红景天有效成分的提取及活性分析
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摘要
红景天具有镇静、强心、抗疲劳、抗炎、抗氧化、抗微波辐射等方面作用,药理作用优于人参、刺五加。本文研究了红景天有效成分超高压提取工艺,使有效成分在常温下提取成为可能;研究了红景天有效成分纤维素酶提取工艺,特别是采用较低浓度乙醇作为反应溶剂,利用乙醇和酶的协同作用使总黄酮浸出率显著增加;对比了不同产地来源红景天提取物的抗氧化活性,完善红景天资源研究;研究了红景天提取物酪氨酸酶抑制活性,完善红景天的药理作用基础研究。
本文完成了国家自然科学基金“中药有效成分常温超高压提取机理研究(30472135)”的部分内容。本文研究结果对天然产物有效成分的提取技术的完善和发展有一定的参考价值,对红景天的深入开发和利用有指导意义。
Rhodiola is a traditional Chinese medicine belonging to the family of Crassulaceae that mainly distributes in north atmosphere with about 90 species recorded in the world and above 70 species found in China. The extracts of Rhodiola exhibits different efficacy from clinical examination such as exciting the central nervous system, anti-virus, anti-lacking of oxygen, anti-inflammatory, anti-cold and anti-tatigue. It was believed that the pharmacological properties of Rhodiola were perior to Panax and Acanthopanax. We employed ultrahigh hydrostatic pressure technique(UHP) and cellulase to obtain bio-active components from Rhodiola, the bioactivity of the extracts also be detailed analyzed.
The UHP treated at room temperature would remarkable increase the leaching-out rates of flavones and salidroside. The optimal conditions of extracting flavones and salidroside were as follows: ethanol concentration was 41% or 60%(v/v), liquid-solid ratio was 70:1ml/g, pressure was 500MPa for 3min, the leaching-out rate of flavones and salidroside was up to 5.233% and 0.411%, respectively. UHP works according to the priciple of static extraction with high hydrostatic pressure. High pressure treatment could enhance the diffusivity of the solvent and the solubility of bio-active components that lead to an increase in mass transfer. Furthermore, in the extraction process the pressure could transfer to the whole material uniformly and instantaneously. So the rate of pressure transfer rapidly with no stress grads, which make the extraction process easy and effective. Therefore, 3min was totally enough to complete the equilibrium. Limited by the energy level, weak bonds, such as the hydrogen bond, the electrostatic bond, the Van der Waals bond and the hydrophobic bond, could be broken by high pressure but the covalent bond could not be broken, which meant the small molecules would not change under UHP. The extraction method could shorten processing time, obtain higher extraction yield, and had no negative effect on the activity and structure of bio-activie components.
Rhodiola was also treated by cellulase and ethanol solution. The optimum conditions of extracting flavones for extraction with cellulase with water or ethanol solution as the extraction system were as follows: amount of cellulose was 1.95% or 2.0%, liquid-solid ratio was 70:1 or 52:1mL/g, pH was 5.5, temperature of enzyme digestion was 40 or 50℃for 5h, the leaching-out rate of flavones was up to 4.385% and 7.144%, respectively. The synergistic effect cellulase and ethanol solution could increase the leaching-out rate of flavones and salidroside, decrease the impurities in extraction solution.
The effects of different extraction methods were compared, the leaching-out rates of flavones to decrease in the order, cellulase-ethanol solution extraction, UHP extraction, cellulase-water extraction, soxhlet extraction, ultrasonic extraction, leaching extraction, and refulx extraction. The leaching-out rates of salidroside to decrease in the order, soxhlet extraction, cellulase-ethanol solution extraction, UHP extraction, cellulase-water extraction, refulx extraction, leaching extraction, and ultrasonic extraction. Cellulase could degrade cellulose and semi-cellulose in the cell wall and cell membrane, so it could destroy the cell structure to a certain extent so that we could obtain more salidroside than leaching extraction method. UHP extraction for 3min gave higher leaching rate of and salidroside than other extractions for several hours, and the extraction efficiency was the highest. Thus, HHP extraction can greatly shorten the extraction time. The DPPH radical scavenging activities of different extracts (0.5 mg/mL water solution) were all beyond 90%.
Rhodiola extracts had showed high DPPH radical scavenging activity, moderate hydroxyl radical scavenging activity, low reducing power, and strong liposome oxidation inhibitory activity which superior to Vc and TBHQ. Rhodiola extract was a potential source of natural anti-oxidant. Rhodiola from Jilin showed higher DPPH radical scavenging activity, hydroxyl radical scavenging activity and liposome oxidation inhibitory activity than those from Shanxi and Yunnan with lower reducing power. Rhodiola from Yunnan had the highest flavones content. It was suggested from the fingerprint curves that Rhodiola from Yunnan and Shanxi contented more flavandiols while Rhodiola from Jilin contented more flavones and flavonols.
Rhodiola extract could inhibit the activity of mushroom tyrosinase obviously, with the inhibitor content leading to 50% activity lost, IC50, as 0.2705mg/mL. Kinetics analyses showed that the extract inhibited the enzyme reversibly and the inhibition belonged to the mix-typed which meant both competitive and uncompetitive type. The inhibitory constants, KI and KES, were 0.1531mmol/L and 0.4303mmol/L, respectively. The value of KIS was greater than KI, indicating that the affinity of the inhibitor for the free enzyme was stronger than for enzyme-substrate complex.
The formula of Rhodiola-black tea health beverages were as Rhodiola extract solution 10%, black tea extract solution 10%, sugar candy 5%, citric acid 0.05%, CMC-Na 0.10%. The formula of Rhodiola-rose health beverages were as Rhodiola extract solution 10%, rose extract solution 15%, sugar candy 6%, citric acid 0.04%, CMC-Na 0.10%.
The innovative points of the thesis were 1) the new extraction process of bio-active components treated by ultrahigh hydrostatic pressure technique under room temperature; 2) the new extraction process treated by cellulase and ethanol solution; 3) the comparison of anti-oxidative activities of the extracts from different regions; 4) the analysis of inhibitory activity of mushroom tyrosinase to complete pharmacological properties projects.
The thesis finished part of project named as“Research on Mechanism of Essential Components Extraction from Herb by High Hydrostatic Pressure at Ambient Temperature (30472135)”that funded by the National Natural Science Foundation of China. The UHP extraction method and cellulase extraction method had showed higher efficiency and effective than traditional methods that created two new process to extract natural products with academic and practical meanings.
本文完成了国家自然科学基金“中药有效成分常温超高压提取机理研究(30472135)”的部分内容。本文研究结果对天然产物有效成分的提取技术的完善和发展有一定的参考价值,对红景天的深入开发和利用有指导意义。
Rhodiola is a traditional Chinese medicine belonging to the family of Crassulaceae that mainly distributes in north atmosphere with about 90 species recorded in the world and above 70 species found in China. The extracts of Rhodiola exhibits different efficacy from clinical examination such as exciting the central nervous system, anti-virus, anti-lacking of oxygen, anti-inflammatory, anti-cold and anti-tatigue. It was believed that the pharmacological properties of Rhodiola were perior to Panax and Acanthopanax. We employed ultrahigh hydrostatic pressure technique(UHP) and cellulase to obtain bio-active components from Rhodiola, the bioactivity of the extracts also be detailed analyzed.
The UHP treated at room temperature would remarkable increase the leaching-out rates of flavones and salidroside. The optimal conditions of extracting flavones and salidroside were as follows: ethanol concentration was 41% or 60%(v/v), liquid-solid ratio was 70:1ml/g, pressure was 500MPa for 3min, the leaching-out rate of flavones and salidroside was up to 5.233% and 0.411%, respectively. UHP works according to the priciple of static extraction with high hydrostatic pressure. High pressure treatment could enhance the diffusivity of the solvent and the solubility of bio-active components that lead to an increase in mass transfer. Furthermore, in the extraction process the pressure could transfer to the whole material uniformly and instantaneously. So the rate of pressure transfer rapidly with no stress grads, which make the extraction process easy and effective. Therefore, 3min was totally enough to complete the equilibrium. Limited by the energy level, weak bonds, such as the hydrogen bond, the electrostatic bond, the Van der Waals bond and the hydrophobic bond, could be broken by high pressure but the covalent bond could not be broken, which meant the small molecules would not change under UHP. The extraction method could shorten processing time, obtain higher extraction yield, and had no negative effect on the activity and structure of bio-activie components.
Rhodiola was also treated by cellulase and ethanol solution. The optimum conditions of extracting flavones for extraction with cellulase with water or ethanol solution as the extraction system were as follows: amount of cellulose was 1.95% or 2.0%, liquid-solid ratio was 70:1 or 52:1mL/g, pH was 5.5, temperature of enzyme digestion was 40 or 50℃for 5h, the leaching-out rate of flavones was up to 4.385% and 7.144%, respectively. The synergistic effect cellulase and ethanol solution could increase the leaching-out rate of flavones and salidroside, decrease the impurities in extraction solution.
The effects of different extraction methods were compared, the leaching-out rates of flavones to decrease in the order, cellulase-ethanol solution extraction, UHP extraction, cellulase-water extraction, soxhlet extraction, ultrasonic extraction, leaching extraction, and refulx extraction. The leaching-out rates of salidroside to decrease in the order, soxhlet extraction, cellulase-ethanol solution extraction, UHP extraction, cellulase-water extraction, refulx extraction, leaching extraction, and ultrasonic extraction. Cellulase could degrade cellulose and semi-cellulose in the cell wall and cell membrane, so it could destroy the cell structure to a certain extent so that we could obtain more salidroside than leaching extraction method. UHP extraction for 3min gave higher leaching rate of and salidroside than other extractions for several hours, and the extraction efficiency was the highest. Thus, HHP extraction can greatly shorten the extraction time. The DPPH radical scavenging activities of different extracts (0.5 mg/mL water solution) were all beyond 90%.
Rhodiola extracts had showed high DPPH radical scavenging activity, moderate hydroxyl radical scavenging activity, low reducing power, and strong liposome oxidation inhibitory activity which superior to Vc and TBHQ. Rhodiola extract was a potential source of natural anti-oxidant. Rhodiola from Jilin showed higher DPPH radical scavenging activity, hydroxyl radical scavenging activity and liposome oxidation inhibitory activity than those from Shanxi and Yunnan with lower reducing power. Rhodiola from Yunnan had the highest flavones content. It was suggested from the fingerprint curves that Rhodiola from Yunnan and Shanxi contented more flavandiols while Rhodiola from Jilin contented more flavones and flavonols.
Rhodiola extract could inhibit the activity of mushroom tyrosinase obviously, with the inhibitor content leading to 50% activity lost, IC50, as 0.2705mg/mL. Kinetics analyses showed that the extract inhibited the enzyme reversibly and the inhibition belonged to the mix-typed which meant both competitive and uncompetitive type. The inhibitory constants, KI and KES, were 0.1531mmol/L and 0.4303mmol/L, respectively. The value of KIS was greater than KI, indicating that the affinity of the inhibitor for the free enzyme was stronger than for enzyme-substrate complex.
The formula of Rhodiola-black tea health beverages were as Rhodiola extract solution 10%, black tea extract solution 10%, sugar candy 5%, citric acid 0.05%, CMC-Na 0.10%. The formula of Rhodiola-rose health beverages were as Rhodiola extract solution 10%, rose extract solution 15%, sugar candy 6%, citric acid 0.04%, CMC-Na 0.10%.
The innovative points of the thesis were 1) the new extraction process of bio-active components treated by ultrahigh hydrostatic pressure technique under room temperature; 2) the new extraction process treated by cellulase and ethanol solution; 3) the comparison of anti-oxidative activities of the extracts from different regions; 4) the analysis of inhibitory activity of mushroom tyrosinase to complete pharmacological properties projects.
The thesis finished part of project named as“Research on Mechanism of Essential Components Extraction from Herb by High Hydrostatic Pressure at Ambient Temperature (30472135)”that funded by the National Natural Science Foundation of China. The UHP extraction method and cellulase extraction method had showed higher efficiency and effective than traditional methods that created two new process to extract natural products with academic and practical meanings.
引文
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[1] 齐峰. 2004 年美国饮料形势报告. 中外食品, 2005, (3): 10-14
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[3] GB 19296-2003, 茶饮料卫生标准