竹子精华水的成分分析及生物学活性研究
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摘要
竹茹,为禾本科竹亚科中青秆竹、大头典竹或淡竹等的茎秆外皮层或次其层,是一种常用的中药材,具有多种药理活性。竹子精华水(WEBS)是竹茹超临界CO2流体萃取时得到的水分级相,性质稳定。本文以WEBS为研究对象,首先采用氯仿、乙酸乙酯、正丁醇等有机溶剂分步萃取其中的有效成分,进行定性和定量分析；利用琼脂平板扩散法研究WEBS对常见食品腐败菌的抑菌能力,同时分析其抑菌成分,并观察常规加热处理方法对其抑菌性能的影响；采用体外无细胞系和黑色素B16细胞两种体系评价了WEBS的美白作用及机制；以熊果苷和市售防晒化妆品为参考研究了WEBS的抗紫外辐射性能；最后对WEBS的皮肤和粘膜安全性进行了评价,现将主要结果归纳如下：
(1)竹子精华水(WEBS)中总黄酮、内酯、酚酸、总糖和蒽醌类化合物的含量分别为0.24 mg/mL,3.65 mg/mL,0.20 mg/mL,0.80 mg/mL和5.18μg/mL;检测出挥发性化合物28种,主要是醛、醇、呋喃、酮类和酚类,C6-C8中等链长的含氧化合物占主导地位。
(2)利用琼脂平板扩散法研究了WEBS对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、黑曲霉、桔青霉菌、啤酒酵母的抑制作用,抑菌效果与浓度呈正相关,最小抑菌浓度(MICs)在4.9～32 mg/mL之间；对细菌的抑制作用明显强于酵母和霉菌,其中对枯草芽孢杆菌的抑制效果最显著；不同的热处理条件对抑菌效果影响不大,甚至121℃、4s的超高温瞬时处理也不影响其抑菌活性,表明WEBS中所含的抑菌成分具有很好的热稳定性。
(3)以小鼠恶性黑色素瘤细胞(B-16细胞)为受试细胞株,分别以从天然植物中提取的公认的美白产品——熊果苷和化学物质氢醌为阳性对照,通过测定黑色素细胞的增殖率、细胞内黑色素含量、细胞内酪氨酸酶活性、细胞毒性以及对体外蘑菇酪氨酸酶活性的抑制等指标,表明WEBS可以抑制B-16细胞增殖,当受试浓度为5mg/mL时,细胞存活率为65%；对细胞酪氨酸酶的抑制率随着试样浓度的增加而增加,当浓度为5mg/mL时,抑制率达75.3%,优于同浓度的熊果苷；WEBS所试浓度均能显著降低黑色素的含量,并呈浓度依赖关系：不同浓度的WEBS对蘑菇酪氨酸酶均有一定的抑制作用,在浓度为16mg/mL时,最大抑制率为89.05%,半抑制浓度(IC50)为6mg/mL。此外,WEBS的细胞毒性低于同浓度的熊果苷和氢醌,表现为细胞密度降低,对细胞形态结构的破坏较小。
(4) WEBS对短波紫外线(295nm左右)有较强吸收,对长波紫外线的吸收能力较弱；吸收紫外线的能力在较低浓度(0.3mg/mL)时已达到防晒剂的防晒指数要求,且随浓度的提高而增强。同时,可以有效保持角质层水分含量,使水分经皮肤散失变小,具有作为护肤因子研究开发的价值。
(5)用小鼠和家兔为实验动物,通过急性毒性试验、多次皮肤刺激试验、急性眼刺激试验等进行安全性评价。结果表明,小鼠经口急性毒性最大耐受量,雌、雄两性均大于5000mg/kg体重,按急性毒性分级标准规定,属于实际无毒类；家兔多次皮肤刺激性试验中,每天每只动物皮肤刺激反应积分均值为0,属于实际无刺激性；家兔急性眼刺激性试验中,受试物对家兔眼刺激性最高积分均值角膜、虹膜、结膜均为0,属实际无刺激性。证明WEBS具有很高的安全性,可用于特殊用途化妆品领域。
综上所述,竹子精华水(WEBS)中富含黄酮、内酯、多糖、有机酸等生物活性物质,具有较强的抗菌、抑菌能力,并能通过多种途径抑制黑色素的生成,同时有一定的防晒和保湿效果,具有作为特殊用途化妆品的功能性成分进行开发的潜力。
Bamboo shavings (Caulis bamfusae in taeniam), which are the intermediate layer of the stems of Bambusa tuldoides Munro, Sinocalamus beecheyana var. pubescens P.F. Li or Phyllostachys nigra (Lodd.) Munro var. henonis (Mitf.) Stapf ex Rendle, are perennial plants of the family Gramineae. WEBS, which is prepared by supercritical carbon dioxide extraction from bamboo shavings. It is water soluble, yellow or light yellow, aroma of bamboo leaves and much stable with no change during two years storage. However, a few scientific researches were performed about it. Based on this background, this study is carried out. First, ethyl acetate, trichloromethane and n-butyl alcohol was used to analyze the volatile components and other bioactive substances were determined. WEBS was evaluated for its antimicrobial action against the range of foodborne and food spoilage pathogens using agar disc diffusion assay in Nutrient Agar and Czapek Dox Agar media. Different heat conditions were selected to treat WEBS to test its antibacterial ability.The inhibitory effects on melanogenesis in cultured B16 melanoma cells were evaluated; The sunscreen ability of WEBS was evaluated with arbutin and market products as comparison. At last, the safety of WEBS was evaluated. Results were summarized as follows:
(i). The results show the flavonoids, triperpenoids, phenolic acids, total sugar and anthraquinone of WEBS was 0.24mg/mL,3.65mg/mL,0.20 mg/mL,0.80mg/mL and 5.18μg/mL respectively.28 volatile compounds were determined and most are ketone, furanone, etc. The oxide compound of middle lenths with C6-C8 was dominated.
(ii). WEBS exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia.coli, Aspergillus niger, Penicilliun citrinum and Saccharomyces cerevisiae with a concentration-dependent relationships. The minimum inhibitory concentrations against the tested bacterial strains were found in the range of 4.9-32 mg/mL using the two-fold dilution method. Different heat treatment conditions have no significant influence on the antibacterial activity especially the heat method of 121℃,4s which shows that the antibacterial components of WEBS was much stable with heat treatment.
(iii). The experiment takes malignant melanoma cells (B—16 cells) of mice as cell strains tested, and takes arbutin (a natural plant extract) and hydroquinone (a chemical substance) as positive control. Research on the whitening effect of WEBS was done through experiments on proliferation rate of melanocytes, content of melanin in cells, activity of tyrosinase in cells, cytotoxicity and inhibition of activity of mushroom tyrosinase in vitro, etc.. The result showed WEBS has strong inhibitory effects against the activity of tyrosinase in B-16 in a dose-dependent manner, and which was higher than that of arbutin at the same dose. The melanin content was significantly inhibited by WEBS with dose dependant and the cytotoxicity of WEBS was lower than that of arbutin and hydroquinone. WEBS in different treatment levels could inhibit mushroom tyrosinase and the maximum was 89.05% at 16mg/mL with IC50 6 mg/mL. The cytotoxicity of EBS was lower than arbutin and hydroquinone with the same concentration; the cell density fell down and the damage to cell morphology was relatively slight.
(iv). UV spectrophotometry of sunscreen and the UV absorbance stability of WEBS were studied through the microbiology experiment. The results show that WEBS has strong ability to absorb UVC. The ability of UV sbsorbance comply with the requirements with a concentration-dependent relationships. The water contents of skin was much higher with less water loss, it is indicated that WEBS was perfect nature sunscreen ingredients.
(v). Activity of each group of animals was all normal with good hair gloss and no toxicity symptom and death in the period of test, there was no abnormality after the animals were anatomized. The LD50 of WEBS to female and male mice was both bigger than 5000mg/kg belonged to the actual non-toxic level. The average score of skin irritation reaction of each animal each day was 0 in the repeated irritation test of tested sample on rabbits and there was no irritation. The average score of irritation to eyes of rabbits by the tested sample was cornea:0; iris:0; conjunctiva:0. under the test condition of no wash and there was no irritation.
In conclusion, WEBS was enriched flavonoids, triperpenoids, total sugar and organic acids and it exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia.coli, Aspergillus niger, Penicilliun citrinum and Saccharomyces cerevisiae with a concentration-dependent relationships.Paticularly, it can inhibit the formation of melanoma through multiple ways, can be exploited in for skin lightening and sucscreen.
(1)竹子精华水(WEBS)中总黄酮、内酯、酚酸、总糖和蒽醌类化合物的含量分别为0.24 mg/mL,3.65 mg/mL,0.20 mg/mL,0.80 mg/mL和5.18μg/mL;检测出挥发性化合物28种,主要是醛、醇、呋喃、酮类和酚类,C6-C8中等链长的含氧化合物占主导地位。
(2)利用琼脂平板扩散法研究了WEBS对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、黑曲霉、桔青霉菌、啤酒酵母的抑制作用,抑菌效果与浓度呈正相关,最小抑菌浓度(MICs)在4.9～32 mg/mL之间；对细菌的抑制作用明显强于酵母和霉菌,其中对枯草芽孢杆菌的抑制效果最显著；不同的热处理条件对抑菌效果影响不大,甚至121℃、4s的超高温瞬时处理也不影响其抑菌活性,表明WEBS中所含的抑菌成分具有很好的热稳定性。
(3)以小鼠恶性黑色素瘤细胞(B-16细胞)为受试细胞株,分别以从天然植物中提取的公认的美白产品——熊果苷和化学物质氢醌为阳性对照,通过测定黑色素细胞的增殖率、细胞内黑色素含量、细胞内酪氨酸酶活性、细胞毒性以及对体外蘑菇酪氨酸酶活性的抑制等指标,表明WEBS可以抑制B-16细胞增殖,当受试浓度为5mg/mL时,细胞存活率为65%；对细胞酪氨酸酶的抑制率随着试样浓度的增加而增加,当浓度为5mg/mL时,抑制率达75.3%,优于同浓度的熊果苷；WEBS所试浓度均能显著降低黑色素的含量,并呈浓度依赖关系：不同浓度的WEBS对蘑菇酪氨酸酶均有一定的抑制作用,在浓度为16mg/mL时,最大抑制率为89.05%,半抑制浓度(IC50)为6mg/mL。此外,WEBS的细胞毒性低于同浓度的熊果苷和氢醌,表现为细胞密度降低,对细胞形态结构的破坏较小。
(4) WEBS对短波紫外线(295nm左右)有较强吸收,对长波紫外线的吸收能力较弱；吸收紫外线的能力在较低浓度(0.3mg/mL)时已达到防晒剂的防晒指数要求,且随浓度的提高而增强。同时,可以有效保持角质层水分含量,使水分经皮肤散失变小,具有作为护肤因子研究开发的价值。
(5)用小鼠和家兔为实验动物,通过急性毒性试验、多次皮肤刺激试验、急性眼刺激试验等进行安全性评价。结果表明,小鼠经口急性毒性最大耐受量,雌、雄两性均大于5000mg/kg体重,按急性毒性分级标准规定,属于实际无毒类；家兔多次皮肤刺激性试验中,每天每只动物皮肤刺激反应积分均值为0,属于实际无刺激性；家兔急性眼刺激性试验中,受试物对家兔眼刺激性最高积分均值角膜、虹膜、结膜均为0,属实际无刺激性。证明WEBS具有很高的安全性,可用于特殊用途化妆品领域。
综上所述,竹子精华水(WEBS)中富含黄酮、内酯、多糖、有机酸等生物活性物质,具有较强的抗菌、抑菌能力,并能通过多种途径抑制黑色素的生成,同时有一定的防晒和保湿效果,具有作为特殊用途化妆品的功能性成分进行开发的潜力。
Bamboo shavings (Caulis bamfusae in taeniam), which are the intermediate layer of the stems of Bambusa tuldoides Munro, Sinocalamus beecheyana var. pubescens P.F. Li or Phyllostachys nigra (Lodd.) Munro var. henonis (Mitf.) Stapf ex Rendle, are perennial plants of the family Gramineae. WEBS, which is prepared by supercritical carbon dioxide extraction from bamboo shavings. It is water soluble, yellow or light yellow, aroma of bamboo leaves and much stable with no change during two years storage. However, a few scientific researches were performed about it. Based on this background, this study is carried out. First, ethyl acetate, trichloromethane and n-butyl alcohol was used to analyze the volatile components and other bioactive substances were determined. WEBS was evaluated for its antimicrobial action against the range of foodborne and food spoilage pathogens using agar disc diffusion assay in Nutrient Agar and Czapek Dox Agar media. Different heat conditions were selected to treat WEBS to test its antibacterial ability.The inhibitory effects on melanogenesis in cultured B16 melanoma cells were evaluated; The sunscreen ability of WEBS was evaluated with arbutin and market products as comparison. At last, the safety of WEBS was evaluated. Results were summarized as follows:
(i). The results show the flavonoids, triperpenoids, phenolic acids, total sugar and anthraquinone of WEBS was 0.24mg/mL,3.65mg/mL,0.20 mg/mL,0.80mg/mL and 5.18μg/mL respectively.28 volatile compounds were determined and most are ketone, furanone, etc. The oxide compound of middle lenths with C6-C8 was dominated.
(ii). WEBS exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia.coli, Aspergillus niger, Penicilliun citrinum and Saccharomyces cerevisiae with a concentration-dependent relationships. The minimum inhibitory concentrations against the tested bacterial strains were found in the range of 4.9-32 mg/mL using the two-fold dilution method. Different heat treatment conditions have no significant influence on the antibacterial activity especially the heat method of 121℃,4s which shows that the antibacterial components of WEBS was much stable with heat treatment.
(iii). The experiment takes malignant melanoma cells (B—16 cells) of mice as cell strains tested, and takes arbutin (a natural plant extract) and hydroquinone (a chemical substance) as positive control. Research on the whitening effect of WEBS was done through experiments on proliferation rate of melanocytes, content of melanin in cells, activity of tyrosinase in cells, cytotoxicity and inhibition of activity of mushroom tyrosinase in vitro, etc.. The result showed WEBS has strong inhibitory effects against the activity of tyrosinase in B-16 in a dose-dependent manner, and which was higher than that of arbutin at the same dose. The melanin content was significantly inhibited by WEBS with dose dependant and the cytotoxicity of WEBS was lower than that of arbutin and hydroquinone. WEBS in different treatment levels could inhibit mushroom tyrosinase and the maximum was 89.05% at 16mg/mL with IC50 6 mg/mL. The cytotoxicity of EBS was lower than arbutin and hydroquinone with the same concentration; the cell density fell down and the damage to cell morphology was relatively slight.
(iv). UV spectrophotometry of sunscreen and the UV absorbance stability of WEBS were studied through the microbiology experiment. The results show that WEBS has strong ability to absorb UVC. The ability of UV sbsorbance comply with the requirements with a concentration-dependent relationships. The water contents of skin was much higher with less water loss, it is indicated that WEBS was perfect nature sunscreen ingredients.
(v). Activity of each group of animals was all normal with good hair gloss and no toxicity symptom and death in the period of test, there was no abnormality after the animals were anatomized. The LD50 of WEBS to female and male mice was both bigger than 5000mg/kg belonged to the actual non-toxic level. The average score of skin irritation reaction of each animal each day was 0 in the repeated irritation test of tested sample on rabbits and there was no irritation. The average score of irritation to eyes of rabbits by the tested sample was cornea:0; iris:0; conjunctiva:0. under the test condition of no wash and there was no irritation.
In conclusion, WEBS was enriched flavonoids, triperpenoids, total sugar and organic acids and it exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia.coli, Aspergillus niger, Penicilliun citrinum and Saccharomyces cerevisiae with a concentration-dependent relationships.Paticularly, it can inhibit the formation of melanoma through multiple ways, can be exploited in for skin lightening and sucscreen.
引文
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