Effect of Delonix elata on Adjuvant Induced Arthritis in Rats - A Radiographic Densitometric Analysis

D Kilimozhi¹, V Parthasarathy^1* and N Amuthavalli²

¹Department of Pharmacy, Annamalai University, Annamalai Nagar-608002, Tamil Nadu, India

²Department of Radiology, Rajah Muthiah Medical College and Hospital, Annamalai University, Annamalai Nagar-608002, Tamil Nadu, India

ABSTRACT

In the present study, the anti-arthritic effect of oral administration of ethanolic extract of Delonix elata on Freund’s adjuvant induced arthritis has been studied in Wistar albino rats. The loss of body weight during the arthritic condition was corrected on treatment with ethanolic extract of Delonix elata at 250 and 500 mg.kg^‑1body weights. The swelling of the paws during the secondary lesions was also markedly reduced on treatment with ethanolic extract of Delonix elata and this results was confirmed using radiographic analysis and the changes in the density of Hind Limb Bone Mass (HLBM) was measured using photodensitometer and aluminium step wedge. The HLBM was significantly reduced on treatment with ethanolic extract (250 and 500 mg.kg^‑1body weight) of Delonix elata and standard drug Indomethacin (10 mg.kg^‑1). From the result we observed that the ethanolic extract of Delonix elata possess potent anti-arthritic activity.

KEY WORDS: Delonix elata, Anti-arthritic, Freund’s complete adjuvant, Photodensitometer, Aluminium step wedge.

INTRODUCTION

Arthritic affects 0.5-1% of the world population with more women being affected than men (Firestein GS et.al.2003). The immune system is a well-organized and well-regulated. The deregulation of the immune system may lead to the development of autoimmune diseases such as Rheumatoidarthritis (RA), is proto-type of such groups of illness with chronic, systemic disorders with destructive inflammatory polyarticular joint potentially resulting in progressive destruction of articular and periarticular structure (Berens DL.et al., 1965). Persistent inflammation produces swollen joints with severe synovitis, decreased nociceptive threshold (Koburova KL et al 1985, Martin W et al., 1980), and massive subsynovial infiltration of mononuclear cells, which is along with angiogenesis leads to pannus formation (Bywaters EGL et al., 1960). Expansion of the pannus induces bone erosion and cartilage thinning, leading to the loss of joint function (Feldmann et al., 1996; Koch et al., 1998). This result in a high degree of morbidity anddisturbed daily life of the patient. Corticosteroids have not been able to fully control the incidence because of its limitations and risk of side effects. Many patients and practitioners are seeking alternative approache to provide an effective cure in the treatment of arthritis and to overcome the serious draw backs such as gastro intestinal bleeding (Sarziputtiniet al., 2000) on treatment with Corticosteroids. Hence there is an urgent need to find safer drug for the management of rheumatoid arthritis.

Delonix elata (L.) Gamble (Caesalpiniaceae) is found in some part of south India and it is a fast growing tree in the footing of rivers and channel banks (Kalpan, et al., 1967; Watsan and Malone 1977; Ali et al., 1995). The height of the tree is 6-9m height, tolerably smooth and ash colored. In the Indian system of medicine the leaves of plant is used for rheumatism, anti-microbial (Ahmed et al., 1999) and flatulence (Shah, 1982, Lechn-Knecht, 1982). Until now no work has been carried out to assess anti-arthritic activity of Delonix elata.

Hence the present investigation was undertaken to study the anti-arthritic activity of the ethanolic extract of Delonix elata due to the fact that any botanical was traditionally used for wound healing, fever, infection, pain, edema or rheumatic disorders is taken as an indicator that the plant should be tested for its anti-arthritic properties (Tunon et al., 1995).

MATERIALS AND METHODS:

Plant Material:

Taxonomic identification of the plant was made from Rapinat Herbarium, St. Joseph’s college of arts and sciences, Trichy, Tamilnadu, India. Whole fresh plant leaves of Delonix elata was collected from Jeyankondam, Perambalur (Dist), Tamilnadu, India. The leaves were dried under shade, segregated, pulverized by a mechanical grinder and passed through 40 mesh sieves.

Preparation of extracts:

The powdered leaves (1000g) were successively extracted with ethanol (70-80ºC) for 24 hrs by continuous hot percolation method using soxhlet apparatus. The fraction was separated from the solvent by distillation under reduced pressure to yield a solid mass (9.6% w/w), stored in refrigerator and used for further studies.

Animals:

The animals for the present study procured after animal ethical clearance from the Institutional Animal Ethical Committee (IAEC) in Annamalai University, Annamalai nagar, Tamilnadu, India. The animal experiments were carried out according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) rules. Inbred wister rats (150-200g) were used for testing anti arthritic activity. The animals were housed at central animal house (Rajah Muthiah Medical College and Hospital, Annamalai University, Tamilnadu, India)

under standard conditions of temperature (23±1°C), relative humidity (55±1%) and 12hrs light and dark cycles. The animals were and fed with standard pellet diet and tap water ad libtum.

Drugs and chemicals:

All the drugs used in this study were of pharmaceutical grade. Freund’s adjuvant was supplied by Sigma Chemicals, Indomethacin is a gift sample from Cadila Pharmaceuticals, Ahamedabad, India.

Acute toxicity studies:

Acute toxicity studies were performed (Ecobichon; 1997) according to OECD-423 guidelines (acute toxic class method). Albino mice (n=3) of either sex selected by random sampling technique were employed in this study. The animals were fasted for 4hrs with free access to water only. The plant extract of Delonix elata was administered orally with an initial dose of 1000 mg.kg^‑1 body weight. The mortality was observed for three days. If mortality was observed in 2/3 or 3/3 of animals, then the dose administered was considered as a toxic dose. However, if the mortality was observed only one mouse out of three animals then the same dose was repeated again to confirm the toxic effect. If mortality was not observed, the procedure was then repeated with higher dose such as 50,300 and 2000 mg.kg^‑1.

Evaluating of paw volume and body weight changes in Freund’s induced arthritic animal:

Freund’s adjuvant induced arthritis model (BB Neobold et al., 1963) was used to assess the anti-arthritic activity of the ethanolic extract of Delonix elata in Wister rats. Animals were randomly divided into five groups of six animals each (n=6). Group I animals received normal saline (5 mg.kg^‑1) served as control, Group II animals received Indomethacine (10 mg.kg^‑1p.o.) served as reference standard, Group III animals received (0.01 ml Freund’s adjuvant) served as an arthritic control and Group IV and V animals received the crude ethanolic extract of Delonix elata (250 and 500 mg.kg^‑1). The paw volume is a indicator of arthritic condition. To asses the anti-inflammatory and anti-arthritic activity of Delonix elata, the extracts were given to the animal 30 minutes before the administration of freund’s adjuvant and continued till 28^th day. Paw volume was measured on 4^th, 8^th, 12^th, 16^th, 20^th , 24^th and 28^th day by using plethismometer and changes in body weight also measured.

Evaluation of radiological changes in the hind limb paw:

Freund’s adjuvant induced Arthritis model (BB Neobold et al., 1963) was used to assess the anti-arthritic activity in Wister rats. Animals were randomly divided into five groups of six animals each (n=6). Group I animals received normal saline (5 mg.kg^‑1) served as control, Group II animals received Indomethacine (10 mg.kg^‑1p.o.) served as reference standard and Group III animals received (0.01 ml Freund’s adjuvant) served as an arthritic control and Group IV and V animals received the crude ethanolic extract of Delonix elata (250 and 500 mg.kg^‑1). The ethanolic extract was administered after 14 days from the day of adjuvant injection for 14 days by intubation and the drug treatment was given until 28^th day by oral route. All radiographs were taken with a Wipro GE X-ray instrument set at 45kV and 4 mAs using Laser Orthochromic film. The film -to- source distance was 100cm. X-ray was taken at the joints of the hind paw for the confirmation and evaluation of the severity of arthritis in FAI induced rats. The X-ray examination of Hind Limb Bone Mass of animals in HLBM of Group I – Group V was carried out continuously for 5 weeks (Esser etal., 1995). On day 1 the animals were subjected to X-ray examination without any treatment and after 3hrs the animals GP I- IV were induced with 0.1ml of Freund’s adjuvant in left hind paw of rats. On 7^th day X-ray examination of animals in all groups were carried out. On day 14^th day X-ray was taken after treatment with ethanolic extract of Delonix elata and indomethacine to the corresponding group of animals. The same treatment was continued until 28^th day but the X-ray examination was carried out on 21^st and 28^th day. Estimation of HLBM using photodensitometer and aluminium step wedge. Densitometer is a device by which we can measure the Optical Density (OD) at a particular area of a film and we can measure the density from 0 to 4 with 0.01 accuracy. The processed X-ray film carries the visible image in terms of metallic silver pattern and in other words the degree of blackness is directly related to the amount of silver present. The film darkness directly depend on intensity of radiation reaching the film which inturn depends on atomic number and density of the tissue through which uniform X-ray beam has passed. By measuring the amount of silver loss or degree of blackening, is indicator of nature of tissue. For radiographic standardization x-ray tube voltage KeV, mAs, developing condition should be as a kept constant. A high level of standardization is required in both projection geometry and image acquisition, which is needed to achieve a precise measurement of density. Radiographic projection geometry is defined by the relative location, and orientation of X-ray source, the object and the film detector. After assessing the nature of tissue, a quantitative measurement was performed using aluminium step wedge. The step of height of aluminium step wedge is 1.5mm to 10.5mm with a width of 3mm was placed on the film cassette and radiograph was exposed. Aluminium was chosen for step wedge since its atomic number is very similar to the effective atomic number of bone. Mineral with similar atomic number will attenuate X-ray in a similar manner. OD of each step of the step wedges was measured and the values were plotted against the corresponding thickness of aluminum. The curve obtained provided the corresponding aluminium equivalent to the measured optical density of the Hind Limb. In this way an indication of the HLBM was obtained.

RESULTS:

Acute toxicity:

The leaf extract of Delonix elata didn’t show any mortality and toxicity even at highest dose of 2000 mg.kg^‑1body weight employed. The present research study was carried out using two different doses (low and high) ethanolic extract of Delonix elata such as 250 and 500mg.kg^‑1 body weight for anti-arthritic activity.

PAW VOLUME:

An oral administration of Delonix elata (250 and 500mg.kg^‑1) showed a marked inhibition of edema of adjuvant induced chronic arthritic rats and the maximum effect was observed on 28^th day and the effect is very similar to the standard drug “Indomethacine” (Fig. 1)

BODY WEIGHT CHANGES:

Delonix elata (250 and 500 mg.kg^‑1) inhibits the loss of body weight on adjuvant induced arthritic animal than compared to vehicle control animals, when the loss of body weight is predominant and indomethacine could ameliorate the weight loss occurred during arthritis (Table.1)

X-RAY Analysis (Densitometry):

Radiographs were taken in the left hind paw once in a week and this procedure was followed for 5 weeks before and after treatment of ethanolic extract of Delonix elata and standard drug indomethacin. Adjuvant – induced group shows severe bone swelling. The bone density changes were evaluated using photodensitometer and quantitative measurements were made using aluminium step wedge and HLBM was calculated. HLBM of rats was increased by Freund’s adjuvant as compared to control group. Interestingly the arthritic animals treated with ethanolic extract of Delonix elata (250 and 500 mg.kg^‑1) showed significant reduction in bone density which is similar to that of standard drug Indomethacin. From the aluminium step wedge we can measure the accurate bone mass density of control and drug treated groups. There was a statistically significant (p<0.001) difference in mean values of densitometry reading was observed and the value was found to be 0.01. The results of present study indicate that the plant extract treatment successfully suppressed the RA induction.

DISCUSSION:

Arthritis is a chronic inflammatory disorder and the inflammation involves the release of mediators like cytokines (IL-IB and TNF-α), GM-CSF, interferons and PGDF. These are responsible for the pain, destruction of bone and cartilage that can lead to severe disability (G.B Eric et al., 1996). The determination of paw swelling is

Table 2. Anti-article effect of ethanolic extract of Delonixelata by X-ray image analysis (optical density Vs Step wedge thickness)

Day – 1

Day – 7

Day – 14

Day – 21

Day – 28

O.D.

Step Wedge Thickness

O.D.

Step Wedge Thickness

O.D.

Step Wedge Thickness

O.D.

Step Wedge Thickness

O.D.

Step Wedge Thickness

2.80±

0.0075

1.7±

0.0248

2.9±

0.236

1.6±

0.00752

2.8±

0.00816

1.8±

0.0075

2.9±

0.0236

1.9±

0.0136

2.7±

0.0075

1.72±

0.0248

2.6±

0.0542

1.6±

0.0075

3.6±

0.0816

2.16±

0.0.00816***

3.4±

0.108

2.13±

0.0116***

2.8±

0.0075

1.89±

0.0089***

2.5±

0.0543

1.68±

0.01505***

2.9±

0.0236

1.76±

0.0150

3.8±

0.0816

2.17±

0.00816***

3.7±

0.516

2.11±

0.0116

3.6±

0.0547

2.18±

0.0081*

3.5±

0.0547

2.18±

0.00816**

2.8±

0.0075

1.81±

0.0075

3.7±

0.0516

2.18±

0.00812***

3.2±.

0109

2.11±

0.0116*

2.9±

0.0236

1.88±

0.0075***

2.6±

0.0547

1.71±

0.0248**

2.8±

0.0.00752

1.72±

0.0248

3.7±

0.00248

2.16±

0.00814***

3.3±

0.108

2.14±

0.0082***

3±

0.1085

1.86±

0.015**

2.4±

0.0150

1.68±

0.01505***

Table.2 The anti-arthritic effect of ethanolic extract of Delonix elata (250 and 500 mg. kg^–1 body weight) was tested by x-ray image analysis technique in rat. The indomethacine (10 mg. kg^–1 body weight) was used as a standard drug. The control animal was treated with saline (5mg. kg^–1 body weight). The arthritis was induced with 0.1 ml of FCA. The anti-arthritic effect was tested in different time interval such as 1,2,3,4 and 5 weeks. Each value represents mean ± S.E.M, n=6. The statistical analysis was carried out usin gone way ANOVA method, where **p<0.001**p<0.001, *p<0.5.

apparently simple, sensitive and quick procedure for evaluating the degree of inflammation and assessing of therapeutic effects of drugs. In the present study, the rat was selected as a animal model since they develop a chronic swelling in multiple joints with an influence of inflammatory cells and followed by erosion of cartilage in joints and destruction of bones. The rat model is a close resemblance to rheumatoid arthritis of human beings (S.Sing et al.,1996). From our study we observed that the ethanolic extract of Delonix elata and indomethacine significantly suppressed the swelling of the paws of rats.

A change in body weight of rats was also measured as one of the parameter to assess the course of the disease and the response to therapy of anti-inflammatory and arthritic drugs (J.K.William et al., 1996). As the incidence and severity of arthritis increased, a decrease in body weights of the rats also occurred during the course of the experimental period and this observation was supported by the findings of C.V.Winder (C.V.Winder et al., 1989) on alterations in the metabolic activities of diseased rats. In addition to the absorption of ¹⁴C- glucose and ¹⁴C- leucine in rat’s intestine was reduced in the case of inflamed rats (D.T.Walls et al., 1981) but on the treatment with anti-arthritic drugs, the decrease in absorption was nullified (S.Somasundaram et al., 1983) and it shows that the anti-arthritic drugs correct the decreased/deranged absorption capacity of intestine during arthritis. The increased body weight during treatment of indomethacine and ethanolic extract of Delonix elata may be due to the restoration of absorption capacity of intestine.

By using image analysis techniques of radiographs, we measured bone swelling, optical density of all groups of tibio tarsal joints of rats using photodensitometer and aluminium step wedge thickness (mm). This method provides a more sensitive and quantitative approach for radiological image analysis as compared with conventional observation. From the study observed a change of bone swelling by measuring OD in terms of aluminium equivalence (mm) after treatment with ethanolic extract of Delonix elata and indomethacine treatment. It has been previously demonstrated that these measurements are positively correlated with the results of conventional radiological and histological evaluation (Esser et al., 1983). The radiographic analysis of the tibio tarsal joint in arthritic and drug treated animals further supported and confirms the potent anti-arthritic effect of Delonix elata in a dose dependent manner and it suppress the pathological changes such as pannus formation and bone destruction (Koburova KL et al., 1965).

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Group	Mean body weight (gm)		Mean changes in body weight (±SEM)
Group	Before induction	After treatment (On 28^th day)	Mean changes in body weight (±SEM)
Control (Normal saline 5 mg.kg^‑1)	158.6	166.4	8.33±1.667
Standard (Indomethacine 10 mg.kg^‑1)	155.1	195.5	40±2.582**
Delonix elata 250 mg.kg^‑1	151.2	174.2	23.33±4.595**
Delonix elata 500 mg.kg^‑1	151.8	162.7	10.83±1.537