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Anti-Aging Activity of Lavandula angustifolia Extract Fermented with Pediococcus pentosaceus DK1 Isolated from Diospyros kaki Fruit in UVB-Irradiated Human Skin Fibroblasts and Analysis of Principal Components
1Seoul National University of Science and Technology, Seoul 01811, Republic of Korea, 2Life Science Research Institute, GFC Life Science Co. Ltd.,Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2019; 29(1): 21-29
Published January 28, 2019 https://doi.org/10.4014/jmb.1809.09037
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
The skin forms the external surface of the human body, and serves as a barrier protecting the internal organs from ultraviolet radiation, toxins, and bacteria, etc. There are two dependent layers of the skin, the epidermis and dermis, and these consist of many cells such as keratinocytes, melanocytes and fibroblasts [1]. The mechanical strength of the skin is contributed by the dermis, which is composed of the extracellular matrix (ECM) where fibroblasts synthesize ECM components such as collagen and elastin to sustain the skin’s elasticity [2]. However, the structure and function of the dermis can be changed by harmful external factors such as oxidative stress, UV exposure and air pollution. These conditions accelerate aging of the skin by collapsing its dermal structure [3, 4].
Ultraviolet light is a significant cause of exogenous skin damage. The ultraviolet rays that reach the Earth are classified as UVA (320-400 nm) and UVB (280-320 nm) [5]. When exposed to UV on the Earth’s surface, the amount of UV radiation that reaches the human skin is known to be 25 J/cm2 under natural sunlight in autumn at 38° N for 4-5 h. It has been reported that this corresponds to ten times the minimum erythema dose in skin [6]. In particular, UVB can penetrate the upper layer of the dermis [7]. UVB exposure increases reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide anion radicals (O•- 2), singlet oxygen (1O2), and hydrogen peroxide (H2O2). To prevent ROS-induced cellular damage, enzymes (superoxide dismutase and catalase) and non-enzymatic antioxidants (L-ascorbic acid and α-tocopherol) are present in skin cells [8, 9]. However, when the balance of this ROS/ antioxidant defense system is upset due to excess ROS generation caused by UVB exposure, skin cells become damaged and skin aging is accelerated [10, 11].
ROS induced by UVB exposure increase the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts, promoting skin photo-aging [12-14]. MMP-1 degrades collagen type 1, which is an ECM component that provides structural support to the skin. This leads to disintegration of the dermis and acceleration of skin aging [15]. Therefore, the development of anti-aging agents to inhibit UVB- induced ROS generation is an essential strategy for suppressing photo-aging [16].
In this study, the anti-aging effects of
Materials and Methods
Reagents and Chemicals
Human skin fibroblasts (HS68 cells) were purchased from Lonza (Basel, Switzerland). Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), trypsin, and penicillin-streptomycin were obtained from Capricorn Scientific (Ebsdorfergrund, Germany).
Fermentation of L. angustifolia Extract
Fermented and non-fermented
Fermentation of L. angustifolia Extract
In order to cultivate the
-
Table 1 . Fermentation conditions of
L. angustifolia extract.Sample Group Fermentation condition L. angustifolia extract Concentration (%)MRS medium Ratio (%) A1 0.1% 0.69% A2 0.1% 1.38% A3 0.1% 2.75% A4 0.1% 5.50% B1 0.5% 0.69% B2 0.5% 1.38% B3 0.5% 2.75% B4 0.5% 5.50%
-
Fig. 7. Preparation of fermented and non-fermented
L. angustifolia extracts.
Cell Culture
Human skin fibroblasts (HS68 cells) were incubated in DMEM medium supplemented with 10% FBS, 100 U/ml, of penicillin and 100 µg/ml of streptomycin. The cells were incubated in medium at 37°C in a humid incubator under 5% CO2 atmosphere.
UVB Radiation
For supply of UVB radiation, a CL-1000 Ultraviolet Crosslinker (UVP, USA), with an emission spectrum of 280–370 nm and a peak at 312 nm, was used. HS68 cells with 70–80% confluence were treated with fermented and non-fermented
Cell Viability
A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) assay was used to analyze cell viability. HS68 cells were seeded in 96-well plates at 1 × 104 cells per well and incubated for 24 h at 37°C. After treatment with various doses of fermented or non-fermented
Quantitative Analysis of MMP-1 Expression
HS-68 fibroblasts were incubated up to 70-80% confluence on 60-mm plates in a humid incubator maintained at 37 C. After treatment with different concentrations of the fermented or non-fermented
HPLC and LC/ESI-MS Analysis
HPLC analysis of the lavender was carried out using a Shimadzu LC-20A HPLC system (Shimadzu, Japan) equipped with a UVD 170s DIONEX detector and Shim-pack VP-ODS C18 column (L: 250 mm, LD: 4.6 mm, 5 µm). The mobile phase was composed of A (2% acetic acid in H2O) and B (0.5% acetic acid in 50% acetonitrile aqueous solution). The working conditions were as follows: 0–25 min, 0% (v/v) of B; 25–40 min, 0–25% (v/v) of B; 40-80 min, 25–35% (v/v) of B; 80–110 min, 35–40% (v/v) of B; 110-120 min, 40–20% (v/v) of B; 120–130 min, 20–10% (v/v) of B; 130-140 min, 10– 0% (v/v) of B; 140–150 min, 0% (v/v) of B. The flow rate was 1.0 ml/min and the samples were observed at 365 nm. The samples were passed through a 0.2-um filter and then 20 µl of the samples with 10,000 µg/ml were injected into the HPLC.
The mass spectrometric analysis was performed using an LCQ Ion Trap Mass Spectrometer (Thermo Finnigan, USA) with an ESI interface and detection was done in positive ion mode by the National Instrumentation Center for Environmental Management College of Seoul National University (Seoul, Korea). The operating conditions were as follows: capillary voltage, 33 V; capillary temperature, 400°C; nebuliser pressure, 10 psi; and drying gas, N2. Compounds within STE were identified by comparing the UV spectra, retention times, and fragment ions of standard materials.
Intracellular ROS Evaluation
The intracellular ROS levels were evaluated using the fluorescence dye 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA, Sigma- Aldrich, USA), which is generated by the conversion of non- fluorescent H2DCF-DA to highly fluorescent 2’,7’-dichlorofluorescein (DCF) by intracellular ROS. HS-68 cells were incubated with 20 µM H2DCFH-DA for 30 min at 37°C. The cells were then washed twice with PBS and irradiated by 80 mJ/cm UVB. Fluorescence signals were detected using a fluorescence ELISA reader (Perkin Elmer, USA; excitation, 490 nm; emission, 530 nm).
Statistical Analysis
All statistical analyses were performed using SPSS 17.0 (SPSS, USA) software. The results were presented as mean ± standard deviation (SD). Statistically significant differences were calculated by one-way ANOVA, where
Results
Effect of Fermented L. angustifolia Extract on UVB- Increased MMP-1 Expression in Human Skin Fibroblasts
UVB-treated fibroblasts secrete a greater amount of matrix metalloproteinase-1 (MMP-1) than untreated fibroblasts. MMP-1 is a metalloproteinase with a zinc ion in its center, and promotes skin aging through degradation of collagen type I and III, which act as support fixtures in the dermis.
We studied the effects of fermented and non-fermented
-
Fig. 1. Effects of UVB irradiation on human skin fibroblasts. The cells were irradiated with various doses of UVB and incubated 72 h. Then, the levels of MMP-1 proteins (A) and cell viability (B) were measured. Data were presented as mean ± SD of three independent experiments. *
p < 0.01 compared with the negative control.
Fermented and non-fermented
In order to select the optimal fermentation conditions for the
-
Fig. 2.
Effect of fermented and non-fermented (A) MMP-1 expression was determined after pre-treatment with 10 μg/ml of fermented and non-fermentedL. angustifolia extracts on UVB-induced MMP-1 expression in human skin fibroblasts.L. angustifolia extracts and irradiation of 80 mJ/cm2 UVB. The cell viability (B) and MMP-1 expression (C) were determined after pre-treatment of fermented and non-fermentedL. angustifolia extracts at indicated concentration and irradiation of 80 mJ/cm2 UVB. Data are presented as the mean ± SD of three independent experiments. *p < 0.01 compared with the UVB-treated control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
As shown in Fig. 2B, non-fermented
Fermented and non-fermented
Effect of Fermented L. angustifolia Extract on Collagen Production
When the skin is exposed to UVB, the levels of type 1 procollagen decrease. Anti-aging agents can protect against the degradation of collagen in UVB-exposed fibroblasts. The effects of fermented and non-fermented
-
Fig. 3.
Effect of fermented and non-fermented Data are presented as the mean ± SD of three independent experiments. *L. angustifolia extracts on UVB-mediated production of procollagen type I in human skin fibroblasts.p < 0.01 compared with the UVB-treated control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
In UVB-irradiated fibroblasts, the level of procollagen type 1 was 42% lower than the levels of collagen in normal cells without UVB irradiation. Fermented and non- fermented
Effect of Fermented L. angustifolia Extract on UVB- Induced ROS Generation
UVB irradiation increases ROS generation in cells and ROS-stimulated fibroblasts increases expression of MMP-1 protein and inhibits collagen production [27]. Therefore, it is important to identify the active compounds that are able to inhibit ROS generation stimulated by UVB for anti-aging activity [28]. H2DCF-DA [29] was used to determine the effect of fermented and non-fermented
As shown in Fig. 4A, fibroblasts irradiated with UVB showed a 421.2% ± 20.4% higher ROS generation than cells without UVB irradiation. At all concentrations (3.1-25
-
Fig. 4.
Effect of fermented and non-fermented The cells were treated with different concentrations of fermented and non-fermentedL. angustifolia extracts on UVB-induced ROS generation in human skin fibroblasts.L. angustifolia extracts for 24 h and then treated with 20 μM H2DCFH-DA for 30 min. Subsequently, the cells were irradiated with 80 mJ/cm2 UVB. ROS generation was measured using a fluorescence reader (A) and fluorescence microscope (B). In Fig. 4B, the cells were treated with (a) non-treatment; (b) 80 mJ/cm2 UVB irradiation; treatment of 6.3 (c) and 25 μg/ml (d) non-fermentedL. angustifolia extract and UVB irradiation; treatment of 6.3 (e) and 25 μg/ml (f) fermentedL. angustifolia extract and UVB irradiation. Scale bar, 50 μm. Data are presented as the mean ± SD of three independent experiments. *p < 0.01 compared with the UVB- treated control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
ROS levels of fermented
Component Analysis of L. angustifolia Extract before and after Fermentation
Changes in the composition of natural products fermented by microorganisms can affect their anti-aging activity [31]. In this study, the anti-aging activity of
We observed the changes in the components of the
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Table 2 . Mass and UV spectrum of identified compounds in
L. angustifolia extract fermented or non-fermented by P.pentosaceus DK1HPLC Peak No. Name of the compound Molecular formula Retention time (min) Measurement λmax (nm) Negative ions ( m/z ) [M+H]-Positive ions ( m/z ) [M+H]+1 Chlorogenic acid C16H18O9 49.754 244, 322 353.4 355.1 2 Luteoln-7- O -glucuronideC21H18O12 69.408 253, 347 - 463.8 3 Luteolin-7- O -glucosideC21H20O11 80.261 254, 346 447.9 449.4 4 Rosmarinic acid C18H15O8 82.956 290, 331 359.2 - 5 Apigenin-7- O -glucosideC21H20O10 92.844 267, 334 431.8 433.8 6 Luteolin C15H10O6 98.003 254.349 285.2 287.4 7 Apigenin C15H10O5 106.566 270, 334 269.1 271.2
-
Fig. 5.
HPLC chromatograms of The peaks were detected at 365 nm of wavelength for 150 min.L. angustifolia extracts fermented (A) and non-fermented (B).
Anti-Aging Effect of the Identified Compounds in UVB-Irradiated Human Skin Fibroblasts
Identified components of the
Luteolin and apigenin reduced 80% and 85.8% of ROS generation increased by UVB irradiation compared to their glycosides (Fig. 6A). Luteolin and apigenin also reduced 26.5% and 22.4% of UVB-enhanced MMP-1 levels less than their glycosides, respectively (Fig. 6B). In UVB-reduced collagen production, luteolin and apigenin increased 9.5% and 12.5% more than their glycosides (Fig. 6C).
-
Fig. 6.
Effect of identified compounds on ROS generation, MMP-1 expression and collagen production in UVB-irradiated human skin fibroblasts. Data are presented as mean ± SD of three independent experiments. *p < 0.01 compared with the negative control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
Taken together, these results suggest that aglyconation of the flavonoids in
Discussion
Recently, fermentation using microorganisms and enzymes has been shown to enhance both the safety and activity of natural products for cosmetics agents [31]. The mechanism of efficacy enhancement of materials by microbial fermentation is as follows: 1) the microorganism breaks down the substance to a small size to increase its absorption into the skin; 2) the microorganism enhances the product’s physiological activities by removing glycoside; 3) favorable nutrients including vitamins and organic acids are produced during microbial cultivation [32, 33]. In addition, heavy metals and pesticide residues in the extracts of raw plants are harmful to the skin, microorganisms can enhance the safety of the extracts through decomposition and/or adsorption [34].
In this study,
In conclusion,
Acknowledgments
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN15C0104).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- MacNeil S. 2007. Progress and opportunities for tissue-engineered skin.
Nature 445 : 874-880. - Hussain SH, Limthongkul B, Humphreys TR. 2013. The biomechanical properties of the skin.
Dermatol. Surg. 39 : 193-203. - D'Orazio J, Jarrett S, Amaro-Ortiz A, Scott T. 2013. UV radiation and the skin.
Int. J. Mol. Sci. 14 : 12222-12248. - Rittie L, Fisher GJ. 2002. UV-light-induced signal cascades and skin aging.
Ageing Res. Rev. 1 : 705-720. - Ramachandran S, Prasad NR. 2008. Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes.
Chem. Biol. Interact. 176 : 99-107. - Packer L. 1994. Ultraviolet radiation(UVA,UVB) and skin antioxidants.
New Compr. Biochem. 28 : 239-255. - Maeda K. 2018. Analysis of ultraviolet radiationwavelengths causing hardening and reduced elasticity of collagen gels in vitro.
Cosmetics 5 : 1-14. - Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. 2012. Oxidative stress and antioxidant defense.
World Allergy Organ. J. 5 : 9-19. - Stanczyk M, Gromadzinska J, Wasowicz W. 2005. Roles of reactive oxygen species and selected antioxidants in regulation of cellular metabolism.
Int. J. Occup. Med. Environ. Health 18 : 15-26. - Nita M, Grzybowski A. 2016. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults.
Oxid. Med. Cell Longev. 2016 : 3164734. - Poljsak B, Suput D, Milisav I. 2013. Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants.
Oxid. Med. Cell Longev. 2013 : 956792. - Khan MN, Mobin M, Abbas ZK, AlMutairi KA, Siddiqui ZH. 2017. Role of nanomaterials in plants under challenging environments.
Plant Physiol. Biochem. 110 : 194-209. - Chekulayeva LV, Shevchuk IN, Chekulayev VA, Ilmarinen K. 2006. Hydrogen peroxide, superoxide, and hydroxyl radicals are involved in the phototoxic action of hematoporphyrin derivative against tumor cells.
J. Environ. Pathol. Toxicol. Oncol. 25 : 51-77. - Moon HJ, Lee SR, Shim SN, Jeong SH, Stonik VA, Rasskazov VA, et al. 2008. Fucoidan inhibits UVB-induced MMP-1 expression in human skin fibroblasts.
Biol. Pharm. Bull. 31 : 284-289. - Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, He T, et al. 2009. Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin.
Am. J. Pathol. 174 : 101-114. - Binic I, Lazarevic V, Ljubenovic M, Mojsa J, Sokolovic D. 2013. Skin ageing: natural weapons and strategies.
Evid. Based Complement Alternat. Med. 2013 : 827248. - Jonganurakkun B, Wang Q, Xu SH, Tada Y, Minamida K, Yasokawa D, et al. 2008. Pediococcus pentosaceus NB-17 for probiotic use.
J. Biosci. Bioeng. 106 : 69-73. - Kwon HK, Jo WR, Park HJ. 2018. Immune-enhancing activity of
C. militaris fermented withPediococcus pentosaceus (GRC-ON89A) in CY-induced immunosuppressed model.BMC Complement. Altern. Med. 18 : 75. - Park SD, Lee DE, Jeong JW, Kim YT, Kim HM, Kim YJ, et al. 2014. Comprising lactic acid fermentation product of
Gelidium amansil extract as an active ingredient for improving skin wrinkle.J. Microbiol Biotechnol. 24 : 1583-1591. - Woronuk G, Demissie Z, Rheault M, Mahmoud S. 2011. Biosynthesis and therapeutic properties of
Lavandula essential oil constituents.Planta Med. 77 : 7-15. - Lopez V, Nielsen B, Solas M, Ramirez MJ, Jager AK. 2017. Exploring pharmacological mechanisms of lavender (
Lavandula angustifolia ) essential oil on central nervous system targets.Front Pharmacol. 8 : 280. - Cavanagh HM, Wilkinson JM. 2002. Biological activities of lavender essential oil.
Phytother. Res. 16 : 301-308. - Hsu CK, Chang CT, Lu HY, Chung YC. 2007. Inhibitory effects of the water extracts of
Lavendula sp. on mushroom tyrosinase activity.on mushroom tyrosinase activity. Food Chem. 105 : 1099-1105. - Kim AA, HA JH, Kim AR, Jeong HJ, Kim KM, Park SN. 2017. Cellular protective effect and active component analysis of lavender (
Lavandula angustifolia ) extracts and fractions.Appl. Chem. Eng. 28 : 479-484. - Spiridon I, Colceru S, Anghel N, Teaca CA, Bodirlau R, Armatu A. 2011. Antioxidant capacity and total phenolic contents of oregano (
Origanum vulgare ), lavender (Lavandula angustifolia ) and lemon balm (Melissa officinalis ) from Romania.Nat. Prod. Res. 25 : 1657-1661. - Ahn YJ, Won BR, Kang MK, Kim JH, Park SN. 2009. Antioxidant activity and component analysis of fermented
Lavandula angustifolia extracts.J. Soc. Cosmet. Scientists Korea 35 : 125-134. - Kim JK, Kim Y, Na KM, Surh YJ, Kim TY. 2007. [6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo.
Free Radic. Res. 41 : 603-614. - Masaki H. 2010. Role of antioxidants in the skin: anti-aging effects.
J. Dermatol. Sci. 58 : 85-90. - Park K, Lee JH. 2008. Protective effects of resveratrol on UVB-irradiated HaCaT cells through attenuation of the caspase pathway.
Oncol. Rep. 19 : 413-417. - Im AR, Song JH, Lee MY, Yeon SH, Um KA, Chae S. 2014. Anti-wrinkle effects of fermented and non-fermented
Cyclopia intermedia in hairless mice.BMC Complement. Altern. Med. 14 : 424. - Gurung N, Ray S, Bose S, Rai V. 2013. A broader view: microbial enzymes and their relevance in industries, medicine, and beyond.
Biomed. Res. Int. 2013 : 329121. - Yu-Ling Wena, Li-Pyng Yan, Chen C-S. 2013. Effects of fermentation treatment on antioxidant and antimicrobial activities of four common Chinese herbal medicinal residues by
Aspergillus oryzae .J. Food Drug Analysis. 21 : 219-226. - Ortiz-Castro R, Contreras-Cornejo HA, Macias-Rodriguez L, Lopez-Bucio J. 2009. The role of microbial signals in plant growth and development.
Plant Signal. Behav. 4 : 701-712. - Antai SP, Obong US. 1992. The effect of fermentation on the nutrient status and on some toxic components of
Icacinia manni .Plant Foods Hum. Nutr. 42 : 219-224. - Sung BK, Chung JW, Bae HR, Choi JS, Kim CM, Kim ND, et al. 2105. Humulus japonicus extract exhibits antioxidative and anti-aging effects via modulation of the AMPK-SIRT1 pathway.
Exp. Ther. Med. 9 : 1819-1826. - Pervin M, Unno K, Nakamura Y, Imai S. 2016. Luteolin suppresses ultraviolet A- and B-induced matrix metalloproteinase 1- and 9 expression in human dermal fibroblast cells.
J. Nutr. Food Sci. 6 : 1-6. - Hwang YP, Oh KN, Yun HJ, Jeong HG. 2011. The flavonoids apigenin and luteolin suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and AP-1- dependent signaling in HaCaT cells.
J. Dermatol. Sci. 61 : 23-31. - Api genin induces dermal collagen synthesis via smad2/3 signaling pathway.
Eur. J. Histochem. 59 : 2467.
Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2019; 29(1): 21-29
Published online January 28, 2019 https://doi.org/10.4014/jmb.1809.09037
Copyright © The Korean Society for Microbiology and Biotechnology.
Anti-Aging Activity of Lavandula angustifolia Extract Fermented with Pediococcus pentosaceus DK1 Isolated from Diospyros kaki Fruit in UVB-Irradiated Human Skin Fibroblasts and Analysis of Principal Components
Ji Hoon Ha 1, A Rang Kim 1, Keon-Soo Lee 1, Song Hua Xuan 1, Hee Cheol Kang 2, Dong Hwan Lee 2, Mi Yeon Cha 2, Hye Jin Kim 2, Mi An 2 and Soo Nam Park 1*
1Seoul National University of Science and Technology, Seoul 01811, Republic of Korea, 2Life Science Research Institute, GFC Life Science Co. Ltd.,Republic of Korea
Correspondence to:Soo Nam Park
snpark@seoultech.ac.kr
Abstract
The effects of Lavandula angustifolia extracts fermented with Pediococcus pentosaceus DK1 on UVB-mediated MMP-1 expression and collagen decrease in human skin fibroblasts Lavandula angustifolia extract fermented with Pediococcus pentosaceus DK1 were determined on UVB-mediated MMP-1 expression and collagen decrease in human skin fibroblasts and the conversion of its components. Fermentation was performed at varying L. angustifolia extract and MRS medium concentrations, and optimal fermentation conditions were selected. L. angustifolia extracts showed decreased cytotoxicity after fermentation in the fibroblasts. UVB-irradiated fibroblasts treated with fermented L. angustifolia extract showed MMP-1 expression 8.2-14.0% lower than that in UVB-irradiated fibroblasts treated with non-fermented extract. This was observed even at fermented extract concentrations lower than those of nonfermented extracts. Fibroblasts treated with fermented L. angustifolia extract showed 20% less reduction in collagen production upon UVB irradiation than those treated with non-fermented extracts. UVB-irradiated fibroblasts treated with fermented L. angustifolia extracts showed 50% higher inhibition of ROS generation than those treated with non-fermented extract. Luteolin and apigenin glycosides of L. angustifolia were converted during fermentation, and identified using RP-HPLC and LC/ESI-MS. Therefore, the effects of L. angustifolia extract were increased through fermentation by P. pentosaceus on MMP-1 expression and collagen decrease in UVBirradiated human skin fibroblasts
Keywords: Pediococcus pentosaceus DK1, fermentation, UVB, matrix metalloproteinase-1, procollagen
Introduction
The skin forms the external surface of the human body, and serves as a barrier protecting the internal organs from ultraviolet radiation, toxins, and bacteria, etc. There are two dependent layers of the skin, the epidermis and dermis, and these consist of many cells such as keratinocytes, melanocytes and fibroblasts [1]. The mechanical strength of the skin is contributed by the dermis, which is composed of the extracellular matrix (ECM) where fibroblasts synthesize ECM components such as collagen and elastin to sustain the skin’s elasticity [2]. However, the structure and function of the dermis can be changed by harmful external factors such as oxidative stress, UV exposure and air pollution. These conditions accelerate aging of the skin by collapsing its dermal structure [3, 4].
Ultraviolet light is a significant cause of exogenous skin damage. The ultraviolet rays that reach the Earth are classified as UVA (320-400 nm) and UVB (280-320 nm) [5]. When exposed to UV on the Earth’s surface, the amount of UV radiation that reaches the human skin is known to be 25 J/cm2 under natural sunlight in autumn at 38° N for 4-5 h. It has been reported that this corresponds to ten times the minimum erythema dose in skin [6]. In particular, UVB can penetrate the upper layer of the dermis [7]. UVB exposure increases reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide anion radicals (O•- 2), singlet oxygen (1O2), and hydrogen peroxide (H2O2). To prevent ROS-induced cellular damage, enzymes (superoxide dismutase and catalase) and non-enzymatic antioxidants (L-ascorbic acid and α-tocopherol) are present in skin cells [8, 9]. However, when the balance of this ROS/ antioxidant defense system is upset due to excess ROS generation caused by UVB exposure, skin cells become damaged and skin aging is accelerated [10, 11].
ROS induced by UVB exposure increase the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts, promoting skin photo-aging [12-14]. MMP-1 degrades collagen type 1, which is an ECM component that provides structural support to the skin. This leads to disintegration of the dermis and acceleration of skin aging [15]. Therefore, the development of anti-aging agents to inhibit UVB- induced ROS generation is an essential strategy for suppressing photo-aging [16].
In this study, the anti-aging effects of
Materials and Methods
Reagents and Chemicals
Human skin fibroblasts (HS68 cells) were purchased from Lonza (Basel, Switzerland). Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), trypsin, and penicillin-streptomycin were obtained from Capricorn Scientific (Ebsdorfergrund, Germany).
Fermentation of L. angustifolia Extract
Fermented and non-fermented
Fermentation of L. angustifolia Extract
In order to cultivate the
-
Table 1 . Fermentation conditions of
L. angustifolia extract..Sample Group Fermentation condition L. angustifolia extract Concentration (%)MRS medium Ratio (%) A1 0.1% 0.69% A2 0.1% 1.38% A3 0.1% 2.75% A4 0.1% 5.50% B1 0.5% 0.69% B2 0.5% 1.38% B3 0.5% 2.75% B4 0.5% 5.50%
-
Figure 7. Preparation of fermented and non-fermented
L. angustifolia extracts.
Cell Culture
Human skin fibroblasts (HS68 cells) were incubated in DMEM medium supplemented with 10% FBS, 100 U/ml, of penicillin and 100 µg/ml of streptomycin. The cells were incubated in medium at 37°C in a humid incubator under 5% CO2 atmosphere.
UVB Radiation
For supply of UVB radiation, a CL-1000 Ultraviolet Crosslinker (UVP, USA), with an emission spectrum of 280–370 nm and a peak at 312 nm, was used. HS68 cells with 70–80% confluence were treated with fermented and non-fermented
Cell Viability
A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) assay was used to analyze cell viability. HS68 cells were seeded in 96-well plates at 1 × 104 cells per well and incubated for 24 h at 37°C. After treatment with various doses of fermented or non-fermented
Quantitative Analysis of MMP-1 Expression
HS-68 fibroblasts were incubated up to 70-80% confluence on 60-mm plates in a humid incubator maintained at 37 C. After treatment with different concentrations of the fermented or non-fermented
HPLC and LC/ESI-MS Analysis
HPLC analysis of the lavender was carried out using a Shimadzu LC-20A HPLC system (Shimadzu, Japan) equipped with a UVD 170s DIONEX detector and Shim-pack VP-ODS C18 column (L: 250 mm, LD: 4.6 mm, 5 µm). The mobile phase was composed of A (2% acetic acid in H2O) and B (0.5% acetic acid in 50% acetonitrile aqueous solution). The working conditions were as follows: 0–25 min, 0% (v/v) of B; 25–40 min, 0–25% (v/v) of B; 40-80 min, 25–35% (v/v) of B; 80–110 min, 35–40% (v/v) of B; 110-120 min, 40–20% (v/v) of B; 120–130 min, 20–10% (v/v) of B; 130-140 min, 10– 0% (v/v) of B; 140–150 min, 0% (v/v) of B. The flow rate was 1.0 ml/min and the samples were observed at 365 nm. The samples were passed through a 0.2-um filter and then 20 µl of the samples with 10,000 µg/ml were injected into the HPLC.
The mass spectrometric analysis was performed using an LCQ Ion Trap Mass Spectrometer (Thermo Finnigan, USA) with an ESI interface and detection was done in positive ion mode by the National Instrumentation Center for Environmental Management College of Seoul National University (Seoul, Korea). The operating conditions were as follows: capillary voltage, 33 V; capillary temperature, 400°C; nebuliser pressure, 10 psi; and drying gas, N2. Compounds within STE were identified by comparing the UV spectra, retention times, and fragment ions of standard materials.
Intracellular ROS Evaluation
The intracellular ROS levels were evaluated using the fluorescence dye 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA, Sigma- Aldrich, USA), which is generated by the conversion of non- fluorescent H2DCF-DA to highly fluorescent 2’,7’-dichlorofluorescein (DCF) by intracellular ROS. HS-68 cells were incubated with 20 µM H2DCFH-DA for 30 min at 37°C. The cells were then washed twice with PBS and irradiated by 80 mJ/cm UVB. Fluorescence signals were detected using a fluorescence ELISA reader (Perkin Elmer, USA; excitation, 490 nm; emission, 530 nm).
Statistical Analysis
All statistical analyses were performed using SPSS 17.0 (SPSS, USA) software. The results were presented as mean ± standard deviation (SD). Statistically significant differences were calculated by one-way ANOVA, where
Results
Effect of Fermented L. angustifolia Extract on UVB- Increased MMP-1 Expression in Human Skin Fibroblasts
UVB-treated fibroblasts secrete a greater amount of matrix metalloproteinase-1 (MMP-1) than untreated fibroblasts. MMP-1 is a metalloproteinase with a zinc ion in its center, and promotes skin aging through degradation of collagen type I and III, which act as support fixtures in the dermis.
We studied the effects of fermented and non-fermented
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Figure 1. Effects of UVB irradiation on human skin fibroblasts. The cells were irradiated with various doses of UVB and incubated 72 h. Then, the levels of MMP-1 proteins (A) and cell viability (B) were measured. Data were presented as mean ± SD of three independent experiments. *
p < 0.01 compared with the negative control.
Fermented and non-fermented
In order to select the optimal fermentation conditions for the
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Figure 2.
Effect of fermented and non-fermented (A) MMP-1 expression was determined after pre-treatment with 10 μg/ml of fermented and non-fermentedL. angustifolia extracts on UVB-induced MMP-1 expression in human skin fibroblasts.L. angustifolia extracts and irradiation of 80 mJ/cm2 UVB. The cell viability (B) and MMP-1 expression (C) were determined after pre-treatment of fermented and non-fermentedL. angustifolia extracts at indicated concentration and irradiation of 80 mJ/cm2 UVB. Data are presented as the mean ± SD of three independent experiments. *p < 0.01 compared with the UVB-treated control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
As shown in Fig. 2B, non-fermented
Fermented and non-fermented
Effect of Fermented L. angustifolia Extract on Collagen Production
When the skin is exposed to UVB, the levels of type 1 procollagen decrease. Anti-aging agents can protect against the degradation of collagen in UVB-exposed fibroblasts. The effects of fermented and non-fermented
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Figure 3.
Effect of fermented and non-fermented Data are presented as the mean ± SD of three independent experiments. *L. angustifolia extracts on UVB-mediated production of procollagen type I in human skin fibroblasts.p < 0.01 compared with the UVB-treated control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
In UVB-irradiated fibroblasts, the level of procollagen type 1 was 42% lower than the levels of collagen in normal cells without UVB irradiation. Fermented and non- fermented
Effect of Fermented L. angustifolia Extract on UVB- Induced ROS Generation
UVB irradiation increases ROS generation in cells and ROS-stimulated fibroblasts increases expression of MMP-1 protein and inhibits collagen production [27]. Therefore, it is important to identify the active compounds that are able to inhibit ROS generation stimulated by UVB for anti-aging activity [28]. H2DCF-DA [29] was used to determine the effect of fermented and non-fermented
As shown in Fig. 4A, fibroblasts irradiated with UVB showed a 421.2% ± 20.4% higher ROS generation than cells without UVB irradiation. At all concentrations (3.1-25
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Figure 4.
Effect of fermented and non-fermented The cells were treated with different concentrations of fermented and non-fermentedL. angustifolia extracts on UVB-induced ROS generation in human skin fibroblasts.L. angustifolia extracts for 24 h and then treated with 20 μM H2DCFH-DA for 30 min. Subsequently, the cells were irradiated with 80 mJ/cm2 UVB. ROS generation was measured using a fluorescence reader (A) and fluorescence microscope (B). In Fig. 4B, the cells were treated with (a) non-treatment; (b) 80 mJ/cm2 UVB irradiation; treatment of 6.3 (c) and 25 μg/ml (d) non-fermentedL. angustifolia extract and UVB irradiation; treatment of 6.3 (e) and 25 μg/ml (f) fermentedL. angustifolia extract and UVB irradiation. Scale bar, 50 μm. Data are presented as the mean ± SD of three independent experiments. *p < 0.01 compared with the UVB- treated control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
ROS levels of fermented
Component Analysis of L. angustifolia Extract before and after Fermentation
Changes in the composition of natural products fermented by microorganisms can affect their anti-aging activity [31]. In this study, the anti-aging activity of
We observed the changes in the components of the
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Table 2 . Mass and UV spectrum of identified compounds in
L. angustifolia extract fermented or non-fermented by P.pentosaceus DK1.HPLC Peak No. Name of the compound Molecular formula Retention time (min) Measurement λmax (nm) Negative ions ( m/z ) [M+H]-Positive ions ( m/z ) [M+H]+1 Chlorogenic acid C16H18O9 49.754 244, 322 353.4 355.1 2 Luteoln-7- O -glucuronideC21H18O12 69.408 253, 347 - 463.8 3 Luteolin-7- O -glucosideC21H20O11 80.261 254, 346 447.9 449.4 4 Rosmarinic acid C18H15O8 82.956 290, 331 359.2 - 5 Apigenin-7- O -glucosideC21H20O10 92.844 267, 334 431.8 433.8 6 Luteolin C15H10O6 98.003 254.349 285.2 287.4 7 Apigenin C15H10O5 106.566 270, 334 269.1 271.2
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Figure 5.
HPLC chromatograms of The peaks were detected at 365 nm of wavelength for 150 min.L. angustifolia extracts fermented (A) and non-fermented (B).
Anti-Aging Effect of the Identified Compounds in UVB-Irradiated Human Skin Fibroblasts
Identified components of the
Luteolin and apigenin reduced 80% and 85.8% of ROS generation increased by UVB irradiation compared to their glycosides (Fig. 6A). Luteolin and apigenin also reduced 26.5% and 22.4% of UVB-enhanced MMP-1 levels less than their glycosides, respectively (Fig. 6B). In UVB-reduced collagen production, luteolin and apigenin increased 9.5% and 12.5% more than their glycosides (Fig. 6C).
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Figure 6.
Effect of identified compounds on ROS generation, MMP-1 expression and collagen production in UVB-irradiated human skin fibroblasts. Data are presented as mean ± SD of three independent experiments. *p < 0.01 compared with the negative control. §p < 0.01 compared with cells treated with non-fermentedL. angustifolia extract.
Taken together, these results suggest that aglyconation of the flavonoids in
Discussion
Recently, fermentation using microorganisms and enzymes has been shown to enhance both the safety and activity of natural products for cosmetics agents [31]. The mechanism of efficacy enhancement of materials by microbial fermentation is as follows: 1) the microorganism breaks down the substance to a small size to increase its absorption into the skin; 2) the microorganism enhances the product’s physiological activities by removing glycoside; 3) favorable nutrients including vitamins and organic acids are produced during microbial cultivation [32, 33]. In addition, heavy metals and pesticide residues in the extracts of raw plants are harmful to the skin, microorganisms can enhance the safety of the extracts through decomposition and/or adsorption [34].
In this study,
In conclusion,
Acknowledgments
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN15C0104).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.
Fig 2.
Fig 3.
Fig 4.
Fig 5.
Fig 6.
Fig 7.
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Table 1 . Fermentation conditions of
L. angustifolia extract..Sample Group Fermentation condition L. angustifolia extract Concentration (%)MRS medium Ratio (%) A1 0.1% 0.69% A2 0.1% 1.38% A3 0.1% 2.75% A4 0.1% 5.50% B1 0.5% 0.69% B2 0.5% 1.38% B3 0.5% 2.75% B4 0.5% 5.50%
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Table 2 . Mass and UV spectrum of identified compounds in
L. angustifolia extract fermented or non-fermented by P.pentosaceus DK1.HPLC Peak No. Name of the compound Molecular formula Retention time (min) Measurement λmax (nm) Negative ions ( m/z ) [M+H]-Positive ions ( m/z ) [M+H]+1 Chlorogenic acid C16H18O9 49.754 244, 322 353.4 355.1 2 Luteoln-7- O -glucuronideC21H18O12 69.408 253, 347 - 463.8 3 Luteolin-7- O -glucosideC21H20O11 80.261 254, 346 447.9 449.4 4 Rosmarinic acid C18H15O8 82.956 290, 331 359.2 - 5 Apigenin-7- O -glucosideC21H20O10 92.844 267, 334 431.8 433.8 6 Luteolin C15H10O6 98.003 254.349 285.2 287.4 7 Apigenin C15H10O5 106.566 270, 334 269.1 271.2
References
- MacNeil S. 2007. Progress and opportunities for tissue-engineered skin.
Nature 445 : 874-880. - Hussain SH, Limthongkul B, Humphreys TR. 2013. The biomechanical properties of the skin.
Dermatol. Surg. 39 : 193-203. - D'Orazio J, Jarrett S, Amaro-Ortiz A, Scott T. 2013. UV radiation and the skin.
Int. J. Mol. Sci. 14 : 12222-12248. - Rittie L, Fisher GJ. 2002. UV-light-induced signal cascades and skin aging.
Ageing Res. Rev. 1 : 705-720. - Ramachandran S, Prasad NR. 2008. Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes.
Chem. Biol. Interact. 176 : 99-107. - Packer L. 1994. Ultraviolet radiation(UVA,UVB) and skin antioxidants.
New Compr. Biochem. 28 : 239-255. - Maeda K. 2018. Analysis of ultraviolet radiationwavelengths causing hardening and reduced elasticity of collagen gels in vitro.
Cosmetics 5 : 1-14. - Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. 2012. Oxidative stress and antioxidant defense.
World Allergy Organ. J. 5 : 9-19. - Stanczyk M, Gromadzinska J, Wasowicz W. 2005. Roles of reactive oxygen species and selected antioxidants in regulation of cellular metabolism.
Int. J. Occup. Med. Environ. Health 18 : 15-26. - Nita M, Grzybowski A. 2016. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults.
Oxid. Med. Cell Longev. 2016 : 3164734. - Poljsak B, Suput D, Milisav I. 2013. Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants.
Oxid. Med. Cell Longev. 2013 : 956792. - Khan MN, Mobin M, Abbas ZK, AlMutairi KA, Siddiqui ZH. 2017. Role of nanomaterials in plants under challenging environments.
Plant Physiol. Biochem. 110 : 194-209. - Chekulayeva LV, Shevchuk IN, Chekulayev VA, Ilmarinen K. 2006. Hydrogen peroxide, superoxide, and hydroxyl radicals are involved in the phototoxic action of hematoporphyrin derivative against tumor cells.
J. Environ. Pathol. Toxicol. Oncol. 25 : 51-77. - Moon HJ, Lee SR, Shim SN, Jeong SH, Stonik VA, Rasskazov VA, et al. 2008. Fucoidan inhibits UVB-induced MMP-1 expression in human skin fibroblasts.
Biol. Pharm. Bull. 31 : 284-289. - Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, He T, et al. 2009. Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin.
Am. J. Pathol. 174 : 101-114. - Binic I, Lazarevic V, Ljubenovic M, Mojsa J, Sokolovic D. 2013. Skin ageing: natural weapons and strategies.
Evid. Based Complement Alternat. Med. 2013 : 827248. - Jonganurakkun B, Wang Q, Xu SH, Tada Y, Minamida K, Yasokawa D, et al. 2008. Pediococcus pentosaceus NB-17 for probiotic use.
J. Biosci. Bioeng. 106 : 69-73. - Kwon HK, Jo WR, Park HJ. 2018. Immune-enhancing activity of
C. militaris fermented withPediococcus pentosaceus (GRC-ON89A) in CY-induced immunosuppressed model.BMC Complement. Altern. Med. 18 : 75. - Park SD, Lee DE, Jeong JW, Kim YT, Kim HM, Kim YJ, et al. 2014. Comprising lactic acid fermentation product of
Gelidium amansil extract as an active ingredient for improving skin wrinkle.J. Microbiol Biotechnol. 24 : 1583-1591. - Woronuk G, Demissie Z, Rheault M, Mahmoud S. 2011. Biosynthesis and therapeutic properties of
Lavandula essential oil constituents.Planta Med. 77 : 7-15. - Lopez V, Nielsen B, Solas M, Ramirez MJ, Jager AK. 2017. Exploring pharmacological mechanisms of lavender (
Lavandula angustifolia ) essential oil on central nervous system targets.Front Pharmacol. 8 : 280. - Cavanagh HM, Wilkinson JM. 2002. Biological activities of lavender essential oil.
Phytother. Res. 16 : 301-308. - Hsu CK, Chang CT, Lu HY, Chung YC. 2007. Inhibitory effects of the water extracts of
Lavendula sp. on mushroom tyrosinase activity.on mushroom tyrosinase activity. Food Chem. 105 : 1099-1105. - Kim AA, HA JH, Kim AR, Jeong HJ, Kim KM, Park SN. 2017. Cellular protective effect and active component analysis of lavender (
Lavandula angustifolia ) extracts and fractions.Appl. Chem. Eng. 28 : 479-484. - Spiridon I, Colceru S, Anghel N, Teaca CA, Bodirlau R, Armatu A. 2011. Antioxidant capacity and total phenolic contents of oregano (
Origanum vulgare ), lavender (Lavandula angustifolia ) and lemon balm (Melissa officinalis ) from Romania.Nat. Prod. Res. 25 : 1657-1661. - Ahn YJ, Won BR, Kang MK, Kim JH, Park SN. 2009. Antioxidant activity and component analysis of fermented
Lavandula angustifolia extracts.J. Soc. Cosmet. Scientists Korea 35 : 125-134. - Kim JK, Kim Y, Na KM, Surh YJ, Kim TY. 2007. [6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo.
Free Radic. Res. 41 : 603-614. - Masaki H. 2010. Role of antioxidants in the skin: anti-aging effects.
J. Dermatol. Sci. 58 : 85-90. - Park K, Lee JH. 2008. Protective effects of resveratrol on UVB-irradiated HaCaT cells through attenuation of the caspase pathway.
Oncol. Rep. 19 : 413-417. - Im AR, Song JH, Lee MY, Yeon SH, Um KA, Chae S. 2014. Anti-wrinkle effects of fermented and non-fermented
Cyclopia intermedia in hairless mice.BMC Complement. Altern. Med. 14 : 424. - Gurung N, Ray S, Bose S, Rai V. 2013. A broader view: microbial enzymes and their relevance in industries, medicine, and beyond.
Biomed. Res. Int. 2013 : 329121. - Yu-Ling Wena, Li-Pyng Yan, Chen C-S. 2013. Effects of fermentation treatment on antioxidant and antimicrobial activities of four common Chinese herbal medicinal residues by
Aspergillus oryzae .J. Food Drug Analysis. 21 : 219-226. - Ortiz-Castro R, Contreras-Cornejo HA, Macias-Rodriguez L, Lopez-Bucio J. 2009. The role of microbial signals in plant growth and development.
Plant Signal. Behav. 4 : 701-712. - Antai SP, Obong US. 1992. The effect of fermentation on the nutrient status and on some toxic components of
Icacinia manni .Plant Foods Hum. Nutr. 42 : 219-224. - Sung BK, Chung JW, Bae HR, Choi JS, Kim CM, Kim ND, et al. 2105. Humulus japonicus extract exhibits antioxidative and anti-aging effects via modulation of the AMPK-SIRT1 pathway.
Exp. Ther. Med. 9 : 1819-1826. - Pervin M, Unno K, Nakamura Y, Imai S. 2016. Luteolin suppresses ultraviolet A- and B-induced matrix metalloproteinase 1- and 9 expression in human dermal fibroblast cells.
J. Nutr. Food Sci. 6 : 1-6. - Hwang YP, Oh KN, Yun HJ, Jeong HG. 2011. The flavonoids apigenin and luteolin suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and AP-1- dependent signaling in HaCaT cells.
J. Dermatol. Sci. 61 : 23-31. - Api genin induces dermal collagen synthesis via smad2/3 signaling pathway.
Eur. J. Histochem. 59 : 2467.