Preparation Of Ledum Extract And Partial Toxicological And Pharmacological Action

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 (O₂^-·) 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.