Effect of Methanolic seed extract of Piper nigrum aganist Acetic acid induced Ulcerative colitis in rats

 

G. Samyuktha^1*, Sahu Nimain Charan²

¹Assistant Professor, Pullareddy Institute of Pharmacy, Domadugu (V), Gummadidala (M), Sangareddy, Telangana

 

ABSTRACT:

To evaluate the effect of methanolic seed extract of Piper nigrum in acetic acid induced ulcerative colitis in rats. Piper nigrum extraction carried out by soxhelation method followed by preliminary phytochemical identification. Acute toxicity test was done by using OECD 423 guidelines. Albino rats were divided into five groups. Sulfasalazine used as a standard drug. Group A received normal saline. Group B received standard drug (Sulfasalazine). Group C received acetic acid. Group D received methanolic seed extract of Piper nigrum 10mg/kg., p.o., Group E received methanolic seed extract of Piper nigrum 30mg/kg., p.o., respectively. Colitis was induced by Intra rectal administration of 4% acetic acid on 8^th day. All animals were sacrificed after 72 hour of colitis induction and distal 10cm of the colon was dissected. Ulcer index, colon weight, colon length ratio, macroscopic score, histological changes were recorded. Intra rectal administration of acetic acid caused enhanced ulcer index, colon weight, colon length ratio. Pretreatment with Piper nigrum for 11d exhibited significant effect in lowering of ulcer index as well as infiltration of epithelial layer at a dose of 30 mg/kg in acetic acid induced colitis. The present investigation demonstrates Piper nigrum have potent therapeutic value in the reduction of experimental ulcerative colitis in rats by inhibiting the inflammatory mediator.

 

 

 

INTRODUCTION:

Inflammatory bowel disease (IBD) comprises of Crohn’s disease (CD) and ulcerative colitis (UC) which can be defined as chronic and relapsing inflammation of the gastrointestinal tract caused by different pathophysiological mechanisms characterized by clinical manifestations including diarrhea, abdominal pain, blood in the stool, and weight loss^[1].

 

Although etiology of IBD still remains poorly understood, complex interactions among genetic, environmental, immunological and reactive oxygen species (ROS) have been implicated in the pathogenesis of IBD^[2,3]. In many studies, it has been reported that antioxidants shows beneficial effects on experimentally induced colitis ^[4,5].

 

Piperine is an alkaloid found in plants belonging to the piperaceae family, such as Piper nigrum L., commonly known as black pepper, and Piper longum L., commonly known as long pepper. Black pepper called as king of spices. It is originated from tropical forest of the Western Ghats of India. It is cultivated in India, Brazil, Indonesia, Sri Lanka, Vietnam and china. Piperine is the major pungent substance in these plants and is isolated from the fruit of the black pepper and long pepper plants. Piperine comprises of 1 to 99% of these plants. It has putative anti-inflammatory activity^[6] and may have activity in promoting digestive processes. There is also preliminary evidence that it may have some anticonvulsant^[7], anti-carcinogenic^[8], hepatoprotective activity^[9] reducing inflammation and pain process, anti ulcer activity^[10]. There is also preliminary evidence that it might interfere with reproductive processes and have negative effects on sperm. Although this Piperine has many useful claims, the mechanism of its therapeutic activities is not understood clearly. The objectives of this study were to evaluate the effect of Piperine as anti-inflammatory activity in acetic acid induced ulcerative colitis in rats.

 

MATERIAL AND METHODS:

Collection of plant material:

The fruits of Piper nigrum were collected from rural areas of East Godavari district, Andhrapradesh in the month of December 2014. Authentication of Plant was carried out by Dr. L. Sunetha, Head of the department, department of botany, Government College, Rajahmundry. The voucher specimen number was 001.

 

Preparation of the extract:

Fruits of Piper nigrum (500gm) were powdered and then the powdered Piper nigrum L. was extracted with methanol. The extract was concentrated and evaporated to obtain crude methanolic extract of Piper nigrum L. The yield was found to be 15%w/w.

 

Preliminary Phytochemical screening:

The Preliminary Phytochemical screening of the above extract of Piper nigrum L. was carried out according to the method described by Khandelwal et al ^[11] and Patil et al ^[12]. Phytochemical analysis of the extract was performed for the identification of Phytochemical like alkaloid, flavonoids, steroid & phenols etc.

 

Animals:

Adult male/female healthy Wister albino rats were obtained from GIET School of pharmacy, Rajahmundry, Andhrapradesh (India). The animals were housed in groups of 5 in solid bottom polypropylene cages. They were maintained at (24 ± 1) ̊C, with relative humidity of 45%-55% and 12:12 h dark/light cycle. The animals were acclimatized for a period of two weeks and were kept under pathogen free conditions. The animals had free access to standard pellet chow (Sai durga feeds & foods, Bangalore, India) throughout the experimental protocol, with the exception of overnight fasting before induction of the ulcer. The animals were provided with filtered water. The pharmacology and acute toxicity protocols were approved by the Institutional Animal Ethics Committee (IAEC) (GSP/IAEC/2015/03/02).

 

Drugs & Chemicals used:

Acetic acid, Anesthetic ether, Methanol, formalin, were purchased from Siri Scientifics, Rajahmundry, India.

 

Acute toxicity studies:

Acute oral toxicity in albino mice was performed according to OECD guidelines 423. Graded doses of the Piper nigrum were made solution in distilled water, administered orally and the animals were observed for 2 week following administration. Body weight, food consumption, fluid intake and psycho-motor activities were recorded daily.

 

EXPERIMENTAL DESIGN:

The freshly prepared aqueous solution of Piper nigrum was administered to animals orally in two different dosages (10mg/kg, 30 mg/kg) for a period of 7 days. On the 8th day of dosage, inflammation was induced using acetic acid by a catheter through intrarectal administration. The drug treatment was continued even after the administration of acetic acid. Standard drug used for ulcerative colitis is Sulfasalazine. Sulfasalazine was not given as pretreatment. Sulfasalazine was administrated before the administration of acetic acid. Sulfasalazine was given in a dose of 100 mg/kg/day orally in rats as suspension containing 0.5% of sodium CMC

 

Twenty five healthy albino rats weighing 150-200g.were used in the study and were divided into five groups with five animals in each group (n=5) as follows.

·       Group A (normal control)-received 0.9% normal saline 10 ml/kg/day p.o.

·       Group B (standard)-received Sulfasalazine 100 mg/kg/p.o.

·       Group C (disease induced)-received acetic acid 4% v/v

·       Group D (test)-received methanolic extract of piper nigrum 10 mg/kg/day p.o.

·       Group E (test)-received methanolic extract of piper nigrum 30 mg/kg/day p.o.

 

Induction of colitis:

Twenty four hours fasted rats were slightly anesthetized with the diethyl ether. Ulcerative colitis was induced by 2 ml of acetic acid solution (4%v/v) was injected into the colon by using a rubber catheter and the tip was 9cm proximal to the anus. To prevent the leakage of colonic solution rat were maintained in a supine trendelenburg position for 30 seconds.

 

Evaluation of the disease:

The disease induced in experimental animals was evaluated based on its macroscopic characteristics, reported by Morris et al ^[13] was used after some modifications.

Table:1. Effect of Piper nigrum on colon weight, colon length, colon weight to length ratio,colon width and macroscopic score of rat in acetic acid induced IBD.

Group	Colon weigth(g)	Colon length(cm)	Colon weight to length ratio	Colon width(cm)	Macroscopic score
Normal	1.1 ± 0.036	8.2 ± 0.10	0.13 ± 0.0019	0.47±0.007	0.0±0.0
Sulfasalazine (100 mg/kg)	1.4 ± 0.057*	11 ± 0.28	0.129±0.0014	0.62±0.008**	1.6±0.085**
Acetic acid	2.6 ± 0.077	8.8 ± 0.04	0.27±0.0070	1.38±0.021	7.76±0.172
Piper nigrum (10 mg/kg)	1.85 ± 0.067	8.0 ± 0.17	0.23±0.011	1.17±0.020*	6.8±0.14
Piper nigrum (30 mg/kg)	1.61 ± 0.079*	7.8 ± 0.05	0.22±0.004	0.93±0.012*	5.35±0.125*

Data are expressed as mean ± SEM from five rats and analyse by one way ANOVA folowed by turkey’s test. P <0.0001 as compared to acetic acid group.

^{* *}significant compared to Acetic acid induced Disease positive control

 

Table:2. Effect of Piper nigrum on ulcer area and ulcer index of rat in acetic acid IBD.

Parameter	Normal	Sulfasalazine	Acetic acid	Piper nigrum (10 mg/kg)	Piper nigrum(30 mg/kg)
Ulcer area (mm2)	0.0 ±0.0	28.17 ± 0.87**	8.0 ± 0.73	21.67 ± 0.49**	17.67 ± 0.66*
Ulcer index	0.0 ±0.0	55.33 ± 1.3**	12.08 ± 0.82	49.33 ± 1.08**	43.83 ± 1.74**

 

Macroscopic scoring:

The macroscopic score of inflammation was calculated by using the no system. Based on the macroscopic scores were given in the following grades. For each animal in the 5 groups, the distal portion of the colon (10cm) was removed and cut into longitudinally and cleaned in 0.9% of saline solution to remove faecal residues.  0=no inflammation; 1= redness, 2= swelling, 3= one/two ulcers, 4=two/five ulcers, 5= more than five ulcers; 6= mild necrosis; 7=severe necrosis, based on the scoring calculate the ulcer activity in each animal.

 

Determination of ulcer area and ulcer index:

Identification of the ulcer index and ulcer area was performed according to the Dengiz et al ^[15] and kandhare et al ^[15]. For the determination of ulcer area, each colon was dissected and sliced and washed with normal saline and image was captured. The images were processed using image J software and adobe Photoshop to determine ulcer area.

 

Histopathological studies:

Colonic specimens were fixed in 10% formalin in phosphate buffered saline, embedded in paraffin and cut into 4 µm sections. Paraffin sections were deparaffinized with xylene, hydrated and stained with hematoxylin and eosin for mucosal damage assessment.

 

Statistical analysis:

For all the above methods, the results were expressed as mean ± SEM statistical analysis was done using one-way analysis of variance (ANOVA), followed by Dunnet’s. p<0.05 was considered significant.

 

RESULTS:

Preliminary Phytochemical screening:

The aqueous extract of Piper nigrum fruit was screened for various chemical tests as per the reported methods and was found to contain alkaloids, flavonoids, steroids, and phenols.

 

 

 

Acute toxicity studies:

Acute toxicity studies of the aqueous extract of Piper nigrum shows no sign and symptoms such as respiratory distress, diarrhea and convulsions it was found safe up to 500 mg/kg.

 

Acetic acid induced colitis:

Intrarectal administration of the acetic acid caused inflammatory reaction in the colon. The inflammation occurred in rectum and distal colon portion. Piper nigrum treated groups showed suppressed inflammatory reaction.

 

Effect of piper nigrum on colon weight:

In the acetic acid control animals colon weight (2.6 ±0.07)g was increased significantly (P<0.0001) as compared to normal group (1.1±0.036) g. Pretreatment of Piper nigrum (30 mg/kg,p.o), for 11d decrease the colon weight (1.61±0.079) significantly (p<0.0001) as compared to acetic acid group.

 

Effect of Piper nigrum on colon width:

The mean colon width of acetic acid control animals was found to be increased (1.38±0.021) significantly (P<0.0001) as compared to normal group (0.47 ±0.007). Pretreatment of Piper nigrum (30 mg/kg, p.o), for 11 d decreased the colon width (0.93 ± 0.012) significantly as compared to acetic acid group.

 

Effect of piper nigrum on colon weight to length ratio:

The ratio of colon weight/length was found to be increased (0.27 ±0.007) significantly (P<0.0001) in acetic acid control group as compared to normal group (0.13±0.001). Pretreatment of Piper nigrum (30 mg/kg,p.o.), for 11 d decreased the colon weight/length ratio(0.22 ± 0.004) was found to be significant as compared to acetic acid group.

 

 

 

 

 

CONTROL                                                                          STD

Fig:1                                                                                    Fig:2

 

Drug Control                                                                 30mg DOSE

Fig:3                                                                                                  Fig:4

 

Effect of Piper nigrum on macroscopic scores:

The colons of the rats were examined macroscopically for signs of hemorrhage and ulceration by an independent observer, in a blinded fashion, using a previously established scoring system. The mean macroscopic score of acetic acid group (7.76 ± 0.172) was found to be significantly (P<0.0001) increased as compared to normal group (0.0 ±0.0). Pretreatment of Piper nigrum (30 mg/kg, p.o), had decreased macroscopic score of colon (5.35 ±0.125) significantly (P<0.0001) as compared to acetic acid group and reduce the inflammation and ulceration of the colon.

 

Effect of piper nigrum on ulcer area:

Rectal administration of 4% acetic acid produced ulcers of colon in acetic acid control and all drug treated animals. The mean ulcer area of acetic acid control group was (8.0 ± 0.73) showed high ulcerogenic effect of acetic acid. Pretreatment of piper nigrum (10, 30 mg/kg, p.o), for 11 days decreased the ulcer area of colon (21.67±0.49), (17.67±0.66),mm² resp. significantly (P<0.0001).

 

Effect of piper nigrum on ulcer index:

The mean ulcer index of acetic acid control group was (12.08 ±0.82) showed high ulcerogenic effect of acetic acid. Pretreatment of Piper nigrum (10 mg/kg, p.o.), for 11 days decreased the ulcer index of the colon (49.33±1.08) significantly (P<0.0001) Piper nigrum (30mg/kg,p.o.), was more effective in reducing ulcer index of colon.(43.83±1.74).

 

Histopathological studies:

We examined the H &E stained sections of ulcerated areas of the colons of the rats for sign of the colitis. The Histopathological features of untreated animals included transmural necrosis, edema and diffuse inflammatory cell infiltration in the mucosa and loss of epithelium, hyperemia and goblet cell hyperplasia. An infiltrate consisted of mixed inflammatory cells was observed. Pretreatment of rats with Piper nigrum (10 mg/kg,p.o.) or treatment with Sulfasalazine significantly attenuated the extent and severity of the histological signs of cell damage. In case of normal rats colon there were no inflammatory cells in the lamina propria and epithelium remained intact.

 

DISCUSSION:

Acetic acid induced colitis in laboratory rats has been used to screen various drugs with proposed disease modifying property. Inflammatory bowel disease (IBD) is chronic relapsing conditions characterized by up-regulated pro inflammatory mediators and dysregulated immune responses resulting in tissue damage, the genetics, immunology and environment are the multiple etiologic theories which are related with IBD.

 

In the present investigation, the aqueous extracts of Piper nigrum L., selected for screening against experimentally induce inflammatory bowel disease. The intestinal mucosa in IBD is characterized by ongoing inflammation that results from activation of resident immune cells and infiltration of inflammatory cells from the circulation. In the present investigation acetic acid was used to induce colonic lesions. The acetic acid causes massive localized erosion of the colonic mucosa leading to sever localized inflammation and hemorrhages.

 

Various parameters such as colonic weight, colonic length, colonic width, colonic length/weight ratio, macroscopic score, ulcer index, body weight were recorded in this animal model. Colonic weight, width, length/weight ratio considered an index for local inflammation along with the other parameters of edema and wall thickening Colons were opened longitudinally and macroscopic damage score was given to each specimen. The results suggest that colon weight, width and weight/length ratio was found to be significantly increased in acetic acid control group as compared to normal group. The elevation in the colon weight and colon weight to length ratio was inhibited by Piper nigrum depicting its healing property. Ulcer area and ulcer index were quantitatively determined and reflect the protective action of Piper nigrum.

 

Alkaloids, steroids, flavonoids, phenols, steroids have been proven anti oxidant, anti-inflammatory and immunomodulatory active principles. These phytoconstituents of Piper nigrum may have synergistically contributed to the attenuation of ulcerative colitis.

 

From the results it could be deduced that piper nigrum possesses potent activity against various pathological changes caused by administration of acetic acid. Piper nigrum was found to be effective in acetic acid induced models. It could be hypothesized that the drug may form a layer over the colonic mucosa and also reduce localize inflammatory processes.

 

The drug possesses the hemorrhagic lesions which were caused by the administration of acetic acid. The results provide an idea to the probable mechanism of action of the drug. However, further studies need to be carried out to enables to zero upon a full proof mechanism of action of Piper nigrum.

 

ACKNOWLEDGMENT:

The authors highly indebted to the management of GIET School of Pharmacy, Chaitanya Knowledge City, Rajahmundry, 533296, Andhra Pradesh, India, for providing the necessary facilities for carrying out this work.

 

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Received on 26.02.2019         Modified on 25.03.2019

Accepted on 04.04.2019       ©A&V Publications All right reserved