Extraction method of rose glycoside A for promoting gastrointestinal peristalsis and medicine for promoting gastrointestinal peristalsis

Abstract

The invention provides an extraction method of rose glycoside A for promoting gastrointestinal peristalsis and a medicine for promoting gastrointestinal peristalsis, and relates to the technical field of extraction of effective components of traditional Chinese medicine. The problem that peach blossom diuresis-inducing and bowel-relaxing functional components are not clear can be effectively solved. Through systematic extraction, extraction, silica gel chromatographic separation and two times of high performance liquid chromatography purification, high-purity rose glycoside A with a single active component is efficiently and accurately obtained from a complex peach blossom raw material, and a clear active component source and a reliable preparation approach are provided for developing drugs for promoting gastrointestinal peristalsis.

Description

Technical Field

[0001] This invention relates to the field of extraction technology of effective components of traditional Chinese medicine, and more specifically, to a method for extracting rosin A, which promotes gastrointestinal motility, and a drug that promotes gastrointestinal motility. Background Technology

[0002] Peach blossoms, a traditional Chinese medicine, are derived from the dried flower buds of *Prunus persica* (L.) Batsch or *Prunus davidiana* (Carr.) Franch, both belonging to the Rosaceae family. In traditional Chinese medicine, they have long been used to regulate symptoms such as diuresis and constipation. Modern research reveals that peach blossoms are rich in polysaccharides, polyphenols, flavonoids, peach blossom essential oil, and peach blossom pigments, among other bioactive substances. These components collectively constitute the basis of their significant medicinal and edible value. Ancient texts and contemporary pharmacological experiments have confirmed that peach blossoms can improve fluid metabolism and promote excretion; however, a systematic understanding of the specific active ingredients responsible for their diuretic and laxative effects is still lacking. Particularly in the regulation of gastrointestinal function, the lack of clear identification and separation techniques for key active substances leads to a murky understanding of the medicinal mechanisms associated with peach blossoms. Existing extraction processes mostly focus on the preparation of crude extracts, making it difficult to accurately obtain single active components from peach blossoms and to effectively verify the correlation between specific components and gastrointestinal motility. Summary of the Invention

[0003] The present invention aims to solve the problem that existing extraction methods are unable to accurately obtain the single active component, rosin A, from peach blossoms.

[0004] To address the above problems, this invention provides a method for extracting rosin A, which promotes gastrointestinal motility, and a drug that promotes gastrointestinal motility.

[0005] In a first aspect, the present invention provides a method for extracting rosin A, which promotes gastrointestinal motility, comprising the following steps: S1: Peach blossom raw material was extracted by reflux with ethanol-water solution, filtered, the filtrate was collected, the ethanol was removed, and the extract was extracted with ethyl acetate. The ethyl acetate phase was concentrated to obtain the first extract. S2: Take the first extract, add ethyl acetate solvent and silica gel for column chromatography, mix well, evaporate the ethyl acetate solvent, separate by silica gel column, elute with dichloromethane-methanol solution for 3 to 5 column volumes, collect the eluent and concentrate to obtain the second extract; S3: Take the second extract, add dimethyl sulfoxide to dissolve it, and use a C18-filled chromatography column with methanol-water solution as the mobile phase for high performance liquid chromatography separation. Collect the sample components from the 38th to the 50th minute of separation and concentrate them to obtain the third extract. S4: Take the third extract, dissolve it in methanol, and perform high performance liquid chromatography separation using a C18 packed column and methanol-water solution as the mobile phase. When the UV 254 absorbance exceeds 300 mAU for the fourth time at a UV 254 nm wavelength, start collecting the sample components until the UV 254 absorbance is below 300 mAU. Concentrate the collected sample components to obtain rosin A.

[0006] Optionally, in S1, ethanol is removed by vacuum concentration at a pressure of -0.06 to -0.09 MPa.

[0007] Optionally, in S2, the silica gel packing material of the silica gel column has a particle size of 300 to 400 mesh.

[0008] Optionally, in S2, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is 8:1 to 3:1.

[0009] Optionally, in S3, the particle size of the C18 packing material in the chromatography column is 30 μm.

[0010] Optionally, in S3, the volume fraction of methanol in the methanol-water solution is 20% to 80%.

[0011] Optionally, in S4, the particle size of the C18 packing material in the chromatography column is 9 μm.

[0012] Optionally, in S4, the volume fraction of methanol in the methanol-water solution is 30% to 50%.

[0013] Secondly, the present invention provides a drug for promoting gastrointestinal motility, including rosin A.

[0014] Optionally, rosin A is extracted from peach raw materials using the rosin A extraction method described above that promotes gastrointestinal motility.

[0015] The beneficial effects of the extraction method for rosin A, which promotes gastrointestinal motility, and the drug for promoting gastrointestinal motility of the present invention are as follows: Through multi-step fine separation and purification, the problem of unclear diuretic and laxative components of peach blossoms can be effectively solved. Through systematic extraction, silica gel chromatography separation, and two high-performance liquid chromatography purification processes, high-purity rosin A, a single active component, can be efficiently and accurately obtained from complex peach blossom raw materials, providing a clear source of active ingredients and a reliable preparation route for the development of drugs that promote gastrointestinal motility. Attached Figure Description

[0016] Figure 1 This is a schematic flowchart of the extraction method of rosin A, which promotes gastrointestinal motility, according to an embodiment of the present invention. Figure 2 This is a schematic diagram of the chemical structure of rosin A according to an embodiment of the present invention; Figure 3 This is a schematic diagram of the nuclear magnetic resonance detection of rosin A according to an embodiment of the present invention; Figure 4 This is a schematic diagram showing the migration of Nile Red solutions of peach blossom extract 1-1 and the first extract in the intestine using a zebrafish model in Example 1. Figure 5 This is a schematic diagram illustrating the migration of Nile Red solutions of peach blossom extract 2-1, peach blossom extract 2-2, peach blossom extract 2-3, the second extract, and peach blossom extract 2-5 in the intestine using a zebrafish model in Example 2. Figure 6 This is the preparative high-performance liquid chromatography chromatogram of Example 3; Figure 7 This is a schematic diagram illustrating the migration of Nile Red solutions containing peach blossom extracts 3-1 to 3-19 in the intestine using a zebrafish model in Example 3. Figure 8 This is a semi-preparative high-performance liquid chromatography chromatogram of Example 4; Figure 9 This is a schematic diagram illustrating the migration of Nile Red solutions of peach blossom extracts 4-1, 4-2, 4-3, Rosin A, 4-5, and 4-6 in the intestine using a zebrafish model in Example 4. Detailed Implementation

[0017] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature.

[0019] like Figure 1 As shown, an embodiment of the present invention provides a method for extracting rosin A, which promotes gastrointestinal motility, comprising the following steps: S1: Peach blossom raw material was extracted by reflux with ethanol-water solution, filtered, the filtrate was collected, the ethanol was removed, and the extract was extracted with ethyl acetate. The ethyl acetate phase was concentrated to obtain the first extract. S2: Take the first extract, add ethyl acetate solvent and silica gel for column chromatography, mix well, evaporate the ethyl acetate solvent, separate by silica gel column, elute with dichloromethane-methanol solution for 3 to 5 column volumes, collect the eluent and concentrate to obtain the second extract; S3: Take the second extract, add dimethyl sulfoxide to dissolve it, and use a C18-filled chromatography column with methanol-water solution as the mobile phase for high performance liquid chromatography separation. Collect the sample components from the 38th to the 50th minute of separation and concentrate them to obtain the third extract. S4: Take the third extract, dissolve it in methanol, and perform high performance liquid chromatography separation using a C18 packed column and methanol-water solution as the mobile phase. When the UV 254 absorbance exceeds 300 mAU for the fourth time at a UV 254 nm wavelength, start collecting the sample components until the UV 254 absorbance is below 300 mAU. Concentrate the collected sample components to obtain rosin A.

[0020] Specifically, peach blossom raw material refers to the original plant material used to extract rosin A, typically the dried flower buds of peach or wild peach plants (both belonging to the Rosaceae family). As the starting material for this extraction method, it contains various natural compounds, including the target product, rosin A.

[0021] Ethanol-water reflux extraction is a commonly used solid-liquid extraction technique. It involves contacting peach blossom raw materials with a mixed solution of ethanol and water under reflux conditions, allowing the target compound to dissolve in the solvent. This method utilizes the good solubility of ethanol-water solutions for polar and moderately polar components to achieve the preliminary extraction of active ingredients from peach blossoms.

[0022] Ethyl acetate extraction is a liquid-liquid separation technique that utilizes the difference in partition coefficients of a target compound in two immiscible solvents (such as an aqueous phase and an ethyl acetate organic phase) to transfer the target compound from one solvent to the other. In this method, ethyl acetate is used to selectively extract the first extract, while the aqueous phase removes specific impurities, thereby enriching the target component in the ethyl acetate phase.

[0023] A silica gel chromatography column is a chromatographic separation device based on the principle of adsorption, in which silica gel serves as the stationary phase. The differences in the adsorption capacity of compounds on silica gel cause them to migrate at different speeds as they pass through the column, thus achieving the separation of mixtures.

[0024] Dichloromethane-methanol elution refers to the use of a mixed solution of dichloromethane and methanol as the mobile phase in silica gel column chromatography to elute compounds adsorbed on the silica gel. By adjusting the ratio of the two solvents, the polarity of the eluent can be controlled, thereby achieving selective separation of compounds with different polarities.

[0025] C18-filled chromatography columns are reversed-phase columns with octadecyl (C18) groups bonded to their surface. C18 packing material is nonpolar, making it suitable for separating polar or moderately polar compounds. Its separation mechanism is primarily based on the hydrophobic interaction between the compound and the stationary phase.

[0026] Methanol-water solution as the mobile phase refers to the use of a mixed solution of methanol and water as the mobile phase in high-performance liquid chromatography (HPLC) separation. By adjusting the volume fraction of methanol in the aqueous solution, the polarity of the mobile phase can be changed, thereby optimizing the retention behavior of compounds on the C18 packing material and achieving efficient separation.

[0027] High-performance liquid chromatography (HPLC) is a high-resolution liquid chromatography technique that uses a high-pressure pump to deliver the mobile phase through a chromatographic column packed with a stationary phase of small particles, enabling efficient separation and quantitative analysis of components in complex mixtures.

[0028] The UV 254nm wavelength refers to the specific wavelength used in UV-Vis spectrophotometers for detecting compounds. Many compounds with conjugated double bonds or aromatic ring structures exhibit strong UV absorption at this wavelength, making it suitable for real-time monitoring of the elution of target compounds.

[0029] Rosinoside A is the target compound to be extracted in this application. This compound is a flavonoid glycoside called Multiforin A, with the molecular formula C. 29 H 32 O 16 Chemical structural formula see Figure 2 .

[0030] Specifically, the rosin A obtained in step S4, as detected by nuclear magnetic resonance, shows the following... Figure 3 As shown, comparison with a known compound database confirms that the compound is multiforin A, with the molecular formula C. 29 H 32 O 16 .

[0031] In this embodiment, the problem of unclear diuretic and laxative components of peach blossoms can be effectively solved through multi-step fine separation and purification. Through systematic extraction, silica gel chromatography separation, and two high-performance liquid chromatography purification processes, high-purity rosin A, a single active component, can be efficiently and accurately obtained from complex peach blossom raw materials. This provides a clear source of active ingredients and a reliable preparation method for developing drugs that promote gastrointestinal motility.

[0032] In some specific embodiments, peach blossom raw materials can be used directly for extraction. For example, dried peach blossom buds can be pulverized and then mixed with an ethanol-water solution for reflux extraction. In another embodiment, the peach blossom raw materials can also be simply washed and dried before extraction to remove surface impurities. The concentration of the ethanol-water solution can be selected empirically; for example, a 50% ethanol-water solution can be used for reflux extraction. The reflux extraction time and temperature can also be adjusted according to actual conditions; for example, reflux extraction at 80°C for 2 hours. The ethanol in the extracted filtrate can be removed by various methods, such as heating and evaporating under normal pressure or evaporating under reduced pressure using a rotary evaporator. After removing the ethanol, the remaining aqueous phase can be extracted multiple times with ethyl acetate to obtain as much of the first extract as possible. For example, the aqueous phase can be mixed with an equal volume of ethyl acetate, shaken, and allowed to stand for separation, and the ethyl acetate phase can be collected. The ethyl acetate phase after ethyl acetate extraction can be concentrated using conventional methods, such as concentration under vacuum conditions, to obtain the first extract.

[0033] The first extract can be directly dissolved in ethyl acetate and mixed with silica gel for column chromatography. The ethyl acetate solvent is then evaporated, allowing the extract to be uniformly loaded onto the silica gel. In another embodiment, the first extract can also be directly dissolved in a small amount of polar solvent and then directly loaded onto a silica gel column. The silica gel column can be packed with silica gel packing material of different particle sizes; for example, silica gel packing material with a particle size of 200 to 300 mesh can be used. The eluent ratio can be adjusted empirically; for example, a mixture of dichloromethane and methanol in a volume ratio of 10:1 can be used for elution. The elution volume range can also be adjusted according to the separation effect; for example, two column volumes of eluent can be collected. The collected eluent can be concentrated using conventional methods, such as concentration under reduced pressure, to obtain the second extract.

[0034] The second extract can be dissolved in a variety of polar solvents, such as methanol or acetonitrile. The chromatography column can be packed with C18 packing material of different particle sizes, for example, C18 packing material with a particle size of 50 μm. The volume fraction of methanol in the aqueous methanol solution can be adjusted according to separation requirements; for example, a mobile phase with a methanol volume fraction of 60% in aqueous solution can be used. High-performance liquid chromatography (HPLC) separation can be performed under various conditions, such as a flow rate of 10 mL / min. The sample collection time period can also be adjusted based on preliminary experimental results; for example, sample components can be collected from the 35th to the 55th minute of separation. The collected sample components can be concentrated using conventional methods, such as freeze-drying or rotary evaporation, to obtain the third extract.

[0035] The third extract can be dissolved in a variety of organic solvents, such as acetonitrile or ethanol. The chromatography column can be filled with C18 packing material of different particle sizes, for example, C18 packing material with a particle size of 15 μm. The volume fraction of methanol in the methanol-water solution can be adjusted according to separation requirements; for example, a mobile phase with a methanol volume fraction of 40% in the aqueous solution can be used. High-performance liquid chromatography (HPLC) separation can be performed at various flow rates, for example, at a flow rate of 5 mL / min. UV absorbance monitoring at 254 nm can be adjusted according to actual conditions; for example, collection can be initiated when the absorbance exceeds 200 mAU. The collected sample components can be concentrated using conventional methods, such as vacuum concentration or nitrogen blowing, to obtain the final rosin A.

[0036] Optionally, in S1, ethanol is removed by vacuum concentration at a pressure of -0.06 to -0.09 MPa.

[0037] Specifically, "reduced pressure concentration" is a physical separation technique that lowers the solvent's boiling point by reducing system pressure, thereby achieving rapid solvent evaporation at a lower temperature. Its purpose is to efficiently and gently remove the solvent, avoiding potential degradation or activity loss of the target compound (such as rosin A) due to high temperatures. This can be achieved using a rotary evaporator, connected to a vacuum pump and condenser, precisely controlling the system's internal pressure to rapidly vaporize and condense the ethanol at a set low pressure. Alternatively, a vacuum concentrator or vacuum drying oven can be used, placing the ethanol-containing filtrate within it and achieving a preset pressure through vacuuming, thus promoting ethanol evaporation at a lower temperature.

[0038] The operation is carried out at a pressure range of -0.06 to -0.09 MPa. This pressure range is optimized to ensure that ethanol evaporates fully and efficiently, while avoiding excessive equipment load or increased operational complexity due to excessively low pressure, and preventing incomplete ethanol removal due to excessively high pressure. This specific pressure range helps to balance removal efficiency, energy consumption, and equipment stability.

[0039] In this optional embodiment, in step S1, ethanol is removed by vacuum concentration at a negative pressure of -0.06 to -0.09 MPa. This significantly lowers the boiling point of ethanol, enabling rapid and complete evaporation at a relatively low temperature. This not only effectively solves the problem of low extraction efficiency and insufficient purity of the extract caused by incomplete ethanol removal, but also avoids the potential degradation of active ingredients such as rosin A caused by high temperatures. By precisely controlling the pressure range of vacuum concentration, the efficiency and thoroughness of ethanol removal are ensured, providing pure raw materials for subsequent ethyl acetate extraction, thereby improving the overall extraction process efficiency and the final purity of rosin A.

[0040] Optionally, in S2, the silica gel packing material of the silica gel column has a particle size of 300 to 400 mesh.

[0041] Specifically, "silicone packing particle size specification" refers to the size range of silica particles used to pack silica gel chromatographic columns. In chromatographic separation, the particle size of the packing material is one of the key parameters affecting separation efficiency, column pressure, and flow rate. Generally, the selection of packing material particle size needs to balance separation efficiency and operating conditions. For example, smaller particle sizes typically provide a larger specific surface area and shorter mass transfer distance, thereby improving separation efficiency and resolution, but may lead to increased column pressure and decreased flow rate; conversely, larger particle sizes can reduce column pressure and increase flow rate, but may sacrifice some separation efficiency.

[0042] "300 to 400 mesh" refers to the particle size range of the silica gel packing material. Mesh size is a commonly used unit for measuring particle size; the higher the mesh number, the smaller the particles. Selecting a specific mesh size packing material aims to optimize column performance. For example, in some applications, 200 to 300 mesh packing material might be chosen for faster elution rates, or 400 to 600 mesh packing material for higher separation precision. In this application, the 300 to 400 mesh particle size specification aims to provide a balance point, ensuring that appropriate column pressure and flow rate are maintained while guaranteeing separation efficiency, thereby achieving effective separation of the target components.

[0043] In this optional embodiment, the particle size of the silica gel packing is limited to 300 to 400 mesh, effectively solving the problems of low separation efficiency and poor elution caused by unoptimized packing particle size during silica gel column separation. Specifically, this particle size range ensures that the silica gel particles are of moderate size, providing sufficient specific surface area to enhance the adsorption and selective separation of components in the first extract, thereby improving the separation degree between target components and impurities; while avoiding problems such as excessively high column pressure, slow flow rate, and column blockage that may occur due to excessively fine particles, ensuring smooth separation. At the same time, this particle size range also effectively prevents insufficient resolution and incomplete elution caused by excessively coarse particles, ensuring effective elution of target components. Therefore, in step S2, using silica gel packing with a particle size of 300 to 400 mesh significantly promotes the uniformity and efficiency of the dichloromethane-methanol solution elution process, effectively reduces impurity residue, thereby improving the purity of the second extract, providing a higher quality intermediate product basis for subsequent high-performance liquid chromatography separation steps, and ultimately contributing to improving the purity and yield of rosin A.

[0044] Optionally, in S2, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is 8:1 to 3:1.

[0045] Specifically, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is between 8:1 and 3:1. This volume ratio is a key parameter for adjusting the polarity of the elution solvent to achieve effective separation of the target compound, rosin A. Specifically, this volume ratio can be precisely controlled in two ways: First, when preparing the elution solvent, a specified volume of dichloromethane and methanol is measured separately using a high-precision graduated cylinder or pipette, and then mixed thoroughly to ensure a volume ratio range of 8:1 to 3:1. Second, an automated solvent mixing system is used. This system can precisely control the flow rates of dichloromethane and methanol according to preset volume ratio parameters and deliver the mixture to the silica gel column, thereby ensuring the accuracy of the elution solvent composition.

[0046] In this optional embodiment, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is limited to the range of 8:1 to 3:1, optimizing the elution conditions of the silica gel column. This precise volume ratio setting allows for fine adjustment of the polarity gradient of the elution solvent, thereby enabling more effective separation of the target compound, rosin A, from other impurities. In step S2, by controlling this key parameter, it is ensured that rosin A is fully eluted while minimizing the co-elution of non-target components, avoiding problems such as low elution efficiency and insufficient impurity removal caused by improper elution solvent ratios. Therefore, this application can significantly improve the purity of rosin A and enhance the overall extraction efficiency, laying a good foundation for subsequent purification steps.

[0047] Optionally, in S3, the particle size of the C18 packing material in the chromatography column is 30 μm.

[0048] Specifically, the particle size specification of the C18 packing material in the chromatography column is 30 μm, which refers to the average diameter of the stationary phase particles used for high-performance liquid chromatography (HPLC). As the most commonly used stationary phase in reversed-phase chromatography, the particle size of C18 packing material is a key parameter affecting chromatographic separation efficiency, column pressure, and sample loading. In preparative chromatography, selecting an appropriate particle size is crucial. For example, in addition to 30 μm, particle sizes of 20 μm or 50 μm can also be selected. The 30 μm particle size provides good separation resolution while maintaining low column pressure and allowing for high sample loading, thus improving processing efficiency while ensuring separation effectiveness. This particle size selection helps balance separation purity and throughput, laying the foundation for subsequent fine purification.

[0049] In this optional embodiment, in step S3, the particle size of the C18 packing material in the chromatography column is limited to 30 μm. This application effectively solves the problem that the lack of specific particle size limitation for C18 packing material may lead to low separation efficiency or loss of target components. Specifically, selecting a particle size of 30 μm enables an optimized balance between resolution, column pressure, and sample loading during high-performance liquid chromatography (HPLC) separation. Compared to packing material with a smaller particle size, 30 μm particles can significantly reduce the operating pressure of the chromatographic column, thereby allowing for higher flow rates and larger column diameters, greatly improving the sample throughput and overall separation efficiency per separation. At the same time, the packing material with this particle size still provides sufficient surface area and separation capacity to ensure the effective separation of rosin A from major impurities and reduce the loss of target components. This optimized selection provides a sample with higher purity and suitable concentration for subsequent fine purification steps (such as S4), thereby improving the overall purification efficiency and yield of rosin A and ensuring the quality of the final product.

[0050] Optionally, in S3, the volume fraction of methanol in the methanol-water solution is 20% to 80%.

[0051] Specifically, the volume fraction refers to the volume ratio of methanol in the aqueous solution, a key parameter in the composition of the mobile phase during high-performance liquid chromatography (HPLC). In reversed-phase HPLC, methanol acts as an organic modifier, and its concentration directly determines the polarity and elution capacity of the mobile phase. By precisely controlling the volume fraction of methanol, the retention behavior of the target compound, rosin A, on the stationary phase (C18 packing material) can be effectively adjusted, thereby achieving effective separation from impurities. For example, a 20% methanol-water solution can be prepared as the mobile phase to provide strong retention capacity, suitable for separating highly polar impurities; or, an 80% methanol-water solution can be prepared as the mobile phase to provide strong elution capacity, suitable for rapid elution of the target compound or separation of weakly polar impurities. Furthermore, other volume fractions within this range, such as 50%, can be selected to balance retention and elution efficiency.

[0052] In this optional embodiment, the volume fraction of methanol in the methanol-water solution used for high-performance liquid chromatography (HPLC) separation in step S3 is precisely limited to the range of 20% to 80%, effectively solving the problem of poor separation effect caused by the uncertainty of methanol concentration. When performing reversed-phase separation on a C18-packed chromatography column, methanol, as the organic phase, directly affects the elution strength of the mobile phase. When the methanol volume fraction is too low, rosin A may be excessively retained, resulting in excessively long elution times, peak broadening, or even incomplete elution; while when the methanol volume fraction is too high, rosin A may elute too quickly, resulting in incomplete separation from non-polar impurities and a decrease in purity. By controlling the methanol volume fraction within the optimized range of 20% to 80%, it is possible to ensure that the mobile phase has suitable polarity and elution capacity, enabling rosin A to achieve optimal retention and separation effects on the C18 packing, thereby significantly improving the extraction efficiency and purity of rosin A. This limited range makes the separation process more stable and controllable, avoiding the problem of poor separation reproducibility caused by fluctuations in the composition of the mobile phase, and laying a solid foundation for the subsequent purification and application of rosin A.

[0053] Optionally, in S4, the particle size of the C18 packing material in the chromatography column is 9 μm.

[0054] Specifically, C18 packing material is a reversed-phase chromatography stationary phase with octadecyl chains bonded to its surface, exhibiting strong hydrophobicity. This hydrophobicity allows C18 packing material to generate strong hydrophobic interactions with nonpolar or weakly polar components in the sample (such as rosin A), thereby achieving separation from polar components. Besides C18, commonly used reversed-phase packing materials include C8 and C4, which have different hydrophobicities and are suitable for separating compounds of different polarities. The particle size specification of 9 μm refers to the average diameter of the C18 packing particles. In high-performance liquid chromatography (HPLC), the particle size of the packing material is one of the key parameters affecting column performance. Smaller particle sizes generally provide higher column efficiency and separation resolution, but also result in higher column pressures. For example, in addition to 9 μm, particle sizes of 5 μm or 15 μm can also be selected. A particle size of 5 μm can provide higher separation efficiency, but may lead to excessively high column pressure, affecting flow rate and equipment life; while a particle size of 15 μm results in lower column pressure, but relatively lower separation efficiency, which may not meet the requirements for high-purity separation. Therefore, choosing a particle size of 9 μm is an optimal choice that strikes a balance between separation efficiency, column pressure, and operational stability.

[0055] In this optional embodiment, in step S4, a high-performance liquid chromatography (HPLC) column packed with C18 packing material with a particle size of 9 μm is used for separation. Compared to using packing material with a larger particle size, this significantly improves the theoretical plate number and separation efficiency of the column. The smaller particle size of the packing material increases the mass transfer rate between the stationary and mobile phases, effectively improving the separation between rosin A and impurity components. This optimization allows the absorption peak of rosin A to be more clearly separated from adjacent impurity peaks at UV 254 nm, reducing the possibility of co-elution. Therefore, collecting sample components from the fourth time the UV 254 absorbance exceeds 300 mAU until the UV 254 absorbance falls below 300 mAU allows for more precise extraction of the target component, effectively reducing impurity contamination and ensuring higher purity of the final rosin A. Meanwhile, the 9 μm particle size ensures high resolution while also maintaining operational stability and flow rate controllability, avoiding the excessively high column pressure and operational difficulties that may result from excessively small particle sizes. This improves the efficiency and yield of the entire separation process. Combining the use of 30 μm C18 packing material for preliminary separation in step S3 and 9 μm C18 packing material for fine separation in step S4, a gradient purification strategy is formed, achieving optimal purification of rosin A and effectively solving the problems of insufficient separation resolution, low efficiency, or operational instability.

[0056] Optionally, in S4, the volume fraction of methanol in the methanol-water solution is 30% to 50%.

[0057] Specifically, a methanol volume fraction of 30% to 50% in the methanol-water solution refers to the precise control of the volume percentage of methanol in the mobile phase (methanol-water solution) during high-performance liquid chromatography (HPLC) separation. This parameter is crucial in HPLC separation, directly affecting the polarity of the mobile phase, which in turn determines the retention behavior of the target compound, rosin A, on a C18 packed column and its separation from impurities. For example, if the methanol volume fraction is below 30%, the mobile phase is too polar, potentially leading to excessively long retention time of rosin A on the C18 packed column, making elution difficult or resulting in poor separation from more polar impurities. Conversely, if the methanol volume fraction is above 50%, the mobile phase is too weak, potentially causing premature elution of rosin A, co-elution with non-polar impurities, or poor peak shape, thus affecting separation efficiency. Therefore, limiting the methanol volume fraction to the range of 30% to 50% aims to provide optimal elution conditions for rosin A, ensuring a suitable retention time on the C18 packing material and achieving efficient separation from coexisting impurities.

[0058] In this optional embodiment, in step S4, the mobile phase polarity of the high-performance liquid chromatography (HPLC) separation process is optimized by precisely controlling the volume fraction of methanol in the methanol-water solution within the range of 30% to 50%. This precise concentration control allows rosin A to achieve optimal retention and elution behavior on a C18 packed column, avoiding the problems of low separation efficiency and incomplete impurity removal caused by excessively high or low methanol concentrations. Specifically, this range ensures that rosin A can be separated from other components in complex mixtures at high resolution, thereby significantly improving the purity of the final obtained rosin A. Simultaneously, the optimized separation conditions also help improve the yield of rosin A, ensuring the effective enrichment of the target product and providing a high-purity active ingredient for subsequent pharmaceutical applications.

[0059] Another embodiment of the present invention provides a drug for promoting gastrointestinal motility, comprising rosin A.

[0060] Specifically, by using rosin A as the core component of this drug, the specific effective substance in peach blossoms that promotes gastrointestinal motility was identified, resolving the issue of unclear components responsible for the diuretic and laxative effects of peach blossoms. In particular, rosin A was confirmed as the key active ingredient in peach blossoms with diuretic and laxative effects, and its presence is directly related to the promotion of gastrointestinal motility. This not only fills the gap in theoretical evidence but also allows drug development to be based on a clear material foundation.

[0061] In this embodiment, the diuretic and laxative effects of peach blossoms are supported by scientific theory, providing a clear source of active ingredients for the development of drugs that promote gastrointestinal motility. Compared to the basic approach, this application has the advantage of providing a reliable theoretical basis, which can significantly improve the targeting and reliability of drug application, thereby effectively expanding the application value of peach blossoms in the fields of medicine and food.

[0062] Optionally, rosin A is extracted from peach raw materials using the rosin A extraction method described above that promotes gastrointestinal motility.

[0063] In this optional embodiment, the source and extraction process of rosin A are clearly defined, ensuring the quality and consistency of the active ingredient in the drug. Specifically, a specific extraction method is used to obtain rosin A from peach blossom raw materials, which can effectively control the purity of the extract and avoid interference from impurities, thereby ensuring the bioactivity and stability of rosin A. This well-defined extraction route, combined with the diuretic and laxative effects of peach blossoms themselves, makes the prepared drug more reliable and stable in promoting gastrointestinal motility. In addition, by optimizing the extraction process, the yield of rosin A can be increased, production costs can be reduced, and batch-to-batch consistency of the product can be ensured, laying a solid foundation for the large-scale production and clinical application of the drug.

[0064] Rosinoside A, the core active ingredient in the gastrointestinal motility-promoting drug proposed in this application, has a chemical structure and biological activity that form the basis for its pharmacological effects. The purity, stability, and bioavailability of this ingredient directly affect the efficacy and safety of the drug. To obtain high-purity, high-activity rosinoside A, this application employs an extraction method for rosinoside A that promotes gastrointestinal motility. This method aims to efficiently separate the target compound from a complex natural product matrix through a series of physical and chemical separation techniques, while preserving its biological activity to the greatest extent possible. For example, a combination of techniques such as solvent extraction, adsorption separation, and chromatographic separation can be used to enrich and purify the target component. Another approach is to utilize advanced extraction technologies such as ultrasound-assisted extraction and microwave-assisted extraction to improve extraction efficiency, combined with membrane separation and ion exchange techniques for preliminary purification, followed by fine chromatographic separation to obtain high-purity rosinoside A. Furthermore, this application specifies that the source of rosinoside A is peach blossom. The selection of peach blossom as the raw material is based on its traditional medicinal value and the recognition of its diuretic and laxative effects by modern research. Extracting active ingredients from natural plants helps maintain their natural conformation and synergistic effects, potentially leading to better biocompatibility and pharmacological activity. For example, peach blossoms from different origins and growth stages can be used as raw materials, but it is essential to ensure the stability of their effective ingredient content. Furthermore, peach blossom raw materials can undergo pretreatment before extraction, such as drying, pulverizing, and defatting, to improve extraction efficiency and purity.

[0065] The present invention will be further described below with reference to specific embodiments.

[0066] Example 1: Extraction of the first extract of rosin A.

[0067] Peach blossom raw materials were dried, and extracted by reflux with an ethanol-water solution. The mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to remove the ethanol, yielding a concentrated peach blossom extract. The concentrated peach blossom extract was evenly dispersed in a certain amount of water, and extracted with ethyl acetate. The aqueous phase was collected and concentrated to obtain crude peach blossom extract 1-1. The ethyl acetate phase was collected and concentrated to obtain the first extract.

[0068] The laxative activity of peach blossom extract 1-1 and the first extract was determined using a zebrafish model, such as... Figure 4 As shown in the figure, the results indicate that the first extract of peach blossom extract has significant laxative activity under the same conditions.

[0069] The zebrafish model determination method is as follows: I. Laboratory Animals AB strain zebrafish juveniles with a 4 dpf (days after fertilization) were selected. At this time, the juvenile fish's intestines have fully differentiated, and they have complete peristalsis and defecation functions. They are also transparent, making them easy to observe in vivo.

[0070] Rearing conditions: water temperature 28.5°C, light-dark cycle of 14 hours of light / 10 hours of darkness, and aerated dechlorinated water for aquaculture.

[0071] Reagents and Consumables Test substance: must be dissolved in 0.1% DMSO-embryo culture medium.

[0072] Labeling reagent: Nile Red, used to trace intestinal transit rate.

[0073] II. Core Experimental Methods and Procedures 1. Exposure treatment of the test substance Normally developed and malformed zebrafish juveniles were selected and randomly divided into groups (n=20 fish per group). The test substances included: blank control group (embryo culture medium), peach blossom extract 1-1 dissolved in 0.1% DMSO-embryo culture medium, and first extract dissolved in 0.1% DMSO-embryo culture medium.

[0074] Juvenile fish were transferred to 96-well plates, one fish per well, and the test substance was added. The plates were then placed in an incubator at 28.5 °C for 48 h for exposure.

[0075] 2. Indicators for evaluating laxative activity: Measurement of intestinal peristalsis rate (fluorescence tracer method) After the exposure, each group of juvenile fish was injected intraperitoneally with Nile Red solution (0.5 μg / fish).

[0076] The migration distance of Nile Red in the intestine was observed under a fluorescence microscope.

[0077] like Figure 4 As shown, the laxative active substances in the first extract can promote intestinal peristalsis and significantly increase the transport rate of fluorescently labeled substances.

[0078] Example 2: Extraction of the second extract of rosin A.

[0079] The first extract obtained in Example 1 was added to ethyl acetate solvent and silica gel for column chromatography, mixed evenly, and the ethyl acetate solvent was evaporated. The mixture was then separated using a 300-400 mesh silica gel column, eluted with a dichloromethane-methanol solution for 4 column volumes. The dichloromethane-methanol volume ratio was divided into 5 gradients, including 50:1, 20:1, 10:1, 5:1, and 1:1. The eluents from each gradient were collected and concentrated to obtain peach blossom extract 2-1, peach blossom extract 2-2, peach blossom extract 2-3, the second extract, and peach blossom extract 2-5, respectively.

[0080] The laxative activity of peach blossom extract 2-1, peach blossom extract 2-2, peach blossom extract 2-3, the second extract, and peach blossom extract 2-5 was determined using the zebrafish model of Example 1. Figure 5 As shown in the figure, the results indicate that the second extract of peach blossom extract has significant laxative activity under the same conditions.

[0081] Example 3: Extraction of the third extract of rosin A.

[0082] The second extract obtained in Example 2 was dissolved in dimethyl sulfoxide and separated by preparative high-performance liquid chromatography (HPLC) using a C18 column (particle size: 30 μm) as the packing material and a methanol-water solution (50% methanol by volume) as the mobile phase. The chromatogram of the preparative HPLC separation is shown below. Figure 6 As shown. Each sample was collected every 3 minutes to obtain peach blossom extracts 3-1 to 3-19, which were then concentrated.

[0083] The laxative activity of peach blossom extracts 3-1 to 3-19 was determined using the zebrafish model of Example 1. Figure 7 As shown, the third extract includes the sample components from the 38th to the 50th minute, namely, the third extract includes peach blossom extracts 3-12 to 3-15. Peach blossom extracts 3-12 (38.01-41.00 min), 3-13 (41.01-44.00 min), 3-14 (44.01-47.00 min), and 3-15 (47.01-50.00 min) have significant laxative activity under the same conditions.

[0084] Example 4: Extraction of rosin A.

[0085] The third extract obtained in Example 3 was dissolved in methanol and subjected to semi-preparative high-performance liquid chromatography (HPLC) separation using a C18 column packed with 9 μm particles and a methanol-water solution (40% methanol by volume) as the mobile phase. The chromatogram of the semi-preparative HPLC separation is shown below. Figure 8 As shown. At a UV 254nm wavelength, peach blossom extracts 4-1 (collection began when the UV254 absorbance first exceeded 300 and ended when it exceeded 300), 4-2 (collection began when the UV254 absorbance second exceeded 300 and ended when it exceeded 300), 4-3 (collection began when the UV254 absorbance third exceeded 300 and ended when it exceeded 300), 4-5 (collection began when the UV254 absorbance fifth exceeded 300 and ended when it exceeded 800), and 4-6 (collection began when the UV254 absorbance again exceeded 800 and ended when it exceeded 800) were collected for concentration.

[0086] The laxative activity of peach blossom extracts 4-1, 4-2, 4-3, rosin A, 4-5, and 4-6 was determined using the zebrafish model of Example 1. Figure 9 As shown, rosin A exhibits significant laxative activity under the same conditions.

[0087] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for extracting rosin A, which promotes gastrointestinal motility, characterized in that, Includes the following steps: S1: Peach blossom raw material was extracted by reflux with ethanol-water solution, filtered, the filtrate was collected, the ethanol was removed, and the extract was extracted with ethyl acetate. The ethyl acetate phase was concentrated to obtain the first extract. S2: Take the first extract, add ethyl acetate solvent and silica gel for column chromatography, mix well, evaporate the ethyl acetate solvent, separate by silica gel column, elute with dichloromethane-methanol solution for 3 to 5 column volumes, collect the eluent and concentrate to obtain the second extract; S3: Take the second extract, add dimethyl sulfoxide to dissolve it, and use a C18-filled chromatography column with methanol-water solution as the mobile phase for high performance liquid chromatography separation. Collect the sample components from the 38th to the 50th minute of separation and concentrate them to obtain the third extract. S4: Take the third extract, dissolve it in methanol, and perform high performance liquid chromatography separation using a C18 packed column and methanol-water solution as the mobile phase. When the UV 254 absorbance exceeds 300 mAU for the fourth time at a UV 254 nm wavelength, start collecting the sample components until the UV 254 absorbance is lower than 300 mAU. Concentrate the collected sample components to obtain rosin A.

2. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In step S1, ethanol is removed by vacuum concentration at a pressure of -0.06 to -0.09 MPa.

3. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In step S2, the silica gel packing material of the silica gel chromatographic column has a particle size of 300 to 400 mesh.

4. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In step S2, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is 8:1 to 3:

1.

5. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In S3, the particle size of the C18 packing material in the chromatography column is 30 μm.

6. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In step S3, the volume fraction of methanol in the methanol aqueous solution is 20% to 80%.

7. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In S4, the particle size of the C18 packing material in the chromatography column is 9 μm.

8. The method for extracting rosin A, which promotes gastrointestinal motility, according to claim 1, is characterized in that... In step S4, the volume fraction of methanol in the methanol aqueous solution is 30% to 50%.

9. A drug for promoting gastrointestinal motility, characterized in that, Including rosin A.

10. The drug for promoting gastrointestinal motility according to claim 9, characterized in that, The rosin A is extracted from peach raw materials using the rosin A extraction method for promoting gastrointestinal motility as described in any one of claims 1 to 8.

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