Evaluation of effect of Piper betel, Centella asiatica and Aristolochia indica extracts on bacterial enzymes in 1, 2-dimethyl hydrazine induced colon cancer in wistar rats

 

V.A. Kangralkar^1,2* and A.R. Kulkarni³

¹Maratha Mandal’s College of Pharmacy, Belgaum Karnataka

²Research Scholar Karpagam University, Coimbatore.

³Department of Pharmacology, SET’s College of Pharmacy, Dharwad- 580 002, India.

*Corresponding Author E-mail: vishnukangralkar@gmail.com

 

The colon enzymes play a very important role in colon carcinogenesis. The different enzymes were initially reported in low compared to high risk colon cancer groups. In the present study the effect of Piper betel, Centella asiatica and Aristolochia indica extracts has been studied on 1, 2-dimethylhydrazine induced colon cancer in wistar rat. A total of 42 rats were randomized into seven groups. Administration of aqueous and alcoholic extracts of Piper betel 200mg/kg b.wt, and alcoholic extracts of Centella asiatica 200mg/kg b.wt and Aristolochia indica 200mg/kg b.wt lowered the activities of colon and fecal bio-transforming enzymes. Results suggest that these extracts may be possible chemopreventive agents against colon cancer.

 

 

 

INTRODUCTION:

The Cancer cases are increasing in every country of the world. It is a leading cause of death in today’s era. It is the uncontrolled cell proliferation. Carcinoma of the large bowel is equally distributed between men and women.¹ metabolically active group of organisms constitute the intestinal microflora, which play a significant role in the pathogenesis of colon cancer ². A number of studies show that gut microbial enzymes play a very important role in the development of colon cancer ³. β-glucosidase, β –glucuronidase, Mucinase, the bacterial enzyme alters in the various pathological conditions and are significant markers in colon cancer. 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 ⁴.

 

Aristolochia indica Linn. a perennial shruby glabrous twiner with a long woody root stock. The roots are anthelmintic, cardiotonic, anti-inflammatory, diuretic and tonic.⁵various extracts and fractions of root showed interceptive activity⁶ and antiestrogenic activity in mice.⁷Extracts or isolates of A.indica possess anticancer activity. It stimulates phagocytic activity in guinea pigs.⁸ The Piper betel Linn. A perennial dioecious root climber, useful in asthma, rheumatism, dyspepsia and laryngitis. ⁹The leaves contain good amount of vitamin B, ascorbic acid and carotene. ¹⁰ The essential oil and extracts of the leaves possess antimicrobial activity, antioxidant activity, cytotoxic activity ¹¹ and anti-inflammatory activity. ¹² It also shows ulcer healing property. Centella asiatca Linn. a slender herbaceous creeping perennial plant. The plant is stomachic, antileprotis, diuretic and febrifuge.¹³It is reported to have wound healing activity,¹⁴neuroprotective property¹⁵, ulcer healing property¹⁶ and cytotoxic activity. The triterpene Madecassoside obtained from Centella asciatica possess anti rheumatoid effect¹⁷, and also reduces ischemia reperfusion injury¹⁸. The present study was planned to explore the effect of Piper betle Linn, Centella asiatca Linn and Aristolochia indica Linn, on colon enzyme in dimethylhydrazine induced colon cancer in Wistar rats.

 

MATERIAL AND METHODS:

Albino Wistar rats of either sex of 4 weeks age 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 seven groups comprising of six animals per group.

 

Preparation of the plant product:

Plant material Collection: Plant material collected from the nearby Belgaum. The leaves of Piper betle Linn, Centella asiatca Linn whole plant and Aristolochia indica Linn roots 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, Piper betle aqueous extract is also prepared by Soxhlet. All the plants were identified and authenticated ICMR and Redekar Ayurvedic College And administered orally at a dosage of 200 mg/kgb.w. everyday till the end of the experiment.

 

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, 6and 7 also received the pellet diet and various 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 ona 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.

 

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 ¹⁹.

 

β-glucuronidase activity was measured by the method of Freeman (1986) . A known 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.The reaction 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. The amount of p-nitrophenol liberated was determined by comparison with a standardnitrophenol curve.

 

β-glucosidase activity was measured by the method of Freeman (1986) ²⁰. The mixture 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 linear with respect to concentration and incubation time to 60 min. The amount of p-nitrophenol liberated was determined by comparison with a standard nitrophenolcurve.

 

Mucinase activity was determined by the method of Shiau and Chang (1983) .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) ²¹ at 520 nm. Values are expressed as mg of glucose liberated/min/mg protein.

 

STATISTICAL ANALYSIS:

Data were analyzed by one-way analysis of variance (ANOVA), and a significant difference 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:

The effects of various extracts on bacterial enzymes are shown in Table1 and 2 The activities of β -glucuronidase, β -glucosidase, and 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 various extracts of Piper betle , Centella asiatica  and Aristolochia indica  DMH treated rats (group 3,4,5,6 and7) as compared to DMH alone treated rats (group 2). The results shown by this study indicates that the various extracts elicit significant effects on colon, fecal and mucosal microbial enzyme activities during dimethylhydrazine induced colon carcinogenesis.

 

As there is colonic permeability to some 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. 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  extracts of Piper betle Linn, Centella asiatica Linn and Aristolochia indica Linn  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 carcinogens. These results may be due to the antioxidant principles present in the plants.

 

 

 

Table no.1Effect on colon enzymes

GROUPS	Β-GLUCURONIDASE	β-GLUCOSIDASE	MUCINASE
	µg of phenolphthalein liberated/g of protein in 45 min incubation time	µg of p-nitrophenol liberated/g of protein in 60 min incubation time	µg of reducing sugar liberated/g of protein in 15 min incubation time
Normal	1.38±0.06	1.48±0.06	1.45±0.05
DMH	7.86±0.12	3.87±0.17	4.37±0.09
Capcetabaine	4.27±0.16***	2.48±0.12***	2.49±0.08***
Aq.Pb	6.41±0.40**	2.92±0.24**	3.65±0.18**
Al.Pb	4.51±0.05***	2.65±0.07***	3.43±0.05***
Al.Ca	6.44±0.34**	2.93±0.21**	3.58±0.16***
Al.Ai	4.51±0.21***	2.23±0.07***	2.57±0.07***

 

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.1Effect on fecal enzymes

GROUPS	Β-GLUCURONIDASE	β-GLUCOSIDASE	MUCINASE
	µg of phenolphthalein liberated/g of protein in 45 min incubation time	µg of p-nitrophenol liberated/g of protein in 60 min incubation time	µg of reducing sugar liberated/g of protein in 15 min incubation time
Normal	6.51±0.11	11.98±0.29	3.41±0.07
DMH	10.01±0.24	15.82±0.28	7.83±0.01
Capcetabaine	8.13±0.15***	13.05±0.16***	4.6±0.039***
Aq.Pb	9.06±0.19*	14.52±0.19*	6.79±0.28**
Al.Pb	7.45±0.11***	12.68±0.08***	5.34±0.04***
Al.Ca	8.85±0.18**	14.18±0.14**	6.98±0.19*
Al.Ai	7.78±0.19***	12.95±0.41***	4.53±0.21***

 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

 

 

CONCLUSION:

The study reveals that various extracts of Piper betle Linn, Centella asiatica Linn and Aristolochia indica Linn lowers β –glucuronidase, β -glucosidase and  mucinase enzymes in colon and fecal homogenates. These extracts can be used as a potential chemopreventive agents in colorectal cancer .

 

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Received on 12.02.2014       Modified on 18.02.2014

Accepted on 25.02.2014      © RJPT All right reserved