Method for extracting and separating securinone from different parts of honeysuckle

Abstract

The present application belongs to the technical field of traditional Chinese medicine active ingredient extraction, and particularly relates to a method for extracting and separating loganic acid from different parts of Lonicera japonica. The present application first extracts a crude product containing loganic acid from different parts of Lonicera japonica, and then separates and purifies the crude product by high-speed counter-current chromatography, with water-saturated n-butanol as the upper phase, n-butanol-saturated water as the lower phase, and the lower phase as the mobile phase, so as to realize separation and purification of loganic acid. The present application has high separation efficiency, large separation amount, no pollution, high safety factor for industrial production, and the recovered solvent can be repeatedly used without affecting the effect. The percentage content of loganic acid separated by the present application is greater than or equal to 95%.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of active ingredients of traditional Chinese medicine, and specifically relates to a method for extracting and separating strychnos nux-vomica acid from different parts of Lonicera japonica. Background Technology

[0002] Honeysuckle (Lonicera japonica Thunb.) is a semi-evergreen climbing vine belonging to the genus Lonicera in the family Caprifoliaceae. According to the Compendium of Materia Medica, honeysuckle is sweet and cold in nature, clearing heat and detoxifying, reducing inflammation and swelling, and is effective against bacterial dysentery and various suppurative diseases. Strychnos nux-vomica acid is an iridoid compound with a hemiacetal hydroxyl group in its molecular structure. It is the main component of iridoid compounds found in honeysuckle flowers and possesses antibacterial, antiviral, antitumor, antioxidant, immune-enhancing, antipyretic, anti-inflammatory, choleretic, and hepatoprotective effects, showing great application potential. However, methods for obtaining strychnos nux-vomica acid are rarely reported.

[0003] High-speed countercurrent chromatography (HSCCC) is a continuous and highly efficient liquid-liquid partition chromatography technique developed in the 1980s. It does not require any solid support or carrier. It utilizes a unique unidirectional hydrodynamic equilibrium established within a high-speed rotating spiral tube, where one phase acts as the stationary phase and the other as the mobile phase. During continuous elution, a large amount of the stationary phase is retained. Because no solid support is needed, the separation of substances is achieved based on their different partition coefficients in the two phases, thus avoiding sample loss, inactivation, and denaturation caused by irreversible adsorption. This allows for complete sample recovery, and the recovered sample better reflects its original characteristics, making it particularly suitable for the separation of natural bioactive components. Furthermore, the sufficient contact between the separated substances and the liquid stationary phase greatly increases the sample yield, making it an ideal preparative separation method. Summary of the Invention

[0004] This invention provides a method for extracting and separating strychnine from different parts of Lonicera japonica. The method first extracts crude products containing strychnine from different parts of Lonicera japonica, and then purifies them by high-speed countercurrent chromatography. Water-saturated n-butanol is used as the upper phase, and water saturated with n-butanol is used as the lower phase. The lower phase is used as the mobile phase to achieve the separation and purification of strychnine. The method has high separation efficiency, large separation volume, high recovery rate, good reproducibility, and is simple and easy to operate. It can be effectively used for the separation of high-purity substances from a large number of traditional Chinese medicines.

[0005] The method for extracting and separating strychnos nux-vomica acid from different parts of Lonicera japonica according to the present invention comprises the following steps:

[0006] (1) Extraction: Honeysuckle was extracted with an extraction solvent, filtered, and the filtrates were combined to obtain the extract;

[0007] (2) Column separation: The solvent of the extract is recovered, concentrated, separated by macroporous adsorption resin chromatography column, concentrated, and dried into a dry paste, which is the crude product containing strychnine.

[0008] (3) High-speed countercurrent chromatography separation and purification: The crude product containing strychnine was separated and purified by high-speed countercurrent chromatography: a water-n-butanol saturated solution was prepared, and the upper phase was used as the stationary phase. The upper phase was filled into the entire column of the countercurrent chromatograph at a flow rate of 20-40 ml / min. The temperature of the constant temperature circulator was set to 15-35℃. After turning on the countercurrent chromatograph at a speed of 600-900 rpm, the column was equilibrated for 5-15 minutes. The lower phase was used as the mobile phase and pumped into the high-speed countercurrent column at a flow rate of 1-5 ml / min until the column was filled with the lower phase. The column was then equilibrated for 5-20 minutes. The crude product containing strychnine was dissolved in the lower phase. After the separation system was equilibrated, the sample was injected through the injection valve. The wavelength was set to 240 nm. The target component was collected according to the detector spectrum. The solvent was recovered and dried to obtain dry powder, i.e., strychnine. If the obtained strychnine content does not meet the requirements, the dry powder can be separated again under the above countercurrent chromatography conditions 1 to 2 times to obtain strychnine that meets the requirements.

[0009] The extraction solvent is a mixed solution of alcohol and water, wherein the volume fraction of alcohol in the mixed solution is 0-40%, and the alcohol is methanol or ethanol.

[0010] During the extraction process, the solid-liquid ratio is 1:5 to 15.

[0011] During column separation, the solution is eluted sequentially with water and 10-30% (v / v) ethanol aqueous solution, and the solution obtained by elution with 20% (v / v) ethanol aqueous solution is collected.

[0012] The temperature of the thermostatic circulator is 25-30℃;

[0013] The mobile phase is pumped into the high-speed countercurrent chromatogram at a flow rate of 2–3 ml / min.

[0014] The main unit of the countercurrent chromatograph rotates at 800 rpm.

[0015] The present invention has the following beneficial effects:

[0016] (1) This is the first time that high-speed countercurrent chromatography has been used to separate and purify strychnine. Compared with other separation and purification methods, it has high separation efficiency, large separation volume, high recovery rate, good reproducibility, simple operation and easy to master. It can be effectively used for the separation of a large number of high-purity substances from traditional Chinese medicine.

[0017] (2) Innovation in the solvent system: This invention uses an unconventional water-saturated n-butanol system, with water saturated with n-butanol as the mobile phase. This allows for reuse, achieving environmental protection, safety, and cost reduction. In existing technologies, high-speed countercurrent chromatography typically uses solvents such as petroleum ether, diethyl ether, ethyl acetate, n-butanol, isopropanol, acetonitrile, methanol, ethanol, and water. Three to six different solvents are mixed in a certain proportion, and after equilibrium, the upper and lower phases are separated for operation. One phase is used as the mobile phase, and the other as the stationary phase. High-speed countercurrent chromatography is used for separation, and the mobile phase is collected and heated for recovery. Because different solvents have different boiling points, the solvent ratio of the recovered mobile phase is inconsistent with the ratio of the mobile phase before separation, resulting in the recovered mobile phase being unusable for high-speed countercurrent chromatography separation. During the experiment, a large amount of mobile phase is needed to meet experimental requirements. Since the recovered phase cannot be reused, this not only increases the separation cost but also incurs extremely high costs for mobile phase storage and harmless treatment, limiting the application of high-speed countercurrent chromatography technology in large-scale production and causing serious environmental pollution. This invention uses water-saturated n-butanol as the upper phase and water-saturated with n-butanol as the lower phase. The lower phase is used as the mobile phase. After separation by high-speed countercurrent chromatography, the mobile phase is collected and recovered by heating. Because the equilibrium system of water and n-butanol is only related to temperature and pressure, the recovered mobile phase, placed under certain temperature and pressure conditions, can have its equilibrium restored by adding a very small amount of water or n-butanol, maintaining the same ratio as the initial mobile phase. The lower solvent layer can then be reused as the mobile phase, thus significantly reducing the amount of organic solvent used and protecting the environment.

[0018] (3) Extensive experiments have shown that the unconventional water-saturated n-butanol system provides better separation for strychnine. Compared to conventional systems such as ethyl acetate-n-butanol-water, ethyl acetate-ethanol-water, ethyl acetate-methanol-water, ethyl acetate-isopropanol-water, n-butanol-ethanol-water, petroleum ether-ethanol-water, and diethyl ether-ethanol-water, the unconventional water-n-butanol solvent system exhibits the best partition coefficient for strychnine and is therefore highly suitable as a high-speed countercurrent chromatography separation solvent system for strychnine, achieving the best separation effect.

[0019] (4) The main solvent in the mobile phase is water, which makes it safer. As is well known, the mobile phase is used in the largest quantity in high-speed countercurrent chromatography. This invention uses water saturated with n-butanol as the mobile phase. The mobile phase is mostly water, and the amount of organic solvent n-butanol used is very small, which has almost no pollution to the environment. At the same time, it is low in cost, easy to operate, and the production process is not likely to cause accidents such as explosions. It has a high safety factor, is suitable for the needs of large-scale production, and has high practical value.

[0020] (5) The present invention uses water and n-butanol to prepare the mobile phase and stationary phase. The two phases separate into layers very quickly, the operation is simple, emulsification is difficult to occur, and even if there is occasional emulsification, it will quickly subside, making it easier to operate in large-scale production.

[0021] (6) Using the above system, high-speed countercurrent chromatography was used to separate strychnine, and the percentage content of strychnine was ≥95% (area normalization method). Attached Figure Description

[0022] Figure 1 For the upper phase, construct the HSCCC spectrum of the stationary phase;

[0023] Figure 2 The liquid chromatography spectrum of strychnine reference standard;

[0024] Figure 3 The liquid phase spectrum of peak 1;

[0025] Figure 4 The liquid phase spectrum of peak 2;

[0026] Figure 5 The liquid phase spectrum of peak 3;

[0027] Figure 6 The total ion flow rate is shown in negative ion mode.

[0028] Figure 7 This is the electrospray ionization source-mass spectrum of strychnine obtained in this invention. Detailed Implementation

[0029] Example 1

[0030] A method for extracting and isolating strychnine reference standard from honeysuckle, the specific steps of which are as follows:

[0031] (1) Extraction: Take 800g of honeysuckle (flower of Lonicera japonica), crush it, pass it through a No. 2 sieve, add extraction solvent to extract, filter it, and combine the filtrates to obtain the extract; the extraction solvent is a mixed solution of ethanol and water, and the volume fraction of ethanol in the mixed solution is 20%; the solid-liquid ratio is 1:10;

[0032] (2) Column separation: The solvent of the extract was recovered, concentrated, and separated using a D101 macroporous resin column. The extract was then concentrated and dried into a dry paste, i.e., the crude product containing strychnine. During column separation, the extract was eluted sequentially with water and 10% and 20% (v / v) ethanol aqueous solutions, and the solution obtained from the 20% (v / v) ethanol aqueous solution was collected.

[0033] (3) High-speed countercurrent chromatography separation and purification: The crude product containing strychnine was separated and purified by high-speed countercurrent chromatography: a water-n-butanol saturated solution was prepared, the upper phase was used as the stationary phase, and the upper phase was filled into the entire column of the countercurrent chromatograph at a flow rate of 30 ml / min. The temperature of the constant temperature circulator was set to 25℃, and the countercurrent chromatograph was turned on at 800 rpm and equilibrated for 10 minutes. The lower phase was used as the mobile phase and pumped into the high-speed countercurrent column at a flow rate of 2.5 ml / min until the lower phase filled the column and equilibrated for 15 minutes. The crude product containing strychnine was dissolved in the lower phase, and after the separation system was equilibrated, it was injected through the injection valve. The wavelength was set to 240 nm, and the target component was collected according to the detector spectrum. The solvent was recovered, dried, and the dry powder, i.e., strychnine, was obtained.

[0034] Example 2: Optimization of Extraction Conditions

[0035] (1) A parallel experimental method was adopted. Honeysuckle (flower of Lonicera japonica) samples were extracted by ultrasonic extraction. The content of strychnine in the dry extracts of different alcohol-water mixtures (ethanol volume fractions of 10%, 20%, 30%, 50%, 75%, and 85%) was investigated, and the extraction solvent was determined by comparing the results. The results showed that the contents of each extract were 17, 15, 15, 14, 14, 11, and 11 mg / g, respectively. However, water extraction and 10% ethanol extraction had too many impurities and contained more polar components such as sugar, which was not conducive to the next step of separation. 30% ethanol extraction required a large amount of ethanol, and the above extraction solvents were not much different from 20% ethanol extraction. Therefore, this invention selected 20% ethanol for extraction.

[0036] (2) A parallel experimental method was used, with 20% ethanol as the extraction solvent to extract samples. The content of strychnine in the dry extract was investigated under different extraction methods (ultrasound, reflux), different solid-liquid ratios (1:4, 1:10, 1:15), different extraction times (45, 60, 90 minutes), and extraction times (1, 2, 3 times). The results were compared to determine the extraction method, solvent volume, and extraction time. The results showed that: ① The extraction efficiency of ultrasound and reflux was not significantly different. ② Extraction time, extraction times, and solid-liquid ratio had a significant impact on the transfer rate of strychnine; strychnine could be extracted under different conditions. Considering factors such as extraction rate and energy saving, the optimal extraction conditions were a solid-liquid ratio of 1:10, 3 extractions, and 30 minutes each time.

[0037] Table 1 Optimization Results of Extracted Parameters

[0038]

[0039]

[0040] Example 3: Pre-selection of column separation conditions

[0041] Take 800g of honeysuckle (flower of Lonicera japonica) medicinal material, crush it, pass it through a No. 2 sieve, and extract it three times by reflux with 10 times the amount of 20% ethanol, each time for 45 minutes. Filter, combine the filtrates, concentrate the filtrate to an appropriate volume, and load it onto a D101 macroporous resin column. Elute with water and ethanol of different concentrations (water solutions of ethanol with volume fractions of 10%, 20%, 30%, 40%, 50%, and 60%) in sequence. Collect the eluted solution, concentrate it, and dry it into a dry extract, which is the crude product containing strychnine. Analyze it by liquid chromatography.

[0042] The results showed that the crude products eluted with 10%, 20%, and 30% ethanol all contained strychnine, with the crude products eluted with 20% and 30% ethanol containing fewer impurities. The experiment compared and analyzed the strychnine content of the crude products eluted with macroporous resin at different concentrations of ethanol. The results are shown in Table 2. The 20% ethanol elution fraction had the highest strychnine content; therefore, 20% ethanol is the optimal elution solvent.

[0043] Table 2 Optimal results of elution solvents

[0044]

[0045] Example 4: Optimization of high-speed countercurrent chromatography separation conditions

[0046] (1) Determination of the distribution coefficient K value

[0047] High-speed countercurrent chromatography is used to efficiently separate strychnine. The solvent system is the most critical factor, as it has a significant impact on the separation effect.

[0048] Strychnos nux-vomica is a strongly polar compound. Experiments were conducted on different polar solvent systems. The appropriate solvent system was selected by measuring the partition coefficient K value of strychnos nux-vomica in the upper and lower phases of different solvent systems. Generally, the permissible range of the partition coefficient K value is 0.2 to 5, and the K value of 0.5 to 2 is the most suitable.

[0049] Table 3. Partition coefficients of strychnos nux-vomica acid in different systems

[0050]

[0051]

[0052] Therefore, in this experiment, we used water-n-butanol as the solvent system for high-speed HSCCC separation of strychnine.

[0053] (2) High-speed countercurrent chromatography separation

[0054] A water-saturated n-butanol upper phase was used as the stationary phase and filled the entire column of the high-speed countercurrent chromatograph at a flow rate of 30 ml / min. The temperature of the circulator was set to 25°C. The countercurrent chromatograph was turned on at 800 rpm and allowed to equilibrate for 10 minutes. A water-saturated n-butanol lower phase was used as the mobile phase and pumped into the high-speed countercurrent column at a flow rate of 2.5 ml / min until the column was filled. After equilibration for 15 minutes, a small amount of the lower phase was used to dissolve the crude strychnine from "Example 3" to prepare a sample solution. The sample solution was rapidly injected into the high-speed countercurrent chromatograph through the injection valve at a wavelength of 240 nm. The target components were collected according to the detector chromatogram. Figure 1 The solvent was recovered under reduced pressure, and the powder was obtained by evaporation to dryness. The powder was dissolved in 10 ml of 20% ethanol and analyzed by HPLC. The content of strychnine in the powder was calculated, and the results are as follows: Figure 3-5 The results showed that peak 2 was the ideal collection peak, and its value was 96.0% when determined by the external standard method.

[0055] Example 5: Investigation on the separation effect of different medicinal parts of honeysuckle

[0056] Take three parallel portions of different parts of honeysuckle stems, leaves, and flowers, each weighing 800g. Crush the powder, pass it through a No. 2 sieve, and extract it three times with 10 times the volume of 20% ethanol for 45 minutes each time. Filter the mixture, combine the filtrates, concentrate the filtrate to an appropriate volume, and load it onto a D101 macroporous resin column. Elute with water and ethanol of different concentrations (water solutions of ethanol with volume fractions of 10% and 20%, respectively). Collect the 20% ethanol eluent, concentrate and dry it into a dry paste to obtain crude strychnine for later use. A water-saturated n-butanol upper phase was used as the stationary phase and filled the entire column of the high-speed countercurrent chromatograph at a flow rate of 30 ml / min. The temperature of the circulator was set to 25°C. After turning on the countercurrent chromatograph at 800 rpm, it was allowed to equilibrate for 10 minutes. A water-saturated n-butanol lower phase was used as the mobile phase and pumped into the high-speed countercurrent column at a flow rate of 2.5 ml / min until the column was filled. After equilibration for 15 minutes, crude strychnine was collected to prepare a sample solution. The sample solution was rapidly injected into the high-speed countercurrent chromatograph through the injection valve at a wavelength of 240 nm. The target components were collected based on the detector chromatogram. Figure 1 The solvent was recovered under reduced pressure, and the powder was obtained by evaporation. The powder was dissolved in 10 ml of 20% ethanol and analyzed by HPLC. The content of strychnine in the powder was calculated. The results showed that this method has a good effect on the separation and purification of strychnine from different medicinal parts of honeysuckle.

[0057] Table 4. Content of Strychnos nux-vomica obtained from different medicinal parts

[0058]

[0059]

[0060] Example 6: Evaluation of Solvent Recovery and Separation Effect

[0061] The solvent recovered under reduced pressure in "Example 4" was allowed to stand at room temperature for 3 hours until the layers were separated. Then, an appropriate amount of water or n-butanol was added, mixed well, and allowed to stand for layer separation. The water-saturated n-butanol system was prepared again and used as the solvent system for high-speed countercurrent chromatography to separate strychnine. The separation effect of reusing the recovered solvent was investigated.

[0062] The crude strychnine from "Example 3" was dissolved in the lower phase of water-saturated n-butanol prepared by recycled solvent. The procedure was the same as that in "Example 4". Peak 2 was collected, and the content of strychnine in the dry powder was determined by external standard method. The results are shown in Table 5.

[0063] Table 5. Content of Strychnos nux-vomica obtained with different solvents

[0064]

[0065] The results showed that the water-saturated n-butanol system prepared by recovering the solvent could be repeatedly used for high-speed countercurrent chromatography to separate strychnine.

[0066] Example 7: Component Identification

[0067] Compound identification was performed using UPLC-MS-MS, with detection conducted in both positive and negative ion modes.

[0068] Positive ion mode: HESI ion source used; capillary voltage 3500V; capillary temperature 350℃; sheath gas 30; auxiliary gas 10; source temperature 350℃; mass spectrometry acquisition range 100–1200 m / z.

[0069] The negative ion mode detection conditions were as follows: HESI ion source; capillary voltage 3000V; capillary temperature 300℃; sheath gas 45℃; auxiliary gas 10℃; source temperature 300℃; mass spectrometry acquisition range 100–1200m / z.

[0070] The results showed that the molecular weight and characteristic fragment peaks of the target compound were consistent with those of strychnine reported in the literature. A comparison of the HPLC chromatograms of the target compound and strychnine revealed that their retention times were consistent. Therefore, the target compound was strychnine.

Claims

1. A method for extracting and isolating secojacowanine from different parts of Lonicera japonica, characterized in that, The solution was obtained by high-speed countercurrent chromatography (HSCLC) using water-saturated n-butanol as the upper phase and water-saturated n-butanol as the lower phase, with the lower phase as the mobile phase. Specifically, a water-n-butanol saturated solution was prepared, and the upper phase was used as the stationary phase. The upper phase was then pumped into the entire column of the HSCLC at a flow rate of 20–40 ml / min. The temperature of the circulator was set to 15–35 °C, and the HSCLC was turned on at 600–900 rpm for 5–15 minutes to equilibrate. The lower phase was then used as the mobile phase and pumped into the HSCLC column at a flow rate of 1–5 ml / min until the column was filled. After equilibration, the column was equilibrated for 5–20 minutes. The crude product containing strychnine was dissolved in the lower phase. After the separation system was in equilibrium, the sample was injected through the injection valve. The wavelength was set to 240 nm. The target component was collected according to the detector spectrum. The solvent was recovered and dried to obtain dry powder, which is strychnine. The process for obtaining the crude product containing strychnine is as follows: (1) Extraction: Extract different parts of honeysuckle with extraction solvent, filter, and combine the filtrates to obtain the extract; (2) Column separation: The solvent of the extract is recovered, concentrated, separated by macroporous adsorption resin chromatography column, concentrated, and dried into a dry paste, which is the crude product containing strychnine. The extraction solvent is a mixed solution of alcohol and water, wherein the volume fraction of alcohol in the mixed solution is 0-40%, and the alcohol is methanol or ethanol; During column separation, the solution is eluted sequentially with water and 10-30% (v / v) ethanol aqueous solution, and the solution obtained by elution with 20% (v / v) ethanol aqueous solution is collected.

2. The method for extracting and separating securin acid from different parts of Lonicera japonica according to claim 1, characterized in that, During the extraction process, the solid-liquid ratio is 1:5 to 15.

3. The method as claimed in claim 1, wherein the method for extracting and isolating seco-loganic acid from different parts of Lonicera japonica is characterized by, The temperature of the thermostatic circulator is 25-30℃.

4. The method as claimed in claim 1, wherein the separation of seco-strychnine acid is carried out from different parts of Lonicera japonica. The mobile phase is pumped into the high-speed countercurrent chromatogram at a flow rate of 2–3 ml / min.

5. The method as claimed in claim 1, wherein the separation of seco-strychnine acid is carried out from different parts of Lonicera japonica. The main unit of the countercurrent chromatograph rotates at 800 rpm.

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