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|Year : 2006 | Volume
| Issue : 3 | Page : 185--189
Anticarcinogenic and antilipidperoxidative effects of Tephrosia purpurea (Linn.) Pers. in 7, 12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinoma
K Kavitha, S Manoharan
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamil Nadu, India
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamil Nadu
Objectives: To investigate the chemopreventive potential and antilipidperoxidative effects of ethanolic root extract of Tephrosia purpurea (Linn.) Pers. (TpEt) on 7,12-dimethylbenz(a)anthracene (DMBA)- induced hamster buccal pouch carcinoma.
Materials and Methods: Oral squamous cell carcinoma was developed in the buccal pouch of Syrian golden hamsters, by painting with 0.5% DMBA in liquid paraffin, thrice a week, for 14 weeks. The tumor incidence, volume and burden were determined. Oral administration of TpEt at a dose of 300 mg/kg, b.w., to DMBA (on alternate days for 14 weeks)- painted animals significantly prevented the incidence, volume and burden of the tumor.
Results: TpEt showed potent antilipidperoxidative effect, as well as enhanced the antioxidant status in DMBA- painted animals.
Conclusion: TpEt has potent chemopreventive efficacy and significant antilipidperoxidative effect, in DMBA-induced oral carcinogenesis. Further studies are needed to isolate and characterize the bioactive principle.
|How to cite this article:|
Kavitha K, Manoharan S. Anticarcinogenic and antilipidperoxidative effects of Tephrosia purpurea (Linn.) Pers. in 7, 12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinoma.Indian J Pharmacol 2006;38:185-189
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Kavitha K, Manoharan S. Anticarcinogenic and antilipidperoxidative effects of Tephrosia purpurea (Linn.) Pers. in 7, 12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinoma. Indian J Pharmacol [serial online] 2006 [cited 2020 Jan 28 ];38:185-189
Available from: http://www.ijp-online.com/text.asp?2006/38/3/185/25805
Oral squamous cell carcinoma, the fifth most common malignancy worldwide, is predominantly a disease of men, in the fifth to eighth decades of life. While it accounts for 3-4% of all malignancies in western countries, India has recorded the highest incidence, accounting about 30 - 40% of all cancers. Cancer of the oral cavity are frequently associated with chewing of betel quid containing tobacco, in addition to smoking and alcohol consumption. 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis, is a well suited model for studying precancerous and cancerous lesions of human oral squamous cell carcinoma, since it is morphologically and histologically similar to human tumors, as well as, it expresses many biochemical and molecular markers that are expressed in humans. 
Lipid peroxidation, a potent reactive oxygen species (ROS)- mediated chain reaction, has been implicated in the pathogenesis of several disorders, including oral carcinoma. Overproduction of reactive oxygen species within tissues can damage DNA and possibly contribute to mutagenesis and carcinogenesis. However organisms have an array of potent adaptive antioxidant defense mechanisms [enzymatic antioxidants: superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and non-enzymatic antioxidants: reduced glutathione (GSH), vitamin C and vitamin E] within the cells, to combat the deleterious effects of reactive oxygen species- mediated oxidative damage.
Cancer chemoprevention is a new promising strategy for prevention, inhibition or reversal of carcinogenesis, induced by specific natural or synthetic chemicals. For a large number of the world's rural population, medicinal plants are the only source for the prevention and treatment of various pathological diseases, from time immemorial. Several medicinal plants and their constituents have been reported, to prevent multistage carcinogenesis.
Tephrosia purpurea is a wild plant known as "Sarapunkha" in Sanskrit, 'Purple tephrosia' or 'Wild indigo' in English and "Avuri" or "Kolinji" in Tamil. T. purpurea has been used for centuries in the Indian traditional medicine, for the treatment of various inflammatory disorders. It is considered beneficial for liver, spleen and kidney disorders. Also, it has the property to cure all types of wounds. Experimental studies suggest that T. purpurea exerts antiulcer and antitumor promoting effects.,
To our best knowledge, there is no scientific report on the chemopreventive efficacy and antilipidperoxidative effects of T. purpurea in DMBA-induced, hamster buccal pouch carcinogenesis. Hence, the present study is designed to evaluate the effectiveness of ethanolic root extract of Tephrosia purpurea (TpEt) in modifying the carcinogenic process, as well as oxidative alterations in DMBA- induced hamster buccal pouch carcinoma.
Materials and Methods
DMBA was obtained from Sigma-Aldrich Chemical Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade.
Male, golden Syrian hamsters 8-10 weeks old, weighing 80-120 g, were purchased from National Institute of Nutrition, Hyderabad, India and maintained in the Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai University. The animals were housed in groups of four or five in polypropylene cages and provided standard pellet diet and water ad libitum and maintained under controlled conditions of temperature and humidity, with a 12 h light/dark cycle.
Tephrosia purpurea was collected in and around Annamalai University, Annamalai Nagar, Tamil Nadu, India, authenticated by Botanist, R. Panneer Selvam, Department of Botany, Annamalai University and a voucher specimen (AUO5102) was also deposited.
Preparation of plant extract
500 g of dried finely powdered T. purpurea roots were soaked with 1500 ml of 95% ethanol, overnight. The residue obtained was again resuspended in equal volume of 95% ethanol for 48 h and filtered again. The above two filtrates were mixed and the solvents were evaporated in a rotovapour at 40-50°C, under reduced pressure. A dark semisolid material (13%) obtained, was stored at -4°C, until use. For experimental studies, a known volume of the extract was suspended in distilled water and orally administered to the experimental animals by gastric intubation, using force-feeding needle.
The institutional animal ethics committee approved the experimental design. The hamsters were randomized into four groups of 10 animals each. Group I served as untreated control. Groups II and III were painted with 0.5% DMBA in liquid paraffin thrice a week, for 14 weeks on the left buccal pouches. Group II received no other treatment. Group III were administered TpEt (300 mg/kg, b.w.) orally starting 1 week before the exposure to the carcinogen and continued on days alternate to DMBA painting, until the animals are sacrified. Group IV received oral TpEt, only throughout the experimental period. The experiment was terminated at the end of 14 weeks and all the animals were sacrificed by cervical dislocation. Biochemical studies were conducted on blood and buccal mucosa of control and experimental animals, in each group. For histopathological examination, buccal mucosal tissues were fixed in 10% formalin and embedded with paraffin, 2-3 µm sections were cut in a rotary microtome and stained with haematoxylin and eosin.
The erythrocyte membrane was prepared by the method of Dodge et al, modified by Quist. Thiobarbituricacid reactive substances (TBARS) was assayed in plasma, erythrocytes and buccal mucosa, according to the methods of Yagi, Donnan and Ohkawa et al, respectively. Reduced glutathione (GSH) was determined by the method of Beutler and Kelley. Vitamin C and E were measured according to the methods of Omaye et al and Desai, respectively. The enzymatic antioxidant activities were estimated by the methods of Kakkar et al , (SOD), Sinha (CAT) and Rotruck et al  (GPx), respectively.
Values are expressed as mean±SD. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT). The values were considered statistically significant, if P was less than 0.05.
[Table 1] depicts the effect of TpEt on the incidence, volume and burden of tumor in DMBA- induced hamster buccal pouch carcinoma. In DMBA- painted hamsters (Group II), a 100% tumor formation with mean tumor volume (472 mm 3) and tumor burden (2029 mm 3), was observed. Oral TpEt (300 mg/kg, b.w.) significantly prevented the incidence, volume and burden of tumor in DMBA- painted hamsters (Group III). No tumor was observed in control (Group I) as well as TpEt alone- treated animals (Group IV).
[Table 2] shows the histopathological features of control and experimental animals in each group. A myriad of histopathological changes (severe keratosis, hyperplasia, dysplasia and squamous cell carcinoma of the epithelium), were observed in hamsters painted with DMBA alone (Group II). A mild to moderate preneoplastic lesions [hyperplasia (++), keratosis (+) and dysplasia (+)], were noticed in Group III animals (DMBA + TpEt).
[Table 3] and [Table 4] show the status of TBARS and antioxidant in plasma and erythrocytes of the control and experimental groups. The concentration of TBARS was increased, whereas the levels of nonenzymatic antioxidants (GSH, Vitamin C and Vitamin E) and activities of enzymatic antioxidants (SOD, CAT and GPx), were significantly decreased in group II (DMBA alone), as compared to control animals. Oral administration of TpEt significantly decreased the levels of TBARS and improved the antioxidants status in DMBA- painted hamsters. TpEt alone- treated hamsters showed no significant difference in TBARS and antioxidants status, as compared to control animals.
[Table 5] indicates the concentration of TBARS and antioxidants status, in the buccal mucosa of all animal groups. Decrease in TBARS concentration and alterations in the antioxidant status (Vitamin E, GSH and GPx were increased; SOD and CAT were decreased), were noticed in cancer animals (Group II) as compared to control (Group I). However oral administration of TpEt (Group III), reverted the concentration of TBARS and antioxidants to near normal range in DMBA- painted animals.Hamsters treated with TpEt alone (Group IV) showed no significant difference in TBARS and antioxidants status, as compared to control animals.
The incidence and mortality rates of oral cancer vary widely across the world, but the highest rates were reported every year from developing countries, particularly from India. Oral cancer is one among the few human cancers, with the vast potential for prevention. A major target of reactive oxygen species is cell membrane, due to the high content of polyunsaturated fatty acids. Reactive oxygen species mediated lipid peroxidation causes damage to cellular DNA, membrane structure and inhibition of functions several enzymes and alterations in the immune system. In cancer, enormous production of free radicals in the system has been reported. A close relationship between free radical activity and neoplastic transformation, has been shown. Antioxidants (enzymatic and non-enzymatic) play a vital role in scavenging reactive oxygen species and protect the cells from oxidative damage. Vitamin E, Vitamin C and reduced glutathione, are primary defense antioxidants. They provide protection against several reactive oxygen species, development of cancer and other oxidative stress- mediated dysfunctions. SOD, CAT and GPx, are important antioxidant enzymes that protect cell and tissue damage from enhanced lipid peroxidation, via elimination of reactive oxygen species.
Elevated lipid peroxidation and poor antioxidant systems, have been reported in oral cancer patients. Sabitha and Shyamaladevi have suggested, that a lack of antioxidant defense is responsible for the elevated lipid peroxidation in erythrocytes. Altered activities of enzymatic antioxidants are reported during carcinogenesis or after tumor formation. Hence, the elevated lipid peroxidation in the circulation of cancer animals, is due to poor antioxidant defense mechanism. Lowered nonenzymatic antioxidants (Vitamin C, Vitamin E and GSH) in the circulation, are probably due to elevated lipid peroxidation or sequestration by the tumor tissues for their rapid growth.
Diminished lipid peroxidation and disturbed antioxidants status (GSH and GPx were increased, whereas SOD and CAT were decreased), were observed in tumor tissues, as compared to normal tissues. Cancer cells showed decreased susceptibility to lipid peroxidation, compared to normal tissues. An inverse relationship between the lipid peroxidation process and the rate of cell proliferation, has been reported. Glutathione, a biologically important tripeptide, is essential for maintaining cell integrity, due to its reducing properties and participation in the cell metabolism. Glutathione peroxidase and its co-substrate glutathione, are reported to have regulatory effects on cell proliferation. Elevated glutathione and GPx activity, have been demonstrated in several tumor tissues, including oral cancer. Subapriya et al  have postulated that diminished lipid peroxidation combined with enhanced glutathione-dependent antioxidant capacity of oral tumors, facilitates cell- proliferation, offering selective growth advantage in tumor cells over their surrounding normal cells. Our results lend credibility to the above observations. Decrease in SOD and catalase activities described in tumors, is regarded as markers of malignant transformation. Lowered activities of SOD and CAT were reported in several cancers, including oral cavity cancer. , Our results corroborate these observations.
The painting of 0.5% DMBA in liquid paraffin thrice a week, for 14 weeks to the hamster buccal pouch, induced oral carcinoma. We have also noticed the precancerous lesions in DMBA- painted hamsters, from the 10th week of the experimental period. In the present study oral administration of TpEt at a dose of 300 mg/kg, b.w., reduced tumor incidence, volume, burden and the number in DMBA- painted hamsters. Our results thus indicate, that TpEt possess significant chemopreventive potential against DMBA- induced buccal pouch carcinoma.
Furthermore, TpEt significantly reduced the levels of TBARS and enhanced the status of antioxidants in the circulation of DMBA- painted hamsters. We also noticed an elevation of TBARS level and improvement in antioxidant defense system in the buccal mucosa of DMBA-painted hamsters, after treatment with TpEt. Johnson reported that the chemopreventive properties of plant anticarcinogens are either due to antilipidperoxidative action, modulating carcinogen detoxification or by improving the antioxidant defense system. Sharma et al, have proposed that several medicinal plants and their constituents, probably exert their chemopreventive effect, by scavenging reactive oxygen species and improving the antioxidant defense systems. The present study reveals, that the chemopreventive effect of TpEt in DMBA- painted animals, is probably due to its antilipidperoxidative and antioxidant properties.
The authors express sincere thanks to Dr. C.R. Ramachandran, Dean, Faculty of Dentistry and Dr. A. Thangavelu, Professor, Faculty of Dentistry, Annamalai University, Annamalai Nagar for their valuable support and help for this study.
|1||Moore SR, Johnson NW, Pierce A.M, Wilson DF. The epidemiology of mouth cancer: A review of global incidence. Oral Dis 2000;6:65-74.|
|2||Boring CC, Squire TS, Tong T, Montgomery S. Cancer statistics. Calif. Cancer J Clin 1994;44:7-26. |
|3||Ray G, Husain SA. Oxidant, antioxidants and carcinogenesis. Indian J Exp Biol 2002;40:1213-32.|
|4||Hans peter W, Deuter M, Imilda E. Chemoprevention of tobacco smoke induced lung carcinogenesis in mice. Carcinogenesis 2000;21:977-82. |
|5||Craig WJ. Health promoting properties of common herbs. Am J Clin Nutr 1999; 70:491-9.|
|6||Joshi SG. Oleaceae. In: Medicinal Plants. New Delhi: Oxford and IBH publishing Co. Pvt. Ltd; 2000. p. 211.|
|7||Saleem M, Ahmed S, Alam A, Sultana S. Tephrosia purpurea alleviates phorbol ester-induced tumor promotion response in murine skin. Pharm Pharmacol Comm 1999;5:455-61. |
|8||Deshpande SS, Shah GB, Parmar NS. Antiulcer activity of Tephrosia purpurea in rats. Indian J Pharmacol 2003;35:631-7.|
|9||Dodge JF, Michell G, Hanahan DJ. The preparation of haemoglobin free ghosts of human red blood cells. Arch Biochem Biophy 1968;110:119-30.|
|10||Quist EH. Regulation of erythrocyte membrane shape by calcium ion. Biochem Biophys Res Commun 1980;92:631-7.|
|11||Yagi K. Lipid peroxides and human disease. Chem Physiol lipids 1978;45: 337-51.|
|12||Donnan SK. The thiobarbituric acid test applied to tissues from rats treated in various way. J Biol Chem 1950;182:415-9.|
|13||Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8.|
|14||Beutler E, Kelley BM. The effect of sodium nitrate on RBC glutathione. Experimentia 1963;29:96-7.|
|15||Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. In: Mc Cormick DB, Wright DC, editors. New York: Methods in enzymology Academic press; 1979. |
|16||Desai FD. Vitamin E analysis methods for animal tissues. Feischer S, Packer L, editors. New York: Methods in enzymology, Academic press; 1984.|
|17||Kakkar D, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophy 1984;21:130-2. |
|18||Sinha KA. Colorimetric assay of catalase. Anal Biochem 1972;47:389-94. |
|19||Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179:588-90. |
|20||Thirunavukkarasu C, Princevijeyasingh J, Selvendiran K, Sakthisekaran D. Chemopreventive efficacy of selenium against DEN-induced hepatoma in albino rats. Cell Biochem Funct 2001;19:265-71. |
|21||McCord JM. The evolution of free radicals and oxidative stress. Am J Med 2000;108:652.|
|22||Sabitha KE, Shyamladevi CS. Oxidant and antioxidant activity changes in patients with oral cancer and treated with radiotherapy. Oral Oncology 1999; 35:273-7.|
|23||Subapriya R, Kumaraguruparan R, Rama chandran CR, Nagini S. Oxidant - antioxidant status in patients with oral squamous cell carcinomas at different intraoral sites. Clin Biochem 2002;35:489-93. |
|24||Johnson IT. Plant anticarcinogens. Eur J cancer Prev 1997;6:515-7. |
|25||Sharma S, Stutzman Id, Kelloff GJ, Steele VE. Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res 1994:54:5848-55.|