Document 13310912

Int. J. Pharm. Sci. Rev. Res., 37(2), March – April 2016; Article No. 02, Pages: 13-21 ISSN 0976 – 044X

Research Article

Evaluation of Anti-arthritic Activity of the Plant Barleria prionitis Linn

.

Preet Kawal Kaur* a,b

, Vaibhav Sharma b

, Maninder Karan a a

University Institute of Pharmaceutical Sciences–UGC Centre for Advanced Studies, Punjab University, Chandigarh, India. b

IEC School of Pharmacy, IEC University, Kalujhanda, Solan, Baddi, Himachal Pradesh, India.

*Corresponding author’s E-mail: preetkawalpu@gmail.com

Accepted on: 30-01-2016; Finalized on: 31-03-2016.

ABSTRACT

Ethnopharmacological relevance – The genus Barleria is distributed in Asian and African countries and is well known traditionally to treat various inflammatory problems. In the present study, the effect of two different extracts namely methanolic (ME, prepared by maceration) and butanolic (BE, obtained after partitioning of ME) of B. prionitis and the isolated three major iridoids viz., acetylbarlerin (AB), barlerin (B) and shanzhiside methyl ester (SME) from the plant using chromatographic techniques were evaluated in a rat model of Complete Freund’s Adjuvant (CFA) induced-arthritis at a single dose of 200 mg/kg for extracts and 1 mg/kg for pure compounds. The results were compared to untreated control and standard (indomethacin, INDO) treated groups.

Test drugs were administered orally for 21 days and body weight changes, reduction in paw swelling and arthritic index were observed on alternate days. On 21 st

day of experiment, the histopathological, radiological and biochemical observations were carried out along with rheumatoid factor. The serum level of cytokines (TNFα and IL-1 β ) were also determined using ELISA kits. The results indicate that Barleria protects rats against the bone loss, body weight changes and haematological perturbations induced by

CFA. Further the histopathological and radiological studies also support the generated observations. Thus, the positive effect of the test samples in controlling the various parameters associated with the progression of arthritis demonstrated their pronounced antiarthritic effects, indicating that B. prionitis would be a potent candidate for treating arthritis.

Keywords: Barleria prionitis , Extracts, Iridoids, Complete Freud’s Adjuvant (CFA).

INTRODUCTION

R heumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease developing over months or years and involves essentially the synovial joints of the body

1

. Bone loss is a common feature of various inflammatory arthritis and its erosion results from the activation of an inflammatory response that increases the number and activity of osteoblasts.

Thus, bone erosions and peri-articular osteopenia constitutes an important signs for the diagnosis of rheumatoid arthritis

2

. Various pro-inflammatory cytokines, such as tumour necrosis factorα (TNFα ) and interleukins (IL-1 β & IL-6) are present abundantly in synovial fluid and joint tissues of RA patients and have revealed the key role in the pathogenesis of RA. These cytokines are produced through continuous activation of

T cells and their interaction with monocytes/macrophages in RA. Moreover, anti-TNF or anti-IL-1 and IL-6 therapies have been reported to be effective in treatment of RA

3

.

The arthritis can be induced with the injection of

Complete Freud’s adjuvant (CFA) in the footpad of rats that serve as a model to test the antiarthritic and antiinflammatory effects of investigational substances

1

. CFA contains heat killed Mycobacterium in water in oil emulsion and CFA induced arthritis model represents a systemic inflammatory disease with bone and cartilage changes similar to those observed in RA. The common pathological features of adjuvant arthritis in rats and RA in human are joint swelling associated with cellular and pannus invasion of the joint space and bone resorption.

The major site of irreversible tissue damage originates from synovium lining the joint capsule with cartilage and bone, which is often termed the “pannus” and this is the characteristic feature of RA

2

.

Although the modern system of medicine provides many drugs that can cure arthritis but these are often associated with side effects. So the urge to look into the natural anti-arthritic agents as viable alternatives to synthetic drugs prompted to explore medicinal plants.

Many such plants having anti-rheumatoid activity and their products are used for the treatment of inflammation associated with arthritis

4

. Barleria is one such plant which enjoys high status for its versatile use in several ailments including inflammation

5,6

. The genus Barleria of family

Acanthaceae comprises of small shrubs or undershrubs, which are distributed in warmer parts of the world. It has about 300 species, of which nearly 32 species are reported to occur in India

7 low hedge plants in gardens

. Many species are grown as and the important medicinal species are B . buxifolia Linn., B . courtallica Nees., B . cristata Linn., B . longifolia Linn., B . prionitis Linn., B . lupulina Lindl. and B . strigosa Willd

6, 8-13

.

The genus is reported to contain iridoids, anthraquinones, flavonoids, sterols, and fatty acids

14

. Iridoids constitute the major class of compounds isolated from Barleria and important bioactive iridoids are acetylbarlerin, barlerin and shanzhiside methyl ester

15,16

. From the bibliography

International Journal of Pharmaceutical Sciences Review and Research

Available online at www.globalresearchonline.net

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13 International Journal of Pharmaceutical Sciences Review and Research


Int. J. Pharm. Sci. Rev. Res., 37(2), March – April 2016; Article No. 02, Pages: 13-21 ISSN 0976 – 044X search, it was found that three reports have been known where Barleria showed antiarthritic activity

17-20

. The earlier comparative inflammation related work done in our lab by us on two species of Barleria viz ., B . prionitis and B . cristata revealed that B . prionitis possess the distinct anti-inflammatory activity and accordingly extract dose of 200 mg/kg was selected to perform anti-arthritic activity

21

. In addition to that isolated pure compounds also exhibited anti-inflammatory activity and the dose selected was 1 mg/kg accordingly for evaluating the antiarthritic activity

22

.

Although reports revealing the anti-arthritic activity have been found but no such detailed work has been reported on the whole plant and the different extracts along with the isolated pure iridoids. Thus the present study was designed to investigate the anti-arthritic profile of the whole plant of B. prionitis only using different prepared active extracts viz ., methanolic (ME) and butanolic (BE) and the isolated three major iridoids viz., acetylbarlerin

(AB), barlerin (B) and shanzhiside methyl ester (SME) at the defined dosage levels. An evaluation system with clinical, hematological, radiological and histopathological parameters was employed in the study and the possible mechanisms in which pro-inflammatory cytokines are predominantly involved were further investigated.

MATERIALS AND METHODS

Plant materials and chemicals

In Sanskrit, B. prionitis is commonly known as ‘ sahacara ’ or ‘ jhinti ’

12,13

. The plant material of B. prionitis Linn. was collected during August to September 2008 from the

Medicinal Plants Garden of University Institute of

Pharmaceutical Sciences, Panjab University, Chandigarh.

The authenticity of the sample was duly confirmed by

National Institute of Science Communication and

Information Resources (NISCAIR), New Delhi (Ref.

NISCAIR/RHMD/Consult/-2008-09/1127/158) and voucher specimens of the same has been deposited in the

Museum-cum-Herbarium of University Institute of

Pharmaceutical Sciences - Centre of Advanced Study,

Panjab University, Chandigarh, India, under the voucher number 1460 for B. prionitis Linn. Indomethacin (IND) was purchased from the local market. Complete Freund’s

Adjuvant (CFA) of Difco, USA and liquid paraffin of Sigma

Aldrich, USA was used for study.

Animals

Female Sprague-Dawley rats (150-180 g) bred in the

Institute’s animal house, were used in the study. Animals were housed under standard conditions (25 ± 2° C, 60-70

% relative humidity and 12 h photoperiod) and were maintained on standard rodent pellet diet (Ashirwad

India Ltd, Chandigarh) and water ad libitum .

The rats were acclimatized to laboratory conditions for 3 days before commencement of the experiment. All experimental procedures were reviewed and approved by

Institutional Animal Ethics Committee (IAEC), vide ref. no.

CAH/09/50 dated 04.06.2009 of Punjab University

Chandigarh.

Acute toxicity study

Acute oral toxicity of methanol extract was performed using Sprague-Dawley rats (n=5) following OECD 425 guidelines (Up and Down Procedure). The p.o

. LD

50

was found to be more than 2000 mg/kg, with no signs of abnormalities or any mortality observed for 14 days after single dose administration.

Treatment

Different extracts (prepared by maceration of whole plant of B. prionitis and partitioning of methanol extract, ME and BE respectively, 200 mg/kg, p.o

.) and iridoids (AB, B and SME, isolated by chromatographic techniques, 1 mg/kg, p.o

.) were evaluated using Indomethacin (3 mg/kg, p.o

.) as a standard drug on female Sprague-

Dawley rats (n=5) in CFA induced arthritic model. All doses were freshly prepared in 2 %, v/v tween 80 in normal saline (0.9%, w/v). The normal (Liquid paraffin injected) and disease control (CFA injected) groups received only vehicle and test groups were given different prepared samples.

Induction of arthritis

Arthritis was induced in rats by the subplantar injection of

0.05 ml of freshly prepared suspension (5 mg/ml, Difco,

USA) of steam killed Mycobacterium tuberculosis in liquid paraffin under light ether anaesthesia. Different doses of the test drug and the vehicle (normal saline) were given p.o. before the injection and then once daily for 21 days.

Total number of eight groups (normal control, disease control, standard, ME, BE, AB, B and SME) were subjected to CFA challenge.

Assessment of the arthritis severity and the effects of the treatment

Rats were inspected daily for the onset of arthritis characterized by oedema and/or erythema in the paws.

The severity of arthritis was evaluated using a system of arthritic scoring, measurement of bi-hind paw volumes

(by means of volume displacement method using a plethysmometer UGO BASILE, 7140) and body weight on alternate days after arthritis induction. Lesions of the four paws of each rat were graded from 0 to 4 according to the extent of both oedema and erythema of the periarticular tissues; 16 was calculated as the maximum of the combined arthritic scores per animal. Intensity of arthritis was scored by grading each paw from 0-4 based on erythema, swelling and deformity of the joint in the following manner:-

0-

1Slight erythema or swelling of one of the toes or

2-

3-

No erythema or swelling fingers

Erythema and swelling of more than one toe or finger

Erythema and swelling of the ankle or wrist







4Complete erythema and swelling of toes or fingers and ankle or wrist and inability to bend the ankle or wrist.

Measurement of haematological parameters and serum cytokine levels

Blood samples were collected by retro-orbital puncture on the 21 st

day and erythrocyte sedimentation rate (ESR) was carried out by the method of Westergren. Other haemotological parameters like Hb, Packed cell volume

(PCV), Total leukocyte count (TLC) and RBC count were also determined. For the estimation of Rheumatoid factor, the blood was centrifuged at 1500 rpm for 10 minutes and the serum was collected into clean dried tubes. Levels of the cytokines TNFα , IL-1 β in the blood serum were measured using commercially available ELISA kits for TNFα and IL-1 β (Pierce Biotechnology, Rockford,

IL) according to the manufacturer’s recommendations.

Radiological and histopathological examinations

At the end of the experiment, rats were anesthetized by ether anaesthesia and imaged on Philips Optimus (Model-

Digital Diagnost, 40-125 KVP, 1000 mA) at PGIMER

(Postgraduate Institute of Medical Education & Research,

Chandigarh). Radiographs of each rat for evaluation of soft tissue swelling and bone erosion were scored blindly, on a scale of 0 (normal), 1 (mild changes), 2 (moderate changes) and 3 (severe changes).

After X-Ray check, rats were sacrificed by diethyl ether asphyxiation and the hind paws were excised and placed in 10 % formalin solution. The fixed tissues of the ankle joints were then decalcified in formic acid, embedded in paraffin, longitudinally cut into 5 µm sections, and stained with hematoxylin and eosin (H and E). Grading of cellular infiltration (polymorphonuclear cells, macrophages or lymphocytes), joint space narrowing, synovial hyperplasia, pannus formation and bone and cartilage erosion of the ankle joints were examined using a semiquantitative scale from 0 (normal), 1 (mild changes),

2 (moderate changes) and 3 (severe changes).

Statistical analysis

All the results were expressed as mean ± SD. Data was analysed using student’s t test and one-way Anova followed by Dunnett Test as post hoc analysis where ever required. The p < 0.05 was considered as the criteria of analyzing statistically significant values.

RESULTS

Therapeutic effects of test samples administered from the day of arthritis onset

The inflammatory and arthritic lesions appeared in the rats inoculated with CFA suspension (Fig No.1), and treated with different test samples viz ., ME, BE and pure compounds showed significant (p < 0.05) attenuation in right hind paw volume from day 7 onwards whereas indomethacin (3 mg/kg) treated rats had significantly (p <

0.05) decreased paw volume from day 3 to 21 as compared to control rats (Table No.1).

As shown in Table 1, test samples at their doses significantly (p < 0.05) reduces the clinical parameters of the arthritic score, hind paw volume and markedly improved the loss of body weight compared to vehicle treated arthritic rats (Table No. 2 & 3).

Similarly, various haematological parameters like

Erythrocyte sedimentation rate (ESR), Total leukocyte count (TLC), Haemoglobin and Packed cell volume (PCV) were also decreased (Table No.4). The reduction in ESR count by extracts/pure compounds at their respective doses was found to be statistically significant. However, the migration of leucocytes into the inflammed area was non-significantly suppressed by the standard drug, pure compounds and test extracts except for methanol extract which showed significant reduction in TLC level. There was significant decrease in haemoglobin level in control rats as compared to normal rats. Rats treated with active extracts and pure compounds showed significant (p <

0.05) increase in haemoglobin level when compared to control. A significant (p < 0.05) increase in packed cell volume was observed in case of rats treated with methanol extract and pure compounds prophylactically.

However, there was a non-significant increase in packed cell volume in butanol extract and standard - indomethacin treated rats.

Protective effects of test samples on cartilage and bone destruction

At the end of the experiment, all animals were subjected to radiological and histopathological examinations.

Localised bone loss in the form of bone erosion and periarticular osteopenia constitutes important signs for the diagnosis of rheumatois arthritis. This is accompanied by soft tissue inflammation and vascularization of the synovium. Representative radiographs of the hind paws from control, negative control and different test samples are shown in Fig No.2. In the present study, radiological analysis revealed that different findings clearly indicate the anti-arthritic activity of extracts and pure compounds, which acts by modulating bone erosion.

The radiological observations were further supported by histopathological examination. Representative histological sections of the ankle joints from different samples are shown in Fig No.3. Histopathological examination of the synovial joints of the vehicle-treated

AIA rats revealed marked cellular infiltration, synovial hyperplasia, and joint space narrowing.

Severe pannus formation, bone, and cartilage destruction were also observed in the synovial joints of vehicletreated rats as compared to the synovial joints of normal rats.

Different sample treated rats manifested these pathological events in the ankle joints to a markedly lesser degree.







Effect of test samples on levels of TNFα and IL-1 β

Levels of different cytokines viz ., TNFα and IL-1 β were determined in the blood serum in different groups of rats.

Consistent with the joint swelling and ESR, the serum levels of TNFα and IL-1 β in the diseased control group

(vehicle treated) were significantly high while prophylactic treatment with active extracts and pure compounds, significantly (p < 0.05) reduced the elevated levels of cytokines (Table No.5 & 6). This reduction in the serum cytokines level by the active extracts and shanzhiside iridoids clearly indicates their protective role.

DISCUSSION

RA is a chronic inflammatory and systemic autoimmune disease characterized by a number of inflammatory and destructive events such as joint pain and swelling, synovial hyperplasia, pannus formation, cartilage and bone destruction, and joint malformation. Many cell populations, various cytokines, and different inflammatory mediators are involved in the generation of the pathological events characteristic of RA

3

. Several herbal plants are available for the treatment of RA and some are under investigation to find out the key moieties responsible for the action. B. prionitis is such plant which was taken up for the evaluation of anti-arthritic potential.

Arthritis is associated with bone loss or loss in bone density. As a result the weight is reduced. In the present findings, the active extracts and shanzhiside iridoids are effective in reducing bone erosion which is shown by the significant effects on increase in rat body weights.

The paw oedema associated with arthritis can be related to PGE

2

which enhances the effects of chemotactic factor mediators, such as leukotriene B and C5a.

One of the potentiating effects of PGs include oedema formation. Interference by test samples (extracts and pure compounds) with the synthesis of PGs, which are reported to stimulate bone resorption and collegenase secretion from macrophages and to diminish proteoglycan synthesis in cartilage, can be claimed to be effective in preventing cartilage and bone pathology in adjuvant induced arthritis in rats.

The arthritic score was found to be reduced in various drug treated groups as there was minimal redness, swelling and blending of claws thereby indicating a significant control on chronicity of inflammation.

The ESR count, which drastically increased in arthritic control group, was remarkably counteracted by the standard drug, extracts and pure compounds of Barleria back to normal, thus justifying its major role in protecting arthritic conditions. Similarly other haematological parameters were also normalized indicating their defensive role in arthritis.

Bone erosion, peri-articular osteopenia accompanied by soft tissue inflammation and vascularization of the synovium was also found to be reduced which clearly indicates the anti-arthritic activity of extracts and pure compounds, which acts by modulating bone erosion.

Table 1: Effect of active extracts and pure compounds on rat paw oedema volume in CFA induced arthritis.

Average paw oedema volume ± SD

Group Day 1

CFA a

11.19 ±

1.16

LP

IND

0.79 ±

0.34*

11.25 ±

2.01

ME

BE

10.17 ±

1.01

10.31 ±

0.67

B

AB

10.22 ±

1.73

10.58 ±

2.31

SME 11.23 ±

2.36

Day 3

13.07 ±

1.04

2.73 ±

2.62*

10.22 ±

1.49

11.24 ±

0.94

11.16 ±

0.92

10.82 ±

2.03

11.31 ±

1.20

11.01 ±

2.54

Day 5

13.2 ±

0.67

3.04 ±

0.94*

10.81 ±

1.78

11.10 ±

2.02

11.23 ±

1.73

11.07 ±

1.15

11.49 ±

1.51

11.56 ±

1.17

Day 7

14.03 ±

0.70

2.25 ±

2.07*

9.58 ±

2.83*

10.54 ±

1.07*

10.77 ±

0.77*

10.11 ±

0.85*

12.75 ±

1.98

12.84 ±

1.68

Day 9

12.86 ±

1.28

1.57 ±

1*

8.37 ±

3.31*

9.81 ±

1.67

9.62 ±

1.59

9.23 ±

0.68*

9.91 ±

2.93

11.54 ±

1.85

Day 11 Day 13 Day 15 Day 17 Day 19 Day 21

12.14 ±

1.64

1.17 ±

0.99*

7.36 ±

1.02*

8.99 ±

1.35*

9.27 ±

1.55

9.02 ±

1.23*

9.07 ±

2.80*

10.35 ±

1.97

12

1.71

1.29 ±

1.19*

5.48 ±

1.77*

7.5 ±

1.07*

8.19 ±

1.12*

8.41 ±

1.26*

9.25 ±

2.47*

9.55 ±

1.32

± 10.67 ±

2.01

1.74

0.85*

4.65

1.30*

7.07

1.29*

7.38

1.47*

7.65

1.67*

9.07

±0.57

8.01

1.90*

±

±

±

±

±

±

9.58

3.59

1.48

1.13*

4.54

1.59*

6.63

0.32

7.28

1.63

7.31

1.82

8.45

1.43

7.48

1.92

±

±

±

±

±

±

±

±

8.91

2.80

1.36

0.86*

4.32

1.41*

6.62

1.39

6.51

0.86

7.00

1.41

7.34

1.22

7.18

2.30

±

±

±

±

±

±

±

±

8.52 ±

2.50

1.27 ±

0.45*

3.76 ±

1.58*

5.64 ±

0.91

5.93 ±

1.25

6.04 ±

1.39

6.56 ±

1.55

7.05 ±

2.72

CFA = Complete freud’s adjuvant, LP = Liquid paraffin, INDO = Indomethacin, ME = Methanol extract, BE = Butanol extract, B=Barlerin,

AB=Acetylbarlerin and SME = Shanzhiside Methyl Ester. All the values are statistically significant at p<0.05 at all dose levels.

Significance vs. control (n=5), * p < 0.05; a

= 2 % v/v tween solution in normal saline







Table 2: Effect of active extracts and pure compounds on rat arthritic score in CFA induced arthritis.

Average arthritic score ± SD

Group

CFA a

LP

IND

Day 1

3.8

0.45

0.6

0.55*

3.4

0.89

±

±

±

Day 3

3.8

0.45

±

0.2 ±

0.45*

Day 5

4.0 ± 0 4.0 ± 0 4.0 ± 0 3.8 ±

0.45

0.2

0.45*

±

4.0 ± 0 3.2 ±

0.45*

Day 7

0 ± 0

3.0 ± 0*

Day 9

0 ± 0

2.6

0.55*

±

Day 11

0 ± 0

2.2

0.45*

±

Day 13

3.8

0.45

0 ± 0

2.2

0.45*

±

±

Day 15

4.4

1.52

0 ± 0

1.8

0.45*

±

±

Day 17

4.6

2.19

0 ± 0

1.2

0.45

±

±

Day 19

5.4

2.19

0 ± 0

1.2

0.45*

±

±

Day 21

5.2

2.05

0 ± 0

0.4

0.55*

±

±

ME

BE

3.8 ±

0.45

3.8 ±

0.45

4.0 ± 0 3.8 ±

0.45

3.8 ±

0.45

3.8 ±

0.45

3.8 ±

0.45

3.4 ±

0.55

3.6 ±

0.55

3.4 ±

0.55

3.2

0.45

3.2

0.55

±

±

2.6

0.55*

3.2

0.55

±

±

2.6 ±

0.55*

2.6 ±

0.55*

2.4 ±

0.89

2.0 ±

0.71*

2.6

0.55

± 2.2

0.45*

±

1.6 ±

0.55*

1.8 ±

0.84*

B

AB

SME

3.2 ±

0.45

3.6 ±

0.55

3.6 ±

0.55

4.0 ± 0 3.8 ±

0.45

4.0 ± 0 3.8 ±

0.45

3.8 ±

0.45

3.8 ±

0.45

3.6 ±

0.55

3.4 ±

0.55

3.6 ±

0.55

3.4 ±

0.55

3.4 ±

0.55

3.6 ±

0.55

3.0 ± 0 2.8 ±

0.45*

3.2 ±

0.45

3.0 ± 0

2.2

0.45*

2.8

0.45*

±

±

3.6 ±

0.55

3.2 ±

0.84

2.6 ±

0.55*

3.0 ±

2.24

2.8 ±

2.39

3.2 ±

2.17

3.0 ±

2.24

3.4 ±

2.07

3.0 ±

2.35

2.2 ±

2.17*

2.6 ±

2.51

2.6 ±

2.51





Table 3: Effect of active extracts and pure compounds on rat body weight in CFA induced arthritis.

Average body weight (g) ± SD

Group Day 1

CFA a

LP

IND

168 ± 8.4

153

9.6*

140

15.8*

±

±

Day 3

164 ± 9.6

153.75 ±

9.5

136 ±

18.2*

Day 5

160 ±

7.1

157.5 ±

8.7

129 ±

15.9*

ME 138

13.0*

BE

B

128

8.4*

146

11.4*

AB 136

15.2*

SME 166

11.4

±

±

±

±

±

138

15.3*

±

120 ±

10.0*

143 ± 9.8

133

10.9*

±

160 ±

14.1

133

17.8*

120 ±

7.9*

137 ±

9.8*

127 ±

14.8*

153

13.5

±

±

Day 7

152 ±

8.4

160 ±

10.8

127 ±

19.9*

129 ±

14.3*

116

8.2*

132

8.4

124

11.4*

±

±

±

150 ±

16.9

Day 9

152 ±

7.6

160 ±

7.1

135 ±

18.7

130

12.3

148

16.4

±

121 ±

14.3*

138 ±

10.9

132 ±

10.9

±

Day 11

149 ±

10.3

162.5 ±

23.6

144.4 ±

15.7

138 ±

10.4

124

13.4*

144

5.5

142

10.9

156

16.4

±

±

±

±

Day 13

148

16.4

165

20.8

151

19.5

143

8.4

162

8.4

±

±

±

±

134 ±

13.4

148 ±

9.1

144 ±

15.2

±

Day 15

144

16.7

165

17.3

157

13.9

±

±

±

Day 17

140

15.8

11.4*

±

171.25 ±

13.2*

164 ±

154 ±

5.5

155 ± 5.0

143 ±

14.8

151 ±

13.4

155 ±

7.1

159 ± 5.5

151 ±

11.4

153 ±

16.4

168 ±

10.9*

177 ±

8.4*

Day 19 Day 21

139 ±

14.3

170 ±

21.6*

171 ±

13.4*

140

22.4

10.8*

±

171.25 ±

14.4*

174 ±

160 ±

7.9

162 ±

14.8

152

19.2

164

5.5

162

16.4

181

12.5*

±

±

±

±

154

16.7

175

10.0*

166

13.9

182

14.8*

±

±

±

±











Figure 1: Antiarthritic effect of active extracts of two Barleria species at 200 mg/kg and pure compounds at 1 mg/kg on morphological appearence of AIA rat paws.

Shown are representative pictures of the hind paws of rats from - Diseased control (DC) - CFA; Negative control (NC) - Liquid paraffin; Standard (Indo) - Indomethacin (3 mg/kg); ME - Methanol extract; BE - Butanol extract; AB -Acetyl barlerin; B - Barlerin; SME - Shanzhiside Methyl Ester; Pictures showing secondary effects - (A.1) Hind right paw swelling, (A.2) Left fore paw swelling, (A.3) Bending of claws, (A.4) Hair fall adjacent to left hind paw

Figure 2: Effect of active extracts and pure compounds on radiological changes of AIA rats. Shown are representative radiographs of the lateral view of the hind paws treated with (A) Diseased control - CFA; (B) Negative control - Liquid paraffin; (C) Standard (Indo) -Indomethacin (3mg/kg); (D) ME - Methanol extract; (E) BE - Butanol extract; (F) Acetyl barlerin; (G) Barlerin; (H) Shanzhiside Methyl Ester.







Figure 3: Effect of active extracts and pure compounds on histopathological changes of AIA rats. Shown are representative haematoxylin & eosin stained histopathological sections of tibiotarsal joints of rats from group

(A) Negative control (NC) - Liquid paraffin 40x; (B) Diseased control (DC) - CFA 40x; (C) Standard (Indo) - Indomethacin

200x; (D) ME - Methanol extract, D.1 - 40x; D.2 - 100x; (E) BE - Butanol extract, E.1 - 40x, E.2 - 100x; (F) B1 - 40x; (G) B2 -

40x; (H) B3 - 40x

Table 4: Effect of active extracts and pure compounds on the haematological parameters in arthritic rats

Changes in the haematological parameters (Mean ± SD)

Treatment

ESR

(mm/1st h)

Hb (g/dl) PCV (%) TLC (Cells /Cu mm)

Complete Freud’s

Adjuvant (CFA)a

Liquid parrafin

Indomethacin

Methanol extract

Butanol extract

B

AB

SME

3.8 ±1.48 10.42 ± 1.46 32.5 ± 6.53 11540 ± 2787

1.75 ± 0.50*

1.25 ± 0.5*

1.2 ± 0.45*

1.4 ± 0.55*

1.0 ± 0*

1.4 ± 0.55*

1.6 ± 0.55*

14.77 ± 0.84*

12.82 ± 1.36*

13.56 ± 1.05*

13.8 ± 1.34*

13.77 ± 0.73*

13.66 ± 1.14*

13.58 ± .08*

40.6 ± 1.88*

36.67 ± 2.78

39.22 ± 3.96*

38.76 ± 3.83

41.08 ± 1.05*

40.9 ± 3.22*

39.4 ± 3.85*

9150 ± 3011

9300 ± 3238

6920 ± 2878*

7400 ± 1574

8579.6 ± 1383

9014 ± 1439

12100 ± 3262


= 2 % v/v tween solution in normal saline.

RBC Count

Millions/ Cu mm

7.144 ± 0.47

7.37 ± 0.38

6.67 ± 0.56

6.94 ± 0.74

7.08 ± 0.69

7.90 ± 0.24

7.59 ± 0.49

7.29 ± 0.51

Table 5: Effect of active extracts and pure compounds on TNFα levels in arthritic rats

Group

Complete Freud’s

Adjuvant (CFA) a

Liquid parrafin (LP)

Indomethacin


Butanol extract

B

AB

SME

Dose (mg/kg)

0.1 ml/100g

0.1 ml/100g

3

200

200

200

200

200

Absorbance

0.163375 ± 0.010

0.057875 ± 0.006*

0.094375 ± 0.010*

0.1035 ± 0.007*

0.113125 ± 0.030*

0.109375 ± 0.050*

0.115 ± 0.006*

0.11975 ± 0.030*

Concentration (pg/ml)

137.59

5.72

51.34

62.75

74.78

70.09

77.12

83.06









Table 6: Effect of active extracts and pure compounds on IL-1β levels in arthritic rats

Group Dose (mg/kg) Absorbance

Concentration

(pg/ml)

Complete Freud’s Adjuvant

(CFA) a

Liquid parrafin (LP)

Indomethacin


Butanol extract

B

AB

SME

0.1 ml/100g

0.1 ml/100g

3

200

200

200

200

200

0.8375 ± 0.150

0.3205 ± 0.020*

0.469125 ± 0.030*

0.541688 ± 0.100*

0.56325 ± 0.030*

0.580875 ± 0.030*

0.572875 ± 0.030*

0.55225 ± 0.020*

1313.39

390.18

655.58

785.16

823.66

804.02

840.85

855.13



It has been speculated that rheumatoid arthritis (RA) could be triggered by a T-cell response to infectious agents. Through cell-cell contact and different cytokines, these stimulated T cells activate monocytes, macrophages, and synovial fibroblasts. The latter then overproduce pro-inflammatory cytokines, mainly TNFα ,

IL-1 β , and IL-6

3

. These soluble molecules, once engaged to their receptors, trigger various signal transduction cascades that lead to the activation of transcription factors and the subsequent induction of genes whose products such as matrix metalloproteinases (MMPs) mediate tissue degradation

3,23

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1876-1888. From the results observed in the current investigation, it may be concluded that Barleria and isolated shanzhiside esters from the same plant can be strongly categorized under potential anti-arthritic drugs since both were active in adjuvant induced arthritic model.

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Source of Support: Nil, Conflict of Interest: None.






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