Ethanol Leaf Extract of Buchholzia coriacea Ameliorates Biochemical Dysregulations Due to Rheumatoid Arthritis in Wistar Albino Rats

Ethanol Leaf Extract of Buchholzia coriacea Ameliorates Biochemical Dysregulations Due to Rheumatoid Arthritis in Wistar Albino Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ethanol Leaf Extract of Buchholzia coriacea Ameliorates Biochemical Dysregulations Due to Rheumatoid Arthritis in Wistar Albino Rats Esther Ugo Alum, Rajapandiyan Krishnamoorthy, Mansour K. Gatasheh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4634515/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Rheumatoid arthritis (RA) is a disease that involves many body organs. In this study, we examined the anti-rheumatoid arthritis effect of ethanol leaf extract of Buchholzia coriacea (ELEBC). Methods Firstly, the chemical compositions of ELEBC were investigated. The in vivo study was further done using 90 female rats weighing 121–146 g. Rats were appropriated into 6 groups ( n = 15). Group 1 was provided with normal saline (1 ml/kg) only. Group 2 was induced with RA and untreated. Group 3 was induced and treated with 5 mg/kg indomethacin™ while Groups 4–6 were induced with RA and treated with ELEBC at varied doses of 200, 400, and 800 mg/kg body weight, respectively. Induction of RA was done using Freund’s adjuvant, whereas the route of administration of the standard drug and the extract was via oral intubation. The study period was 31 days. Results The chemical composition analysis revealed that ELEBC has a high level of various chemical constituents. The adjuvant injection caused a significant increase in paw sizes plus a reduction in body weight. Levels of creatinine, uric acid, total protein, white blood cell, and total and conjugated bilirubin were significantly elevated in the arthritic rats. There were significant elevations in alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase activities while the levels of red blood cells, packed cell volume, and hemoglobin were lowered significantly. Treatment with ELEBC markedly reduced the paw sizes and caused weight gain. Conclusion Other biochemical dysregulations were also ameliorated. Thus, ELEBC may be useful in the control of RA. Rheumatoid arthritis Buchholzia coriacea indomethacin adjuvant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction RA is an autoimmune disease marked by body-wide inflammation, persistent synovitis, and the production of autoantibodies ( 1 , 2 ). It is characterized by some complications like joint damage, disability, cardiovascular and other comorbidities. It also has emotional upset and can lead to decreased quality of life ( 3 , 4 ). RA is a very common rheumatic disease, with a prevalence of about 1% worldwide ( 5 , 6 ). The ratio of male-to-female prevalence is about 1:3 ( 7 ). The cause of RA is still poorly understood. However, the role of genetics in RA presentation cannot be ignored as evidence of a family history of RA abounds ( 8 ). Environmental and hormonal factors can also trigger inflammatory processes culminating in tissue damage and joint destruction ( 9 , 10 ). Most commonly prescribed drugs in RA management include non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin( 11 , 12 ). Some other drugs are corticosteroids and disease-modifying anti-rheumatic drugs (DMARDs)( 13 , 14 ). Regrettably, NSAIDs are expensive, and reports of unfriendly effects like gastric and intestinal tract disturbances are documented ( 15 , 16 ). Due to the shortcomings of conventional drugs, research is in progress to discover better therapeutic alternatives ( 17 , 18 ). The use of herbal medicine especially by the poor rural dwellers has been an ancient alternative treatment regime. B. coriacea may be an example of such alternative treatment for inflammation. B. coriacea belongs to the family of Capparidaceae . It is popularly referred to as “wonderful kola”. It has assorted medicinal values. It is credited with hypoglycemic, hypolipidemic, and lipid peroxidation-reducing properties ( 19 – 21 ). Its anti-microbial, antihelmintic, and antifungal properties have been documented ( 22 , 23 ). The seed extract has anti-ulcer and gastric anti-secretory activities ( 24 ). Ground-dried leaves and seeds of B. coriacea are used mixed with warm water or alcohol and taken to alleviate pain and for detoxification by some rural dwellers. Also, ground seed is added to soups and other prepared foods to reduce blood sugar. The leaf is commonly employed in the traditional treatment of inflammation. We have previously revealed the anti-inflammatory and antioxidant effects of B. coriacea ethanol leaf extract and ethyl acetate fraction( 25 ). As a follow-up, this present study, we investigated other biochemical makers that are dysregulated in rheumatoid arthritis and how this could be ameliorated using ethanol leaf extract of Buchholzia coriacea (ELEBC). Materials and Methods 2.1 Sample Collection, Identification, and Preparation B. coriacea leaves (BCL) were harvested from Ngodo community in Afikpo North, Ebonyi State, Nigeria. BCL was named and authenticated by a Taxonomist in the Department of Applied Biology, Ebonyi State University, and deposited in the department herbarium for reference purposes (EBSU/APB/HB/2016/101). The method was used for the extraction was as previously reported( 26 ). Briefly, B. coriacea leaves were extensively washed, dried under shade, and further ground. The ground sample was further sieved out of which 800 g were immersed in 2000 ml of ethanol for 48 hours. Thereafter, it was filtered and the filtrate was evaporated to dryness at 35 0 C. The extract was stored in a sealed box and used for the investigation. 2.2. Chemicals and Reagents Freund’s adjuvant and other chemicals used were bought from Sigma Aldrich Company (USA). All the chemicals used were of the standard grade and purchased from reputable dealers. 2.3 Determination of Phytochemical, Vitamins, and Mineral Compositions of Ethanol leaf Extract (ELEBC). The method of Ugbaja ( 27 ) was employed to quantify saponin and terpenoid concentrations. Other phytochemicals such as flavonoids, alkaloids, and glycoside compositions were assessed according to the method outlined by Harborne ( 28 ) whereas tannins concentration was quantified by Trease and Evans ( 29 ) method. Vitamins and mineral compositions were quantified using AOAC method as reported by Md Noh et al ( 30 ). 2.4 Animal Study and Experimental Design For the animal study, 90 female Wistar albino rats (121–146 g) were purchased from the University of Nigeria Nsukka Enugu, Nigeria, and used for the study. The animals were fed with chow and clean water al libitum in the laboratory for about a week for acclimatization to take place. After acclimatization, the animals were randomly apportioned into 6 groups ( n = 15). Group 1 was normal control without induction of RA and treatment. Rats in this group received normal saline (1 ml/kg). Group 2 rats were induced with RA but no treatment was offered rather normal saline was given (1 ml/kg). Animals in Group 3 were induced and treated with 5 mg/kg indomethacin™. Animals in Groups 4–6 were arthritic rats treated with ELEBC at varied doses of 200, 400, and 800 mg/kg body weight, respectively. The study period was 31 days. The drug, extract, and fractions were administered orally. Variations in body weight and biochemical parameters of the rats in various groups were analyzed. Global approved standard procedure for Experimental Animal Handling as adopted by the Ethical Committee of the Biochemistry Department, Ebonyi State University, Nigeria was strictly followed after due approval (Approval No: EBSU/BCH/ET/17/005). This study was conducted and reported in accordance with Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. 2.4.1 Acute Toxicity Study The modified Lorke ( 31 ) style was employed to check acute toxicity as described in our previous study ( 32 ). 2.4.2 Induction of Rheumatoid Arthritis Pearson’s method ( 33 ) was used to induce Rheumatoid Arthritis in rats. Complete Freund’s adjuvant (CFA) (0.1 ml) was injected intradermally into the left hind paws of the rats. CFA is made up of heat-killed Mycobacterium tuberculosis and sterile paraffin oil (10 mg/ml). The paw sizes of all the rats were measured using a calibrated automatic vernier caliper. The body weights of the animals were also measured across groups. On the 10th day, arthritis had fully commenced. Graded doses of ELEBC were administered to rats from day 10 and lasted for 21 days. On days 10, 17, 24, and 31, through a cardiac puncture, blood samples were gotten from three rats from each test group and transferred into plain bottles and later centrifuged for various analyses. 2.5 Determination of Liver, Renal, and Hematological Indices ALT and AST activities were checked with the Randox test kits using the method of Reitman and Frankel and Reitmann ( 34 ). ALP activity was assayed using Schumann method ( 35 ). Total protein, albumin, uric acid, and creatinine concentration were estimated following Janssen et al ( 36 ), procedures. Bain et al ( 37 ) method was used to assay PCV, RBC, and WBC while Hb and Platelet levels were estimated by da silva Pereira ( 38 ) method. 2.6 Result Analysis Data generated were analyzed statistically using Graph Pad Prism 5.00. Data were expressed, as Mean ± SD. Two-way ANOVA with multiple variances was used for the statistical tests. In general, p < 0.05 was accepted as the statistical significance. RESULTS 3.1 Chemical Composition of ELEBC The quantitative phytochemical constituents of ELEBC are displayed below. The results revealed that the quantities of phenols, alkaloids, terpenoids and flavonoids were very high in the extract, whereas steroids and glycoside were found to be low (Table 1). The high presence of the antioxidant phytochemical suggests that ELEBC may have antioxidant potentials. Table 1: Phytochemical Constituents of Ethanol Leaf Extract of BC. Figure 1.0 shows the vitamin compositions of ELEBC. The result revealed that vitamin C was found to be the highest in concentration in ELEBC followed by Vitamin B 1 and B 2 . Vitamins A were amongst the lowest in concentrations in the ELEBC. Furthermore, Fig. 1.1 shows the mineral composition of ELEBC. The result revealed a very high level of Iron in the extract while calcium, magnesium, and phosphorus were in commensurable amounts. Copper and sodium were the least in concentration in ELEBC. 3.2 Acute Toxicity Result Administration of the extracts and fractions did not cause any demonstrable toxic effect even at a 5,000 mg/kg dose. 3.3 Effect of ELEBC on Paw Size in Adjuvant-induced Arthritic Rats. Injection of Freund’s adjuvant led to significant elevation in the paw sizes of the animals across the groups beginning from day 10 as shown in Fig. 2.0 . More so, redness of the swollen part was visually observed during the period of the study further indicating possible inflammation. Interestingly, administration of the graded doses of ELEBC resulted in a reduction of the paw sizes of the animals to levels similar to those treated with the standard drug. More interestingly, as the days of administration increased, the paw sizes of the animals further reduced and tended towards the paw sizes of the normal rats suggesting a possible complete reversal of the physiological and biochemical dysregulations caused due to arthritis induction in the study rats. 3.4 Impact of ELEBC on Body Weight of Rats Induced with Rheumatoid Arthritis Figure 3.0 reveals the impact of ELEBC on the body weight of rats induced with rheumatoid arthritis. The result revealed that induction of rheumatoid arthritis caused a significant abnormal reduction in the body weights of the rats over the study period. Interestingly, oral administration of ELEBC normalized this distortion in body weight and maintained the body weights of the animals similar to the effect of standard drugs against the impact of rheumatoid arthritis. 3.5 Effect of ELEBC Administration on Enzymatic Markers of Liver Dysfunction in Rheumatoid Arthritis-Induced Rats. Figure 4.0 (A-C) reveals the effect of ELEBC administration on enzymatic markers of liver dysfunction due to rheumatoid arthritis. The result shows that induction of rheumatoid arthritis caused a significant elevation in the activities of Alanine aminotransferase (A) and Aspartate aminotransferase (B) and irregularity in the activity of alkaline phosphatase (C) especially in 24th and 31st days of study respectively. Interestingly, in all the enzymes investigated, administration of ELEBC reversed these trends. 3.6 Effect of ELEBC Administration on Non-Enzymatic Markers of Liver Dysfunction in Rheumatoid Arthritis-Induced Rats. Figure 4.1 (A-D) shows the effect of ELEBC administration on (A) Total Bilirubin (B) Conjugated Bilirubin (C) Total Protein (D) Albumin levels respectively of rheumatoid arthritis-induced rats. The result revealed that induction of RA caused slight distortions in the serum levels of the markers although not significant across the days of the studies. Administration of ELEBC however normalized the dysregulation and prevented significant distortions in the serum levels of the markers. 3.7 Effect of ELEBC Administration on Markers of Renal Dysfunction in Rheumatoid Arthritis-Induced Rats. Figure 5.0 shows the effect of ELEBC administration on (A) Creatinine and (B) Uric acid levels respectively, of rheumatoid arthritis-induced rats. The results revealed that induction of rheumatoid arthritis led to a sustained elevation in the serum level of creatinine across the days of the study an indication of severe damage to the nephritic system in addition to destruction of muscle mass. Uric acid levels were also distorted as seen in Fig. 5B . 3.8 Effect of ELEBC Administration on Hematological Markers in Rheumatoid Arthritis-Induced Rats. Figure 6.0 shows the effect of ELEBC administration on (A) PCV, (B) RBC, and (C) Hb, respectively of rheumatoid arthritis-induced rats. Rheumatoid arthritis led to a significant decrease in PCV, RBC, and Hb levels respectively. Interestingly, the administration of various doses of ELEBC significantly prevented this abnormal decrease in the levels of these important markers of hematological dysregulations. 3.9 Effect of ELEBC Administration on other Hematological Markers in Rheumatoid Arthritis Induced Rats. Figures 7.0 shows the effect of ELEBC administration on (A) Erythrocyte Sedimentation Rate (B) white blood cells of rheumatoid arthritis induced rats. In this study, induction of rheumatoid arthritis produced elevation in the concentrations of these markers, an indication of severe inflammation. Conversely, oral administration of ELEBC significantly prevented the elevation of these markers of inflammation to levels comparable with the standard treatment and normal control groups respectively. DISCUSSION This study investigated the impact of ethanol leaf extract of Buchholzia coriacea (ELEBC) on biochemical dysregulations due to rheumatoid arthritis induction in rats. First, we investigated the chemical composition of the leaves of Buchholzia coriacea to ascertain whether it has relevant bioactive components. The result of quantitative Vitamin, Phytochemical and Mineral analysis of Buchholzia coriacea leaves (BCL) revealed that BCL has important compounds in varying concentrations, which could have impacted the positive ameliorative trend observed in the in vivo studies. There were high levels of Vitamin C, B 1, and B 2 in the extract in addition to high levels of phenols, flavonoids, alkaloids, and terpenoids. Iron was highest in the mineral constituents present in ELEBC. The above bioactive compounds are known to have various physiological potentials ranging from antioxidant to anti-inflammatory potentials respectively. Furthermore, the result from the in vivo studies revealed that administration of ELEBC at varied doses of 200, 400 and 800 mg/kg displayed anti-rheumatoid arthritis potentials in the albino rats. The ELEBC reduced the inflamed paw volumes and also led to significant elevations in lowered body weight of the arthritic rats. Rheumatoid arthritis further caused some biochemical abnormalities and rats administered with varied doses of ELEBC displayed a reversal of these deleterious abnormalities. This indicates that ELEBC has ameliorating potentials in the rheumatoid arthritis-induced liver, renal and hematological dysfunctions. Rheumatoid arthritis is a disease that involves many body organs like the liver, heart, and kidney ( 39 ). Some functions of the liver include the metabolism of drugs, detoxification, synthesis, and storage of some vital biomolecules. The liver is an essential modulatory organ whose roles during autoimmune and other chronic inflammatory diseases are indispensable ( 40 ). Hepato-protective effects of medicinal plants are reported in our earlier studies ( 41 ). Severe rheumatoid arthritis is marked by the over-production of autoantibodies, inflammatory cytokine release, and immune complex deposition ( 42 ). RA, like other inflammation disorders, can lead to changes in the hematopoietic system ( 43 ). Thus, the assessment of hematologic indices could be helpful in evaluating disease severity or the extent of recovery of arthritic patients. RA being a systemic disorder can also affect kidney adversely ( 44 ). Creatinine and uric acid are well-known markers of glomerular filtration rate (GFR). Elevated creatinine and uric acid levels could be due to lowered capacity of nephrons culminating in a decrease in the kidney’s filtering capacity and subsequent accumulation of waste products within the system ( 44 ). In this study, some of the biochemical markers of the liver (ALT, AST, ALP, total protein, albumin, total and conjugated bilirubin), hematological (Hb, PCV, RBC, WBC, and ESR) and renal (creatinine and uric acid) functions were investigated. Our study revealed a bifold increase in paw size of the arthritic rats. Tang et al . ( 44 )) made a similar observation. We observed a reduced paw size after administering standard drugs and various doses of ELEBC to rats. Previous studies have also documented the paw size-reducing properties of some medicinal plants in arthritic rats ( 32 ). Edema is a key attribute of acute inflammation and hence always monitored when examining anti-inflammatory potential of any compound ( 45 ). Manifestation of inflammation causes vasodilation and increased blood flow culminating in increased heat and redness. Inflamed rat paws heighten extra vascular protein infiltration and subsequent edema. Extravascular protein infiltration is aided by enhanced vascular permeability of blood vessels, culminating in the discharge of plasma proteins and fluids into the tissue, and consequent edema and swelling. It has been reported that hind paw swelling at the site of injection of Freund’s adjuvant connotes inflammation which is the initial presentation of RA ( 46 ). Markedly, at the first week of induction of RA, there was an obvious reduction in body weight. This trend was however reversed when the animals received the standard drug and various doses of ELEBC. A continuous reduction of weight was noticed in rats that received no drug or ELEBC. This result conforms to the study of other authors ( 47 ). Perhaps this reduction in body weights of the rats may be owing to elevated leptin amount as suggested by Mariam et al . ( 48 ). Leptin is a hormone made by fat cells which subdue hunger and also influences the immune system. An increase in leptin level can lead to the generation of pro-inflammatory cytokines culminating in chronic inflammation if not arrested ( 48 ). The presence of other inflammatory mediators can also lead to increased leptin levels ( 48 ). It is pertinent to note that raised concentration of pro-inflammatory cytokines could impair energy metabolism and also enhance muscle protein breakdown. Enhanced catabolism accelerates resting energy expenditure leading to weight loss and reduced lean body mass ( 49 ). The increase in body weight in rats that received standard drug and extract could be attributed to the decline of the inflammatory cytokines coupled with a decline in protein and muscle breakdown. Inflammation can also cause a reduction in the absorption ability of the intestine( 49 ). According to Zhang et al. ( 50 ), the absorption power of the intestine was restored when anti-inflammatory drugs were administered and weight gain was observed. Therefore, the observed significant gain in body weight of rats treated with standard drug and extract could be due to enhanced absorption capacity of the intestine. Additionally, our results showed high activities of liver enzymes (ALT, AST, and ALP) as well as raised total and conjugated bilirubin in arthritic rats while lowered albumin level was observed. Administration of indomethacin and samples to rats reversed this trend in a time and dose-dependent approach except for AST activity which had no change on the administration of ELEBC. Usually, AST and ALT are domiciled in the liver. However, when hepatocellular integrity is impaired, these enzymes leak into the general circulation ( 51 ). The fact that AST activity remained unchanged after standard drug and sample administration shows that the samples produced no harmful effect on the liver of rats nor did it ameliorate the high activity of AST seen as a consequence of induction of arthritis. Administration of standard drug and ELEBC to arthritic rats significantly lowered the raised ALP level. ALP is commonly present in the liver, bile, and other organs. Increased activity of ALP could portend biliary obstruction, gallstone, and other liver diseases. ALP activity can also rise when there is a bone disease where osteoblastic activity is enhanced ( 52 ). Adjuvant administration caused a significant increase in uric acid and creatinine concentrations. Treatment did not cause any alteration in uric acid level even at dose 800 mg/kg in the arthritic rats. More so, treatment with standard drug, and ELEBC yielded no effect on creatinine level on days 10 and 17. However, ELEBC treatment produced highest reduction on creatinine level on days 24 and 31 of the investigation. Plasma creatinine is an established marker of glomerular filtration rate (GFR). Elevated creatinine and uric acid concentration could signify impairment in filtering capacity of kidney and this could result to pileup of unwanted products within various systems ( 53 ). Perhaps, the observed non-alteration in uric acid and creatinine concentrations in treated groups portends that the administration of ELEBC probably did not alter renal function indices. Furthermore, concentrations of Hb, PCV, and RBCs in rats were significantly reduced on induction with rheumatoid arthritis. Interestingly, the administration of standard drugs and varied doses of ELEBC reversed this trend. Several authors have also reported marked improvement in Hb, PCV, and RBCs in rats administered with medicinal plant extracts ( 54 – 56 ). Anemia is a very recurrent feature observed in RA patients. The commonest one is anemia of chronic disease ( 57 ). It is mostly a result of chronic blood loss from gastritis (induced by NSAID S ), peptic ulcer, or gastroesophageal reflux. The reduction in the Hb concentration could have been caused by the reduced lifespan of RBCs and impaired iron absorption and utilization ( 58 ). Inflammatory mediators released during anemia in RA patients can induce excess production of hepcidin. Hepcidin is a hormone that regulates iron metabolism. It hinders iron exportation from cells by reducing the activity of ferroportin ( 59 , 60 ). WBCs are biological soldiers recruited against foreign attacks. Induction of RA resulted in a rise in WBC counts. The stimulation of the immune system by the adjuvant could have led to the generation of WBCs. Reduction in WBC levels was remarkable during treatment. This is suggestive of the immune system suppressing effect and consequent WBCs lowering the ability of the samples. We also observed that induction of RA caused a significant increase in the erythrocyte sedimentation rate in the arthritic rats, which suggests an increase in inflammation. This result corroborates the findings of previous authors( 32 ). During periods of RA, cytokines like TNF-α, IL-6, and IL-1β play contribute to disease severity. Lymphocytes are affiliated with arthritic diseases since their signaling could elicit autoimmunity. Lymphocyte activation could result in chronic inflammation, damage to structure and function ( 39 , 61 ) CONCLUSION This study has revealed the possible use of ethanol extract from B. coriacea leaves for treating RA based on the observed effects in arthritic rats and its non-toxic effect on the liver and kidney. We also observed that the extract lowered RA symptoms by ameliorating inflammatory, liver, and hematological markers. This could be attributed to the vitamins, minerals, and phytochemicals present in B. coriacea leaves. Since some medicinal plants and synthetic drugs used in the treatment of RA and pain have been shown to have many side effects, especially on the liver and kidney, coupled with high cost, the use of B. coriacea could go a long way in managing RA. Declarations Acknowledgment The authors are thankful to the Researchers Supporting Project (RSP2024R393), King Saud University, Riyadh, Saudi Arabia. Conflict of Interest: None Funding: None Ethics approval: All protocols for the safe use of animals were adhered to stringently. The Research Ethics Unit of the Biochemistry Department, Ebonyi State University Abakaliki, approved this study approval (Approval No: EBSU/BCH/ET/17/005). The protocols subscribe to the global conventional use of animal models in research. Authors contributions: Conceptualization: Alum and Awoke. Methodology: Alum and Awoke. 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Aloke C, Ibiam UA, Obasi NA, Orji OU, Ezeani NN, Aja PM, et al. Effect of ethanol and aqueous extracts of seed pod of Copaifera salikounda (Heckel) on complete Freund’s adjuvant-induced rheumatoid arthritis in rats. J Food Biochem. 2019 Jul;43(7):e12912. Pearson D. The Chemical Analysis of Foods. Churchill Livingstone; 1976. 600 p. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957 Jul;28(1):56–63. Schumann G, Dominick HC, Hellmann D, Klauke R, Möckesch M, Stekel H, et al. Alkaline Phosphatase Activity: New Assay for the Reflotron® System. Results of the Evaluation in Eight Clinical Laboratories. 2001 Feb 21;39(1):71–8. Janssen GM, Degenaar CP, Menheere PP, Habets HM, Geurten P. Plasma urea, creatinine, uric acid, albumin, and total protein concentrations before and after 15-, 25-, and 42-km contests. Int J Sports Med. 1989 Oct;10 Suppl 3:S132-138. Bain BJ, Bates I, Laffan MA, Lewis SM. Dacie and Lewis Practical Haematology: Twelfth Edition. Dacie and Lewis Practical Haematology: Twelfth Edition. 2016. 1 p. da Silva Pereira A, de Castro IRR, Bezerra FF, Nogueira Neto JF, da Silva ACF. Reproducibility and validity of portable haemoglobinometer for the diagnosis of anaemia in children under the age of 5 years. J Nutr Sci. 9:e3. Ding Q, Hu W, Wang R, Yang Q, Zhu M, Li M, et al. Signaling pathways in rheumatoid arthritis: implications for targeted therapy. Signal Transduct Target Ther. 2023 Feb 17;8:68. Zhang S, Lu S, Li Z. Extrahepatic factors in hepatic immune regulation. Front Immunol. 2022 Aug 16;13:941721. Offor CE, Anyanwu C, Alum EU, Egwu C. Effect of Ethanol Leaf-Extract of Ocimum basilicum on Plasma Cholesterol Level of Albino Rats. 2013; Jang S, Kwon EJ, Lee JJ. Rheumatoid Arthritis: Pathogenic Roles of Diverse Immune Cells. Int J Mol Sci [Internet]. 2022 Jan [cited 2024 Apr 8];23(2). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8780115/ Kondo N, Kuroda T, Kobayashi D. Cytokine Networks in the Pathogenesis of Rheumatoid Arthritis. Int J Mol Sci [Internet]. 2021 Oct [cited 2024 Apr 8];22(20). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8539723/ Tang Y, Varavko Y, Aringazina R, Menshikova I. Changes in renal function and morphological variations of kidney diseases in rheumatoid arthritis patients. Asian J Urol [Internet]. 2022 Sep 13 [cited 2024 Apr 8]; Available from: https://www.sciencedirect.com/science/article/pii/S2214388222001035 Patil KR, Mahajan UB, Unger BS, Goyal SN, Belemkar S, Surana SJ, et al. Animal Models of Inflammation for Screening of Anti-inflammatory Drugs: Implications for the Discovery and Development of Phytopharmaceuticals. Int J Mol Sci [Internet]. 2019 Sep [cited 2024 Apr 8];20(18). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770891/ Noh ASM, Chuan TD, Khir NAM, Zin AAM, Ghazali AK, Long I, et al. Effects of different doses of complete Freund’s adjuvant on nociceptive behaviour and inflammatory parameters in polyarthritic rat model mimicking rheumatoid arthritis. PLoS ONE [Internet]. 2021 [cited 2024 Apr 8];16(12). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8654228/ Kim JS, Kearns DN. Reduced Ethanol Self-administration in Rats Produced by the Introduction of a High Value Non-drug Alternative Reinforcer. Pharmacol Biochem Behav. 2019 Sep;184:172744. de Git KCG, den Outer JA, Wolterink‐Donselaar IG, Luijendijk MCM, Schéle E, Dickson SL, et al. Rats that are predisposed to excessive obesity show reduced (leptin‐induced) thermoregulation even in the preobese state. Physiol Rep. 2019 Jul 24;7(14):e14102. Dombrecht D, Van Daele U, Van Asbroeck B, Schieffelers D, Guns P, Gebruers N, et al. Molecular mechanisms of post‐burn muscle wasting and the therapeutic potential of physical exercise. J Cachexia Sarcopenia Muscle. 2023 Feb 9;14(2):758–70. Zhang M, Xia F, Xia S, Zhou W, Zhang Y, Han X, et al. NSAID-Associated Small Intestinal Injury: An Overview From Animal Model Development to Pathogenesis, Treatment, and Prevention. Front Pharmacol. 2022 Feb 9;13:818877. Mihajlovic M, Vinken M. Mitochondria as the Target of Hepatotoxicity and Drug-Induced Liver Injury: Molecular Mechanisms and Detection Methods. Int J Mol Sci. 2022 Mar 18;23(6):3315. Shu J, Tan A, Li Y, Huang H, Yang J. The correlation between serum total alkaline phosphatase and bone mineral density in young adults. BMC Musculoskelet Disord. 2022 May 18;23:467. Ephraim RKD, Awuku YA, Numekevor P, Botchway F, Adoba P, Dadzie EK, et al. Serum Uric acid is a better indicator of kidney impairment than serum uric acid to creatine ratio ; a cross sectional study of type 2 diabetes mellitus patients. J Diabetes Metab Disord. 2021 Feb 10;20(1):313–20. Ekpono EU, Aja PM, Ibiam UA, Alum EU, Ekpono UE. Ethanol Root-extract of Sphenocentrum jollyanum Restored Altered Haematological Markers in Plasmodium berghei-infected Mice. Earthline J Chem Sci. 2019 Jul 31;2(2):189–203. Alum E, P.C. U. Nutritional Strategies for Rheumatoid Arthritis: Exploring Pathways to Better Management 1,2. 2024 Jan 5; Alum E, Ibiam U, Ugwu O, P.C. U. A Comprehensive Review of Treatment Approaches and Perspectives for Management of Rheumatoid Arthritis 1,2. 2024 Jan 5; Khalaf W, Al-Rubaie HA, Shihab S. Studying anemia of chronic disease and iron deficiency in patients with rheumatoid arthritis by iron status and circulating hepcidin. Hematol Rep. 2019 Mar 12;11(1):7708. Abbaspour N, Hurrell R, Kelishadi R. Review on iron and its importance for human health. J Res Med Sci Off J Isfahan Univ Med Sci. 2014 Feb;19(2):164–74. Nemeth E, Ganz T. Hepcidin and Iron in Health and Disease. Annu Rev Med. 2023 Jan 27;74:261–77. Matsuoka T, Abe M, Kobayashi H. Iron Metabolism and Inflammatory Mediators in Patients with Renal Dysfunction. Int J Mol Sci. 2024 Jan;25(7):3745. Elemam NM, Hannawi S, Maghazachi AA. Role of Chemokines and Chemokine Receptors in Rheumatoid Arthritis. ImmunoTargets Ther. 2020 Mar 9;9:43–56. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4634515","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":331404895,"identity":"cb65ec04-a47f-4157-bc90-8d9363eec932","order_by":0,"name":"Esther Ugo Alum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3SIQvCQBTA8TcGz3K4eseJfoVbsajsqygDDSJotA2EWQTrPoYimAwnAy2CdTYtNkHBYDC4iRoM52yC9w+PKz/eCweg0/1meJ9ZAGPniVTCxCc07a8JMi8NsKz1YnuaQQFps94N2iFYfYnsrCAscDN2sAfbp63pZiRCoKsq8pyCiMhETiQYd7KNCUSAnCqIsw6RXyU4CekkpPCJCHCRg4RaQozkMBETdlQQGrlFNpDU9clhwgLRIPaq1ispBFjD+Z5eZLkyzLTGp8G1lM8vw/nmojKPZa8XiX+BZ5LP5C0jxRadTqf7n24GG0XskW7gQQAAAABJRU5ErkJggg==","orcid":"","institution":"Kampala International University","correspondingAuthor":true,"prefix":"","firstName":"Esther","middleName":"Ugo","lastName":"Alum","suffix":""},{"id":331404896,"identity":"ae35413a-5399-4266-93d7-4023c74b970b","order_by":1,"name":"Rajapandiyan Krishnamoorthy","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Rajapandiyan","middleName":"","lastName":"Krishnamoorthy","suffix":""},{"id":331404897,"identity":"939b16de-a85f-4f9c-98ff-2e8caffb006e","order_by":2,"name":"Mansour K. Gatasheh","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mansour","middleName":"K.","lastName":"Gatasheh","suffix":""},{"id":331404898,"identity":"266e50c6-f569-40ff-9a82-3aeba7e45fb3","order_by":3,"name":"Shanthi Subbarayan","email":"","orcid":"","institution":"Kampala International University","correspondingAuthor":false,"prefix":"","firstName":"Shanthi","middleName":"","lastName":"Subbarayan","suffix":""},{"id":331404899,"identity":"d755c4ea-8e80-413b-b3e3-de9cbddfc62d","order_by":4,"name":"Periyasamy Vijayalakshmi","email":"","orcid":"","institution":"Holy Cross College In","correspondingAuthor":false,"prefix":"","firstName":"Periyasamy","middleName":"","lastName":"Vijayalakshmi","suffix":""},{"id":331404900,"identity":"c3a3318f-fa17-4fcd-8d01-cf364a7eef97","order_by":5,"name":"Joshua N. Awoke","email":"","orcid":"","institution":"Ebonyi State University","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"N.","lastName":"Awoke","suffix":""}],"badges":[],"createdAt":"2024-06-25 07:47:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4634515/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4634515/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61116541,"identity":"8bf552dc-4cdd-4284-9269-149d94737127","added_by":"auto","created_at":"2024-07-25 19:55:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLegend not included with this version\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/b4ada01eff1ce3c8efdecefa.png"},{"id":61116543,"identity":"73d2e04d-77e9-4c47-ad81-5d35a49dd6bd","added_by":"auto","created_at":"2024-07-25 19:55:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1.1. Mineral Constituents of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecoriacea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Ethanol leaf extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/90d4918443767edd518cdb78.png"},{"id":61116540,"identity":"248d421b-d65b-4d6f-888f-81606a06f45e","added_by":"auto","created_at":"2024-07-25 19:55:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":89027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2.0. \u003c/strong\u003eEffect of ELEBC on Paw Size in Adjuvant-induced Arthritic Rats. Multiple variances were used in the comparison and statistically significant levels were indicated with asterisks.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/cce4e0a3eff1dcbff316d907.png"},{"id":61116542,"identity":"dd1f0182-c07e-4004-9899-0896de133092","added_by":"auto","created_at":"2024-07-25 19:55:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3.0 \u003c/strong\u003eEffect of ELEBC on Body Weight of Rats Induced with Rheumatoid Arthritis. Multiple variances were used in the comparison and statistically significant levels were indicated with asterisks.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/187b6189b1f70d4f34f3cab5.png"},{"id":61116675,"identity":"cd0d0514-c0cc-4ac4-bc47-3ea4770b3271","added_by":"auto","created_at":"2024-07-25 20:04:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.0. \u003c/strong\u003eEffect of ELEBC Administration on Enzymatic Markers of Liver Dysfunction in Rheumatoid Arthritis Induced Rats. Two-way ANOVA was used in the comparison and statistically significant levels were indicated with asterisks.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/31155b4bf856aa33a3391510.png"},{"id":61116545,"identity":"f5c005ec-bf29-4790-a2b8-ed273ed8697f","added_by":"auto","created_at":"2024-07-25 19:55:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":207397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.1 (A-D). \u003c/strong\u003eEffect of ELEBC Administration on Non-Enzymatic Markers of Liver Dysfunction in Rheumatoid Arthritis Induced Rats. Two-way ANOVA were used in the comparison and statistical significant levels indicated with asterisks.\u003c/p\u003e","description":"","filename":"4.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/3cd662591d0261bd0542821b.png"},{"id":61116548,"identity":"978223ad-e97b-4a02-b3e4-b32323ca6748","added_by":"auto","created_at":"2024-07-25 19:56:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":29850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5.0 (A-B). \u003c/strong\u003eEffect of ELEBC Administration on Parameters of Renal Dysfunction in Rheumatoid Arthritis Induced Rats. Two-way ANOVA were used in the comparison and statistical significant levels were indicated with asterisks.\u003c/p\u003e","description":"","filename":"55.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/7ebad2d7e4cec84ce31ae522.png"},{"id":61116673,"identity":"be492e8f-c94e-418a-8564-72572f3c9bb6","added_by":"auto","created_at":"2024-07-25 20:03:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":193452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigures 6.0 (A-C). \u003c/strong\u003eEffect of ELEBC Administration on Hematological Markers in Rheumatoid Arthritis Induced Rats. Two-way ANOVA were used in the comparison and statistical significant levels indicated with asterisks.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/bb56f8f94b42d93b6ea541c8.png"},{"id":61116975,"identity":"a9f95f51-3d9f-45d2-ad2c-16775296af79","added_by":"auto","created_at":"2024-07-25 20:11:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":31673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigures 7.0 (A-B). \u003c/strong\u003eEffect of ELEBC Administration on other Hematological Markers in Rheumatoid Arthritis Induced Rats. Two-way ANOVA were used in the comparison and statistical significant levels indicated with asterisks.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/db1192be5a2a0c618e1188f5.png"},{"id":64356626,"identity":"90aca5a9-5aee-4658-9897-bf7530936d4e","added_by":"auto","created_at":"2024-09-12 06:02:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1609710,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4634515/v1/f8abab25-2883-4802-9eeb-be6fbeabddef.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ethanol Leaf Extract of Buchholzia coriacea Ameliorates Biochemical Dysregulations Due to Rheumatoid Arthritis in Wistar Albino Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRA is an autoimmune disease marked by body-wide inflammation, persistent synovitis, and the production of autoantibodies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). It is characterized by some complications like joint damage, disability, cardiovascular and other comorbidities. It also has emotional upset and can lead to decreased quality of life (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). RA is a very common rheumatic disease, with a prevalence of about 1% worldwide (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The ratio of male-to-female prevalence is about 1:3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The cause of RA is still poorly understood. However, the role of genetics in RA presentation cannot be ignored as evidence of a family history of RA abounds (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Environmental and hormonal factors can also trigger inflammatory processes culminating in tissue damage and joint destruction (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Most commonly prescribed drugs in RA management include non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Some other drugs are corticosteroids and disease-modifying anti-rheumatic drugs (DMARDs)(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Regrettably, NSAIDs are expensive, and reports of unfriendly effects like gastric and intestinal tract disturbances are documented (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Due to the shortcomings of conventional drugs, research is in progress to discover better therapeutic alternatives (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The use of herbal medicine especially by the poor rural dwellers has been an ancient alternative treatment regime. \u003cem\u003eB. coriacea\u003c/em\u003e may be an example of such alternative treatment for inflammation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. coriacea\u003c/em\u003e belongs to the family of \u003cem\u003eCapparidaceae\u003c/em\u003e. It is popularly referred to as \u0026ldquo;wonderful kola\u0026rdquo;. It has assorted medicinal values. It is credited with hypoglycemic, hypolipidemic, and lipid peroxidation-reducing properties (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Its anti-microbial, antihelmintic, and antifungal properties have been documented (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The seed extract has anti-ulcer and gastric anti-secretory activities (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Ground-dried leaves and seeds of \u003cem\u003eB. coriacea\u003c/em\u003e are used mixed with warm water or alcohol and taken to alleviate pain and for detoxification by some rural dwellers. Also, ground seed is added to soups and other prepared foods to reduce blood sugar. The leaf is commonly employed in the traditional treatment of inflammation. We have previously revealed the anti-inflammatory and antioxidant effects of \u003cem\u003eB. coriacea\u003c/em\u003e ethanol leaf extract and ethyl acetate fraction(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). As a follow-up, this present study, we investigated other biochemical makers that are dysregulated in rheumatoid arthritis and how this could be ameliorated using ethanol leaf extract of \u003cem\u003eBuchholzia coriacea\u003c/em\u003e (ELEBC).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample Collection, Identification, and Preparation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. coriacea\u003c/em\u003e leaves (BCL) were harvested from Ngodo community in Afikpo North, Ebonyi State, Nigeria. BCL was named and authenticated by a Taxonomist in the Department of Applied Biology, Ebonyi State University, and deposited in the department herbarium for reference purposes (EBSU/APB/HB/2016/101). The method was used for the extraction was as previously reported(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Briefly, \u003cem\u003eB. coriacea\u003c/em\u003e leaves were extensively washed, dried under shade, and further ground. The ground sample was further sieved out of which 800 g were immersed in 2000 ml of ethanol for 48 hours. Thereafter, it was filtered and the filtrate was evaporated to dryness at 35\u003csup\u003e0\u003c/sup\u003eC. The extract was stored in a sealed box and used for the investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Chemicals and Reagents\u003c/h2\u003e \u003cp\u003eFreund\u0026rsquo;s adjuvant and other chemicals used were bought from Sigma Aldrich Company (USA). All the chemicals used were of the standard grade and purchased from reputable dealers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of Phytochemical, Vitamins, and Mineral Compositions of Ethanol leaf Extract (ELEBC).\u003c/h2\u003e \u003cp\u003eThe method of Ugbaja (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) was employed to quantify saponin and terpenoid concentrations. Other phytochemicals such as flavonoids, alkaloids, and glycoside compositions were assessed according to the method outlined by Harborne (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) whereas tannins concentration was quantified by Trease and Evans (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) method. Vitamins and mineral compositions were quantified using AOAC method as reported by Md Noh et al (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Animal Study and Experimental Design\u003c/h2\u003e \u003cp\u003eFor the animal study, 90 female Wistar albino rats (121\u0026ndash;146 g) were purchased from the University of Nigeria Nsukka Enugu, Nigeria, and used for the study. The animals were fed with chow and clean water \u003cem\u003eal libitum\u003c/em\u003e in the laboratory for about a week for acclimatization to take place. After acclimatization, the animals were randomly apportioned into 6 groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15). Group 1 was normal control without induction of RA and treatment. Rats in this group received normal saline (1 ml/kg). Group 2 rats were induced with RA but no treatment was offered rather normal saline was given (1 ml/kg). Animals in Group 3 were induced and treated with 5 mg/kg indomethacin\u0026trade;. Animals in Groups 4\u0026ndash;6 were arthritic rats treated with ELEBC at varied doses of 200, 400, and 800 mg/kg body weight, respectively. The study period was 31 days. The drug, extract, and fractions were administered orally. Variations in body weight and biochemical parameters of the rats in various groups were analyzed. Global approved standard procedure for Experimental Animal Handling as adopted by the Ethical Committee of the Biochemistry Department, Ebonyi State University, Nigeria was strictly followed after due approval (Approval No: EBSU/BCH/ET/17/005). This study was conducted and reported in accordance with Animal Research: Reporting \u003cem\u003eIn Vivo\u003c/em\u003e Experiments (ARRIVE) guidelines.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Acute Toxicity Study\u003c/h2\u003e \u003cp\u003eThe modified Lorke (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) style was employed to check acute toxicity as described in our previous study (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Induction of Rheumatoid Arthritis\u003c/h2\u003e \u003cp\u003ePearson\u0026rsquo;s method (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) was used to induce Rheumatoid Arthritis in rats. Complete Freund\u0026rsquo;s adjuvant (CFA) (0.1 ml) was injected intradermally into the left hind paws of the rats. CFA is made up of heat-killed \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and sterile paraffin oil (10 mg/ml). The paw sizes of all the rats were measured using a calibrated automatic vernier caliper. The body weights of the animals were also measured across groups. On the 10th day, arthritis had fully commenced. Graded doses of ELEBC were administered to rats from day 10 and lasted for 21 days. On days 10, 17, 24, and 31, through a cardiac puncture, blood samples were gotten from three rats from each test group and transferred into plain bottles and later centrifuged for various analyses.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of Liver, Renal, and Hematological Indices\u003c/h2\u003e \u003cp\u003eALT and AST activities were checked with the Randox test kits using the method of Reitman and Frankel and Reitmann (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). ALP activity was assayed using Schumann method (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Total protein, albumin, uric acid, and creatinine concentration were estimated following Janssen et al (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), procedures. Bain et al (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) method was used to assay PCV, RBC, and WBC while Hb and Platelet levels were estimated by da silva Pereira (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Result Analysis\u003c/h2\u003e \u003cp\u003eData generated were analyzed statistically using Graph Pad Prism 5.00. Data were expressed, as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Two-way ANOVA with multiple variances was used for the statistical tests. In general, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was accepted as the statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.1 Chemical Composition of ELEBC\u003c/h2\u003e\n \u003cp\u003eThe quantitative phytochemical constituents of ELEBC are displayed below. The results revealed that the quantities of phenols, alkaloids, terpenoids and flavonoids were very high in the extract, whereas steroids and glycoside were found to be low (Table\u0026nbsp;1). The high presence of the antioxidant phytochemical suggests that ELEBC may have antioxidant potentials.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1: Phytochemical Constituents of Ethanol Leaf Extract of BC.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1721921572.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 1.0\u003c/strong\u003e shows the vitamin compositions of ELEBC. The result revealed that vitamin C was found to be the highest in concentration in ELEBC followed by Vitamin B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e. Vitamins A were amongst the lowest in concentrations in the ELEBC.\u003c/p\u003e\n \u003cp\u003eFurthermore, Fig. \u003cspan\u003e1.1\u003c/span\u003e shows the mineral composition of ELEBC. The result revealed a very high level of Iron in the extract while calcium, magnesium, and phosphorus were in commensurable amounts. Copper and sodium were the least in concentration in ELEBC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.2 Acute Toxicity Result\u003c/h2\u003e\n \u003cp\u003eAdministration of the extracts and fractions did not cause any demonstrable toxic effect even at a 5,000 mg/kg dose.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.3 Effect of ELEBC on Paw Size in Adjuvant-induced Arthritic Rats.\u003c/h2\u003e\n \u003cp\u003eInjection of Freund\u0026rsquo;s adjuvant led to significant elevation in the paw sizes of the animals across the groups beginning from day 10 as shown in Fig. \u003cspan\u003e2.0\u003c/span\u003e. More so, redness of the swollen part was visually observed during the period of the study further indicating possible inflammation. Interestingly, administration of the graded doses of ELEBC resulted in a reduction of the paw sizes of the animals to levels similar to those treated with the standard drug. More interestingly, as the days of administration increased, the paw sizes of the animals further reduced and tended towards the paw sizes of the normal rats suggesting a possible complete reversal of the physiological and biochemical dysregulations caused due to arthritis induction in the study rats.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e3.4 Impact of ELEBC on Body Weight of Rats Induced with Rheumatoid Arthritis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan\u003e3.0\u003c/span\u003e reveals the impact of ELEBC on the body weight of rats induced with rheumatoid arthritis. The result revealed that induction of rheumatoid arthritis caused a significant abnormal reduction in the body weights of the rats over the study period. Interestingly, oral administration of ELEBC normalized this distortion in body weight and maintained the body weights of the animals similar to the effect of standard drugs against the impact of rheumatoid arthritis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e3.5 Effect of ELEBC Administration on Enzymatic Markers of Liver Dysfunction in Rheumatoid Arthritis-Induced Rats.\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan\u003e4.0\u003c/span\u003e \u003cstrong\u003e(A-C)\u003c/strong\u003e reveals the effect of ELEBC administration on enzymatic markers of liver dysfunction due to rheumatoid arthritis. The result shows that induction of rheumatoid arthritis caused a significant elevation in the activities of Alanine aminotransferase (A) and Aspartate aminotransferase (B) and irregularity in the activity of alkaline phosphatase (C) especially in 24th and 31st days of study respectively. Interestingly, in all the enzymes investigated, administration of ELEBC reversed these trends.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e3.6 Effect of ELEBC Administration on Non-Enzymatic Markers of Liver Dysfunction in Rheumatoid Arthritis-Induced Rats.\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan\u003e4.1\u003c/span\u003e \u003cstrong\u003e(A-D)\u003c/strong\u003e shows the effect of ELEBC administration on (A) Total Bilirubin (B) Conjugated Bilirubin (C) Total Protein (D) Albumin levels respectively of rheumatoid arthritis-induced rats. The result revealed that induction of RA caused slight distortions in the serum levels of the markers although not significant across the days of the studies. Administration of ELEBC however normalized the dysregulation and prevented significant distortions in the serum levels of the markers.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.7 Effect of ELEBC Administration on Markers of Renal Dysfunction in Rheumatoid Arthritis-Induced Rats.\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan\u003e5.0\u003c/span\u003e shows the effect of ELEBC administration on (A) Creatinine and (B) Uric acid levels respectively, of rheumatoid arthritis-induced rats. The results revealed that induction of rheumatoid arthritis led to a sustained elevation in the serum level of creatinine across the days of the study an indication of severe damage to the nephritic system in addition to destruction of muscle mass. Uric acid levels were also distorted as seen in \u003cstrong\u003eFig.\u0026nbsp;5B\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.8 Effect of ELEBC Administration on Hematological Markers in Rheumatoid Arthritis-Induced Rats.\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan\u003e6.0\u003c/span\u003e shows the effect of ELEBC administration on (A) PCV, (B) RBC, and (C) Hb, respectively of rheumatoid arthritis-induced rats. Rheumatoid arthritis led to a significant decrease in PCV, RBC, and Hb levels respectively. Interestingly, the administration of various doses of ELEBC significantly prevented this abnormal decrease in the levels of these important markers of hematological dysregulations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.9 Effect of ELEBC Administration on other Hematological Markers in Rheumatoid Arthritis Induced Rats.\u003c/h2\u003e\n \u003cp\u003eFigures \u003cspan\u003e7.0\u003c/span\u003e shows the effect of ELEBC administration on (A) Erythrocyte Sedimentation Rate (B) white blood cells of rheumatoid arthritis induced rats. In this study, induction of rheumatoid arthritis produced elevation in the concentrations of these markers, an indication of severe inflammation. Conversely, oral administration of ELEBC significantly prevented the elevation of these markers of inflammation to levels comparable with the standard treatment and normal control groups respectively.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study investigated the impact of ethanol leaf extract of \u003cem\u003eBuchholzia coriacea\u003c/em\u003e (ELEBC) on biochemical dysregulations due to rheumatoid arthritis induction in rats. First, we investigated the chemical composition of the leaves of \u003cem\u003eBuchholzia coriacea\u003c/em\u003e to ascertain whether it has relevant bioactive components. The result of quantitative Vitamin, Phytochemical and Mineral analysis of \u003cem\u003eBuchholzia coriacea\u003c/em\u003e leaves (BCL) revealed that BCL has important compounds in varying concentrations, which could have impacted the positive ameliorative trend observed in the \u003cem\u003ein vivo\u003c/em\u003e studies. There were high levels of Vitamin C, B\u003csub\u003e1,\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e in the extract in addition to high levels of phenols, flavonoids, alkaloids, and terpenoids. Iron was highest in the mineral constituents present in ELEBC. The above bioactive compounds are known to have various physiological potentials ranging from antioxidant to anti-inflammatory potentials respectively.\u003c/p\u003e \u003cp\u003eFurthermore, the result from the \u003cem\u003ein vivo\u003c/em\u003e studies revealed that administration of ELEBC at varied doses of 200, 400 and 800 mg/kg displayed anti-rheumatoid arthritis potentials in the albino rats. The ELEBC reduced the inflamed paw volumes and also led to significant elevations in lowered body weight of the arthritic rats. Rheumatoid arthritis further caused some biochemical abnormalities and rats administered with varied doses of ELEBC displayed a reversal of these deleterious abnormalities. This indicates that ELEBC has ameliorating potentials in the rheumatoid arthritis-induced liver, renal and hematological dysfunctions.\u003c/p\u003e \u003cp\u003eRheumatoid arthritis is a disease that involves many body organs like the liver, heart, and kidney (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Some functions of the liver include the metabolism of drugs, detoxification, synthesis, and storage of some vital biomolecules. The liver is an essential modulatory organ whose roles during autoimmune and other chronic inflammatory diseases are indispensable (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Hepato-protective effects of medicinal plants are reported in our earlier studies (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Severe rheumatoid arthritis is marked by the over-production of autoantibodies, inflammatory cytokine release, and immune complex deposition (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). RA, like other inflammation disorders, can lead to changes in the hematopoietic system (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Thus, the assessment of hematologic indices could be helpful in evaluating disease severity or the extent of recovery of arthritic patients.\u003c/p\u003e \u003cp\u003eRA being a systemic disorder can also affect kidney adversely (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Creatinine and uric acid are well-known markers of glomerular filtration rate (GFR). Elevated creatinine and uric acid levels could be due to lowered capacity of nephrons culminating in a decrease in the kidney’s filtering capacity and subsequent accumulation of waste products within the system (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In this study, some of the biochemical markers of the liver (ALT, AST, ALP, total protein, albumin, total and conjugated bilirubin), hematological (Hb, PCV, RBC, WBC, and ESR) and renal (creatinine and uric acid) functions were investigated. Our study revealed a bifold increase in paw size of the arthritic rats. Tang \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)) made a similar observation. We observed a reduced paw size after administering standard drugs and various doses of ELEBC to rats. Previous studies have also documented the paw size-reducing properties of some medicinal plants in arthritic rats (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEdema is a key attribute of acute inflammation and hence always monitored when examining anti-inflammatory potential of any compound (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Manifestation of inflammation causes vasodilation and increased blood flow culminating in increased heat and redness. Inflamed rat paws heighten extra vascular protein infiltration and subsequent edema. Extravascular protein infiltration is aided by enhanced vascular permeability of blood vessels, culminating in the discharge of plasma proteins and fluids into the tissue, and consequent edema and swelling. It has been reported that hind paw swelling at the site of injection of Freund’s adjuvant connotes inflammation which is the initial presentation of RA (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMarkedly, at the first week of induction of RA, there was an obvious reduction in body weight. This trend was however reversed when the animals received the standard drug and various doses of ELEBC. A continuous reduction of weight was noticed in rats that received no drug or ELEBC. This result conforms to the study of other authors (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Perhaps this reduction in body weights of the rats may be owing to elevated leptin amount as suggested by Mariam \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Leptin is a hormone made by fat cells which subdue hunger and also influences the immune system. An increase in leptin level can lead to the generation of pro-inflammatory cytokines culminating in chronic inflammation if not arrested (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). The presence of other inflammatory mediators can also lead to increased leptin levels (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is pertinent to note that raised concentration of pro-inflammatory cytokines could impair energy metabolism and also enhance muscle protein breakdown. Enhanced catabolism accelerates resting energy expenditure leading to weight loss and reduced lean body mass (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The increase in body weight in rats that received standard drug and extract could be attributed to the decline of the inflammatory cytokines coupled with a decline in protein and muscle breakdown. Inflammation can also cause a reduction in the absorption ability of the intestine(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). According to Zhang \u003cem\u003eet al.\u003c/em\u003e (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), the absorption power of the intestine was restored when anti-inflammatory drugs were administered and weight gain was observed. Therefore, the observed significant gain in body weight of rats treated with standard drug and extract could be due to enhanced absorption capacity of the intestine.\u003c/p\u003e \u003cp\u003eAdditionally, our results showed high activities of liver enzymes (ALT, AST, and ALP) as well as raised total and conjugated bilirubin in arthritic rats while lowered albumin level was observed. Administration of indomethacin and samples to rats reversed this trend in a time and dose-dependent approach except for AST activity which had no change on the administration of ELEBC. Usually, AST and ALT are domiciled in the liver. However, when hepatocellular integrity is impaired, these enzymes leak into the general circulation (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The fact that AST activity remained unchanged after standard drug and sample administration shows that the samples produced no harmful effect on the liver of rats nor did it ameliorate the high activity of AST seen as a consequence of induction of arthritis.\u003c/p\u003e \u003cp\u003eAdministration of standard drug and ELEBC to arthritic rats significantly lowered the raised ALP level. ALP is commonly present in the liver, bile, and other organs. Increased activity of ALP could portend biliary obstruction, gallstone, and other liver diseases. ALP activity can also rise when there is a bone disease where osteoblastic activity is enhanced (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdjuvant administration caused a significant increase in uric acid and creatinine concentrations. Treatment did not cause any alteration in uric acid level even at dose 800 mg/kg in the arthritic rats. More so, treatment with standard drug, and ELEBC yielded no effect on creatinine level on days 10 and 17. However, ELEBC treatment produced highest reduction on creatinine level on days 24 and 31 of the investigation. Plasma creatinine is an established marker of glomerular filtration rate (GFR). Elevated creatinine and uric acid concentration could signify impairment in filtering capacity of kidney and this could result to pileup of unwanted products within various systems (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Perhaps, the observed non-alteration in uric acid and creatinine concentrations in treated groups portends that the administration of ELEBC probably did not alter renal function indices.\u003c/p\u003e \u003cp\u003eFurthermore, concentrations of Hb, PCV, and RBCs in rats were significantly reduced on induction with rheumatoid arthritis. Interestingly, the administration of standard drugs and varied doses of ELEBC reversed this trend. Several authors have also reported marked improvement in Hb, PCV, and RBCs in rats administered with medicinal plant extracts (\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e–\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Anemia is a very recurrent feature observed in RA patients. The commonest one is anemia of chronic disease (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). It is mostly a result of chronic blood loss from gastritis (induced by NSAID\u003csub\u003eS\u003c/sub\u003e), peptic ulcer, or gastroesophageal reflux. The reduction in the Hb concentration could have been caused by the reduced lifespan of RBCs and impaired iron absorption and utilization (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Inflammatory mediators released during anemia in RA patients can induce excess production of hepcidin. Hepcidin is a hormone that regulates iron metabolism. It hinders iron exportation from cells by reducing the activity of ferroportin (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWBCs are biological soldiers recruited against foreign attacks. Induction of RA resulted in a rise in WBC counts. The stimulation of the immune system by the adjuvant could have led to the generation of WBCs. Reduction in WBC levels was remarkable during treatment. This is suggestive of the immune system suppressing effect and consequent WBCs lowering the ability of the samples. We also observed that induction of RA caused a significant increase in the erythrocyte sedimentation rate in the arthritic rats, which suggests an increase in inflammation. This result corroborates the findings of previous authors(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). During periods of RA, cytokines like TNF-α, IL-6, and IL-1β play contribute to disease severity. Lymphocytes are affiliated with arthritic diseases since their signaling could elicit autoimmunity. Lymphocyte activation could result in chronic inflammation, damage to structure and function (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e)\u003c/p\u003e "},{"header":"CONCLUSION","content":"\u003cp\u003eThis study has revealed the possible use of ethanol extract from \u003cem\u003eB. coriacea\u003c/em\u003e leaves for treating RA based on the observed effects in arthritic rats and its non-toxic effect on the liver and kidney. We also observed that the extract lowered RA symptoms by ameliorating inflammatory, liver, and hematological markers. This could be attributed to the vitamins, minerals, and phytochemicals present in \u003cem\u003eB. coriacea\u003c/em\u003e leaves. Since some medicinal plants and synthetic drugs used in the treatment of RA and pain have been shown to have many side effects, especially on the liver and kidney, coupled with high cost, the use of \u003cem\u003eB. coriacea\u003c/em\u003e could go a long way in managing RA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the Researchers Supporting Project \u003cstrong\u003e(RSP2024R393),\u003c/strong\u003e King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e None\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e All protocols for the safe use of animals were adhered to stringently. The Research Ethics Unit of the Biochemistry Department, Ebonyi State University Abakaliki, approved this study approval (Approval No: EBSU/BCH/ET/17/005). The protocols subscribe to the global conventional use of animal models in research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eAlum and\u0026nbsp;Awoke.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u0026nbsp;\u003c/strong\u003eAlum\u0026nbsp;and Awoke.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u0026nbsp;\u003c/strong\u003eKrishnamoorthy,\u0026nbsp;Gatasheh, and\u0026nbsp;Subbarayan\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e Subbarayan and\u0026nbsp;Vijayalakshmi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u0026nbsp;\u003c/strong\u003eAlum and Awoke\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e Alum and Awoke\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting \u0026ndash; original draft:\u0026nbsp;\u003c/strong\u003eAlum,\u0026nbsp;Krishnamoorthy and\u0026nbsp;Gatasheh\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting \u0026ndash; review \u0026amp; editing:\u0026nbsp;\u003c/strong\u003eAlum,\u0026nbsp;Krishnamoorthy,\u0026nbsp;Gatasheh, Awoke,\u0026nbsp;Vijayalakshmi,\u0026nbsp;and\u0026nbsp;Subbarayan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All Authors read and approved the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing statement:\u003c/strong\u003e Data are available from the corresponding author (EU Alum), upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuo Q, Wang Y, Xu D, Nossent J, Pavlos NJ, Xu J. 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ImmunoTargets Ther. 2020 Mar 9;9:43\u0026ndash;56.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rheumatoid arthritis, Buchholzia coriacea, indomethacin, adjuvant","lastPublishedDoi":"10.21203/rs.3.rs-4634515/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4634515/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRheumatoid arthritis (RA) is a disease that involves many body organs. In this study, we examined the anti-rheumatoid arthritis effect of ethanol leaf extract of \u003cem\u003eBuchholzia coriacea\u003c/em\u003e (ELEBC).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFirstly, the chemical compositions of ELEBC were investigated. The \u003cem\u003ein vivo\u003c/em\u003e study was further done using 90 female rats weighing 121\u0026ndash;146 g. Rats were appropriated into 6 groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15). Group 1 was provided with normal saline (1 ml/kg) only. Group 2 was induced with RA and untreated. Group 3 was induced and treated with 5 mg/kg indomethacin\u0026trade; while Groups 4\u0026ndash;6 were induced with RA and treated with ELEBC at varied doses of 200, 400, and 800 mg/kg body weight, respectively. Induction of RA was done using Freund\u0026rsquo;s adjuvant, whereas the route of administration of the standard drug and the extract was via oral intubation. The study period was 31 days.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe chemical composition analysis revealed that ELEBC has a high level of various chemical constituents. The adjuvant injection caused a significant increase in paw sizes plus a reduction in body weight. Levels of creatinine, uric acid, total protein, white blood cell, and total and conjugated bilirubin were significantly elevated in the arthritic rats. There were significant elevations in alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase activities while the levels of red blood cells, packed cell volume, and hemoglobin were lowered significantly. Treatment with ELEBC markedly reduced the paw sizes and caused weight gain.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOther biochemical dysregulations were also ameliorated. Thus, ELEBC may be useful in the control of RA.\u003c/p\u003e","manuscriptTitle":"Ethanol Leaf Extract of Buchholzia coriacea Ameliorates Biochemical Dysregulations Due to Rheumatoid Arthritis in Wistar Albino Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 19:55:55","doi":"10.21203/rs.3.rs-4634515/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb7801bf-a9d3-48ad-aa4c-4dc5758b65f0","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-12T05:54:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-25 19:55:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4634515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4634515","identity":"rs-4634515","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}