Artemisia Princeps) Extracts Against Staphylococcus Aureus and Cutibacterium Acnes
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❙ ISSN(Print)：1738-7248, ISSN(Online)：2287-7428 ❙ Korean J. Food Preserv. ❙ 26(4), 381-390 (2019) ❙ https://doi.org/10.11002/kjfp.2019.26.4.381 Antimicrobial activities of Korean mugwort (Artemisia iwayomogi and Artemisia princeps) extracts against Staphylococcus aureus and Cutibacterium acnes Eun Jeong Park, Jun-Hyun Oh* Department of Plant and Food Sciences, Sangmyung University, Cheonan 31066, Korea Abstract Cutibacterium acnes and Staphylococcus aureus are recognized as pus-forming bacteria that trigger skin inflammation in acne. The goal of this research was to determine the antimicrobial activity of Korean mugworts including In-jin-ssuk (Artemisia iwayomogi) and Yak-ssuk (Artemisia princeps). Dried mugwort powders were extracted using water, ethanol and methanol. The antimicrobial activities of the extracts were evaluated by determining minimum inhibitory concentrations (MICs) and squares of inhibition zone widths. The chemical compositions of the chloroform fractions were analyzed by GC/MS. The inhibitory effects of the ethanol and methanol extracts of In-jin-ssuk (A. iwayomogi) (20 mg/mL) against C. acnes were significantly greater than the inhibitory effect of the water extract (p<0.05). Among various extracts, the ethanol extract of A. iwayomogi exhibited the greatest antimicrobial activity against S. aureus. The MICs of the chloroform fractions of ethanol- and methanol-extracted A. iwayomogi were determined to be 15 and 10 mg/mL, respectively, against both C. acnes and S. aureus. The chloroform fraction of the methanol extract exhibited squares of zone widths of 44 and 41 mm2 against C. acnes and S. aureus, respectively, which were the highest among the tested fractions. Scopoletin (484 mg/100 g) was found to be a major phenolic compound in the chloroform extract, while 2-furanmethanol (61 mg/100 g) and isofraxidin (55 mg/100 g) were minor phenolic compounds. Key words：antimicrobial activity, mugwort, Staphylococcus aureus, Cutibacterium acnes Introduction plays a critical role in the development of inflammatory acne 1) when it overgrows and colonizes the pilosebaceous unit (3). Acne vulgaris, ordinarily known as acne, is a common C. acnes is a gram-positive non-spore-forming anaerobic skin disease that affects most adolescents and adults at some bacterium; it is pleomorphic and rod shaped, and can produce time in their lives (1). Acne is a chronic inflammatory disease propionic acid. The organism forms units in the normal flora of the pilosebaceous unit. The lesions are localized in “acne of the oral cavity, large intestine, conjunctiva, external ear prone areas”, where sebaceous follicles are most common, canal and skin, where it dominates over other normal flora which includes the surface skin of the face, neck, chest, and components in pilosebaceous follicles (4). back. Acne generally develops in young people due to several Staphylococcus aureus is a common foodborne pathogen factors including food, stress, the use of cosmetics, hormonal capable of causing a variety of human infections (5). In man, imbalance, or bacterial infection (2). It is accepted that S. aureus causes two main infection types: (1) cutaneous or inflammation in acne vulgaris is mainly induced by an mucosal infections and (2) septicemic infections that are immunological response to Cutibacterium acnes (once generally associated with visceral (abscesses, endocarditis, referred to Propionibacterium acnes) (2). This bacterium lung) or bone (osteomyelitis) infections (6). S. aureus has the capacity to readily acquire antimicrobial resistance, which *Corresponding author. E-mail：[email protected] makes remedy difficult. Treatment of acne invloves the use Phone：82-41-550-5295; Fax：82-41-550-5296 of topical or systemic antibiotics; however, gradual increase Received 11 December 2018; Revised 01 February 2019; in incidences of antibiotic resistance in the causative Accepted 28 February 2019. bacterium has led to a serious need for effective, Copyright ⓒ The Korean Society of Food Preservation. All rights reserved. non-antibiotic treatments (5). - 381 - 382 한국식품저장유통학회지 제26권 제4호 (2019) Many researchers have worked toward the development determined by measuring the optical density at 650 nm with of remedial agents for acne that do not contribute to resistance a spectrophotometer (UVmini 1240, Shimadzu, Kyoto, but still have high antibacterial activities (1). In recent years, Japan). there has been increasing interest in healthy lifestyles and aging using products of plant origin. A large number of Extraction of mugworts aromatic, spicy, medicinal, and other plants that belong to Korean native mugworts including In-jin-ssuk (Artemisia the Asteraceae family contain chemical compounds that iwayomogi) and Yak-ssuk (Artemisia princeps) were exhibit antimicrobial and antioxidant properties (7). purchased from a local market located in Daegu, South Korea. Mugwort (Artemisia) species are part of the Asteraceae The leaves and stems of the mugworts were harvested in family and have been used in traditional medicine to treat the Kangwha area of South Korea during the summer of 2012 serious diseases, including microbial and viral infections (8). and sun-dried immediately after harvesting. Each mugwort Above all, Artemisia iwayomogi Kiramura is a well-known was ground into a fine powder prior to extraction. Ground herbal hepatotherapeutic drug that has been traditionally used mugwort (50 g) was separately added to 500 mL of distilled in Korea and/or China over long periods of time for antitumor, water, 500 mL of 80% ethanol, and 500 mL of 80% methanol, immunomodulating, antimutagenic, antioxidant, antibacterial, and stirred in a water bath at 60℃ for 12 h. Each extract antifungal, liver-defense and choleretic purposes (8,9). was then filtered through Whatman No.2 filter paper, and Artemisia iwayomogi has attracted substantial attention for the extracted residue was re-extracted twice (12). The solvent its potential use as a chemopreventative agent (10). from each extract was completely removed by rotary Pharmaceutical researchers have identified a number of evaporation. The extracts were freeze-dried and stored at 4℃ terpenoid acids, polyenes, phenols, coumarins, chromones, prior to use. The extraction yield of each sample was and flavonoids as the ingredients of the crude drug. There determined using the following equation: Extraction yield (%) are also numerous reports on the biological activities of = [weight of extract (g)/weight of dried mugwort (g)]×100. Artemisia against human pathogens comprising viruses, fungi, and yeasts (11); however, there are only a few research reports Fractionation of the organic solvent extracts that examine the effects of A. iwayomogi against C. acnes, The methanolic or ethanolic mugwort extracts were further one of the major acne-causing bacteria. Therefore, the fractionated using organic solvents, including n-hexane, objectives of this research are 1) to examine the antimicrobial chloroform, ethyl acetate, and n-butanol. n-Hexane (200 mL) activities of mugworts against C. acnes and S. aureus, and and water were added to the alcohol extract in a separating 2) to identify the major compounds responsible for funnel to obtain the n-hexane fraction. The n-hexane phase antimicrobial activity. was separated, and the remaining aqueous phase was successively treated with various solvents. Chloroform, ethyl acetate and n-butanol fractions were obtained using similar Materials and Methods procudures (13). The overall fractionation process is illustrated in Fig 1. Following fractionation, each solvent was Preparation of bacterial cultures completely removed by rotary evaporation and each fraction Cutibacterium acnes KCTC 3314 and Staphylococcus was freeze-dried and stored at 4℃ prior to further use. aureus KCTC 3881 were cultured in Reinforced Clostridial Medium (Difco Laboratories, Detroit, MI, USA) and Nutrient Determination of the antimicrobial activity of broth (Difco Laboratories, Detroit, MI, USA), respectively. mugwort extracts The C. acnes culture was incubated at 37℃ for 72 h under The alcoholic mugwort extracts were incorporated into anaerobic conditions in a candle gas jar (GasPak EZ Container cultural broths at concentrations of 0, 5, 10, 15, and 20 mg/mL. Systems, Franklin Lakes, N.J., USA). The S. aureus culture Streptomycin (5 mg/mL) (Dufhefa, Biochemie, Netherlands) was incubated in an incubation shaker at 37℃ and 200 rpm was used as the positive control. Test bacterial cultures were for 24 h. After incubation, both cultures were washed twice inoculated at concentrations of 106 to 108 CFU/mL, and with Butterfield’s phosphate buffer (BPB) by centrifuging incubated at 37℃ for 48 h while shaken at 200 rpm. An at 5,000 rpm for 5 min at 5℃. The collected bacteria were aliquot of 1 mL culture was washed twice with BPB by re-suspended in BPB and the bacterial population was centrifugation at 5,000 rpm for 5 min using a centrifuge Antimicrobial activities of Korean mugwort extracts 383 (Microcentrifuge 1730R, Labogene, Seoul, Korea). The removed from the extract by rotary evaporation. Each residual washed bacteria were re-suspended in 1 mL of BPB, after extract was dissolved in water for further fractionation with which 100 μL of the bacterial suspension was transferred chloroform. GC/MS was performed using a 6,890 GC gas in a 96-well microplate (BioTek, Seoul, Korea), and the chromatograph coupled to a 5,975 mass spectrometer (HP, optical density at 650 nm was measured using a plate reader Palo Alto, CA, USA). Compounds were