Studies On Extraction, Purification Of Alkaloid And Flavonoids And Bioactivity Of Flavonoids From Mistletoe

Mistletoe, the dried culms of Viscum coloratum(Kom.) Nakai of Loranthaceae family, is a well-known tradition herbal, medicine in our country. It is used from the Qin and Han Dynasties. But the name usage of V. coloratum(Kom.)Nakai and Loranthus parasiticus(L.)Merr, are comparatively disordered. Furthermore, due to their efficacy and usage being similar, both of these are confusedly used at clinic. According to the Chinese Pharmacopeia (1985), Loranthaceae and Viscum coloratum(Kom.)Nakai are separated, and only Viscum coloratum (Kom.) Nakai is embodied. The species of mistletoes are single in the northern provinces of our country and are Viscum coloratum (Kom.) Nakai. Traditionally, it is considered that mistletoe has an effect on dispelling chronic rheumatism, nourishing liver and kidney, boosting up physique, reducing blood pressure, and protecting fetus. In recent years, it is considered that mistletoe has an effect on anti-cancer, anti-bacteria, anti-consenescence, therapy cardiovascular disease and immunoregulation.The main effective components of mistletoe are flavonoids, alkaloid, lectins and viscotoxins, but only its herbal or rough extract is used at clinic. In this way, its utilization is limited. It is very important that effective components in mistletoe are extracted, purified while their structures are characterized. At present, the researches on the effective anti-cancer protein component and its action mechanism are always one of the hotspots in the correlative field. Up to now, the alkaloid and flavonoids of mistletoe are studied, no systematic and detailed studies are found on its extraction, separation, purification and activity. Our focus in this dissertation is on the studying of flavonoids and alkaloid, which can enrich application of mistletoe. It offers scientific data for the ulterior study on mistletoe. The main contents and conclusions in this dissertation are summarized as follows:1. Using modern analytical technologies, the nutrients of mistletoe are analyzed. The results are shown as follows (g/100g, dry mass):â‘ water 9.03, ash 6.07, coarse fibers 19.65, fats 14.69, total sugar 5.53, reduced sugar 1.98, protein 11.29,â‘¡Vitamin E (μg/g) 127.63. Trace elements of mistletoe are simultaneously determined by ICP-AES. The results showed that K, Ca, Mg and Na are rich in the mistletoe. The content change of trace elements is a little between once decoction solution and twice decoction solution. So, once decoction solution is satisfied to trace elements.2. During the study of alkaloid of mistletoe, the weight method is used to determine the content. Mistletoe alkaloid is extracted by reflux, SFE, ultrasonic methods. These methods are compared and the experimental conditions are optimized by orthogonal design. The results indicated that ultrasonic extraction is better than reflux and SFE methods. Orthogonal test is used to evaluate the influence factors, that 90% ethanol, extracting 30min, the rate of solid and liquid 20:1 are the optimum extraction conditions. The rate of total alkaloid is 1.73%. Therefore, ultrasonic extraction is an easy, simple and effective method.3. The purification technique of alkaloid is studied. The macroporous resin is much fitter for the separation and purification of alkaloid than ion exchange resin. Four macroporous resins are used for the separation of total alkaloid. Adopting the sorption and desorption, the different macroporous resins and its capacity are studied. The results showed that D 101 resin possesses much higher adsorption and desorption capacity. Under optimum condition (initial solution 0.288mg/mL, 4BV/h and pH10), the ratio of adsorption is more that 3.15mg/g dry resin. Under desorption optimal condition (50%ethanol, 2BV/h), the ratio of desorption can reach 94%.4. Mistletoe flavonoids are main active components. It is studied amply in this dissertation The spectrophotometry is used to analyze flavonoids content. The recovery is 99.73%. Flavonoids are extracted by conventional extraction, SFE and ultrasonic extraction. Meanwhile, several experimental designs, such as the orthogonal experimental design and the orthogonal & rotable experimental design, together with Statistical analysis and response surface graph analysis are also performed to get the upmost extraction efficiency and optimal condition. It is indicated that ultrasonic extraction is better than others. Its optimal extraction conditions are as follows: the concentration of alcohol is 84.72% (V/V), the ratio of material and alcohol is 1:32 (W/V), and the time need of ultrasonic extraction is 32.78 min. The ratio of flavonoids is 1.32% under this condition.5. The purification of flavonoids is studied after extracting it from mistletoe, and AB-8 resin is selected to separate and purify flavonoids. Based on the research of eluent solvent, concentration and pH of initial solution, the velocity of initial solution and eluent, the optimal conditions are as follows: when the concentration of initial solution is 5.21mg/mL, pH is 4.0, the sample solution speed is 2.0BV/h, the elution solvent 4.0BV of 40% ethanol, the flow rate is 1.0BV/h, and the purity of flavonoids could reach the height of 69.63%, which is approximately 5.73 times higher than the previous 12.16%, while the yield of flavonoids could reach 81.36%.6. The flavonoids from mistletoe detected by color reaction and UV spectrum analysis belong to flavone and flavonol.7. Flavonoids get primary separation after grads desorption by AB-8 resin. In the outflow of 10% ethanol to 30% ethanol, there are a lot of compounds and the separation effect is dissatisfactory. But the eluent is 40% ethanol to 60% ethanol, and the compounds of outflow separate satisfactorily. The flavonoids components of fraction of 40% ethanol eluent are further investigated. Two fractions are collected after the flavonoids are further separated and purified by Prep-HPLC. Analyzed by UV, IR, ¹H NMR/¹³C NMR and LC/M, then their molecular structures have been determined. Monomer A is homoeriodictyol-7-O-β-D-glycoside-4'-O-β-D-apiosyl and B may be homoeriodictyol-7-O-β-D-glycoside respectively.8. Finally, the antioxidant activity of mistletoe flavonoids is studied by four different systems (oil, DPPH·, O₂^·and·OH). The result showed that the purified samples have satisfying the antioxidant activity in the oil system.. Their antioxidant activity is enhanced with the adding of concentration. At certain concentration, their antioxidant activity reduced. In the system of oil, citric acid and Vc have synergic anti-oxidative activities for purified samples. In the system of radicals, the abilities of scavenging DPPH·, O₂^-·and·OH of the purified samples are detected by UV spectrophotometry and chemiluminescence. The results showed that the antioxidant activity of the extract is excellent. The order of scavenging abilities is DPPH·＞·OH＞O₂^·.9. Homoeriodictyol-7-O-β-D-glycoside-4'-O-β-D-apiosyl shows good activities against Platelet Activiting Factor (PAF) for the first time. The inhibition effect on PAF-induced rabbit platelet aggregation is dose-dependent.