Effect of Annona squamosa, Bacopa monneri and Baliospermum montanum alcoholic extracts on bacterial enzymes in 1, 2-dimethyl hydrazine induced Colon Cancer in rats.
Vinut S. Nandagaon1*, Dr. A.R. Kulkarni2
1K.L.E. U’s College of Pharmacy, Belgaum Karnataka.
1Research Scholar Karpagam University, Coimbatore.
2SETs College of Pharmacy Dharwad, Karnataka
*Corresponding Author E-mail: scburli@gmail.com
ABSTRACT:
The intestinal microflora may play a significant role in the pathogenesis of colon cancer. Differences in composition and concentrations of some fecal and colon bacterial enzymes were initially reported in low compared to high risk colon cancer groups. Activity of colon biotransforming enzymes were considered to be hallmarks of colon carcinogenesis. Annona squamosa, Bacopa monneri and Baliospermum montanum show various pharmacological and biological activities. Our present study shows the effect of alcoholic extracts of Annona squamosa, Bacopa Monneri and Baliospermum montanum administration on 1, 2-dimethylhydrazine induced colon cancer in rat colon, and fecal and mucosal biotransforming enzyme activities. A total of 42 rats were randomized into seven groups. Administration of alcoholic extracts of Annona squamosa 300mg/kg b.wt, Bacopa monneri 300mg/kg b.wt and Baliospermum montanum 200mg/kg b.wt lowered the activities ofcolon, fecal and mucosal bio-transforming enzymes. Our findings suggest that these extracts may be possible chemopreventive agents against colon cancer.
KEYWORDS: Colon cancer, Bacterial enzymes, DMH, Mucinase, β-glucuronidase, β-glucosidase.
INTRODUCTION:
The incidence of colon cancer is low in India and lesser developed areas such as South America and Africa Colorectal cancer is the third most common malignant neoplasm in the world. It is the second leading cause of cancer death in USA 1. Colon cancer is thought to arise from the accumulation of mutations in a single epithelial cell of the colon and rectum 2. Metabolically active group of organisms constitute the intestinal microflora, which play a significant role in the pathogenesis of colon cancer 3. A number of studies show that gut microbial enzymes play a very important role in the development of colon cancer 4. b-glucuronidase is present in virtually all tissues and is involved in a stepwise degradation of glucosamine-glucuronides5. Moreover, b-glucuronidase enzyme has been considered as a key enzyme for the activation of DMH metabolites to carcinogens in the colonic lumen 6. Bacterial b-glucuronidase is a hydrolase enzyme that catalyzes the cleavage of terminal glucuronic acid, which is believed to be largely responsible for the hydrolysis of glucuronide conjugates in the gut and thus to be important in the generation of toxic and carcinogenic substances 7.
Bacterial b-glucosidase hydrolyses plant glycosides to release aglycones, many of which are mutagenic, although some also have anticarcinogenic activity8 Mucinase, a bacterial hydrolase enzyme, hydrolysis the mucinous layer of the gastrointestinal tract, is of interest since it may alter the permeability and barrier function of the colon9.
1,2-dimethylhydrazine (DMH), used as a carcinogen in our present study is frequently used to induced experimental colon carcinogenesis in rodents. DMH is an alkylating agent that targets DNA and induces the formation of methyl adducts with DNA bases, point mutations, micronuclei, and sister chromatid exchanges 10.
Several possible colon cancer chemopreventive agents are found in edible plants, including fruits and vegetables 11.
Annona squamosa Linn is a small ever green tree is cultivated throughout India for its fruits, different parts of Annona squamosa Linn. are used in folkloric medicine for the treatment of various disease 12. This plant is commonly called custard apple in English and sharifa in hindi and sitaphalam in telugu in India. Annona squamosa Linn, belonging to family Annonaceaeis commonly found in India It is considered beneficial for cardiac disease, diabetes hyperthyroidism and cancer. The root is considered as a drastic purgative 13. An infusion of the leaves is considered efficacious in prolapsusani of children, the crushed leaves are sniffed to overcome hysteria and fainting spells, they are also applied on ulcer and wounds. The ripe fruits of this plant are applied to malignant tumors to hasten suppuration. The dried unripe fruit powder is used to destroy vermin the seeds are acrid andpoisonous. Powdered seeds serve as fish poison and insecticides. A paste of seed powder has been applied to the head to kill lice. It is also used for destroying worm in the wound of cattles 14.
Bacopa monnieri Linn. (Family-Scrophulariaceae) a small, common, amphibious plant growing in marshy areas throughout the Indian subcontinent. Bacopais also called Brahmi, a name derived from Brahma, the creator god of the Hindu pantheon of deities. The triterpenoid saponins and their bacosides are responsible for Bacopa’s ability to enhance nerve impulse transmission. Of synaptic activity, and ultimately nerve impulse transmission15.Traditionally, it was used as a brain tonic to enhance memory development, learning, and concentration, and to provide relief to patients with anxiety or epileptic disorders 16. Research on anxiety, epilepsy, bronchitis and asthma, irritable bowel syndrome, and gastric ulcers also supports the Ayurvedic uses of Bacopa17 .Bacopa’s antioxidant properties may offer protection from free radical damage in cardiovascular disease and certain types of cancer. It helps prevent induced lipid peroxidation 18. In-vitro research suggests an anticancer effect for Bacopa extracts, possibly due to inhibition of DNA replication in cancer cell lines 19.
Baliospermum montanum (Willd.) Muell is an important medicinal plant, which is commonly called as Danti. The plant is a stout monoecious under shrub with many shoots from the base. The various parts of the plant like roots, leaves, and seeds are used traditionally for the treatment of various ailments. In Ayurveda, root are used to cure jaundice, leucoderma, skin diseases, wounds, and as an anthelmintic .The solvents and aqueous extract of only roots have been studied and found to posses anticancer, antimicrobial, free radical scavenging, immunomodulatory, hepatoprotective and anthelmintic properties20. Leaves are found to be useful in asthma, bronchitis21 and in treating abdominal tumor22.
In the present study, our aim was to evaluate the potential chemopreventive properties of morin on colon biotransforming enzymes in experimental colon cancer induced by DMH.
MATERIAL AND METHODS:
Animals and diet Albino Wister rats of either sex aged4 weeks weighing about 80-100 g were housed at room temperature (23 ± 1ċ) and humidity (55 ± 5%) with a 12-h light/dark cycle. Animals were maintained as per the CPCSEA guidelines. Water was given ad libitum to all the animals. The animals were randomly divided into six groups comprising of six animals per group.
Chemicals:
Alcoholic extracts of plants, DMH, p-nitrophenyl-b-D-glucopyranoside, p-nitrophenyl-b-D-glucoside, p-nitrophenyl-b-D-galactopyranoside, p-nitrocatecholsulfate, p-nitrobenzoic acid and porcine gastric mucin were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals used were of analytical grade.
Preparation of the plant product:
Plant material Collection: Collected from the Savadatti nearby Belgaum.
The aerial part of Annona squamosa Linn were dried in shade, under normal environmental conditions an d then subjected to size reduction to get coarse powder and charged into the Soxhlet apparatus and extraction was carried out with 95% ethanol. And administered orally at a dosage of 300 mg/kgb.w. everyday till the end of the experiment.
Collected from the ayurvedic shops in Belgaum .The whole plant Bacopa monnieri Linn were dried in shade, under normal environmental conditions and then subjected to size reduction to get coarse powder and charged into the Soxhlet apparatus and extraction was carried out with 95% ethanol and administered orally at a dosage of 300 mg/kgb.w. everyday till the end of the experiment.
Collected from the ayurvedic shops in Belgaum The bark was subjected to size reduction to get coarse powder and charged into the Soxhlet apparatus and extraction was carried out with 95% ethanol, and administered orally at a dosage of 200 mg/kgb.w. everyday till the end of the experiment.
Plants and bark is identified and authenticated in Ghatprapha Ayurvedic College, Ghataprapha.
DMH (20 mg/kg b.w.) was dissolved in 1 mM EDTA and the pH adjusted to 6.5 with 1mMNaOH, prior to subcutaneous injection, once a week for 4 weeks.
Experimental protocol:
Group 1 received normal pellet diet and served as control, Group 2 received DMH (20 mg/kg b.w. s.c.) once a week for the first four weeks. Group 3 received Capacetabaine 359mg/kg bw, 4, 5, and 6 also received the modified pellet diet and alcoholic extracts of Annona squamosa (300 mg/kg b.w), Bacopa monneri (300 mg/kg b.w) and Baliospermun montanum (200 mg/kg b.w) respectively everyday orally for the entire of 12 weeks. After completion of the experimental period of 16 weeks, the animals were sacrificed.
Measurement of bacterial enzyme activity:
The colon was flushed gently with saline, cut open longitudinally and placed on a flat surface.
Preparation of colon homogenate:
The colons of the six rats were (1gm of colon) homogenized in an ice cold phosphate buffer saline (0.02M pH 7.4) in 1:10 ratio, centrifuged at 5000 x g for 15 minutes and used for assays.
Table no.1
Groups |
β-GLUCURONIDASE µg of phenolphthalein liberated/g of protein in 45 min incubation time. / |
β- β-GLUCOSIDASE µg of p-nitrophenol liberated/g of protein in 60 min incubation time |
MUCINASE µg of reducing sugar liberated/g of protein in 15 min incubation time. |
Normal |
1.49±0.0280 |
1.327±0.0353 |
1.438±0.0498 |
DMH (20mg/kg b.wt) |
7.467±0.0987 |
3.897±0.1173 |
4.287±0.0786 |
DMH+Capcetabaine (359mg/kg) |
3.913±0.1955*** |
1.992±0.0730*** |
1.928±0.0359*** |
DMH+A.al (300mg/kg) |
5.168±0.0441*** |
3.453±0.0870** |
3.538±0.1177*** |
DMH+B.al (300mg/kg) |
5.133±0.0709*** |
2.687±0.0312*** |
2.632±0.0878*** |
DMH+Bl.al (200mg/kg) |
5.432±0.1084** |
3.372±0.1218** |
3.855±0.0319** |
Values are mean ± S.E.M of six rats from each group. One Way ANOVA followed Bonferroni's Multiple Comparison Test P<0.05, where compare to normal group and compare to DMH group.
Table no.2
Groups |
Β-G β-GLUCURONIDASE µg of phenolphthalein liberated/g of protein in 45 min incubation time |
β- β-GLUCOSIDASE µg of p-nitrophenol liberated/g of protein in 60 min incubation time |
MUCINASE µg of reducing sugar liberated/g of protein in 15 min incubation time |
Normal |
6.097±.0792 |
11.96±0.1293 |
3.232±0.0386 |
DMH (20mg/kg |
10.64±0.1366 |
16.4±0.0422 |
7.167±0.1582 |
DMH+Capcetabaine (359mg/kg) |
7.335±0.0863*** |
13.23±0.1084*** |
4.695±0.1271*** |
DMH+A.al (300mg/kg) |
9.55±0.2552** |
15.75±0.1211** |
6.242±0.1282** |
DMH+B.al (300mg/kg) |
8.315±0.1137*** |
13.92±0.1416*** |
5.324±0.1185*** |
DMH+Bl.al (200mg/kg) |
9.887±0.1374* |
15.65±0.1341** |
6.177±0.2783** |
Values are mean ± S.E.M of six rats from each group. One Way ANOVA followed Bonferroni's Multiple Comparison Test P<0.05 wherecompare to normal group and compare to DMH group
Graph 1.β-glucuronidase activity in colon of various groups.
Preparation of faecal homogenate:
The fecal pellets were weighed and then mixed with 0.01 M sodium phosphate buffer (pH 7.4, 0.02 M) in a ratio of 1:10 (w/v) and allowed to sit in test tubes on ice for approximately 20 minutes to promote softening of the pellet. The pellets were then homogenized at 2000 x g for 2 minutes 23.
β-glucuronidase activity was measured by the method of Freeman (1986) [24]. Aknown volume of 0.02 M phosphate-buffered saline (pH 7.0), 0.1 mM EDTA, 3.0mM p-nitrophenyl-b-D-glucopyranoside and the enzyme supernatant was madeup to a final volume of 1 ml, and the mixture was incubated at 37ºC for 15 min.Thereaction was arrested with 0.2 M glycine buffer (pH 10.4) and the amount of p-nitrophenolreleased was read at 540 nm with a spectrophotometer. All reactionswere linear with respect to concentration and incubation time to 45 min. Theamount of p-nitrophenol liberated was determined by comparison with a standardnitrophenol curve.
Graph 2. β-glucosidase activity in colon of various groups.
Graph 3. Mucinase activity in colon of various groups.
Graph 4. β-glucuronidase activity in faecal of various groups.
Graph 5.β-glucosidase activity in faecal of various groups.
β-glucosidase activity was measured by the method of Freeman (1986) 24. Themixture of samples and substrate (p-nitrophenyl-b-D-glucoside) were incubatedwith 37ºC for 60 min. After incubation 0.2 M Na2CO3, was added to arrest the reaction.The released p-nitrophenol was measured at 400 nm. All reactions were linearwith respect to concentration and incubation time to 60 min. The amount of p-nitrophenolliberated was determined by comparison with a standard nitrophenolcurve.
Mucinase activity was determined by the method of Shiau and Chang (1983) [23].The assay mixture contained 0.2 M porcine gastric mucin with a known amount of fecal suspension made up to 1 ml with distilled water. The mixture was then incubated at 37ºC for 25 min. The amount of reducing sugar was measured by the method of Nelson (1944) 25 at 520 nm. Values are expressed as mg of glucose liberated/min/mg protein.
Graph 6. Mucinase activity in faecal of various groups.
STATISTICAL ANALYSIS:
Data were analyzed by one-way analysis of variance (ANOVA), and a significantdifference among treatment groups was evaluated by Bonferroni's Multiple Comparison Test. The results were considered statistically significant at p < 0.05. All statistical analysis were made using Graph pad prism 5 software package.
RESULTS AND DISCUSSION:
Table 1 and 2 shows the activity of bacterial enzymes of control and experimental rats on colon and fecal enzymes respectively. The activities of b-glucuronidase, b-glucosidase, b-galactosidaseand mucinase were significantly(p < 0.05) increased in DMH treated rats as compared to control, where as the activities of the colon and fecal bacterial enzymes was significantly lowered on administration of alcoholic extracts of Annona squamosa, Bacopa monneri and Baliospermum montanum to DMH treated rats (group 3,4,5 and6) as compared to DMH alone treated rats (group 2). The results obtained in this study indicates that the alcoholic extracts of Annona squamosa, Bacopa monneri and Baliospermum montanum elicits significant effects on colon, fecal and mucosal microbial enzyme activities during chemically induced colon carcinogenesis.
DMH is a colon carcinogen which is detoxified by glucuronidation in liver and secreted via bile into the intestine. When the conjugated carcinogen reaches the colon, it is hydrolyzed by bacterial enzymes and the colon gets exposed to free carcinogen. In the present study, elevated levels of faucal and colonic β-glucuronidase and β-glucosidase were observed in DMH induced group which confirms that bacterial enzyme activities got enhanced in the presence of pro-carcinogen like DMH 26. Enhanced level of these enzymes hydrolyze glucuronides of DMH as well as activates DMH metabolites to toxic carcinogens in the colon, a prerequisite for colon carcinogenesis thereby eliciting the conversion of normal epithelial cells to neoplastic cells. Treatment with alcoholic extracts of Annona squamosa, Bacopa monneri and Baliospermum montanum in DMH induced group reduced the activities of these enzymes in fecal as well as in colon homogenate. Thus, may prevent the colon from the toxic effects of DMH by lowering the hydrolysis of glucouronide conjugates 26. This is similar to the previous reported study where, inhibition of bacterial β-glucuronidase and β-glucosidase caused a reduction in the appearance of carcinogen-induced colon tumors27.
Colonic permeability is an important factor in the etiology of colon cancer. Chemicals probably need to reach the colonic mucosa before they can induce tumour formation. Mucinase is an enzyme present in the intestinal microflora, which hydrolyzes the protective mucins in the colon. Mucins are glycoproteins consisting of a larger number of carbohydrate side chains attached to a protein core. They also form gels coating the intestinal mucosa and function as a lubricant and probably as a chemical and mechanical barrier against bacteria, viruses, and toxins. In the present study, the fecal and colonic activity of mucinase was significantly elevated in the DMH induced groups. This increased mucinase activity leads to decreased protection of the underlying tissues. In addition, the carcinogenic metabolites that are released by glucuronidase activity may also induce microbial mucinase in order to interact with the colonocytes resulting in DNA damage and cell proliferation. Treatment with alcoholic extracts of Annona squamosa, Bacopa monneri and Baliospermum montanum in DMH induced group was found to decrease this activity both in the colon and in the fecal contents thereby, exerting protective effect on colonic mucosa and decreasing the susceptibility of the colonic mucosa to attack by carcinogens23.
CONCLUSION:
Our study reveals that alcoholic extracts of Annona squamosa, Bacopa monneri and Baliospermum montanum administration on 1,2-dimethylhydrazine induced colon cancer in rat can be as a potential chemopreventive agents in colorectal cancer as these extracts lowers b-glucuronidase b-glucosidase and mucinase enzymes in colon and fecal homogenates.
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Received on 22.01.2013 Modified on 10.02.2013
Accepted on 10.03.2013 © RJPT All right reserved
Research J. Pharm. and Tech. 6(4): April 2013; Page 379-383