Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities of Rhodomyrtus tomentosa (Aiton) Hassk. Leaf extract

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol. 58, No. 9, pp. 123-131 This paper is available online at 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. 1. 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. Content 2.1. 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 0C, 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 0C 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 2 cm from the edge, using capillary tubes. Solvent including toluene, ethyl acetate, acetone and formic acid 5 : 3 : 1 : 1 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 1 mg/mL and standard solutions were from 0 - 0.25 mg/mL. Sample or standard solution (25 µL) was mixed with Folin-Ciocalteu reagent (500 µL). After 5 min, 500 µL of 10% sodium carbonate was added. The mixture was kept at room temperature for 90 min. 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 1 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 min 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 5 - 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 min. The control was prepared with ethanol instead of extracts. Ascorbic acid was used for comparison with extracts. The reduction of DPPH free radicals was measured at 517 nm. DPPH scavenging activity was calculated using the following formula: DPPH scavenging activity (%) = [(Acontrol – Asample)/(Acontrol)]× 100 where Acontrol represents the absorbance of the control and Asample is the absorbance of the test sample. * Antimicrobial activity 125 Le Thi Phuong Hoa and Hoang Thi Nga The antimicrobial activity was tested against Bacillus subtilis, Staphylococcus aureus, Pseudomonas sp., Escherichia coli, and Candida sp. by using the agar well diffusion method. The 24 hrs culture broth of the test microorganisms (approximately 1 × 108 CFU/mL) was spread onto petri plates containing MPA (meat-peptone-agar) for bacteria and glucose yeast extract agar for fungi. Wells of 10 mm diameter were made aseptically in the inoculated plates. Each extract was dissolved in ethanol to a final concentration of 10 mg/mL. Ethanol served as a negative control and 0.4% chloramphenicol was used as the positive control. Aliquots of 100 µL of the extracts and controls were added into the respectively labeled wells. The plates were incubated at 30 0C for 24 hrs for bacteria and 36 hrs for fungi in an upright position. Antimicrobial activity was determined by measuring the diameter of the inhibition zone formed around the well. * Antimelanogenic activity Antimelanogenic activity was estimated by observing tyrosinase inhibitory activity in a cell-free system according to the procedure of Yagi et al. [14] using L-DOPA as the substrate. Kojic acid was used for comparison. One hundred µL of each test sample (kojic acid or extract solutions at concentrations of 1, 1.5 and 2 mg/mL in a 0.175 M phosphate buffer at pH 6.8) was mixed with 20 µL of phosphate buffer at pH of 6.8 and 40 µL of 5 mM L-DOPA before being combined with 40 µL of 110 U/mL mushroom tyrosinase. The reaction mixture was incubated at 30 oC for 2 min. The amount of DOPAchrome was determined at 475 nm. The percent inhibition of tyrosinase activity was calculated as follows: Tyrosinase inhibition (%) = [(A− B)/A]× 100 where A stands for the absorbance at 475 nm without the test sample, and B is the absorbance at 475 nm with the test sample. 2.2. Results and discussion 2.2.1. Thin layer chromatography The crude methanol extract and fractions of Rhodomyrtus tomentosa (Aiton) Hassk. were subjected to thin layer chromatographic analysis to find the presence of a number of chemical constituents. TLC chromatogram of different extracts, developed using a toluene, ethyl acetate, acetone, formic acid solution 5 : 3 : 1 : 1 as a solvent system and visualized using 10% H2SO4, is shown in Figure 1. TLC of R. tomentosa leaf extracts allowed the identification of various compounds. The dominant compounds are terpenoids, revealed by pink and purple bands, chrolophylls (green) and flavonoids (yellow and orange). The ethyl acetate fraction had the highest number of bands (15 bands) with different Rf values (data not shown). The crude extract and the n-hexane fraction gave 13 and 12 bands, respectively, while the water fraction showed only one band. The ethyl acetate fraction had a thick yellow band, suggesting the presence in high content of flavonoids, as compared to the n-hexane and the water fraction which requires further characterization. 126 Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities... Figure 1. TLC chromatogram of Rhodomyrtus tomentosa leaf extracts in a toluene/ethyl acetate/acetone/formic acid (5 : 3 : 1 : 1) solvent system 30: crude methanolic extract, Et: ethyl acetate fraction, HE: n-hexane and H2O: water fraction 2.2.2. Total phenolic and flavonoid content Phenolic compounds are commonly found in various parts of all sorts of plants. They have been widely investigated in manymedicinal plants and plant foods because they are responsible for multiple biological effects [9,13]. The level of phenolic compounds and flavonoids in the crude methanolic extract of R. tomentosa leaves and its three fractions are shown in Table 1. Table 1. Total phenolic and flavonoid contents of R. tomentosa leaf extract Sample Phenolic content Flavonoid content (mg GAE/g) (mg QE/g) Crude extract 151,98 ± 10,87 63,04 ± 2,68 Ethyl acetate fraction 253,09 ± 12,59 171,67 ± 5,99 n-Hexane fraction 76,22 ± 4,44 68,10 ± 4,01 Water fraction 20,11 ± 7,69 7,23 ± 2,15 GAE: gallic acid equivalents, QE: quercetin equivalents Total phenolic and flavonoid contents in the ethyl acetate fraction were the highest in all samples. It seems that phenolic compounds and flavonoids of R. tomentosa leaves were most distributed in this fraction. The total content of phenolics and flavonoids in the ethyl acetate fraction was approximately three times more than that in the n-hexane fraction. The water fraction had very low amount of phenolics and flavonoids. The result confirms the chromatogram analysis on biochemical constituents of three fractions. The level of phenolic compounds in R. tomentosa leaves was much higher than in the fruit (24 ± 0.4 mg GAE/g) as compared to the previous report [3]. Lai et al. [5] identified 19 different phenolic compounds from mature R. tomentosa fruits. Stilbenes and ellagitannins predominate, followed by anthocyanins, flavonols, and gallic acid. Phenolics exhibit a wide variety of beneficial biological activities including antiviral, antibacterial, antihypertensive, antilipoperoxidant, hepatoprotective, anti-inflammatory and anti-carcinogenic actions. The result showed a high content of phenolics in the ethyl acetate fraction of R. tomentosa leaf extract, among which flavonoids are important components. Some of its biological effects could be attributed to the presence of these valuable constituents. 127 Le Thi Phuong Hoa and Hoang Thi Nga 2.2.3. Antioxidant activity Table 2. DPPH scavenging activities of R. tomentosa leaf extracts Sample (µg/mL) DPPH scavenging activity (%) IC50 (µg/mL)5 10 50 100 500 EtoAc 6.27±0.61 8.24±4.04 36.10±5.15 59.80±2.57 87.27±1.83 69.50±1.55 n-Hexane 2.19±1.07 4.37±0.49 8.72±3.95 18.41±1.49 57.24±6.81 - Water 1.59±0.33 2.94±11.99 4.83±1.74 5.65±2.45 25.13±1.79 - Ascorbic acid 12.19±2.01 25.02±0.39 89.89±0.26 93.80±0.35 95.06±0.31 21.71±0.79 EtoAc: ethyl acetate, (-): not determined Antioxidants are believed to be highly effective in the management of tissue impairment caused by reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals [3, 9]. DPPH free radical scavenging assay is an easy, rapid and sensitive method which is widely used for antioxidant screening of plant extracts. In the presence of an antioxidant, DPPH radicals obtain one more electron, decolorized, and the absorbance decreases as a result [11]. Table 2 shows the DPPH scavenging activity of extracts from R. tomentosa leaves in different concentrations and their IC50 values. It was observed that extracts of R. tomentosa leaves had a dose-dependent DPPH scavenging potential. The ethyl acetate fraction of the leaf methanolic extract showed higher activity than the n-hexane and water fractions. At a concentration of 500 µg/mL, the scavenging activity of the ethyl acetate fraction was nearly 90%, while at the same concentration that of the other fractions were much lower (57.24% and 25.24%). The free radical scavenging capacity of the ethyl acetate fraction reached half-maximal inhibition level at 69.50± 1.55 µg/mL. The antioxidant activity of R. tomentosa leaf extracts showed a tight relationship to their phenolic and flavonoid content. Free radicals contribute to many forms of human illness such as aging, cancer, atherosclerosis, coronary heart ailment, diabetes, Alzheimer’s disease and other neurodegenerative disorders. They are chemical species containing one or more unpaired electrons that makes them highly unstable and able to cause damage to other molecules as they extract electrons from them in order to attain stability [3, 6, 9]. The DPPH scavenging capacity of the ethyl acetate fraction of R. tomentosa leaves may be due to their reducing actions which might donate hydrogen to a free radical, reducing it to a nonreactive species. Acetone 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 diffusionmethod. 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 3. Table 3. Antimicrobial activity of R. tomentosa leaf extracts Sample Zone of inhibition (mm) B. subtilis S. aureus E. coli Pseudomonas sp. Candida sp. Control (+) 39.00±2.00 44.00±1.73 31.67±2.08 15.57±3.99 34.67±2.08 Control (-) - - - - - Crude extract 11.67±1.15 7.67±0.58 9.33±0.58 - 9.00±1.00 EtoAc 10.33±1.15 7.00±1.00 10.33±1.53 - 6.67±1.53 n-Hexane 15.00±1.73 15.67±0.58 9.33±2.51 4.33±0.58 10.67±1.53 Water - 12.67±1.15 7.00±1.41 - - (-): 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