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Genotoxicity of Sennosides on the Bone Marrow Cells of Mice
Food and Chemical Toxicology 36 (1998) 937±940
Genotoxicity of Sennosides on the Bone Marrow Cells of Mice M. J. MUKHOPADHYAY1, A. SAHA1, A. DUTTA2, B. DE2 and A. MUKHERJEE1*
1 Centre for Advanced Studies on Cell and Chromosome Research, Department of Botany, University of Calcutta and 2Pharmacognosy Research Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta-700019, India
(Accepted 20 November 1997) AbstractÐPreparations of a number of plants which contain hydroxyanthraquinones as active constitutents are used worldwide for their laxative eect. Anthraquinone glycosides of Cassia angustifolia and C. ®stula were investigated for their ability to induce a clastogenic eect on the bone marrow cells of Swiss albino mice. The endpoints screened were chromosomal aberrations and frequency of aberrant cells. Oral exposure to doses of these anthraquinones and their equivalent amount in leaf and pod extracts did not induce signi®cant numbers of chromosomal aberrations or aberrant cells. The results indicate that anthraquinone sennoside B and rhein are weakly genotoxic. # 1998 Elsevier Science Ltd. All rights reserved Keywords: sennoside B; chromosomal aberrations; genotoxicity. Abbreviations: ANOVA=analysis of variance; CA/cell=chromosomal aberration/cell; % DC=percentage of aberrant cells; MMC=mitomycin C.
INTRODUCTION
Cassia angustifolia, a small shrub indigenous to Somaliland and Arabia, commonly called senna, is valued in medicine for its cathartic properties. The shrub cultivated in south India is recognized by the British and American pharmacopoeias and is widely used as a source of natural laxative. Almost all senna produced in India is exported; the US and UK are the two main import markets. Senna is reported to increase peristaltic movement in the colon and is contraindicated in spastic constipation and in cases of colitis. The laxative principles in senna are mainly two glycosides, sennoside A and sennoside B, both having the formula C21H20O10 and diering principally in the matter of linkage of glucose to one aglycone fraction. C. ®stula, a related species of the genus, also has a high content of sennosides and rhein (Wealth of India, 1950). It is recognized by the British Pharmacopoeia. This deciduous tree, indigenous to India, can be exploited as an alternative to senna. The genotoxicity of anthraquinone-containing laxatives is of particular interest because of the possibility of induction of colon cancer by these widely *Author for correspondence.
used compounds; 20±30% of people over the age of 60 yr in the UK are reported to take laxatives once a week or more, and approximately 80% of patients chronically abusing laxatives use anthraquinonecontaining drugs (Cooke, 1981; May, 1982). The pharmacology and toxicology of the anthraquinone glycosides and their aglycones have been described by Van Os (1976), who concluded that in normal doses these substances are not toxic. In the present study, pure sennoside B and its equivalent amount in leaf and pod extract of C. angustifolia and C. ®stula were tested in vivo on the bone marrow cells of mice.
MATERIALS AND METHODS
Animals Male Swiss albino mice, 8±10 wk old and weighing 20±25 g, were obtained from the Departmental Animal House. They were housed six per cage under standard husbandry and feeding schedules for clean conventional colonies (temperature 25 2 28C, relative humidity 60 2 5%, 12-hr light/ dark photoperiod). Animals were allowed to access ad lib. to standard rodent pellet diet (Gold Mohar, Lipton India, India) and water.
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00049-0
Sennoside (mg)
Rhein glycoside (mg)
5 5
0.0032 0.0001
5
0.087
2 2 7 63 Ð Ð Ð Ð Ð Ð Ð 14 2 2 8 136 1 Ð 1 4 4 2 2 22 150 150 150 200
150 150 150
8 9 8
1 1 0
5 6 6
Ð Ð Ð
Ð Ð Ð
4 5 6
0.013 2 0.006 0.020 2 0.010 0.023 2 0.006 0.040 2 0.010 0.047 2 0.008 3.004* 0.033 2 0.006 0.040 2 0.016 0.040 2 0.016 1.24 NS 0.013 2 0.006 0.013 2 0.006 0.053 2 0.035 0.750 2 0.120 2 3 7 10 12 Ð Ð Ð Ð Ð Ð Ð Ð 4 2 2 3 7 8 12 Ð 1 Ð 1 Ð Ð 3 4 5 5 150 300 300 300 300
Ca/cell X 2SEM DC RR B0 B'
Total CA
G', G0 = chromatid and chromosome gap; B', B0 = chromatid and chromosome break; RR = chromosomal rearrangements; CA = chromosomal aberrations; DC = damaged cells with at least one CA (excluding gaps); *Signi®cant Z value (P = 0.05, Cochran±Armitage test of linear trend). SEN = sennoside B; RN = rhein; CFL, CFP = C. ®stula leaf extract, pod extract; CAL = C. angustifolia leaf extract; MMC = mitomycin C; NS = not signi®cant.
Cassia ®stula Leaf Pod Cassia angustifolia Leaf
CFL (SEN) CFP (SEN) CAL (SEN) MMC
Species
RN
Table 1. Sennoside and rhein content in the extracts of leaves and pods of Cassia sp.
0 1.25 2.50 5.00 20.00 Z value 0.50 1.00 2.00 Z value 5.00 5.00 5.00 2.00
The Cochran±Armitage one-tailed trend test (Margolin et al., 1986) was used to determine whether a treatment-related increase occurred. Oneway analysis of variance (ANOVA) followed by Duncan's multiple range test (Sokal and Rohlf, 1981) were carried out to observe signi®cant dierences between individual groups. The level of signi®cance was established as P = 0.05.
Control SEN
Statistical analysis
G0
Bone marrow cells were ¯ushed in 0.075 M KCl, incubated at 378C for 30 min and ®xed in cold glacial acetic acid±methanol (1:3). Slides were prepared by ¯ame-drying and stained in Giemsa (Preston et al., 1987). 50 well-spread metaphase plates were scanned per animal per treatment except for sennoside B (pure), where 100 metaphases were scored per animal. The types of aberrations were scored according to the method of Tice et al. (1987). Thus, the percentage of aberrant cells (% DC) and chromosomal aberration/cell (CA/cell) were calculated. In all cases, gaps were scored, but not included for calculation. For mitomycin C (MMC), earlier laboratory data were used.
G'
Chromosomal aberration assay
No. of metaphase scored
In all cases treatments were by oral administration in volumes of 0.01 ml/10 g body weight. The animals were killed by cervical dislocation 18 hr after administration (McFee and Tice, 1990). 90 min prior to sacri®ce the animals were injected ip with 0.04% colchicine. Three animals were used for each group of treatment and as controls.
Dose (mg/kg)
Treatment
Table 2. In vivo eects of sennoside B and rhein in extracts of leaf and pod of C. ®stula and C. angustifolia leaf
Doses of sennoside and rhein were selected on the basis of the sennoside content of available drug (Pursennid), namely 18 mg/90 mg tablet. The normal dose of the drug is two tablets/60 kg human. The leaves and pods of C. ®stula and leaves of C. angustifolia were collected, weighed (2.571 g, 3.33 g and 403 mg, respectively) and extracted with 50% methanol. After evaporation of methanol, the aqueous extracts were freeze-dried and the sennoside and rhein content were determined spectrophotometrically (Habib and El-Sebakhy, 1980) (see Table 1). Sennoside B was a kind gift from Sandoz Pharma AG, Basel, Switzerland. Rhein was purchased from Aldrich Chemical Co., Milwaukee, US.
% DC X2 SEM
Chemicals and dose
1.33 20.667 2.00 21.000 2.33 21.330 3.33 21.760 4.00 22.000 2.557* 2.67 21.760 3.33 20.670 4.00 21.160 1.25 NS 1.33 20.670 1.33 20.660 4.57 22.530 31.5 23.400
M. J. Mukhopadhyay et al.
Test substance
938
Genotoxicity of sennosides
939
Table 3. One-way ANOVA showing signi®cant dierences (if any) among dierent treatment groups Source of variationa % DC A SEN B RN CA/cell A SEN B RN
Degrees of freedom
Sum of squares
Mean sum of squares
F ratio
Among groups (treatments) Within groups (error, replicates) Among groups (treatments)
3 8 3 8
61.336 77.334 14.67 37.33
20.44 9.66 4.89 4.66
2.114
Among groups (treatments) Within groups (error, replicates) Among groups (treatments) Within groups (error, replicates)
3 8 3 8
24.67 19.33 4.25 6.66
8.22 2.41 1.42 0.83
3.403
1.047
1.71
a F0.05 (3.8) = 4.07. Abbreviations as given in Table 2.
RESULTS
The results of the chromosome analysis in bone marrow cells are shown in Table 2. The Cochran± Armitage trend test indicated a linear trend in dose±response (P = 0.05) for the pure compound sennoside B. For all compounds there were no signi®cant dierences in CA/cell or % DC. The aberrations were mainly chromatid in type. The CA/cell and damaged cell scores showed a maximum three to ®vefold increase over control values; the data on the positive control MMC (2 mg/kg body weight) show a higher frequency of aberrations and aberrant cells. Treatment with leaf and pod extracts of C. ®stula containing sennoside B and rhein (see Table 1) induced weak clastogenic eects with values nearly the same as the vehicle control. Only leaf extract of C. angustifolia (senna) gave a higher frequency of CA/cell and damage which were, however, not statistically signi®cant. Table 3 represents the ANOVA % DC and CA/cell, showing non-signi®cant dierences among the dierent treatment groups (sennoside B and rhein).
demonstrated that senna and cascara glycosides might behave as weak promoters in rat colon carcinogenesis, suggesting that they do not behave as genotoxic carcinogens. The results of the present study are in line with those reported by Heidemann et al. (1993). Pure sennoside B and rhein were weakly clastogenic. Crude extracts of C. augustifolia (leaves) and C. ®stula (leaves and pods) each containing sennoside B and rhein were also weak clastogens. The CA/cell and % DC were lower than those induced by an equivalent amount of pure sennoside B. Therefore, these phytolaxatives to not behave as potent clastogens and pods or leaves of C. ®stula can be used as an alternative source of sennosides. AcknowledgementsÐThe authors are grateful to the Programme Coordinator, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, for the laboratory facilities. M.J. Mukhopadhyay acknowledges CSIR (13(6978-A/96-Pool)) for ®nancial support.
REFERENCES DISCUSSION
In recent years evidence has been given that several natural and synthetic hydroxyanthraquinones are mutagenic in the Ames/Salmonella microsome system (Blomeke et al., 1992; Krivobok et al., 1992; Sandness et al., 1992; Westendorf et al., 1990). Some can induce genotoxic eects in cultured mammalian cells (Kawai et al., 1987; Westendorf et al., 1990) and behave as possible tumour promoters (Wol¯e et al., 1990). Heidemann et al. (1993) carried out an extensive study on the genotoxicity status of senna and showed that the sennosides, rhein and aloe-emodin, were non-genotoxic and nonmutagenic in both in vivo and in vitro test systems. Based on the fact that the tumorigenic eect of anthraquinone has been observed only after longterm exposure to high toxic doses (Mori et al., 1985, 1986 and 1990), a property typical of carcinogen acting through the induction of cell proliferation, Mereto et al. (1996) have elegantly
Blomeke B., Poginsky B., Schmutte C., Marquardt H. and Westendorf J. (1992) Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L. Mutation Research 265, 263±272. Cooke W. T. (1981) Laxative abuse. Acta Gastroenterologica Belgica 44, 448±458. Habib A. M. and El-Sebakhy N. A. (1980) Spectroscopic estimation of sennosides and rhein glycoside in senna and its preparation. Journal of Natural Products 43, 452±457. Heidemann A., Miltonburger H. G. and Mengs U. (1993) The genotoxicity status of senna. Pharmacology 47, 78± 86. Kawai K., Mori H., Sugie S., Yoshimi N., Inoue T., Nakamura T., Nozawa Y. and Matsushima T. (1987) Genotoxicity in the hepatocyte/DNA repair test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dihydroxyanthraquinones. Cell Biology and Toxicology 2, 457±468. Krivobok S., Seigle Murandi F., Steiman R., Marzin D. R. and Betina V. (1992) Mutagenicity of substituted anthraquinones in the Ames/Salmonella microsome system. Mutation Research 279, 1±8. McFee A. F. and Tice R. R. (1990) In¯uence of treatment to sacri®ce time and the presence of BrdUrd on
940
M. J. Mukhopadhyay et al.
chemically induced aberration rates in mouse marrow cells. Mutation Research 241, 95±108. May H. (1982) Missbrauch von AbfuÈhrmi Heln, nachweisbare schaden an Kolon, Anus and Stowechsel. Arztezeitschrift fuÈr Naturheilverhafen 7, 365±371. Margolin B. H., Resnick M. A., Rimpo J. Y., Archer P., Galloway S. M., Bloom A. D. and Zeiger E. (1986) Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells. Environmental Mutagenesis 8, 181±204. Mereto E., Ghia M. and Brambilla G. (1996) Evaluation of the potential carcinogenic activity of senna and cascara glycosides for the rat colon. Cancer Letters 101, 79±83. Mori H., Sugie S., Niwa K., Takahashi M. and Kawai K. (1985) Induction of intestinal tumours in rats by chrysazin. British Journal of Cancer 52, 781±783. Mori H., Sugie S., Niwa K., Yoshimi N., Tanaka T. and Hirono I. (1986) Carcinogenicity of chrysazin in large intestine and liver of mice. Japanese Journal of Cancer Research 77, 871±876. Mori H., Yoshimi N., Iwata H., Mori Y., Hara A., Tanaka T. and Kawai K. (1990) Carcinogenicity of naturally occurring 1±hydroxy-anthraquinone in rats: induction of large bowel, liver and stomach neoplasma. Carcinogenesis 11, 799±802.
Preston R. J., Dean B. J., Galloway S., Holden H., McFee A. and Shelby M. (1987) Mammalian in vivo cytogenetic assays of chromosome aberrations in bone marrow cells. Mutation Research 189, 157±165. Sandness D., Johansen T., Teien G. and Ulsaker G. (1992) Mutagenicity of crude senna and senna glycosides in Salmonella typhimurium. Pharmacology and Toxicology 71, 165±172. Sokal R. R. and Rohlf F. J. (1981) Biometry, 3rd Ed. Freeman, San Francisco. Tice R. R., Luke C. A. and Shelby M. D. (1987) Methyl isothiocyanate: an evaluation of in vivo cytogenetic activity. Environmental Mutagenesis 9, 37±58. The Wealth of India, Raw Materials, Vol. II (1950) CSIF, Delhi. Van Os F. H. L. (1976) Some aspects of the pharmacology and anthraquinone drugs. Pharmacology 14 (Suppl. 1), 18±29. Westendorf J., Marquardt H., Poginsky B., Dominiak M., Schmidt J. and Marquardt H. (1990) Genotoxicity of naturally occurring hydroxyanthraquinones. Mutation Research 240, 1±12. Wol¯e D., Schmutte C., Westendorf J. and Marquardt H. (1990) Hydroxyanthraquinones as tumor promoters: enhancement of malignant transformation of C3H mouse ®broblasts and growth stimulation of primary rat hepatocytes. Cancer Research 50, 6540±6544.
Genotoxicity of Sennosides on the Bone Marrow Cells of Mice M. J. MUKHOPADHYAY1, A. SAHA1, A. DUTTA2, B. DE2 and A. MUKHERJEE1*
1 Centre for Advanced Studies on Cell and Chromosome Research, Department of Botany, University of Calcutta and 2Pharmacognosy Research Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta-700019, India
(Accepted 20 November 1997) AbstractÐPreparations of a number of plants which contain hydroxyanthraquinones as active constitutents are used worldwide for their laxative eect. Anthraquinone glycosides of Cassia angustifolia and C. ®stula were investigated for their ability to induce a clastogenic eect on the bone marrow cells of Swiss albino mice. The endpoints screened were chromosomal aberrations and frequency of aberrant cells. Oral exposure to doses of these anthraquinones and their equivalent amount in leaf and pod extracts did not induce signi®cant numbers of chromosomal aberrations or aberrant cells. The results indicate that anthraquinone sennoside B and rhein are weakly genotoxic. # 1998 Elsevier Science Ltd. All rights reserved Keywords: sennoside B; chromosomal aberrations; genotoxicity. Abbreviations: ANOVA=analysis of variance; CA/cell=chromosomal aberration/cell; % DC=percentage of aberrant cells; MMC=mitomycin C.
INTRODUCTION
Cassia angustifolia, a small shrub indigenous to Somaliland and Arabia, commonly called senna, is valued in medicine for its cathartic properties. The shrub cultivated in south India is recognized by the British and American pharmacopoeias and is widely used as a source of natural laxative. Almost all senna produced in India is exported; the US and UK are the two main import markets. Senna is reported to increase peristaltic movement in the colon and is contraindicated in spastic constipation and in cases of colitis. The laxative principles in senna are mainly two glycosides, sennoside A and sennoside B, both having the formula C21H20O10 and diering principally in the matter of linkage of glucose to one aglycone fraction. C. ®stula, a related species of the genus, also has a high content of sennosides and rhein (Wealth of India, 1950). It is recognized by the British Pharmacopoeia. This deciduous tree, indigenous to India, can be exploited as an alternative to senna. The genotoxicity of anthraquinone-containing laxatives is of particular interest because of the possibility of induction of colon cancer by these widely *Author for correspondence.
used compounds; 20±30% of people over the age of 60 yr in the UK are reported to take laxatives once a week or more, and approximately 80% of patients chronically abusing laxatives use anthraquinonecontaining drugs (Cooke, 1981; May, 1982). The pharmacology and toxicology of the anthraquinone glycosides and their aglycones have been described by Van Os (1976), who concluded that in normal doses these substances are not toxic. In the present study, pure sennoside B and its equivalent amount in leaf and pod extract of C. angustifolia and C. ®stula were tested in vivo on the bone marrow cells of mice.
MATERIALS AND METHODS
Animals Male Swiss albino mice, 8±10 wk old and weighing 20±25 g, were obtained from the Departmental Animal House. They were housed six per cage under standard husbandry and feeding schedules for clean conventional colonies (temperature 25 2 28C, relative humidity 60 2 5%, 12-hr light/ dark photoperiod). Animals were allowed to access ad lib. to standard rodent pellet diet (Gold Mohar, Lipton India, India) and water.
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00049-0
Sennoside (mg)
Rhein glycoside (mg)
5 5
0.0032 0.0001
5
0.087
2 2 7 63 Ð Ð Ð Ð Ð Ð Ð 14 2 2 8 136 1 Ð 1 4 4 2 2 22 150 150 150 200
150 150 150
8 9 8
1 1 0
5 6 6
Ð Ð Ð
Ð Ð Ð
4 5 6
0.013 2 0.006 0.020 2 0.010 0.023 2 0.006 0.040 2 0.010 0.047 2 0.008 3.004* 0.033 2 0.006 0.040 2 0.016 0.040 2 0.016 1.24 NS 0.013 2 0.006 0.013 2 0.006 0.053 2 0.035 0.750 2 0.120 2 3 7 10 12 Ð Ð Ð Ð Ð Ð Ð Ð 4 2 2 3 7 8 12 Ð 1 Ð 1 Ð Ð 3 4 5 5 150 300 300 300 300
Ca/cell X 2SEM DC RR B0 B'
Total CA
G', G0 = chromatid and chromosome gap; B', B0 = chromatid and chromosome break; RR = chromosomal rearrangements; CA = chromosomal aberrations; DC = damaged cells with at least one CA (excluding gaps); *Signi®cant Z value (P = 0.05, Cochran±Armitage test of linear trend). SEN = sennoside B; RN = rhein; CFL, CFP = C. ®stula leaf extract, pod extract; CAL = C. angustifolia leaf extract; MMC = mitomycin C; NS = not signi®cant.
Cassia ®stula Leaf Pod Cassia angustifolia Leaf
CFL (SEN) CFP (SEN) CAL (SEN) MMC
Species
RN
Table 1. Sennoside and rhein content in the extracts of leaves and pods of Cassia sp.
0 1.25 2.50 5.00 20.00 Z value 0.50 1.00 2.00 Z value 5.00 5.00 5.00 2.00
The Cochran±Armitage one-tailed trend test (Margolin et al., 1986) was used to determine whether a treatment-related increase occurred. Oneway analysis of variance (ANOVA) followed by Duncan's multiple range test (Sokal and Rohlf, 1981) were carried out to observe signi®cant dierences between individual groups. The level of signi®cance was established as P = 0.05.
Control SEN
Statistical analysis
G0
Bone marrow cells were ¯ushed in 0.075 M KCl, incubated at 378C for 30 min and ®xed in cold glacial acetic acid±methanol (1:3). Slides were prepared by ¯ame-drying and stained in Giemsa (Preston et al., 1987). 50 well-spread metaphase plates were scanned per animal per treatment except for sennoside B (pure), where 100 metaphases were scored per animal. The types of aberrations were scored according to the method of Tice et al. (1987). Thus, the percentage of aberrant cells (% DC) and chromosomal aberration/cell (CA/cell) were calculated. In all cases, gaps were scored, but not included for calculation. For mitomycin C (MMC), earlier laboratory data were used.
G'
Chromosomal aberration assay
No. of metaphase scored
In all cases treatments were by oral administration in volumes of 0.01 ml/10 g body weight. The animals were killed by cervical dislocation 18 hr after administration (McFee and Tice, 1990). 90 min prior to sacri®ce the animals were injected ip with 0.04% colchicine. Three animals were used for each group of treatment and as controls.
Dose (mg/kg)
Treatment
Table 2. In vivo eects of sennoside B and rhein in extracts of leaf and pod of C. ®stula and C. angustifolia leaf
Doses of sennoside and rhein were selected on the basis of the sennoside content of available drug (Pursennid), namely 18 mg/90 mg tablet. The normal dose of the drug is two tablets/60 kg human. The leaves and pods of C. ®stula and leaves of C. angustifolia were collected, weighed (2.571 g, 3.33 g and 403 mg, respectively) and extracted with 50% methanol. After evaporation of methanol, the aqueous extracts were freeze-dried and the sennoside and rhein content were determined spectrophotometrically (Habib and El-Sebakhy, 1980) (see Table 1). Sennoside B was a kind gift from Sandoz Pharma AG, Basel, Switzerland. Rhein was purchased from Aldrich Chemical Co., Milwaukee, US.
% DC X2 SEM
Chemicals and dose
1.33 20.667 2.00 21.000 2.33 21.330 3.33 21.760 4.00 22.000 2.557* 2.67 21.760 3.33 20.670 4.00 21.160 1.25 NS 1.33 20.670 1.33 20.660 4.57 22.530 31.5 23.400
M. J. Mukhopadhyay et al.
Test substance
938
Genotoxicity of sennosides
939
Table 3. One-way ANOVA showing signi®cant dierences (if any) among dierent treatment groups Source of variationa % DC A SEN B RN CA/cell A SEN B RN
Degrees of freedom
Sum of squares
Mean sum of squares
F ratio
Among groups (treatments) Within groups (error, replicates) Among groups (treatments)
3 8 3 8
61.336 77.334 14.67 37.33
20.44 9.66 4.89 4.66
2.114
Among groups (treatments) Within groups (error, replicates) Among groups (treatments) Within groups (error, replicates)
3 8 3 8
24.67 19.33 4.25 6.66
8.22 2.41 1.42 0.83
3.403
1.047
1.71
a F0.05 (3.8) = 4.07. Abbreviations as given in Table 2.
RESULTS
The results of the chromosome analysis in bone marrow cells are shown in Table 2. The Cochran± Armitage trend test indicated a linear trend in dose±response (P = 0.05) for the pure compound sennoside B. For all compounds there were no signi®cant dierences in CA/cell or % DC. The aberrations were mainly chromatid in type. The CA/cell and damaged cell scores showed a maximum three to ®vefold increase over control values; the data on the positive control MMC (2 mg/kg body weight) show a higher frequency of aberrations and aberrant cells. Treatment with leaf and pod extracts of C. ®stula containing sennoside B and rhein (see Table 1) induced weak clastogenic eects with values nearly the same as the vehicle control. Only leaf extract of C. angustifolia (senna) gave a higher frequency of CA/cell and damage which were, however, not statistically signi®cant. Table 3 represents the ANOVA % DC and CA/cell, showing non-signi®cant dierences among the dierent treatment groups (sennoside B and rhein).
demonstrated that senna and cascara glycosides might behave as weak promoters in rat colon carcinogenesis, suggesting that they do not behave as genotoxic carcinogens. The results of the present study are in line with those reported by Heidemann et al. (1993). Pure sennoside B and rhein were weakly clastogenic. Crude extracts of C. augustifolia (leaves) and C. ®stula (leaves and pods) each containing sennoside B and rhein were also weak clastogens. The CA/cell and % DC were lower than those induced by an equivalent amount of pure sennoside B. Therefore, these phytolaxatives to not behave as potent clastogens and pods or leaves of C. ®stula can be used as an alternative source of sennosides. AcknowledgementsÐThe authors are grateful to the Programme Coordinator, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, for the laboratory facilities. M.J. Mukhopadhyay acknowledges CSIR (13(6978-A/96-Pool)) for ®nancial support.
REFERENCES DISCUSSION
In recent years evidence has been given that several natural and synthetic hydroxyanthraquinones are mutagenic in the Ames/Salmonella microsome system (Blomeke et al., 1992; Krivobok et al., 1992; Sandness et al., 1992; Westendorf et al., 1990). Some can induce genotoxic eects in cultured mammalian cells (Kawai et al., 1987; Westendorf et al., 1990) and behave as possible tumour promoters (Wol¯e et al., 1990). Heidemann et al. (1993) carried out an extensive study on the genotoxicity status of senna and showed that the sennosides, rhein and aloe-emodin, were non-genotoxic and nonmutagenic in both in vivo and in vitro test systems. Based on the fact that the tumorigenic eect of anthraquinone has been observed only after longterm exposure to high toxic doses (Mori et al., 1985, 1986 and 1990), a property typical of carcinogen acting through the induction of cell proliferation, Mereto et al. (1996) have elegantly
Blomeke B., Poginsky B., Schmutte C., Marquardt H. and Westendorf J. (1992) Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L. Mutation Research 265, 263±272. Cooke W. T. (1981) Laxative abuse. Acta Gastroenterologica Belgica 44, 448±458. Habib A. M. and El-Sebakhy N. A. (1980) Spectroscopic estimation of sennosides and rhein glycoside in senna and its preparation. Journal of Natural Products 43, 452±457. Heidemann A., Miltonburger H. G. and Mengs U. (1993) The genotoxicity status of senna. Pharmacology 47, 78± 86. Kawai K., Mori H., Sugie S., Yoshimi N., Inoue T., Nakamura T., Nozawa Y. and Matsushima T. (1987) Genotoxicity in the hepatocyte/DNA repair test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dihydroxyanthraquinones. Cell Biology and Toxicology 2, 457±468. Krivobok S., Seigle Murandi F., Steiman R., Marzin D. R. and Betina V. (1992) Mutagenicity of substituted anthraquinones in the Ames/Salmonella microsome system. Mutation Research 279, 1±8. McFee A. F. and Tice R. R. (1990) In¯uence of treatment to sacri®ce time and the presence of BrdUrd on
940
M. J. Mukhopadhyay et al.
chemically induced aberration rates in mouse marrow cells. Mutation Research 241, 95±108. May H. (1982) Missbrauch von AbfuÈhrmi Heln, nachweisbare schaden an Kolon, Anus and Stowechsel. Arztezeitschrift fuÈr Naturheilverhafen 7, 365±371. Margolin B. H., Resnick M. A., Rimpo J. Y., Archer P., Galloway S. M., Bloom A. D. and Zeiger E. (1986) Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells. Environmental Mutagenesis 8, 181±204. Mereto E., Ghia M. and Brambilla G. (1996) Evaluation of the potential carcinogenic activity of senna and cascara glycosides for the rat colon. Cancer Letters 101, 79±83. Mori H., Sugie S., Niwa K., Takahashi M. and Kawai K. (1985) Induction of intestinal tumours in rats by chrysazin. British Journal of Cancer 52, 781±783. Mori H., Sugie S., Niwa K., Yoshimi N., Tanaka T. and Hirono I. (1986) Carcinogenicity of chrysazin in large intestine and liver of mice. Japanese Journal of Cancer Research 77, 871±876. Mori H., Yoshimi N., Iwata H., Mori Y., Hara A., Tanaka T. and Kawai K. (1990) Carcinogenicity of naturally occurring 1±hydroxy-anthraquinone in rats: induction of large bowel, liver and stomach neoplasma. Carcinogenesis 11, 799±802.
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