Isolation and purification and content determination method of jingtaiji glycolipid compound

Abstract

The application discloses a glycosphingolipid compound from the origin of Trichosanthin and a separation and purification method and a content determination method thereof. The compound is HexCer 18:1;3O / 26:1;(2OH), and a molecular formula of the compound is C 50 H 95 NO 10 The compound is obtained by adopting a method of separation by means of a dextran gel chromatographic column, combined with petroleum ether degreasing and methanol recrystallization. The content determination adopts a high performance liquid chromatography triple quadrupole mass spectrometry, mass spectrometry detection is carried out in a multiple reaction monitoring mode, quantitative ion pairs are m / z 870.631→708.600, a de-clustering potential is 221.00, and a collision energy is 23.00. Chromatographic conditions adopt a C8 chromatographic column, methanol and 0.05-0.2% formic acid water are used as mobile phases to carry out gradient elution. After the sample is ultrasonically extracted by methanol, the sample is filtered to obtain a test sample solution. The method has strong specificity, high accuracy and good reproducibility, can accurately determine the content of HexCer 18:1;3O / 26:1;(2OH) in Trichosanthin, and provides a reliable technical means for quality control of Trichosanthin medicinal materials and related products.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the separation, extraction and quality control of effective components of traditional Chinese medicine, and particularly to a method for separating and purifying glycosphingolipid compounds in Euphorbia pekinensis and a method for determining their content based on high performance liquid chromatography-triple quadrupole mass spectrometry. Background Technology

[0002] Euphorbia pekinensis, a traditional Chinese medicinal herb, was first recorded in the Shennong Bencao Jing (Shennong's Classic of Materia Medica). It is bitter, cold, and toxic, and enters the lung, spleen, and kidney meridians. Clinically, it is often used to purge water retention and reduce swelling. Guided by traditional Chinese medicine theory, Euphorbia pekinensis is frequently used to treat edema, abdominal distension, ascites, and phlegm accumulation, demonstrating its strong purgative and diuretic effects. However, its definite toxic side effects are also recorded in numerous medical texts throughout history, mainly manifesting as gastrointestinal reactions such as nausea, vomiting, abdominal pain, and severe watery diarrhea after administration. This, to some extent, limits its widespread clinical application and in-depth development. Therefore, elucidating the specific material basis of the purgative effect of Euphorbia pekinensis is not only crucial for controlling its clinical medication risks and achieving safe and effective use, but also an important scientific issue for identifying key active ingredients from the complex system of traditional Chinese medicine, thereby providing lead compounds for the development of related new drugs.

[0003] In recent years, modern research on *Euphorbia pekinensis* has increasingly focused on its toxic substances and mechanisms of action. Previous studies have found a close correlation between the toxicity of *Euphorbia pekinensis* and the inflammatory response it induces. For example, triterpenoids isolated from *Euphorbia pekinensis* can significantly stimulate macrophages to release inflammatory factors such as tumor necrosis factor-α and interleukin-1β, which is considered one of the main mechanisms of its inflammatory toxicity. These studies have provided clues to understanding the immunotoxicity of *Euphorbia pekinensis*, but mainly focus on the inflammatory process. However, the attribution and pathway of action of the substances that cause diarrhea most typically and directly in clinical applications of *Euphorbia pekinensis* remain unclear. Although significant diarrheal effects can be observed in animal experiments with the whole decoction or crude extract of *Euphorbia pekinensis*, it is still inconclusive which chemical component plays a dominant role—whether it directly affects intestinal smooth muscle and intestinal motility, or affects intestinal mucosal secretion, or both. Current research on the chemical constituents of *Euphorbia pekinensis* mainly focuses on triterpenes and diterpenes. While patent literature reports diterpenoids with antitumor activity and their preparation methods, less attention has been paid to its potential glycosphingolipid components, and there are no reports clearly linking these components to the purgative effect of *Euphorbia pekinensis*. In the field of screening active ingredients in traditional Chinese medicine, whole animal models are commonly used to evaluate purgative effects; however, these models are complex and subject to many interfering factors, making them difficult to directly track and identify single active ingredients. While in vitro intestinal experiments and other methods for activity-directed separation can more directly reflect the impact on intestinal function, their application in *Euphorbia pekinensis* research has not yet been systematically combined with the separation and identification of specific chemical components. Furthermore, studies on the content determination of specific components in *Euphorbia pekinensis* mostly focus on known triterpenes, lacking specific and sensitive quality control methods for potential purgative active ingredients. Therefore, the existing technology still has obvious shortcomings. First, it has failed to isolate the characteristic glycosphingolipid components with purgative activity from the complex system of Euphorbia pekinensis. Second, there is no research on establishing a reliable content determination method for this specific purgative component, which cannot provide a basis for the quality evaluation of medicinal materials and related products. These shortcomings urgently need to be addressed. Summary of the Invention

[0004] Technical Problem Solved: To address the technical problem that existing technologies have not successfully isolated characteristic glycosphingolipid components from complex extracts of Euphorbia pekinensis, and lack reliable quantitative detection methods for this specific component, this invention provides a method for the separation and purification of glycosphingolipid compounds derived from Euphorbia pekinensis, as well as a method for content determination. This method enables the first-ever isolation of high-purity HexCer 18:1;3O / 26:1;(2OH) compounds from Euphorbia pekinensis, and establishes a highly specific and sensitive content determination method based on high-performance liquid chromatography-triple quadrupole mass spectrometry. This allows for accurate quantification of this key component in Euphorbia pekinensis and related products, providing technical support for the quality control and rational clinical use of Euphorbia pekinensis.

[0005] Technical solution: A glycosphingolipid compound derived from Euphorbia pekinensis, wherein the compound is HexCer 18:1;3O / 26:1;(2OH), and the molecular formula is C 50 H 95 NO 10 .

[0006] A method for preparing glycosphingolipid compounds includes the following steps: separating Euphorbia pekinensis extract by dextran gel chromatography, collecting the fraction containing the target compound, defatting with petroleum ether, and then recrystallizing with methanol to obtain HexCer 18:1;3O / 26:1;(2OH).

[0007] A method for detecting the content of the glycosphingolipid compound in a sample, comprising high performance liquid chromatography-triple quadrupole mass spectrometry, wherein the mass spectrometry detection adopts multiple reaction monitoring mode, and the ion pair used for quantitative analysis is m / z 870.631→708.600.

[0008] The chromatographic conditions were as follows: a C8 column was used, with methanol as mobile phase A and 0.05–0.2% formic acid aqueous solution as mobile phase B, and gradient elution was performed.

[0009] The C8 column mentioned above is a ZORBAX XDB-C8 with a size of 150×4.6mm.

[0010] The declustering potential for mass spectrometry detection is 221.00, and the collision energy is 23.00.

[0011] The above samples are Euphorbia pekinensis or its products. Before the test, a test solution needs to be prepared: take the sample powder, add methanol to extract, filter, and take the filtrate as the test solution.

[0012] The above extraction was performed by ultrasonic extraction or reflux extraction, with an extraction time of 10 to 60 minutes and a methanol dosage of 50 to 300 times the weight of the sample powder.

[0013] The above extraction was performed by ultrasonic extraction for 30 minutes, with 200 times the amount of methanol used and a methanol concentration of 100%.

[0014] The above method also includes the step of preparing a reference solution: take HexCer 18:1;3O / 26:1;(2OH) reference standard, dissolve and dilute it with methanol to prepare a series of concentration solutions, inject them into a chromatography-mass spectrometry instrument for analysis, and establish a standard curve.

[0015] Beneficial Effects: This invention utilizes a dextran gel chromatography column combined with petroleum ether-methanol recrystallization to isolate and purify a glycosphingolipid compound, HexCer 18:1;3O / 26:1;(2OH), from *Euphorbia pekinensis*, filling a gap in the research on this type of component in *Euphorbia pekinensis*. Based on this, a high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS) method for the content determination of this compound was established. Through systematic investigation of the extraction solvent, solvent volume, extraction method, and extraction time, the optimal sample preparation conditions were determined: methanol as the extraction solvent, ultrasonic extraction at 20 times the solvent volume for 30 minutes, ensuring complete and stable extraction of the target component. In chromatographic separation, a ZORBAX XDB-C8 column was used with gradient elution of methanol and 0.1% formic acid-water as the mobile phase, overcoming the problem of dead adsorption of long-chain glycosphingolipids by the C18 column. Mass spectrometry detection employed multiple reaction monitoring (MRM) mode. Optimized ion pairs (m / z 870.631→708.600), a declustering potential of 221.00, and a collision energy of 23.00 significantly improved detection sensitivity. The synergistic optimization of the sample pretreatment, chromatographic separation, and mass spectrometry detection conditions ensured that the methodological results met the requirements. The relative standard deviations (RSDs) of precision, repeatability, and stability were 2.4%, 3.2%, and 2.7%, respectively. The recoveries ranged from 98.4% to 104.1%, with a RSD of 2.4%, indicating that the method is highly specific, accurate, and reproducible, and can accurately determine the content of HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis. This invention provides a reliable technical means for the quality control of Euphorbia pekinensis medicinal materials and related products, which is of great significance for ensuring the safety and efficacy of clinical medication. Attached Figure Description

[0016] Figure 1 Investigation on solvent extraction from Euphorbia pekinensis.

[0017] Figure 2 Investigation on the amount of solvent used in the extraction of Euphorbia pekinensis.

[0018] Figure 3 An investigation into extraction methods of Euphorbia pekinensis.

[0019] Figure 4 Time was investigated when extracting from Euphorbia pekinensis. Detailed Implementation

[0020] Example 1 The present invention provides a screening and separation method for HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis, comprising the following steps: (1) separation and purification of the effect component HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis; (2) determination of the content of HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis.

[0021] Example 2 A screening and separation method for HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis (1) The fraction of ethyl acetate:methanol = 8:2 was fractionated and recrystallized to obtain a compound: HexCer 18:1;3O / 26:1;(2OH) by gel chromatography column.

[0022] The process for preparing the effect flow fraction is as follows: Ten kilograms of Euphorbia pekinensis slices were pulverized using a pulverizer and extracted twice with 10 times the volume of 95% ethanol under reflux, 1 hour each time. The extract was concentrated under reduced pressure, suspended in 20 L of water, and extracted three times with 20 L of dichloromethane. The dichloromethane fractions of the extracts were combined, concentrated under reduced pressure, and mixed with silica gel. Elution was then performed sequentially with solvents (petroleum ether-ethyl acetate: 9-1, 8-2, 7-3, 6-4, 5-5, 4-6, 3-7, 2-8, 1-9, 0-10; ethyl acetate-methanol: 9-1, 8-2, 7-3, 5-5, 0-10). Monitoring was performed using isolated rabbit intestinal segments, revealing that fraction 12 exhibited good intestinal motility stimulating activity. Liquid chromatography and liquid mass spectrometry analysis showed that the components in the fraction had no UV absorption and exhibited strong baseline noise interference during evaporation, affecting the preparative liquid chromatography separation process. Mass spectrometry revealed that the main components were glycosphingolipids and interfering fatty acids. Considering the structural differences between these components, gel chromatography was used for further separation. The dry gel powder was added to an excess of methanol:dichloroethylene (1:1) and swollen overnight at room temperature. The supernatant was discarded, and the suspension-sedimentation cycle was repeated three times to remove fine particles. The column was vertically fixed with a bottom sieve plate. The gel slurry (gel and buffer solution mixed 1:1) was slowly poured into the column, avoiding air bubbles. After natural sedimentation, the column was filled to the desired height. Equilibrate with 3-5 column volumes of elution buffer. Fraction 12 was reconstituted with buffer, filtered through a 0.45 μm microporous membrane, and slowly loaded. Elution was performed at a flow rate of 1 column volume / hour. The resulting fraction was dried under nitrogen. A large amount of solid precipitate was observed in tube 22. Considering the structural differences between fatty acids and glycosphingolipids, the fatty components were first removed with a large amount of petroleum ether, followed by repeated dissolution with methanol, centrifugation, and removal of insoluble components. Finally, the fraction was dried under nitrogen. NMR and high-resolution mass spectrometry were used to detect the solids after dissolution in deuterated methanol.

[0023] Example 3 Systematic experiments revealed that HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis mediates the opening of TRPA1 ion channels on enterochromaffin cells, promoting the influx of calcium ions and the release of serotonin, thus producing both dynamic and secretory diarrhea. Therefore, a method for determining the content of HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis was established.

[0024] A method for determining the content of HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis was established.

[0025] 3.1 Establishment of chromatographic conditions 3.1.1 Investigation of the stationary phase The C18 and C8 packing materials were investigated. The results showed that the carbon chains in the C18 packing material interacted too strongly with the carbon chains in HexCer 18:1;3O / 26:1;(2OH), resulting in dead adsorption of the components. Only the C8 packing material could be eluted. Therefore, ZORBAXXDB-C8 was used as the stationary phase.

[0026] 3.1.2 Investigation of the mobile phase Elution was performed using acetonitrile and methanol respectively. It was found that the hydrogen bonds in methanol could better bind to the hydrogen bonds on the sugar in HexCer 18:1;3O / 26:1;(2OH) compared with acetonitrile, and thus had a stronger elution ability. Therefore, methanol was used for elution, with methanol as mobile phase A and 0.05-0.2% formic acid water as mobile phase B, and gradient elution was performed according to the specifications in Table 5.

[0027] Table 1 Liquid Phase Conditions

[0028] 3.2 Sample preparation method of HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis 3.2.1 Investigation of Extraction Solvents Weigh approximately 0.1 g of the test sample powder accurately, and add 5 mL of 100% and 75% methanol respectively for ultrasonic extraction for 30 min. Cool, make up the weight loss, shake well, filter, and collect the supernatant. Inject the sample, record the peak area of ​​each component, and calculate the content. The results showed that 100% methanol extraction had the best effect. The results are shown in Table 1. Figure 1 .

[0029] Table 2. Extraction content results of different extraction solvents

[0030] 3.2.2 Investigation on the amount of extraction solvent used Weigh approximately 0.1 g of the test sample powder accurately, and add 2 mL, 5 mL, 10 mL, and 20 mL of 75% methanol respectively for ultrasonic extraction for 30 min. Cool, make up the weight loss, shake well, filter, and collect the supernatant. Inject the sample, record the peak area of ​​each component, and calculate the content. The results showed that a solvent concentration of 200 times the concentration yielded the best extraction effect. The results are shown in Table 2. Figure 2 .

[0031] Table 3. Results of extraction content at different amounts of extraction solvent

[0032] 3.2.3 Examination of Extraction Methods Weigh approximately 0.1 g of the sample powder accurately, add 5 mL of methanol, and extract by ultrasonication and reflux for 30 min each. Cool, make up the weight loss, shake well, filter, and collect the supernatant. Inject the sample, record the peak area of ​​each component, and calculate the content. The results showed that ultrasonication and reflux were equally effective. Considering the extraction efficiency, ultrasonication was chosen as the extraction method. The results are shown in Table 3. Figure 3 .

[0033] Table 4. Content results of different extraction methods

[0034] 3.2.4 Examination of extraction time Weigh approximately 0.1 g of the test sample powder accurately, add 10 mL of 75% methanol, and extract by ultrasonication for 10, 30, and 60 min respectively. Cool, make up for weight loss, shake well, filter, and collect the supernatant. Inject the sample, record the peak area of ​​each component, and calculate the content. The results showed that a solvent concentration of 200 times the concentration yielded the best extraction effect. The results are shown in Table 4. Figure 4 .

[0035] Table 5. Content results at different extraction times

[0036] The sample preparation method for HexCer 18:1;3O / 26:1;(2OH) in Euphorbia pekinensis was determined as follows: Approximately 0.1 g of raw Euphorbia pekinensis powder was accurately weighed and placed in a stoppered conical flask. 20 mL of methanol was accurately added, weighed, and extracted ultrasonically for 30 min. After cooling to room temperature, the mass was measured again, and the missing mass was made up with methanol. The mixture was shaken well and filtered through a 0.45 μm microporous membrane. The filtrate was collected as the final product.

[0037] 3.3 Preparation of HexCer 18:1;3O / 26:1;(2OH) standard solution Accurately weigh an appropriate amount of HexCer and add an appropriate amount of methanol to prepare a reference standard stock solution with a concentration of 100 μg / mL. For linearization, perform stepwise dilution. 3.4 Mass spectrometric parameters of HexCer 18:1;3O / 26:1;(2OH) The mass spectrometry parameters and linearity of HPLC-QQQ-MS for the determination of HexCer in Euphorbia pekinensis are shown in Table 6 below. Table 6 Mass Spectrometry Parameters and Linearity of HexCer

[0038] 3.5 Methodological Results for the Determination of HexCer 18:1;3O / 26:1;(2OH) Content in Euphorbia pekinensis The experiment showed that the precision RSD for HexCer content determination was 2.4%, the repeatability RSD was 3.2%, and the stability RSD was 2.7%. The recovery rate ranged from 98.4% to 104.1%, with an RSD of 2.4%. All methodological results met the requirements, indicating that the instrument had good precision, the method had good repeatability, and the sample stability was good.

[0039] Table 7. Methodological Results for HexCer Content Determination

[0040] 3.6 Determination of HexCer 18:1;3O / 26:1;(2OH) content in Euphorbia pekinensis The content of HexCer 18:1;3O / 26:1;(2OH) in three batches of Euphorbia pekinensis was determined, and the results are shown in Table 8. Table 8. Results of HexCer 18:1;3O / 26:1;(2OH) content determination in Euphorbia pekinensis.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glycosphingolipid compound derived from Euphorbia pekinensis, characterized in that, The compound is HexCer 18:1;3O / 26:1;(2OH), with the molecular formula C. 50 H 95 NO 10 .

2. A method for preparing the glycosphingolipid compound of claim 1, characterized in that, The procedure includes the following steps: separating the Euphorbia pekinensis extract by dextran gel chromatography, collecting the fraction containing the target compound, defatting with petroleum ether, and then recrystallizing with methanol to obtain HexCer 18:1;3O / 26:1;(2OH).

3. A method for detecting the content of the glycosphingolipid compound of claim 1 in a sample, characterized in that, The determination was performed using high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS), with multiple reaction monitoring (MRM) mode used for mass spectrometry detection. The ion pairs used for quantitative analysis were m / z 870.631→708.

600.

4. The method according to claim 3, characterized in that, The chromatographic conditions were as follows: a C8 column was used, with methanol as mobile phase A and 0.05–0.2% formic acid aqueous solution as mobile phase B, and gradient elution was performed.

5. The method according to claim 4, characterized in that, The C8 column is a ZORBAX XDB-C8 with a size of 150×4.6mm.

6. The method according to claim 3, characterized in that, The declustering potential for mass spectrometry detection is 221.00, and the collision energy is 23.

00.

7. The method according to claim 3, characterized in that, The sample is Euphorbia pekinensis or its products. Before the test, the test solution needs to be prepared: take the sample powder, add methanol to extract, filter, and take the filtrate as the test solution.

8. The method according to claim 7, characterized in that, The extraction is performed by ultrasonic extraction or reflux extraction, with an extraction time of 10 to 60 minutes, and the amount of methanol used is 50 to 300 times the weight of the sample powder.

9. The method according to claim 8, characterized in that, The extraction was performed by ultrasonic extraction for 30 minutes, with 200 times the amount of methanol used and a methanol concentration of 100%.

10. The method according to claim 3, characterized in that, It also includes the step of preparing the reference solution: take HexCer18:1;3O / 26:1;(2OH) reference standard, dissolve and dilute it with methanol to prepare a series of concentration solutions, inject them into the chromatography-mass spectrometry instrument for analysis, and establish a standard curve.

Images