Evaluation of the inhibitory potential of flavonoid-rich fraction of Myrica esculenta against DSS-induced colonic inflammation in mice

Evaluation of the inhibitory potential of flavonoid-rich fraction of Myrica esculenta against DSS-induced colonic inflammation in mice | 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 Research Article Evaluation of the inhibitory potential of flavonoid-rich fraction of Myrica esculenta against DSS-induced colonic inflammation in mice Monika Joshi, Manju Pandey, Akash Ved This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3832022/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 Myrica esculenta (family Myricaceae) is a plant species valued in India and China for the management of gut disorders. Scientific validation of its anti-ulcerative colitis activity was aimed. Methods and Results The ethyl acetate fraction of Myrica esculenta (MeEa) was prepared and evaluated for its potency against DSS-induced ulcerative colitis (UC) in mice at 200 and 400 mg/kg BW oral dose. The effective dose of MeEa was determined through its effect on DSS-induced UC and was further analyzed through its effects on disease activity index (DAI), colon length, colon weight/length ratio, spleen weight, serum and colon tissue cytokine level, cell count (total WBC, lymphocytes, monocytes, granulocytes, RBC and platelet) and hemoglobin content. Furthermore, the effect was determined through histopathology and FITC-dextran-induced membrane permeability assay. Between the two doses MeEa at 400 mg/kg BW was found to be the most effective dose in terms of reduced DAI scores, which were increased due to DSS administration, protected colon length from shortening, decreased colon weight/length ratio, reduced spleen weight, decreased pro-inflammatory cytokine (IL6, IL8, TNF α and IFN γ) level and stabilized the anti-inflammatory cytokine (IL10) level in serum and colon tissue. MeEa 400 reduced cell counts and increased hemoglobin content and platelet count. Furthermore, MeEa 400 prevented the colon by protecting epithelial cells and crypts. MeEa 400 provided significant protection from intestinal leakage and reduced FITC dextran level in serum. Conclusions MeEa 400 possesses significant anti-inflammatory potential and acts via attenuation of DSS-induced UC and inhibition of DAI scores. It reduces pro-inflammatory cytokines and stabilizes anti-inflammatory cytokine levels, reduces cell count, and protects epithelial tissue and crypts in the colon as well as intestinal membrane leakage that occurred due to FITC-dextran administration in mice. Myrica esculenta ulcerative colitis inflammatory bowel disease colon DSS FITC etc Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Morella esculenta , synonym Myrica esculenta Buch.-Ham.ex D. Don and Myrica nag belongs to Myricaceae family, is found worldwide in tropical and sub-tropical regions and is known for its nutritive value and health advantages [ 1 ]. Approximately 12 to 15 meters high, this medium-sized to large woody, evergreen dioecious tree has a trunk girth of 92.5cm, light brown to black bark, green lanceolate leaves and bracteate pistillate flowers [ 2 ]. The plant is commonly referred to as Kaphal or Soh-Phie. Myrica esculenta ( M. esculenta ) is extensively spread in the mid Himalayas, Kasia hills, Sylhet in India and China, Japan, Australia, Taiwan, South Africa, North America, Brazil, Ethiopia and Nepal [ 3 , 4 ]. The bark of M. esculenta exhibits extreme potential and is used for treating a variety of ailments like asthma, cough, fever, inflammation, ulcers, diarrhoea, nose, ear and throat infections. The hydrolyzable tannin castalagin is present in M. esculenta bark along with gallic acid, myricanol, myricanone, and epigallocatechin 3-O-gallate, as well as two prodelphinidin dimers, namely epigallocatechin-(4β→8)-epigallocatechin 3-O-gallate and 3-O-galloyl epigallocatechin-(4β→8)-epigallocatechin 3-O-gallate .The presence of flavonoids and steroids in M. esculenta has contributed to its pharmacological activity against inflammation [ 5 ]. An inflammatory illness that affects the colon and rectum to varying degrees is ulcerative colitis (UC). UC is a type of Inflammatory Bowel Disease (IBD) directing various gastrointestinal diseases [ 6 ]. UC is a chronic condition leading to 5 million cases globally in 2023 and the incidence is still rising. People with abnormalities in the gut epithelial barrier, the microbiota, the irregulated response of the immune system and genetic predisposition are more likely to develop UC. Symptoms of the disorder include pain in the abdomen, bloating, diarrhoea, blood in stool leading to fatigue, fever, anaemia and weight loss. 5-aminosalicylic acid medications, thiopurines, biologics (such as anti-integrins and anti-cytokines), and small molecules (such as sphingosine-1-phosphate receptor modulators and Janus kinase inhibitors) are examples of maintenance therapy. These treatment approaches have high costs and are associated with several side effects like metabolic changes, lymphomas, increased susceptibility to infections and a higher rate of mortality [ 7 ]. Mesalamines are the first choice of therapy for the UC and are available in different dosage forms like oral (AsacolTM, PentasaTM, AprisoTM, ColazalTM), suppository (CanasaTM) and liquid enema (RowasaTM) [ 8 ]. However, its administration has resulted in acute intolerance syndrome, symptoms of which are similar to the exacerbation of UC making it difficult to differentiate [ 9 ]. Complementary and alternative medicine (CAM) like herbal therapy targets multiple factors and offers more patient acceptability and fewer side effects. In recent years use of CAM has increased because of its natural origin [ 10 ]. Numerous individual herbs (Indigo naturalis, Lycium ruthenicum Murray, Abelmoschus manihot, Centella asiatica) , Chinese herbal formulas (Baweixilei-San, Huanglian Jiedu Decoction, Qingchang Huashi Formula etc.) have shown protective role against UC by affecting intestinal immunity and gut flora [ 11 ]. Thus M. esculenta bark contains several active constituents making it a potential herb for numerous inflammatory disorders. However, none of the literature has reported its activity for the UC. In the current research, we have explored the ME bark for the UC using DSS induced IBD model. The primary aim of the treatment is to improve the remission rates, decrease side effects and improve the patient's quality of life. M. esculenta is an herbal therapy that may serve the current needs of the patient. 2. Material and Methods 2.1. Chemicals and reagents The reagents and chemicals used in the present study were of analytical grade. The DSS (colitis grade) was purchased from MP Biomedicals, California. Mesalazine was purchased from Himedia, India. All the biochemical kits were purchased from Agappe Diagnostic Ltd., Kerala, India. ELISA kits were purchased from Krishgen Biosystems, Mumbai, India. 2.2. Plant material The stem bark of Myrica esculenta ( M. esculenta ) was purchased from the local herbal market in Lucknow (U.P.). India. The plant material was authenticated and obtained a voucher specimen number, i.e., NIScPR/RHMD/Consult/2022/4414-15, by the Head, Raw Material Herbarium and Museum (RHMD), CSIR-National Institute of Science Communication and Policy Research (NIScPR), Delhi, India. 2.3. Extraction Procedure The stem bark of M. esculenta was cleaned, shade-dried, and converted into a coarse powder using a milling machine, Milcent Appliances Pvt. Ltd., Gujarat, India. The 200 g of coarse powder was further put into the soxhlet apparatus and kept for 6 h on heating mental for extraction using ethyl acetate as solvent. The filtrate was then concentrated in a rotary evaporator (R-210 BUTCHI, Switzerland), dried in a water bath, weighed, and stored in a refrigerator for further evaluation. The extractive yields of the extract was calculated and found to be 21% [ 12 ]. 2.4. High performance liquid chromatography (HPLC) The HPLC profiling of the ethyl acetate fraction of M. esculenta (MeEa) was done and characterizes the presence of certain flavonoid compounds such as gallic acid (GA), myricetin (My), and quercetin (Q) following the previously published literature with slight modifications [ 13 ]. The quantification of GA, My and Q was done using the HPLC platform Shimadzu, Japan; Prominence (Pump: LC-20AD, Autosampler: SIL-20AC, Oven: CTO-10ASvp) connected with a photodiode array detector (PDA: SPD-M20A), software (LabSolutions 6.72 SP1) and a C18 column (ShimadzuRP-C-18, 250 x 4.6 mm, 5µm pore size). The method comprised solvent A (1% acetic acid) in water and solvent B (acetonitrile) in isocratic elution mode with a flow rate of 0.6 ml/min. The injection volume was 20 µl, and the detector wavelength was set at 254 nm. The stock solutions (1 mg/ml) of standards (GA, My, and Q) were prepared in HPLC-grade methanol. For the identification and quantification of flavonoids in MeEa, UV-absorbance and retention time were compared with those of the standards. 2.5. In vivo studies Animal experiments in the study were performed following the guidelines of the Organization for Economic Co-operation and Development (OECD) (1987) for the care and use of laboratory animals. Adult (6–7 weeks old) Swiss albino mice (20–25 g) were used in the study. All the experimental animals were acclimatized for a period of 1 week prior to the experiment and maintained at normal laboratory conditions (25 ± 1°C, 55 ± 5% relative humidity and an automatic 12 h light/12 h dark cycle). Animals were provided with the commercial standard diet and water ad libitum . The experiments on ulcerative colitis activity were carried out under the protocol CCSEA/IAEC/SLSRPL/18/07/2023-02, which was approved by the Institutional Animal Ethics Committee (IAEC) constituted under the Committee for Control and Supervision of Experiments on Animals (CCSEA), Govt. of India. 2.5.1. DSS-induced UC Thirty Swiss albino mice were randomly divided into five groups (n = 6/group). UC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%). Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively [ 14 , 15 ]. In Groups 3, 4, and 5, oral doses of mesalazine, MeEa 200 and MeEa 400 were started in parallel with the DSS (3%) induction from day one, once a day for seven days. A daily record of body weight and disease activity index (DAI) was kept throughout the seven days of the experiment. On the eighth day prior to the animal sacrifice, blood collection was done; thereafter, collection of organs (colon and spleen) and measurement of colon weight and length were also performed [ 16 ]. 2.5.2. Evaluation of disease activity index (DAI) The DAI scores in all the animals were evaluated on parameters such as visible stool consistency, presence of blood in stool and body weight loss on a 4-point scale (0–4). Where 0 signifies formed and hard stool, 1 shows formed but soft stool, 2 for loose stool and 3 signifies runny and watery stool. The percent (%) weight loss in animals was calculated relative to the percent body weight of day one. Further, the traces of the occult blood in the stool were analyzed by using the Hemoccult SENSA single-slide rapid diagnostic kit based on the following criteria: 0 shows hemooccult negative (-), 1 for hemooccult positive (+), 2 signifies hemoocult positive (++) and 3 reports bleeding through the anus site (+++) [ 17 ]. 2.5.3. Analysis of colon weight to length ratio After the sacrifice of each mouse, their colons were carefully removed, opened longitudinally and washed with normal saline to remove faecal matter present in the colon. Thereafter, the wet weight of the colon, approximately 10 cm long, was measured. The wet weight of each colon was then divided by the colon length to calculate the colon weight-to-length ratio [ 18 ]. 2.5.4. Estimation of inflammatory cytokine level in serum and colon tissues Serum and colon tissue samples were analyzed to determine the concentration of inflammatory cytokines (IL-6, IL-8, IL-10, TNF-α, and IFN-γ) by using ELISA kits from Krishgen Biosystems, Mumbai, India. The ELISA tests were performed according to the manufacturer’s instructions. 2.5.5. Hematological and biochemical analysis The blood was withdrawn from the retro-orbital plexus of each mouse into two tubes. The first tube was the heparinized tube that contained whole blood to assay the complete blood count (CBC) in a hematology analyzer, Mindray BC 3000 Plus, Agappe, India. The second tube was a non-heparinized tube that contained the blood from which serum was separated to perform the ELISA assays (as mentioned above, section 2.5.5 ) and biochemical tests. The biochemical tests were performed using biochemical kits from Agappe Diagnostic Ltd., Kerala, India, for the estimation of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphates (ALP), bilirubin, creatinine, urea, uric acid HDL, and LDL. The biochemical assays were done as per the manufacturer’s protocol. 2.5.6. Histopathology and inflammatory score Colon tissues were fixed with 10% neutral-buffered formalin and embedded in paraffin wax. The 5 µm sections were cut and stained with hematoxylin and eosin (H&E) for histopathological evaluation. The sections were observed under a high-resolution microscope (at 400X), Lieca, Germany, to determine the degree of colonic inflammation resulted due to DSS administration, which was scored on a subjective scale of 0–4, whereby 0 denoted normal colonic mucosa (nil inflammation); 1 denoted loss of one-third of the crypts; 2 denoted loss of two-thirds of the crypts; 3 denoted a single disruptive epithelial layer covering the lamina propia; 4 erosions and significant inflammatory cell infiltration [ 19 ]. 2.6. Assessment of intestinal membrane permeability using FITC dextran The assessment of intestinal permeability was done by using FITC (Fluorescein isothiocyanate-dextran sulfate sodium salt; 4000 kDa, Sigma-Aldrich, USA) following the method published earlier [ 20 ]. Swiss albino mice were administered with DSS for fourteen days thereafter all the mice were allowed to fast for 12h. Mice were then administered with FITC-dextran (60 mg/100 g BW) via intragastric infusion. After four hours blood was collected from all the mice and centrifuged at 3500 rpm for 10 min., The obtained serum was further diluted with an equal volume of phosphate buffer saline (PBS). The concentration of FITC-dextran in serum was determined by using a fluorescence spectrophotometer (Eppendorf, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The concentration of FITC-dextran in the serum was calculated from the standard curve of diluted FITC-dextran obtained in non-treated serum. 2.7. Statistical analysis Results are demonstrated as the mean + SEM (n = 6) of the number of independent experiments. Statistical significance between the experimental groups was determined by the one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Data with a p-value < 0.05 were considered significant. Analyses of the data obtained were done using GraphPad Prism software version 5.01. 3. Results 3.1. HPLC analysis of MeEa The chemical profiling of MeEa indicated the presence of targeted flavonoids (GA, My, and Q), which were expressed in terms of area%, i.e., GA-8.564, My-2.486 and Q-0.167. The findings obtained were comparable with those of the standard mix, whose area% was found to be GA-12.155, My-41.549 and Q-46.296 respectively (Fig. 1 ). 3.2. Effect of MeEa against DSS-induced UC in mice After DSS (3%) induction in Groups 2–5, there was a development of symptoms of acute UC in mice, which were revealed by the alteration in certain parameters such as a decrease in body weight (Fig. 2 A), reduced average food intake (Fig. 2 B), a significant change in stool consistency (Fig. 2 C), and the presence of blood in the stool (Fig. 2 D). The above parameters were extremely altered in the NC group as compared to the NC group. Further, a significant difference was observed in terms of protection in the PC group than the NC group. The PC group was administered with the standard drug mesalazine at 100 mg/kg BW. The treatment with MeEa 200 did not provide significant protection against DSS-induced damage. It was noticeable that the group treated with MeEa 400 was found to be effective in normalizing the diseased condition induced by the DSS. A significant difference was observed after the administration of MeEa at 400 mg/kg BW in terms of % weight loss, average food intake, mean stool consistency, and mean stool blood score, especially on days 5 and 7 than the NC group. 3.3. Effect of MeEa on colon and spleen tissues To determine the extent of inflammation that occurred due to the induction of DSS, the measurement of colon length was performed ( Fig. 3 ) . As observed in Fig. 3 A and Fig. 3 B there was a significant reduction in the length of the colon in the NC group as compared to the VC group. In the PC group, it was observed that after treatment with mesalazine at 100 mg/kg BW the colon was significantly protected which was observed by its normal structure and length as compared to the NC group whereby the length of the colon was shortened and prominent tissue damage occurred. In the group treated with MeEa 200 there was slight to negligible protection was seen due to the lower dose but at a higher dose i.e. MeEa 400 there was significant protection to the colon tissue was observed than in the NC group ( Fig. 3 C). In Fig. 3 C the colon weight/length ratio (mg cm-1) was significantly increased in the NC group as compared to the the VC group. In the PC group, a significant reduction in colon weight/length ratio was observed as compared to the NC group. Treatment with MeEt 400 also showed significant protection that resulted in a decreased colon weight/length ratio than the NC group. MeEa 200 showed lesser protection against colon weight/length ratio than the NC group. It is well accepted that the increased weight of the spleen is markedly associated with the increased risk of UC. In Fig. 3 D there was a significant increase in the weight of the spleen in the NC group due to DSS administration as compared to the VC group. A significant reduction in the weight of the spleen was recorded in the PC group than in the NC group. Treatment with MeEa 400 significantly inhibited the increased weight of the spleen in the NC group. The effect of MeEa 400 was comparable with that of the PC group. Furthermore, there was no significant protection was observed in the case of MeEa 200 treatment. 3.4. Effect of MeEa on serum and colon tissue cytokines The inflammatory responses in UC begin with an increased level of inflammatory cytokines (IL6, IL8, TNF-α and IFN-γ) and a decreased level of anti-inflammatory cytokine (IL10) in serum (left panel) and colon tissue (right panel) samples. The resultant effects of MeEa in serum and colon tissue cytokines were examined by using the ELISA technique ( Fig. 4 ) . Serum and colon tissue samples showed similar patterns of cytokine levels in both cases. In the NC group, there was a significant increase in inflammatory cytokines levels and a decrease in anti-inflammatory cytokine levels due to the administration of DSS (3%) as compared to the VC group. Groups treated with mesalazine (PC group) or MeEa 400 significantly reduced the level of increased inflammatory cytokine level and normalized the level of anti-inflammatory cytokine in serum as well as colon tissue than the NC group respectively. Further treatment with MeEa 200 moderately reduced the level of elevated inflammatory cytokines and enhanced the level of anti-inflammatory cytokine in serum and colon tissues than the NC group. It was also observed that the effect exerted by MeEa 200 was not statistically significant as compared to the PC group and MeEa 400 group. 3.5. Effect of MeEa on hematological parameters The whole blood samples were used to determine cell counts ( Fig. 5 ) . A significant increase in total WBC count was observed in the NC group due to DSS administration as compared to the VC group (Fig. 5 A). Treatment with 100 mg/kg BW mesalazine (PC group) or MeEa 400 significantly decreased the total WBC count as compared to the NC group. Similarly, the level of lymphocytes, monocytes, granulocytes and RBC counts increased significantly in the NC group as compared to the VC group. Treatment with mesalazine or MeEa 400 significantly attenuated the increased level of lymphocytes, monocytes, granulocytes and RBC as compared to the NC group (Fig. 5 B, 5 C, 5 D and 5 E). After the administration of DSS, there was a significant reduction in the level of hemoglobin content and platelet count in the NC group as compared to the VC group. Groups treated with mesalazine or MeEa 400 significantly normalize the level of hemoglobin content and platelet count as compared to the NC group. It was also observed that mesalazine performed marginally better than MeEa 400 (Fig. 5 F and 5 G). Further, treatment with MeEa 200 did not markedly attenuate the increased level of cell count, hemoglobin content and platelet count as compared to the NC group. 3.6. Effect of MeEa on serum biochemical parameters Studies have shown that there is a marked imbalance in biochemical parameters in UC that leads to kidney and liver disorders. Thus biochemical tests such as SGOT, SGPT, ALP, bilirubin, creatinine, urea, uric acid, HDL and LDL were assessed. It was found that due to the administration of DSS (3%) there was an aberrant increase in the level of certain enzymes in blood-serum such as SGOT, SGPT, bilirubin, creatinine, urea, uric acid, and LDL followed by a decrease in the level of HDL and ALP. Treatment with mesalazine at 100 mg/Kg BW (PC group) or MeEa 400 exhibited a significant reduction in the level of these enzymes ( Table 1 ) than the NC group. Treatment with MeE 200 did not significantly reduce the level of these enzymes as compared to the NC group. Table 1 Evaluation of MeEa 200 and MeEa 400 on serum biochemical parameters in DSS (3%)-induced ulcerative colitis (UC). Biochemical Tests VC NC PC MeEa 200 MeEa 400 SGOT (U/L) 35.2 ± 3.8 81.9 ± 6.3 ### 48.2 ± 5.2 *** 57.3 ± 3.2 * 51.1 ± 5.0 ** SGPT (U/L) 45.6 ± 4.5 86.1 ± 5.0 ### 54.0 ± 4.5 ** 63.6 ± 7.1 * 54.8 ± 2.9 ** ALP (U/L) 362.3 ± 38.0 193.9 ± 26.8 ## 383.4 ± 36.6 ** 248.7 ± 26.1 346.7 ± 29.1 * Bilirubin (mg/dL) 1.4 ± 0.3 5.0 ± 0.6 ### 2.3 ± 0.3 *** 3.0 ± 0.3 * 2.7 ± 0.3 ** Creatinine (mg/dL) 13.1 ± 2.0 31.8 ± 5.0 ## 13.8 ± 2.5 * 23.8 ± 4.5 14.5 ± 2.7 * Urea (mg/dL) 2.1 ± 0.4 8.6 ± 0.6 ### 3.6 ± 0.3 *** 7.3 ± 0.7 5.5 ± 0.8 ** Uric acid (mg/dL) 2.8 ± 0.4 7.4 ± 0.5 ### 3.4 ± 0.3 *** 5.8 ± 0.5 4.6 ± 0.4 ** HDL (mg/dL) 89.4 ± 7.4 31.1 ± 3.7 ### 54.1 ± 3.4 * 51.7 ± 4.4 57.2 ± 6.5 * LDL (mg/dL) 16.2 ± 1.9 39.9 ± 4.5 ### 17.6 ± 3.2 ** 33.7 ± 3.9 21.3 ± 3.3 ** Values are demonstrated as mean + SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. # p < 0.05, ## p < 0.01, ### p < 0.001 VC vs. NC group; * p < 0.05, ** p < 0.01, *** p < 0.001 NC vs. treatment groups. 3.7. Histopathology of colon tissue Colon histology indicated the pathological alteration occurred due to the accumulation of inflammatory cells and damage to the intestinal epithelium induced by DSS (3%) administration and the protective effects of mesalazine, MeEa 200 and MeEa 400 ( Fig. 6 ) . Colon tissue samples were stained with H&E and examined under a high-resolution microscope at 40X. In the VC group, the epithelial cells were intact, and no sign of inflammation was observed ( Fig. 6 A ) . In the NC group, the epithelial cells and structure of crypts were severely injured due to damage induced by DSS (3%) ( Fig. 6 B ) . However, treatment with mesalazine at 100 mg/kg BW and MeEa 400 significantly reduced the damage to colon tissue than the NC group ( Figs. 6 C and 6 D ) . Treatment with MeEa 200 provides less protection to the epithelial cells and crypts than the NC group ( Fig. 6 E ) . The degree of colonic inflammation was scored semi-quantitatively on a 0–6 grading scale ( Fig. 6 E ) , whereby the level of colonic inflammation in the VC group was the lowest (~ 0), showing that mice in that group were free from signs of UC. The inflammation score in the NC group was ~ 5, indicating significantly higher inflammatory cell infiltration as compared to the VC group. The inflammation score was ~ 2 in the PC group, which demonstrated the significant protective effect of mesalazine in normalizing inflammatory cell infiltration and colonic epithelium integrity as compared to the NC group. The inflammation score was ~ 3 in the MeEa 400 treatment group, thereby indicating significant protection offered by the MeEa 400 in normalizing the colonic inflammation as compared to the NC group. Treatment with MeEa 200 provides comparatively less protection than mesalazine and MeEa 400; the resulting inflammation score was found to be ~ 3.5 as compared to the NC group (Supplementary Table 1 ). 3.8. Effect of MeEa against FITC dextran-induced intestinal membrane damage Intestinal barrier dysfunction is a prominent feature of UC. In this study assessment of intestinal permeability was performed in mice using FITC-dextran (Fig. 7 ). In the NC group, there was huge damage occurred to the intestinal barrier due to FITC-dextran induction as compared to the VC group. Treatment with mesalazine and MeEa 400 provided significant protection to the intestinal membrane from leakage as well as reduced the elevated levels of FITC dextran in the serum than the NC group. The effect of mesalazine was marginally better than the MeEa 400. Treatment with MeEa 200 did not provide significant protection against intestinal membrane damage as compared to the NC group. 4. Discussion Inflammatory bowel disease or IBD includes ulcerative colitis (UC) and Crohn’s disease (CD), characterized by the inflammation of the intestine Interestingly, over the past few decades, the medical therapeutic armamentarium now approved for the management of UC has exploded which continues to expand [ 21 ]. Therefore, highlighting the controversial aspects of currently available treatment is the basis for emerging novel herb-based therapies. Myrica esculenta ( M. esculenta ) is a well-known medicinal plant, that possesses a wide range of pharmacological properties For a reason since long time it has been used as a folk medicine for the treatment of various diseases. Previously published phytochemical studies prove that M. esculenta contains distinct bioactive compounds including alkaloids, triterpenoids, flavonoids, tannins, glycosides, saponins, volatile oils etc. As an ethnomedicine the stem bark, of M. esculenta has been widely used by local tribes in India and China for the treatment of conditions like Inflammation- Meghalaya, India (Khasi tribe), Vietnam, South China; Diarrhea, dysentery, stomach problem in Meghalaya, India (Khasi tribe), Almora, Uttarakhand, India; As a carminative-Meghalaya, India (Khasi tribe), Mizoram, India. Earlier studies have reported that myricetin obtained from M. esculenta plant [ 22 , 23 ] is commonly used as a food additive and possesses multiple pharmacological potential such as anti-inflammatory, anticancer, antidiabetic and strong antioxidant properties [ 24 ]. Our HPLC analysis has confirmed that the ethyl acetate fraction of M. esculenta (MeEa) indicated the presence of targeted flavonoids such as gallic acid (GA), myricetin (My), and quercetin (Q) (Fig. 1 ). DSS-induced UC is the most common and frequently used model that perfectly mimics the human UC symptoms in mice. Commonly 2.5-5% of dextran sodium sulfate (DSS) is mixed with drinking water induces UC symptoms in mice owing to its ease, controllability and reproducibility. DSS administration resulted in an alteration of the disease activity index (DAI) that upregulated the DAI scores. Parameters such as body weight loss in mice, reduction in average food intake, mean stool consistency score and mean stool blood score were elevated. Oral administration of MeEa 400 significantly downregulated the DAI scores as compared to the disease control (NC) group ( Fig. 2 ). UC is majorly associated with colonic histological changes in which there is a shortening of colon length, an increase in colon weight/length ratio and an increased weight of spleen. Upon administration, MeEa 400 protects the colon from shortening and inhibits the increased colon weight/length ratio as well as reduces spleen weight than the NC group (Fig. 3 ). After DSS administration there is an increase in pro-inflammatory cytokine levels (IL6, IL8, TNF-α and IFN-γ) and a decrease in anti-inflammatory cytokine levels (IL10) in blood-serum and colon tissue was observed. IL6, IL8, TNF-α and IFN-γ are the major pro-inflammatory cytokines in UC. Where the level of IL6 is increased by lamina propria and a CD4 + T cell, it promotes the survival of T-cell and apoptosis resistance in the lamina propria at the site of inflammation. The accumulation of CD4 + T cells in lamina propria to regulate inflammatory responses [ 25 ]. IL-8 cytokine is produced by distinct tissue and blood cells that act as a chemoattractant and activate neutrophil accumulation at the site of inflammation [ 26 ]. It has been reported that patients with UC showed elevated levels of IL-8 gene expression which is markedly associated with the inflammatory responses in the colon [ 27 ]. TNF α is secreted by Th1 cells causing the accumulation of immune cells in UC including intestinal fibroblasts, neutrophils, and macrophages in the gut [ 28 ]. Moreover, intestinal epithelial cell damage is also regulated by TNF α signalling pathways via myosin light chain kinase (MLCK) activation. The increased level of IFN-γ promotes intestinal permeability by disrupting the vascular endothelial-cadherin junctions. These are associated with an increased risk of inflammation in bowel disease [ 29 ]. Studies have reported that untreated IL10 gene-deficient mouse leads to gradual histological alterations, elevated colon weight/length ratios and increased levels of inflammatory cytokines. Therapeutic options for the treatment of UC can be provided by modulating IL10 gene expression. In this way, our molecular findings advocated the protective effect of MeEa, 400, as its treatment decreased the level of IL6, IL8, TNF-α and IFN-γ and also stabilised the level of IL10 in both mouse serum and colon tissues in a similar fashion (Fig. 4 ). In blood, there is an aberrant increase in the number of the various cell types such as total WBC, lymphocytes, monocytes, granulocytes and RBC due to the administration of DSS. In contrast, DSS administration reduces platelet counts and hemoglobin content significantly. Total WBC count is a crucial parameter to predict the extent of severity in UC, it is usually associated with severe clinical and mucosal disorders [ 30 ]. In UC there is an abnormal increase in lymphocyte count in the intestinal linings which is a sign of activated immune system to combat infection [ 31 ]. It has been reported that absolute peripheral blood monocyte level was increased in UC consequently associated with the severity of UC [ 32 ]. The red cell distribution width (RDW) or RBC count is significantly increased in the case of UC [ 33 ]. The hemoglobin content becomes low in conditions like UC due to iron deficiency anaemia secondary to severe blood loss and reduced iron absorption caused by tissue inflammation [ 34 ]. The platelet counts are decreased in UC due to the presence of auto-antibodies against platelet membrane antigens that cause immune-mediated platelet destruction. It is considered a rare extraintestinal indication of UC [ 35 ]. Thus, treatment with MeEa 400 significantly reduced the number of cell counts like total WBC, lymphocytes, monocytes, granulocytes and RBC followed by an increase in platelet count and hemoglobin content (Fig. 5 ). Studies have shown that the severity of UC is associated with the extent of colonic injury. DSS induction caused a significant degree of inflammation and colonic tissue injury in the mice, which is signified by the inflammatory cell infiltration epithelial disruption, oedema, and haemorrhage [ 36 ]. Findings of the present study have shown that mice of the negative control group (NC) resulted in a thicker and more erupted mucosal layer as a consequence of DSS (3%) administration. Treatment with MeEa 400 provided significant protection exhibited by the reduction in inflammatory scores and increased crypts count in the intestinal mucosa ( Fig. 6 ). The intestinal paracellular permeability and severity of intestinal mucosal barrier dysfunction in mice can be assessed by using 4-kDa FITC-dextran (non-digestible polysaccharides). Upon oral administration 4-kDa FITC-dextran does not cross the intestinal epithelial barrier unless the intestinal barrier becomes injured. Conditions like inflammation and injury to the intestinal epithelium cause mucosal barrier dysfunction. This results in barrier defects that promote the passage of 4-kDa FITC-dextran molecules towards the intestinal serous layer. Subsequently moves into the the systemic circulation, where it can be quantified in blood serum. Results obtained showed that at a higher dose treatment MeEa 400 provided significant protection from intestinal membrane leakage, thereby reduced level of FITC dextran in the serum was observed as compared to the untreated NC group ( Fig. 7 ) . Thus, the present study reports the beneficial effects of MeEa in the management of UC that may be due to the presence of certain types of phytomolecules in it like GA, My and Q as characterized in HPLC analysis. 5. Conclusions HPLC fingerprinting of the ethyl acetate fraction of M. esculenta (MeEa) showed the presence of gallic acid (GA), myricetin (My) and quercetin (Q) therein. Upon pharmacological evaluation, MeEa demonstrated suppressive effects against DSS (3%)-induced UC in mice. Treatment with MeEa 400 significantly reduced the DAI scores, protected colon length from shortening following a decreased colon weight/length ratio and also reduced spleen weight, respectively. Both in serum and colon tissues, MeEa 400 treatment down-regulated the elevated levels of pro-inflammatory cytokines (IL6, IL8, TNF-α, and IFNγ) and up-regulate the IL10 gene expression. Cell counts such as total WBC, lymphocyte, monocyte, granulocyte, and RBC were increased after DSS administration. Treatment with MeEa 400 significantly reduced the cell counts. Further, oral administration of MeEa 400 markedly reversed histological changes raised due to DSS-induction and also reduced histopathological scores. Our future research must be directed towards the in vivo investigation of the effect of MeEa on microbiota alteration in cases of UC. Abbreviations IBD, Inflammatory bowel disease; UC, Ulcerative colitis; M. esculenta, Myrica esculenta ; MeEa, Myrica esculenta ethyl acetate fraction, DSS, Dextran sodium sulphate; IL, Interleukin; TNF-α, Tumor necrosis factor alpha; INF-γ, Interferon gamma; RBC, Red blood cell; WBC, White blood cell; HGB, Haemoglobin; H&E, Hematoxylin & eosin; SGOT, Serum glutamate oxaloacetate transaminase; SGPT, Serum glutamate pyruvate transaminase; ALP, Alkaline phosphatase; HDL, High density lipoprotein; LDL, Low density lipoprotein. Declarations Conflict of interest The authors declare no conflict of interests. Ethical approval Animal experiments in the study were performed following the guidelines of the Organization for Economic Co-operation and Development (OECD) (1987) for the care and use of laboratory animals. Adult (6-7 weeks old) Swiss albino mice (20–25 g) were used in the study. All the experimental animals were acclimatized for a period of 1 week prior to the experiment and maintained at normal laboratory conditions (25 ± 1°C, 55 ± 5% relative humidity and an automatic 12 h light/12 h dark cycle). Animals were provided with the commercial standard diet and water ad libitum . The experiments on ulcerative colitis activity were carried out under the protocol CCSEA/IAEC/SLSRPL/18/07/2023-02, which was approved by the Institutional Animal Ethics Committee (IAEC) constituted under the Committee for Control and Supervision of Experiments on Animals (CCSEA), Govt. of India. Authors Contributions: All authors contributed equally to the concept, experimental study, and data analysis. MJ has planned the experimental study and written the manuscript. Further data analysis and manuscript review were done by MP. The final manuscript was reviewed, evaluated and edited by AV. Data availability statement The data that supports the findings of this study are available in the manuscript and supplementary material of this article. Statements & Declarations “The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.” Competing Interests “The authors have no relevant financial or non-financial interests to disclose.” References Clara E, Sawian AM, Susngi B, Manners, Jasmine T, Sawian (2023) Chap. 28- Myrica esculenta Editor(s). Tarun Belwal, Indra Bhatt, Hari Devkota, Himalayan Fruits and Berries. Academic Press, pp 287–303 Khan Y, Sagrawat H, Upmanyu N, Siddique S (2008) Anxiolytic properties of Myrica nagi bark extract. 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PLoS ONE 12(9):e0184709 Liu J, Teng PY, Kim WK, Applegate TJ (2021) Assay considerations for fluorescein isothiocyanate-dextran (FITC-d): an indicator of intestinal permeability in broiler chickens. Poult Sci 100(7):101202 Sudirman S, Hsu YH, He JL, Kong ZL (2018) Dietary polysaccharide-rich extract from Eucheuma cottonii modulates the inflammatory response and suppresses colonic injury on dextran sulfate sodium-induced colitis in mice. PLoS ONE 13(10):e0205252 Lau-Cam CA, Chan HH (1973) Flavonoids from Comptonia peregrine . Phytochem 12:1829 Jones JR, Lebar MD, Jinwal UK, Abisambra JF, Koren J, Blair L et al (2011) The diarylheptanoid (+)-aR,11S-myricanol and two flavones from bayberry ( Myrica cerifera ) destabilize the microtubule-associated protein tau. J Nat Prod 74:38–44 Zhang X, Zhang K, Wang Y, Ma R (2019) Biological effects study of Myricitrin and relevant molecular mechanisms. Curr Stem Cell Res Ther 2019:14 Mudter J, Neurath MF (2007) Il-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance. Inflamm Bowel Dis 13(8):1016–1023 Bickel M (1993) The role of interleukin-8 in inflammation and mechanisms of regulation. J Periodontol 64:456–460 Okada T, Kanda T, Ueda N, Ikebuchi Y, Hashiguchi K, Nakao K et al (2020) IL-8 and LYPD8 expression levels are associated with the inflammatory response in the colon of patients with ulcerative colitis. Biomed Rep 12(4):193–198 Jang DI, Lee AH, Shin HY, Song HR, Park JH, Kang TB et al (2021) The role of tumor necrosis factor alpha (TNF-α) in autoimmune disease and current TNF-α inhibitors in therapeutics. Inter J Mol Sci 22(5):2719 Langer V, Vivi E, Regensburger D, Winkler TH, Waldner MJ, Rath T et al (2019) IFN-γ drives inflammatory bowel disease pathogenesis through VE-cadherin–directed vascular barrier disruption. J Clin Investig 129(11):4691–4707 Mack DR, Saul B, Boyle B, Griffiths A, Sauer C, Markowitz J et al (2020) Analysis of using the total white blood cell count to define severe new-onset ulcerative colitis in children. J Pediatr Gastroenterol Nutr 71(3):354–360 Yen EF, Pardi DS (2011) Review article: microscopic colitis-lymphocytic, collagenous and ‘mast cell’ colitis. Aliment Pharmacol Ther 34:21–32 Furukawa S, Ikeda Y, Yagi S, Miyake T, Shiraishi K, Tange K et al (2021) Association between peripheral blood monocyte count and mucosal healing in Japanese patients with ulcerative colitis. Clin Translational Gastroenterol 12(11) Yeşil A, Şenateş E, Bayoğlu İV, Erdem ED, Demirtunç R, Övünç AO (2011) Red cell distribution width: a novel marker of activity in inflammatory bowel disease. Gut Liver 5(4):460–467 Kaitha S, Bashir M, Ali T (2015) Iron deficiency anemia in inflammatory bowel disease. World J Gastrointest Pathophysiol 6(3):62 Dutra DMR, Almeida LR, Sinkos C, Muraro Bonini AC, Saad-Hossne R, Baima JP (2022) Immune thrombocytopenic purpura as an extraintestinal manifestation in a patient with ulcerative colitis. Case Rep Gastroenterol 16(1):140–147 Deguchi Y, Andoh A, Inatomi O, Yagi Y, Bamba S, Araki Y et al (2007) Curcumin prevents the development of dextran sulfate sodium (DSS)-induced experimental colitis. Digest Dis Sci 52:2993–2998 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx 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. 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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-3832022","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265152020,"identity":"1c50c0ab-5132-4a37-9c92-ebb14f6347cc","order_by":0,"name":"Monika Joshi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACxgYGNoYEIIMNyH4gUQFkMTM3EK2F2cDiDEgLI34tEMVQhkRlG9QYfIC5vf3Zg4c5Nnl87IefSdycVxvN3w7U8qNiG26H9ZwxN0jcllbMxpNmbDlz2/HcGYcZG4Cit3FrmZHDJpG47XBimwSD4W3JbcdyG4BamBnb8GlJfwbVwv5B+u+cY7nzCWtJMINq4TGSkGyoyd1AUEvPGZCWtMQ2npxiA4ljB3I3ArUcxOcXQ2CISf7cZpM4v/34xgcSNXW5884fPvjgRwUeLQ2o/MNg8gBO9UAgj8avw6d4FIyCUTAKRigAADtPXJc49zYKAAAAAElFTkSuQmCC","orcid":"","institution":"Sri Ramswaroop Memorial University","correspondingAuthor":true,"prefix":"","firstName":"Monika","middleName":"","lastName":"Joshi","suffix":""},{"id":265152022,"identity":"2cf4aad2-05c1-4b05-84fc-5137d62fcdbe","order_by":1,"name":"Manju Pandey","email":"","orcid":"","institution":"Sri Ramswaroop Memorial University","correspondingAuthor":false,"prefix":"","firstName":"Manju","middleName":"","lastName":"Pandey","suffix":""},{"id":265152025,"identity":"b8771527-97ad-4bd2-b6bd-a35c43ff19ef","order_by":2,"name":"Akash Ved","email":"","orcid":"","institution":"Dr. A.P.J Abdul Kalam Technical University","correspondingAuthor":false,"prefix":"","firstName":"Akash","middleName":"","lastName":"Ved","suffix":""}],"badges":[],"createdAt":"2024-01-03 14:14:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3832022/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3832022/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49330362,"identity":"e5f6bb32-dd9a-4f86-9b22-fdb2e001ec24","added_by":"auto","created_at":"2024-01-08 18:53:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30199,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC profiling of the ethyl acetate fraction of \u003cem\u003eMyrica esculenta\u003c/em\u003e (MeEa) \u003cstrong\u003e[A]\u003c/strong\u003eRepresentative chromatogram of the standard mixture: (1) gallic acid (GA); (2) myricetin (My); and (3) querecetin (Q). \u003cstrong\u003e[B]\u003c/strong\u003e Chromatogram of MeEa, indicating the presence of (1) GA; (2) My; and (3) Q, at 254 nm.\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/b0bf28e75d387b04925a9e78.jpeg"},{"id":49330529,"identity":"f25ed86e-ccfd-4da7-8b03-fa7fdb2bd460","added_by":"auto","created_at":"2024-01-08 19:01:58","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50348,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of MeEa 200 and MeEa 400 on the body weight and disease activity index (DAI) against DSS-induced ulcerative colitis (UC). UC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%), Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively. \u003cstrong\u003e(A)\u003c/strong\u003e %\u0026nbsp;of initial body weight; \u003cstrong\u003e(B)\u003c/strong\u003e average\u0026nbsp;food intake per mouse (g); \u003cstrong\u003e(C) \u003c/strong\u003emean\u0026nbsp;stool consistency score; and \u003cstrong\u003e(D) \u003c/strong\u003emean\u0026nbsp;stool blood score. Values are demonstrated as mean\u0026nbsp;+ SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 NC vs. treatment groups.\u003c/p\u003e","description":"","filename":"groupimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/932db7f8cd40a7ce40ac7c8e.jpeg"},{"id":49330711,"identity":"1c7859db-2033-4ec8-8f4e-7b662d14892e","added_by":"auto","created_at":"2024-01-08 19:09:58","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81884,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of MeEa 200 and MeEa 400 on the colon and spleen tissues against DSS-induced ulcerative colitis (UC). UC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%), Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively. All the mice were sacrificed after 24 hours of the last dose, and their colon and spleen tissues were harvested carefully and stored accordingly. \u003cstrong\u003e(A)\u003c/strong\u003e colon tissues from all the groups, harvested on day 7; \u003cstrong\u003e(B)\u003c/strong\u003e colon length of all the groups; \u003cstrong\u003e(C)\u003c/strong\u003e colon weight/length ratio (mg/cm) of all the groups; and \u003cstrong\u003e(D)\u003c/strong\u003e spleen weight (g) of all the groups. Values are demonstrated as mean\u0026nbsp;+ SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 NC vs. treatment groups.\u003c/p\u003e","description":"","filename":"groupimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/897026f50375ad91599c6397.jpeg"},{"id":49330530,"identity":"69bf18e2-313c-46fc-a90d-5682edd10253","added_by":"auto","created_at":"2024-01-08 19:01:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101080,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of MeEa 200 and MeEa 400 on the production of inflammatory cytokines in the serum (left panel) and the colon tissues (right panel) against DSS-induced ulcerative colitis (UC). UC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%), Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively. All the mice were sacrificed on day 7, and their blood collection and colon tissue harvesting were done for the measurement of the following cytokines: \u003cstrong\u003e(A)\u003c/strong\u003e IL6 (pg/mL); \u003cstrong\u003e(B)\u003c/strong\u003e IL8 (pg/mL); \u003cstrong\u003e(C)\u003c/strong\u003e IL10 (pg/mL); \u003cstrong\u003e(D)\u003c/strong\u003e TNFα (pg/mL); and \u003cstrong\u003e(E)\u003c/strong\u003e IFNγ (pg/mL). Values are demonstrated as mean\u0026nbsp;+ SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 NC vs. treatment groups.\u003c/p\u003e","description":"","filename":"groupimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/b27f941dc2b1ed4420653991.jpeg"},{"id":49330366,"identity":"8ac069fc-e198-4e56-b09d-0d2f3d365afd","added_by":"auto","created_at":"2024-01-08 18:53:58","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82800,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of MeEa 200 and MeEa 400 on hematological parameters against DSS-induced ulcerative colitis (UC). UC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%), Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively. All the mice were sacrificed on day 7, and their blood was collected for the measurement of the following parameters: \u003cstrong\u003e(A)\u003c/strong\u003e Total WBC (x 10\u003csup\u003e3\u003c/sup\u003e/µL); \u003cstrong\u003e(B)\u003c/strong\u003e RBC (x 10\u003csup\u003e6\u003c/sup\u003e/µL); \u003cstrong\u003e(C)\u003c/strong\u003e Hemoglobin (g/dL); \u003cstrong\u003e(D)\u003c/strong\u003e Lymphocytes (x 10\u003csup\u003e3\u003c/sup\u003e/µL); \u003cstrong\u003e(E)\u003c/strong\u003e Monocytes (x 10\u003csup\u003e3\u003c/sup\u003e/µL); \u003cstrong\u003e(F)\u003c/strong\u003e Granulocytes (x 10\u003csup\u003e3\u003c/sup\u003e/µL); and \u003cstrong\u003e(G)\u003c/strong\u003e Platelets (x 10\u003csup\u003e3\u003c/sup\u003e/µL). Values are demonstrated as mean\u0026nbsp;+ SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 NC vs. treatment groups.\u003c/p\u003e","description":"","filename":"groupimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/25b873e3319f85db27b5314a.jpeg"},{"id":49330531,"identity":"bc6e3720-42a3-454d-ba14-89519e7f1f25","added_by":"auto","created_at":"2024-01-08 19:01:59","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":195246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHistopathological evaluation of the colon tissues after the administration of\u0026nbsp;\u003c/em\u003eMeEa 200 and MeEa 400 against DSS-induced ulcerative colitis (UC). Colon tissue sections were stained with hematoxylin and eosin (H\u0026amp;E).\u003cem\u003e\u0026nbsp;\u003c/em\u003eUC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%), Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively. All the mice were sacrificed on day 7, and their colon tissues were harvested for histopathological studies.\u0026nbsp;\u003cstrong\u003e(A)\u003c/strong\u003e VC group showed intact architecture of colon tissue with no infiltration of inflammatory cells;\u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e NC group showed degenerated and abnormal intestinal epithelial layer due to the damage by the DSS (3%) administration;\u0026nbsp;\u003cstrong\u003e(C)\u003c/strong\u003e PC group administered with mesalazine showed significant protection of the intestinal epithelium and also reduced the inflammatory cell infiltration as compared to the NC group;\u0026nbsp;\u003cstrong\u003e(D)\u003c/strong\u003e group treated with MeEa 200 did not provide significant protection to the intestinal epithelial layer as compared to other treatment groups i.e. PC and MeEa 400;\u0026nbsp;\u003cstrong\u003e(E)\u003c/strong\u003e \u003cstrong\u003egroup treated with\u0026nbsp;\u003c/strong\u003eMeEa 400 exhibited moderate to significant protection against inflammatory cell accumulation as compared to the NC group; and \u003cstrong\u003e(F)\u003c/strong\u003e histopathological evaluation expressed in terms of colonic histopathological score,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewhereby the degree of colonic inflammation scored on a semi-quantitative scale. Values are demonstrated as mean\u0026nbsp;+ SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 NC vs. treatment groups.\u003c/p\u003e","description":"","filename":"groupimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/bce88a1beb5ade8064b64b2d.jpeg"},{"id":49330368,"identity":"103506b0-0005-4ed0-95ea-ef020b8fe80d","added_by":"auto","created_at":"2024-01-08 18:53:59","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24083,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of MeEa 200 and MeEa 400 on translocation of FITC-dextran across the intestinal membrane into the blood of mice. Values are demonstrated as mean\u0026nbsp;+ SEM (n = 6). A one-way ANOVA with Tukey's multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001 NC vs. treatment groups.\u003c/p\u003e","description":"","filename":"groupimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/3a63ad30d468849e160f9300.jpeg"},{"id":49332073,"identity":"ea89e06c-232d-4722-ac78-aef507ee550f","added_by":"auto","created_at":"2024-01-08 19:26:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":910245,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/5173e480-1f33-4728-a757-2d20659bc6ed.pdf"},{"id":49330361,"identity":"1d4c4763-3773-4a5f-b3f5-6371f794165f","added_by":"auto","created_at":"2024-01-08 18:53:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13537,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3832022/v1/92297123549151043051e773.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the inhibitory potential of flavonoid-rich fraction of Myrica esculenta against DSS-induced colonic inflammation in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eMorella esculenta\u003c/em\u003e, synonym \u003cem\u003eMyrica esculenta\u003c/em\u003e Buch.-Ham.ex D. Don and \u003cem\u003eMyrica nag\u003c/em\u003e belongs to Myricaceae family, is found worldwide in tropical and sub-tropical regions and is known for its nutritive value and health advantages [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 12 to 15 meters high, this medium-sized to large woody, evergreen dioecious tree has a trunk girth of 92.5cm, light brown to black bark, green lanceolate leaves and bracteate pistillate flowers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The plant is commonly referred to as Kaphal or Soh-Phie. \u003cem\u003eMyrica esculenta\u003c/em\u003e (\u003cem\u003eM. esculenta\u003c/em\u003e) is extensively spread in the mid Himalayas, Kasia hills, Sylhet in India and China, Japan, Australia, Taiwan, South Africa, North America, Brazil, Ethiopia and Nepal [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The bark of \u003cem\u003eM. esculenta\u003c/em\u003e exhibits extreme potential and is used for treating a variety of ailments like asthma, cough, fever, inflammation, ulcers, diarrhoea, nose, ear and throat infections. The hydrolyzable tannin castalagin is present in \u003cem\u003eM. esculenta\u003c/em\u003e bark along with gallic acid, myricanol, myricanone, and epigallocatechin 3-O-gallate, as well as two prodelphinidin dimers, namely epigallocatechin-(4β\u0026rarr;8)-epigallocatechin 3-O-gallate and 3-O-galloyl epigallocatechin-(4β\u0026rarr;8)-epigallocatechin 3-O-gallate .The presence of flavonoids and steroids in \u003cem\u003eM. esculenta\u003c/em\u003e has contributed to its pharmacological activity against inflammation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn inflammatory illness that affects the colon and rectum to varying degrees is ulcerative colitis (UC). UC is a type of Inflammatory Bowel Disease (IBD) directing various gastrointestinal diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. UC is a chronic condition leading to 5\u0026nbsp;million cases globally in 2023 and the incidence is still rising. People with abnormalities in the gut epithelial barrier, the microbiota, the irregulated response of the immune system and genetic predisposition are more likely to develop UC. Symptoms of the disorder include pain in the abdomen, bloating, diarrhoea, blood in stool leading to fatigue, fever, anaemia and weight loss. 5-aminosalicylic acid medications, thiopurines, biologics (such as anti-integrins and anti-cytokines), and small molecules (such as sphingosine-1-phosphate receptor modulators and Janus kinase inhibitors) are examples of maintenance therapy. These treatment approaches have high costs and are associated with several side effects like metabolic changes, lymphomas, increased susceptibility to infections and a higher rate of mortality [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMesalamines are the first choice of therapy for the UC and are available in different dosage forms like oral (AsacolTM, PentasaTM, AprisoTM, ColazalTM), suppository (CanasaTM) and liquid enema (RowasaTM) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, its administration has resulted in acute intolerance syndrome, symptoms of which are similar to the exacerbation of UC making it difficult to differentiate [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Complementary and alternative medicine (CAM) like herbal therapy targets multiple factors and offers more patient acceptability and fewer side effects. In recent years use of CAM has increased because of its natural origin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Numerous individual herbs \u003cem\u003e(Indigo naturalis, Lycium ruthenicum Murray, Abelmoschus manihot, Centella asiatica)\u003c/em\u003e, Chinese herbal formulas (Baweixilei-San, Huanglian Jiedu Decoction, \u003cem\u003eQingchang Huashi Formula etc.)\u003c/em\u003e have shown protective role against UC by affecting intestinal immunity and gut flora [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus \u003cem\u003eM. esculenta\u003c/em\u003e bark contains several active constituents making it a potential herb for numerous inflammatory disorders. However, none of the literature has reported its activity for the UC. In the current research, we have explored the ME bark for the UC using DSS induced IBD model. The primary aim of the treatment is to improve the remission rates, decrease side effects and improve the patient's quality of life. \u003cem\u003eM. esculenta\u003c/em\u003e is an herbal therapy that may serve the current needs of the patient.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eThe reagents and chemicals used in the present study were of analytical grade. The DSS (colitis grade) was purchased from MP Biomedicals, California. Mesalazine was purchased from Himedia, India. All the biochemical kits were purchased from Agappe Diagnostic Ltd., Kerala, India. ELISA kits were purchased from Krishgen Biosystems, Mumbai, India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant material\u003c/h2\u003e \u003cp\u003eThe stem bark of \u003cem\u003eMyrica esculenta\u003c/em\u003e (\u003cem\u003eM. esculenta\u003c/em\u003e) was purchased from the local herbal market in Lucknow (U.P.). India. The plant material was authenticated and obtained a voucher specimen number, i.e., NIScPR/RHMD/Consult/2022/4414-15, by the Head, Raw Material Herbarium and Museum (RHMD), CSIR-National Institute of Science Communication and Policy Research (NIScPR), Delhi, India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction Procedure\u003c/h2\u003e \u003cp\u003eThe stem bark of \u003cem\u003eM. esculenta\u003c/em\u003e was cleaned, shade-dried, and converted into a coarse powder using a milling machine, Milcent Appliances Pvt. Ltd., Gujarat, India. The 200 g of coarse powder was further put into the soxhlet apparatus and kept for 6 h on heating mental for extraction using ethyl acetate as solvent. The filtrate was then concentrated in a rotary evaporator (R-210 BUTCHI, Switzerland), dried in a water bath, weighed, and stored in a refrigerator for further evaluation. The extractive yields of the extract was calculated and found to be 21% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. High performance liquid chromatography (HPLC)\u003c/h2\u003e \u003cp\u003eThe HPLC profiling of the ethyl acetate fraction of \u003cem\u003eM. esculenta\u003c/em\u003e (MeEa) was done and characterizes the presence of certain flavonoid compounds such as gallic acid (GA), myricetin (My), and quercetin (Q) following the previously published literature with slight modifications [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The quantification of GA, My and Q was done using the HPLC platform Shimadzu, Japan; Prominence (Pump: LC-20AD, Autosampler: SIL-20AC, Oven: CTO-10ASvp) connected with a photodiode array detector (PDA: SPD-M20A), software (LabSolutions 6.72 SP1) and a C18 column (ShimadzuRP-C-18, 250 x 4.6 mm, 5\u0026micro;m pore size). The method comprised solvent A (1% acetic acid) in water and solvent B (acetonitrile) in isocratic elution mode with a flow rate of 0.6 ml/min. The injection volume was 20 \u0026micro;l, and the detector wavelength was set at 254 nm. The stock solutions (1 mg/ml) of standards (GA, My, and Q) were prepared in HPLC-grade methanol. For the identification and quantification of flavonoids in MeEa, UV-absorbance and retention time were compared with those of the standards.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. In vivo studies\u003c/h2\u003e \u003cp\u003e Animal experiments in the study were performed following the guidelines of the Organization for Economic Co-operation and Development (OECD) (1987) for the care and use of laboratory animals. Adult (6\u0026ndash;7 weeks old) Swiss albino mice (20\u0026ndash;25 g) were used in the study. All the experimental animals were acclimatized for a period of 1 week prior to the experiment and maintained at normal laboratory conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity and an automatic 12 h light/12 h dark cycle). Animals were provided with the commercial standard diet and water \u003cem\u003ead libitum\u003c/em\u003e. The experiments on ulcerative colitis activity were carried out under the protocol CCSEA/IAEC/SLSRPL/18/07/2023-02, which was approved by the Institutional Animal Ethics Committee (IAEC) constituted under the Committee for Control and Supervision of Experiments on Animals (CCSEA), Govt. of India.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. DSS-induced UC\u003c/h2\u003e \u003cp\u003eThirty Swiss albino mice were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;6/group). UC was induced in all the groups except Group 1 (Vehicle Control, VC) by administering DSS (3%) in the drinking water of mice for seven consecutive days. For the dosing schedule, the VC group received distilled water throughout the experiment. Group 2 (Negative Control, NC), which received distilled water and DSS (3%). Group 3 (Positive Control, PC) was administered with the standard drug, mesalazine, at a dose of 100 mg/kg BW orally. Groups 4 and 5 (MeEa 200 and MeEa 400, treatment groups) were treated with MeEa at doses of 200 and 400 mg/kg BW orally once a day for seven days, respectively [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In Groups 3, 4, and 5, oral doses of mesalazine, MeEa 200 and MeEa 400 were started in parallel with the DSS (3%) induction from day one, once a day for seven days. A daily record of body weight and disease activity index (DAI) was kept throughout the seven days of the experiment. On the eighth day prior to the animal sacrifice, blood collection was done; thereafter, collection of organs (colon and spleen) and measurement of colon weight and length were also performed [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Evaluation of disease activity index (DAI)\u003c/h2\u003e \u003cp\u003eThe DAI scores in all the animals were evaluated on parameters such as visible stool consistency, presence of blood in stool and body weight loss on a 4-point scale (0\u0026ndash;4). Where 0 signifies formed and hard stool, 1 shows formed but soft stool, 2 for loose stool and 3 signifies runny and watery stool. The percent (%) weight loss in animals was calculated relative to the percent body weight of day one. Further, the traces of the occult blood in the stool were analyzed by using the Hemoccult SENSA single-slide rapid diagnostic kit based on the following criteria: 0 shows hemooccult negative (-), 1 for hemooccult positive (+), 2 signifies hemoocult positive (++) and 3 reports bleeding through the anus site (+++) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Analysis of colon weight to length ratio\u003c/h2\u003e \u003cp\u003eAfter the sacrifice of each mouse, their colons were carefully removed, opened longitudinally and washed with normal saline to remove faecal matter present in the colon. Thereafter, the wet weight of the colon, approximately 10 cm long, was measured. The wet weight of each colon was then divided by the colon length to calculate the colon weight-to-length ratio [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Estimation of inflammatory cytokine level in serum and colon tissues\u003c/h2\u003e \u003cp\u003eSerum and colon tissue samples were analyzed to determine the concentration of inflammatory cytokines (IL-6, IL-8, IL-10, TNF-α, and IFN-γ) by using ELISA kits from Krishgen Biosystems, Mumbai, India. The ELISA tests were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5. Hematological and biochemical analysis\u003c/h2\u003e \u003cp\u003eThe blood was withdrawn from the retro-orbital plexus of each mouse into two tubes. The first tube was the heparinized tube that contained whole blood to assay the complete blood count (CBC) in a hematology analyzer, Mindray BC 3000 Plus, Agappe, India. The second tube was a non-heparinized tube that contained the blood from which serum was separated to perform the ELISA assays (as mentioned above, section \u003cspan refid=\"Sec12\" class=\"InternalRef\"\u003e2.5.5\u003c/span\u003e) and biochemical tests. The biochemical tests were performed using biochemical kits from Agappe Diagnostic Ltd., Kerala, India, for the estimation of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphates (ALP), bilirubin, creatinine, urea, uric acid HDL, and LDL. The biochemical assays were done as per the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.6. Histopathology and inflammatory score\u003c/h2\u003e \u003cp\u003eColon tissues were fixed with 10% neutral-buffered formalin and embedded in paraffin wax. The 5 \u0026micro;m sections were cut and stained with hematoxylin and eosin (H\u0026amp;E) for histopathological evaluation. The sections were observed under a high-resolution microscope (at 400X), Lieca, Germany, to determine the degree of colonic inflammation resulted due to DSS administration, which was scored on a subjective scale of 0\u0026ndash;4, whereby 0 denoted normal colonic mucosa (nil inflammation); 1 denoted loss of one-third of the crypts; 2 denoted loss of two-thirds of the crypts; 3 denoted a single disruptive epithelial layer covering the lamina propia; 4 erosions and significant inflammatory cell infiltration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Assessment of intestinal membrane permeability using FITC dextran\u003c/h2\u003e \u003cp\u003eThe assessment of intestinal permeability was done by using FITC (Fluorescein isothiocyanate-dextran sulfate sodium salt; 4000 kDa, Sigma-Aldrich, USA) following the method published earlier [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Swiss albino mice were administered with DSS for fourteen days thereafter all the mice were allowed to fast for 12h. Mice were then administered with FITC-dextran (60 mg/100 g BW) via intragastric infusion. After four hours blood was collected from all the mice and centrifuged at 3500 rpm for 10 min., The obtained serum was further diluted with an equal volume of phosphate buffer saline (PBS). The concentration of FITC-dextran in serum was determined by using a fluorescence spectrophotometer (Eppendorf, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The concentration of FITC-dextran in the serum was calculated from the standard curve of diluted FITC-dextran obtained in non-treated serum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eResults are demonstrated as the mean\u0026thinsp;+\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;6) of the number of independent experiments. Statistical significance between the experimental groups was determined by the one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test. Data with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. Analyses of the data obtained were done using GraphPad Prism software version 5.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. HPLC analysis of MeEa\u003c/h2\u003e\n \u003cp\u003eThe chemical profiling of MeEa indicated the presence of targeted flavonoids (GA, My, and Q), which were expressed in terms of area%, i.e., GA-8.564, My-2.486 and Q-0.167. The findings obtained were comparable with those of the standard mix, whose area% was found to be GA-12.155, My-41.549 and Q-46.296 respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Effect of MeEa against DSS-induced UC in mice\u003c/h2\u003e\n \u003cp\u003eAfter DSS (3%) induction in Groups 2\u0026ndash;5, there was a development of symptoms of acute UC in mice, which were revealed by the alteration in certain parameters such as a decrease in body weight (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), reduced average food intake (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), a significant change in stool consistency (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the presence of blood in the stool (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). The above parameters were extremely altered in the NC group as compared to the NC group. Further, a significant difference was observed in terms of protection in the PC group than the NC group. The PC group was administered with the standard drug mesalazine at 100 mg/kg BW. The treatment with MeEa 200 did not provide significant protection against DSS-induced damage. It was noticeable that the group treated with MeEa 400 was found to be effective in normalizing the diseased condition induced by the DSS. A significant difference was observed after the administration of MeEa at 400 mg/kg BW in terms of % weight loss, average food intake, mean stool consistency, and mean stool blood score, especially on days 5 and 7 than the NC group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Effect of MeEa on colon and spleen tissues\u003c/h2\u003e\n \u003cp\u003eTo determine the extent of inflammation that occurred due to the induction of DSS, the measurement of colon length was performed \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. As observed in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB there was a significant reduction in the length of the colon in the NC group as compared to the VC group. In the PC group, it was observed that after treatment with mesalazine at 100 mg/kg BW the colon was significantly protected which was observed by its normal structure and length as compared to the NC group whereby the length of the colon was shortened and prominent tissue damage occurred. In the group treated with MeEa 200 there was slight to negligible protection was seen due to the lower dose but at a higher dose i.e. MeEa 400 there was significant protection to the colon tissue was observed than in the NC group \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). In Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC the colon weight/length ratio (mg cm-1) was significantly increased in the NC group as compared to the the VC group. In the PC group, a significant reduction in colon weight/length ratio was observed as compared to the NC group. Treatment with MeEt 400 also showed significant protection that resulted in a decreased colon weight/length ratio than the NC group. MeEa 200 showed lesser protection against colon weight/length ratio than the NC group. It is well accepted that the increased weight of the spleen is markedly associated with the increased risk of UC. In Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD there was a significant increase in the weight of the spleen in the NC group due to DSS administration as compared to the VC group. A significant reduction in the weight of the spleen was recorded in the PC group than in the NC group. Treatment with MeEa 400 significantly inhibited the increased weight of the spleen in the NC group. The effect of MeEa 400 was comparable with that of the PC group. Furthermore, there was no significant protection was observed in the case of MeEa 200 treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.4. Effect of MeEa on serum and colon tissue cytokines\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe inflammatory responses in UC begin with an increased level of inflammatory cytokines (IL6, IL8, TNF-\u0026alpha; and IFN-\u0026gamma;) and a decreased level of anti-inflammatory cytokine (IL10) in serum (left panel) and colon tissue (right panel) samples. The resultant effects of MeEa in serum and colon tissue cytokines were examined by using the ELISA technique \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. Serum and colon tissue samples showed similar patterns of cytokine levels in both cases. In the NC group, there was a significant increase in inflammatory cytokines levels and a decrease in anti-inflammatory cytokine levels due to the administration of DSS (3%) as compared to the VC group. Groups treated with mesalazine (PC group) or MeEa 400 significantly reduced the level of increased inflammatory cytokine level and normalized the level of anti-inflammatory cytokine in serum as well as colon tissue than the NC group respectively. Further treatment with MeEa 200 moderately reduced the level of elevated inflammatory cytokines and enhanced the level of anti-inflammatory cytokine in serum and colon tissues than the NC group. It was also observed that the effect exerted by MeEa 200 was not statistically significant as compared to the PC group and MeEa 400 group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Effect of MeEa on hematological parameters\u003c/h2\u003e\n \u003cp\u003eThe whole blood samples were used to determine cell counts \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. A significant increase in total WBC count was observed in the NC group due to DSS administration as compared to the VC group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Treatment with 100 mg/kg BW mesalazine (PC group) or MeEa 400 significantly decreased the total WBC count as compared to the NC group. Similarly, the level of lymphocytes, monocytes, granulocytes and RBC counts increased significantly in the NC group as compared to the VC group. Treatment with mesalazine or MeEa 400 significantly attenuated the increased level of lymphocytes, monocytes, granulocytes and RBC as compared to the NC group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). After the administration of DSS, there was a significant reduction in the level of hemoglobin content and platelet count in the NC group as compared to the VC group. Groups treated with mesalazine or MeEa 400 significantly normalize the level of hemoglobin content and platelet count as compared to the NC group. It was also observed that mesalazine performed marginally better than MeEa 400 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). Further, treatment with MeEa 200 did not markedly attenuate the increased level of cell count, hemoglobin content and platelet count as compared to the NC group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Effect of MeEa on serum biochemical parameters\u003c/h2\u003e\n \u003cp\u003eStudies have shown that there is a marked imbalance in biochemical parameters in UC that leads to kidney and liver disorders. Thus biochemical tests such as SGOT, SGPT, ALP, bilirubin, creatinine, urea, uric acid, HDL and LDL were assessed. It was found that due to the administration of DSS (3%) there was an aberrant increase in the level of certain enzymes in blood-serum such as SGOT, SGPT, bilirubin, creatinine, urea, uric acid, and LDL followed by a decrease in the level of HDL and ALP. Treatment with mesalazine at 100 mg/Kg BW (PC group) or MeEa 400 exhibited a significant reduction in the level of these enzymes \u003cstrong\u003e(\u003c/strong\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e than the NC group. Treatment with MeE 200 did not significantly reduce the level of these enzymes as compared to the NC group.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEvaluation of MeEa 200 and MeEa 400 on serum biochemical parameters in DSS (3%)-induced ulcerative colitis (UC).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBiochemical Tests\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMeEa 200\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMeEa 400\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSGOT (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSGPT (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eALP (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e362.3\u0026thinsp;\u0026plusmn;\u0026thinsp;38.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e193.9\u0026thinsp;\u0026plusmn;\u0026thinsp;26.8\u003csup\u003e\u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e383.4\u0026thinsp;\u0026plusmn;\u0026thinsp;36.6\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e248.7\u0026thinsp;\u0026plusmn;\u0026thinsp;26.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e346.7\u0026thinsp;\u0026plusmn;\u0026thinsp;29.1\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBilirubin (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCreatinine (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003csup\u003e\u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUrea (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUric acid (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHDL (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e89.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDL (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eValues are demonstrated as mean\u0026thinsp;+\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;6). A one-way ANOVA with Tukey\u0026apos;s multiple comparison tests was used to analyze the significant differences among the experimental groups. \u003csup\u003e#\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001 VC vs. NC group; \u003csup\u003e*\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001 NC vs. treatment groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. Histopathology of colon tissue\u003c/h2\u003e\n \u003cp\u003eColon histology indicated the pathological alteration occurred due to the accumulation of inflammatory cells and damage to the intestinal epithelium induced by DSS (3%) administration and the protective effects of mesalazine, MeEa 200 and MeEa 400 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. Colon tissue samples were stained with H\u0026amp;E and examined under a high-resolution microscope at 40X. In the VC group, the epithelial cells were intact, and no sign of inflammation was observed \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. In the NC group, the epithelial cells and structure of crypts were severely injured due to damage induced by DSS (3%) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. However, treatment with mesalazine at 100 mg/kg BW and MeEa 400 significantly reduced the damage to colon tissue than the NC group \u003cstrong\u003e(\u003c/strong\u003eFigs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e. Treatment with MeEa 200 provides less protection to the epithelial cells and crypts than the NC group \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. The degree of colonic inflammation was scored semi-quantitatively on a 0\u0026ndash;6 grading scale \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e, whereby the level of colonic inflammation in the VC group was the lowest (~\u0026thinsp;0), showing that mice in that group were free from signs of UC. The inflammation score in the NC group was ~\u0026thinsp;5, indicating significantly higher inflammatory cell infiltration as compared to the VC group. The inflammation score was ~\u0026thinsp;2 in the PC group, which demonstrated the significant protective effect of mesalazine in normalizing inflammatory cell infiltration and colonic epithelium integrity as compared to the NC group. The inflammation score was ~\u0026thinsp;3 in the MeEa 400 treatment group, thereby indicating significant protection offered by the MeEa 400 in normalizing the colonic inflammation as compared to the NC group. Treatment with MeEa 200 provides comparatively less protection than mesalazine and MeEa 400; the resulting inflammation score was found to be ~\u0026thinsp;3.5 as compared to the NC group \u003cstrong\u003e(Supplementary Table\u0026nbsp;1\u003c/strong\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8. Effect of MeEa against FITC dextran-induced intestinal membrane damage\u003c/h2\u003e\n \u003cp\u003eIntestinal barrier dysfunction is a prominent feature of UC. In this study assessment of intestinal permeability was performed in mice using FITC-dextran (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). In the NC group, there was huge damage occurred to the intestinal barrier due to FITC-dextran induction as compared to the VC group. Treatment with mesalazine and MeEa 400 provided significant protection to the intestinal membrane from leakage as well as reduced the elevated levels of FITC dextran in the serum than the NC group. The effect of mesalazine was marginally better than the MeEa 400. Treatment with MeEa 200 did not provide significant protection against intestinal membrane damage as compared to the NC group.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eInflammatory bowel disease or IBD includes ulcerative colitis (UC) and Crohn\u0026rsquo;s disease (CD), characterized by the inflammation of the intestine Interestingly, over the past few decades, the medical therapeutic armamentarium now approved for the management of UC has exploded which continues to expand [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, highlighting the controversial aspects of currently available treatment is the basis for emerging novel herb-based therapies. \u003cem\u003eMyrica esculenta\u003c/em\u003e (\u003cem\u003eM. esculenta\u003c/em\u003e) is a well-known medicinal plant, that possesses a wide range of pharmacological properties For a reason since long time it has been used as a folk medicine for the treatment of various diseases. Previously published phytochemical studies prove that \u003cem\u003eM. esculenta\u003c/em\u003e contains distinct bioactive compounds including alkaloids, triterpenoids, flavonoids, tannins, glycosides, saponins, volatile oils etc. As an ethnomedicine the stem bark, of \u003cem\u003eM. esculenta\u003c/em\u003e has been widely used by local tribes in India and China for the treatment of conditions like Inflammation- Meghalaya, India (Khasi tribe), Vietnam, South China; Diarrhea, dysentery, stomach problem in Meghalaya, India (Khasi tribe), Almora, Uttarakhand, India; As a carminative-Meghalaya, India (Khasi tribe), Mizoram, India. Earlier studies have reported that myricetin obtained from \u003cem\u003eM. esculenta\u003c/em\u003e plant [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] is commonly used as a food additive and possesses multiple pharmacological potential such as anti-inflammatory, anticancer, antidiabetic and strong antioxidant properties [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our HPLC analysis has confirmed that the ethyl acetate fraction of \u003cem\u003eM. esculenta\u003c/em\u003e (MeEa) indicated the presence of targeted flavonoids such as gallic acid (GA), myricetin (My), and quercetin (Q) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). DSS-induced UC is the most common and frequently used model that perfectly mimics the human UC symptoms in mice. Commonly 2.5-5% of dextran sodium sulfate (DSS) is mixed with drinking water induces UC symptoms in mice owing to its ease, controllability and reproducibility. DSS administration resulted in an alteration of the disease activity index (DAI) that upregulated the DAI scores. Parameters such as body weight loss in mice, reduction in average food intake, mean stool consistency score and mean stool blood score were elevated. Oral administration of MeEa 400 significantly downregulated the DAI scores as compared to the disease control (NC) group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). UC is majorly associated with colonic histological changes in which there is a shortening of colon length, an increase in colon weight/length ratio and an increased weight of spleen. Upon administration, MeEa 400 protects the colon from shortening and inhibits the increased colon weight/length ratio as well as reduces spleen weight than the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After DSS administration there is an increase in pro-inflammatory cytokine levels (IL6, IL8, TNF-α and IFN-γ) and a decrease in anti-inflammatory cytokine levels (IL10) in blood-serum and colon tissue was observed. IL6, IL8, TNF-α and IFN-γ are the major pro-inflammatory cytokines in UC. Where the level of IL6 is increased by lamina propria and a CD4\u0026thinsp;+\u0026thinsp;T cell, it promotes the survival of T-cell and apoptosis resistance in the lamina propria at the site of inflammation. The accumulation of CD4\u0026thinsp;+\u0026thinsp;T cells in lamina propria to regulate inflammatory responses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. IL-8 cytokine is produced by distinct tissue and blood cells that act as a chemoattractant and activate neutrophil accumulation at the site of inflammation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It has been reported that patients with UC showed elevated levels of IL-8 gene expression which is markedly associated with the inflammatory responses in the colon [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. TNF α is secreted by Th1 cells causing the accumulation of immune cells in UC including intestinal fibroblasts, neutrophils, and macrophages in the gut [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, intestinal epithelial cell damage is also regulated by TNF α signalling pathways via myosin light chain kinase (MLCK) activation. The increased level of IFN-γ promotes intestinal permeability by disrupting the vascular endothelial-cadherin junctions. These are associated with an increased risk of inflammation in bowel disease [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Studies have reported that untreated IL10 gene-deficient mouse leads to gradual histological alterations, elevated colon weight/length ratios and increased levels of inflammatory cytokines. Therapeutic options for the treatment of UC can be provided by modulating IL10 gene expression.\u003c/p\u003e \u003cp\u003eIn this way, our molecular findings advocated the protective effect of MeEa, 400, as its treatment decreased the level of IL6, IL8, TNF-α and IFN-γ and also stabilised the level of IL10 in both mouse serum and colon tissues in a similar fashion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In blood, there is an aberrant increase in the number of the various cell types such as total WBC, lymphocytes, monocytes, granulocytes and RBC due to the administration of DSS. In contrast, DSS administration reduces platelet counts and hemoglobin content significantly. Total WBC count is a crucial parameter to predict the extent of severity in UC, it is usually associated with severe clinical and mucosal disorders [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In UC there is an abnormal increase in lymphocyte count in the intestinal linings which is a sign of activated immune system to combat infection [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It has been reported that absolute peripheral blood monocyte level was increased in UC consequently associated with the severity of UC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The red cell distribution width (RDW) or RBC count is significantly increased in the case of UC [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The hemoglobin content becomes low in conditions like UC due to iron deficiency anaemia secondary to severe blood loss and reduced iron absorption caused by tissue inflammation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The platelet counts are decreased in UC due to the presence of auto-antibodies against platelet membrane antigens that cause immune-mediated platelet destruction. It is considered a rare extraintestinal indication of UC [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, treatment with MeEa 400 significantly reduced the number of cell counts like total WBC, lymphocytes, monocytes, granulocytes and RBC followed by an increase in platelet count and hemoglobin content (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Studies have shown that the severity of UC is associated with the extent of colonic injury. DSS induction caused a significant degree of inflammation and colonic tissue injury in the mice, which is signified by the inflammatory cell infiltration epithelial disruption, oedema, and haemorrhage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Findings of the present study have shown that mice of the negative control group (NC) resulted in a thicker and more erupted mucosal layer as a consequence of DSS (3%) administration. Treatment with MeEa 400 provided significant protection exhibited by the reduction in inflammatory scores and increased crypts count in the intestinal mucosa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe intestinal paracellular permeability and severity of intestinal mucosal barrier dysfunction in mice can be assessed by using 4-kDa FITC-dextran (non-digestible polysaccharides). Upon oral administration 4-kDa FITC-dextran does not cross the intestinal epithelial barrier unless the intestinal barrier becomes injured. Conditions like inflammation and injury to the intestinal epithelium cause mucosal barrier dysfunction. This results in barrier defects that promote the passage of 4-kDa FITC-dextran molecules towards the intestinal serous layer. Subsequently moves into the the systemic circulation, where it can be quantified in blood serum. Results obtained showed that at a higher dose treatment MeEa 400 provided significant protection from intestinal membrane leakage, thereby reduced level of FITC dextran in the serum was observed as compared to the untreated NC group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Thus, the present study reports the beneficial effects of MeEa in the management of UC that may be due to the presence of certain types of phytomolecules in it like GA, My and Q as characterized in HPLC analysis.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eHPLC fingerprinting of the ethyl acetate fraction of \u003cem\u003eM. esculenta\u003c/em\u003e (MeEa) showed the presence of gallic acid (GA), myricetin (My) and quercetin (Q) therein. Upon pharmacological evaluation, MeEa demonstrated suppressive effects against DSS (3%)-induced UC in mice. Treatment with MeEa 400 significantly reduced the DAI scores, protected colon length from shortening following a decreased colon weight/length ratio and also reduced spleen weight, respectively. Both in serum and colon tissues, MeEa 400 treatment down-regulated the elevated levels of pro-inflammatory cytokines (IL6, IL8, TNF-α, and IFNγ) and up-regulate the IL10 gene expression. Cell counts such as total WBC, lymphocyte, monocyte, granulocyte, and RBC were increased after DSS administration. Treatment with MeEa 400 significantly reduced the cell counts. Further, oral administration of MeEa 400 markedly reversed histological changes raised due to DSS-induction and also reduced histopathological scores. Our future research must be directed towards the in vivo investigation of the effect of MeEa on microbiota alteration in cases of UC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIBD, Inflammatory bowel disease; UC, Ulcerative colitis;\u0026nbsp;\u003cem\u003eM. esculenta, Myrica esculenta\u003c/em\u003e;\u0026nbsp;MeEa, \u003cem\u003eMyrica esculenta\u003c/em\u003e ethyl acetate fraction, DSS, Dextran sodium sulphate; IL, Interleukin; TNF-\u0026alpha;, Tumor necrosis factor alpha; INF-\u0026gamma;, Interferon gamma; RBC, Red blood cell; WBC, White blood cell; HGB, Haemoglobin; H\u0026amp;E, Hematoxylin \u0026amp; eosin; SGOT, Serum glutamate oxaloacetate transaminase; SGPT, Serum glutamate pyruvate transaminase; ALP, Alkaline phosphatase; HDL, High density lipoprotein; LDL, Low density lipoprotein.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments in the study were performed following the guidelines of the Organization for Economic Co-operation and Development (OECD) (1987) for the care and use of laboratory animals. Adult (6-7 weeks old) Swiss albino mice (20\u0026ndash;25 g) were used in the study. All the experimental animals were acclimatized for a period of 1 week prior to the experiment and maintained at normal laboratory conditions (25 \u0026plusmn; 1\u0026deg;C, 55 \u0026plusmn; 5% relative humidity and an automatic 12 h light/12 h dark cycle). Animals were provided with the commercial standard diet and water \u003cem\u003ead libitum\u003c/em\u003e. The experiments on ulcerative colitis activity were carried out under the protocol CCSEA/IAEC/SLSRPL/18/07/2023-02, which was approved by the Institutional Animal Ethics Committee (IAEC) constituted under the Committee for Control and Supervision of Experiments on Animals (CCSEA), Govt. of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed equally to the concept, experimental study, and data analysis. MJ has planned the experimental study and written the manuscript. Further data analysis and manuscript review were done by MP. The final manuscript was reviewed, evaluated and edited by AV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that supports the findings of this study are available in the manuscript and supplementary material of this article.\u003c/p\u003e\n\u003ch3\u003eStatements \u0026amp; Declarations\u003c/h3\u003e\n\u003cp\u003e\u0026ldquo;The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;The authors have no relevant financial or non-financial interests to disclose.\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eClara E, Sawian AM, Susngi B, Manners, Jasmine T, Sawian (2023) Chap. 28-\u003cem\u003eMyrica esculenta\u003c/em\u003e Editor(s). 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Poult Sci 100(7):101202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSudirman S, Hsu YH, He JL, Kong ZL (2018) Dietary polysaccharide-rich extract from \u003cem\u003eEucheuma cottonii\u003c/em\u003e modulates the inflammatory response and suppresses colonic injury on dextran sulfate sodium-induced colitis in mice. PLoS ONE 13(10):e0205252\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLau-Cam CA, Chan HH (1973) Flavonoids from \u003cem\u003eComptonia peregrine\u003c/em\u003e. Phytochem 12:1829\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones JR, Lebar MD, Jinwal UK, Abisambra JF, Koren J, Blair L et al (2011) The diarylheptanoid (+)-aR,11S-myricanol and two flavones from bayberry (\u003cem\u003eMyrica cerifera\u003c/em\u003e) destabilize the microtubule-associated protein tau. 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Gut Liver 5(4):460\u0026ndash;467\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaitha S, Bashir M, Ali T (2015) Iron deficiency anemia in inflammatory bowel disease. World J Gastrointest Pathophysiol 6(3):62\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDutra DMR, Almeida LR, Sinkos C, Muraro Bonini AC, Saad-Hossne R, Baima JP (2022) Immune thrombocytopenic purpura as an extraintestinal manifestation in a patient with ulcerative colitis. Case Rep Gastroenterol 16(1):140\u0026ndash;147\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeguchi Y, Andoh A, Inatomi O, Yagi Y, Bamba S, Araki Y et al (2007) Curcumin prevents the development of dextran sulfate sodium (DSS)-induced experimental colitis. Digest Dis Sci 52:2993\u0026ndash;2998\u003c/span\u003e\u003c/li\u003e\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":"Myrica esculenta, ulcerative colitis, inflammatory bowel disease, colon, DSS, FITC etc","lastPublishedDoi":"10.21203/rs.3.rs-3832022/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3832022/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMyrica esculenta\u003c/em\u003e (family Myricaceae) is a plant species valued in India and China for the management of gut disorders. Scientific validation of its anti-ulcerative colitis activity was aimed.\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eThe ethyl acetate fraction of \u003cem\u003eMyrica esculenta\u003c/em\u003e (MeEa) was prepared and evaluated for its potency against DSS-induced ulcerative colitis (UC) in mice at 200 and 400 mg/kg BW oral dose. The effective dose of MeEa was determined through its effect on DSS-induced UC and was further analyzed through its effects on disease activity index (DAI), colon length, colon weight/length ratio, spleen weight, serum and colon tissue cytokine level, cell count (total WBC, lymphocytes, monocytes, granulocytes, RBC and platelet) and hemoglobin content. Furthermore, the effect was determined through histopathology and FITC-dextran-induced membrane permeability assay. Between the two doses MeEa at 400 mg/kg BW was found to be the most effective dose in terms of reduced DAI scores, which were increased due to DSS administration, protected colon length from shortening, decreased colon weight/length ratio, reduced spleen weight, decreased pro-inflammatory cytokine (IL6, IL8, TNF α and IFN γ) level and stabilized the anti-inflammatory cytokine (IL10) level in serum and colon tissue. MeEa 400 reduced cell counts and increased hemoglobin content and platelet count. Furthermore, MeEa 400 prevented the colon by protecting epithelial cells and crypts. MeEa 400 provided significant protection from intestinal leakage and reduced FITC dextran level in serum.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMeEa 400 possesses significant anti-inflammatory potential and acts via attenuation of DSS-induced UC and inhibition of DAI scores. It reduces pro-inflammatory cytokines and stabilizes anti-inflammatory cytokine levels, reduces cell count, and protects epithelial tissue and crypts in the colon as well as intestinal membrane leakage that occurred due to FITC-dextran administration in mice.\u003c/p\u003e","manuscriptTitle":"Evaluation of the inhibitory potential of flavonoid-rich fraction of Myrica esculenta against DSS-induced colonic inflammation in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 18:53:54","doi":"10.21203/rs.3.rs-3832022/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":"e436d87c-2234-4b56-b9f3-8fde9034b27a","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-08T18:53:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 18:53:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3832022","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3832022","identity":"rs-3832022","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}