Introduction
Interest in the study of various medicinal plants has increased globally during last few decades, may be due to their antibacterial and antioxidant activities (Chew et al., 2012; Farjana et al., 2014). Plants produce secondary metabolites that possess effective pharmacological activity (Raja and Sreenivasulu, 2015). Phytochemicals, derived from secondary metabolites, help controlling active oxygen in the body through pharmacological actions including antioxidant and anti-inflammatory effects (Asensi et al., 2011). The most important of these bioactive compounds are flavonoids, tannins, alkaloids, and phenolic compounds (Woo et al., 2001). Thus, various experiments using natural products are being conducted in many foods, cosmetics, and pharmaceutical industries (Oh et al., 2002; Hassimotto et al., 2005; Kim et al., 2007).
Abeliophyllum distichum Nakai (AdN), a Korean native plants, is known to exhibit various effects on the leaves (Xiong et al., 1996; He et al., 2000). Especially, it has been reported that the antioxidant effect and antibacterial effect are extraordinary due to the large amounts of polyphenol and flavonoid (Ji et al., 1993; Ko et al., 2019). Also, some researchers reported that the extract of AdN has the effect of improving skin wrinkles, skin aging, and whitening (You et al., 2004; Kim and Lee, 2015; Jang and Park, 2017; Chang et al., 2018). Due to effect of AdN, various industries are trying to develop products using AdN. But there is a lack of scientific evidence and research. Therefore, the objectives of this study were: (1) verification of antibacterial and antioxidative effects of AdN extract; (2) verification of the collagen and adipocyte proliferation effect of AdN extract.
Materials and Methods
Plants materials
AdN extract was supplied by Our Tree Farming Association (Geosan, Korea). Foreign matter and soil derived from the leaves of the AdN were washed with distilled water and then dried to retain a moisture content of less than 15% using an agricultural product dryer at 40℃. Dried AdN leaves were immersed and extracted for 15 minutes at 121℃ and 1.2 atm in an autoclave.
Antibacterial activity
Antibacterial activity was measured using the disc diffusion method to compare the growth of pathogenic microorganisms ((E.coli, Salmonella)). A colony of each strain was immersed in liquid medium, activated for 24 - 36 hours and subcultured three times. Bacteria were cultured to an optical density (OD) of 0.9 at 660 nm, suspended at a concentration of 1 × 106, and plated on agar medium and with a paper disc (6 mm, Toyo Roshi Kaicha Ltd., Tokyo, Japan). The sample concentrations were 0.0125, 0025, 0.05, 0.1, and 0.5%, which were absorbed completely into the disc following incubation at 37℃ for 24 to 36 hours. The antibacterial activity was evaluated by measuring the diameter of the clear zone (clear zone: -, no inhibition; +, very slight inhibition (6 - 10 mm); ++ slight inhibition (10 - 15 mm); +++ moderate inhibition (15 - 20 mm); and ++++, clear inhibition (15 - 20 mm).
Scavenging ability of active oxygen
Oxidation of xanthine to uric acid by xanthine oxidase generated free active oxygen. Nitro blue tetrazolium (NBT) was reduced to formazan by active oxygen, and the absorbance was measured at 560 nm using a UV spectrophotometer (Shimadzu, Kyoto, Japan) (Aruoma et al., 1993). After adding 690 μL of 0.1 M potassium phosphate buffer (pH 7.8) and 100 μL of 10 mM xanthine and 5 mM NBT solution each, a 10 μL sample of a specific concentration and 100 μL of 0.3 units·mL-1 xanthine oxidase solution were added to initiate the reaction. The products were left for 10 minutes at room temperature and the absorbance at 560 nm was measured to calculate the active oxygen scavenging ability.
Collagen proliferation
The cytotoxicity of AdN extract against human dermal fibroblasts was determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, based on the conversion of MTS to formazan by mitochondrial dehydrogenases. Human dermal fibroblasts (1.0 × 105 cells·well-1) were dispersed into a 96-well plate, followed by the addition of AdN extract for 18 hours according to the concentration, and incubation for 72 hours. Following the addition of 20 μL of MTS solution, and incubation for 4 hours in a CO2 incubator, the absorbance was measured at 450 nm. Fibroblasts were dispensed in a 96-well plate at a density of 1 × 105 cells·well-1. The amount of procollagen was measured by procollagen type 1 C-peptide EIA KIT (MK101, Takara, Japan).
Adipocytes
The cytotoxicity of AdN extract against animal adipocytes (backfat cells of Hanwoo cattle) was determined using the MTS assay, to measure the conversion of MTS to formazan by mitochondrial dehydrogenases. Human dermal fibroblasts (1.0 × 103 cells·well-1) were dispensed into a 96-well plate and treated with the AdN extract for 18 hours according to the concentration, followed by incubation for 72 hours. Following addition of 20 μL of MTS solution, and incubation for 4 hours in a CO2 incubator, the absorbance was measured at 450 nm.
Statistical analysis
All data were subjected to statistical analysis in a completely randomized design using mixed procedures of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) in the experimental unit. Orthogonal comparisons were conducted using polynomial regression to determine linear and quadratic effects at different graded levels of 0, 0.125, 0.025, 0.05, 0.1, and 0.5% of AdN extract. Differences among treatment means were determined using Tukey’s multiple range test with a p < 0.05 indicating statistical significance.
Results
Antibacterial activity
In the concentration of 0.125 - 0.250 mg·disc-1, AdN extract showed higher antibacterial activity than other treatment groups. While the antibacterial activity against E.coli and Salmonella was verified at a concentration of 0.5 - 1.0 mg·disc-1, no antibacterial activity against Salmonella was detected at the concentration of 5.0 mg·disc-1 (Table 1; Fig. 1).
Fig. 1. Antimicrobial activities of Abeliophyllum distichum Nakai extract against two strains. The inoculum (about 1 × 106) was spread on the different media then, were grown at 37℃ for 24 h (E.coli) and 48 h (Salmonella). The antimicrobial activity was evaluated by measuring the zone (mm) of clear zone against the test microorganisms. CON, no additive; T1, 0.0125 mg·disc-1 ; T2, 0.25 mg·disc-1; T3, 0.50 mg·disc-1; T4, 1.0 mg·disc-1; T5, 5.0 mg·disc-1.
Measurement of scavenging ability
Allopurinol and 0.125 mg·mL-1 of AdN extract showed higher active oxygen scavenging activity compared to the treatment group (p < 0.05), and 5.0 mg·mL-1 of AdN extract showed lower than the concentration of 0.25 - 1.0 mg·mL-1 (Fig. 2).
Collagen
The Control group showed significantly higher collagen production than other treatment groups. Collagen production increased linearly (p = 0.040) with increased AdN. AdN did not affect the morphological changes in human dermal fibroblasts. (Table 2; Fig. 3).
Adipocyte
Viability decreased linearly (p = 0.040) with increased AdN (Table 3). The amount of fat cells decreased with increased AdN (Fig. 4).
Discussion
AdN extract (0.125 - 0.250 mg·disc-1) exhibited higher antibacterial effect than the other treatments. However, no antibacterial effect against Salmonella was detected using 5.0 mg·disc-1 of AdN extract. In a similar study, a methanol extract of mulberry resulted in a low antibacterial effect against Bacillus cereus and Staphylococcus aureus at concentrations of 10,000 μg·disc1. However, the antibacterial effect of the mulberry extracts obtained by methanol and water was not measured at concentrations ranging between 1,000 and 10,000 μg·disc-1 (Kim and Kang, 2012). Antibacterial effects observed in our study might be attributed to flavonoid compounds. Extracts of various medicinal plants containing flavonoids and phenols have been reported to possess antibacterial effect (Rahman et al., 2007; Ayaz et al., 2008). Verbascoside contained in the AdN exhibits various physiological activities (Schlesier et al., 2002). Verbascoside showed a significant antibacterial effect, especially against Gram-positive bacteria (Avila et al., 1999; Nazemiyeh et al., 2008). Despite the availability of antibacterial tests for plant extracts known to contain physiologically active substances, no antibacterial activity was established compared with the physiological activity.
Xanthine oxidase, a non-specific enzyme associated with purine metabolism, generates uric acid from xanthine or hypoxanthine via oxidation (Duke et al., 1973). Xanthine oxidase generates hydroxyl radical (OH-), a type of reactive oxygen species (ROS), from hypoxanthine as an oxidizing substrate (Warner and Wispé, 1992). The large amounts of ROS are known to cause skin pigmentation or aging (Chin et al., 2011). The findings suggest that the active oxygen scavenging activity of AdN was comparable to that of allopurinol, which is widely known for its antioxidant effect.
Fibroblasts in the dermal layer contribute to the expression of collagen and elastin. The number and function of fibroblasts in the dermal layer are decreased with age and the decreased moisture content in skin cells reduces the synthesis of proteins such as collagen, elastin, and changes in the stratum corneum (Jeon and Yi, 2014). In a previous study, HFEA (ethyl acetate fraction of H. fusiformis) was effective as an anti-aging compound when used on skin due to its antioxidant activity mediated via protection against natural aging or photoaging, and increasing the amount of procollagen type I (Cui et al., 2019). In our experiment, morphological changes and cell viability in the MTS assay following the addition of AdN extract showed no difference; however, the control group showed significantly higher collagen synthesis than the other groups. Further studies are needed to investigate the decrease in collagen synthesis. Certain compounds derived from plants such as polyphenols are prescribed to combat obesity by suppressing adipocyte differentiation and regulating lipid metabolism in vitro (Rayalam et al., 2008; Gourineni et al., 2012; Lu et al., 2012). For example, caffeine, a xanthine alkaloid, reduces the accumulation of lipids in adipocytes and enhances lipolysis by inhibiting phosphodiesterase activity (Herman and Herman, 2013). Similarly, AdN was shown to reduce the viability of adipocytes in the MTS assay in our experiment, suggesting that AdN extracts are useful in prevention or treatment of obesity.
Conclusion
In our experiment, Abeliophyllum distichum Nakai extract was verified the antibacterial activity and antioxidant. 0.125 mg·mL-1 of AdN extract showed the highest active oxygen scavenging activity and antibacterial activity. So, Abeliophyllum distichum Nakai seems to be worth using as a functional cosmetic material for skin aging and wrinkle improvement.
Acknowledgements
This result was supported by "Regional Innovation Strategy (RIS)" through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE).
Authors Information
Dong Cheol Song, https://orcid.org/0000-0002-5704-603X
Ji Hwan Lee, https://orcid.org/0000-0001-8161-4853
Han Jin Oh, https://orcid.org/0000-0002-3396-483X
Yong Ju Kim, https://orcid.org/0000-0002-0960-0884
Jae Woo An, https://orcid.org/0000-0002-5351-6970
Se Yeon Chang, https://orcid.org/0000-0002-5238-2982
Young Bin Go, https://orcid.org/0000-0002-5351-6970
Hyun Ah Cho, https://orcid.org/0000-0003-3469-6715
Jin Ho Cho, https://orcid.org/0000-0001-7151-0778
