Background: Melastoma malabathricum L. Smith (family Melastomaceae) is a shrub that has been used by the Malay practitioners of traditional medicine to treat various types of ailments. The present study aimed to determine the chemopreventive activity of methanol extract of M. malabathricum leaves (MEMM) using the standard 7,12-dimethylbenz(α)anthracene (DMBA)/croton oil-induced mouse skin carcinogenesis model. 
Materials and Methods: In the initiation phase, the mice received a single dose of 100µl/100 µg DMBA (group I-V) or 100µl acetone (group VI) topically on the dorsal shaved skin area followed by the promotion phase involving treatment with the respective test solutions (100 μl of acetone, 10 mg/kg curcumin or MEMM (30, 100 and 300mg/kg)) for 30 min followed by the topical application of tumour promoter (100µl croton oil). Tumors were examined weekly and the experiment lasted for 15 weeks. 
Results: MEMM and curcumin significantly (

Cheah, Yee Ling

Kooi, Ooi Kah

Mohtaruddin, Nurhafizah

Othman, Fezah

Rodzi, Roihanah

Suhaili, Zarizal

Susanti, Deny

Taher, Muhammad

Tohid, Siti Farah

Zakaria, Zainul Amiruddin

ATHMSI Journals

Ooi et al., Afr J Tradit Complement Altern Med. (2014) 11(4):66-70http://dx.doi.org/10.4314/ajtcam.v11i4.1166CHEMOPREVENTIVE ACTIVITY OF METHANOL EXTRACT OF MELASTOMA MALABATHRICUMLEAVES IN DMBA-INDUCED MOUSE SKIN CARCINOGENESISOoi Kah Kooi1, Cheah Yee Ling1, Roihanah Rodzi1, Fezah Othman1, Norhafizah Mohtarrudin3, ZarizalSuhaili4, and Zainul Amiruddin Zakaria1,2,*1Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400UPM Serdang, Selangor, Malaysia. 2Halal Product Research Institute, Universiti Putra Malaysia, 43400 UPMSerdang, Selangor, Malaysia. 3Department of Pathology, Faculty of Medicine and Health Sciences, Universiti PutraMalaysia, 43400 UPM Serdang, Selangor, Malaysia. 4Faculty of Agriculture and Biotechnology, Universiti SultanZainal Abidin, Kampus Kota, Jalan Sultan Mahmud, 20400 KualaTerengganu, Malaysia.*E-mail: dr_zaz@yahoo.comAbstractBackground: Melastoma malabathricum L. Smith (family Melastomaceae) is a shrub that has been used by the Malay practitioners oftraditional medicine to treat various types of ailments. The present study aimed to determine the chemopreventive activity of methanol extractof M. malabathricum leaves (MEMM) using the standard 7,12-dimethylbenz(α)anthracene (DMBA)/croton oil-induced mouse skin carcinogenesis model.Materials and Methods: In the initiation phase, the mice received a single dose of 100µl/100 µg DMBA (group I-V) or 100µl acetone(group VI) topically on the dorsal shaved skin area followed by the promotion phase involving treatment with the respective test solutions(100 μl of acetone, 10 mg/kg curcumin or MEMM (30, 100 and 300mg/kg)) for 30 min followed by the topical application of tumour promoter (100µl croton oil). Tumors were examined weekly and the experiment lasted for 15 weeks. Results: MEMM and curcuminsignificantly (p<0.05) reduced the tumour burden, tumour incidence and tumour volume, which were further supported by thehistopathological findings.Conclusion: MEMM demonstrated chemoprevention possibly via its antioxidant and anti-inflammatory activities, and the action offlavonoids like quercitrin.Key words: Melastomaceae; skin cancer; anti-carcinogenic activityIntroductionCancer is one of the major public health problem areas and remains a major cause of mortality and morbidity in developing as wellas in developed countries (Arya, and Kumar, 2011). One of the commercial ways of providing treatments for cancer patients is chemotherapy- using antineoplastic drugs to kill rapid dividing cells. However, these drugs effectiveness have been overshadowed by its associated sideeffects, which include harm to the rapid dividing cells (i.e. bone marrow, digestive tract and hair follicles) that later lead toimmunosuppression, mucositis and hair loss (Nouri, 2008).Interestingly, the phyto-chemical constituents of some herbs and medicinal plants contain bioactive compounds that displayedpotent anti-tumor or anti-carcinogenesis activity and are, therefore, important in the cancer preventive strategy in order to inhibit, delay orreverse carcinogenesis (Surh, 2002). One of the plants that are being studied in our laboratory is a small shrub belonging to the familyMelastomaceae known as Melastoma malabathricum L. Smith. This medicinal plant has been scientifically reported to possess variouspharmacological activities including the antioxidant, cytotoxic and, anti-inflammatory activities (Zakaria et al., 2011) that have beenassociated as parts of the mechanism of anti-carcinogenesis (Arya and Kumar, 2011). It is eminent that there is association between themechanisms of oxidation inflammation, and cancer and the capability to hamper any of the mechanisms will definitely lead to the inhibitionof the others. Nitric oxide (NO), for instance, is generated /liberated under the action of inflammatory stimuli such as the reactive oxygenspecies (ROS). Hence, inhibition of ROS results in decrease production of NO, which, in turn, triggered the antioxidant, anti-inflammatory,and anticancer processes (Robak and Gryglewski 1988; Middleton et al. 2000; Olszanecki et al. 2002). Interestingly, M. malabathricum hasbeen reported to exert antioxidant and anti-inflammatory activities and, therefore, is believed to also possess anticancer activity. Moreover,the free radical scavenging effect is suggested to play important role through which this plant might exert its anticancer activity. However,since these association have not been proven scientifically, the present study was carried out to study the anti-carcinogenesis activity ofmethanol extract of M. malabathricum leaves (MEMM) using the 7,12-dimethylbenz(α)anthracene (DMBA)/crotton oil-induced mouse skin carcinogenesis model.Materials and MethodPlant leaves collection and preparation of methanol extractThe leaves of M. malabathricum were collected around Serdang, Selangor, Malaysia between September and October, 2011 and avoucher specimen (SK 1986/11) was deposited at the herbarium of Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM), Serdang,Selangor, Malaysia. The MEMM was prepared according to the method described in detail by Zakaria et al. (Zakaria et al., 2011). Basically,from 410 g of dried leaves soaked in methanol (Fischer Scienctific, UK) three times (1:20 (w/v); room temperature for 72 hr) yieldedapproximately 108.34 g of dried MEMM. The concentrated MEMM was further dried in an oven (40oC) to eliminate excess methanol residue.Prior to use, the MEMM was dissolved in acetone (Mallinckrodt Chemicals, USA) to make up the concentrations of 30, 100 and 300 mg/kg.Forty eight (48) ICR strain female mice (6-7 weeks old; 20-28 g) were used in this study (Abel et al., 2009). The animals were keptin the Animal House, Faculty of Medicine and Health Sciences, UPM and cared according to the standard procedure described elsewhereOoi et al., Afr J Tradit Complement Altern Med. (2014) 11(4):66-70http://dx.doi.org/10.4314/ajtcam.v11i4.1167(Zimmermann,1983). An ethical approval was received from the Animal Care and Use Committee of UPM (UPM/FPSK/PADS/BR-UUH/00432). The animals were divided into six groups (n=8) prior to the experimentation.Three days before the application of DMBA, an area with 2 cm x 2 cm of dorsal skin area of mice was shaved for application ofchemicals. Briefly, in the initiation phase, each mouse in group II, III, IV, V and VI received a single dose of 100 µl/100 µg DMBA (Sigma-Aldrich Co, USA) on the dorsal shaved skin area and each animal was held for a few seconds to ensure the chemical distributed evenly on theshaved area before being released back into the cage. On the other hand, all mice in group I only received 100 µl acetone.Briefly, during the promotion phase, all mice in group I (vehicle control) received only 100 µl of acetone throughout promotionphase. On the other hand, the respective mice in group II (carcinogen control) and III (positive control) received 100 μl of acetone or 10 mg/kg curcumin (Sigma-Aldrich Co, USA) followed 30 min later by the application of 100 µl of croton oil (Sigma-Aldrich Co, USA)throughout the promotion phase. Lastly, the respective mice in group IV, V and VI were treated with 100 µl of 30, 100 and 300 mg/kgMEMM, 30 minutes before the topical application of 100 µl croton oil throughout the promotion phase. All treatments were applied twiceweekly for fifteen weeks of promotion period.Throughout the 15 weeks of experiment, the dorsal skin area was observed carefully for any papilloma growth as described in detailby (Abel et al., 2009). Briefly, several important parameters such as body weight, latency period of tumor formation, percentage of tumorincidence, tumor burden and tumor volume were also recorded weekly. The experiment was terminated on the 15th week, and all animals weresacrificed by using diethyl ether and the dorsal skin was shaved and dissected out for histo-pathological analysis. The dissected skin tissuesthen underwent the standard Haematoxylin and Eosin (H&E) staining procedure as described by Abel et al.All data are presented as Mean  Standard Error of Mean (S.E.M.). The data were statistically analyzed using the one-way analysisof variance (ANOVA) followed by LSD multiple comparison test. Values with p ≤ 0.05 were considered as statistically significant.   ResultsThe body weight of mice receiving acetone, 10 mg/kg curcumin or, 30 and 100 mg/kg MEMM increased gradually until week 15th(data not shown). The initial and final body weight of mice in Group I to VI is shown in Table 1. Comparison between the initial (beforepromotion) and final body weight (15th week after promotion) demonstrated significant (p<0.05) increased in body weight in all groupsexcept for the group receiving 300 mg/kg MEMM. This finding was supported by data for percentage of tumor incidence obtained at the endof experimental weeks (15th week) wherein group II showed the highest tumor incidence (75.0%) in comparison with Group I, III, IV, V andI II IIIIV V VIFigure 1. The gross appearance of the skin papilloma on the shaved dorsal skin of DMBA/croton oil-treated mice following thetopical administration of MEMM or curcumin on week 15th. Representative photographs for each experimental group captured at the end ofstudy. (I) Group I: vehicle control only applied with acetone; (II) Group II: carcinogen control only applied with 1% croton oil at thepromotion stage and without any pre-treatment; (III) Group III: positive control treated with 10 mg/kg body weight curcumin; (IV) Group IV:treated with 30 mg/kg body weight MEDL; (V) Group V: treated with 100 mg/kg body weight MEDL, and; (VI) Group VI: treated with 300mg/kg body weight MEDL.Ooi et al., Afr J Tradit Complement Altern Med. (2014) 11(4):66-70http://dx.doi.org/10.4314/ajtcam.v11i4.1168I II III IV V VIFigure 2:. Microphotographs of skin tissue from different groups after pretreatment with acetone, croton oil, curcumin or MEMM. (40x magnification). I) Microphotographs of skin tissue from acetone (vehicle control)showed well-defined histology of skin with intact basement membrane. The basement membrane is well defined, with no signs of invasions into dermis layer.; II) Microphotographs of skin tissue from pre-treatment with 1%croton oil (carcinogen control) showed the keratin pearls (KP) found within the papilloma (yellow arrow) with mild hyperchromatism (HC) (black arrow) surrounding the pearls. III) Microphotographs of skin tissue from pre-treatment with 10 mg/kg curcumin (positive control) showing the papilloma and hyperplasia (red arrow) with no disruption of basement membrane (black arrow); IV) Microphotographs of skin tissue from pre-treatment with30 mg/kg MEMM showing a clear structure of papilloma and hyperplasia of epidermis (EH) ( red arrow) with defined basement membrane; V) Microphotographs of skin tissue from pre-treatment with 100 mg/kg MEMMshowing the papilloma was protruding from the epidermis (red arrow) with hyperplastic changes of epidermis (black arrow), the structure inside the papilloma are hair follicle (hf); and VI) Microphotographs of skin tissuefrom pre-treatment with 300 mg/kg MEMM showing the papilloma has growth outwards the skin with hyperplasia on epidermis. Abbreviation: HK - Hyperchromatins ; EH – Epidermis hyperplasia; KP – Keratin pearls; S -EHHKKPSEHHKEHKP EHHK KPHCEHHKKPOoi et al., Afr J Tradit Complement Altern Med. (2014) 11(4):66-70http://dx.doi.org/10.4314/ajtcam.v11i4.1169VI (tumor incidence range between 0-62.5%) (Table. 1). There are significant (p < 0.05) increased in tumor incidence with increase in periodof exposure to carcinogenic agent, which were significantly (p<0.05) reduced following pre-treatment with curcumin or MEMM (100 and300 mg/kg). Groups receiving curcumin and MEMM exerted significant (p<0.05) reduction in tumor burden and tumor volume at the end of15th week with the latter occurred in a dose-dependent manner (Table 1). The tumor burden increased from Group I to Group VI followed byGroup III, V, IV and II. The largest tumor volume was found in Group VI followed by Group V, IV, II and III. Group I has no tumor volume.As for the latency period of tumor formation, curcumin and MEMM (30, 100 and 300 mg/kg) delayed the formation of tumor until week 10th,8th, 9th and 14th, respectively in comparison to the negative control group (week 7th) (data not shown). The overall gross appearance of theshaved dorsal skin of a representative mouse from all the treatment groups on week 15 prior to dissection supported the ability of curcuminand MEMM to exert anti-carcinogenic activity (Figure 1) and supported by the microscopic examination (Figure 2).DiscussionResults obtained from the present study confirmed the novel anti-carcinogenic activity of MEMM in DMBA/croton oil-inducedmice. The DMBA initiates skin tumor formation through the generation ROS that are responsible for genetic materials damage (Das et al.,2010). Therefore, the chemoprevention activity of MEMM could be attributed to the extract’s antioxidant activity, which, in turn, could beassociated with the extract’s phytochemical constituents (e.g. flavonoids, triterpenes, tannins and saponins) (Surh, 2002; Zakaria et al., 2011).MEMM has demonstrated high antioxidant and free radical scavenging potential when assessed by the DPPH radical scavenging andsuperoxide scavenging assays suggesting these effects direct or indirect contribution to the extract’s observed anti-carcinogenic activity (Li etal. 2008). Compounds like flavonoids (Ferguson et al. 2004), saponins (Lemeshko et al. 2006), triterpenes (Kimura et al. 2002) and tannins(Ait Mbarek et al. 2007) have all been reported to possess antioxidant and anticancer activities and are, therefore, believed to actsynergistically within the respective extract to cause the observed anti-carcinogenic activity.In term of the mechanisms of anti-carcinogenesis that might be modulated by MEMM, several pathways could be suggested basedon the flavonoids detected in MEMM. Flavonoids are important in the impediment of various chronic maladies involving oxidative stress anddemonstrated antioxidant, anti-inflammatory and anticancer activities via the in vivo and in vitro models (Lee et al. 2003; Middleton et al.2000; Robak and Gryglewski, 1988; Calixto et al. 2003; 2004). Moreover, flavonoids also regulated the expression of pro-inflammatorygenes (i.e. nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2)) (Dawson and Snyder, 1994; Kim et al., 2004).Interestingly, we have also detected quercitrin in the MEMM (Mamat et al., 2013), which have been earlier reported to exert in vitroanti-carcinogenic activity against mouse JB6 cells and antioxidant activities (Ding et al., 2010). Moreover, quercitrin down-regulatestransactivation of AP-1 and NF-κB induced by UVB or TPA. Quercitrin was also shown to inhibit several biochemical pathways (i.e.  ERKs, p38 kinase, and JNKs) related to cancer via the inhibition of MAPKs phosphorylation. In addition, quercitrin stimulated the activation of NF-E2-related factor (Nrf2) and GST ARE-luciferase activity.On the other hand, curcumin had been chosen as the positive control in comparison to the MEMM since curcumin contains anti-inflammatory properties which lead to its effectiveness as a chemo-preventive agent (Sonavane et al., 2012). In conclusion, MEMM exhibitedchemoprevention activity, which is attributed, partly, to its antioxidant activity and phyto-constituents, particularly, quercitrin.Conflict of interest: The authors declare that there is no conflict of interest.AcknowledgementThe authors thanked the Ministry of Science, Technology and Innovation (MOSTI), Malaysia for providing the Science Fundresearch grant (Ref. no.: 06-01-04-SF1127) and the University Putra Malaysia (UPM), Selangor, Malaysia for providing the ResearchUniversity Grant Schemes 2012 (Ref. no.: 04-02-12-2019RU) to carry out the experiment. The authors also thanked the Faculty of Medicineand Health Sciences, UPM, Selangor, Malaysia for providing the facilities to carry out the experiments.References1. Abel EL, Angel JM, Kiguchi K, DiGiovanni J. Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications. NatProtoc 2009; 4: 1350-1362.2. Ait Mbarek L, Ait Mouse H, Elabbadi N, Bensalah M, Gamouh A, Aboufatima R et al. Anti-tumor properties of blackseed (Nigellasativa L.) extracts. Braz J Med Biol Res 2007; 40: 839–847.3. Arya P, Kumar M. Chemoprevention by Triticum Aestivum of mouse skin carcinogenesis induced by DMBA and croton oil –Association with oxidative status. Asian Pacific J Cancer Prev 2011; 12: 143-148.4. Calixto JB, Otuki MF, Santos ARS. Anti-inflammatory compounds of plant origin. Part I. Action on arachidonic acid pathway, nitricoxide and nuclear factor -B (NFB). Planta Med 2003; 69: 973–983.5. Calixto JB, Campos MM, Otuki MF, Santos ARS. Anti-inflammatory compounds of plant origin. Part II. Modulation of pro-inflammatory cytokines, chemokines and adhesion molecules. Planta Med 2004; 70: 93–103.6. Das I, Das S, Saha T. Saffron suppresses oxidative stress in DMBA-induced skin carcinoma: A histopathological study. Acta Histochem2010; 12: 317-327.7. Dawson TM, Snyder SH. Gases as biological messengers: Nitric oxide and carbon monoxide in the brain. J Neurosci 1994; 14: 5147–5159.8. Ferguson PJ, Kurowska E, Freeman DJ, Chambers AF, Koropatnick DJ. A flavonoid fraction from cranberry extract inhibitsproliferation of human tumor cell lines. J Nutr 2004; 134: 1529-1535.9. Kim HP, Son KH, Chang HW, Kang SS. Anti-inflammatory plant flavonoids and cellular action mechanisms. J Pharmacol Sci 2004; 96:229–245.10. Kimura Y, Taniguchi M, Baba K. Antitumor and antimetastatic effects on liver of triterpenoid fractions of Ganoderma lucidum:Mechanism of action and isolation of an active substance. Anticancer Res 2002; 22: 3309–3318.11. Lee KW, Kim YJ, Lee HJ, Lee CY. Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than teas and red wine. JAgric Food Chem 2003; 51: 7292–7295.Ooi et al., Afr J Tradit Complement Altern Med. (2014) 11(4):66-70http://dx.doi.org/10.4314/ajtcam.v11i4.117012. Lemeshko VV, Haridas V, Quijano Perez JC, Gutterman JU. Avicins, natural anticancer saponins, permeabilize mitochondrialmembranes. Arch Biochem Biophys 2006; 454: 114–122.13. Li F, Wang F, Yu F, Fang Y, Xin Z, Yang F, Xu J, Zhao L, Hu Q. In vitro antioxidant and anticancer activities of ethanolic extract ofselenium-enriched green tea. Food Chem 2008; 111: 165–170.14. Mamat SS, Kamarolzaman MFF, Yahya F, Mahmood ND, Shahril MS, Jakius KF et al. Methanol extract of Melastoma malabathricumleaves exerted antioxidant and liver protective activity in rats. BMC Comp Alternat Med 2013; 13: 326.15. Middleton Jr. E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation,heart disease, and cancer. Pharmacol. Rev 2000; 52: 673–751.16. Nouri K. Skin Cancer. 2nd ed. New York : McGraw-Hill; 2008; pp. 1-17.17. Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 1988; 37: 837–841.18. Sonavane K, Phillips J, Ekshyyan O, Moore-Medlin T, Gill JR, Rong X et al. Topical curcumin-based cream is equivalent to dietarycurcumin in a skin cancer model. J Skin Cancer 2012; 2012, Article ID147863; doi: 10.1155/2012/147863.19. Surh YJ. Anti-tumor promoting potential of selected spice ingredients with antioxidative and anti-inflammatory activities: A short review.Food Chem Toxicol 2002; 40: 1091-1097.20. Zakaria ZA, Rofiee MS, Mohamed AM, Teh LK, Salleh MZ. In vitro antiproliferative and antioxidant activities and total phenoliccontents of the extracts of Melastoma malabathricum leaves. J Acupunct Meridian Stud, 4: 248-256.21. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 6: 109–110.

English

CHEMOPREVENTIVE ACTIVITY OF METHANOL EXTRACT OF MELASTOMA MALABATHRICUM LEAVES IN DMBA-INDUCED MOUSE SKIN CARCINOGENESIS

https://journals.athmsi.org/index.php/ajtcam/article/download/1978/1983

CHEMOPREVENTIVE ACTIVITY OF METHANOL EXTRACT OF MELASTOMA MALABATHRICUM LEAVES IN DMBA-INDUCED MOUSE SKIN CARCINOGENESIS

Abstract

Similar works

Full text

Available Versions

ATHMSI Journals