CN108355115A - A kind of method of continuous extraction purification ginger polyphenol - Google Patents

Abstract

The invention discloses a method for continuously extracting and purifying ginger polyphenols. Firstly, ginger polyphenols are extracted jointly by enzyme-ultrasonic method, purified by ultrafiltration, and then adsorbed by AB-8 macroporous resin, and then polyamide column, silica gel column, Sephadex LH-20 gel column to continue chromatography purification, and freeze-dry the sample to obtain a purified sample. The ginger polyphenol purification sample prepared by the method of the present invention is analyzed by ultraviolet scanning and GC-MS to obtain a maximum absorption wavelength of polyphenols of 281nm, and GC-MS analysis confirms that the purified product mainly contains 66.38% of 6-gingerol, 8-gingerol 3.12%, Z 6 shogaol 2.06%, 8 shogaol 1.93%, citral 2.53%, decanal 9.35% (for thermal degradation products of gingerol), the ginger polyphenol purity prepared by the method of the present invention Up to 85.37%.

Description

A method for continuous extraction and purification of ginger polyphenols

technical field

The invention relates to a technique for jointly extracting multiple functional components of ginger, which belongs to the fields of light industry and agriculture.

Background technique

Ginger is not only a common cooking ingredient and spice seasoning in daily diet, but also a warming and tonic material commonly used in traditional Chinese medicine. Modern medical research shows that ginger has the functions of protecting the digestive system such as protecting the liver and gallbladder, invigorating the stomach and antiemetic, improving the body's immunity, promoting blood circulation, regulating the central nervous system, anti-inflammatory, antibacterial, and insecticidal; ginger extract can inhibit the activity of The generation of oxygen and the oxidation of linoleic acid can reduce serum and liver cholesterol, lower blood sugar, lower blood fat, anti-aging, anti-tumor, regulate body temperature and metabolism, and the research on its related physiologically active ingredients is also quite active. Among them, the most important physiologically active ingredients are gingerol, gingerol, curcumin, ginger protease, etc., which have been proved by modern medicine to be the basis of various physiological and pharmacological effects of ginger, and can be used as the main ingredients of natural flavors, functional foods, and seasonings. Binder. The active ingredients of ginger have been used abroad to make anti-stunning agents, cardiotonic agents, anti-allergic agents and anti-tumor agents.

There are more than 100 chemical components that have been discovered in ginger, and ginger polyphenols are one of the main components. Gingerol is a yellow oily liquid and is a mixture of 6-gingerol, 8-gingerol, 10-gingerol and other substances, of which 6-gingerol has the highest content. The properties and structures of these components are similar, and the molecular structures all contain C ₃ -carbonyl and C ₅ -hydroxyl groups, which make the chemical properties of gingerol active. The phenolic hydroxyl group, hydroxyl group and carbonyl group contained in the gingerol molecule make it have the common properties of ordinary phenols, secondary alcohols and ketones, and can undergo the general chemical reactions of the above compounds, and the methoxyl group and phenolic hydroxyl group contained on the aromatic ring All can affect the biological activity of gingerol.

The separation and purification methods of polyphenols are mainly based on the difference in molecular polarity and solubility, the difference in molecular shape and size between different substances, the difference in the adsorption properties of substances, the difference in ionization of molecules between substances, and the differences in the ligands of substances. Selected with different specificities.

At present, the extraction and purification methods of ginger polyphenols mainly include solvent extraction, precipitation, microwave extraction, supercritical fluid extraction (SFE), ultrafiltration, ultrasonic extraction, biological enzymatic hydrolysis, column chromatography, etc. These methods have their own advantages and disadvantages, but there are many disadvantages in single application, such as long time-consuming, high labor intensity, high energy consumption for pretreatment, large solvent consumption, serious damage to the structure of phenolic substances, and low extraction efficiency. The cost of the process is more expensive and the maintenance requirements are higher.

Contents of the invention

In order to improve the extraction rate and purity, the invention provides a method for continuously extracting and purifying ginger polyphenols. In the present invention, ginger polyphenols are firstly extracted by combined enzyme-ultrasonic method, and after being purified by ultrafiltration, the polyphenols are adsorbed by AB-8 macroporous resin, and then the chromatography is continued by using polyamide column, silica gel column, and Sephadex LH-20 gel column. For purification, the sample was freeze-dried to obtain a purified sample. It is confirmed by UV scanning and GC-MS that it mainly contains 6-gingerol and 8-gingerol. Technical scheme of the present invention is as follows:

1. Enzyme-ultrasonic method combined extraction of ginger polyphenols, the method steps are as follows:

(1) Ginger is shredded, cellulase is added, enzymatically hydrolyzed, and the supernatant is collected;

(2) adding 80% ethanol solvent to the remaining ginger residue in step (1), ultrasonically extracting twice, collecting the supernatant, and recovering the solvent under reduced pressure;

(3) merge two supernatants;

In the above steps, the mass volume ratio of ginger and cellulase is 100:1; the enzymolysis condition is 35°C for 50 minutes; the mass volume ratio of ginger residue and ethanol solvent is 1:2, and the ultrasonic power is 400W. Ultrasound for 1.5h.

2. Purification by ultrafiltration, the method steps are as follows:

Adjust the concentration of the supernatant obtained in the step of extracting ginger polyphenols by enzyme-ultrasonic method to 0.5mg/mL, use a 30KD ultrafiltration membrane to carry out ultrafiltration purification, and collect ultrafiltrates less than 30KD; the conditions for ultrafiltration purification are : The temperature is 30°C, the pressure is 0.15MPa, and the membrane flux is 33.34mL*m-2*s-1.

3. Column chromatography continuous purification, including macroporous resin adsorption, polyamide column purification, silica gel column purification, SephadexLH-20 gel column chromatography purification, the specific method steps are as follows:

(1) Macroporous resin adsorption: 10g AB-8 macroporous resin was packed into a column, and ginger polyphenols were taken for adsorption under the condition of a loading flow rate of 1mL/min, and eluted with 70% ethanol solution;

The adsorption conditions of the macroporous resin are as follows: the adsorption temperature is 30° C., the adsorption time is 1.5 h; the eluting time with ethanol solution is 6 h.

(2) Polyamide column purification: the eluent obtained by macroporous resin adsorption is introduced into the polyamide column to continue chromatographic purification, the mobile phase is 50% ethanol, slowly added, and the elution is complete, and the polyamide column is purified ginger polyphenol liquid, concentrated;

(3) Silica gel column purification: the obtained ginger polyphenol polyamide concentrated solution is carried out to silica gel column chromatography, with sherwood oil and ethyl acetate as eluent elution, obtains ginger polyphenol silica gel purified liquid; Described sherwood oil and ethyl acetate The volume ratio of ethyl acetate is 7:3.

(4) Purification by Sephadex LH-20 gel column chromatography: the ginger polyphenol silica gel purification solution is further purified by SephadexLH-20 gel column chromatography, and eluted with chloroform and ethyl acetate as eluents. The eluent was removed by rotary evaporation to obtain a purified ginger polyphenol; the volume ratio of chloroform to ethyl acetate was 3:1.

4. Freeze-drying: freeze-drying the purified ginger polyphenols obtained above to obtain purified ginger polyphenols.

Beneficial effect:

The combined enzyme-ultrasound method can rapidly change the structure and permeability of the cell wall, and improve the extraction efficiency and purity of active ingredients. Then, it is further purified by ultrafiltration, which has the advantages of no interphase change, low energy consumption, easy operation, and small footprint, and then column chromatography for purification. Commonly used polyphenolic substance column chromatography packing materials include macroporous resin, polyamide, silica gel, gel, etc., each with its own characteristics. The macroporous resin has stable physical and chemical properties and good selectivity, and can be used for crude extraction; polyamide molecules are rich in amide groups, which can form hydrogen bonds with phenolic compounds and be adsorbed, and compounds that cannot form hydrogen bonds Separation; the adsorption capacity of silica gel is mainly related to the number of silanol groups. Silica gel chromatography has dual properties of adsorption chromatography and partition chromatography, and even has a very weak ion exchange effect; Sephadex LH-20 dextran gel has molecular sieve It is especially suitable for the separation of polyphenols containing hydroxyl groups. Therefore, these fillers are respectively used for continuous column chromatography purification, and finally high-purity gingerol is obtained.

The ginger polyphenol purification sample prepared by the method of the present invention is analyzed by ultraviolet scanning and GC-MS to obtain a maximum absorption wavelength of polyphenols of 281nm, and GC-MS analysis confirms that the purified product mainly contains 66.38% of 6-gingerol and 8-gingerol 3.12%, Z-6-shogaol 2.06%, 8-shogaol 1.93%, citral 2.53%, decanal 9.35% (heating degradation products of gingerol). The ginger polyphenols prepared by the method of the present invention have a purity of 85.37%, far higher than the highest 75% in current literature (Zhang Yingfeng et al., 2012).

Description of drawings

Fig. 1 is the influence of enzyme addition amount on polyphenol yield;

Fig. 2 is the influence of enzymolysis temperature on polyphenol yield;

Fig. 3 is the influence of enzymolysis time on polyphenol yield;

Fig. 4 is the impact of ultrasonic extraction solvent on polyphenol yield;

Fig. 5 is the inhibitory rate of different solvent extracts to HMG-CoA reductase;

Fig. 6 is the impact of ultrasonic extraction material-liquid ratio on polyphenol yield;

Fig. 7 is the impact of ultrasonic extraction ethanol concentration on the rate of polyphenols;

Fig. 8 is the influence of ultrasonic extraction time on polyphenol yield;

Fig. 9 is the impact of ultrasonic power on polyphenol yield;

Figure 10 is the influence of ultrafiltration purification temperature on membrane flux;

Fig. 11 is the influence of ultrafiltration purification temperature on decay rate;

Figure 12 is the influence of ultrafiltration purification pressure on membrane flux;

Fig. 13 is the influence of ultrafiltration purification pressure on decay rate;

Figure 14 is the impact of ultrafiltration purified feed liquid concentration on membrane flux;

Fig. 15 is the impact of ultrafiltration purified feed liquid concentration on decay rate;

Fig. 16 is the determination of the static adsorption time of AB-8 macroporous resin;

Fig. 17 is the determination of static adsorption temperature of AB-8 macroporous resin;

Fig. 18 is the determination of AB-8 macroporous resin resin quality;

Figure 19 is the influence of macroporous resin dynamic adsorption sample loading flow rate on leakage rate;

Figure 20 is the influence of macroporous resin dynamic adsorption sample concentration on leakage rate;

Figure 21 is the influence of macroporous resin dynamic adsorption eluent volume on desorption rate;

Figure 22 is the dynamic adsorption and elution curve of ginger polyphenol macroporous resin;

Figure 23 is a polyamide purification dynamic elution curve;

Figure 24 is the determination of polyamide purification elution volume;

Figure 25 is a dynamic elution curve for silica gel purification;

Figure 26 is the dynamic elution curve of Sephadex LH-20;

Figure 27 is a UV scan of ginger polyphenols;

Figure 28 is a GC-MS chromatogram of ginger polyphenols;

Detailed ways

The present invention will be further described below in conjunction with the experimental examples and examples, but it should not be construed as a limitation of the present invention.

Experimental example: selection of process parameters

The raw material used in this experiment is Anqiu ginger provided by Shandong Datang Biotechnology Co., Ltd.

(1) Selection of enzymatic extraction process

① Enzyme dosage selection

According to experiments, when the amount of cellulase added is between 0.5% and 1%, the yield of polyphenols increases with the increase of enzyme amount, and reaches the maximum value at 1%. After more than 1%, the yield basically remained the same, and the difference was not significant. As shown in Figure 1. Therefore, the optimal amount is 1%.

②Enzymolysis temperature selection

According to experiments, within 50°C, as the temperature increases, the yield of polyphenols increases gradually, and reaches the maximum at 50°C. After 50°C, the yield gradually decreased with the increase of temperature. as shown in picture 2. Therefore, the optimum enzymatic hydrolysis temperature is 50°C.

③Selection of enzymatic hydrolysis time

According to experiments, within 30 minutes, the yield of polyphenols gradually increased with time, and reached the maximum value at 30 minutes. After 30 minutes, with the prolongation of time, the yield of polyphenols gradually decreased, and the increase of time was not conducive to the extraction of polyphenols. As shown in Figure 3. Therefore, the optimal extraction time is 30min.

④ Orthogonal experiment of enzymatic extraction

Taking the yield of ginger polyphenols as an index, the factors and levels of the orthogonal experiment are shown in Table 1, the results of the orthogonal experiment are shown in Table 2, and the variance analysis is shown in Table 3.

Table 1 Factor level table of orthogonal test of ginger extracted by enzymatic method

Table 2 Orthogonal test design and results of enzymatic extraction L ₉ (3 ⁴ )

Table 3 enzymatic extraction analysis of variance table

It can be seen from Table 2 that the order of the influence of each factor on the extraction effect is: temperature>time>enzyme amount, the optimal combination is A ₂ B ₂ C ₃ D ₁ , that is: enzyme amount 1%, time 50min, temperature 35 °C, the yield was 0.522%.

It can be seen from Table 3 that the results of variance analysis show that there is no significant difference (p>0.05), and they are all insignificant factors.

(2) Ultrasonic-assisted extraction process selection

① Selection of extraction solvent

Water, methanol, ethanol, acetone, and ethyl acetate of different polarities were used to extract polyphenols in ginger, and the yields were significantly different, as shown in Figure 4. The extraction effect of water is the worst, that of acetone is the highest, and that of ethanol is second. From the economic point of view, ethanol is more economical and cheaper; in the case of no significant difference between ethanol and acetone (P>0.05), because of the high toxicity of acetone, ethanol was selected as the extraction solvent from the point of view of safety.

Ginger polyphenols extracted by different solvents had different inhibition rates on HMG-CoA reductase. As shown in Figure 5, ethanol extraction has the best effect, and the enzyme activity inhibition rate is as high as 51.39%, which is the same as the extraction solvent for polyphenol yield. The second is ethyl acetate with an inhibition rate of 48.39%, and water has the lowest inhibition rate with an inhibition rate of 17.51%. Therefore, it is reasonable to choose ethanol as the extraction solvent.

② Effect of solid-liquid ratio on yield of ginger polyphenols

The solid-liquid ratio is the ratio of the mass of ginger to the volume of the extraction solvent, and the solid-liquid ratio affects the dissolution effect of the extraction solvent on polyphenols. If the amount of solvent is too much, the greater the solid-to-liquid ratio, the dissolution rate of polyphenols will remain basically the same, which will not only waste solvent but also increase the time for subsequent solvent removal. It can be seen from Figure 6 that when the solid-liquid ratio is before 1:3, the yield of polyphenols increases with the increase of the solid-liquid ratio, and when the solid-liquid ratio is 1:3, the yield reaches a maximum value of 0.457%. When the ratio of solid to liquid was more than 1:3, the yield of polyphenols showed a downward trend with the increase of solid to liquid ratio. Therefore, the solid-liquid ratio of ginger and solvent is selected as 1:3.

③The effect of ethanol concentration on the yield of ginger polyphenols

The way polyphenols form compounds in plants is through hydrogen bonds and hydrophobic interactions. Ethanol can break hydrogen bonds, which is conducive to the dissolution of phenolic substances and the separation of polyphenols. The yield of polyphenols will be different with the concentration of ethanol.

As can be seen from Figure 7, before the ethanol concentration was 80%, as the ethanol concentration increased, the polyphenol content continued to increase, and the yield increased significantly (p<0.05) at 70% to 80%; at 80% Later, with the increase of ethanol concentration, the dissolution of other alcohol-soluble impurities and pigments in ginger increased, resulting in a decrease in the yield of polyphenols. Therefore, choose an ethanol concentration of 80%.

④ Effect of extraction time on the yield of ginger polyphenols

If the extraction time is long, the solvent can penetrate into the plant for a long time, thereby improving the yield, but if the extraction time is too long, the extracted phenolic substances are easily oxidized.

As shown in Figure 8, the extraction time is between 0.5 and 2 hours: on the one hand, polyphenols are gradually infiltrated as time goes on; The yield of phenol gradually increased. If it exceeds 2 hours, the extraction effect will be affected due to oxidation loss of partially leached polyphenols due to too long time, and the yield of polyphenols will decrease. Therefore, the extraction time is selected as 2h.

⑤Effect of ultrasonic power on yield of ginger polyphenols

As shown in Figure 9, when the ultrasonic power is lower than 400W, the yield of polyphenols tends to increase, and reaches the maximum at 400W; when it exceeds 400W, the yield of polyphenols decreases. Therefore, it is advisable to choose an ultrasonic power of about 400W.

⑥ Orthogonal test of ultrasonic-assisted extraction process

Taking the yield of ginger polyphenols as an index, the factors and levels of the orthogonal experiment are shown in Table 4, the results of the orthogonal experiment are shown in Table 5, and the variance analysis is shown in Table 6.

Table 4 Ultrasonic Extraction Ginger Orthogonal Test Factor Level Table

Table 5 Ultrasonic extraction L ₉ (3 ⁴ ) orthogonal test design and results

Table 6 Ultrasonic Extraction Analysis of Variance Table

It can be seen from Table 5 that the order of the influence of each factor on the extraction effect is: A>C>B>D, and the optimal combination is A ₁ B ₂ C ₁ D ₂ , that is: the ratio of solid to liquid is 1:2, and the concentration of ethanol is 80%, the extraction time is 1.5h, the ultrasonic power is 400W, and the yield is 0.501%.

It can be seen from Table 6 that the result of variance analysis is F(A)>F(C)>F(B)>F(D), and the material-liquid ratio has the greatest influence on the extraction effect, and there is a significant difference (p<0.05).

Under optimal conditions, repeated experiments were carried out three times, and the average value was 0.503%, which was greater than the treatment value of the orthogonal experiment, indicating that the extraction process was stable, the results were reasonable, and the reproducibility was good.

(3) Selection of ginger polyphenol extraction method

According to the enzymatic extraction method, the enzyme dosage is 1%, the time is 50 min, and the temperature is 35° C. to extract and collect the supernatant. The filter residue was extracted twice according to the ultrasonic-assisted extraction method, the ratio of solid to liquid was 1:2, the concentration of ethanol was 80%, the extraction time was 1.5 hours, and the ultrasonic power was 400W. All the extracts were combined, the solvent was recovered under reduced pressure, and the yield of polyphenols was determined. It can be seen from Table 7 that, compared with ultrasonic and enzymatic methods, the combined extraction of polyphenols by ultrasonic-enzymatic method has the highest yield. Therefore, ginger polyphenols were extracted by ultrasonic-enzyme method.

Table 7 Comparison of yields of different extraction methods

(4) Ultrafiltration purification process of ginger polyphenols

①Effect of ultrafiltration on the retention rate of ginger polyphenols

Pre-treat the polyphenol polyphenol extract, pass through ultrafiltration membranes of 50KD, 30KD, and 10KD respectively, perform ultrafiltration, and collect the retentate and permeate. It can be seen from Table 8 that the rejection rates of the three membranes for polyphenols are 4.8%, 13.2%, and 20.2%, respectively, and the rejection rates of the three membranes for impurities are 57.89%, 73.94%, and 80.06%, respectively. The polyphenol rejection rate of the 50KD membrane is the smallest, that is, the transmission rate is the largest, but the retention rate of impurities is also the smallest. The 10KD membrane has the largest rejection rate of impurities, but the smallest polyphenol permeability and loss. It can be seen that the 30KD ultrafiltration membrane has a higher protein retention rate and a higher polyphenol permeability, which is an ideal model. The permeated end of the 30KD ultrafiltration membrane was collected for enrichment of polyphenols, pending the next experiment.

Table 8 ultrafiltration rejection rate table

② Effect of temperature on membrane flux and decay rate

Keep the pressure and concentration constant during the test, and measure the membrane flux at 20, 30, and 40°C, respectively.

As shown in Figure 10, the membrane flux increases with the increase of temperature, and the membrane flux reaches the maximum when the temperature is 40°C. As shown in Figure 11, as the temperature increases, the decay is slow, further illustrating that 40°C is the best. Since the maximum temperature of the film is 40°C, in order not to affect the use of the film, 40°C is selected as the optimum temperature.

③ Effect of pressure on membrane flux and decay rate

In this experiment, the temperature and concentration were kept constant, and the membrane fluxes at 0.05, 0.1, and 0.15 MPa were measured respectively.

It can be seen from Figure 12 that as the pressure increases, the membrane flux increases. In the first 7 minutes, at 0.15MPa, the membrane flux is relatively large. As time goes on, the membrane flux at 0.1MPa is higher than that at 0.15MPa. . It can be seen from Figure 13 that as the pressure increases, the attenuation rate increases accordingly. Considering the above factors, a pressure of 0.1 MPa is selected for subsequent tests.

④ Effect of concentration on membrane flux and decay rate

In this experiment, the temperature and pressure were kept constant, and the membrane fluxes at 0.5, 1, and 1.5 mg/mL were measured respectively.

As shown in Figure 14, when the concentration is 1.5mg/mL, the membrane flux is the lowest. In the first 6 minutes, the membrane flux with a concentration of 1mg/mL is higher than that of 0.5mg/mL. After this time, the flux of 0.5mg/mL is higher than that of 1mg /mL membrane flux is high. The feed solution concentration of 1mg/mL is moderate, and the membrane flux is relatively large at first. As time goes by, a gel layer begins to form on the membrane surface, and the concentration polarization is serious, resulting in the attenuation of the membrane flux. The feed solution concentration of 0.5mg/mL is small, and the membrane flux is not much different from that of 1mg/mL feed solution. The concentration is lower, and the concentration polarization formed on the membrane surface is slower. It can be seen from Figure 15 that the attenuation rate of the 0.5 mg/mL feed liquid film is significantly lower than 1 mg/mL, so the feed liquid concentration is selected as 0.5 mg/mL.

⑤ Orthogonal experiment of ultrafiltration purification process

Taking the membrane flux as the index, the factors and levels of the orthogonal experiment are shown in Table 9, the results of the orthogonal experiment are shown in Table 10, and the variance analysis is shown in Table 11.

It can be seen from Table 10 that the order of the influence of various factors on the membrane flux is: C>B>A, and the optimal combination is A ₂ B ₃ C ₁ , that is, the temperature is 30°C, the pressure is 0.15MPa, and the ginger polyphenol solution The concentration is 0.5mg/mL, and the membrane flux is 33.34mL*m ^-2 *s ^-1 .

It can be seen from Table 11 that the result of variance analysis is F(C)>F(B)>F(A), and the concentration has the greatest influence on the ultrafiltration effect, and there is a significant difference (p<0.05).

Table 9 Ginger ultrafiltration orthogonal test factor level table

Table 10 L9(34) Orthogonal Experimental Design and Results

Table 11 variance analysis table

(5) macroporous resin purification of ginger polyphenols

① Screening of macroporous resin

In order to select a suitable resin, the adsorption rate was measured respectively as shown in Table 12 below. It can be seen from Table 12 that the five macroporous resins with different polarities all have adsorption rates, indicating that the macroporous resins all have different degrees of adsorption, and the AB-8 macroporous resin has the highest adsorption rate compared to other resins. It is related to the weak polarity of ginger polyphenols, which is more likely to be adsorbed by weakly polar macroporous resins. So choose AB-8 macroporous resin.

Table 12 Comparison of adsorption results of five resins

② Static adsorption of macroporous resin

A. Determination of static adsorption time

It can be seen from Figure 16 that the adsorption rate of AB-8 macroporous resin increases with the prolongation of time (P<0.05), the adsorption rate rises rapidly before 1.5h, and basically remains unchanged after 1.5h, the difference is not significant (P > 0.05). Therefore, the optimal adsorption time is 1.5h.

B. Determination of static adsorption temperature

It can be seen from Figure 17 that the adsorption rate of AB-8 macroporous resin increases significantly with the increase of temperature, reaches the maximum at 30°C, and then decreases significantly. Therefore, the optimum adsorption temperature is 30 °C.

C. Determination of resin quality

It can be seen from Figure 18 that with the increase of resin mass, the adsorption rate increases gradually (P<0.05), and reaches the maximum value when the mass is 10 g. After 10 g, it remained basically unchanged, and the difference was not significant (P>0.05). Therefore, the resin mass is 10g.

③Dynamic adsorption of macroporous resin

A. Determination of sample loading rate

It can be seen from Figure 19 that when the sample volume is lower than 80mL, the leakage rate of the sample solution at different flow rates remains basically the same, and when it exceeds 100mL, the leakage rate increases with the increase of the flow rate, because the flow rate is too fast. When fully in contact with the resin, it will flow out, causing the leakage point to advance to 80ml. Slowing down the flow rate can make the polyphenol fully contact with the resin, thereby absorbing more amount, and the leakage rate of 1mL/min is lower than 2mL/min under the same sample volume. min, in the case of the same amount of polyphenols, if the sample loading rate is lower than 1mL/min, the time consumption will increase and the efficiency will decrease. Based on the above factors, the optimal sample loading rate was determined to be 1 mL/min, and the optimal sample loading volume was 100 mL.

B. Determination of loading concentration

It can be seen from Figure 20 that when the sample volume is lower than 60mL, the leakage rate of the sample solution with different concentrations is basically unchanged, and when the sample volume is 80mL, the leakage rate of the 2mg/mL sample solution increases significantly, and when it exceeds 100mL, the leakage rate increases with the increase of the concentration. Big and increase. Under the same leakage conditions, the leakage rates of 1mg/mL and 0.5mg/mL are not much different. Considering the efficiency of adsorption, the concentration of 1mg/mL is more suitable for production requirements. Therefore, the optimal loading concentration was chosen to be 1 mg/mL.

C. Determination of eluent concentration

Commonly used eluents in experiments are methanol, ethanol, acetone, etc., because methanol and acetone are more toxic, while ethanol is non-toxic and has better solubility, and ginger polyphenols are mainly used in food and medicine fields. Consider, choose non-toxic ethanol as desorbent. It can be seen from Table 13 that with different concentrations of ethanol as the eluent, the desorption rate increases significantly with the increase of the concentration, reaches the maximum at the concentration of 70%, and then decreases. Because the ethanol concentration is too high, the volatility will increase, so 70% ethanol is selected as the elution solvent.

The influence of table 13 eluent concentration on desorption rate

D. Determination of eluent volume

It can be seen from Figure 21 that the desorption rate increases gradually (P<0.05) as the eluent volume increases when the volume is lower than 150 mL. When it exceeds 150mL, the desorption rate is basically balanced, and the difference is not significant (P>0.05). Considering comprehensively, on the premise that ginger polyphenols are fully eluted, the amount of eluent should be saved as much as possible. Therefore, the optimum eluent volume is 150mL.

E. Chromatogram

100mL of ginger polyphenols with a loading concentration of 1mg/mL was adsorbed under the condition of a loading flow rate of 1mL/min, and eluted with a volume of 150mL of 70% ethanol solution, and a dynamic elution curve was drawn by a chromatographic spectrometer. As shown in Figure 22. With the prolongation of time, the concentration of polyphenols in the desorbed effluent continued to increase, mainly concentrated at 2.5h-3.5h. Take off completely.

(6) Polyamide purification of ginger polyphenols

① Polyamide gradient elution

It can be seen from Figure 23 that there are mainly four components after polyamide column chromatography, and the chromatographic peak shape is relatively narrow, and the degree of separation is good. The components in the four time periods are collected, combined, and concentrated. The desorption rate of each section is shown in Table 14, and the content eluted with 50% ethanol is relatively high, and the desorption rate is 80.3%. However, water, 30% ethanol, and 70% ethanol elute less, and it is not convenient to collect, so the eluent of 50% ethanol is selected.

The influence of table 14 eluent concentration on desorption rate

②Determination of elution volume

It can be seen from Figure 24 that when eluted with 50% ethanol, as the volume of ethanol increases, the absorbance first increases and then decreases, and the content of polyphenols eluted increases. When the elution volume is 160mL, polyphenols have been basically washed out. Take off completely. Therefore, an elution volume of 160 mL was chosen.

(7) Silica gel purification of ginger polyphenols

Under different ratios of petroleum ether and ethyl acetate, the measured absorbance is shown in Figure 25. As can be seen from the figure, 7 components are obtained after silica gel column chromatography, wherein the elution content is the highest when sherwood oil:ethyl acetate=7:3, and the content of other ratios is less, and it is also inconvenient to collect, so this experiment only The third fraction is collected for subsequent processing.

(8) Sephadex purification of ginger polyphenols

① Sephadex LH-20 gel dynamic elution

It can be seen from Figure 26 that after Sephadex LH-20 gel column chromatography, two fractions were obtained, and the two fractions were collected. The content of fraction 2 was relatively low, and the fraction of fraction 1 was relatively high. Group 1 was concentrated and evaporated. , to determine the next experiment.

②UV scan

It can be seen from Figure 27 that there is an absorption peak at 281nm, which is generated by the n-π* transition, which belongs to the R absorption band, which is the characteristic absorption peak of the benzene ring; there is an absorption near 230nm, which belongs to the K absorption band, which is formed by the π-π* transition Produced, this is the characteristic absorption of the conjugated double bond.

③Gas chromatography to detect ginger polyphenol components experiment

The chromatogram obtained through GC-MS analysis of the purified ginger polyphenols is shown in Figure 28, and the mass spectral data obtained by automatic searching of the NIST08 mass spectral library is shown in Table 15. It can be seen from Figure 28 that there are mainly 10 components, among which hexanal, octanal, and zingerone are relatively high in content. But zingerone is not a natural component of ginger, it is an anti-aldol cracking product of 6-gingerol, which can be generated under the conditions of gas chromatography. When the inlet temperature of GC-MS reaches or exceeds 200 ° C, 6-gingerol will be Instantly split into zingerone and hexanal and enter the chromatographic column. In GC-MS analysis, 8-gingerol undergoes complete or partial pyrolysis and McFarland rearrangement reaction, and its products are the corresponding octanal and zingerone. Gingerol compounds do not exist in fresh ginger, and gingerol will be dehydroxylated and transformed into shogaol under conditions such as long-term storage and high temperature. Therefore, 6-gingerol and 8-gingerol mainly exist in the purified product.

Table 15 Composition Analysis

Embodiment: the preparation of ginger polyphenol

Add 1g cellulase according to every 100g shredded ginger, enzymolysis 50min at 35°C, collect the supernatant, add 2 times (mass: volume) 80% ethanol solvent to the remaining ginger slag, and ultrasonically extract twice under 400W power. After 1.5 hours, the supernatants were collected twice and combined, and the yield of ginger polyphenols extracted by the enzyme-ultrasonic method was 0.551%;

Adjust the concentration of ginger polyphenols in the supernatant extracted by enzyme-ultrasonic method to 0.5mg/mL, and perform ultrafiltration purification. The conditions are 30KD ultrafiltration membrane, 30°C, 0.15MPa pressure, and the membrane flux is 33.34mL *m ^-2 *s ^-1 , collect ultra-filtered ginger polyphenols less than 30KD;

The ultrafiltration ginger polyphenol solution was purified by the AB-8 macroporous resin adsorption method. The static adsorption conditions were: the optimal adsorption time was 1.5 h, the adsorption temperature was 30°C, and the resin mass was 10 g; the dynamic adsorption conditions were: the ginger polyphenols The concentration was adjusted to 1mg/mL, and the adsorption was carried out under the condition of sample loading flow rate of 1mL/min, and eluted with 70% ethanol solution. Under this condition, the desorption rate was 85.9%, and the purity was 56.26%;

Above-mentioned eluent utilizes polyamide column chromatography to continue to purify, and mobile phase adopts 50% ethanol elution, and elution completes calculation ginger polyphenol resolution rate reaches 80.3%; Elution part continues to be sherwood oil: ethyl acetate= in volume ratio Carry out silica gel column chromatography under the eluent proportioning ratio of 7:3; The chromatographic liquid obtained is chloroform again with volume ratio: ethyl acetate=3:1 is eluent, utilizes Sephadex LH-20 gel column layer Analyze and continue to purify, and remove the eluent by rotary evaporation to obtain ginger polyphenol purified liquid;

The purified ginger polyphenols obtained from the above purification were freeze-dried to obtain purified ginger polyphenols with a purity of 85.37%, far higher than the highest 75% in current literature (Zhang Yingfeng et al., 2012). Through ultraviolet scanning and GC-MS analysis, the maximum absorption wavelength of ginger polyphenols obtained is 281nm, and GC-MS analysis mainly contains 66.38% of 6-gingerol, 3.12% of 8-gingerol, 2.06% of Z-6-shogaol, 1.93% of 8-shogaol, 2.53% of citral, and 9.35% of decanal (the thermal degradation product of gingerol).

Claims (8)

1. A method for continuous extraction and purification of ginger polyphenols, characterized in that, comprising the steps of: enzyme-ultrasonic method combined extraction of ginger polyphenols; ultrafiltration purification; column chromatography continuous purification; freeze-drying to obtain ginger polyphenols purification material; the specific steps are as follows: (1) described enzyme-ultrasonic method is combined to extract ginger polyphenols, and method is as follows: (1) Ginger is shredded, cellulase is added, enzymatically hydrolyzed, and the supernatant is collected; (2) adding 80% ethanol solvent to the remaining ginger residue in step (1), ultrasonically extracting twice, collecting the supernatant, and recovering the solvent under reduced pressure; (3) Merge the two supernatants. (2) described ultrafiltration purification, the method is as follows: Adjust the concentration of the supernatant obtained in the step of combined enzyme-ultrasonic extraction of ginger polyphenols to 0.5 mg/mL, use a 30KD ultrafiltration membrane for ultrafiltration purification, and collect ginger polyphenol ultrafiltrates less than 30KD; (3) The continuous purification of the column chromatography method includes macroporous resin adsorption, polyamide column purification, silica gel column purification, Sephadex LH-20 gel column chromatography purification, and the specific methods are as follows: (1) Macroporous resin adsorption: 10 g of AB-8 macroporous resin was packed into a column, ginger polyphenols were taken for adsorption at a flow rate of 1 mL/min, and eluted with 70% ethanol solution; (2) Polyamide column purification: the eluent obtained by macroporous resin adsorption is introduced into the polyamide column to continue chromatographic purification, the mobile phase is 50% ethanol, slowly added, and the elution is complete, and the polyamide column is purified ginger polyphenol liquid, concentrated; (3) Silica gel column purification: the obtained ginger polyphenol polyamide concentrated solution is subjected to silica gel column chromatography, and petroleum ether and ethyl acetate are used as eluent for elution to obtain ginger polyphenol silica gel purified solution; (4) Purification by Sephadex LH-20 gel column chromatography: the ginger polyphenol silica gel purification solution was further purified by Sephadex LH-20 gel column chromatography, and eluted with chloroform and ethyl acetate as eluents. Remove the eluent by rotary evaporation to obtain the purified ginger polyphenols; (4) Freeze-drying: freeze-dry the purified ginger polyphenols obtained above to obtain purified ginger polyphenols. 2. the method for continuously extracting and purifying ginger polyphenols according to claim 1, is characterized in that, in the step of extracting ginger polyphenols in conjunction with the enzyme-ultrasonic method, the mass-to-volume ratio of ginger and cellulase is 100:1; The enzymatic hydrolysis condition was 50 min at 35°C. 3. The method for continuously extracting and purifying ginger polyphenols according to claim 1, characterized in that, in the step of combined extraction of ginger polyphenols by the enzyme-ultrasound method, the ultrasonic power is 400W, and each ultrasonic wave is 1.5h. 4. The method for continuously extracting and purifying ginger polyphenols according to claim 1, characterized in that, in the combined extraction of ginger polyphenols by the enzyme-ultrasonic method, the mass-to-volume ratio of ginger slag and ethanol solvent is 1:2. 5. The method for continuously extracting and purifying ginger polyphenols according to claim 1, wherein the conditions for purification by ultrafiltration are: temperature is 30°C, pressure is 0.15MPa, and membrane flux is 33.34mL*m -2*s-1. 6. The method for continuously extracting and purifying ginger polyphenols according to claim 1, characterized in that, the adsorption conditions of the macroporous resin are: the adsorption temperature is 30° C., and the adsorption time is 1.5 h; ethanol solution eluting time for 6h. 7. the method for continuous extraction and purification of ginger polyphenols according to claim 1, is characterized in that, in the silica gel column purification, the volume ratio of sherwood oil and ethyl acetate is 7:3. 8. the method for continuous extraction and purification of ginger polyphenols according to claim 1, is characterized in that, in the described SephadexLH-20 gel column chromatography purification, the volume ratio of chloroform and ethyl acetate is 3:1.