Studies On Extraction, Isolation, Characterization And Anti-oxidant Activities Of Flavonoids From Podocarpus Nagi

Podocarpus nagi belongs to the family podocarpeace,genus Podocarpus.As one kind of ancient gymnosperms,Podocarpus nagi originated in the Mesozoic cretaceous period,which was known as "living fossil".Podocarpus nagi was widely distributed over the provinces south of China,for example,Fujian province,Hunan province,Sichuan province and et al.Podocarpus nagi has extremely unique ornamental value with graceful shape,which is one kind of precious trees for landscaping.In addition,Podocarpus nagi is popularly used as traditional folk medicines in the Yao nationality to treat sorts of ailments,for instance,the pronounced function on fracture with leaves,rheumatism with barks,and polio with roots and et al.It is found that Podocarpus nagi is packed with a variety of compounds including flavonoids,terpenoids,volatile oils together with other functional compounds,which were reported to perform well anti-inflammatory,anti-oxidant,anti-tumor and anti-virus activities.What is more,Flavonoids,one kind of significant natural compound,plays an important role in the regulation of plant growth with extensive pharmacological activities.As a common ornamental foliage plant,Podocarpus nagi with a large annual increment should be pruned regularly.In order to find an economic and effective means to make the best use of Podocarpus nagi,waste branches obtained by manual pruning can be used as a newly raw resource for the researches on flavonoids in this study.This thesis elaborated the studies on the extraction,isolation,purification and structural identification of the flavonoids in Podocarpus nagi,besides,antioxidant capacity was evaluated in vitro.The main results are as bellow:1.The extraction process of flavonoids from Podocarpus nagi.On the basis of one single factor experiments,orthogonal experiments has been adopted to optimize parameters of reflux extraction and ultrasonic extraction conditions,and the yield of total flavonoids from Podocarpus nagi was investigated by UV.The results showed that the optimum conditions of reflux extraction were confirmed as follows: the ratio of liquid to material is 30:1?mL:g?,70% EtOH as extracting solvent,refluxing for 1.0 h at 80?,and the extraction yield of flavonoids is 15.52%.The optimum ultrasonic conditions were determined as below: the ratio of liquid to material 25:1?mL:g?,ultrasonicing for 30 min at 60? with 70% EtOH,and the extraction yield of flavonoids is 13.64%.2.The enrichment and purification process of amentoflavone in Podocarpus nagi.A brief survey of the optimization of the load and wash steps for amentoflavone from Podocarpus nagi with macroporous adsorption resin is given in this study.Based on the comparison of several common resins,AB-8 macroporous adsorption resin with the preferable adsorption and desorption performances was elected to the enrichment of amentoflavone in the following tests.And an optimized process for AB-8 macroporous adsorption resin was recommended as follows: The concentration,load rate and volume of loading solution were determined with 0.0324 mg/mL,1 BV/h and 200 mL,respectively;Desorption was sequentially operated with 70 mL 80% ethanol-water?v/v?eluent at the load rate of 1 BV/h.the content of amentoflavone has increased from 0.1% to 2.0%.Then,Different chromatographies including silica gel,Sephadex LH-20 gel and preparative thin layer chromatography were used to the separation and purification of amentoflavone.Eventually,4.0 mg amentoflavone was obtained with high purity.3.By means of different kinds of chromatographies over various materials such as AB-8 macroporous adsorption resin,silica gel,Sephadex LH-20,C18,YMC and et al,six flavonoids were isolated from Podocarpus nagi.Furthermore,their structures were identified mainly on the foundation of spectroscopic studies like IR,1H-NMR,13C-NMR,et al.And the compounds are catechin?I?,epicatechin?II?,amentoflavone?III?,vitexin?IV?,vitexin rhamnoside?V?,apigenin?VI?.4.Anti-oxidant capacity in vitro was evaluated by total antioxidant capacity assay kit with FRAP method,and researches on the scavenging rate of the well-known free radicals DPPH·,O₂^-· and ·OH has been carried on in this paper.Meanwhile,Vitamin C was considered as the positive control.At a certain degree,it appeared that flavonoids from Podocarpus nagi performed significant antioxidant capacity.The scavenging rate on DPPH· can reach 89.28 percent at 0.078 mg/mL,and the scavenging rate on O₂^-· and ·OH can reach 64.35 percent and 83.72 percent at 0.039 mg/mL,respectively.They all performed a dose-dependent manner.For a further investigation on the assessment of the antioxidant activity,another value represented the concentration of the flavonoids needed for scavenging 50 percent free radicals was caculated by a formula obtained from the linear fitting curves on the foundation of the scavenging capacity with different concentrations.The results showed that there are 0.0165,0.0127 and 0.0020 mg/mL flavonoids of the sample at least to scaveng half of the DPPH·,O₂^-· and ·OH contained in the solution.