2023 Volume 29 Issue 2 Pages 155-161
Glycation reactions between proteins and sugars or their metabolites produce advanced glycation end-products, and the glycation of collagen in normal human dermal fibroblasts (NHDF) causes skin spots and freckles. UV exposure induces oxidative stress in NHDF, which overexpresses enzymes that degrade collagen, resulting in dry skin and wrinkling. Herein, we produced a novel polyphenolic decoction of Chaga mushroom extracted with a fermentation medium. Chaga polyphenol decoction (CPD) inhibited the glycation of albumin and collagen gel 3 to 4 times more than 2-aminoguanidine. The antioxidant effects of CPD were investigated using the fluorescence of an intracellular reactive oxygen species (ROS) scavenger, and NHDF exposed to UV-A for 60 min (9.5 J/cm2) after pre-treatment with 190 jig/mL of CPD suppressed ROS scavenger emission by 50 % compared to treatment with phosphate buffer saline. These results suggest that CPD might be a promising glycation inhibitor and ROS scavenger.
Chaga mushroom (Inonotus obliquus), a fungus belonging to the Hymenochaetaceae family of the Basidiomycetes phylum, preferentially grows on the trunks of mature live birch trees in colder northern climates (Park et al., 2004; Lee et al., 2008; Agnestisia et al., 2019). Previous studies reported that Chaga mushroom contains many bioactive compounds with anti-fungal (Kahlos and Tikka, 1994), anti-thrombotic (Hyun et al., 2006), hypoglycemic (Balandaykin and Zmitrovich, 2015), anti-viral (Shibnev et al., 2011), anti-inflammatory (Choi et al., 2010), anti-oxidant (Cui et al., 2005) and anti-tumor activities (Song et al., 2013). These beneficial effects are often caused by an isolated component and while organic solvents are used to extract and isolate pharmacological components from Chaga, aqueous Chaga decoctions with no organic solvents would be nontoxic and also easy to formulate for prolonged oral administration. Chemical studies have also shown that Chaga contains significant levels of polyphenols such as lignin, inonoblins, phelligridins (Shashkina et al., 2006, Lee et al., 2007) and small phenolic compounds such as syringol and vanillic acid (Mazurkiewicz, 2006). Polyphenols are well-known to act as antioxidants, which scavenge reactive oxygen species (ROS). However, the anti-oxidant effects of Chaga aqueous decoctions on intracellular ROS induced by ultraviolet (UV) exposure have not yet been investigated.
In human nutrition, sugars are an essential source of energy. A high intake of sugars such as glucose leads to a high concentration in the blood and can cause diabetes mellitus. If metabolism of glucose in the intracellular glycolysis system becomes saturated, unusual glucose metabolism such as the polyol pathway that produces glyoxal and aldehyde derivatives (Lindstad and McKinley-McKee, 1993) via the hexosamine pathway can occur, and glucose itself or the resulting glucose metabolites easily react with proteins and enzymes. Glycation can produce advanced glycation end products (AGEs) that change the chemical structure of proteins and enzymes by reacting with glucose metabolites. Impairment of protein and enzyme activity by the production of AGEs causes various diseases and adverse effects, such as diabetic complications (Goh and Cooper, 2008), Alzheimer's disease (Ko et al., 2015), hypertension (Vasdev et al., 2007), arteriosclerosis (Liu et al., 2015) and skin aging (Gkogkolou and Böhm, 2012). Acidic polyphenols would be useful for protecting proteins or enzymes to suppress the glycation, but because many proteins and enzymes are composed of basic amino acids such as lysine, electrostatic interactions with acidic polyphenols can be expected to occur.
Firmness and moisturization of human skin are maintained by the composition of polysaccharides and proteins in the dermis including hyaluronic acid, collagen and elastin between the skin fibroblasts. Collagen is the most abundant protein in the body and is fundamentally involved in the formation of skin through establishment of an extracellular matrix. The turnover rate of collagen is very slow at about 15 years (Verzijl et al., 2000). Glycated collagen with irreversible crosslinked structure also has slow metabolism and absorption of visible light, resulting skin blotches. Therefore, it is important to suppress glycation of skin collagen from a cosmetic perspective.
Exposure to UV-A or UV-B light would produce ROS in skin fibroblasts, and intracellular ROS may induce overexpression of mRNA, resulting in the production of collagenase such as matrix metalloproteinase-1, which degrades collagen in the skin (Lee et al., 2021). The activation of collagenase causes the extracellular matrix in the dermis to be distorted, resulting in wrinkles and decreased moisturization of the skin. In order to suppress degradation of collagen in the skin, it is important to protect skin fibroblasts from exposure to UV. Application of sunscreen containing UV absorbers is often used to shield the skin from UV light. However, as UV absorbers are also excited and decomposed by prolonged UV exposure, frequent application is needed. The question thus arises whether Chaga polyphenol decoction (CPD) permeated into human skin fibroblasts can capture the intracellular ROS produced by UV light irradiation and thus suppress the decomposition of collagen in the skin. To ensure that various polyphenols can penetrate into the dermal fibroblasts to efficiently capture ROS, fermentation medium containing bacteria that produce polyphenols can be used to extract polyphenols in Chaga mushroom (Fig. 1).
Scheme of assumed anti-glycation and antioxidant effects of Chaga polyphenol decoction.
This study aimed to reveal the anti-glycation activity of CPD extracted with a fermentation medium by conducting anti-glycation tests on albumin and collagen as representative proteins. The intracellular ROS scavenging activity of CPD was also investigated using a fluorescent compound that fluoresces upon a specific reaction with intracellular ROS.
Preparation of Chaga polyphenol decoction (CPD) To an aqueous medium of plant-derived starch (corn cob, coffee, tofu meal, crushed bamboo and grass) of approximately 65 % (w/v), lactic acid bacteria (Lactobacillus, Bacillus subtilis) was added at 3 % (v/v), and the medium was fermented at room temperature for 1 month. Each mushroom fungi (shiitake, enokitake, maitake, shimeji and king trumpet mushrooms) was added to the fermentation medium at 3 % (v/v), and fermentation was continued at room temperature for 3 months. Chaga mushroom (Inonotus obliquus) collected in the Irkutsk region of Russia was extracted under optimal extraction conditions with the prepared fermentation medium. Crushed dry Chaga mushroom was added to fermentation medium at 20 % (w/v), and then the solution was boiled for 24 h at 100 °C. The resulting CPD was cooled to room temperature and filtered (Fig. S1).
Anti-glycation activity of CPD for albumin Albumin glycation was determined by fluorometry of glycated albumin, which emits fluorescence at 440 nm following excitation at 370 nm (Ramkissoon et al., 2013). Briefly, 50 uL of 10 % (v/v) heat-inactivated fetal bovine serum (FBS; E.U. Origin, Biowest, Nuaill, France) aqueous solutions, 10 uL of CPD as final concentrations from 12.5 to 200 ug/mL and 10 uL of 500 mM glyceraldehyde in DMSO solution was poured into 96-well black plates and then incubated at 37 °C for 24 h in a humidified atmosphere without CO2 to prevent water evaporation from the samples. Fluorescence of the reactants was assessed by emission at 440 nm following excitation at 370 nm with a GloMax®-Multi Detection System (Promega, Fitchburg, WI, USA). The inhibition of albumin glycation was calculated as follows:
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Fluorescence was completely inhibited in albumin in pH 7.4 PBS without glyceraldehyde, which was the negative control. 2-Aminoguanidine aqueous solution was used as a positive control.
Collagen gelation and the characterization Type I Collagen, extracted with acid or digested with pepsin, forms fibrils under physiological conditions (Suzuki et al., 1999). Pepsin-solubilized type I collagen from bovine dermis (3 mg/mL in 5 mM acetic acid aqueous solution) was purchased from Nippi, Inc. (Tokyo, Japan). Collagen gels were prepared with collagen solutions and pH 7.4 PBS. Briefly, 25 to 42 μL of collagen solutions at 3 mg/mL and 25 μL of 20 mM PBS or 8 to 25 μL of 100 mM PBS were added into 96-well black plates and incubated at 37 °C in a humidified atmosphere without CO2. The kinetics of collagen gelation were monitored by measuring turbidity, which is defined as the absorbance at 405 nm. Collagen gels produced using 42 μL of collagen solution and 8 μL of 100 mM PBS were used in the following experiments.
Anti-glycation activity of CPD for collagen gel The anti-glycation effects of CPD on collagen gels were evaluated using the same procedure as for albumin. Briefly, 50 μL of collagen gel, 40 μL of CPD as final concentrations from 50 to 800 μg/mL, and 10 μL of 500 mM glyceraldehyde solutions in DMSO were added into 96-well black plates and incubated at 37 °C for 24 h in a humidified atmosphere without CO2. The fluorescence of the reactants was measured. The inhibition of the glycated collagen gel was calculated using Eq. 1.
Cell culture Primary normal human dermal fibroblasts (NHDF) were purchased from Zen-Bio, Inc. (Durham, NC, USA). NHDF were grown in DMEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin and 2 mM L-glutamine at 37 °C (under 5 % CO2 in a humidified incubator). NHDF were passaged by trypsinization with 0.25 % trypsin-EDTA every 3 to 5 days, and they were used in the following experiments.
Evaluation of cell viability of NHDF exposed to UV-A Aliquots (100 iL) of NHDF suspension (2.0 x 105 cell/mL) were seeded in 96-well plates, and then incubated for 24 h at 37 °C. Plates were directly irradiated with UV-A light (FL10BL light, 9.5 μW/cm2, NEC HotaluX Ltd., Tokyo, Japan) for 20 min (11.4 mJ/cm2), 40 min (22.8 mJ/cm2), 60 min (34.2 mJ/cm2), 90 min (51.3 mJ/cm2) or 180 min (102.6 mJ/cm2). After the UV-A irradiation, the medium was replaced with 100 μL of fresh medium. The following solutions were prepared: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium sodium salt (WST-1) dissolved in 20 mM pH 7.4 HEPES buffer (5.5 mM WST-1 solution), and 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) was dissolved in sterile water (2 mM 1-methoxy PMS solution). A 19:1 ratio solution of 5.5 mM WST-1 and 2 mM 1-methoxy PMS was mixed and then filtered through a 0.22 μm sterilized membrane filter. After adding 10 μL of WST-1 reagent solution to the 96-well plate, the plate was incubated at 37 °C for 2 h. The absorbance of each well was measured at 450 nm. The percentage of cell viability was calculated using Eq. 2:
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where Abs. Experimental is the absorbance of each sample, Abs. Negative control is the absorbance without cells, and Abs. Positive control is the absorbance of the confluent cells.
Antioxidant effects of CPD on the intracellular ROS of NHDF exposed to UV-A Aliquots (100 L) of NHDF suspension (2.0 × 105 cell/mL) were seeded in 96-well plates and then incubated at 37 °C for 24 h. The medium was removed, and 100 μL of CPD diluted with DMEM was added. The UV-A light was placed 60 mm above the 96-well plates to provide irradiation for 60 min.
2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA; Funakoshi Co., Ltd., Tokyo, Japan) was used to detect the intracellular ROS. Non-fluorescent DCFH-DA would be deacetylated by intracellular esterase to non-fluorescent DCFH, which is then quickly oxidized by intracellular ROS to fluorescent DCF. DCF has an excitation wavelength of 485 nm and emission between 500 and 600 nm. UV irradiation is known to rapidly auto-oxidize DCFH-DA or hydrolyze DCFH (Rastogi et al., 2010); therefore, DCFH-DA aqueous solution must be prepared at the time of use and cannot be added to cells before UV irradiation. The medium was removed after UV-A irradiation, and 100 μL of DCFH-DA (20 μM as final concentration) solubilized in DMSO and pH 7.4 PBS was added to 96-well plates, and then incubated in the dark at 37 °C for 1 h. Aliquots (100 μL) of cell lysate (Cell Lysis Buffer M, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were also added and incubated at 37 °C for 1 h. The fluorescence of each well was measured by emission at 510–570 nm with excitation at 490 nm. The antioxidant activities of CPD for NHDF exposed to UV-A were calculated using Eq. 3:
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Anti-glycation effects of Chaga polyphenol decoction (CPD) Albumin is an important protein responsible for transporting water in the human body. When sugar saturates in a cell, the fructose metabolic pathway produces sugar metabolites such as glyceraldehyde that readily promote the glycation of proteins. AGEs with cross-linked structures are well-known to emit fluorescence at 440 nm upon excitation at 370 nm. The formation of AGEs would be reduced if CPD protects albumin through electrostatic interactions, and this anti-glycation effect of CPD would be observed as suppressed emission fluorescence. The polyphenol concentration in CPD was 2.0 mg/mL as determined by the Folin-Ciocalteu method. The polyphenol concentrations of the boiled water extract of Chaga mushroom alone and with fermentation medium were 0.9 and 1.1 mg/mL, respectively. CPD showed higher polyphenol concentration than Chaga mushroom alone, and better anti-glycation effect from CPD could thus be expected.
Figure 2 shows that the fluorescence of the AGEs in bovine serum albumin treated with CPD was obviously suppressed compared to the PBS blank. Significant differences in the concentration dependence effects of inhibition were observed between 200 and 12.5 μg/mL CPD, with the inhibitory effect of 200 ug/mL of CPD producing an effect 4fold greater than that of 880 μg/mL of 2-aminoguanidine, a standard compound used for anti-glycation activity. The IC50 on the inhibition of albumin glycation was 72.5 μg/mL. Therefore, CPD exhibited effective anti-glycation activity and would be expected to greatly inhibit the glycation of blood proteins.
Effect of Chaga polyphenol decoction concentration on the inhibition of albumin glycation. *AG: 2-Aminoguanidine. Data are shown as the mean ± SD (n = 6). Statistical significance was measured between two groups using unequal variances Welch's t-test. *p < 0.05, **p < 0.01
Preparation and evaluation of gelatinized collagen mimicking skin collagen To evaluate the anti-glycation effects of CPD on the collagen in skin, we prepared and evaluated collagen gels. When basic collagen dissolves in acidic water and is neutralized, intramolecular interactions of collagen based mainly on hydrogen bonds are triggered, which induces the self-assembly and gelation of collagen. Since the size of collagen gels is generally greater than 400 nm, the absorbance of these solutions would increase because light at a wavelength of 400 nm could be scattered by the collagen gels.
Figure 3 (a) shows that the absorbance of the neutralized aqueous collagen solutions increased with time under each neutralization condition with pH 7.4 PBS. The acidic aqueous solution of collagen exhibited no absorbance throughout the incubation. The concentration of collagen was almost constant from 4 to 10 h after neutralization, followed by a gradual decrease. This decreasing behavior of the absorbance may have been caused by the gradual reduction in the pH of the collagen aqueous solutions because of the dissolution of carbon dioxide absorbed from the air and repulsion between the ionic amino acids of collagen causing a gradual dissociation of the collagen gel. The collagen gel exhibiting the highest absorbance was used to evaluate the anti-glycation activity of CPD.
(a) Changes in absorbance of the collagen gel with incubation time. (b) Effect of Chaga polyphenol decoction concentration on the inhibition of collagen gel glycation. *AG: 2-Aminoguanidine. Both data are shown as the mean ± SD (n = 6). Statistical significance was measured between two groups using unequal variances Welch's t-test. *p < 0.05, **p < 0.01
Evaluation of anti-glycation activity of CPD on skin-mimicking collagen gel The anti-glycation activity of CPD on the collagen gel was examined using the same procedure as for albumin. Figure 3 (b) shows that the fluorescence intensity of the glycated collagen gel was suppressed by CPD. CPD at 800 μg/mL showed greater anti-glycation activity than did 2 490 μg/mL of 2-aminoguanidine. The AGEs-derived absorbance was decreased by 50 % compared to the blank at 100 μg/mL CPD for collagen gels. The IC50 of 126 μg/mL for collagen gel was higher than that of 72.5 μg/mL for albumin. This result may be due to differences in coating efficiency of polyphenols on proteins of different sizes.
Cell viability of human skin fibroblasts after UV-A exposure UV-A light has wavelengths between 320 and 400 nm and is known to kill bacteria. Prolonged exposure of cells to UV light is thought to induce apoptosis. The amount of ROS produced in cells and cellular apoptosis are considered to depend on UV intensity and exposure time. Thus, it is important to determine the UV exposure conditions that cause no cell death prior to evaluating the antioxidant effects of CPD. The viability of NHDF at various durations of UV-A exposure was examined.
Figure 4 (a) shows the viability of NHDF with increasing UV-A exposure time. No NHDF death occurred until 60 min (34.2 mJ/cm2) of UV-A exposure, and an exposure time of more than 90 min (51.3 mJ/cm2) markedly reduced the viability of NHDF. Therefore, to evaluate the antioxidant effects of CPD, NHDF were irradiated with UV-A for 60 min in the following experiments.
(a) Effect of UV-A exposure time on the cell viability of human dermal fibroblasts (NHDF). (b) Effect of Chaga polyphenol decoction concentration on antioxidant activity for UVA-irradiated NHDF. Both data are shown as mean ± SD (n = 6). Statistical significance was measured between two groups using unequal variances Welch's t-test. *VC: Vitamin C. *p < 0.05, **p < 0.01
Antioxidant effects of CPD on NHDF exposed to UV-A The antioxidant effects of CPD on ROS generated in NHDF exposed to UV were investigated using DCFH-DA as an intracellular ROS detection agent. DCFH-DA does not fluoresce in its unchanged form, but it undergoes deacetylation by intracellular esterase in DCFH after uptake by cells. The generated DCFH changes to fluorescent DCF (λex.: 485 nm, λem.: 530 nm) upon oxidation by ROS, and therefore the amount of intracellular ROS production can be evaluated by measuring the fluorescence intensity of DCF. It has also been reported that polyphenols can reliably penetrate cell membranes within 2 h by pre-incubating the cells (Hong et al., 2002).
Figure 4 (b) shows that the DCF-derived fluorescence decreased in a concentration-dependent manner with increasing CPD. A significant difference in DCF-derived fluorescence was observed between 800 and 12.5 μg/mL in the study. The 50 % inhibition of oxidation compared to the blank was observed at a concentration of 190 μg/mL. CPD at 800 μg/mL exhibited a higher antioxidation effect than for 350 μg/mL of ascorbic acid (vitamin C), which is known to be easily retained in the cytoplasm, indicating efficient intracellular antioxidant activity. Therefore, CPD would be expected to be retained in the cytoplasm and to play a role in scavenging ROS in the cytoplasm. These findings suggest that CPD would suppress the expression of enzymes that degrade components of skin tissue through reducing the oxidative stress of UV-exposed cells. Thus, CPD should be useful as a skin anti-aging agent and for maintaining the cosmetic beauty of the skin.
Polyphenol decoction extracted from Chaga mushrooms with a fermentation medium exhibited higher anti-glycation activity on albumin and collagen gels than a standard anti-glycation agent, 2-aminoguanidine. CPD also inhibited ROS production by human skin fibroblasts exposed to UV-A in a concentration-dependent manner. These findings suggest that the anti-glycation and antioxidant effects of CPD may have health and cosmetic benefits for the skin.
Acknowledgements We thank Philip Creed, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Conflict of interest There are no conflicts of interest to declare.
HPLC chromatogram of the filtrate and residue of filtered Chaga polyphenol decoction extracted with the fermented medium.
The filtrate and the residue of Chaga polyphenol decoction extracted with fermentation medium were also analyzed by high-performance liquid chromatography (HPLC). Figure S1 (Supplementary materials) shows that no difference was observed between the HPLC results from the filtrate and residue, with Chaga mushroom having 5 main components from the HPLC results.
