Effect of Aqueous Extract of Trigonella Foenum-Graecum L. Seeds On Acetic Acid- Induced Ulcerative Colitis In Rats

Aqsa Fathima Kasturba Medical College Manipal Shivaprakash Gangachannaiah (  shiva.g@manipal.edu ) Kasturba Medical College Manipal https://orcid.org/0000-0002-6359-4024 Ujjal Bose Melaka Manipal Medical College Shama Prasada K SLS: Manipal Academy of Higher Education School of Life Sciences Rituparna Chakraborty Melaka Manipal Medical College Praveen Kumar S E ManipalTATA Medical College, Jamshedpur Padmanabha E G Udupa Kasturba Medical College Manipal Rachagolla Sai Pratap Yadav Kasturba Medical College Manipal Vidya Monappa Kasturba Medical College Manipal


Introduction
In ammatory bowel disease (IBD) is a chronic in ammatory disease of the gastrointestinal tract (S. V. Joshi et al., 2011). There are two main subtypes of IBD: Crohn's disease (CD) and Ulcerative colitis (UC) having a combined prevalence of 150-250/100000 population (Parkes & Jewell, 2001). The reported hospitalization was found to be 50.1 and 50.6 per 1 lakh population in CD and UC, respectively (Button et al., 2010).
Previous reports on the disease pathology of UC suggests that chronic in ammatory response in the colonic mucosa leads to tissue damage as a result of overproduction of reactive oxygen species (ROS) (Lih-Brody et al., 1996). The in ammatory response is ampli ed by the release of cytokines like TNF-α, IL-6 and IL-1β, which triggers the pathological response. The disease manifests as disabling intestinal and extra intestinal symptoms affecting the quality of life (Sartor, 1997).
Current medications for Ulcerative colitis are 5-aminosalicylic acid, corticosteroids, azathioprine, methotrexate, in iximab etc. These drugs are not speci c and are associated with intolerable adverse effects as many of them are cytotoxic immunosuppressants and were given for prolonged periods owing to the chronic nature of the disease. Therefore, the development of newer drugs are essential to treat this disorder which has a long and complex pathogenetic history. Treatment with herbal products are believed to be safer and economical.
Trigonella foenum-graecum L. (TFG) is widely distributed throughout the world and belongs to the family Fabacecae. Traditionally in Ayurveda, this plant was found to be bene cial in many digestive conditions.
Fenugreek seed is composed of biologically active compounds including avanoids, amino acids, vitamins and alkaloids like, choline and trigonelline (Li et al., 2008). TFG is well known for its multiple pharmacological properties including antidiabetic, antioxidative, hypocholesterolemic, antineoplastic, anti-in ammatory, antiulcerogenic, antipyretic, immunomodulatory and antitumor activity (Yadav & Baquer, 2014). Till date there are not enough studies to prove the bene cial effects of TFG in UC. The present study was designed to evaluate its potential bene ts in UC model.

Materials And Methods
Drugs and chemicals Sulfasalazine (SAZO, 500mg), was procured from Wallace Pharmaceuticals Private, Limited, India. Trigonella foenum greacum L. seeds was obtained from the local market in Udupi, Karnataka, India, which was authenticated by a botanist. All the Chemicals Sodium carbonate (Na2CO3), Sodium hydroxide (NaOH), Sodium-potassium alloy (NaK), Copper sulphate (CuSO4), Trichloro acetic acid (TCA), DTNB [5-5'- Reverse: 5'GGCATGGACTGTGGTCATGA 3', cDNA synthesis kit and GoTaq Green mater mix were procured from Gennext Scienti c and IT solutions, Mulky, India. All the solutions were freshly prepared and all the reagents used were of analytical grade.

Animals and ethics approval
The study was approved by institutional Animal Ethics Committee (IAEC/KMC/93/2018). Wistar rats were procured from the Central Animal Facility of the institute in accordance with the committee for the purpose of Control and supervision of experimentation on animal guidelines (CPCSEA). Thirty male albino wistar rats of weight ranging 150-250gm, aged around 8-10 weeks were used in the study. The animals were housed under standard condition, 12:12 light-dark cycle, 50% humidity and 28°C temperature and provided with standard food granules and water ad libitum.

Preparation of extract
Trigonella foenum-graecum L. aqueous extract was prepared as described by Noor (2007). Stock solution was prepared by boiling 10 gm of dried, grounded fenugreek seeds in 250 mL of double distilled water for 1 h. The extract was left all night and then ltered and made to 250 mL by double distilled water.

Experimental design
Thirty adult male rats were randomly allocated into ve groups: The dose of Trigonella foenum greacum L. seed extract was selected based on the anti-in ammatory property and toxicity study which demonstrated that there was no mortality rate till 5g/kg of Trigonella foenum greacum L. seeds (Muralidhara et al., 1999). Based on this we have selected two doses of Trigonella foenum greacum L. seed (500mg/kg and 1000mg/kg).

Induction of Ulcerative colitis
The drugs were administered per orally for 7 days, along with diet. The volume of drugs were kept constant at 5 ml/kg. Control group received distilled water. On day 8 following overnight fasting, UC was induced by administration of 1 ml of 4% acetic acid (AA) transrectally using an 8Fr (2.7 mm) soft paediatric catheter coated with lignocaine anaesthetic. The catheter was passed till 6-8 cm from the anus under low dose ether anaesthesia. The rats were maintained in head down position (trendelenburg position) after AA administration for 10 seconds to prevent any leakage. The acidic solution was aspirated out and it was followed by transrectal colon wash with 2 ml of phosphate buffer saline (PBS) at pH 7. Ulcerative colitis was induced in groups II to V. The normal control group received only normal saline transrectally. Twenty-four hours following induction of colitis, animals were sacri ced under anaesthesia.
Colon weight/ length ratio Colon weight was measured in grams and length in centimetres. The ratio of weight in grams to length in cm was calculated by Aleisa et al. (2014).
Disease Activity Index (DAI) DAI was assessed by Cooper et al. (1993) method. The changes in growth rate, stool consistency, and presence of gross bleeding or occult blood in feces was scored from 0~4 for each animal with a score 0 as normal stool consistency and no occult/gross rectal bleeding while the maximum score 4 with diarrhea, gross bleeding along signi cant decrease in growth.

Macroscopic Scoring
Colon from each animal was separated and dissected out for macroscopic scoring. Scoring was done according to Morris et al. (1989). A colon of length 4 cm extending proximally 2 cm above the rectum was dissected, split longitudinally on a piece of paper, and the colon was scored macroscopically with score 0 as normal gross morphology and 5 as intensive in ammation and ulceration [13].

Histopathology
The colonic tissues stored in 10% formalin, xed in para n wax and washed in graded alcohol series. The tissue sections of 5µm thickness were taken and mounted on the slides. The staining was done by depara nization using xylene at different time intervals followed by hydration using a series of graded alcohol (100%, 90%, 80%, 50%). After hydration, the tissues were stained with hematoxylin, and the slides were subjected to washing by slow running water. Differentiation and blueing were done after the hematoxylin stain and the tissues were counterstained with 1% eosin. Later the slides were dehydrated with absolute alcohol and cleared with pure xylene. Following this, the tissue was mounted with Dibutylphthalate Polystyrene Xylene, and coverslip was placed. The slides were left for air drying at room temperature and examined under light microscope.
The microscopic colon damage was scored as described by Gálvez et al. (2001) with a score 0 being normal colon and a maximal score of 5 with transmural in ammation and intense ulceration.

Preparation of Homogenate
For the determination of tissue Total protein (TP), antioxidants and Lipid peroxidation (LP), 10% homogenate of colon was prepared with ice cold KCL (150mM) using homogenizer (Model:Yamato L.S.GL.H-21, Japan) and centrifuged at 3000 rpm for 10 min at 4°C.

Total protein
The total protein estimation is based on the reduction of Cu2+ to Cu+ by protein in an alkaline medium.
Chelation of copper (cupric ion) with protein in alkaline environment form a light blue chelate complex between peptides of three or more amino acids with cupric ion and results in formation of cuprous ion (biuret reaction). The Bicinchoninic acid (BCA) reagents reacts with the cuprous ions to form a strong purple colour, with maximum absorbance at 540nm (Assay et al., 2000).

Reduced Glutathione
Tissue glutathione was estimated as described by Laboratorien & Jenapharm (1962). The non-protein compound containing the sulfhydryl group in its structure is known as Glutathione reductase. It reduces 5,5'-dithiobis-2-nitrobenzoic acid to a deep yellow colored compound. The GSH activity was measured by spectrophotometer.

Catalase
Tissue catalase was estimated as described by Aebi (1984). The colon was homogenized and mixed with 50 mmol/L PBS (pH 7.0) and 20 mmol/L hydrogen peroxide. The catalase activity was measured by spectrophotometer.

Superoxide dismutase
Superoxide radicals present in supernatant oxidizes adrenaline bitartarate to form adrenochrome. Estimating the amount of adrenochrome formed is related to SOD activity as it will inhibit oxidation of adrenaline bitartarate by eliminating superoxide radicals (Kuninaka et al., 2000).
Lipid peroxidase by MDA assay Polyunsaturated fatty acid breaks down to form malondialdehyde, which helps to determine the extent of peroxidation reaction. Pink color is formed by the reaction between thiobarbituric acid and malondialdehyde which is measured at 532nm (Ohkawa et al., 1979).

Method of RT-PCR
The total RNA was extracted from rat tissue biopsies homogenized in TRI-Reagent (T9424, Merck, India) following the manufacturer's protocol. The extracted RNA was carefully assessed for its quality, purity and integrity using the 260/280 ratio, 260/230 ratio obtained from BioSpectrometer basic, (6135000009, Eppendorf, India) and agarose gel electrophoresis (intact gel bands corresponding to 28S and 18S RNA) respectively. The extracted RNA from each group was diluted to 180 ng/μl using nuclease free water to ensure a known amount of starting mRNA concentration from every group for cDNA synthesis. High-Capacity Reverse Transcriptase cDNA synthesis kit (ThermoFisher Scienti c India Pvt. Ltd., India) was used to prepare cDNA ( nal volume of 20 μl) from the extracted total RNA using thermocycler (T100, BioRad, India). Synthesis of cDNA involved the use of random primers from the cDNA synthesis kit with internal controls as +RT/-RT(with or without reverse transcriptase) to reduce experimental errors and track speci city of primers in case of genomic DNA contamination. . PCR was performed with a total volume of 25 μL. PCR mixture comprised: cDNA -4 μL, forward and reverse primers -4 μL each, GoTaq Green master mix -12.5 μL and nuclease free water -4.5 μL. The reaction mixture was then subjected to 33 cycles which consists of 5 minutes pre-apo morphosis at 95°C, Then 10 μL of the ampicon was added to 1.5% agarose gel for electrophoresis. Gel doc (Gel Doc EZ Imager,BioRad, India) was used to capture the image of the gel followed by analysis using the Image-J software (Fiji) (Perera et al., 2010).

Statistical analysis
Statistical analysis was done using Graph Pad Prism 5.03 Demo Version (Graph Pad Software Inc., La Jolla, CA, USA) by one-way analysis of variance (Tukey test). Results were expressed as Mean ± SEM, and p≤0.05 was considered signi cant. The relative mRNA expression of TNFα was analyzed by Image-J 1.51f software (Wayne Rasband, National Institutes of Health, USA) using ANOVA (Dunnett's test).

Results
In UC group, the colonic weight ( Figure 1) was increased compared to the control group. Following seven days of pre-treatment with Sulfasalazine in standard and TFG in test groups, marked reduction in colon weights was observed in them compared to UC-group. There was a signi cant reduction in DAI, macroscopic and microscopic score in standard and TFG treated groups compared to UC-group. In the UC group, there was intense hyperaemia, ulcerations, and in ammation. In standard and TFG groups, there was a reduction in ulceration and in ammation compared to UC group (Table 1).

Histopathological changes
In normal Control group, the colon sections had normal architecture with no signs of ulceration and in ammation (Figure 2A). In UC group, there was extensive ulceration and in ammation with a maximal microscopic damage indicating focal necrosis of mucosal and submucosal region with involvement of serosa. Diffuse leukocyte in ltration and crypt damage was observed ( Figure 2B). In the groups treated with sulfasalazine, TFG-I (500mg/kg) and TFG-II (1000mg/kg), the degree of in ammation was lesser compared to UC group and there were no signs of crypt damage or ulceration. (Figure 2C, 2D, 2E).

Biochemical analysis
The total protein level (Figure 3a) was decreased (P< 0.05) in UC group. The total protein levels signi cantly increased in standard, and TFG treated groups compared to the UC group. CAT, SOD and GSH activity (Figure 3b, 3c and 3d) was signi cantly (P < 0.05) decreased in colon tissues of UC rats compared to normal control. Pre-treatment with TFG at both doses increased CAT, SOD and GSH activity in colon as compared to UC group. The lipid peroxidase levels decreased signi cantly in standard, and TFG treated groups compared to the UC group (Figure 3e).
Relative mRNA expression of TNF α As shown in Figure 4, the mRNA levels of TNF α in UC group showed a signi cantly high expression compared to control group (p < 0.0001). The mRNA expressions of TNF α were inhibited in animals treated with Sulfasalazine and TFG (500mg/kg and 1000mg/kg).

Discussion
The present original study has demonstrated the bene cial effect of TFG in UC in rats. The histological changes observed with AA induced colitis in rats is similar to UC seen in humans. AA affects the distal colon and causes in ammation, edema and ulceration of mucosal and submucosal layers. The aqueous extract of TFG has shown protection against colitis in rats, as evidenced by colon length, weight, DAI, gross, histological and biochemical evaluations.
The colon weight/length ratio indicates the intestinal in ammation with the consequent shortening of the colon and increase in weight. There was a signi cant reduction in the colon weight/length ratio in groups treated with TFG, indicating its anti-in ammatory property.
DAI assess disease severity of ulcerative colitis as described by Cooper et al. (1993). It is a combined score of rectal bleeding and stool consistency and was assessed after colitis induction. The DAI score was signi cantly decreased in groups treated with TFG indicating its ability to diminish the intensity of in ammation in ulcerative colitis (Maheshwari et al., 2015).
Previous reports have demonstrated the anti-in ammatory activity of TFG in rat model (Vyas et al., 2008).
In the present study, the macroscopic score and microscopic scores which quantitatively measures severity by assessing TNFα is an in ammatory marker and is implicated in the pathogenesis of UC. Studies have shown its production is upregulated in colon tissue in IBD and the levels closely correlate with mayo endoscopic score (severity of UC). Its importance is proven by the robust improvement in symptoms observed in refractory or immunosuppressive intolerant cases receiving anti-TNFα antibody therapy (Hendrickson et al., 2002), (

Limitations
The study did not assess other proin ammatory cytokines apart from TNF-alfa. Also, the TNF-alfa expression was investigated at mRNA level and not the proteins.
The acetic acid induced colitis may not mimic exactly the in ammatory features of chronic in ammation in humans, though it is an established model for UC in animals.
Further, molecular studies on in ammatory signalling are required to precisely delineate the protective mechanisms of TFG in UC. However, this study gives rst-hand assertion of bene cial effects of TFG in UC model.

Conclusion
Our study con rms the bene cial effects of Trigonella foenum greacum L. at both the doses in attenuating the acetic acid induced colitis in a rat model. The study con rms the underlying bene ts due to its antioxidant and anti-in ammatory properties. Both the doses of Trigonella foenum greacum L. seeds extract were equally effective. The study suggests the potential of Trigonella foenum greacum L. seed extract in treating UC patients. However, the clinical bene ts needs to be con rmed by further clinical studies. Figure 1 Effect of TFG on colon weight/length in rats. Data represented as mean ± SEM and analysed using with * p≤0.05 vs. UC group.