A method for extracting and separating lycopene A and lycopene B from Lycium bark.

Abstract

This invention provides a method for the simultaneous extraction and separation of lycopene A and lycopene B from Lycium chinense root bark. Based on obtaining an effective eutectic solvent as the extractant, ultrasonic-assisted extraction is employed. Response surface methodology is used to optimize three factors significantly affecting the extraction process of lycopene A and lycopene B: extraction time, extraction temperature, and water content, thus obtaining optimal extraction conditions. Subsequently, the Lycium chinense root bark extract is passed through an ion exchange resin to remove the eutectic solvent. Finally, high-speed countercurrent chromatography (HSCLC) separation and preparative liquid chromatography (PCLC) purification are used to further obtain lycopene A and lycopene B with high purity, which are then detected and analyzed using high-performance liquid chromatography (HPLC).

Description

Technical Field

[0001] This invention belongs to the field of extraction and separation technology of active ingredients in traditional Chinese medicine, and specifically relates to a method for simultaneously extracting and separating Lycium chinense root bark extract A and Lycium chinense root bark B. Background Technology

[0002] Lycium chinense root bark is the dried root bark of Lycium chinense Mill. or Lycium barbarum L., both belonging to the Solanaceae family. It is sweet and cold in nature, and enters the lung, liver, and kidney meridians. It has the effects of cooling the blood and relieving fever, clearing the lungs and reducing heat, and is mainly used to treat yin deficiency with tidal fever, night sweats, lung heat cough, hemoptysis, epistaxis, and internal heat with thirst. Currently, there is no method for determining the content of active ingredients in Lycium chinense root bark, resulting in an incomplete quality evaluation system. Lycium chinense A and B are considered the main active biomarkers of Lycium chinense root bark, possessing activities such as lowering blood pressure, lowering blood sugar, lowering blood lipids, antipyretics, antibacterial, and antiviral properties. However, the difficulty in separating and preparing Lycium chinense A and B leads to a lack of corresponding standard substances, and consequently, a lack of quantitative detection methods for Lycium chinense root bark, affecting its quality evaluation. Currently, the extraction of Lycium chinense A and B extracts mainly relies on ultrasonic or reflux extraction with methanol, ethanol, or n-butanol. Traditional extraction methods consume a lot of solvents and are cumbersome, easily causing alterations to the target substances. Therefore, seeking and developing a green, efficient, and simple extraction technology is crucial. Based on this, further separation of Lycium chinense A and B extracts will provide a physical standard for the quality control of Lycium chinense root bark. Summary of the Invention

[0003] The purpose of this invention is to provide a method for the simultaneous extraction and separation of lycopene A and lycopene B from Lycium chinense root bark, thereby effectively improving the extraction efficiency and separation effect of lycopene A and lycopene B from Lycium chinense root bark.

[0004] This invention first provides a method for extracting Lycium bark extract A and Lycium bark B from Lycium bark. The extraction solvent is one of the following reagents: water, methanol, ethanol, acetonitrile, dichloromethane, eutectic solvent, etc., or different ratios of 2-3 reagents, to obtain an extract of Lycium bark containing Lycium bark extract A and Lycium bark B.

[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] In the method described, the mass-to-volume ratio of Lycium chinense root bark to eutectic solvent is 1:10 to 1:20 (g / ml).

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

[0008] The present invention also provides a eutectic solvent for extracting Lycium chinense A and Lycium chinense B, comprising 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-propanediol, 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 an application of the aforementioned eutectic solvent for the extraction of products containing Lycium chinense root extract A and Lycium chinense root extract B;

[0012] Furthermore, the extraction and separation method also includes the step of purifying Lycium bark extract A and Lycium bark B. This involves passing the above-mentioned Lycium bark extract through an ion exchange resin, eluting with water of different pH values, concentrating the eluent by rotary evaporation, and drying under reduced pressure to obtain enriched fractions of Lycium bark extract A and Lycium bark B. These enriched fractions are then separated by high-speed countercurrent chromatography. The obtained Lycium bark extract A and Lycium bark B fractions are further purified by preparative liquid chromatography to obtain Lycium bark extract A and Lycium bark B with a purity >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 styrene.

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

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

[0016] The vacuum drying temperature is 45–60°C, and the vacuum degree is >1.00 MPa.

[0017] 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.

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

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

[0020] The purity of the Lycium chinense root extract A and Lycium chinense root extract B samples obtained by this invention was determined by high performance liquid chromatography, and the content was determined by external standard method.

[0021] The high-performance liquid chromatography (HPLC) method was described as follows: Samples were filtered through a 0.45 μm filter membrane and analyzed using HPLC. A chromatographic column (4.6 mm × 250 mm, 5 μm) was used as the packing material. The mobile phase consisted of 0.1% trifluoroacetic acid (A) and acetonitrile (B). The elution gradient was: 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. Attached Figure Description

[0022] Figure 1 A coordinate graph showing the effects of different types of DES on the content of Lycium barbarum glycoside A and B;

[0023] Figure 2 A coordinate graph showing the effect of extraction time on the content of Lycium barbarum alpha and alpha-12-in-1.

[0024] Figure 3 A coordinate graph showing the effect of extraction temperature on the content of Lycium chinense root bark A and B.

[0025] Figure 4 A coordinate graph showing the effect of water content on the content of Lycium barbarum glycoside A and B;

[0026] Figure 5 A graph showing the effect of the material-liquid ratio on the content of Lycium bark extract A and B;

[0027] Figure 6 Response surface plot of the effects of extraction temperature, extraction time and moisture content on the yields of KuA and KuB;

[0028] Figure 7 High-speed countercurrent chromatograms of Lycium Root Extract A and Lycium Root Extract B;

[0029] Figure 8 High-performance liquid chromatogram of Lycium chinense root extract;

[0030] Figure 9 This is a high-performance liquid chromatogram of Lycium bark extract. Detailed Implementation

[0031] In this invention specification, DES is a eutectic solvent, HBA is a hydrogen bond acceptor, and HBD represents a hydrogen bond donor; the conditions for HPLC determination of the contents of lycium styracin and lycium styracin in the embodiments of this invention are as follows:

[0032] 1. HPLC Conditions: 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 system, with analysis conducted at 30 °C using a SinoChrom ODS-BP C18 column (5 μm, 4.6 mm × 250 mm). The mobile phase consisted of water containing 0.05% trifluoroacetic acid (mobile phase A) and acetonitrile containing 0.05% trifluoroacetic acid (mobile phase B). The gradient was: 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 Lycium chinense A and B are expressed in mg / g. The standard curves for Lycium chinense A and B are Y = 5267x + 23896(R²). 2 =0.9921,n=7), Y=6355367x+270224(R 2 =0.9910, n=7), where the contents of Lycium bark extract A and Lycium bark extract B are calculated according to the following formula:

[0034] Lycium chinense root extract content (mg / g) = (mass of lycium chinense root extract / mass of lycium chinense root bark)

[0035] Lycium bark extract content (mg / g) = (mass of Lycium bark extract / mass of Lycium bark)

[0036] 3. Data Processing

[0037] All experiments were repeated three times. The Dsign-Expert response surface methodology was used to analyze and optimize the relevant factor data extracted from Lycium chinense root extract (Lycium chinense root extract A and B), and SPSS 25.0 statistical software and Origin 2020 software were used for analysis and graphing.

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] Example 1: Screening of Eutectic Solvents

[0040] This embodiment provides a method for ultrasound-assisted extraction of lycopene A and lycopene B from Lycium chinense root bark using a DES. The DES is composed of HBA and HBD in a molar ratio of 1:2. The extraction of lycopene A and lycopene B from Lycium chinense root bark is performed using ultrasound-assisted DES. HBA is either choline chloride or levulinic acid; the hydrogen bond donor HBD is one of levulinic acid, lactic acid, n-butyric acid, ethylene glycol, 1,2-propanediol, glycerol, 1,4-butanediol, 1,6-hexanediol, or urea.

[0041] Non-medicinal parts and impurities in the dried Lycium chinense root bark raw material were removed, and the remaining part was crushed. 0.1g of Lycium chinense root bark powder was accurately weighed and added to 2ml of DES (30% water content) into a 5ml centrifuge tube for mixing. The mixture was ultrasonically extracted for 30min (ultrasonic power of 500W and temperature of 40℃). The contents of Lycium chinense root bark A and B were determined by high performance liquid chromatography.

[0042] According to Table 1, HBA and HBD were magnetically stirred at 80°C in a molar ratio of 1:2 until homogeneous and clear.

[0043] Table 1: Components and Molar Ratios of Different Types of DES

[0044]

[0045] As shown in Table 1, Lycium bark was extracted using 12 different types of DES. The extraction effects of the 12 DES on Lycium bark A and B were analyzed based on the content of Lycium bark A and B. The DES with the best extraction effect was selected for subsequent single-factor experiments.

[0046] 1) Single-factor experiment

[0047] Extraction of Lycium chinense root bark A and B was conducted under the following conditions: ultrasonic power 500W, extraction temperature 40℃, extraction time 30 min, and water content 30%. The effects of different liquid-to-solid ratios (1:20, 1:30, 1:40, 1:50, 1:60) on the content of Lycium chinense root bark A and B were compared. With the liquid-to-solid ratio kept constant at 1:20 (g / ml), extraction temperature 40℃, and extraction time 30 min, the effects of different water contents (10%, 20%, 30%, 40%, 50%) on the content of Lycium chinense root bark A and B were compared. The effects of different extraction times (10 min, 20 min, 30 min, 40 min, and 50 min) on the content of lycium bark extract A and B were compared, while keeping the liquid-to-solid ratio at 1:20 (g / ml), the extraction temperature at 40℃, and the water content at 30%. The effects of different extraction temperatures (20℃, 30℃, 40℃, 50℃, and 60℃) on the content of lycium bark extract A and B were also compared, while keeping the liquid-to-solid ratio at 1:20 (g / ml), the extraction time at 30 min, and the water content at 30%.

[0048] Based on the results of the single-factor experiments, an analysis was conducted using extraction temperature (A), extraction time (B), and moisture content (C) as independent variables, and lycium bark extract and lycium bark extract (g) as response values, following a BDD design with three factors and three levels. The experimental design factors and levels are shown in Table 2.

[0049] Table 2: Factors and Levels in Response Surface Design Experiments

[0050]

[0051] according to Figure 1 It can be seen that among the 12 DES types affecting the content of lycopene A and lycopene B, DES-2 (choline chloride-lactic acid) showed the best extraction effect, with lycopene A content at 2.41±0.04 mg / g and lycopene B content at 17.41±0.10 mg / g. Therefore, the eutectic solvent of choline chloride-lactic acid was chosen for subsequent experiments.

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

[0053] 1) Effect of extraction time on the content of Lycium chinense root bark extract A and B

[0054] Ultrasonic extraction time is a significant factor affecting extraction efficiency, and this condition needs to be optimized to minimize extraction time and reduce costs. In this experiment, the extraction time was between 10 and 50 minutes. Figure 2 The results showed that within 10–30 minutes, the extraction rates of Lycium chinense dermatitis A increased from 1.74±0.10 mg / g to 2.49±0.04 mg / g, and those of Lycium chinense dermatitis B increased from 9.62±0.34 mg / g to 17.12±0.39 mg / g. This indicates that the longer the contact time between the solvent and solute, the stronger the destructive effect of ultrasound on the solute, allowing the solvent to rapidly penetrate into the solid and dissolve the components in the solvent as completely as possible. The combined content of Lycium chinense dermatitis A and B reached its maximum at 30 minutes, and there was no significant change in content after 30 minutes. Therefore, 20, 30, and 40 minutes were selected for subsequent experiments.

[0055] 2) Effect of extraction temperature on the content of Lycium chinense root bark extract A and B.

[0056] High temperatures can increase the kinetic energy of DES molecules, reduce the viscosity of the eutectic solvent, and enhance the interaction between the solvent and solid particles, thereby improving extraction efficiency. However, excessively high temperatures can lead to the degradation of bioactive components and reduce extraction efficiency. Therefore, the effect of extraction temperatures ranging from 20 to 60°C on the content was investigated. Figure 3 As shown, the contents of lycopene A and B increase with increasing temperature, reaching a maximum at 40℃. However, once the temperature exceeds 40℃, the contents of lycopene A and B decrease slightly. Increased temperature enhances the cavitation effect, and the combination of these two effects leads to a relative increase in extraction efficiency. However, further increases in temperature have the opposite effect on cavitation, as the intensity of cavitation decreases with increasing temperature. Therefore, 30℃, 40℃, and 50℃, which exhibit significant variations, were selected as the response surface methodology experimental conditions.

[0057] 3) Effect of moisture content on the content of Lycium barbarum glycoside A and B

[0058] The presence of hydroxyl groups in DES generates more hydrogen bonds, leading to increased intermolecular attraction and higher solvent viscosity. High viscosity reduces solution flowability, decreases the mass transfer rate of the target component in DES, and hinders the formation of the target component-DES complex, thus reducing the content of the target component. Adding an appropriate amount of water can reduce solvent viscosity and improve extraction efficiency. Figure 4 As shown, the contents of KuA (lycium styraxein) and KuB (lycium styraxein B) continuously increase with increasing water content. At a water content of 30%, the maximum contents of KuA and KuB are 2.41±0.04 mg / g and 17.41±0.10 mg / g, respectively. When the water content exceeds 30%, the contents of KuA and KuB gradually decrease. It is speculated that the increased water content weakens the interaction between KuA, KuB, and DES, leading to a decrease in extraction rate. Therefore, water contents of 20%, 30%, and 40% were selected for further experiments.

[0059] 4) Effect of the material-to-liquid ratio on the content of Lycium barbarum glycoside A and B.

[0060] Investigate the effect of a material-liquid ratio of 1:10–1:60 (g / ml). Figure 5 The results showed that the contents of lycium chinense glycoside A and B did not increase significantly as the solid-liquid ratio increased from 1:20 to 1:60. Excessive solvent will lead to resource waste and may also have adverse effects on the extraction process. Taking all factors into consideration, a solid-liquid ratio of 1:20 was selected for further research.

[0061] Example 2: Results of Response Surface Methodology Optimization of Extraction Conditions for Lycium Root Extract A and B

[0062] 1. Results and Analysis of Variance of Response Surface Optimization Experimental Design

[0063] Based on the results of the single-factor experiments, extraction temperature (A), extraction time (B), and moisture content (C) were selected as the optimal parameters affecting extraction. The total content of lycium chinense glycoside A and B (Y) was used as the response value. Response surface analysis was conducted based on the BBD design principle, and the results are shown in Table 3.

[0064] Table 3: Response Surface Experimental Design and Results

[0065]

[0066] As shown in Table 4, the coefficient of determination R of the regression model is... 2 = 0.9947, Corrected coefficient of determination R 2The regression equation (Adj) = 0.9878, indicating that the model is highly significant (P < 0.0001), demonstrating its good agreement with reality and reliability. The lack-of-fit term was not significant (P = 0.4656 > 0.05), indicating that non-experimental factors had a relatively small impact on the content of Lycium chinense glycosides and Lycium chinense glycosides, with minimal abnormal errors. The single-factor effects of extraction temperature (A), extraction time (B), and moisture content (C) were all significant (P < 0.05). The interaction between extraction temperature and extraction time (AB) was significant (P < 0.05), while the interactions between extraction temperature and moisture content (AC) and extraction time and moisture content (BC) were not significant (P > 0.05). Based on the F-values, the influence of each factor on the content of Lycium chinense glycosides and Lycium chinense glycosides was B > A > C, i.e., extraction time > extraction temperature > moisture content.

[0067] Table 4: Regression Model and Analysis of Variance

[0068]

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

[0070] 2. Response Surface Analysis

[0071] The response surface variance analysis and regression equation model of Lycium barbarum extract A and B were established using Design-expert software, such as... Figure 6 As shown.

[0072] Depend on Figure 6 This study analyzes the effects of various factors on the content of Lycium chinense glycoside A and B, as well as the interactions between these factors. It maintains one of the extraction factors (A), extraction time (B), and moisture content (C) at a zero level, and examines the interaction between the other two factors and their effects on Lycium chinense glycoside A and B. Figure 6 The results show that, judging from the trend and slope of the response surface, Figure 6 The surface curves are steep, and the high lines are all elliptical in shape with dense curves, indicating that the interaction between extraction time and extraction temperature is significant, and their 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 using Design-expert software. Based on the optimal conditions for extracting Lycium chinense root bark predicted by the regression model, and considering the operability and convenience of the process, the modified process parameters for extracting Lycium chinense root bark A and B are: extraction temperature 43℃, extraction time 33min, water content 30.5%, and the total content of Lycium chinense root bark A and B reaches a maximum of 19.83mg / g.

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

[0076] The method determined in Examples 1 and 2 was used to extract an extract containing lycopene A and lycopene B from Lycium chinense root bark. The specific method is as follows:

[0077] Accurately weigh 0.1g of Lycium chinense root bark powder, add 2ml of DES (hydrogen bond acceptor is choline chloride, hydrogen bond donor is lactic acid, molar ratio is 1:2) to a 5ml centrifuge tube and mix. Extract by sonication for 30min (ultrasonic power is 500W, temperature is 45℃) to obtain Lycium chinense root bark extract.

[0078] The extract of Lycium chinense root bark was passed through an ion exchange resin, eluted with water, and the eluent was concentrated by rotary evaporation and dried under reduced pressure to obtain the enriched fractions of Lycium chinense root bark A and B. The ion exchange resin was one of the following: gel-strong acid styrene, gel-strong base styrene, macroporous strong base styrene, or macroporous weak acid styrene; the elution flow rate was 2–5 BV / h.

[0079] The rotary evaporation concentration water bath temperature is 45-60℃, and the vacuum degree is >0.09Mpa; the reduced pressure vacuum drying temperature is 45-60℃, and the vacuum degree is >1.00Mpa.

[0080] Take 0.3 g of the enriched fractions of Lycium chinense A and B collected by ion exchange resin and dissolve them in 5 mL of solvent (2.5 mL each of the upper and lower phases). The solvent system is ethyl acetate: n-butanol: water: methanol = 0.5:4.5:5:0.2, K value 0.7, flow rate 2 mL / min, retention rate 40%, wavelength 214 nm, rotation speed 896.5 rpm, FWD mode, positive inlet and positive outlet (In in, Out out), automatic collector automatically collects one tube every 4 min, one tube contains approximately 8 mL. The high-speed countercurrent chromatogram is shown below. Figure 9 .

[0081] Example 4: High Performance Liquid Chromatography Analysis

[0082] Lycium chinense A and B, separated by high-speed countercurrent chromatography and purified by preparative liquid chromatography, were dissolved and filtered through a 0.45 μm filter membrane before analysis using high-performance liquid chromatography (HPLC). A chromatographic column (4.6 mm × 250 mm, 5 μm) packed with octadecylsilane-bonded silica gel was used. The mobile phase was 0.1% trifluoroacetic acid (A) and acetonitrile (B). The elution gradient was: 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 lycium chinense A was 96.32%, and the purity of lycium chinense B was 98.55%.

[0083] This invention is not limited to the preferred embodiments described above. Any other products that are the same as or similar to this invention, derived by anyone under the guidance of this invention, fall within the protection scope of this invention.

Claims

1. A method for simultaneous extraction of oxymatrine and oxysophoramine from Cortex Lycii, characterized in that, The method involves using an eutectic solvent for ultrasonic-assisted extraction to obtain a Lycium bark extract containing Lycium bark extract A and Lycium bark extract B. The extract of Lycium chinense root bark was passed through an ion exchange resin and eluted with water of different pH values. The eluent was concentrated by rotary evaporation and dried under reduced pressure to obtain the enriched fractions of Lycium chinense root bark A and Lycium chinense root bark B. The enriched fractions of Lycium chinense root bark A and Lycium chinense root bark B were then separated by high-speed countercurrent chromatography to obtain Lycium chinense root bark A and Lycium chinense root bark B, respectively. The hydrogen bond acceptor of the eutectic solvent is choline chloride, and the hydrogen bond donor is lactic acid, with a molar ratio of 1:2 and a water content of 30%.

2. The method as described in claim 1, characterized in that, In the method described, the mass-to-volume ratio of Lycium bark to eutectic solvent is 1:10 to 1:20; the ultrasonic power is 400W to 600W, the temperature is 40℃ to 50℃, and the time is 20min to 60min.

3. The method as described in claim 1, characterized in that, The ion exchange resin is one of the following: gel-strong acid styrene, gel-strong base styrene, macroporous strong base styrene, and macroporous weak acid styrene.

4. The method as described in claim 1, 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℃, and the vacuum degree is >0.09 Mpa.

5. The method as described in claim 1, characterized in that, The solvents used in the high-speed countercurrent chromatography are 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.

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