Method for preparing pure ABS (Acrylonitrile Butadiene Styrene) of white cortex of citrus fruit

Abstract

The invention discloses a method for preparing pure ABS (Acrylonitrile Butadiene Styrene) of a citrus fruit white cortex layer, which comprises the following steps: extracting fruit'blue stain 'white cortex layer dry powder by using a hydrochloric acid ethanol solution, and adjusting the pH value of an extracting solution to 7.0; the method comprises the following steps: sequentially introducing an HLB column, an MCX column and a Sephadex LH-20 column, then purifying by using a semi-preparative liquid chromatograph, collecting ABS absorption peak effluent at 570nm, and drying to obtain an ABS pure product. The ABS pure product in the citrus white cortex layer is separated by a series of separation and purification technologies, and the purity is greater than or equal to 99%.

Description

Technical Field

[0001] The invention relates to a method for separating and purifying organic matter from citrus fruits, in particular to a method for preparing pure ABS products from the white peel of citrus fruits. Background Art

[0002] Since the 21st century, China's citrus industry has developed rapidly. With the rapid development of China's citrus industry, the white skin of fruits has been found to be "blue". "Blue change" is also called "browning change", "blue-brown change", "mildew" and "internal mold". Initially, the white skin tissue near the orange peel of the fruit changes to light brown-blue. When the disease is mild, it is limited to the white skin near the orange peel. When the disease is more serious, the brown-blue color extends to the entire white skin. The normal light yellow or light yellow white skin turns light brown-blue or dark brown-blue, and turns to black-blue in severe cases. "Blue change" not only affects the appearance of the fruit, but also causes the flavor of the fruit to deteriorate or even produce obvious odors when the disease is serious, which seriously affects the commerciality of the fruit and causes consumers to have strong concerns about its food safety. It has become a major concern for the citrus industry.

[0003] The problem of "blue discoloration" of the white cortex of citrus fruits was first reported in the United States in 1944, and was observed in oranges, tangerines, and grapefruits in Arizona, Florida, California, and Texas. In China, the "blue discoloration" problem has occurred in varieties such as Gonggan, Wogan, Mashuiju, Maogugan, Qianjinju, and Shatangju. Researchers believe that the "blue discoloration" of fruits is not a pathological change caused by pathogens, but a physiological lesion caused by abiotic stress. The "blue discoloration" substance (abedo bluing substance, ABS) was initially thought to be a natural water-soluble dye similar to anthocyanins, but no related structural identification reports have been reported. In recent years, researchers have found that there is no characteristic absorption peak of anthocyanins in the extract of the "blue discoloration" white cortex, and no related substances have been found by LC-MS detection.

[0004] Since ABS is difficult to separate and purify, its chemical structure is still unclear, which makes it difficult to clarify the factors and mechanisms that cause the "blue change" and prevent and control it in production. Summary of the invention

[0005] In view of this, the present invention aims at the problem that citrus ABS is difficult to separate and purify, and provides a method for preparing pure ABS from the white peel of citrus fruit.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a method for preparing a pure ABS product of the white peel layer of citrus fruit, comprising the following steps:

[0008] S1: preparing a dry powder of the "blue-turned" white cortex of the fruit, extracting the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adjusting the pH of the extract to 6.5-7.5, standing and filtering, and obtaining an ABS crude extract;

[0009] S2: dissolving the ABS crude extract with formic acid, injecting into an HLB column, eluting impurities with water and 30-40% methanol in sequence, and eluting ABS with 0.04-0.2% hydrochloric acid methanol to obtain sample solution 1;

[0010] S3: injecting the sample solution 1 into the MCX column, eluting impurities with 1% hydrochloric acid and methanol, and eluting ABS with 1-10% ammonia methanol to obtain sample solution 2;

[0011] S4: subjecting the sample solution 2 to Sephadex LH-20 column chromatography, using 25-35% methanol as the eluent, collecting the blue band fractions, and concentrating;

[0012] S5: The obtained concentrated solution is purified by semi-preparative liquid chromatography, 2-4 mL of ABS absorption peak effluent is manually collected at 570 nm, and dried to obtain pure ABS.

[0013] As an embodiment, the preparation method of the dry powder of the "blue-turned" white skin layer of the fruit is to freeze the fresh sample of the "blue-turned" white skin layer with liquid nitrogen and then grind it into powder with a pulverizer.

[0014] Preferably, the volume concentration of the hydrochloric acid ethanol solution is 0.5-2%, and the solid-liquid ratio of the extraction is 1:5-25.

[0015] Preferably, the extraction is performed twice.

[0016] As an embodiment, the pH of the extract is adjusted with 5% by volume NaHCO3 or ammonia water.

[0017] As an embodiment, the ABS crude extract is dissolved and then filtered through a 0.22 μm filter membrane.

[0018] As an embodiment, in step S2, the amount of water, 30-40% methanol, and 0.04-0.2% hydrochloric acid methanol for elution is 6 ml respectively.

[0019] As an embodiment, in step S3, the elution amount of the 1% hydrochloric acid and methanol is 6 ml respectively, and the elution amount of the 1-10% ammonia methanol is 12 mL.

[0020] As an embodiment, in step S4, the flow rate is 0.4-0.6 mL / min, and one fraction is collected every 10 minutes.

[0021] As an embodiment, the chromatographic conditions of the semi-preparative liquid chromatograph are: C18 column, mobile phase A is acetonitrile, phase B is water, and the gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm.

[0022] Beneficial effects of the present invention:

[0023] The invention discloses a method for preparing pure ABS from the white peel of citrus fruit, wherein the pure ABS from the white peel of citrus fruit is separated by a series of separation and purification technologies, and the purity is ≥99%. The separation and purification method of the invention can be applied to the separation of ABS from Wogan and Mashuiju, and a fast and efficient separation and purification method for ABS from the white peel of citrus fruit is established, laying a foundation for subsequent structural research on ABS. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Visible light spectra of the white cortex in acidic and alkaline extracts.

[0025] Figure 2 : Effects of extraction time (left) and solid-liquid ratio (right) on ABS extraction efficiency.

[0026] Figure 3 : HPLC analysis of ABS at different purification stages, including: (A) hydrochloric acid ethanol extract of dry powder; (B) ABS crude extract; (C) HLB; (D) MCX.

[0027] Figure 4 : UV-Vis and HPLC analysis during the separation and purification of ABS; wherein, (A) UV-Vis spectrum of hydrochloric acid ethanol extract of dry powder; (B) HPLC analysis (570nm) of hydrochloric acid ethanol extract of dry powder; (C) HPLC analysis (240nm) of pure ABS and its UV-Vis spectrum.

[0028] Figure 5 : From top to bottom, they are UPLC-QTOF-MS analysis of pure Gonggan ABS, UPLC-QTOF-MS analysis of sample solution 1 of Mashuiju and Wogan after HLB solid phase extraction pretreatment. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a pure ABS product of the white peel layer of citrus fruit, comprising the following steps:

[0030] S1: preparing a dry powder of the "blue-turned" white cortex of the fruit, extracting the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adjusting the pH of the extract to 6.5-7.5, standing and filtering, and obtaining an ABS crude extract;

[0031] S2: dissolving the ABS crude extract with formic acid, injecting into an HLB column, eluting impurities with water and 30-40% methanol in sequence, and eluting ABS with 0.04-0.2% hydrochloric acid methanol to obtain sample solution 1;

[0032] S3: injecting the sample solution 1 into the MCX column, eluting impurities with 1% hydrochloric acid and methanol, and eluting ABS with 1-10% ammonia methanol to obtain sample solution 2;

[0033] S4: subjecting the sample solution 2 to Sephadex LH-20 column chromatography, using 25-35% methanol as the eluent, collecting the blue band fractions, and concentrating;

[0034] S5: Purify the concentrated solution by semi-preparative liquid chromatography, collect the ABS absorption peak effluent at 570 nm, and dry to obtain pure ABS.

[0035] The "blue-changed" white skin layer samples of the fruit of the present invention are preferably "blue-changed" fresh samples of the white skin layer of citrus fruits such as Wogan, Gonggan, and Mashuiju. Subsequent experiments have shown that the ABS substances of the above-mentioned different citrus fruits are the same chemical substance. In the present invention, the preparation method of the "blue-changed" white skin layer dry powder of the fruit is to freeze the "blue-changed" white skin layer fresh sample with liquid nitrogen, and then grind it into powder with a pulverizer to facilitate the separation and extraction of ABS.

[0036] During the concentration of ABS crude extract by rotary evaporator, we found that when the rotary evaporation temperature was too high, the crude extract quickly faded and ABS may have changed; when the rotary evaporation temperature was too low, the solvent could not be completely removed, which was not conducive to subsequent separation and purification. Therefore, we could only concentrate and enrich ABS by other means. In the early extraction test, we found that neutral or alkaline ethanol could not effectively extract ABS, while under acidic conditions, the solubility of ABS in ethanol would increase significantly (see Figure 1 ). Based on this discovery, the present invention extracts the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adds a 5% sodium bicarbonate solution or ammonia water to the extract, adjusts the pH of the extract to 6.5-7.5, and filters it to obtain an ABS crude extract. Preferably, by adjusting the pH of the solvent to 7, the solubility difference of ABS at different pH values ​​is utilized to precipitate ABS from ethanol, thereby achieving the purpose of enrichment.

[0037] When the present invention uses organic solvents of different polarities and concentrations to extract the ABS of the white cortex, it is found that the ABS can be effectively extracted only under acidic conditions, and then the ABS extraction method is optimized: 0.5-2% hydrochloric acid ethanol (v / v) extraction, preferably 1% hydrochloric acid ethanol (v / v) extraction; extraction time 0.5-4h, preferably 2h; solid-liquid ratio 1:5-25, preferably 1:15 (w / v) (see Figure 2 ). Under the above conditions, the extraction rate of ABS is relatively high. As a preferred embodiment, the extraction times are twice.

[0038] To determine whether ABS changes before and after extraction, we performed HPLC analysis on the crude ABS extract and its corresponding blue precipitate. ABS was detected simultaneously at 210nm and 570nm, where 570nm was used as the characteristic absorption wavelength of ABS to eliminate the interference of impurities and accurately locate ABS; 210nm was used as the terminal absorption wavelength to detect impurities as much as possible and was used to characterize the separation of ABS. Figure 3 It can be seen from A and B that at 570nm, the peak time and type of each ABS before and after enrichment did not change; at 210nm, there were multiple shoulder peaks before enrichment, and the shoulder peaks were significantly reduced after enrichment. The above results show that the enrichment method of the present invention can well retain ABS and has a certain purification effect on ABS.

[0039] The present invention uses formic acid to dissolve the ABS crude extract, and solid phase extraction is performed after passing through a 0.22 μm filter membrane. The volume concentration of the formic acid is preferably 20%. Selecting a suitable adsorbent and a correct elution procedure are the key to solid phase extraction. When further separating and purifying the ABS crude extract, chromatographic fillers such as silica gel, macroporous resin, and polyamide have been tried. However, ABS is very easy to tail or form dead adsorption on these fillers, resulting in very low ABS recovery rate and separation effect. HLB is not easy to form dead adsorption with ABS. The present invention uses an HLB solid phase extraction column to effectively remove residual salt and some hydrophilic impurities in the sample, and adding a trace amount of hydrochloric acid to the eluent can effectively inhibit the dead adsorption of ABS, thereby increasing its recovery rate to 95.2%. Therefore, the present invention uses HLB as an adsorbent, water, and 30-40% methanol (v / v) as elution solvents, and removes a portion of impurities as much as possible on the basis of retaining ABS. Subsequently, ABS is eluted with 0.04-0.2% hydrochloric acid methanol (v / v). As an embodiment, water, 35% methanol, and 0.04% hydrochloric acid methanol are used for elution in an amount of 6 ml respectively. The purification effect is as follows Figure 3 As shown in C, it can be seen that HLB removes a large amount of impurities while retaining ABS.

[0040] The present invention selects MCX as an adsorbent to further purify ABS. As an embodiment, 1% hydrochloric acid (v / v) and methanol are used as eluents to remove impurities, and then 1-10% ammonia methanol is used to elute ABS. As an embodiment, the elution amount of 1% hydrochloric acid and methanol is 6 ml respectively, and the elution amount of 5% ammonia methanol is 12 mL. Figure 3 D It can be seen that MCX can further remove a large number of impurities, and all of them are sharp single peaks at 210nm.

[0041] Sephadex LH-20 is a filler that combines molecular sieve and reverse phase distribution chromatography, which can further separate ABS from impurities according to molecular weight. Considering that ABS is a water-soluble pigment, the present invention uses 25-35% methanol as the mobile phase and a flow rate of 0.4-0.6mL / min to separate ABS. As an embodiment, a fraction collector is used to collect the blue band, and 1 fraction is collected every 10 minutes. The collected liquid is concentrated using a rotary evaporator and a freeze dryer in turn.

[0042] The final purification was performed using a semi-preparative liquid chromatograph. The chromatographic conditions of the semi-preparative liquid chromatograph were: C18 column, mobile phase A was acetonitrile, phase B was water, and the gradient elution conditions were as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min. The detection wavelength was 570nm, and 2-4ml of ABS absorption peak effluent was manually collected. The collected liquid was subjected to rotary evaporation and freeze-dried to obtain pure ABS.

[0043] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Take 9958.0g of fresh sample of the white skin of Gonggan "Blue Change", freeze it with liquid nitrogen and grind it into powder with a pulverizer. Then add 1% hydrochloric acid ethanol (v / v) at a solid-liquid ratio of 1:15 (w / v), extract with magnetic stirring for 2h at room temperature, repeat twice, and combine the two extracts after filtration. Slowly add 5% NaHCO3 (v / v) to the extract, adjust the pH of the extract to 7.0, let it stand, wait for the blue floccules to fully precipitate, and filter to obtain the ABS crude extract.

[0046] The HLB solid phase extraction column (500 mg / 6 mL, Waters, Milford, USA) was activated and balanced. The crude ABS extract was dissolved in 20% formic acid (v / v), filtered through a 0.22 μm PES filter (Bickman Bio, Changde, China), and then the sample solution was adsorbed on the balanced HLB column. The impurities were eluted with 6 mL of water and 35% methanol (v / v), and the ABS was eluted with 6 mL of 0.04% hydrochloric acid and methanol (v / v) to obtain sample solution 1. Then, sample solution 1 was adsorbed on the balanced MCX column, and the impurities were eluted with 6 mL of 1% hydrochloric acid (v / v) and methanol, and the ABS was eluted with 12 mL of 5% ammonia and methanol (v / v) to obtain sample solution 2. ABS sample solution 2 was further separated by Sephadex LH-20 (Cytiva, Sweden) column chromatography (40×600 mm). The eluent was 30% methanol (v / v) at a flow rate of 0.5 mL / min. The blue band was collected using a BSZ-100 fraction collector (Qingpu Luxi Instrument, Shanghai, China), and one fraction was collected every 10 min. The collected solution was concentrated using a rotary evaporator and a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., Beijing, China).

[0047] The concentrated ABS was purified using a NP7000 semi-preparative liquid chromatograph (Hanbang Technology, Jiangsu, China). Chromatographic conditions: Waters BEH C18 column (10mm×250mm, 5μm, Waters, Milford, USA), mobile phase A is acetonitrile, phase B is water, gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm, manually collect 2mL ABS absorption peak effluent. The collected liquid was rotary evaporated and freeze-dried to obtain 3.7mg of pure ABS with a purity of ≥99%.

[0048] Example 2

[0049] Take 20.0g of fresh sample of the white skin of the "blue change" Ma Shui tangerine, freeze it with liquid nitrogen and grind it into powder with a pulverizer. Then add 0.7% hydrochloric acid ethanol (v / v) at a solid-liquid ratio of 1:25 (w / v), extract with magnetic stirring for 4h at room temperature, repeat twice, and combine the two extracts after filtration. Slowly add 5% NaHCO3 (v / v) to the extract, adjust the pH of the extract to 6.5, let it stand, wait for the blue floccules to fully precipitate, and filter to obtain the ABS crude extract.

[0050] The HLB solid phase extraction column (500 mg / 6 mL, Waters, Milford, USA) was activated and balanced. The crude ABS extract was dissolved in 20% formic acid (v / v), filtered through a 0.22 μm PES filter (Bickman Bio, Changde, China), and then the sample solution was adsorbed on the balanced HLB column. The impurities were eluted with 6 mL of water and 30% methanol (v / v), and the ABS was eluted with 6 mL of 0.1% hydrochloric acid and methanol (v / v) to obtain sample solution 1. Then, sample solution 1 was adsorbed on the balanced MCX column, and the impurities were eluted with 6 mL of 1% hydrochloric acid (v / v) and methanol, and the ABS was eluted with 12 mL of 8% ammonia and methanol (v / v) to obtain sample solution 2. ABS sample solution 2 was further separated by Sephadex LH-20 (Cytiva, Sweden) column chromatography (40×600 mm). The eluent was 25% methanol (v / v) at a flow rate of 0.4 mL / min. The blue band was collected using a BSZ-100 fraction collector (Qingpu Luxi Instrument, Shanghai, China), and one fraction was collected every 10 min. The collected solution was concentrated using a rotary evaporator and a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., Beijing, China).

[0051] The concentrated ABS was purified using a NP7000 semi-preparative liquid chromatograph (Hanbang Technology, Jiangsu, China). Chromatographic conditions: Waters BEH C18 column (10mm×250mm, 5μm, Waters, Milford, USA), mobile phase A is acetonitrile, phase B is water, gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm, manually collect 4mL ABS absorption peak effluent. The collected liquid was rotary evaporated and freeze-dried to obtain pure ABS, with a purity of ≥99%.

[0052] Example 3

[0053] Take 20.0g of fresh sample of the white skin of the "blue change" mandarin orange, freeze it with liquid nitrogen and grind it into powder with a pulverizer. Then add 2% hydrochloric acid ethanol (v / v) at a solid-liquid ratio of 1:5 (w / v), extract with magnetic stirring for 1h at room temperature, repeat twice, and combine the two extracts after filtration. Slowly add 5% ammonia water (v / v) to the extract, adjust the pH of the extract to 7.5, let it stand, wait for the blue floccules to fully precipitate, and filter to obtain the ABS crude extract.

[0054] The HLB solid phase extraction column (500 mg / 6 mL, Waters, Milford, USA) was activated and balanced. The crude ABS extract was dissolved in 20% formic acid (v / v), filtered through a 0.22 μm PES filter (Bickman Bio, Changde, China), and then the sample solution was adsorbed on the balanced HLB column. The impurities were eluted with 6 mL of water and 40% methanol (v / v), and the ABS was eluted with 6 mL of 0.2% hydrochloric acid and methanol (v / v) to obtain sample solution 1. Then, sample solution 1 was adsorbed on the balanced MCX column, and the impurities were eluted with 6 mL of 1% hydrochloric acid (v / v) and methanol, and the ABS was eluted with 12 mL of 2% ammonia and methanol (v / v) to obtain sample solution 2. ABS sample solution 2 was further separated by Sephadex LH-20 (Cytiva, Sweden) column chromatography (40×600 mm). The eluent was 35% methanol (v / v) at a flow rate of 0.6 mL / min. The blue band was collected using a BSZ-100 fraction collector (Qingpu Luxi Instrument, Shanghai, China), and one fraction was collected every 10 min. The collected solution was concentrated using a rotary evaporator and a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., Beijing, China).

[0055] The concentrated ABS was purified using a NP7000 semi-preparative liquid chromatograph (Hanbang Technology, Jiangsu, China). Chromatographic conditions: Waters BEH C18 column (10mm×250mm, 5μm, Waters, Milford, USA), mobile phase A is acetonitrile, phase B is water, gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm, manually collect 3mL ABS absorption peak effluent. The collected liquid was rotary evaporated and freeze-dried to obtain pure ABS with a purity of ≥99%.

[0056] Example 4

[0057] UPLC-QTOF-MS / MS Analysis of ABS

[0058] ACQUITY-UPLC I-Class-G2-XS-QTOF (Waters, Milford, USA) was used to analyze the molecular weights of the three varieties of ABS in Examples 1-3. Chromatographic conditions: ACQUITY UPLC BEHC18 (2.1 mm×100 mm, 1.7 μm, Waters, Milford, USA); mobile phase A was acetonitrile, B was 0.1% formic acid (v / v), gradient elution program: 0-8 min, 10%-30% A; 8-9 min, 30%-100% A; 9-10 min, 100%-10% A; 10-12 min, 10% A, flow rate 0.4 mL / min, column temperature 35°C, injection volume 1 μL. Mass spectrometry conditions: electrospray ion source (ESI), positive ion mode detection, capillary voltage 3 kV, cone voltage 40 V, collision voltage 20 V at low energy, collision voltage 40 V at high energy, desolvation temperature 250 °C, ion source temperature 100 °C, desolvation gas flow rate 600 L / h, cone gas flow rate 50 L / h.

[0059] from Figure 5 It can be seen that at around 2.4 min, the molecular ion peaks m / z of Gonggan, Mashuiju and Wogan ABS were 642.1135, 642.1134 and 642.1134, respectively, indicating that the ABS of the three varieties are the same chemical substance.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing pure ABS from the white peel of citrus fruit, characterized in that: The following steps are involved: S1: preparing dry powder of the "blue-turned" white cortex of the fruit, extracting the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adjusting the pH of the extract to 6.5-7.5, standing and filtering, and obtaining an ABS crude extract; S2: dissolving the ABS crude extract with formic acid, injecting into an HLB column, eluting impurities with water and 30-40% methanol in sequence, and eluting ABS with 0.04-0.2% hydrochloric acid methanol to obtain sample solution 1; S3: injecting the sample solution 1 into the MCX column, eluting impurities with 1% hydrochloric acid and methanol, and eluting ABS with 1-10% ammonia methanol to obtain sample solution 2; S4: subjecting the sample solution 2 to Sephadex LH-20 column chromatography, using 25-35% methanol as the eluent, collecting the blue band fractions, and concentrating; S5: Purify the concentrated solution by semi-preparative liquid chromatography, manually collect the ABS absorption peak effluent at 570 nm, and dry to obtain pure ABS.

2. The method according to claim 1, characterized in that: The preparation method of the "blue-changed" white skin layer dry powder of the fruit is as follows: a fresh sample of the "blue-changed" white skin layer is frozen with liquid nitrogen, and then ground into powder with a pulverizer.

3. The method according to claim 1, characterized in that: The volume concentration of the hydrochloric acid ethanol solution is 0.5-2%, and the solid-liquid ratio of the extraction is 1:5-25.

4. The method according to claim 1 or 3, characterized in that: The extraction times are twice.

5. The method according to claim 1, characterized in that: The pH of the extract was adjusted with 5% volume concentration of NaHCO3 or ammonia water.

6. The method according to claim 1, characterized in that: The ABS crude extract was dissolved and passed through a 0.22 μm filter membrane.

7. The method according to claim 1, characterized in that: In step S2, the amount of water, 30-40% methanol, and 0.04-0.2% hydrochloric acid-methanol for elution is 6 ml respectively.

8. The method according to claim 1, characterized in that: In step S3, the elution amount of the 1% hydrochloric acid and methanol is 6 ml respectively, and the elution amount of the 1-10% ammonia methanol is 12 mL.

9. The method according to claim 1, characterized in that: In step S4, the flow rate is 0.4-0.6 mL / min, and one fraction is collected every 10 min.

10. The method according to claim 1, characterized in that: The chromatographic conditions of the semi-preparative liquid chromatograph are as follows: C18 column, mobile phase A is acetonitrile, mobile phase B is water, and gradient elution conditions are as follows: 0-14 min, 10%-22.5% A; 14-17 min, 22.5%-100% A; 17-18 min, 100%-10% A; 18-23 min, 10% A; flow rate 4 mL / min; detection wavelength 570 nm.

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