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Preliminary screening to assess the antimicrobial activities of extracts of evergreen woody species from South Korea against Staphylococcus aureus
J Plant Biotechnol 2020;47:90-99
Published online March 31, 2020
© 2020 The Korean Society for Plant Biotechnology.

Bo Kook Jang · Lai Won Chi · Ju Sung Cho · Cheol Hee Lee

Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju, 28644, Korea
Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju, 28644, Korea
LOY Culture & Art College, Incheon, 21366, Korea
Correspondence to: e-mail: leech@chungbuk.ac.kr
Received January 20, 2020; Revised March 11, 2020; Accepted March 11, 2020.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
This study aimed to screen for plants with antimicrobial potential among the evergreen woody species of South Korea that are used for horticulture and landscaping and to provide basic information about plants with proven antimicrobial activity to underpin future research. The plant materials were extracted under various conditions, and the antimicrobial activities of the extracts were evaluated by agar diffusion assay. The screening tests demonstrated that the crude extracts of 43 species had inhibitory effects against S. aureus. The inhibitory activities of four species (Elaeocarpus sylvestris, Camellia japonica, Cleyera japonica, and Quercus salicina) were relatively higher than that of the synthetic antimicrobial agents methylparaben and phenoxyethanol. The highest inhibitory activity was observed with the leaf extracts (extracted with methanol for 30 minutes) of E. sylvestris, based on induction of the largest inhibition zone of 23.3 mm in size. In addition, solvent fractions of E. sylvestris were evaluated. The largest inhibitory zone of 23.1 mm was observed for the n-butanol fraction, which is likely to contain effective compounds that exhibit inhibitory activity against S. aureus. In contrast, n-hexane and residual aqueous fractions showed no antimicrobial activity. Overall, our findings confirm that evergreen woody plants native to South Korea have potential antimicrobial activity.
Keywords : Agar diffusion assay, Elaeocarpus sylvestris var. ellipticus, Methylparaben, n-butanol fraction, Phenoxyethanol, Ultrasonic extraction
Introduction

Disease has affected the survival of humans throughout their history, but it has been controlled by the development of medicines (Ríos and Recio 2005). Materials obtained from the natural environment have been the source for many medicines, and antibiotics and drugs have been derived directly or indirectly from endophytic microbes or plants (Newman et al. 2003). Although antibiotics obtained from many microorganisms have been developed and used, only a few plant materials with antimicrobial potential have been revealed, and these may possibly be used for antibiotic production (Newman and Cragg 2016).

The number of plant-derived antimicrobial products is increasing, but there are still many synthetic antimicrobials. Synthetic antimicrobials or antibiotics can effectively control microorganisms, but the development of antimicrobial resistance in microorganisms owing to the continued use of antibiotics has raised considerable issues. This has promoted research into plant-derived natural products (Chandra et al. 2017; Iwu et al. 1999; Lee and Shin 2010). The antimicrobial activity of plant-derived extracts has been demonstrated to be attributed to essential oils (Haloui et al. 2015), alkaloids (Altameme et al. 2015; Deng et al. 2011), terpenes (Moghrovyan et al. 2019), and flavonoids (Cushnie and Lamb 2005; Górniak et al. 2019). Microorganisms are less resistant to these plant-derived compounds than to synthetic compounds, and, thus, they can be used safely without causing side effects.

Staphylococcus aureus is a dangerous and versatile pathogen that can cause a multitude of diseases in addition to food poisoning (Otto 2014). Most frequently, it causes skin infections and infections of the respiratory tract; it also causes diseases, such as pneumonia, toxic shock syndrome, scalded skin syndrome, and osteomyelitis. S. aureus has been controlled by the development of antibiotics, such as penicillin, but the emergence of its resistance to several antibiotics has created new problems (Lowy 1998; Lowy 2003). Typically, resistance to penicillin, methicillin, quinolone, and vancomycin is observed. The use of antibiotics against S. aureus is still effective, but the number of antibiotic-resistant S. aureus strains is predicted to increase. It is, thus, essential to find new drugs that can avoid microbial resistance.

Evergreen species native to South Korea have been planted in gardens, parks, and urban green spaces; along city streets; and for landscaping and windbreak forests since the late 1970s (Lee and Kim 2017). Such species have been planted for ornamental, horticultural, and landscaping purposes, and every year, many pruning by-products (mostly twigs and leaves) are produced. It is necessary to recycle these by-products to reduce processing costs and create a new added value. Some woody species have been used in traditional herbal medicines (Fenglin et al. 2004); extracts of Elaeocarpus sylvestris var. ellipticus are antimicrobial (Piao et al. 2009; Taguri et al. 2006) and extracts of Quercus species have shown antioxidant activity (Tuyen et al. 2016). In addition, the antimicrobial activity of extracts of several woody species have been reported in South Korea (Cho et al. 2018; Jang et al. 2018). Therefore, the present study was carried out to evaluate the potential for antimicrobial activity in 64 evergreen woody species native to South Korea. Our findings provide information on the antimicrobial activity of Korean evergreen woody plants and contribute to the development of plant-derived natural products.

Materials and Methods

Plant collection

Leaves and twigs of 64 evergreen woody plants were collected in January 2013. The collection location for each sample is listed in Table 1. Forty-three species were collected from Wando, Jeollanam-do, South Korea and 21 species were collected from Jeju-si, Jeju-do.

List of plants used in this study

Sample number Family Speciesa Korean nameb Collectionregion
(1) Apocynaceae Nerium oleander L. Hyeop-juk-do Jeju-si
(2) *Trachelospermum asiaticum var. majus Makino Baek-hwa-deung Wando
(3) Trachelospermum asiaticum (Siebold & Zucc.) Nakai Ma-sak-jul Wando
(4) Aquifoliaceae Ilex cornuta Lindl. & Paxton Ho-rang-ga-si-na-mu Wando
(5) *Ilex Xwandoensis C.F.Mill. & M.Kim Wan-do-ho-rang-ga-si-na-mu Wando
(6) Ilex crenata Thunb. Kkwang-kkwang-na-mu Wando
(7) Ilex rotunda Thunb. Meon-na-mu Jeju-si
(8) Araliaceae Dendropanax morbiferus H.Lév. Hwang-chil-na-mu Wando
(9) Fatsia japonica (Thunb.) Decne. & Planch. Pal-son-i Wando
(10) Hedera rhombea (Miq.) Siebold ex Bean Song-ak Wando
(11) Caprifoliaceae Viburnum odoratissimum Ker Gawl. ex Rümpler var. awabuki (K.Koch) Zabel A-wae-na-mu Wando
(12) Cephalotaxaceae *Cephalotaxus koreana Nakai Gae-bi-ja-na-mu Wando
(13) Cupressaceae Chamaecyparis obtusa (Siebold & Zucc.) Endl. Pyeon-baek Wando
(14) Juniperus rigida Siebold & Zucc. No-gan-ju-na-mu Wando
(15) Daphniphyllaceae Daphniphyllum macropodum Miq. Gul-geo-ri-na-mu Wando
(16) Elaeagnaceae Elaeagnus glabra Thunb. Bo-ri-jang-na-mu Jeju-si
(17) Elaeagnus umbellata Thunb. Bo-ri-su-na-mu Wando
(18) Elaeocarpaceae Elaeocarpus sylvestris (Lour.) Poir. Dam-pal-su Jeju-si
(19) Ericaceae *Rhododendron brachycarpum auct. Man-byeong-cho Wando
(20) Vaccinium bracteatum Thunb. Mo-sae-na-mu Wando
(21) Vaccinium oxycoccos L. Neon-chul-wol-gyul Jeju-si
(22) Fagaceae Castanopsis sieboldii (Makino) Hatus. Gu-sil-jat-bam-na-mu Wando
(23) Quercus acuta Thunb. Buk-ga-si-na-mu Wando
(24) Quercus gilva Blume Gae-ga-si-na-mu Jeju-si
(25) Quercus glauca Thunb. Jong-ga-si-na-mu Wando
(26) Quercus myrsinifolia Blume Ga-si-na-mu Wando
(27) Quercus salicina Blume Cham-ga-si-na-mu Wando
(28) Flacourtiaceae *Xylosma japonica A.Gray San-yu-ja-na-mu Jeju-si
(29) Gramineae *Phyllostachys pubescens J.Houz. Juk-sun-dae Jeju-si
(30) Sasa quelpaertensis Nakai Je-ju-jo-rit-dae Jeju-si
(31) Hamamelidaceae Distylium racemosum Siebold & Zucc. Jo-rok-na-mu Jeju-si
(32) Illiciaceae *Illicium anisatum L. But-sun-na-mu Jeju-si
(33) Lardizabalaceae *Stauntonia hexaphylla (Thunb.) Decne. Meol-kkul Wando
(34) Lauraceae *Actinodaphne lancifolia (Blume) Meisn. Yuk-bak-na-mu Wando
(35) *Cinnamomum loureirii Nees Yuk-gye-na-mu Jeju-si
(36) Cinnamomum yabunikkei H. Ohba Saeng-dal-na-mu Wando
(37) Cinnamomum camphora (L.) J. Presl Nok-na-mu Jeju-si
(38) Laurus nobilis L. Wol-gye-su Wando
(39) Litsea japonica (Thunb.) Juss. Kka-ma-gwi-jjok-na-mu Wando
(40) Machilus japonica Siebold & Zucc. Sen-dal-na-mu Wando
(41) Machilus thunbergii Siebold & Zucc. Hu-bak-na-mu Wando
(42) Neolitsea aciculata (Blume) Koidz. Sae-deok-i Jeju-si
(43) Neolitsea sericea (Blume) Koidz. Cham-sik-na-mu Jeju-si
(44) Loganiaceae *Gardneria insularis Nakai Yeong-ju-chi-ja Wando
(45) Magnoliaceae Magnolia grandiflora L. Tae-san-mok Jeju-si
(46) Moraceae *Ficus oxyphylla Miq. Mo-ram Wando
(47) Myricaceae Myrica rubra (Lour.) Siebold & Zucc. So-gwi-na-mu Jeju-si
(48) Myrsinaceae Ardisia crenata Sims Baek-ryang-geum Jeju-si
(49) Oleaceae Ligustrum lucidum W.T.Aiton Dang-gwang-na-mu Wando
(50) Osmanthus fragrans Lour. Mok-seo Wando
(51) Osmanthus fragrans var. aurantiacus Makino Geum-mok-seo Wando
(52) Pinaceae Pinus thunbergii Parl. Gom-sol Wando
(53) Rosaceae Eriobotrya japonica (Thunb.) Lindl. Bi-pa-na-mu Wando
(54) Rhaphiolepis indica var. umbellata (Thunb.) H.Ohashi Da-jeong-keum-na-mu Wando
(55) Rutaceae *Citrus xjunos Siebold ex Yu. Tanaka Yu-ja-na-mu Wando
(56) Schisandraceae Kadsura japonica (L.) Dunal Nam-o-mi-ja Wando
(57) Taxaceae Torreya nucifera (L.) Siebold & Zucc. Bi-ja-na-mu Wando
(58) Taxodiaceae Cryptomeria japonica (Thunb. ex L.f.) D.Don Sam-na-mu Wando
(59) Theaceae Camellia japonica L. Dong-baek-na-mu Wando
(60) Cleyera japonica Thunb. Bi-jju-gi-na-mu Jeju-si
(61) Eurya emarginata (Thunb.) Makino U-muk-sa-seu-re-pi Jeju-si
(62) Eurya japonica Thunb. Sa-seu-re-pi-na-mu Wando
(63) Ternstroemia gymnanthera (Wight & Arn.) Sprague Hu-pi-hyang-na-mu Wando
(64) Thymelaeaceae Daphne odora Thunb. Seo-hyang Jeju-si

*Indicates unresolved name or synonym (TPL, 2013).

aIndicates accepted name, refer to The Plant List (TPL), 2013.

bRefers to Korea National Arboretum, 2017.


Preparation of plant extracts

The samples were freeze dried (-70°C to -85°C, FD8512, Ilshin Biobase, Dongducheon, Korea) for 48 hours after washing in running tap water, except that part of each sample was retained for moisture content measurement as described below. The dry samples were finely ground (Hood mixer FM-681C, Hanil, Seoul, Korea) and stored in a deep freezer at -70°C (MDF-U73V, Sanyo Electric Co., Ltd., Osaka, Japan) as powder for use in our experiments. To screen the samples for antimicrobial activity, 1 g of dry leaf samples (powder) was ultrasonically extracted with methanol solvent for 30 minutes, and the inhibition zone was measured by agar diffusion assay.

Part of the fresh sample for each of the 64 species was used to measure moisture content. Fresh weight (W1) was measured before oven drying at 60°C for 48 hours. After drying, the weight (W2) was measured again, and the moisture content (%) was calculated as follows: (W1 - W2) / W1. The content of soluble solids was also measured. The fresh weight of leaves and twigs was measured before freeze drying for 48 hours. A 5 ml sample of each 50-fold concentrated extract (methanol) was placed in an evaporating dish and dried at 60°C in an oven for 48 hours. After transferring it to a desiccator, the soluble solid content (g·g-1) of the extract was measured.

Microorganisms

The microorganisms used in the experiment were provided by the Korean Collection for Type Cultures (KCTC) (Staphylococcus aureus KCTC 1927). A culture medium in which Mueller-Hinton broth (275730, Difco Laboratories, Sparks, MD, USA) was mixed with 1.2% agar powder was prepared and cultured at 3~5-week intervals to maintain S. aureus activity. Two weeks before the experiment, 100 µl of S. aureus culture was activated in 10 ml broth at intervals of 3~5 days.

Extraction method

All powder samples were extracted with an ultrasonic cleaner (300 × 240 × 145 mm, 5510-DTH, Bransonic, Danbury, CT, USA) for 30 minutes, and then 1 g of the powder was added to a 200-ml glass bottle, and 30 ml methanol was added until the powder was immersed. The sample was extracted in the ultrasonic cleaner for 30 minutes. The extraction was repeated twice for each sample, and finally, two extracts were mixed and used. The resultant was filtered under reduced pressure with a vacuum pump using 55-mm quantitative filter paper. The filtered extract was quantified at 50-fold concentration (1 g / 50 ml) by adding a solvent (methanol). The extract was then concentrated under reduced pressure again and dissolved in dimethyl sulfoxide to adjust the final concentration to 50 mg·ml-1. The extract was stored in a -70°C deep freezer until use in the experiment. In all experiments, the concentration of the crude extract was diluted to 50 mg·ml-1. The concentration of the extract to be used in the agar diffusion assay was diluted to 2.00 mg/disc.

Agar diffusion assay

The agar diffusion assay was performed following the method of Shin (2010) with minor modifications. Preparation of the agar diffusion plate used in the experiment was as follows: 1) The broth medium with 0.7% soft agar was sterilized and stored at 60°C in a constant temperature water bath. 2) The optical density of the activated S. aureus culture was adjusted to 1.0 using a visible spectrophotometer. 3) The adjusted S. aureus culture was added to the sterilized broth medium at 1% (v/v) ratio and stirred before pipetting 12 ml per petri dish.

On the surface of the agar diffusion plate, an 8-mm paper disk injected with the extract (2 mg / disc (40 µl)) was attached. The paper-disk-attached medium was cooled for 1 hour at 4°C and then transferred to a growth chamber at 37°C and incubated for 24 hours. After incubation, the inhibition zone, including the diameter of the paper disc, was measured. As controls, phenoxyethanol (0H2108, Junsei Chemical Co., Ltd., Tokyo, Japan) and methylparaben (8K5015, Junsei Chemical Co.) at concentrations of 0.40, 1.00, 2.00, and 4.00 mg / disc were used. The same method was used for all experiments.

Antimicrobial activities according to the extraction conditions of four species

Four species (E. sylvestris, Camellia japonica, Cleyera japonica, and Quercus salicina), the extracts of which induced large inhibition zones, were selected from the 64 species screened (Table 2). The antimicrobial activity of the extracted parts (leaf and twig), extraction solvents (distilled water, 80% ethanol, and methanol), and extraction time (15, 30, and 45 minutes) were compared.

Antimicrobial activities of extracts obtained from the leaves of 64 species against Staphylococcus aureus

Samplenumber Concentration (mg/disc) Inhibition zone (mm) Sample number Inhibition zone (mm)
Methylparabena 0.40 -b (29) -
1.00 - (30) 10.10 ± 2.40 f
2.00 11.20 ± 2.90 ef (31) 15.45 ± 1.35 c
4.00 13.80 ± 2.30 d (32) 10.35 ± 0.45 f

Phenoxyethanola 0.4 - (33) 8.55 ± 0.35 h
1 - (34) 10.35 ± 0.30 f
2 8.50 ± 0.70 f (35) 9.95 ± 0.05 g
4 9.80 ± 1.80 g (36) -

(1) 2.00 - (37) 10.00 ± 0.00 f
(2) 2.00 - (38) 9.40 ± 0.10 g
(3) 2.00 - (39) -
(4) 2.00 - (40) 9.20 ± 1.00 g
(5) 2.00 - (41) -
(6) 2.00 - (42) -
(7) 2.00 - (43) -
(8) 2.00 15.75 ± 0.85 c (44) -
(9) 2.00 - (45) 14.85 ± 0.95 cd
(10) 2.00 - (46) 8.75 ± 0.15 gh
(11) 2.00 12.45 ± 0.35 e (47) 11.70 ± 0.40 ef
(12) 2.00 - (48) 9.10 ± 0.20 g
(13) 2.00 11.50 ± 0.40 ef (49) 10.45 ± 0.35 f
(14) 2.00 10.40 ± 0.20 f (50) -
(15) 2.00 8.35 ± 0.15 h (51) -
(16) 2.00 - (52) 12.50 ± 0.50 e
(17) 2.00 - (53) 12.50 ± 0.00 e
(18) 2.00 23.30 ± 0.80 a (54) 11.30 ± 0.00 ef
(19) 2.00 12.65 ± 0.85 e (55) 8.05 ± 0.05 h
(20) 2.00 12.15 ± 0.05 e (56) 10.15 ± 0.15 f
(21) 2.00 11.90 ± 0.10 ef (57) 9.00 ± 0.10 g
(22) 2.00 13.80 ± 1.60 d (58) 12.65 ± 0.55 e
(23) 2.00 12.40 ± 0.60 e (59) 16.70 ± 1.30 bc
(24) 2.00 9.35 ± 0.15 g (60) 18.95 ± 0.15 b
(25) 2.00 11.00 ± 0.50 f (61) 10.40 ± 0.40 f
(26) 2.00 9.50 ± 0.50 g (62) 12.30 ± 0.02 e
(27) 2.00 18.65 ± 2.30 b (63) 16.00 ± 2.10 bc
(28) 2.00 8.95 ± 0.15 gh (64) 12.35 ± 0.45 e

Mean ± S.E. (n = 10). Different lowercase letters indicate a significant difference at p < 0.05 based on Duncan's multiple range test.

aPositive control.

bNot detected.


Antimicrobial activities according to solvent fractions of E. sylvestris

Solvent fractionation of the final E. sylvestris extract was carried out to determine inhibitory activity. Fractions were obtained using a ratio of powdered sample:distilled water: n-hexane (v:v:v) at 1:9:10. The n-hexane aqueous layer was sequentially partitioned into chloroform, ethyl acetate, and n-butanol (Fig. 1). Each solvent fraction was concentrated under reduced pressure and freeze dried to obtain a powdered sample, which was dissolved in distilled water and used in the antimicrobial experiment in the same manner as the agar diffusion method.

Fig. 1. Schematic diagram of the solvent fractionation of the MeOH extract using n-hexane, chloroform, ethyl acetate, and n-butanol

Statistical analysis

For the agar diffusion assay, treatments included three replicates. In the case of the extraction conditions of the four species and solvent fractions of E. sylvestris, six replicates were performed twice. SAS software version 9.3 (SAS Institute Inc., Cary, NC, USA) was used to calculate the mean ± standard error for each treatment, and a factorial analysis was performed using Duncan’s multiple range test with a significance level of p < 0.05.

Results

Screening for antimicrobial activity of 64 evergreen woody species

The antimicrobial activity against S. aureus was observed in crude extracts of 43 of the 64 evergreen woody species used in this study (Table 2). Forty-three leaf extracts exhibited inhibition zones of 8.05~23.30 mm. The E. sylvestris leaf extract had the largest inhibition zone (23.30 mm), followed by Cleyera japonica (18.95 mm), Q. salicina (18.65 mm), and Camellia japonica (16.70 mm).

Soluble solid and moisture contents of the 64 evergreen woody species are shown in Table 3. The moisture content ranged from 41.58 to 79.33% for leaf samples and 32.80 to 65.68% for twig samples. Soluble solid content ranged from 50 to 400 mg·g-1 for leaf samples and 100 to 340 mg·g-1 for twig samples.

Soluble solid and moisture contents of extracts obtained from leaves and twigs of 64 species

Sample number Soluble solids content (mg·g-1) Moisture content (%) Sample number Soluble solids content (mg·g-1) Moisture content (%)




Leaf Twig Leaf Twig Leaf Twig Leaf Twig
(1) 160 140 51.54 56.89 (33) 290 150 63.09 59.62
(2) 150 130 58.16 46.72 (34) 160 180 71.54 52.55
(3) 100 270 56.05 32.80 (35) 120 270 57.97 40.74
(4) 190 240 46.63 47.75 (36) 270 320 47.39 50.28
(5) 240 180 65.49 40.72 (37) 200 260 53.15 51.77
(6) 280 340 57.77 55.80 (38) 160 150 58.31 49.43
(7) 180 220 64.98 51.81 (39) 320 210 57.00 57.13
(8) 270 250 71.19 59.83 (40) 260 310 55.21 52.72
(9) 260 300 60.31 60.28 (41) 150 220 50.24 52.49
(10) 230 270 54.77 59.14 (42) 200 340 51.20 53.49
(11) 210 180 65.86 65.42 (43) 210 220 55.08 54.90
(12) 330 210 69.21 64.67 (44) 400 250 49.81 51.49
(13) 270 150 50.17 33.72 (45) 220 300 63.79 50.76
(14) 270 180 52.52 46.83 (46) 50 270 48.99 46.20
(15) 150 320 67.40 62.34 (47) 200 140 65.86 52.38
(16) 260 340 65.66 56.90 (48) 360 240 51.75 65.68
(17) 140 200 60.27 54.32 (49) 240 180 46.64 48.72
(18) 350 140 63.91 58.39 (50) 270 340 79.33 41.64
(19) 240 240 54.44 48.09 (51) 270 220 48.03 45.21
(20) 370 220 63.86 52.63 (52) 190 250 58.21 52.13
(21) 210 220 57.76 53.86 (53) 220 180 57.08 57.69
(22) 190 330 55.00 51.74 (54) 270 210 59.59 53.82
(23) 220 270 49.10 51.20 (55) 220 150 49.72 47.64
(24) 180 150 52.25 55.08 (56) 290 180 74.47 64.52
(25) 330 100 44.59 40.53 (57) 320 270 60.87 56.24
(26) 120 190 44.14 42.63 (58) 340 320 67.17 52.37
(27) 190 240 54.83 43.10 (59) 200 260 54.16 42.60
(28) 200 250 49.76 43.97 (60) 140 150 59.73 48.94
(29) 230 300 55.84 53.87 (61) 240 200 48.71 51.26
(30) 150 270 51.37 54.87 (62) 220 210 61.35 43.97
(31) 260 180 41.58 52.25 (63) 220 310 59.94 53.87
(32) 280 210 66.94 59.32 (64) 330 220 75.46 53.30

Antimicrobial activities according to extraction conditions of four species

The extracts of the four plants exhibited different levels of inhibitory activity against S. aureus depending on the extraction conditions used (Table 4). The leaf extracts of E. sylvestris and Cleyera japonica were found to have a stronger inhibitory effect on S. aureus than the twigs. However, for Camellia japonica, the twig extract was more effective. Leaf extracts of E. sylvestris exhibited strong inhibitory effects regardless of the solvent used: methanol (23.3 mm, 30 min), 80% ethanol (21.8 mm, 15 min), and distilled water (21.4 mm, 45 min). In the extracts of Cleyera japonica, the inhibition zone against S. aureus was observed in methanol (21.3 mm, 30 min) and 80% ethanol (20.6 mm, 15 min), but no inhibition zone was formed in distilled water. Camellia japonica twigs showed the highest inhibitory activity with 80% ethanol for 45 minutes (18.5 mm). Antimicrobial activity against S. aureus was observed in both Q. salicina leaf (19.7 mm) and twig (19.3 mm) extracts. In these plants, strong inhibition zones were observed at 45 min extraction with 80% ethanol regardless of the extraction part.

Antimicrobial activities of extracts obtained from leaves and twigs of four species gainst Staphylococcus aureus at varying concentrations according to extraction conditions, compared with those of chemical controls

Part Concentration (mg/disc) Inhibition zone (mm)

Sample (18) Sample (59) Sample (60) Sample (27)
Methylparabena 0.40 -b - - -
1.00 - - - -
2.00 11.0 ± 0.41 gh 11.0 ± 0.41 i 11.0 ± 0.41 g 11.0 ± 0.41 hi
4.00 13.7 ± 0.26 fg 13.7 ± 0.26 f-h 13.7 ± 0.26 e 13.7 ± 0.26 de

Phenoxyethanola 0.40 - - - -
1.00 - - - -
2.00 8.7 ± 0.22 i 8.7 ± 0.22 j 8.7 ± 0.22 i 8.7 ± 0.22 j
4.00 9.9 ± 0.10 hi 9.9 ± 0.10 i 9.9 ± 0.10 gh 9.9 ± 0.10 i
Solvent Extraction time (min)
Leafc MeOH 15 19.5 ± 0.62 b-d 13.4 ± 2.05 f-h 17.5 ± 0.20 c 17.6 ± 0.03 b
30 23.3 ± 0.79 a 13.4 ± 0.56 f-h 21.3 ± 0.64 a 16.6 ± 0.20 bc
45 18.6 ± 0.55 c-e 13.8 ± 0.27 f-h 17.1 ± 0.41 c 17.3 ± 0.58 b

80% EtOH 15 21.8 ± 0.55 ab 12.6 ± 1.80 h 20.6 ± 0.74 ab 16.1 ± 0.50 c
30 21.2 ± 0.55 a-c 13.8 ± 0.58 f-h 19.8 ± 0.54 b 17.2 ± 0.28 bc
45 18.9 ± 0.20 c-e 16.9 ± 0.19 bc 21.3 ± 0.46 a 19.7 ± 0.17 a

DW 15 18.0 ± 0.46 de 14.5 ± 2.14 ef - 12.7 ± 0.75 ef
30 19.6 ± 0.64 b-d 13.9 ± 0.09 fg - 10.8 ± 0.54 hi
45 21.4 ± 1.03 a-c 13.0 ± 0.49 gh - 12.1 ± 0.44 fg
Twigc MeOH 15 11.5 ± 0.50 gh 16.2 ± 0.09 b-d 10.3 ± 0.25 gh 11.5 ± 0.33 gh
30 14.3 ± 0.50 f 15.6 ± 0.56 de 12.1 ± 0.25 f 10.8 ± 0.33 hi
45 18.8 ± 0.18 c-e 16.0 ± 0.31 cd 15.2 ± 0.64 d 14.3 ± 0.19 d

80% EtOH 15 16.4 ± 0.14 ef 17.2 ± 0.22 b 9.4 ± 0.23 hi 14.6 ± 0.32 d
30 14.5 ± 0.14 f 18.5 ± 0.22 a 8.7 ± 0.10 i 18.6 ± 0.10 a
45 18.7 ± 0.29 c-e 17.4 ± 0.40 b 9.3 ± 0.29 hi 19.3 ± 0.18 a

DW 15 - 10.2 ± 0.28 i - 16.1 ± 0.50 c
30 - - - 16.8 ± 0.25 bc
45 - - - 13.8 ± 0.23 de
Part (A) *** *** *** *
Solvent (B) NS *** ** ***
Time (C) * *** ** ***
A × B NS *** *** ***
A × C *** NS *** **
B × C ** *** *** ***
A × B × C NS ** *** ***

Mean ± S.E. (n = 10). Different lowercase letters indicate a significant difference at p < 0.05 based on Duncan's multiple range test.

NS, *, **, ***; nonsignificant or significant at p < 0.05, 0.01, or 0.001, respectively.

aPositive control.

bNot detected.

cConcentration of 2.00 mg/disc was used for all plant extracts


Antimicrobial activities according to solvent fractions of E. sylvestris

The inhibition zone of each solvent fraction was measured (Table 5). The n-butanol fraction showed the largest inhibition zone at 23.1 mm (Fig. 2), whereas no inhibition zone was observed for the residual aqueous or n-hexane fractions. Inhibition zones of 13.5 and 11.4 mm were observed for the chloroform and ethyl acetate fractions, respectively. Therefore, substances that exhibit antimicrobial activity against S. aureus are most likely to be found in the n-butanol fraction.

Antimicrobial activities of solvent fractions obtained from leaves of Elaeocarpus sylvestris (18) against Staphylococcus aureus

Control/fraction Concentration (mg/disc) Inhibition zone (mm)
Methylparabena 0.40 -b
1.00 10.0 ± 0.12 e
2.00 13.9 ± 0.07 c
4.00 15.0 ± 0.12 b

Phenoxyethanola 0.40 -
1.00 -
2.00 -
4.00 11.9 ± 0.52 d

Whole extract 2.00 23.3 ± 0.79 a

n-hexane fraction 2.00 -
Chloroform fraction 2.00 13.5 ± 0.23 c
Ethyl acetate fraction 2.00 11.4 ± 0.13 d
n-butanol fraction 2.00 23.1 ± 0.25 a
Residual aqueous fraction 2.00 -

Mean ± S.E. (n = 10). Different lowercase letters indicate a significant difference at p < 0.05 based on Duncan's multiple range test.

aPositive control.

bNot detected.



Fig. 2. Antimicrobial activities of different solvent fractions of Elaeocarpus sylvestris (18) MeOH extract against Staphylococcus aureus represented by inhibition zones. 1, n-hexane; 2, chloroform; 3, ethyl acetate; 4, n-butanol; 5, residual aqueous

Discussion

We screened evergreen woody plants in South Korea for antimicrobial activity. The plant groups used in the study were more effective than the synthetic antimicrobial agents used as controls, and the results suggested that they have potential for new plant-derived natural products. The leaf extracts of four species strongly inhibited S. aureus, the causative agent of skin problems. However, antimicrobial activity differed in the different parts (leaves and twigs), and the soluble solid content examined was also different. In previous reports, it was demonstrated that there were differences in the compounds of essential oils in the different extracted parts (Hafsé et al. 2013; Haloui et al. 2015). The results of the present study indicate that the leaf extracts exhibited stronger antimicrobial activity than twig extracts, suggesting that they contain compounds with high antimicrobial activity.

The active ingredient of the plant-derived extract shows different effects depending on the extraction conditions, even from the same plant. Depending on the solvent used, extractions can be used to extract water-soluble or water-insoluble compounds. In the present study, the crude extracts of four species extracted with organic solvents exhibited higher overall antimicrobial activity than those extracted with distilled water. Previous reports on Achillea millefolium subsp. (Candan et al. 2003), Hypericum capitatum and H. scabrum (Sokmen et al. 1999) demonstrated that water-insoluble extracts contain more antimicrobial compounds than water-soluble extracts. These results suggest that compounds that exhibit inhibitory activity against S. aureus may be water-insoluble rather than water-soluble.

Non-polar extraction with organic solvents is more effective for extracting compounds from plants, including non-polar compounds (Webster et al. 2008). The crude extract of E. sylvestris, which showed the strongest antimicrobial activity, contained a mixture of various chemical compounds (i.e., 1,2,3,4,6-penta-O-galloyl-β-d-glucose, coniferyl alcohol, umbelliferone, scopoletin, β-sitosterol, and daucosterol). To isolate the active component, fractionation according to solvent polarity was performed. Different solvent fractions exhibited different levels of antibiotic activity, and the strongest inhibitory activity against S. aureus was observed for the n-butanol fraction.

According to a recent study, the leaves of E. sylvestris contain gallotannin precursors 1,2,3,4,6-penta-O-galloyl-β-d-glucose, which show high antioxidant potential (Piao et al. 2009). Taguri et al (2006) reported that E. sylvestris extracts inhibited bacterial growth. Furthermore, 70% ethanol extract of E. sylvestris inhibited cytomegalovirus and varicella-zoster virus (Bae et al. 2017; To et al. 2014). It is considered that the leaves of E. sylvestris may contain a variety of inhibitory compounds, especially the n-butanol fraction, which may contain compounds that exhibit potent antimicrobial activity against S. aureus. This inhibitory activity was consistent across the results of two screening experiments using crude extracts.

In conclusion, 43 of the 64 evergreen woody species in this study showed potential antimicrobial activity against S. aureus. In particular, the strongest antimicrobial activity was observed for the n-butanol fraction of E. sylvestris leaf crude extract. It is expected that this fraction can be developed as a plant-derived natural product in the future. Our study also indicates that extracts from Korean evergreen woody plants have antimicrobial activity against S. aureus.

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