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Influence of spices on the bacterial (enzyme) activity in experimental colon cancer
Journal of Ethnopharmacology 62 (1998) 15 – 24
Influence of spices on the bacterial (enzyme) activity in experimental colon cancer N. Nalini a, K. Sabitha a, P. Viswanathan b, V.P. Menon a,* b
a Department of Biochemistry, Annamalai Uni6ersity, Annamalainagar 608 002, Tamilnadu, India Di6ision of Pathology, Rajah Muthiah Medical College, Annamalai Uni6ersity, Annamalainagar 608 002, Tamilnadu, India
Received 1 December 1997; received in revised form 5 January 1998; accepted 6 January 1998
Abstract In the presence of a known colon carcinogen, 1,2-dimethyl hydrazine (DMH), the activity of b-glucuronidase was found to be significantly increased in the distal colon, distal intestine, liver and colon contents and the activity of mucinase was increased in both the colon and fecal contents when compared to control rats. Chilli (Capsicum annum L., Solanaceae) administration also showed an increase when compared to control rats, whereas supplementation with cumin (Cuminum cyminum L., Apiaceae) and black pepper (Piper nigrum L., Piperaceae) in the presence of DMH, showed more or less similar values as that of the control rats. The increase in b-glucuronidase activity may increase the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Thus cumin and black pepper may protect the colon by decreasing the activity of b-glucuronidase and mucinase. Histopathological studies also showed lesser infiltration into the submucosa, fewer papillae and lesser changes in the cytoplasm of the cells in the colon in cumin and black pepper groups when compared to the DMH and chilli treated animals. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Black pepper; Cumin; Dimethyl hydrazine; b-Glucuronidase; Mucinase; Red chilli
1. Introduction Dietary factors can modify the metabolic activity of the intestinal microflora which in turn may play an important role in the conversion of bile * Corresponding author. Tel.: + 91 4144 20154; fax: + 91 4144 22987/23080.
acids and neutral sterols to reactive carcinogens (Reddy et al., 1974; Weisburger and Williams, 1975; Goldin and Gorbach, 1976; Reddy et al., 1977). The major components of this flora are both anaerobes and aerobes, though the amount of anaerobes is 1000-fold more than the aerobes. Several ways by which the intestinal microflora could play a significant role are: (i) it could con-
0378-8741/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. PII S0378-8741(98)00007-5
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N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
vert dietary components into cocarcinogens or carcinogens (or both), (ii) it could metabolize endogenous secretions, themselves controlled by diet, into different compounds and (iii) it could produce carcinogenic toxins from suitable precursors. These reactions are catalysed by bacterial enzymes, the measurement of the appropriate bacterial enzymes in the intestine gives an indication of the ability of the microflora to support these transformations. Two important activities of the colon microflora are mucinase and b-glucuronidase. Mucinase is the enzyme system responsible for degrading the protective mucins in the colon. b-Glucuronidase is of interest as it is the enzyme that hydrolyses biliary glucuronides when they reach the colon. Many of the glucuronides are conjugated products of detoxification by the liver and their hydrolysis may liberate free toxins, which may then act on the colonocytes or may be absorbed by the intestines (Hill, 1975; Goldin and Gorbach, 1977; Reddy et al., 1977; Goldin et al., 1978). When conjugated carcinogen reaches the colon, it may be hydrolysed and the colon mucosa exposed to free carcinogens. If glucuronide hydrolysis is a rate limiting step in this process, then the activity of microbial b-glucuronidase in the colon may influence the risk of colon carcinogenesis. Spices and condiments comprise the most important products used for flavouring foods. They are also extensively used in medicine, pharmaceuticals, perfumery and cosmetics. Spices are well known appetizers and help to cool the body by increasing the rate of perspiration (Lee, 1954; Jiffry and Udipihille, 1979). In addition they possess several other properties like antioxidant and anti-inflammatory properties (Srimal and Dhawan, 1973; Sharma, 1976; Toda et al., 1985; Satoskar et al., 1986; Tonnesan, 1991). Curcumin, the active principle of turmeric is known to have an antimutagenic effect (Nagabhushan et al., 1987). Oral administration of diallylsulphide (DAS), a major garlic component has also been shown to inhibit 1,2-dimethyl hydrazine (DMH) induced nuclear injury in the mouse colon (Ip et
al., 1992) whereas the active principle of Capsicum annum, capsaicin is known to be mutagenic, carcinogenic and a tumor promoting agent (Nagabhushan and Bhide, 1985). The effect of spices on the intestinal microflora is yet to be unravelled, so we studied the effect of red chilli (Capsium annum L., Solanaceae), cumin seeds (Cuminum cyminum L., Apiacea) and black pepper (Piper nigrum L., Piperaceae) (Sambamurty and Subrahmanyam, 1989) in combination with or without DMH (a known colon carcinogen) on b-glucuronidase and mucinase activity.
2. Materials and methods Wistar male rats bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, weighing 120–150 g were divided into 9 groups of 10 rats each. They were all fed commercial diet (Lipton Lever Limited). Water was given ad libitum. Except for the low fat control rats, the rats in the rest of the groups were given 20% peanut oil. Low fat control High fat control DMH Chilli Chilli +DMH Cumin Cumin +DMH Pepper Pepper +DMH
rats fed commercial diet rats fed commercial diet (defatted)+20% peanut oil rats administered DMH rats fed red chilli (8 mg/day per 100 g body weight in water) rats fed red chilli and also administered DMH rats fed cumin seeds (1.25%) rats fed cumin and also administered DMH rats fed black pepper (0.5%) rats fed black pepper and also administered DMH
Red chilli (Capsicum annum L., Solanacea), cumin seeds (Cuminum cyminum L., Apiacea) and black pepper (Piper nigrum L., Piperaceae) (as authenticated by Sambamurty and Subrahmanyam, 1989), were purchased from the local
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
17
Fig. 1. The rat digestive system removed from the body.
market, powdered and mixed with the feed along with 20% peanut oil. DMH, p-nitrophenyl b-Dglucuronide and mucin were purchased from Sigma (St. Louis, MO). The lower dose of spices used here is with reference to the average daily intake by human beings, learnt from a survey conducted in India (Srinivasan and Sambaiah, 1991). 20 mg/kg Body weight of DMH was dissolved in 1 mmol/1 EDTA, just prior to use and the pH adjusted to 6.5 with 1 mmol/1 NaHCO3 and administered subcutaneously once a week (Marianne et al., 1990; Tsunoda et al., 1992). After 15 weeks the DMH injection was discontinued and the rats were given only the commercial diet. At the end of 30 weeks, fresh fecal pellets were collected and the activity of mucinase was estimated by the method of Shiau and Chang (1983). They were then sacrificed by cervical decapitation after administering anesthesia. The neoplasms in the intestine and colon were counted after cutting open the tissues longitudinally, taking care not to disturb the tumors. Part of the tissue was immediately fixed in 10% formalin for 24 h. Then the tissue was cut open at the anti mesentric border, processed and embedded on paraffin wax. 3 – 5 mm Sections were sliced, stained with Hematoxylin and Eosin and then the cell morphology
as a whole was studied. The normal mucosa was compared with the abnormal mucosa. The rest of the tissues and colon contents were transferred to ice-cold containers for measuring the activity of b-glucuronidase and mucinase. The method by which we divided the intestine and colon of the rats are represented in Fig. 1. b-Glucuronidase activity was measured by the method of Kawai and Anno (1971), using p-nitrophenyl b-D-glucuronide as the substrate and measuring the amount p-nitrophenol liberated. Protein was estimated by the method of Lowry et al. (1951). The results obtained were expressed as mean + S.D. from 6 rats in each group. The statistical significance of the difference in means was analysed by Student’s t-test. A one way analysis of variance (ANOVA) was also determined (Chou, 1975).
3. Results The weight gain at the end of the experimental period was 329 g in cumin, 316 g in cumin+ DMH, 335 g in black pepper and 305 g in pepper+ DMH. These were comparatively greater than the rats in the DMH (210 g), chilli (218 g) and chilli+ DMH (238 g) groups. The number of
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N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
Fig. 2. High fat: a normal mucosal fold with exaggerated inflammatory cell infiltrate in the submucosa. Hematoxylin and Eosin staining, magnification ×20.
Fig. 4. Chilli: a polyp filled with glands of varying sizes and some of them reveal pseudostratification of the lung columnar cells. Foci and dysplasia also noted. Hematoxylin and Eosin staining, magnification × 10.
rats with tumors per total number of rats in the chilli group was 25/30 (incidence 83.3%), DMH group 27/30 (incidence 90%), chilli+ DMH 28/30 (93.3%), cumin+DMH 14/30 (46.7%) and pepper+DMH 25/30 (83.3%). The macroscopic and light microscopic observations (histopathological) of the colon of rats in the different groups are given in Table 1. Low fat and high fat control rats showed a similar microscopic pattern except that the latter had exaggerated inflammatory cell infiltrates. When cumin and black pepper were supplemented in the diet the animals showed fewer papillae, no infiltration into the submucosa and lesser changes in the cell
morphology compared to the DMH, chilli and chilli+ DMH groups (Figs. 2–7). The activity of b-glucuronidase showed a slight increase in all the tissues and the colon contents of the high fat and DMH groups when compared to the control animals (Table 2). The chilli treated group showed a significant increase in the proximal colon, distal intestine, proximal intestine and in the colon contents compared to the control rats. In the chilli + DMH group a significant increase was noted in the distal colon, proximal colon, distal intestine, proximal intestine, liver, and the colon contents (bacterial microflora). In
Fig. 3. DMH: a picture showing a papillary adenocarcinoma with ( ) several papillae lined by pleomorphic cells. Some of the glands are filled with mucinous material. Hematoxylin and Eosin staining, magnification × 20.
Fig. 5. Chilli +DMH: papillary processes lined by stratified columnar epithelium. Hematoxylin and Eosin staining, magnification × 20.
—
—
— —
Cytoplasm Mitotic figures
Others Vascular granulation and vascular congestion
—
—
Vascular congestion
— —
—
—
Focal areas of fibroblastic reaction
Scanty Numerous
Prominent
Marked
Observed
Not observed
—
—
Mixed population
2 cm
DMH
Mixed inflammation
—
High fat control
Cell morphology Nuclear pleomorphism Nucleoli
Infiltration into — the submucosa
Microscopy Inflammatory — cell infiltrates into the mucosa Lymphoid aggre- — gates in the submucosa
Macroscopy Size
Low fat control
Table 1 Pathological changes in the colon
Not seen
Less prominent Moderate Present
Less severe
—
Not observed
Mixed population
1 cm
Chilli
—
Cumin
Vascular granulation not very prominent
Scanty Numerous
Prominent
Marked
—
— —
—
—
Rare lymphoid Prominent aggregate lymphoid aggregates in the submucosa along with collection of lymphocytes in the mucosa Several areas — of infiltration
Mixed popula- Mixed inflamtion mation
2 cm
Chilli+DMH
Observed
Scanty Present
Prominent
Observed
No infiltration
0.5 cm
Pepper+DMH
Vascular congestion
— —
—
—
—
Vascular granulation
Scanty Observed
Prominent
Marked
Not observed
Not seen
Predominantly Mixed eosinophils 10/HPF
—
Pepper
Lymphoid ag- — gregates deeper to the tumor seen
Mixed population
1 cm
+ DMH
Cumin
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24 19
85.77 912.80
54.26 9 9.50 51.01 97.00
53.66 9 6.25 53.96 9 7.64
40.649 3.30 43.2494.02
45.03 9 1.69 53.85 95.04b 40.83 9 2.09 49.10 9 4.20NS
65.34 9 4.96a
40.85 9 5.14b
40.33 9 4.47a
43.19 9 3.05a 42.84 9 3.56b
29.40* 46.71*
24.46*
31.09*
60.03* 14.09*
Pepper+DMH F-ratio
75.929 4.44 85.31 9 5.55a 80.659 11.32 96.57 9 8.58b
34.569 7.84
30.159 4.69
31.759 3.05 34.399 4.19
Cumin+DMH Pepper
48.28 9 5.13 50.35 9 5.98a 40.06 9 6.08 54.15 9 3.26a
Cumin
80.8898.47
83.40 9 14.50a 56.91 97.27b
Chilli+DMH
92.6593.66 99.95 98.29 145.12 912.98 96.08 912.60 131.63 9 23.79NS 61.31 9 3.56 93.36 9 6.24a 62.889 2.81 102.1398.24 112.18 9 13.73 149.00 924.39 173.98 915.33a 87.95 9 7.19 77.36 9 7.19a
79.53912.59
44.76 94.36 50.97 95.45
Chilli
55.84 9 6.92 108.73 99.30 47.269 3.78 47.36 93.73
DMH
43.299 4.72 42.249 2.82
High fat control
Values are mean 9S.E. (mg of p-nitrophenol liberated/h per g protein) from 6 rats in each group. DMH has been compared with chilli+DMH, cumin+DMH and pepper+DMH groups. a PB0.01; b PB0.05; NS, not significant; ANOVA, significant at the 1% level.
Distal colon Proximal colon Distal intestine Proximal intestine Liver Colon contents
Low fat control
Table 2 b-Glucuronidase
20 N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
Fig. 6. Cumin+ DMH: () a lymphoid aggregate extending up to the mucosa with an occasional mucosal gland exhibiting dysplastic changes. Hematoxylin and Eosin staining, magnification × 10.
the cumin group the b-glucuronidase activity showed a significant increase only in the distal colon, distal intestine and the colon contents, when compared to the control rats. In the cumin + DMH group the b-glucuronidase activity was slightly increased in the colon and intestines compared to the control rats but the values were less than that of the DMH group (except for the proximal colon). In the black pepper group b-glucuronidase activity was less than the control animals in all the tissues expect the colon contents and in the black pepper+DMH group the values except for the liver and colon contents were more or less comparable with the control rats.
Fig. 7. Black pepper +DMH: focal areas of mucinous secretion in which the tumor cells appear floating. Hematoxylin and Eosin staining, magnification ×10.
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Mucinase activity was slightly less in the high fat group when compared with the control rats (Table 3). When DMH was administered the increase was very significant, chilli administration showed a similar increase, though it was less than that of the DMH group. When chilli was supplemented with DMH the increase in the activity was more than even that of the DMH group. The cumin group showed a slight increase and the cumin+DMH group a significant increase in the mucinase activity, though the values were less than that of the DMH group. The black pepper group showed a slight fall in the mucinase activity and in the black pepper+ DMH group the values were comparable to that of the control rats.
4. Discussion The above results indicate that C. annum, C. cyminum and P. nigrum bring about profound alterations in the activity of b-glucuronidase and mucinase, both in the presence and/or absence of DMH in high fat fed rats. Pathological studies showed a great degree of variation in the different groups. Vascular congestion was seen in the high fat fed rats. In the DMH group, the size of the tumor was around 2 cm, pedunculated having a well-defined margin with a large number of papillae and invasive adenocarcinoma, which showed marked pleomorphism. The nucleoli were very prominent with scanty cytoplasm and numerous mitotic figures. In the chilli groups, the size of the tumor was about 1 cm pedunculated with a defined margin. Histopathology exhibited areas of dysplasia which was less severe, with a less prominent nucleoli, moderate cytoplasm and few mitotic figures. In the chilli+ DMH group, the size of the tumor was more than 2 cm. There was a transitional zone with areas of marked dysplasia and infiltrating adenocarcinoma. The nucleoli were also prominent. The mucosa of the cumin group had ulceration. Lymphoid aggregates were seen both in the submucosa and mucosa. The cumin+ DMH group showed the size of the tumor to be 1 cm, sessile, with no transitional zone of dysplasia, though there were a considerable number of
7.360 9 0.92a
4.300 9 0.65
5.580 90.63
1.92190.26
2.2409 0.22
4.518 9 0.66a
2.693 90.33
3.06590.42
1.997 90.11
2.06090.15
Chilli+DMH
Chilli
DMH
High fat control
Low fat
2.554 90.13
2.821 90.06
Cumin
Values are mean 9 S.E. (mg of glucose liberatedl/min per mg protein) from 6 rats in each group. DMH has been compared with chilli+DMH, cumin+DMH and pepper+DMH groups. a PB0.01; b PB0.05; NS, not significant. ANOVA, significant at the 1% level.
Colon contents Fecal contents
Table 3 Mucinase
3.341 9 0.26a
3.051 9 0.11NS
Cumin+DMH
1.0829 0.06
1.0839 0.05
Pepper
2.15690.43a
2.12490.09a
Pepper+DMH
107.17*
36.50*
F-ratio
22 N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
papillae which were lined by anaplastic cells. Lymphoid aggregates deeper to the tumor were also seen. In black pepper +DMH group the size of the tumor was less than 0.5 cm with a defined margin, sessile with focal areas of dysplasia. Nuclear pleomorphism was noted with prominent nucleoli, scanty cytoplasm and few mitotic figures. The vascular granulation and desmoplastic reaction are some of the common reactions executed by the normal tissue towards a malignant neoplasm. In our observation we also saw a similar reaction in the DMH, chill+ DMH and pepper + DMH groups. In the case of chilli + DMH group such a reaction was present but not very prominent. Vascular granulation was present in the pepper + DMH group and this may explain the lower virulence and lower infiltration of the tumor. Thus chilli intake clearly shows that it acts as a promoter of carcinogenesis, whereas the inclusion of cumin and black pepper inhibits carcinogenesis as evident by the decreased number of tumors, comparatively, in both the cumin+ DMH and black pepper+ DMH groups. The liver is the major site of detoxification of many drugs, toxins, and hormones. One of the main functions of the liver is to conjugate these drugs, toxins and hormones with glucuronides, thus making these soluble and at the same time detoxifying them. They are then excreted via the bile to the intestine. When they reach the colon, normally more than 90% is excreted. But when the activity of these colonic microflora are stimulated as in the presence of the procarcinogen DMH, these glucuronides may be hydrolyzed, which may lead to the liberation of drugs and toxins that can be harmful to the colonocytes (Goldin and Gorbach, 1977; Goldin et al., 1978). This may be one of the ways by which DMH is said to enhance the conversion of normal epithelial cells of the colon into cancerous ones. Chilli, as noted from the results, is shown to increase the activity of b-glucuronidase by enhancing the hydrolysis of glucuronides, while cumin and black pepper on the other hand have the opposite effect. Thus, cumin and black pepper may preserve the colon from the toxic effects of drugs and toxins. This are confirmed by the increased incidence of tumors in the chilli+ DMH group and the de-
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creased incidence in the cumin+ DMH and black pepper+ DMH groups. Mucins are glycoproteins consisting of a large number of carbohydrate side chains attached to a protein core. They form gels coating the intestinal mucosa and also function as a lubricant and probably as a chemical or mechanical barrier against bacteria, viruses and toxins. Mucinase is an enzyme present in the intestinal microflora which hydrolyses these protective mucins in the colon (Reddy and Wynder, 1973). Once the mucins are hydrolysed, the underlying cells are exposed to a number of toxic intermediates (Hoskins and Zancheck, 1968; Salvers et al., 1977). The activity of mucinase was significantly elevated in the DMH and/or chilli treated groups. This increased mucinase activity leads to decreased protection of the underlying tissues (Wynder and Reddy, 1974; Miller and Hoskins, 1981). Whereas the inclusion of cumin and black pepper in the diet was found to decrease it’s activity both in the colon microflora and in the fecal content. If this change in enzyme activity reflects the change in the rate of mucin degradation and the shift in the balance between mucin secretion and degradation, then it may also be accompanied by a change in the susceptibility of the intestinal mucosa to pathogenic organisms, toxins and carcinogens.
5. Conclusion Thus, treatment with cumin and black pepper was shown to protect the colon in the presence of the procarcinogen 1,2-dimethyl hydrazine as a possible result of the decreased activity of b-glucuronidase and mucinase, whereas chilli supplementation had the opposite effect in the proximal intestine and proximal colon.
Acknowledgements The authors wish to thank Mr Arumuga Perumal and Mr Krishnaswamy, for their technical assistance.
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Influence of spices on the bacterial (enzyme) activity in experimental colon cancer N. Nalini a, K. Sabitha a, P. Viswanathan b, V.P. Menon a,* b
a Department of Biochemistry, Annamalai Uni6ersity, Annamalainagar 608 002, Tamilnadu, India Di6ision of Pathology, Rajah Muthiah Medical College, Annamalai Uni6ersity, Annamalainagar 608 002, Tamilnadu, India
Received 1 December 1997; received in revised form 5 January 1998; accepted 6 January 1998
Abstract In the presence of a known colon carcinogen, 1,2-dimethyl hydrazine (DMH), the activity of b-glucuronidase was found to be significantly increased in the distal colon, distal intestine, liver and colon contents and the activity of mucinase was increased in both the colon and fecal contents when compared to control rats. Chilli (Capsicum annum L., Solanaceae) administration also showed an increase when compared to control rats, whereas supplementation with cumin (Cuminum cyminum L., Apiaceae) and black pepper (Piper nigrum L., Piperaceae) in the presence of DMH, showed more or less similar values as that of the control rats. The increase in b-glucuronidase activity may increase the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Thus cumin and black pepper may protect the colon by decreasing the activity of b-glucuronidase and mucinase. Histopathological studies also showed lesser infiltration into the submucosa, fewer papillae and lesser changes in the cytoplasm of the cells in the colon in cumin and black pepper groups when compared to the DMH and chilli treated animals. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Black pepper; Cumin; Dimethyl hydrazine; b-Glucuronidase; Mucinase; Red chilli
1. Introduction Dietary factors can modify the metabolic activity of the intestinal microflora which in turn may play an important role in the conversion of bile * Corresponding author. Tel.: + 91 4144 20154; fax: + 91 4144 22987/23080.
acids and neutral sterols to reactive carcinogens (Reddy et al., 1974; Weisburger and Williams, 1975; Goldin and Gorbach, 1976; Reddy et al., 1977). The major components of this flora are both anaerobes and aerobes, though the amount of anaerobes is 1000-fold more than the aerobes. Several ways by which the intestinal microflora could play a significant role are: (i) it could con-
0378-8741/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. PII S0378-8741(98)00007-5
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N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
vert dietary components into cocarcinogens or carcinogens (or both), (ii) it could metabolize endogenous secretions, themselves controlled by diet, into different compounds and (iii) it could produce carcinogenic toxins from suitable precursors. These reactions are catalysed by bacterial enzymes, the measurement of the appropriate bacterial enzymes in the intestine gives an indication of the ability of the microflora to support these transformations. Two important activities of the colon microflora are mucinase and b-glucuronidase. Mucinase is the enzyme system responsible for degrading the protective mucins in the colon. b-Glucuronidase is of interest as it is the enzyme that hydrolyses biliary glucuronides when they reach the colon. Many of the glucuronides are conjugated products of detoxification by the liver and their hydrolysis may liberate free toxins, which may then act on the colonocytes or may be absorbed by the intestines (Hill, 1975; Goldin and Gorbach, 1977; Reddy et al., 1977; Goldin et al., 1978). When conjugated carcinogen reaches the colon, it may be hydrolysed and the colon mucosa exposed to free carcinogens. If glucuronide hydrolysis is a rate limiting step in this process, then the activity of microbial b-glucuronidase in the colon may influence the risk of colon carcinogenesis. Spices and condiments comprise the most important products used for flavouring foods. They are also extensively used in medicine, pharmaceuticals, perfumery and cosmetics. Spices are well known appetizers and help to cool the body by increasing the rate of perspiration (Lee, 1954; Jiffry and Udipihille, 1979). In addition they possess several other properties like antioxidant and anti-inflammatory properties (Srimal and Dhawan, 1973; Sharma, 1976; Toda et al., 1985; Satoskar et al., 1986; Tonnesan, 1991). Curcumin, the active principle of turmeric is known to have an antimutagenic effect (Nagabhushan et al., 1987). Oral administration of diallylsulphide (DAS), a major garlic component has also been shown to inhibit 1,2-dimethyl hydrazine (DMH) induced nuclear injury in the mouse colon (Ip et
al., 1992) whereas the active principle of Capsicum annum, capsaicin is known to be mutagenic, carcinogenic and a tumor promoting agent (Nagabhushan and Bhide, 1985). The effect of spices on the intestinal microflora is yet to be unravelled, so we studied the effect of red chilli (Capsium annum L., Solanaceae), cumin seeds (Cuminum cyminum L., Apiacea) and black pepper (Piper nigrum L., Piperaceae) (Sambamurty and Subrahmanyam, 1989) in combination with or without DMH (a known colon carcinogen) on b-glucuronidase and mucinase activity.
2. Materials and methods Wistar male rats bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, weighing 120–150 g were divided into 9 groups of 10 rats each. They were all fed commercial diet (Lipton Lever Limited). Water was given ad libitum. Except for the low fat control rats, the rats in the rest of the groups were given 20% peanut oil. Low fat control High fat control DMH Chilli Chilli +DMH Cumin Cumin +DMH Pepper Pepper +DMH
rats fed commercial diet rats fed commercial diet (defatted)+20% peanut oil rats administered DMH rats fed red chilli (8 mg/day per 100 g body weight in water) rats fed red chilli and also administered DMH rats fed cumin seeds (1.25%) rats fed cumin and also administered DMH rats fed black pepper (0.5%) rats fed black pepper and also administered DMH
Red chilli (Capsicum annum L., Solanacea), cumin seeds (Cuminum cyminum L., Apiacea) and black pepper (Piper nigrum L., Piperaceae) (as authenticated by Sambamurty and Subrahmanyam, 1989), were purchased from the local
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
17
Fig. 1. The rat digestive system removed from the body.
market, powdered and mixed with the feed along with 20% peanut oil. DMH, p-nitrophenyl b-Dglucuronide and mucin were purchased from Sigma (St. Louis, MO). The lower dose of spices used here is with reference to the average daily intake by human beings, learnt from a survey conducted in India (Srinivasan and Sambaiah, 1991). 20 mg/kg Body weight of DMH was dissolved in 1 mmol/1 EDTA, just prior to use and the pH adjusted to 6.5 with 1 mmol/1 NaHCO3 and administered subcutaneously once a week (Marianne et al., 1990; Tsunoda et al., 1992). After 15 weeks the DMH injection was discontinued and the rats were given only the commercial diet. At the end of 30 weeks, fresh fecal pellets were collected and the activity of mucinase was estimated by the method of Shiau and Chang (1983). They were then sacrificed by cervical decapitation after administering anesthesia. The neoplasms in the intestine and colon were counted after cutting open the tissues longitudinally, taking care not to disturb the tumors. Part of the tissue was immediately fixed in 10% formalin for 24 h. Then the tissue was cut open at the anti mesentric border, processed and embedded on paraffin wax. 3 – 5 mm Sections were sliced, stained with Hematoxylin and Eosin and then the cell morphology
as a whole was studied. The normal mucosa was compared with the abnormal mucosa. The rest of the tissues and colon contents were transferred to ice-cold containers for measuring the activity of b-glucuronidase and mucinase. The method by which we divided the intestine and colon of the rats are represented in Fig. 1. b-Glucuronidase activity was measured by the method of Kawai and Anno (1971), using p-nitrophenyl b-D-glucuronide as the substrate and measuring the amount p-nitrophenol liberated. Protein was estimated by the method of Lowry et al. (1951). The results obtained were expressed as mean + S.D. from 6 rats in each group. The statistical significance of the difference in means was analysed by Student’s t-test. A one way analysis of variance (ANOVA) was also determined (Chou, 1975).
3. Results The weight gain at the end of the experimental period was 329 g in cumin, 316 g in cumin+ DMH, 335 g in black pepper and 305 g in pepper+ DMH. These were comparatively greater than the rats in the DMH (210 g), chilli (218 g) and chilli+ DMH (238 g) groups. The number of
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N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
Fig. 2. High fat: a normal mucosal fold with exaggerated inflammatory cell infiltrate in the submucosa. Hematoxylin and Eosin staining, magnification ×20.
Fig. 4. Chilli: a polyp filled with glands of varying sizes and some of them reveal pseudostratification of the lung columnar cells. Foci and dysplasia also noted. Hematoxylin and Eosin staining, magnification × 10.
rats with tumors per total number of rats in the chilli group was 25/30 (incidence 83.3%), DMH group 27/30 (incidence 90%), chilli+ DMH 28/30 (93.3%), cumin+DMH 14/30 (46.7%) and pepper+DMH 25/30 (83.3%). The macroscopic and light microscopic observations (histopathological) of the colon of rats in the different groups are given in Table 1. Low fat and high fat control rats showed a similar microscopic pattern except that the latter had exaggerated inflammatory cell infiltrates. When cumin and black pepper were supplemented in the diet the animals showed fewer papillae, no infiltration into the submucosa and lesser changes in the cell
morphology compared to the DMH, chilli and chilli+ DMH groups (Figs. 2–7). The activity of b-glucuronidase showed a slight increase in all the tissues and the colon contents of the high fat and DMH groups when compared to the control animals (Table 2). The chilli treated group showed a significant increase in the proximal colon, distal intestine, proximal intestine and in the colon contents compared to the control rats. In the chilli + DMH group a significant increase was noted in the distal colon, proximal colon, distal intestine, proximal intestine, liver, and the colon contents (bacterial microflora). In
Fig. 3. DMH: a picture showing a papillary adenocarcinoma with ( ) several papillae lined by pleomorphic cells. Some of the glands are filled with mucinous material. Hematoxylin and Eosin staining, magnification × 20.
Fig. 5. Chilli +DMH: papillary processes lined by stratified columnar epithelium. Hematoxylin and Eosin staining, magnification × 20.
—
—
— —
Cytoplasm Mitotic figures
Others Vascular granulation and vascular congestion
—
—
Vascular congestion
— —
—
—
Focal areas of fibroblastic reaction
Scanty Numerous
Prominent
Marked
Observed
Not observed
—
—
Mixed population
2 cm
DMH
Mixed inflammation
—
High fat control
Cell morphology Nuclear pleomorphism Nucleoli
Infiltration into — the submucosa
Microscopy Inflammatory — cell infiltrates into the mucosa Lymphoid aggre- — gates in the submucosa
Macroscopy Size
Low fat control
Table 1 Pathological changes in the colon
Not seen
Less prominent Moderate Present
Less severe
—
Not observed
Mixed population
1 cm
Chilli
—
Cumin
Vascular granulation not very prominent
Scanty Numerous
Prominent
Marked
—
— —
—
—
Rare lymphoid Prominent aggregate lymphoid aggregates in the submucosa along with collection of lymphocytes in the mucosa Several areas — of infiltration
Mixed popula- Mixed inflamtion mation
2 cm
Chilli+DMH
Observed
Scanty Present
Prominent
Observed
No infiltration
0.5 cm
Pepper+DMH
Vascular congestion
— —
—
—
—
Vascular granulation
Scanty Observed
Prominent
Marked
Not observed
Not seen
Predominantly Mixed eosinophils 10/HPF
—
Pepper
Lymphoid ag- — gregates deeper to the tumor seen
Mixed population
1 cm
+ DMH
Cumin
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24 19
85.77 912.80
54.26 9 9.50 51.01 97.00
53.66 9 6.25 53.96 9 7.64
40.649 3.30 43.2494.02
45.03 9 1.69 53.85 95.04b 40.83 9 2.09 49.10 9 4.20NS
65.34 9 4.96a
40.85 9 5.14b
40.33 9 4.47a
43.19 9 3.05a 42.84 9 3.56b
29.40* 46.71*
24.46*
31.09*
60.03* 14.09*
Pepper+DMH F-ratio
75.929 4.44 85.31 9 5.55a 80.659 11.32 96.57 9 8.58b
34.569 7.84
30.159 4.69
31.759 3.05 34.399 4.19
Cumin+DMH Pepper
48.28 9 5.13 50.35 9 5.98a 40.06 9 6.08 54.15 9 3.26a
Cumin
80.8898.47
83.40 9 14.50a 56.91 97.27b
Chilli+DMH
92.6593.66 99.95 98.29 145.12 912.98 96.08 912.60 131.63 9 23.79NS 61.31 9 3.56 93.36 9 6.24a 62.889 2.81 102.1398.24 112.18 9 13.73 149.00 924.39 173.98 915.33a 87.95 9 7.19 77.36 9 7.19a
79.53912.59
44.76 94.36 50.97 95.45
Chilli
55.84 9 6.92 108.73 99.30 47.269 3.78 47.36 93.73
DMH
43.299 4.72 42.249 2.82
High fat control
Values are mean 9S.E. (mg of p-nitrophenol liberated/h per g protein) from 6 rats in each group. DMH has been compared with chilli+DMH, cumin+DMH and pepper+DMH groups. a PB0.01; b PB0.05; NS, not significant; ANOVA, significant at the 1% level.
Distal colon Proximal colon Distal intestine Proximal intestine Liver Colon contents
Low fat control
Table 2 b-Glucuronidase
20 N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
Fig. 6. Cumin+ DMH: () a lymphoid aggregate extending up to the mucosa with an occasional mucosal gland exhibiting dysplastic changes. Hematoxylin and Eosin staining, magnification × 10.
the cumin group the b-glucuronidase activity showed a significant increase only in the distal colon, distal intestine and the colon contents, when compared to the control rats. In the cumin + DMH group the b-glucuronidase activity was slightly increased in the colon and intestines compared to the control rats but the values were less than that of the DMH group (except for the proximal colon). In the black pepper group b-glucuronidase activity was less than the control animals in all the tissues expect the colon contents and in the black pepper+DMH group the values except for the liver and colon contents were more or less comparable with the control rats.
Fig. 7. Black pepper +DMH: focal areas of mucinous secretion in which the tumor cells appear floating. Hematoxylin and Eosin staining, magnification ×10.
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Mucinase activity was slightly less in the high fat group when compared with the control rats (Table 3). When DMH was administered the increase was very significant, chilli administration showed a similar increase, though it was less than that of the DMH group. When chilli was supplemented with DMH the increase in the activity was more than even that of the DMH group. The cumin group showed a slight increase and the cumin+DMH group a significant increase in the mucinase activity, though the values were less than that of the DMH group. The black pepper group showed a slight fall in the mucinase activity and in the black pepper+ DMH group the values were comparable to that of the control rats.
4. Discussion The above results indicate that C. annum, C. cyminum and P. nigrum bring about profound alterations in the activity of b-glucuronidase and mucinase, both in the presence and/or absence of DMH in high fat fed rats. Pathological studies showed a great degree of variation in the different groups. Vascular congestion was seen in the high fat fed rats. In the DMH group, the size of the tumor was around 2 cm, pedunculated having a well-defined margin with a large number of papillae and invasive adenocarcinoma, which showed marked pleomorphism. The nucleoli were very prominent with scanty cytoplasm and numerous mitotic figures. In the chilli groups, the size of the tumor was about 1 cm pedunculated with a defined margin. Histopathology exhibited areas of dysplasia which was less severe, with a less prominent nucleoli, moderate cytoplasm and few mitotic figures. In the chilli+ DMH group, the size of the tumor was more than 2 cm. There was a transitional zone with areas of marked dysplasia and infiltrating adenocarcinoma. The nucleoli were also prominent. The mucosa of the cumin group had ulceration. Lymphoid aggregates were seen both in the submucosa and mucosa. The cumin+ DMH group showed the size of the tumor to be 1 cm, sessile, with no transitional zone of dysplasia, though there were a considerable number of
7.360 9 0.92a
4.300 9 0.65
5.580 90.63
1.92190.26
2.2409 0.22
4.518 9 0.66a
2.693 90.33
3.06590.42
1.997 90.11
2.06090.15
Chilli+DMH
Chilli
DMH
High fat control
Low fat
2.554 90.13
2.821 90.06
Cumin
Values are mean 9 S.E. (mg of glucose liberatedl/min per mg protein) from 6 rats in each group. DMH has been compared with chilli+DMH, cumin+DMH and pepper+DMH groups. a PB0.01; b PB0.05; NS, not significant. ANOVA, significant at the 1% level.
Colon contents Fecal contents
Table 3 Mucinase
3.341 9 0.26a
3.051 9 0.11NS
Cumin+DMH
1.0829 0.06
1.0839 0.05
Pepper
2.15690.43a
2.12490.09a
Pepper+DMH
107.17*
36.50*
F-ratio
22 N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
N. Nalini et al. / Journal of Ethnopharmacology 62 (1998) 15–24
papillae which were lined by anaplastic cells. Lymphoid aggregates deeper to the tumor were also seen. In black pepper +DMH group the size of the tumor was less than 0.5 cm with a defined margin, sessile with focal areas of dysplasia. Nuclear pleomorphism was noted with prominent nucleoli, scanty cytoplasm and few mitotic figures. The vascular granulation and desmoplastic reaction are some of the common reactions executed by the normal tissue towards a malignant neoplasm. In our observation we also saw a similar reaction in the DMH, chill+ DMH and pepper + DMH groups. In the case of chilli + DMH group such a reaction was present but not very prominent. Vascular granulation was present in the pepper + DMH group and this may explain the lower virulence and lower infiltration of the tumor. Thus chilli intake clearly shows that it acts as a promoter of carcinogenesis, whereas the inclusion of cumin and black pepper inhibits carcinogenesis as evident by the decreased number of tumors, comparatively, in both the cumin+ DMH and black pepper+ DMH groups. The liver is the major site of detoxification of many drugs, toxins, and hormones. One of the main functions of the liver is to conjugate these drugs, toxins and hormones with glucuronides, thus making these soluble and at the same time detoxifying them. They are then excreted via the bile to the intestine. When they reach the colon, normally more than 90% is excreted. But when the activity of these colonic microflora are stimulated as in the presence of the procarcinogen DMH, these glucuronides may be hydrolyzed, which may lead to the liberation of drugs and toxins that can be harmful to the colonocytes (Goldin and Gorbach, 1977; Goldin et al., 1978). This may be one of the ways by which DMH is said to enhance the conversion of normal epithelial cells of the colon into cancerous ones. Chilli, as noted from the results, is shown to increase the activity of b-glucuronidase by enhancing the hydrolysis of glucuronides, while cumin and black pepper on the other hand have the opposite effect. Thus, cumin and black pepper may preserve the colon from the toxic effects of drugs and toxins. This are confirmed by the increased incidence of tumors in the chilli+ DMH group and the de-
23
creased incidence in the cumin+ DMH and black pepper+ DMH groups. Mucins are glycoproteins consisting of a large number of carbohydrate side chains attached to a protein core. They form gels coating the intestinal mucosa and also function as a lubricant and probably as a chemical or mechanical barrier against bacteria, viruses and toxins. Mucinase is an enzyme present in the intestinal microflora which hydrolyses these protective mucins in the colon (Reddy and Wynder, 1973). Once the mucins are hydrolysed, the underlying cells are exposed to a number of toxic intermediates (Hoskins and Zancheck, 1968; Salvers et al., 1977). The activity of mucinase was significantly elevated in the DMH and/or chilli treated groups. This increased mucinase activity leads to decreased protection of the underlying tissues (Wynder and Reddy, 1974; Miller and Hoskins, 1981). Whereas the inclusion of cumin and black pepper in the diet was found to decrease it’s activity both in the colon microflora and in the fecal content. If this change in enzyme activity reflects the change in the rate of mucin degradation and the shift in the balance between mucin secretion and degradation, then it may also be accompanied by a change in the susceptibility of the intestinal mucosa to pathogenic organisms, toxins and carcinogens.
5. Conclusion Thus, treatment with cumin and black pepper was shown to protect the colon in the presence of the procarcinogen 1,2-dimethyl hydrazine as a possible result of the decreased activity of b-glucuronidase and mucinase, whereas chilli supplementation had the opposite effect in the proximal intestine and proximal colon.
Acknowledgements The authors wish to thank Mr Arumuga Perumal and Mr Krishnaswamy, for their technical assistance.
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