|Year : 2015 | Volume
| Issue : 1 | Page : 28-33
Evaluation of anticancer activity of Asteracantha longifolia in 7,12-Dimethylbenz(a)anthracene-induced mammary gland carcinogenesis in Sprague Dawley rats
Dhanya Venugopalan Nair1, NB Shridhar2, K Jayakumar2
1 Department of Pre-clinical, Venus Medicine?? Research Centre, Venus Remedies Ltd, Hill Top Industrial Estate, Baddi, Himachal Pradesh, India
2 Department of Veterinary Pharmacology and Toxicology, Veterinary College, Karnataka Veterinary, Animal and Fisheries Sciences University, Bangalore, Karnataka, India
|Date of Submission||23-Apr-2014|
|Date of Acceptance||14-Jun-2014|
|Date of Web Publication||27-Jan-2015|
Dhanya Venugopalan Nair
Venus Medicine Research Centre, Venus Remedies Ltd, Hill Top Industrial Estate, Jharmajri EPIP, Phase - I (Extension), Bhatoli Kalan, Baddi, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Ethnopharmacological Relevance: Asteracantha longifolia Nees. (Syn. Hygrophila spinosa, Hygrophila auriculata) is a common weed growing in marshy and water logged areas. It is an important medicinal herb, widely distributed in India, and used by the local population for different medicinal purposes. In Ayurveda, the plant parts are used for the treatment of rheumatism, urinary tract infection, malaria, inflammation, diabetes, dysentery, jaundice, hepatic obstruction, pain, and decreased libido. Aim: The objective of the study is to explore the anticancer activity of the methanolic extract of A. longifolia on 7,12-Dimethylbenz (a) anthracene (DMBA)-induced mammary carcinogenesis in rats. Materials and Methods: The anticancer activity of the methanolic whole plant extract of A. longifolia was evaluated in Sprague Dawley rats in the mammary tumor induced by DMBA. The doses administered were 100, 200, and 400 mg/kg body weight by oral route. The extracts were administered for 20 consecutive days. The tumor size was determined before and after the administration of the plant extract and compared with the antitumor effect of the standard drug cyclophosphamide (CYC). After the experiment, the rats were sacrificed and subjected to histopathology and biochemical analysis. Results: A. longifolia extracts showed a significant (P < 0.05) decrease in the tumor size of DMBA-induced mammary tumor in mice. The extracts significantly (P < 0.05) decreased the levels of lipid peroxidation and significantly (P < 0.05) increased the levels of superoxide dismutase and catalase. Phytochemical screening revealed the presence of flavanoids, which are potent antioxidants. Conclusion: The results showed that the methanolic extract of A. longifolia was more effective in inhibiting the tumor growth and safe in DMBA-induced mammary tumor in rats as compared to the standard drug, cyclophosphamide.
Keywords: Antioxidant, Asteracantha longifolia, Dimethylbenz (a) anthracene, mammary tumor
|How to cite this article:|
Nair DV, Shridhar N B, Jayakumar K. Evaluation of anticancer activity of Asteracantha longifolia in 7,12-Dimethylbenz(a)anthracene-induced mammary gland carcinogenesis in Sprague Dawley rats. Int J Nutr Pharmacol Neurol Dis 2015;5:28-33
|How to cite this URL:|
Nair DV, Shridhar N B, Jayakumar K. Evaluation of anticancer activity of Asteracantha longifolia in 7,12-Dimethylbenz(a)anthracene-induced mammary gland carcinogenesis in Sprague Dawley rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2020 Jan 24];5:28-33. Available from: http://www.ijnpnd.com/text.asp?2015/5/1/28/150072
| Introduction|| |
Breast cancer is the most frequent malignancy among women worldwide. It is a highly heterogeneous disease represented by tumors that have a diverse natural history, complex histology, and a variable response to therapy. One of the significant features of breast cancer is the high level of oxidative stress, partly due to the pro-oxidant effects of estrogen metabolism and various other interlinked mechanisms. As a result, the detection of oxidative DNA adducts in breast cancer tissue, Nowsheen, et al.  and a significant rise in oxidative stress markers in the plasma of breast cancer patients Hussien,  Sener et al.,  Khambete and Kumar  are observed. As it is a well-known fact that oxygen radicals are associated with the activation of carcinogen as well as in the promotion of an initiated cell Cerutti,  a therapeutic approach can be explored toward developing antioxidant-based therapies, which can heal cancer by combating oxidative stress.
A. longifolia Nees. (Syn. Hygrophila spinosa, Hygrophila auriculata) is a wild herb of medicinal value, widely distributed in India and used by the local population for different medicinal purposes. It possesses potent antioxidant and erythropoietic activities Pawar et al and is used in 'Ayurveda' as a 'Rasayana' drug for treatment of various disorders Vijaykumar, et al.  Ahmed et al.  reported the anti-tumor promoting potential of the methanol fraction of the A. longifolia seed extract against chemically-induced hepatocarcinogenesis in Wistar rats. However, there are no studies reported on the effect of A. longifolia whole plant extract against chemically induced mammary carcinogenesis in rats. Hence, this study is an attempt to explore the possibilities of the anticancer property of A. longifolia in DMBA-induced mammary carcinogenesis in Sprague Dawley rats.
| Materials and methods|| |
7, 12- dimethylbenz (a) anthracene was purchased from Sigma Chemicals Co. (St. Louis, MO, USA) and all the other reagents were of analytical grade. CYC was obtained from Apollo Medical, Chennai, India.
Fresh plants of A. longifolia were collected from the marshy areas of the village Patnayanakanahalli, near Tumkur district, Karnataka state, in India during the month of November. The plant material was dried under the shade for 15 days. The dried plant was finely powdered and stored in an air-tight container until the preparation of the plant extract.
Phytochemical analysis of the A. longifolia plant extract was carried out using the High performance thin layer chromatography (HPTLC) technique Wagner et al. 
Preparation of extract
One hundred grams of powder from the whole dried plant of A. longifolia was taken, to which 1000 ml of methanol was added, mixed, and kept for two days. The contents were periodically shaken using an electric shaker. After two days, the contents were filtered through a Buchner funnel in a conical flask and it was further concentrated by evaporation by keeping the filtrate in a round-bottomed flask kept in a rotary flash evaporator, maintained at 39-40 0 C, till the solvent completely evaporated and the extract settled down to the bottom. The residues were weighed after concentration and their respective percentage yield was determined.
Young female rats, 45 days old, of the Sprague-Dawley strain, were procured from a reputed breeder and were used for the present study. The animals were weighed and kept separately in cages and were allowed to acclimatize to the experimental conditions for one week before the commencement of the study. The rats were housed in polypropylene rat cages during the whole experiment. They were kept under the standard hygienic laboratory conditions, providing the standard laboratory animal feed and water ad libitum. Prior permission was obtained from the Institutional Animal Ethics Committee before the start of the experiment (No. 71 LPM/IAEC/2011).
Acute oral toxicity studies were performed according to OECD 421 (Organization for Economic Co-operation and Development). The general behavior, such as, motor activity, tremors, convulsions, aggressiveness, piloerection, loss of lighting reflex, sedation, muscle relaxation, hypnosis, analgesia, ptosis, lacrimation, diarrhea, and skin color were observed for the first hour and 24 hours after administration of the test drug.
Evaluation of in vivo antitumor property
Chemical induction of mammary tumor with DMBA (7, 12-Dimethylbenz (a) anthracene) (DMBA) is a powerful organ-specific laboratory carcinogen, which is widely used in many research laboratories studying cancer. DMBA serves as a tumor-initiator by making the necessary mutations Miyata et al. Tumor induction was carried out as per the method described by Samy et al.,  wherein, 25 mg of DMBA was dissolved in 1 ml of vehicle (0.5 ml of sunflower oil plus 0.5 ml of saline) and injected into the rats, by subcutaneous injection, beneath the mammary gland on either side. The tumor yield and size were stabilized after three months with the initiation of DMBA and these animals were further used for the experiment.
Grouping of animals and dosing
The animals were divided into six groups, with each group having six animals. Group I served as the normal negative control group, where the animals were not induced with cancer and were provided normal feed and water. Group II animals served as the DMBA-control group, where the animals were induced with cancer, but did not receive any treatment. Group III animals, served as the standard positive control group, wherein, the standard drug cyclophosphamide was given at the rate of 10 mg/kg weight i.p Samy et al.  Group IV served as the test group, where the methanolic extract of A. longifolia was administered at the rate of 100 mg/kg. Group V served as the test group, where methanolic extract of A. longifolia was administered at the rate of 200 mg/kg. Group VI served as the test group, where the methanolic extract of A. longifolia was administered at the rate of 400 mg/kg. The standard drug and the plant extract were administered for a period of 20 days. The doses were selected based on the acute oral toxicity of the plant. The methanolic extract of A. longifolia was dissolved in 0.4%(w/v) carboxy methyl cellulose (CMC) to make the solution of required quantity and 1 ml of suspension was administered to each rat. The biochemical findings were studied and later the animals were humanely sacrificed and the organs were subjected to histopathology. The results obtained from the experimental trial were compared with those of the DMBA control group (Group II) and standard group (Group III). The data were statistically analyzed and the results were interpreted.
Determination of the percent decrease in tumor size
The tumor size was measured using a vernier calliper before and after the experiment. The percent decrease in tumor size was calculated as per the formula given below:
On day 20, that is, at the end of the study all the animals were starved overnight and humanely sacrificed. Immediately after sacrificing the animals, the tissues were processed for the estimation of the activity of antioxidant enzymes, such as, thiobarbituric acid reactive substances (TBARS) as described by Ramanarayan et al.,  superoxide dismutase (SOD) as per described by Marklund and Marklund,  and catalase (CAT) as described by Caliborne 1985. 
The subcutaneous tumors were excised and the size was measured. Organs like the liver, kidney, mammary tissues, and tumor growths were collected in 10% neutral buffered formalin for histopathology and were further stained with hematoxylin and eosin Chhabra et al. 
The data obtained from the present study were analyzed by using the one-way analysis of variance (ANOVA) by Bonferroni's Multiple Comparison Test using the Graph Pad prism software (Graph Pad Prism, 2007).
| Results|| |
The methanolic extract of A. longifolia was subjected to HPTLC for the presence of flavanoids. Without chemical treatment, fluorescence quenching on the TLC plates was observed at UV-254 nm and light blue fluorescence appeared on the TLC plates at UV-366 nm. With natural products - polyethylene glycol intense blue fluorescence appeared on the TLC plate at UV-366 nm and orange bands appeared in visible light. With Fast blue salt B, a blue fluorescence appeared on the TLC plates at UV-366 nm. Thus, it was concluded that A. longifolia was positive for the presence of flavonoids .
The methanolic extract of A. longifolia was evaluated for acute toxicity in rats. The extract did not alter their general behavior and failed to produce any mortality even at the highest dose (2000 mg/kg, p.o.). On the basis of acute toxicity, three doses, that is, 100, 200, and 400 mg/kg were used for further pharmacological analysis.
The methanolic extract of A. longifolia showed potent antitumor activity against DMBA-induced mammary carcinogenesis in rats. The activity was measured by the percent decrease in tumor size. The percent decrease in tumor size was significant in all the three doses administered as compared to the DMBA control group. The antitumor activity of the extract exhibited a dose-dependent activity [Table 1].
|Table 1: Effect of methanol extract of Asteracantha longifolia on in tumour volume and biochemical (antioxidant) parameters |
Click here to view
The administration of the extract caused significant decreases in the levels of the antioxidant enzymes SOD and CAT while significantly increasing the levels of TBARS in the DMBA administered animals, when compared to the normal group. However, treatment with methanol extract of A. longifolia reversed these changes to the normal levels [Table 1].
The antitumor activity was further confirmed by histopathological findings. In case of animals treated with DMBA and not given any treatment, that is, Group II, the kidneys showed swollen and necrosed tubular epithelial cells resulting in tubular destruction [Plate 1a].The liver exhibited swollen and degenerating hepatocytes, with infiltration of the mononuclear cells [Plate 1b]. In addition, congestion of the vessels and sinusoids along with hemorrhage was observed in this section. A section of the mammary gland revealed proliferation of neoplastic cells and these neoplastic cells were characterized by basophilic vesicular nucleus and cellular pleomorphism arranged in a trabecular pattern [Plate 1c]. In the cyclophosphamide-treated group, that is, group III, the liver revealed normal architecture. The kidney showed slight congestion and infiltration of mononuclear cells in the interstitium [Plate 2a] and the mammary gland revealed neoplastic cell proliferation, which was arranged in a glandular pattern [Plate 2b]. In the animals administered with plant extract at 400 mg/kg dose of A. Longifolia, the kidney and liver apparently revealed normal architecture, whereas, the mammary gland revealed degeneration and necrosis of the neoplastic cells [Plate 3a].
| Discussion|| |
The reactive oxygen species (ROS) leading to oxidative stress are involved in a variety of important pathophysiological conditions including mutagenesis and carcinogenesis (Aruoma, 1994). Oxidative stress plays an important role in both the treatment response of the patient and the clinical outcome in general Ozben.  Synthetic drugs used for the systemic treatment of breast cancer cause large amounts of oxidative stress and interfere with the treatment effectiveness and enhance its toxic side effects Conklin.  The natural endogenous antioxidants are seldom able to cope up with the cumulative free radical overload, thus worsening the condition. Thus, oxidative stress caused by chemotherapy is added to the oxidative stress that is inherent in the tumor. Aggressive breast tumors adapt to this high oxidative environment and reinforce the activation of signalling cascades that contribute to tumor growth, angiogenesis, and metastasis. Hence, investigation and development of novel phytotherapeutic agents to detect anti-tumor and free radical scavenging activities becomes an unmet need Ioset et al 2001. 
Thus, the present study was carried out to primarily evaluate the anticancer property, phytochemical screening, and antioxidant effect of the methanolic extract of the A. longifolia whole plant extract. Phytochemical analysis of the A. longifolia plant extract, using the HPTLC technique, revealed the presence of flavanoids. These findings are in accordance with Sawadogo et al.  and various other studies, which state that the methanolic extract of the leaves of the A. longifolia plant contains various polyphenols and flavonoids.
Furthermore, the extract of the A. longifolia plant exhibited a potent anticancer activity on DMBA-induced mammary tumors in rats as the percent decrease in the tumor size was statistically more significant (P < 0.05) than the standard anticancer drug cyclophosphamide. These results corroborate with the studies that reported the antitumor activities of A. Longifolia in hepatocarcinogenesis Ahmed et al.  These findings were further strengthened by histopathological revelations. Nephrotoxicity was clearly evidenced in the cyclophosphamide-treated group, thus indicating the toxic side effects of the synthetic chemotherapeutic agents, which have also been vastly reported in literature Sinanoglu et al. 
The antioxidant status of the plant extract showed significantly increased lipid peroxidation (LPO) (TBARS) in DMBA-induced experimental rats [Table 1], indicating the production of freer radicals. The elevation of lipid peroxidation is already known to be associated with cancer Navarro et al.  Decrease in the activities of the antioxidant enzymes SOD and CAT noted in the tumor group [Table 1] is regarded as a marker of malignant transformation Kavitha and Manoharan.  Therefore, the significant elevation of SOD and CAT and significant reduction in LPO by the extract treatment confirms the potent antioxidant activity and free radical scavenging property of A. Longifolia. These results are in accordance with the studies conducted by Vijaykumar et al.  and Dasgupta,  which emphasize the potent antioxidant activity of A. Longifolia.
Thus from the present study, it can be anticipated that the anticancer property of A. longifolia can be attributed to the presence of flavanoids, as these essential photochemicals inhibit tumor promotion through their effects on signal transduction in cell proliferation and angiogenesis Chahar et al,  Subash and Subramanian. 
| Conclusion|| |
The present study clearly demonstrates the potent anticancer properties of the A. longifolia extract. The treatment of the methanolic extract of A. longifolia in tumor-affected rats reversed the changes in the biochemical parameters to a normal level, when compared with the control DMBA-induced rat. The phytochemical revelation of the presence of natural antioxidants such as flavanoids has further strengthened the evidence for the anticancer property of this plant extract. Further studies and clinical trials will help evaluate its clinical implications.
| Acknowledgments|| |
This research article was made possible through help and support from everyone, including: The parents, teachers, family, and friends, and all sentient beings, who believed that herbals have a vast potential in treating a dreaded diseases like cancer.
Please allow me to especially dedicate my acknowledgment of gratitude to Late. Dr. K. Jayakumar and Dr. N.B. Shridhar, for the support and encouragement they extended to me during this study.
Second, I would like to thank Mr. Anil Kumar Singhal for kindly reading my article and offering his invaluable detailed advice on the grammar, organization, and theme of the article.
Finally, I sincerely thank the Karnataka Veterinary, Animal and Fisheries University, Bidar, for the financial support.
The product of this research article would not have been possible without all of them.
| References|| |
Nowsheen S, WukovichRL, Aziz K, Kalogerinis PT, Richardson CC, Panayiotidis MI, et al
. Accumulation of oxidatively induced clustered DNA lesions in human tumor tissues. Mutat Res 2009;674:131-6.
Hussien MM, McNulty H, Armstrong N, Johnston PG, Spence RA, Barnett Y. Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients. Br J Cancer 2005;92:1524-30.
Sener DE, Gönenç A, Akinci M, Torun M. Lipid peroxidation and total antioxidant status in patients with breast cancer. Cell Biochem Funct 2007;25:377-82.
Khambete N, Kumar R. Carcinogens and cancer preventors in diet. Int J Nutr Pharmacol Neurol Dis 2014;4:4-10.
Cerutti PA. Prooxidant states and tumor promotion. Science 1985;227:375-81.
Pawar RS, Jain AP, Lodhi S, Singhai AK. Erythropoietic activity of Asteracantha longifolia (Nees.) in rats. J Ethnopharmacol 2010;129:280-2.
Vijaykumar M, Govindrajan R, Rao GM, Rao Ch V, Shirwalkar A, Mehrotra S, et al.
Action of hygrophila auriculata against streptozotocin-induced oxidative stress. J Ethnopharmacol 2006;104:356-61.
Ahmed S, Rahman A, Mathur M, Athar M, Sultana S. Anti-tumor promoting activity of Asteracantha longifolia against experimental hepatocarcinogenesis in rats. Food Chem Toxicol 2001;39:19-28.
Wagner H, Bladt S, Zgainski EM. Plant Drug Analysis: A Thin Layer Chromatography Atlas. 2 nd
ed. New York: Springer Publications; 1984.
Miyata M, Furukawa M, Takahashi K, Gonzalez FJ, Yamazoe Y. Mechanism of 7, 12-dimethylbenz [a] anthracene-induced immunotoxicity: Role of metabolic activation at the target organ. Jpn J Pharmacol 2001;86:302-9.
Samy RP, Gopalakrishnakone P, Ignacimuthu S. Anti-tumor promoting potential of luteolin against 7, 12-dimethylbenz (a) anthracene-induced mammary tumors in rats. Chem Biol Interact 2006;164:1-14.
Ramanarayan K, Bhat A, Shripathi V, Swamy GS, Rao KS. Triacontanol inhibits both enzymatic and nonenzymatic lipid peroxidation. Phytochemistry 2000;55:59-66.
Marklund SL, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469-74.
Caliborne AL. Assay of Catalase. In: Handbook of methods for Oxygen Radical Research. In: Greenward RA, editor. Baco-Raton: CRC Press; 1985. p. 283-4.
Chhabra N, Buzarbaruah S, Singh R, Kaur J. Silibinin: A promising anti-neoplastic agent for the future? A critical reappraisal. Int J Nutr Pharmacol Neurol Dis 2013;3:206-18.
Ozben T. Oxidative stress and apoptosis: Impact on cancer therapy. J Pharm Sci 2007;96:2181-96.
Conklin KA. Chemotherapy-associated oxidative stress: Impact on chemotherapeutic effectiveness. Integr Cancer Ther 2004;3:294-300.
Ioset JR, Marston A, Gupta MP, Hostettman K. A methyfl avan with free radical scavenging properties from Pancratium littorale. Fitoterapia 2001;72:35-9.
Sawadogo WR, Aline M, Lamien CE, Martin K, Guissou IP, Nacoulma OG. Phenolic content and antioxidant activity of six acanthaceae from Burkina faso. J Biol Sci 2006;6:249-52.
Sinanoglu O, Yener AN, Ekici S, Midi A, Aksungar FB. The protective effects of spirulina in cyclophosphamide induced nephrotoxicity and urotoxicity in rats. Urology 2012;80:1392.e1-6.
Navarro J, Obrador E, Pellicer JA, Aseni M, Viña J, Estrela JM. Blood glutathione as an index of radiation-induced oxidative stress in mice and humans. Free Radic Biol Med 1997;22:1203-9.
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;38:185-9.
Dasgupta N. Antioxidant activity of some leafy vegetables in India: A comparative study. Food Chem 2007;101:471-4.
Chahar MK, Sharma N, Dobhal MP, Joshi YC. Flavonoids: A versatile source of anticancer drugs. Pharmacogn Rev 2011;5:1-12.
Subash S, Subramanian P. Effect of N-phthaloyl gamma-aminobutyric acid on lipid peroxidation, antioxidants and liver markers in constant light exposed rats. Int J Nutr Pharmacol Neurol Dis 2011;1:163.