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Abstract Extraction process of Cordyceps militaris polysaccharide (PS) was optimized with single factor experiment and orthogonal test. Then, the anti-UVB ability of the polysaccharide was investigated to develop anti-UV sun-screening agent through determining the cell viability after UVB irradiation. The results showed that the optimum were solid-liquid ratio at 1∶40 (g/ml) at 80 ℃ for 4 h, and the extract ratio was 27.4 % according to the verification test. The PS showed no cytotoxicity to HaCaT cells and pretreatment with the PS significantly decreased UVB-induced cytotoxicity in a dose-dependent relationship. It suggests that the PS has strong anti-UVB irradiation ability and has a good application prospect in developing sunscreen agent.
Key words Cordyceps militaris; Polysaccharide; Optimization of extraction conditions; Anti-UV radiation
Received: January 23, 2021 Accepted: March 4, 2021
Supported by College Students Practice Innovation Training Project in Henan Province (No.201911834019); Henan Provincial Department of Education (No.21B350001).
Duofei WANG (1998- ), female, P. R. China, major: clinical medicine.
*Corresponding author. E-mail: liyanli0921@163.com.
As food and medicine fungus, research on Cordyceps militaris has attracted much attention recent years because it replaced scarce Cordyceps sinensis. It has been reported that C. militaris is rich in nutrients, including a variety of trace elements, proteins, vitamins and amino acids, as well as cordyceps polysaccharide, cordycepin and cordyceps acid, which are medicinal ingredients, showing the functions such as anti-tumor, anti-aging, enhancing immunity and liver protection［1-4］, while the anti-UV effect of C. militaris PS has not been reported.
Over-exposure of human skin to environmental factors such as ultraviolet radiation (UV) leads to different skin diseases. Particularly UVB (280-315 nm) is more genotoxic and about 1 000 times more capable of causing sunburn than UVA (315-400 nm)［5］. Chronic exposure to UVB results in erythema, skin aging, inflammation, oxidative damage, and immune-suppression, sometimes results in skin cancer［6-12］. Recently, attention has been focused on developing topically administered natural materials from dietary and medicinal origins for reducing the amount of UV penetrating skin and reinforcing skins own protective mechanisms, such as Codium fragile extract, Prunella vulgaris extract, green tea polysaccharide and so on［13-15］, while the skin protecting potentials of polysaccharide from C. militaris have not been reported. Therefore, we aimed to investigate the protective effects of the PS on UVB-irradiated HaCaT cell line after optimization of extraction condition of the PS. Materials and Methods
Chemicals and reagents
Fruiting body of C. militaris was purchased from Yihongtang Pharmaceutical Co. Ltd of Bozhou, Anhui. Modified Eagles medium (MEM), penicillin-streptomycin solution, and non-animal L-glutamine and trypsin-EDTA solution was purchased from Life Technologies Company. Fetal bovine serium (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. HaCaT cells were purchased from Cell Resource Center of Chinese Union Medical College. CCK-8 kit was purchased from Beijing Sorlabio Biological Co., Ltd. All other reagents were analytical pure.
Glucose standard curve
The phenol and sulfuric acid method was used to draw a glucose standard curve. Glucose standard solutions with different concentrations were mixed with sulfuric acid and the absorbance was determined at 490 nm. The standard curve was drawn, and the regression equation was obtained.
Extract of polysaccharide of C. militaris
Effect of solid-liquid ratio on the extraction yield of polysaccharide
The fruiting body of C. militaris was dried and crushed into 200 meshes. The powder was soaked with water at ratios of 1 g∶10 ml, 1 g∶20 ml, 1 g∶30 ml and 1 g∶40 ml at 60 ℃ for 1 h, respectively. The extracts were diluted with water to 50 ml, and determined for the absorbance at 490 nm. Each experiment was repeated three times.
Effect of temperature on extraction yield of polysaccharide
1 g of the dried powder mentioned above was soaked with 20 ml of water and incubated in 50, 60, 70, 80 and 90 ℃ for 1 h, respectively. The absorbance was determined according to the method mentioned above. Each experiment was repeated three times.
Effect of time on extraction yield of polysaccharide
1 g of the dried powder mentioned above was soaked with 20 ml of water and incubated in 70 ℃ for 0.5, 1, 2, 3, 4 h, respectively. The absorbance was determined according to the method mentioned above. Each experiment was repeated three times.
Optimization of extract condition of polysaccharide
Orthogonal test was done according to the orthogonal factor level table (Table 1) and Table 2 to optimize the extract condition of the PS. The K value was used to verify the optimum level, and R value was used to verify the optimum factor for extract condition optimization of the PS.
1.5 Cell culture
HaCaT cells were cultured in MEM supplemented with 10% FBS, 5 mM non-animal L-glutamine, 1% penicillin-streptomycin solution, in a humidified incubator aerated with 5% CO2, at 37 ℃. Cytotoxicity of polysaccharide
HaCaT cells were exposed for 48 h to polysaccharide of C. militaris (0.5-4.0 mg/ml) in MEM , and CCK-8 method was used to determine the cytotoxicity of the polysaccharide later.
Cell viability after UVB irradiation
HaCaT cells were seeded in 96-well plates and were exposed to the polysaccharide (0.25-2.00 mg/ml) for 2 h after proliferating to 80% confluence. The UV irradiation system included one UVB lamp (peak 312 nm), the irradiance of which was 230 μw/cm2. The emitted intensity was measured using the UV340B radiometer (Sanpometer Company, China). Before irradiation, culture medium was removed. Cells were rinsed once with phosphate-buffered saline (PBS), covered with a thin PBS layer, and irradiated with different doses of UVB (30-120 mJ/cm2). To prevent the cells overheating, plates were kept on ice. Control cells were treated in the same way but were not exposed to UVB rays. The cells were incubated for another 24 h in culture medium after irradiation. 10% CCK-8 solution was added to the medium and incubated 1 h and measured spectrophotometrically on a microplate reader at 450 nm (Multimode Plate Reader, Envision, PerkinElmer, USA). Viable cells were calculated as a percentage of the negative control cells set at 100%.
Statistical analysis
Values were expressed as mean±SEM. The analysis of variance (AVONA) and the Tukey test were used to assess biological activity data, with P<0.05 established as statistically significant.
Results and Discussion
Extract of polysaccharide of C. militaris
Glucose standard curve
According to the glucose standard curve, the linear regression equation is y=0.088 9x-0.077 7, R2=0.998 8.
Effect of solid-liquid ratio on extraction yield of polysaccharide
The effect of solid-liquid ratio on the extraction yield of the polysaccharide is shown in Fig. 2. The yield rate increased with the increase of the extract volume until 1∶30 (g/ml) and decreased slightly at 1∶40 (g/ml). The yield rate was the maximum when the solid-liquid ratio was 1∶30 (g/ml).
Effect of temperature on extraction yield of polysaccharide
The effect of temperature on the extraction yield of the polysaccharide is shown in Fig. 3. The yield ratio increased with the extraction temperature increasing, and reached the largest value at 80 ℃.
Effect of time on extraction yield of polysaccharide
The effect of time on the extraction yield of the PS is shown in Fig.4. When other extraction conditions were fixed, with the extraction time increasing, the yield rate of the PS in fruit body of C. mystaris increased gradually, and then decreased after extraction time of 3 h. The reason might be that the prolonged high temperature extraction caused certain damage to polysaccharide structure. Optimization of extract condition of polysaccharide
According to the result of orthogonal test, the optimum extraction conditions were solid-liquid ratio at 1∶40 (g/ml) and at 80 ℃ for 4 h. Temperature is the most important influencing factor on polysaccharide yield, followed by time and solid-liquid ratio. A verification test was done with the best extraction condition and the extract ratio was 27.4%.
Cytotoxicity of polysaccharide
HaCaT cells were exposed to different concentrations of the PS and cytotoxicity was assessed immediately after the end of exposure and 48 h later. Results are shown as percentage of viable cells (% cell viability) compared to negative control cell (Ctr) set on 100%. Each data point is the mean of three independently experiments.
The cell viability after drug administration was shown in Fig.5. The cell viability was 115%, 119%, 100%, 94.95% and 92.11% at a serial concentration. It suggested that the PS showed almost no cytotoxicity and promoted the cell proliferation at low concentration. Therefore, 2 mg/ml was used as the maximal concentration for the further experiments.
Cell viability after UVB irradiation
UVB significantly reduced HaCaT viability and showed in a dose-dependent manner. The cell viability was 56% with the UVB dose of 30 mJ/cm2 and decreased to 29.94% with UVB dose of 120 mJ/cm2. It suggests that UVB ray has a delayed effect on cell damage. However, pretreatment with CMPS significantly decreased UVB-induced cytotoxicity and also showed a dose-dependent relationship (Fig. 6). For each UV irradiation group, the cell viability increased with the extract concentrations increasing (P<0.05, vs control group).
The results of anti-UVB experiment suggest that the PS is not only non-cytotoxicity but also promotes the growth of HaCaT cells. Pretreatment with the PS significantly decreased UVB-induced cytotoxicity in a dose-dependent relationship. It means the PS has strong anti-UVB irradiation ability. Also, cordyceps polysaccharide showed some anti-aging, anti-oxidant ［16］ and enhancing immunity activity, which will be much more beneficial to the PS as a sunscreen additive to be developed. However, the anti-UV mechanisms of the PS need to be further studied in future.
Conclusions and Discussion
It is worth mentioning that, in present research, the phenol and sulfuric acid method was used to determine the content of PS of C. militaris according to the glucose standard curve. The extract conditions were optimized and the extract ratio could reach to 27.4%, which was a theoretical value. Ethanol precipitation method is always used to get PS, and different volume fraction of ethanol could precipitate different fragment sizes of polysaccharide, some small molecular segments cannot be precipitated, so the actual yield by ethanol precipitation method will be lower that the theoretical value by phenol and sulfuric acid method. References
［1］ SUN Y. Extraction and synergistic antioxidant effect analysis of Cordyceps militaris polysaccharide［D］. Harbin: Northeast Forestry University, 2013. (in Chinese)
［2］ ZHU ZY, LIU N, SI CL, et al. Structure and anti-tumor activity of a high-molecular-weight polysaccharide from cultured mycelium of Cordyceps gunnii［J］. Carbohydrate Polymers, 2012, 88(3): 1072-1076.
［3］ GUO LX, ZHONG W, JIANG L. Effects of Cordyceps militaris polysaccharide on antioxidant level and Bcl-2, Bax mRNA expression in H22 tumor tissues of aging rats［J］. Journal of Guizhou Medical University, 2019, 44(2): 173-177.
［4］ LI XT, LI HC, LI CB, et al. Protective effects on mitochondria and anti-aging activity of polysaccharides from cultivated fruiting bodies of Cordyceps militaris［J］. American Journal of Chinese Medicine, 2010, 38(6): 1093-1106.
［5］ SVOBODOV A, PSOTOV J, WALTEROV D. Natural phenolics in the prevention of UV-induced skin damage［J］. A review, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub., 2003(147): 137-145.
［6］ ARMSTRONG BK, KRICKER A. The epidemiology of UV induced skin cancer［J］. J.Photochem. Photobiol., 2001(B63): 8-18.
［7］ DAZARD JE, GAL H, AMARIGLIO N, et al. Genome-wide comparison of human keratinocyte and squamous cell carcinoma responses to UVB irradiation: implications for skin and epithelial cancer［J］. Oncogene, 2003(22): 2993-3006.
［8］ GUO W, AN Y, JIANG L, et al. The protective effects of hydroxytyrosol against UVB-induced DNA damage in HaCaT cells［J］. Phytother Res, 2010, 24(3): 352-359.
［9］ AFAQ F, KATIYAR SK. Polyphenols: skin photoprotection and inhibition of photocarcinogenesis［J］. Mini Rev Med Chem, 2011, 11(14): 1200-1215.
［10］ HSEU YC, CHOU CW, SENTHIL KUMAR KJ, et al. Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes［J］. Food Chem Toxicol, 2012, 50(5): 1245-1255.
［11］ KIM JK, KIM Y, NA KM, et al. Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo［J］. Free Radic Res, 2007(41): 603-614.
［12］ TANAKA K, HASEGAWA J, ASAMITSU K, et al. Prevention of the ultraviolet B-mediated skin photoaging by a nuclear factor kappaB inhibitor, parthenolide［J］. J Pharmacol Exp Ther, 2005(315): 624-630.
［13］ LEE C, PARK GH, AHN EM, et al. Protective effect of Codium fragile against UVB-induced pro-inflammatory and oxidative damages in HaCaT cells and BALB/c mice［J］. Fitoterapia, 2013(86): 54-63.
［14］ JITKA VOSTLOV, ADLA ZDAILOV, ALENA SVOBODOV. Prunella vulgaris extract and rosmarinic acid prevent UVB-induced DNA damage and oxidative stress in HaCaT keratinocytes［J］. Arch. Dermatol. Res., 2010(302): 171-181.
［15］ CRAIG A. ELMETS, DIVYA SINGH, KAREN TUBESING, et al. Cutaneous photoprotection from ultraviolet injury by green tea polyphenols［J］. J. Am. Acad. Dermatol, 2001(44): 425-432.
［16］ WU YY, QI XN, XIE M, et al. Study on optimization of extraction process and antioxidant activity of Cordyceps militaris polysaccharides［J］. Food Research And Development, 2020, 41(9): 103-109.
Key words Cordyceps militaris; Polysaccharide; Optimization of extraction conditions; Anti-UV radiation
Received: January 23, 2021 Accepted: March 4, 2021
Supported by College Students Practice Innovation Training Project in Henan Province (No.201911834019); Henan Provincial Department of Education (No.21B350001).
Duofei WANG (1998- ), female, P. R. China, major: clinical medicine.
*Corresponding author. E-mail: liyanli0921@163.com.
As food and medicine fungus, research on Cordyceps militaris has attracted much attention recent years because it replaced scarce Cordyceps sinensis. It has been reported that C. militaris is rich in nutrients, including a variety of trace elements, proteins, vitamins and amino acids, as well as cordyceps polysaccharide, cordycepin and cordyceps acid, which are medicinal ingredients, showing the functions such as anti-tumor, anti-aging, enhancing immunity and liver protection［1-4］, while the anti-UV effect of C. militaris PS has not been reported.
Over-exposure of human skin to environmental factors such as ultraviolet radiation (UV) leads to different skin diseases. Particularly UVB (280-315 nm) is more genotoxic and about 1 000 times more capable of causing sunburn than UVA (315-400 nm)［5］. Chronic exposure to UVB results in erythema, skin aging, inflammation, oxidative damage, and immune-suppression, sometimes results in skin cancer［6-12］. Recently, attention has been focused on developing topically administered natural materials from dietary and medicinal origins for reducing the amount of UV penetrating skin and reinforcing skins own protective mechanisms, such as Codium fragile extract, Prunella vulgaris extract, green tea polysaccharide and so on［13-15］, while the skin protecting potentials of polysaccharide from C. militaris have not been reported. Therefore, we aimed to investigate the protective effects of the PS on UVB-irradiated HaCaT cell line after optimization of extraction condition of the PS. Materials and Methods
Chemicals and reagents
Fruiting body of C. militaris was purchased from Yihongtang Pharmaceutical Co. Ltd of Bozhou, Anhui. Modified Eagles medium (MEM), penicillin-streptomycin solution, and non-animal L-glutamine and trypsin-EDTA solution was purchased from Life Technologies Company. Fetal bovine serium (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. HaCaT cells were purchased from Cell Resource Center of Chinese Union Medical College. CCK-8 kit was purchased from Beijing Sorlabio Biological Co., Ltd. All other reagents were analytical pure.
Glucose standard curve
The phenol and sulfuric acid method was used to draw a glucose standard curve. Glucose standard solutions with different concentrations were mixed with sulfuric acid and the absorbance was determined at 490 nm. The standard curve was drawn, and the regression equation was obtained.
Extract of polysaccharide of C. militaris
Effect of solid-liquid ratio on the extraction yield of polysaccharide
The fruiting body of C. militaris was dried and crushed into 200 meshes. The powder was soaked with water at ratios of 1 g∶10 ml, 1 g∶20 ml, 1 g∶30 ml and 1 g∶40 ml at 60 ℃ for 1 h, respectively. The extracts were diluted with water to 50 ml, and determined for the absorbance at 490 nm. Each experiment was repeated three times.
Effect of temperature on extraction yield of polysaccharide
1 g of the dried powder mentioned above was soaked with 20 ml of water and incubated in 50, 60, 70, 80 and 90 ℃ for 1 h, respectively. The absorbance was determined according to the method mentioned above. Each experiment was repeated three times.
Effect of time on extraction yield of polysaccharide
1 g of the dried powder mentioned above was soaked with 20 ml of water and incubated in 70 ℃ for 0.5, 1, 2, 3, 4 h, respectively. The absorbance was determined according to the method mentioned above. Each experiment was repeated three times.
Optimization of extract condition of polysaccharide
Orthogonal test was done according to the orthogonal factor level table (Table 1) and Table 2 to optimize the extract condition of the PS. The K value was used to verify the optimum level, and R value was used to verify the optimum factor for extract condition optimization of the PS.
1.5 Cell culture
HaCaT cells were cultured in MEM supplemented with 10% FBS, 5 mM non-animal L-glutamine, 1% penicillin-streptomycin solution, in a humidified incubator aerated with 5% CO2, at 37 ℃. Cytotoxicity of polysaccharide
HaCaT cells were exposed for 48 h to polysaccharide of C. militaris (0.5-4.0 mg/ml) in MEM , and CCK-8 method was used to determine the cytotoxicity of the polysaccharide later.
Cell viability after UVB irradiation
HaCaT cells were seeded in 96-well plates and were exposed to the polysaccharide (0.25-2.00 mg/ml) for 2 h after proliferating to 80% confluence. The UV irradiation system included one UVB lamp (peak 312 nm), the irradiance of which was 230 μw/cm2. The emitted intensity was measured using the UV340B radiometer (Sanpometer Company, China). Before irradiation, culture medium was removed. Cells were rinsed once with phosphate-buffered saline (PBS), covered with a thin PBS layer, and irradiated with different doses of UVB (30-120 mJ/cm2). To prevent the cells overheating, plates were kept on ice. Control cells were treated in the same way but were not exposed to UVB rays. The cells were incubated for another 24 h in culture medium after irradiation. 10% CCK-8 solution was added to the medium and incubated 1 h and measured spectrophotometrically on a microplate reader at 450 nm (Multimode Plate Reader, Envision, PerkinElmer, USA). Viable cells were calculated as a percentage of the negative control cells set at 100%.
Statistical analysis
Values were expressed as mean±SEM. The analysis of variance (AVONA) and the Tukey test were used to assess biological activity data, with P<0.05 established as statistically significant.
Results and Discussion
Extract of polysaccharide of C. militaris
Glucose standard curve
According to the glucose standard curve, the linear regression equation is y=0.088 9x-0.077 7, R2=0.998 8.
Effect of solid-liquid ratio on extraction yield of polysaccharide
The effect of solid-liquid ratio on the extraction yield of the polysaccharide is shown in Fig. 2. The yield rate increased with the increase of the extract volume until 1∶30 (g/ml) and decreased slightly at 1∶40 (g/ml). The yield rate was the maximum when the solid-liquid ratio was 1∶30 (g/ml).
Effect of temperature on extraction yield of polysaccharide
The effect of temperature on the extraction yield of the polysaccharide is shown in Fig. 3. The yield ratio increased with the extraction temperature increasing, and reached the largest value at 80 ℃.
Effect of time on extraction yield of polysaccharide
The effect of time on the extraction yield of the PS is shown in Fig.4. When other extraction conditions were fixed, with the extraction time increasing, the yield rate of the PS in fruit body of C. mystaris increased gradually, and then decreased after extraction time of 3 h. The reason might be that the prolonged high temperature extraction caused certain damage to polysaccharide structure. Optimization of extract condition of polysaccharide
According to the result of orthogonal test, the optimum extraction conditions were solid-liquid ratio at 1∶40 (g/ml) and at 80 ℃ for 4 h. Temperature is the most important influencing factor on polysaccharide yield, followed by time and solid-liquid ratio. A verification test was done with the best extraction condition and the extract ratio was 27.4%.
Cytotoxicity of polysaccharide
HaCaT cells were exposed to different concentrations of the PS and cytotoxicity was assessed immediately after the end of exposure and 48 h later. Results are shown as percentage of viable cells (% cell viability) compared to negative control cell (Ctr) set on 100%. Each data point is the mean of three independently experiments.
The cell viability after drug administration was shown in Fig.5. The cell viability was 115%, 119%, 100%, 94.95% and 92.11% at a serial concentration. It suggested that the PS showed almost no cytotoxicity and promoted the cell proliferation at low concentration. Therefore, 2 mg/ml was used as the maximal concentration for the further experiments.
Cell viability after UVB irradiation
UVB significantly reduced HaCaT viability and showed in a dose-dependent manner. The cell viability was 56% with the UVB dose of 30 mJ/cm2 and decreased to 29.94% with UVB dose of 120 mJ/cm2. It suggests that UVB ray has a delayed effect on cell damage. However, pretreatment with CMPS significantly decreased UVB-induced cytotoxicity and also showed a dose-dependent relationship (Fig. 6). For each UV irradiation group, the cell viability increased with the extract concentrations increasing (P<0.05, vs control group).
The results of anti-UVB experiment suggest that the PS is not only non-cytotoxicity but also promotes the growth of HaCaT cells. Pretreatment with the PS significantly decreased UVB-induced cytotoxicity in a dose-dependent relationship. It means the PS has strong anti-UVB irradiation ability. Also, cordyceps polysaccharide showed some anti-aging, anti-oxidant ［16］ and enhancing immunity activity, which will be much more beneficial to the PS as a sunscreen additive to be developed. However, the anti-UV mechanisms of the PS need to be further studied in future.
Conclusions and Discussion
It is worth mentioning that, in present research, the phenol and sulfuric acid method was used to determine the content of PS of C. militaris according to the glucose standard curve. The extract conditions were optimized and the extract ratio could reach to 27.4%, which was a theoretical value. Ethanol precipitation method is always used to get PS, and different volume fraction of ethanol could precipitate different fragment sizes of polysaccharide, some small molecular segments cannot be precipitated, so the actual yield by ethanol precipitation method will be lower that the theoretical value by phenol and sulfuric acid method. References
［1］ SUN Y. Extraction and synergistic antioxidant effect analysis of Cordyceps militaris polysaccharide［D］. Harbin: Northeast Forestry University, 2013. (in Chinese)
［2］ ZHU ZY, LIU N, SI CL, et al. Structure and anti-tumor activity of a high-molecular-weight polysaccharide from cultured mycelium of Cordyceps gunnii［J］. Carbohydrate Polymers, 2012, 88(3): 1072-1076.
［3］ GUO LX, ZHONG W, JIANG L. Effects of Cordyceps militaris polysaccharide on antioxidant level and Bcl-2, Bax mRNA expression in H22 tumor tissues of aging rats［J］. Journal of Guizhou Medical University, 2019, 44(2): 173-177.
［4］ LI XT, LI HC, LI CB, et al. Protective effects on mitochondria and anti-aging activity of polysaccharides from cultivated fruiting bodies of Cordyceps militaris［J］. American Journal of Chinese Medicine, 2010, 38(6): 1093-1106.
［5］ SVOBODOV A, PSOTOV J, WALTEROV D. Natural phenolics in the prevention of UV-induced skin damage［J］. A review, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub., 2003(147): 137-145.
［6］ ARMSTRONG BK, KRICKER A. The epidemiology of UV induced skin cancer［J］. J.Photochem. Photobiol., 2001(B63): 8-18.
［7］ DAZARD JE, GAL H, AMARIGLIO N, et al. Genome-wide comparison of human keratinocyte and squamous cell carcinoma responses to UVB irradiation: implications for skin and epithelial cancer［J］. Oncogene, 2003(22): 2993-3006.
［8］ GUO W, AN Y, JIANG L, et al. The protective effects of hydroxytyrosol against UVB-induced DNA damage in HaCaT cells［J］. Phytother Res, 2010, 24(3): 352-359.
［9］ AFAQ F, KATIYAR SK. Polyphenols: skin photoprotection and inhibition of photocarcinogenesis［J］. Mini Rev Med Chem, 2011, 11(14): 1200-1215.
［10］ HSEU YC, CHOU CW, SENTHIL KUMAR KJ, et al. Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes［J］. Food Chem Toxicol, 2012, 50(5): 1245-1255.
［11］ KIM JK, KIM Y, NA KM, et al. Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo［J］. Free Radic Res, 2007(41): 603-614.
［12］ TANAKA K, HASEGAWA J, ASAMITSU K, et al. Prevention of the ultraviolet B-mediated skin photoaging by a nuclear factor kappaB inhibitor, parthenolide［J］. J Pharmacol Exp Ther, 2005(315): 624-630.
［13］ LEE C, PARK GH, AHN EM, et al. Protective effect of Codium fragile against UVB-induced pro-inflammatory and oxidative damages in HaCaT cells and BALB/c mice［J］. Fitoterapia, 2013(86): 54-63.
［14］ JITKA VOSTLOV, ADLA ZDAILOV, ALENA SVOBODOV. Prunella vulgaris extract and rosmarinic acid prevent UVB-induced DNA damage and oxidative stress in HaCaT keratinocytes［J］. Arch. Dermatol. Res., 2010(302): 171-181.
［15］ CRAIG A. ELMETS, DIVYA SINGH, KAREN TUBESING, et al. Cutaneous photoprotection from ultraviolet injury by green tea polyphenols［J］. J. Am. Acad. Dermatol, 2001(44): 425-432.
［16］ WU YY, QI XN, XIE M, et al. Study on optimization of extraction process and antioxidant activity of Cordyceps militaris polysaccharides［J］. Food Research And Development, 2020, 41(9): 103-109.