Evaluation Of Active Ingredients From Lycium Barbarum L.Inhibiting Angiotensin Converting Enzyme Inhibition

Hypertension is a frequently-occurring common disease, which is characterized as "three high" and "three low", that is, high morbidity, high mortality and high disability, low awareness, low treatment and low control rate. Although there are many synthetic antihypertensives with high efficacyin market, their side effects such as short action time, dependency strong, and significant toxicity prevent their uses. Therefore, more and more researches are focused on the development of new medicine with low toxicity and high efficancy. It is well-known that Traditional Chinese medicines (TCM) have rich resources and long history in China. Screening active ingredients from TCMs is one of effective ways to obtain antihypertensive drugs. Reports showed that Lycium barbarum L. possessed vital biological activities, including anti-hypertension, antihyperglycemia, immunomodulation, anti-fatigue/endurance, anti-tumor activity and anti-oxidant properties, and so on. However, the mechanism and active ingredient with anti-hypertension are not still clear.In this study, the anti-hypertensive activity of Lycium barbarum L.was evaluated using angiotensin converting enzyme (ACE). The active fractions were further separated by column chromatography, and high speed counter-current chromatography (HSCCC).The main contents of this paper are as follows:(1) Screening of ACE inhibitor from32kinds of traditional Chinese medicines.(2) Extraction of active compounds with inhibiting ACE from Lycium barbarum L. extracts using D-101macroporous adsorption resins.(3) Isolation and separation of active compounds with inhibiting ACE from Lycium barbarum L. extracts by silica column chromatography and SephadexG-20column.(4) Purification of active compounds from Lycium barbarum L. extracts by D-101macroporous resin column, and by high speed counter-current chromatography (HSCCC), using chloroform-methanol-water (10:7:3, v/v/v) as solvent system. Scopoletin was successfully separated from50%ethanol eluted fraction.The results of these experiments were shown as follows:(1) Using density gradient frozen centrifugation method to extract ACE from fresh rabbit lung, which showed high activity. Mensurating content of hippuric acid (Hip) with substrate N-hippuryl-L-histidyl-L-leucine (HHL)-ACE system, Hip peak area with ACE concentration showed a good linear relationship:r=0.99. It explained this method could accurately mensurate the activity of ACE, it could determine suitable dose of ACE.(2) Evaluation of the angiotensin converting enzyme (ACE) IR of32kinds of traditional Chinese medicines. The experimental results showed:Rhizoma Alismatis, Semen Cassiae, Rhizoma Anemarrhenae, Crataegus Pinnatifida Bge, Fruclus Schisandrae, Radix Astragali, Fructus Citri Sarcodactylis and Lycium barbarum L., their inhibitory activities to ACE all were greater than60%, in addition, Lycium barbarum L. have exhibited the highest IR, and the IR was up to92.30%. So, Lycium barbarum L. was further researched as anti-hypertension through ACE assay.(3) The Lycium barbarum L. extracts were separated with using D-101macroporous adsorption resins, then six fractions were obtained. The experimental results showed:the fraction which eluted by50%ethylalcohol exhibited the maximal inhibitory effect to35.46%.(4) Using column chromatography to do further isolation and purify active ingredients, it obtained seven fractions by silica column chromatography, the fraction which eluted by Ethyl acetate/MeOH (6:4) exhibited the maximal inhibitory effect to56.26%. This fraction was further purified and six compounds were obtained, the compound of scopoletin glycoside exhibited the maximal inhibitory effect to96.67%. So, we preliminary thought, the compound of scopoletin glycoside from Lycium barbarum L. was the active compound of ACE.(5) The thin layer chromatograph coupling with fluorometric (TLC-F) method was used to screen solvent system of HSCCC. The scopoletin were successfully separated from Lycium barbarum L. by successive sample injection for the first time through HSCCC, using chloroform-methanol-water (10:7:3, v/v/v) as the solvent system.