Evaluation of Anti-arthritic Activity of Ethanolic Extract of Pisonia grandis R.Br

 

Elumalai A.*, Prakash Yoganandam G.

Department of Pharmacognosy, SRM College of Pharmacy, Kattankulathur, Kancheepuram Dt, Tamil Nadu, India, 603 319.

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

 

ABSTRACT:

The present study was carried out to evaluate the anti-arthritic activity of ethanolic of extract Pisonia grandis R.Br leaf (EPGL) in adjuvant induced arthritis in rats. Loss in body weight during arthritis condition was corrected on treatment with EPGL and standard drug, indomethacin. Biochemical parameters such as hemoglobin content, total WBC, RBC, erythrocyte sedimentation rate were also estimated. The ethanolic extract at the dose of 300 mg/kg body weight inhibited the rat paw edema by 68.32% which is comparable with standard drug indomethacin 75.87% inhibition of rat paw edema after 21 days. The results of the current investigation concluded that EPGL possess a significant anti-arthritic activity against freund's adjuvant induced arthritis and justifying its therapeutic role in arthritic condition. The observed antiarthritic activity may be due to the presence of phytoconstituents such as alkaloids, phenolic compounds and flavonoids.

 

KEYWORDS: Arthritis, Freund’s adjuvant, Indomethacin, EPGL-Ethanolic Extract of Pisonia grandis leaf.

 

INTRODUCTION:

Pisonia grandis R.Br is an important plant belonging to the family Nyctaginaceae and is one of the most widespread large shrubs in the forests of India, usually occurring in deciduous forests. The whole plant possesses medicinal properties, useful in the treatment of skin diseases, inflammatory diseases, arthritis, gonorrhea, rheumatism, anorexia and jaundice. Leaves also consumed as vegetable and salad, also fed to cattle1-4. The phytochemical study reveals that the presence of steroids likes octocosanol, betositosterol, alphaspinosterol, dulcitol and flavonoids in the leaves of the plant1. It is an important Ayurvedic medicinal herb and its synonym is Pisonia morindaefolia R.Br. It is popularly known as “Leechai kottai keerai” in Tamil. According to traditional use, the different parts of the plants of Pisonia grandis are used as diuretics and purgative. The anti inflammatory, antifungal, wound healing and diuretic activities of Pisonia grandis were reported earlier5-9.

 

Most of the plant parts of Pisonia grandis R.Br have been used in the traditional system of medicine in India to treat various infectious diseases. Adjuvant induced arthritis is a chronic crippling, skeleton-muscular disorder having nearest approximation to human rheumatoid arthritis for which there is presently no medicine available effecting a permanent cure. The modern drugs both steroidal and non-steroidal anti-inflammatory drugs are used for the amelioration of the symptoms of the disease, however they offer only temporary relief and also produce severe side effects. In the Ayurvedic system of medicine, this plant has been used in the treatment of rheumatoid arthritis, Literature survey revealed that there is no systematic study regarding the anti-arthritic activity of the Pisonia grandis R.Br, leaf. Hence in the present study, an attempt has been made to evaluate the anti-arthritic activity of the EPGL using adjuvant induced arthritis in rats.

 

MATERIALS AND METHODS:

Plant material

Fresh leaf of Pisonia grandis R.Br was collected in and around the place of SRM College of Pharmacy, Chennai, Tamil Nadu, India. The plant was identified and authenticated by Botanist, Dr. P. Jayaraman, Plant Anatomical Research Centre (PARC), Tambaram, Chennai and the voucher specimen (PARC/2010/654) have been kept in the Department of Pharmacognosy, SRM College of Pharmacy, Chennai, for future reference. Care was taken to select the healthy plants.

 

Extraction

The freshly collected leaves was chopped, shade dried and coarsely powdered (40 mesh size). The powder was defatted with petroleum ether (60- 80°C) and then extracted with 90% ethanol in a soxhlet extractor. The extract was dried under reduced pressure using a rotary vacuum evaporator and the percentage yield was 20.90% w/w. Standard methods (Trease and Evans, 1989; Harborne, 1994) were used for preliminary phytochemical screening of the ethanolic extract to know the nature of phytoconstituents present in it. The obtained ethanol extract was suspended in 5% gum acacia for the pharmacological screening.

 

Animals
For acute toxicity and anti-arthritic activities, Wistar albino rats weighing between 150-200 gm were selected. The animals were acclimatized to the standard laboratory conditions (temperature 25 ± 2°C) and maintained on 12 hr light, 12 hr dark cycle. The animals were fed with standard diet and water ad libitum. The animals were maintained as per the norms of CPCSEA and the experiments were cleared by CPSEA and the institutional ethics committee. The voucher number is IAEC/135/2010.

 

Acute toxicity studies

Acute toxicity study of Ethanolic extract of Pisonia grandis was carried out in rats according to OECD (423) guidelines. Different doses of EPG were administered up to 5000mg/Kg (p.o) and the animals observed for a period of 72 hr for behavioral changes, toxic reactions and mortality10.

 

Anti-arthritic activity

Freund's adjuvant induced arthritis model (Newbould 1963) was used to assess the anti-arthritic activity in albino rats. Animals were divided into three groups of six animals each. Group I served as control, which received 5% gum acacia suspension, Group II served as reference standard, which received 10 mg/kg body weight IP of indomethacin, and Group III served as test, which received the EPGL at the dose of 300mg/kg body weight PO, respectively. Arthritis was induced by injecting 0.05 ml of suspension of killed Mycobacterium tuberculosis bacteria (0.5% w/w) homogenized in liquid paraffin into the left hind paw. Drug treatment was started from the initial day i.e. from the day of adjuvant injection (0 day) and continued till 21st day.

Paw volume was measured on 4th, 8th, 14th and 21st day with the help of Plethysmometer. The mean changes in injected paw edema with respect to initial paw volume, were calculated on respective days and percentage inhibition of paw edema with respect to untreated group (control) was calculated using the formula.

                                                               

 

 

                                          Mean change in paw volume of treated rats

Percentage inhibition of paw edema = ­------------------------------ x 100

                                        Mean change in paw volume of untreated rats

 

Statistical analysis

Results were expressed as mean ± SD. The significance of difference among the groups was assessed using One way analysis of variance (ANOVA) followed by Dunnet's test. P<0.05, P<0.01 was considered significant.

 

RESULTS:

The results of the preliminary phytochemical screening of the EPGL revealed the presence of phytoconstituents such as alkaloids, steroids, flavonoids, phenolic compounds, tannins and glycosides. In acute toxicity studies, the EPGL did not produce any toxic symptoms or mortality up to the dose level of 2000 mg/kg body weight in rats, and hence the extract was considered to be safe and non-toxic for further pharmacological screening. In adjuvant induced arthritis model, rats developed a chronic swelling in multiple joints with the influence of inflammatory cells, erosion of joint cartilage and bone destruction and remodeling. These inflammatory changes ultimately result in the complete destruction of joint integrity and functions in the affected animal. The ethanol extract inhibited the rat paw edema by 68.32%, which is comparable with standard drug, indomethacin 75.87% inhibition of rat paw edema after 21 days (Tables 1 and 2). As shown in table 3 standard drug, indomethacin and the ethanol extract have shown to increase the hemoglobin content when compared to control group. The total WBC counts were remarkably increased in adjuvant-induced rats (Table3: control group). However, EPGL and the standard drug treated groups significantly decreased (p<0.01) the total WBC count. The ESR count, which drastically increased in arthritic control group, has been remarkably counteracted by the standard drug; indomethacin and ethanol extract, restoring it back to normal, thus justifying its significant roles in the severe arthritic conditions. The loss of body weight observed during the arthritis condition, which is in the arthritic control group, the standard drug and the ethanol extract treatment has significantly increased the body weight further confirming the significant anti-arthritic activity of the EPGL and it is shown in the table 4.

 

Table 1: Mean changes in paw volume using Plethysmometer in adjuvant induced arthritis in rats

Treatment

4th day

8th day

14th day

21st day

Group I (Arthritic control, 5% Gum acacia)

5.76±0.26

5.84±0.20

5.76±0.21

5.63±0.22

Group II (Standard drug- Indomethacin,10 mg/kg )

5.13±0.15*

4.45±0.25*

3.63±.29*

1.81±0.18*

Group III (EPGL300mg/kg)

5.19±0.22*

4.6±0.24*

3.69±0.29*

2.11±0.11*

n=6, values are expressed as mean ± SEM, p<0.05 - significant, *p<0.01 - more significant when compared to the control

 

Table 2: Percentage inhibition of paw volume in adjuvant- induced arthritis in rats.

Treatment

4th day

8th day

14th day

21st day

Group I (Arthritic control, 5% Gum acacia)

 0

 0

 0

 0

Group II (Standard drug- Indomethacin,10 mg/kg )

 12.41

 25.84

 41.59

75.87

Group III (EPGL300mg/kg)

11.91

23.30

38.74

68.32

 

Table 3: Effect on hematological parameters in adjuvant-induced arthritis in rats

Treatment

4th day

8th day

14th day

21st day

Group I (Arthritic control, 5% Gum acacia)

 7.95±0.08

 5.3±0.1

12.7 ±0.2

 4.15±0.1

Group II (Standard drug- Indomethacin,10 mg/kg )

7.25 ±0.1*

5.42 ±0.1*

14.28±0.1*

3.45 ±0.1*

Group III (EPGL300mg/kg)

 7.15±0.1*

5.68 ±0.1*

14.6 ±0.1*

3.31 ±0.1 *

n=6, values are expressed as mean ± SEM, p<0.05 - significant, *p<0.01 - more significant when compared to the control.  

 

Table 4: Changes in the body weight in adjuvant-induced arthritis in rat

Groups

Before induction (gm)

On 21st day (gm)

Mean changes in the body weight

Group I (Arthritic control, 5% Gum acacia)

160

168

8.56±1.56

Group II (Standard drug- Indomethacin,10 mg/kg )

157

196

38.6±2.5*

Group III (EPGL300mg/kg)

155

172

22.7±1.6*

n=6, values are expressed as mean ± SEM, p<0.05 - significant, *p<0.01 - more significant when compared to the control.

 

 

DISCUSSION:

In the present study, the rats were selected to induce arthritis because rats develop a chronic swelling in multiple joints with the influence of inflammatory cells, erosion of joint cartilage and bone destruction. It has close similarities to human rheumatoid disease11. The determination of rat paw swelling is apparently simple, sensitive and one of the quick procedures for evaluating the degree of inflammation and the therapeutic effects of drugs. The chronic inflammation involves the release of number of mediators like cytokines, GM-CSF, interferons and PGDF. These mediators are responsible for the pain, destruction of bone and cartilage that can leads to severe disability. However, the standard drug, indomethacin and the EPGL significantly suppressed the swelling of the rat paws. The study is further extended to identify and characterize the exact active phytoconstituents and to elucidate the exact mechanism of action, which is responsible for the observed significant anti-arthritic activity against adjuvant induced arthritis in rats.

 

ACKNOWLEDGEMENT:

The author thanks to Mrs. Chandramathi Arunachalam for providing the support during this project.

 

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Received on 31.05.2012       Accepted on 26.06.2012     

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Asian J. Pharm. Res. 2(3): July-Sept. 2012; Page 91-93