The Korean Society For Biotechnology And Bioengineering
[ Research Paper ]
Korean Society for Biotechnology and Bioengineering Journal - Vol. 33, No. 2, pp.76-82
ISSN: 1225-7117 (Print) 2288-8268 (Online)
Print publication date 30 Jun 2018
Received 31 Jan 2018 Revised 11 Apr 2018 Accepted 12 Apr 2018
DOI: https://doi.org/10.7841/ksbbj.2018.33.2.76

In vitro Screening of Jeju Island Plants for Cosmetic Ingredients

Mi Jin Kim1 ; Taek Kyu Jung1 ; Moo-Han Kim1 ; Kyung-Sup Yoon2, *
1Skin Science R&D Center, Saimdang Cosmetics Co., Ltd.
2Department of Chemistry and Cosmetics, College of Natural Science, Jeju National University
화장품 소재 개발을 위한 제주 식물 탐색
김미진1 ; 정택규1 ; 김무한1 ; 윤경섭2, *
1(주)사임당화장품 피부과학연구소
2제주대학교 자연과학대학 화학·코스메틱스학과

Correspondence to: Department of Chemistry and Cosmetics, College of Natural Science, Jeju National University, Korea Tel: +82-64-754-3541, e-mail: ksyoonjh@jejunu.ac.kr


© 2018 The Korean Society for Biotechnology and Bioengineering

Abstract

To identify new active cosmetic ingredients of natural origin, we screened the biological activities of 50 different kinds of plants from Jeju Island, the southernmost island of the Korean Peninsula. In this study, plant extracts were screened for inhibitory effects on elastase, nitric oxide, and melanin production, and for free radical scavenging effects. Eight plants, Viburnum erosum Thunb., Trachelospermum asiaticum var. intermedium Nakai, Sageretia theezans Brongn., Sorbus alnifolia var. hirtella T. Lee, Staphylea bumalda DC., Pourthicea villosa var. brunnea Nak., Illicium religiosum S. et Z., and Castanopsis cuspidata var. sieboldii Nakai were potent inhibitors of elastase (over 80% inhibition at 1 mg/mL). Elaeocarpus sylvestris var. Ellipticus (Thunb.) Hara (IC50: 13.99 μg/mL), Sapium sebiferum (L.) ROXB. (IC50: 14.27 μg/mL), and Castanopsis cuspidata var. sieboldii Nakai (IC50: 16.46 μg/mL) showed good antioxidant effects. Eurya japonica Thunb., Quercus myrsinaefolia Bl., Caesalpinia decapetala var. japonica (S. et Z.) Ohashi, Weigela subsessilis L. H. Bailey, and Crinum asiaticum var. japonicum dosedependently inhibited LPS-induced NO production on RAW 264.7 cells (at 50-200 μg/mL). Furthermore, Elaeocarpus sylvestris var. Ellipticus (Thunb.) Hara, Ilex integra Thunb., and Agastache rugosa (Fisch. et Meyer) O. Kuntze dose-dependently inhibited α-MSH stimulated melanin synthesis on B16 F10 cells (at 25-100 μg/mL). These results suggest that Jeju Island plants possess several biological activities and may be potent anti-aging, anti-inflammatory, skin-whitening, and antioxidative ingredients as functional cosmetic materials.

Keywords:

anti-oxidative, elastase, melanin, anti-inflammation, Jeju plants

1. INTRODUCTION

The aging of the skin is a complex process that renders several morphological and chemical changes to human skin. Skin aging is generally divided into an intrinsic aging and a photo-aging [1]. Intrinsic aging occurs naturally as time passes, while photoaging is induced by UV irradiation of skin. The conspicuous clinical signs accompanying photo-aging are optically hyperpigmentation and wrinkles of skin. Wrinkle formation is a striking feature of intrinsic and photo-induced skin aging. In both cases, it is association with oxidative stress and inflammatory responses. UV irradiated photo-aging induces the formation of free radicals and related reactive oxygen species, which injure the DNA and extracellular matrix in skin cells [2]. Inflammation is one of the defense mechanisms against viral infections and physical or chemical stimulation. In addition, it is a mechanism for regenerating or recovering damaged organs. Aging, and especially human aging, can be explained by the emerging concept of parainflammation-driven inflammaging, (i.e., a combination of inflammation and aging). Inflammaging posits that aging, either physiologically or pathologically, can be driven by pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β produced by the immune system [3].

Melanin is very important in protecting the skin against UV light. Also, it determines skin color and several aspects of phenotypic appearance. However, hyperpigmentation, which usually presents as age spots, uneven color, freckles, and sometimes melasma, has become an object of public concern. Thus, skin whitening agents, such as retinoic acid, hydroquinone, arbutin, and so on, are developed for the prevention and treatment of irregular hyperpigmentation. Tyrosinase is the rate-limiting enzyme that controls melanin biosynthesis. Melanogenesis is initiated by tyrosine that is metabolized into DOPA and then dopaquinone by tyrosinase. So, the majority of whitening agents are tyrosinase inhibitors. But recently, various mechanisms for or approaches to skin whitening agents are being studying, such as the inhibition of melanosome transfers, acceleration of epidermal turnover and desquamation, antioxidants, and so on [4].

Natural products, mostly from plants, have begun to gain worldwide interest for promoting healthcare and have been used as conventional or complementary ingredients, due to the toxicity and side effects of synthetic materials. Indeed, plant extracts have received considerable attention as potential candidate ingredients in several cosmetic applications. Previous studies have reported the use of plant extracts, either singly or in combinations, as cosmetic ingredients, such as extracts of Carthamus tinctorius (safflower), Forsythia viridissima, Astragalus membranaceus (anti-aging), Acanthopanax sessiliflorum, Corn Bran, Hoechunyangkyeok-San (whitening activity), and Yeongyoseungma-Tang (anti-irritant) [5-11].

Jeju Island, the southernmost island of the Korean Peninsula, is known as a crossroads for several migration routes [12]. In addition, its geographical position, elevation, and topography, as well as the vertical distribution of temperatures and pressures that range from subtropical to subarctic, allow for the generation of an unique ecosystem on Jeju Island [13,14]. So far, over 1,990 species, including 719 species of edible plants, have been classified [15,16]. This indicates that Jeju Island has a rich diversity of plants.

To identify new active cosmetics ingredients of natural origin, we screened the biological activities of 50 plants collected from Jeju Island. The natural products were investigated for their anti-oxidant, anti-inflammatory, anti-melanogenic, and antiwrinkle effects.


2. MATERIALS AND METHODS

2.1. Preparation of plant extracts

Fifty kinds of plant extracts from Jeju Island were purchased in the Jeju Technopark Biodiversity Research Institute Extract Bank. Plant extracts were extracted with 75% (v/v) ethanol.

2.2. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the plant extracts was measured using the previously described method [17]. Briefly, 1 mL of DPPH (Sigma, USA) solution in 0.2 mM methanol was mixed with 2 mL of the sample in methanol at the indicated concentration and incubated in the dark at 25°C for 10 min. Then, the absorbance was measured at 517 nm using a microplate reader (Synergy, BioTek, USA). The DPPH radical scavenging activity of the sample was calculated by using the following equation:

Scavenging activity (%) = [1 − (Abs sample − Abs blank) / Abs control] × 100

2.3. Elastase inhibition assay

The activity of porcine pancreatic elastase (Sigma, USA) was examined using N-Suc-(Ala)3-nitroanilide as the substrate, and the release of nitroaniline at 405 nm was measured. The reaction was carried out in a 200 mM Tris-HCl buffer (pH 8.0) containing 8.8 mM N-Suc-(Ala)3-nitroanilide (Sigma, USA) and 10 μg/mL elastase (Sigma, USA). Plant extracts were added to the reaction mixture to reach a final concentration of 1 mg/mL, and elastase inhibition was assessed at 25°C. There action mixture was pre-incubated for 10 min before adding the substrate. The change in absorbance was measured at 405 nm microplate reader. The percent of inhibition of elastase was calculated as follows:

Inhibition (%) = [1 − (Abs sample − Abs blank) / Abs control] × 100

2.4. Tyrosinase inhibition assay

Tyrosinase activity was measured using the previously described method [18]. Briefly, the test reaction mixture comprised of each plant extract 0.9 mL, mushroom tyrosinase (1,500 unit/mL, Sigma, USA) 0.1 mL and 1.5 mM L-tyrosine (Sigma, USA) 1.0 mL in 0.1M potassium phosphate buffer (pH 6.8). The reaction mixture was incubated at 37oC for 10 min, and the absorbance was measured at 475 nm using microplate reader (Synergy, BioTek, USA). The absorbance of the same mixture without tyrosinase was used as the control. The percent of the inhibition of the tyrosinase activity was calculated as follows:

Inhibition (%) = [1 − (Abs sample − Abs blank) / Abs control] × 100

2.5. Cytotoxicity assay

2.5.1. LDH release assay

Murine macrophage RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA) supplemented with penicillin (100 U/mL)/streptomycin (100 μg/mL) and 10% FBS (Gibco, USA). The cells were seeded at a density of 1×104 cells/well in a 96-well plate and incubated at 37˚ C with 5% CO2. The cells cultured in the 96-well plates were treated with or without the sample for 24 h. Lactate dehydrogenase (LDH) was released into the cell culture medium upon damage of the plasma membrane. The CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA) was used. The absorbance was measured at 490 nm using microplate reader (Synergy, BioTek, USA).

2.5.2. MTT assay

Murine melanoma cells, B16F10 cells were obtained from the ATCC and cultured in DMEM supplemented with penicillin (100 U/mL)/streptomycin (100 μg/mL) and 10% FBS. The cells were seeded at a density of 5×103 cells/well in a 96-well plate and incubated at 37oC with 5% CO2. The cells cultured in the 96-well plates were treated with or without the sample for 72 h. Following incubation, the cells were treated with the MTT solution for 4 h at 37oC. The supernatants were aspirated, and DMSO was added to each well. After incubation for 20 min, the absorbance was measured at 540 nm using a microplate reader (Synergy, BioTek, USA). Cytotoxicity is presented as a percentage of the optical density of the vehicle group.

2.6. NO (Nitric Oxide) assay

Murine macrophage RAW 264.7 cells were obtained from the ATCC and cultured in DMEM supplemented with penicillin (100 U/mL)/streptomycin (100 μg/mL) and 10% FBS. RAW 264.7 cells were plated at a density of 5×105 cells/well of 24-well plate and incubated overnight. The cells were treated with LPS (1 μg/mL) at 50-200 μg/mL concentrations of the plant extracts. After incubation for 24 h, the supernatant was analyzed for the levels of NO using an assay kit (iNtRON Biotechnology, Korea), according to the manufacturer’s protocol.

2.7. Melanin content assay

Murine melanoma cells, B16F10 cells were obtained from the ATCC and cultured in DMEM supplemented with penicillin (100 U/mL)/streptomycin (100 μg/mL) and 10% FBS. The cellular melanin content was measured using a slight modification of a previously reported method [10]. Briefly, B16F10 cells were incubated at a density of 5×104 cells/well in a 6-well plate overnight. The B16F10 cells, pretreated with the indicated concentrations of plant extracts for 1 h, were treated for 72 h with α-MSH (50 nM, Sigma, USA). In order to measure the melanin content, cells were detached by incubation in trypsin/EDTA. After precipitation, the color of the cell pellets was evaluated visually, and pellets were solubilized in 1 N NaOH containing 10% DMSO. Spectrophotometric analysis of the cellular melanin content was performed by quantifying the absorbance at 405 nm.

2.8. Statistical analysis

The values in the figures are expressed as the mean ± standard deviation. Statistical analyses were performed using an unpaired Student’s t-test. The differences were considered significant if p values were < 0.05 (indicated by *p<0.05, **p<0.01).


3. RESULTS AND DISCUSSION

3.1. Antioxidant activity

The principle of the antioxidant activity of a substance depends on the availability of electrons to neutralize any free radicals. The antioxidant activity of the plant extracts was evaluated by using the DPPH radical scavenging assay. DPPH, a stable radical, is widely accepted as a suitable method for screening the free radical-scavenging ability. The results of the radical scavenging assays for all the extracts are presented in Table 1 as the IC50 (μg/mL), which indicates the concentration of the test sample that efficiently scavenged half of the DPPH radicals. The most active plants examined were E. sylvestris, S. sebiferum, and C. cuspidata with IC50 values of 14.0, 14.3, and 16.5 μg/mL, respectively. The results imply that these active extracts may contain constituents with strong proton-donating abilities.

Anti-oxidant, anti-elastase, and anti-tyrosinase activities of Jeju Island plant extracts

3.2. Elastase and tyrosinase inhibition assay

As shown in Table 1, 17 of 50 plant extracts showed no inhibition (<30% inhibition at 1 mg/mL concentration) of elastase activity. In contrast, T. asiaticum, I. religiosum, V. erosum, P. villosa, S. alnifolia, S. bumalda, S. theezans, and C. cuspidata showed more than 80% inhibition of elastase activity at 1 mg/mL. The most active plants examined were T. asiaticum, V. erosum, and S. theezans, with values of 89.9, 92.7, and 88.7 %, respectively (Table 1). S. theezans extract had anti-elastase activity similar to that of the positive control oleanolic acid (23.4 μg/mL) of IC50 value is 28.5 μg/mL. These results suggest that topical application of plant-based inhibitors of nonspecific elastase in cosmetics may provide beneficial effects for UV-irradiated and dry skin.

Tyrosinase plays an essential role in melanin production. Therefore, the modulation of melanogenesis through the regulation of tyrosinase and its related proteins is an important strategy for treating abnormal skin pigmentation. Table 1 summarized the results of the assessment of mushroom tyrosinase inhibition by the plant extract. In this study, three of the 50 plants extracts (C. jessoensis, A. rugose, and C. cuspidata) had higher antityrosinase activity than the positive control, arbutin, which had a value of 33.9%, while the three plant extracts mentioned had values of 38.4, 46.2, and 37.9%, respectively.

3.3. Anti-inflammation effect

NO overproduction is harmful and results in various chronic inflammatory skin conditions. Severe inflammation is an aspect of many aging-related diseases, and the life-long accumulation of molecular damage resulting from chronic inflammation has been suggested to serve as a major contributor to the process of aging. Therefore, pharmacological blockade of NO production offers promising strategies for therapeutic intervention in skin inflammatory conditions. Consequently, we evaluated the effect of the 50 plant extracts on NO synthesis in activated macrophages. Fig. 1 shows the inhibitory activity of the plant extracts against NO production by LPS-treated RAW 264.7 cells. Of the 50 extracts, 11 showed a greater than 50% inhibition of NO production at a concentration of 100 μg/mL in the culture media. Furthermore, three of the 11 extracts (A. rugosa, C. cuspidata, and C. asiaticum) showed the most potent effect, with NO production inhibition values of 74.3, 63.2, and 75.9%, respectively (Fig. 1, p<0.05).

Fig. 1.

Inhibitory effects of Jeju Island plant extracts on nitric oxide (NO) production in RAW 264.7 cells. NO production was assayed in cultured medium of LPS-stimulated cells and treated with extracts. Cytotoxicity was determined using LDH assay. Values are mean±standard deviation of duplicate experiments. *p<0.05 and **p<0.01.

3.4. Melanin synthesis inhibitory effect

Melanin, as a main pigment to determine a skin color, prevents skin damage from UV rays and removes ROS to protect skin. Chronic exposure to sunlight facilitates production of melanin in the base layer of the epidermis and thereby triggers melasma or excess pigmentation in the dermis and epidermis. To examine the whitening effect of the plant extracts, this study used B16F10 cells and measured melanin inhibition efficacy. Fig. 2 shows the melanin contents of the plant extracts on B16F10 cells. Of the 50 extracts, two showed a greater than 50% inhibition of melanin production at a concentration of 100 μg/mL. Two of the 50 plant extracts (E. sylvestris and I. integra) had higher anti-melanogenesis activity compared to the positive control arbutin (45.5% at 500 μg/mL). Their inhibition values are 50.5 and 51.2% (at 100 μg/mL), respectively (p<0.01).

Fig. 2.

Inhibitory effects of Jeju Island plant extracts on melanin production in B16F10 cells treated with α-MSH. The cells were treated with α-MSH and plant extracts for 72 h. Cytotoxicity was determined using MTT assay. Values are meanᄆstandard deviation of triplicate experiments. *p<0.05 and **p<0.01.


4. CONCLUSION

In conclusion, 50 plant extracts were investigated in the present study for their potential effectiveness as skin-care agents. Eight plants, V. erosum, T. asiaticum, S. theezans, S. alnifolia, S. bumalda, P. villosa, I. religiosum, and C. cuspidate, were potent inhibitors of elastase, S. sebiferum and C. cuspidata showed good antioxidant effects. In addition to E. japonica, Q. myrsinaefolia, C. decapetala, W. subsessilis, and C. asiaticum dose-dependently inhibited LPS-induced NO production on RAW 264.7 cells. Furthermore, E. sylvestris, I. integra, and A. rugosa dose-dependently inhibited α-MSH stimulated melanin synthesis on B16F10 cells. These results demonstrate that Jeju Island plants exhibit several biological activities that may be potentially efficacious inhibitors of skin aging, melanogenesis, and inflammation. Further studies will be focused on identifying the major active components mediating these biological activities.

Acknowledgments

This work was supported by the Chungcheong Institute for Regional Program Evaluation Promotion Project (R0002893) of the Ministry of Trade, Industry and Energy Republic of Korea.

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Fig. 1.

Fig. 1.
Inhibitory effects of Jeju Island plant extracts on nitric oxide (NO) production in RAW 264.7 cells. NO production was assayed in cultured medium of LPS-stimulated cells and treated with extracts. Cytotoxicity was determined using LDH assay. Values are mean±standard deviation of duplicate experiments. *p<0.05 and **p<0.01.

Fig. 2.

Fig. 2.
Inhibitory effects of Jeju Island plant extracts on melanin production in B16F10 cells treated with α-MSH. The cells were treated with α-MSH and plant extracts for 72 h. Cytotoxicity was determined using MTT assay. Values are meanᄆstandard deviation of triplicate experiments. *p<0.05 and **p<0.01.

Table 1.

Anti-oxidant, anti-elastase, and anti-tyrosinase activities of Jeju Island plant extracts

Voucher
specimen
Scientific name Part DPPH
scavenging
Elastase
inhibition (%)
Tyrosinase
inhibition (%)
IC50 (μg/mL) 1 mg/mL 300 μg/mL
Abbreviations: Entire plants (Wh), Leaves (Le), Stems (St), Fruits (Fr). Nd, not determined.
JBRI-10002 Euonymus japonica Thunb. Le - 13.1 ± 3.1 -
JBRI-10024 Eurya japonica Thunb. Le - 19.4 ± 2.4 9.0 ± 12.5
JBRI-10055 Ftsia japonica Decne. et Planch. Le - - 23.7 ± 3.2
JBRI-10058 Trachelospermum asiaticum var. intermedium Nakai Le 35.2 ± 1.6 89.9 ± 1.5 5.9 ± 2.1
JBRI-10090 Elaeocarpus sylvestris var. Ellipticus (Thunb.) Hara Le 14.0 ± 0.5 11.1 ± 1.1 23.5 ± 2.5
JBRI-10092 Illicium religiosum S. et Z. Le - 82.7 ± 0.5 1.2 ± 1.3
JBRI-10100 Ilex integra Thunb. Le - 17.4 ± 0.2 18.5 ± 4.2
JBRI-10103 Quercus myrsinaefolia Bl. Le 44.9 ± 1.4 20.1 ± 1.9 25.3 ± 6.3
JBRI-10124 Daphniphyllum macropodum D. glaucescens Blume Le - 7.4 ± 0.9 20.9 ± 1.6
JBRI-10128 Aucuba japonica Thunb. Le - 16.3 ± 1.1 1.7 ± 4.6
JBRI-10146 Euonymus japonica Thunb. Le - 1.9 ± 2.0 -
JBRI-10153 Vicia angustifolia var. segetilis K. Koch. Wh - - -
JBRI-10175 Cerastium holosteoides var. hallaisanense Mizushima Wh - - 9.7 ± 1.2
JBRI-10229 Sonchus oleraceus L. Wh - - 25.5 ± 3.8
JBRI-10231 Limonium tetragonum (Thunb.) A.A.Bullock Wh - 18.5 ± 0.7 8.0 ± 2.3
JBRI-10289 Suaeda malacosperma H.Hara Wh - - -
JBRI-10297 Elaeagnus umbelellata Thunb. Le - - 4.7 ± 3.9
JBRI-10303 Sambucus sieboldiana Bl. Le - 15.9 ± 1.3 20.8 ± 1.9
JBRI-10304 Quercus serrata Thunb. Le 26.0 ± 0.2 68.5 ± 1.2 16.8 ± 4.6
JBRI-10309 Viburnum erosum Thunb. Le 43.3 ± 1.4 92.7 ± 2.1 2.9 ± 1.2
JBRI-10328 Calystegia soldanella (Linnaeus) Roemer & Schultes Wh - 2.7 ± 1.3 19.9 ± 4.1
JBRI-10339 Caesalpinia decapetala var. japonica (S. et Z.) Ohashi Le - 30.7 ± 0.5 10.6 ± 6.2
JBRI-10340 Plantago lanceolata L. Wh - - 18.7 ± 0.7
JBRI-10343 Ranunculus japonicus Thunb. Wh - - 0.1 ± 2.4
JBRI-10347 Pourthicea villosa var. brunnea Nak. Le 52.6 ± 2.2 83.2 ± 2.1 -
JBRI-10386 Lilium longiflorum Thunb. Wh - 6.8 ± 2.9 10.7 ± 0.7
JBRI-10399 Sedum oryzifolium Makino Wh - - 5.1 ± 1.2
JBRI-10409 Stephanandra incisa Zabel Le 28.5 ± 0.3 74.4 ± 1.8 21.7 ± 4.0
JBRI-10417 Boehmeria pannosa Nakai et Satake Wh 49.2 ± 2.3 72.1 ± 2.0 -
JBRI-10425 Wistaria floribunda A.P. DC Le - 47.8 ± 3.5 13.9 ± 1.8
JBRI-10429 Celtis jessoensis Koidz Le - 46.7 ± 0.9 38.4 ± 0.5
JBRI-10450 Rhamnella frangulioides (Max) Weberb Le - 27.0 ± 3.2 31.9 ± 0.8
JBRI-10451 Picrasma quassioides (D. Don) Benn Le 31.2 ± 0.4 11.9 ± 1.5 28.5 ± 3.6
JBRI-10452 Weigela subsessilis L. H. Bailey Le - 74.8 ± 4.0 9.1 ± 1.2
JBRI-10469 Abelia mosanensis T.H.Chung Le - 23.1 ± 3.0 25.0 ± 3.8
JBRI-10479 Sorbus alnifolia var. hirtella T. Lee Le 30.6 ± 0.2 87.0 ± 2.6 9.3 ± 1.0
JBRI-10487 Styrax obassia S. et Z. Le 30.2 ± 1.0 35.5 ± 1.1 23.6 ± 0.9
JBRI-10488 Staphylea bumalda DC. Le - 86.4 ± 1.6 7.8 ± 2.2
JBRI-10503 Sapium sebiferum (L.) ROXB. Le 14.3 ± 1.4 22.4 ± 2.0 30.9 ± 1.8
JBRI-10599 Agastache rugosa (Fisch. et Meyer) O. Kuntze Wh - 16.9 ± 1.7 46.2 ± 3.5
JBRI-10605 Sageretia theezans Brongn. Le 40.3 ± 1.5 88.7 ± 2.5 4.3 ± 1.2
JBRI-10606 Vicia unijuga A. Br. Le - 2.6 ± 1.5 21.0 ± 4.0
JBRI-10611 Geum aleppicum Jacq. Le 37.4 ± 3.7 73.6 ± 0.3 10.0 ± 1.5
JBRI-10614 Ficus stipulata Thunb. Fr - 15.1 ± 1.8 -
JBRI-10628 Brassica juncea var. integrifolia Sinsk. Wh - 12.0 ± 4.0 1.7 ± 1.8
JBRI-10635 Castanopsis cuspidata var. sieboldii Nakai Le 16.5 ± 1.0 80.7 ± 0.2 37.9 ± 3.0
JBRI-10699 Crinum asiaticum var. japonicum Nd - 5.2 ± 1.6 2.6 ± 2.3
JBRI-10708 Clinopodiumt gracile (Benth.) O. Kuntze Nd - 21.7 ± 2.2 -
JBRI-20078 Setaria viridis (L.) Beauvois Le, St - 6.0 ± 0.1 16.9 ± 1.3
JBRI-20178 Sueada japonica Makino Wh - - -
L-ascorbic acid 2.8 ± 0.0 - -
Oleanolic acid - 67.5 ± 1.4 (at 50 μg/mL) -
Arbutin - - 33.9 ± 1.4