Extraction and quantitative detection method for quassin components in sample

Abstract

The invention relates to the technical field of biological medicine and chemical analysis, in particular to an extraction and quantitative detection method for quassin components in a sample. The extraction method comprises a sample pretreatment step, in the step, an extraction solvent is firstly adopted for extraction, matrix interference is reduced, and then a final extract is prepared. According to the quantitative detection method for the quassin component in the sample, the detection limit is 0.01 mu g / ml, the quantification limit is 0.04 mu g / ml, the content of the quassin component in the sample can be accurately reflected, and quality control of a preparation or a pharmaceutical composition containing a brucea javanica extract is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and chemical analysis, and in particular to a method for extracting and quantitatively detecting quassin components in a sample. Background Art

[0002] Brucea javanica oil emulsion is an anti-tumor drug that has been on the market for many years. It is mainly used to treat lung cancer, brain metastasis of lung cancer and digestive tract tumors. It has a wide range of clinical applications. Its preparation formula includes Brucea javanica oil, refined soybean lecithin, and glycerol. The main components of Brucea javanica oil include oleic acid and linoleic acid, which have multiple biological activities such as anti-tumor, anti-inflammatory, and antibacterial. Currently, the quality standards of Brucea javanica medicinal materials and their preparations only stipulate the oleic acid content, but recent studies have found that the quasin-like components in Brucea javanica have strong anti-tumor activity, such as the IC of brucein in pancreatic cancer cells. 50 The concentration of quassin in Brucea javanica oil and Brucea javanica oil emulsions is 34 nM, and this type of component can produce a synergistic effect with oleic acid components. However, the analysis of quassin components in Brucea javanica oil and Brucea javanica oil emulsions faces two major challenges: First, the concentration of quassin components is low, only at the ng / g level, making it difficult to completely extract them using conventional methods, let alone accurately detect their content; second, the cost of common samples is complex, and they significantly interfere with the extraction and detection of quassin components. For example, preparations or pharmaceutical compositions containing Brucea javanica extracts contain a large number of excipients, and oleic and linoleic acid lipids have strong interference. Therefore, existing technologies cannot quantitatively detect the quassin content in Brucea javanica oil and Brucea javanica oil emulsions.

[0003] Therefore, developing a method for extracting and quantitatively detecting quasin-like components, which can extract quasin-like components from samples containing lower content and then reliably detect their content, especially being able to extract quasin-like components from preparations or pharmaceutical compositions of Brucea javanica extracts and quantitatively detect the content of quasin-like components therein with high sensitivity, is of great significance for controlling the quality of drugs and improving the efficacy of drugs. Summary of the Invention

[0004] Traditional Chinese medicine preparations contain complex ingredients. When formulating quality standards for Brucea javanica oil emulsions, oleic acid, a large, physiologically active chemical component, is generally used as a quality evaluation and control standard. However, methods for determining the content of highly active trace active ingredients remain to be developed. Commonly used methods for determining the content of active ingredients in traditional Chinese medicines include UV-visible spectrophotometry, high-performance liquid chromatography, liquid chromatography-mass spectrometry, infrared spectroscopy, gas chromatography-mass spectrometry, thin-layer chromatography scanning, and nuclear magnetic resonance spectroscopy. However, since Brucea javanica oil is composed of more than 90% fatty oil compounds, and Brucea javanica oil emulsions contain auxiliary ingredients such as phospholipids and glycerol, these seriously interfere with the quantitative detection of the small but highly active Brucea javanica quasin compounds. Therefore, a method for extracting and quantifying quasin components has been developed. This method can extract quasin components from samples and quantify their content in the detector, which is beneficial for quality control of preparations or pharmaceutical compositions containing Brucea javanica oil.

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0006] To this end, the first aspect of the present invention provides a method for extracting quassin components from a sample, the method comprising:

[0007] Step A) Sample pretreatment: pretreating the sample with extraction solvent 1 to obtain a primary extract;

[0008] Step B) preparing a final extract: extracting the primary extract described in step A) with extraction solvent 2 to obtain a final extract;

[0009] The extraction solvent 1 or 2 is at least one of ethyl acetate, oleic acid, methanol, ethanol, propylene glycol, n-butanol, Tween 80, and DMSO.

[0010] Preferably, the extraction solvent 1 includes at least one of ethyl acetate, oleic acid, methanol, ethanol, Tween 80, and DMSO, and the extraction solvent 2 includes at least one of methanol, ethanol, propylene glycol, n-butanol, DMSO, and ethyl acetate.

[0011] The method for extracting quassin-like components provided by the present invention includes a sample pretreatment step, wherein extraction is performed using an extraction solvent to reduce interference from excipients, followed by preparation of a final extract. The extraction method of the present invention offers high stability and good reproducibility, providing a foundation for content determination in preparations or pharmaceutical compositions containing quassin-like components.

[0012] According to a specific embodiment of the present invention, the content of quassin components in the sample is at least 0.01 μg / ml, preferably 0.04 μg / ml.

[0013] According to a specific embodiment of the present invention, the sample is at least one of a preparation or pharmaceutical composition containing a Brucea javanica extract; preferably, the sample includes at least one of a Brucea javanica oil emulsion injection, a Brucea javanica oil oral emulsion, and a Brucea javanica oil soft capsule.

[0014] According to a specific embodiment of the present invention, the quassin-like component includes at least one of brucein, brucein A, brucein D, and brucein.

[0015] According to a specific embodiment of the present invention, the number of extractions in step A) and / or step B) is at least two times, and can also be three times, four times, etc. The number of times is not limited here, but when the number of extractions is two, the content of the extracted quassin component is sufficient to reflect the content of the quassin-like components in the sample itself.

[0016] According to an embodiment of the present invention, the extraction method includes any one of ultrasonic extraction, mechanical stirring extraction, and heating extraction.

[0017] According to an embodiment of the present invention, the ultrasonic time is not less than 10 minutes, preferably, the ultrasonic time is not less than 20 minutes; when the ultrasonic time is 20 minutes, the content of the extracted quassin component is sufficient to reflect the content of the quassin-like components in the sample itself.

[0018] A second aspect of the present invention provides a method for quantitatively detecting the content of quassin-like components in a sample, comprising:

[0019] a) Establishing a standard curve: Take a quassin standard of known concentration for testing, and draw a standard curve with the concentration of the standard as the horizontal axis and the peak area as the vertical axis;

[0020] b) Preparation of test solution: A solution of quassin components extracted by any of the above extraction methods is used as the test sample;

[0021] c) Determination: Perform quantitative detection on the test solution.

[0022] The inventors of this application have developed a method for quantitatively detecting quasin-like components in samples through a large number of experiments. The detection limit of this method is 0.01μg / ml and the quantification limit is 0.04μg / ml. This method breaks through the difficulty in the existing technology that the target component cannot be extracted and detected in samples with low content of quasin-like compounds. It is of great significance for controlling the quality of medicines containing quasin-like components and improving the efficacy of drugs.

[0023] According to an embodiment of the present invention, the method for detecting the test solution in step c) includes any one of ultraviolet-visible spectrophotometry, chromatography, liquid chromatography-mass spectrometry, infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy.

[0024] According to a specific embodiment of the present invention, the detection method is preferably liquid chromatography-mass spectrometry (LC-MS), which combines the high separation capability of chromatography for complex samples with the advantages of mass spectrometry, which offers high selectivity, high sensitivity, and the ability to provide molecular mass and structural information. This method provides accurate and reliable detection results, with a detection limit as low as 0.01 μg / ml and a quantification limit as low as 0.04 μg / ml.

[0025] According to an embodiment of the present invention, the chromatographic conditions of the chromatography are as follows:

[0026] The detection conditions of the chromatography method include: an octadecyl bonded silica gel chromatography column; a flow rate of 0.2-0.4 mL / min; a column temperature of 35-45°C; an injection volume of 3-10 μL; a mobile phase system of mobile phase A and mobile phase B, wherein mobile phase A is formic acid-water solution and mobile phase B is acetonitrile; and an elution method of gradient elution.

[0027] The gradient elution conditions include:

[0028] The initial ratio is that the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase;

[0029] 0.01min~5min, the volume of the formic acid-water accounts for 30%-40% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 60%-70% of the total volume of the mobile phase;

[0030] 5.01 min to 5.50 min, the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase;

[0031] 5.51 min to 8 min, the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase.

[0032] The detection conditions of the liquid chromatography-mass spectrometry method include: the ion source is an electrospray ion source; the detection mode is ion monitoring; the scanning mode is a positive ion mode; the mass spectrometer dryer temperature is 250°C, the nebulizer pressure is 30 psi, the dryer flow rate is 7 L / min, the sheath gas temperature is 325°C, and the sheath gas flow rate is 11 L / min.

[0033] According to an embodiment of the present invention, the quassin standard includes any one of brucein, brucein A, brucein D, and brucein.

[0034] The third aspect of the present invention provides the use of the method described in the first aspect or the method described in the second aspect in the quality detection, quality evaluation or quality control of a preparation or pharmaceutical composition containing Brucea javanica extract.

[0035] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0037] Figure 1 Shows the chromatogram of the methanol blank solution in the embodiment of the present invention;

[0038] Figure 2 The chromatogram of the 1 μg / mL brucein working standard solution in Example 3 of the present invention is shown;

[0039] Figure 3 The chromatogram of the test solution extracted in Example 2 of the present invention is shown;

[0040] Figure 4 The chromatogram of the test solution extracted from Example 1 of the present invention is shown;

[0041] Figure 5 Shows the chromatogram of the negative sample in Example 3 of the present invention;

[0042] Figure 6 The linear regression equation of Brucea javanica oil in Example 3 of the present invention is shown;

[0043] Figure 7 The linear regression equation of the Brucea javanica oil emulsion in Example 3 of the present invention is shown. DETAILED DESCRIPTION

[0044] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0045] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0046] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0047] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0048] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0049] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0050] The present invention provides a method for extracting quassin components in a sample, the method comprising:

[0051] Step A) Sample pretreatment: pretreating the sample with extraction solvent 1 to obtain a primary extract;

[0052] Step B) preparing a final extract: extracting the primary extract described in step A) with extraction solvent 2 to obtain a final extract;

[0053] Optionally, the extraction solvent 1 or 2 is at least one of ethyl acetate, oleic acid, methanol, ethanol, propylene glycol, n-butanol, Tween 80, and DMSO.

[0054] According to a specific embodiment of the present invention, the extraction solvent is miscible with Brucea javanica oil and can dissolve quassin components but is insoluble in water.

[0055] According to a specific embodiment of the present invention, the extraction is performed at least twice in step A) and / or step B).

[0056] According to a specific embodiment of the present invention, a method for extracting quassin components from Brucea javanica oil emulsion of the present invention specifically comprises:

[0057] ① extracting the Brucea javanica oil emulsion with ethyl acetate to obtain a primary extract;

[0058] ② extracting the primary extract with methanol to obtain a final extract containing quassin components.

[0059] According to a specific embodiment of the present invention, the number of extractions in step ① and / or step ② is at least two times, and can also be three times, four times, etc. The number of times is not limited here, but when the number of extractions is two times, it is sufficient to reflect the content of quassin components in the sample itself.

[0060] It should be noted that the volume of the extractant ethyl acetate in step ① can vary according to the amount of sample, and the amount of ethyl acetate added for the first time can be the same as or different from the amount added for the second time, as long as the sample is completely dissolved in ethyl acetate.

[0061] It should be noted that the volume of methanol as the extractant in step ② can vary depending on the amount of sample, and the amount of methanol added the first time can be the same as or different from the amount added the second time, as long as the sample is completely dissolved in methanol.

[0062] According to a more specific embodiment of the present invention, when extracting the Brucea javanica oil emulsion, the present invention first pre-treats the Brucea javanica oil emulsion. The preferred treatment method is: take 20.0 g of the Brucea javanica oil emulsion sample, accurately weigh it, place it in a 50 ml centrifuge tube, accurately add 20 ml of ethyl acetate, ultrasonically extract for 20 minutes (power 250 W, frequency 20 Hz), centrifuge at 4000 r / min for 5 minutes, draw the upper clear solution into an evaporating dish, accurately add 20 ml of ethyl acetate, ultrasonically extract for 20 minutes (power 250 W, frequency 20 Hz), centrifuge at 4000 r / min for 5 minutes, combine the two extracts and concentrate under reduced pressure to obtain a primary extract. It should be noted that the volume of the extractant ethyl acetate here can vary according to the amount of sample, and the amount of ethyl acetate added for the first time can be the same or different from the amount added for the second time. It is only necessary to ensure that the sample is completely dissolved in ethyl acetate. For example, when the sample of Brucea javanica oil emulsion is 20.0g, the first amount of ethyl acetate added can be 30ml, and the second amount can be 20ml or 40ml, without limitation. At the same time, the best method is to add ethyl acetate for extraction twice, but this does not mean that the one-time extraction method cannot be used for extraction, but that the extraction rate of the two-time extraction method is higher.

[0063] In some specific embodiments, the inventors of the present application have screened out a reagent that can reduce the interference of excipients and almost does not lose the quasin component in the Brucea javanica oil emulsion after a large number of experiments. The reagent is insoluble in water. Since water accounts for a large proportion in the Brucea javanica oil emulsion, when the reagent is dissolved in water, complete separation and extraction cannot be achieved, resulting in the loss of the extracted quasin compounds. The reagent can simultaneously dissolve with the Brucea javanica oil and the quasin components and fully extract them.

[0064] According to a more specific embodiment of the present invention, the present invention begins to prepare the ultimate extract after eliminating the interference of auxiliary materials in the pretreatment stage. According to one embodiment of the present invention, the preferred preparation method of the ultimate extract is: take 2.0g of the sample (Brucea javanica oil) from which the auxiliary material components have been removed, accurately weigh it, put it in a centrifuge tube, accurately add 20ml of methanol, weigh it, ultrasonically extract it for 20min (power 250W, frequency 20Hz), centrifuge it at 4000r / min for 5 minutes, draw the upper clear solution into an evaporating dish, accurately add 20ml of methanol again, weigh it, ultrasonically extract it for 20min (power 250W, frequency 20Hz), centrifuge it at 4000r / min for 5 minutes, combine the two extracts and concentrate them under reduced pressure to near dryness, redissolve them with 2ml of methanol, centrifuge them at 4000r / min for 5 minutes, draw the upper clear solution, filter it, and take the filtrate. It should be noted that the volume of the extractant methanol here can vary according to the amount of sample, and the amount of methanol added for the first time can be the same or different from the amount added for the second time. It is only necessary to ensure that the sample is completely dissolved in methanol. For example, when the sample of Brucea javanica oil is 2.0g, the methanol added for the first time can be 30ml, and the second can be 20ml or 40ml, without limitation. At the same time, the best method is to add methanol for extraction twice, but this does not mean that the single extraction method cannot be extracted, but the extraction rate of the two extraction methods is higher. According to a more specific embodiment of the present invention, the extraction method includes any one of ultrasonic extraction, mechanical stirring extraction, and heating extraction. When the extraction method is ultrasonic extraction, the ultrasonic power is 250W, the frequency is 20Hz, and the ultrasonic time is not less than 5min. Preferably, the ultrasonic time is not less than 10min. More preferably, the ultrasonic time is 20min. After exceeding 20min, the ultrasonic time does not affect the extraction quality. It should be noted that the ultrasonic time is related to the amount of sample. The more samples, the longer the ultrasonic time. The intensity of the ultrasound is also related to the ultrasonic power. If the ultrasonic power is large, the ultrasonic time can be shortened accordingly.

[0065] The present invention provides a method for quantitatively detecting quassin components in a sample, which is characterized by comprising the following steps:

[0066] a) Establishing a standard curve: Take a quassin standard of known concentration for testing, and draw a standard curve with the concentration of the standard as the horizontal axis and the peak area as the vertical axis;

[0067] b) Preparation of test solution: A solution of quassin components extracted by the method of any one of claims 1 to 4 is used as the test sample;

[0068] c) Determination: Perform quantitative detection on the test solution.

[0069] According to a specific embodiment of the present invention, the present invention provides a method for quantitatively detecting the content of brucein in a Brucea javanica oil emulsion, comprising the following steps: (1) establishing a standard curve: taking a brucein standard of known concentration for detection, and drawing a standard curve with the concentration of the standard as the horizontal axis and the peak area as the vertical axis; (2) preparing a test solution: taking an appropriate amount of Brucea javanica oil emulsion and performing appropriate treatment to extract the quassin components therein to prepare a test solution; (3) separating and detecting the extracted brucein components by HPLC-MS to obtain the content of brucein. The treatment method described in step (2) includes, but is not limited to, appropriate chemical or physical methods such as extraction, purification, and concentration.

[0070] According to the embodiment of the present invention, the present invention utilizes chromatography to detect the content of quassin components in the extracted solution, wherein the standard substance is brucein. It should be noted that the quassin components extracted by the method of the present invention include not only brucein, but also brucein A, brucein D, and brucein. When other quassin components need to be detected, the standard substance will also be replaced accordingly. In addition, the use of chromatography for detection is one of the optional schemes of the present invention and should not be a limiting scheme of the present invention.

[0071] The detection method includes any one of ultraviolet-visible spectrophotometry, chromatography, liquid chromatography-mass spectrometry, infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy;

[0072] The detection conditions of the chromatography method of the present invention include: the chromatographic column is an octadecyl bonded silica gel chromatographic column; the flow rate is 0.2-0.4 mL / min; the column temperature is 35-45°C; the injection volume is 3-10 μL; the mobile phase system comprises mobile phase A and mobile phase B, wherein the mobile phase A is formic acid-water solution and the mobile phase B is acetonitrile; and the elution mode is gradient elution.

[0073] Preferably, the gradient elution conditions include:

[0074] The initial ratio is that the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase;

[0075] 0.01min~5min, the volume of the formic acid-water accounts for 30%-40% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 60%-70% of the total volume of the mobile phase;

[0076] 5.01 min to 5.50 min, the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase;

[0077] 5.51 min to 8 min, the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase.

[0078] The mass spectrometry detection conditions described in the present invention include: the ion source is an electrospray ion source; the detection mode is ion monitoring; the scanning mode is a positive ion mode; the mass spectrometry dryer temperature is 250°C, the nebulizer pressure is 30 psi, the dryer flow rate is 7 L / min, the sheath gas temperature is 325°C, and the sheath gas flow rate is 11 L / min.

[0079] The quantitative detection method provided by the present invention has low detection limit and quantification limit, good reproducibility, high stability and good repeatability, provides a basis for the component content detection method of preparations or pharmaceutical compositions containing Brucea javanica extract, and is beneficial to the quality control of existing products.

[0080] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0081] Example 1

[0082] Screening of extraction solvent 1 in sample pretreatment:

[0083] Since the Brucea javanica oil emulsion is a milky white uniform emulsion, during the extraction process, it is impossible to directly observe which reagents are suitable for the extraction of quasin components in the Brucea javanica oil emulsion. The prescription of the Brucea javanica oil emulsion includes water, Brucea javanica oil (oleic acid, linoleic acid, quasin components, etc.), auxiliary materials (phospholipids, glycerol), etc. After a large number of experiments, the inventors of the present application screened out pretreatment extraction reagents that are miscible with Brucea javanica oil and can dissolve quasin components. Some of the screened reagents are shown in Table 1.

[0084] Table 1

[0085]

[0086] The qualified pretreatment extraction reagents (DMSO, methanol, ethyl acetate) selected in Table 1 were used to extract the quassin components in the Brucea javanica oil emulsion:

[0087] Step A) Sample pretreatment: Take 4 20.0g samples of Brucea javanica oil emulsion (Brucea javanica oil emulsion injection, Shenyang Yaoda Leiyunshang Pharmaceutical Co., Ltd. batch number: 0230211), accurately weigh them, and place them in 50ml centrifuge tubes respectively, marked as sample 1, sample 2, sample 3 and sample 4. Accurately add 20ml of DMSO, methanol and ethyl acetate to sample 1, sample 2 and sample 3 respectively, and do not add any extraction reagent to sample 4. All four samples were ultrasonically extracted for 20min (power 250W, frequency 20Hz), centrifuged at 4000r / min for 5 minutes, and the upper clear solution was aspirated into an evaporating dish, and then accurately added 20ml of DMSO, methanol and ethyl acetate respectively, and do not add any extraction reagent to sample 4. The four samples were ultrasonically extracted for a second time for 20min (power 250W, frequency 20Hz), centrifuged at 4000r / min for 5 minutes, and the two extracts were combined and concentrated under reduced pressure to obtain DMSO primary extract, methanol primary extract, ethyl acetate primary extract and extractant-free primary extract;

[0088] Step B) Preparation of the final extract: 20 ml of methanol was precisely added to the primary extract obtained in step A), ultrasonic extraction was performed for 20 min (power 250 W, frequency 20 Hz), and centrifugation was performed at 4000 r / min for 5 minutes. The upper clear solution was aspirated into an evaporating dish, and 20 ml of methanol was precisely added again, and the weight was determined. Ultrasonic extraction was performed for 20 min (power 250 W, frequency 20 Hz), and centrifugation was performed at 4000 r / min for 5 minutes. The two extracts were combined and concentrated under reduced pressure to near dryness, re-dissolved with 2 ml of methanol, centrifuged at 4000 r / min for 5 minutes, the upper clear solution was aspirated, filtered, and the filtrate was taken to obtain a methanol solution containing quassin components. The content of the extracted quassin components was detected according to the method described in Example 3 of the present invention. The results are shown in Table 2.

[0089] Table 2

[0090] Experimental samples Extraction agent in step (A) Quassin content (μg / g) Sample 1 DMSO 0.025 Sample 2 Methanol 0.03 Sample 3 Ethyl acetate 0.045 Sample 4 - -

[0091] The content of brucea javanica alcohol in the sample measured by ethyl acetate extraction was about 0.045 μg / g, the content of brucea javanica alcohol in the sample measured by DMSO extraction was about 0.025 μg / g, and the content of brucea javanica alcohol in the sample measured by methanol extraction was about 0.030 μg / g. The content of brucea javanica alcohol in the sample without extraction agent could not be detected, and the content of brucea javanica alcohol could not be detected by skipping step A). ​​Therefore, ethyl acetate is preferably used as the pretreatment extraction reagent for extracting the quassin component in the brucea javanica oil emulsion.

[0092] Example 2

[0093] Screening of the final extract extraction step:

[0094] Take 3 parts of the sample (Brucea javanica oil, batch number 023001) without auxiliary ingredients, accurately weigh, and place in 50 ml centrifuge tubes, labeled as sample 1, sample 2, and sample 3. Sample 1 and sample 2 are accurately added with 20 ml of methanol, weighed, ultrasonic extracted for 20 min (power 250 W, frequency 20 Hz), centrifuged at 4000 r / min for 5 min, the upper clear solution is absorbed in an evaporating dish, and then accurately added with 20 ml of methanol, weighed, ultrasonic extracted for 20 min (power 250 W, frequency 20 Hz), centrifuged at 4000 r / min for 5 min, and the two extraction solutions are combined, concentrated to near dryness under reduced pressure, redissolved with 2 ml of methanol, centrifuged at 4000 r / min for 5 min, and the upper clear solution is absorbed and filtered, and the filtrate is obtained, which is the methanol solution containing picrasinoid components; sample 3 is accurately added with 40 ml of methanol, weighed, ultrasonic extracted for 40 min (power 250 W, frequency 20 Hz), centrifuged at 4000 r / min for 10 min, the upper clear solution is absorbed in an evaporating dish, filtered, and the filtrate is obtained, which is the methanol solution containing picrasinoid components. The content of the extracted picrasinoid components (javanicin) is detected according to the method in Example 3, and the specific values are shown in Table 3.

[0095] Table 3

[0096]

[0097] As shown in Table 3, the extraction rate of twice methanol extraction is higher than that of once methanol extraction (the average content of twice methanol extraction is 1.1 μg / g, and the average content of once methanol extraction is 0.8 μg / g), so it is determined that the sample is extracted twice. The content determination results of the sample with a sample amount of 2.0 g are basically equivalent to those of the sample with a sample amount of 1.0 g, but considering the response value, the sample amount of 2.0 g is selected, and the extraction method is twice methanol extraction, 20 ml each time.

[0098] The picrasinoid components are extracted according to the method of twice methanol extraction, and the content of the extracted picrasinoid components is detected, the ultrasonic power and frequency after each methanol extraction are unchanged, and different ultrasonic time lengths are set, and the specific values are shown in Table 4.

[0099] Table 4

[0100]

[0101] After comparing different ultrasonic times, the extraction rate of ultrasonic extraction for 10 min is low (the content is about 0.9 μg / g), the extraction rates of ultrasonic extraction for 20 min, 40 min, and 60 min are similar (the content is about 1.1 μg / g), in order to simplify the operation in the experiment, the ultrasonic time of the sample is determined to be 20 min.

[0102] Example 3: Detection of picrasinoid compounds in samples

[0103] The methanol solution, 1 μg / mL brucein working standard solution, the solution extracted by the method in Example 2, the solution extracted by the method in Example 1, and the negative control (soybean lecithin and glycerol mixture solution) were chromatographed. The chromatograms are shown as follows: Figure 1-Figure 5 shown.

[0104] The retention time of Brucea bruceiol is 4.158min. As shown in the figure, in the negative control chromatogram, no chromatographic peak with the same retention time as the chromatographic peak of Brucea bruceiol was detected. Figure 5 ) and methanol solvent ( Figure 1 ) had no interference, while bruceiol was detected in the 1 μg / mL bruceiol working standard solution in Example 1 of the present invention, the test solution extracted in Example 2 of the present invention, and the test solution extracted in Example 1 of the present invention, with good peak shape.

[0105] The chromatographic conditions used were as follows:

[0106] Column: Aglient XDB-C18, 2.1 × 100 mm, 1.8 μm;

[0107] Column temperature: 40°C; flow rate: 0.3 mL / min;

[0108] Mobile phase A: 0.1% formic acid-water solution;

[0109] Mobile phase B: acetonitrile;

[0110] The gradient elution program was:

[0111] The initial ratio is that the volume of formic acid-water accounts for 95% of the total volume of the mobile phase, and the volume of acetonitrile accounts for 5% of the total volume of the mobile phase;

[0112] 0.01 min to 5 min, the volume of the formic acid-water accounts for 36% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 64% of the total volume of the mobile phase;

[0113] 5.01 min to 5.50 min, the volume of the formic acid-water accounts for 95% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 5% of the total volume of the mobile phase;

[0114] 5.51 min to 8 min, the volume of the formic acid-water accounts for 95% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 5% of the total volume of the mobile phase.

[0115] Injection volume: 5 μL;

[0116] The mass spectrometry detection conditions used were:

[0117] Ion source: electrospray ionization source (AJS-ESI);

[0118] Scanning mode: positive ion scanning;

[0119] Detection method: selected ion monitoring (SIM);

[0120] Source parameters: Set according to the provisions in Table 5;

[0121] Table 5

[0122]

[0123] In the liquid phase conditions of the present invention, the column temperature, mobile phase, flow rate, etc. can be appropriately adjusted without affecting the technical effects of the present invention. For example, the same effect can be achieved when the flow rate is 0.2-0.4 mL / min; the column temperature is 35-45°C; and the injection volume is 3-10 μL.

[0124] The mass spectrometry parameters are shown in Table 6;

[0125] Table 6

[0126] Component name parent ion Dwell time Transmission voltage Accelerating voltage model Brucea javanica 543.4 400 280 3 Positive mode

[0127] Specificity: The retention time of bruceiol was determined to be 4.158 min.

[0128] Detection limit and quantification limit:

[0129] The brucea javanica reference solution was diluted with methanol and determined according to the instrumental method. The signal-to-noise ratio (S / N) of the smaller peak of brucea javanica was used for calculation. When the signal-to-noise ratio (S / N) = 3:1, it was determined as the detection limit of brucea javanica (0.01 μg / ml). When the signal-to-noise ratio (S / N) = 10:1, it was determined as the quantification limit of brucea javanica (0.04 μg / ml).

[0130] Linearity and Range:

[0131] Take an appropriate amount of javanica pyrol reference substance and dilute it with methanol in sequence to a series of standard working solutions of 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 5 μg / mL. 1 is the standard working series solution of javanica oil, and a series of standard working solutions of 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL are the standard working series solution of javanica oil emulsion. Accurately pipette 5 μL of the above solutions and inject them into liquid chromatography-mass spectrometry for determination. Draw a standard curve with the concentration of the reference substance (μg / mL) as the abscissa and the peak area as the ordinate. Calculate the regression equation and correlation coefficient r (r≥0.99). The results are as follows: Figure 6 、 Figure 7 shown.

[0132] Figure 6 、 Figure 7 The results showed that the correlation coefficient r of Brucea javanica oil was 0.9968, and the correlation coefficient r of Brucea javanica oil emulsion was 0.9975, both satisfying r≥0.99, and the Brucea javanica oil showed a good linear relationship in the range of 0.05μg / mL to 5μg / mL, and the Brucea javanica oil emulsion showed a good linear relationship in the range of 0.02μg / mL to 2μg / mL.

[0133] Instrument precision:

[0134] A 1 μg / mL working standard solution of brucein was prepared and the needle was inserted six times continuously. The peak area and retention time of brucein were recorded. The RSD of the peak area of ​​brucein in javanica oil was 1.2%, and the RSD of the retention time was 0.1%. The RSD of the peak area of ​​brucein in javanica oil emulsion was 1.5%, and the RSD of the retention time was 0%.

[0135] Repeatability:

[0136] Six parallel sample solutions were prepared and injected twice in parallel. The peak area and retention time of bruceiol were recorded. The RSD of bruceiol content in the six brucei oil sample solutions was 1.2%, and the RSD of retention time was 0.05%. The RSD of bruceiol content in the brucei oil emulsion was 1.2%, and the RSD of retention time was 0%. This method has good reproducibility and meets the requirements.

[0137] Accuracy:

[0138] Accurately weigh Brucea javanica oil and Brucea javanica oil emulsion, add a certain amount of Brucea javanica bitter alcohol standard, and calculate the recovery rate. After adding the reference substance, the total amount of bitter alcohol measured is a, the amount of bitter alcohol in the preparation without the reference substance is b, and the amount of the added reference substance is c. The recovery rate = (ab) / c*100%. The recovery rate of Brucea javanica oil is 86.3% to 89.7%; the recovery rate of Brucea javanica oil emulsion is 73.7% to 74.5%, which meets the recovery limit analysis requirements of the "Guidelines for Validation of Analytical Methods" of the 2020 edition of the Chinese Pharmacopoeia. See Table 7.

[0139] Table 7 Accuracy calculation table

[0140]

[0141]

[0142] Solution stability:

[0143] Brucea javanica oil and Brucea javanica oil emulsion samples were prepared and tested on a spectrophotometer at 0, 2, 4, 8, 12, and 24 hours to investigate the stability of brucein in the sample solutions. The chromatographic peak retention time and content of brucein in Brucea javanica oil and Brucea javanica oil emulsion samples within 24 hours all met RSDs ≤ 2.0, demonstrating the method's good solution stability.

[0144] Experimental results show that the extraction method provided by the present invention has good reproducibility, high stability and good repeatability, which provides a basis for the detection method of the content of quassin components in Brucea javanica oil and Brucea javanica oil emulsion in drugs or pharmaceutical compositions, and is beneficial to the quality control of existing products.

[0145] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0146] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for extracting quassin components from a sample, characterized in that: The following steps are involved: Step A) Sample pretreatment: pretreating the sample with extraction solvent 1 to obtain a primary extract; Step B) preparing a final extract: extracting the primary extract described in step A) with extraction solvent 2 to obtain a final extract; Optionally, the extraction solvent 1 or 2 is at least one of ethyl acetate, oleic acid, methanol, ethanol, propylene glycol, n-butanol, Tween 80, and DMSO.

2. The method according to claim 1, characterized in that The content of quassin components in the sample is at least 0.01 μg / ml, preferably 0.04 μg / ml.

3. The method according to claim 2, characterized in that The sample is at least one of a preparation or pharmaceutical composition containing a Brucea javanica extract; preferably, the sample includes at least one of a Brucea javanica oil emulsion injection, a Brucea javanica oil oral emulsion, and a Brucea javanica oil soft capsule.

4. The method according to claim 1, wherein The extraction is performed at least twice in step A) and / or step B); Optionally, the extraction method includes any one of ultrasonic extraction, mechanical stirring extraction, and heating extraction; Optionally, the ultrasound duration is not less than 10 min; more preferably, the ultrasound duration is not less than 20 min.

5. A method for quantitatively detecting quassin components in a sample, characterized in that: The following steps are involved: a) Establishing a standard curve: Take a quassin standard of known concentration for testing, and draw a standard curve with the concentration of the standard as the horizontal axis and the peak area as the vertical axis; b) Preparation of test solution: A solution of quassin components extracted by the method of any one of claims 1 to 4 is used as the test sample; c) Determination: Perform quantitative detection on the test solution.

6. The method according to claim 5, characterized in that The detection method includes any one of ultraviolet-visible spectrophotometry, chromatography, liquid chromatography-mass spectrometry, infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy; Optionally, the detection conditions of the chromatography method include: the chromatographic column is an octadecyl bonded silica gel chromatographic column; the flow rate is 0.2-0.4 mL / min; the column temperature is 35-45° C.; the injection volume is 3-10 μL; the mobile phase system is mobile phase A and mobile phase B, the mobile phase A is formic acid-water solution, and the mobile phase B is acetonitrile; the elution method is gradient elution; Preferably, the gradient elution conditions include: The initial ratio is that the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase; 0.01min~5min, the volume of the formic acid-water accounts for 30%-40% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 60%-70% of the total volume of the mobile phase; 5.01 min to 5.50 min, the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase; 5.51 min to 8 min, the volume of the formic acid-water accounts for 90%-97% of the total volume of the mobile phase, and the volume of the acetonitrile accounts for 3%-10% of the total volume of the mobile phase; Preferably, the detection conditions of the liquid chromatography-mass spectrometry method include: the ion source is an electrospray ion source; the detection mode is ion monitoring; the scanning mode is a positive ion mode; the mass spectrometer dryer temperature is 250°C, the nebulizer pressure is 30psi, the dryer flow rate is 7L / min, the sheath gas temperature is 325°C, and the sheath gas flow rate is 11L / min.

7. The method according to claim 5, characterized in that The quassin standard in the test solution includes any one of brucein, brucein A, brucein D, and brucein.

8. Use of the method according to any one of claims 1 to 7 in quality detection, quality evaluation or quality control of a preparation or pharmaceutical composition comprising a Brucea javanica extract.

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