Anti-arthritic Effects of Bark Extracts of Holoptelea integrifolia (Roxb). Planch in a Chronic Inflammatory Model

Rheumatoid arthritis is an autoimmune disorder characterized by synovial proliferation, inflammation, cartilage destruction and deformity of the joints and a major cause of disability. A wide range of drugs are being used in the treatment, which causes severe side effects in the long run. Hence the current study was undertaken to explore the anti-arthritic effect of aqueous and ethanolic bark extracts of Holoptelea integrifolia, at different doses (200 mg/kg and 400 mg/kg), in comparison with the standard drug Diclofenac sodium (1mg/Kg) in adjuvant induced arthritic model in rats. The treatment was assessed by measuring the paw volume and by using various hematological parameters like haemoglobin, total red blood cell count, white blood cell count and C reactive protein. All the extracts of Holoptelea integrifolia reduced severity FCA induced arthritis extract, with significant effect at 400 mg /Kg.


INTRODUCTION
Rheumatoid arthritis (RA) belongs to a group of disorders broadly termed as rheumatic diseases. Articular involvement is manifested clinically by joint pain, stiffness, limitation of motion and signs of inflammation (Rindfleisch & Muller, 2005). Arthritis being a chronic inflammatory condition, its treatment takes a long course, which can cause serious side effects to the health of the individual. Novel drugs from alternate system of medicine could provide a suitable replacement, being economical, locally available and devoid of side effects.
Holoptelea integrifolia (Roxb) also called as Indian elm is a very popular traditional plant in India. It is used traditionally for the treatment of inflammation, gastritis, dyspepsia, colic, intestinal worms, vomiting, wound healing, leprosy, diabetes, hemorrhoids, dysmenorrhoea and rheumatism (Sharma & Singh, 2012).

Experimental design
Animals are divided into seven groups with ten animals in each group. The rats were acclimatized for a period of seven days before the start of the experiment. Group I -untreated control, Group II-Arthritic control, Group III-Arthritis + Diclofenac (1 mg/kg), Group IV-Arthritis + Holoptelea integrifolia ( aqueous extract -200mg/kg), Group V-Arthritis + Holoptelea integrifolia ( aqueous extract -400mg/kg), Group VI-Arthritis + Holoptelea integrifolia ( ethanolic extract -200mg/kg), group VII-Arthritis + Holoptelea integrifolia ( ethanolic extract -400mg/kg) Induction of adjuvant arthritis in rats Freund's complete adjuvant (Chondrex, Inc. USA) was suspended in mineral oil with a final concentration of 10 mg/ ml. Arthritis was induced by injecting about 0.05 ml of Freund's complete adjuvant below the plantar aponeurosis of the right hind paw of the rats (Newbould, 1963, Whitehouse, 1998 on day 8 after acclimatization for a period of seven days. Paraffin oil (0.05ml) oil was injected into the right hind paw of another group of rats which served as untreated control.

Treatment of animals
All the animals in groups III, IV, V, VI and VII were treated continuously from day 10 after induction of AIA for a period of 20 days by oral dosing. Diclofenac (Himedia laboratories Pvt). Ltd., Mumbai and HI aqueous extract were dissolved in distilled water, whereas ethanolic extract was administered as a 0.1% solution(prepared in dimethyl sulfoxide (DMSO) (upto 10% of 10 ml solution). No treatment was given to the rats in untreated control and arthritic control.

Measurement of paw swelling
The changes in the oedema of right hind paw was measured on 8 th ,12 th , 16 th , 21 st , 26 th , 31 st and 36 th day using digital plethysmometer (Panlab digital plethysmometer). The initial level of the fluid and final level after dipping the foot is noted. The difference between the initial and final volumes indicated the oedema volume. Calculation of percent inhibition of paw volume [ ] where, -oedema volume of arthritic control group and Vt-oedema volume of treatment group.

Haematological and Biochemical assays
Whole blood was collected from animals on 36 th day and was used to assess haematological parameters such as Packed Cell Volume (PCV), Haemoglobin, RBC, WBC, platelets count. C-Reactive Protein levels in serum was estimated quantitatively which is a turbidimetric immunoassay.

Statistical analysis
The data were statistically analysed by using non parametric Duncan's one way analysis of variance.

Phytochemical Analysis
Phytochemistry of the plant had revealed the presence of several phytoconstituents such as tannins, saponins, flavonoids, alkaloids, quinones, terpenoids, phenols, coumarins and proteins in aqueous extracts whereas tannins, saponins, proteins were absent in ethanolic extract. The chromatogram obtained from GC-MS analysis of the aqueous and ethanolic extracts indicate the presence of at-least eleven peaks for aqueous and ten peaks for ethanolic extract suggesting the presence of multiple phytoconstituents.
Analysis of the chromatogram has shown the presence of an active principle, Friedelan-3-one in the ethanolic extract.

Changes in paw volume
In arthritic control (Group II) rats, swelling and redness developed over 24 hour period in the right hind paw injected with adjuvant and reached maximum intensity after 9-14 days from the day of induction. Paw volume of all the rats of both control and treatment groups were measured on days 8, 12, 16, 21, 26, 31, 36 using digital plethysmometer. The paw volumes of the arthritic control group kept increasing gradually till day 31 and then decreased slightly whereas paw volumes of all treatment groups (Groups IV to VII) has shown a considerable reduction in paw volume (Fig. 1) compared to diclofenac group (Group III). (Table 1) Haematological and Biochemical changes A significant decrease in haemoglobin and total erythrocyte was seen in arthritic control group. But those changes were reversed by the treatment and no significant changes were noticed among the treatment groups in comparison with the standard drug group. An increase in TLC and C reactive protein was seen in arthritic control group. The increases in WBC count, serum C-reactive protein level were significantly suppressed in the extractadministered arthritic group. (Table 2).

DISCUSSION
Freund's complete adjuvant (FCA)-induced arthritis is a model that have been extensively used to study the pathogenesis of RA for therapeutics testing. Adjuvant induced arthritis is thought to occur through cell mediated auto immunity by structural mimicry between mycobacteria and cartilage proteoglycans in rats (Van Eden, Holoshitz, Nevo, Frenkel, Klajman, & Cohen, 1985). In AIA, heat killed mycobacterium tuberculosis cells do not degrade but remain in the synovium as perpetual antigens (Arend, 1997) and can initiate an inflammatory cascade mimicking RA.
Paw swelling is one of the major factors in assessing the degree of inflammation and curative efficacy of drugs. On treatment with extracts of Holoptelea integrifolia significant reduction in paw swelling has been observed. This reduced edema formation may be due to its inhibitory action on prostaglandin synthesis (Williams, 1979). Triterpenoids are able to significantly reduce the paw swelling in AIA model (Agarwal & Rangari, 2003) and the reduction in paw swelling in this study may be due to the presence of terpenoids which possess anti-inflammatory action.
Anaemia is commonly noted in patients with chronic arthritis which may be due depression of bone marrow due to formation of immune complexes The results of the study concur with the findings of Malik, Manvi1, Nanjware, Dwivedi, Purohit, & Chouhan (2010). Mowat (1971) suggested that during arthritis, iron is not released as well as incorporated in haemoglobin. The reversal of these effects to normalcy after treatment may be due to the erythropoietic effect of the plant.
An elevated TLC was seen in arthritic control which shows that inflammation persists. The reversal of these effects to normalcy after treatment may be due to the erythropoietic effect as well the antiinflammatory effect of the plant.
An indicator of infectious and inflammatory diseases the WBC count was increased in arthritic rats. The migration of leukocytes to the inflamed area was significantly suppressed by this plant extract, as indicated by the significant decrease in the WBC count which shows its antiinflammatory effect.
C-reactive protein is a member of the class of acute phase reactants -its levels rise dramatically during inflammatory processes. C-Reactive protein is elevated in response to stress or inflammation that occurs after infection, injury, surgery and tissue damage (Blake & Swift, 2004). Elevated C-reactive protein in arthritic suggests an active inflammation. The concentration of C-reactive protein was found to be significantly reduced in the plant-treated as well as the diclofenactreated groups.
Among the extracts, both aqueous and ethanolic are equally efficacious in reducing the paw swelling. However the parameters that determine the progression of arthritis suggest that ethanolic extract at the dose of 400 mg /kg was able to produce significant effects This may be due to the difference in the concentration of phytoconstituents in the extracts. A better result was seen at the higher dose of 400 mg / kg.

CONCLUSION
Hence Holoptelea integrifolia could be considered as a potential for the treatment of RA. However identification of the active principle that is exactly responsible for the anti-arthritic activity and the elucidation of its mechanism of action will help validate its utility in the therapy of arthritis.