CN107727763B - Rapid qualitative and quantitative detection method of seven phenethyl glycosides in Cistanche deserticola - Google Patents

Abstract

The invention discloses a rapid qualitative and quantitative detection method of seven phenylethanoid glycoside components in cistanche medicinal materials, which can be used for content determination of the active components of the cistanche medicinal materials.

Description

Rapid qualitative and quantitative detection method of seven phenethyl glycosides in Cistanche deserticola

technical field

The invention relates to the field of determination of the content of active ingredients in traditional Chinese medicines, in particular to a method for rapid qualitative and quantitative detection of seven active ingredients of phenylethanoid glycosides in the medicinal material of Cistanche deserticola.

Background technique

Cistanche is a traditional kidney-tonifying Chinese medicine in China. The 2015 edition of "Chinese Pharmacopoeia" records that it is the fleshy stem of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight, which is mainly distributed in Inner Mongolia, Ningxia, Gansu, Qinghai and Xinjiang. Cistanche Cistanche and Cistanche Cistanche have extremely high medicinal value. They enter the kidney and large intestine meridians. "Compendium of Materia Medica" records that Cistanche Cistanche is "tonic but not harsh, so it has the name of Cistanche", "It is warm and moisturizing, nourishing. Not dry, slippery but not diarrhea", has the effect of tonifying kidney yang, nourishing essence and blood, moistening bowel and laxative, traditionally used for deficiency of kidney yang, deficiency of essence and blood, impotence, infertility, soreness of waist and knees, weakness of muscles and bones, intestinal dryness and constipation , is the most frequently used medicine for kidney yang in the past dynasties. Modern pharmacological studies have shown that Cistanche deserticola has many functions such as improving sexual function, anti-aging, improving learning and memory ability, anti-senile dementia, and laxative, and is widely used in traditional Chinese medicine clinical prescriptions, Chinese patent medicines and health care products.

The main chemical components of Cistanche are phenylethanol glycosides, iridoids and their glycosides, lignans and polysaccharides. Modern pharmacological studies have shown that phenethyl glycosides are the most important active substances in Cistanche deserticola, and have obvious anti-senile dementia, Parkinson's disease and myocardial ischemia effects. At present, there are many quality control methods for the active ingredients of Cistanche deserticola. The pharmacopoeia has established a content determination method for echinacoside and verbasicin in Cistanche deserticola. The literature also established a multi-evaluation method for echinacoside and verbascoside in Cistanche deserticola. However, the two components are difficult to fully reflect and control the quality of Cistanche deserticola. In recent years, although there are reports of using HPLC to measure the content of four phenethyl glycosides in Cistanche deserticola simultaneously, multiple reference substances are needed, which increases the experimental cost and application. At present, the quantitative determination of seven phenethyl glycosides in Cistanche deserticola has not been reported at the same time, especially the method for determining the content of Cistanche deserticola with one standard and multiple determinations based on the percentage absorption coefficient to establish a relative correction factor has not been reported in literature and patent applications. .

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a method for rapid qualitative and quantitative detection of seven active ingredients of phenylethanoid glycosides in a medicinal material of Cistanche deserticola

The technical scheme adopted by the present invention to solve its technical problems is:

A method for rapid qualitative and quantitative detection of seven phenethyl glycosides in a medicinal material of Cistanche deserticola, comprising the following steps:

Preparation of reference solution: weigh echinacoside, verbascoside, isoverbasidin, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverrascoside reference substances dried to constant weight, respectively, Add solvent to prepare echinacoside reference substance stock solution, verbascoside reference substance stock solution, isoverascoside reference substance stock solution, cistanoside C reference substance stock solution, cistanoside A reference substance stock solution, 2'-acetyl Verbasil Glycoside reference substance stock solution and 6'-acetylverascoside reference substance stock solution, namely seven reference substance stock solutions with known concentrations are obtained;

Determination of the relative correction factor of each reference substance: after adding the obtained seven reference substance stock solutions, add solvent to dilute, measure the absorbance values of the seven reference substance dilutions, and calculate the percentage absorption coefficient of the seven reference substances respectively; The product is the reference substance, and the ratio of the percent absorption coefficient of the seven reference substances to the percent absorption coefficient of the reference substance is the relative correction factor of the seven reference substances, that is, the relative correction factors of the seven reference substances are obtained respectively;

Preparation of sample solution: take at least one of Cistanche Cistanche, Cistanche Cistanche, an item containing Cistanche Cistanche or an item containing Cistanche Cistanche as a sample to prepare a sample solution;

Sample determination: The obtained sample solution and the seven reference substance stock solutions were tested by liquid chromatography respectively; the retention time of the reference substance chromatographic peak was taken as the reference retention time, and the retention time of the seven reference substance chromatographic peaks and the reference substance chromatographic The ratio of the retention times of the peaks was calculated, and the relative retention times of the seven reference substances were calculated, so as to determine the echinacoside, verbascoside, isoverascoside, cistanoside C, cistanoside A, 2'-acetylverascoside in the chromatographic peaks of the sample solution and the attribution of the chromatographic peaks of the seven components of 6'-acetylverbasoside, and read the peak areas of the chromatographic peaks of the seven components at the same time;

和

分别计算得到样品中松果菊苷、 毛蕊花糖苷、异毛蕊花糖苷、肉苁蓉苷C、肉苁蓉苷A、2’-乙酰毛蕊花糖苷和 6’-乙酰毛蕊花糖苷七种待测成分的含量，公式中C_k为待测成分的浓度，A_k为 待测成分的色谱峰的峰面积，C_s为参照物贮备液的浓度，f_s/k是相对校正因子， A_s为参照物的色谱峰的峰面积，W_k为待测成分的含量，V为样品溶液的体积，m 为样品的称样量。Calculation of the content of the seven components: the relative correction factors of the obtained seven reference substances, the peak area of the chromatographic peaks of the seven components in the chromatographic peaks of the sample solution, the concentration of the reference substance stock solution, and the peak area of the reference substance stock solution, according to formula

and

The contents of seven components to be tested, including echinacoside, verbascoside, isoverascoside, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverbasic glycoside, in the sample were calculated respectively. In the formula, C _k is The concentration of the component to be measured, A _k is the peak area of the chromatographic peak of the component to be measured, C _s is the concentration of the reference substance stock solution, f _s/k is the relative correction factor, A _s is the peak area of the chromatographic peak of the reference substance, W _k is the content of the component to be tested, V is the volume of the sample solution, and m is the weighing amount of the sample.

Optimally, the wavelength when the absorbance value is measured in the relative correction factor determination step of each reference substance is the same as the detection wavelength when liquid chromatography is performed in the sample determination step.

Optimally, the wavelength when the absorbance value is measured in the relative correction factor determination step of each reference substance is consistent with the detection wavelength when liquid chromatography is performed in the sample measurement step, and the wavelength is in the range of 200 to 400 nm. any wavelength.

Optimally, in the step of determining the relative correction factor of each reference substance, the absorbance values of the seven reference substances at a wavelength of 333 nm are measured; according to Lambert's law A=ECL, the percentage absorption of the seven reference substances is calculated respectively. coefficient, wherein A is the absorbance value of each reference substance stock solution, C is the concentration of each reference substance stock solution, L is the measured liquid film thickness; the detection wavelength when liquid chromatography is detected in the sample measurement step is 333 nm.

Most preferably, in the preparation step of the reference substance solution, the solvent is at least one of methanol, ethanol or water; in the step of determining the relative correction factor of each reference substance, the solvent is at least one of methanol, ethanol or water; The specific preparation steps of the sample solution are: taking at least one of Cistanche cistanche, Cistanche pipiensis, articles containing Cistanche cistanche or articles containing Cistanche cistancheae as a sample, adding a solvent to the sample after accurately weighing the sample, and performing reflux, ultrasonic or Cold soaking treatment, filtering to obtain a sample solution in which the sample is completely dissolved, wherein the solvent is at least one of methanol, ethanol or water.

Most preferably, in the sample determination step, the chromatographic conditions for liquid chromatography detection are as follows: the filler is a C18 chromatographic column, the inner diameter of the chromatographic column is 2.1-5 mm, and the length is 100-300 mm, and the particle size of the filler is 1.7-4.6 mm. μm, the optimal elution conditions are: eluent A-eluent B isocratic elution or gradient elution, wherein the gradient elution is: 0 ~ 2min The concentration of eluent A is 85 ~ 83%, 2 ~ 5min The concentration of eluent A is 83-80%, the concentration of eluent A is 80-77% in 5-10 minutes, and the concentration of eluent A in 10-12 minutes is 77-85%; the flow rate is 0.1-0.6ml/min; sample injection Amount: 0.1μl～10μl; column temperature: 0℃～40℃; wherein eluent A-eluent B is methanol-water, or acetonitrile-water, or methanol-acetic acid, or methanol-formic acid, or acetonitrile-acetic acid , or acetonitrile-formic acid.

Optimally, in the sample determination step, the eluent A-eluent B are 0.5% acetic acid water-acetonitrile, the flow rate is 0.4 ml/min; the injection volume: 1 μl; the column temperature: 40°C.

Optimally, in the step of determining the relative correction factor of each reference substance, the measured liquid film thickness is 1 cm.

As can be seen from the above technical solutions, the method for rapid qualitative and quantitative detection of seven phenethyl glycosides in the medicinal materials of Cistanche deserticola provided by the present invention is calculated according to the percentage absorption coefficient of each component and the percentage absorption coefficient of echinacoside. Once the relative correction factor is established, in the subsequent determination of the active ingredients of Cistanche cistanche medicinal material phenylethanoid glycosides, only echinacoside, a reference substance, can be simultaneously determined The contents of seven constituents of verbascoside, cistanoside C, cistanoside A, 2'-acetylverbasicanoside and 6'-acetylverbasicanoside, the content determination results have no significant difference with the external standard method commonly used for drug analysis, the method is simple and accurate High degree of accuracy, and greatly save the experimental cost.

Description of drawings

Accompanying drawing 1 is the ultraviolet full wavelength scanning diagram of echinacoside reference solution.

Accompanying drawing 2 is the ultraviolet full-wavelength scanning diagram of Verbasin reference substance solution.

Fig. 3 is a UV full wavelength scanning diagram of the isoverbasin reference solution.

Accompanying drawing 4 is the UV full-wavelength scanning diagram of Cistanche glucoside C reference solution.

Accompanying drawing 5 is the ultraviolet full-wavelength scanning diagram of Cistanche A side reference solution.

Accompanying drawing 6 is the ultraviolet full-wavelength scanning diagram of 2'-acetylverbasin reference substance solution.

Accompanying drawing 7 is the ultraviolet full wavelength scanning diagram of 6'-acetylverbasin reference substance solution.

Accompanying drawing 8 is the HPLC chromatogram of echinacoside reference substance solution.

Figure 9 is the HPLC chromatogram of the Verbasin reference solution.

Figure 10 is the HPLC chromatogram of the isoverbasin reference solution.

Accompanying drawing 11 is the HPLC chromatogram of Cistanche glycoside C reference substance solution.

Accompanying drawing 12 is the HPLC chromatogram of Cistanche glycoside A reference substance solution.

Accompanying drawing 13 is the HPLC chromatogram of 2'-acetylverbasin reference substance solution.

Accompanying drawing 14 is the HPLC chromatogram of 6'-acetylverbasin reference substance solution.

Figure 15 is the HPLC chromatogram of Cistanche deserticola medicinal material.

Figure 16 is the relative correction factor f _s/k (n=6) of each component in Table 1 with echinacoside as the reference.

Figure 17 is the relative retention time of each component in Table 2 with echinacoside as the reference substance (n=6).

Figure 18 shows the simultaneous determination of the contents of seven phenethyl glycosides in the medicinal materials of Cistanche Cistanche and Cistanche Cistanche (mg·g ^-1 ) (n=4) by the external standard method in Table 3 and this method.

Detailed ways

With reference to the accompanying drawings of the present invention, the technical solutions of the embodiments of the present invention are further elaborated.

The rapid qualitative and quantitative detection method of seven phenylethanoid glycosides in Cistanche deserticola medicinal materials includes the following steps:

S1: Preparation of reference solution: weigh echinacoside, verbascoside, isoverbasidin, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverbasidin glycoside controls that were dried to constant weight. The product was accurately weighed, and a solvent was added, wherein the solvent was at least one of methanol, ethanol or water, to prepare echinacoside reference substance stock solution, verbascoside reference substance stock solution, isaveraside reference substance stock solution, and Cistanche deserticola respectively. Glycoside C reference substance stock solution, Cistanche glycoside A reference substance stock solution, 2'-acetylverascoside reference substance stock solution and 6'-acetylverascoside reference substance stock solution, namely seven reference substance stock solutions with known concentrations are obtained.

S2: Determination of the relative correction factor of each reference substance: after adding the obtained seven reference substance stock solutions, adding a solvent to dilute, the solvent is at least one of methanol, ethanol or water, and measuring the seven reference substances in the wavelength range of 200-400nm. The absorbance value at any wavelength in the, respectively, calculates the percentage absorption coefficient of seven reference substances, according to Lambert Beer's law A=ECL, wherein A is the absorbance value of each reference substance stock solution, C is each reference substance The concentration of the stock solution, L is the measured liquid film thickness; one reference substance is selected as the reference substance, and the ratio of the percentage absorption coefficient of the seven reference substances to the percentage absorption coefficient of the reference substance is the relative correction factor of the seven reference substances , that is, the relative correction factors of the seven reference substances are obtained respectively. The relative correction factors are calculated according to the formula f _s/k =E _s /E _k , where f _s/k is the relative correction factor of each component, and E _s is the reference substance The percent absorption coefficient of , E _k is the percent absorption coefficient of the component to be tested.

S3: Liquid chromatography system adaptability test: The chromatographic conditions for liquid chromatography detection are: a chromatographic column with a filler of C18, the inner diameter of the chromatographic column is 2.1-5mm, the length is 100-300mm, and the particle size of the filler is 1.7-4.6μm , the optimal elution conditions are: eluent A-eluent B isocratic elution or gradient elution, wherein gradient elution is: 0 ~ 2min eluent A concentration of 85 ~ 83%, 2 ~ 5min wash Dehydration A concentration is 83-80%, 5-10min eluent A concentration is 80-77%, 10-12min eluent A concentration is 77-85%; flow rate is 0.1-0.6ml/min; injection volume : 0.1μl～10μl; column temperature: 0℃～40℃; wherein eluent A-eluent B is methanol-water, or acetonitrile-water, or methanol-acetic acid, or methanol-formic acid, or acetonitrile-acetic acid, or acetonitrile-formic acid.

S4: Preparation of sample solution: Take at least one of Cistanche Cistanche, Cistanche Cistanche, articles containing Cistanche Cistanche, or articles containing Cistanche Cistanche as a sample, accurately weigh the sample, add solvent to the sample, and undergo reflux, ultrasonic or cold soaking treatment , filter to obtain a sample solution in which the sample is completely dissolved, wherein the solvent is at least one of methanol, ethanol or water.

S5: Sample determination: The obtained sample solution and the seven reference substance stock solutions are respectively subjected to liquid chromatography detection, and the detection wavelength during liquid chromatography detection and the wavelength when the absorbance value is determined in the relative correction factor determination step of each reference substance It is consistent. The chromatographic conditions for liquid chromatography detection are as follows: the packing is a C18 chromatographic column, the inner diameter of the chromatographic column is 2.1-5 mm, the length is 100-300 mm, the particle size of the packing is 1.7-4.6 μm, and the elution conditions are optimal. It is: eluent A-eluent B isocratic elution or gradient elution, wherein the gradient elution is: the concentration of eluent A in 0-2min is 85-83%, and the concentration of eluent A in 2-5min is 83% ～80%, 5～10min eluent A concentration is 80～77%, 10～12min eluent A concentration is 77～85%; flow rate is 0.1～0.6ml/min; injection volume: 0.1μl～10μl; Column temperature: 0℃～40℃; wherein eluent A-eluent B are methanol-water, or acetonitrile-water, or methanol-acetic acid, or methanol-formic acid, or acetonitrile-acetic acid, or acetonitrile-formic acid. Taking the retention time of the chromatographic peak of the reference substance as the reference retention time, according to the ratio of the retention time of the chromatographic peak of the reference substance to the retention time of the chromatographic peak of the reference substance, calculate the relative retention time of the seven reference substance, thereby determining the sample solution chromatogram. The assignment of the chromatographic peaks of the seven components of echinacoside, verbascoside, isoverbasidin, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverbasic glycoside in the peak, and read seven components at the same time The peak area of the chromatographic peak.

和

分别计算得到样品中松果菊 苷、毛蕊花糖苷、异毛蕊花糖苷、肉苁蓉苷C、肉苁蓉苷A、2’-乙酰毛蕊花糖 苷和6’-乙酰毛蕊花糖苷七种待测成分的含量，公式中C_k为待测成分的浓度， A_k为待测成分的色谱峰的峰面积，C_s为参照物贮备液的浓度，f_s/k是相对校正因 子，A_s为参照物的色谱峰的峰面积，W_k为待测成分的含量，V为样品溶液的体 积，m为样品的称样量。S6: Calculation of the content of the seven components: the relative correction factors of the obtained seven reference substances, the peak areas of the chromatographic peaks of the seven components in the chromatographic peaks of the sample solution, the concentration of the reference substance stock solution, and the peak area of the reference substance stock solution , according to the formula

and

The contents of seven components to be tested, including echinacoside, verbascoside, isoverascoside, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverbasic glycoside, were calculated respectively. In the formula, C _k is The concentration of the component to be measured, A _k is the peak area of the chromatographic peak of the component to be measured, C _s is the concentration of the reference substance stock solution, f _s/k is the relative correction factor, A _s is the peak area of the chromatographic peak of the reference substance, W _k is the content of the component to be tested, V is the volume of the sample solution, and m is the weighing amount of the sample.

Example 1:

The rapid qualitative and quantitative detection method of seven phenylethanoid glycosides in Cistanche deserticola medicinal materials includes the following steps:

S1: Preparation of reference solution: weigh echinacoside, verbascoside, isoverbasidin, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverbasidin glycoside controls that were dried to constant weight. Precisely weighed, add methanol to prepare echinacoside reference substance stock solution, verbascoside reference substance stock solution, isoverascoside reference substance stock solution, cistanoside C reference substance stock solution, and cistanoside A reference substance stock solution solution, 2'-acetylverbasic glycoside reference substance stock solution and 6'-acetylverbasoside reference substance stock solution, namely seven reference substance stock solutions with known concentrations are obtained.

S2: Determination of the relative correction factor of each reference substance: After adding methanol to dilute the obtained seven reference substance stock solutions, measure the absorbance values of the seven reference substances at a wavelength of 333 nm, and calculate the percentage absorption coefficients of the seven reference substances respectively. , according to Lambert's law A=ECL, where A is the absorbance value of each reference substance stock solution, C is the concentration (g/ml) of each reference substance stock solution, L is the measured liquid film thickness, and the liquid film thickness is 1cm. The UV full-wavelength scanning diagrams of the seven reference substance stock solutions are shown in Figures 1 to 7.

Select echinacoside as the reference substance, the ratio of the percent absorption coefficient of the seven reference substances to the percentage absorption coefficient of the reference substance is the relative correction factor of the seven reference substances, wherein the relative correction factor is based on the formula f _s/k =E _s /E _k is calculated, where f _s/k is the relative correction factor of each component, Es is the percent absorption coefficient of the reference object, and E _k is the percent absorption _coefficient of the component to be measured.

The calculated relative correction factors of verbascoside, isoverascoside, cistanoside C, cistanoside A, 2'-acetylverbasoside and 6'-acetylverrascoside and echinacoside are shown in Table 1 in Figure 16 .

As shown in Fig. 16, the relative correction factor f echinacoside/cistanoside A of _{Cistanche A is 1.4416, the relative correction factor f echinacoside/} verascoside of _verbascoside is 0.9628, and the relative correction factor of isaveraside is 0.9628. f echinacoside _/ isoverascoside is 0.7403, relative correction factor f echinacoside _{/cistanoside C} is 0.8727, relative correction factor f echinacoside _/2'- The relative correction factor f echinacoside _{/6'- acetylverbasoside} is 0.7797 for acetylverbasoside, the relative correction factor f _echinacoside /echinacoside for echinacoside is 1.0205 1.

S3: Liquid chromatography system adaptability test: The chromatographic conditions for liquid chromatography detection are: a chromatographic column with a C18 filler, the inner diameter of the chromatographic column is 2.1mm, the length is 100mm, the particle size of the filler is 1.7μm, and the elution conditions are the best. The best is: 0.5% acetic acid water (A)-acetonitrile (B) gradient elution: 0～2min A concentration is 85～83%, 2～5min A concentration is 83～80%, 5～10min A concentration is 80～77%, 10 The concentration of A in ~12min is 77~85%; the flow rate is 0.4ml/min; the detection wavelength is 333nm; the injection volume: 1 μl; the column temperature: 40°C.

S4: Preparation of the sample solution: Take at least one of Cistanche Cistanche, Cistanche Cistanche, articles containing Cistanche Cistanche or articles containing Cistanche Cistanche as a sample, after accurately weighing 1.0g of the sample, add 10ml of methanol to the sample, extract by ultrasonic for 30min, After cooling, methanol was added to make up for the lost weight, filtered, and 1 ml of the subsequent filtrate was taken and diluted 50 times to obtain a sample solution in which the sample was completely dissolved.

S5: Sample determination: The obtained sample solution and the seven reference substance stock solutions are respectively subjected to liquid chromatography detection, and the detection wavelength during liquid chromatography detection and the wavelength when the absorbance value is determined in the relative correction factor determination step of each reference substance It is consistent. The chromatographic conditions for liquid chromatography detection are as follows: the packing is a C18 chromatographic column, the inner diameter of the chromatographic column is 2.1 mm, the length is 100 mm, the particle size of the packing is 1.7 μm, and the optimal elution condition is: 0.5% acetic acid Water (A)-acetonitrile (B) gradient elution: 0～2min A concentration of 85～83%, 2～5min A concentration of 83～80%, 5～10min A concentration of 80～77%, 10～12min A concentration of 77～80% 85%; flow rate is 0.4ml/min; detection wavelength is 333nm; injection volume: 1μl; column temperature: 40°C. The results of liquid chromatography are shown in Figures 8 to 15.

Taking the retention time of the reference substance echinacoside chromatographic peak as the reference retention time, according to the ratio of the retention time of the six reference substance chromatographic peaks except the reference substance and the retention time of the reference substance echinacoside chromatographic peak, the calculated The relative retention time of the six reference substances except the reference substance echinacoside, so as to determine the echinacoside, verbascoside, isoverascoside, cistanoside C, cistanoside A, 2'-acetylverrascoside in the chromatographic peaks of the sample solution and the chromatographic peaks of these seven components of 6'-acetylverbasidin, and then obtain the peak area of the chromatographic peaks of the seven components, wherein the relative retention time of each component and echinacoside reference peak is shown in Table 2 in accompanying drawing 17. Show.

As shown in Figure 17, the ratio of the retention time of the chromatographic peak of Cistanche glycoside A to the chromatographic peak of echinacoside is 1.7017, the ratio of the retention time of the chromatographic peak of verbascoside to the chromatographic peak of echinacoside is 2.2278, the chromatographic peak of isaveraside and the chromatographic peak of echinacoside are 2.2278. The ratio of the retention time of echinacoside chromatographic peak is 2.7858, the ratio of the chromatographic peak retention time of cistanoside C to that of echinacoside is 3.6098, and the ratio of the retention time of 2'-acetylverascoside chromatographic peak to that of echinacoside chromatographic peak is 3.6098. The ratio was 4.1488, the ratio of the retention time of the 6'-acetylverbasin chromatographic peak to the echinacoside chromatographic peak was 5.1285, and the ratio of the retention time of the echinacoside chromatographic peak to the echinacoside chromatographic peak was 1. In the subsequent determination of the content of the above seven components in the medicinal material of Cistanche deserticola, according to the ratio of the retention time of the chromatographic peaks, it is possible to determine which component each chromatographic peak is.

分别计算得到样品中松果菊苷、毛蕊花糖 苷、异毛蕊花糖苷、肉苁蓉苷C、肉苁蓉苷A、2’-乙酰毛蕊花糖苷和6’-乙酰毛 蕊花糖苷七种待测成分的含量，公式中C_k为待测成分的浓度，A_k为待测成分的 色谱峰的峰面积，C_s为参照物贮备液的浓度，f_s/k是相对校正因子，A_s为参照物 的色谱峰的峰面积，W_k为待测成分的含量，V为样品溶液的体积，m为样品的 称样量。S6: Calculation of the content of seven components: the relative correction factors of the obtained seven reference substances, the peak areas of the chromatographic peaks of the seven components in the chromatographic peaks of the sample solution, and the concentration of the reference substance echinacea stock solution are calculated according to formula C _k =A _k ×C _s ×f _s/k /A _s and

The contents of seven components to be tested, including echinacoside, verbascoside, isoverascoside, cistanoside C, cistanoside A, 2'-acetylverbasic glycoside and 6'-acetylverbasic glycoside, were calculated respectively. In the formula, C _k is The concentration of the component to be tested, A _k is the peak area of the chromatographic peak of the component to be tested, C _s is the concentration of the reference substance stock solution, f _s/k is the relative correction factor, A _s is the peak area of the chromatographic peak of the reference substance, W _k is the content of the component to be tested, V is the volume of the sample solution, and m is the weighing amount of the sample.

The contents of the seven phenethyl glycoside components in the medicinal materials of Cistanche Cistanche and Cistanche Cistanche from different sources are shown in Table 3 in accompanying drawing 18:

As shown in Figure 18, the method is used to measure the contents of seven phenethyl glycosides in each medicinal material of Cistanche deserticola and compared with the results obtained by the external standard method commonly used in drug analysis, there is no significant difference, that is, the results are close, indicating the results obtained by this method. reliable.

The method for rapid qualitative and quantitative detection of seven phenethyl glycosides in the medicinal materials of Cistanche deserticola provided by the invention is a relative correction factor calculated according to the percentage absorption coefficient of each component and the percentage absorption coefficient of echinacoside; Once the correction factor is established, in the subsequent determination of the active ingredients of Cistanche deserticola medicinal material phenylethanoid, only echinacoside, a reference substance, can be used to simultaneously determine echinacoside, verbascoside, isoverbasin glycoside, and Cistanche glycoside C. , Cistanche glycosides A, 2'-acetylverbasic glycosides and 6'-acetylverbasic glycosides, the content of the determination results has no significant difference with the external standard method commonly used in drug analysis, the method is simple, high accuracy, and greatly saves experiment cost.

Claims (3)

1. A quick qualitative and quantitative detection method for seven phenylethanoid glycoside components in cistanche deserticola medicinal materials is characterized by comprising the following steps:

preparation of control solutions: weighing echinacoside, acteoside, isoacteoside, cistanoside C, cistanoside A, 2 '-acetylacteoside and 6' -acetylacteoside reference substances which are dried to constant weight respectively, adding a solvent to prepare an echinacoside reference substance storage solution, a acteoside reference substance storage solution, an isoacteoside reference substance storage solution, a cistanoside C reference substance storage solution, a cistanoside A reference substance storage solution, a 2 '-acetylacteoside reference substance storage solution and a 6' -acetylacteoside reference substance storage solution, namely obtaining seven reference substance storage solutions with known concentrations;

each control was measured against the correction factor: adding a solvent into the seven control stock solutions for dilution, measuring absorbance values of the seven control solutions at the wavelength of 333nm, and respectively calculating the percentage absorption coefficients of the seven control solutions according to the Lambert-beer law, namely ECL, wherein A is the absorbance value of each control stock solution, C is the concentration of each control stock solution, and L is the measured liquid film thickness; selecting a reference substance as a reference substance, wherein the ratio of the percentage absorption coefficient of the seven reference substances to the percentage absorption coefficient of the reference substance is the relative correction factor of the seven reference substances, namely the relative correction factor of the seven reference substances is obtained respectively;

preparation of sample solution: taking at least one of cistanche, cistanche tubulosa containing articles or cistanche tubulosa containing articles as a sample, and preparing a sample solution;

and (3) sample determination: respectively carrying out liquid chromatography detection on the obtained sample solution and seven kinds of reference stock solutions at a detection wavelength of 333 nm; calculating the relative retention time of the seven reference substances according to the ratio of the retention time of chromatographic peaks of the seven reference substances to the retention time of chromatographic peaks of the reference substances by taking the retention time of chromatographic peaks of the reference substances as reference retention time, thereby determining the attribution of chromatographic peaks of seven components, namely echinacoside, verbascoside, isoacteoside, cistanoside C, cistanoside A, 2 '-acetyl acteoside and 6' -acetyl acteoside in chromatographic peaks of a sample solution, and simultaneously reading the peak areas of the chromatographic peaks of the seven components; wherein, the chromatographic conditions for carrying out the liquid chromatographic detection are as follows: the packing material is a chromatographic column of C18, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the grain diameter of the packing material is 1.7 mu m, and the elution conditions are as follows: the eluent A-the eluent B are 0.5% acetic acid water-acetonitrile, the concentration of the eluent A is 85-83% in 0-2 min, the concentration of the eluent A is 83-80% in 2-5 min, the concentration of the eluent A is 80-77% in 5-10 min, and the concentration of the eluent A is 77-85% in 10-12 min; the flow rate is 0.4 ml/min; sample introduction amount: 1 mul; column temperature: 40 ℃;

calculating the content of seven components: the relative correction factors of the seven obtained reference substances, the peak areas of the chromatographic peaks of the seven components in the chromatographic peaks of the sample solution, the concentration of the reference substance storage solution and the peak area of the reference substance storage solution are calculated according to the formula

And

respectively calculating the contents of seven components to be detected, namely echinacoside, verbascoside, isoacteoside, cistanoside C, cistanoside A, 2 '-acetylacteoside and 6' -acetylacteoside in the sample, wherein Ck is the concentration of the component to be detected, Ak is the peak area of the chromatographic peak of the component to be detected, Cs is the concentration of a reference storage solution, fs/k is a relative correction factor, As is the peak area of the chromatographic peak of the reference, Wk is the content of the component to be detected, V is the volume of the sample solution, and m is the sample weighing amount of the sample.

2. The method for rapidly qualitatively and quantitatively detecting seven phenylethanoid glycoside components in cistanche drugs according to claim 1, which is characterized in that: in the step of preparing the reference solution, the solvent is at least one of methanol, ethanol or water; in the step of measuring the relative correction factor of each control, the solvent is at least one of methanol, ethanol or water; the preparation steps of the sample solution are as follows: taking at least one of cistanche, cistanche tubulosa containing articles or cistanche tubulosa containing articles as a sample, precisely weighing the sample, adding a solvent into the sample, and performing reflux, ultrasonic or cold soaking treatment and filtration to obtain a sample solution in which the sample is completely dissolved, wherein the solvent is at least one of methanol, ethanol or water.

3. The method for rapidly qualitatively and quantitatively detecting seven phenylethanoid glycoside components in cistanche deserticola as claimed in claim 1, which is characterized in that: in the step of measuring the relative correction factor of each control, the thickness of the liquid film measured is 1 cm.

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