CN113588839A

CN113588839A - Method for detecting periploca forrestii schltr, preparation of periploca forrestii schltr control extract and application thereof

Info

Publication number: CN113588839A
Application number: CN202110931125.6A
Authority: CN
Inventors: 刘刚; 刘育辰; 安兰兰; 周环娟
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2021-11-02
Anticipated expiration: 2041-08-13
Also published as: CN113588839B

Abstract

The invention discloses a method for detecting periploca forrestii schltr, and preparation and application of a periploca forrestii schltr control extract; the method comprises the steps of extracting the caulis periplocae to obtain an extracted solution, wherein the detection method comprises a content determination method, and the content determination method comprises the content detection of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the extracted solution. The detection method disclosed by the invention is accurate, high in sensitivity, good in repeatability and reliable in result, and the detection cost is reduced; the content of a plurality of components can be simultaneously measured by using the reference extract as a standard substance, and the idea of the overall quality control of the traditional Chinese medicine is met; the invention also provides application of the caulis et folium piperis nigri control extract in preparation of a medicine for resisting rheumatoid arthritis, and has important significance in researching index components of the caulis et folium piperis nigri for resisting rheumatoid arthritis.

Description

Method for detecting periploca forrestii schltr, preparation of periploca forrestii schltr control extract and application thereof

Technical Field

The invention relates to a method for detecting periploca forrestii schltr, and preparation and application of a periploca forrestii schltr control extract, and belongs to the technical field of medicines.

Background

The traditional Chinese medicine is a treasure of Chinese nationality, and the establishment of perfect quality standards is a precondition for ensuring the quality and the curative effect of the traditional Chinese medicine, and determines the development prospect of the traditional Chinese medicine. The Chinese medicine standard substance is an organic component of Chinese medicine quality standard and its evaluation system, and is a main means for controlling Chinese medicine production, improving and ensuring quality. The Chinese medicinal standard substance mainly comprises 3 types of Chinese medicinal chemical reference substances, reference medicinal materials and reference extracts. The chemical reference substances of the traditional Chinese medicine are extremely difficult to obtain, expensive and extremely high in detection cost, so that the method for analyzing the components of the multiple indexes in the medicinal materials is especially important for solving the shortage of the reference substances and establishing a simple, convenient and feasible method for analyzing the components of the multiple indexes in the medicinal materials. The traditional Chinese medicine control extract is a non-monomer component control, and the stability of an unstable monomer component in a mixture is improved. Since the 2005 edition of the Chinese pharmacopoeia was filed to date, the Chinese pharmacopoeia has a small number of varieties but has a wide application prospect, relates to identification, content determination and inspection items, and plays an increasingly important role in ensuring the quality of traditional Chinese medicines. The reference extract is easy to prepare, low in price, good in stability and simple in preparation operation, and can be used as a standard substance to greatly reduce the use of a monomer reference substance, so that the rare resources of the traditional Chinese medicine are saved, and the inspection cost is reduced; the content of a plurality of components can be simultaneously measured by using the reference extract as a standard substance, and the idea of controlling the overall quality of the traditional Chinese medicine is reflected. In addition, systematic pharmacological experiments are adopted to verify the index components of the caulis et folium piperis nigri for resisting rheumatoid arthritis, and the method also has important significance.

Disclosure of Invention

The invention aims to provide a method for detecting periploca forrestii schltr, and preparation and application of a periploca forrestii schltr control extract. The detection method disclosed by the invention is accurate, high in sensitivity, good in repeatability and reliable in result, and reduces the detection cost; the content of a plurality of components can be measured simultaneously by using the reference extract as a standard substance, which accords with the idea of overall quality control of traditional Chinese medicines. The invention also provides application of the periploca forrestii ethyl acetate part in preparing a medicament for resisting rheumatoid arthritis.

The technical scheme of the invention is as follows: a preparation method of a periploca forrestii schltr control extract comprises the following steps: accurately weighing medicinal powder, adding 60-80% ethanol solution according to a material-liquid ratio of 1:20-30, heating and reflux extracting for 2-4 times, each time for 50-70min, mixing extractive solutions, evaporating to dryness in 70 deg.C water bath, adding water to obtain suspension, respectively extracting with equal amount of petroleum ether, chloroform, and ethyl acetate for 3 times, mixing 3 times of ethyl acetate extractive solutions, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding into powder to obtain HEIGTENG ethanol extract ethyl acetate extraction part HGT-C powder; weighing ethyl acetate part of caulis et folium Periplocae Forrestii, mixing with PRP-512B type resin, loading into column, and gradient eluting with 20%, 30% and 50% methanol sequentially, wherein the 20% sections are combined to form a first section a1, the middle is combined to form a second section a2, and then a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

The preparation method of the periploca forrestii schltr control extract comprises the following steps: accurately weighing medicinal powder, adding 70% ethanol solution according to a material-liquid ratio of 1:25, heating and reflux-extracting for 3 times, each time for 60min, mixing extractive solutions, evaporating to dryness in 70 deg.C water bath, adding water to obtain suspension, respectively extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, mixing 3 times of ethyl acetate extractive solutions, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding to powder to obtain HEIGETA HERBA ethyl acetate extractive part HGT-C powder; weighing 10g of periploca forrestii ethyl acetate part, mixing the sample with the same amount of PRP-512B type resin, loading the sample into a column, and performing gradient elution by using 20%, 30% and 50% of methanol sequentially, wherein 4000mL of each gradient elution is obtained, 1000mL of the sample before the 20% section is combined into a first section a1, 2500mL of the sample in the middle is combined into a second section a2, and 500mL of the sample in the later section is a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

The caulis et folium Periplocae Forrestii control extract can be used for preparing medicine for treating rheumatoid arthritis.

A method for detecting the periploca forrestii schltr comprises the following steps: extracting the periploca forrestii schltr to obtain an extracted solution, wherein the detection method comprises a content determination method, and the content determination method comprises content detection of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the extracted solution.

In the foregoing detection method of caulis et folium Periplocae Forrestii, the content detection methods of the neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C are as follows:

a ZORBAX Eclipse plus C18 column 2.1mm × 100mm, 1.8 μm; taking 0.0.05% -0.15% formic acid A-acetonitrile B as a mobile phase, carrying out gradient elution, wherein the detection wavelength is 327nm, the flow rate is 0.2ml/min, and the column temperature is 30 ℃;

preparation of mixed control solution: precisely weighing 5.06 parts of neochlorogenic acid, 25.05 parts of chlorogenic acid, 8.04 parts of cryptochlorogenic acid, 5.05 parts of chlorogenic acid methyl ester, 5.02 parts of isochlorogenic acid B, 5.00 parts of isochlorogenic acid A and 7.05 parts of isochlorogenic acid C, putting the mixture into a 50mL measuring flask, adding 50% methanol to a constant volume, preparing a mixed reference solution, and shaking uniformly for later use; sucking 1mL of the mixed reference solution, placing the mixed reference solution in a 100mL measuring flask, fixing the volume by 50% methanol, shaking up to prepare a mixed reference solution II, and shaking up for later use;

preparation of control extract solution: weighing 10g of periploca forrestii ethyl acetate part, mixing the sample with PRP-512B type resin in equal amount, and packing the sample into a column; sequentially carrying out gradient elution by using 20%, 30% and 50% methanol, wherein 4000mL of gradient elution is carried out; wherein the first 1000ml of the 20% section is combined into a first section a1, the middle 2500ml is combined into a second section a2, and the last 500ml is a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain control extract; precisely weighing 14mg of caulis et folium Periplocae Forrestii control extract, placing in a 10mL measuring flask, adding 2mL of 50% methanol, performing ultrasonic treatment for 1min, cooling, adding 50% methanol to constant volume, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate to obtain control extract solution;

preparation of a test solution: accurately weighing 10-20g of medicinal material sample powder, adding 400mL of 70% ethanol solution according to the volume ratio of the material liquid to the sample powder of 1:20-30, heating, refluxing and extracting for 2-4 times, each time for 50-70min, combining the extracting solutions, evaporating to dryness in a water bath at 70 ℃, adding water to obtain a suspension, respectively extracting with equivalent petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain powder of an ethyl acetate extraction part HGT-C of the periploca forrestii alcoholic extract; precisely weighing 25mg of HGT-C powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain sample solution;

the determination method comprises the following steps: respectively sucking mixed reference solution, reference extract solution and test solution, injecting into liquid chromatograph, and measuring.

In the method for detecting periploca forrestii schltr, the mobile phase is 0.1% of formic acid A-acetonitrile B.

In the method for detecting periploca forrestii schltr, the elution process comprises: 0-3.4 min; 7% -9% of B; 3.4-5.4 min; 9% -11% of B; 5.4-7 min; 11% -13% of B; 7-8.3 min; 13% -13.5%; 8.3-10 min; 13.5% -15% of B; 10-14.5 min; 15% -15.1% of B; 14.5-15 min; 15.1% -16% of B; 15-17 min; 16% -18% of B; 17-21 min; 18% -21% of B; 21-23 min; 21% -26% of B; 23-25 min; 26% -100% of B.

In the method for detecting periploca forrestii schltr, the preparation of the test solution: precisely weighing 16 parts of medicinal material sample powder, adding 400mL of 70% ethanol solution according to a material-liquid ratio of 1:25, heating and refluxing for 3 times, each time for 50-70min, combining extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding water to obtain a suspension, respectively and sequentially extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain powder of an ethyl acetate extraction part HGT-C of the periploca forrestii alcohol extract; precisely weighing 25mg of HGT-C powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain the sample solution.

The inventors carried out a number of experiments, and the following were part of the experimental studies:

experimental example 1: caulis et folium Periplocae Forrestii control extract calibration and its application in medicinal material quality control

1. Instruments and reagents

An Agilent 1290 definition ii ultra performance liquid chromatograph (Agilent inc.) comprising a binary gradient pump, a high energy autosampler, a DAD detector and a chromatographic workstation; a ten thousandth electronic balance model FA2204B (shanghai tianmei balance instruments ltd); a one-hundred-ten-thousandth electronic balance model MS205DU (mertlettoduo instruments (china) ltd., milu, zurich, beijing). DZ-2BCIV vacuum drying oven (Tester instruments, Inc. of Tianjin).

Neochlorogenic acid (batch number CHB190217), chlorogenic acid (batch number CHB190121), cryptochlorogenic acid (batch number CHB1809055), chlorogenic acid methyl ester (batch number CHB190117), isochlorogenic acid B (batch number CHB180923), isochlorogenic acid a (batch number CHB180921), and isochlorogenic acid C (batch number CHB180925) were all purchased from kyropoma biotechnologies ltd, and the mass fractions were all greater than 98%. The caulis et folium Piperis Futokadsurae is purchased from Guiyang city flower orchard medicinal material market, and identified as plant Periploca forrestii Schltr of Periploca of Asclepiadaceae (Asclepiadaceae) by professor Liuyuchen of Guizhou traditional Chinese medicine university.

Acetonitrile is chromatographically pure (TEDIA, USA), other reagents are analytically pure, and HPLC water is purified water from Waha, Inc.

2. Method and results

2.1 study of caulis Periplocae Forrestii control extract

2.1.1 preparation of control extract

Weighing 10g of the periploca forrestii ethyl acetate part, mixing the sample with PRP-512B type resin in equal amount, and packing the mixture into a column. The column was eluted sequentially with 20%, 30%, 50% methanol gradients of 4000mL each. Wherein the first 1000ml of the 20% stage is combined into the first stage (a1), the middle 2500ml is combined into the second stage (a2), and the last 500ml is combined into the first stage (a 3). The 30% and 50% sections are respectively combined as a4 and a 5.2 g of the a5 fragment was sampled and packed with an equal amount of PRP-512B type resin. The elution was carried out with a gradient of 20%, 30% and 50% methanol in this order. Respectively combined as b1, b2 and b 3. Mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

2.1.2 calibration of control extracts

The content of the 7 index components in the caulis et folium Periplocae Forrestii control extract is determined by taking reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C as controls.

2.2 chromatographic conditions

ZORBAX Eclipse plus C was used₁₈Chromatography column (2.1 mm. times.100 mm, 1.8 μm); gradient elution was performed with 0.1% formic acid (a) -acetonitrile (B) as mobile phase, the elution procedure was: 0-3.4 min; 7% -9% of B; 3.4-5.4 min; 9% -11% of B; 5.4-7 min; 11% -13% of B; 7-8.3 min; 13 to 13.5 percent; 8.3-10 min; 13.5 to 15 percent of B; 10-14.5 min; 15% -15.1% of B; 14.5-15 min; 15.1% -16% of B; 15-17 min; 16% -18% of B; 17-21 min; 18% -21% of B; 21-23 min; 21% -26% of B; 23-25 min; 26 to 100 percent of B. The flow rate is 0.2ml/min, the detection wavelength is 327nm, the column temperature is 30 ℃, the sample injection amount is 2 μ L, and the chromatogram of the mixed reference substance and the chromatogram of the reference extract are respectively shown in figure 1 and figure 2 under the chromatographic conditions.

2.3 preparation of Mixed control solutions

Respectively and precisely weighing 5.06mg of new chlorogenic acid, 25.05mg of chlorogenic acid, 8.04mg of cryptochlorogenic acid, 5.05mg of chlorogenic acid methyl ester, 5.02mg of isochlorogenic acid, 5.00mg of isochlorogenic acid A and 7.05mg of isochlorogenic acid C, putting the weighed materials into a 50mL measuring bottle, adding 50% methanol to a constant volume, preparing a mixed reference solution, and shaking uniformly for later use. Sucking 1mL of the mixed reference solution, placing in a 100mL measuring flask, fixing the volume with 50% methanol, shaking up to prepare a mixed reference solution II, and shaking up for later use.

2.4 preparation of control extract test solution

Precisely weighing 14mg of caulis et folium Periplocae Forrestii control extract, placing in a 10mL measuring flask, adding 2mL of 50% methanol, performing ultrasonic treatment for 1min, cooling, adding 50% methanol to constant volume, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate to obtain sample solution.

2.5 methodological considerations

2.5.1 Linear relationship

Precisely sucking mixed reference substance solution I and solution II under the item '2.3', filtering through a 0.22 mu m microporous filter membrane, taking a subsequent filtrate and mixing the reference substance solution I, respectively injecting 1 mu L, 2 mu L, 3 mu L, 4 mu L and 5 mu L of the reference substance solution according to the chromatographic condition under the item '2.2', respectively injecting 1 mu L, 3 mu L and 5 mu L of the mixed reference substance solution II according to the chromatographic condition under the item '2.2', and measuring the peak areas of various chromatographic peaks. Taking the sample amount X (mug) of the reference substance as a horizontal coordinate (X) and the peak area Y of a chromatographic peak as a vertical coordinate, and performing linear regression to obtain a regression equation; the limit of detection (LOD) is the concentration of the control solution with a signal-to-noise ratio (S/N) of about 3:1, and the limit of quantitation (LOQ) is the concentration of the control solution with a signal-to-noise ratio (S/N) of about 10: 1. The results are shown in Table 1.

Table 17 regression equation and linear range for index components

Table 1 The regression equations and liner ranges of the seven marker components

2.5.2 precision test

Precisely sucking a mixed reference solution under the item of 2.3, continuously injecting samples for 6 times according to the chromatographic condition under the item of 2.2, and respectively measuring the RSD (n is 6) of the chromatographic peak areas of the new chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C to be 0.1%, 0.2% and 0.2%, thereby indicating that the instrument has good precision.

2.5.3 repeatability test

Taking the caulis et folium Periplocae Forrestii control extract, preparing 6 parts of control extract test solution in parallel according to the method under item 2.4, injecting sample respectively, determining contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, calculating RSD of 1.1%, 0.3%, 0.5%, 1.2%, 1.1%, 1.2% and 2.1%, respectively, and all less than 3%, thus indicating that the method has good repeatability.

2.5.4 stability test

Preparing 1 part of a reference extract test solution according to the method under the item '2.4', and performing sample injection measurement according to the chromatographic condition under the item '2.2' at 0, 2, 4, 8, 12, 20 and 24h after preparation respectively, wherein the results of RSD of the chromatographic peak areas of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are respectively 0.4%, 0.1%, 0.4%, 0.3%, 2.3%, 0.3% and 0.2%, which indicates that the test solution is stable within 24 h.

2.5.5 recovery rate of sample

Taking 7mg of a caulis et folium Periplocae Forrestii control extract with known content, precisely weighing, adding 6 parts in parallel, precisely adding different concentrations of neochlorogenic acid (0.1794mg/mL), chlorogenic acid (2.59mg/mL), cryptochlorogenic acid (0.667mg/mL), chlorogenic acid methyl ester (0.3984mg/mL), isochlorogenic acid B (0.01466mg/mL), isochlorogenic acid A (0.02744mg/mL) and isochlorogenic acid C (0.0519mg/mL) into each 1mL of control, preparing control extract sample solution according to the method under item "2.4", measuring according to the chromatographic condition under item "2.2", and calculating the average sample adding recovery rate and RSD value of each component. The results show that the average sample adding recovery rates of the new chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are 102.51%, 100.85%, 99.05%, 97.84%, 100.33%, 99.49% and 97.70% respectively, and the RSDs are 0.7%, 1.3%, 1.5%, 2.4%, 1.6%, 1.5% and 1.3% respectively, which indicates that the established content determination method has good accuracy. The results are shown in Table 2.

Table 2 sample application recovery test results (n ═ 6)

Table 1.2 Results of recovery rates(n＝6)

3. Application of control extract in quality control of caulis et folium Periplocae Forrestii

Taking the caulis et folium Periplocae Forrestii control extract as control, determining the content of 7 components including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in caulis et folium Periplocae Forrestii, comparing with the result of monomer control, and examining the scientificity and feasibility of the control extract for controlling the quality of medicinal materials. The source of the medicinal materials is shown in Table 3.

TABLE 3 sample Source information

Table 3 Source information of samples

3.1 preparation of control extract solution

Accurately weighing caulis et folium Periplocae Forrestii control extract 40.03mg, placing in 5ml measuring flask, adding 50% methanol 2ml, ultrasonic treating for 1min, cooling, adding 50% methanol to constant volume, shaking, dissolving, and making into control extract solution A with chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C mass concentrations of 0.2050, 2.9588, 0.7633, 0.4552, 0.0168, 0.0313, and 0.0592mg/ml respectively; diluting the control extract solution A by 10 times; 100 times of the total weight of the powder; 250 times as the control extract solution B, C, D.

3.2 preparation of the test solution of caulis et folium Periplocae Forrestii

Precisely weighing 16.0g of medicinal material sample powder, adding 400mL of 70% ethanol solution according to a material-liquid ratio of 1:25, heating and refluxing for 3 times, each time for 1h, combining extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding water to obtain a suspension, sequentially extracting with equivalent amounts of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain an ethyl acetate extraction part (HGT-C) of the periploca forrestii ethanol extract. Precisely weighing 25mg of HGT-C part powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to constant volume to scale, shaking, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain the sample solution.

3.3 chromatographic conditions

The chromatographic conditions were the same as those under "2.2". Chromatogram of the control extract and caulis et folium Periplocae Forrestii is shown in FIG. 3 and FIG. 4.

3.4 methodological considerations

3.4.1 Linear relationship

Precisely sucking caulis et folium Periplocae Forrestii control extract solutions of different multiples under item "3.1", respectively adding control extract A into 1 μ L, 3 μ L, and 5 μ L; control extract B was added to 1. mu.L, 2. mu.L, and 3. mu.L, respectively; control extract C was obtained in 5. mu.L; the control extract D was injected into an ultra high performance liquid chromatograph at 1. mu.L and 3. mu.L, respectively, and the chromatographic peak area was measured under the chromatographic conditions of "2.2". Taking the sample amount X (mug) of a reference substance as a horizontal coordinate, taking the peak area Y of a chromatographic peak as a vertical coordinate, performing linear regression to obtain a regression equation, and finally taking the reference extract B, the reference extract C and the reference extract D to obtain a linear regression equation of the new chlorogenic acid, the cryptochlorogenic acid and the chlorogenic acid methyl ester; the results of the linear regression equation of isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C obtained by comparing the extract A and the extract B are shown in Table 4.

TABLE 4 control extract linearity study

Tab 4 The regression equations and linear ranges of the reference extracts

3.4.2 precision test

Preparing 1 part of reference extract test solution according to the method under the item 2.4, continuously injecting samples for 6 times under the chromatographic condition under the item 2.2, and respectively measuring RSD (n is 6) of the chromatographic peak areas of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C to be 0.4%, 0.1%, 0.3%, 1.5%, 0.7% and 0.3%, which indicates that the instrument has good precision.

3.4.3 repeatability test

Taking the same batch of powder of the HGT-C part of the periploca forrestii medicinal material (S9), preparing 6 parts of periploca forrestii medicinal material test solution in parallel according to the method under the item of '2.2', respectively injecting samples, measuring the peak area, calculating the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, calculating the RSD of the chlorogenic acid A, the isochlorogenic acid A and the isochlorogenic acid C to be respectively 0.2%, 1.0%, 1.1%, 1.6%, 0.7%, 1.2% and 1.1%, and respectively less than 3%, thus indicating that the method has good repeatability.

3.4.4 stability test

Preparing 1 part of a caulis et folium Periplocae Forrestii test solution according to the method of '3.2', and performing sample injection determination on 0, 2, 4, 8, 12, 20 and 24h according to chromatographic conditions, wherein the results of the RSDs of the chromatographic peak areas of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are respectively 0.3%, 0.5%, 1.1%, 0.9%, 0.5% and 0.2%, which indicates that the test solution is stable within 24 h.

3.4.5 sample recovery

Taking 12.5mg of a test sample powder at the HGT-C part of the same batch of caulis et folium Periplocae Forrestii sample (S9), precisely weighing, placing in a 10mL measuring flask, precisely adding 1mL of each of neochlorogenic acid (0.03372mg/mL), chlorogenic acid (0.852mg/mL), cryptochlorogenic acid (0.1139mg/mL), methyl chlorogenic acid (0.04568mg/mL), isochlorogenic acid B (0.0559mg/mL), isochlorogenic acid A (0.1142mg/mL) and isochlorogenic acid C (0.5648mg/mL) reference substances with different concentrations, performing ultrasonic treatment for 2min, adding methanol to fix the volume to the scale, shaking up, and preparing 6 parts in parallel. According to the chromatographic condition under the item of '2.2.3', sample injection and measurement are carried out, the peak areas of all the components are recorded, the recovery rates are respectively calculated to be 98.52%, 98.44%, 97.44%, 26%, 100.1%, 99.54% and 102.09%, the RSD values are respectively 2.0%, 2.5%, 2.3%, 2.8%, 1.6% and 1.4%, and the established content measurement method is good in accuracy. The results are shown in Table 5.

TABLE 5 sample recovery test results (n ═ 6)

Table5 Results of recovery rates(n＝6)

3.5 sample analysis

Taking 12 batches of caulis et folium Periplocae Forrestii, precisely weighing, preparing caulis et folium Periplocae Forrestii test solution according to the method under item 3.2, and determining contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, methyl chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C by caulis et folium Periplocae reference extract method and monomer reference method respectively, the results are shown in Table 6.

TABLE 612 batch samples determination results (mg/g)

Table 6 Determination results of 12 batches of samples

4. And (3) knotting: the application compares chromatographic peaks of test solution extracted by 70% methanol and 70% ethanol in sequence. From the chromatogram, the number of peaks of 50% methanol and 50% ethanol is small, and particularly the peak area of methyl chlorogenic acid is small, so that after 70% ethanol is adopted for reflux extraction, solvents (petroleum ether, chloroform and ethyl acetate) with different polarities are used for extraction, and the method can determine 7 caffeoyl quinic acid components (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, methyl chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C) in the same way.

The invention prepares a control extract with definite content on the basis of the prior research on the preparation method of caffeoyl quinic acids and a control extract thereof, establishes a method for measuring the content of the caulis kadsurae medicinal material based on the caffeoyl quinic acid control extract, and respectively adopts a control extract method and a control method to analyze and measure the content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, methyl chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the caulis kadsurae medicinal material, and the result shows that the measurement result of the control extract is basically consistent with that obtained by the control method, the comparison extract can replace a monomer comparison product to realize the quality control of the periploca forrestii medicinal material, and the quality control can better meet the actual requirement and better accord with the characteristics of multiple components of the traditional Chinese medicine. And simultaneously, the problems of instability and difficulty in preparation of the components are solved.

Experimental example 2: validity verification of anti-rheumatoid arthritis effect of periploca forrestii control extract

1. Laboratory animal

SPF grade SD rats, body weight (200 ± 20 g); license number: SCXK (Xiang) 2019-. During pharmacological test, the temperature control and the relative humidity of an animal room are respectively controlled at 25 +/-2 ℃; 60 +/-10 percent. The lighting time is alternated day and night, the animals can drink water and eat food freely, the padding materials are kept dry, and the rat model is established after adaptive feeding for 3 d. All experimental studies are in accordance with the guidelines of the Chinese ethics committee on animal research.

2. Medicinal materials and reagents

The medicinal materials are purchased in the market of Guiyang city orchard medicinal materials in 2018, and are identified as asclepiadaceae (Asclepiadacea) Periploca (Periploca) plant black dragon bone (P.forrestii Schltr.) by professor Liuyuchen of Guizhou university of traditional Chinese medicine; tripterygium glycosides tablets (batch No. 1450003, Guizhou Han prescription pharmaceutical Co., Ltd.); the chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are purchased from Chengdu Croma Biotech Co., Ltd and have mass fractions of more than 98%. Bovine type ii collagen solution (Chondrex, usa); freund's complete adjuvant (Chondrex, USA); freund's incomplete adjuvant (Chondrex, USA), etc. EDTA decalcification solution (type: B0317-11, Sichuan Poxter technologies, Inc.); 7122 hematoxylin (thermo Fisher, usa); eosin (thermo Fisher, usa).

3. Laboratory apparatus

TD5A-WS type centrifuge (Hunan vertical science instruments, Inc. in Hunan province), JE1002 type electronic balance (Shanghai Puchun measuring instruments, Inc.), MNT-150 type digital display caliper (Zhejiang Dexintai electronic science and technology, Inc.). One hundred thousand electronic balances (beijing zhongfeng switzerland mettlerthropo instruments (china) ltd.), Rayto RT-6100 enzyme-labeled analyzer, RM2235 paraffin slicer (Leica, germany), CX22 optical microscope (OLMPUS, japan), DM1000 Leica microscopic imaging system (Leica, germany), and the like, of type MS205 DU.

4. Experimental methods

4.1 preparation of test drugs

The periploca forrestii control extract was enriched as described in 2.1.1 of Experimental example 1. The content of caffeoylquinic acid components is determined to be 448.01mg/g in total.

4.2 establishment of CIA rat model

Under the ice bath condition, fully emulsifying the bovine type II collagen solution and Freund's complete adjuvant in equal volume by a homogenizer (fully emulsifying the emulsion by dripping the emulsion on the water surface to form a cluster without dispersing) to prepare the collagen emulsion, and using the collagen emulsion as the preparation. After the rats were bred adaptively for 3 days, the rats were fixed by a fixator, the hair at the tail roots was shaved off, and the roots of the first immunized tail were injected intradermally with 0.2mL of collagen emulsion. After 7 days of the initial immunization, the preparation method is the same as that of the collagen emulsion prepared by fully emulsifying a bovine type II collagen solution and an equal volume of Freund's incomplete adjuvant, and 0.1mL of the collagen emulsion is injected into the tail root of the patient in the intradermal mode (the injection point of the initial immunization is avoided). Normal group was injected with normal saline. After 18 days of primary immunization, the success of model making is divided into the point that the Arthritis Index (AI) of the rat is more than or equal to 4. AI is the sum of the four limb arthritis index scores, with a maximum of 16 points.

TABLE 7 AI scoring criteria

4.3 grouping and administration

After 18d of primary immunization, rats successfully modeled were randomly divided into Tripterygium Glycosides (TG) groups (10mg/kg), model groups, control extract administration groups (36, 144mg/kg), control substance administration groups (low and high dose groups (calculated according to the ratio of the components in the control extract), and blank control groups, with 8 rats in each group.

The low and high dosages of the caulis et folium Periplocae Forrestii reference extract are respectively 12.6 and 50.4 times of the clinical crude drug dosage of human. The medicines are dispersed by physiological saline solution, the normal group and the model group are given with the physiological saline solution, the administration volume is 10mL/kg, and the stomach is perfused for 28 days, and the administration is carried out once a day.

4.4 AI value score and toe thickness measurement

The first day of dosing was scored at 0d, interval 6d, once, interval 7d, and the toe thickness of the rats was measured and photographed. When the thickness of toes is measured, the average value is obtained by parallel measurement for three times.

4.5 serum factor assay and synovial pathological change observation

After administration for 28 days, fasting is carried out for more than 12 hours, water is not forbidden, a rat is anesthetized by newly configured 10% chloral hydrate 0.4mL/100g, abdominal aorta is adopted for blood sampling, a blood sample is placed on an ice bath for standing for 30min, then the blood sample is placed in a centrifuge for centrifugation at 4000rpm/min for 20min, a liquid transfer gun is used for taking supernatant, and the supernatant is placed at-80 ℃ for standby. The procedures were according to the ELISA kit instructions. After blood is taken out, neck is removed and killed, fur of the left hind ankle joint is removed, the ankle joint is removed by using animal bone scissors and is fixed by using 4 percent paraformaldehyde neutral fixing liquid, tissue blocks are embedded in paraffin after conventional decalcification and are cut into thin slices with the thickness of 5 mu m, and after dewaxing, eosin staining is carried out, and histopathological changes are observed under a microscope.

4.6 statistical analysis

SPSS 24.0 and Graphpad Prism 8.0.2 statistical software are used for analysis and mapping, data are expressed by mean standard deviation +/-standard deviation (mean +/-SD), one-factor variance analysis is carried out, LSD (least squares) test is adopted for pairwise comparison among groups when the variances are uniform, Tamhance's T2 test is adopted for pairwise comparison among groups when the variances are not uniform, and P <0.05 is used for difference to have statistical significance.

5. Results of the experiment

5.1 Effect of caulis Periplocae Forrestii control extract on CIA rats

5.1.1 Effect of caulis Periplocae Forrestii control extract on thickness of toe of right hind paw of CIA rat

Before administration, compared with a model group, each drug group has no significant difference, a normal group has a very significant difference (P <0.01) and a very significant difference (P <0.01) is still remained after 28d administration, which indicates that the model is successfully made and the model is relatively stable; after 28d administration, TG groups were compared to model groups; the control extract has very significant difference (P <0.01) in the low and high dose groups. See table 8.

TABLE 8 Effect of caulis et folium Periplocae Forrestii control extract on the thickness of the right hind paw of CIA rats

mm

Note: in comparison with the set of models,^*P<0.05，^**P<0.01 (the same below)

5.1.2 Effect of caulis Periplocae Forrestii control extract on toe thickness of the left hind paw of CIA rat

After 28d administration, TG groups were compared to model groups; the control extract has very significant difference (P <0.01) in the low and high dose groups. See table 9.

TABLE 9 Effect of caulis et folium Periplocae Forrestii control extract on the thickness of the left hind toe of CIA rats

mm

5.1.3 Effect of the caulis Periplocae Forrestii control extract on the AI value of CIA rats

Before administration, compared with a model group, each drug group has no significant difference; after 28d administration, TG and control extracts were low compared to the model group, with significant differences in the high dose group (P < 0.01). See table 10. According to fig. 10, there was no significant difference between the model groups 0d to 28d, and there was a significant difference between the control extract high and the control extract low after 13d administration, and between the TG group and the control extract low after 20d administration.

TABLE 10 Effect of caulis et folium Periplocae Forrestii control extract on AI values in CIA rats

5.1.4 Effect of caulis Periplocae Forrestii control extract on the expression of proinflammatory cytokines in serum of CIA rat

Compared with the normal group, the content of TNF-alpha in the serum of the rat in the model group is obviously increased (P <0.01) and the content of TNF-alpha in the TG group is obviously increased (P < 0.05); compared with the model group, the TNF-alpha content of the control extract in the low and high dose groups is obviously reduced (P <0.01, P < 0.05); the TNF-alpha content of the control extract low-dose group and the TG group is obviously reduced (P < 0.05). See table 11.

TABLE 11 Effect of control extracts on the expression of proinflammatory cytokines in serum of CIA rats

pg/mL

5.1.5 Effect of the caulis Periplocae Forrestii control extract on pathological changes of synovial tissue of CIA rat

Rat joint tissue samples were observed under a microscope for histopathological changes after HE staining of paraffin-embedded sections. The main lesion features include: proliferative disorders characterized by proliferation of synovial lining cells and connective tissue of the joints; inflammatory reaction, which is again manifested by a varying degree of inflammatory cell infiltration in synovial tissue, with a large number of inflammatory cells and erythrocytes found in some joint cavities. See FIG. 12 for details: (Unit: Bar ═ 100 μm)

5.2 Effect of Mixed controls on CIA rats

5.2.1 Effect of Mixed control on the thickness of the right hind paw of CIA rats

Before administration, compared with a model group, each drug group has no significant difference, a normal group has a very significant difference (P <0.01) and a very significant difference (P <0.01) is still remained after 28d administration, which indicates that the model is successfully made and the model is relatively stable; after 28d administration, TG groups were compared to model groups; the control extract has very significant difference (P <0.01) in the low and high dose groups. See table 12.

TABLE 12 Effect of the Mixed control on the thickness of the right hind paw of CIA rats

mm

5.2.2 Effect of Mixed control on the thickness of the left hind paw of CIA rats

After 28d administration, TG groups were compared to model groups; the control extract has very significant difference (P <0.01) in the low and high dose groups. See table 13.

TABLE 13 Effect of the Mixed control on the thickness of the left hind toe of CIA rats

mm

5.2.3 Effect of Mixed control on AI values in CIA rats

Before administration, compared with a model group, each drug group has no significant difference; after 28d administration, TG and control extracts were low compared to the model group, with significant differences in the high dose group (P < 0.01). See table 14.

TABLE 14 Effect of the Mixed control on AI values in CIA rats

5.2.4 Effect of Mixed controls on the expression of proinflammatory cytokines in serum from CIA rats

Compared with the normal group, the content of the TNF-alpha in the serum of the rat in the model group is obviously increased (P <0.01), and the content of the TNF-alpha in the serum of the low dose of the TG group and the mixed control is obviously increased (P < 0.05); compared with the model group, the content of IL-6, IL-1 beta and TNF-alpha in the serum of the mixed control high-dose group is obviously reduced (P is less than 0.01); the content of TNF-alpha in the serum of the low dose of the mixed control is obviously reduced (P is less than 0.05); compared with TG group, the content of IL-6, IL-1 beta and TNF-alpha in the serum of high dose of the mixed control is obviously reduced (P is less than 0.05, P is less than 0.01); the content of IL-6, IL-1 beta and TNF-alpha in serum is obviously reduced compared with the low dose of the mixed control substance (P is less than 0.01, and P is less than 0.01). See table 15.

TABLE 15 Effect of Mixed controls on the expression of proinflammatory cytokines in serum from CIA rats

pg/mL

5.2.5 Effect of Mixed control on pathological changes in synovial tissue of CIA rats

Rat joint tissue samples were observed under a microscope for histopathological changes after HE staining of paraffin-embedded sections. See FIG. 19 for details: (Unit: Bar ═ 100 μm)

6 small knot

Experimental results show that the total 7 caffeoyl quinic acid components of the periploca forrestii control extract and the mixed control can effectively relieve toe swelling, reduce arthritis score, improve histopathological changes and effectively reduce the level of proinflammatory cytokines, especially TNF-alpha, in the serum of a CIA rat. The caffeoylquinic acid component contained in the ethyl acetate part of the periploca forrestii has good anti-RA activity.

RA is an autoimmune disease that causes chronic inflammation of synovial tissue and joints, leading to joint dysfunction and ultimately to disability. The CIA model shares immunological and pathological features with human RA and is therefore commonly used for preliminary pharmacological studies of new or other therapeutic drugs [34 ]. The pathology of RA involves a variety of lymphocytes, e.g., T cells, B cells, etc., with synovitis being the most typical feature of RA. TNF- α plays a key role in the development and progression of RA, and it can enhance the innate immune response, stimulating the release of other inflammatory cytokines, ultimately leading to synovial matrix degradation and synovial hyperplasia. Elevated levels of IL-6 have been generally demonstrated in the serum of RA patients and are directly correlated with clinical indicators of disease severity. anti-IL-1 β treatment of type II CIA mice is effective in preventing cartilage and bone destruction, and TNF- α antagonists can improve synovitis. TNF-alpha, IL-6 and IL-1 beta are important indicators reflecting the symptoms of pain and inflammation in patients.

The invention adopts systematic pharmacological experiments to verify whether the caffeoylquinic acid components in the ethyl acetate part of the caulis et folium piperis nigri are index components of the caulis et folium piperis nigri for resisting rheumatoid arthritis. The anti-RA effect of the control extract is slightly superior to that of a caffeoyl quinic acid component mixed control, which shows that other chemical components in the caulis et folium piperis nigri control extract also have activity on resisting RA, and research results also provide theoretical basis for determining the anti-RA drug effect substance basis of the caulis et folium piperis nigri medicinal material and establishing a comprehensive and scientific caulis et folium piperis nigri medicinal material quality evaluation system.

Compared with the prior art, the detection method is accurate, high in sensitivity, good in repeatability and reliable in result, and reduces the detection cost; the content of a plurality of components can be measured simultaneously by using the reference extract as a standard substance, which accords with the idea of overall quality control of traditional Chinese medicines. The invention also provides application of the caulis et folium piperis nigri control extract in preparation of a medicine for resisting rheumatoid arthritis, and has important significance in researching index components of the caulis et folium piperis nigri for resisting rheumatoid arthritis.

Drawings

FIG. 1 is a UPLC chromatogram of 2.2 portions of the mixed control of Experimental example 1; in the figure, 1-new chlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-methyl chlorogenic acid, 5-isochlorogenic acid B, 6-isochlorogenic acid A and 7-isochlorogenic acid C are marked;

FIG. 2 is a UPLC chromatogram of a 2.2 portion of the control extract of Experimental example 1; in the figure, 1-new chlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-methyl chlorogenic acid, 5-isochlorogenic acid B, 6-isochlorogenic acid A and 7-isochlorogenic acid C are marked;

FIG. 3 is a UPLC chromatogram of a portion 3.3 of the control extract of Experimental example 1; in the figure, 1-new chlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-methyl chlorogenic acid, 5-isochlorogenic acid B, 6-isochlorogenic acid A and 7-isochlorogenic acid C are marked;

FIG. 4 is a UPLC chromatogram of a 3.3 portion of the periploca forrestii sample from Experimental example 1; in the figure, 1-new chlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-methyl chlorogenic acid, 5-isochlorogenic acid B, 6-isochlorogenic acid A and 7-isochlorogenic acid C are marked;

FIG. 5 is a timeline of 4.6 fractions of caulis et folium Periplocae Forrestii in Experimental example 2 against rheumatoid arthritis;

FIG. 6 is a graph showing the effect of 5.1.1 parts of the control extract on the thickness of the right hind toe of CIA rats in Experimental example 2;

FIG. 7 is a graph showing the effect of 5.1.1 parts of the control extract on the right hind paw of CIA rats in Experimental example 2;

FIG. 8 is a graph showing the effect of 5.1.2 parts of the control extract on the thickness of the left hind toe of CIA rats in Experimental example 2;

FIG. 9 is the effect of 5.1.2 parts of the control extract on the left hind paw of CIA rats in Experimental example 2;

FIG. 10 Effect of part 5.1.3 of the control extract in Experimental example 2 on the AI values of CIA rats;

FIG. 11 is a graph of the effect of a portion 5.1.4 of the control extract on the expression of pro-inflammatory cytokines in CIA rat serum in Experimental example 2;

FIG. 12 is the effect of 5.1.4 parts of the periploca forrestii control extract on pathological changes of synovial tissue of CIA rats in Experimental example 2;

FIG. 13 is a graph showing the effect of 5.2.1 parts of the mixed control on the thickness of the right hind toe of a CIA rat in Experimental example 2;

FIG. 14 is a graph showing the effect of 5.2.1 parts of the mixed control on the right hind paw of a CIA rat in Experimental example 2;

FIG. 15 is a graph showing the effect of 5.2.2 parts of the mixed control on the thickness of the left hind toe of CIA rats in the experimental examples;

FIG. 16 is a photograph of the left hind paw of a CIA rat of part 5.2.2 of the mixed control in Experimental example 2;

FIG. 17 is a graph showing the effect of 5.2.3 parts of the mixed control on the AI values of CIA rats in Experimental example 2;

FIG. 18 is a graph showing the effect of 5.2.4 parts of the mixed control on the expression of proinflammatory cytokines in CIA rat serum in experimental examples;

FIG. 19 shows the effect of 5.2.5 portions of the mixed control substance in Experimental example 2 on pathological changes of synovial tissue of CIA rats.

Detailed Description

The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.

Example 1. A preparation method of a periploca forrestii schltr control extract comprises the following steps: accurately weighing medicinal powder, adding 70% ethanol solution according to a material-liquid ratio of 1:25, heating and reflux-extracting for 3 times, each time for 60min, combining the extracts, evaporating to dryness in 70 deg.C water bath, adding 24-26 times of water to obtain suspension, respectively extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracts, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding to powder to obtain HEIGETA HERIGINSIS ETHANA ETIC EXTRACT HGT-C powder; weighing 10g of periploca forrestii ethyl acetate part, mixing the sample with the same amount of PRP-512B type resin, loading the sample into a column, and performing gradient elution by using 20%, 30% and 50% of methanol sequentially, wherein 4000mL of each gradient elution is obtained, 1000mL of the sample before the 20% section is combined into a first section a1, 2500mL of the sample in the middle is combined into a second section a2, and 500mL of the sample in the later section is a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

The efficacy is as follows: the caulis et folium Periplocae Forrestii control extract can be used for treating rheumarthritis.

Example 2. A preparation method of a periploca forrestii schltr control extract comprises the following steps: accurately weighing medicinal powder, adding 80% ethanol solution according to a material-liquid ratio of 1:30, heating and reflux-extracting for 4 times, each time for 70min, combining the extracts, evaporating to dryness in 70 deg.C water bath, adding 20-24 times of the mixture to obtain suspension, respectively extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracts, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding to obtain powder of HEIGETA HERIGINSIS ETRA (HERICIMA HERICINENSIS) extract ethyl acetate extraction part HGT-C; weighing 10g of periploca forrestii ethyl acetate part, mixing the sample with the same amount of PRP-512B type resin, loading the sample into a column, and performing gradient elution by using 20%, 30% and 50% of methanol sequentially, wherein 4000mL of each gradient elution is obtained, 1000mL of the sample before the 20% section is combined into a first section a1, 2500mL of the sample in the middle is combined into a second section a2, and 500mL of the sample in the later section is a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

Example 3. A preparation method of a periploca forrestii schltr control extract comprises the following steps: accurately weighing medicinal powder, adding 60% ethanol solution according to a material-liquid ratio of 1:20, heating and reflux-extracting for 2 times, each time for 50min, combining the extracting solutions, evaporating to dryness in 70 ℃ water bath, adding 26-30 times of the obtained suspension, respectively extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding to powder to obtain the periploca forrestii ethanol extract ethyl acetate extraction part HGT-C powder.

Embodiment 4. a method for detecting caulis et folium piperis nigri, which comprises a method for detecting the content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, methyl chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in an extract solution, wherein the specific detection method comprises the following steps:

a ZORBAX Eclipse plus C18 column 2.1mm × 100mm, 1.8 μm; performing gradient elution by using 0.0.05% -0.15% of formic acid A-acetonitrile B as a mobile phase, wherein the elution process comprises the following steps: 0-3.4 min; 7% -9% of B; 3.4-5.4 min; 9% -11% of B; 5.4-7 min; 11% -13% of B; 7-8.3 min; 13% -13.5%; 8.3-10 min; 13.5% -15% of B; 10-14.5 min; 15% -15.1% of B; 14.5-15 min; 15.1% -16% of B; 15-17 min; 16% -18% of B; 17-21 min; 18% -21% of B; 21-23 min; 21% -26% of B; 23-25 min; 26% -100% of B; the detection wavelength is 327nm, the flow rate is 0.2ml/min, and the column temperature is 30 ℃;

preparation of control extract solution: weighing the ethyl acetate part of the periploca forrestii schltr, mixing the sample with PRP-512B type resin in equal amount, and packing the sample into a column; sequentially carrying out gradient elution by using 20%, 30% and 50% methanol, wherein 4000mL of gradient elution is carried out; wherein the first 1000ml of the 20% section is combined into a first section a1, the middle 2500ml is combined into a second section a2, and the last 500ml is a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain control extract; precisely weighing 14mg of caulis et folium Periplocae Forrestii control extract, placing in a 10mL measuring flask, adding 2mL of 50% methanol, performing ultrasonic treatment for 1min, cooling, adding 50% methanol to constant volume, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate to obtain control extract solution;

preparation of a test solution: precisely weighing 16 parts of medicinal material sample powder, adding 400mL of 70% ethanol solution according to a material-liquid ratio of 1:25, heating and refluxing for extraction for 3 times, each time for 50-70min, combining the extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding 24-26 times of water to obtain a suspension, respectively extracting with equivalent amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain powder of an ethyl acetate extraction part HGT-C of the periploca forrestii alcohol extract; precisely weighing 25mg of HGT-C powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain sample solution;

Embodiment 5. a method for detecting caulis et folium piperis nigri, comprising the steps of detecting the content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, methyl chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the extracted solution, wherein the specific detection method comprises the following steps:

a ZORBAX Eclipse plus C18 column 2.1mm × 100mm, 1.8 μm; performing gradient elution by using 0.15% formic acid water A-acetonitrile B as a mobile phase, wherein the elution process comprises the following steps: 0-3.4 min; 7% -9% of B; 3.4-5.4 min; 9% -11% of B; 5.4-7 min; 11% -13% of B; 7-8.3 min; 13% -13.5%; 8.3-10 min; 13.5% -15% of B; 10-14.5 min; 15% -15.1% of B; 14.5-15 min; 15.1% -16% of B; 15-17 min; 16% -18% of B; 17-21 min; 18% -21% of B; 21-23 min; 21% -26% of B; 23-25 min; 26% -100% of B; the detection wavelength is 327nm, the flow rate is 0.2ml/min, and the column temperature is 30 ℃;

preparation of a test solution: precisely weighing 20g of medicinal material sample powder, adding 400mL of 70% ethanol solution according to the volume ratio of the material liquid to the material liquid of 1:30, heating and refluxing for extraction for 2-4 times, each time for 50-70min, combining the extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding 25-27 times of water to obtain a suspension, respectively extracting with equivalent petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain the powder of the ethyl acetate extraction part HGT-C of the periploca forrestii alcohol extract; precisely weighing 25mg of HGT-C powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain sample solution;

Embodiment 6. a method for detecting caulis et folium piperis nigri, comprising the steps of detecting the content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, methyl chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the extracted solution, wherein the specific detection method comprises the following steps:

a ZORBAX Eclipse plus C18 column 2.1mm × 100mm, 1.8 μm; performing gradient elution by using 0.0.05% formic acid water A-acetonitrile B as a mobile phase, wherein the elution process comprises the following steps: 0-3.4 min; 7% -9% of B; 3.4-5.4 min; 9% -11% of B; 5.4-7 min; 11% -13% of B; 7-8.3 min; 13% -13.5%; 8.3-10 min; 13.5% -15% of B; 10-14.5 min; 15% -15.1% of B; 14.5-15 min; 15.1% -16% of B; 15-17 min; 16% -18% of B; 17-21 min; 18% -21% of B; 21-23 min; 21% -26% of B; 23-25 min; 26% -100% of B; the detection wavelength is 327nm, the flow rate is 0.2ml/min, and the column temperature is 30 ℃;

preparation of a test solution: precisely weighing 10g of medicinal material sample powder, adding 400mL of 70% ethanol solution according to the volume ratio of the material liquid to the material liquid of 1:20, heating and refluxing for extraction for 2-4 times, each time for 50-70min, combining the extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding 23-25 times of water to obtain a suspension, respectively extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain the powder of the ethyl acetate extraction part HGT-C of the periploca forrestii alcohol extract; precisely weighing 25mg of HGT-C powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain sample solution;

Claims

1. A preparation method of a periploca forrestii schltr control extract is characterized by comprising the following steps: the method comprises the following steps: accurately weighing medicinal powder, adding 60-80% ethanol solution according to a material-liquid ratio of 1:20-30, heating and reflux extracting for 2-4 times, each time for 50-70min, mixing extractive solutions, evaporating to dryness in 70 deg.C water bath, adding water to obtain suspension, respectively extracting with equal amount of petroleum ether, chloroform, and ethyl acetate for 3 times, mixing 3 times of ethyl acetate extractive solutions, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding into powder to obtain HEIGTENG ethanol extract ethyl acetate extraction part HGT-C powder; weighing ethyl acetate part of caulis et folium Periplocae Forrestii, mixing with PRP-512B type resin, loading into column, and gradient eluting with 20%, 30% and 50% methanol sequentially, wherein the 20% sections are combined to form a first section a1, the middle is combined to form a second section a2, and then a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

2. The method for preparing the periploca forrestii control extract as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps: accurately weighing medicinal powder, adding 70% ethanol solution according to a material-liquid ratio of 1:25, heating and reflux-extracting for 3 times, each time for 60min, mixing extractive solutions, evaporating to dryness in 70 deg.C water bath, adding water to obtain suspension, respectively extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, mixing 3 times of ethyl acetate extractive solutions, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, and grinding to powder to obtain HEIGETA HERBA ethyl acetate extractive part HGT-C powder; weighing 10g of periploca forrestii ethyl acetate part, mixing the sample with the same amount of PRP-512B type resin, loading the sample into a column, and performing gradient elution by using 20%, 30% and 50% of methanol sequentially, wherein 4000mL of each gradient elution is obtained, 1000mL of the sample before the 20% section is combined into a first section a1, 2500mL of the sample in the middle is combined into a second section a2, and 500mL of the sample in the later section is a section a 3; the 30% and 50% sections are respectively combined into a4 and a 5; taking 2g of the a5 section, mixing the sample with an equal amount of PRP-512B type resin, and packing the sample into a column; gradient elution is sequentially carried out by 20 percent, 30 percent and 50 percent of methanol; respectively combined as b1, b2 and b 3; mixing the above a2 and b3 at a ratio of 5:2 to obtain caulis et folium Periplocae Forrestii control extract.

3. A method for detecting the caulis periplocae is characterized by comprising the following steps: extracting the periploca forrestii schltr to obtain an extracted solution, wherein the detection method comprises a content determination method, and the content determination method comprises content detection of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, chlorogenic acid methyl ester, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the extracted solution.

4. The method for detecting caulis et folium piperis nigri of claim 3, wherein: the content detection method of the new chlorogenic acid, the cryptochlorogenic acid, the chlorogenic acid methyl ester, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C comprises the following steps:

5. The method for detecting caulis et folium piperis nigri of claim 4, wherein: the mobile phase was 0.1% formic acid water A-acetonitrile B.

6. The method for detecting caulis et folium piperis nigri of claim 4, wherein: the elution process comprises the following steps: 0-3.4 min; 7% -9% of B; 3.4-5.4 min; 9% -11% of B; 5.4-7 min; 11% -13% of B; 7-8.3 min; 13% -13.5%; 8.3-10 min; 13.5% -15% of B; 10-14.5 min; 15% -15.1% of B; 14.5-15 min; 15.1% -16% of B; 15-17 min; 16% -18% of B; 17-21 min; 18% -21% of B; 21-23 min; 21% -26% of B; 23-25 min; 26% -100% of B.

7. The method for detecting caulis et folium piperis nigri of claim 4, wherein: preparing a test solution: precisely weighing 16 parts of medicinal material sample powder, adding 400mL of 70% ethanol solution according to a material-liquid ratio of 1:25, heating and refluxing for 3 times, each time for 50-70min, combining extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding water to obtain a suspension, respectively and sequentially extracting with equal amount of petroleum ether, chloroform and ethyl acetate for 3 times, combining the 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, and grinding to powder to obtain powder of an ethyl acetate extraction part HGT-C of the periploca forrestii alcohol extract; precisely weighing 25mg of HGT-C powder, placing in a 10mL measuring flask, adding 2mL of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with 0.22 μm microporous membrane, and collecting the filtrate to obtain the sample solution.

8. Use of the periploca forrestii control extract as defined in claim 1 or 2 for the preparation of a medicament against rheumatoid arthritis.