viva presentation

M.pharm Viva Voce

by

SYEDA TANVEER

(109F1S0115)

under the supervision of

Dr.Vasudha Bakshi, M.pharm, Ph.D,

Associate professor,

School of pharmacy, Anurag group of institutions,

Ghatkesar, Hyderabad.

Anti-Inflammatory activity of Operculina turpethum(L.) in chronic inflammation

Inflammation is a spontaneous biological response to a variety of noxious stimuli

of varied nature including local injury.

The gross pathology of inflammation includes leakage of serum proteins and

phagocytic cells, complex interaction of various chemical mediators.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by

swelling of multiple joints due to accumulation of inflammatory cells.

INTRODUCTION

Fig no 1 : INFLAMMATORY PROCESS MECHANISM OF INFLAMMATION

Fig no:2 DIFFERENCES BETWEEN NORMAL AND ARTHRITIC JOINTS

About 1.3 million of world’s population has arthritis.

Most frequently occurs b/w ages of 40 and 50.

There are nearly 3 times as many females as males effected

with the disease.

As many as 300,000 children are diagnosed with distinct but

related form of inflammatory arthritis called juvenile arthritis.

The disease occurs in all ethnic groups and in every part of

the world.

Statistics of RA

NSAIDS

CORTICOSTEROIDS

DMARDs

BIOLOGICAL RESPONSE MODIFIERS

GOLD SALTS

A DEVICE CALLED PROSORBA COLUMN.

Current therapeutic approaches for RA

This study was designed to investigate the Anti- inflammatory

effect of OTE on CFA induced arthritis in rats.

To study the effect of OTE on RA by determining behavioural

and biochemical parameters.

AIMS & OBJECTIVES

CFA consist of 1 mg/ml of heat killed Mycobacterium

tuberculosis in paraffin oil and mannide mono-oleate.

CFA mainly prolong [action of] the presence of antigens at the

site of injection,

Transport of the antigens to the lymphatic system & lungs,

where it promotes the accumulation of cells concerned with the

immune response.

Complete fruend’s adjuvant (CFA)

fig no 3: Mechanism of CFA

After Injecting CFA at sub-plantar region

mycobacterial components targets MPC & DC’s

induction of cytokines-TNFα, IL- 12, 6, INF-γ etc…

IL-12 induces NK cells TNF α induces other cytokines

produces INF –γ IL-6 & chemokines

excess production of t-cells

TH-1 profile Swelling of paw

Operculina turpethum (L.) Opercuina turpethum is a

perennial herb belonging to family convolvulaceace.

Fig no :3 flower & dried roots

Anti-secretory and ulcer protective property (Rajashekar M, et

al., 2006).

Anti-inflammatory activity using Formalin (Rajashekar M, et al.,

2006)

Hepatoprotective activity (Suresh kumar ,et al., 2006)

Anti-microbial activity (Md. Harun-ur-rashid et al.,2002)

Anti cancer and antioxidant acitivites (c. Anbuselvam et al;

2007)

Cytotoxic activity (Alluri V. krishnarajau et al 2005)

Reported pharmacological studies on OTE

Operculina turpethum

Extraction with ethanol

Induction of Arthritis

low dose (100mg/kg)

Treatment with OTE

high dose(200mg/kg)

Measurement of paw edema

Pharmacological activity

Behavioural Biochemical

parameters

Materials and methods

Male Wistar rats weighing 250-320g were used and were

divided into four groups of six each.

(IAEC) I/IAEC/LCP/012/2012/WR/30♂.

Group-I: Animals treated with normal saline as control.

Group-II: CFA: Animals were injected with CFA

subcutaneously on day 1 of the experiment..

Group-III: CFA + OTE dose-1: Oral administration of OTE

low dose (100mg/kg) for 14 days after injecting CFA on day 1.

Group-IV: CFA + OTE dose-2: Oral administration of OTE

high dose (200mg/kg) for 14 days after injecting CFA on day 1.

Grouping, Induction of arthritis & Treatment

Behavioural parameters :

Measurement of paw edema: Paw edema of rats of different

groups were measured using digital plethysmometer.

Rota–rod test: The duration of time that each rat stays on

this rotating rod is noted.

Eddy’s hot plate: Pain threshold was determined by the

latency for nociceptive response (withdrawl of any paw) using

eddy’s hot plate.

In-vivo pharmacological evaluation

Time(days) control CFA CFA+OTE100 CFA+OTE200

PAW VOLUME OF INDUCED LEFT PAW(ml) 

DAY 0 1.35 ± 0.60 1.35 ± 0.17 1.18 ± 0.94 1.21 ± 0.26 

1 1.17 ± 0.06 2.36 ± 0.22*** 2.58 ± 0.96 2.37 ± 0.35 

4 1.23 ± 0.10 2.37 ± 0.93*** 2.29 ± 0.76 2.04 ± 0.14

7 1.35 ± 0.09 2.30 ± 0.19** 1.91 ± 0.26 1.78± 0.03 

10 1.18 ± 0.01 2.35±0.17** 1.84 ±0.95# 1.65± 0.23$$ 

13 1.35 ± 0.15 2.24± 0.56** 1.79 ±0.43# 1.55 ± 0.92$$

16 1.25 ± 0.08 2.23± 0.48*** 1.72 ±0.86# 1.49 ± 0.36$$

19 1.20 ±0.73 2.28 ±0.83*** 1.65 ±0.72## 1.41 ±0.45$$$

21 1.28 ±0.15 2.12 ±0.57*** 1.55±0.43## 1.32 ±0.18$ 

 Values are expressed as mean ± SEM of 6 animals. Superscript represents the statistical

significance done by ANOVA, followed by Tukey’s multiple comparison tests. Data are expressed

as Mean ± S.E. n=6, ***p<0.001 Control v/s CFA ###p<0.001 CFA vs CFA+ OTE100, #p<0.05 CFA

vs CFA+ OTE100 ,$$$p<0.001 CFA vs CFA+ OTE200, $$p<0.01 CFA vs CFA+ OTE200

Table 1: Inhibition of edema of induced paw by OTE

Fig no 4 : Diagrammatic representation of inhibition of paw by OTE

Morphological representations of rat paw after subplantar administration of Freund’s complete adjuvant indicated production of inflammation A) Normal paw, B) Paw injected with CFA, C) paw treated with OTE 200mg/kg, D) paw treated with OTE 100mg/kg.

Groups Rota-rod (sec) Eddy’s hot plate (sec)

Control

CFA

CFA+OTE100

CFA+OTE200

10.40±0.78

4.11±0.47***

6.11±0.27#

8.46±0.30$$$

9.05±0.26

2.5±0.23***

4.87±0.14###

6.95±0.08$$$

Table 2: Effect of OTE on pain threshold and motor in-coordination activity

Values are expressed as mean ± SEM of 6 animals. Superscript represents the

statistical significance done by ANOVA, followed by Tukey’s multiple comparison tests.

Data are expressed as Mean ± S.E. n=6, ***p<0.001 Control v/s CFA ###p<0.001 CFA

vs CFA+ OTE100, #p<0.05 CFA vs CFA+ OTE100 ,$$$p<0.001 CFA vs CFA+ OTE200, $

$p<0.01 CFA vs CFA+ OTE200.

Fig no 5, 6, 7: Effect of OTE on paw volume, eddy’s hot plate and rota-rod

Estimation of AST/SGOT: AST/SGOT were estimated in plasma using biochemical kits using semi auto analyzer.

Estimation of ALT/SGPT: ALT/SGPT were estimated in plasma using biochemical kits using semi auto analyzer.

Biochemical estimations

GROUPS AST/SGOT ALT/SGPT (U/L) (U/L)

CONTROL 56±1.52 67.6±1.45 CFA 71.3±1.45*** 128.3±2.9*** CFA+OTE 100 64.6±0.89# 87±2.08###

CFA+OTE 200 61±1.58$$ 75±3.05$$$

Table 2: Effect of OTE on SGOT and SGPT in arthritic rats

Values are expressed as mean ± SEM of 6 animals. Superscript represents the statistical significance done by ANOVA, followed by Tukey’s multiple comparison tests. Data are expressed as Mean ± S.E. n=6, ***p<0.001 Control v/s CFA, ###p<0.001 CFA vs CFA+ OTE100, #p<0.05 CFA vs CFA+ OTE100, $$$p<0.001 CFA vs CFA+ OTE200, $$p<0.01 CFA vs CFA+ OTE200

Fig 8, 9: Effect of OTE on SGOT and SGPT in arthritic rats

Assay of super oxide dismutase: Superoxide dismutase

(SOD) activity was assayed by pyrogallol oxidation.

Assay of Catalase: CAT activity was determined with

ammonium molybdate solution and absorbance change was

measured at 405 nm.

Lipid peroxidation: it was estimated in terms of

thiobarbituric acid reactive species (TBARS), using

malondialdehyde (MDA) as a standard by method of Buege

and Aust.

Estimation of anti-oxidant enzymes

GROUPS

SOD

CATALASE

MDA

CONTROL CFA CFA+OTE 100 CFA+OTE 200

9.73±0.23

4.13±0.06***

7.2±0.17###

5.36±0.29$$$

9.09±0.55

7.37±0.18***

8.47 ±0.26#

9.05±0.16$$$

2.90±0.67

6.03±0.38***

4.39±0.49##

3.37±0.25$$$

Table 3: Effect of OTE on anti-oxidants enzyme in arthritic rats

Values are expressed as mean ± SEM of 6 animals. Superscript represents the statistical significance done by ANOVA, followed by Tukey’s multiple comparison tests. Data are expressed as Mean ± S.E. n=6, ***p<0.001 Control v/s CFA, ###p<0.001 CFA vs CFA+ OTE100, ##p<0.01 CFA vs CFA+ OTE100, #p<0.05 CFA vs CFA+ OTE100, $$$p<0.001 CFA vs CFA+ OTE200.

Fig 10,11,12: Effect OTE on SOD, CAT and MDA in arthritic rats

Treatment with OTE significantly decreased the thickness of paw indicating its anti-inflammatory potential in CFA induced arthritis.

Oral administration of OTE has shown improvement in nociceptive threshold latency of arthritic rats on eddy’s hot plate.

Oral administration of OTE has improved the motor coordination which is evident by improved performance on rota rod.

Discussion

The presence of flavanoids in OTE is responsible for its anti-inflammatory and analgesic activty.

Accumulation of MDA on plasma membrane results in inflammatory response. Treatment with OTE has significantly reduced the elevated level of MDA.

OTE increased the enzyme activities of SOD and CAT, suggested that OTE prevented the oxidative damage due to ROS overproduction from rheumatoid arthritis response.

Cont’d

OTE contains substantial amount of phenolics and flavonoids responsible for its marked antioxidant activity.

Serum ALT/SGPT and AST/SGOT have been reported with vital role in the formation chemical mediators such as bradykinins in inflammatory process.

Treatment with OTE has significantly reduced the elevated levels of ALT/SGPT and AST/SGOT.

Cont’d

The present study confirms anti-inflammatory activity of OTE.

The present study has shown the effect of OTE on improvement of behavioural parameters.

Further more results reveal that the action might be achieved by inhibition of synovial inflammation and through regulating the expressions of pro-inflammatory cytokines TNF-α, IL-2.

These findings justify the traditional use of the plant in the treatment of chronic inflammatory and arthritic conditions.

Conclusion

Anderson, G.D., Hauser, S.D., McGarity, K.L., Bremer, M.E., Isakson, P.C., Gregory, S.A. (1996). Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J Clin Invest. 97, 2672–9.

  Aebi, H. (1974). Catalase: Methods of Enzymatic Analysis. Academic Press, New

York. 2, 673-678.

Buege, J.A., Aust, S.D. (1978). Microsomal lipid peroxidation methods. Enzymol. 52, 302 - 310.

  Calkins, E., Black, R.L. (1960).The evaluation of treatment in rheumatoid arthritis.

Arthritis rheum. 3,101.

Calvino, B., Bernard, M.O., Bars, D.L. (1987). Parallel clinical and behavioural studies of adjuvant induced arthritis in the rats: possible relationship with chronic pain. Behav Brain Res. 24, 11–29.

References

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Md. Harun-or-Rashid, M.A. Gafur, Md. Golam Sadik, Md. & Aziz Abdur Rahman, Antibacterial and Cytotoxic Activities of Extracts and Isolated Compounds of Ipomoea turpethum. Pakistan Journal of Biological Sciences, 5(5): 597-599, (2002).

Rajashekar M. Bhande, Laakshmayya, Pramod Kumar, Nitin K. Mahurkar, & S.Ramachandra Setty, Pharmacological Screening of Root of Operculina turpethum and its Formulations. Acta Pharmaceutical Sciencia, 48: 11-17, (2006).

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Cont’d

C. Anbuselvam, K. Vijayavel, & M. P. Balsubramaniyan, Protective effect of Operculina turpethum against 7,12 dimethylbenz(a)anthracene induced oxidative stress with reference to breast cancer in experimental rats. Chemico Biological Interactions, 168: 229-236, (2007).

Alluri V. Krishnarajua, Tayi V. N. Raoa, Dodda Sundararajua, Mulabagal Vanisreeb, Hsin-Sheng Tsayb, & Gottumukkala V. Subbarajua,Assessment of Bioactivity of Indian Medicinal Plants Using Brine Shrimp (Artemia salina) Lethality Assay. International Journal of Applied Science and Engineering, 3(2): 125- 34, (2005).

Cont’d

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