| Abstract|| |
The aim of this study is to evaluate the effect of the ethanolic extract of the leaves of Psidium guajava (PGE) on experimentally induced colitis in animal models. Fresh tender leaves of the plant were collected, air-dried, powdered and percolated in 95% ethanol. Acute toxicity test was done according to Organization for Economic Cooperation and Development guidelines. Five groups of animals of the species Rattus norvegicus, of either sex, weighing 150-200 g, were taken for the study (n=6). Group A and Group B consisted of normal and experimental control animals (3% gum acacia, 10 ml/kg body weight), respectively. Group C and Group D were test groups (PGE 250 mg/kg body weight and PGE 500 mg/kg body weight, respectively) and Group E was administered standard [5-amino salicylic acid (5-ASA) 100 mg/kg body weight]. All animals were pretreated with the respective drugs for 5 days. Colitis was induced next morning in Groups B, C, D and E by administration of 1 ml of 4% acetic acid transrectally. All the animals were sacrificed 48 hours after colitis induction and distal 10 cm of the colon was resected. Colon was weighed for Disease Activity Index (DAI), and scored macroscopically and microscopically. Biochemical assessment included myeloperoxidase (MPO), tissue catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD) estimation. Statistical analysis was done by one way analysis of variance, followed by multiple comparison tests. Groups C, D, and E showed a significant (P<0.05) decrease in DAI, and macroscopic and microscopic lesion score, as well as a significant improvement (P<0.05) in MPO, CAT, GSH and SOD levels as compared to Group B. The ethanolic extract of P. guajava leaves showed significant amelioration of experimentally induced colitis, which may be attributed to its anti-inflammatory and anti-oxidant property.
Keywords: Acetic acid, antioxidant, colitis, Psidium guajava
|How to cite this article:|
Dutta S, Das S. Effect of the leaves of Psidium guajava Linn. on experimentally induced colitis in animal models. Int J Green Pharm 2011;5:55-60
| Introduction|| |
Inflammatory Bowel Disease (IBD) is a set of chronic inflammatory conditions resulting from inappropriate persistent activation of the mucosal immune system, driven by the process of intestinal flora. Crohn's disease is an autoimmune disease which may affect any portion of the gastrointestinal tract from oesophagus to anus, but most often involves the distal small intestine and colon. Ulcerative colitis is a chronic inflammatory disease limited to the colon and rectum.  Despite the fact that aetiology 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. , In many studies, it has been reported that antioxidants show beneficial effects on experimental colitis. 
Psidium guajava Linn. (family: Myrtaceae, common name: Guava), known as the poor man's apple of the tropics, has a long history of traditional use.  Some of the ethnomedicinal uses of P. guajava plant are for the treatment of various disease conditions like diarrhoea, dysentery, gastroenteritis, ulcers, wounds, boils, inflamed gums and soft tissue infections. Guava is rich in flavonoids, tannins, phenols, triterpenes, quercetins, sterols, carotenoids, vitamins, fibre and fatty acids.  The leaves of guava are rich in flavonoids, particularly quercetin. Guava also has antioxidant properties, which is attributed to the polyphenols found in the leaves. 
Effect of various herbal drugs on IBD has been studied before concluding the antioxidant potential as the main mechanism of action against IBD. , As the leaves of P. guajava possess anti-inflammatory and antioxidant properties, this study has been undertaken to evaluate the effect of P. guajava in experimentally induced IBD and to find its probable mechanism of action including its antioxidant potential.
| Materials and Methods|| |
Collection, Identification and Extraction of Plant Materials
Approximately 1 kg of fresh tender leaves of P. guajava collected during April-May 2009 was used for the study. The plant was authenticated by Dr. M. Islam, Professor of Life Science, Dibrugarh University, Assam, India. The plant material was air-dried at room temperature. The dried leaves were grounded to a fine powder and stored in an air tight container.
Preparation of the Extract
Two hundred and fifty grams of the dry powder obtained was soaked in 95% ethanol for 24 hours in a percolator. After 24 hours, it was allowed to percolate slowly and the extract was collected in Petri dish More Detailses.  The extract was concentrated in vacuum using a rotary flash evaporator. There was a net yield of 22.6 g of the concentrated extract (9.12%).
The experiments were carried out in albino rats of the species Rattus norvegicus of either sex, weighing 150-200 g. The animals were procured from Chakraborty Enterprise, Kolkata. The study was conducted in accordance with Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA) guidelines and the study was approved by the Institutional Animal Ethical Committee (Registration no.-634/02/a/CPCSEA). The animals were acclimatised for 1 week under laboratory conditions. They were fed with standard diet, and water was provided ad libitum.
Acute Toxicity Studies
Acute oral toxicity test for the ethanolic extract of leaves of P. guajava was carried out as per Organization for Economic Cooperation and Development (OECD) Guidelines 425.  Two arbitrary doses of 250 and 500 mg/kg were selected for the study, as the extract was found safe even at doses more than 2000 mg/kg without any sign of toxicity or mortality.
Thirty healthy albino rats of the species R. norvegicus, weighing 150-200 g, were used in the study and were divided into five groups with six animals in each group (n=6) as follows:
Induction of colitis
- Group A (normal control) - received 3% gum acacia 10 ml/kg/day p.o.
- Group B (experimental control) - received 3% gum acacia 10 ml/kg/day p.o.
- Group C (test) - received P. guajava extract 250 mg/kg/day p.o.
- Group D (test) - received P. guajava extract 500 mg/kg/day p.o.
- Group E (standard) - received 5-aminosalicylic acid (5-ASA) 100 mg/kg/day p.o.
The experiment was performed using acetic acid for inducing colitis.  All the animals were pretreated with the respective drugs (volume of drugs was kept constant at 10 ml/kg) for 5 days, along with the normal diet. On the fifth day, animals were fasted for 12 hours (overnight) and IBD was induced the next morning in Groups B, C, D and E by administration of 1 ml of 4% acetic acid solution transrectally (TR). Group A (normal control) received 0.9% normal saline TR instead. 
IBD induction was done using an 8-mm soft paediatric catheter which was advanced 6 cm from the anus under low-dose ether anaesthesia. Rats were in Trendelenburg position during this process and 1 ml of 4% acetic acid or 0.9% normal saline solution was slowly administered TR. The rats were maintained in head-down position for 30 seconds to prevent leakage. After this process, 2 ml of phosphate buffer solution of pH 7 was administered TR. 
All the animals were sacrificed after 48 hours of IBD induction, by ether overdose. Abdomens were opened and colons were exposed. Distal 10 cm of colon was excised and opened by a longitudinal incision. After washing the mucosa with saline solution, mucosal injury was assessed macroscopically using the scale of Morris et al. - no damage (score 0); localised hyperaemia but no ulceration (score 1); linear ulcer without significant inflammation (score 2); linear ulcer with significant inflammation at one site (score 3); two or more sites of ulceration and inflammation (score 4) and two or more sites of ulceration and inflammation or one major site of inflammation and ulcer extending >1 cm along the length of colon (score 5). Disease activity index (DAI) was also measured, and the ratio of colon weight to body weight, which was used as a parameter to assess the degree of tissue oedema and reflects the severity of colonic inflammation, was measured. 
A 6-8 mm sample block of the inflamed colonic tissue with full thickness was excised from a region of grossly visible damage for histological analysis. Formalin-fixed tissue samples were embedded in paraffin and stained with haematoxyllin-eosin (HE). Colonic tissues were scored for histological damage using the criteria of Wallace and Keenan:  0=intact tissue with no apparent damage; 1=damage limited to surface epithelium; 2=focal ulceration limited to mucosa; 3=focal, transmural inflammation and ulceration; 4=extensive transmural ulceration and inflammation bordered by normal mucosa; 5=extensive transmural ulceration and inflammation involving entire section. 
Preparation of the sample
The proximal 5 cm of the dissected colon specimen was used for biochemical analysis  of myeloperoxidase (MPO), tissue catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD). The colonic samples were minced and homogenised using a Polytron homogenizer. The supernatant was obtained by centrifuging at 3000 rpm for 20 minutes.
The minced colonic samples were homogenised in 10 ml of ice-cold 50 mM potassium phosphate buffer (pH 6) containing 0.5% hexadecyl trimethyl ammonium bromide (HETAB). The homogenates were then sonicated and centrifuged for 20 minutes at 12,000 rpm. MPO activity was measured spectrophotometrically as follows. Exactly 0.1 ml of supernatant was combined with 2.9 ml of 50 mM phosphate buffer containing 0.0005% H 2 O 2 . The change in absorbance was measured spectrophotometrically at 460 nm. One unit of MPO activity is defined as the change in absorbance per minute at room temperature, in the final reaction. MPO activity (U/g)=X/weight of the piece of tissue taken, where X=10×change in absorbance per minute/volume of supernatant taken in the final reaction. 
Assessment of antioxidant status in colonic tissue
CAT was measured by the method of Beers and Sizer.  Phosphate buffer (2.5 ml, pH 7.8) was added to the supernatant and incubated at 25°C for 30 minutes. After transferring into the cuvette, the absorbance was measured at 240 nm spectrophotometrically. Hydrogen peroxide (650 μl) was added and change in absorbance was measured for 3 minutes. Values were expressed as μmol/min/mg of proteins.
GSH level was determined according to the method of Beutler.  The reaction mixture contained supernatant, phosphate buffer and 5,5′-dithio-bis 2-nitrobenzoic acid (DTNB) in a final volume of 10 ml. A blank was also prepared. The absorbance was immediately read spectrophotometrically at 412 nm before and after the addition of DTNB. The values were determined from standard curve.
SOD was measured according to the method of Fridovich.  Assay medium consisted of 0.01 M phosphate buffer, 3-cyclohexilamino-1-propanesulfonic acid (CAPS), saturated NaOH at pH 10.2, solution of substrate (0.05 mM xanthine, 0.025 mM p-iodonitrotetrazlium violet) and 80 ml xanthine oxidase. Absorbance was read spectrophotometrically at 505 nm. SOD was expressed as U/mg of proteins.
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 and Bonferroni's multiple comparison test. P<0.05 was considered significant.
| Results|| |
As observed from this study, acetic acid administration to the experimental control group caused significant macroscopic ulcerations and inflammations (P<0.05) in rat colon along with significant mucosal injury microscopically (P<0.05), when compared to the normal control group [Table 1], [Figure 1] and [Figure 2]. Also, there was significant derangement of biochemical parameters including tissue levels of MPO, CAT, GSH and SOD (P<0.05), indicating oxidative stress due to colon damage and colonic inflammation [Figure 3], [Figure 4], [Figure 5] and [Figure 6].
|Figure 2: Group B (experimental control): Extensive necrosis with transmural infiltration|
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|Figure 3: Group C (PGE 250): Focal, transmural inflammation and ulceration|
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|Figure 5: Group E (Standard): Near normalization of architecture with mucosal infiltration only|
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|Figure 6: Effect of P. guajava leaves on MPO activity. Values expressed asmean±SEM (n=6). aP<0.05 when compared to normal control; bP<0.05 when compared to experimental control and cP<0.05 when compared to standard; ANOVA followed by Dunnet's test and Bonferroni's test|
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Ethanolic extract of P. guajava leaves has shown significant activity against experimentally induced IBD when compared to that of the experimental control (P<0.05) animals, with an improved picture of colon architecture both macroscopically as well as microscopically [Table 1], [Figure 7], [Figure 8] and [Figure 9]. There is reduction of oxidative stress with significant improvement in tissue levels of CAT, GSH, SOD (P<0.05), showing its antioxidant potential [Figure 4], [Figure 5] and [Figure 6]. There is also significant improvement in the levels of MPO, showing its potential anti-inflammatory activity (P<0.05) [Figure 3]. However, the effects are better at a higher dose (PGE 500) than that at a lower dose (PGE 250) [Table 2].
|Figure 7: Effect of P. guajava leaves on tissue CAT activity. Values expressed as mean±SEM (n=6). aP<0.05 when compared to normal control; bP<0.05 when compared to experimental control and cP<0.05 when compared to standard; ANOVA followed by Dunnet's test and Bonferroni's test|
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|Figure 8: Effect of P. guajava leaves on tissue GSH activity. Values expressed as mean±SEM (n=6). aP<0.05 when compared to normal control; bP<0.05 when compared to experimental control and cP<0.05 when compared to standard; ANOVA followed by Dunnet's test and Bonferroni's test|
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|Figure 9: Effect of P. guajava leaves on tissue SOD activity. Values expressed as mean±SEM (n=6), aP<0.05 when compared to normal control; bP<0.05 when compared to experimental control and cP<0.05 when compared to standard; ANOVA followed by Dunnet's test and Bonferroni's test|
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As for the standard drug 5-ASA, its activity against IBD was significantly better than that of P. guajava extract with regard to all the parameters (P<0.05). The 5-ASA showed near normalisation of DAI and macroscopic and microscopic score as compared to normal control [Table 1], [Figure 5].
| Discussion|| |
The results showed that ethanolic extract of fruit extract of P. guajava has got a significant protective activity against experimental colitis in rats, as indicated by DAI, macroscopic, microscopic and biochemical evaluations.
Acetic acid induced colitis model is similar to human ulcerative colitis in terms of histological features. It affects the distal colon portion and induces non-transmural inflammation, massive necrosis of mucosal and submucosal layers, mucosal oedema, neutrophil infiltration of the mucosa and submucosal ulceration. The protonated form of the acid liberates protons within the intracellular space and causes massive intracellular acidification resulting in massive epithelial damage. Inflammation is the pathogenesis of IBD, and several pathways are associated with inflammatory response in IBD due to mucosal intestinal flora.  The inflammatory response initiated by acetic acid includes activation of cyclooxygenase and lipooxygenase pathways. ,
5-ASA is an anti-inflammatory drug commonly used in the treatment of IBD.  5-ASA acts on and is metabolised by intestinal epithelial cells. Consequently, ulcerative colitis is more susceptible to treatment by 5-ASA.  Most currently used therapeutic drugs for IBD, in particular sulfasalazine and its active moiety 5-ASA, control the disease partly by their property as potent reactive oxygen metabolite (ROM) scavengers. 
The leaves of guava are rich in flavonoids, in particular, quercetin. Much of guava's therapeutic activity is attributed to tannins, phenols and flavonoids. ,
Plant flavonoids show anti-inflammatory activity in vitro and in vivo.  Also, flavonoids possess anti-proliferative activity which is found to cause a decrease in the weight and volume of contents of granuloma in inflammation. 
Oxidative stress is believed to play a key role in the pathogenesis of IBD-related intestinal damage.  As a matter of fact, intestinal mucosal damage in the IBD, including Crohn's disease and ulcerative colitis, is related to both increased free radical production and a low concentration of endogenous antioxidant defence.  Guava leaf showed antioxidant and free radical scavenging capacity.  Flavonoids are phenolic substances which have antioxidant property.  As proved by the above study and as also described in literature, leaf extract of P. guajava possesses significant antioxidant property, proving its role in the management of experimentally induced IBD.
| Conclusion|| |
From the above study, it can be concluded that ethanolic extract of leaves of P. guajava has a potent activity against experimentally induced IBD due to its anti-inflammatory and antioxidant properties. However, further studies are required to establish and elaborate the molecular mechanism for proper clinical utility.
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Department of Pharmacology, Assam Medical College & Hospital, Dibrugarh - 786 002, Assam
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2]