抗光敏中药活性成分的筛选及主要功效研究
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摘要
紫外线来自于太阳辐射中的200-400nm波段,对人体皮肤具有双重影响,一方面能够促进人体内维生素D的合成,另一方面,UVB波段(280-320nm)照射到表皮,容易引起晒斑和色素沉着等急性损伤。UVA波段(320-400nm)具有较高的能量和穿透性,长期累积会对皮肤真皮层产生伤害,导致皮肤光老化,UVB与UVA共同作用则容易引起皮肤光敏反应的发生。针对光敏反应的发生机制,本文对20种具有抗光敏功效的中药及天然植物提取物进行了筛选,对其主要功效成分的含量、提取及工艺优化、作用途径和作用机理进行了研究。
本文建立了长波紫外线诱导光敏药物致使红细胞溶血模型,通过检测红细胞损伤后上清液中的血红蛋白含量,计算溶血率,从而反映细胞损伤程度。实验考察了紫外线或光敏药物单独存在条件下以及不同照射时间和不同药物浓度条件对模型的影响。结果表明,紫外线或氯丙嗪单一条件存在时,并不能引起红细胞溶血;当氯丙嗪含量为1mg/3mL,紫外线照射30分钟可致红细胞50%溶血,60分钟可使红细胞100%溶血。此模型可应用于光敏物质的检测及抗光敏物质的筛选。
采用紫外线扫描图谱对20种中药及植物提取液进行了初步筛选,银杏叶、葡萄籽、茶多酚、五味子等提取液紫外吸收效果优异。通过红细胞溶血实验对几种提取液刺激性进行检测,红细胞的溶血率均在10%以下,证明以上几种提取液对皮肤无刺激。稳定性实验筛选出银杏叶与抗过敏作用良好的仙人掌提取液,其中银杏叶提取液紫外吸收最强。化学成分分析表明银杏叶提取液主要功效成分为黄酮类物质。
根据黄酮类物质在水中的溶解度及工业化生产的需要,选择水作为活性成分的提取溶剂。经单因素考察,液料比10~20,浸提温度80~100℃,提取时间2~4h,总黄酮得率较高。采用Box-Benhnken中心组合试验及响应面和实际操作分析,确定了黄酮提取优化参数:液料比15:1、浸取时间4h;浸取温度90℃,此时提取液中黄酮的平均含量为6.22mg/mL。
采用离子交换树脂对抗光敏复配提取液进行脱盐处理。离子柱采用001×7-D309离子交换树脂,上样量5:1,样品流速为4BV·h-1,结果将原液大于8000μs/cm的电导率降至100μs/cm以下,脱盐效果显著。最终复配液电导率低于800μs/cm,可溶性固形物含量大于30%,颜色、pH值均达到要求,可作为外用制剂或化妆品添加剂使用。
实验所得的抗敏抗刺激复配液在4.0%的使用量时,其透明质酸酶抑制率、O2-、?OH和DPPH自由基的清除能力分别为63.2%、80.7%、80.5%和56.7%,表明具有很好的抗敏和清除自由基功效。光溶血实验表明UV照射60min之内,添加10.0%复配液可保护RBC红细胞悬液不发生溶血。UV照射30min之内,添加5.0%复配液可保护RBC红细胞悬液不发生溶血,表明抗光敏复配液具有良好的抑制UV辐射损伤细胞膜功效。
通过紫外线导致DNA损伤模型检测抗光敏复配液对紫外线损伤DNA的抑制效果。结果表明复配液添加量质量浓度在0.1g/mL时,即对UVA导致的DNA损伤具有良好的抑制作用。当浓度在0.2g/mL时,对UVA致DNA损伤的抑制作用更加显著。凝胶电泳图显示,UVA照射10min时,可见DNA被损伤降解,条带变暗且出现拖尾;UVA照射20min时,DNA降解部分逐渐扩大、拖尾更加严重。而添加抗光敏复配液时DNA损伤不明显,DNA条带清晰且无拖尾。实验证明抗光敏复配液具有抑制UVA导致DNA损伤的作用。
UV radiation comes from the sun in the 200-400nm band and it has the dual effect to human skin. On the one hand the appropriate ultraviolet radiation can accelerate the synthesis of vitamin D of body, On the other hand, UVB band (280-320nm) radiation to the skin, prone to sunburn and pigmentation and other acute injury; UVA band (320-400nm) with high energy and penetrating, long-term cumulative will damage the dermal layer of skin, resulting in the formation of skin photoaging; UVB and UVA interaction was likely to cause skin photosensitive reactions. According to the mechanisms of photosensitivity , 20 plant extracts with anti-photosensitive effect were screened. The main functional ingredient content, extraction and process optimization, function pathway and mechanism were also studied.
In this paper, a relative less time and money consuming convenient method was developed to detect the damage of UV-A by red blood cell hemolysis, the hemoglobin of the injured red blood cell, and then calculate hemolytic rate. The experiments seeing about the effects on the model under the conditions of ultraviolet light or photo sensitizer existence alone and the different exposure time and the different drug concentration. The results show that red blood cell hemolysis cannot happen only under ultraviolet light radiation without Chlorpromazine nor only Chlorpromazine without ultraviolet light radiation. Under the condition of the chlorpromazine content is 1mg/3mL in red blood cell, 50% hemolysis can be resulted after they were radiated by ultraviolet for about 30min, and 100% hemolysis after 60min. This model can be applied to photosensitive material testing and screening of anti-photosensitive material.
20 kinds of Chinese medicines and plant extracts were screened initially by UV scanning profiles, ginkgo biloba, grape seed, tea polyphenols, schisandra extract and so on had excellent UV absorption. The stimulation of several extract were detected by hemolysis experiments, hemolytic rate below 10%, proving the several extract have no stimulation. Ginkgo biloba and cactus extract which good anti-allergic were screened by stability test, ginkgo biloba extract in which the strongest UV absorption. Chemical composition analysis revealed that flavonoids was the major components.
According to flavonoid solubility in water and industrial production needs, deionized water was selected as solvent extraction of the active ingredient. In the case of single factor, water to solid ratio 10~20, temperature 80~100℃, time 2~4h, the higher flavonoids rate was obtained. Based on the single factor experiments results and practical operation, the extraction procedure was optimized using Box-Behnken design and Surface Response Methodology (RSM).The obtained optimal parameters of the extraction procedure of flavonoids were: water to solid ratio 15:1, time 4 h, temperature 90℃, and the average extract content of flavonoids 6.22 mg/mL.
Ion exchange resin was used to desalinize mixture extract. Ionic columns are 001×7-D309 ion exchange resin, the sample volume 5:1, sample flow rate was 4BV ? h -1 , the results was reduce the conductivity of liquid more than 8000μs/cm to 100μs/cm below a n d desalting effect was remarkable. The compound final electrical conductivity lower than 800μs/cm, more than 30% soluble solids, color, pH value both to meet the requirement, can be used as a cosmetic additive.
The experimental anti-allergy and anti-irritation compound solution had good anti-allergic and free radicals clearance effects. Under 4.0% dosage, the hyaluronic acid enzyme inhibition rate, O 2- , ? OH and DPPH radicals clearance rates were 63.2%, 80.7%, 80.5% and 56.7%, respectively. The light hemolysis experiments show that adding 10.0% compound solution can protect red blood cells do not hemolysis within UV irradiation 60 min. 30min UV irradiation within the mixture add 5.0% solution can protect red blood cells hemolysis do not occur. Results showed that the anti-photosensitive liquid compound has good inhibition effect of UV radiation damage the cell membrane.
By the model of DNA damaged detect the inhibitory effect of UV-damage DNA of anti-photosensitive compound solution. The results show that good inhibition on the DNA damage caused by UVA when the volume of compound solution in the concentration of added 0.1g/mL. When the concentration of 0.2g/mL, the inhibitory effect of UVA induced DNA damage was more obvious. Gel electrophoresis showed that the DNA was degradated and damaged w i t h i n 10min UVA radiation and the bands ghosting; When UVA irradiation 20min, DNA degradation was gradual expansion and more severe tailing; And the DNA bands added anti-photosensitive compound liquid significantly more clear and no tail bands than the damaged DNA. Experimental results showed anti-photosensitive compound liquid can inhibit the UVA caused DNA damage.
本文建立了长波紫外线诱导光敏药物致使红细胞溶血模型,通过检测红细胞损伤后上清液中的血红蛋白含量,计算溶血率,从而反映细胞损伤程度。实验考察了紫外线或光敏药物单独存在条件下以及不同照射时间和不同药物浓度条件对模型的影响。结果表明,紫外线或氯丙嗪单一条件存在时,并不能引起红细胞溶血;当氯丙嗪含量为1mg/3mL,紫外线照射30分钟可致红细胞50%溶血,60分钟可使红细胞100%溶血。此模型可应用于光敏物质的检测及抗光敏物质的筛选。
采用紫外线扫描图谱对20种中药及植物提取液进行了初步筛选,银杏叶、葡萄籽、茶多酚、五味子等提取液紫外吸收效果优异。通过红细胞溶血实验对几种提取液刺激性进行检测,红细胞的溶血率均在10%以下,证明以上几种提取液对皮肤无刺激。稳定性实验筛选出银杏叶与抗过敏作用良好的仙人掌提取液,其中银杏叶提取液紫外吸收最强。化学成分分析表明银杏叶提取液主要功效成分为黄酮类物质。
根据黄酮类物质在水中的溶解度及工业化生产的需要,选择水作为活性成分的提取溶剂。经单因素考察,液料比10~20,浸提温度80~100℃,提取时间2~4h,总黄酮得率较高。采用Box-Benhnken中心组合试验及响应面和实际操作分析,确定了黄酮提取优化参数:液料比15:1、浸取时间4h;浸取温度90℃,此时提取液中黄酮的平均含量为6.22mg/mL。
采用离子交换树脂对抗光敏复配提取液进行脱盐处理。离子柱采用001×7-D309离子交换树脂,上样量5:1,样品流速为4BV·h-1,结果将原液大于8000μs/cm的电导率降至100μs/cm以下,脱盐效果显著。最终复配液电导率低于800μs/cm,可溶性固形物含量大于30%,颜色、pH值均达到要求,可作为外用制剂或化妆品添加剂使用。
实验所得的抗敏抗刺激复配液在4.0%的使用量时,其透明质酸酶抑制率、O2-、?OH和DPPH自由基的清除能力分别为63.2%、80.7%、80.5%和56.7%,表明具有很好的抗敏和清除自由基功效。光溶血实验表明UV照射60min之内,添加10.0%复配液可保护RBC红细胞悬液不发生溶血。UV照射30min之内,添加5.0%复配液可保护RBC红细胞悬液不发生溶血,表明抗光敏复配液具有良好的抑制UV辐射损伤细胞膜功效。
通过紫外线导致DNA损伤模型检测抗光敏复配液对紫外线损伤DNA的抑制效果。结果表明复配液添加量质量浓度在0.1g/mL时,即对UVA导致的DNA损伤具有良好的抑制作用。当浓度在0.2g/mL时,对UVA致DNA损伤的抑制作用更加显著。凝胶电泳图显示,UVA照射10min时,可见DNA被损伤降解,条带变暗且出现拖尾;UVA照射20min时,DNA降解部分逐渐扩大、拖尾更加严重。而添加抗光敏复配液时DNA损伤不明显,DNA条带清晰且无拖尾。实验证明抗光敏复配液具有抑制UVA导致DNA损伤的作用。
UV radiation comes from the sun in the 200-400nm band and it has the dual effect to human skin. On the one hand the appropriate ultraviolet radiation can accelerate the synthesis of vitamin D of body, On the other hand, UVB band (280-320nm) radiation to the skin, prone to sunburn and pigmentation and other acute injury; UVA band (320-400nm) with high energy and penetrating, long-term cumulative will damage the dermal layer of skin, resulting in the formation of skin photoaging; UVB and UVA interaction was likely to cause skin photosensitive reactions. According to the mechanisms of photosensitivity , 20 plant extracts with anti-photosensitive effect were screened. The main functional ingredient content, extraction and process optimization, function pathway and mechanism were also studied.
In this paper, a relative less time and money consuming convenient method was developed to detect the damage of UV-A by red blood cell hemolysis, the hemoglobin of the injured red blood cell, and then calculate hemolytic rate. The experiments seeing about the effects on the model under the conditions of ultraviolet light or photo sensitizer existence alone and the different exposure time and the different drug concentration. The results show that red blood cell hemolysis cannot happen only under ultraviolet light radiation without Chlorpromazine nor only Chlorpromazine without ultraviolet light radiation. Under the condition of the chlorpromazine content is 1mg/3mL in red blood cell, 50% hemolysis can be resulted after they were radiated by ultraviolet for about 30min, and 100% hemolysis after 60min. This model can be applied to photosensitive material testing and screening of anti-photosensitive material.
20 kinds of Chinese medicines and plant extracts were screened initially by UV scanning profiles, ginkgo biloba, grape seed, tea polyphenols, schisandra extract and so on had excellent UV absorption. The stimulation of several extract were detected by hemolysis experiments, hemolytic rate below 10%, proving the several extract have no stimulation. Ginkgo biloba and cactus extract which good anti-allergic were screened by stability test, ginkgo biloba extract in which the strongest UV absorption. Chemical composition analysis revealed that flavonoids was the major components.
According to flavonoid solubility in water and industrial production needs, deionized water was selected as solvent extraction of the active ingredient. In the case of single factor, water to solid ratio 10~20, temperature 80~100℃, time 2~4h, the higher flavonoids rate was obtained. Based on the single factor experiments results and practical operation, the extraction procedure was optimized using Box-Behnken design and Surface Response Methodology (RSM).The obtained optimal parameters of the extraction procedure of flavonoids were: water to solid ratio 15:1, time 4 h, temperature 90℃, and the average extract content of flavonoids 6.22 mg/mL.
Ion exchange resin was used to desalinize mixture extract. Ionic columns are 001×7-D309 ion exchange resin, the sample volume 5:1, sample flow rate was 4BV ? h -1 , the results was reduce the conductivity of liquid more than 8000μs/cm to 100μs/cm below a n d desalting effect was remarkable. The compound final electrical conductivity lower than 800μs/cm, more than 30% soluble solids, color, pH value both to meet the requirement, can be used as a cosmetic additive.
The experimental anti-allergy and anti-irritation compound solution had good anti-allergic and free radicals clearance effects. Under 4.0% dosage, the hyaluronic acid enzyme inhibition rate, O 2- , ? OH and DPPH radicals clearance rates were 63.2%, 80.7%, 80.5% and 56.7%, respectively. The light hemolysis experiments show that adding 10.0% compound solution can protect red blood cells do not hemolysis within UV irradiation 60 min. 30min UV irradiation within the mixture add 5.0% solution can protect red blood cells hemolysis do not occur. Results showed that the anti-photosensitive liquid compound has good inhibition effect of UV radiation damage the cell membrane.
By the model of DNA damaged detect the inhibitory effect of UV-damage DNA of anti-photosensitive compound solution. The results show that good inhibition on the DNA damage caused by UVA when the volume of compound solution in the concentration of added 0.1g/mL. When the concentration of 0.2g/mL, the inhibitory effect of UVA induced DNA damage was more obvious. Gel electrophoresis showed that the DNA was degradated and damaged w i t h i n 10min UVA radiation and the bands ghosting; When UVA irradiation 20min, DNA degradation was gradual expansion and more severe tailing; And the DNA bands added anti-photosensitive compound liquid significantly more clear and no tail bands than the damaged DNA. Experimental results showed anti-photosensitive compound liquid can inhibit the UVA caused DNA damage.
引文
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