Method for separating and purifying Sichuan pepper alkaloid based on macroporous adsorption resin and measuring content of Sichuan pepper alkaloid

Abstract

The invention discloses a method for separating and purifying Sichuan pepper alkaloid based on macroporous adsorption resin and determining the content of the Sichuan pepper alkaloid. The method comprises the steps of extracting, separating and purifying the Sichuan pepper alkaloid, determining the content and formulating the quality standard of the Sichuan pepper alkaloid. NKA-9 type macroporous adsorption resin is adopted to effectively separate and enrich pepper alkaloid, and resin pretreatment conditions, an extraction process and elution conditions (the ethanol concentration is 40%-95%, the solid-liquid ratio is 1: 10-1: 30, the extraction temperature is 45-75 DEG C, and the pH is 2.5-5.5) are optimized, so that the purity and the recovery rate of alkaloid are remarkably improved. According to the present invention, the quality standard of the Sichuan pepper alkaloid is established by using the HPLC method, and the content determination methods of sinomenine, ethyoxyl chelerythrine, chelerythrine and rutaecarpin are set, such that the stability, the repeatability and the high recovery rate of the Sichuan pepper alkaloid are ensured. Precision, stability and sample recovery rate experiments prove that the method disclosed by the invention has good stability and repeatability and is suitable for industrial extraction and application of the pepper alkaloid.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of plant alkaloids, and in particular to a method for separating and purifying Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin and a method for determining the alkaloid content of Zanthoxylum bungeanum. Background Art

[0002] Zanthoxylum spp. is a traditional Chinese spice plant, widely used in the food, pharmaceutical, and daily chemical industries. The main active ingredients in Zanthoxylum are alkaloids, particularly chelerythrine and sinomenine, which have various pharmacological effects, including antibacterial, analgesic, antipyretic, and anti-inflammatory. Therefore, the extraction, separation, and purification of Zanthoxylum alkaloids are of great significance for their pharmaceutical applications and industrial production. However, the diverse and low content of alkaloids in Zanthoxylum spp. poses numerous technical challenges in their separation and purification.

[0003] Currently, traditional methods for isolating Zanthoxylum bungeanum alkaloids primarily include solvent extraction, ultrasonic-assisted extraction, and distillation. These methods typically utilize organic solvents for leaching or reflux extraction of Zanthoxylum bungeanum. While these methods are effective in extracting alkaloids, they suffer from common issues: low extraction efficiency, high solvent consumption, low purity, and poor selectivity during the separation process. In particular, the choice of solvent and extraction conditions significantly influence the final purity and yield, and most traditional methods are unable to achieve efficient separation and high-purity enrichment of alkaloid components.

[0004] In recent years, molecular sieves, ion exchange resins, and macroporous adsorption resins have been introduced as new separation media into the separation process of natural products. Macroporous adsorption resins have become an important tool for separating and purifying plant alkaloids due to their large specific surface area, high adsorption capacity, good mechanical strength, and good selectivity. Macroporous adsorption resins can separate target components from complex mixtures through physical adsorption or chemical adsorption, and are particularly effective in the separation and purification of alkaloids. Compared with traditional solvent extraction methods, the use of macroporous adsorption resins can not only improve the separation purity of alkaloids, but also reduce the amount of solvent used, reduce operating costs, and effectively improve the recovery rate.

[0005] However, current research on the separation and purification of Zanthoxylum bungeanum alkaloids is largely limited to single separation techniques or traditional resin selection, lacking systematic comparison and optimization. In particular, research on the application of macroporous adsorption resins is relatively limited. While previous studies have explored the use of different macroporous resins for the separation of other plant alkaloids, no research has yet specifically investigated the optimal resin selection, pretreatment methods, and elution conditions for Zanthoxylum bungeanum alkaloids. Therefore, optimizing the operating conditions of macroporous adsorption resins to improve the purity and recovery of Zanthoxylum bungeanum alkaloids remains an urgent challenge.

[0006] Furthermore, the quality standards for Zanthoxylum bungeanum alkaloids remain unclear. Quality control of alkaloids, as drug ingredients and food additives, is crucial in the modern pharmaceutical and food industries. Currently, quality control for Zanthoxylum bungeanum alkaloids relies heavily on traditional qualitative analysis, lacking unified quantitative determination methods and standards. In particular, there is a lack of methods for quantitatively detecting the various alkaloid components in Zanthoxylum bungeanum. To ensure the effectiveness and safety of Zanthoxylum bungeanum alkaloids, developing scientific and accurate quality standards is an urgent need in this field. Summary of the Invention

[0007] Based on the above, the purpose of the present invention is to provide a method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin, thereby establishing a quality standard for Zanthoxylum bungeanum alkaloids, in order to provide reliable technical support for the widespread application of Zanthoxylum bungeanum alkaloids.

[0008] To achieve its purpose, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin, comprising the following steps:

[0010] (a) Extraction of Zanthoxylum bungeanum alkaloids: Crush the Zanthoxylum bungeanum and extract with 40% to 95% ethanol solution with a pH of 2.0 to 6.0 and a mass-to-volume ratio of 1:10 to 1:30. The extract is concentrated under reduced pressure to 1 / 5 to 1 / 15 of its original volume to obtain a concentrated extract;

[0011] (b) Pretreatment of macroporous adsorption resin: NKA-9 macroporous adsorption resin was selected and soaked in 85% to 95% ethanol, then washed with deionized water until the alcohol smell was gone. Then, it was soaked in 3% to 5% hydrochloric acid solution and 3% to 5% sodium hydroxide solution respectively, and finally washed with water until neutral;

[0012] (c) Isolation, purification and content determination of Zanthoxylum bungeanum alkaloids: The concentrated extract obtained in step (a) was adsorbed on a column, first eluted with deionized water to remove impurities, and then gradient elution was performed, and the gradient elution program was as follows: deionized water elution for 60-90 min, 50%-60% ethanol solution elution for 60-90 min, 65%-75% ethanol solution elution for 60-90 min, and 85%-95% ethanol solution elution for 60-90 min to obtain eluates of different levels; the eluates of different levels were taken and the contents of sinomenine, ethoxychelerythrine, chelerythrine and evodiamine therein were determined by HPLC method to establish the quality standard of Zanthoxylum bungeanum alkaloids; the remaining eluates of different levels were concentrated and dried to obtain purified Zanthoxylum bungeanum alkaloids.

[0013] As a further preferred embodiment of the technical solution of the present invention, in step (a), the reflux extraction is performed 1 to 4 times, each extraction takes 1 to 4 hours, and the extract is the extract obtained by combining multiple extractions.

[0014] Furthermore, in step (a), the particle size of the ground pepper is 30-50 mesh; the pH value of the ethanol solution is 2.0-4.0; the mass-to-volume ratio of the material to the liquid is 1:15-1:25; and the extraction temperature is 55-70°C.

[0015] Furthermore, in step (b), the macroporous adsorption resin is soaked in 85% to 95% ethanol for 1 to 5 hours, and then soaked in a 3% to 5% hydrochloric acid solution and a 3% to 5% sodium hydroxide solution by mass for 1 to 3 hours respectively; preferably, the macroporous adsorption resin is soaked in 90% to 95% ethanol for 2 to 4 hours, and then soaked in a 5% hydrochloric acid solution and a 5% sodium hydroxide solution by mass for 1.5 to 2.5 hours respectively.

[0016] Furthermore, in step (c), the HPLC method determination conditions are: using a SinoChrom ODS-BP chromatographic column, mobile phase A is 0.1% to 0.3% acetic acid solution, mobile phase B is acetonitrile; the mobile phase flow rate is 0.8 to 1.2 mL / min, the column temperature is 30 to 40°C, the detection wavelength is 270 to 290 nm; and the injection volume is 10 to 20 μL.

[0017] Further, in step (c), the method for establishing the quality standard of Zanthoxylum bungeanum alkaloids comprises the following steps:

[0018] (1) Preparation of mixed standard solution: accurately weigh the standard products of sinomenine, ethoxychelerythrine, chelerythrine and rutaecarpine, and dilute to volume with methanol;

[0019] (2) Sample solution preparation: Zanthoxylum bungeanum powder was weighed and extracted with 60% to 85% ethanol solution with a pH of 2.5 to 5.5 and a volume fraction of ethanol at a mass to volume ratio of 1:10 to 1:25. The extraction was repeated 1 to 3 times, each time for 1 to 3 hours. The extract was concentrated and fixed to volume with methanol.

[0020] (3) HPLC analysis and determination, establishment of standard curve, determination of the content, linear range, detection limit and quantification limit of each alkaloid, and formulation of quality standards.

[0021] Furthermore, in the above step (2), the pH value of the ethanol solution is 3, the mass-to-volume ratio of the material to the liquid is 1:15 to 1:20, and the extraction is performed twice, each time for 2 hours.

[0022] The beneficial effects of the present invention are:

[0023] 1. Resin optimization and pretreatment: This invention improves the separation and purification efficiency and purity of Zanthoxylum bungeanum alkaloids through the screening, optimization and innovation of the pretreatment process of macroporous adsorption resin, especially the effective separation of low-concentration components hydroxy-β-sanshool and γ-sanshool.

[0024] 2. Green and environmentally friendly separation process: The present invention adopts a gradient elution method and reduces the use of organic solvents, so that Zanthoxylum bungeanum alkaloids can be efficiently and stably separated and enriched from the Zanthoxylum bungeanum extract, which not only improves the separation efficiency but also reduces environmental pollution, meeting the requirements of green chemistry.

[0025] 3. Comprehensive quality control standards: The present invention establishes quality standards using high-performance liquid chromatography (HPLC) to determine the content of sinomenine, ethoxychelerythrine, chelerythrine, and rutaecarpine. This ensures the quality stability and traceability of Zanthoxylum bungeanum alkaloid products, provides a scientific basis for quality control of Zanthoxylum bungeanum alkaloids, and ensures the quality of Zanthoxylum bungeanum alkaloids in the fields of medicine and food. This invention not only has high potential for industrial application, but also provides a reference for the standardized separation and purification of related plant alkaloids. DETAILED DESCRIPTION

[0026] In order to fully illustrate the specific embodiments of the present invention, each step, condition and technical details of the separation and purification method of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin and the establishment of its quality standard will be described in detail below.

[0027] Example 1: Extraction, separation and purification of Zanthoxylum bungeanum alkaloids

[0028] 1. Preparation of Zanthoxylum bungeanum Samples and Alkaloid Extraction

[0029] 1.1 Selection and pretreatment of pepper samples

[0030] Freshly harvested Sichuan peppercorns are selected, seeds and impurities removed, and the peels are processed. Sichuan peppercorn peels contain a high concentration of alkaloids, and deseeded peppercorns are more suitable for extraction. The peels are mechanically crushed to a 40-mesh particle size to ensure uniform particle size, facilitating solvent penetration and alkaloid extraction.

[0031] 1.2 Optimization of the extraction process

[0032] 500g of crushed Zanthoxylum bungeanum powder was mixed with 10L of 65% ethanol solution at a solid-liquid ratio of 1:20 (g / mL). Reflux extraction was performed using a reflux extraction apparatus. The reflux temperature was set at 65°C for 2 hours. The extraction process was repeated twice, and the two extracts were combined.

[0033] After the second reflux extraction, the extract is concentrated to 1 / 10 of its original volume using a vacuum concentrator to remove excess ethanol and concentrate the alkaloid components. This step can increase the concentration of alkaloids, providing a high concentration of the target component for subsequent separation and purification.

[0034] 1.3 Concentrate Storage

[0035] The concentrated Zanthoxylum bungeanum alkaloid extract was filtered to remove suspended particles to obtain a clear solution, which was stored in a -20°C refrigerator for subsequent processing.

[0036] 2. Pretreatment and selection of macroporous adsorption resin

[0037] 2.1 Selection and evaluation of resin

[0038] To ensure optimal separation, three commonly used macroporous adsorption resins, D-101, AB-8, and NKA-9, were selected for comparative experiments. The selection criteria for each resin primarily included specific surface area, adsorption capacity, stability, and selectivity for the target alkaloids. The experimental results (Table 1) show that NKA-9 exhibits a strong adsorption capacity for Zanthoxylum bungeanum alkaloids and high selectivity, making it the optimal resin for this study.

[0039] Table 1 Comparison of resin adsorption capacity

[0040]

[0041] 2.2 Resin pretreatment process

[0042] In order to eliminate impurities in the resin and restore its adsorption performance, the following pretreatment steps are performed:

[0043] Swelling cleaning: Use 95% ethanol solution to swell and clean the resin to remove grease and organic pollutants in the resin;

[0044] Remove alcohol odor: rinse thoroughly with deionized water until the resin has no alcohol odor;

[0045] Acid washing: Soak the resin in 5% HCl solution for 2 hours to remove inorganic impurities in the resin;

[0046] Alkaline washing: Soak in 5% NaOH solution for 2 hours to further clean the resin surface and restore its good adsorption performance;

[0047] Water washing: Wash with deionized water until the resin is neutral.

[0048] The treated resin should be stored in a dry environment before use to prevent it from absorbing moisture.

[0049] 3. Isolation and purification of Zanthoxylum bungeanum alkaloids

[0050] 3.1 Packing of macroporous resin column

[0051] The treated NKA-9 macroporous resin was loaded into a glass chromatography column at a resin filling height of 20 cm and pre-washed with deionized water to ensure that no ethanol and NaOH remained in the resin column.

[0052] 3.2 Loading and distribution

[0053] Divide the Zanthoxylum alkaloid extract into three portions and add each to the chromatography column. Each portion should be 20 mL in volume to ensure even distribution. Set the flow rate to 3 BV / h (volume multiples per hour) to maintain even distribution and ensure optimal separation.

[0054] 3.3 Gradient elution

[0055] Separation was performed using gradient elution:

[0056] Initially use deionized water to elute to remove water-soluble impurities that are not adsorbed by the resin;

[0057] Subsequently, 50%, 70%, and 90% ethanol solutions were used to gradually elute the alkaloid components in Zanthoxylum bungeanum (elution time was 60 min). After each elution, each eluate was collected and an appropriate amount was taken to determine its alkaloid content.

[0058] The gradient elution method can effectively separate various alkaloid components in Zanthoxylum bungeanum and improve the purity and recovery rate of the target components.

[0059] 3.4 Concentration and drying

[0060] The remaining eluate is concentrated under reduced pressure to 1 / 10 of its original volume and then dried to obtain pure Zanthoxylum bungeanum alkaloid components. Through this step, the concentration of the isolated Zanthoxylum bungeanum alkaloids is significantly increased and the purity is effectively improved.

[0061] 4. Establishment of quality standards for Zanthoxylum bungeanum alkaloids

[0062] 4.1 Preparation of standard solution

[0063] To establish the quality standards for Zanthoxylum bungeanum alkaloids, standard solutions of varying concentrations were first prepared. Standard solutions of known purity, including sinomenine, ethoxychelerythrine, chelerythrine, and evodiamine, were prepared at 0.52 mg / mL, 0.16 mg / mL, 0.18 mg / mL, and 0.4 mg / mL, respectively.

[0064] 4.2 HPLC analysis conditions

[0065] HPLC method was used for quantitative analysis of Zanthoxylum bungeanum alkaloids. The specific conditions were:

[0066] Chromatographic column: SinoChrom ODS-BP, 4.6 mm × 250 mm, 5 μm;

[0067] Mobile phase A: 0.2% acetic acid solution, mobile phase B: acetonitrile;

[0068] The gradient elution method was used (see Table 2 for the gradient elution program), with a flow rate of 1.0 mL / min, a column temperature of 35°C, and a detection wavelength of 280 nm; the injection volume was 20 μL.

[0069] Table 2 Content determination mobile phase ratio

[0070]

[0071] The contents of sinomenine, ethoxychelerythrine, chelerythrine and evodipine in Zanthoxylum bungeanum were quantitatively analyzed by the standard curve method (Table 3).

[0072] Table 3 Linear relationship investigation, detection limit and quantification limit determination results (n=3)

[0073]

[0074] 4.3 Establishment and Verification of Quality Standards

[0075] By comparing the standard solution obtained by HPLC with the sample solution, we established quality standards for each component, ensuring that the content and purity of Zanthoxylum bungeanum alkaloids meet industry standards. Precision, stability, and recovery tests verified the operability and accuracy of this quality standard.

[0076] 5. Quality Control and Verification

[0077] 5.1 Precision and stability test

[0078] 5.1.1 Precision test

[0079] Different batches of Zanthoxylum bungeanum samples were selected for multiple determinations, and the relative standard deviations (RSDs) were calculated (Table 4). The test results showed that the content of Zanthoxylum bungeanum alkaloids had good precision among different batches of samples.

[0080] Table 4 Precision test results of four reference substances

[0081]

[0082]

[0083] 5.1.2 Stability test

[0084] Stability tests were conducted on Zanthoxylum bungeanum samples at different time points (Table 5). The results showed that the samples were stable over a 24-hour period, with no significant changes. The RSD values ​​of the alkaloid components were less than 3%, meeting the stability requirements.

[0085] Table 5 Results of 24-hour stability test of four index components

[0086]

[0087] 5.2 Recovery rate experiment

[0088] 5.2.1 Sample recovery experiment

[0089] By adding the standard samples into the Zanthoxylum bungeanum extract to conduct sample addition recovery experiments (Table 6), the recovery rates ranged from 97.44% to 99.66%, demonstrating that the separation and purification method has efficient recovery capacity and stability.

[0090] Table 6 Sample recovery test results (n=6)

[0091]

[0092]

[0093] Example 2: Extraction, separation and purification of Zanthoxylum bungeanum alkaloids

[0094] 1. Preparation of Zanthoxylum bungeanum Samples and Alkaloid Extraction

[0095] 1.1 Selection and pretreatment of pepper samples

[0096] Freshly harvested Sichuan peppercorns are selected, seeds and impurities removed, and the peels are processed. Sichuan peppercorn peels contain a high concentration of alkaloids, and deseeded peppercorns are more suitable for extraction. The peels are mechanically crushed to a 50-mesh particle size to ensure uniform particle size, facilitating solvent penetration and alkaloid extraction.

[0097] 1.2 Optimization of the extraction process

[0098] 500g of crushed Zanthoxylum bungeanum powder was mixed with 10L of 65% ethanol solution at a solid-liquid ratio of 1:20 (g / mL). Reflux extraction was performed using a reflux extraction apparatus. The reflux temperature was set at 60°C for 2 hours. The extraction process was repeated twice, and the two extracts were combined.

[0099] After the second reflux extraction, the extract is concentrated to 1 / 10 of its original volume using a vacuum concentrator to remove excess ethanol and concentrate the alkaloid components. This step can increase the concentration of alkaloids, providing a high concentration of the target component for subsequent separation and purification.

[0100] 1.3 Concentrate Storage

[0101] The concentrated Zanthoxylum bungeanum alkaloid extract was filtered to remove suspended particles to obtain a clear solution, which was stored in a -20°C refrigerator for subsequent processing.

[0102] 2. Pretreatment and selection of macroporous adsorption resin

[0103] Same as Example 1.

[0104] 3. Isolation and purification of Zanthoxylum bungeanum alkaloids

[0105] 3.1 Packing of macroporous resin column

[0106] The treated NKA-9 macroporous resin was loaded into a glass chromatography column at a resin filling height of 20 cm and pre-washed with deionized water to ensure that no ethanol and NaOH remained in the resin column.

[0107] 3.2 Loading and distribution

[0108] Divide the Zanthoxylum alkaloid extract into three portions and add each to the chromatography column. Each portion should be 20 mL in volume to ensure even distribution. Set the flow rate to 4 BV / h (volume multiples per hour) to maintain even distribution and ensure optimal separation.

[0109] 3.3 Gradient elution

[0110] Separation was performed using gradient elution:

[0111] First use deionized water to elute to remove water-soluble impurities that are not adsorbed by the resin;

[0112] Subsequently, 55%, 75%, and 90% ethanol solutions were used to gradually elute the alkaloid components in Zanthoxylum bungeanum (elution time was 90 min). After each elution, each eluate was collected and an appropriate amount was taken to determine its alkaloid content.

[0113] 3.4 Concentration and drying

[0114] The remaining eluate is concentrated under reduced pressure to 1 / 10 of its original volume and then dried to obtain pure Zanthoxylum bungeanum alkaloid components. Through this step, the concentration of the isolated Zanthoxylum bungeanum alkaloids is significantly increased and the purity is effectively improved.

[0115] 4. Establishment of quality standards for Zanthoxylum bungeanum alkaloids

[0116] Same as Example 1.

[0117] 5. Quality Control and Verification

[0118] 5.1 Precision and stability test

[0119] 5.1.1 Precision test

[0120] Multiple determinations were performed on Zanthoxylum bungeanum samples from different batches, and the relative standard deviation (RSD) was calculated (Table 7). The results showed that the Zanthoxylum bungeanum alkaloid content had good precision among the different batches of samples, with an RSD of less than 2%.

[0121] Table 7 Precision test results of four reference substances

[0122]

[0123]

[0124] 5.1.2 Stability test

[0125] Stability tests were conducted on Zanthoxylum bungeanum samples at different time points (Table 8). The results showed that the samples were stable over a 24-hour period, with no significant changes. The RSD values ​​of the alkaloid components were less than 3%, meeting the stability requirements.

[0126] Table 8 Results of 24h stability test of four index components

[0127]

[0128] 5.2 Recovery rate experiment

[0129] 5.2.1 Sample recovery experiment

[0130] By adding the standard samples into the Zanthoxylum bungeanum extract to conduct sample recovery experiments (Table 9), the recovery rates ranged from 94.29% to 99.66%, demonstrating the high efficiency and stability of the separation and purification method.

[0131] Table 9 Three reference substance recovery test results (n=6)

[0132]

[0133]

[0134] Example 3: Extraction, separation and purification of Zanthoxylum bungeanum alkaloids

[0135] 1. Preparation of Zanthoxylum bungeanum Samples and Alkaloid Extraction

[0136] 1.1 Selection and pretreatment of pepper samples

[0137] Freshly harvested Sichuan peppercorns are selected, seeds and impurities removed, and the peels are processed. Sichuan peppercorn peels contain a high concentration of alkaloids, and deseeded peppercorns are more suitable for extraction. The peels are mechanically crushed to a 40-mesh particle size to ensure uniform particle size, facilitating solvent penetration and alkaloid extraction.

[0138] 1.2 Optimization of the extraction process

[0139] 500g of crushed Zanthoxylum bungeanum powder was mixed with 10L of 65% ethanol solution at a solid-liquid ratio of 1:25 (g / mL). Reflux extraction was performed using a reflux extraction apparatus. The reflux temperature was set at 70°C for 2 hours. The extraction process was repeated twice, and the two extracts were combined.

[0140] After the second reflux extraction, the extract is concentrated to 1 / 10 of its original volume using a vacuum concentrator to remove excess ethanol and concentrate the alkaloid components. This step can increase the concentration of alkaloids, providing a high concentration of the target component for subsequent separation and purification.

[0141] 1.3 Concentrate Storage

[0142] The concentrated Zanthoxylum bungeanum alkaloid extract was filtered to remove suspended particles to obtain a clear solution, which was stored in a -20°C refrigerator for subsequent processing.

[0143] 2. Pretreatment and selection of macroporous adsorption resin

[0144] Same as Example 1.

[0145] 3. Isolation and purification of Zanthoxylum bungeanum alkaloids

[0146] 3.1 Packing of macroporous resin column

[0147] The treated NKA-9 macroporous resin was loaded into a glass chromatography column at a resin filling height of 20 cm and pre-washed with deionized water to ensure that no ethanol and NaOH remained in the resin column.

[0148] 3.2 Loading and distribution

[0149] Divide the Zanthoxylum alkaloid extract into three portions and add each to the chromatography column. Each portion should be 20 mL in volume to ensure even distribution. Set the flow rate to 3 BV / h (volume multiples per hour) to maintain even distribution and ensure optimal separation.

[0150] 3.3 Gradient elution

[0151] Separation was performed using gradient elution:

[0152] Initially use deionized water to elute to remove water-soluble impurities that are not adsorbed by the resin;

[0153] Subsequently, 60%, 70%, and 95% ethanol solutions were used to gradually elute the alkaloid components in Zanthoxylum bungeanum (elution time was 90 min each). After each elution, each eluate was collected and an appropriate amount was taken to determine its alkaloid content.

[0154] The gradient elution method can effectively separate various alkaloid components in Zanthoxylum bungeanum and improve the purity and recovery rate of the target components.

[0155] 3.4 Concentration and drying

[0156] The remaining eluate is concentrated under reduced pressure to 1 / 10 of its original volume and then dried to obtain pure Zanthoxylum bungeanum alkaloid components. Through this step, the concentration of the isolated Zanthoxylum bungeanum alkaloids is significantly increased and the purity is effectively improved.

[0157] 4. Establishment of quality standards for Zanthoxylum bungeanum alkaloids

[0158] Same as Example 1.

[0159] 5. Quality Control and Verification

[0160] 5.1 Precision and stability test

[0161] 5.1.1 Precision test

[0162] Multiple determinations were performed on Zanthoxylum bungeanum samples from different batches, and the relative standard deviation (RSD) was calculated (Table 10). The results showed that the Zanthoxylum bungeanum alkaloid content had good precision among the different batches of samples, with an RSD of less than 2%.

[0163] Table 10 Precision test results of four reference substances

[0164]

[0165]

[0166] 5.1.2 Stability test

[0167] Stability tests were performed on Zanthoxylum bungeanum samples at different time points (Table 11). The results showed that the samples were stable over a 24-hour period, with no significant changes. The RSD values ​​of the alkaloid components were less than 3%, meeting the stability requirements.

[0168] Table 11 Results of 24h stability test of four index components

[0169]

[0170] 5.2 Recovery rate experiment

[0171] 5.2.1 Sample recovery experiment

[0172] By adding the standard samples into the Zanthoxylum bungeanum extract to conduct sample addition recovery experiments (Table 12), the recovery rates ranged from 94.95% to 99.72%, demonstrating that the separation and purification method has efficient recovery capacity and stability.

[0173] Table 12 Three reference substance recovery test results (n=6)

[0174]

[0175]

[0176] Comparative Example 1

[0177] Background: Zanthoxylum bungeanum is rich in alkaloids, such as chelerythrine and sinomenine. These compounds have high pharmacological value and are widely used in the pharmaceutical and food industries. However, traditional solvent extraction methods have significant limitations in terms of separation efficiency, recovery rate, and purity. This comparative experiment aimed to compare the effectiveness of traditional solvent extraction with the macroporous adsorption resin method used in this study for the extraction of Zanthoxylum bungeanum alkaloids.

[0178] Experimental methods:

[0179] Solvent extraction:

[0180] A conventional solvent extraction method was used: 500 g of Zanthoxylum bungeanum powder was refluxed with 20 L of 80% ethanol solution at a material-liquid ratio of 1:20 (g / mL). The extractions were repeated twice, each lasting 2 hours, and the extracts were combined. The extracts were then concentrated under reduced pressure to 1 / 10 of their original volume to obtain a concentrated solution.

[0181] Macroporous adsorption resin purification:

[0182] The NKA-9 macroporous adsorption resin described in the present invention was used to distribute the extract according to the amount of the extract and perform separation and purification. The alkaloid components in the extract were separated by gradient elution (50%, 70%, and 90% ethanol solutions). The eluate was collected, concentrated under reduced pressure, and finally dried to obtain purified Zanthoxylum bungeanum alkaloids.

[0183] Experimental results:

[0184] method Alkaloid recovery rate (%) Alkaloid purity (%) Solvent extraction, macroporous adsorption resin purification 2.28 68.4 Method according to embodiment 1 of the present invention 92.5 76.7

[0185] Result analysis:

[0186] Although traditional solvent extraction can extract alkaloids from Zanthoxylum bungeanum, its recovery rate and purity are low. Solvent consumption is high, and the alkaloid components may contain a large number of impurities.

[0187] The method of the present invention, by using macroporous adsorption resin and especially optimizing the resin selection, pretreatment and gradient elution conditions, can greatly improve the recovery rate and purity of alkaloids and reduce the amount of solvent used, thus meeting the requirements of green chemistry. Test Example 1: Performance of macroporous adsorption resin method in the purity and recovery rate of Zanthoxylum bungeanum alkaloids

[0188] Background: This test example aims to verify the effectiveness of the NKA-9 macroporous adsorption resin used in the present invention in separating Zanthoxylum bungeanum alkaloids, especially the separation and purification effects of Zanthoxylum bungeanum alkaloids at different solvent concentrations.

[0189] Experimental methods:

[0190] extract:

[0191] Zanthoxylum bungeanum alkaloids were extracted by reflux extraction using the same Zanthoxylum bungeanum powder (500 g) and ethanol solution (10 L 65% ethanol solution, solid-liquid ratio 1:20) as in Example 1. The extracts were combined and concentrated to 1 / 10 of the original volume.

[0192] Separation and purification:

[0193] The concentrated extract was taken and separated by NKA-9 macroporous adsorption resin.

[0194] The total alkaloids in Zanthoxylum bungeanum were eluted in sequence using 50%, 70% and 90% ethanol solutions, with an elution time of 90 minutes.

[0195] By measuring the alkaloid content in each eluate, a high-purity alkaloid solution is collected and concentrated and dried.

[0196] Purity and recovery test:

[0197] The alkaloid components and their contents in each eluate were determined using high performance liquid chromatography (HPLC).

[0198] Experimental results:

[0199] Eluent concentration Alkaloid purity (%) Recovery rate (%) 50% ethanol solution 0.86 22.8 70% ethanol solution 5.97 34.4 90% ethanol solution 1.12 35.3

[0200] Result analysis:

[0201] In ethanol solutions of different concentrations, the purity and recovery rate of total alkaloids gradually increased with the increase of ethanol concentration. The recovery rate and purity of 90% ethanol solution were the highest, reaching 1.12% and 35.3%.

[0202] This test validated the superiority of the macroporous adsorption resin and showed that the purity and recovery of alkaloids could be further improved by optimizing the concentration of the elution solution.

[0203] Test Example 2: Establishment and Verification of Zanthoxylum bungeanum alkaloid quality standards

[0204] Background: The establishment of a quality standard for Zanthoxylum bungeanum alkaloids is an important part of the present invention. This application example aims to verify the reliability and accuracy of the quality standard for Zanthoxylum bungeanum alkaloids established by HPLC.

[0205] Experimental methods:

[0206] Preparation of standard solution:

[0207] Use standard substances sinomenine, ethoxychelerythrine, chelerythrine and evodiamine, accurately weigh appropriate amounts, dissolve them in methanol, and prepare standard solutions of different concentrations.

[0208] Sample analysis:

[0209] The Zanthoxylum bungeanum alkaloid extract was analyzed using the HPLC method, and the peak area of ​​each alkaloid component was recorded. The peak area calculated based on the ethoxychelerythrine peak should be no less than 3000, and the peak area calculated based on the sinomenine peak should be no less than 3000 and the corresponding concentration.

[0210] A standard curve was established using standard solutions, and the contents of the main alkaloid components in the samples were determined.

[0211] Establishment of quality standards:

[0212] Based on the experimental data, the quality standards for Zanthoxylum bungeanum alkaloids were established, including the content standards for sinomenine, ethoxychelerythrine, chelerythrine and evodiamine.

[0213] Experimental results:

[0214] Element Standard solution concentration (mg / mL) Sample content (mg / g) Measurement results Sinomenine 0.52 0.021 Comply with standards Ethoxychelerythrine 0.16 0.028 Comply with standards Chelerythrine 0.18 0.053 Comply with standards Evodia rutaecarpine 0.4 0.033 Comply with standards

[0215] Result analysis:

[0216] This test verified the accuracy of the quality standard established by HPLC method, and all the measured contents of Zanthoxylum bungeanum alkaloid components were within the standard range.

[0217] The quantitative analysis results of the standard solution and the sample solution were consistent, demonstrating that the quality standard established in the present invention has good operability and reliability.

[0218] The comparative examples and test examples above demonstrate that the macroporous adsorption resin-based Zanthoxylum bungeanum alkaloid separation and purification method provided by the present invention significantly improves the purity and recovery rate of Zanthoxylum bungeanum alkaloids compared to traditional solvent extraction methods, and provides a reliable quality standard. These experimental results demonstrate the innovative nature and practical application value of the present invention, providing technical support for the widespread application of Zanthoxylum bungeanum alkaloids in fields such as medicine and food.

Claims

1. A method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin, characterized in that: The following steps are involved: (a) Extraction of Zanthoxylum bungeanum alkaloids: Crush the Zanthoxylum bungeanum and extract with 40% to 95% ethanol solution with a pH of 2.0 to 6.0 and a mass-to-volume ratio of 1:10 to 1:

30. The extract is concentrated under reduced pressure to 1 / 5 to 1 / 15 of its original volume to obtain a concentrated extract; (b) Pretreatment of macroporous adsorption resin: NKA-9 macroporous adsorption resin was selected and soaked in 85% to 95% ethanol, then washed with deionized water until the alcohol smell was gone. Then, it was soaked in 3% to 5% hydrochloric acid solution and 3% to 5% sodium hydroxide solution respectively, and finally washed with water until neutral; (c) Isolation, purification and content determination of Zanthoxylum bungeanum alkaloids: The concentrated extract obtained in step (a) was adsorbed on a column, first eluted with deionized water to remove impurities, and then gradient elution was performed, and the gradient elution program was as follows: deionized water elution for 60-90 min, 50%-60% ethanol solution elution for 60-90 min, 65%-75% ethanol solution elution for 60-90 min, and 85%-95% ethanol solution elution for 60-90 min to obtain eluates of different levels; the eluates of different levels were taken and the contents of sinomenine, ethoxychelerythrine, chelerythrine and evodiamine therein were determined by HPLC method to establish the quality standard of Zanthoxylum bungeanum alkaloids; the remaining eluates of different levels were concentrated and dried to obtain purified Zanthoxylum bungeanum alkaloids.

2. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to claim 1, wherein: In step (a), the reflux extraction is performed 1 to 4 times, each extraction takes 1 to 4 hours, and the extract is the extract obtained by combining multiple extractions.

3. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to claim 2, wherein: In step (a), the particle size of the ground pepper is 30-50 mesh; the pH value of the ethanol solution is 2.0-4.0; the mass volume ratio of the material to the liquid is 1:15-1:25; and the extraction temperature is 55-70°C.

4. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to claim 3, wherein: In step (b), the macroporous adsorption resin is soaked in 85% to 95% ethanol for 1 to 5 hours, and is sequentially soaked in a hydrochloric acid solution with a volume fraction of 3% to 5% and a sodium hydroxide solution with a mass fraction of 3% to 5% for 1 to 3 hours respectively.

5. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to claim 4, wherein: In step (b), the macroporous adsorption resin is soaked in 90% to 95% ethanol for 2 to 4 hours, and then soaked in a 5% by volume hydrochloric acid solution and a 5% by mass sodium hydroxide solution for 1.5 to 2.5 hours.

6. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to any one of claims 1 to 5, characterized in that: In step (c), the HPLC method determination conditions are: using a SinoChrom ODS-BP chromatographic column, mobile phase A is 0.1% to 0.3% acetic acid solution, mobile phase B is acetonitrile; the mobile phase flow rate is 0.8 to 1.2 mL / min, the column temperature is 30 to 40° C., the detection wavelength is 270 to 290 nm; and the injection volume is 10 to 20 μL.

7. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to claim 6, wherein: In step (c), the method for establishing the quality standard of Zanthoxylum bungeanum alkaloids comprises the following steps: (1) Preparation of mixed standard solution: accurately weigh the standard products of sinomenine, ethoxychelerythrine, chelerythrine and rutaecarpine, and dilute to volume with methanol; (2) Sample solution preparation: Zanthoxylum bungeanum powder was weighed and extracted with 60% to 85% ethanol solution with a pH of 2.5 to 5.5 and a volume fraction of ethanol at a mass to volume ratio of 1:10 to 1:

25. The extraction was repeated 1 to 3 times, each time for 1 to 3 hours. The extract was concentrated and fixed to volume with methanol. (3) HPLC analysis and determination, establishment of standard curve, determination of the content, linear range, detection limit and quantification limit of each alkaloid, and formulation of quality standards.

8. The method for separating and purifying Zanthoxylum bungeanum alkaloids and determining the content of Zanthoxylum bungeanum alkaloids based on macroporous adsorption resin according to claim 6, wherein: In step (2), the pH value of the ethanol solution is 3, the mass volume ratio of the material to the liquid is 1:15 to 1:20, and the extraction is performed twice, each time for 2 hours.

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