Effects of aqueous and ethanol root extracts of Olax subscorpioidea on inflammatory parameters in complete Freund’s adjuvant-collagen type II induced arthritic albino rats

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R heumatoid arthritis (RA) is an auto-immune disease that primarily affects joints especially the wrist and hands resulting in warm, swollen, and painful joints (1). Pain and stiffness associated with RA often worsens after rest. Rheumatoid arthritis may also affect other parts of the body, resulting in a low red blood cell count, inflammation around the lungs, and inflammation around the heart (2). Prevalence data on arthritis in Africa and Nigeria in particular remain scarce however rheumatoid arthritis is affecting up to one million and three hundred thousand people in the United States alone with a low occurrence in some parts of Africa, according to current census data. The ratio of women to men having the disease is between 3: 1 and 2: 1 (3). The disease onset is usually after 40 years of age and before 60 years of age, but it can also start at any age and even affects children (juvenile rheumatoid arthritis) (4). The genetic basis of the disorder suggests that multiple members can be affected in some families. There is no cure for RA yet. Over the years, the treatment of rheumatoid arthritis optimally involves a combination of patient education, rest and exercise, joint protection, medications, and occasionally surgery (5). Some of the Medications used in the treatment of rheumatoid arthritis include Non-steroidal antiinflammatory drug (NSAIDs) such as methotrexate, hydroxychloroquine, sulfasalazine, disease modifying anti-rheumatic drugs (DMARDs) such as TNF alpha inhibitors, IL-6 inhibitors, T-cell activation inhibitors, B-cell depletes, JAK inhibitors, immunosuppressants, and steroids (etanercept, adalimumab, certolizumab etc). these drugs show common adverse effects such as disorders of the gastro-intestinal tracks, loss of appetite, sore mouth, diarrhea, headaches and hair loss (6,7) mild skin reactions at the site of injections, infections, nausea, a rise in temperature and headaches (8). Many arthritis patients resort to the consumption of locally available herbs to manage or treat their condition cause of the high cost of orthodox drugs. They claim that these local herbs are effect in alleviating their arthritis, therefore it is becoming the general practice in our society today. Olax subscoripoidea, Oliv is among the plants that have been successfully used for the treatment of arthritis by the rural dwellers (9).
Olax subscorpioidea is a tree up to 10 m high, bole to 60 cm girth with long thin, drooping branches, but sometimes a many-stemmed shrub (10). Its leaf is used in as traditional medicines as pain-killers, its leaf, twig and bark are used in the treatment of arthritis, rheumatism, liver diseases, venereal diseases, febrifuges, antidotes (venomous stings, bites, etc.) The root is applied as medicine in the treatment of cutaneous, subcutaneous parasitic infection, genital stimulants/depressants. It is also used as chewing-sticks (11). There are animal disease models that exhibit the pathology of human rheumatoid disease and are therefore employed as vehicles for research on potential therapeutics designed for the management and treatment of RA (12). In a bid to evaluate the safety, effectiveness, and toxicity of new potential RA therapeutics, rodent populations which mimics human RA disease are created by collagen-induced arthritis (CIA) and complete Freund's adjuvant (CFA). They both exhibit similar effect as is seen in rheumatoid arthritis within the cells, creating synovitis and erosions (13). Olax subscorpioidea plants has been used over the years by majorly rural dwellers in the management of arthritis, however, no scientific base for this has been established or compared the effects of ethanol and aqueous extracts of the plant. Therefore the study is aimed at determining the effects of aqueous and ethanol root extracts of Olax subscorpioidea on inflammatory parameters such as weight, paw size, Erythrocyte Sedimentation Rate, C-reactive protein and Cytokines such as interleukine 1-Β, interleukine-6, tumor necrosis factor, rheumatoid factor in complete Freund's Adjuvant-Collagen Type II induced arthritic rat.

Materials
The roots of Olax subscorpioidea was harvested from Ndi-Nwali Village in Izzi Local Government Area of Ebonyi State in South-Eastern Nigeria. A botanist, Dr. (Mrs.) Kate Nnamani in the Department of Biological Sciences, Ebonyi State University, Abakaliki, authenticated the plant. Some of the plant root samples were preserved in the Department of Biological Science Herbarium. A total of one hundred and thirty-five female albino rats were purchased from the Department of Animal Science, University of Nigeria Nsukka, Enugu State, Nigeria and were acclimatized for a period of two weeks at the Animal House of the Department of Biochemistry, Ebonyi State University, Abakaliki. The animals were kept in cages and fed on commercial rat feed and were allowed access to clean water. The weight of the test rats were measured daily ( Figure 1).

Preparation of the plant extracts
The roots of Olax subscoipioidea were washed under tap water and air dried in a suitable environment. The roots were ground using laboratory milling machine and sifted using 0.25 mm sieve. Exactly 800 g of the powdered root sample of Olax suscorpioidea were soaked in 2 L each of ethanol and deionized water for 48 hours, filtered with a clean white cloth and concentrated using a water bath at 35°C until the solvents were completely removed. Extracts obtained were used for various analyses.

Induction of arthritis in albino rats
Pearson's method of induction of arthritis in rats was applied (1956), (14). Briefly, 0.1 ml of chicken type II collagen-complete Freund's adjuvant (CFA) was injected into the left hind paw of the rats (The constituents of the CFA are heat killed Mycobacterium tuberculosis and sterile paraffin oil (10 mg/ml). The degree of hind paw swelling in each animal was determined using a calibrated automated veneer caliper twice weekly throughout the duration of the study prior to and after the administration of the adjuvant. Those rats with elevated level of inflammatory biomarkers when compared to the control were therefore considered to have arthritis and were used for subsequent experiments. The weights of rats were taken daily throughout the study duration ( Figure 2).

Treatment of arthritic rats with plant extracts
Female albino rats weighing between 150-200g were used for this work. The animals were sub divided into 9 groups with 15 animals in each group. Group I served as negative control (without induction of arthritis and treatment) and received 5 ml/kg normal saline; Group 2 was induced with arthritis but without treatment. It served as the positive control and received 5 ml/kg normal saline; Group 3 was administered 10 mg/kg indomethacin (standard control), Groups 4-6 were induced with arthritis and were treated with Olax subscopioidea aqueous root extract at doses of 400, 600 and 800 mg/kg body weight, respectively, from day ten after induction till the end of the study; Groups 7-9 were induced with arthritis and received Olax subscorpioidea ethanol root extract at 400, 600 and 800 mg/kg body weight, respectively from day 10 after induction till the end of the study. Various changes in the body weight and inflammatory index were measured daily. Three albino rats from each group were sacrificed on days 10, 18, 25 and 32 and blood samples were collected in plain tubes for serum separation.

Statistical analysis
The basic statistics, means, standard deviation and ranges of the measured parameters were estimated using Statistical Analysis System (SAS) windows version 9.0. Data expressed was done as means ± SD of 15 replicates. Values were considered statistically significant at p<0.05.

RESULTS
The results of the percentage yield of ethanol and aqueous root extracts of the plants Table 1 showed that the percentage Olax subscorpiodea ethanol extract yield was 9.5% while its aqueous extract yield was 10.68%.
The effect of the administration of Olax subscorpiodea ethanol and aqueous root extracts on the weight of rats as presented in Table 2 shows the ameliorator effect of indomethacin and Olax subscorpoidea aqueous and ethanol root extracts on the weight of the treated arthritic rats when compared to the arthritic but untreated group and the normal control. The weight of the arthritic rats reduced significantly (p<0.05). the administration of Olax subscorpioidea aqueous and ethanol root extracts on the arthritic rats however reversed the effect of adjuvant on the weight of the rats to normal while the weight of untreated arthritic rats continued to decrease significantly (p<0.05) until the end of the study period.
The effect of Olax subscorpioidea ethanol and aqueous root extracts on paw size (inflammation) of rats Table 3 shows the anti-inflammatory effect of indomethacin and Olax subscorpioidea ethanol and aqueous root extracts at

TABLE 2
Percentage of phytochemical constituent of ethanol and aqueous extracts of Rauwolfia vomitoria roots 400, 600 and 800 mg/kg bd wt respectively, on the changes in paw edema of untreated arthritic rats and treated animals. There paw size increased significantly (p<0.05) and redness developed within 48 hr period in the feet pad of the animals administered with the adjuvant. The administration of Olax subscorpioidea aqueous and ethanol root extracts at different doses showed a marked reduction in paw size while the paw size of arthritic untreated albino rats increased significantly (p<0.05) ( Figure 3).

Figure 3) (A-D): Photomicrograph of joint of normal control rats (GP1) H&E. mag. 100x. (A)Thickened stratum corneum (SC) layer (white curve) and stratum granulosum (SG) (black curve), a clear cell layer (black arrow) and thick collagen fiber (star) (day 10). (B)Synovial lining cells (black arrow) (day 18). (C) A few number of multiple synoviocytes and giant cells (yellow arrow) at day 25 without evidence of inflammation (day 25). (D) Thick collagen fiber (white star) (day 32)
The result of C-reactive protein is presented in Table 4. The C-reactive protein levels in the arthritic untreated rats significantly (p<0.05) increased with the highest value observed on day 32. However, treatment of the levels close to the values found in the indomethacine treated group; thus at extract dose of 800 mg/kg b.w, the effect was similar to that of the standard drug ( Figure 7).

DISCUSSION
Plants have been used in the preparation of traditional medicines all over the World for so many years now and yet remain as the major source of important plant chemicals for drug development (22). The application of medicinal plants like Olax subscopioidea in the management of rheumatoid arthritis is associated with its use by rural dwellers in Nigeria (23). Rheumatoid arthritis is an autoimmune disease in which the immune system which usually fights infections now attacks the cells that surround the joints causing swelling of the joints, joint stiffness and pain, leading to damage to the joint itself, the cartilage and nearby bones (24).
There is yet no cure for rheumatoid arthritis (25). Presently, NSAIDs as well as DMARDs are used to manage rheumatoid arthritis patients. These drugs lower the associated pain and inflammation and also decrease the progression of the disease (26). Due to the adverse effects of NSAIDS and DEMARDS use and the cost of the drugs, more number of arthritic patients demand and prefer alternative medicines, hence the use of herbal preparations (27). The systemic injection of chicken type II collagen CFA in experimental animals like rats, models human arthritis, resulting in polyarthritis and systemic disease that mimics transformations as is found in human rheumatoid arthritis (28). Some inflammatory parameters were determined to ascertain the effectiveness of this plant. The results presented in Table 1 showed that rheumatoid arthritis was induced in rats within a period of 10 days by administering chicken type 11 collagen-complete Freund's adjuvant. The report by Bendele (29) is in agreement with our findings. He reported that polyarthritis results from the administration of chicken type 11 collagen-complete Freund's adjuvant. was characterized by marked cartilage destruction the deposition of immune complex on articular surfaces, bone resorption and periosteal proliferation, and moderate to marked synovitis. Induction of arthritis condition caused significant (p<0.05) weight loss in rats. Our finding is agreement with the report of mondal et al. (30). This weight loss could be associated with reduction in food intake by the experimental animals and with increased synthesis of TNFa and IL-1 by splenocytes. Ronen (31). Weight loss has been reported to be a marker for an upsurge in the synthesis of TNFα and interleukin-1 which are pro-inflammatory cytokines. The association of cytokines such as TNFa and IL-1 synthesis with loss of weight in adjuvant arthritis suggests that inflammatory cachexia is cytokine-driven Table 8. TNF-α and IL-1 production by peripheral blood mononuclear cells (PBMC) in humans was associated with increased resting energy expenditure (31). It has also been reported body weight loss in adjuvant induced arthritic rats may be caused by muscle wasting in experimental arthritis which is seen as an increased breakdown of protein by the ubiquitin-proteosome proteolytic pathway (32). A rapid increase in leptin level just within twenty-four hours of injecting of CFA in rats leading to anorexia and loss of weight is seen in CFA induced arthritic rats (32). The administration of Olax subscorpioidea aqueous and ethanol root extracts at different doses ameliorated the effect of the adjuvant on the weight of the arthritic rats ( Figure 8). The ameliorative effect of these extracts may have been due to the presence of phytochemical constituents like alkaloids, flavonoids and terpenoids. These phytochemicals may have exerted this effect by inhibiting enzymes such as tyrosine protein kinase that induces anti-proliferative effects on M-CSF-activated macrophages. These enzymes are involved in signaling transduction and cell activation processes (T cell, B lymphocyte) or cytokine production (33). Increase in paw size is a physical indicator of the inflammation in the onset as well as in the chronic phase of the disease (34) In this study, the arthritis induced rats developed a chronic swelling in multiple joints as a result of influence of inflammatory cells, erosion of joint cartilage, and bone destruction. It exhibited close similarities with rheumatoid disease seen in man. An increase in the paw volume after intra-dermal administration of the adjuvant reveals the status of arthritis (30). Effect of adjuvant-induced arthritis on Paw size rats Table 4 shows the anti-inflammatory effect of Olax subscorpioidea ethanol and aqueous root extracts at 400, 600 and 800 mg/kg bd wt respectively, on the changes in paw edema of induced untreated rats and treated animals. There was increase in paw size and redness developed over a 48 hr period in the feet of experimental animals injected with chicken type 11 collagen complete Freund's adjuvant. Swelling of the hind paw of adjuvant induced arthritic rats is seen in adjuvant induced arthritis rats. The arthritic rats are often relatively immobile due to severity of paw swelling (29). The Olax subscorpoidea aqueous and ethanol root extracts at different doses showed a marked reduction in paw size. However, the extracts decreased the paw arthritic rats with root extracts of Olax subscorpioidea aqueous and 800 mg/ kg b.w doses, caused a significant (p<0.05) reduction of C-RP levels relative to the levels found in the normal rats. The effect was both dose and timedependent. There was no significantly difference between the effects due to the solvent used ( Figure 4).
The result of the effect of Olax subscorpioidea ethanol and aqueous root extracts on Rheumatoid factor (RF) of rats is presented in Table 2. RF count was found to be significantly (p<0.05) higher in the arthritic rat groups. Treatment with Olax subscorpiodea ethanol and aqueous root extracts at 400, 600 and 800 mg/kg bd wt however caused a significant reversal of RF value back to normal as is found in the normal rat group.  Figure 5).
The results of cytokine levels are presented in Tables 4-6. The result shows the levels of Serum cytokines measured in the arthritic and non-arthritic samples ( Figure 6). In comparison to the normal control group, the arthritic animal groups that were not administered the plant extracts had a significant (p<0.05) higher values of TNF-α, IL-1β and IL-6 levels. The measured cytokine concentration were observed to have reduced (p<0.05) following the administration of plant extracts to the arthritic rats. This ameliorative effect of the plant extracts was comparable to those of the normal control group Table 7. The values of TNF-α, IL-1B and IL-6 in the untreated arthritic rats continued to increase significantly (P <0.05) till the end of the study period. Within the treatment groups, GP 10 rats which were administered 800 mg/kg Olax subscorpiodea ethanol extract, were found to contain cytokine   volume by inhibiting the release of inflammatory mediators, showing its antiinflammatory effect in adjuvant induced arthritis. Increase in the size of the paw is a parameter used in the measurement of the anti-arthritic activity of different drugs administered in the treatment of inflammations. Increase in paw foot pad and tibio tarsal joint diameters in adjuvant induced arthritis rats could possibly be due to the delayed immunological upsurge in the disease. The influx of inflammatory cells in rats causes chronic swelling in many joints. The increase in swelling of the hind paw decreased significantly (p<0.05) in the days that followed arthritis induction when compared to the untreated arthritic group. It can therefore be said that the likely mechanism for the reduction in paw size may be as a result of the suppressive effect of indomethacin and Olax subscoripoidea aqueous and ethanol root extracts. The phytochemical components present in the extracts such as alkaloids hinder antigen presentation and immune cells release while terpenoids regulate

(B) The oedema appeared reduced (star) with increased proliferation inflammatory cells mainly macrophages (yellow arrow) at the end of day 18. (C-D) The stratum corneum (black arrow) and granulosum (yellow arrow) appeared intact (Day 25 and 32)
of inflammation. Ismail et al., observed maximum inhibition at 400 mg/ kg. They related the effect of Olax subscorpioidea to their phytochemical constituents such as flavonoids, anthraquinones, saponins, cardiac glycoside and polyphenols. According to this report, saponins have before now reported to display anti-inflammatory activities (36). The C-reactive protein (C-RP) level in serum samples were found to be significantly (p<0.05) higher in all arthritic rats than the normal control rat group as is seen in Table  4. C-reactive protein level is used to determine the rate of progression of disease. C-RP is an acute phase protein which is synthesized in the liver under conditions of systemic inflammation (37). The synthesis of CRP is activated by the influx of synovial macrophages and fibroblasts by inflammatory cytokines including TNF-α, IL-1, and especially IL-6. These cytokines are found to be synthesized in large amount in rheumatoid arthritis (38)     some cell to cell information transfer pathways that play a role in the course of inflammation for instance, nuclear transcription factor-kappaB (NF-kappaB) (35). These actions of these and more phytochemical components may have caused the reversal in the continued increase in paw size of the arthritic rats. Our result agrees with another report where it was discovered that Olax subscorpioidea produced significant dose dependent inhibition J Pharm chem Pharmacol Vol 2 No 1 August 2018 and 800 mg/kg bd wt caused a significant (p<0.05) reversal of the C-reactive proteins to levels close to that found in the normal control rats (Figure 7). The phytochemical and other chemical constituents of the plant extract may have modulated the synthesis of other pro-inflammatory molecules thereby inhibiting the pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α from different sources. A rise in RF level in adjuvant diseases study of rheumatoid arthritis has been observed. The high titer values of rheumatoid factor (RF) was observed in the sera from individuals with rheumatoid arthritis (RA) (39). In this study, the arthritic rats had significantly (p<0.05) elevated level of rheumatoid factor. However, treatment with varied doses of Olax subscorpioidea significantly (p<0.05) decreased the level close to normal as is found in the normal control ( Figure 9).
The effect of the extract was both time and dose dependent. Our result showed that the anti-inflammatory effects of Olax subscorpioidea could be as a result of the inhibition of B-cell action. B-cells activation is through tolllike receptors and other genetic factors that predispose organisms like the rats used in this study, leading to rheumatoid factor generation in arthritis. Rheumatoid factor (RF) is an antibody directed to determinants in the Fc portion of immunoglobin G molecule. The ameliorative effect of our extracts could be attributed to the action of flavonoids, tannins, saponins and other phytochemical constituents present in their plants (40). Erythrocyte sedimentation rate is an indicator of a state of chronic inflammatory (41). In acute tissue damage, several factors such as tissue damage, inflammation and infection are increased which gives rise to chronic inflammation and chronic infection that may have played an important role in increased erythrocyte aggregation (42) raised CRP levels in rheumatoid arthritis plays an important role in the induction and maintenance of increased erythrocyte aggregation as is found in the blood of rheumatoid arthritis patients Table 9.
Treatment of the arthritic rats with the plant extracts resulted in a timedependent reduction in ESR level Table 6, with effect being more pronounced on day 32 of the study. Our result is in agreement with the result of the study done by Patel and Shar (41). An increased disease processes is indicated by an upsurge in ESR. In this study, the decrease in ESR denotes the anti-arthritic activity of the plant extracts and the standard drug. Cytokines levels were measured in the arthritic and non-arthritic serum samples. In comparison to the normal control group, the levels of TNF-α, IL-1β and IL-6 were found to be significantly (p<0.05) higher the arthritic rat groups in contrast to the normal control group. It is has been established that TNF-α increases proliferation and cytokine (IL-1β and IL-6) productions in immune cells infiltrating the joints ( Figure 10). The presence of IL-6 activates the synthesis of acute phase proteins like C-reactive protein, fibrinogen and serum amyloid in the joints of arthritic patients. This elevation of level of IL-6 is correlated with clinical variables such as morning joint stiffness, number of joints affected and other laboratory variables such as erythrocyte sedimentation rate, C-reactive protein and rheumatoid factor titer. This action turns on the expression of the CRP-gene. This leads to the synthesis of C-reactive protein (43) Tables 8-10. The Cytokine levels in the arthritic rats decreased (p<0.05) significantly following treatment with the plant extract whereas the cytokine levels in arthritic untreated rats continued to increase significantly (p<0.05) till day 32 when the study was terminated. Within the treatment arthritic rats administered 800 mg/kg Olax subscorpiodea ethanol extract were found to be as effective as the indomethacin standard treated rats also observed that rheumatoid arthritis is caused by a number of inflammatory molecules released by macrophages and fibroblasts (Figure 9). Such molecules include IL-1β, IL-6 and TNFα, prostaglandins and reactive oxygen are responsible for the initiation of pain along with swelling of the limbs and joints, bone deformations and disability of joint function (44) pro inflammatory cytokines produced by the inflamed synovium as well as by chondrocytes in the arthritic joints attributes of arthritic joints is the tend to persist, hence enforcing the continued inflammation (45).

Histological analysis of adjuvant induced arthritic rat joints
Histopathological studies of the paw joints in the negative control group (normal rats) revealed normal joint structure ( Figure 3) with an intact cartilage and no distortions. However, the untreated arthritic rats Figure  4 showed moderate to severe hyperplasia of synovium with focal cartilage destruction. It also showed a marked damage of articular structure which shows that there exist joint damage and inflammation. The arthritic treated groups ( Figure 6-11) also showed moderate to severe rheumatoid tissue damage. But treatment of arthritic rats with Olax subscorpoidea (aqueous and ethanol) root extracts at 400, 600 and 800 mg/kg body weight and standard drugs (indomethacine) showed significant reduction in hyperplasia of synovium when compared to the induced but not treated group rats ( Figure 5) during the course of treatment. The effectiveness of the plant extracts could be compared to that of the standard drug. This result shows that bones can re-form when treated with Olax subscorpoidea aqueous and ethanol root extracts and standard drugs (indomethacine). The potential of Olax subscorpoidea aqueous, ethanol root extracts and standard drug (indomethacine) to exhibit such therapeutic ability may imply that it is able to counter inflammation by suppressing the process, interfere with synovitis and protect the joint as desired in rheumatoid arthritis therapy. Both plants possess anti-arthritic potentials. The anti-arthritic effect of the plant extracts studied here could be due to the presence of the phytochemical constituents in the plants (46) (Figure 11).

CONCLUSION
In conclusion, the study of plants product remains one of the main means of discovering pharmaceutical chemical components. this study has provided evidence of the anti-arthritic potentials of root extracts (ethanol and aqueous) of Olax subscorpioidea by reversing the effect of adjuvant on the rats by reducing the arthritic markers such as C-RP, ESR, RF and cytokine levels (TNF-α, IL-1β and IL-6) in adjuvant induced arthritic rats. This effect was  time and concentration dependent as the levels of these parameters analyzed depreciated significantly (p<0.05) as the days of the study progressed. The observed effects are attributed to the presence of some active principles of Olax subscorpioidea such as flavonoids, alkaloids and phenols which have anti-inflammatory, anti-pyretic and anti-oxidant properties. However, it is paramount that further studies be carried out to identify and characterize the lead active compounds and evaluation its anti-arthritis activities on human clinical trials.