Exploring Anti-inflammatory effect of Holarrhena antidysenterica and Coriandrum sativum Linn in the Digestive System disease IBD in Rats

Kiran Bala¹, Ajeet Pal Singh¹, Indu Melkani², Amar Pal Singh³

¹St. Soldier Institute of Pharmacy, Jalandhar Near Amritsar Road, Behind NIT, Jalandhar, Punjab.

²Assistant Professor, Amity Institute of Pharmacy, Amity University, Lucknow, U.P., India.

³Lovely Professional University, Phagwara, Kapurthala, Punjab, India.

*Corresponding Author E-mail: kiranbala814626610@gmail.com, ajeetakarpuria@gmail.com, ikshitamelkani@gmail.com, amar.psingh75@gmail.com

ABSTRACT:

Background: Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory gastrointestinal disease that includes Crohn's disease and ulcerative colitis. Symptoms include diarrehoea, stomach discomfort, blood in the stool and other similar issues. Since there are no safe and effective treatments for gut inflammation, new treatments developed to efficiently treat the disease's symptoms and implications. Methods: The development of collitus was promoted by the injection of 3% acetic acid into the rat colon through the rectum. Macroscopic, biochemical, histological and disease activity scoring of the colon were performed after 5 days of infusion to evaluate colonic damage. A study on the activity of Holarrhena antidysenterica and coriander sativum Linn was investigated in collitus-induced 36 albino rats randomized into five groups (n=6). Control Group I given the normal saline, Group II give the Acetic acid 3% through rectum, Group III give the Budesonide (0.5mg/kg) through oral. Group IV give the aqueous extract of Holarrhena antidysenterica (HA) (40mg/kg) through oral, Group V give the aqueous extract of Coriander sativum Linn. (CS) (40mg/kg) through oral and in Group VI given the combination of both extract HA and CS. Both treatment drugs given 2 days before induction of collitus. Results: A significant colon inflammatory response to acetic acid infusion is observed macroscopically and histopathologically. Additionally, it results in a decrease in GSH, SOD and an increase in MPO level. Thus in current study aq. extracts of Holarrhena antidysenterica and Coriandrum sativum were administered for 7 days i.e. commencing 2 days prior to the induction of acetic acid and continuing for 5 days after. Compared to disease control there was a significantly reduced amount of macroscopic damage and disease activity score. Additionally, it increased intestinal levels of reduced GSH, SOD and significantly decreased MPO levels. Conclusion: Inflammation is inhibited by coriander sativum while the primary symptoms of IBD are inhibited by Holarrhena antidysenterica. Inflammatory bowel disease can be treated more effectively with a combination of these two medications which also eliminates the side effects of glucocorticoids (Budesonide).

KEYWORDS: IBD, Ulcerative collitus, Glutathione, Holarrhena antidysenterica, Coriander sativum.

INTRODUCTION:

Inflammation is a targeted response to cellular injury that includes vascular dilation, leukocytic aggregation, redness, heat, discomfort, swelling, and frequent loss of function. It also serves as a trigger to the removal of toxic substances and abnormal cells. Inflammatory bowel disease (IBD) is an Idiopathic chronic inflammatory gastrointestinal illness that includes Crohn's disease and ulcerative colitis with unknown etiology.¹ Ulcerative Colitis (UC) is the inflammation of large intestine, colon and the rectum. It includes Genetic variables, infection with mycobacterium, yeast, E.coli and other pathogens. The distinction between diseased and normal mucosa is significant in ulcerative collitus.² Crohn’s disease (CD) includes Patchy and transmural lesions. It can affect any area of the GIT from the mouth to the anus. It also includes extra intestinal problems such as fever, mouth ulcer and dermatitis which may develop in 50% of cases. The cause of IBD is unknown. Symptoms include diarrhoea, stomach discomfort, blood in the stool, and other similar issues. The current theories relates a variety of factors including immune dysregulation (caused by genetic or environmental factors), abnormal GI tract luminal factors (such as microorganisms making up the GI tract flora), oxidative stress.^3,4 The patho physiology involves multiple factors including NOD gene, TLR receptors, interleukin-1, interleukin-6 and TNF.⁵ A homozygous mutation in the CARD15 gene (NOD gene) which encodes NOD2 coding was discovered in a large number of Crohn's disease patients. Around 10-15% of patients with this condition involves.^6,7

The Children’s shows various types of symptoms depending on the disease's region, complexity and number of days and include acute severe colitis (ASC) that is characterized by recurrent bloody bowel movements, pain and systemic symptoms like tachycardia can develop in some children with UC.⁸ Children under the age of six are more likely to develop ulcerative colitis (31-47%) but children over six are more likely to get Crohn's disease (60-66%). Young children seemed to have a higher incidence of localized colonial disease (41-69%) compared to older children (24-46%).⁹

Fig. 1: Inflamed Intestine.

MATERIALS AND METHODS:

Procurement of animal:

Male Wistar Rats of 6-8 weeks with 200-250g were procured from Punjab University Chandigarh. They were kept in the central animal house facility at St. Soldier group of Institutions. They were maintained in controlled sterile environment. The animals were housed in conditions of ambient temperature and humidity with a cycle of 12hours of light and 12hours of dark. They were group housed in cages with not more than three animals per cage. The experimental protocol approved by the Institutional Animal Ethics Committee.

Drugs and chemicals:

Holarrhena antidysenterica, Coriander sativum, Budesonide, Acetic acid, Potassium Phosphate buffer, Hexadecyl trimethyl ammonium bromide, EDTA, Disodium Hydrogen Phosphate, Tris acetate, Bovine serum albumin, HEPES buffer.

Inducing models for Inflammatory Bowel Disease (IBD): The commonly employed models are the Acetic acid induced collitus, Trinitrobenzene sulfonic acid (TNBS)-induced colitis, dextran sodium sulphate induced colitis model (DSS), Oxazolone-induced colitis, Nonsteroidal anti-inflammatory drugs for colitis (NSAIDs), peptidoglycan-polysaccharide (PGPS)-induced colitis model and Carrageenan-induced colitis.

Acetic acid induced collitus model: Through the rectum, acetic acid (3% v/v) was injected into a rat's colon to create ulcerative colitis.^10,11 A polyethylene tube (2 mm in diameter) was inserting into the colon through the rectum up to 8cm and used to infuse two millilitres of acetic acid (3% v/v, in 0.9% saline) for 30 seconds. The animals were kept for five days following the onset of ulcerative colitis. Subsequently, Rats were sacrificed, and colonic samples were taken for biochemical analyses including myeloperoxidase (MPO), total protein, and glutathione scoring, histological analysis, and macroscopical evaluation (GSH).¹²

Note: Animal should not feed for 24hrs and they were given free access to water.

Experimental design: Five days after receiving an acetic acid infusion anaesthetized rats were euthanized on the seventh day of the experiment and the distal colon of the animal were transversely cut up to 8cm from the anus. The tissue was carefully clean in regular saline solution and then fastened to a rat board. The severity of the mucosal injury was graded macroscopically. The outcomes are as follows: No ulceration: 0, Mucosal erythema Only:1, Mild Bleeding or Small Erosion:2, Moderate Edema, Bleeding Ulcers or Erosion:3, Severe Edema, and Tissue Necrosis:4.¹⁰

Figure 2: Experimental protocol

Table 1: study design for treatment

Group	Study design	Treatment Schedule	Dose	Animal Required
Group I	Control group	Normal saline	-	6
Group II	Diseased group	Acetic acid	3 % v/v/rectal	6
Group III	Standard group	Budesonide	0.5mg/kg/per oral	6
Group IV	Treatment group I	Aqueous extract of Holarrhena antidysenterica	40 mg/kg/per oral	6
Group V	Treatment group II	Aqueous extract of Coriander sativum	40 mg/kg/per oral	6
Group VI	Combination of Treatment I and Treatment II	Aqueous extract of Holarrhena antidysenterica and Coriander sativum	40 mg/kg + 40 mg/kg/per oral	6

Rats were giving colonic infusions of acetic acid (3% v/v in 0.9% saline) to cause ulcerative colitis. Two days before to the induction of colitis, Holarrhena antidysenterica and coriander sativum were giving and continued for five consecutive days. On the fifth day (a total of 7 days of treatment) after the acetic acid infusion, macroscopical, histopathological and biochemical examinations was carried out. Rats are sacrifice on the seventh day of treatment and the morphological, histopathological and a biochemical examination on the colonic tissue were performed.

Behavioral/ Observational parameter:

Body Weight analysis: On the third day after collitus induction, a body weight study was carried out. And the changes are occur in body weight of each group of animals.¹³

Stool consistency: From the first day following collitus induction until the seventh day, the consistency of the stools was assessed. It was display daily consistency changes to demonstrate the precise function of the intestine.¹³

Biochemical parameters:

Determination of MPO activity: Colonic tissues were taken into small pieces, cleaned with ice-cold normal saline and dried before weighed and cut into small 0.5g pieces and homogenized in 10 volumes of ice-cold potassium phosphate buffer (pH 7.4). At 4°C, the homogenate was centrifuged for 30minutes at 3500rpm. The supernatant was discarded and add 10ml of ice-cold 50mM potassium phosphate buffer (pH 6.0), 0.5% hexadecyl trimethyl ammonium bromide and l0mM EDTA to the pellet. After that it was proceed through one cycle of freezing and thawing, and was subjected to sonication for only 15 seconds. The solution will again centrifuge after sonication for 20min at 15,000rpm at         4°C. And the MPO activity measures spectrophotometrically at 460nm.^14,15

Determination of GSH: The GSH concentration in the homogenate was measure spectrophotometrically by combining 0.5ml of tissue homogenate with 2ml of (0.3 M) disodium hydrogen phosphate solution and 0.25ml of (0.001M) freshly made 5,5′-dithiobis (2-nitrobenzoic acid) DTNB solution to produce a yellow chromogen. And the GSH activity (10-100μm) as a standard was determined spectrophotometrically at 412nm.¹¹

Determination of Protein content: The determination of total protein content in the homogenate was done by Lowry using a standard bovine serum albumin.¹⁶

Determination of Superoxide Dismutase (SOD) activity: For determining the SOD, the sample of colonic mucosa was select and homogenizes in a cold 20mM HEPES buffer and centrifuge for 5minutes. The supernatant was collect and assayed using colorimetric assay.¹⁷

Histopathology: At the end of the study colon segments from each rat were collected and preserved in the fixative solution (10% formalin). Hematoxylin and eosin were used for the staining process. And the sections were examined under a light microscope.

Statistics: The results were expressed as mean ± and standard error of six readings. Tukey's multiple comparison tests have been used since an analysis of variance (ANOVA) to determine statistical significance. 𝑃 < 0.05 was considered to be significant.

RESULTS:

Morphological evaluations:

Figure 3: Morphological evaluations

G-I Normal, G-II Acetic Acid, G-III Budesonide, G-IV HA, G- V Coriander Sativum, G-VI combination of Holarrhena antidysenterica And Coriandersativum

Stool consistency:

The rat's stools were thick on the first day of the acetic acid infusion, but after two days they became soft. And following the administration of acetic acid, blood is also found in stools (fig.4).

Figure 4: Stool consistency

Body weight:

Starting on the fourth day following the intracolonic administration of acetic acid until the seventh day which is the day of sacrifice, the body 0 weights decreased. Compared to control there was a significant weight loss in AA group. In the groups give the HA and CS there was no significant weight loss. Also, a significant difference between the test drugs (HA and CS) and the control drug were discovered.

Histopathology:

Figure 5: Histopathology of colon- In group I exhibit the normal group and Group II shown the acetic acid induced colitis in rat colon, group III shows the effect of standard drug i.e. Budesonide, group IV shows the activity of Holarrhena antidysenterica, group V shows activity of Coriander sativum, Group VI shows the activity of Combination of coriander sativum and Holarrhena antidysenterica.

Effect on Biochemical parameters:

Acetic acid induced collitus in Rats:

Acetic acid insertion (through the intra-rectal route) led to an inflammatory response in the colon. The rectum and distal colon were also affected by the inflammation. Severe epithelial necrosis and ulcerated mucosa were the alterations.

Table 2: effect of drugs on MPO, GSH and SOD biochemical parameter

Groups	MPO	GSH	SOD
Normal	150.68± 30.25	298.75± 29.47	13.46± 2.91
Acetic acid	200.61± 21.76	98.79± 20.33	4.29± 1.90
Budesonide	169.50± 3.48*	240.8± 25.44*	11.20± 1.60*
Holarrhena antidysenterica	174.83± 3.33***	190.30± 24.22***	8.85± 1.20***
Coriander sativum	173.95± 2.66***	200± 24.29***	9.25± 1.10***
Combination (HA and CS)	171.52± 2.15**	218± 21.23**	10.11± 1.30**

Values are expressed as mean ± S.E.M. (n=6).

* Significantly different from Normal control group at p < 0.001. **Different from acetic acid group at p<0.01. ***Different from acetic acid group at p<0.05.

MPO- Myeloperoxidase, GSH- Glutathione level, SOD, Superoxide dismutase, HA-Holarrhena antidysenterica, CS- coriander sativum

Effect on MPO level:

According to the results of the biochemical tests, the animals in the control group with colitis were significantly different from those in the normal group. MPO parameter was increased when compared to that of normal group. The treatment given with Budesonide, Holarrhena antidysenterica and Coriandrum sativum decreases the level of MPO as compared with the normal group.

Figure 6: Effect of different drugs on Colonic myeloperoxidase (MPO) level in acetic acid induced ulcers in rats

Effect on GSH level:

The levels of GSH in colonic tissue significantly decreased after acetic acid infusion as comparison with the control group. Furthermore Budesonide, Coriandrum sativum and Holarrhena antidysenterica reduced the level of GSH caused by acetic acid in colonic tissue.

Figure 7: Effect of different drugs on Colonic Glutathione (GSH) level in acetic acid induced ulcers in rats

Effect on SOD:

It has been suggested that superoxide dismutase (SOD), which is broadly distributed in cells with high oxidative metabolism can protect these cells from the harmful effects of superoxide anion. When compared to the normal control group animals, the levels of SOD and GSH are lower, and treatment with an aqueous extract of Holarrhena antidysenterica and coriander was found to increase SOD.

Figure 8: Effect of different drugs on Superoxide dismutase (SOD) level in acetic acid induced ulcers in rats

DISCUSSION:

In this present study, the infusion of acetic acid produces a ulcerative collitus or Crohn's disease in the rat colon. Before the two days of induction of colitis, Holarrhena antidysenterica and coriander sativum were administered into the body of rats and continued for five consecutive days. On the fifth day (a total of 7 days of treatment) after the acetic acid infusion, macroscopical, histopathological and biochemical examinations was carried out. Rats are sacrifice on the seventh day of treatment and the morphological, histopathological and a biochemical examination on the colonic tissue were performed. H. antidysenterica produces gastrointestinal tract activation by activating histamine receptors and relaxes the gastrointestinal tract by blocking the Ca ++ channel, which gives usefulness in gastrointestinal disorders such as constipation and diarrhoea.¹⁸ Coriander reduces the MPO level directly by inhibiting the inflammatory mediators.¹⁹ The various biochemical parameters i.e. MPO level, GSH level, SOD level were performed during the seven days. Furthermore, acetic acid produced collitus model is widely used to induce the ulceration in experimental animals because it produces resemblance with human’s ulcers. It shows that the level of MPO was increased or reduced the level of GSH. It has been suggested that superoxide dismutase (SOD), which is broadly distributed in cells with high oxidative metabolism can protect these cells from the harmful effects of superoxide anion. The treatment given with Budesonide, Holarrhena antidysenterica and Coriandrum sativum for seven days, changes the levels of MPO, GSH and SOD as compared with the diseased groups. After the scarification of animal at the last day the histopathology has been done (figure no. 5). The histological changes shows that the effect of drugs into the animals. Holarrhena antidysenterica and coriander sativum decreases the level of MPO which is increased by the acetic acid in ulcer (figure no. 6). Both the drugs increase the level of GSH and SOD as reduced by the acetic acid (figure no. 7, 8). It shows that the effect of combination of drugs is better than the drugs given individually. Therefore, it may be proposed that the effect of Holarrhena antidysenterica and coriander sativum diminished the acetic acid induced ulcerative collitus in the view of fact that it’s anti-inflammatory activity and antioxidant activity.

CONCLUSION:

It may be concluded that the Holarrhena antidysenterica and Coriandrum sativum decreases the level of acetic acid induced ulcers in rats due to its anti-inflammatory action, antioxidant action.

CONFLICT OF INTEREST:

All the authors have no conflict of interest.

ACKNOWLEDGMENT:

The authors are grateful to the St. Soldier Institute of Pharmacy and the respected Chairman Mr. Anil Chopra, Vice Chairperson Ms. Sangeeta Chopra, St. Soldier Educational Society, Jalandhar for providing the necessary facilities to complete this research word.

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Received on 04.12.2024      Revised on 22.03.2025 Accepted on 26.04.2025 Published on 22.07.2025 Available online from July 26, 2025 Res.J. Pharmacology and Pharmacodynamics.2025;17(3):159-164. DOI: 10.52711/2321-5836.2025.00026 ©A and V Publications All right reserved
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