阅读说明：本技术 一种测定小白菊中含α,β-不饱和酮的倍半萜内酯总量的试剂和方法 (Reagent and method for measuring total amount of alpha, beta-unsaturated ketone-containing sesquiterpene lactone in feverfew ) 是由 陈雏 吴燕 李彬 杜蕾蕾 于 2021-05-31 设计创作，主要内容包括：本发明公开了一种测定小白菊中含α,β-不饱和酮的倍半萜内酯总量的试剂和方法,属于药物分析技术领域,所述检测试剂为4-氯-N-(2-巯基乙基)苯甲酰胺,其检测方法是将小白菊提取液与4-氯-N-(2-巯基乙基)苯甲酰胺在设定条件下反应一定时间,检测反应液中4-氯-N-(2-巯基乙基)苯甲酰胺的剩余量,通过计算得到小白菊中含α,β-不饱和酮的倍半萜内酯总量；本发明不依赖被测化合物的对照品,为测定小白菊中含α,β-不饱和酮的倍半萜内酯总量提供了一种简便易行的分析方法。(The invention discloses a reagent and a method for measuring the total amount of alpha, beta-unsaturated ketone-containing sesquiterpene lactone in feverfew, belonging to the technical field of drug analysis, wherein the detection reagent is 4-chloro- N - (2-mercaptoethyl) benzamide, which is detected by mixing the feverfew extract with 4-chloro- N Reacting (2-mercaptoethyl) benzamide for a certain time under set conditions, and detecting 4-chloro- N The residual amount of the- (2-mercaptoethyl) benzamide is calculated to obtain the total amount of sesquiterpene lactone containing alpha, beta-unsaturated ketone in the feverfew; the invention does not depend on a reference substance of a tested compound, and provides a simple and easy analysis method for determining the total amount of the sesquiterpene lactone containing alpha, beta-unsaturated ketone in the feverfew.)

1. A reagent for measuring the content of sesquiterpene lactone containing alpha, beta-unsaturated ketone is characterized in that: the reagent is 4-chloro-N- (2-mercaptoethyl) benzamide.

2. A method for determining the total amount of alpha, beta-unsaturated ketone-containing sesquiterpene lactones in feverfew using the reagent of claim 1, wherein: mixing the extract of feverfew with 4-chloro-NAfter the reaction of (2-mercaptoethyl) benzamide, detecting 4-chloro-substituted benzene in the reaction liquidNThe residual amount of the- (2-mercaptoethyl) benzamide is calculated to obtain the total amount of sesquiterpene lactone containing alpha, beta-unsaturated ketone in the feverfew.

3. The method of claim 2, wherein the formula for the calculation is:

C = 0.0082x+0.1458, wherein,Cis 4-chloro-NThe clearance rate of the- (2-mercaptoethyl) benzamide,Cthe calculation method is (4-chloro-NAmount of- (2-mercaptoethyl) benzamide-4-chloro-substituted after reactionNAmount of- (2-mercaptoethyl) benzamide)/4-chloro-substituted benzene before reactionN(2-mercaptoethyl) benzamide in an amount of 100%;xthe parthenolide concentration in the initial reaction solution is expressed in μ M.

4. The method of claim 2, wherein: the reaction temperature is 25-50 ℃.

5. The method of claim 2, wherein: the reaction time is 90-150 minutes.

6. The method of claim 2, wherein: sesquiterpene lactone containing alpha, beta-unsaturated ketone and 4-chloro-NThe molar ratio of the (2-mercaptoethyl) benzamide to the (75: 100) benzamide during the reaction is 5:100 to 75: 100.

7. The method of claim 2, wherein: the reaction was carried out in a mixed solution of phosphate buffer and methanol.

8. The method of claim 7, wherein: the concentration of the phosphate buffer solution is 10mM, the pH value is 7.4, and the volume ratio of the phosphate buffer solution to the methanol mixed solution is 1: 1.

Technical Field

The invention relates to a method for measuring the total amount of alpha, beta-unsaturated ketone-containing sesquiterpene lactone in feverfew, belonging to the technical field of pharmaceutical analysis.

Background

Feverfew, also known as Tanacetum parthenium (L.) Schultz Bip, is a medicinal plant of asteraceae originally produced in europe and has the effects of preventing and treating migraine, diminishing inflammation, relieving stomachache, etc. Research shows that the feverfew contains various sesquiterpene lactones, and most of the sesquiterpene lactones have the effects of resisting inflammation, resisting tumors, regulating immunity and the like. The components have the common characteristic of having structural fragments of alpha, beta-unsaturated ketone, so that the components can be covalently combined with cysteine residues of some proteins in vivo, and further regulate and control the functions of the proteins. Sesquiterpene lactones are considered to be the most prominent group of pharmacodynamic substances in feverfew, and a representative compound thereof is parthenolide (parthenolide).

The structure of parthenolide is shown below:

the content of parthenolide in feverfew medicinal material, extract or preparation is determined by high performance liquid chromatography, and parthenolide is used as reference. However, the content of other sesquiterpene lactones containing α, β -unsaturated ketones in feverfew has been reported due to the lack of corresponding commercial controls.

Considering that sesquiterpene lactone containing alpha, beta-unsaturated ketone in feverfew generally has similar pharmacodynamic activity and action mechanism, and the quality of feverfew raw materials and products thereof is only reflected by the content of the parthenolide, the total content of the ingredients needs to be measured, which has important significance for the quality control of the related products taking the feverfew as the raw material.

Disclosure of Invention

The invention aims to provide a reagent for measuring the total content of sesquiterpene lactones containing alpha, beta-unsaturated ketone in feverfew.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a reagent for determining the content of sesquiterpene lactone containing alpha, beta-unsaturated ketone is 4-chloro-N- (2-mercaptoethyl) benzamide, and the structure of the reagent is shown as follows:

the inventor of the application proves that the compound can be used for simple determination of the total amount of the sesquiterpene lactone containing alpha, beta-unsaturated ketone in the feverfew through a large number of experimental researches.

The invention also aims to provide a method for measuring the total amount of alpha, beta-unsaturated ketone-containing sesquiterpene lactones in feverfew by using the reagent, which comprises the following specific steps:

and (3) reacting the feverfew extract with 4-chloro-N- (2-mercaptoethyl) benzamide, detecting the residual amount of the 4-chloro-N- (2-mercaptoethyl) benzamide in the reaction solution, and calculating to obtain the total amount of the alpha, beta-unsaturated ketone-containing sesquiterpene lactone in the feverfew.

The above feverfew extract is obtained by ultrasonic extraction of feverfew raw medicinal materials with 75% methanol, and may be obtained by other extraction solvents and methods.

As a preferred technical solution, the formula of the calculation is:

c ═ 0.0082x +0.1458, where C is the clearance of 4-chloro-N- (2-mercaptoethyl) benzamide, calculated as: (amount of 4-chloro-N- (2-mercaptoethyl) benzamide before reaction-amount of 4-chloro-N- (2-mercaptoethyl) benzamide after reaction)/amount of 4-chloro-N- (2-mercaptoethyl) benzamide before reaction x 100%; x is the parthenolide concentration in the initial reaction solution in. mu.M (. mu. mol/L).

As known to those skilled in the art, the sesquiterpene lactones comprising α, β -unsaturated ketones according to the present invention have the formula:

the mechanism of the reaction of 4-chloro-N- (2-mercaptoethyl) benzamide with the compounds is as follows:

first, the reaction equation of parthenolide with 4-chloro-N- (2-mercaptoethyl) benzamide is:

since the molar ratio of the reaction of other sesquiterpene lactones containing α, β -unsaturated ketones with 4-chloro-N- (2-mercaptoethyl) benzamide is "the same as the" molar ratio of the reaction of parthenolide 4-chloro-N- (2-mercaptoethyl) benzamide ", for example:

based on the principle, after the feverfew extract is reacted with 4-chloro-N- (2-mercaptoethyl) benzamide for 90-150 minutes, the total amount of the alpha, beta-unsaturated ketone-containing sesquiterpene lactone in the feverfew can be obtained by detecting the variable quantity of the 4-chloro-N- (2-mercaptoethyl) benzamide in the reaction solution and then calculating by a formula.

With respect to the aforementioned reaction time of 90 to 150 minutes, it should be noted that the quantitative determination is not affected by the completion of the reaction. In the embodiment of the invention, the reaction time is 120 minutes, which is a time point selected in the test process, and a calibration curve drawn at the time point is used for detecting the total amount of sesquiterpene lactones containing alpha, beta-unsaturated ketone in feverfew, wherein the clearance rate of 4-chloro-N- (2-mercaptoethyl) benzamide and the concentration of parthenolide in the initial reaction liquid are linear in the range of 5-75 mu M; since the reaction between the lactone and the thiol proceeds at a specific rate under the reaction conditions set by us (the extent of reaction at a certain point in time is relatively constant), the extent of reaction of the total lactone with 4-chloro-N- (2-mercaptoethyl) benzamide can be determined after establishing a standard curve with parthenolide (parthenolide with 4-chloro-N- (2-mercaptoethyl) benzamide), and the total lactone content can be calculated by comparing with the standard curve.

As a preferred technical scheme: the reaction temperature is 25-50 ℃.

As a preferred technical scheme: the reaction time is 90-150 minutes.

As a preferred technical scheme: the molar ratio of the alpha, beta-unsaturated ketone-containing sesquiterpene lactone to the 4-chloro-N- (2-mercaptoethyl) benzamide is 5: 100-75: 100.

As a preferred technical scheme: the reaction was carried out in a mixed solution of phosphate buffer and methanol.

As a preferred technical scheme: the concentration of the phosphate buffer solution is 10mM, the pH value is 7.4, and the volume ratio of the phosphate buffer solution to the methanol mixed solution is 1: 1. This is a preferred reaction condition, and those skilled in the art will know that the present invention can be realized by appropriately adjusting and changing the conditions.

Compared with the prior art, the invention has the beneficial effects that: the method can realize quantitative determination without depending on a reference substance of a detected compound, and provides a simple, convenient and feasible analysis method for determining the total amount of the alpha, beta-unsaturated ketone-containing sesquiterpene lactone in the feverfew.

Drawings

FIG. 1 is a UPLC diagram for the detection of 4-chloro-N- (2-mercaptoethyl) benzamide;

FIG. 2 is a hydrogen spectrum of 4-chloro-N- (2-mercaptoethyl) benzamide;

FIG. 3 is a carbon spectrum of 4-chloro-N- (2-mercaptoethyl) benzamide;

FIG. 4 is a calibration curve for content measurement.

Detailed Description

Example 1

Synthesis of 4-chloro-N- (2-mercaptoethyl) benzamide

4-chlorobenzoyl chloride (1.00g,5.71mmol) was added slowly to 0 deg.C dichloromethane (30ml), followed by β -mercaptoethylamine (0.45g,5.86mmol) and heated at reflux for 2 hours. The reaction mixture was washed with saturated sodium bicarbonate (10mL) and concentrated brine (10mL) and then with Na₂SO₄Dried and concentrated under reduced pressure as a yellowish solid. The resulting crystals were repeatedly crystallized from a mixed solvent of n-hexane and dichloromethane (1:1, v/v) to obtain a white solid (0.62 g, yield 50.3%). ESI-MS M/z 214.0087[ M-H ]]^-(calculated value m/z 214.0093, C₉H₉ClNOS)；¹H NMR(600MHz,CDCl₃)δ7.64(2H,d,J＝8.6Hz),7.31(2H,d, J＝8.6Hz),6.80(1H,br s),3.53(2H,d,J＝6.4Hz),2.69(2H,dt,J＝8.4Hz,6.4 Hz),1.38(H,t,J＝8.4Hz)；¹³C NMR (150MHz, CDCl3) δ 166.6,137.9,132.6, 128.7,128.4,42.9,24.6, wherein the hydrogen and carbon spectra are shown in fig. 2 and 3.

Example 2

Detection method of 4-chloro-N- (2-mercaptoethyl) benzamide

Adopts ACQUITYUPLC ultra-high performance liquid chromatography with chromatographic column Waters BEH C18(50 × 2.1mm,1.7 μm), mobile phase acetonitrile-water (35:65, v/v), flow rate of 0.4mL/min, column temperature of 35 deg.C, detection wavelength of 238nm, and chromatogram shown in figure 1. Under these conditions, the regression equation for 4-chloro-N- (2-mercaptoethyl) benzamide is y-2284.6 x-1474.9 (R)²0.9999), y is the peak area, x is the concentration of 4-chloro-N- (2-mercaptoethyl) benzamide (μ M), R²The linear range is 2.5-200. mu.M for the correlation coefficient.

Example 3

Creation of calibration curves

Taking a proper amount of parthenolide, precisely weighing, dissolving with phosphate buffer (10mM, pH7.4) -methanol mixed solution (1:1, v/v), and making into series of control solutions with concentration of 10,20,60,80,100,150,200 μ M. An appropriate amount of 4-chloro-N- (2-mercaptoethyl) benzamide was weighed precisely, and dissolved in a mixed solution of phosphate buffer (10mM, pH7.4) and methanol (1:1, v/v) to prepare a solution having a concentration of 200. mu.M. The parthenolide solutions with different concentrations are respectively mixed with 4-chloro-N- (2-mercaptoethyl) benzamide solution with the same volume, and the mixture is placed at the temperature of 37 ℃ for reaction for 120 minutes. The remaining amount of 4-chloro-N- (2-mercaptoethyl) benzamide in the solution after 120 minutes of reaction was measured and the clearance thereof was calculated. A calibration curve was drawn with the concentration of parthenolide in the initial reaction liquid as the abscissa and the clearance of 4-chloro-N- (2-mercaptoethyl) benzamide after 120 minutes of reaction as the ordinate, as shown in FIG. 4.

The results show that the clearance of 4-chloro-N- (2-mercaptoethyl) benzamide (17.6% -74.8%) and the concentration of parthenolide (5-75. mu.M) are in a good linear relationship, and the regression equation is C ═ C0.0082x+ 0.1458(R²0.9982). Wherein C is the clearance (%), x is the concentration of parthenolide (. mu.M) in the initial reaction solution, and R is²Is the correlation coefficient. Therefore, under the set conditions, when the total concentration of the parthenolide and other alpha, beta-unsaturated ketone-containing sesquiterpene lactones in the parthenolide extract is between 5 and 75 mu M, the total amount of the alpha, beta-unsaturated ketone-containing sesquiterpene lactones in the parthenolide can be calculated according to the clearance rate of 4-chloro-N- (2-mercaptoethyl) benzamide (based on the parthenolide).

Example 4

Determination of total amount of alpha, beta-unsaturated ketone-containing sesquiterpene lactones in feverfew

Taking 0.2g of dry powder of a raw medicinal material sample of the feverfew, precisely weighing, and ultrasonically extracting for 2 times with 8mL of 75% methanol for 15 minutes each time. And combining the two extracting solutions, transferring the extracting solutions into a 25mL volumetric flask, and metering the volume to the scale by using 75% methanol to obtain a sample stock solution. The sample stock solution was diluted 10-fold with a mixed solution of phosphate buffer (10mM, pH7.4) -methanol (1:1, v/v), mixed with an equal volume of 4-chloro-N- (2-mercaptoethyl) benzamide solution (200. mu.M), reacted at 37 ℃ for 120 minutes, and the remaining amount of 4-chloro-N- (2-mercaptoethyl) benzamide was measured to calculate its clearance rate. The total amount of sesquiterpene lactones containing α, β -unsaturated ketones (based on parthenolide) in a sample of feverfew was obtained by substituting the clearance into the regression equation in example 3. The results showed that the total amount of sesquiterpene lactones containing α, β -unsaturated ketones in the flowers and 76-98 μmol/g in the leaves of the three batches of Feverfew tested, and the results are shown in Table 1.

Table 1. total amount of sesquiterpene lactones containing α, β -unsaturated ketones in samples of feverfew (μmol/g dry weight, n-3).

Sample number	Plant parts	Total amount of
			FF-1	Flower (A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A	107.97±2.82
FF-1	Leaf of Chinese character	76.40±1.09
			FF-2	Flower (A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A	67.38±1.17
FF-2	Leaf of Chinese character	78.53±2.70
			FF-3	Flower (A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A	75.07±2.54
FF-3	Leaf of Chinese character	95.09±2.34

Example 5

Method verification

5.1 precision parthenolide solutions (20, 80, 150 μ M) were mixed with 4-chloro-N- (2-mercaptoethyl) benzamide (200 μ M) in equal volume, and the mixture was reacted at 37 ℃ for 120 minutes, and the remaining amount of 4-chloro-N- (2-mercaptoethyl) benzamide was measured and the clearance was calculated to obtain an intra-day precision of 1.02% (N ═ 3) and an inter-day precision of 3.54% (N ═ 3), indicating that the precision of the method was good.

5.2 sample application recovery rate six portions of feverfew samples, each 0.1g, were added with feverfew lactone in an amount of 80%, 100% and 120% of the total amount of sesquiterpene lactones known to contain α, β -unsaturated ketones, and extracted and measured according to the given method. The result shows that the recovery rate of the method is 111.3% -112.4%, which indicates that the method has good accuracy.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.