Abstract:
The present invention discloses a method for separating flavonoid substances in  Camellia nitidissima  Chi based on a magnetic nanoparticles-PAMAM nano composites, which comprises the following steps: preparing PAMAM dendrimer, then using the PAMAM dendrimer to prepare the magnetic nanoparticles-PAMAM nano composites, then adding the obtained magnetic nanoparticles-PAMAM nano composites in a  Camellia nitidissima  Chi extract, extracting and performing magnetic separation on the flavonoid substances in  Camellia nitidissima  Chi under ultrasound or microwave condition. According to the present invention, flavonoid substances with faintly acid characteristics are extracted and adsorbed in a plant concentrate such as  Camellia nitidissima  Chi or  Hedyotis diffusa  etc. based on the magnetic nanoparticles-PAMAM nano composites, in a successive step, high efficiency separation of the flavonoid substances can be realized by the technologies such as magnetic separation and microwave-assisted extraction.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation in part of, and claims priority to, Chinese Patent Application No. 201410639271.1 with a filing date of Nov. 13, 2014. The content of the aforementioned application, including any intervening amendments thereto, is incorporated herein by reference. 
       TECHNICAL FIELD 
       [0002]    The present invention refers to the field of biotechnology, more particularly, to a method for separating flavonoid substances in  Camellia  nitidissima Chi based on a magnetic nanoparticles-PAMAM nano composites. 
       BACKGROUND OF THE PRESENT INVENTION 
       [0003]      Camellia nitidissima  Chi (or name  Camellia nitidissima ) is a precious and rare plant in the world, which is as famous as  Cathaya argyrophylla, Alsophila spinulosa, Davidia involucrate  and other precious living plant fossils. Founded in 1993 by Chinese botanist Jinglie Zuo in Fangcheng of Guangxi Province,  Camellia nitidissima  Chi is one of eight national first-class protective plants and included in plant species in Annex II of  Convention on International Trade in Endangered Species of Wild Fauna and Flora . At present, 40 breeds and 5 variants of  Camellia nitidissima  Chi are well known to the world, mainly distributed in Vietnam and southern edge of subtropical zone and northern edge of tropical zone within the borders of Guangxi Province in China. 
         [0004]      Camellia nitidissima  Chi, Inula nervosa Wall, Noni Puree, Yeast β-glucan, and Tissue culture of  Saussurea involucrata  are new resources food approved by the No. 9 document in 2010 of Ministry of Health of China. The leaves and flowers of  Camellia nitidissima  Chi are enriched in total flavones (isoflavonoids, biflavonoids, anthocyanin and neoflavonoids etc.), tea polyphenols, multiple amino acids and microelement etc. by studies. 
         [0005]    As one kind of compounds widely existed in the nature, flavonoids are the derivative of chromane, characterized in that it is with basic framework of C6-C3-C6. Flavonoids can be divided into dozens of categories: flavones, flavanols, isoflavones, flavanones, flavanonols, aurones, flavanones, anthocyanidins, chalcones and chromones etc., over 4000 kinds of flavonoids are found at present, mainly existed in the leaves, fruits, roots and skin of plants, experimental results show it is of extensive physiological and pharmacological activity (including antioxidant, scavenging oxygen free radicals, antivirus, anticancer, anti-inflammatory, anti-aging etc.), so the study on flavonoids has become a hot topic in Chinese and foreign medical profession. Traditional extracting methods mainly include ultrafiltration, enzymolysis, adsorption by coarse pored resin, supercritical fluid extraction, ultrasonic method and microwave field extraction etc. (Rui Zhang, Yaqin Yu, Yang Shi.  Study on Flavonoids Extracting Technology. Food and Machinery , 2003, 01, 21-22), while they are always restricted by extracting efficiency or cost. 
         [0006]    At present, most studies on active ingredients of flavonoid substances in  Camellia nitidissima  Chi are confined to traditional ultraviolet and chromatographic detection or solvent extraction, membrane separation etc., like Zhihua Zhan performed qualitative analysis on the chemical component in leaves of  Camellia nitidissima  Chi. The flavonoids in  Camellia nitidissima  Chi are occupied about 2.33% by qualitative analysis. (Zhihua Zhan,  Extraction and Separation of Flavones in Leaves of Camellia nitidissima Chi [D], Guangxi Normal University, 2006); repeated silica gel column chromatography, Sephadex LH 20 gel chromatography, ODS chromatography and repeated recrystallization and other methods are adopted by Xiao Peng and co-workers to perform separation and purification on chemical component in flowers of  Camellia nitidissima  Chi, and the structure is identified on the basis of physicochemical constants and spectrum analysis. 13 compounds including flavonoids are obtained from the separation of ethanol extract in flowers of  Camellia nitidissima  Chi. (Xiao Peng, Dayong Yu, Baomin Feng, Ling Tang, Yongqi Wang, Liying Shi.  Study on Chemical Component in Flowers of Camellia nitidissima  Chi. Guangxi Plant, 2011, 31(04), 550-553). The content of total saponins, total polyphenol (tannin) and total flavones in different plant parts of  Camellia nitidissima  Chi,  Camellia pubipetala, Camellia impressinervis, Camellia chrysantha  and  Camellia chrysanthoides  is detected through ultraviolet spectrophotometry by Qian Tang. The content of total saponins, total polyphenol(tannin) and total flavones of alcohol extract in flowers of  Camellia nitidissima  Chi is respectively 21.30%, 6.56% and 21.76%. (Qian Tang, Yanying Luo, Yongqi Wang etc.  Quantitative Analysis on Chemical Component of Camellia nitidissima Chi Section , Lishizhen Med Mater Med Res, 2009, 20(4), 769-771); while Quanbin Chen and co-workers take following method, the leaves of  Camellia nitidissima  Chi are used as the material to extract flavones, high purification aglycone is obtained by hydrolyzing the flavones, solvent extraction, column chromatography and other methods; structure characterization is performed by melting point, TLC, liquid chromatography, infrared spectroscopy, nuclear magnetic resonance method and other analysis methods to confirm the leaves of  Camellia nitidissima  Chi contain flavonoid glycosides, the aglycon of which is quercetin and kaempferol. (Quanbin Chen, Zhihua Zhan, Qiaoyun Zhang, Na Liao.  Separation and Purification of Flavones Aglucone in Leaves of Camellia nitidissima  Chi and Characterization. Guangxi Tropical Agriculture, 2005, 6, 10-11). 
         [0007]    Flavonoids in  Hedyotis diffusa  are extracted by microwave-assisted technology by Caixia Zhang and co-workers, and the extraction processes are optimized, the primary and secondary sequence of affecting flavones extraction ratio is as follows on the basis of identification of chemical structure of extract, extracting temperature, ethanol concentration, extracting time and solid to liquid ratio, the best extraction process is ethanol concentration 80%, extraction time of 40 min, solid to liquid ratio of 1:100 under 40° C., the extraction ratio of flavones in  Hedyotis diffusa  reaches to 2.86% under this condition. (Caixia Zhang, Dingjian Cai, Wenying Fang.  Optimization of Flavones Extracting Process and Structure Identification . Anhui Agricultural Sciences, 2011, 39(01), 131-133); Jialin Li and co-workers discussed the effect of extraction temperature, extraction time, solid to liquid ratio and ethanol concentration on the total flavones extraction ratio of  Rhus chinensis . The extraction ratio is the best under extraction time of 10 min, extraction temperature of 60° C., solid to liquid ratio of 1:60 and ethanol concentration of 70%. Experimental results show the extraction ratio of total flavones reaches to 6.65% under the best conditions. (Jialin Li, Suzhen Wu, Zhanyu Bei.  Study on Extraction Process of Total Flavones in Rhus Chinensis ). 
         [0008]    Dendrimer is a new class of three-dimensional and highly ordered macromolecules in recent years. PAMAM is one of the most widely and deeply studied dendrimer, with highly branched and highly symmetrical structure and a large number functional groups on the surface and other unique structural features, PAMAM is widely used in drug carrier, surface active agent, catalyst, nano materials, membrane materials and other fields. One generation of obtained product will be increased every time the structural unit of dendrimer is repeated and multiplied, at present, 10.0 G divergent method is the leading and most mature synthetic method in the synthesis of PAMAM dendrimer, this method is of mild reaction condition, rapid reaction and high selectivity. (Huimin Tan, Yunjun Luo.  Dendritic Polymer  [M]. Beijing: Chemistry Industry Press, 2001). 
         [0009]    PAMAM dendrimer is of rich amine terminated structure and inner secondary amine and quaternary amine structure, so PAMAM dendrimer presents faintly alkalinity. With phenolic hydroxyl group, most flavonoids present faintly acidity, it is easy to be dissolved into water containing soda, and it can be precipitated out after acidification. The first reason is the acidity of flavone phenolic hydroxyl group, and the second is the ring opening of mother nucleus of flavone under alkaline conditions, so 2′-hydroxychalcone is formed, with the rise of polarity, it is dissolved. (Xuefeng Guo, Yongde Yue.  Research Progress of Extraction, Separation and Purification of Flavonoids and Content Measuring Method. Anhui Agricultural Sciences , 2007, 35(26), 8083-8086). According to the present invention, the high efficiency separation of the flavonoid substances in  Camellia nitidissima  Chi is realized by the extraction and adsorption effect of PAMAM dendrimer with faintly alkalinity and flavonoid substances with faintly acidity. 
         [0010]    For better separation of the flavonoid substances adsorbed by PAMAM, the magnetic nanoparticles-PAMAM nano composites of the present invention are prepared by compounding PAMAM and magnetic nanoparticles, like ferrites (Fe 3 O 4 , γ-Fe 2 O 3  and MeFe 2 O 4 , wherein Me=Co, Ni, Mn), Fe, Co, Ni, alloy particles and iron nitrides (FeN, Fe 2 N, ε-Fe 3 N and Fe 16 N 2 ) and other particles, separating the magnetic nanoparticles-PAMAM nano composites extracted and adsorbed with faintly acidity flavonoid substances from extract by magnetic effect; and finally performing separation on the adsorbed flavonoid substances with magnetic nanoparticles-PAMAM nano composites by ultrasound-assisted extraction. 
       SUMMARY OF THE PRESENT INVENTION 
       [0011]    The purpose of the present invention is to solve problems above mentioned by offering a method for separating flavonoid substance in  Camellia nitidissima  Chi based on a magnetic nanoparticles-PAMAM nano composites. 
         [0012]    To achieve purpose, the present invention employs the following technical solution: 
         [0013]    A method for separating flavonoid substances in  Camellia nitidissima  Chi based on the magnetic nanoparticles-PAMAM nano composites, which comprises the following steps: 
         [0014]    1) Preparing PAMAM Dendrimer 
         [0015]    Ethylenediamine is used as the centronucleus to prepare PAMAM dendrimer, the first step is performing Michael addition reaction between ethylenediamine and methyacrylate to obtain generation 0.5 polyamidoamine dendrimer, namely PAMAM G0.5, the reaction has high selectivity under 25° C.; the second step is reacting the obtained PAMAM G0.5 with excessive ethylenediamine under 25° C. to obtain generation 1.0 polyamidoamine dendrimer, namely PAMAM G1.0; one generation will be increased every time above two steps reaction are repeated, PAMAM of different generations are obtained by constantly repeating above two steps under proper conditions. 
         [0016]    2) Preparing the Magnetic Nanoparticles-PAMAM Nano Composites 
         [0017]    The magnetic nanoparticles are dispersed into methanol through magnetic separation to obtain solution with 0.0128 mol/L concentration; diluting 25 mL methanol solution of magnetic nanoparticles prepared above to 150 mL by methanol and ultrasonication is performed for 30 min; then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h; performing magnetic separation on the obtained solution after washing it by methanol for 5 times, then the solution is dispersed in methanol to obtain 5 wt % solution for standby. 
         [0018]    50 mL 5 wt % magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, the volume concentration of the methyacrylate is 20%, performing ultrasonication to the mixture for 7 h in water bath at room temperature; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, the volume concentration of the ethylenediamine is 20%, performing ultrasonication for 3 h at room temperature; then washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, higher generations of magnetic nanoparticles modified by dendrimer will be obtained for each cycle; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the magnetic nanoparticles modified by PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0019]    3) Extracting and Performing Magnetic Separation on the Flavonoid Substances in  Camellia nitidissima  Chi 
         [0020]    Adding the obtained magnetic nanoparticles-PAMAM nano composites in the  Camellia nitidissima  Chi extract, extracting 0.5-3 h under ultrasound or microwave condition, after extraction, separating the nanoparticles-PAMAM nano composites with flavonoid substances adsorbed and extracted through magnetic separation, the flavonoid substances adsorbed by magnetic nanoparticles are extracted and separated by organic solvents from separated nanoparticles-PAMAM nano composites, the nanoparticles-PAMAM nano composites can be recycled again after ablution and activation. 
         [0021]    As a further plan of the present invention, the magnetic nanoparticles include Fe, Co, Ni as well as alloy particles, ferrites and iron nitrides; ferrites include Fe 3 O 4 , γ-Fe 2 O 3  and MeFe 2 O 4 , wherein Me=Co, Ni, Mn; iron nitrides include FeN, Fe 2 N, ε-Fe 3 N and Fe 16 N 2 . 
         [0022]    As a further plan of the present invention, the organic solvents include ethanol, methanol, DMSO and acetone. 
         [0023]    As a further plan of the present invention, the method also applies to plant leaves, flowers, fruits and roots enriched in flavonoid substances. 
         [0024]    Compared with the prior art, the advantage of the present invention is that flavonoid substances with faintly acidity are extracted and adsorbed in a plant concentrate such as  Camellia nitidissima  Chi and  Hedyotis diffusa  based on the magnetic nanoparticles-PAMAM nano composites, in a successive step, high efficiency separation of the flavonoid substances can be realized by the technologies such as magnetic separation and microwave-assisted extraction. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0025]      FIG. 1  is a classical synthetic route of PAMAM dendrimer. 
           [0026]      FIG. 2  shows a schematic diagram of Fe 3 O 4  magnetic particle-PAMAM composites synthesis and flavonoid substances separation in  Camellia nitidissima  Chi and other plants. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0027]    The technical proposals of embodiments are described below dearly and completely with reference to the accompanying drawings. Obviously, it merely shows several specific embodiments of the present invention, rather than the whole embodiments. Other embodiments obtained by one of ordinary skilled in the art without creative work based on the embodiments of the present invention are all included within the protection scope of the present invention. 
         [0028]    The flavonoid substances separation steps of the present invention are as follows, for better high efficient separation of the flavonoid substances adsorbed by PAMAM, the magnetic nanoparticles-PAMAM nano composites of the present invention is prepared by compounding PAMAM dendrimer and magnetic nanoparticles, like ferrites (Fe 3 O 4 , γ-Fe 2 O 3  and MeFe 2 O 4 , wherein Me=Co, Ni, Mn), Fe, Co, Ni, alloy particles and iron nitrides (FeN, Fe 2 N, ε-Fe 3 N and Fe 16 N 2 ) and other particles as well as generation 1.0-10.0 PAMAM dendrimer. Because the end of its ball structure is with rich RNH 2  functional group, and the middle part is with quantities of R 2 NH, R 3N , quaternary ammonium and others, with faintly alkalinity and 10-30 grams solubility for every 100 grams of water, this kind of composites has strong adsorption effect on flavonoid substances with faintly acidity, which is good for the separation of flavonoid substances. The magnetic nanoparticles-PAMAM nano composites extracted and adsorbed with flavonoid substances with faintly acidity are separated from the extract through magnetic separation, and then the extracted and adsorbed flavonoid substances are separated with magnetic nanoparticles-PAMAM nano composites through solvent extraction technology. 
       Embodiment 1 
       [0029]    Preparation of leaves of  Camellia nitidissima  Chi concentrate: picking fresh leaves of  Camellia nitidissima  Chi in spring; weighting it with electronic scale; performing screen for the picked fresh leaves of  Camellia nitidissima  Chi as per the standard of  Pharmacopoeia of China  (2010), smashing the optimized leaves of  Camellia nitidissima  Chi after washing; adding 50 L acetone (over 95%) into 20 kilograms of smashed leaves of  Camellia nitidissima  Chi and extracting for 5-6 h by Soxhlet extractor to obtain extract A; adding about 40 L acetone (over 95%) into extracted residue and performing ultrasonic wave processing for about 1.5 h under 40-60° C. to obtain extract B; mixing extract A and extract B and evaporating most acetone solvent by rotary evaporation of rotary evaporators, and finally obtaining about 2.0 L  Camellia nitidissima  Chi concentrate in organic phase. 
         [0030]    Preparation process of Fe 3 O 4  magnetic particles-4.0 G PAMAM composites: preparing the mixture of 0.085 mol/L ferric chloride solution and 0.05 mol/L ferrous sulfate, then adding 1.5 mol/L ammonia solution into the mixture and stir vigorously until pH=9. Washing the obtained Fe 3 O 4  magnetic nanoparticles for 5 times immediately and washing it with methanol for 3 times, the Fe 3 O 4  magnetic nanoparticles is dispersed in methanol by magnetic separation, then the solution with 0.0128 mol/L concentration is obtained. Diluting the above prepared 25 mL methanol solution of Fe 3 O 4  magnetic nanoparticles into 150 mL with methanol and performing ultrasonic processing for 30 min. Then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h. After washing the obtained solution for 5 times with methanol, the magnetic separation is performed, the solution is dispersed in methanol to obtain 5 wt % solution for standby. 50 mL 5 wt % of magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, performing ultrasonication to the mixture for 7 h in water bath at room temperature; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, performing ultrasound processing for 3 h at room temperature; then washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; the methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, the Fe 3 O 4  magnetic nanoparticles modified by 4.0 G dendrimer will be obtained after four times of cycles; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the Fe 3 O 4  magnetic nanoparticles modified by 4.0 G PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0031]    Extracting and separating flavonoid substances in  Camellia nitidissima  Chi: adding the obtained 10 g magnetic particles-4.0 G PAMAM composites in the 2.0 L  Camellia nitidissima  Chi extract prepared in the first step, ultrasonic extracting 0.5 h under 400 W, after extraction, separating the Fe 3 O 4 -PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the magnetic nanoparticles adsorbed with flavonoid substances several times with ethanol to extract the adsorbed flavonoid substances. Mixing the extract, removing ethanol by rotary evaporation and drying the anhydrous sodium sulfate for the night, then the flavonoid substances in  Camellia nitidissima  Chi will be obtained. Based on the detection method of total flavones in health food in  Inspection and Evaluation Technical Specification of Health Food  (2003), the content of flavonoid substances is more than 85% according to the separation and purification method of the present invention. The Fe 3 O 4  magnetic particles-4.0 G PAMAM magnetic nano composites can be recycled again after being washed by water and ethanol for several times, drying and activating. 
       Embodiment 2 
       [0032]    Preparation of flowers of  Camellia nitidissima  Chi concentrate: picking fresh flowers of  Camellia nitidissima  Chi in autumn: weighting it with electronic scale; performing screen for the picked fresh flowers of  Camellia nitidissima  Chi as per the standard of  Pharmacopoeia of China  (2010), smashing the optimized leaves of  Camellia nitidissima  Chi after washing; adding 20-30 L acetone (over 95%) into 10 kilograms of smashed leaves of  Camellia nitidissima  Chi and extracting for 5-6 h by Soxhlet extractor to obtain extract A; adding about 20 L acetone (over 95%) into extracted residue and performing ultrasonication for about 1.5 h under 40-60° C. to obtain extract B; mixing extract A and extract B and evaporating most acetone solvent by rotary evaporation of rotary evaporators, and finally obtaining about 1.0 L  Camellia nitidissima  Chi concentrate in organic phase. 
         [0033]    Preparation of γ-Fe 2 O 3  magnetic particles-5.0 G PAMAM composites: adding 2 mol/L sodium hydroxide solution in the 1 mol/L ferrous sulfate solution, then the white floc precipitation Fe(OH) 2  will be generated, and it becomes grayish green precipitation Fe 6 (SO 4 ) 2 (OH) 4 O 3  rapidly. Dropping right amount of hydrogen peroxide into above solution with precipitation with dropper until it becomes from green to black. The above solution with precipitation is separated and precipitated by vacuum filter, then it is put into drying oven under 80° C. for an hour. The dried solution is calcined at 100-240° C. in muffle furnace to obtain γ-Fe 2 O 3  red powder. The prepared magnetic nanoparticles are dispersed in the methanol to obtain the solution with 0.0128 mol/L concentration. Diluting the above prepared methanol solution of γ-Fe 2 O 3  magnetic nanoparticles 25 mL into 150 mL with methanol and performing ultrasonic processing for 30 min. Then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h. After washing the obtained solution for 5 times with methanol, the magnetic separation is performed, the solution is dispersed in methanol to obtain 5 wt % solution for standby. 50 mL 5 wt % of γ-Fe 2 O 3  magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, immersing the mixture into water bath at room temperature and performing ultrasonic processing for 7 h; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, performing ultrasonic processing for 3 h at room temperature, then washing the magnetic nanoparticles for 5 times by methanol and performing magnetic separation; the methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, the magnetic nanoparticles modified by 5.0 G dendrimer will be obtained after five times of cycles; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the γ-Fe 2 O 3  magnetic nanoparticles modified by 5.0 G PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0034]    Extracting and separating flavonoid substances in flowers of  Camellia nitidissima  Chi: adding the obtained γ-Fe 2 O 3  magnetic particles-5.0 G PAMAM composites in the 1.0 L  Camellia nitidissima  Chi flowers extract prepared in the first step, ultrasonic extracting 1 h under 400 W, after extraction, separating the γ-Fe 2 O 3 -5.0 G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the γ-Fe 2 O 3 -5.0 G PAMAM nano composites adsorbed with flavonoid substances several times with ethanol to extract the adsorbed flavonoid substances. Mixing the extract, removing methanol solvent by rotary evaporation and drying the anhydrous magnesium sulfate, then the flavonoid substances in  Camellia nitidissima  Chi will be obtained. Based on the detection method of total flavones in health food in Inspection and Evaluation Technical Specification of Health Food (2003), the content of flavonoid substances is more than 85% according to the separation and purification method for flavonoid substances in flowers of  Camellia nitidissima  Chi of the present invention. The γ-Fe 2 O 3  magnetic particles-5.0 G PAMAM composites can be recycled again after being washed several times, drying and activating. 
       Embodiment 3 
       [0035]    Preparation of fruits of  Camellia nitidissima  Chi concentrate: picking fresh fruits of  Camellia nitidissima  Chi in November and December; weighting it with electronic scale; performing screen for the picked fruits of  Camellia nitidissima  Chi as per the standard of  Pharmacopoeia of China  (2010), smashing the optimized fruits of  Camellia nitidissima  Chi after washing, and vacuum drying at 50° C. to remove the moisture; adding 45 L ethanol (over 95%) into vacuum dried 15 kilograms fruits of  Camellia nitidissima  Chi and extracting for 5-6 h by Soxhlet extractor to obtain extract A; adding about 30 L ethanol (over 95%) into extracted residue and performing ultrasonication for about 1.5 h under 40-60° C. to obtain extract B; mixing extract A and extract B and evaporating most ethanol solvent by rotary evaporation of rotary evaporators, and finally obtaining about 1.5 L  Camellia nitidissima  Chi concentrate in organic phase. 
         [0036]    Preparation of Co x Ni 1 -xFe 2 O 4  nano Ni—Co ferrite magnetic particles-3.0 G PAMAM composites: x means 0.1, 0.3, 0.5, 0.7, 0.9 etc. weighing the cobalt nitrate, nickel nitrate, ferric nitrate and citric acid by stoichiometric ratio, and utilizing distilled water to dissolve them into transparent color solution. The ammonia is dropped for the PH value of solution is between 7 and 8. The solution is heated in 90° C. water bath for 4-5 h to be sticky colloidal solution, and dried to drying gel at 120° C. The drying gel is heated in the muffle furnace from 120° C./h to 850° C./h, then after preserving the heat for 1 h, turning off the power supply for nature cooling. The prepared magnetic nanoparticles are ultrasonic dispersed in the methanol to obtain the solution with 0.0128 mol/L concentration. Diluting the above prepared methanol solution of Co x Ni 1 -xFe 2 O 4  magnetic nanoparticles 25 mL into 150 mL with methanol and performing ultrasonic processing for 30 min. Then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h. After washing the obtained solution for 5 times with methanol, the magnetic separation is performed, the solution is dispersed in methanol to obtain 5 wt % solution for standby. 50 mL 5 wt % Co x Ni 1-x Fe 2 O 4  magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, immersing the mixture into water bath at room temperature and performing ultrasonication for 7 h; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, performing ultrasonication for 3 h at room temperature; then washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; the methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, the magnetic nanoparticles modified by 3.0 G dendrimer will be obtained after three times of cycles; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the Co x Ni1-xFe2O4 magnetic nanoparticles modified by 3.0 G PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0037]    Extracting and separating flavonoid substances in fruits of  Camellia nitidissima  Chi: adding the obtained 7.5 g Co x Ni 1-x Fe 2 O 4 -3.0 G magnetic particles-PAMAM composites (faintly alkalinity) in the 1.5 L  Camellia nitidissima  Chi fruits extract prepared in the first step, ultrasonic extracting 1.5 h under 400 W, after extraction, separating the Co x Ni 1-x Fe 2 O 4 -3.0 G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the Co x Ni 1-x Fe 2 O 4 -3.0 G PAMAM nano composites adsorbed with flavonoid substances (faintly acidity) several times with ethanol to extract the adsorbed flavonoid substances. 
         [0038]    Mixing the extract, removing ethanol solvent by rotary evaporation and drying the room empty sulfate, then the flavonoid substances in  Camellia nitidissima  Chi will be obtained. Based on the detection method of total flavones in health food in  Inspection and Evaluation Technical Specification of Health Food  (2003), the content of flavonoid substances is more than 85% according to the separation and purification method for flavonoid substances in flowers of the present invention. The Co x Ni 1-x Fe 2 O 4 -3.0 G magnetic particles-3.0 G PAMAM composites can be recycled again after being washed several times, drying and activating. 
       Embodiment 4 
       [0039]    Preparation of leaves of  Camellia nitidissima  Chi concentrate: picking fresh leaves of  Camellia nitidissima  Chi in spring; weighting it with electronic scale; performing screen for the picked fresh leaves of  Camellia nitidissima  Chi as per the standard of  Pharmacopoeia of China  (2010), smashing the optimized leaves of  Camellia nitidissima  Chi after washing; adding 15 L methanol (over 95%) into 5 kilograms smashed leaves of  Camellia nitidissima  Chi and extracting for 5-6 h by Soxhlet extractor to obtain extract A; adding about 10 L methanol (over 95%) into extracted residue and performing ultrasonic processing for about 1.5 h under 40-60° C. to obtain extract B; mixing extract A and extract B and evaporating most methanol solvent by rotary evaporation of rotary evaporators, and finally obtaining about 0.5 L  Camellia  nitidissima Chi concentrate in organic phase. 
         [0040]    Preparation process of γ-Fe 4 N-6.0 G PAMAM composites: preparing iron powder 10 g with 0.1 micron of particle size, the ammonia and hydrogen. Diluting the above prepared methanol solution of γ-Fe 4 N magnetic nanoparticles 25 mL into 150 mL with methanol and performing ultrasonication for 30 min. Then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h. After washing the obtained solution for 5 times with methanol, the magnetic separation is performed, the solution is dispersed in methanol to obtain 5 wt % solution for standby. 50 mL 5 wt % of γ-Fe 4 N magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, immersing the mixture into water bath at room temperature and performing ultrasonication for 7 h; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, performing ultrasonication for 3 h at room temperature; then washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; the methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, the γ-Fe 4 N magnetic nanoparticles modified by 6.0 G dendrimer will be obtained after six times of cycles; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the γ-Fe 4 N magnetic nanoparticles modified by 6.0 G PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0041]    Extracting and separating flavonoid substances in  Camellia nitidissima  Chi: adding the obtained γ-Fe 4 N magnetic particles-PAMAM composites (faintly alkalinity) in the 0.5 L  Camellia nitidissima  Chi leaves extract prepared in the first step, ultrasonic extracting 2 h under 400 W, after extraction, separating the Fe 3 O 4 -6.0 G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the Fe 3 O 4 -6.0 G PAMAM nano composites adsorbed with flavonoid substances several times by organic solvent. Mixing the extract, removing solvent by rotary evaporation and drying at room temperature, then the flavonoid substance in  Camellia nitidissima  Chi will be obtained. Based on the detection method of total flavones in health food in  Inspection and Evaluation Technical Specification of Health Food  (2003), the content of flavonoid substance is more than 85% according to the separation and purification method for flavonoid substances. The Fe 3 O 4  magnetic particles-6.0 G PAMAM composites can be recycled again after being washed several times, drying and activating. 
       Embodiment 5 
       [0042]    Preparation of  Hedyotis diffusa  concentrate: picking fresh  Hedyotis diffusa  in autumn and removing root; weighting it with electronic scale; performing screen for the picked fresh  Hedyotis diffusa  as per the standard of  Pharmacopoeia of China  (2010), smashing the optimized  Hedyotis diffusa  after washing; adding over 95% acetone into 20 kilograms of smashed  Hedyotis diffusa  and extracting for 5-6 h by Soxhlet extractor to obtain extract A; adding over 95% acetone into extracted residue and performing ultrasonic processing for about 1.5 h under 40-60° C. to obtain extract B; mixing extract A and extract B and evaporating most acetone solvent by rotary evaporation of rotary evaporators, and finally obtaining about 2.0 L  Hedyotis diffusa  concentrate in organic phase. 
         [0043]    Preparation processes of Fe 3 O 4  magnetic particles-4.0 G PAMAM composites: preparing the mixture of 0.085 mol/L ferric chloride solution and 0.05 mol/L ferrous sulfate, then adding 1.5 mol/L ammonia solution into the mixture and stir vigorously until pH=9. Washing the obtained Fe 3 O 4  magnetic nanoparticles for 5 times immediately and washing it by methanol for 3 times, the Fe 3 O 4  magnetic nanoparticles is dispersed in methanol by magnetic separation, then the solution with 0.0128 mol/L concentration is obtained. Diluting the above prepared methanol solution of Fe 3 O 4  magnetic nanoparticles 25 mL into 150 mL with methanol and performing ultrasonic processing for 30 min. Then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h. After washing the obtained solution for 5 times with methanol, the magnetic separation is performed, the solution is dispersed in methanol to obtain 5 wt % solution for standby. 50 mL 5 wt % Fe 3 O 4  magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, immersing the mixture into water bath at room temperature and performing ultrasonication for 7 h; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, performing ultrasonication for 3 h at room temperature; then washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; the methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, the Fe 3 O 4  magnetic nanoparticles modified by 4.0 G dendrimer will be obtained after four times of cycles; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the Fe 3 O 4  magnetic nanoparticles modified by 4.0 G PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0044]    Extracting and separating flavonoid substances in  Hedyotis diffusa : adding the obtained Fe 3 O 4  magnetic particles-4.0 G PAMAM composites (faintly alkalinity) in the 2.0 L  Camellia nitidissima  Chi extract prepared in the first step, ultrasonic extracting 0.5 h under 400 W, after extraction, separating the Fe 3 O 4 -PAMAM nano composites adsorbed and extracted with flavonoid substances (faintly acidity) through magnetic separation, after the completion of Fe 3 O 4 -PAMAM separation, extracting the magnetic nanoparticles adsorbed with flavonoid substances several times with ethanol to extract the adsorbed flavonoid substances. Mixing the extract, removing ethanol by rotary evaporation and drying the anhydrous sodium sulfate for the night, then the flavonoid substances in  Hedyotis diffuse  will be obtained. Based on the detection method of total flavones in health food in  Inspection and Evaluation Technical Specification of Health Food  (2003), the content of flavonoid substance is more than 85% according to the separation and purification method of the present invention. The Fe 3 O 4  magnetic particles-4.0 G PAMAM magnetic nano composites can be recycled again after being washed by water and ethanol for several times, drying and activating. 
       Embodiment 6 
       [0045]    Preparation of leaves of  Rhus chinensis  concentrate: picking fresh leaves of  Rhus chinensis  weighting it with electronic scale; performing screen for the picked fresh leaves of  Rhus chinensis  as per the standard of  Pharmacopoeia of China  (2010), smashing the optimized leaves of  Rhus chinensis  after washing; adding 20-30 L ethanol (over 95%) into 10 kilograms smashed leaves of  Rhus chinensis  and extracting for 5-6 h by Soxhlet extractor to obtain extract A; adding about 20 L ethanol (over 95%) into extracted residue and performing ultrasonic processing for about 1.5 h under 40-60° C. to obtain extract B; mixing extract A and extract B and evaporating most ethanol solvent by rotary evaporation of rotary evaporators, and finally obtaining about 1.0 L leaves of  Rhus chinensis  concentrate in organic phase. 
         [0046]    Preparation of γ-Fe 2 O 3  magnetic particles-5.0 G PAMAM composites: adding 2 mol/L sodium hydroxide solution in the 1 mol/L ferrous sulfate solution, then the white floc precipitation Fe(OH) 2  will be generated, and it becomes grayish green precipitation Fe 6 (SO 4 ) 2 (OH) 4 O 3  rapidly. Dropping right amount of hydrogen peroxide into above solution with precipitation with dropper until it becomes from green to black. The above solution with precipitation is separated and precipitated by vacuum filter, and then it is put into drying oven under 80° C. for an hour. The dried solution is calcined at 100-240° C. in muffle furnace to obtain γ-Fe 2 O 3  red powder. The prepared magnetic nanoparticles are dispersed in the methanol to obtain the solution with 0.0128 mol/L concentration. Diluting the above prepared methanol solution of γ-Fe 2 O 3  magnetic nanoparticles 25MI into 150 mL with methanol and performing ultrasonic processing for 30 min. Then adding 10 mL 3-aminopropyltriethoxysilane, the solution is stirred strongly together with ultrasonication for 7 h. After washing the obtained solution for 5 times with methanol, the magnetic separation is performed, the solution is dispersed in methanol to obtain 5 wt % solution for standby. 50 mL 5 wt % of γ-Fe 2 O 3  magnetic nanoparticles methanol solution is used as the initial solution, adding 200 mL methanol solution with methyacrylate, immersing the mixture into water bath at room temperature and performing ultrasonication for 7 h; then collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; after washing, adding 40 mL methanol solution with ethylenediamine, performing ultrasonication for 3 h room temperature; then washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; the methanol solution with methyacrylate and methanol solution with ethylenediamine are added repeatedly, the magnetic nanoparticles modified by 5.0 G dendrimer will be obtained after five times of cycles; washing the solution after cycle with 25 mL methanol for 3 times and with 25 mL water for 5 times, the γ-Fe 2 O 3  magnetic nanoparticles modified by 5.0 G PAMAM dendrimer is collected and obtained by magnetic separation. 
         [0047]    Extracting and separating flavonoid substances in flowers of  Rhus chinensis : adding the obtained γ-Fe 2 O 3  magnetic particles-5.0 G PAMAM composites (faintly alkalinity) in the 1.0 L  Camellia nitidissima  Chi flowers extract prepared in the first step, ultrasonic extracting 1 h under 400 W, after extraction, separating the γ-Fe 2 O 3 -5.0 G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the γ-Fe 2 O 3 -5.0 G PAMAM nano composites adsorbed with flavonoid substances several times with ethanol to extract the adsorbed flavonoid substance. Mixing the extract, removing methanol solvent by rotary evaporation and drying the anhydrous magnesium sulfate, then the flavonoid substances in  Rhus chinensis  will be obtained. Based on the detection method of total flavones in health food in  Inspection and Evaluation Technical Specification of Health Food  (2003), the content of flavonoid substances is more than 85% according to the separation and purification method of flavonoid substances in flowers of  Camellia nitidissima  Chi of the present invention. The γ-Fe 2 O 3  magnetic particles-5.0 G PAMAM composites can be recycled again after being washed several times, drying and activating. 
         [0048]    The technology of the present invention also applies to other leaves, flowers, fruits and roots of plants enriched in flavonoid substances, such as tea,  chrysanthemum indicum, ginkgo biloba , mulberry leaf,  artemsia argyi , honeysuckle, grapefruit, orange, soybean, ginger, star anise, celery, pineapple, guava, corn stigma,  patrinia scaniosaefolia , clover,  houttuynia cordata  thunb, licorice,  Rhus chinensis, Phyllanthus ruinaria , Cactus,  Selaginella tamariscina, Hedyotis diffuse , Burdock,  Mahonia bealei, Pteris semipinnata, Apocynum venetum, Lespedeza dunnii, Cynomorium songaricum rupr, Sarcandra glabra  etc., the extraction and separation method for flavonoid substances in above plants is similar with the magnetic particles-PAMAM nano magnetic separation technology. 
         [0049]    It is obvious that the present invention is not limited to above embodiments for those skilled in the art, and the present invention can be realized by other embodiments without departing from the spirit or basic feature of the present invention. So the embodiments are demonstrative and non-restrictive, it is intended that the invention be limited only in terms of the appended claims, rather than above embodiments. So any variations that equivalent to the content and scope of claims shall fall into the protection scope of the present invention. 
         [0050]    Besides, while the specification has described embodiments, it should be understood that not each embodiment merely include one independent technical proposal, it is for clearly description, the skilled in the art shall regard the specification as a whole, the technical proposals of each embodiments can be combined to form other embodiments.