Antiarthritic potential of ethanolic extract of Ixora coccinea leaves on complete Freund’s adjuvant-induced arthritis in animal model

Abstract

CONTEXT:

Ixora coccinea leaves possess antioxidant, anti-inflammatory, antinociceptive, antimutagenic, and gastroprotective properties. On this background, its antiarthritic potential was evaluated.

AIMS:

The objective is to evaluate the effect of Ethanolic extract of Ixora coccinea leaves (EEICL) on complete Freund’s adjuvant-induced arthritis in rats.

SETTINGS AND STUDY DESIGN:

PG research laboratory, Pharmacology Department, MKCG Medical College, Berhampur, Odisha.

SUBJECTS AND METHODS:

Thirty-six Wistar albino rats were randomly distributed into sixgroups (n = 6) as follows: Gr 1 (normal control)-DW p.o, Gr-2 (disease control [DC] - Tween 80 p.o), Gr-3 (piroxicam 0.9 mg/kg p.o), Gr-4 (EEICL-1 g/kg, p.o, Gr 4-EEICL-1.5 g/kg p.o, Gr 5-ED50 (0.82 g/kg) + piroxicam (0.45 mg/kg) p.o. After induction of arthritis, drugs, and vehicles were administered daily from 5^th to 25^th day. On 0, 5^th, 10^th, 15^th, and 25^th day, parameters like body weight, rotarod fall time, paw volume displacement, and arthritis index were measured. On the last day, Erythrocyte sedimentation rate (ESR), tissue malondialdehyde (MDA), and histopathological analysis were done.

STATISTICAL ANALYSIS USED:

Analysis of parametric data was done by one-way ANOVA and nonparametric data by Kruskal–Wallis test using graph pad prism 7.0. P < 0.05 was considered statistically significant.

RESULTS:

EEICL (1.5 mg/kg) showed anti-arthritic effect compared with DC. Rotarod fall-off time 137.5 ± 2.5 sec and body weight (139 ± 12.74 g) were increased significantly. The percentage inhibition of paw volume was increased(52%) whereas arthritic score(0.33), ESR(3.51mm/hr), synovial tissue MDA level (0.62±0.13µmol/gm) and Mankin score(2) were reduced significantly as compared to disease control.

CONCLUSIONS:

EEICL has anti-arthritic potential in rat model.

Introduction

Rheumatoid arthritis (RA), an autoimmune and inflammatory disease, is presenting as polyarthritis affecting joints of the hands, wrists, and knees. In this condition, the joint lining becomes inflamed and causes damage to joint tissue and chronic pain, unsteadiness, and deformity which affects a person’s quality of life severely. RA can also affect other tissues in the body such as lungs, heart, and eyes.[1] An Indian study reported that females were more affected, i.e. 81.5%, 26% had a family history and 39.1% with high disease activity of the total study population. Mostly metacarpophalangeal joints were involved and lead to joint deformity.[2] Nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) are effective costlier treatment options but cause multiple adverse effects. Many patients are dependent on indigenous medicines. Hence, researchers are in search of the most safe and effective medicine from plant source.

Ixora coccinea, an evergreen Indian shrub and the extracts of various parts of this plant possess significant anti-oxidant, analgesic, anti-inflammatory, gastroprotective, and anti-mutagenic activity.[3] However, its antiarthritic property has not been studied as per various literature reviews done by us.

Phytochemicals present in plant extracts possess anti-inflammatory effects by inhibiting lipoxygenase, cyclooxygenase (COX), phospholipase A2, and other pro-inflammatory cytokines, for example, interleukin (IL)-1, tumor necrosis factor-α (TNF-α) which play an important role in pathogenesis of RA.[4]

In this context, the present study aimed at evaluating the anti-arthritic potential of ethanolic extract of I. coccinea leaves in complete Freund’s adjuvant (CFA)-induced RA in Wistar albino rats with the following objectives:

1. Pathophysiological markers

   • Body weight (BW)
   • Arthritis Index
   • Rota rod fall time
   • Paw volume displacement and % inhibition of paw volume.

2. Inflammatory and oxidative stress marker

   • ESR for anti-inflammatory property
   • Tissue MDA level for antioxidant property.

3. Histopathological examination.

Mankin scoring of histopathology of ankle joint.

Subjects and Methods

Study subjects

Thirty-six female Wistar albino rats of 120–150 g b.w were procured from a registered laboratory animal supplier, Chakraborty Enterprise, Kolkata, (Regd No. 1443/PO/Bt/s/11/CPCSEA). Animals were maintained in the departmental animal house, Pharmacology, MKCG Medical College, Berhampur, Odisha, and fed with a standard pellet diet and water ad libitum. They were kept at a temperature 24°C ± 2°C, relative humidity 70%–85%, and 12 h of light: Dark cycle. The animals were acclimatized for 7 days in the laboratory before the beginning of experiments.

Animals were randomly distributed into six groups (n = 6) by the process of computer-generated random numbers.

• Group I – 0.5 mL DW p.o

• Group-II – Tween -80, 2% v/v p.o

• Group-III – Piroxicam -0.9 mg/kg p.o

• Group IV – EEICL- 1 g/kg p.o

• Group V – EEICL- 1.5 g/kg p.o

• Group VI – ED50 of EEICL + piroxicam (0.45 mg/kg).

Induction of rheumatoid arthritis

0.1 mL of CFA (heat-killed Mycobacterium butyricum suspended in heavy paraffin oil prepared 6 mg/mL) was injected subcutaneously into the foot pad of the left hind paw. The primary response on the left hind paw was observed on the 5^th day and secondary response seen on 12^th day in the right hind paw.[5]

Drug administration

The drugs and vehicles in respective groups were administered daily orally for 20 days after the 5^th day of CFA administration up to the 25^th day. All the experimental work was carried out during that time. The following parameters in all groups were observed on day 0, 5, and 25.

Assessment of different parameters

Body weight

The BW of all groups of rats were measured using rat weighing machine and compared between groups.

Paw volume

The paw volume of left hind paws (injected paws) was measured using a digital plethysmometer (PP160) before and after treatment. The changes in paw volume on the 5^th day of induction were measured. The paw volume of uninjected paws (right hind paws) was measured for secondary response on the 15^th day after drug treatment and compared with disease control (DC).

The percentage of inhibition of paw volume of injected left hind paw over vehicle control was measured on the 25^th day.

Formula of % inhibition-

Arthritic score

The severity of arthritis was scored by macroscopic inspection on the 25^th day. The grading of inflammation of each paw was done by a person who was not aware (blinding) of the study. The scoring was done for 3 uninjected paws separately and the final score was obtained by adding the scores of all 3 paws of each rat with a maximum score of 12. The scoring was done as follows:

No change = 0, mild erythema/swelling digits = 1, moderate swelling and erythema = 2, severe swelling and erythema involving the ankle = 3, and ankylosis and inability to bend the ankle = 4.[6]

Fall off time on rota rod apparatus

The rotarod test is performed using Rotarod apparatus (INCO Company). The test measures endurance based on grip strength, motor coordination of animals.[7] In this test, 4 rats were placed simultaneously on rod for 1 min rotating at a speed 20 r.p.m. The mean fall-off time of different groups during 1 min period was recorded at 5^th, 15^th, 20^th, and 25^th days after drug treatment and compared with DC.

Inflammatory and oxidative stress parameter assessment

The rats were sacrificed by cervical dislocation method under injection xylazine and ketamine aesthesia (i.p) on 25^th day. By retro-orbital puncture, 2–3 mL of blood in EDTA tubes was collected for ESR estimation. The ankle joint was dissected out and was collected for MDA estimation and some of it was preserved with 10% formalin solution for histopathological test.

ESR estimation (Wintrobe’s method)

The blood was collected in an EDTA-containing vial. The Wintrobe’s tube was filled with blood up to “0” mark with the help of a Pasteur’s pipette. The blood reached the top by capillary action. The Wintrobe’s tube was placed vertically on the stand. After 1 h, the layers were noted in Wintrobe’s tube as follows:

1. Uppermost layer of plasma

2. Middle whitish layer of platelets with leucocytes

3. Lower most layer of red blood cell (RBC).

ESR is calculated as the lowermost height of packed RBC, expressed as mm/h.[7]

MDA estimation

The ankle joint synovial tissue was collected and weighed by an electronic weighing balance (Merck), added to phosphate buffer saline, and homogenized by a homogenizer (REMI Lab World). The homogenate was used to measure MDA through spectrophotometer (thermo scientific genesis).

Histopathology of joint

Synovial tissue was taken from the left ankle joint, weighed, and fixed with 24 h in 10% formalin. The pathologist unaware of the experiment evaluated the H and E stained sections under light microscope at ×40 magnifications for synovial hyperplasia, inflammatory cells infiltration, and articular injury. Histopathological changes were studied using Mankin scoring system.

Statistical analysis

The parametric data such as BW, rota rod fall-off time (RRFT), paw volume displacement, MDA, and ESR were analyzed by one-way ANOVA. Nonparametric data such as arthritic score and Mankin score were analyzed by Kruskal–Wallis test using graph pad prism 7.0. P < 0.05 was considered statistically significant.

Results

Effect on body weight

Table 1 depicts that on day 0 mean BW (MBW) of all groups were not statistically different from each other. On day 5, MBW of groups (2–4) were reduced significantly compared with normal control (NC) (126 ± 1.2 g). In DC, BW (99.17 ± 2.3 g) was significantly decreased over that of NC. EEICL (1.5 g/kg) and with combination with piroxicam increased MBW significantly on the 25^th day, i.e. 137.5 ± 2.5 g and 143.3 ± 5.4 g, respectively, compared with DC (78.3 ± 1.6 g).

Table 1.

Effect of drugs on body weight of complete Freund’s adjuvant-induced arthritic rats

Group	Body weight (g)
	Day-0	Day-5	Day-15	Day-20	Day-25
NC DW-0.5 mL	126.0±1.2	126.8±2.0	130.8±1.53	135.8±1.5	141.7±2.1
DC (Tween 80-2% v/v)	125.0±1.8	99.17±2.3###	82.5±2.8###	78.33±1.6###	78.3±1.6###
Standard piroxicam (0.9 mg/kg)	125.0±1.4	100.8±2.7###	124.2±1.5***	135.8±1.5***	147.5±1.1***
EEICL (1 g/kg)	125.0±1.8	100.8±2.7###	110.0±2.2***	115.0±2.2***	120.8±2.7***
EEICL (1.5 g/kg)	128.0±1.6	97.5±1.7###	115.8±2.0***	127.5±2.5***	137.5±2.5***
EEICL (0.82 mg/kg) + piroxicam (0.45 mg/kg)	130.0±1.8	99.17±2.3###	120.8±3.0***	132.5±3.8***	143.3±5.4***

***P<0.001, comparision of DC versus all, ^###P<0.001 comparision of DC versus NC; treatment groups. Data were analyzed by one-way analysis of variance followed by Bonferroni’s multiple comparisons tests. Data expressed as the mean±SEM (n=6). SEM=Standard error of mean, NC=Normal control, DC=Disease control, EEICL=Ethanolic extract of Ixora coccinea leaves

Effect on paw volume and % of inhibition

Data presented in Table 2 showed, on day 0, before induction of arthritis, no significant difference was found in paw volume among different groups. On 5^th day, all arthritis-induced groups (Gr-2-4) produced statistically significant increase in paw volume displacement (P < 0.001) compared to NC (0.80 ± 0.12 mL). The paw volume displacement was 1.78 ± 0.21 mL in DC.

Table 2.

Effect of drugs on rota rod fall-off time and paw volume displacement of complete Freund’s adjuvant-induced arthritic rats

Group	Rota rod fall time (s)			Paw volume displacement (mL) and percentage of inhibition of edema
	Day-0	Day-5	Day-25	Day-0	Day-5	Day-25
NC Dw-0.5 mL	108.3±2.5	120.5±2.3	164.21.44	0.78±0.16	0.80±0.12	0.75±0.07
DC (Tween 80-2% v/v)	112.0+1.06	67±1.48###	11.33±1.14	0.87±0.24	1.78±0.21###	1.97±0.09###
Standard-piroxicam (0.9 mg/kg)	111.5±0.99	69.33±2.10###	196.3±1.66	0.72±0.12	1.69±0.20###	0.28±0.11*** (62%)
EEICL (1 g/kg)	111.3±0.99	69.5±1.45###	146.3±2.8	0.76±0.12	1.61±0.33###	0.50±0.04*** (33.3%)
EEICL (1.5 g/kg)	112.0±1.05	72.00±1.5###	179.3±4.16	0.81±0.14	1.59±0.22###	0.36±0.09*** (52%)
EEICL (0.82 mg/kg) + piroxicam (0.45 mg/kg)	109.2±1.06	72.17±1.30###	198.3±0.73	0.84±0.11	1.65±0.33###	0.31±0.04*** (58%)

^###P<0.001 indicates comparison of DC versus NC, ***P<0.001 indicates comparison of DC versus each treatment groups. Data expressed as the mean±SEM (n=6). Data were analyzed by one-way analysis of variance followed by Bonferroni’s multiple comparisons test. SEM=Standard error of mean, NC=Normal control, DC=Disease control, EEICL=Ethanolic extract of Ixora coccinea leaves

EEICL (1 g/kg, 1.5 g/kg) and combined treatment (ED50 EEICL + half effective dose of piroxicam) showed a reduction of paw volume displacement (P < 0.001) on 25 days, i.e. 0.36 ± 0.09 mL compared to DC (1.97 ± 0.09 mL). Percentage inhibition of paw edema was also increased significantly in drug-treated groups which was 52% with EEICL (1.5 g/kg) alone and 58% in combination.

Effect on rotarod fall-off time

Table 2 shows that on day 0, before induction of arthritis, no statistically significant difference was recorded between groups in RRFT. On day 5, all arthritis-induced group (Gr 2-4) showed a significant reduction (P < 0.001) of RRFT (11.33 ± 1.14 s in DC) compared to NC (164.2 ± 1.44 s). EEICL (1 g/kg, 1.5 g/kg) and combined treatment groups showed significant improvement, i.e. 198.3 ± 0.73 s (P < 0.001) in motor coordination and increased RRFT on day 25^th compared to DC. The effect of test groups produced a similar effect as piroxicam treated group.

Effect on arthritic score

Figure 1 clearly shows arthritic score in the DC group went on increasing and reached maximum on 25^th day, i.e. 6.6.

Figure 1.

Effect of drugs on morphological changes and arthritic score of complete Freund’s adjuvant-induced arthritis in rats. (a) Image of morphological changes, (b) – Bar diagram depicting arthritic score

On days 20^th and 25^th, treatment with both extracts of EEICL (1 g/kg, 1.5 g/kg) and combined treatment showed significant (P < 0.01, P < 0.001) suppression of arthritic index (0.33, 0.16), respectively, compared to DC which was similar to that of piroxicam-treated group.

Effect on ESR

Figure 2 clearly depicts that arthritic control showed significantly (P < 0.001) increased ESR (9.33 mm/h) compared to that of NC (5.5 mm/h). EEICL (1.5 g/kg) dose and in combination showed decreased ESR value significantly (P < 0.001) to 3.51 mm/h and 3.66 mm/h respectively compared to arthritic control whereas piroxicam showed similar results with EEICL as well as in combination.

Figure 2.

Effect of drugs on tissue MDA level and ESR of complete Freund’s adjuvant-induced arthritic rats. (a) Effect of drugs on MDA (μm/g synovial tissue). (b) Effect of drugs on ESR (mm/h) 25^th day. DC: Disease control, NC: Normal control, MDA: Malondialdehyde, ESR: Erythrocyte sedimentation rate

Effects on tissue MDA of injected paw

Figure 2 shows that, DC produced two-fold increase in oxidative stress marker MDA which was significantly different (P < 0.001) from NC.

However, treatment with EEICL (1 g/kg, 1.5 g/kg) as well as in combination with piroxicam produced a significant reduction (P < 0.001) of MDA value compared to DC which was not significantly different from that of piroxicam.

Effect of drugs on morphological changes in injected paw on 25^th day

Figure 1a (A, B, C, D, E, F) depicts the result as follows:

1A – NC showed normal morphology of ankle joint without any edema, induration, and swelling.

1B – DC showed the highest swelling of ankle joint on the injected paw with the highest paw edema, swelling erythema, and increased thickness.

1C – Piroxicam-treated group did not show any swelling of ankle joint and paw edema, paw thickness, erythema, and induration compared to DC.

1D – EEICL (1.0 g/kg) produced medium swelling of ankle joint and medium paw edema, paw thickness, erythema, and induration compared to DC.

1E – (1.0 g/kg) and 2F (combination) showed minimum edema, paw thickness, induration, and swelling of paw as well as ankle joint which are closer to NC.

Effect of drugs on histopathology of ankle joint of rats on complete Freund’s adjuvant-induced arthritis

Figure 3 (A, B, C, D, E, F) depicts the result as follows:

Figure 3.

Effect of drugs on histopathology of the ankle joint of complete Freund’s adjuvant-induced arthritic rats. EEICL: Ethanolic extract of Ixora coccinea leaves

3A – NC presented with normal connective tissue and no sign of inflammation such as edema, necrosis, angiogenesis, and inflammatory cells.

3B – DC showed maximum destruction of connective tissue and lymphocytic cell infiltrate sign of chronic inflammtion, granuloma formation, a giant cell in the field, and synovial inflammation.

3C – Piroxicam-treated group was found to have normal connective tisuue, no inflammatory infiltrate, less edema, and no necrosis.

3D – EEICL 1g/kg had inflammatory infiltration, no necrosis, and synovial hyperplasia.

3E – EEICL 1.5g/kg produced significantly less inflammatory infiltration, no necrosis, less connective tissue destruction, and synovial hyperplasia.

3F – Combined effect had normal connective tissue, nearly no inflammatory infiltrate, no necrosis, and no synovial hyperplasia.

All of the histological features of different groups were scored by the Mankin scoring system.[8]

Effect on Mankin scoring of histopathology of joints

DC rats displayed severe degree of inflammation depicted by inflammatory cell infiltration and edema with articular cartilage injury, synovial hyperplasia of the ankle joint. The Mankin scoring of histopathological changes is shown in Figure 4. It was observed that DC having Manikin’s score of 7–8 with all parameters such as inflammatory infiltrate (8), synovial hyperplasia (7), and articular injury (8). On treatment of both doses of EEIC reduced the score significantly which was highest at 25^th day which was 1–5 with EEICL.

Figure 4.

Effect of drugs on Mankin’s scoring of histopathology of the ankle joint of complete Freund’s adjuvant-induced arthritic rats. DC: Disease control, EEIC: Ethanolic extract of Ixora coccinea leaves

One (1.5 g/kg) and 2 in all cases with a combined effect of EEICL ED50 and half effective dose of piroxicam.

Discussion

The study was undertaken to establish the anti-arthritic potential of ethanolic extract of I. coccinea linn (EEICL) leaves on CFA-induced arthritis in animal model of Wistar albino rats. The effects of EEIC were studied on BW, paw volume displacement, % inhibition of paw volume, arthritic score, RRFT, tissue MDA level, and ESR. The histopathology of ankle joint was studied to evaluate the effect of drugs on joint pathology and assessed by Mankin scoring. A comparison of the results was done with that of the standard drug piroxicam.

The joint pathology is more prominent in females than males in RA[9] and Wistar albino rats were used for adjuvant arthritis in a previous study.[10] Hence, female Wistar albino rats were used in this study.

CFA-induced arthritis has many clinical similarities with humans by activating both cellular and humoral immune systems (CD4 lymphocytes), play a significant role in inflammation.[11] Similar studies done in the recent past have shown the role of different pro-inflammatory mediators such as TNF-α, IL-1β, and PDGF which played a significant role in the pathogenesis of RA.[12,13]

Piroxicam was taken as reference standard drug in this experiment. Because various previous studies used NSAIDs such as diclofenac/indomethacin/meloxicam as standard drugs which are commonly prescribed PG synthesis inhibitors.[14]

Effect of drugs on body weight(g)

Disease progression and response to anti-arthritic medications are indirectly linked to decrease in BW in RA where severity being termed as rheumatoid cachexia, caused by inflammatory cytokines.[15] This might be caused due to reduced intestinal absorption of C-glucose and C-leucine, destruction of muscle proteins by proteolysis caused by lysosomal proteases, and mediated by PGE2. An anti-arthritic drug increase in BW by increase absorption through intestine and lysosomal membrane stabilization.[16] This study result showed that EEICL in both the doses (1 g/kg, 1.5 g/kg) and in combined treatment produced significant improvement (P < 0.001) of BW on the 25^th day.

Effect of drugs on paw volume displacement and % inhibition of paw volume

Table 2 shows that CFA produce increase in swelling of soft tissues which increased paw volume displacement by plethysmometer in diseased rats which was reduced significantly by EEICL in both the doses (1 g/kg, 1.5 g/kg) and in combination with piroxicam (P < 0.001). In other study reported that arthritic rats treated with tocotrienol-rich fraction found in palm oil containing antioxidant Vitamin E with potent antioxidant and anti-inflammatory activities showed decreased paw volumes compared with untreated rats. These effects may be possibly due to the suppression of various mediators of inflammation and free radicals.[13] This is similar to other studies that plant-derived polyphenolic compounds inhibited the inflammatory reactions by modulating the mitogen-activated protein kinase, arachidonic acid pathways, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Polyphenols also produce anti-inflammatory action by inhibiting phosphatidylinositide 3-kinases/protein kinase B, kappa kinase/c-Jun aminoterminal kinases, and mammalian target of Rapamycin complex 1 which controls protein synthesis, and JAK/STAT. They also suppress toll-like receptor and pro-inflammatory gene expression.[17]

Effect of drugs on arthritic score of rats on complete Freund’s adjuvant induced arthritis

The degree of severity of arthritis was measured by arthritic score that is an index of joint inflammation. EEIC (1 g/kg, 1.5 g/kg) and combined treatment with piroxicam produced a significant reduction of arthritic score compared to DC.

Stevioside, a plant glycoside, significantly ameliorated the adjuvant-induced arthritic scoring by normalizing the endogenous antioxidant (SOD, CAT, GSH, and GST) activities, reducing the CFA-induced pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and protein expressions (iNOS, COX-2, NF-κB (p65), and pIκB/IκB ratio). It restored the anti-inflammatory cytokine (IL-10) in paw tissues. As EEIC contains glycoside its ability to decrease arthritic score may be similar to this study.[18]

Effect of drugs on Rotarod fall-off time of rats on complete Freund’s adjuvant-induced arthritis

As Rotarod test is a performance test used to evaluate the muscle and grip strength of animals, is used to assess the motor coordination by determining the mean fall-off time. Roubenoff et al. observed that there was reduced physical activity, muscle strength, and daily performance by rats in RA.[15]

Treatment with both doses of EEIC (1 g/kg, 1.5 g/kg) and combination with piroxicam showed significant improvement (P < 0.001) over vehicle treated group in motor coordination as mean fall of time was increased. This may be due to the presence of glycosides and flavonoids in EEICL which ameliorates the inflammation and oxidative stress.[12,18]

Effect of drugs on ESR (mm/h) of rats on complete Freund’s adjuvant-induced arthritis

During inflammation, there is increased proteins (fibrinogen, α-and β-globulins) production leading to increase in ESR value which is an inflammatory marker of RA.[19] EEIC in individual dose of 1 g/kg/1.5 g/kg and in combination with piroxicam decreased ESR value significantly (P < 0.001) compared to arthritic control due to its anti-inflammatory effect.

Effect of drugs on MDA level of synovial tissue in complete Freund’s adjuvant-induced arthritic rats

Free radicals have crucial roles as secondary messengers in inflammation and they can exacerbate joint damage.[15] MDA, an important marker of oxidative stress[20] and it is formed due to lipid peroxidation and ROS generation. Our study showed MDA level in DC group was increased significantly and reduced by EEIC alone and in combination with standard drug due to the presence of antioxidant phytochemicals such as flavonoids and glycosides.[11,16]

Effect of drugs on histopathology of synovial tissue of ankle joint (Mankin scoring)

The histopathological findings of ankle joints such as infiltration of inflammatory cells, and synovial hyperplasia were measured by Mankin scoring[7] which was significantly increased in arthritic control and reduced by EEIC alone and in combination. Many researcher suggested that histological modification such as angiogenesis, cartilage damage, and synovial hyperplasia occur as the synovial membrane is infiltrated by macrophages, activated B and T cells, mast cells, and plasma cells[21] and also bone and cartilage destruction participate in an aggravating inflammation.

This study result proved antiarthritic effect of EEIC on CFA-induced arthritis in rats. Hence, it can be concluded that further preclinical study in genetic species and clinical trial in human can through some light on this regard and it may be a safe and effective future antiarthritic drug.

Conclusion

Ethanolic extract of Ixora coccinea leaves was found to have potential anti-arthritic effect alone as well as in combination with low dose Piroxicam in CFA induced arthritic rats. Further studies may be conducted to prove its role for clinical use.

Limitations of the study

1. Important inflammatory parameters like C-reactive protein, IL-1, IL-6, TNF-α, and IFN-γ could not be done. Antioxidant activity of the body like SOD could not be measured

2. DMARDs could be selected as standard drug

3. Immunohistochemistry and radiological study of ankle joint might provide better arthritic measurement.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.