杜香提取物的制备及部分毒理药理作用
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摘要
杜香(Ledum palustre L.),主要分布于我国东北的大小兴安岭和长白山区,资源十分丰富,含有多种化学成分,如三萜类化合物(熊果酸、熊果醇、熊果苷等)、黄酮类化合物(槲皮素、儿茶素、金丝桃苷等),具有抗炎杀菌、美容等多种药理作用。本文首先以熊果酸为标的物,以工业化生产为目的,用乙醇回流法对杜香提取物的制备工艺进行了系统的研究,并优化出以熊果酸为标的物的杜香提取物的最佳工艺条件。利用在此条件下制备的杜香提取物,进行了初步的毒理学研究,包括急性毒性试验、细胞毒性试验,生殖毒性试验、遗传毒性试验等,对杜香提取物的安全性进行考察;并开展了部分药理学研究,包括抗氧化作用、抗皮肤衰老作用、对酪氨酸酶活性的影响、肝细胞保护作用和抗肿瘤作用等研究。结果发现杜香提取物最大耐受量大于30 g/kg(小鼠,经口),属于无毒级物质,且杜香提取物不仅无细胞毒性、生殖毒性和遗传毒性,反而具有肝细胞保护、生殖系统保护、抗突变、抗氧化、抗皮肤衰老、增强酪氨酸酶活性和抗肿瘤等作用,为杜香提取物的进一步开发和应用提供了理论依据。
Ledum palustre L. is an evergreen shrub, belonging to Ericaceae, Ledum palustre. Mainly distributes in our country Northeast's size of Mt. Xingan and the Changbai Mountain. The resource of Ledum palustre L.is very rich. Ledum palustre L.have many kinds of chemical compositions, such as triterpenoid compound (ursolic acid, arbutin alcohol, arbutin), flavonoid (Quercetin, catechin, hyperin ), Coumarin, tanning substances and so on, and have many kinds of pharmacological actions, such as cough- relieveing,phlegm-dispelling, breathe-calming, blood vesselexpanding,voltage-dropping and fungus-suppressing effects. Ursolic acid belongs toα-amyrin (α-amyrin) non-polar triterpenoid pentacyclic compounds.Its molecular formula is C30H48O3 and molecular weight is 456.68.It is one of the main active substances with a variety of pharmacological effects including anti-tumor, anti-mutagenic, antioxidant,liver cell protection effects.
In this article, we firstly using alcohol reflux carried out a systematic study on Ledum ursolic extraction process, for the purpose of extraction process optimization and industrial production. Ursolic acid is the subject matter. The optimum conditions are: 85℃, pure alcohol: Ledum =10:1, reflux extraction 2 times, 1 time 1 hour. The detection conditions of Ledum Ursolic Acid Extract are: ODS column (250 mm×4 ? 6 mm. I d., 5μm), mobile phase: methanol - water (volume ratio of 90:10), flow rate of 1mL / min, detection wavelength: 210 nm, injection volume: 10μL, detection sensitivity of 1.0, column temperature: 30℃, injection volume: 0.14-2.20 g/L. Under the detection conditions, sample concentration and peak area have a good linear relationship.The regression equation was A = 228006.17C +3961.05, r = 0.9984. Ursolic acid content is 1.34%.
We conducted a preliminary toxicology studies, including acute toxicity, cell toxicity, reproductive toxicity, genetic toxicity tests, etc to evaluate the safety of Ledum extract. We also carried out some pharmacology research, including antioxidant effect, anti-skin aging effect, and the impact of tyrosinase activity, liver cell protection role and the role of anti-tumor effect. Results of acute toxicity test show that Ledum extract is non-toxic material and the maximal tolerance dose is more than 30 g/kg (mice, mouth). Reproductive toxicity test in mice showed that Ledum extract at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can not influencd the genital organ coefficient. The incidence of sperm abnormality in mice and mouse testis rate of chromosomal abnormalities had no significant changed after giving Ledum extract. Ledum extract at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can reduce cyclophosphamide-induced mouse sperm abnormality rates. Especially the dose of 10 g/kg, 5 g/kg. Mouse genetic toxicity test showed that extracts from Ledum at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can not influence the mouse bone marrow polychromatic erythrocytes micronuclei incidence and mouse bone marrow cells chromosomal abnormalities. Ledum extracts at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can inhibit the mouse bone marrow polychromatic erythrocytes micronucleus and chromosome aberration of bone marrow cells induced by cyclophosphamide. 10g/kg dose has significantly inhibitory effects. Toxicity test in vitro showed that Ledum extract at the dose of 0.05 g/mL, 0.005 g /mL, 0.0005 g/mL has no cytotoxicity on mouse liver cells, but has a role in cell promoting, and plays a protective role in cyclophosphamide and ethanol-induced liver cell injury. Cosmetology studies showed that extracts from Ledum at the dose of 0. 05 g/mL, 0.005 g /mL, 0.0005 g/mL have antioxidant activity and can be an effective way to remove superoxide anion radical (O_2~-·) and hydroxyl radical (·OH).Ledum extracts at the dose of 0.5 g/mL, 0.25 g/mL, 0.125 g/mL, 62.5 mg/mL, 31.25 mg/mL, 15.625 mg/mL, 7.81 mg/mL, 3.9 mg/mL, 1.95 mg/mL can promote tyrosinase activity, increase the concentration of dopa-quinone in the reaction system. Ledum extract at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can enhance the content of L-HYP and collagen in D-galactose aging mouse skin.Anti-tumor experimental results showed that Ledum extract at the dose of 0.05 g/mL, 0.005 g/mL, 0.0005 g/mL can inhibit proliferation and division of liver cancer cells BEL-7404 and SMMC-7721, also can promote apoptosis of liver cancer cells, It has anti-tumor effect.
In short, Ledum extract is safe and non-toxic. It has reproductive protection, genetic conservation, beauty, liver cells protection and anti-tumor effect. It is worthy of in-depth development and application.
Ledum palustre L. is an evergreen shrub, belonging to Ericaceae, Ledum palustre. Mainly distributes in our country Northeast's size of Mt. Xingan and the Changbai Mountain. The resource of Ledum palustre L.is very rich. Ledum palustre L.have many kinds of chemical compositions, such as triterpenoid compound (ursolic acid, arbutin alcohol, arbutin), flavonoid (Quercetin, catechin, hyperin ), Coumarin, tanning substances and so on, and have many kinds of pharmacological actions, such as cough- relieveing,phlegm-dispelling, breathe-calming, blood vesselexpanding,voltage-dropping and fungus-suppressing effects. Ursolic acid belongs toα-amyrin (α-amyrin) non-polar triterpenoid pentacyclic compounds.Its molecular formula is C30H48O3 and molecular weight is 456.68.It is one of the main active substances with a variety of pharmacological effects including anti-tumor, anti-mutagenic, antioxidant,liver cell protection effects.
In this article, we firstly using alcohol reflux carried out a systematic study on Ledum ursolic extraction process, for the purpose of extraction process optimization and industrial production. Ursolic acid is the subject matter. The optimum conditions are: 85℃, pure alcohol: Ledum =10:1, reflux extraction 2 times, 1 time 1 hour. The detection conditions of Ledum Ursolic Acid Extract are: ODS column (250 mm×4 ? 6 mm. I d., 5μm), mobile phase: methanol - water (volume ratio of 90:10), flow rate of 1mL / min, detection wavelength: 210 nm, injection volume: 10μL, detection sensitivity of 1.0, column temperature: 30℃, injection volume: 0.14-2.20 g/L. Under the detection conditions, sample concentration and peak area have a good linear relationship.The regression equation was A = 228006.17C +3961.05, r = 0.9984. Ursolic acid content is 1.34%.
We conducted a preliminary toxicology studies, including acute toxicity, cell toxicity, reproductive toxicity, genetic toxicity tests, etc to evaluate the safety of Ledum extract. We also carried out some pharmacology research, including antioxidant effect, anti-skin aging effect, and the impact of tyrosinase activity, liver cell protection role and the role of anti-tumor effect. Results of acute toxicity test show that Ledum extract is non-toxic material and the maximal tolerance dose is more than 30 g/kg (mice, mouth). Reproductive toxicity test in mice showed that Ledum extract at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can not influencd the genital organ coefficient. The incidence of sperm abnormality in mice and mouse testis rate of chromosomal abnormalities had no significant changed after giving Ledum extract. Ledum extract at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can reduce cyclophosphamide-induced mouse sperm abnormality rates. Especially the dose of 10 g/kg, 5 g/kg. Mouse genetic toxicity test showed that extracts from Ledum at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can not influence the mouse bone marrow polychromatic erythrocytes micronuclei incidence and mouse bone marrow cells chromosomal abnormalities. Ledum extracts at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can inhibit the mouse bone marrow polychromatic erythrocytes micronucleus and chromosome aberration of bone marrow cells induced by cyclophosphamide. 10g/kg dose has significantly inhibitory effects. Toxicity test in vitro showed that Ledum extract at the dose of 0.05 g/mL, 0.005 g /mL, 0.0005 g/mL has no cytotoxicity on mouse liver cells, but has a role in cell promoting, and plays a protective role in cyclophosphamide and ethanol-induced liver cell injury. Cosmetology studies showed that extracts from Ledum at the dose of 0. 05 g/mL, 0.005 g /mL, 0.0005 g/mL have antioxidant activity and can be an effective way to remove superoxide anion radical (O_2~-·) and hydroxyl radical (·OH).Ledum extracts at the dose of 0.5 g/mL, 0.25 g/mL, 0.125 g/mL, 62.5 mg/mL, 31.25 mg/mL, 15.625 mg/mL, 7.81 mg/mL, 3.9 mg/mL, 1.95 mg/mL can promote tyrosinase activity, increase the concentration of dopa-quinone in the reaction system. Ledum extract at the dose of 10 g/kg, 5 g/kg, 2.5 g/kg can enhance the content of L-HYP and collagen in D-galactose aging mouse skin.Anti-tumor experimental results showed that Ledum extract at the dose of 0.05 g/mL, 0.005 g/mL, 0.0005 g/mL can inhibit proliferation and division of liver cancer cells BEL-7404 and SMMC-7721, also can promote apoptosis of liver cancer cells, It has anti-tumor effect.
In short, Ledum extract is safe and non-toxic. It has reproductive protection, genetic conservation, beauty, liver cells protection and anti-tumor effect. It is worthy of in-depth development and application.
引文
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