Separation Of Flavonoids From Several Salt Tolerant Plants Using Macroporous Resin And Evaluation Of Their Antialgal Activities

The red tide is a worldwide marine disaster,which not only destroyed marine ecological environment and resulted in heavey economic loss in marine fisheries,but also had great harmful-ness to human health.Therefore,it is very necessary to find effective measures for preventing and treating the red tide.In recent years,the plant allelopathy has shown a good application prospect in the prevention and control of red tide,and provides a new way for finding efficient and environmentally friendly method of algae suppression.In order to enhance stress resistance,salt tolerant plants often contain special chemical components,which are important resources for finding antialgal allelochemicals.Flavonoids are a kind of allelochemicals widely existed in plants.Recently,the application of flavonoids in inhibiting the growth of harmful algae has attracted more and more attention.Therefore,it is particularly important to study the large-scale preparation of flavonoids.In this paper,one kind of macroporous resin which can be used for the separation of flavonoids in different plants was screened.At the same time,the adsorptive behaviour of flavonoids on macroporous resin was studied preliminarily,and the purification technology of flavonoids in several salt tolerant plants had been established by macroporous resin.Finally,the antialgal activities of the isolated flavonoids were evaluated,which provided a basis for finding environmentally friendly natural algaecide agent,and also provided a theoretical basis for the application of salt tolerant plants to the prevention and control of red tide.The results of the study are as follows:1.Study on screening and adsorption behavior of macroporous resin.The macroporous resin with good adsorption and desorption properties of flavonoids in plants was screened using the hawthorn leaves as model plant,which have high contents of known structural flavonoids.The HPD-300 and HPD-400 resins had the best adsorption performance.The results showed that the larger the specific surface area of the resin,the higher the adsorption capacity is.The smaller the pore size of the resin is,the higher the adsorption capacity is.Scanning electron microscopy?SEM?was used to observe the microstructure of macroporous resin before and after adsorption.It was found that the surfaces of HPD-300 and HPD-400resins were porous and rough before adsorption,and became smooth and dense after adsorption,indicating that their porous surface are favorable for adsorption.It was observed that the sample had been adsorbed by the resin using infrared spectroscopy.The absorption peak of functional group of resin had no obvious change,indicating that the sample was not chemically combined with resin.The adsorption kinetics results showed that the adsorption speed of total flavonoids of hawthorn leaves on the HPD-400 resin was faster than that of the HPD-300 resin.It indicated that flavonoids received different resistance when it diffused inside the pore of different resins.The resin with large pore size had faster adsorption speed due to its smaller resistance.The kinetic data of the total flavonoids of hawthorn leaves on two resins could be fitted well by the pseudo-second-order model.The adsorption process was controlled by not only particle diffusion but also liquid film diffusion.Langmuir adsorption isotherm model could better describe the adsorption behavior of hawthorn leaves flavonoids on HPD-300 resin,indicating that it is monolayer adsorption.The adsorption is exothermic and spontaneous physical process.The technology of the purification of hawthorn leaves flavonoids by the HPD-300 resin was optimized.After treated by the HPD-300 resin,the content of total flavonoids of hawthorn leaves was increased from 23.75%to 58.05%,with a recovery yield of 81.88%.The method for separating vitexin-4?-O-glucoside and vitexin-2?-O-rhamnoside in hawthorn leaves hawthorn leaves was established using the HPD-400 resin followed by preparative HPLC.After one run treatment with HPD-400 resin,the contents of vitexin-4?-O-glucoside and vitexin-2?-O-rhamnoside in the product were increased 8.44-fold and 8.43-fold from 0.72%,2.63%to 6.08%and 22.18%,with recovery yields of 79.14%and 81.17%,respectively.After separation by the preparative HPLC,the purities of vitexin-4?-O-glucoside and vitexin-2?-O-rhamnoside were more than 96%.2.Purification of flavonoids from Poacynum hendersonii leaves using macroporous resins.The resin screening results of the model plants were verified with the salt tolerant plant Poacynum hendersonii.The HPD-300 resin had also the best static adsorption capacity,implying that the specific surface area was an important factor affecting the adsorption capacity of the resin.The adsorption capacities of flavonoids on the resin was mainly related to its content in the plant.The higher the content,the greater the adsorption capacity.The relative adsorption capacity?adsorption rate?was related to the structure of flavonoids,including the structure of aglycone and the number of sugar groups.The number of hydroxyl groups on the aglycone has a great influence on the adsorption rate of flavonoids on the resin,which indicates that the flavonoids are mainly combined with the polystyrene framework of the resin by hydrophobic aglycone.Additionally,the adsorption capacities of chlorogenic acid on all the test resins to were very poor and the adsorption rate was less than 10%,indicating that?-?effect also had influence on the adsorption process between the phenyl ring of the resin and the aromatic ring of the flavonoid aglycone.A suitable method to separate quercetin-3-O-sophoroside and isoquercitrin from Poacynum hendersonii leaves was developed using the HPD-300 resin followed by Sephadex LH-20 column chromatography.The contents of quercetin-3-O-sophoroside and isoquercitrin in the product separated by the HPD-300 resin were increased from2.16%and 1.26%to 21.34%and 10.70%with recovery yields of 82.1%and 77.3%,respectively.After the purification by Sephadesx LH-20 column chromatography,the purities of quercetin-3-O-sophoroside and isoquercitrin achieved 93.5%and 95.6%,respectively.The pseudo-second-order kinetic model fitted the adsorption kinetics data well,while the fitting of intraparticle diffusion model demonstrated that the adsorption process was controlled by liquid film diffusion and particle diffusion.The adsorption isotherms were well represented by the Langmuir isotherm model,indicating the adsorption was monolayer adsorption.In addition,the adsorption process is exothermic and spontaneous physical process.The above results showed that HPD-300 resin also had good separation effect on flavonoids from salt tolerant plants.3.Application of HPD-300 resin in the separation of flavonoids from Tamarix chinensis branches and leaves and lotus leaves.Several kinds of flavonoid sulfate were identified from salt tolerant plant Tamarix chinensis by HPLC-MS technology for the first time.An efficient method was established for separating kaempferide and quercetin-3',4'-dimethyl ether using acid extraction and the HPD-300 resin followed by silica gel column chromatography.After separation,the purities of two compounds achieved 97%.The experimental results showed that not only flavonoid glycosides but also free flavonoids could be separated by the HPD-300 resin.A suitable method to separate quercetin-3-O-glucuronide with a purity greater than 97%from wetland plant lotus leaves using the HPD-300 resin combined with silica gel column chromatography was developed.It is further indicated that the HPD-300 resin is suitable for the separation of flavonoids from different plants.4.Evaluation of antialgal activities of flavonoids.Skeletonema costatum is a kind of typical red tide algae with high eruption frequency in the near coast of China.In this paper,the inhibitory effects on the growth of Skeletonema costatum of islated flavonoids were studied.Kaempferide from Tamarix chinensis branches and leaves showed the strongest antialgal activities and EC₅₀ was 16.70?M after a 3-d culture.The antialgal activity of quercetin-3',4'-dimethyl ether is relatively weak,and EC₅₀ was 99.76?M,which may be related to its poor solubility in seawater.Quercetin and its oxyglucoside derivatives,including quercetin-3-O-sophoroside,isoquercitrin and quercetin-3-O-glucuronide also showed strong antialgal activities,while their EC₅₀0 were 35.56?M,19.98?M,24.16?M and34.18?M,respectively.Among them,the antialgal activities of isoquercitrin,quercetin-3-O-sophoroside and kaempferide were stronger than that of positive control copper sulphate.Two flavonoid C-glycosides,vitexin-4?-O-glucoside and vitexin-2?-O-rhamnoside showed weak antialgal activities,and the highest inhibition rates were only about 30%-40%.In conclusion,the specific surface area and pore size of the resin have great influence on the adsorption capacity.The resin mainly adsorbed the hydrophobic part of flavonoid compounds,and the?-?effect between flavonoid aglycone and resin benzene ring also contributed to the adsorption process.The selected HPD-300 resin was suitable for separating flavonoids from different plants.The adsorption kinetics data of flavonoids on the HPD-300 resin were fitted well by the pseudo-second-order model,and the adsorption rate was controlled by particle diffusion and liquid film diffusion.The adsorption of flavonoids on HPD-300 resin is exothermic and spontaneous monolayer physical adsorption.The separation technique of flavonoids from different plants established using HPD-300 resin in this article was reliable,simple,environmentally friendly and inexpensive,which provided a basis for the separation of flavonoids from plants.Flavonoids isolated from Poacynum hendersonii leaves and Tamarix chinensis branches and leaves could significantly inhibit the growth of Skeletonema costatum,and were expected to be environmentally friendly algaecide agent.