灵芝三萜的超声波提取工艺及其指纹图谱检测方法的研究
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摘要
灵芝是我国珍贵的药食两用保健药用真菌,灵芝三萜具有丰富的生物活性。本文通过对灵芝三萜超声波提取工艺的研究,为灵芝三萜的理化性质、生理活性等方面的进一步研究提供了高效的提取途径,也为灵芝三萜的开发利用、高效利用灵芝资源提供理论根据和实验方法。本文以提高灵芝三萜提取率为目的,研究赤芝和紫芝在不同生长期的三萜含量,然后通过单因素实验研究各种因素(包括乙醇浓度、料液比、超声时间、超声温度、超声功率)对灵芝三萜含量的影响。在此基础上,再用正交实验,进行4因素3(L34)水平分析,探讨各因素的交互作用,最后得出赤灵芝和紫芝三萜类物质超声波提取的最佳工艺条件。研究表明,提取灵芝三萜的最适工艺条件为:样品量500mg,赤灵芝在超声波功率为250W,乙醇浓度为75%,料液比为1:15,提取时间为40 min,提取温度为60℃的条件下,赤灵芝三萜一次提取得率为1.941%;紫灵芝在超声功率为100W,温度为80℃,乙醇浓度为80%,料液比为1:35,提取时间为30min的条件下,紫灵芝三萜的一次提取收率为0.934%。
利用指纹图谱分析技术,建立了赤芝三萜酸成分的指纹图谱分析方法,拟定赤芝指纹图谱共有模式,并用于评价赤芝与紫芝的质量差异,为灵芝药材与灵芝制剂质量控制提供一定的参考依据。采用HPLC法测定,色谱柱为A型号C18柱,流动相为乙腈-2%冰醋酸(梯度洗脱),检测波长为252nm,流速为1mL?min-1,柱温为35℃。结果表明,在18批赤芝、7批紫芝中共标示出29个共有峰,对比对照品鉴定了其中三个成分(灵芝酸A、C2、G),其中灵芝酸A为最强峰;本研究确定了紫芝的三萜酸成分指纹图谱,虽然紫芝与赤芝的共有峰类似,但研究表明紫芝的单位生药峰面积只有赤芝的1/10~1/20;不同入土时间的指纹图谱表明,赤芝入土60天、紫芝入土60-70天,三萜酸含量相对较高,为灵芝的最佳采收期。
Ganoderma lucidum is a precious health-care edible medicinal fungus in China. Ganoderma lucidum triterpenoids are rich in biological activities. This paper was to provide practical and efficient way of extraction of Ganoderma lucidum triterpenoids for further research on its physical and chemical properties and the physiological activities, and to provide a theoretical basis and experimental methods for the development and utilization of triterpenoids and the efficient use of the resource by studying on ultrasonic extraction of triterpenoids from Ganoderma lucidum. In this paper, in order to enhance the yield of Ganoderma lucidum triterpenoids,firstly, we studied on the triterpenes content during different growth periods of Ganoderma lucidum. Secondly, we studied effects of the factors (included concentration of ethanol, material-liquid ratio, extraction time, extraction temperature and ultrasonic power) on triterpenes contents through single factor experiments. Basing on these, Orthogonal experiments were used to run 4 factors 3 levels analysis to explore the interaction of various factors. Finally, the optimization process of the ultrasonic extraction of Ganoderma lucidum triterpenoids was found out. The experimental results showed that the optimization condition was: 500mg of test sample and 250W of the ultrasonic power, 75% of the ethanol concentration, 1:15 of the material-liquid ratio, 40 min of extraction time, 60℃of extraction temperature for Ganoderma lucidum. The extraction rate was 1.941% under the optimal condition. And when the temperature was 80℃, the optimal extraction condition for Ganoderma sinense was:80% of the ethanol concentration, 1:35 of the material-liquid ratio, 30 min of extraction time, 100W of ultrasonic power. The extraction rate was 0.934% under the optimal condition.
By using fingerprint analysis technology, the fingerprint analysis method of Ganoderma lucidum triterpenoid acid was established, fingerprint common model of Ganoderma lucidum was studied out, and this was used to estimate quality differences of Ganoderma lucidum and Ganoderma sinense and to provide a frame of reference of quality of Ganoderma lucidum and Ganoderma lucidum preparation. The HPLC condition was: A type C18 column of chromatogram column, the mobile phase was acetonitrile and 2% acetic acid (gradient elution), detection wavelength was 252nm, the velocity of flow was 1mL?min-1, column temperature was 35℃. Results indicated that 29 common peak areas of 18 batches of Ganoderma lucidum and 7 batches of Ganoderma sinense were marked and three components (ganodenic acid A, C2 and G) of them were identified by contrast with the reference substance. The peak of ganodenic acid A was the highest peak. Fingerprint of ganodenic acid from Ganoderma sinense was confirmed in this research. Though, the common peaks of Ganoderma lucidum and Ganoderma sinense were similar, the unit peak area of crude drug of Ganoderma sinense was 1/10~1/20 of Ganoderma lucidum. Fingerprints of different times in the dust indicated that the contents of ganodenic acid were the highest for harvest on the sixtieth day for Ganoderma lucidum and on the sixtieth to seventieth day for Ganoderma sinense.
利用指纹图谱分析技术,建立了赤芝三萜酸成分的指纹图谱分析方法,拟定赤芝指纹图谱共有模式,并用于评价赤芝与紫芝的质量差异,为灵芝药材与灵芝制剂质量控制提供一定的参考依据。采用HPLC法测定,色谱柱为A型号C18柱,流动相为乙腈-2%冰醋酸(梯度洗脱),检测波长为252nm,流速为1mL?min-1,柱温为35℃。结果表明,在18批赤芝、7批紫芝中共标示出29个共有峰,对比对照品鉴定了其中三个成分(灵芝酸A、C2、G),其中灵芝酸A为最强峰;本研究确定了紫芝的三萜酸成分指纹图谱,虽然紫芝与赤芝的共有峰类似,但研究表明紫芝的单位生药峰面积只有赤芝的1/10~1/20;不同入土时间的指纹图谱表明,赤芝入土60天、紫芝入土60-70天,三萜酸含量相对较高,为灵芝的最佳采收期。
Ganoderma lucidum is a precious health-care edible medicinal fungus in China. Ganoderma lucidum triterpenoids are rich in biological activities. This paper was to provide practical and efficient way of extraction of Ganoderma lucidum triterpenoids for further research on its physical and chemical properties and the physiological activities, and to provide a theoretical basis and experimental methods for the development and utilization of triterpenoids and the efficient use of the resource by studying on ultrasonic extraction of triterpenoids from Ganoderma lucidum. In this paper, in order to enhance the yield of Ganoderma lucidum triterpenoids,firstly, we studied on the triterpenes content during different growth periods of Ganoderma lucidum. Secondly, we studied effects of the factors (included concentration of ethanol, material-liquid ratio, extraction time, extraction temperature and ultrasonic power) on triterpenes contents through single factor experiments. Basing on these, Orthogonal experiments were used to run 4 factors 3 levels analysis to explore the interaction of various factors. Finally, the optimization process of the ultrasonic extraction of Ganoderma lucidum triterpenoids was found out. The experimental results showed that the optimization condition was: 500mg of test sample and 250W of the ultrasonic power, 75% of the ethanol concentration, 1:15 of the material-liquid ratio, 40 min of extraction time, 60℃of extraction temperature for Ganoderma lucidum. The extraction rate was 1.941% under the optimal condition. And when the temperature was 80℃, the optimal extraction condition for Ganoderma sinense was:80% of the ethanol concentration, 1:35 of the material-liquid ratio, 30 min of extraction time, 100W of ultrasonic power. The extraction rate was 0.934% under the optimal condition.
By using fingerprint analysis technology, the fingerprint analysis method of Ganoderma lucidum triterpenoid acid was established, fingerprint common model of Ganoderma lucidum was studied out, and this was used to estimate quality differences of Ganoderma lucidum and Ganoderma sinense and to provide a frame of reference of quality of Ganoderma lucidum and Ganoderma lucidum preparation. The HPLC condition was: A type C18 column of chromatogram column, the mobile phase was acetonitrile and 2% acetic acid (gradient elution), detection wavelength was 252nm, the velocity of flow was 1mL?min-1, column temperature was 35℃. Results indicated that 29 common peak areas of 18 batches of Ganoderma lucidum and 7 batches of Ganoderma sinense were marked and three components (ganodenic acid A, C2 and G) of them were identified by contrast with the reference substance. The peak of ganodenic acid A was the highest peak. Fingerprint of ganodenic acid from Ganoderma sinense was confirmed in this research. Though, the common peaks of Ganoderma lucidum and Ganoderma sinense were similar, the unit peak area of crude drug of Ganoderma sinense was 1/10~1/20 of Ganoderma lucidum. Fingerprints of different times in the dust indicated that the contents of ganodenic acid were the highest for harvest on the sixtieth day for Ganoderma lucidum and on the sixtieth to seventieth day for Ganoderma sinense.
引文
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[2]神农本草经[M].上海:群联出版社,1955
[3]李时珍.本草纲目[M].北京:人民卫生出版社,1978
[4]章灵华,王会贤,王立为,肖培根.灵芝孢子粉提取物在体内外的免疫效应[J].中国免疫学杂志,1994,3(10):169-172
[5] Wang SY, Hsu ML, Hsu HC, Tzeng CH, Lee SS, Shiao MS, Ho CK. The anti-tumor effect of Ganoderma lucidum is mediated by cytokine released from activated macrophages and Tlymphocytes [J]. Int J Cancer,1997,70(6): 699-705
[6]赵东旭,王利波,杨新林,朱鹤荪.灵芝子实体抗肿瘤成分提取的研究[J].北京理工大学学报,1999,6(19):782-786
[7]林东星.灵芝辅助治癌探讨[J].中国食用菌,2000,1(19):3-5
[8]郭春沅.灵芝的药理研究综述[J].中国食用菌,1998,3(17):34-35
[9]国家药典委员会.中华人民共和国药典[M].北京:化学工业出版社,2000
[10]湖南医学工业研究所201组.灵芝有效成分的研究[J].中草药通讯,1979,(5):1-5
[11] Mizuno, et al. Host-mediated antitumor triterpenoids. NFractional,structural study and antitumor activity of hetergalactans isolated from the fruit bodies of Ganoderma applanatum and Fomitopisis pinicola [J]. 1982, 96
[12]陈国良,陈晓清.灵芝有效成分研究综述[J].中国食用菌,1995,4(14):5
[13] Jorge Toth, et al. Cytotoxic triterpenes from Ganoderma Lucidum: Structures of Ganoderma acid U-Z [J]. J Chem Researc, 1983, 99
[14]李开本,陈体强,何修金,陈莎莎,徐洁,陈则树.灵芝营养成分测试分析初报[J].营养学报,1994,1(16):95-98
[15]周赓超.灵芝的药效和食效[J].生命的化学,1987,1(7):17
[16] Takashi Mizuno, Shin’ichi Ohtahara and Jingxuau Li. Mineral composition and germanium contents of several medicinal mushrooms [J]. Bull. Fac. Agr. Shizuoka Univ., 1989,(38): 37-46
[17]余竟光.薄盖灵芝深层发酵菌丝体化学成分的研究[J].药学学报,1979,6(14):374-376
[18]刘耕陶.灵芝的药理研究[J].药学论文摘要,1978,191-192
[19]湖南医学工业研究所201组.灵芝有效成分灵芝生物碱的研究[J].中草药通讯,1979,6:1
[20]余竟光.薄盖灵芝深层发酵菌丝体化学成分的研究(III) [J].药学学报,1983,10(14):5
[21]何来英.灵芝的研究及进展(I)[J].中国食品卫生杂志,1997,2 (9):32
[22]林志彬.灵芝药理研究的新进展[J].食用菌.1981,1:10-12
[23]罗立新.灵芝菌深层培养及灵芝多糖分离纯化的研究[D].广州:华南理工大学,1996
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