A Study of Physiological Activities for Cosmeceutical Ingredient from Fermented Aroniamelanocarpa Extract
Jeong Ran Kang1, Dong-Soon Oh1, Xiao Xiao Huang2, Jong-Hwa Kim1, Kap-Hoon Han1*
1Professor, Derpartment of Pharmaceutical Engineering, Woosuk University
2Ph. D. Candidate, Derpartment of Pharmaceutical Cosmetics Engineering, Woosuk University
화장품 소재로서 아로니아 발효추출물의 생리활성 연구
강정란1, 오동순1, 황소소2, 김종화1, 한갑훈1*
1우석대학교 제약공학과 교수, 2우석대학교 일반대학원 제약·화장품 공학과 박사수료
Abstract Due to the unique sourness and sweet taste of Aronia, it is necessary to develop it for processing rather than raw. The antioxidant activity and cytotoxin about the aronia powder fermentation extract using lactobacillus are verified. In the case of total polyphenol content, the content of non-fermented extract was 32.15 μg/mg fermentation extract, 43.08 μg/mg after fermentation, and the flavonoid content was 0.47 μg/mg in non-fermented extract and 0.44 μg/mg in fermented extract, which was similar to that of non-fermented extract. In the DPPH radical inhibition assay, 77.5% of the non-fermented extract and 89.1% of the fermented extract showed better activity than the non-fermented extract. Nitric oxide (NO) measurement showed a concentration-dependent inhibitory effect. From the above results, it was confirmed that the fermentation of Aronia powder could be utilized based on some antioxidant activities and the possibility of using it as a vegetable extract and fermented cosmetic material in the future.
Key Words : Aronia melanocarpa, Ferment, Polyphenol, Antioxidant activity, Cytotoxicity
요 약 아로니아는 특유의 신맛과 떫은 맛 때문에 생과보다는 가공용으로 개발할 필요성이 대두되고 있다. 유산균 을 이용한 아로니아분말 발효추출물에 대한
항산화 활성 및 세포독성을 검정한 결과 총 폴리페놀 함량의 경우 발효전
32.15μg/mg, 발효 후 43.08 μg/mg으로 증가하였고, 플라보노이드 함량은 발효전 0.47 μg/mg, 0.44 μg/mg 으로
조금 높거나 유사하게 나타내었다. DPPH radical 억제 활성은 발효전 77.5% 에서 89.1%로 발효전 보다 조금더 높은 활성을 나타냈으며, Nitric oxide (NO) 측정은 농도 의존적인 억제효능이 나타났다. 이상의 결과로부터 아로니아분말을 발효할 경우 일부 항산화 활성을 기반으로한 활용가능성과 향후 식물성 천연 추출물 과 발효 화장품 소재로서의 이용가능성을 확인할 수 있었다.주제어 : 아로니아, 발효, 폴리페놀, 항산화활성, 세포독성
*This paper is a research conducted by Woosuk University's Industry-Academy Collaboration Foundation and the Small and Medium Business Administration's (2016) joint research and development project (Project No: C0453887).
*Corresponding Author : khhan(@woosuk.ac.kr) Received November 28, 2019
Accepted January 20, 2020
Revised January 1, 2020 Published January 28, 2020
1. Introduction
Aronia (Aronia melanocarpa) is a genus of berries belonging to the rosaceae, originally
native to northern America[1]. The color of the fruit is black purplish red, and the color of the pulp is somewhat weaker than the skin but it is
also called black chokeberry with intense red purple color. Aronia is a kind of functional cattle, and berry, which is one of fruits, contains a large amount of anthocyanin pigment, and red anthocyanin is widely present in fruit, flower, fruit, stem, Phenolic compounds[2]. Berries containing a large amount of anthocyanin pigment are attracting attention as a substance effective for various physiological activities such as reduction of cardiovascular disease, increase of visual function, anticancer, and immunity[3]. Aronia contains 7.2 to 8 g of anthocyanin per kg and contains more anthocyanins than other berry plants[4]. Aronia contains physiologically active substances such as phenolic acids and flavonoids[5] and it is necessary to develop it for processing rather than as a raw material because of its unique sour taste and bitter taste.
Until recently, studies on the function of Aronia in Korea have been reported on antioxidant effect, anti-diabetic effect, anti-inflammatory effect, immunomodulatory activity, gastric protective effect and the like as raw materials of health and functional foods and cosmetics[6-8]. Bioconversion is a technology that transforms the components of existing materials by utilizing the biological catalytic reactions of microorganisms or enzymes. Since natural plant extracts have side effects due to toxicity, interest in natural organic and fermented extracts that can minimize skin toxicity and side effects is increasing, Recently, many researches have been conducted to increase the functionality, bioavailability, and safety of bioactive materials of natural materials utilizing bioconversion, and they are being activated in various fields such as foods, medicines, and cosmetics[9]. So far, researches on the main function and product development of cosmetic composition for Aronia extract
have been going on, but research data on the physiological activity and efficacy of Aronia fermented extract as a cosmetic material are not yet available. The purpose of this study was to examine the feasibility of using the Aronia fermented extract as a cosmetic material.
Therefore, we intend to provide the basic data on the vitalization of research on the natural extracts of plant and fermented cosmetic materials.
2. Materials and methods
2.1 Production of experimental material and fermented extract
The Aronia cultivar used in this experiment is the national seed source application number 2015-399 of the black choke berry variety registered in the national seed source, and it is produced in Jinan County agricultural products processing center in February, Highland aronia fruit powder (100%) was purchased and used.
The composition of the fermentation medium was sterilized at 121 ℃and 1.5 psi for 20 minutes in a high-temperature high-pressure sterilizer after adding 50 g of Aronia powder, 5 g of yeast extract and 1 L of distilled water Agar powder (2%) was added. L. acidophilus, L. casei, and L. plantarum were cultured in MRS and TSA medium for 2 to 3 days at 37 ℃ incubator (IB-450M, Jeiotech, Daejeon, Korea), S.
cerevisiae was cultured on YEPD medium for 2 to 3 days at 37 ℃, and then inoculated on a fermentation broth of 1 x 109ml per ml of each strain on a fermentation broth (VS-8480SR, Scientific, Daejeon, Korea) for 2 ~ 3 days at 150 rpm and 37 ℃ for mixed fermentation. This was placed in a freeze dryer (FD8508, Ilsin lab, Dongduchen, Korea) at -80 ℃ for 12 hours in a Deepfreezer (8963, Fisher Scientific, USA), lyophilized and stored frozen at -20 ± 3 ℃.
2.2 DPPH radical inhibitory activity
DPPH radical scavenging activity was measured by changing the Blois MS method [10]
to determine the electron donating ability by 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma Chemical Co., St. Louis, donating ability, EDA) were measured. That is, the solution was added to a 96-well plate in an amount equivalent to 180 μl, and 20 μL of the sample dissolved in the second distilled water was added to the well containing 0.1 mM DPPH.
After reacting at 37 ℃. After reacting for 30 minutes, the absorbance was measured at 517 nm. Vit. C was used as a control.
2.3 Total polyphenol content
The total polyphenol content of the samples was measured by Singleton and Rossiet[11]. 1 mL of sample solution (DMSO reconstituted) and 1 mL of gallic acid standard (20, 40, 60, 80, 100 mg / L) were added to a 25 mL flask containing 9 mL of tertiary distilled water and 1 mL of Folin & Ciocalteu's phenol reagents were added. After 5 minutes, 10 mL of 7% Na2CO3
solution was added and stirred well. Then 25 L of distilled water was added to the mixture, and the mixture was reacted at 37 ℃ for 90 minutes.
Then, using a UV / Vis-spectrometer (DU- The absorbance was measured using a blank as a control. At this time, the total polyphenol content was calculated from the standard calibration curve obtained with gallic acid having different concentration as a reference material.
2.4 Total flavonoid content
Total flavonoid contents in the samples were determined by [12] was modified and measured.
The mixture was mixed with 1:1 ratio of DMSO reconstituted and quercetin standard (5, 10, 20, 40, 80 mg / L) and 10% AlCl3 (in MeOH) The
absorbance of each solution was measured using a Spectrometer (DU-730, Bekman) at the reference wavelength of 415 nm. Total flavonoid contents were calculated from the standard calibration curves prepared with quercetin as a standard.
2.5 Cell culture
RAW264.7 (TIB-71; ATCC, USA) macrophages were purchased from American type cell collection (ATCC, USA), the medium used was prepared by adding 10% FBS (Fetal Bovine Serum; Gibco, USA) and 1% Penicillin (100 U / mL) / Streptocycin (100 μg / mL) to Dulbecco's Modified Eagle's Medium After sufficient proliferation in 75 ㎠ flask (SPL, Korea), the cells were washed three times with phosphate buffered saline solution at 3-day intervals, and trypsin solution was added. After incubation at 37 ℃ in a 5% CO 2incubator for 3 minutes, the cells were desorbed and subcultured.
2.6 Cytotoxicity assay
The survival rate of the sample was tested by MTT, Sigma Chemical Co., St. Louis MO, USA).
Cells were seeded in 96-well plates at a density of 5 × 103 cells / well and cultured for 24 hours. To the wells, 100 μL of fresh DMEM (10%
FBS, 1% Penicillin (100 U / mL) Streptocycin (100 μg / mL) was added to each well and cultured at 37 ℃ and 5% CO2 for 4 hours. After removing the medium and adding 100 μL / well of dimethylsulfoxide (DMSO; Sigma Chemical Co., St. Louis MO, USA), formazan produced in the cells was stirred for 15 minutes, absorbance was measured at 590 nm using an ELISA reader.
Cell viability was calculated by the following formula.
Cell viability(%) = 100 × AC / AT AC : absorbance of control
AT : absorbance of tested extract solution
2.7 Nitric oxide (NO) assay
RAW 264.7 cells were seeded in 96-well plates at a concentration of 1 × 105 cells / well, and
then cultured for 24 hours. The samples were diluted in fresh DMEM (10% FBS, 1%
Penicillin (100 U / mL) / Streptocycin (100 μg / mL) for 1 hour and treated with 1 μg / mL lipopolysaccharide And incubated for 24 h at 37
℃
in a 5% CO
2incubator. Twenty-four hours after the sample treatment, 50 μL of the cell culture supernatant and 50 μL of Griess reagent (1% sulfanilamide + 0.1% α-naphthylamide in 2.5% phosphoric acid) were mixed and reacted on 96-well plates for 10 minutes nm absorbance was measured. The yield of nitrite, the NO metabolite, was calculated from the calibration curve of sodium nitrite.
3. Results and Discussion
3.1 Free Radical Inhibitory Activity by DPPH Method
The DPPH method converts to non-radical when electrons or hydrogen are supplied from antioxidants[13]. Hydrogen donating antioxidants are characterized by a ring containing one or more hydroxyl groups and a nonpolar group such as a methyl or nonpolar hydrocarbon chain structure[14]. Free radicals in the body are known to accelerate the aging of living organisms by reacting with lipids and proteins, and studies on natural substances capable of removing such free radicals have been actively conducted. Fig. 1 showed free radial inhibition activity of fermented extract and non-fermented Aroniamelanocarpa extract. The fermented extracts showed higher free radical inhibitory activity than non - fermented extracts (77.5%
and 89.1%, respectively) than the non - fermented extracts (90.4%). These antioxidant activities are known to be mainly due to
phenolic compounds[15]. In the case of the golden fermented products, the fermented products reported about 1.5 times more DPPH radical scavenging activity than before fermentation[16].
Fig. 1. DPPH Radical Scavenging Activity of non-fermented and fermented Aroniamelanocarpa Extract. NFAE:
non-fermented Aroniamelanocarpa Extract. FAE: fermented
Aroniamelanocarpa Extract.
3.2 Total polyphenol content analysis
The phenolic compound is contained in plants and is known to have various antioxidative and physiological functions such as cholesterol lowering action, suicide action, anti-cancer and antioxidant action as one of the secondary metabolites. Fig. 2 showedtotal phenol contents of fermented extracts were 32.15 μg / mg and 43.08 μg / mg, respectively, which were higher than non fermented extracts[17]. Phenolic compounds are mainly phenolic compounds and flavonoids, and especially caffeic acid, chlorogenic acid, and gentistic acid have been reported to have strong antioxidant effects. Therefore, it is thought that the enzymes such as protease, amylase, and lipase secrete during fermentation and increase phenolic substances[18].
Fig. 2. Total polyphenol contents in non-fermentes and fermented Aroniamelanocarpa Extract.
NFAE: non-fermented Aroniamelanocarpa Extract. FAE: fermented Aroniamelanocarpa Extract.
3.3 Total flavonoid content analysis
Flavonoids present in large amounts in plants have been reported to exhibit antioxidant activity, prevention of circulatory diseases, antiinflammatory, antiallergic, antibacterial, antiviral, lipid lowering, immune enhancement and capillary strengthening[19-20]. It is mainly composed of anthocyanidins, flavonoids, flavonoes, cathechins and flavanones. Depending on its structure, certain flavonoids are known to have antioxidant and antimicrobial properties.
Fig. 3 showed the total flavonoid contents were 0.47 and 0.44 μg / mg, respectively, when fermented and fermented extracts were compared. The total flavonoid contents of non -
fermented and fermented extracts were similar.
These results suggest that there is no difference in the physiological activity of the herbal extracts obtained by mixing the mushroom with the mushroom mycelium[21]. According to a recent
report of total flavonoids in fermented products, The total flavonoid contents of non-fermented and fermented extracts of acid garlic were 57.77 mg / g and 62.27 mg / g, respectively[22]. This difference is considered to be related to the material material and the efficacy of the strain.Fig. 3. Total flavonoid contents in non-fermentes and fermented AroniamelanocarpaExtract.
NFAE: non-fermented Aroniamelanocarpa Extract. FAE: fermented
Aroniamelanocarpa Extract.
3.4 Cell viability rate
In order to be applied as a cosmetic material, stability of material is important. The safety tests of cosmetic materials used animal tests such as single-dose citrus test, skin irritation test, and mucosal test, An animal-based safety test has been banned since 2013, and MTT assay is widely used as an alternative cytotoxicity test[23]. Fig. 4 showed cell viability of RAW 264.7 cells was measured by MTT method after treatment of fermented organisms with 0.01, 0.025, 0.05, 0.1 and 0.25%, respectively. As a result, it was confirmed that the cell survival rate of more than 80% was shown up to 0.25% concentration of aronia fermentation extract compared to the control group. Nitric oxide assay was performed at the concentration without cytotoxicity.
Fig. 4. Percentage of fibroblast cell viability at various concentration of Fermented Aroniamelanocarpa Extract.
3.5 Nitric oxide (NO) inhibitory effect
Generally, NO is produced from L-arginine by NO synthase (NOS) due to microbial invasion or inflammatory cytokine stimulation.
Overexpressed NO by iNOS in inflammatory conditions is known to induce inflammation, edema, and tissue damage along with other inflammatory mediators, causing mutations in the cells and tumorigenesis[24]. It has been reported that excessive NO production causes inflammation, destruction of tissues and abnormalities of the immune system[25]. Fig. 5 showed RAW 264.7 cells were cultured in the fermented RAW 264.7 cells at concentrations of 0.05, 0.1 and 0.25%, respectively, in order to determine the inhibitory effect of LPS (lipopolysaccharide) The results are shown. The fermented extracts of Aronia showed a concentration-dependent inhibition effect, blocking nitric oxide (NO) production from the concentration of 0.1% to less than 50%. It was confirmed that the fermented Ahnonia fermented extracts through yeast and lactic acid bacteria had an inhibitory effect on NO production.
Fig. 5. Percentage of NO production at a various cultural concentration of Fermented Aroniamelanocarpa Extract.
4. Conclusion
Although aronia melanocarpa contains physiologically active substances such as
phenolic acids (flavonoids), there is a need to develop it for processing rather than as a raw material because of its unique sour taste and bitter taste. Antioxidant activity and cytotoxicity of the fermented extract of Aronia powder using lactic acid bacterium were tested. The content of total polyphenols was increased in fermented extracts of 32.15 μg/mg and 43.08 μ g/mg in non-fermented extracts. The content of flavonoids was 0.47 μg/mg in non-fermented extracts and 0.44 μg/mg. In the DPPH radical inhibition assay, 77.5% of non-fermented extracts and 89.1% of fermented extracts were slightly lower than the control group of vitamin C (90.4%). In the nitric oxide (NO) measurement, NO production was reduced to less than 50% from 0.1% concentration, and concentration-dependent inhibitory effect was shown. From the above results, it was found that the fermentation of the Aronia powder increased the antioxidative activity and the radical scavenging ability, and it could be confirmed that it could be used as a plant natural extract and a fermented cosmetic material.
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강 정 란(Jeong-Ran kang) [정회원]
․ 2004년 8월 : 중앙대학교 의약식 품대학원 향장미용학(향장학석사)
․ 2009년 2월 : 건국대학교 대학원 향장생물학(이학박사)
․ 2018월 2월 ~ 현재 : 우석대학교 조교수
․ 관심분야 : 피부미용, 화장품
․ E-Mail : jrkang@woosuk.ac.kr
오 동 순(Dong-Soon Oh) [정회원]
․ 2004년 2월 : 우석대학교 대학원 생명공학(이학석사)
․ 2012년 8월 : 우석대학교 대학원 생명공학(이학박사)
․ 2016년 3월 ~ 현재 :우석대학교 제약공학과 겸임조교수
․ 관심분야 : 발효, 미용식품 ․ E-Mail : eastbowl@hanmail.net 황 소 소 (Xiao Xiao Huang) [정회원]
․ 2015년 2월 : 우석대학교 대학원 화학공학 (이학석사)
․ 2015년 3월 : 우석대학교 대학원 제약화장품공학(박사수료)
․ 관심분야 : 화장품, 미용식품
․ E-Mail : hxx89@naver.com
김 종 화(Jong-Hwa Kim) [정회원]
․ 1983년 2월 :서울대학교 농화학과 (이학사)
․ 1985년 2월 : 한국과학기술원 유 전공학과(이학석사)
․ 1999년 8월 : 한국과학기술원 유 전공학과(이학박사)
․ 1991년 3월 ∼ 현재 : 우석대학교 제약공학과 교수
․ 관심분야 : 유전공학
․ E-Mail : jhkim@woosuk.ac.kr
한 갑 훈(Kap-Hoon Han) [정회원]
․ 1993년 2월 : 원광대학교 분자생 물학과(이학사)
․ 1996년 2월 : 원광대학교 대학원 생물학과(이학석사)
․ 1999년 8월 : 원광대학교 대학원 생물학과(이학박사)
․ 2005년 3월 ∼ 현재 : 우석대학교 제약공학과 교수
․ 관심분야 : 미생물, 향장미생물
․ E-Mail : khhan@woosuk.ac.kr
