Emerging intervention of antidepressant with <i>DMARD</i> in non-cancerous nociceptive persistent pain associated depression in <i>FCA</i> induced rheumatoid arthritic rats

Pachiappan, Sudhakar; Murugaiyan, Sudaroli; Chandrasekar, Sabarinath

doi:10.1590/s2175-97902020000318852

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Brazilian Journal of Pharmaceutical Sciences

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Article • Braz. J. Pharm. Sci. 56 • 2020 • https://doi.org/10.1590/s2175-97902020000318852 copy

Emerging intervention of antidepressant with DMARD in non-cancerous nociceptive persistent pain associated depression in FCA induced rheumatoid arthritic rats

Authorship SCIMAGO INSTITUTIONS RANKINGS

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that causes pain, systemic complications and premature mortality. Depression has also been identified as a problem for persons with RA. This association remaining significant even after the degree of disease activity is controlled. In the present study, the efficacy of combination therapeutic effect of antidepressant (amitriptyline) with Disease Modifying Anti rheumatoid drug (leflunomide) was determined in rheumatoid arthritis pain associated depression in Freund’s complete adjuvant (FCA) induced arthritic rats. Drug treatment was started 9 days after induction of FCA induced arthritis in rats. The antiarthritic activity was assessed by measuring paw volume, weight-bearing, hematological, biochemical, serological parameters, Radiographic analysis and Histopathology of tibiotarsal joints. The antidepressant activity was assessed by Forced swimming test, Rota-rod test and confirmed by estimation of brain neuro transmitters (serotonin and norepinephrine) level. Results of this study revealed that leflunomide and amitriptyline combination showed more significant (p<0.001) antiarthritic and antidepressant action and leflunomide alone treatment showed significant (p<0.001) antiarthritic activity only as compared to arthritic control. The leflunomide and low dose amitriptyline combination found to be more effective in pain associated depression in rheumatoid arthritic rats.

Keywords:
Arthritis associated depression; Freund’s complete adjuvant; Amitriptyline; Leflunomide

Score	Flexion pain test score	Mobility score	Stance score
0	No squeaking and no leg withdrawal	Normal	_
1	Either squeaking or leg withdrawal	Limping	Paw lift continuously
2	Both squeaking or leg withdrawal	Walking with difficulty	Paw touching but with no weight bearing
3	_	Walking without touching the injected paw	Some weight bearing on the paw
4	_	_	Normal

Post insult time in days	Mean changes in swelling volume (ml) ± SEM on injected paw
Post insult time in days	Group-I (Vehicle con.)	Group-II (Arth con.)	Group-III (LEF)	Group- IV (LELA)	Group- V (LEHA)
0	0.42± 0.01	0.40± 0.12	0.41± 0.02	0.40± 0.01	0.40± 0.01
1	0.41± 0.01	0.81± 0.03 a^* ***** p< 0.001,	0.80± 0.03 a^* ***** p< 0.001,	0.87± 0.09 a^* ***** p< 0.001,	0.87± 0.06 a^* ***** p< 0.001,
3	0.43± 0.01	0.71± 0.02 a^* ***** p< 0.001,	0.68± 0.01 a^* ***** p< 0.001,	0.71± 0.02 a^* ***** p< 0.001,	0.73± 0.02 a^* ***** p< 0.001,
5	0.44± 0.01	0.77± 0.03 a^* ***** p< 0.001,	0.74± 0.01 a^* ***** p< 0.001,	0.75± 0.03 a^* ***** p< 0.001,	0.76± 0.02 a^* ***** p< 0.001,
7	0.45± 0.01	0.88± 0.02 a^* ***** p< 0.001,	0.87± 0.04 a^* ***** p< 0.001,	0.87± 0.02 a^* ***** p< 0.001,	0.83± 0.05 a^* ***** p< 0.001,
9	0.45± 0.001	0.94± 0.03 a^* ***** p< 0.001,	0.89± 0.04 a^* ***** p< 0.001,	0.92± 0.02 a^* ***** p< 0.001,	0.88± 0.03 a^* ***** p< 0.001,
14	0.48± 0.01	0.85± 0.02 a^* ***** p< 0.001,	0.81± 0.02 a^* ***** p< 0.001,	0.72± 0.03 a^* *** p< 0.001, b^ ** p< 0.01,	0.75± 0.02 a^* ***** p< 0.001, b^* * p< 0.05
19	0.49± 0.003	0.78± 0.04 a^* ***** p< 0.001,	0.72± 0.01 a^* ***** p< 0.001,	0.63± 0.02 a^* *** p< 0.001, b^ ** p< 0.01,	0.67± 0.01 a^* ***** p< 0.001, b^* * p< 0.05
21	0.49± 0.01	0.82± 0.02 a^* ***** p< 0.001,	0.68± 0.02 a^* *** p< 0.001, b^* ******* p< 0.001,	0.54± 0.01 b^* ***** p< 0.001,	0.60± 0.01 a^ p< 0.01, b^* ***** p< 0.001,

Post insult time in days	Mean changes in swelling volume (ml) ± SEM on injected paw
Post insult time in days	Group-I (Vehicle con.)	Group-II (Arth. con.)	Group-III (LEF)	Group- IV (LELA)	Group-V (LEHA)
1	0.41± 0.01	0.43± 0.003	0.40± 0.02	0.40± 0.02	0.41± 0.01
5	0.43± 0.01	0.45± 0.02	0.48± 0.02	0.48± 0.01	0.46± 0.01
7	0.44± 0.01	0.65± 0.02 a^* ***** p< 0.001,	0.65± 0.02 a^* ***** p< 0.001,	0.64± 0.02 a^* ***** p< 0.001,	0.63± 0.01 a^* ***** p< 0.001,
9	0.45± 0.01	0.70± 0.02 a^* ***** p< 0.001,	0.69± 0.02 a^* ***** p< 0.001,	0.67± 0.02 a^* ***** p< 0.001,	0.70± 0.02 a^* ***** p< 0.001,
14	0.45± 0.02	0.73± 0.01 a^* ***** p< 0.001,	0.66± 0.02 a^* ***** p< 0.001,	0.59± 0.02 a^* *** p< 0.001, b^ ** p< 0.01,	0.62± 0.02 a^* *** p< 0.001, b^ ** p< 0.01,
19	0.48± 0.01	0.75± 0.02 a^* ***** p< 0.001,	0.64± 0.03 a^* ***** p< 0.001, b^* * p< 0.05	0.53± 0.02 b^* ***** p< 0.001,	0.60± 0.02 a^ p< 0.01, b^ p< 0.01,
21	0.48± 0.01	0.78± 0.01 a^* ***** p< 0.001,	0.63± 0.03 a^* *** p< 0.001, b^* ******* p< 0.001,	0.50± 0.01 b^* ***** p< 0.001,	0.58± 0.03 a^ p< 0.01, b^* ***** p< 0.001,

Treatment Groups	Arthritis score	Flexion pain test score	Mobility score	Stance score
Arthritic control	11.5 (9, 13)	2.0 (1, 2)	3.0 (2, 3)	1.0 (1, 2)
LEF	5.5 (4, 8)^ p< 0.01,	1.5 (1, 2)	2.5 (1, 3)	2.5 (2, 4)
LELA	3.0 (2, 5)^* ***** p< 0.001,	1 (0, 1) ^* * p< 0.05	1.0 (0, 1)^ p< 0.01,	4.0 (2, 4) ^ p< 0.01,
LEHA	4.0 (3, 6)^* ***** p< 0.001,	1.25 (0, 2)	1.5 (1, 2) ^* * p< 0.05	3.0 (2, 4)^* * p< 0.05

Groups	Paw withdrawal threshold (g)
Groups	Day 0	Day 3	Day 9	Day 14	Day 21
Group I (Vehicle.con)	280.44 ± 4.77	275.47 ± 4.88	250 ± 4.23	238.92 ± 7.10	240.41 ± 5.79
Group II (Arth.con)	276.75 ± 8.29	171.04 ± 9.15 a^* ***** p< 0.001,	113.34 ± 6.42 a^* ***** p< 0.001,	99.28 ± 6.72 a^* ***** p< 0.001,	96.35 ± 4.71 a^* ***** p< 0.001,
Group III (LEF)	280.02 ± 4.94	168.92 ± 11.4 a^* ***** p< 0.001,	110.67 ± 8.22 a^* ***** p< 0.001,	120 ± 7.47 a^* ***** p< 0.001,	146.78 ± 4.87 a^* *** p< 0.001, b^ ** p< 0.01,
Group IV (LELA)	281.56 ± 7.35	167.24 ± 10.05 a^* ***** p< 0.001,	115.42 ± 6.51 a^* ***** p< 0.001,	138 ± 5.87 a^* *** p< 0.001, b^ ** p< 0.01,	203.02 ± 8.15 a^ p< 0.01, b^* ***** p< 0.001,
Group V (LEHA)	277.38 ± 8.67	171.41 ± 12.80 a^* ***** p< 0.001,	108.42 ± 4.66 a^* ***** p< 0.001,	125 ± 3.25 a^* ***** p< 0.001, b*	188.44 ± 10.42 a^* *** p< 0.001, b^* ******* p< 0.001,

Group	Paw withdrawal threshold (g)
Group	Day 5	Day 9	Day 14	Day 21
Group I (Vehicle.con)	260.71 ± 6.01	255.14 ± 4.11	247.26 ± 4.21	242.78 ± 6.67
Group II (Arth.con)	204.67 ± 7.59	138.56 ± 5.20 a^* ***** p< 0.001,	120.64 ± 5.17 a^* ***** p< 0.001,	107.13 ± 9.19 a^* ***** p< 0.001,
Group III (LEF)	207.24 ± 11.44	136.45 ± 5.94 a^* ***** p< 0.001,	142.14 ± 7.75 a^* ***** p< 0.001,	157.24 ± 5.34 a^* *** p< 0.001, b^ ** p< 0.01,
Group IV (LELA)	207.53 ± 7.79	141.02 ± 6.65 a^* ***** p< 0.001,	176.41 ± 7.47 a^* *** p< 0.001, b^* ******* p< 0.001,	206.48 ± 9.47 a* b^* ***** p< 0.001,
Group V (LEHA)	202.47 ± 12.31	137.12 ± 5.33 a^* ***** p< 0.001,	155.06 ± 4.59 a^* *** p< 0.001, b^ ** p< 0.01,	180.03 ± 7.16 a^* *** p< 0.001, b^* ******* p< 0.001,

Group	Riding time (sec)
Group	Day 0	Day 1	Day 9	Day 14	Day 21
Group I (Vehicle.con)	58.8 ± 1.37	65.8 ± 1.11	62.5 ± 1.71	63.25 ± 1.25	63.5 ± 1.94
Group II (Arth.con)	59.3 ± 1.93	46.3 ± 1.49 a^* ***** p< 0.001,	25.0 ± 1.08 a^* ***** p< 0.001,	22.5 ± 1.19 a^* ***** p< 0.001,	18.8 ± 1.65
Group III (LEF)	62.5 ± 1.7	49.0 ± 2.04 a^* ***** p< 0.001,	25.5 ± 1.44 a^* ***** p< 0.001,	28.0 ± 1.68 a^* ***** p< 0.001,	29.3 ± 1.11 a^* ***** p< 0.001, b^* * p< 0.05
Group IV (LELA)	61.3 ± 3.30	50.0 ± 1.08 a^* ***** p< 0.001,	24.8 ± 1.49 a^* ***** p< 0.001,	45.3 ± 1.49 a^* *** p< 0.001, b^* ******* p< 0.001,	52.8 ± 3.43 a^* * p< 0.05 b^* ***** p< 0.001,
Group V (LEHA)	59.5 ± 1.3	46.3 ± 1.38 a^* ***** p< 0.001,	26.3 ± 1.25 a^* ***** p< 0.001,	42.5 ± 1.04 a^* *** p< 0.001, b^* ******* p< 0.001,	47.0 ± 1.47 a^* *** p< 0.001, b^* ******* p< 0.001,

Group	Duration of immobility (sec)
Group	Day 0	Day 3	Day 9	Day 14	Day 21
Group I (vehicle.con)	155.22± 2.86	158.40 ± 2.10	154.20 ± 2.71	151.32 ± 2.86	169.21 ± 4.34
Group II (Arth.con)	150.24 ± 5.01	114.40 ± 3.97 a^* ***** p< 0.001,	205.62 ± 2.50 a^* ***** p< 0.001,	196.24 ± 4.61 a^* ***** p< 0.001,	203.11 ± 3.71 a^* ***** p< 0.001,
Group III (LEF)	156.41 ± 3.43	115.14 ± 2.27 a^* ***** p< 0.001,	199.43 ± 5.18 a^* ***** p< 0.001,	182.74 ± 1.89 a^* ***** p< 0.001, b^* * p< 0.05	185.23 ± 5.01 a^* * p< 0.05 b^* * p< 0.05
Group IV (LELA)	157.34 ± 3.01	121.64 ± 2.5 a^* ***** p< 0.001,	204.12 ± 2.10 a^* ***** p< 0.001,	160.70 ± 3.23 b^* ***** p< 0.001,	134.57 ± 2.10 a^* *** p< 0.001, b^* ******* p< 0.001,
Group V (LEHA)	156.21 ± 4.64	121.42 ± 4.78 a^* ***** p< 0.001,	204.47 ± 4.29 a^* ***** p< 0.001,	135.00 ± 2.38 a^ p< 0.01, b^* ***** p< 0.001,	117.12 ± 3.24 a^* *** p< 0.001, b^* ******* p< 0.001,

Group	Body weight gain (g)	Spleen weight (g/100 g body weight)	Thymus weight (g/100 body weight)
Group I (vehicle.con)	15.2 ± 1.63	0.251 ± 0.006	0.079 ± 0.002
Group II (Arth.con)	3.1 ± 1.16 a^* ***** p< 0.001,	0.54 ± 0.009 a^* ***** p< 0.001,	0.115 ± 0.012 a^ p< 0.01,
Group III (LEF)	8.3 ± 2.45 a^* * p< 0.05	0.47 ± 0.02 a^* *** p< 0.001, b^ ** p< 0.01,	0.090 ± 0.004 b^* * p< 0.05
Group IV (LELA)	12.2 ± 0.71 b^ p< 0.01,	0.29 ± 0.007 b^* ***** p< 0.001,	0.079 ± 0.001 b^ p< 0.01,
Group V (LEHA)	8.4 ± 0.41 a^* * p< 0.05	0.309 ± 0.007 a^ p< 0.01, b^* ***** p< 0.001,	0.084 ± 0.004 b^* * p< 0.05

Hematological parameters	Group-I (Vehicle.con)	Group-II (Arth.con)	Group-III (LEF)	Group-IV (LELA)	Group-V (LEHA)
RBC (x106/µl)	7.02 ± 0.09	5.77 ±0.07 a^* ***** p< 0.001,	6.97 ± 0.05 b^* ***** p< 0.001,	7.08 ± 0.05 b^* ***** p< 0.001,	7.01 ± 0.03 b^* ***** p< 0.001,
WBC(x103/µl)	7.35 ± 0.25	12.80 ± 0.2 a^* ***** p< 0.001,	10.98 ± 0.08 a^* ***** p< 0.001, b^* * p< 0.05	8.40 ± 0.2 b^* ***** p< 0.001,	9.25 ± 0.45 a^ p< 0.01, b^ p< 0.01,
Hb(g/dL)	12.51 ± 0.4	10.40 ± 0.1 a^ p< 0.01,	10.85 ± 0.05 a^ p< 0.01,	13.34 ± 0.2 b^* ***** p< 0.001,	11.82 ± 0.1 b^ p< 0.01,
ESR (mm/1sthr)	3.51 ± 0.5	11.50 ± 0.5 a^* ***** p< 0.001,	9.50 ± 0.5 a^ p< 0.01,	3.52 ± 0.5 b^* ***** p< 0.001,	4.52 ± 0.5 b^ p< 0.01,
PCV (%)	54.70 ± 0.6	34.75 ± 0.37 a^* ***** p< 0.001,	40.57 ± 0.57 a^* *** p< 0.001, b^ ** p< 0.01,	48.64 ± 0.49 a^ p< 0.01, b^* ***** p< 0.001,	42.41 ± 0.45 a^* *** p< 0.001, b^* ******* p< 0.001,
Lympho(x103/µl)	6.23 ± 0.2	16.12 ± 0.7 a^* ***** p< 0.001,	11.90 ± 0.7 a^ p< 0.01, b^* * p< 0.05	4.90 ± 0.3 b^* ***** p< 0.001,	5.95 ± 0.15 b^* ***** p< 0.001,
MCHC	36.25 ± 0.15	32.50 ± 0.20 a^* ***** p< 0.001,	34.42 ± 0.1 a^ p< 0.01, b^ p< 0.01,	36.95 ± 0.15 b^* ***** p< 0.001,	36.05 ± 0.25 b^* ***** p< 0.001,

Group	RF (IU/mL)		CRP (mg/dL)
Group	Day 14	Day 21	Day 14	Day 21
Arthritic control	54.34 ± 3.76	67.72 ± 2.03	5.74 ± 0.05	6.56 ± 0.25
LEF	46.01 ± 1.73	36.67 ± 2.03 ^ p< 0.01,	5.1 ± 0.1 ^* * p< 0.05	4.45 ± 0.25 ^ p< 0.01,
LELA	30.32 ± 2.03^* ***** p< 0.001,	23.33 ± 1.45 ^* ***** p< 0.001,	3.12 ± 0.07 ^* ***** p< 0.001,	1.70 ± 0.1 ^* ***** p< 0.001,
LEHA	37.32 ± 2.33 ^ p< 0.01,	32.67 ± 1.20 ^* ***** p< 0.001,	4.54 ± 0.19 ^* ***** p< 0.001,	3.25 ± 0.15 ^* ***** p< 0.001,

Biochemical parameters	Group-I (Vehicle.con)	Group-II (Arth.con)	Group-III (LEF)	Group-IV (LELA)	Group-V (LEHA)
AST (IU/L)	44.33 ± 0.88	92.33 ± 1.45 a^* ***** p< 0.001,	66.67 ± 1.2 a^* *** p< 0.001, b^* ******* p< 0.001,	51.3 ± 0.88 a^* * p< 0.05 b^* ***** p< 0.001,	67.7 ± 1.2 a^* *** p< 0.001, b^* ******* p< 0.001,
ALT (IU/L)	40.2 ± 1.53	77.0 ± 1.73 a^* ***** p< 0.001,	56.7 ± 0.88 a^* *** p< 0.001, b^* ******* p< 0.001,	51.3 ± 1.45 a^ p< 0.01, b^* ***** p< 0.001,	57.1 ± 2.08 a^* *** p< 0.001, b^* ******* p< 0.001,
ALP (IU/L)	237.67 ± 3.48	351.67 ±0.38 a^* ***** p< 0.001,	296.32 ± 2.52 a^* *** p< 0.001, b^* ******* p< 0.001,	251.01 ± 1.45 a^* * p< 0.05 b^* ***** p< 0.001,	297.67 ±1.76 a^* *** p< 0.001, b^* ******* p< 0.001,
Serum cholesterol (mg/dL)	111.15 ± 3.97	171.58 ± 5.3 a^* ***** p< 0.001,	139.9 ± 8.3 a^* * p< 0.05 b^* ***** p< 0.001,	115.16 ± 3.1 b^* ***** p< 0.001,	123.66 ± 6.8 b^* ***** p< 0.001,
Serum triglyceride (mg/dL)	109.21 ± 5.1	174.12 ± 4.3 a^* ***** p< 0.001,	151.55 ± 6.2 a^* ***** p< 0.001, b^* * p< 0.05	122.47 ± 2.9 b^* ***** p< 0.001,	138.89 ± 4.9 a^* * p< 0.05 b^* ***** p< 0.001,
Serum albumin (mg/dL)	2.90 ± 0.03	2.19 ± 0.05 a^* ***** p< 0.001,	2.34 ± 0.05 a^* ***** p< 0.001, b^* * p< 0.05	2.84 ±0.05 b^* ***** p< 0.001,	2.68 ± 0.03 a^ p< 0.01, b^* ***** p< 0.001,
Serum urea (mg/dL)	30.33 ± 2.4	42.67 ± 1.2 a^* ***** p< 0.001,	33.67 ± 0.88 b^* ***** p< 0.001,	27.67 ± 0.88 b^* ***** p< 0.001,	31.02 ± 0.58 b^* ***** p< 0.001,
Serum creatinine (mg/dL)	0.72 ± 0.02	0.85 ± 0.01 a^ p< 0.01,	0.55 ± 0.03 b^* ***** p< 0.001,	0.41 ± 0.01 b^* ***** p< 0.001,	0.64 ± 0.02 b^* ***** p< 0.001,

Group	NA (ng/g Wet tissue)	5-HT (ng/g Wet tissue)
Group I (vehicle.con)	4.22 ± 0.05	1.89 ± 0.06
Group II (Arth.con)	3.12 ± 0.07 a^* ***** p< 0.001,	0.80 ± 0.03 a^* ***** p< 0.001,
Group III (LEF)	3.30 ± 0.07 a^* ***** p< 0.001,	0.93 ± 0.04 a^* ***** p< 0.001,
Group IV (LELA)	3.94 ± 0.03 a^ p< 0.01, b^* ***** p< 0.001,	1.61 ± 0.05 a^* *** p< 0.001, b^* ******* p< 0.001,
Group V (LEHA)	4.15 ± 0.03 b^* ***** p< 0.001,	1.73 ± 0.04 b^* ***** p< 0.001,

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br

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[1] *Correspondence: S. Pachiappan. Department of Pharmacology, Swamy Vivekanandha College of Pharmacy, Tamil Nadu, India, Namakkal-637 205. Mobile: +919600616196. E-mail: sudhakar00pharma@gmail.com

Post insult time in days	Mean changes in swelling volume (ml) ± SEM on injected paw
Post insult time in days	Group-I (Vehicle con.)	Group-II (Arth con.)	Group-III (LEF)	Group- IV (LELA)	Group- V (LEHA)
0	0.42± 0.01	0.40± 0.12	0.41± 0.02	0.40± 0.01	0.40± 0.01
1	0.41± 0.01	0.81± 0.03 a^* ***** p< 0.001,	0.80± 0.03 a^* ***** p< 0.001,	0.87± 0.09 a^* ***** p< 0.001,	0.87± 0.06 a^* ***** p< 0.001,
3	0.43± 0.01	0.71± 0.02 a^* ***** p< 0.001,	0.68± 0.01 a^* ***** p< 0.001,	0.71± 0.02 a^* ***** p< 0.001,	0.73± 0.02 a^* ***** p< 0.001,
5	0.44± 0.01	0.77± 0.03 a^* ***** p< 0.001,	0.74± 0.01 a^* ***** p< 0.001,	0.75± 0.03 a^* ***** p< 0.001,	0.76± 0.02 a^* ***** p< 0.001,
7	0.45± 0.01	0.88± 0.02 a^* ***** p< 0.001,	0.87± 0.04 a^* ***** p< 0.001,	0.87± 0.02 a^* ***** p< 0.001,	0.83± 0.05 a^* ***** p< 0.001,
9	0.45± 0.001	0.94± 0.03 a^* ***** p< 0.001,	0.89± 0.04 a^* ***** p< 0.001,	0.92± 0.02 a^* ***** p< 0.001,	0.88± 0.03 a^* ***** p< 0.001,
14	0.48± 0.01	0.85± 0.02 a^* ***** p< 0.001,	0.81± 0.02 a^* ***** p< 0.001,	0.72± 0.03 a^* *** p< 0.001, b^ ** p< 0.01,	0.75± 0.02 a^* ***** p< 0.001, b^* * p< 0.05
19	0.49± 0.003	0.78± 0.04 a^* ***** p< 0.001,	0.72± 0.01 a^* ***** p< 0.001,	0.63± 0.02 a^* *** p< 0.001, b^ ** p< 0.01,	0.67± 0.01 a^* ***** p< 0.001, b^* * p< 0.05
21	0.49± 0.01	0.82± 0.02 a^* ***** p< 0.001,	0.68± 0.02 a^* *** p< 0.001, b^* ******* p< 0.001,	0.54± 0.01 b^* ***** p< 0.001,	0.60± 0.01 a^ p< 0.01, b^* ***** p< 0.001,

Hematological parameters	Group-I (Vehicle.con)	Group-II (Arth.con)	Group-III (LEF)	Group-IV (LELA)	Group-V (LEHA)
RBC (x106/µl)	7.02 ± 0.09	5.77 ±0.07 a^* ***** p< 0.001,	6.97 ± 0.05 b^* ***** p< 0.001,	7.08 ± 0.05 b^* ***** p< 0.001,	7.01 ± 0.03 b^* ***** p< 0.001,
WBC(x103/µl)	7.35 ± 0.25	12.80 ± 0.2 a^* ***** p< 0.001,	10.98 ± 0.08 a^* ***** p< 0.001, b^* * p< 0.05	8.40 ± 0.2 b^* ***** p< 0.001,	9.25 ± 0.45 a^ p< 0.01, b^ p< 0.01,
Hb(g/dL)	12.51 ± 0.4	10.40 ± 0.1 a^ p< 0.01,	10.85 ± 0.05 a^ p< 0.01,	13.34 ± 0.2 b^* ***** p< 0.001,	11.82 ± 0.1 b^ p< 0.01,
ESR (mm/1sthr)	3.51 ± 0.5	11.50 ± 0.5 a^* ***** p< 0.001,	9.50 ± 0.5 a^ p< 0.01,	3.52 ± 0.5 b^* ***** p< 0.001,	4.52 ± 0.5 b^ p< 0.01,
PCV (%)	54.70 ± 0.6	34.75 ± 0.37 a^* ***** p< 0.001,	40.57 ± 0.57 a^* *** p< 0.001, b^ ** p< 0.01,	48.64 ± 0.49 a^ p< 0.01, b^* ***** p< 0.001,	42.41 ± 0.45 a^* *** p< 0.001, b^* ******* p< 0.001,
Lympho(x103/µl)	6.23 ± 0.2	16.12 ± 0.7 a^* ***** p< 0.001,	11.90 ± 0.7 a^ p< 0.01, b^* * p< 0.05	4.90 ± 0.3 b^* ***** p< 0.001,	5.95 ± 0.15 b^* ***** p< 0.001,
MCHC	36.25 ± 0.15	32.50 ± 0.20 a^* ***** p< 0.001,	34.42 ± 0.1 a^ p< 0.01, b^ p< 0.01,	36.95 ± 0.15 b^* ***** p< 0.001,	36.05 ± 0.25 b^* ***** p< 0.001,

Biochemical parameters	Group-I (Vehicle.con)	Group-II (Arth.con)	Group-III (LEF)	Group-IV (LELA)	Group-V (LEHA)
AST (IU/L)	44.33 ± 0.88	92.33 ± 1.45 a^* ***** p< 0.001,	66.67 ± 1.2 a^* *** p< 0.001, b^* ******* p< 0.001,	51.3 ± 0.88 a^* * p< 0.05 b^* ***** p< 0.001,	67.7 ± 1.2 a^* *** p< 0.001, b^* ******* p< 0.001,
ALT (IU/L)	40.2 ± 1.53	77.0 ± 1.73 a^* ***** p< 0.001,	56.7 ± 0.88 a^* *** p< 0.001, b^* ******* p< 0.001,	51.3 ± 1.45 a^ p< 0.01, b^* ***** p< 0.001,	57.1 ± 2.08 a^* *** p< 0.001, b^* ******* p< 0.001,
ALP (IU/L)	237.67 ± 3.48	351.67 ±0.38 a^* ***** p< 0.001,	296.32 ± 2.52 a^* *** p< 0.001, b^* ******* p< 0.001,	251.01 ± 1.45 a^* * p< 0.05 b^* ***** p< 0.001,	297.67 ±1.76 a^* *** p< 0.001, b^* ******* p< 0.001,
Serum cholesterol (mg/dL)	111.15 ± 3.97	171.58 ± 5.3 a^* ***** p< 0.001,	139.9 ± 8.3 a^* * p< 0.05 b^* ***** p< 0.001,	115.16 ± 3.1 b^* ***** p< 0.001,	123.66 ± 6.8 b^* ***** p< 0.001,
Serum triglyceride (mg/dL)	109.21 ± 5.1	174.12 ± 4.3 a^* ***** p< 0.001,	151.55 ± 6.2 a^* ***** p< 0.001, b^* * p< 0.05	122.47 ± 2.9 b^* ***** p< 0.001,	138.89 ± 4.9 a^* * p< 0.05 b^* ***** p< 0.001,
Serum albumin (mg/dL)	2.90 ± 0.03	2.19 ± 0.05 a^* ***** p< 0.001,	2.34 ± 0.05 a^* ***** p< 0.001, b^* * p< 0.05	2.84 ±0.05 b^* ***** p< 0.001,	2.68 ± 0.03 a^ p< 0.01, b^* ***** p< 0.001,
Serum urea (mg/dL)	30.33 ± 2.4	42.67 ± 1.2 a^* ***** p< 0.001,	33.67 ± 0.88 b^* ***** p< 0.001,	27.67 ± 0.88 b^* ***** p< 0.001,	31.02 ± 0.58 b^* ***** p< 0.001,
Serum creatinine (mg/dL)	0.72 ± 0.02	0.85 ± 0.01 a^ p< 0.01,	0.55 ± 0.03 b^* ***** p< 0.001,	0.41 ± 0.01 b^* ***** p< 0.001,	0.64 ± 0.02 b^* ***** p< 0.001,