achieving full remission is uncommon, and most patients experience mild to moderate improvements.[2]

Historical Aspects of Rheumatoid Arthritis:

Archaeological evidence suggests RA may have originated in the Western world around 200 years ago.[3] American-Indian skeletons with RA dating back to 4500 BC have been found Literary references date back to 123 AD in the Indian text "Charak Samhita" The condition "rheumatoid arthritis was first used in 1859 by Dr. Alfred Baring Garrod.[4]

Risk Factors for RA: Genetic factors contribute about 60% to RA risk, with HLA-DRB1-shared epitope alleles being significant Environmental factors like smoking, silica dust exposure, mineral oils, dietary factors, coffee consumption, and infections are associated with increased risk RA incidence is higher in females (2.1to 3:1 ratio) Autoantibodies, particularly ACPA, are important in RA pathology.

Stages of RA: Monocyclic or Self-limited, Polycyclic or Persistent, Progressive [5]

Comorbidities: RA, a systemic disease, can lead to cardiovascular, renal, and respiratory issues. Delayed or inadequate treatment can result in joint deformity. disability, and a reduced lifespan.

Stages of RA:

Stage I: Synovitis causes joint swelling and pain, with no visible joint damage on X-rays.

Stage II: Inflammation affects joint cartilage, leading to cartilage failure.

Stage III: Pannus development in the synovium, visible joint deformities, and erosions around joints.

Stage IV: End stage, joint function ceases due to fibrous tissue or bone fusion.

Criteria for Classification: Revised ACR/EULAR (2010) criteria use a scoring system to identify early-onset RA patients, considering factors like serology, joint distribution, acute-phase reactions and symptom duration.[6]

Clinical Features: Rheumatoid Arthritis affects small and large joints, causing morning stiffness, joint swelling, and tenderness. Common deformities include ulnar deviation, boutonniere, and swan-neck deformities. [7]

Pathophysiology: Immune cell activation, antigen presentation, cytokine release, and interactions with synovial cells contribute to RA pathogenesis, Pro-inflammatory, cytokines, TNF-4, IL-1, and IL-6, play crucial roles. [8]

Pathways in Pathogenesis: MAPK, Syk, JAK-STAT, and NF-KB pathways are involved in RA pathogenesis, influencing inflammation, cytokine production, and immune cell regulation [9,10]

RA Therapy: Early aggressive therapy and treat-to-target strategies guide RA treatment. DMARDs, including synthetic and biologic options, aim to reduce inflammation and prevent joint damage.[11]

Biosimilars: The advent of biosimilars for biologics may impact drug pricing offering alternatives with similar efficacy.[12]

MATERIALS AND METHODS

Collection and Isolation of raw extract:

Source of Material: Kale leaves from Urbana Superfoods and Farms Ventures.

Identification: Authenticated as curly kale (Brassica oleracea var sabellica) by taxonomist.

Authentication: Accompanied by an authentication letter with reference number.

Phytochemical Tests: [11,12]

Alkaloids Identification:

Dragendroff's Kraut's test: Mixture of few ml filtrate 1-2 ml Dragendroff's reagents will form a reddish-brown precipitate.

Mayer's/ Bertrand's /Valser's test: Mixture of few ml filtrate 1-2 drops of Maver's reagent (Along the sides of test tube) will form a creamy white/yellow precipitate
Wagner's test Mixture of few ml filtrate 1-2 drops of Wagner’s reagent. Along the sides of test tube will form brown/reddish precipitate

Carbohydrates Identification:

Barfoed's test: mixture of 1ml filtrate and 1ml of Barfood’s test red precipitate will appear after 2mins of heating.
Molish's test: A violet ring will form along the test tube’s sides when two ml of filtrate are combined with two drops alcoholic naphthol and one millilitre of concentrated H2SO4.

Tannins Identification:

The gelatin test: five ml of distilled water are used to dissolve the plant extract. A whitish precipitate of 10% NaCl.

Braymer's test: 3ml of distilled water with 1ml of filtrated water and also 3 drops of ferric chloride solution 10%.it shows blue green
10% test with NaOH + 4ml of plant extract. 10%NaOH with a good shake -Formation of emulsion.

Flavonoids Identification:

In Alkaline reagent test:1 millilitre of extract and 2 millilitres of 2% NaOH solution will mature to a yellow colour before turning colourless when a few drops of dil HCL are added.
Ferric chloride test: Aqueous extract solution combined with a few drops of 10% ferric chloride green precipitate will form from the solution.
Ammonia test: The filtrate and five ml diluted Ammonia solution mixture along with H2SO4 produce yellow colour.
Conc.H2SO4 test: The plant extract and conc. H2SO4 mixture will form orange colour

Terpenoids Identification:

Terpenoids can be detected by combining 2 millilitres of chloroform with 5 millilitres of plant extract that has evaporated in a water bath and 3milliliters of con H2SO4 that has been heated in water bath to create a grey solution.

Identification of Cardine Glycosides:

Keller-Killani test: A blue coloured solution will form in the acetic acid when 1.5ml of glacial acetic acid layer,1.5ml of filtrate, a drop of 5% ferric chloride, and concentrated H2SO4 are mixed along the test tube’s side.
Cardenolide test: extract, pyridine, sodium nitroprusside, and 20% NaOH mixture initially crimson, it eventually turns brownish yellow.
Test for bromide water: A yellow precipitate will appear when a mixture of plant extract and few millilitres of bromine water are combined.

Experimental Animals:

Species: Albino Wistar rats (200-250g).

Ethical Approval: The Institutional Animal Ethics Committee (IAEC) granted ethical approval. Approval No: KCP/IAEC/PCOL/103/2022.

Acute Oral Toxicity Studies: The doses of Curly kale leaves were selected as per the previous toxicity studies conducted and given by oral route.[13]

Doses: Selected based on previous toxicity studies.

Groups:

Normal Group
Positive Group (Complete Freund's adjuvant induced)
Low Dose Model [250mg/kg of curly kale extract] (13)
High Dose Model [500mg/kg of curly kale extract] (13)
Standard Drug Group [13mg/kg of Indomethacin] (15)

Physical Parameters:

Body Weight Measurement: Periodic measurements from day 0 to day 22^.(13,15)
Paw Volume measurement: Determined on particular days using a plethysmometer.
Motor Coordination Test: Evaluated using a rotating rota-rod. (17)
Haematological Parameters:
RBC Estimation: Using Neubauer's chamber. (18)
Haemoglobin Estimation: Sahli's Hemoglobinometer method. (19)
WBC Estimation: Using Neubauer's chamber. (18)
Platelet Count: Hematocytometry method. (20)
ESR Determination: Wintrobe method. (21)

Freund's adjuvant induced rheumatoid arthritis in rat:

Animals after 30 min of oral administration of test & standard (Indomethacin 13 mg/kg body weight) was treated with Complete Freund's adjuvant (0.1ml) in sub-plantar surface of the hind paw region. This was designated as day 0. Drug treatment was continued for duration of 22 days. [14]

Histopathological Examination:

Histopathological findings of bone & joint of rat revealed that in RA-induced Control group, there is a moderate distortion of areolar tissue with cartilage cell hyperplasia and blood vessel congestion were observed. The lesions manifested proliferation of cartilage cells and distortion of areolar tissues with narrowing of joint space, congestion of blood vessels at the arteriole level in the RA induced Control rats were evident which were reduced drastically to near normal morphology with healing and protective response in treated groups. (17)

Statistical Analysis: Data were analysed using one-way ANOVA, followed by Dunnett's test for multiple comparisons.

Radiographical Analysis:

The sample images presented in Fig.7 illustrated morphological alterations in each group rats' hind paws on day 22. When compared to the control group, the model group's arthritic paws showed clear signs of inflammatory swelling and erythema. Additionally, rats given varying dosages of indomethacin and curly kale extract showed a substantial reduction in arthritic symptoms. When compared to the rats in the normal group, the CFA-induced rats showed clear soft tissue enlargement, bone degradation, and joint deformity, according to an X-radiographic examination of their hind paws. Treatment with 250 mg/kg and 500 mg/kg of curly kale extract significantly reduced soft tissue oedema and inhibited joint erosion and distortion.[22]

DISUSSION:

An autoimmune condition known as rheumatoid arthritis (RA) affects 0.3-1% of people worldwide and has no known cure. Timely diagnosis is essential for targeted treatment. Natural products are becoming more and more popular because of their affordability and safety. The rat model of arthritis caused by FCA mirrors the symptoms of RA. The anti-rheumatoid potential of curly kale leaf extract, which is high in flavonoids, was investigated. (23) FCA-induced arthritis is a widely used experimental model that closely mimics the clinical and pathological features of human rheumatoid arthritis (RA), including rapid onset, polyarticular inflammation, joint swelling, lymphocyte infiltration, and cartilage degradation (26). Symptoms typically emerge within 10–14 days of induction, often progressing to permanent joint deformities such as ankylosis. FCA (Complete Freund’s Adjuvant) is composed of inactivated mycobacteria that strongly stimulate cell-mediated immunity and immunoglobulin production. In this model, rats develop persistent joint inflammation, tissue edema, bone remodelling, and degeneration—hallmarks also seen in human RA. (27) The present study aimed to evaluate the anti-rheumatic effects of Curly kale leaf extract on FCA-induced arthritis in rats. Rich in bioactive flavonoids such as quercetin, kaempferol, epicatechin, and apigenin, Curly kale offers potent antioxidant properties capable of protecting against oxidative stress, DNA damage, and erythrocyte injury. The extract was tested in vivo at doses of 250 mg/kg, 500 mg/kg, and 1000 mg/kg, with results compared to the standard NSAID, Indomethacin. One of the indicators of disease severity, body weight loss due to rheumatoid cachexia, was notably lower in the groups treated with Curly kale extract and Indomethacin, suggesting a restoration of metabolic activity and intestinal absorption. The treated groups also exhibited reduced paw volume, indicating significant anti-inflammatory activity. Notably, the highest dose (1000 mg/kg) showed effects comparable to Indomethacin (26). The extract inhibited paw swelling in a dose-dependent manner and alleviated typical RA symptoms such as redness, joint pain, and limited mobility. Haematological parameters showed that FCA-induced rats developed anaemia, evidenced by reduced RBC counts and haemoglobin levels, potentially due to iron mismanagement and impaired bone marrow response. These changes were mitigated in the extract and Indomethacin-treated groups. Additionally, elevated leukocyte counts in arthritic rats were reduced following treatment, indicating immunomodulatory effects. The erythrocyte sedimentation rate (ESR), a marker of systemic inflammation, was also significantly lowered in the treatment groups. Radiographic analysis revealed severe joint changes in untreated arthritic rats, including soft tissue swelling and narrowing of joint spaces—indications of cartilage and bone damage. In contrast, rats treated with Curly kale extract or Indomethacin displayed less joint space narrowing and reduced soft tissue swelling, reflecting protective effects on joint structure. Histopathological examination further confirmed these findings. While CFA-only rats showed increased vascularization, synovial distortion, and cartilage loss, treated rats had better-preserved joint architecture, reduced inflammation, and nearly normal synovial tissue.

CONCLUSION:

Finally, this investigation showed that the application of extract from curly kale leaves was effective. When used in conjunction with Complete Freund's adjuvant to develop rheumatoid arthritis, indomethacin dramatically increased anti-rheumatoid action. The extract is rich in flavonoids, polyphenolic chemicals, and anthraquinone glycoside, and their high concentration may have contributed to its antioxidant activity. This is because phenolic compounds possess direct antioxidant qualities as a result of their hydroxyl groups' capacity to donate hydrogen. The capacity of a chemical to decrease can serve as a helpful indicator of its potential antioxidant action. It would also be worthwhile to study the effects of Curly kale leaves extract with different drugs used in rheumatoid arthritis treated patients, in the hope of reducing morbidity.

RESULTS:

The study investigated the phytochemical composition of curly kale leaves and evaluated its effects on body weight, paw volume, fall of time, and haematological parameters in a Freund's Complete adjuvant (CFA)-induced model. The results are summarized as follows:

Phytochemical Investigation (Table 1):

Compounds Identified: Alkaloids, flavonoids, cardiac glycosides, tannins, and carbohydrates.
Alkaloids Test: Positive results for Dragendroff, Wanger's, and Mayer's reagent tests.
Flavonoids Test: Positive results for alkaline reagent, ferric chloride, conc, H2SO4 and ammonia tests.
Tannins Test: Positive results for gelatine, Braymer's, and 10% NaOH tests.
Terpenoids Test: Negative results for various tests.
Cardiac Glycoside Test: Positive results for tests related to cardenolide and bromine water.
Carbohydrate Test: Positive results for Barford's and Molisch's tests.

In Vivo Study:

Effect on Body Weight (Tables 2): CFA caused a significant decrease in body weight.

Curly kale extract and Indomethacin restored body weight to near-normal levels.

Effect on Paw Volume (Tables 3): CFA induced a significant increase in paw volume.

Curly kale extract and Indomethacin significantly decreased paw volume

Effect on Fall of Time (Tables 4): CFA caused a significant decrease in tall of time

Curly kale extract and Indomethacin increased fall of time.

Haematological Parameters (Tables 5): CFA caused a decrease in Hb and RBC levels, while increasing the ESR count and WBC.

Curly kale extract and indomethacin helped normalize Hb and RBC levels, while lowering the ESR count and WBC.

Overall Conclusion:

Curly kale leaves extract demonstrated potential in mitigating CPA-induced changes in body weight, paw volume, fall of time, and haematological parameters, suggesting its anti-inflammatory and immune-modulating effects.

Figures:

Refer to Figures 1-4 for visual representation of the effects observed in the study.
Note: The abbreviations used are LD represents (Low Dose), HD stands for (High Dose), and NS refers to (Normal Saline).
The symbols ,,,**** indicate the level of significance in statistical comparisons.
Histopathological Analysis: Histopathological findings of bone & joint of rat revealed that In RA-induced Control group, there is a moderate distortion of areolar tissue with cartilage cell hyperplasia and blood vessel congestion were observed.
The lesions manifested proliferation of cartilage cells and distortion of areolar tissues with narrowing of joint space, congestion of blood vessels at the arteriole level in the RA induced Control rats were evident which were reduced drastically to near normal morphology with healing and protective response in treated groups. A quantitative phytochemical screening on curly kale Leaf indicates the existence of cardiac glycosides, alkaloids, flavonoids, carbohydrates and tannins.

Table 1: Phytochemical investigation for various compounds.

Test	Observation	Result
	Test for alkaloids
Dragendroff test	Reddish brown colour	+ve
Wagner’s reagent test	Brown precipitate	+ve
Mayer’s reagent	Creamy white precipitate	+ve
	Test for flavonoids
Alkaline reagent	When dil is added, a bright yellow colour turns colourless. HCL	+ve
Ferric chloride test	A green ppt	+ve
Conc. H2SO4 test	Orange colour	+ve
Ammonia test	Yellow colour	+ve
	Test for tannins
Gelatine test	Five millilitres of distilled water, 1% gelatin solution, and 10% NaC are used to dissolve the plant extract.	+ve
Braymer’s test	Filtered water (1 ml), distilled water (3 ml), and three drops of 10% ferric chloride solution	+ve
10% of NaOH test	0.4 ml plant extract, 4 ml 10% NaOH, and an adequate shake	+ve
	Test for terpenoids:
Test for terpenoids	Grey coloured solution	-ve
Salkowski’s test	Grey coloured solution	-ve
Libermann -barchord	Brown ring formed at junction of two layer	-ve
Sulphur powder test	Sulphur sink at bottom	-ve
Test for terpenoids	Grey coloured solution	-ve
	Test for cardiac glycoside
Keller Killani	No blue colour	-ve
Test for cardenolide	Reddish yellow	+ve
Bromine water test	Yellow ppt	+ve
	Test for carbohydrate
Barford’s test	No red ppt	+ve
Molisch’s test	A violet ring	+ve

Table 2: Effect of Curly Kale leaves extract and Indomethacin on CFA induced changes in body weight on 1^th,7^th,13^th ,17^th and 22^nd day.

	1^st Day	7^th Day	13^th Day	17^th Day	22^nd Day
Normal Saline	155.3±1.66	161±1.26	167.66±2.57	174.33±2.38	182.1±2.70
Positive (Cfa Induced)	175.5±3.10	174.8±2.51	168.7±3.7	169 ± 2.95	171.3±3.12
LOW DOSE (250 Mg)	157.8±1.21	165.5±1.45	174.83±2.38	185.83±2.37	179.0±2.63
HIGH DOSE (500mg)	151.5±0.9	160.2±1.27	166.33±2.38	183.00±2.08	189.3±1.9
STANDARD (13mg/Kg)	167.7±1.05	185.2±2.73	190.16±4.51	199.16±3.53	208.8±2.44

Effects of curly kale leaves (250mg / kg and 500mg / kg) on body weight in CFA induced model. Values are plotted as the mean plus/minus S * EM n-6 in each group; analyzed by ANOVA followed by Dunnett's test.

Where,

*Indicates P < 0.05 bullet p < 0.01, *** p < 0.001 p < 0.0001 when control group is compared with normal saline group and NS indicates non-significant, a indicates P ^ 0.05, bp < 0.01 c p < 0.001 dp < 0.0001 when LD, HD and standard group is compared with control group. Effect of Curly kale leaves extract and Indomethacin on paw volume Complete Freund's adjuvant (CFA) caused significant (P < 0.001) increase in the paw volume when compared to control rats. Treatment with Curly kale leaves extract and Indomethacin showed a significant decrease in paw volume to near-normal levels. Immunization with sub-plantar administration of CFA produced an increase in the volume of paw compared to vehicle treated group. There was a maximum paw volume on day 7 and after that there was a slight decrease and this was continued till the end of the study. On treatment with Curly kale leaves extract (250mg / kg and 500mg / kg) a significant (p < 0.05) and dose dependent decrease in paw volume was seen.

Table 3: Effect of Curly kale leaves extract and lndomethacin on CFA induced changes in paw volume on 1^st, 7^th, 13^th, 17^th and 22^nd day.

	1^st day	7^th day	13^th day	17^th day	22^nd day
Normal Saline	0.30±0.20	0.33±0.024	0.32±0.03	0.32±0.03	0.31±0.03
Positive (CFA Induced)	1.20±0.09	1.38±0.09	1.81±0.09	1.85±0.1	1.71±0.09
Low Dose (250mg)	1.21±0.03	1.41±0.06	1.2±0.05	1.1±0.03	0.93±0.03
HIGH DOSE (500mg)	1.4±0.14	1.48±0.06	1.2±0.05	1.0±0.04	0.83±0.04
Standard (13mg/Kg)	1.1±0.07	1.2±0.06	1.03±0.02	0.91±0.03	0.81±0.04

Effects of Curly kale leaves (250mg/kg and 500mg/kg) on paw volume in CFA induced model. Values are plotted as the mean ±SEM, n-6 in each group: analyzed by ANOVA followed by Dunnett's test.

Where,

*Indicates P < 0.05 bullet p < 0.01, *** p < 0.001 **** p < 0.0001 when control group is compared with normal saline group and ns indicates non-significant, a indicates p<0.05, bp < 0.01 c p < 0.001 1p < 0.0001 when LD, HD and standard group is compared with control group. Effect of Curly kale leaves extract and Indomethacin in fall of time Complete Freund's adjuvant (CFA) caused significant (p<0.05) decrease when compared to control rats. Treatment with Curly kale leaves extract and Indomethacin showed increase in Fall of time. Mean fall of time in rotarod test was determined for the assessment of motor coordination. Administration of CFA results in the decrease in the fall of time as compared to the vehicle treated group. Rats treated with Curly kale leaves extract (250mg/kg and 500mg/kg) and Indomethacin (13mg/kg) significantly increased (p<0.05) fall of time as compared to CFA control group.

Table 4: Effect of Curly kale leaves extract and lndomethacin on CFA induced changes in fall of time on 1^st, 7^th, 13^th, 17^th and 22^nd day.

	1^st day	7^th day	13^th day	17^th day	22^nd day
Normal Saline	81.5± 4.32	74.6 ± 6 .07	69.6 ± 4 4	74 ± 3.11	81 ± 4 54
Positive (CFA INDUCED)	48.0± 4.18	34.3± 3 .95	26.3 ± 2. 4	32.5 ± 5 57	35 ± 5 49
Low Dose (250mg)	47.3± 2.49	48.8 ± 2 .98	46.5 ± 3. 1	46.5 ± 2 62	50.8 ± 3.05
High Dose (500mg)	52.8±2.07	54.8±2.62	60.3±2.9	64.0±2.00	63.6±2.76
Standard (13mg/kg)	62.0±1.89	63.6±4.7	67.8±3.0	72.0±2.87	79.3±1.42

Effects of Curly kale (250mg / kg and 500mg / kg) on fall of time in CFA induced model Values are plotted as the mean±SEM, n-6 in each group; analyzed by ANOVA followed by Dunnett's test. Indicates P < 0.05 bullet p < 0.01 *** p < 0.001 **** p < 0.0001 when control group is

Where,

compared with normal saline group and ns indicates non-significant, a indicates p 0.05, b p < 0.01 c p < 0.001 1p < 0.0001 when LD, HD and standard group is compared with control group. Effect of Curly kale leaves extract and Indomethacin on Complete Freund's Adjuvant (CFA) induced changes in RBC, WBC, Hb and ESR A significant (p < 0.01) reduction in the levels of HB and RBC was observed in the CFA treated when compared with normal rats. Administration of Curly kale leaves extract and Indomethacin to diseased rats enhanced the levels of RBC & Hb to normal levels. The raise in WBC count and ESR were significantly (p<0,05) overcome in the extract treated groups.

Table 5: Effect of Curly kale leaves extract and lndomethacin on CFA induced changes in RBC, WBC, HB and ESR on 1^st, 7^th, 13^th, 17^th and 22^nd day.

	RBCRBC (106×µL)	WBC (103×µL)	Hb (gm/dl)	ESR (mm/hr)
Normal Saline	1.5±4.32	9.41±0.8	13.18±0.3	2.39±0.5
Positive (CFA Induced)	48.0±4.18	18.7±0.3	6.32±0.8	18.58±2.8
Low Dose (250mg)	47.3±2.49	18.0± 0.5	8.03±0.8	15.34±2.3
High Dose (500mg)	52.8±2.07	13.2±0.8	9.4±0.8	10.73±1.2
Standard (13mg/Kg)	62.0±1.89	11.4±0.25	10.67±0.6	8.61±0.27

Effects of Curly kale leaves extract (250mg / k g and 500mg / kg) on RBC, WBC, Hb. ESR count in CFA induced model. Values are plotted as the mean ±SEM. n=6 in each group; analyzed by ANOVA followed by Dunnett's test.

Where,

"Indicates P < 0.05 ** p < 0.01 p < 0.001 ** p < 0.0001 when control group is compared with normal saline group and NS indicates non-significant, a indicates P<0.05. b p^ ∞ * 0.01, c p < 0.001 d p < 0.0001 when L.D, HD and standard group is compared with control group.

Histopathological analysis:

A. Normal bone structure: the typical (areolar) Normal histology of the synovium normal 45 is observed - NAD+ (X50).

B. Normal bone structure: The normal cells that line the cartilage of the synovium (areolar) normal histology is observed - NAD+ (X100)

A. Moderate (3+) proliferation of cartilage cells Joint space narrowing and deformation of the synovium's areolar tissue (arrow) blood vessel congestion - moderate (3+)

B. Cartilage cell hyperplasia - moderate (3+) is observed (X100)

A. Normal bone structure: Normal histology of follicles and normal (areolar) synovial lining cells are seen, as indicated by NAD+ (X50).

B. Normal bone structure. NAD+ (X100) shows the typical (areolar) synovial lining cells with follicles and normal histology.

Normal bone structure: Normal (areolar) synovial cells with normal follicular histology are seen, as is NAD+ (X50).

B. Normal bone structure: normal (areolar) synovial cells with normal histology of follicles are seen; NAD+ (X100)

Normal bone structure: NAD+ (X50) shows normal (areolar) synovial cells with follicles and normal histology.
Normal bone structure: normal histology is seen in the synovial lining cells (cartilage) and areolar cells (NAD+) (X100).

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