A method for extracting, separating and detecting active ingredients in safflower medicinal materials

Abstract

The present application is suitable for the technical field of active ingredient processing of medicinal materials, and particularly relates to a method for extracting, separating and detecting active ingredients in safflower medicinal materials, which comprises selecting a proper amount of target safflower medicinal materials, fully crushing them, and performing several times of reflux extraction on the crushed target safflower medicinal materials with ethanol solution. Through the whole process, the active ingredients such as safflowerin in safflower are fully extracted and enriched by the systematic extraction, separation and detection means, and finally the content and purity thereof are accurately detected by HPLC. The beneficial effects of the scheme lie in that the purity of safflowerin active ingredients is significantly improved by optimizing the extraction and separation steps, the interference of impurities is reduced, and the stability of the components and the reliability of the detection results in the detection process are improved by using the strategy of preventing oxidation by ascorbic acid.

Description

Technical Field

[0001] This invention relates to the field of active ingredient processing technology for medicinal materials, specifically a method for extracting, separating and detecting active ingredients in safflower. Background Technology

[0002] Safflower (Carthamus tinctorius L.) is a commonly used traditional Chinese medicine with various effects such as promoting blood circulation, removing blood stasis, relieving pain, and anti-oxidation. Its main active components include safflower tinctorius, flavonoids, and phenolic compounds. Safflower tinctorius, as a key active component in safflower, has high pharmacological value, especially in promoting blood circulation, anti-inflammation, and anti-oxidation. To fully develop the medicinal value of safflower, traditional extraction, separation, and detection techniques have always been a focus of research and industrial production. In existing technologies, ethanol or water is usually used as a solvent to extract safflower. Safflower tinctorius components are extracted from the medicinal material through solvent reflux or ultrasonic extraction, followed by separation by liquid-liquid extraction, column chromatography, etc. Finally, high-performance liquid chromatography (HPLC) and other analytical methods are used for quantitative detection of safflower tinctorius components. However, the efficiency and separation accuracy of traditional methods are often limited by the extraction process, resulting in insufficient extraction of active components and affecting the realization of its medicinal value.

[0003] Currently, the extraction of active ingredients from safflower is hampered by technical shortcomings such as low extraction rates, poor separation effects, and severe interference from impurities. Due to the polarity differences of safflowerin and other active ingredients, it may be impossible to completely extract all effective components, especially during multiple extractions where active ingredients are easily lost. Furthermore, traditional separation techniques, such as macroporous adsorption resins or liquid-liquid extraction, often fail to effectively remove impurities due to their low selectivity, making it difficult to obtain high-purity safflowerin-like components in subsequent detection processes. Regarding detection techniques, although HPLC is widely used for quantitative analysis, under current technological conditions, the instability of components and oxidative degradation often affect the accuracy of detection results, thus limiting the assessment of the medicinal value of extracts and standardized production. Summary of the Invention

[0004] A method for extracting, separating, and detecting active ingredients in safflower, comprising the following steps:

[0005] Step S100: Select an appropriate amount of target safflower medicinal material, pulverize it thoroughly, and use an ethanol solution to perform several reflux extractions on the pulverized target safflower medicinal material.

[0006] Step S200: Filter the mixed solution of the target safflower medicinal material that has undergone several reflux extractions, combine the extracts, and concentrate them until alcohol-free to obtain a concentrated solution;

[0007] Step S300: The obtained concentrate is adsorbed by macroporous adsorption resin, and impurities are first eluted with ethanol solution of the first preset concentration, and then eluted with ethanol solution of the second preset concentration to separate safflowerin and obtain the target eluent.

[0008] Step S400: The target eluent is concentrated and dried to obtain an enrichment of safflower glycosides.

[0009] Step S500: The safflower glycosides enrichment obtained in step S400 is detected by HPLC under the following conditions:

[0010] The chromatographic column was a Waters Symmetry C18 column;

[0011] Column temperature 25-35℃;

[0012] Mobile phase: Mobile phase A is 0.02%-0.05% formic acid, of which mobile phase A contains 0.1%-0.2% ascorbic acid; mobile phase B is methanol.

[0013] The elution method is gradient elution.

[0014] Furthermore, in step S100, the pulverized target safflower material is subjected to reflux extraction with an ethanol solution at least three times.

[0015] Furthermore, in step S300, the first preset concentration is 0-10%, and the second preset concentration is 20-30%.

[0016] Furthermore, in step S400, when concentrating the target eluent, low-temperature reduced pressure conditions are used, and the temperature is below 40°C; when drying the target eluent, vacuum conditions are used.

[0017] Furthermore, in step S500, the chromatographic column is selected as a C18 chromatographic column with a particle size of 3 μm or smaller, including the Waters Symmetry C18 3 μm column.

[0018] Furthermore, in step S500, the column temperature is set to a constant 30°C. The temperature is kept stable by using an automatic temperature control system to avoid differences in separation effect and detection reproducibility due to temperature fluctuations.

[0019] Furthermore, in step S500, the gradient elution program is set as follows: the proportion of mobile phase B (methanol or acetonitrile) is 20% in the initial stage, and gradually increased to 50% at a rate of 2% per minute in the first 10 minutes;

[0020] Next, increase the concentration to 80% at a rate of 3% per minute over 10 to 20 minutes; finally, maintain the 80% concentration and perform isocratic elution for 5 minutes.

[0021] The present invention provides a method for the extraction, separation and detection of active ingredients in safflower, which has the following beneficial effects:

[0022] The entire process utilizes systematic extraction, separation, and detection methods to fully extract and enrich active ingredients such as safflower oleracea. Finally, HPLC is used to accurately determine their content and purity. The beneficial effects of this method lie in significantly improving the purity of safflower oleracea active ingredients and reducing interference from impurities by optimizing extraction and separation steps. Simultaneously, the use of ascorbic acid to prevent oxidation improves the stability of components and the reliability of detection results during the detection process. Compared to traditional extraction and detection methods, this technology offers higher extraction efficiency and separation accuracy, providing a more efficient technical means for the development and utilization of safflower medicinal materials, demonstrating significant technological advancement and application prospects. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0025] In one embodiment of the present invention, a method for extracting, separating and detecting active ingredients in safflower includes the following steps:

[0026] Step S100: Select an appropriate amount of target safflower medicinal material, pulverize it thoroughly, and use an ethanol solution to perform several reflux extractions on the pulverized target safflower medicinal material.

[0027] Step S200: Filter the mixed solution of the target safflower medicinal material that has undergone several reflux extractions, combine the extracts, and concentrate them until alcohol-free to obtain a concentrated solution;

[0028] Step S300: The obtained concentrate is adsorbed by macroporous adsorption resin, and impurities are first eluted with ethanol solution of the first preset concentration, and then eluted with ethanol solution of the second preset concentration to separate safflowerin and obtain the target eluent.

[0029] Step S400: The target eluent is concentrated and dried to obtain an enrichment of safflower glycosides.

[0030] Step S500: The safflower glycosides enrichment obtained in step S400 is detected by HPLC under the following conditions:

[0031] The chromatographic column was a Waters Symmetry C18 column;

[0032] Column temperature 25-35℃;

[0033] Mobile phase: Mobile phase A is 0.02%-0.05% formic acid, of which mobile phase A contains 0.1%-0.2% ascorbic acid; mobile phase B is methanol.

[0034] The elution method is gradient elution.

[0035] In one embodiment of the present invention, a systematic design and optimization were carried out for the extraction, separation, and detection of active ingredients in safflower. First, in step S100, an appropriate amount of target safflower is selected and thoroughly pulverized to increase its surface area. This process aims to better expose the effective components within the cells, thereby improving the efficiency of subsequent extraction. The pulverized safflower is then subjected to several reflux extractions using an ethanol solution. Ethanol, as a commonly used organic solvent, has strong dissolving power and can effectively extract active ingredients from safflower, such as safflowerin and its flavonoids. Multiple reflux extractions maximize the extraction of effective components, ensuring sufficient extraction.

[0036] In step S200, the mixed solution that has undergone multiple reflux extractions is filtered to remove solid residues and ensure the purity of the liquid in subsequent processing. The extracts are then combined and concentrated under reduced pressure until alcohol-free, yielding a concentrated solution. The purpose of this concentration process is to remove excess ethanol solvent and increase the concentration of the active ingredient, preparing for subsequent separation steps.

[0037] Next, in step S300, the concentrate is subjected to adsorption treatment with a macroporous adsorption resin. The purpose of this process is to utilize the resin's porous structure to adsorb and enrich safflower-like active ingredients while removing other irrelevant impurities. First, elution is performed with a first preset concentration (0-10%) of ethanol solution to remove low-polarity impurities. Then, safflower-like ingredients are eluted with a second preset concentration (20-30%) of ethanol solution. This fractional elution method effectively separates safflower-like ingredients and their related active components, thereby obtaining the target eluent.

[0038] In step S400, the obtained target eluent is concentrated under low-temperature reduced pressure and then vacuum dried to finally obtain the enriched safflowerin components. The purpose of concentration and drying is to remove residual solvent, further increase the concentration of safflowerin components, and maintain the stability of the active ingredients of safflowerin by controlling the drying conditions, thus preventing their degradation at high temperatures.

[0039] In step S500, the safflower extract concentrate obtained in step S400 is detected using high-performance liquid chromatography (HPLC). The HPLC detection conditions are as follows: a Waters Symmetry C18 column is used, and the column temperature is controlled between 25-35℃ to ensure optimal separation. Mobile phase A is 0.02%-0.05% formic acid, containing 0.1%-0.2% ascorbic acid, to maintain an acidic environment and prevent oxidation of safflower extract during detection. Mobile phase B is methanol, using a gradient elution method to better separate the target compounds and ensure the accuracy and precision of the detection results.

[0040] The entire process utilizes systematic extraction, separation, and detection methods to fully extract and enrich active ingredients such as safflower oleracea. Finally, HPLC is used to accurately determine their content and purity. The beneficial effects of this method lie in significantly improving the purity of safflower oleracea active ingredients and reducing interference from impurities by optimizing extraction and separation steps. Simultaneously, the use of ascorbic acid to prevent oxidation improves the stability of components and the reliability of detection results during the detection process. Compared to traditional extraction and detection methods, this technology offers higher extraction efficiency and separation accuracy, providing a more efficient technical means for the development and utilization of safflower medicinal materials, demonstrating significant technological advancement and application prospects.

[0041] In this embodiment, in step S100, the pulverized target safflower material is subjected to at least three reflux extractions using an ethanol solution.

[0042] The step S100, which involves at least three reflux extractions of the pulverized safflower herb with an ethanol solution, is to ensure the full extraction of active ingredients. Firstly, pulverizing the safflower herb increases its surface area, making it easier for the internal active ingredients to contact the solvent, thereby improving extraction efficiency. The pulverized safflower herb is then placed in an appropriate amount of ethanol solution. Ethanol, as the extraction solvent, has good polarity matching and can effectively dissolve the active ingredients in safflower, such as safflowerin, flavonoids, and phenolic compounds.

[0043] Reflux extraction is an extraction method that involves heating to evaporate the solvent and then returning it to the sample after cooling in a condenser. This method allows for the repeated use of the same solvent with a relatively low solvent volume, ensuring continuous impregnation of the medicinal material and thus achieving higher extraction efficiency. At least three reflux extractions ensure that the active ingredients in safflower are fully extracted, reducing the likelihood of incomplete extraction. The amount of active ingredients in the solution gradually decreases after each extraction; after the third reflux extraction, almost all of the target components can usually be obtained, avoiding incomplete extraction or waste of medicinal material.

[0044] Meanwhile, triple reflux extraction ensures that components with different polarities and solubilities are fully dissolved in the solvent. This is crucial for extracting various active ingredients from safflower, especially safflower oleoresins, which may require longer solvent contact times and more thorough reflux treatment. The extract after multiple reflux extractions maximizes the content of safflower's effective components, providing a high concentration of extract for subsequent separation and purification steps.

[0045] In summary, the operation of reflux extraction with ethanol solution at least three times not only improved the extraction efficiency of safflower active ingredients, but also ensured the sufficiency and stability of the extraction, laying a good foundation for subsequent separation and detection steps.

[0046] In this embodiment, in step S300, the first preset concentration is 0-10%, and the second preset concentration is 20-30%.

[0047] In step S300, safflowerin is separated by macroporous adsorption resin, and elution is performed using ethanol solutions of different preset concentrations. Specifically, the first preset ethanol concentration is 0-10% for eluting impurities, and the second preset concentration is 20-30% for separating safflowerin.

[0048] The first preset concentration of 0-10% ethanol solution is mainly used to remove highly polar impurities, such as water-soluble sugars and inorganic salts. These components have weak adsorption to the resin and can be easily eluted in a low-concentration ethanol environment. This process effectively removes most unwanted components from the sample, preparing for subsequent fine separation. Simultaneously, controlling the concentration within the 0-10% range ensures that active ingredients are not eluted along with impurities, guaranteeing the retention of safflower-like components.

[0049] The second preset ethanol solution, with a concentration of 20-30%, is specifically used to separate safflower-like components. Safflower-like components, as the main active ingredient in safflower, have a certain degree of lipophilicity, thus requiring a higher concentration of ethanol solution for elution. A 20-30% ethanol solution can effectively dissolve and elute safflower-like components without carrying away excessive amounts of other non-target components, ensuring selective and efficient separation. This fractional elution method can effectively remove impurities while obtaining high-purity safflower-like components.

[0050] Using ethanol solutions of different concentrations for segmented elution helps to precisely control the separation process of impurities and target components. The first preset low-concentration ethanol solution preferentially removes ineffective impurities, while the second preset high-concentration ethanol solution ensures the full separation and recovery of target components, thereby maximizing the purity and yield of safflower-like components.

[0051] In this embodiment, in step S400, when concentrating the target eluent, low-temperature reduced pressure conditions are used and the temperature is below 40°C; when drying the target eluent, vacuum conditions are used.

[0052] The target eluent concentration and drying process in step S400 employs optimized conditions to maximize the stability of the safflower-like active ingredients. During the concentration of the target eluent, low-temperature vacuum conditions are used, with the temperature controlled below 40°C. This optimized design is to avoid damage to heat-sensitive components caused by high temperatures. Safflower-like active ingredients are relatively fragile and may degrade or lose their biological activity under high-temperature conditions. Therefore, low-temperature vacuum concentration effectively removes the solvent while lowering its boiling point, thus preventing the loss of heat-sensitive components.

[0053] During the drying process, vacuum drying conditions are employed to further reduce the impact of temperature on the active ingredients. In a vacuum environment, the solvent evaporation temperature is further reduced, allowing the solvent to evaporate rapidly at a relatively low temperature, ensuring that safflower pigments are not destroyed by high temperatures. Furthermore, vacuum drying effectively avoids the presence of oxygen, preventing oxidation reactions, which is crucial for maintaining the color and bioactivity of safflower pigments.

[0054] The combined application of low-temperature vacuum concentration and vacuum drying ensures the efficient recovery and stability of safflowerin components. Low-temperature vacuum concentration helps maintain the integrity of the components, while vacuum drying further improves drying efficiency and component purity. This optimized processing method preserves the medicinal value of safflowerin active ingredients while improving the purity and stability of the final product, providing high-quality safflowerin concentrates for subsequent applications.

[0055] In this embodiment, in step S500, the chromatographic column is selected as a C18 chromatographic column with a particle size of 3 μm or smaller, including the Waters Symmetry C18 3 μm column.

[0056] In step S500, high-performance liquid chromatography (HPLC) was used to detect safflower-like components. Specifically, a C18 column with a particle size of 3 μm or smaller, including the Waters Symmetry C18 3 μm column, was selected. This choice aimed to improve separation efficiency and resolution, ensuring accurate separation and detection of the active safflower-like components.

[0057] C18 chromatographic columns with smaller particle sizes, such as 3 μm or even smaller, offer a larger surface area and higher column efficiency. Compared to columns with larger particle sizes, smaller particle sizes provide finer separation, allowing for better resolution of structurally similar compounds. For example, safflower compounds have relatively complex molecular structures and may exist in multiple isomers or similar compounds. Using a C18 column with a particle size of 3 μm can yield clearer chromatographic peaks, thereby improving detection accuracy.

[0058] Furthermore, the Waters Symmetry C18 column, due to its stable packing material and high separation efficiency, is widely used for the separation and analysis of complex natural products. Combined with its 3μm particle size, this column not only enhances the resolution of target components but also completes the analysis process in a short time, improving detection efficiency. Therefore, this column is better suited to the separation requirements of safflowerin compounds, ensuring the effective identification and quantification of safflowerin in complex extracts.

[0059] By selecting more efficient chromatographic columns, the sensitivity, resolution, and stability of HPLC detection can be significantly improved. This optimization helps solve the problem of separating safflower-like components from complex extracts and ensures high reliability and repeatability of the final detection results, thus providing strong technical support for the entire extraction, separation, and detection process.

[0060] In this embodiment, in step S500, the column temperature is set to a constant 30°C. By using an automatic temperature control system, the temperature stability is maintained, avoiding differences in separation effect and detection reproducibility caused by temperature fluctuations.

[0061] In step S500, the column temperature is set to a constant 30°C, and an automatic temperature control system is used to maintain temperature stability. The purpose of this temperature setting is to optimize the HPLC separation process and ensure that safflower-like active ingredients can be separated and detected under stable conditions throughout the entire detection process.

[0062] Column temperature has a significant impact on chromatographic separation. Temperature fluctuations can cause changes in the viscosity of the mobile phase in the column, which in turn affects the flow rate of the mobile phase and the retention time of the solute. If the temperature is unstable, it may lead to changes in chromatographic peak shape, decreased separation efficiency, and even affect the repeatability and accuracy of the detection. By keeping the column temperature constant at 30℃, these adverse factors caused by temperature fluctuations can be avoided, ensuring the repeatability of the separation process.

[0063] The chosen temperature of 30℃ strikes a balance between column efficiency and the stability of the target component. At this temperature, the mobile phase viscosity is moderate, ensuring good separation of the target component, safflowerin, in the chromatographic column, while also preventing degradation or inactivation of the active ingredient that may occur due to excessively high temperatures. Furthermore, the use of an automatic temperature control system ensures minimal temperature fluctuations throughout the analysis process, further improving the accuracy of separation and the consistency of detection results.

[0064] This optimization scheme ensures the stable separation of safflower components by maintaining a stable temperature environment, thereby improving the reliability of the detection results. Especially when precise quantification and repeated experiments are required, constant temperature can effectively guarantee the reproducibility and accuracy of the experimental results.

[0065] In this embodiment, in step S500, the gradient elution program is set as follows: the proportion of mobile phase B (methanol or acetonitrile) is 20% in the initial stage, and gradually increased to 50% at a rate of 2% per minute in the first 10 minutes;

[0066] Next, increase the concentration to 80% at a rate of 3% per minute over 10 to 20 minutes; finally, maintain the 80% concentration and perform isocratic elution for 5 minutes.

[0067] The gradient elution procedure in step S500 is set to optimize the separation of safflower-like components. Specifically, the initial proportion of mobile phase B (methanol or acetonitrile) is 20%, which is gradually increased to 50% at a rate of 2% per minute over the first 10 minutes; then, it is increased to 80% at a rate of 3% per minute over 10 to 20 minutes; finally, isocratic elution is performed for 5 minutes while maintaining 80% proportion. The purpose of this design is to progressively optimize the separation process of safflower-like active ingredients under different proportions of mobile phases with varying polarities.

[0068] The initial low proportion (20%) of mobile phase B ensures that more polar impurities are eluted first, preventing them from interfering with subsequent separations. As the proportion of mobile phase B gradually increases, the elution capacity enhances, allowing safflower extracts to be separated under appropriate polarity conditions. By controlling a gentle gradient increase of 2% per minute over the first 10 minutes, low-polarity compounds are gradually eluted, effectively avoiding peak overlap and ensuring refined separation.

[0069] Within 10 to 20 minutes, the proportion of mobile phase B was increased to 80% at a rate of 3% per minute, accelerating the separation rate and ensuring complete elution of the target active ingredient, safflowerin, under suitable mobile phase conditions. This rapid gradient ascent shortened the overall analysis time while maintaining sufficient resolution to ensure clear and accurate separation of safflowerin from other components.

[0070] Finally, isocratic elution at 80% mobile phase B for 5 minutes ensures complete elution of all target components without residue. This isocratic elution stage also further removes unseparated highly polar impurities from the column, providing a stable baseline for subsequent sample analysis.

[0071] This gradient elution procedure, through reasonable control of the proportion of mobile phase B and optimization of time and rate, ensures efficient separation of safflower glycosides, greatly improving separation efficiency and detection accuracy. Simultaneously, the entire procedure, while guaranteeing separation effectiveness, also considers the rationality of analysis time, thereby improving overall experimental efficiency.

[0072] The following are targeted experiments conducted by those skilled in the art using the technical solution of this invention.

[0073] Experiment Report

[0074] 1. Experiment Name

[0075] A method for extraction, separation and detection of active ingredients in safflower.

[0076] 2. Experimental Objective

[0077] This experiment aims to optimize the extraction efficiency and purity of safflower active ingredients in safflower through systematic extraction, separation and detection methods, reduce the interference of impurities, and use high performance liquid chromatography (HPLC) to accurately detect the content of safflower, thus verifying the effectiveness and reliability of the method.

[0078] 3. Experimental Materials and Equipment

[0079] Materials: Safflower medicinal material, ethanol solution (70%), formic acid, ascorbic acid, methanol, acetonitrile (for HPLC mobile phase).

[0080] Equipment: reflux extraction device, low temperature vacuum concentrator, macroporous adsorption resin separation device, vacuum dryer, high performance liquid chromatograph (HPLC, equipped with Waters Symmetry C183μm column).

[0081] 4. Experimental Methods

[0082] 4.1. Step S100: Extraction of safflower medicinal material

[0083] First, select an appropriate amount of safflower and grind it thoroughly to increase the surface area, making it easier for the active ingredients to dissolve in the solvent. Then, use a 70% ethanol solution for reflux extraction of the ground safflower. To ensure sufficient extraction, perform three reflux extractions, each lasting one hour. The extracted solutions are then collected and combined.

[0084] 4.2. Step S200: Filtration and concentration of the extract

[0085] The solution after three extractions was filtered through filter paper to remove solid medicinal residues, yielding a clear extract. Next, a low-temperature vacuum concentration device was used to concentrate the extract, with the temperature controlled below 40°C to ensure that heat-sensitive components (such as safflower extract) were not destroyed. After concentration until no ethanol remained, a viscous concentrate was obtained.

[0086] 4.3. Step S300: Separation with macroporous adsorption resin

[0087] The concentrated solution was separated using a macroporous adsorption resin. The resin's ability to adsorb different components was utilized for fractional elution. First, elution was performed with a 0-10% ethanol solution to remove impurities; then, elution was performed with a 20-30% ethanol solution to collect the target safflowerin components. This fractional elution method effectively separates high-purity safflowerin.

[0088] 4.4. Step S400: Concentration and drying of safflower pigment components

[0089] The target eluent was concentrated under low-temperature reduced pressure, with the temperature controlled below 40°C to avoid thermal degradation of safflowerin. Subsequently, the remaining solvent in the concentrate was completely removed under vacuum drying conditions, finally yielding a dry powder of enriched safflowerin components.

[0090] 4.5. Step S500: HPLC detection

[0091] The obtained safflowerin-like components were detected by high-performance liquid chromatography (HPLC). The detection conditions were as follows:

[0092] Column: Waters Symmetry C183μm column

[0093] Column temperature: constant at 30℃

[0094] Mobile phase A: 0.02%-0.05% formic acid solution containing 0.1%-0.2% ascorbic acid to prevent oxidation of active ingredients.

[0095] Mobile phase B: Methanol or acetonitrile

[0096] Gradient elution: The initial proportion of mobile phase B is 20%, increasing by 2% to 50% per minute for the first 10 minutes; then increasing by 3% to 80% per minute for the next 10 to 20 minutes; finally, maintaining 80% isocratic elution for 5 minutes.

[0097] Detection wavelength: 280nm

[0098] 5. Experimental Results

[0099] 5.1 Extraction and Separation Effects

[0100] Through three ethanol reflux extractions, the main active ingredients in safflower were fully extracted, and the extract was deep yellow, indicating a high concentration of safflower-like components. After separation treatment with macroporous adsorption resin, high-purity safflower-like components were successfully separated using a 20-30% ethanol solution. The impurities were significantly reduced after elution, proving the effectiveness of the separation method.

[0101] 5.2. HPLC Detection Results

[0102] HPLC results showed clear, high-resolution, and symmetrical peaks for the safflowerin components, indicating good separation. The addition of ascorbic acid effectively prevented oxidation of safflowerin during detection, ensuring the stability of the data. The final results showed that the separated safflowerin content reached over 95%, with significantly reduced impurities, and the detection results were accurate and reliable.

[0103] 6. Experimental Analysis

[0104] Experimental results showed that three-stage reflux extraction with ethanol solution, combined with fractional elution using macroporous adsorption resin, significantly improved the extraction efficiency and separation purity of safflower active ingredients. Low-temperature reduced-pressure concentration and vacuum drying prevented thermal degradation of safflower components, ensuring the stability and purity of the active ingredients. During HPLC detection, the addition of ascorbic acid prevented oxidation, making the detection process more stable and ultimately yielding highly accurate quantitative results.

[0105] 7. Conclusion

[0106] This experiment successfully achieved the efficient extraction and separation of safflower-like active ingredients from safflower through a systematic extraction, separation, and detection method, obtaining high-purity safflower-like compounds. HPLC analysis showed that the safflower-like compound content was over 95%, verifying the superiority of this method in terms of extraction efficiency, separation purity, and detection accuracy. This method provides an efficient and reliable technical means for the development and application of safflower, possessing high application value and industrialization potential.

[0107] 8. References

[0108] Wang Wei et al. Study on extraction and purification process of safflower oleracea extract [J]. Chinese Traditional and Herbal Drugs, 2012, 43(5): 865-868.

[0109] This paper presents a detailed study on the extraction and purification process of safflower oleracea. By optimizing the extraction conditions and separation process, the extraction efficiency and purity of safflower oleracea were significantly improved, providing an important theoretical basis for the extraction scheme of this invention.

[0110] Li Xiaohong, Liu Mingjie. Application of high performance liquid chromatography in the analysis of components of traditional Chinese medicine [J]. Analytical Chemistry, 2018, 46(3):343-348.

[0111] This article introduces the application of high performance liquid chromatography (HPLC) in the detection of active ingredients in traditional Chinese medicine, especially the advantages of C18 column in the separation of complex components. Combined with the use of antioxidants, it provides an important reference for the HPLC detection steps of this invention.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting, separating, and detecting active ingredients in safflower, characterized in that, Includes the following steps: Step S100: Select an appropriate amount of target safflower medicinal material, pulverize it thoroughly, and use an ethanol solution to perform several reflux extractions on the pulverized target safflower medicinal material. Step S200: Filter the mixed solution of the target safflower medicinal material that has undergone several reflux extractions, combine the extracts, and concentrate them until alcohol-free to obtain a concentrated solution; Step S300: The obtained concentrate is adsorbed by macroporous adsorption resin, and impurities are first eluted with ethanol solution of the first preset concentration, and then eluted with ethanol solution of the second preset concentration to separate safflowerin and obtain the target eluent. Step S400: The target eluent is concentrated and dried to obtain an enrichment of safflower glycosides. Step S500: The safflower glycosides enrichment obtained in step S400 is detected by HPLC under the following conditions: The chromatographic column was a Waters Symmetry C18 column; Column temperature 25-35℃; Mobile phase: Mobile phase A is 0.02%-0.05% formic acid, of which mobile phase A contains 0.1%-0.2% ascorbic acid; mobile phase B is methanol. The elution method is gradient elution.

2. The method for extraction, separation and detection of active ingredients in safflower medicinal material according to claim 1, characterized in that, In step S100, the pulverized target safflower material is subjected to reflux extraction with ethanol solution at least three times.

3. The method for extraction, separation and detection of active ingredients in safflower medicinal material according to claim 1, characterized in that, In step S300, the first preset concentration is 0-10%, and the second preset concentration is 20-30%.

4. The method for extraction, separation and detection of active ingredients in safflower medicinal material according to claim 1, characterized in that, In step S400, when concentrating the target eluent, low-temperature reduced pressure conditions are used and the temperature is below 40°C. When drying the target eluent, vacuum conditions are used.

5. The method for extraction, separation and detection of active ingredients in safflower according to claim 1, characterized in that, In step S500, the chromatographic column is selected as a C18 column with a particle size of 3 μm or smaller, including the Waters Symmetry C18 3 μm column.

6. The method for extraction, separation and detection of active ingredients in safflower medicinal material according to claim 1, characterized in that, In step S500, the column temperature is set to a constant 30°C. The temperature is kept stable by using an automatic temperature control system to avoid differences in separation effect and detection reproducibility due to temperature fluctuations.

7. The method for extraction, separation and detection of active ingredients in safflower medicinal material according to claim 1, characterized in that, In step S500, the gradient elution program is set as follows: the proportion of mobile phase B (methanol or acetonitrile) is 20% in the initial stage, and gradually increased to 50% at a rate of 2% per minute in the first 10 minutes; Next, increase the concentration to 80% at a rate of 3% per minute over 10 to 20 minutes; finally, maintain the 80% concentration and perform isocratic elution for 5 minutes.