Method for separating flavonoid substances in Camellia nitidissima Chi based on a magnetic nanoparticles-PAMAM nano composites

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.

Claims

1. A method for separating flavonoid substances in Camellia nitidissima Chi based on magnetic nanoparticle-PAMAM nano composites, comprising: preparing PAMAM dendrimer: performing Michael addition reaction between ethylenediamine and methyacrylate to obtain generation 0.5 polyamidoamine dendrimer, namely PAMAM G0.5; wherein the Michael addition reaction is performed under 25 C.; reacting the obtained PAMAM G0.5 with excessive ethylenediamine under 25 C. to obtain generation 1.0 polyamidoamine dendrimer, namely PAMAM G1.0; repeating the above two steps to obtain PAMAM with different generation; preparing magnetic nanoparticle-PAMAM nano composites dispersing magnetic nanoparticles into methanol through magnetic separation to obtain methanol solution with 0.0128 mol/L concentration; diluting 25 mL methanol solution containing magnetic nanoparticles therein to 150 mL by methanol and performing ultrasonication for 30 min; adding 10 mL 3-aminopropyltriethoxysilane, stirring and performing ultrasonication for 7 hours; performing magnetic separation on the obtained solution after washing it by methanol for 5 times; dispersing the washed solution in methanol to obtain 5 wt % magnetic nanoparticle methanol solution; adding 200 mL methanol solution with methyacrylate into 50 mL 5 wt % magnetic nanoparticle methanol solution to obtain a mixed solution, wherein a volume concentration of the methyacrylate is 20%; performing ultrasonication to the mixed solution for 7 hrs in water bath at room temperature; collecting magnetic nanoparticles by magnet and washing it for 5 times by methanol and performing magnetic separation; adding 40 mL methanol solution with ethylenediamine, the volume concentration of the ethylenediamine is 20%, performing ultrasonication for 3 hrs at room temperature; washing the magnetic nanoparticles for 5 times with methanol and performing magnetic separation; repeatedly adding the methanol solution with methyacrylate and methanol solution with ethylenediamine; obtaining higher generations of magnetic nanoparticles modified by dendrimer for each cycle; washing the solution after cycle with 25 mL methanol for 3 times and washing with 25 mL water for 5 times; and obtaining the magnetic nanoparticles modified by PAMAM dendrimer by magnetic separation; and extracting and performing magnetic separation on the flavonoid substances in Camellia nitidissima Chi: adding the obtained magnetic nanoparticle-PAMAM nano composites in a Camellia nitidissima Chi extract; extracting for 0.5-3 hrs under ultrasound or microwave condition; separating the nanoparticle-PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation after the extraction, the flavonoid substances adsorbed by magnetic nanoparticles are extracted and separated by using an organic solvent from separated nanoparticle-PAMAM nano composites.

2. The method of claim 1, wherein the magnetic nanoparticles are Fe.sub.3O.sub.4.

3. The method of claim 1, wherein the organic solvent is methanol.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a classical synthetic route of PAMAM dendrimer.

(2) FIG. 2 shows a schematic diagram of Fe.sub.3O.sub.4 magnetic particle-PAMAM composites synthesis and flavonoid substances separation in Camellia nitidissima Chi and other plants.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(3) 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.

(4) 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.sub.3O.sub.4, -Fe.sub.2O.sub.3 and MeFe.sub.2O.sub.4, wherein Me=Co, Ni, Mn), Fe, Co, Ni, alloy particles and iron nitrides (FeN, Fe.sub.2N, -Fe.sub.3N and Fe.sub.16N.sub.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.sub.2 functional group, and the middle part is with quantities of R.sub.2NH, R.sub.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

(5) 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.

(6) Preparation process of Fe.sub.3O.sub.4 magnetic particles-4.0G 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.sub.3O.sub.4 magnetic nanoparticles for 5 times immediately and washing it with methanol for 3 times, the Fe.sub.3O.sub.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.sub.3O.sub.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.sub.3O.sub.4 magnetic nanoparticles modified by 4.0G 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.sub.3O.sub.4 magnetic nanoparticles modified by 4.0G PAMAM dendrimer is collected and obtained by magnetic separation.

(7) Extracting and separating flavonoid substances in Camellia nitidissima Chi: adding the obtained 10 g magnetic particles-4.0G 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.sub.3O.sub.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.sub.3O.sub.4 magnetic particles-4.0G PAMAM magnetic nano composites can be recycled again after being washed by water and ethanol for several times, drying and activating.

Embodiment 2

(8) 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.

(9) Preparation of -Fe.sub.2O.sub.3 magnetic particles-5.0G PAMAM composites: adding 2 mol/L sodium hydroxide solution in the 1 mol/L ferrous sulfate solution, then the white floc precipitation Fe(OH).sub.2 will be generated, and it becomes grayish green precipitation Fe.sub.6(SO.sub.4).sub.2(OH).sub.4O.sub.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 -Fe.sub.2O.sub.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 -Fe.sub.2O.sub.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 -Fe.sub.2O.sub.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 5.0G 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 -Fe.sub.2O.sub.3 magnetic nanoparticles modified by 5.0G PAMAM dendrimer is collected and obtained by magnetic separation.

(10) Extracting and separating flavonoid substances in flowers of Camellia nitidissima Chi: adding the obtained -Fe.sub.2O.sub.3 magnetic particles-5.0G 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 -Fe.sub.2O.sub.3-5.0G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the -Fe.sub.2O.sub.3-5.0G 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 -Fe.sub.2O.sub.3 magnetic particles-5.0G PAMAM composites can be recycled again after being washed several times, drying and activating.

Embodiment 3

(11) 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.

(12) Preparation of Co.sub.xNi.sub.1-xFe.sub.2O.sub.4 nano NiCo ferrite magnetic particles-3.0G 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.sub.xNi.sub.1-xFe.sub.2O.sub.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.sub.xNi.sub.1-xFe.sub.2O.sub.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 3.0G 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.sub.xNi1-xFe2O4 magnetic nanoparticles modified by 3.0G PAMAM dendrimer is collected and obtained by magnetic separation.

(13) Extracting and separating flavonoid substances in fruits of Camellia nitidissima Chi: adding the obtained 7.5 g Co.sub.xNi.sub.1-xFe.sub.2O.sub.4-3.0G 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.sub.xNi.sub.1-xFe.sub.2O.sub.4-3.0G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the Co.sub.xNi.sub.1-xFe.sub.2O.sub.4-3.0G PAMAM nano composites adsorbed with flavonoid substances (faintly acidity) several times with ethanol to extract the adsorbed flavonoid substances. 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.sub.xNi.sub.1-xFe.sub.2O.sub.4-3.0G magnetic particles-3.0G PAMAM composites can be recycled again after being washed several times, drying and activating.

Embodiment 4

(14) 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.

(15) Preparation process of -Fe.sub.4N-6.0G 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 -Fe.sub.4N 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 -Fe.sub.4N 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.sub.4N magnetic nanoparticles modified by 6.0G 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 -Fe.sub.4N magnetic nanoparticles modified by 6.0G PAMAM dendrimer is collected and obtained by magnetic separation.

(16) Extracting and separating flavonoid substances in Camellia nitidissima Chi: adding the obtained -Fe.sub.4N 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.sub.3O.sub.4-6.0G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the Fe.sub.3O.sub.4-6.0G 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.sub.3O.sub.4 magnetic particles-6.0G PAMAM composites can be recycled again after being washed several times, drying and activating.

Embodiment 5

(17) 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.

(18) Preparation processes of Fe.sub.3O.sub.4 magnetic particles-4.0G 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.sub.3O.sub.4 magnetic nanoparticles for 5 times immediately and washing it by methanol for 3 times, the Fe.sub.3O.sub.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.sub.3O.sub.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.sub.3O.sub.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.sub.3O.sub.4 magnetic nanoparticles modified by 4.0G 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.sub.3O.sub.4 magnetic nanoparticles modified by 4.0G PAMAM dendrimer is collected and obtained by magnetic separation.

(19) Extracting and separating flavonoid substances in Hedyotis diffusa: adding the obtained Fe.sub.3O.sub.4 magnetic particles-4.0G 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.sub.3O.sub.4-PAMAM nano composites adsorbed and extracted with flavonoid substances (faintly acidity) through magnetic separation, after the completion of Fe.sub.3O.sub.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.sub.3O.sub.4 magnetic particles-4.0G PAMAM magnetic nano composites can be recycled again after being washed by water and ethanol for several times, drying and activating.

Embodiment 6

(20) 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.

(21) Preparation of -Fe.sub.2O.sub.3 magnetic particles-5.0G PAMAM composites: adding 2 mol/L sodium hydroxide solution in the 1 mol/L ferrous sulfate solution, then the white floc precipitation Fe(OH).sub.2 will be generated, and it becomes grayish green precipitation Fe.sub.6(SO.sub.4).sub.2(OH).sub.4O.sub.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 -Fe.sub.2O.sub.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 -Fe.sub.2O.sub.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 -Fe.sub.2O.sub.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 5.0G 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 -Fe.sub.2O.sub.3 magnetic nanoparticles modified by 5.0G PAMAM dendrimer is collected and obtained by magnetic separation.

(22) Extracting and separating flavonoid substances in flowers of Rhus chinensis: adding the obtained -Fe.sub.2O.sub.3 magnetic particles-5.0G 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 -Fe.sub.2O.sub.3-5.0G PAMAM nano composites adsorbed and extracted with flavonoid substances through magnetic separation, after separation, extracting the -Fe.sub.2O.sub.3-5.0G 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 -Fe.sub.2O.sub.3 magnetic particles-5.0G PAMAM composites can be recycled again after being washed several times, drying and activating.

(23) 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.

(24) 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.

(25) 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.