In vitro and in vivo Anti-arthritic and Antioxidant potential of Glycosmis pentaphylla in Complete Freund’s Adjuvant model

 

M. Sivakumar1, V.S. Chandrasekaran2*, B. Pushpa Kumari3

1Professor, HOD, Department of Pharmacognosy, Faculty of Pharmacy, 

Sree Balaji Medical College and Hospital, Bharat Institute of Science and Technology (DU), Chennai.

2Associate Professor, Department of Pharmaceutical Biotechnology,

Krishna Teja Pharmacy College, Tirupati 517506.

3Professor, Department of Pharmacology, Krishna Teja Pharmacy College, Tirupati.

*Corresponding Author E-mail: vschandru610@gmail.com

 

ABSTRACT:

Rheumatoid arthritis (RA) is a systemic, chronic inflammatory and auto immune disorder. RA is provoked by genetical predisposition and environmental factors. Oxidative stress makes the human liable to diseases like diabetes, RA, cancer, atherosclerosis etc. Hence the study investigated the anti-arthritic and oxidative activity of ethanolic extract of Glycosmis pentaphylla (EEGP) as it has not been pharmacologically tested. Anti-arthritic potential was evaluated in vitro using protein denaturation by bovine serum albumin and in vivo by Complete Freund Adjuvant (CFA) models at 200 and 400mg/kg doses. Also, in vitro and in vivo antioxidant ability was appraised by DPPH scavenging and assessment of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and lipid peroxidation (LPO). The results exhibited concentration dependent inhibition of albumin denaturation with maximum results obtained at 800μg/ml. The results of CFA model depicted better protection against arthritic lesions and body weight alterations. Also G. pentaphylla remarkably ameliorated histopathological changes. Additionally, plant exhibited remarkable anti-oxidant activity.

 

KEYWORDS: Arthritis, Therapeutic, Prophylactic, Antioxidative activity.

 

 


INTRODUCTION: 

Medicinal plants in particular have added advantage of possessing tremendous natural healing power.1,2,3.The use of plants as food and medicine has started ever since man was born in this universe. In traditional system of medicine the practitioners use various indigenous plants for the treatment of different types of arthritic conditions.4,5 One such plant drug used by siddha practitioners is Glycosmis pentaphylla (Rutaceae) commonly called bala or atibala is claimed by folklore for various ailments like rheumatism, seminal weakness, and diarrhoea6,7. Since no scientific validation is carried out on the anti rheumatic activity on the roots, the roots were selected for the study.

 

Rheumatoid arthritis is a systemic disease and it involves rheumatoid nodules, vasculitis, eye inflammation, cardio pulmonary disease as manifestations along with genetic susceptibility.

 

MATERIALS AND METHODS:

Animals:

Healthy adult female Wistar Albino rats, weighing about 150-180g were procured from Biogen, Bangalore. The study was approved by the Institutional Animal Ethical Committee, Sri Ramachandra University, Chennai (IAEC/XXXIV/ SRU/294/2013Version-I). Animals were housed individually in a well ventilated polypropylene cage. 12-hr light/12-hr dark artificial photo period was maintained. Room temperature of 25°C (±3°C) and relative humidity of 50–70% were maintained in the room.8,9 Animals had free access to pelleted feed (Nutrilab rodent, Tetragon Chemie Pvt Ltd., India) and reverse osmosis (Rios, USA) purified water ad libitum.

 

 

Acute oral toxicity study:

Three healthy, Wistar Albino rats weighing 150-180g were randomly selected for the study. The rats were fasted over-night and provided with water and libitum. Following the period of fasting, a dose limit at 2000 mg/kg body weight of ethanol extract dissolved in olive oil was administered orally to the animals22. Following administration, the animals were closely observed during the first 3 hr, and 48 hr, thereafter, 14 days, for toxic signs and symptoms such as convulsions, salivation, diarrhea, lethargy, sleep, coma and death. The weight of the animals was monitored throughout the experimental period.

 

DPPH:

DPPH (1, 1-Diphenyl-2-picrylhydrazyl) assay is the most widely reported method for the measurement of free radical scavenging capacity. To ethanolic solution of DPPH (200μl), 0.1ml of the extracts dissolved in methanol were added at different concentrations (50-1000μg/ml). An equal amount of methanol was added to control. Butylated hydroxyl toluene (1mg/ml) was used as standard for comparison.10,11.12 All the test tubes including blank were incubated at room temperature under dark condition for about 30 min. After 30 min the decrease in the absorbance of test mixtures was read at 517nm using methanol as blank. The percentage inhibition of DPPH radical was calculated by comparing the results of the test with those of the control using the formula.

 

                   (Absorbance of control - absorbance of test)

% inhibition = --------------------------------------------100

                              Absorbance of control

 

Inhibition of protein denaturation – in vitro anti arthritic activity:

Protein denaturation study was performed by using bovine serum albumin (BSA). When BSA is heated it undergoes denaturation and express antigens associated with type-III hypersensitivity reaction. 0.5ml of test solution, test control solution, standard solution was prepared. Various concentrations (50, 100, 200, 400, 800 and 1000μg/ml) of test drugs chloroform, ethyl acetate and ethanol extracts and standard drug diclofenac sodium were prepared. 1N HCl was used to adjust the pH to 6.3 for all the above solutions.13,14,15 The samples were incubated at 37°C for 20 minutes and the temperature was increased to keep the samples at 57°C for 3 minutes.16,17 After cooling, 2.5ml of phosphate buffer was added to the above solutions. The absorbance was measured at 416nm. The control represents 100% protein denaturation. The percentage inhibition of protein denaturation can be calculated as

 

   (Optical density of control - Optical density of sample)

% Inhibition = 100 ------------------------------------ X 100

                                 Optical density of control

In vivo anti arthritic activity:

Animals were injected with 0.1ml (i.p) of Complete Freund’s Adjuvant (CFA) into the right hind paw of each rat. Animals were assigned to groups of 6 animals.18,19 Arthritic control group received CFA, but non-arthritic control (IFA group) received 0.1ml Incomplete Freunds Adjuvant (IFA) (Sterile Paraffin Oil). EEGP (200mg/kg and 400mg/kg), Methotrexate (0.1ml/kg) were administered to rats in the various groups respectively. Paw volume was measured by water displacement by using plethysmograph for both the ipsilateral (injected paw) and the contralateral paw (non- injected paw) before and after injection of CFA (day 0) and every week. The edema component of inflammation was quantified by measuring the difference in foot volume between day 0 and day 28. Two sets of experiments were carried out as therapeutic protocol and prophylactic protocol.

 

Immunohistochemistry (IHC):

IHC is also widely used in basic research to understand the distribution and localization of biomarkers and detecting antigens in different parts of a biological tissue20,21. The protocols in IHC as follows, sections deparaffinized in xylene and hydrated through descending grades of alcohol. Heat-induced antigen retrieval was carried out by microwaving at HI-90 for 20 minutes using citrate buffer (pH 6.8) for TNF-alpha or Tris-EDTA buffer (pH 8.0) for CD4, IL-2 and TGF-beta. Each step herein is preceded by three washings in PBST (Phosphate buffered saline with 0.1% tween 20) then endogenous peroxidase quenching by incubation with 3% H2O2 for 30 minutes, blocking with 5% goat serum in 1% BSA for 30 minutes and primary antibody incubation at 4°C overnight, Biotinylated secondary antibody incubation for 30minutes at room temperature. The rats from each group at the end of the experimental period were euthanized using anesthetic ether for histopathological evaluation of joints and paw tissues for arthritis and other associated lesions.22,23,24

 

Statistical Analysis:

The data obtained was presented as mean±standard error. Clinical edema volumes measured in different treatment groups were compared with adjuvant non- treated control group (group II) and adjuvant control group (group I) by one way ANOVA and Dunnett's Multiple Comparison Test. Significance was calculated at (p˂0.05).

 

RESULTS AND DISCUSSION:

The air dried roots of G. pentaphylla were extracted by maceration extraction. Free radical scavenging of DPPH by the ethanol extract (Table 2) was found to be more significant than the rest of the extracts and the significance was concentration dependent. From the results of the present study, all the extracts of G. pentaphylla roots significantly controlled the production of auto antigen involved in the inhibition of the denaturation of proteins (Table 3). The result also revealed that a concentration dependent impediment of protein denaturation by G. pentaphylla as well as diclofenac sodium throughout the concentration range (10–1000μg/ml). EEGP demonstrated 90.12% inhibition of protein denaturation at 1000μg/ml, which was parallel to aspirin i.e., 94.02% at 1000μg/ml. EEGP had shown significant inhibition of protein denaturation, may be by alteration of the mechanisms involved in it, like variation in electrostatic, hydrogen, hydrophobic and disulphide bonding.

 

The results of arthritic index score for the therapeutic and prophylactic groups (Table 4) shows that CFA induced rats resulted in increase in arthritic score index compared with normal group. Rats treated with EEGP and diclofenac sodium significantly decreased in the arthritic score in comparison to that of the adjuvant control group. The results also revealed a significant (p < 0.05) decrease in paw diameter of treatment groups as compared to CFA control group. Redness and swelling of affected joints were significantly less in treated as compared to arthritic control animals (Table 5 and 6). EEGP significantly inhibited arthritis in chronic stage and protected the joints as clear from histopathology results, which possibly could be due to its impeding action on inflammatory mediators i.e., IFN α, PDGF and cytokines (IL-1, IL 6 and TNF-α) as well as the result of immunological protection rendered by plant when compared with the CFA treated group.

 

The effect of biochemical parameters in blood and effect of antioxidant parameters in liver were estimated (Table 7 and 8) respectively. The number of cells of CD4 (Figure 1.1 to 1.5), IL 2 (Figure 2.1 to 2.5), TGF β1 (Figure 3.1 to 3.5) and TGF β1 (Figure 4.1 to 4.2) in case of the group induced with CFA was found to be high compared with the groups treated with the standard drug and the extract treated groups thereby confirming the efficacy of the extract at the higher dose as well as dose dependent effect. Thus, the reduction in the joint inflammation and significant protection of G. pentaphylla against oxidative stress may be attributed to the presence of flavonoids and phenols in it.

 

The H&E section of the left ankle joints and paw tissue from normal control group (Figure 5.1 and 5.6) revealed normal histology, CFA induced group (Figure 5.2 and 5.7) revealed severe degree of dermal infiltration by polymorphonuclear cells and panniculitis with mononuclear cells infiltration, tissues of the reference control (methotrexate) (Figure 5.3 and 5.8) has reduced the severity of synovitis and inflammatory cells infiltration, muscular necrosis, panniculitis and articular cartilage erosions and tissue of arthritic group treated with Glycosmsis pentaphylla at the dose level of 400 mg/kg b.wt (Figure 5.5 and 5.9) (Both prophylactic and therapeutic dose) has revealed mild to moderate degree of the panniculitis, moderate dermis and subcutaneous infiltration of polymorphonuclear cell in the skin, minimal to mild degree of synovitis , no articular erosions and decreased proliferation of connective tissues, lymphocytic infiltration around the blood vessels, decreased inflammatory cells infiltrationin the subcutaneous paw tissues when compared to the arthritic rats treated with Glycosmsis pentaphylla at the dose of 200mg/kg b.wt (Figure 5.4 and 6.0) (both prophylactic and therapeutic dose)

 


 
 

Table 1.Treatment Schedule: Therapeutic and Prophylactic protocol

Group

Therapeutic protocol

Prophylactic protocol

Group1

Non Control/IFA (Intraperitoneal injection of 0.1ml of IFA)

Non Control/IFA (Intraperitoneal injection of 0.1ml of IFA)

Group 2

Arthritic control/CFA (Intraperitoneal injection of 0.1ml CFA)

Arthritic control/CFA (Intraperitoneal injection of 0.1ml CFA)

Group 3 and 4

ETGP 200mg/kg and 400mg/kg respectively from day 9

ETGP 200mg/ kg and 400 mg/kg respectively from day 0

Group 5

Methotrexate (0.1ml/ kg)

Methotrexate (0.1ml/ kg)

 
Table 2. Free radical scavenging activity by DPPH method

Concentration

µg/ ml

Percentage inhibition

Standard

(Ascorbic acid)

Petroleum Ether extract

Chloroform

Ethyl acetate

Ethanol

10

12.640 ±0.94

14.26 ±1.37

15.12 ±1.22

21.86 ±3.04

34.87 ±3.34

50

14.220 ± 0.96

17.93 ±4.24

19.040 ± 0.58

28.83 ± 2.28

44.76 ±1.98

100

17.160 ± 1.80

20.06 ±1.06

26.540 ± 2.08

42.65 ± 2.43

59.78 ±2.74

200

19.280 ± 2.14

17.21 ±2.59

28.120 ± 3.84

51.33 ± 3.71*

69.65 ±1.50

400

32.160 ± 3.18

38.92 ±5.18

46.540 ± 4.16

69.42 ± 5.79

81.86 ±4.56

800

49.140 ± 1.08

54.07 ±0.76

59.140 ± 2.16

83.58 ± 1.41*

91.89 ±4.26

1000

32.120 ± 0.48

37.89 ±1.95

53.260 ± 3.20

90.79 ± 2.85*

94.65 ±1.50

*Each values represents mean±SEM n=3

 

Table 3. In-vitro anti arthritic activity of G. pentaphylla roots by protein denaturation method

Concentration µg/ml

Chloroform

Ethyl acetate

Ethanol

Diclofenac Sodium

10

38.420 ±1.80

40.20 ± 2.0

47.420 ± 3.10

51.20 ± 0.4

50

49.280 ± 1.90

51.17 ± 2.5

55.280 ± 4.00

59.68 ± 0.5

100

55.020 ± 2.10

60.74 ± 2.1

62.020 ± 2.50

63.03 ± 0.9

200

67.670 ± 2.60

67.07 ± 0.9

70.670 ± 3.00

79.52 ± 3.5

400

73.290 ± 3.00

74.84 ± 3.5

79.290 ± 2.40

85.02 ± 0.8

800

72.120 ± 2.60

78.37 ± 2.5

88.120 ± 3.30

92.87 ± 1.5

1000

79.120 ± 3.20

82.93 ± 3.0

90.120 ± 2.20

94.02 ± 2.1

*Each values represents mean ± SEM n=6

 
Table 4. Effect on arthritic score in adjuvant induced arthritis

 

Groups

Arthritic Index Score

Prophylactic Model

Therapeutic Model

Normal control

0 ± 0

0 ± 0

Arthritic control

4 ± 0.16

4 ± 0.16

EEGP 200 mg/ kg

2.4 ± 0.14

2.4 ± 0.14

EEGP 400 mg/ kg

1.9 ± 0.17*

1.7 ± 0.16*

Methotrexate 0.1 mg/ kg

1.6 ± 0.14**

1.4 ± 0.13**

 

Table 5 Effect of root of ETGP on change in body weight

Groups

% Body Weight Change

Prophylactic Model

Therapeutic Model

 

Days

Day 7

Day14

Day 21

Day 28

Day 7

Day 14

Day 21

Day 28

Normal control

3.2

3.8

4.87

5.28

3.2

3.4

4.17

4.88

Arthritic control

3

3.92

6.16

7.29

3

3.92

6.16

7.29

EEGP 200 mg/ kg

2.8

3.76

4.14

4.2

2.99

3.46

4.47

4.96

EEGP 400 mg/ kg

2.61

3.84

4.24

4.8

1.27

2.58

4.58

5.2

Methotrexate 0.1mg/ kg

1.95

2.51

2.89

3.22

2.2

2.84

3.2

3.1

 
Table 6. Effect of root of ETGP on paw volume on complete Freund’s adjuvant induced arthritic in rats

Groups

Paw volume

Prophylactic model

Therapeutic model

Days

0

7

14

21

28

0

7

14

21

28

 

Normal control

1.22 ±0.04

1.21 ±0.04

1.32 ± 0.04

1.45 ± 0.03

1.24 ± 0.02

1.22 ±0.03

1.2 ± 0.11

1.24 ± 0.01

1.24 ±0.11

1.28 ±0.12

 

Arthritic control

1.37 ±0.03

3.15 ±0.06*

3.54 ± 0.06**

4.01 ± 0.02

4.14 ± 0.06**

1.37 ±0.03

2.55 ± 0.15*

3.03 ± 0.08**

4.12 ± 0.18**

4.4 ± 0.02**

 

EEGP 200 mg/kg

1.24 ±0.02

3.39 ± 0.09

2.18 ± 0.05**

2.63 ± 0.03**

2.02 ± 0.06**

1.24 ±0.03

3.29 ±0.19*

2.3 ± 0.33**

2.03 ± 0.26**

1.9 ± 0.03**

 

EEGP 400 mg/kg

1.31 ±0.03

3.52 ± 0.06*

1.77 ± 0.05**

2.02 ± 0.02**

1.85 ± 0.06**

1.31 ±0.03

3.34 ± 0.19*

1.72 ± 0.24**

1.98 ± 0.02**

1.76 ± 0.03**

 

Methotrexate 0.1 mg/kg

1.31 ±0.03

2.94 ± 0.11*

1.52 ± 0.05**

1.77 ± 0.02**

1.66 ± 0.06**

1.47 ±0.02

3.39 ± 0.03*

1.95 ± 0.02**

1.65 ± 0.33**

1.53 ± 0.03**

 

Values are expressed as mean ± SEM; n=6 rats in each group, *p< 0.05 compared with normal control, **p< 0.01 compared with arthritic control.

 

Table 7. Effect on biochemical parameters in blood

Parameters

Normal control

Arthritic control

Prophylactic Model

Therapeutic Model

EEGP

200 mg/Kg

EEGP

400 mg/Kg

Metho

threxate 0.1mg/ kg

EEGP

200 mg/ Kg

EEGP 400

mg/Kg

Metho

threxate

0.1mg/ kg

BUN

37.75±1.18

45.50±5.17**

40.50±0.50**

39.00±1.15*

41.75±1.31**

35.50±3.20**

34.00±2.38**

39.25±2.06**

CR(mg/dl)

0.61±0.05

0.48±0.11**

0.50±0.014**

0.65±0.03*

0.57±0.06*

0.45±0.04*

0.45±0.09*

0.51±0.05*

TP (g/dl)

7.21±0.22

7.25±0.38*

6.48±0.08*

7.15±0.10*

6.85±0.19

5.98±0.34*

6.38±0.44*

6.53±0.19

ALT(U/L)

96.50±5.72

99.75±6.16**

85.75±9.75*

92.00±3.32

71.75±13.26

83.25±4.57*

79.50±7.46

71.25±11.33

AST(U/L)

142.50±

3.57

186.25±30.92*

146.50±7.50**

127.00±6.04**

115.00±10.68*

139.0±4.08**

115.00±5.46**

107.50±12.6*

ALP(U/L)

607.25±7.98

685.00±52.42**

823.50±27.50*

518.00±85.7

567.75±13.14

813.50±27.50**

498.00±85.78

560.25±13.99

CRP-hs (mg/L)

4.23±0.49

3.74±0.09**

3.64±0.49*

3.48±0.53*

3.74±0.37

3.11±0.42*

3.33±0.54

2.99±0.35

LDH(U/L)

278.00±32.5

232.00±2.26*

250.00±24.00

265.00±35.6

274.00±20.31

248.50±19.97

260.00±36.29

266.50±17.85

Values are mean ± SEM, n=6 rats per group.. When compared with normal control group, *P<0.05, **P<0.01

ALP- Alkaline phosphatase, ALT- Alanine aminotransaminase, AST- Aspartate aminotransaminase, CR-Creatinine, LDH- Lactate dehydrogenase, TP- Total protein, CRP- hs- High sensitive C- reactive Protein

Table 8. Effect on antioxidant parameters in liver

Parameter

Normal control

Arthritic control

Prophylactic Model

Therapeutic Model

ETGP

200

mg/ Kg

ETGP

400

mg/ Kg

Metho

threxate

 0.1mg/ kg

ETGP

200

mg/ Kg

ETGP

400

mg/ Kg

Metho

threxate

0.1mg/ kg

SOD

0.35±0.02

0.11±0.006**

0.26±0.005*

0.25±0.005*

0.24±0.006

0.26±0.006

0.26±0.005*

0.25±0.02*

CAT

3.13±0.008

1.03±0.15**

2.41±0.29*

2.4±0.07*

2.4±0.09*

2.49 ± 0.13

2.48 ± 0.04

2.46 ± 0.05*

LPO

0.11±0.008

0.24±0.04**

0.14±0.02**

0.12±0.02*

0.12±0.03*

0.16±0.02*

0.132±0.01*

0.11±0.005*

GPX

8.55±0.32

17.93±0.65**

11.28±0.66*

10.12±0.06

10.02±0.39

10.66±0.59*

10.51±0.32

10.26±0.53

Values are mean ± SEM.When compared with vehicle control group, *P<0.05, **P<0.01

SOD- Superoxide dismutase, CAT- catalase,  LPO- Lipid peroxidase, GPX- Glutamate peroxidase.

 

IHC Inflammatory markers - CD4

 

 

 

 

 

Fig 1.1 Normal Control

Fig 1.2 CFA Induced

Figure 1.3 CFA+ Methothrex

Figure 1.4 CFA+ ETGP 200 mg

Figure 1.5 CFA+ ETGP400 mg

IHC Inflammatory markers – IL 2

 

 

 

 

 

Figure 2.1 Normal control

Figure 2.2 CFA Induced

Figure 2.3 CFA+ Methothrexate

Figure 2.4 CFA+ETGP 200 mg

Figure 2.5 CFA+ ETGP400 mg

IHC (Tissue) Inflammatory markers – TGFβ1

 

 

 

 

 

Figure 3.1 Normal control

Figure 3.2 CFA Induced

Figure 3.3 CFA+ Methothrexate

Figure 3.4 CFA+ETGP 200 mg

Figure 3.5 CFA+ ETGP400 mg

IHC Inflammatory markers – TNF-α

 

 

 

 

 

Figure 4.1 Normal control

Figure 4.2 CFA Induced

Figure 4.3 CFA+ Methothrexate

Figure 4.4 CFA+ETGP 200 mg

Figure 4.5 CFA+ ETGP400 mg

Histopathology of synovial joint in in-vivo anti-arthritic activity of GP

 

 

 

 

 

Figure 5.1 Normal control

Figure 5.2 CFA Induced

Figure 5.3 CFA+ Methothrexate

Figure 5.4 CFA+ETGP 200 mg

Figure 5.5 CFA+ ETGP400 mg

Histopathology of paw tissues in vivo antiarthritic activity of GP

 

 

 

 

 

Figure 5.6 Normal control

Figure 5.7 CFA Induced

Figure 5.8 CFA+ Methothrexate

Figure 5.9 CFA+ETGP 200 mg

Figure 6.0 CFA+ ETGP400 mg


CONCLUSION:

In the present investigation, it has been observed that G. pentaphylla has exerted significant anti-arthritic activity in the experimental model. Its valuable effects on RA could possibly be correlated with the presence of phenols and flavonoids in G. pentaphylla. However, further research is required for appraisal of exact mechanism of action of G. pentaphylla, determination of pro-inflammatory cytokines level, isolation of active constituents and cellular characterization that could conclusively establish it as a potentially safer disease modifying agent in the treatment of RA.

 

REFERENCES:

1.     Ariful, H.M et al Ethnomedicinal uses of some medicinal plants for prevention against all forms of cancer by the traditional healers in Gazipur district of Bangladesh. AACR International conference on frontiers in cancer prevention research. Cancer Prev Res. 2010;Vol 3.  DOI: 10.1155/2016/7832120.

2.     Anonymous, In., The Wealth of India A dictionary of Indian Raw materials and Industrial Products Products Publication & Information Directorate, Vol. IV, CSIR, New Delhi. 2003; PMCID: PMC5189551.

3.     Shyama S. Kumar et al  Indian Medicinal Plants Used for Treatment of Rheumatoid Arthritis, Research J. Pharm. and Tech.2015; 8(5), 597-610. DOI: 10.5958/0974-360X.2015.000992

4.     Srivastava S, Patel et al  Rheumatoid Arthritis: An Autoimmune Disease Prevalent in Females. Research J. Pharm. and Tech. 2016; 9(2), 170-172. DOI: 10.5958/0974-360X.2016.000305

5.     Nandgude Tanaji et al Clinical features and treatment of Rheumatoid Arthritis; A review. Research J. Pharm. and Tech.2018; 11(12), 5701-5706. DOI: 10.5958/0974-360X.2018.010326.

6.     Bhattacharyya, P, et al Glycozolidol, an antibacterial carbazole alkaloid from Glycosmis pentaphylla.Phytochemistry.1985; 24(4), 882-883. doi:10.3390/molecules24020293

7.     Namita, A, et al Pharmacognostic and Preliminary phytochemical studies on the leaves of Glycosmis pentaphylla (Retz.) Corr. J. pharm. Res. 2011;4: 1800-1801. DOI: 10.13040/IJPSR.0975-8232.12(2).1135-42.

8.     McCartney-Francis, N., et al Suppression of arthritis by an inhibitor of nitric oxide synthase. The Journal  of  experimental  medicine, 1983;178(2), 749-754. doi: 10.1084/jem.178.2.749.

9.     McCartney-Francis, et al Inflammatory Joint Disease. In Inflammation Protocols (pp. 147-159). Humana Press.2013; doi: 10.4314/ajtcam.v9i1.2

10.   Praveen D et al  Antioxidant and Analgesic Activity of Leaf Extracts of Artocarpus heterophyllus. Research J. Pharm. and Tech. 2016;9(3); 257-261. doi: 0.5958/0974-360X.2016.00047.0.

11.   B. Premkuma Antioxidant Defense and Disease activity in Rheumatoid Arthritis. Research J. Pharm. and Tech. 2018;11(5):1810-1814. doi: 10.5958/0974-360X.2018.00336.0.

12.   Pavithra. S, N. Banu Free Radical Scavenging Activity and Total Antioxidant Capacity of Tin Chlorophyllin from Morinda citrifolia L. Research J. Pharm. and Tech.2017; 10(2): 453-455. doi: 10.5958/0974-360X.2017.00091.9.

13.   Sripradha and lakshmi T In vitro Anti arthritic activity of Sesbania grandiflora ethyl acetate extract. Research J. Pharm. and Tech.2015; 8(11); 1509-1511. DOI: 10.5958/0974-360X.2015.00269.3.

14.   S.B. Prabha, et al Evaluation of in vitro Antioxidant, Antibacterial and Anticancer activities of leaf extracts of Cleome rutidosperma. Research J. Pharm. and Tech.2017; 10(8): 2492-2496. doi: 10.5958/0974-360X.2017.00440.1.

15.   V. Chitra, J. Narayanan. In vitro Screening for Anti-Cholinesterase and Anti Oxidant Activity of Extract of Garcinia hanburyi. Research J. Pharm. and Tech 2018; 11(7): 2918-2921. doi: 10.5958/0974-360X.2018.00538.3.

16.   Clatworthy, A. L, et al. Role of peri-axonal inflammation in the development of thermal hyperalgesia and guarding behavior in a  rat  model  of  neuropathic  pain. Neuroscience letters,1985;184(1), 5-8. DOI: 10.1016/0304-3940(94)11154-b .

17.   David. B.L, et al.  Anti-inflammatory effects of ABT-702, a novel non-nucleoside adenosine kinase inhibitor, in rat adjuvant arthritis.Journal of Pharmacology and Experimental Therapeutics, 2001;296(2), 495-500. PMID: 11160636.

18.   Anderson, G. D, et al.  Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in  rat  adjuvant  arthritis. Journal  of  Clinical Investigation, 1996;97(11), 2672-2679. doi: 10.1172/JCI118717.

19.   Fereidoni, M, et al. An accurate and simple method for measurement of paw edema. Journal of Pharmacological and Toxicological Methods,2000; 43(1), 11-14. DOI: 10.1016/s1056-8719(00)00089-7.

20.   Gutteridge, J. M.  Antioxidant properties of the proteins caeruloplasmin, albumin and transferrin. A study of their activity inserum   and   synovial   fluid   from   patients   with    rheumatoid arthritis. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1986;869(2), 119-127. DOI: 10.1016/0167-4838(86)90286-4 .

21.   Kannan, S ―Free radical theory of autoimmunity‖. Theror Biol Med Model, 2006;3: 22. doi: 10.1186/1742-4682-3-22.

22.   Alamgeer* , Ambreen Malik Uttra and Umme Habiba Hasan  Anti-arthritic activity of aqueous-methanolic extract and various fractions of Berberis orthobotrys Bien ex Aitch. BMC Complementary and Alternative Medicine .2017; 17:371 DOI 10.1186/s12906-017-1879-9.

23.   Patil MVK, Kandhare AD, Bhise Anti-arthritic and anti-inflammatory activity of Xanthium srtumarium L. ethanolic extract in Freund’s complete adjuvant induced arthritis. Biomed Aging Pathol.;2012;2:6–15 DOI: 10.1016/j.jep.2014.05.023.

24.   Philippe, L et al.  Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 1997;273(4), R1550-R1556. https://doi.org/10.1152/ajpregu.1997.273.4.R1550.

 

 

 

 

 

Received on 01.11.2021          Modified on 10.02.2022

Accepted on 08.04.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):621-626.

DOI: 10.52711/0974-360X.2023.00106