阅读说明：本技术 一种广东紫珠多糖的提取方法 (Extraction method of callicarpa kwangtungensis chun polysaccharide ) 是由 王振辉 牛超 张真真 杨兰萍 孔继川 周德军 翟洋洋 于 2021-01-20 设计创作，主要内容包括：本发明涉及植物提取分析技术领域,具体来说是一种广东紫珠多糖的提取方法,先将广东紫珠采用微波醇提方法进行有效成分提取,再进行大分子的分离,继续采用高效逆流色谱分离,最后采用柱层析洗脱分离技术得到广东紫珠多糖。本发明先进行有效成分的提取,得到浸膏,再将浸膏内的鞣质、蛋白质、树脂、蜡纸等胶体不稳定成分去除,又经由高效逆流色谱处理后将不溶于水的有机物去除,保留的水溶性物质中含有水溶性多糖、水溶性氨基酸和多肽,最后采用柱层析洗脱分离技术将广东紫珠多糖进行有效分离,得到纯度高的广东紫珠多糖。(The invention relates to the technical field of plant extraction and analysis, in particular to a method for extracting Callicarpa kwangtungensis Chun polysaccharide. The invention firstly extracts effective components to obtain an extract, removes colloid unstable components such as tannin, protein, resin, wax paper and the like in the extract, removes water-insoluble organic matters after high-efficiency countercurrent chromatography treatment, effectively separates the callicarpa kwangtungensis chun polysaccharide by adopting column chromatography elution separation technology to obtain the callicarpa kwangtungensis chun polysaccharide with high purity, wherein the reserved water-soluble substances contain water-soluble polysaccharide, water-soluble amino acid and polypeptide.)

1. The extraction method of callicarpa kwangtungensis chun polysaccharide is characterized by comprising the following steps:

(1) extracting effective components of Callicarpa kwangtungensis Chun by microwave alcohol extraction method to obtain extract:

(2) separation of macromolecules:

the component A of the natural clarifying agent is as follows: dissolving a natural clarifying agent in distilled water to prepare a 1 wt% solution A;

and the natural clarifying agent B comprises the following components: dissolving a natural clarifying agent in 1 wt% of glacial acetic acid solution to prepare 1 wt% of solution B;

dissolving the extract obtained in the step (1) in water to prepare 1-2 wt% solution, and mixing the solution at 90-110 ℃ according to a volume ratio of 100: 2-3, dropwise adding the solution B, mixing for 1-2h, and then mixing according to the volume ratio of 100: 6-10, dripping the solution A, standing for 2-3h, taking out, centrifuging, and taking supernatant to obtain a primary purified product;

(3) high-efficiency countercurrent chromatographic separation:

water is taken according to the following volume ratio: carrying out efficient countercurrent chromatographic separation on the primary purified product obtained in the step (2) to obtain a secondary purified product, wherein the ethyl acetate is 8-10:2-3, and the secondary purified product is obtained by fully and uniformly mixing and then layering the mixture to obtain an upper phase and a lower phase, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase;

(4) and (3) column chromatography elution separation:

and (3) completely dissolving the secondary purified product in the step (3) in deionized water, passing through a DEDA cellulose DE-52 ion exchange column, sequentially carrying out gradient elution by using 0.1-1.0mol/L ammonium sulfate solution, collecting eluent, and carrying out reduced pressure concentration to obtain the callicarpa kwangtungensis chun polysaccharide.

2. The extraction method of callicarpa kwangtungensis chun polysaccharide as claimed in claim 1, wherein the microwave alcohol extraction method in step (1) comprises: cleaning and drying Callicarpa kwangtungensis Chun, pulverizing, adding into 50-70 wt% ethanol solution 4-6 times of the volume, refluxing for 2-3h, further heating with microwave under power of 150-.

3. The extraction method of Callicarpa kwangtungensis polysaccharide as claimed in claim 1, wherein the natural clarifier of step (2) is type II ZTC1+1 natural clarifier or clarifier KBT-ZTC.

4. The extraction method of callicarpa kwangtungensis chun polysaccharide as claimed in claim 1, wherein during the separation by high performance counter current chromatography in step (3), the flow rate when pumping the stationary phase is 40-60mL/min, and the flow rate when pumping the mobile phase is 5-8 mL/min.

5. The method as claimed in claim 1, wherein the rotation speed of the main machine is 2500-3500rpm during the separation process of the high performance countercurrent chromatography in step (3).

Technical Field

The invention relates to the technical field of plant extraction and analysis, in particular to an extraction method of callicarpa kwangtungensis chun polysaccharide.

Background

Callicarpa kwangtungensis is Larix Gmelini of Callicarpa of Verbenaceae, and is about 2m high; oval to oval shape, tapered to short tip at the top, wedge-shaped base, fine sawtooth at the edge, gray-brown back, dense dark red or red fine-grained glandular spots on both sides, distributed in Henan, Jiangsu, Anhui, Zhejiang, Jiangxi, Hunan, China,North of a lakeGuangdong, Guangxi, Sichuan, Guizhou, Yunnan and the like and Vietnam; it is used in forest, forest margin and shrub at elevation of 200-.

The prior art mainly focuses on the discovered micromolecular chemical components in the basic research of pharmacodynamic substances of the Callicarpa kwangtungensis Chun medicinal material, and along with the deepening of the research, the research on macromolecules in the Callicarpa kwangtungensis Chun is gradually valued, wherein the research comprises the extraction of water-soluble polysaccharide of Callicarpa kwangtungensis Chun, and the polysaccharide extracted by the method in the prior art has more impurities and complex components, so that the efficient application of the Callicarpa kwangtungensis Chun polysaccharide is difficult to realize.

Disclosure of Invention

In order to overcome the technical defects, the invention aims to provide an extraction method of callicarpa kwangtungensis chun polysaccharide.

In order to solve the technical problems, the invention adopts the following technical scheme:

the extraction method of callicarpa kwangtungensis chun polysaccharide comprises the following steps:

(1) extracting effective components of the Guangdong purple beautyberry by adopting a microwave alcohol extraction method to obtain an extract:

(2) separation of macromolecules:

the component A of the natural clarifying agent is as follows: dissolving a natural clarifying agent in distilled water to prepare a 1 wt% solution A;

and the natural clarifying agent B comprises the following components: dissolving a natural clarifying agent in 1 wt% of glacial acetic acid solution to prepare 1 wt% of solution B;

dissolving the extract obtained in the step (1) in water to prepare 1-2 wt% solution, and mixing the solution at 90-110 ℃ according to a volume ratio of 100: 2-3, dropwise adding the solution B, mixing for 1-2h, and then mixing according to the volume ratio of 100: 6-10, dripping the solution A, standing for 2-3h, taking out, centrifuging, and taking supernatant to obtain a primary purified product;

(3) high-efficiency countercurrent chromatographic separation:

water is taken according to the following volume ratio: carrying out efficient countercurrent chromatographic separation on the primary purified product obtained in the step (2) to obtain a secondary purified product, wherein the ethyl acetate is 8-10:2-3, and the secondary purified product is obtained by fully and uniformly mixing and then layering the mixture to obtain an upper phase and a lower phase, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase;

(4) and (3) column chromatography elution separation:

and (3) completely dissolving the secondary purified product in the step (3) in deionized water, passing through a DEDA cellulose DE-52 ion exchange column, sequentially carrying out gradient elution by using 0.1-1.0mol/L ammonium sulfate solution, collecting eluent, and carrying out reduced pressure concentration to obtain the callicarpa kwangtungensis chun polysaccharide.

Preferably, the microwave alcohol extraction method in the step (1) comprises the following steps: cleaning and drying Callicarpa kwangtungensis Chun, pulverizing, adding into 50-70 wt% ethanol solution 4-6 times of the volume, refluxing for 2-3h, further heating with microwave under power of 150-.

Preferably, the natural clarifying agent in the step (2) is a type II ZTC1+1 natural clarifying agent or a clarifying agent KBT-ZTC.

Preferably, in the separation process of the high performance counter current chromatography in the step (3), the flow rate when the fixed phase is pumped is 40-60mL/min, and the flow rate when the mobile phase is pumped is 5-8 mL/min.

Preferably, in the separation process of the high performance counter current chromatography in the step (3), the rotation speed of the host is 2500-.

Compared with the prior art, the invention has the beneficial effects that:

1. the organic components in the callicarpa kwangtungensis are extracted by adopting a microwave alcohol extraction mode, and the organic components in the callicarpa kwangtungensis are formed into an extract after reduced pressure distillation; extracting by using a natural clarifier, wherein the natural clarifier mainly removes colloid unstable components such as tannin, protein, resin, wax paper and the like, does not influence effective components such as flavone, alkaloid, glycosides, saponins, terpenes, polysaccharide, amino acid, polypeptide, vitamin, mineral substances and the like, and after treatment by using a natural clarifier A solution and a natural clarifier B solution, the removal rate of the colloid unstable components reaches more than 90 percent;

separating the rest effective components such as flavone, alkaloid, glycosides, saponins, terpenes, polysaccharide, amino acid, polypeptide, vitamins, minerals, etc. by high performance countercurrent chromatography, effectively separating water soluble compounds from organic compounds in the Callicarpa kwangtungensis Chun extract, further purifying the Callicarpa kwangtungensis Chun extract, wherein the purified water soluble polysaccharide also contains water soluble amino acid and polypeptide, separating the Callicarpa kwangtungensis Chun polysaccharide by column chromatography elution separation technology, wherein the Callicarpa kwangtungensis Chun polysaccharide comprises mannose, rhamnose, glucose, galactose, arabinose, etc., separating by column chromatography elution separation, salting out, separating with amino acid and polypeptide to obtain high purity Callicarpa kwangtungensis Chun polysaccharide.

2. Compared with the prior art, the extraction method comprises the steps of extracting effective components to obtain an extract, removing colloid unstable components such as tannin, protein, resin, wax paper and the like in the extract, removing water-insoluble organic matters after high-efficiency countercurrent chromatography treatment, keeping water-soluble substances containing water-soluble polysaccharide, water-soluble amino acid and polypeptide, and finally effectively separating the callicarpa kwangtungensis chun polysaccharide by adopting a column chromatography elution separation technology to obtain the callicarpa kwangtungensis chun polysaccharide with high purity.

Drawings

FIG. 1 is a graph of a standard curve obtained using glucose as a standard.

Detailed Description

The following description is provided for the best mode of carrying out the invention.

Example 1

The extraction method of callicarpa kwangtungensis chun polysaccharide comprises the following steps:

(1) extracting effective components of Callicarpa kwangtungensis Chun by microwave alcohol extraction method, cleaning, drying and pulverizing Callicarpa kwangtungensis Chun, adding into 70 wt% ethanol solution 4 times the volume of the Callicarpa kwangtungensis Chun, refluxing for 2h, continuously heating with microwave under the condition of power of 150W for 15min, cooling to room temperature, and concentrating under reduced pressure to obtain extract:

(2) separation of macromolecules:

the component A of the natural clarifying agent is as follows: dissolving a II type ZTC1+1 natural clarifying agent in distilled water to prepare a 1 wt% solution A;

and the natural clarifying agent B comprises the following components: dissolving a II type ZTC1+1 natural clarifying agent in 1 wt% glacial acetic acid solution to prepare 1 wt% solution B;

dissolving the extract obtained in the step (1) in water to prepare a 1 wt% solution, and mixing the solution at 90 ℃ according to a volume ratio of 100: 3, dropwise adding the solution B, mixing for 1h, and then mixing according to the volume ratio of 100: 10, dropwise adding the solution A, standing for 2 hours, taking out, centrifuging, and taking supernatant to obtain a primary purified substance;

(3) high-efficiency countercurrent chromatographic separation:

water is taken according to the following volume ratio: and (3) fully and uniformly mixing ethyl acetate 8:3, layering to obtain an upper phase and a lower phase, taking the upper phase as a stationary phase and the lower phase as a mobile phase, and performing high-efficiency countercurrent chromatographic separation on the primary purified product obtained in the step (2) to obtain a secondary purified product; the flow rate when the fixed phase is pumped is 40/min, the flow rate when the mobile phase is pumped is 8mL/min, and the rotating speed of the main machine is 2500 rpm;

(4) and (3) column chromatography elution separation:

and (3) completely dissolving the secondary purified product in the step (3) in deionized water, passing through a DEDA cellulose DE-52 ion exchange column, sequentially carrying out gradient elution by using 0.1-1.0mol/L ammonium sulfate solution, collecting eluent, and carrying out reduced pressure concentration to obtain the callicarpa kwangtungensis chun polysaccharide.

Example 2

The extraction method of callicarpa kwangtungensis chun polysaccharide comprises the following steps:

(1) extracting effective components of Callicarpa kwangtungensis Chun by microwave alcohol extraction method, cleaning, drying and pulverizing Callicarpa kwangtungensis Chun, adding into 5 times volume of 60 wt% ethanol solution, refluxing for 2.5h, further heating for 10min under 200W microwave, cooling to room temperature, and concentrating under reduced pressure to obtain extract:

(2) separation of macromolecules:

the component A of the natural clarifying agent is as follows: dissolving a clarifying agent KBT-ZTC in distilled water to prepare a 1 wt% solution A;

and the natural clarifying agent B comprises the following components: dissolving a clarifying agent KBT-ZTC in 1 wt% glacial acetic acid solution to prepare 1 wt% solution B;

dissolving the extract obtained in the step (1) in water to prepare 1.5 wt% solution, and mixing the solution at 100 ℃ according to a volume ratio of 100: 2.5 dropwise adding the solution B, mixing for 1.5h, and then mixing according to a volume ratio of 100: 8, dropwise adding the solution A, standing for 2.5h, taking out, centrifuging, and taking supernatant to obtain a primary purified product;

(3) high-efficiency countercurrent chromatographic separation:

water is taken according to the following volume ratio: carrying out efficient countercurrent chromatographic separation on the primary purified product obtained in the step (2) to obtain a secondary purified product, wherein the ethyl acetate is 9:2.5, and the secondary purified product is obtained by fully and uniformly mixing and then layering the mixture to obtain an upper phase and a lower phase, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase; the flow rate when the fixed phase is pumped is 50mL/min, the flow rate when the mobile phase is pumped is 6mL/min, and the rotating speed of the main machine is 3000 rpm;

(4) and (3) column chromatography elution separation:

and (3) completely dissolving the secondary purified product in the step (3) in deionized water, passing through a DEDA cellulose DE-52 ion exchange column, sequentially carrying out gradient elution by using 0.1-1.0mol/L ammonium sulfate solution, collecting eluent, and carrying out reduced pressure concentration to obtain the callicarpa kwangtungensis chun polysaccharide.

Example 3

The extraction method of callicarpa kwangtungensis chun polysaccharide comprises the following steps:

(1) extracting effective components of Callicarpa kwangtungensis Chun by microwave alcohol extraction method, cleaning, drying and pulverizing Callicarpa kwangtungensis Chun, adding into 50 wt% ethanol solution 6 times the volume of the Callicarpa kwangtungensis Chun, refluxing for 3h, continuously heating with microwave under the condition of 250W power for 5min, cooling to room temperature, and concentrating under reduced pressure to obtain extract:

(2) separation of macromolecules:

the component A of the natural clarifying agent is as follows: dissolving a II type ZTC1+1 natural clarifying agent in distilled water to prepare a 1 wt% solution A;

and the natural clarifying agent B comprises the following components: dissolving a II type ZTC1+1 natural clarifying agent in 1 wt% glacial acetic acid solution to prepare 1 wt% solution B;

dissolving the extract obtained in the step (1) in water to prepare a 2 wt% solution, and mixing the solution at 110 ℃ according to a volume ratio of 100: 2, dropwise adding the solution B, mixing for 2 hours, and then mixing according to the volume ratio of 100: 6, dropwise adding the solution A, standing for 3 hours, taking out, centrifuging, and taking supernatant to obtain a primary purified substance;

(3) high-efficiency countercurrent chromatographic separation:

water is taken according to the following volume ratio: carrying out efficient countercurrent chromatographic separation on the primary purified product obtained in the step (2) to obtain a secondary purified product, wherein the ethyl acetate is 5:1, and the secondary purified product is obtained by fully and uniformly mixing and then layering the mixture to obtain an upper phase and a lower phase, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase; the flow rate when the stationary phase is pumped is 60mL/min, the flow rate when the mobile phase is pumped is 5mL/min, and the rotating speed of the main machine is 3500 rpm;

(4) and (3) column chromatography elution separation:

and (3) completely dissolving the secondary purified product in the step (3) in deionized water, passing through a DEDA cellulose DE-52 ion exchange column, sequentially carrying out gradient elution by using 0.1-1.0mol/L ammonium sulfate solution, collecting eluent, and carrying out reduced pressure concentration to obtain the callicarpa kwangtungensis chun polysaccharide.

Results and analysis

Measuring standard curve charts of the concentrations of 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL and 0.05mg/mL respectively by using glucose as a standard substance and adopting a High Performance Liquid Chromatograph (HPLC);

preparing 0.03mg/mL Callicarpa kwangtungensis Chun polysaccharide of example 2, injecting 1 μ L into High Performance Liquid Chromatograph (HPLC), and obtaining absorbance, wherein a standard curve chart obtained by using glucose as a standard substance is shown in FIG. 1;

and respectively taking mannose, rhamnose, galactose and arabinose as standard substances, respectively obtaining different standard curve graphs by adopting the same method, and calculating to obtain the content ratio of glucose, mannose, rhamnose, galactose and arabinose in Callicarpa kwangtungensis polysaccharide of 23.66 according to the absorbance value in the standard curve graphs: 0.981: 20.14: 4.52: 1.47.

the formula for calculating the polysaccharide extraction rate is as follows: the measured absorbance a was substituted into the regression equation to calculate the extraction rate and polysaccharide yield of callicarpa kwangtungensis chun, and the extraction rate and polysaccharide yield of callicarpa kwangtungensis chun in examples 1 to 3 are shown in table 1:

polysaccharide extraction ratio (%). ratio of sample concentration x volume of sample after constant volume/mass of raw material

Polysaccharide yield (%) [ polysaccharide dilution concentration (mg/mL) × dilution factor × 250mL]/[ dry weight (g) of Guangdong purple pearl powder X10³]

TABLE 1 Callicarpa kwangtungensis polysaccharide extraction and polysaccharide yield study

The result shows that the polysaccharide yield of the invention reaches more than 40 percent, and compared with the polysaccharide yield of the prior art, the polysaccharide yield of the invention is obviously improved.

It will be apparent to those skilled in the art that various changes and modifications may be made in the invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.