Phytochemical constituents and antioxidant, antimicrobial and antimelanogenic activities of Rhodomyrtus tomentosa (aiton) HASSK. Leaf 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.

This paper is available online at http://stdb.hnue.edu.vn

PHYTOCHEMICAL CONSTITUENTS AND ANTIOXIDANT,

ANTIMICROBIAL AND ANTIMELANOGENIC ACTIVITIES

OFRhodomyrtus 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.

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

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 tomentosaleaves in relation to their phytochemical constituents

* Materials

Bacillus subtilis, Staphylococcus aureus, Pseudomonas sp., Escherichia coli, and

Candidasp 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 400C, 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 00C for further use

* 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 (500µL) 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 (100µL) were mixed with 20 µL 10% Al(NO3)3, 20µL1M 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 20µL of extract solutions

at various concentrations ranging from 5 - 500µg/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

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 hundredµL 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 20µL of phosphate buffer at pH of 6.8 and 40 µL of

5 mM L-DOPA before being combined with 40µL 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

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 many medicinal 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

Ethyl acetate fraction 253,09 ± 12,59 171,67 ± 5,99

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

2.2.3 Antioxidant activity

Table 2 DPPH scavenging activities of R tomentosa leaf extracts

Sample

(µg/mL)

DPPH scavenging activity (%) IC50

(µg/mL)

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 IC50values

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 500µg/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 ± 1.55µg/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

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 3

Table 3 Antimicrobial activity of R tomentosa leaf extracts

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

-Crude

n-Hexane 15.00±1.73 15.67±0.58 9.33±2.51 4.33±0.58 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

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 4 Antimelanogenic activity of R tomentosa leaf extracts

Sample ( µg/mL) Tyrosinase inhibition activity (%)

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 2 mg/mL concentration) followed by the water fraction and the ethyl acetate fraction

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)

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