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Effect of Caralluma tuberculata on the cytological and biochemical changes induced by cyclophosphamide in mice
Fd Chem. Toxic. Vol. 30, No. 8, pp. 719-722, 1992 Printed in Great Britain.All rights reserved
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
EFFECT OF CARALLUMA TUBERCULATA ON THE CYTOLOGICAL A N D BIOCHEMICAL CHANGES INDUCED BY CYCLOPHOSPHAMIDE IN MICE A. M. AL-BEKAIRI*,S.QuRESHI*, M. M. AHMED*, N. S. QAZIt, Z. A. KHANt and A. H. SHAH~§ *Quality Control and Research Laboratory, Experimental Animal Care Centre, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, tGomal University, D. I. Khan, Pakistan and :~Central Laboratory for Drug and Food Analysis, M.O.H., PO Box 59082, Riyadh-l1525, Saudi Arabia (Accepted 27 March 1992)
Abstract--Treatment with Caralluma tuberculata extract induced complex biochemical and cytological changes in mice. Its cytotoxicity in the bone marrow cells of mice was comparable with that of the standard drug cyclophosphamide (CP); however, unlike CP, C. tuberculata was not clastogenic (as shown by the micronucleus assay). A dose-dependent decrease in the RNA content of liver and testes was produced by C. tuberculata treatment whereas there was no effect on the content of nucleic acid and protein in the brain. In the extract-treated animals there was a significant and dose-dependent increase in the DNA content of the liver, with a negligible effect on the protein content. Combined treatment with C. tuberculata and CP showed that C. tuberculata diminished the effect of CP on DNA levels; however, RNA levels were further suppressed, resulting in increased cytotoxicity. Pretreatment with C. tuberculata extract significantly reduced the clastogenicity of CP. These results indicated the involvement of different phytoconstituents acting by different routes.
INTRODUCTION
MATERIALSAND METHODS
Recent reports have suggested that plants used as food and spices may have various types of pharmacological activity and toxicity (Akira et al., 1986; AI-Bekairi et al., 1991b; Kutten et al., 1985; Vincenzi and Dessi, 1991). Caralluma tuberculata N.E. Brown (Asclepiadaceae) is a plant widely grown in the Punjab, North-West Frontier and Balochistan Provinces of Pakistan. It is popularly known as 'Chungan' and is consumed as food. For medicinal purposes C. tuberculata is widely used in the treatment of rheumatism, diabetes, leprosy and blood disorders, and as an antipyretic alternative and anthelmintic (Ahmad and Shaikh, 1989; Ali, 1983; Chopra et al., 1956). This plant has been reported to contain flavonoids and much saponin (Ahmad et al., 1988; Rizwani et al., 1990; Tanaka et al., 1990). However, there is limited evidence in support of its claims in popular medicine. Bearing in mind the widepsread use of C. tuberculata by the local population the present study was designed to evaluate some of its cytological and biochemical effects in mice. This study also deals with the effect of C. tuberculata on cyclophosphamide (CP) treatment in mice.
Plant extract. Caralluma tuberculata N.E. Brown was collected fresh from the local vegetable market in Dera Ismail Khan, Pakistan and a voucher specimen was kept on record in the Department of Pharmacognosy, Faculty of Pharmacy, Gomal University, Pakistan. The plant was cut into small pieces and extracted with 96% ethanol in a soxhlet apparatus. The solvent was removed under reduced pressure; the dark green semi-solid residue thus obtained was refrigerated until required (Mohsin et al., 1989). Animal stock. A total of 40 male mice (SWR, home bred), aged 6-8 wk weighing 25-30 g were used. The animals were randomly assigned to different control and treatment groups (five animals in each group) that were maintained under standard conditions of humidity, temperature and light (12hr light/12hr dark). The animals were fed with purina chow diet and had free access to water. A dose of 500 mg/kg body weight was used as the highest dose of the extract (Tanira et al., 1988). Unless otherwise specified, a fresh aqueous suspension of the plant extract was prepared and administered orally (gavage) to the animals for 7 days. The experimental groups of mice were as follows: (1) untreated control (distilled water); (2) 125 mg C. tuberculata/kg/day; (3) 250mg C. tuberculata/kg/day; (4) 500mg C. tuberculata/kg/day; (5) 100mg CP/kg given ip (6) pretreatment (7 days) with 125mg C. tuberculata followed by 100 mg CP ip/kg; (7) pretreatment (7 days) with 250 mg C. tuberculata followed by 100 mg
§To whom correspondence should be addressed. Abbreviations: CP = cyclophosphamide; NCE = norrnochromatic erythrocyte; PCE = polychromatic erythrocyte.
719
720
A.M. AL-BEKAIR!et al.
CP ip/kg; (8) pretreatment with 500 mg C. tuberculata (7 days) followed by 100 mg CP ip/kg. CP (100 mg/kg body weight, group 5) was injected 30hr before animals were killed. In groups 6, 7 and 8, CP was injected simultaneously with the last dose of C. tuberculata. In each case animals were killed 30 hr after the last treatment and the femoral cells were collected in foetal calf serum. After centrifugation the cells were spread on slides and air-dried. Coded slides were fixed in methanol and stained with May--Gruenwald solution followed by Giemsa staining. The polychromatic erythrocytes (PCE, 1000 per mouse) were screened for micronuclei and reduction of the mitotic index was assessed on the basis of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio) (A1-Bekairi et al., 1991a). Biochemical procedures. The protein and nucleic acid contents of the liver, testes and brain were determined as follows. From each animal, the liver, testes and brain were rapidly excised, frozen in liquid nitrogen and stored at - 2 0 ° C until analysis of total protein and nucleic acid (RNA, DNA) content. Total protein was determined by the method of Lowry et al. (1951); the method described by Bregman (1983) was used to determine the levels of nucleic acids. Tissues were homogenized and the homogenate was suspended in ice-cold trichloroacetic acid (TCA). After centrifugation, the pellet was extracted with ethanol. D N A was determined by treating the nucleic acid extract with diphenylamine reagent and reading the intensity of blue colour at 600 rim. For quantification of RNA, the nucleic acid extract was treated with orcinol and the green colour was read at 660 nm. Standard curves were used to determine the amounts of nucleic acids present. The statistical analyses of the data was by Student's t-test. RESULTS
The results of this study are summarized in Tables 1 and 2. Treatment with C. tuberculata extract (groups 2, 3 and 4) resulted in a dose-related increase
in the percentage of micronuclei in the PCE of mice; however, there was not a statistically significant difference between these groups and the control (group 1). CP treatment (group 5) significantly increased (P < 0.001) the number of micronucleated PCE, but this effect of CP was significantly reduced in a dose-dependent manner by pretreatment with C. tuberculata (groups 6, 7 and 8). Treatment with C. tuberculata was found to cause a dose-dependent increase in mitodepression in the bone marrow cells of the animals (groups 2, 3 and 4) compared with the control. CP treatment (group 5) caused a significant reduction in the PCE/NCE ratio. The cytotoxicity of CP was further increased by pretreatment with C. tuberculata (groups 6, 7 and 8) (Table 1). The level of D N A in the liver was increased in a dose-dependent manner by treatment with C. tuberculata (groups 2, 3 and 4) whereas the R N A content was reduced; the effect on proteins was not significant. The R N A content of the testes was also significantly affected and reduced by C. tuberculata treatment; other effects were negligible. There was no effect on brain tissues. CP treatment (group 5) significantly reduced the DNA, R N A and protein contents of the liver, with a relatively insignificant effect on the testes and brain tissues, compared with the control. Pretreatment with C. tuberculata gave dose-dependent protection against the depletion of liver D N A and protein that was attributable to CP. However, the reduction in R N A content that was induced by CP was further increased in these groups in a dosedependent manner (groups 6, 7 and 8) (Table 2). DISCUSSION
In the study described here, treatment with C. tuberculata had a dose-dependent weakly mutagenic effect that was statistically non-significant. However, a highly significant and dose-dependent cytotoxic effect was observed in the extract-treated groups, compared with the control. These findings clearly indicated that C. tuberculata extract is highly cyto-
Table 1. Effect of Caralluma tuberculata on the frequency of micronuclei in bone marrow cells of mice
Group 1 2 3 4
5 6 7 8
Treatment and dose (mg/kg body weight) Control (distilled water) C. tuberculata (125) C. tuberculata (250) C. tuberculata (500) Cyclophosphamide (CP, 100) ip C. tuberculata (125) + C P (100) ip C. tuberculata (250) + C P (100) ip C. tuberculata (500) + C P (100) ip
Polychromatic erythrocytes screened
Micronucleated polychromatic erythrocytes (%) (mean _+SE)
Normochromatic erythrocytes screened
5996 5343 4793 4042
0.43 _+0.04 0.48 __.0.10 0.54 _+0.19 0.66 _ 0.14
5690 6119 7692 8000
1.10 _+0.09 0.88 + 0.03* 0.66 + 0.07** 0.51 _+0.05***
5053
6.72 + 0.73***
7085
0.73 + O.lO*
5171
4.20 + 0.67*
9407
0.56 + 0.08
4118
3.49 + 0.69*
13346
0.35 + 0.09*
5552
2.20 _+ 0.23***
14930
0.39 + 0.05*
PCE/NCE
Five mice were used in each group (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test). Groups 2, 3, 4 and 5 were statisticallycomparedwith group I; groups 6, 7 and 8 were statisticallycomparedwith group 5.
Effects of Caralluma tuberculata in mice
+I
+I
+I
+I
8
I+I oo
+I
~3 "d
Ie~
=.$ Vl-+l ~
-Iv-~ ~ I*
~ ~
I ~ - ~ ~, ~ I v ~i=I ~o =0
721
toxic but not clastogenic; CP treatment, on the other hand, showed significant cytotoxicity and was found to be highly clastogenic. The known cytotoxic potential of CP (AI-Bekairi et al., 1991) is thus confirmed by this study and the observed clastogenicity of CP may be attributed to its mutagenic and pro-oxidant properties (A1-Bekairi et al., 1991; Oleinik, 1985 and 1986). Pretreatment with C. tuberculata was found to increase the cytotoxicity of CP, apparently through the additive effect of both treatments. However, such pretreatment significantly reduced the clastogenicity of CP. Unlike treatment with CP (group 5), treatment with C. tuberculata significantly increased liver DNA levels in a dose-dependent manner whereas RNA contents were reduced and there was no significant effect on protein content. Pretreatment with C. tuberculata reduced the effect of CP on the DNA and protein contents of liver in a dose-dependent manner. However, the reduction of RNA content caused by CP treatment was further enhanced by pretreatment with C. tuberculata and increased cytotoxicity was noted. Among the chemical constituents of C. tuberculata are flavonoids and saponins (Ahmad et al., 1988; Rizwani et al., 1990; Tanaka et al., 1990). Dietary flavonoids are known to be involved in reducing RNA and increasing cytotoxicity and mutagenicity (Alldrick et al., 1989; Qureshi et al., 1991). With regard to the complex biochemical and cytotoxic effects of C. tuberculata described here, flavonoids may be implicated, at least in part. Cytotoxicity is usually associated with a large-scale breakdown of nucleic acids (Kaplan and Szabo, 1983). However, the results of the study reported here have indicated that, besides cytotoxic compounds, C. tuberculata contains some other phytoconstituents that may be involved in the protection of DNA and protein biosynthesis• C. tuberculata is a rich source of saponins (Ahmad et aL, 1988; Tanaka et al., 1990), which may be the cause of the effects observed because such compounds have been found to possess strong antimutagenic potential and are often believed to be responsible for the efficacy of herbal drugs (Elias et al., 1990; Guevara et al., 1990; Hiller, 1987; Shah et al., 1989; Shibata, 1977). It seems probable that the flavonoids and saponins of C. tuberculata affect the balance of the toxic species generated by CP (Al-Bekairi et al., 1991a) during its metabolism in liver, and their detoxification. Based on the fact that cytotoxicity and DNA damage may operate by independent mechanisms (Ochi and Cerutti, 1989), the cytotoxicity of C. tuberculata may not be associated with DNA synthesis. The results of this study show that pretreatment with C. tuberculata inhibits the clastogenicity induced by CP without impairing its cytotoxic potential. Further studies are required to elucidate the exact mode of action of its chemical constituents and to explore the protective role of C. tuberculata against genotoxic agents in the environment.
A.M. AL-BEKAIRIet al.
722 REFERENCES
Ahmad M. M. and Shaikh M. M. (1989) Improvement in glucose tolerance by Caralluma tuberculata, Acacia nilotica and Papaversomniferum. Pakistan Journal of Zoology 21, 325-332. Ahmad V. U., Usmanghani K. and Rzwani G. H. (1988) New pregnane glycosides from Caralluma tuberculata. Journal of Natural Products 51, 1092-1097. Akira T., Tanaka S. and Tabata M. (1986) Pharmacological studies on the anti-ulcerogenic activity of Chinese Cinnamon. Planta Medica 6, 440-443. A1-Bekairi A. M., Qureshi S., Chaudhry M. A. and Shah A. H. (1991a) Uric acid as inhibitor of cyclophosphamideinduced micronuclei in mice. Mutation Research 262, 115-118. AI-Bekairi A. M., Qureshi S. and Shah A. H. (1991b) Toxicity studies on Allium cepa, its effect on estradioltreated mice and epididymal spermatozoa. Fitoterapia 62, 301-306. Ali S. I. (1983) Asclepiadaceae. In Flora of Pakistan. Vol. 150. Edited by E. Nasir and S. I. Ali. pp. 48-49. Shamim Printing Press, Karachi. Alldrick A. J., Lake B. G. and Rowland I. R. (1989) Modification of heterocyclic amine genotoxicity by dietary fiavonoids. Mutagenesis 4, 365-370. Bregman A. A. (1983) Laboratory Investigations and Cell Biology. pp. 51~0. John Wiley & Sons, New York. Chopra R. N., Nayar S. L. and Chopra I. C. (1956) Glossary of Indian Medicinal Plants. p. 51. Council of Scientific & Industrial Research, New Delhi. Elias R., Meo M. De., Vidal-Ollivier E., Laget M., Balansard G. and Dumenil G. (1990) Antimutagenic activity of some saponins isolated from Calandula o~cinalis L., C. arvensis L. and Hedera helix L. Mutagenesis 5, 327-331. Guevara A. P., Lim-Sylianco C., Dayrit F. and Finch P. (1990) Antimutagens from Momordica charantia. Mutation Research 230, 121-126. Hiller K. (1987) New results on the structure and biological activity of terpenoid saponins. In Annual Proceedings of the Phytochemical Society of Europe. Biologically Active Natural Products. Edited by K. Hostettman and P. J. Lea. pp. 167-184. Clarendon Press, Oxford. Kaplan A. and Szabo L. L. (1983) Clinical Chemistry: Interpretation and Techniques. 2nd Ed. pp. 138-145. Lea and Febiger, Philadelphia.
Kutten R., Bhanumathy P., Nirmala K. and George M. C. (1985) Potential anticancer activity of turmeric (Curcuma longa). Cancer Letters 29, 197-202. Lowry O. H., Rosebrough N. S., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Mohsin A., Shah A. H., Al-Yahya M. A., Tariq M., Tanira M. O. M. and Ageel A. M. (1989) Analgesic, antipyretic activity and phytochemical screening of the plants used in the traditional Arab system of medicine. Fitoterapia 2, 174-179. Ochi T. and Cerutti P. A. (1989) Differential effects of glutathione depletion and metallothionein induction on the induction of DNA in cultured mammalian cells. Chemico-Biological Interactions 72, 335-345. Oleinik A. V. (1985) Effect of cyclophosphane on lipid peroxidation. Voprosy Onkologie 31, 97-101. Oleinik A. V. (1986) Effect of cyclophosphane on bile formation and lipid peroxidation in the liver. Farmakologiya i Toksikologiya 49, 79-91. Qureshi S., Shah A. H., AI-Yahya M. A. and Ageel A. M. (1991) Toxicity of Achillea fragrantissima and Thymus vulgaris in mice. Fitoterapia 62, 319-323. Rizwani G. H., Usmanghani K., Ahmad M. and Ahmad V. U. (1990) Flavone glycosides of Caralluma tuberculata N.E. Brown. Pakistan Journal of Pharmaceutical Science 3, 27-32. Shah A. H., A1-Bekairi A. M., Qureshi S. and Ageel A. M. (1989) Zizyphus sativa fruits: evaluation of some biological activities and toxicity. Phytotherapy Research 3, 232-236. Shibata S. (1977) Saponins with biological and pharmacological activity. In New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutic Activity. Edited by H. Wagner and P. Wolf. pp. 177-196. Springer-Verlag, Berlin. Tanaka T., Tsukamoto S. and Hayashi K. (1990) Pregnane glycosides of Boucerosia aucheriana. Phytochemistry 29, 229-237. Tanira M. O. M., Ageel A. M., Tariq M., Mohsin A. and Shah A. H. (1988) Evaluation of some pharmacological, antimicrobial and physical properties of Zizyphus spinachristi. International Journal of Crude Drug Research 26, 56~0. Vincenzi M. De and Dessi M. R. (1991) Botanical flavouring substances used in foods: proposal of classification. Fitoterapia 62, 39-46.
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
EFFECT OF CARALLUMA TUBERCULATA ON THE CYTOLOGICAL A N D BIOCHEMICAL CHANGES INDUCED BY CYCLOPHOSPHAMIDE IN MICE A. M. AL-BEKAIRI*,S.QuRESHI*, M. M. AHMED*, N. S. QAZIt, Z. A. KHANt and A. H. SHAH~§ *Quality Control and Research Laboratory, Experimental Animal Care Centre, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, tGomal University, D. I. Khan, Pakistan and :~Central Laboratory for Drug and Food Analysis, M.O.H., PO Box 59082, Riyadh-l1525, Saudi Arabia (Accepted 27 March 1992)
Abstract--Treatment with Caralluma tuberculata extract induced complex biochemical and cytological changes in mice. Its cytotoxicity in the bone marrow cells of mice was comparable with that of the standard drug cyclophosphamide (CP); however, unlike CP, C. tuberculata was not clastogenic (as shown by the micronucleus assay). A dose-dependent decrease in the RNA content of liver and testes was produced by C. tuberculata treatment whereas there was no effect on the content of nucleic acid and protein in the brain. In the extract-treated animals there was a significant and dose-dependent increase in the DNA content of the liver, with a negligible effect on the protein content. Combined treatment with C. tuberculata and CP showed that C. tuberculata diminished the effect of CP on DNA levels; however, RNA levels were further suppressed, resulting in increased cytotoxicity. Pretreatment with C. tuberculata extract significantly reduced the clastogenicity of CP. These results indicated the involvement of different phytoconstituents acting by different routes.
INTRODUCTION
MATERIALSAND METHODS
Recent reports have suggested that plants used as food and spices may have various types of pharmacological activity and toxicity (Akira et al., 1986; AI-Bekairi et al., 1991b; Kutten et al., 1985; Vincenzi and Dessi, 1991). Caralluma tuberculata N.E. Brown (Asclepiadaceae) is a plant widely grown in the Punjab, North-West Frontier and Balochistan Provinces of Pakistan. It is popularly known as 'Chungan' and is consumed as food. For medicinal purposes C. tuberculata is widely used in the treatment of rheumatism, diabetes, leprosy and blood disorders, and as an antipyretic alternative and anthelmintic (Ahmad and Shaikh, 1989; Ali, 1983; Chopra et al., 1956). This plant has been reported to contain flavonoids and much saponin (Ahmad et al., 1988; Rizwani et al., 1990; Tanaka et al., 1990). However, there is limited evidence in support of its claims in popular medicine. Bearing in mind the widepsread use of C. tuberculata by the local population the present study was designed to evaluate some of its cytological and biochemical effects in mice. This study also deals with the effect of C. tuberculata on cyclophosphamide (CP) treatment in mice.
Plant extract. Caralluma tuberculata N.E. Brown was collected fresh from the local vegetable market in Dera Ismail Khan, Pakistan and a voucher specimen was kept on record in the Department of Pharmacognosy, Faculty of Pharmacy, Gomal University, Pakistan. The plant was cut into small pieces and extracted with 96% ethanol in a soxhlet apparatus. The solvent was removed under reduced pressure; the dark green semi-solid residue thus obtained was refrigerated until required (Mohsin et al., 1989). Animal stock. A total of 40 male mice (SWR, home bred), aged 6-8 wk weighing 25-30 g were used. The animals were randomly assigned to different control and treatment groups (five animals in each group) that were maintained under standard conditions of humidity, temperature and light (12hr light/12hr dark). The animals were fed with purina chow diet and had free access to water. A dose of 500 mg/kg body weight was used as the highest dose of the extract (Tanira et al., 1988). Unless otherwise specified, a fresh aqueous suspension of the plant extract was prepared and administered orally (gavage) to the animals for 7 days. The experimental groups of mice were as follows: (1) untreated control (distilled water); (2) 125 mg C. tuberculata/kg/day; (3) 250mg C. tuberculata/kg/day; (4) 500mg C. tuberculata/kg/day; (5) 100mg CP/kg given ip (6) pretreatment (7 days) with 125mg C. tuberculata followed by 100 mg CP ip/kg; (7) pretreatment (7 days) with 250 mg C. tuberculata followed by 100 mg
§To whom correspondence should be addressed. Abbreviations: CP = cyclophosphamide; NCE = norrnochromatic erythrocyte; PCE = polychromatic erythrocyte.
719
720
A.M. AL-BEKAIR!et al.
CP ip/kg; (8) pretreatment with 500 mg C. tuberculata (7 days) followed by 100 mg CP ip/kg. CP (100 mg/kg body weight, group 5) was injected 30hr before animals were killed. In groups 6, 7 and 8, CP was injected simultaneously with the last dose of C. tuberculata. In each case animals were killed 30 hr after the last treatment and the femoral cells were collected in foetal calf serum. After centrifugation the cells were spread on slides and air-dried. Coded slides were fixed in methanol and stained with May--Gruenwald solution followed by Giemsa staining. The polychromatic erythrocytes (PCE, 1000 per mouse) were screened for micronuclei and reduction of the mitotic index was assessed on the basis of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio) (A1-Bekairi et al., 1991a). Biochemical procedures. The protein and nucleic acid contents of the liver, testes and brain were determined as follows. From each animal, the liver, testes and brain were rapidly excised, frozen in liquid nitrogen and stored at - 2 0 ° C until analysis of total protein and nucleic acid (RNA, DNA) content. Total protein was determined by the method of Lowry et al. (1951); the method described by Bregman (1983) was used to determine the levels of nucleic acids. Tissues were homogenized and the homogenate was suspended in ice-cold trichloroacetic acid (TCA). After centrifugation, the pellet was extracted with ethanol. D N A was determined by treating the nucleic acid extract with diphenylamine reagent and reading the intensity of blue colour at 600 rim. For quantification of RNA, the nucleic acid extract was treated with orcinol and the green colour was read at 660 nm. Standard curves were used to determine the amounts of nucleic acids present. The statistical analyses of the data was by Student's t-test. RESULTS
The results of this study are summarized in Tables 1 and 2. Treatment with C. tuberculata extract (groups 2, 3 and 4) resulted in a dose-related increase
in the percentage of micronuclei in the PCE of mice; however, there was not a statistically significant difference between these groups and the control (group 1). CP treatment (group 5) significantly increased (P < 0.001) the number of micronucleated PCE, but this effect of CP was significantly reduced in a dose-dependent manner by pretreatment with C. tuberculata (groups 6, 7 and 8). Treatment with C. tuberculata was found to cause a dose-dependent increase in mitodepression in the bone marrow cells of the animals (groups 2, 3 and 4) compared with the control. CP treatment (group 5) caused a significant reduction in the PCE/NCE ratio. The cytotoxicity of CP was further increased by pretreatment with C. tuberculata (groups 6, 7 and 8) (Table 1). The level of D N A in the liver was increased in a dose-dependent manner by treatment with C. tuberculata (groups 2, 3 and 4) whereas the R N A content was reduced; the effect on proteins was not significant. The R N A content of the testes was also significantly affected and reduced by C. tuberculata treatment; other effects were negligible. There was no effect on brain tissues. CP treatment (group 5) significantly reduced the DNA, R N A and protein contents of the liver, with a relatively insignificant effect on the testes and brain tissues, compared with the control. Pretreatment with C. tuberculata gave dose-dependent protection against the depletion of liver D N A and protein that was attributable to CP. However, the reduction in R N A content that was induced by CP was further increased in these groups in a dosedependent manner (groups 6, 7 and 8) (Table 2). DISCUSSION
In the study described here, treatment with C. tuberculata had a dose-dependent weakly mutagenic effect that was statistically non-significant. However, a highly significant and dose-dependent cytotoxic effect was observed in the extract-treated groups, compared with the control. These findings clearly indicated that C. tuberculata extract is highly cyto-
Table 1. Effect of Caralluma tuberculata on the frequency of micronuclei in bone marrow cells of mice
Group 1 2 3 4
5 6 7 8
Treatment and dose (mg/kg body weight) Control (distilled water) C. tuberculata (125) C. tuberculata (250) C. tuberculata (500) Cyclophosphamide (CP, 100) ip C. tuberculata (125) + C P (100) ip C. tuberculata (250) + C P (100) ip C. tuberculata (500) + C P (100) ip
Polychromatic erythrocytes screened
Micronucleated polychromatic erythrocytes (%) (mean _+SE)
Normochromatic erythrocytes screened
5996 5343 4793 4042
0.43 _+0.04 0.48 __.0.10 0.54 _+0.19 0.66 _ 0.14
5690 6119 7692 8000
1.10 _+0.09 0.88 + 0.03* 0.66 + 0.07** 0.51 _+0.05***
5053
6.72 + 0.73***
7085
0.73 + O.lO*
5171
4.20 + 0.67*
9407
0.56 + 0.08
4118
3.49 + 0.69*
13346
0.35 + 0.09*
5552
2.20 _+ 0.23***
14930
0.39 + 0.05*
PCE/NCE
Five mice were used in each group (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test). Groups 2, 3, 4 and 5 were statisticallycomparedwith group I; groups 6, 7 and 8 were statisticallycomparedwith group 5.
Effects of Caralluma tuberculata in mice
+I
+I
+I
+I
8
I+I oo
+I
~3 "d
Ie~
=.$ Vl-+l ~
-Iv-~ ~ I*
~ ~
I ~ - ~ ~, ~ I v ~i=I ~o =0
721
toxic but not clastogenic; CP treatment, on the other hand, showed significant cytotoxicity and was found to be highly clastogenic. The known cytotoxic potential of CP (AI-Bekairi et al., 1991) is thus confirmed by this study and the observed clastogenicity of CP may be attributed to its mutagenic and pro-oxidant properties (A1-Bekairi et al., 1991; Oleinik, 1985 and 1986). Pretreatment with C. tuberculata was found to increase the cytotoxicity of CP, apparently through the additive effect of both treatments. However, such pretreatment significantly reduced the clastogenicity of CP. Unlike treatment with CP (group 5), treatment with C. tuberculata significantly increased liver DNA levels in a dose-dependent manner whereas RNA contents were reduced and there was no significant effect on protein content. Pretreatment with C. tuberculata reduced the effect of CP on the DNA and protein contents of liver in a dose-dependent manner. However, the reduction of RNA content caused by CP treatment was further enhanced by pretreatment with C. tuberculata and increased cytotoxicity was noted. Among the chemical constituents of C. tuberculata are flavonoids and saponins (Ahmad et al., 1988; Rizwani et al., 1990; Tanaka et al., 1990). Dietary flavonoids are known to be involved in reducing RNA and increasing cytotoxicity and mutagenicity (Alldrick et al., 1989; Qureshi et al., 1991). With regard to the complex biochemical and cytotoxic effects of C. tuberculata described here, flavonoids may be implicated, at least in part. Cytotoxicity is usually associated with a large-scale breakdown of nucleic acids (Kaplan and Szabo, 1983). However, the results of the study reported here have indicated that, besides cytotoxic compounds, C. tuberculata contains some other phytoconstituents that may be involved in the protection of DNA and protein biosynthesis• C. tuberculata is a rich source of saponins (Ahmad et aL, 1988; Tanaka et al., 1990), which may be the cause of the effects observed because such compounds have been found to possess strong antimutagenic potential and are often believed to be responsible for the efficacy of herbal drugs (Elias et al., 1990; Guevara et al., 1990; Hiller, 1987; Shah et al., 1989; Shibata, 1977). It seems probable that the flavonoids and saponins of C. tuberculata affect the balance of the toxic species generated by CP (Al-Bekairi et al., 1991a) during its metabolism in liver, and their detoxification. Based on the fact that cytotoxicity and DNA damage may operate by independent mechanisms (Ochi and Cerutti, 1989), the cytotoxicity of C. tuberculata may not be associated with DNA synthesis. The results of this study show that pretreatment with C. tuberculata inhibits the clastogenicity induced by CP without impairing its cytotoxic potential. Further studies are required to elucidate the exact mode of action of its chemical constituents and to explore the protective role of C. tuberculata against genotoxic agents in the environment.
A.M. AL-BEKAIRIet al.
722 REFERENCES
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