This fraction showed the highest antioxidant activity by dose-dependent free radical scavenging action (IC50 69.50 ± 1.55 µg/mL). R. tomentosa leaf extracts, especially the n-hexane fraction, also exhibited strong antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi as well as moderate inhibitory effect on L-DOPA (L-3,4-dihydroxyphenylalanine) oxidase activity of tyrosinase in the melanin biosynthesis pathway.
JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol 58, No 9, pp 123-131 This paper is available online at http://stdb.hnue.edu.vn PHYTOCHEMICAL CONSTITUENTS AND ANTIOXIDANT, ANTIMICROBIAL AND ANTIMELANOGENIC ACTIVITIES OF Rhodomyrtus tomentosa (AITON) HASSK LEAF EXTRACT Le Thi Phuong Hoa and Hoang Thi Nga Faculty of Biology, Hanoi National University of Education Abstract The antioxidant, antimicrobial and antimelanogenic activities of various leaf extracts of Rhodomyrtus tomentosa (Aiton) Hassk were investigated together with their phytochemical constituents Among the extracts, the ethyl acetate fraction had the highest level of phenolics and flavonoids (253.09 ± 12.59 mg gallic acid equivalent/g of extract and 171.67 ± 5.99 mg quercetin equivalent/g of extract) This fraction showed the highest antioxidant activity by dose-dependent free radical scavenging action (IC50 69.50 ± 1.55 µg/mL) R tomentosa leaf extracts, especially the n-hexane fraction, also exhibited strong antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi as well as moderate inhibitory effect on L-DOPA (L-3,4-dihydroxyphenylalanine) oxidase activity of tyrosinase in the melanin biosynthesis pathway Further work is suggested to characterize bioactive compounds from ethyl acetate and n-hexane fractions for use in pharmaceutical applications, especially in skin care Keywords: Rhodomyrtus tomentosa, antioxidant, antimicrobial, antimelanogenic Introduction In recent years, the search for natural sources for bioactivities, such as antioxidant and antimicrobial properties, has been rising with the global concern for preventive healthcare and the problem of drug-resistant bacteria In Vietnam, as in other tropical South East Asian countries, there is high diversity of plants, among which a number have traditionally been used to treat ailments and as food Rhodomyrtus tomentosa (Aiton) Hassk., commonly known as rose myrtle or sim in Vietnamese, is an evergreen shrub, abundant in the midlands, with dark purple edible bell-shaped fruits Ripe fruits have been utilized in wine production, to treat anemia during pregnancy, to reduce hemorrhoids and for gynaecopathy The buds and leaves have been used to treat diarrhea, hemostasis, gastritis and enteritis [2] Received November 19, 2013 Accepted December 23, 2013 Contact Le Thi Phuong Hoa, e-mail address: lephhoa@yahoo.com 123 Le Thi Phuong Hoa and Hoang Thi Nga R tomentosa has been reported to be a good source of antibiotics Extract from this plant exhibited strong inhibitory activity against Gram-positive bacteria with low MIC (minimum inhibitory concentration) such as Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Streptococcus pyogenes and Propionibacterium acnes [7, 8, 12] Rhodomyrtone, an acylphloroglucinol component from this plant, has been recently reported to be an effective antibacterial agent against various bacterial pathogens that cause skin and respiratory tract infection [8, 12] A range of compounds, including stilbenes, ellagitannins, anthocyanins, flavonols and gallic acid, have been identified as components of this plant [5] An acetone extract of R tomentosa leaves is a proven potential antioxidant capable of strong lipid peroxidation inhibition and reducing ability in vitro, and effectively reducing lipid peroxidation and balance of free radical scavenging enzymes in experimental mice undergoing CCl4-induced oxidative stress [6] Recently, Jeong et al [4] reported the in vitro and in vivo anti-inflammatory activity of a methanolic extract from R tomentosa leaves, acting to inhibit the production of nitric oxide and prostaglandin E2 in lipopolysaccharide-activated cells and peritoneal macrophages and ameliorating both gastritis and colitis symptoms in mice Although R tomentosa extracts have been extensively investigated for their antibacterial activity, there is still limited data regarding its gram-negative antibacterial, antifungal activity as well as other bioactivities like skin depigmentation Therefore, this study aims to evaluate the antimicrobial, antioxidant and antimelanogenic activities of methanol extract from Rhodomyrtus tomentosa leaves in relation to their phytochemical constituents 2.1 Content Material and methods * Materials Bacillus subtilis, Staphylococcus aureus, Pseudomonas sp., Escherichia coli, and Candida sp were obtained from the National Institute of Hygiene and Epidemiology Quercetin, 1,1-diphenyl-2-picryl hydrazyl (DPPH), ascorbic acid, mushroom tyrosinase, L-3,4-dihydroxyphenylalanine (L-DOPA) and kojic acid was purchased from Sigma Chemicals (MO, USA) Gallic acid and Folin-Ciocalteu reagent were obtained from Merck Chemicals (Darmstadt, Germany) * Sample preparation Fresh leaves were washed with distilled water to remove adhering debris and dust, and then freeze dried to constant weights The dried tissues were ground to powder and then extracted with methanol in an ultrasonic bath for 30 mins at room temperature The extraction was performed in three replicates The extracts were mixed and concentrated in a rotary evaporator at 40 C, and then freeze dried The crude extract was further fractionated in distilled water, n-hexane and ethyl acetate The three fractions were concentrated by vacuum evaporation and freeze dried All of the extracts were stored at 0 C for further use 124 Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities * Thin layer chromatography The extracts were prepared at the concentration of 10 mg/mL in absolute ethanol Each extract was applied as a single spot in a row along one side of the precoated silica gel aluminum plate 60F254 , about cm from the edge, using capillary tubes Solvent including toluene, ethyl acetate, acetone and formic acid : : : was used as the mobile phase The plate was sprayed with 10% sulfuric acid, heat dried, and observed under visible light A qualitative evaluation of the plate was done by determining the migration behavior of the separated substances given in the form of Rf value * Determination of total phenolic content The total phenolic content was estimated employing the method of Sapkota et al [11], using Folin-Ciocalteu reagent with gallic acid as the standard Sample solutions were prepared in ethanol at a concentration of mg/mL and standard solutions were from - 0.25 mg/mL Sample or standard solution (25 µL) was mixed with Folin-Ciocalteu reagent (500 µL) After min, 500 µL of 10% sodium carbonate was added The mixture was kept at room temperature for 90 The absorbance was then measured at 725 nm The amounts of total phenolics were calculated using a gallic acid calibration curve The results were expressed as mg gallic acid equivalents (GAE) per g dry weight of each extract * Determination of total flavonoid content The total flavonoid content of each extract was determined making used of the method described by Sapkota et al [11] using quercetin as the standard Extracts were diluted with 80% aqueous ethanol to arrive at a concentration of mg/mL Quercetin solutions were prepared in the same manner to the range of 0, 0.05, 0.1, 0.2 and 0.3 mg/mL Different quercetin solutions and extracts (100 µL) were mixed with 20 µL 10% Al(NO3 )3 , 20 µL1M K – acetate and 860 µL 80% ethanol After standing for 40 at room temperature, the absorbance of the mixture was determined spectrophotometrically at 415 nm The results were expressed in mg quercetin/g dry weight by comparison with the quercetin standard curve * Antioxidant activity Antioxidant activity was evaluated through free radical scavenging potential using DPPH according to Blois [1] The reaction mixture contained 20 µL of extract solutions at various concentrations ranging from - 500 µg/mL in ethanol and 180 µL of 0.3 mM DPPH solution The samples were allowed to stand in a dark place at room temperature for 30 The control was prepared with ethanol instead of extracts Ascorbic acid was used for comparison with extracts The reduction of DPPH free radicals was me extract of R tomentosa leaves was reported to have strong ferric reducing ability, 2.7 - 3.0-fold higher than gallic acid and ellagic acid [6] Although the DPPH radical scavenging activity of the ethyl acetate fraction was less than that of ascorbic acid, the result showed that the extract has a proton-donating ability and might act as primary antioxidant Furthermore, it is likely that the activity of the ethyl acetate fraction is due to the high content of phenolic compounds, which have redox properties, adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides [9,13] Previous research has revealed the highly positive 128 Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities correlation between total phenolic content and antioxidant activity [3,11] Many previous reports also showed that phenolic compounds were major antioxidant constituents in medicinal herbs, vegetables, fruits and spices [9] 2.2.4 Antimicrobial activity Methanolic extract of R tomentosa leaves and its three fractions were subjected to a screening of antimicrobial activity on two Gram-positive bacterial strains (B subtilis and S aureus), two Gram-negative bacterial strains (E coli and Pseudomonas sp.) and a fungal strain (Candida sp.) by the agar well diffusion method The results were recorded as the absence or presence and the diameter of zones of microbial growth inhibition around the wells, as shown in Table Table Antimicrobial activity of R tomentosa leaf extracts Sample Control (+) Control (-) Crude extract EtoAc n-Hexane Water B subtilis 39.00±2.00 - Zone of inhibition (mm) S aureus E coli Pseudomonas sp 44.00±1.73 31.67±2.08 15.57±3.99 - Candida sp 34.67±2.08 - 11.67±1.15 7.67±0.58 9.33±0.58 - 9.00±1.00 10.33±1.15 15.00±1.73 - 7.00±1.00 15.67±0.58 12.67±1.15 10.33±1.53 9.33±2.51 7.00±1.41 4.33±0.58 - 6.67±1.53 10.67±1.53 (-): no inhibition The results of antibacterial activity of R tomentosa leaf extracts are consistent with previous reports regarding Gram-positive bacteria [7, 8, 12] All of the extracts showed antibacterial activity to two Gram-positive bacteria except the water fraction had an effect only on S aureus However, the extracts also exhibited antimicrobial activity against Gram-negative bacteria and fungi The n-hexane fraction showed stronger activity, followed by the ethyl acetate fraction and then the water fraction It is the only extract having an inhibitory effect on the growth of Pseudomonas sp although the inhibition is expressed at a modest level The antibacterial action of R tomentosa leaf extracts is still unknown However, it is supposed that it is related to the action of phenolic compounds like flavonoids and terpenoids, which were reported to have antiviral and antibacterial activities [13] Rhodomyrtone, an acylphloroglucinol component purified from ethyl acetate extract of R tomentosa leaves, is thought to contribute to the antibacterial activity of R tomentosa leaf extracts [7] It has been recently reported as a natural antibiotic against a range of Gram-positive bacteria including those in mediated infections of skin and respiratory tracts and even some antibiotic-resistant bacteria [7,8,12] Further chemical characterization of the n-hexane fraction from R tomentosa leaves may reveal new compounds with antibacterial and antifungal activities Our results for the antibacterial assays support the popular usage of R tomentosa leaves in treating diarrhea, hemostasis, gastritis and enteritis 129 Le Thi Phuong Hoa and Hoang Thi Nga 2.2.5 Antimelanogenic activity Previous reports showed the potential use of R tomentosa leaf extract as a natural antioxidant [6] as well as a topical therapeutic agent useful in treating skin disease like acne due to its antibacterial activity against skin pathogens, its low toxicity to human fibroblast [12] and its anti-inflammatory effect [4] In order to further characterize its effects on skin, we attempted to test the antimelanogenic activity of R tomentosa leaf extract using a tyrosinase inhibition assay Table Antimelanogenic activity of R tomentosa leaf extracts Tyrosinase inhibition activity (%) Sample (µg/mL) 1.5 EtoAc 6.88 ± 13.56 35.83 ± 5.68 39.30 ± 7.40 n-Hexane 34.13 ± 16.94 43.07 ± 8.51 54.21 ± 15.71 Water 34.07 ± 12.31 44.19 ± 8.02 48.43 ± 6.86 Melanin synthesis in human skin is regulated by melanogenic enzymes such as tyrosinase, the enzyme catalyzing the two first reactions in the biosynthesis pathway of melanin One is the hydroxylation of tyrosine to form L-DOPA, and the next is the oxidation of L-DOPA to dopaquinone which leads to the polymerizing of brown pigments [10] Hence, the tyrosinase inhibitory assay has commonly been used to investigate potential depigmentation agents As shown in Table 4, R tomentosa leaf extracts had inhibitory effects on the DOPA oxidase activity of mushroom tyrosinase at a moderate level The activity was concentration dependent The inhibitory activity increased with the increase in concentration of the extracts The n-hexane fraction showed highest tyrosinase inhibition activity (54.21% at mg/mL concentration) followed by the water fraction and the ethyl acetate fraction Conclusion The results of this study indicated that R tomentosa leaf extracts, especially the ethyl acetate fraction, had good antioxidant activity by radical scavenging in correlation with high phenolic content This fraction also possesses the highest flavonoid content and a number of phytochemical constituents R tomentosa leaf extracts exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi as well as tyrosinase inhibitory activity The present findings encourage further characterization of bioactive compounds in the ethyl acetate and n-hexane fractions and their mechanism of action for better application as natural antioxidants and antibiotics, especially in skin care Acknowledgements This work was supported by Ministry of Education and Training, Vietnam through Hanoi National University of Education (Project number B2013-17-40) 130 Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities REFERENCES [1] Blois M.S., 1958 Antioxidant determination by the use of a stable free radical Nature, Vol 181, pp 1199-1200 [2] Do Tat Loi, 2004 Vietnamese medicinal plants and remedies Medical Publishing House, Hanoi, p 434 [3] Huang W.Y., Cai Y.Z., Corke H., and Sun M., 2010 Survey of antioxidant capacity and nutritional quality of selected edible and medicinal fruit plants in Hong Kong Journal of food composition and analysis, Vol 23, pp 510-517 [4] Jeong D., Yang W.S., Yang Y., Nama G., Kim J.H., Yoon D.H., Noh H.J., Lee S., Kim T.W., Sung G.H., Cho J.Y., 2013 In vitro and in vivo anti-inflammatory effectof Rhodomyrtus tomentosa methanol extract Journal of Ethnopharmacology, Vol 146, pp 205-213 [5] Lai T.N.H., Herent M.F, Quetin-Leclercq J., Nguyen T.B.T., Rogez H., Larondelle Y., and André C.M., 2012 Piceatannol, a potent bioactice stilbene, as major phenoic component in Rhodomyrtus tomentosa Food Chemistry, Vol 138, pp 1421-1430 [6] Lavanya G., Voravuthikunchai S.P., and Towatana N.H., 2012 Acetone ectract from Rhodomyrtus tomentosa: a potent natural antioxidant Evidence-Based Complementary and Alternative Medicine, Vol 2012 (2012), p [7] Limsuwan S., Trip E.N., Kouwen T.R.H.M., Piersma S., Hiranrat A., Mahabusarakam W., Voravuthikunchai S.P., van Dijl J.M., and Kayser O., 2009 Rhodomyrtone: A new candidate as natural antibacterial drug from Rhodomyrtus tomentosa Phytomedicine, Vol.16, pp 645–651 [8] Limsuwan S., Kayser O., and Voravuthikunchai S.P., 2012 Antibacterial activity of Rhodomyrtus tomentosa (Aiton) Hassk Leaf extract against clinical isolates of Streptococcus pyogenes Evidence-Based Complementary and Alternative Medicine, Vol 2012 (2012), p [9] Jan Pokorný, 1991 Natural antioxidants for food use Trends in Food Science & Technology, Vol 2, pp 223-227 [10] Parvez S., Kang M., Chung H.S., Cho C., Hong M.C., Shin M.K., and Bae H., 2006 Survey and mechanism of skin depigmenting and lightening agents Phytotherapy research, Vol 20, pp 921-934 [11] Sapkota K., Park S.E., Kim J.E., Kim S., Choi H.S., Chun H.S., and Kim S.J., 2010 Antioxidant and antimelanogenic properties of chestnut flower extract Bioscience, Biotechnology and Biochemistry, Vol 74 (8), pp 1527-1533 [12] Saising J., and Voravuthikunchai S.P., 2012 Anti Propionibacterium acnes activity of rhodomyrtone, an effective compound from Rhodomyrtus tomentosa (Aiton) Hassk leaves Anaerobe, Vol 18, pp 400-404 [13] Svobodová A., Psotová J and Walterová D., 2003 Natural phenolics in the prevention of UV-induced skin damage, a review Biomedical Papers, Vol 147 (2), pp 137-135 [14] Yagi A., Kanbara T., and Morinobu N., 1987 Inhibition of mushroom-tyrosinase by aloe extract Planta Med, Vol 53 (6), pp 515-517 131 ... dried All of the extracts were stored at 0 C for further use 124 Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities * Thin layer chromatography The extracts... highest flavonoid content and a number of phytochemical constituents R tomentosa leaf extracts exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi as well as... Ministry of Education and Training, Vietnam through Hanoi National University of Education (Project number B2013-17-40) 130 Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic