Method for extracting and separating kukoamine A and kukoamine B in cortex lycii radicis

Abstract

The invention provides a method for synchronously extracting and separating kukoamine A and kukoamine B. On the basis of obtaining an effective eutectic solvent as an extracting agent, ultrasonic-assisted extraction is adopted; three factors, namely extraction time, extraction temperature and water content, which have relatively great influence on the kukoamine A and kukoamine B extraction process are optimized by applying a response surface method, so that optimal extraction conditions are obtained. Then, the cortex lycii radicis extracting solution passes through ion exchange resin, and the eutectic solvent in the extracting solution is removed; finally, high-speed counter-current chromatography separation and preparative liquid chromatography purification are adopted to further obtain kukoamine A and kukoamine B with higher purity, and high performance liquid chromatography is utilized to detect and analyze the kukoamine A and kukoamine B.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and separation of active ingredients of traditional Chinese medicines, and particularly relates to a method for synchronously extracting and separating lycopodione A and lycopodione B from the root of Chinese wolfberry. Background Art

[0002] The root bark of Lycium chinense Mill. or Lycium barbarum L. of the Solanaceae family is a dry root bark. It is sweet and cold in nature and belongs to the lung, liver, and kidney meridians. It has the effects of cooling blood and removing steam, clearing the lungs and reducing fire. It is mainly used to treat yin deficiency, night sweats due to bone steaming, cough due to lung heat, hemoptysis, epistaxis, and internal heat and thirst. At present, there is no method for determining the content of the active ingredients of the root bark, resulting in an imperfect quality evaluation system for the root bark. Lycium bark A and Lycium bark B are considered to be the main active markers of the root bark, with activities such as lowering blood pressure, lowering blood sugar, lowering blood lipids, relieving fever, and antibacterial and antiviral activities. However, the nature of Lycium bark A and Lycium bark B that are difficult to separate and prepare leads to a lack of corresponding standard substances, and then a lack of a quantitative detection method for the root bark, which affects the quality evaluation of the root bark. At present, the extraction of lycopodione and lycopodione is mainly based on methanol, ethanol, n-butanol ultrasonic or reflux extraction. The traditional extraction method and extraction solvent consume a lot and the operation is cumbersome, which can easily change the target substance. Therefore, it is very important to seek and develop a green, efficient, simple and easy extraction technology. On this basis, the samples of lycopodione and lycopodione are further separated to provide a physical standard for the quality control of Lycopodione. Summary of the invention

[0003] The purpose of the present invention is to provide a method for synchronously extracting and separating lycopodione and lycopodione from the cortex lycopodione, thereby effectively improving the extraction efficiency and separation effect of lycopodione and lycopodione from the cortex lycopodione.

[0004] The present invention first provides a method for extracting lycopodione and lycopodione from the cortex of Lycium barometz, wherein the extraction solvent is one of reagents such as water, methanol, ethanol, acetonitrile, dichloromethane, a low eutectic solvent, or different ratios of 2-3 reagents, to obtain a cortex of Lycium barometz extract containing lycopodione and lycopodione;

[0005] Furthermore, the ratio of the two reagents is 1-10:1-10, and the ratio of the three reagents is 1-10:1-10:1-10;

[0006] The method described herein, wherein the mass volume ratio of the cortex lycii to the low eutectic solvent is 1:10 to 1:20 (g / ml).

[0007] The extraction is ultrasound-assisted extraction, wherein the power of the ultrasound is 400W-600W, the temperature is 40°C-50°C, and the time is 20min-60min.

[0008] The present invention also provides a low eutectic solvent for extracting lycopodiol and lycopodiol, comprising a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is choline chloride or levulinic acid; the hydrogen bond donor is levulinic acid, lactic acid, n-butyric acid, ethylene glycol, 1,2-propylene glycol, glycerol, 1,4-butanediol, 1,6-hexanediol or urea;

[0009] Furthermore, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is lactic acid;

[0010] Preferably, the molar ratio of choline chloride to lactic acid is 1:2.

[0011] The present invention also provides a use of the low eutectic solvent, which is used to extract products of lycopodine and lycopodine;

[0012] Furthermore, the extraction and separation method also includes the step of purifying lycopoein and lycopoein ethyl, which comprises passing the above-mentioned lycopoein extract through ion exchange resin, eluting with water of different pH values, concentrating the eluate by rotary evaporation, and drying under reduced pressure and vacuum to obtain lycopoein and lycopoein ethyl enriched fractions; then separating the lycopoein and lycopoein ethyl enriched fractions through high-speed countercurrent chromatography, and further purifying the obtained lycopoein and lycopoein ethyl fractions through preparative liquid phase to obtain lycopoein and lycopoein ethyl with a purity of >95%.

[0013] The ion exchange resin is one of gel strong acid styrene, gel strong base styrene, macroporous strong base styrene, and macroporous weak acid benzoic acid;

[0014] The elution flow rate is 2 to 5 BV / h;

[0015] The rotary evaporation concentration water bath temperature is 45-60°C, and the vacuum degree is >0.09Mpa;

[0016] The reduced pressure vacuum drying temperature is 45-60° C., and the vacuum degree is greater than 1.00 MPa.

[0017] The solvent used in the high-speed countercurrent chromatography includes ethyl acetate, n-butanol, water, and methanol, wherein the ratio of ethyl acetate: n-butanol: water: methanol is 1-10:1-10:1-10:0.1-5;

[0018] The K value of the countercurrent chromatography solvent system is 0.5 to 2;

[0019] The countercurrent chromatography rotation speed is 700-900 rpm.

[0020] The lycopene A and lycopene B samples obtained by the invention are tested for purity by high performance liquid chromatography and for content by external standard method.

[0021] The high performance liquid chromatography method: After the sample is filtered through a 0.45 μm filter membrane, it is analyzed using a high performance liquid chromatography, using a chromatographic column (4.6 mm×250 mm, 5 μm) filled with octadecylsilane bonded silica gel, and the mobile phase is 0.1% trifluoroacetic acid (A) and acetonitrile (B). Elution gradient: 0-10 min, 12%-14.5% B; 10-11 min, 14.5%-12% B; 11-16 min, 12% B. The flow rate is 1 mL / min, and the injection volume is 20 μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a coordinate diagram showing the effects of different types of DES on the contents of lycopodin A and B;

[0023] Figure 2 This is a coordinate diagram showing the effect of extraction time on the contents of lycopodin A and B;

[0024] Figure 3 This is a coordinate diagram showing the effect of extraction temperature on the contents of lycopodin A and B;

[0025] Figure 4 This is a coordinate diagram showing the effect of water content on the contents of lycopodin A and B;

[0026] Figure 5 This is a coordinate diagram showing the effect of the solid-liquid ratio on the contents of lycopodiella A and B;

[0027] Figure 6 The response surface diagram of the effects of extraction temperature, extraction time and water content on the yields of KuA and KuB;

[0028] Figure 7 This is a high-speed countercurrent chromatogram of lycopodipine and lycopodipine;

[0029] Figure 8 This is the HPLC chromatogram of lycopodiella;

[0030] Fig. 9 This is the high performance liquid chromatogram of dichotoma euphrasiaticum. DETAILED DESCRIPTION

[0031] In the present specification, DES is a low eutectic solvent, HBA is a hydrogen bond acceptor, and HBD represents a hydrogen bond donor; the conditions for HPLC determination of the contents of lycopodin A and lycopodin B in the present embodiment are as follows:

[0032] 1. HPLC conditions: After the extracted sample was diluted with methanol and filtered through a 0.45 μm filter membrane, HPLC analysis was performed using an Agilent 1260 HPLC and analyzed on a SinoChrom ODS-BP C18 column (5 μm, 4.6 mm×250 mm) at 30°C. The mobile phases were water containing 0.05% trifluoroacetic acid (mobile phase A) and acetonitrile containing 0.05% trifluoroacetic acid (mobile phase B), with a gradient of: 0-10 min, 12%-14.5% B; 10-11 min, 14.5%-12% B; 11-16 min, 12% B. The flow rate was 1 mL / min, and the injection volume was 20 μL.

[0033] 2. Content determination: The contents of lycopodione and lycopodione are expressed in mg / g. The standard curves of lycopodione and lycopodione are Y=5267x+23896(R 2 =0.9921,n=7), Y=6355367x+270224(R 2 =0.9910, n=7), wherein the contents of lycopodiol A and lycopodiol B were calculated as follows:

[0034] Content of Lycium barbarum (mg / g) = (mass of Lycium barbarum / mass of Lycium barbarum)

[0035] Content of lycopodiol (mg / g) = (mass of lycopodiol / mass of lycopodiol)

[0036] 3. Data processing

[0037] All experiments were repeated three times. The Dsign-Expert response surface software was used to analyze and optimize the data of relevant factors of the extraction of lycopodiella and lycopodiella, and SPSS25.0 statistical software and Origin 2020 software were used for analysis and drawing.

[0038] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0039] Example 1: Deep eutectic solvent screening

[0040] The present embodiment provides a method for extracting lycopodione and lycopodione from the cortex of Lycium barbarum by using ultrasound-assisted DES, wherein HBA and HBD with a molar ratio of 1:2 form DES, and ultrasound-assisted DES is used to extract lycopodione and lycopodione from the cortex of Lycium barbarum. HBA is choline chloride or levulinic acid; the hydrogen bond donor HBD is one of levulinic acid, lactic acid, n-butyric acid, ethylene glycol, 1,2-propylene glycol, glycerol, 1,4-butanediol, 1,6-hexanediol, and urea.

[0041] The non-medicinal parts and impurities in the dried Radix Rehmanniae raw materials were sorted out, and the remaining parts were crushed. 0.1 g of Radix Rehmanniae powder was accurately weighed, 2 ml of DES (water content 30%) was added to a 5 ml centrifuge tube for mixing, and ultrasonic extraction was performed for 30 minutes (ultrasonic power of 500 W, temperature of 40°C), and the contents of Radix Rehmanniae A and B were determined by high performance liquid chromatography.

[0042] According to Table 1, HBA and HBD were mixed at a molar ratio of 1:2 at 80°C with magnetic stirring until homogeneous and clear.

[0043] Table 1: Components and molar ratios of different types of DES

[0044]

[0045] As shown in Table 1, the cortex lychnophorae was extracted with 12 different types of DES, and the extraction effects of the 12 DES on lychnophorae acetone and acetone were analyzed according to the contents of lychnophorae acetone and acetone, and the DES with the best extraction effect was selected for subsequent single factor experiments.

[0046] 1) Single factor experiment

[0047] Extraction of Lycium barbarum A and Lycium barbarum B, extraction conditions: ultrasonic power 500W, extraction temperature 40°C, extraction time 30min, water content 30% factors remain unchanged, and the effects of different liquid-to-solid ratios (1:20, 1:30, 1:40, 1:50, 1:60) on the contents of Lycium barbarum A and Lycium barbarum B are compared; keeping the liquid-to-solid ratio 1:20 (=g / ml), extraction temperature 40°C, extraction time 30min factors unchanged, and comparing the effects of different water contents (10%, 20%, 30%, 40%, 50%) on the contents of Lycium barbarum A and Lycium barbarum B. The influence of A and B content on the content of Lycium barbarum; keeping the liquid-to-solid ratio at 1:20 (g / ml), the extraction temperature at 40°C and the water content at 30% unchanged, and comparing the influence of different extraction times (10min, 20min, 30min, 40min, 50min) on the content of A and B in Lycium barbarum; keeping the liquid-to-solid ratio at 1:20 (g / ml), the extraction time at 30min and the water content at 30% unchanged, and comparing the influence of different extraction temperatures (20°C, 30°C, 40°C, 50°C, 60°C) on the content of A and B in Lycium barbarum.

[0048] Based on the results of the single factor experiment, with extraction temperature (A), extraction time (B), and water content (C) as independent variables, and lycopodiol A and B (g) as response values, a three-factor three-level experiment was designed based on the BDD design. The experimental design factors and levels are shown in Table 2.

[0049] Table 2: Response surface design experimental factors and levels

[0050]

[0051] according to Figure 1 It can be seen that among the effects of the 12 DES on the contents of lycopodione and lycopodione, DES-2 (choline chloride-lactic acid) has the best extraction effect on lycopodione and lycopodione, with lycopodione content of 2.41±0.04mg / g and lycopodione content of 17.41±0.10mg / g. Therefore, choline chloride-lactic acid deep eutectic solvent was selected for subsequent experiments.

[0052] The results of the single factor experiment are as follows:

[0053] 1) Effect of extraction time on the contents of lycopodione and lycopodione

[0054] Ultrasonic time is an important factor affecting extraction efficiency. It is necessary to optimize the conditions to shorten the time and reduce the cost. The extraction time in this experiment is between 10min and 50min. Figure 2 The results showed that within 10-30 minutes, the extraction rate of lycopodiol increased from 1.74±0.10mg / g to 2.49±0.04mg / g, and the extraction rate of lycopodiol increased from 9.62±0.34mg / g to 17.12±0.39mg / g. This shows that the longer the contact time between the solvent and the solute, the stronger the destructive effect of ultrasound on the solute, allowing the solvent to quickly enter the solid and dissolve the components contained in the substance as completely as possible in the solvent. At 30 minutes, the sum of the contents of lycopodiol and lycopodiol reached the maximum value, and there was no significant change in the content after 30 minutes. Therefore, 20 minutes, 30 minutes, and 40 minutes were selected for subsequent experiments.

[0055] 2) Effect of extraction temperature on the contents of lycopodione and lycopodione

[0056] High temperature can increase the kinetic energy of DES molecules, reduce the viscosity of the low eutectic solvent, and enhance the interaction between the solvent and the solid particles, thereby improving the extraction efficiency. Too high a temperature will cause the degradation of the bioactive components and reduce the extraction efficiency. Therefore, the effect of the extraction temperature between 20 and 60 °C on the content was investigated. Figure 3 As shown in the figure, with the increase of temperature, the contents of lycopodione and lycopodione are increasing, reaching the maximum value at 40℃, but once the temperature exceeds 40℃, the contents of lycopodione and lycopodione decrease slightly. The increase of temperature enhances the cavitation effect, and the combination of the two effects leads to a relative improvement in the extraction efficiency. However, the further increase of temperature has the opposite effect on cavitation, because the intensity of cavitation decreases with the increase of temperature. Therefore, 30℃, 40℃, and 50℃ with large changes were selected as the response surface experimental conditions.

[0057] 3) Effect of water content on the content of lycopodiella A and B

[0058] The presence of hydroxyl groups in DES will produce more hydrogen bonds, resulting in increased attraction between molecules and increased solvent viscosity. High viscosity will reduce solution fluidity, reduce the mass transfer rate of the target component in DES, hinder the formation of the target component-DES complex, and thus reduce the content of the target component. Adding an appropriate amount of water can reduce solvent viscosity and improve extraction efficiency, such as Figure 4 As shown in the figure, with the increase of water content, the content of KuA (lycopodione) and KuB (lycopodione B) increases continuously; when the water content is 30%, the maximum content of KuA and KuB is 2.41±0.04mg / g and 17.41±0.10mg / g respectively; when the water content exceeds 30%, the content of KuA and KuB will gradually decrease. It is speculated that the increase of water content may weaken the interaction between KuA, KuB and DES, resulting in a decrease in the extraction rate. Therefore, 20%, 30%, and 40% water contents were selected for the next experiment.

[0059] 4) Effect of material-liquid ratio on the content of lycopodiella A and B

[0060] The effect of the solid-liquid ratio of 1:10–1:60 (g / ml) was investigated. Figure 5 The results in the results show that as the solid-liquid ratio increases from 1:20 to 1:60, the content of lycopodiol A and B does not increase significantly. Excessive solvent will lead to waste of resources and may also have an adverse effect on the extraction process. After comprehensive consideration, a solid-liquid ratio of 1:20 was selected for the next study.

[0061] Example 2: Response surface methodology to optimize the extraction conditions of lycopodiella serrata and lycopodiella serrata

[0062] 1. Response surface optimization experimental design results and variance analysis

[0063] According to the results of the single factor experiment, the extraction temperature (A), extraction time (B) and water content (C) were taken as the optimization parameters affecting the extraction, and the total content of lycopodiella and lycopodiella (Y) was taken as the response value. The response surface analysis was carried out according to the BBD design principle. The results are shown in Table 3.

[0064] Table 3: Response surface experimental design and results

[0065]

[0066] As shown in Table 4, the regression model determination coefficient R 2 =0.9947, Corrected determination coefficient R 2(Adj) = 0.9878, the regression equation model is extremely significant (P < 0.0001), indicating that the model is consistent with the actual situation and is reliable. The lack of fit term is not significant (P = 0.4656 > 0.05), indicating that non-experimental factors have little effect on the content of lycium barbarum and lycium barbarum, and there are few abnormal errors. The single factor extraction temperature (A), extraction time (B), and water content (C) have significant effects (P < 0.05), the interaction effect of the extraction temperature and extraction time interaction term (AB) is significant (P < 0.05), and the interaction effect of the extraction temperature and water content interaction term (AC) and the extraction time and water content interaction term (BC) is not significant (P > 0.05). According to the size of the F value, the influence of each factor on the content of lycium barbarum and lycium barbarum is B> A> C, that is, extraction time> extraction temperature> water content.

[0067] Table 4: Regression model and variance analysis table

[0068]

[0069] Note: p<0.01 is extremely significant, p<0.05 is significant

[0070] 2. Response surface plot analysis

[0071] The response surface variance analysis and regression equation model were established for lycopodin A and lycopodin B using Design-expert software. Figure 6 shown.

[0072] Depend on Figure 6 The influence of various factors on the content of lycium barbarum A and B and the interaction between the factors can be analyzed. If any one of the factors, extraction temperature (A), extraction time (B) and water content (C), is kept at zero level, the interaction between the other two factors and their influence on lycium barbarum A and B can be analyzed. Figure 6 The results show that from the trend and slope of the response surface, Figure 6 The surface trend is steep, the high lines are elliptical and the curves are dense, indicating that the interaction effect between extraction time and extraction temperature is significant, and the interaction effect has a significant impact on the response value.

[0073] 3. Optimal conditions and verification experiments

[0074] The regression equation was obtained by analysis of Design-expert software. Based on the regression model predicting the best optimization conditions for extracting Lycium bark, and in view of the operability and convenience of the process flow, the revised process parameters for the extraction of Lycium bark A and Lycium bark B were: extraction temperature 43°C, extraction time 33 min, water content 30.5%, and the maximum total content of Lycium bark A and Lycium bark B reached 19.83 mg / g.

[0075] Example 3: High-speed countercurrent chromatography separation

[0076] The method determined in Example 1 and Example 2 is used to extract an extract containing lycopodione and lycopodione from the cortex lycopodione, and the specific method is as follows:

[0077] Accurately weigh 0.1 g of Radix Lycii powder, add 2 ml of DES (hydrogen bond acceptor is choline chloride, hydrogen bond donor is lactic acid, molar ratio is 1:2) to a 5 ml centrifuge tube for mixing, and perform ultrasonic extraction for 30 min (ultrasonic power is 500 W, temperature is 45°C) to obtain the Radix Lycii extract.

[0078] The extract of Lycium bark is passed through an ion exchange resin, eluted with water, the eluate is concentrated by rotary evaporation, and dried under reduced pressure and vacuum to obtain the enriched part of Lycium bark A and Lycium bark B. The ion exchange resin is one of gel strong acid styrene, gel strong base styrene, macroporous strong base styrene, and macroporous weak acid phenyl acrylate; the elution flow rate is 2-5 BV / h.

[0079] The rotary evaporation concentration water bath temperature is 45-60° C., and the vacuum degree is greater than 0.09 MPa; the reduced pressure vacuum drying temperature is 45-60° C., and the vacuum degree is greater than 1.00 MPa.

[0080] Take 0.3g of the enriched part of lycopodipine and lycopodipine collected by ion exchange resin, and dissolve it with 5mL solvent (take 2.5mL of each upper and lower phase). The solvent system is ethyl acetate: n-butanol: water: methanol = 0.5:4.5:5:0.2, K value is 0.7, flow rate is 2mL / min, retention rate is 40%, wavelength is 214nm, rotation speed is 896.5rpm, FWD mode, positive connection and positive output (In in Out out), the automatic collector automatically collects one tube every 4min, and one tube contains about 8mL. High-speed countercurrent chromatogram is shown in Fig. 9 .

[0081] Example 4: High performance liquid chromatography analysis

[0082] The lycopodione and lycopodione obtained by high-speed countercurrent chromatography separation and preparative liquid chromatography purification were dissolved and filtered through a 0.45 μm filter membrane, and analyzed by high-performance liquid chromatography, using a chromatographic column (4.6 mm×250 mm, 5 μm) filled with octadecylsilane bonded silica gel, and the mobile phase was 0.1% trifluoroacetic acid (A) and acetonitrile (B). Elution gradient: 0-10 min, 12%-14.5% B; 10-11 min, 14.5%-12% B; 11-16 min, 12% B. The flow rate was 1 mL / min, and the injection volume was 20 μL. The purity of lycopodione was 96.32%, and the purity of lycopodione was 98.55%.

[0083] The present invention is not limited to the above-mentioned optimal implementation mode. Any other products identical or similar to the present invention obtained by anyone under the inspiration of the present invention shall fall within the protection scope of the present invention.

Claims

1. A deep eutectic solvent for extracting lycopodine and lycopodine, characterized in that: The low eutectic solvent contains a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is choline chloride or levulinic acid; and the hydrogen bond donor is levulinic acid, lactic acid, n-butyric acid, ethylene glycol, 1,2-propylene glycol, glycerol, 1,4-butanediol, 1,6-hexanediol or urea.

2. The deep eutectic solvent according to claim 1, characterized in that The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is lactic acid, and the molar ratio thereof is 1:

2.

3. Use of the deep eutectic solvent according to claim 1 or 2 in the preparation of a product for extracting lycopodine and lycopodine ethyl.

4. A method for extracting lycopodione and lycopodione from the cortex lycopodione, characterized in that: The method is to use the low eutectic solvent according to claim 1 or 2 for extraction to obtain a cortex lycopersicum extract containing cortex lycopersicum acetate and cortex ethyl lycopersicum acetate.

5. The method according to claim 4, characterized in that In the method described, the mass volume ratio of the Chinese wolfberry bark to the low eutectic solvent is 1:10-1:20; and ultrasound-assisted extraction is used, wherein the power of the ultrasound is 400W-600W, the temperature is 40°C-50°C, and the time is 20min-60min.

6. The method according to claim 4, characterized in that The method also includes the step of purifying lycopodipine and lycopodipine, which comprises the steps of passing the lycopodipine extract through an ion exchange resin, eluting with water of different pH values, concentrating the eluate by rotary evaporation, and drying under reduced pressure and vacuum to obtain lycopodipine and lycopodipine enriched parts; and then separating the lycopodipine and lycopodipine enriched parts through high-speed countercurrent chromatography to obtain lycopodipine and lycopodipine, respectively.

7. The method according to claim 6, characterized in that The ion exchange resin is one of gel strong acid styrene, gel strong base styrene, macroporous strong base styrene and macroporous weak acid benzoic acid.

8. The method according to claim 6, characterized in that The elution flow rate in the method is 2-5 BV / h; the rotary evaporation concentration water bath temperature is 45-60°C, and the vacuum degree is greater than 0.09Mpa; the reduced pressure vacuum drying temperature is 45-60°C, and the vacuum degree is greater than 1.00Mpa.

9. The method according to claim 6, characterized in that The solvents used in the high-speed countercurrent chromatography include ethyl acetate, n-butanol, water and methanol, wherein the ratio of ethyl acetate: n-butanol: water: methanol is 1-10:1-10:1-10:0.1-5.

10. A method for determining the purity of lycopodine and lycopodine samples, characterized in that: The method described is to use high performance liquid chromatography to determine the purity: after the sample is filtered through a 0.45 μm filter membrane, it is analyzed using high performance liquid chromatography, a chromatographic column with octadecylsilane bonded silica gel as a filler, and a mobile phase of 0.1% trifluoroacetic acid A and acetonitrile B; elution gradient: 0-10 min, 12%-14.5% B; 10-11 min, 14.5%-12% B; 11-16 min, 12% B; the flow rate is 1 mL / min, and the injection volume is 20 μL.

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