Cytotoxic effect of methanol extract of Conyza bonariensis on DMBA-induced skin carcinogenesis: An in vivo study
Abstract
In the present study, we examined the cytotoxic effect of Conyza bonariensis (methanolic extract). The skin carcinogenesis was induced in two stages, first, applying tumor initiator, 7-12-dimethyl benz(α)antheracene and thereafter applying croton oil, a tumor promotor in Swiss albino mice. The morphological alterations observed and measured during the induction of skin ulceration, included; cumulative number of papilloma, tumor yield and tumor burden. C. bonariensis extract (300 and 600 mg/kg/day) was applied locally on mice skin for 16 weeks. The higher dose (600 mg/kg/day) inhibited the tumor formation up to 40% and showed a significant decline in cumulative number of papilloma of continuous group. The results indicated that extract increased the reduced glutathione, superoxide dismutase and catalase, and decreased lipid peroxidation compared to carcinogen group. Histopathological changes showed papilomatosis and ulceration in carcinogen control group. HPLC analysis indicated the presence of flavonoid i.e. quercetin which may be responsible for the cytotoxic action of C. bonariensis methanol extract.
Introduction
Cancer is emerging as one of the most horrific disease and almost all of the anticancer drugs available in the market have serious side effects. So, it is direly needed to explore new anticancer agents from plants which can effectively kill cancer cells without damaging normal body cells.
Many plants like Convolvulous arvensis (Saleem at al, 2014), Catharanthus roseus (Cragg and Newman, 2005; Okouneva et al., 2003; Simeons et al., 2008), Podophyllum peltatum and Podophyllum emodi (Shoeb et al., 2006), Taxus brevifolia Nutt, Taxus baccata (Kingston, 2007; Hait et al., 2007), Camptotheca acuminate (Zhang et al., 2004, Fuchs et al., 2006), Berberis amarensis (Xie et al., 2009; Xu et al., 2006), Hvdrastis canadensis L (Wang et al., 2011; Patil et al., 2010), Tabebuia avellanedae (Li et al., 2000; De Almeida, 2009), Betula alba (Fulda, 2008), Colchicum autumnale (Dubey et al., 2008), Curcuma longa (Sa et al., 2010; Goel et al., 2008), Wikstroemia indica (Lu et al. 2011; Diogo et al., 2009), Psoralea corylifolia (Moon et al., 2006; Dixon and Ferreira, 2002), Ochrosia elliptica (Kuo et al., 2006), Amoora rohituka and Dysoxylum binectariferum (Mans et al., 2000), Euphorbia peplus L. (Hampson et al., 2005), Ipomoeca batatas L (Ancuceanu and Istudor, 2004), Salvia prionitis (Deng et al., 2011), Centaurea schischkinii (Shoeb et al., 2005) have been known to possess anticancer activity.
In the present study Conyza bonariensis was selected to evaluate its cytotoxic efects on DMBA induced skin carcinogenesis. C. bonariensis is a cosmopolitan plant and belongs to family Asteraceae (Compositeae). It is used in fungal and bacterial infections (Chaudhry et al., 2001), hepatic toxicity and gastro enteritis, diarrhea, leucorrhoea, menorrhagia. It possesses anticoagulant (Favila and Antonio, 2006), antioxidant (Shahwar et al., 2012), homeostatic, tonic, astringent (Ahmad, 2007), cholinergic (Khan et al., 2006), anti-inflammatory and antimitotic activities (Santana et al., 2011). The following study indicated that methanol extract of C. Bonariensisis possesses protective effects against skin carcinogenesis in Swiss albino mice.
Materials and Methods
Collection of plant
Whole plants of C. bonariensis were collected during April and May from both sides of motorway M2 from Lahore to Islamabad and were identified by a plant taxonomist Dr. Mansoor Hameed, head of Botany Department, Agriculture University, Faisalabad.
Preparation of plant extract
The aerial parts were washed, chopped and dried under shade at room temperature for several days until fully dried, ground by electric grinder, powdered and sieved. The powdered material was macerated in methanol for 7 days with frequent shaking every day, filtered out by using Whatman filter paper. Finally, the solvent from solid material was removed by using a rotary evaporator at 45-55°C and residues obtained were stored in small amber jars at 4°C.
Drug/chemical
Carcinogen, 7-12-dimethyl benz(a)antheracene (DMBA) and croton oil obtained from Sigma-Aldrich Chemical Company USA. Acetone was used as a vehicle for all topically applied carcinogens and dilution of plant extract and methanol (analytical grade) were purchased from Asian scientific store, Jinnah colony, Faisalabad.
Experimental animals
The male mice, 6-8 weeks old, weighing 20-30 g were obtained from National Institute of Health Islamabad and kept in animal house of department of Pharmacology, Government College, University Faisalabad under controlled conditions of temperature (25 ± 1°C) and humidity (50 ± 5°C). They were given standard diet and water ad libitum. Mice were acclimatized to environment for one week prior to commencement of experiment (Roslida et al., 2011).
Experimental plan
Dorsal skin of albino mice was shaved with an electric clipper for approximately 2 x 2 cm area and marked with permanent marker (Arya and Kumar, 2011). A total number of 50 animals were selected and divided into 5 subgroups (Figure 1): (Carcinogen control group): 10 mice were applied topically with a single dose of DMBA as a tumor initiator on the shaved area of skin of mice and two weeks later croton oil was applied as tumor promoter thrice a week till the end of 16th week. (Pre group): This group was subdivided into two sub groups, each group consisting of 5 mice and 300 mg/kg and 600 mg/kg C. bonariensis (methanol) extract was given topically to these two subgroups for consecutive 7 days. DMBA single topical dose was applied at 8th day and two weeks later croton oil was applied thrice a week till the end of 16th week. (Peri group): Ten mice were divided into two sub groups each having 5 mice and were given DMBA single dose topically, then 300 mg/kg and 600 mg/kg C. bonariensis (methanol) extract topically for consecutive 15 days. After that, croton oil was applied thrice a week till the end of 16th week. (Post group): Initially single dose of DMBA was applied to this group. Then the group was subdivided into two subgroups of 5 mice in each group and after 2 week, first subgroup received 300 mg/kg and second subgroup received 600 mg/kg C. bonariensis (methanol) extract topically and half an hour, croton oil was applied thrice a week till the end of 16th week. (Continuous group): Two subgroups, each having 5 mice received 300 mg/kg and 600 mg/kg C. bonariensis (methanol) extract topically throughout the experimental period daily and at 8th day, DMBA single topical dose was applied and two weeks later croton oil was applied thrice a week till the end of 16th week.
Preparation of stock solutions
1M DMBA was dissolved in acetone at 100 μg/100 μL (w/v) and croton oil at 1 μg/100 μL to make 1% (v/v) dilution, prepared just before its use and kept in amber glass bottle at about 20°C. A stock solution of extract was prepared by dissolving 10 mg extract in 1.0 mL acetone. Serial dilution of 300 mg/kg and 600 mg/kg was made (Roslida et al., 2011).
Preliminary phytochemical screening
Preliminary phytochemical analysis of C. bonariensis was performed according to protocol previously described (Mojab et al., 2003; Khan et al., 2011).
Determination of cytotoxic activity
Cytotoxic effect of the methanol extract of C. bonariensis was evaluated by considering various morphological parameters like 1- Tumor incidence: number of tumor bearing mice, 2- Cumulative number of papilloma: Total number of papillomas, 3- Tumor yield: average number of tumor per mouse and 4- Tumor burden: number of tumor per tumor bearing mice (Roslida et al., 2011).
Morphological studies
Skin of each mice was weekly observed for loss of hair, redness, ulceration and outgrowths. These were counted and measured by digital vernire caliper till the end of 16th weeks.
Biochemical studies
Mice were sacrificed and shaved ulcerated skin were removed, washed with cold normal saline and kept in formalin bottles. Excised skin was used to prepare 10% tissue homogenate in 0.15 molar tris potassium chloride having a pH of 7.4. Then centrifuge for ten mints at 2000 rpm. reduced glutathione, superoxide dismutase, catalase and lipid peroxidation level were determined by the methods previously described (Marklund and Marklund, 1974; Moron et al., 1979; Ohkhawa et al., 1979; Aebi, 1984).
Histopathological study
Specimens of mice ulcerated skin were excised, washed with normal saline and fixed in 10% formalin for a day. Again fixed with paraffin wax, cut 5 µm portion of each specimen and observed the histopathology (Parmar et al., 2011).
Preliminary phytochemical analysis
Preliminary phytochemical screening methanol extract of C. bonariensis was performed according to the procedure described elsewhere (Saleem et al, 2014).
Chromatogram by HPLC for identification of active constituent
High performance liquid chromatography (HPLC) was performed to identify various compounds present in C. bonariensis methanol extract (Ali et al., 2013). The sample was dissolved in 5 mL distilled water and 12 mL methanol, kept for 5 min, again added 6 mL distilled water, waited for 5 min and added 10 mL 5M HCl in this solution. Placed in oven for 2 hours and filtered the solution by syringe filter. Isocratic: dichloromethane: methanol (60:20:20) was used as the mobile phase with the flow rate of 1 mL/min. The column was ODS 250 mm x 4.6 mm and UV detector was used to obtain chromatogram at 280 nm at room temperature (Saleem et al., 2014).
Statistical analysis
All the obtained results were statistically analyzed by one way analysis of variance (ANOVA). Minitab 16.0 software was used for calculation and all values were represented as mean ± standard deviation (STD). Values were taken as p<0.001 (significant).
Results
Results obtained from present study had shown that single topical application of carcinogen DMBA followed by a thrice/week repeated application of 1% croton oil till 16th week produced 100% skin ulceration in the carcinogen control group. 18.2 ± 1.6, 3.6 ± 0.3 and 3.6 ± 0.3 were calculated as the cumulative number of papilloma, tumor yield and tumor burden respectively (Table I). Ulcerated skin specimens were observed under microscope after fixation with 10% formalin, shown in Figure 2.
Figure 1: Flow diagram of methodology
Figure 2: Lactobacillus plantarum YML009 tolerance in artificial gastric acid (A) and bile juice (B)
Table I: Inhibition of morphological parameters of DMBA/croton oil-induced skin tumors by C. bonariensis methanol extract
| Control | Pre group | Peri group | Post group | Continuous group | |||||
|---|---|---|---|---|---|---|---|---|---|
| 300 mg/kg | 600 mg/kg | 300 mg/kg | 600 mg/kg | 300 mg/kg | 600 mg/kg | 300 mg/kg | 600 mg/kg | ||
| Cumulative number of papilloma | 18.2 (1.6) |
16.8 (3.6) |
15.8 (2.4) |
12.0 (2.6) |
10.6 (6.2) |
10.0 (6.0) |
8.6 (5.0) |
7.4 ± 4.9c | 4.8 ± 6.6c |
| Tumor yield | 3.6 (0.3) |
3.4 (0.7) |
3.2 (0.5) |
2.4 (0.5) |
2.1 (1.2) |
2.0 (1.2) |
1.7 (1.0) |
1.5 ± 1.0c | 1.0 ± 1.3c |
| Tumor burden | 3.6 (0.3) |
3.4 (0.7) |
3.6 (0.3) |
2.4 (0.5) |
2.7 (1.6) |
2.5 (1.5) |
1.2 (1.7) |
1.9 ± 1.2c | 1.2 ± 1.7c |
| Tumor incidence | 5.0 (0.0) |
5.0 (0.0) |
5.0 (0.0) |
4.0 (2.2) |
4.0 (2.2) |
4.0 (2.2) |
3.0 (2.2) |
4.0 ± 2.2c | 3.0 ± 2.2c |
bonariensis (methanol) extract was applied locally to pre, peri, post and continuous groups. Significant decline in cumulative number of papillomas in 300 mg/kg and 600 mg/kg from pre to continuous group 16.8 ± 3.6 to 7.4 ± 4.9 and 15.8 ± 2.4 to 4.8 ± 6.6 respectively. 300 mg/kg extract showed tumor yield from pre to continuous groups 3.4 ± 0.7 to 1.5 ± 1.0 but 600 mg/kg showed 3.2 ± 0.5 to 1.0 ± 1.3. Tumor burden in 300 mg/kg was 3.4 ± 0.7 to 1.9 ± 1.2 and in 600 mg/kg was 3.6 ± 0.3 to 1.2 ± 1.7 shown in Table I. The tumor incidence in continuous group was compared (p<0.001) with carcinogen group decreased up to 20% with 300 mg/kg but 40% with 600 mg/kg.
Results obtained from present study had shown that topical application of DMBA and croton oil produced 100% skin ulceration in carcinogen control group and decreased the oxidative stress parameters, i.e. reduced glutathione, SOD and catalase level to 3.3 ± 0.2 μmol/g, 1.7 ± 0.1 μmol/g and 13.6 ± 0.8 μmol of H2O2 reduction/mg protein/min respectively and increased the lipid peroxidation level as 7.7 ± 0.2 nmol/mg (Table II).
bonariensis (methanol) extract in 300 mg/kg caused reduced glutathione, superoxide dismutase and catalase increased up to 9.7 ± 0.7 μmol/g, 7.9 ± 0.6 μmol/g and 18.8 ± 0.7 μmol of H2O2 reduction/mg protein/min but lipid peroxidation decreased up to 4.2 ± 0.6 nmol/mg levels in the continuous group. While 600 mg/kg caused an induction in reduced glutathione 12.1 ± 1.1 μmol/g, superoxide dismutase 9.1 ± 0.8 μmol/g and catalase 25.5 ± 1.0 μmol of H2O2 reduction/mg protein/min and increased the lipid peroxidation level to 1.4 ± 0.4 nmol/mg as compared with the carcinogen control group (p<0.001) shown in Table II.
Table II: Induction of reduced glutathione, superoxide dismutase, catalase and inhibition of lipid peroxidase level by C. bonariensis methanol extract
| Control | Pre group | Peri group | Post group | Continuous group | |||||
|---|---|---|---|---|---|---|---|---|---|
| 300 mg/kg | 600 mg/kg | 300 mg/kg | 600 mg/kg | 300 mg/kg | 600 mg/kg | 300 mg/kg | 600 mg/kg | ||
| Reduced glutathione | 3.3 (0.2) |
3.7 (0.4) |
7.6 (0.6) |
4.1 (0.6) |
8.7 (0.9) |
7.9 (0.7) |
10.3 (1.0) |
9.7 (0.7)c |
12.1 (1.1)c |
| Superoxide dismutase | 1.7 (0.1) |
2.0 (0.6) |
4.2 (0.4) |
5.5 (0.5) |
6.7 (0.7) |
7.0 (0.5) |
8.8 (1.0) |
7.9 (0.6)c |
9.1 (0.8)c |
| Catalase | 13.6 (0.8) |
13.8 (0.6) |
16.0 (0.3) |
16.5 (0.7) |
18.3 (1.1) |
19.6 (1.0) |
21.2 (1.3) |
18.8 (0.7)c |
25.5 (1.0)c |
| Lipid peroxidation | 7.7 (0.2) |
6.2 (0.4) |
5.8 (0.2) |
4.9 (0.5) |
3.5 (0.8) |
4.1 (0.5) |
2.9 (0.6) |
4.2 (0.6)c |
1.4 (0.4)c |
The phytochemical analysis showed the presence of reducing sugars, alkaloids, tannins, saponins, terpinoids, flavonoids, anthraquinones and glycosides and detection of quercetin by HPLC analysis is shown in Figure 3.
Figure 3: Hemolytic phenomenon test. (A) Test strain: Lactobacillus plantarum YML009. (B) β control: Streptococcus agalactiae KCCM 40417. (C) α Control: Lactobacillus paracasei KCTC 3510
Discussion
Cancer chemoprevention by phytochemicals or herbal medicines is grabbing high interest now a day. These phytochemical exert their anticancer potential due to chemical constituents such as flavonoids, polyphenols, carotenoids, terpinoids and tannins which have been obtained from our daily dietary agents. Flavonoids are potent anti-inflammatory, antioxidant and cytotoxic anti tumor agent. They have ability to reverse the process of carcinogenesis and inhibit the development of persistent tumor (Sengupta et al., 2004).
When tumor initiator, 7-12-dimethyl benz(a)antheracene (DMBA) and tumor promoter, croton oil (active constituent: 12-O-tetradecanoylphorbol-13-acetate) was applied on the mice skin, they produced inflammation and reactive oxygen species (ROS). These ROS including O2-, OH-, H2O2 have ability to move from site of formation to the other healthy cells. DMBA with its active metabolites cause mutation in healthy cells via diol epoxide induction. Increased ROS disturb the balance of oxidation/reduction reaction, oxidative stress parameters and take part in chemical carcinogenesis by changing the gene expression and destructing the cellular components. TPA along with ROS, increase the epidermal ornithine decarboxylase, COX-2 and nitric oxide synthase level (Shakilur et al., 2008).
Similarly, enzymatic oxidative stress parameters including superoxide dismutase and catalase and non enzymatic reduced glutathione help to play important role in enzymatic defense system and their lower level promote the tumor in healthy cells. Reduced glutathione helps to protect the body from xenobiotics, toxic metabolites and ROS (Lu, 1999). Superoxide dismutase and CAT capture the reactive oxygen species and minimize their carcinogenic and mutagenic potential, balance the hydrogen/oxygen per oxide level by causing alteration in O2 and H2O2 radical (Dasgupta et al., 2004). In carcinogen control group, level of reduced glutathione, superoxide dismutase and CAT were significantly decreased and lipid peroxidation increased along with the tumor incidence, tumor yield and tumor burden due to the presence of increased ROS.
bonariensis methanol extract decreased the tumor incidence, tumor yield, tumor burden, cumulative number of papilloma and lipid peroxidation level as compare to carcinogen control group. The plant extract increased the level reduced glutathione, superoxide dismutase and catalase in continuous group in which plant extract was applied throughout the experimental period (16 weeks) with higher effects at 600 mg/kg/b.wt as compare to 300 mg/kg. The phytochemical analysis had shown the presence of flavonoids, saponins, tannis and terpinoids and HPLC analysis indicated querectin i.e a flavonoid. It is a potent bioactive molecule that possess anticarcinogenic potential since it can interfere with the initiation, development and progression of cancer by the modulation of cellular proliferation, differentiation, apoptosis, angiogenesis and metastasis (Kumar et al., 2011). Flavonoids have potential as chemopreventive agent for cancer treatment due to their ability to induce apoptosis (Ramos, 2007) by arresting cell cycle at G1, S, G2 and M phases of cell cycle. Also, previously, it is known that quercetin has ability to capture the ROS, superoxide anions, hydroxyl and lipid peroxy radicals, inhibit cyclooxygenase, lipooxigenase, monoxygenase, phospholipase A2, protein kinase and NADH-oxidative pathways (Kumar et al., 2011).
Conclusion
C. bonariensis methanol extract possesses significant cytotoxic activity which may due to the presence of quercetin (a flavonoid) against DMBA induced skin carcinogenesis.
References
Aebi H. Catalase: In vitro. In: Method in enzymology. Colowick SP, Kaplan NO (eds). Vol. 105. New York, Academic Press, 1984, pp 121-26.
Ahmad S. Medicinal wild plants from Lahore-Islamabad motorway (M-2). Pakistan J Bot. 2007; 39: 355-75.
Ali M, Qadir MI, Saleem M, Janbaz KH, Gul H, Hussain L, Ahmad B. Hepatoprotective potential of Convolvulus arvensis against paracetamol-induced hepatotoxicity. Bangladesh J Pharmacol. 2013; 8: 300-04.
Ancuceanu RV, Istudor V. Pharmacologically active natural compounds for lung cancer. Altern Med Rev. 2004; 9: 402-19.
Arya P, Kumar M. Chemoprevention by Triticum aestivum of mouse skin carcinogenesis induced by DMBA and croton oil: Association with oxidative status. Asi Pacific J Can Prev. 2011; 12: 143-48.
Chaudhry A, Janbaz KH, Uzair M, Ejaz AS. Biological studies of Conyza and Euphorbia species. J Res Sci. 2001; 12: 85-88.
Cragg GM, Newman DJ. Plants as a source of anticancer agents. J Ethnopharmacol. 2005; 100: 72-79.
Dasgupta T, Yadava PK, Rao AR. Chemomodulatory efficiency of basil leaf (Ocimum basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen induced skin and forestomach papillomagenesis. J Mol Cell Biochem. 2004; 226: 27-38.
De Almeida ER. Preclinical and clinical studies of lapachol and betalapachone. Open Nat Prod J. 2009; 2: 42-47.
Deng F, Lu JJ, Liu HY, Lin LP, Ding J, Zhang JS. Synthesis and antitumor activity of novel salvicine analogues. Chin Chem Lett. 2011; 2: 25-28.
Diogo CV, Felix L, Vilela S, Burgeiro A, Barbosa IA, Carvalho M, Oliveira P, Peixoto F. Mitochondrial toxicity of the phyotochemicals daphnetoxin and daphnoretin: Relevance for possible anticancer application. Toxicol in vitro. 2009; 23: 772-79.
Dixon RA, Ferreira D. Molecules of interest: Genistein. Phytochemistry 2002; 60: 205-11.
Dubey K, Ray A, Behera B. Production of demethylated colchicine through microbial transformation and scale-up process development. Process Biochem. 2008; 43: 251-57.
Favila C, Antonio M. Chemical and biological study of Conyza bonariensis (L.) Cronquist (Asteraceae). Thesis dissertation. 2006.
Fuchs C, Mitchell EP, Hoff PM. Irinotecan in the treatment of colorectal cancer. Cancer Treat Rev. 2006; 32: 491-503.
Fulda S. Betulinic acid for cancer treatment and prevention. Int J Mol Sci. 2008; 9: 1096-107.
Goel A, Kunnumakkara AB, Aggarwal B. Curcumin as ‘‘Curecumin’’: From kitchen to clinic. Biochem Pharmacol. 2008; 75: 787-809.
Hait WN, Rubin E, Alli E, Goodin S. Tubulin targeting agents. Update Cancer Ther. 2007; 2: 1-18.
Hampson P, Wang K, Lord J. Treatment of actinic keratoses, acute myeloid leukemia therapy treatment of basal cell carcinoma protein kinase C activator. Drugs Fut. 2005; 30: 1003-05.
Khan FA, Hussain I, Farooq S, Ahmad M, Arif M, Rehman IU. Phytochemical screening of some Pakistanian medicinal plants. Middle-East J Sci Res. 2011; 8: 575-78.
Khan RA, Bukhari IA, Nawaz SA, Choudhary MI. Acetylcholinesterase and butyrylcholinesterase inhibitory potential of some Pakistani medicinal plants. J Basic Appl Sci. 2006; 2: 07-10.
Kingston DG. The shape of things to come: Structural and synthetic studies of taxol and related compounds. Phytochemistry. 2007; 68(14): 1844-54.
Kumar B, Sandhar H, Prasher S, Tiwari P, Salhan M, Sharma P. A review of phytochemistry and pharmacology of flavonoids. Int J Pharmaceut Sciencia. 2011; 1: 25-41.
Kuo Y, Kuo P, Hsu Y, Cho C, Lin C. Ellipticine induces apoptosis through p53-dependent pathway in human hepatocellular carcinoma HepG2 cells. Life Sci. 2006; 78: 2550-57.
Li Y, Li C, Yu D, Pardee AB. Potent induction of apoptosis by β-lapachone in human multiple myeloma cell lines and patient cells. Mol Med. 2000; 6: 1008-15.
Lu L, Li YM, Fu GQ, Yang L, Jiang JG, Zhu L, Lin FL, Chen J, Lin QS. Extraction optimisation of daphnoretin from root bark of Wikstroemia indica (L.) C.A. and its antitumour activity tests. Food Chem. 2011; 124: 1500-06.
Lu SC. Regulation of hepatic glutathione synthesis: Current concepts and controversies. FASEB J. 1999; 13: 1169-73.
Mans, D, Da Rocha A, Schwartsmann G. Anticancer drug discovery and development in Brazil: Targeted plant collection as a rational strategy to acquire candidate anticancer compounds. Oncologist 2000; 5: 185-98.
Marklund S, Marklund G. Involvement of the superoxide anion radical in autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Euo J Biochem. 1974; 47: 469-74.
Mojab F, Kamalinejad M, Ghaderi N, Vahidipour HR. Phytochemical screening of some species of Iranian plants. Iran J Pharmac Res. 2003; 4: 77-82.
Moon YJ, Wang X, Morris ME. Dietary flavonoids: Effects on xenobiotic and carcinogen metabolism. Toxicol in vitro. 2006; 20: 187-210.
Moron MA, Depierre JW, Mannervick B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochem Biophys Acta. 1979; 582: 67-78.
Ohkhawa H, Ohishi N, Yogi K. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Analy. Biochem. 1979; 95: 351-58.
Okouneva T, Hill BT, Wilson L, Jordan MA. The effects of vinflunine, vinorelbine, and vinblastine on centromere dynamics. Mol Cancer Ther. 2003; 2: 427-36.
Parmar J, Sharma P, Verma P, Goyal PK. Modulation of DMBA-induced biochemical and histopathological changes by Syzygium cumini seed extract during skin carcinogenesis. Int J Curr Biomed Pharmac Res. 2011; 1: 24-30.
Patil JB, Kim J, Jayaprakasha GK. Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway. Eur J Pharmacol. 2010; 645: 70-78.
Ramos, S. Effects of dietary flavonoids on apoptic pathways related to cancer chemoprevention. J Nut Biochem. 2007; 18: 427-42.
Roslida AH, Fezah O, Yeong LT. Suppression of DMBA/croton oil-induced mouse skin tumor promotion by Ardisia crispa root hexane extract. Asi Pacific J Can Prev. 2011; 12: 665-69.
Sa G, Das T, Banerjee S, Chakraborty J. Curcumin: From exotic spice to modern anticancer drug. Al Ameen J Med Sci. 2010; 3: 21-37.
Saleem M, Ahmed B, Qadir MI, Rafiq M, Ahmad M, Ahmad B. Hepatoprotective effect of Chenopodium murale in mice. Bangladesh J Pharmacol. 2014; 9: 124-28.
Santana PM, Miranda M, Gutiérrez Y, GarcÃa G, Orellana T, Orellana-Manzano A. Anti-inflammatory and antimitotic effect of the alcoholic extract and chemical composition of the oil from Conyza bonariensis (L.) Cronquist (deer shinbone) leaves. Rev Cubana Plant Med. 2011; 16: 13-23.
Sengupta A, Ghosh S, Bhattacharjee S. Indian food ingredients and cancer prevention: An experimental evaluation of anticarcinogenic effects of garlic in rat colon. Asian Pacific J Can Prev. 2004; 5: 126-32.
Shahwar D, Raza MA, Saeed A, Riasat M, Chattha F, Javaid M, Ullah S, Ullah S. Antioxidant potential of the extracts of Putranjiva roxburghii, Conyza bonariensis, Woodfordia fruiticosa and Senecio chrysanthemoids. Afr J Biotech. 2012; 11: 4288-95.
Shakilur R, Kanchan B, Abdul QK. Topically applied vitamin E prevents massive cutaneous inflammatory and oxidative stress responses induced by double application of 12-o-tetradecanoylphorbol-13-acetate (TPA) in mice. Chemico-Biologi Intera. 2008; 172: 195-205.
Shoeb M, MacManus SM, Jaspars M, Trevidadu J, Nahar L, Thoo-Lin PK, Sarker SD. Montamine, a unique dimeric indole alkaloid, from the seeds of Centaurea montana (Asteraceae), and its in vitro cytotoxic activity against the CaCo2 colon cancer cells. Tetrahedron 2006; 62: 11172-77.
Shoeb M, Celik S, Jaspars M, Kumarasamy Y, MacManus S, Nahar L, Kong T, Sarker S. Isolation, structure elucidation and bioactivity of schischkiniin, a unique indole alkaloid from the seeds of Centaurea schischkinii. Tetrahedron. 2005; 61: 9001-06.
Simoens C, Lardon F, Pauwels B, De Pooter CM, Lambrechts HA, Pattyn GG, Breillout F, Vermorken JB. Comparative study of the radiosensitising and cell cycle effects of vinflunine and vinorelbine, in-vitro. BMC Cancer. 2008; 8: 65.
Wang F, Gao Y, Gao L, Xing T. Study on the electrochemical behavior of the anticancer herbal drug berberine and its analytical application. J Chin Chem Soc. 2011; 58.
Xie J, Ma T, Gu Y, Zhang X, Qiu X, Zhang L, Xu R, Yu Y. Berbamine derivatives: A novel class of compounds for anti-leukemia activity. Eur J Med Chem. 2009; 44: 3293-98.
Xu R, Dong Q, Yu Y, Zhao X, Gan X, Wu D, Lu Q, Xu X, Yu XF. Berbamine: A novel inhibitor of bcr/abl fusion gene with potent anti-leukemia activity. Leuk Res. 2006; 30: 17-23.
Zhang JA, Xuan T, Parmar M, Ma L, Ugwu S, Ali S, Ahmad I. Development and characterization of a novel liposome-based formulation of SN-38. Int J Pharm. 2004; 270: 93-107.
