刺梨不同提取成分对B16细胞增殖及酪氨酸酶活性的影响
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摘要
[目的]评价刺梨不同提取成分对小鼠黑色素瘤B16细胞增殖和细胞内酪氨酸酶活性的影响,为刺梨在美白化妆品领域的开发应用奠定基础。[方法]分别采用水提法、醇提法和超声提取法获得刺梨多糖、黄酮和多酚,分别采用苯酚-硫酸法、亚硝酸钠-硝酸铝-氢氧化钠法和Folin-Ciocaltean法检测其纯度。以刺梨多糖(200、400、800、1 600μg·mL-1)、刺梨黄酮(30、60、120、240μg·m L-1)、刺梨多酚(40、80、160、320μg·mL-1)及维生素C干预B16细胞,采用MTT法检测B16细胞增殖活性,倒置显微镜观察B16细胞形态和数量,多巴氧化法检测酪氨酸酶活性。[结果]刺梨多糖的提取率最高,可达45.33%;刺梨黄酮的纯度最高,可达52.50%。刺梨不同提取成分对B16细胞增殖和酪氨酸酶活性均有一定的抑制作用,且呈现显著的浓度依赖性,与正常组比较,具有统计学差异(P<0.05)。刺梨不同提取成分各剂量组均可抑制B16细胞的增殖,增加细胞碎片,且呈显著的浓度依赖性;药物处理48h后,刺梨多糖、刺梨黄酮和刺梨多酚抑制B16细胞增殖的IC50分别为649.83、105.87、134.16μg·m L-1,抑制酪氨酸酶活性的IC50分别为376.13、57.51、82.42μg·mL-1,其中刺梨黄酮(60、120、240μg·mL-1)和刺梨多酚(80、160、320μg·mL-1)对B16细胞酪氨酸酶的抑制作用优于阳性对照维生素C,具有统计学差异(P<0.05)。[结论]刺梨的不同提取成分对小鼠黑色素瘤B16细胞增殖和酪氨酸酶活性均有一定的抑制作用,其中刺梨黄酮的活性最佳,刺梨多酚次之,提示刺梨在皮肤美白等方面具有潜在的研究价值。
[Objective] To evaluate the effects of different Rosa roxburghii extractions on the proliferation, morphology, and tyrosinase activity of mouse melanoma B16 cells, which could lay a foundation for the development and application of Rosa roxburghii in the field of whitening cosmetics. [Methods]Polysaccharides, flavonoids and polyphenols of Rosa roxburghii were extracted with hot water, ethanol, and ultrasonic respectively, and the purity were determined. The effects of different concentrations of these compounds on B16 cells were detected after detecting their purity. B16 Cells were treated with different concentration of polysaccharides(200, 400, 800, 1 600μg·mL-1), flavonoids(30, 60, 120, 240μg·mL-1), polyphenols(40, 80, 160, 320μg·mL-1) of Rosa roxburghii and VC(positive control group). MTT assay was used to evaluated the effects of different extracts of Rosa roxburghii on the proliferation of B16 cells, and the effects on cell morphology and activity of tyrosinase activity were detected by the dopa oxidation method respectively. [Results] The extraction rate of polysaccharides from Rosa roxburghii was the highest, and the purity of flavonoids extracted from Rosa roxburghii was the highest. The different concentrations of Rosa roxburghii extracts had certain inhibitory effects on the proliferation and tyrosinase activity of B16 cells, which were significantly dependent on the concentration, compared with normal group(P<0.05). Different dosage groups of Rosa roxburghii extracts could significantly inhibit the proliferation of B16 cells and increase the cell debris, which were significantly dependent on the concentration and after 48 hours of drug treatment, the IC50 values of proliferation of the three extracts were 649.83, 105.87 and 134.16 μg·mL-1, respectively. The IC50 values of tyrosinase activity were 376.13, 57.51 and 82.42μg·mL-1, respectively.The inhibitory effect of Rosa roxburghii flavonoids(60,120,240μg·m L-1) and Rosa roxburghii polyphenols(80,160,320μg·m L-1) on the tyrosinase activity in B16 cells was better than VC(P<0.05). [Conclusion] The different Rosa roxburghii different extracts had certain inhibitory effects on the proliferation and tyrosinase activity of mouse melanoma B16 cells, and that of flavonoids was the strongest,followed by the polyphenols extractions, suggesting that Rosa roxburghii has a potential development prospect in whitening.
[Objective] To evaluate the effects of different Rosa roxburghii extractions on the proliferation, morphology, and tyrosinase activity of mouse melanoma B16 cells, which could lay a foundation for the development and application of Rosa roxburghii in the field of whitening cosmetics. [Methods]Polysaccharides, flavonoids and polyphenols of Rosa roxburghii were extracted with hot water, ethanol, and ultrasonic respectively, and the purity were determined. The effects of different concentrations of these compounds on B16 cells were detected after detecting their purity. B16 Cells were treated with different concentration of polysaccharides(200, 400, 800, 1 600μg·mL-1), flavonoids(30, 60, 120, 240μg·mL-1), polyphenols(40, 80, 160, 320μg·mL-1) of Rosa roxburghii and VC(positive control group). MTT assay was used to evaluated the effects of different extracts of Rosa roxburghii on the proliferation of B16 cells, and the effects on cell morphology and activity of tyrosinase activity were detected by the dopa oxidation method respectively. [Results] The extraction rate of polysaccharides from Rosa roxburghii was the highest, and the purity of flavonoids extracted from Rosa roxburghii was the highest. The different concentrations of Rosa roxburghii extracts had certain inhibitory effects on the proliferation and tyrosinase activity of B16 cells, which were significantly dependent on the concentration, compared with normal group(P<0.05). Different dosage groups of Rosa roxburghii extracts could significantly inhibit the proliferation of B16 cells and increase the cell debris, which were significantly dependent on the concentration and after 48 hours of drug treatment, the IC50 values of proliferation of the three extracts were 649.83, 105.87 and 134.16 μg·mL-1, respectively. The IC50 values of tyrosinase activity were 376.13, 57.51 and 82.42μg·mL-1, respectively.The inhibitory effect of Rosa roxburghii flavonoids(60,120,240μg·m L-1) and Rosa roxburghii polyphenols(80,160,320μg·m L-1) on the tyrosinase activity in B16 cells was better than VC(P<0.05). [Conclusion] The different Rosa roxburghii different extracts had certain inhibitory effects on the proliferation and tyrosinase activity of mouse melanoma B16 cells, and that of flavonoids was the strongest,followed by the polyphenols extractions, suggesting that Rosa roxburghii has a potential development prospect in whitening.
引文
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[9]郑殿钦.刺梨美白茶及其生产工艺:CN 105360448 A[P].2016.ZHENG Dianqin.Whitening tea of Rosa roxburghii and its production technology:CN 105360448 A[P].2016.
[10]王慧,黄聪,杜薇.均匀设计法优化刺梨多糖的提取工艺[J].中国药业,2012,21(13):41-43.WANG Hui,HUANG Cong,DU Wei.Optimization of extraction process of polysaccharide from Rosa roxburghii by uniform design[J].China Pharmaceuticals,2012,21(13):41-43.
[11]阎力君,洪涛,王福玲,等.黄芪多糖水提工艺的优化及其体外抗肿瘤活性[J].中成药,2017,39(10):2045-2049.YAN Lijun,HONG Tao,WANG Fuling,et al.Optimization of aqueous extraction for polysaccharides from Astragali Radix and the in vitro antitumor activity[J].Chinese Traditional Patent Medicine,2017,39(10):2045-2049.
[12]张汇慧.刺梨黄酮的提取、分离及抗氧化活性研究[D].南京:南京林业大学,2015:16-17.ZHANG Huihui.Study on extraction,separation and antioxidant activity of flavonoids from Rosa roxburghii[D]Nanjing:Nanjing Forestry University,2015:16-17.
[13]党艺航,吕家乐,任艳,等.苦竹枝总黄酮提取工艺的优化及其体外抗炎活性[J].中成药,2018,40(2):331-335.DANG Yihang,LYU Jiale,REN Yan,et al.Extraction optimization for total flavonoids from Pleioblastus amarus branches and the in vitro anti-inflammatory activity[J]Chinese Traditional Patent Medicine,2018,40(2):331-335.
[14]孙红艳,胡凯中,郭志龙,等.超声波法提取刺梨多酚的工艺优化及体外抑菌活性研究[J].中国食品添加剂,2016,144(2):57-61.SUN Hongyan,HU Kaizhong,GUO Zhilong,et al.Study on the extraction technology and antibacterial activity of polyphenols from fruits of Rosa roxburghii Tratt by ultrasonic assistance[J].China Food Additives,2016,144(2):57-61.
[15]Li W,Sun H,Zhou J,et al.Antibacterial activities,antioxidant contents and antioxidant properties of three traditional Chinese medicinal extracts[J].Bangl J Pharmacol2015,10(1):131-137.
[16]王煜,王梅,王香兰,等.曲酸寡肽对小鼠B16黑素瘤细胞酪氨酸酶活性及黑色素合成的影响[J].中国皮肤性病学杂志2013,27(12):1221-1224.WANG Yu,WANG Mei,WANG Xianglan,et al,Effects of kojic acid oligopeptide on tyrosinase activity and melanin synthesis in mouse B16 melanoma cells[J].The Chinese Journal of Dermatovenereology,2013,27(12):1221-1224.
[17]黄少丹,吴宗泽,宋凤兰,等.中药美白成分的筛选及美白霜的制备[J].宜春学院学报,2014,36(12):17-19.HUANG Shaodan,WU Zongze,SONG Fenglan,et al Screening of Chinese medicines whitening ingredients and preparation of whitening cream[J].Journal of Yichun College,2014,36(12):17-19.
[18]李红艳,刘艳杰,王倩,等.红花中酪氨酸酶抑制成分的快速筛选和分析[J].现代药物与临床,2013,28(2):170-174.LI Hongyan,LIU Yanjie,WANG Qian,et al.Quick screening and analysis on components from Carthami Flos with tyrosinase inhibitory activity[J].Drugs&Clinic,2013,28(2):170-174.
[19]盛晓笑,王春丽.蔷薇花总黄酮对酪氨酸酶的抑制作用及其动力学行为[J].中成药,2018,40(1):97-100.SHENG Xiaoxiao,WANG Chunli.Inhibitory effect of total flavonoids from Rose multiflora on tyrosinase and the kinetic behaviors[J].Chinese Traditional Patent Medicine,2018,40(1):97-100.
[20]严航,唐婷,干丽,等.薏苡仁提取物对酪氨酸酶抑制作用[J].中成药,2013,35(4):696-699.YAN Hang,TANG Ting,GAN Li,et al.Inhibition of Coicis Semen extract on tyrosinase[J].Chinese Traditional Patent Medicine,2013,35(4):696-699.
[21]王国林,达热卓玛,徐德平.洋甘菊抑制酪氨酸酶活性成分研究[J].食品与生物技术学报,2018,37(2):191-194.WANG Guolin,DARE Zhuoma,XU Deping.Inhibition of tyrosinase by Anthemis nobile L.extract[J].Journal of Food Science and Biotechnology,2018,37(2):191-194.
[22]赵京霞,张蕾,李萍,等.滋补肝肾方含药血清对小鼠黑素瘤细胞酪氨酸酶活性及表达的调节作[J].中国中药杂志,2011,36(18):2567-2570.ZHAO Jingxia,ZHANG Lei,LI Ping,et al.Effect of zibuganshenfang on the expression and activity of tyrosinase on B16 cells[J].China Journal of Chinese Materia Medica,2011,36(18):2567-2570.
[23]王加利,潘纪红,袁将,等.驱虫斑鸠菊化学成分及酪氨酸酶抑制活性研究[J].中药材,2017,40(8):1833-1837.WANG Jiali,PAN Jihong,YUAN Jiang,et al.Chemical constituents and tyrosinase inhibition activities of the fruits from Vernonia anthelmintica[J].Journal of Chinese Medicinal Materials,2017,40(8):1833-1837.
[24]付小华.甘草黄酮对酪氨酸酶的抑制作用研究[J].中南药学,2017,15(4):436-439.FU Xiaohua.Study on the inhibitory effect of Glycyrrhiza flavonoids on tyrosinase[J].Central South Pharmacy,2017,15(4):436-439.
[25]Nquyen H X,Nguyen N T,Nquyen M H,et al.Tyrosinase inhibitory activity of flavonoids from Artocarpus heterophyllous[J].Chem Cent J,2016,10(1):2.
[26]Hao M H,Zhang F,Liu X X,et al.Qualitative and quantitative analysis of catechin and quercetin in flavonoids extracted from Rosa roxburghii Tratt[J].Trop JPharm Res,2018,17(1):71.
[2]Yu F,Pan Z,Qu B,et al.Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin[J].Gene,2018,656(1):1-2.
[3]吕静.简述美白祛斑类化妆品[J].科技创新与应用,2012(13):32-33.LYU Jing.A brief introduction to whitening and freckling Cosmetics[J].Science Technology Innovation and Application,2012(13):32-33.
[4]Wang H,Li Y,Ren Z,et al.Optimization of the microwave-assisted enzymatic extraction of Rosa roxburghii Tratt.polysaccharides using response surface methodology and its antioxidant andα-d-glucosidase inhibitory activity[J].Int J Biol Macromol,2018,112:473-482.
[5]胡斯杰,佟长青,郑鲁平,等.刺梨的化学成分药理作用与性味归经[J].农产品加工,2017(3):48-50.HU Sijie,TONG Changqing,ZHENG Luping,et al.Chemical composition pharmacological effects and property taste and meridian tropism of Rosa roxburghii Tratt[J].Academic Periodical of Farm Products Processing,2017(3):48-50.
[6]张汇慧,吴彩娥,范龚健,等.刺梨黄酮的精制及其抗氧化活性比较[J].南京林业大学学报(自然科学版),2015,39(3):101-105.ZHANG Huihui,WU Cai’e,FAN Gongjian,at al.Refining and antioxidant activity of flavonoids from Rosa roxburghii Tratt[J].Journal of Nanjing Forestry University(Natural Science Edition),2015,39(3):101-105.
[7]何伟平,滕建文,朱晓韵,等.刺梨Vc和多酚成分稳定性及抗氧化性研究[J].广西轻工业,2011,27(9):1-3.HE Weiping,TENG Jianwen,ZHU Xiaoyun,et al.Studies on the stability and antioxidant of VC and polyphenols in Rosa roxburghii[J].Guangxi Journal of Light Industry,2011,27(9):1-3.
[8]李根林,孙静雅,吴宿慧,等.常用美白祛斑中药抗氧化活性比较[J].中医学报,2015,30(10):1467-1469.LI Genlin,SUN Jingya,WU Suhui,et al.The comparision of antioxidant activities on skin-whitening and antifreckling Chinese herbs[J].Journal of Chinese Medicine,2015,30(10):1467-1469.
[9]郑殿钦.刺梨美白茶及其生产工艺:CN 105360448 A[P].2016.ZHENG Dianqin.Whitening tea of Rosa roxburghii and its production technology:CN 105360448 A[P].2016.
[10]王慧,黄聪,杜薇.均匀设计法优化刺梨多糖的提取工艺[J].中国药业,2012,21(13):41-43.WANG Hui,HUANG Cong,DU Wei.Optimization of extraction process of polysaccharide from Rosa roxburghii by uniform design[J].China Pharmaceuticals,2012,21(13):41-43.
[11]阎力君,洪涛,王福玲,等.黄芪多糖水提工艺的优化及其体外抗肿瘤活性[J].中成药,2017,39(10):2045-2049.YAN Lijun,HONG Tao,WANG Fuling,et al.Optimization of aqueous extraction for polysaccharides from Astragali Radix and the in vitro antitumor activity[J].Chinese Traditional Patent Medicine,2017,39(10):2045-2049.
[12]张汇慧.刺梨黄酮的提取、分离及抗氧化活性研究[D].南京:南京林业大学,2015:16-17.ZHANG Huihui.Study on extraction,separation and antioxidant activity of flavonoids from Rosa roxburghii[D]Nanjing:Nanjing Forestry University,2015:16-17.
[13]党艺航,吕家乐,任艳,等.苦竹枝总黄酮提取工艺的优化及其体外抗炎活性[J].中成药,2018,40(2):331-335.DANG Yihang,LYU Jiale,REN Yan,et al.Extraction optimization for total flavonoids from Pleioblastus amarus branches and the in vitro anti-inflammatory activity[J]Chinese Traditional Patent Medicine,2018,40(2):331-335.
[14]孙红艳,胡凯中,郭志龙,等.超声波法提取刺梨多酚的工艺优化及体外抑菌活性研究[J].中国食品添加剂,2016,144(2):57-61.SUN Hongyan,HU Kaizhong,GUO Zhilong,et al.Study on the extraction technology and antibacterial activity of polyphenols from fruits of Rosa roxburghii Tratt by ultrasonic assistance[J].China Food Additives,2016,144(2):57-61.
[15]Li W,Sun H,Zhou J,et al.Antibacterial activities,antioxidant contents and antioxidant properties of three traditional Chinese medicinal extracts[J].Bangl J Pharmacol2015,10(1):131-137.
[16]王煜,王梅,王香兰,等.曲酸寡肽对小鼠B16黑素瘤细胞酪氨酸酶活性及黑色素合成的影响[J].中国皮肤性病学杂志2013,27(12):1221-1224.WANG Yu,WANG Mei,WANG Xianglan,et al,Effects of kojic acid oligopeptide on tyrosinase activity and melanin synthesis in mouse B16 melanoma cells[J].The Chinese Journal of Dermatovenereology,2013,27(12):1221-1224.
[17]黄少丹,吴宗泽,宋凤兰,等.中药美白成分的筛选及美白霜的制备[J].宜春学院学报,2014,36(12):17-19.HUANG Shaodan,WU Zongze,SONG Fenglan,et al Screening of Chinese medicines whitening ingredients and preparation of whitening cream[J].Journal of Yichun College,2014,36(12):17-19.
[18]李红艳,刘艳杰,王倩,等.红花中酪氨酸酶抑制成分的快速筛选和分析[J].现代药物与临床,2013,28(2):170-174.LI Hongyan,LIU Yanjie,WANG Qian,et al.Quick screening and analysis on components from Carthami Flos with tyrosinase inhibitory activity[J].Drugs&Clinic,2013,28(2):170-174.
[19]盛晓笑,王春丽.蔷薇花总黄酮对酪氨酸酶的抑制作用及其动力学行为[J].中成药,2018,40(1):97-100.SHENG Xiaoxiao,WANG Chunli.Inhibitory effect of total flavonoids from Rose multiflora on tyrosinase and the kinetic behaviors[J].Chinese Traditional Patent Medicine,2018,40(1):97-100.
[20]严航,唐婷,干丽,等.薏苡仁提取物对酪氨酸酶抑制作用[J].中成药,2013,35(4):696-699.YAN Hang,TANG Ting,GAN Li,et al.Inhibition of Coicis Semen extract on tyrosinase[J].Chinese Traditional Patent Medicine,2013,35(4):696-699.
[21]王国林,达热卓玛,徐德平.洋甘菊抑制酪氨酸酶活性成分研究[J].食品与生物技术学报,2018,37(2):191-194.WANG Guolin,DARE Zhuoma,XU Deping.Inhibition of tyrosinase by Anthemis nobile L.extract[J].Journal of Food Science and Biotechnology,2018,37(2):191-194.
[22]赵京霞,张蕾,李萍,等.滋补肝肾方含药血清对小鼠黑素瘤细胞酪氨酸酶活性及表达的调节作[J].中国中药杂志,2011,36(18):2567-2570.ZHAO Jingxia,ZHANG Lei,LI Ping,et al.Effect of zibuganshenfang on the expression and activity of tyrosinase on B16 cells[J].China Journal of Chinese Materia Medica,2011,36(18):2567-2570.
[23]王加利,潘纪红,袁将,等.驱虫斑鸠菊化学成分及酪氨酸酶抑制活性研究[J].中药材,2017,40(8):1833-1837.WANG Jiali,PAN Jihong,YUAN Jiang,et al.Chemical constituents and tyrosinase inhibition activities of the fruits from Vernonia anthelmintica[J].Journal of Chinese Medicinal Materials,2017,40(8):1833-1837.
[24]付小华.甘草黄酮对酪氨酸酶的抑制作用研究[J].中南药学,2017,15(4):436-439.FU Xiaohua.Study on the inhibitory effect of Glycyrrhiza flavonoids on tyrosinase[J].Central South Pharmacy,2017,15(4):436-439.
[25]Nquyen H X,Nguyen N T,Nquyen M H,et al.Tyrosinase inhibitory activity of flavonoids from Artocarpus heterophyllous[J].Chem Cent J,2016,10(1):2.
[26]Hao M H,Zhang F,Liu X X,et al.Qualitative and quantitative analysis of catechin and quercetin in flavonoids extracted from Rosa roxburghii Tratt[J].Trop JPharm Res,2018,17(1):71.