Quality standard detection method of Chinese herbal medicine pluchea indica

Abstract

The invention discloses a quality standard detection method of Chinese herbal medicine pluchea indica, and relates to the technical field of Chinese herbal medicine pluchea indica. The quality standard detection method of the Chinese herbal medicine pluchea indica comprises the following specific steps: collecting pluchea indica leaves; establishing a high performance liquid chromatography content determination method for the isochlorogenic acid A, the isochlorogenic acid B and the isochlorogenic acid C in the koelreuteria pluchea leaves, and performing content determination; performing verification; measuring the content of the collected pluchea flower leaves; the judgment quality is excellent; and the acquisition area standard is established. On the basis of condition investigation and methodology verification, the result of the quality standard detection method is further reliable, and a reasonable and accurate basis and detection method are provided for development and utilization of subsequent pluchea indica resources in the production area and quality evaluation.

Description

Technical Field

[0001] The invention relates to the technical field of Chinese herbal medicine Luanxiong, and in particular to a quality standard detection method of the Chinese herbal medicine Luanxiong. Background Art

[0002] Koelreuteria paniculata is a dicotyledonous plant belonging to the genus Asteraceae, Asteraceae, Asteraceae, and Asteraceae. It is also known as broad-bract chrysanthemum, Geza tree, crucian carp gall, stinky tablets, five-spice incense, and holly tree. Koelreuteria paniculata first appeared in "Records of Collecting Medicinal Herbs in Lingnan" written by Xiao Budan. Koelreuteria paniculata is mainly distributed in the central and southern parts of Taiwan, the coastal areas of southern provinces of China, and some islands. It is also distributed in India, Myanmar, Indochina, Malaysia, Indonesia, and the Philippines. Koelreuteria paniculata likes light, tolerates shade, resists strong winds, has few diseases and insect pests, and has a strong ability to adapt to the environment. It often grows in patches on the edge of mangroves, wetlands on both sides of ditches, sandy land, and fish pond embankments. It is a typical semi-mangrove plant.

[0003] In the Lingnan region, Luanxi is a traditional Chinese medicine for both medicine and food. As a Chinese medicine, it tastes sweet, is slightly warm in nature, and enters the stomach and liver meridians. It has the effects of softening and dispersing lumps, warming the middle and dispersing cold, warming the stomach and eliminating accumulation, and removing rheumatism. It is mainly used to treat scrofula, phlegm nodules, food accumulation in children, epigastric pain, and rheumatic bone pain. In Foshan, Zhongshan, Zhuhai, Guangdong, as well as Macau and Hong Kong, the custom of eating Luanxi cakes on the day of the mother-in-law's birthday is still preserved.

[0004] Modern research has found that Luan Osmanthus contains chemical components such as thiophene, sesquiterpenes, sterols, quinic acid, glucoside, phenolic acid, flavonoids, etc. It has pharmacological activities such as antioxidant, anti-inflammatory, antibacterial, anti-cancer, hypoglycemic, and anti-diabetic. It has high medicinal value and huge development potential.

[0005] However, most of the current research on Luanwujia is about the extraction and separation of chemical components and the study of pharmacological activity. There is no comprehensive literature report on its quality evaluation, nor are there national and local standards, making it difficult to further develop and utilize it. Therefore, it is urgent to establish a standardized quality inspection method for Luanwujia. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a quality standard detection method for the Chinese herbal medicine Luanxiong, which solves the problems raised in the above background technology.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a quality standard detection method of the Chinese herbal medicine Luanxiong, comprising the following specific steps:

[0008] S1: Collection of Koelreuteria serrata leaves; multiple groups of Koelreuteria serrata leaves were collected, each group of Koelreuteria serrata leaves were collected from different locations, and the collection time of the collected Koelreuteria serrata leaves covered the whole year;

[0009] S2: Establish a high performance liquid chromatography content determination method for isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Luanqiu leaves and perform content determination; according to the original characteristics of Luanqiu leaves collected in S1, complete the establishment of the test sample preparation method and chromatographic conditions, prepare the test sample solution and the reference solution, and finally complete the determination and record the chromatogram;

[0010] S3: Verification: Methodological verification of S2 was performed, and the content determination results of Luanxiong leaf samples were calculated;

[0011] S4: Determination of the content of the collected Koelreuteria osmanthus leaves; moisture determination, total ash determination and extract determination are performed based on the collected Koelreuteria osmanthus leaves.

[0012] S5: Determine whether the quality is excellent and establish the standard for the collection area; determine the quality of the Luan osmanthus leaves and establish the standard for the sampling area based on the content of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, moisture content, total ash content and extract content in the collected Luan osmanthus leaves.

[0013] A further improvement of the technical solution of the present invention is that: the establishment of the test sample preparation method in S2 completes the selection of the extraction method based on the original characteristics of the Luanqiu leaves, and selects the ideal extraction time and extraction solvent, and finally establishes the preparation of the test sample solution; for the content detection of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Luanqiu leaves, multiple extraction methods, multiple extraction solvents, and multiple extraction times are required. Only after extraction can the test sample solution for quality inspection be prepared, and the selection of the ideal extraction time, extraction solvent, and extraction method needs to be determined in combination with the original characteristics of the Luanqiu leaves.

[0014] The further improvement of the technical scheme of the present invention lies in: the selection of the extraction method; it is determined by the spectrum separation effect in the chromatogram of different extraction methods, the peak height of the chromatogram of the measured component, and the peak area of ​​the chromatogram of the measured component; the extraction method mainly adopts ultrasonic extraction method, heating reflux method and cold soaking method, the above method is used to treat the Luanxiong leaf sample, and the chromatogram is compared to judge the spectrum separation effect in the chromatogram, the peak height of the chromatogram of the measured component, and the peak area of ​​the chromatogram of the measured component to select a suitable extraction method; the selection of the extraction solvent; it is determined by the number of components with high polarity in the chromatogram of different extraction solvents, the peak shape of the chromatogram, the peak height of the chromatogram of the measured component, the peak area of ​​the chromatogram of the measured component, and whether the component is completely extracted; the extraction solvent is mainly water, ethanol, 50% methanol, and toluene. Alcohol, the extraction solvent is used to treat the Luanxiong leaf sample, and the chromatograms are compared to determine the number of components with high polarity in the chromatogram, the peak shape of the chromatogram, the peak height of the chromatogram of the measured component, the peak area of ​​the chromatogram of the measured component, and whether the component is completely extracted to determine the extraction solvent; the extraction time is selected; the peak height of the chromatogram of the indicator component in the chromatogram at different extraction times, the peak area of ​​the chromatogram of the indicator component, and comprehensive consideration is given to economic and environmental protection, experimental operation and required time; the extraction time is mainly heated and refluxed for 30 minutes, 60 minutes, and 120 minutes. After treating the Luanxiong leaf sample, the chromatograms are compared to determine the peak height of the chromatogram of the indicator component in the chromatogram, the peak area of ​​the chromatogram of the indicator component, and the extraction time is selected by comprehensive consideration of economic and environmental protection, experimental operation and required time.

[0015] A further improvement of the technical solution of the present invention is that a preparation of a test solution is also provided, comprising the following specific steps:

[0016] Q1: Accurately weigh 0.5 g of Luanxiong leaf powder and pass it through a No. 2 sieve, then place it in a stoppered conical flask;

[0017] Q2: Accurately pipette the extraction solvent selected in S2, seal it, weigh it, and carry out the treatment method selected in S2 at 87°C for the extraction time selected in S2;

[0018] Q3: Remove the conical flask, quickly seal it, let it cool, weigh it again, accurately pipette the extraction solvent selected in S2 to make up the weight, shake it well, filter it with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0019] The further improvement of the technical solution of the present invention is that: the establishment of the chromatographic conditions in S2 includes the optimization of the mobile phase conditions and the selection of the column temperature and flow rate;

[0020] Optimization of mobile phase conditions; determined by the retention time of the component to be determined and other components in the chromatogram of different mobile phase systems, whether the component to be determined is eluted too early or too late, spectral information, separation effect, chromatographic peak separation effect of the component to be determined and retention time; the mobile phase systems are mainly acetonitrile-water, methanol-water, acetonitrile-phosphoric acid aqueous solution, methanol-phosphoric acid aqueous solution; selection of column temperature; determined by the chromatographic peak separation degree and chromatographic separation effect of the component to be determined in the chromatogram under different column temperature conditions; selection of flow rate; determined by the retention time of the chromatographic peak in the chromatogram under different flow rates.

[0021] A further improvement of the technical solution of the present invention is that the preparation of the reference solution also includes the following specific steps: accurately weighing appropriate amounts of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances, adding the extraction solution selected in S2, ultrasonically dissolving for 3 minutes, and fixing the volume to prepare mixed reference solutions of different concentrations.

[0022] A further improvement of the technical solution of the present invention is that: the determination of the test solution and the reference solution in S2 is carried out based on the established chromatographic conditions, and the chromatogram is recorded, and a methodological verification is carried out after the determination results are obtained. The methodological verification includes linear range verification, precision test, stability test, repeatability test, and sample recovery test, which aims to judge the precision of the instrument used in the method, the stability of the test solution, the repeatability of the content measurement of the same sample, and the recovery rate.

[0023] A further improvement of the technical solution of the present invention is that the specific steps taken for the moisture determination in S4 are: accurately weigh 2g of Luanxiong leaf powder, pass it through a No. 2 sieve and spread it flat in a flat weighing bottle that has been dried to constant weight, with a thickness not exceeding 10mm, accurately weigh it, open the bottle cap and dry it at 100-105°C for 5h, cover the bottle cap and quickly transfer it to a dryer, cool it to room temperature, accurately weigh it, then dry it at the above temperature for 1h, cool it for 30min, and weigh it until the difference between two consecutive weighings does not exceed 5mg; calculate the water content in the Luanxiong leaf sample based on the lost weight.

[0024] A further improvement of the technical solution of the present invention is that the specific steps taken for determining the total ash content in S4 are: accurately weigh 2-3 g of Koelreuteria osmanthus leaf powder, pass it through a No. 2 sieve, and place it in a crucible that has been incinerated to a constant weight, accurately weigh the weight, slowly heat it on an electric furnace to avoid burning, and after complete carbonization, gradually increase the temperature to 550°C to achieve complete ash conversion and constant weight, and after cooling, accurately weigh the crucible and the total ash weight; calculate the total ash content in the sample based on the weight of the residue.

[0025] A further improvement of the technical solution of the present invention is that: the specific steps taken for the determination of the extract in S4 are: accurately weigh 4g of Luanxiong leaf powder, pass it through a No. 2 sieve, place it in a 150ml stoppered conical flask, accurately add 50ml of 50% ethanol, seal it, weigh it, let it stand for 1h, connect a reflux condenser, heat it to boiling, and keep it slightly boiling for 1h; after cooling, remove the conical flask, quickly seal it, weigh it again, add 50% ethanol to make it heavy, shake it well, filter it with a drying filter, accurately measure 10ml of the filtrate, place it in an evaporating dish that has been dried to constant weight, evaporate it to dryness on a water bath, dry it at 105°C for 3h, cool it in a desiccator for 30min, and quickly and accurately weigh it; calculate the content of the extract in the test sample based on the dried product.

[0026] Compared with the prior art, the beneficial effects of the present invention are: accurately and efficiently determining the contents of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in Luan osmanthus leaves collected from different origins and harvesting times, obtaining the minimum and maximum contents of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C, and reasonably lowering the average content of each origin as the minimum limit, providing a basis for rapid quality evaluation for subsequent collection;

[0027] And on the basis of condition investigation and methodological verification, the results of this quality standard detection method are further made reliable, providing a reasonable and accurate basis and detection method for the subsequent development and utilization of Luanxiong resources and quality evaluation in the collection area. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flowchart of the quality standard detection method in the quality standard detection method of the Chinese herbal medicine Luanxiong;

[0029] Figure 2 This is a flow chart for the preparation of the test solution in the quality standard test method of the Chinese herbal medicine Luanxiong;

[0030] Figure 3 The chromatograms of different extraction methods in Example 2 of the quality standard detection method of the Chinese herbal medicine Luanxiong are shown in FIG.

[0031] Figure 4 The chromatograms of different extraction solvents in Example 2 of the quality standard detection method of the Chinese herbal medicine Luanxiong are shown in FIG.

[0032] Figure 5 The chromatograms are at different extraction times in Example 2 of the quality standard detection method of the Chinese herbal medicine Luanxiong;

[0033] Figure 6 The chromatograms of different mobile phase systems in Example 2 of the quality standard detection method of the Chinese herbal medicine Luanxiong;

[0034] Figure 7The chromatograms of different concentrations of phosphoric acid in Example 2 of the quality standard detection method of the Chinese herbal medicine Luanxiong;

[0035] Figure 8 The chromatograms are under different column temperature conditions in Example 2 of the quality standard detection method of the Chinese herbal medicine Luanxiong;

[0036] Fig. 9 The chromatogram is a HPLC content determination chart of Luanxiong leaves in the test solution and the reference solution in Example 3 of the quality standard detection method of the Chinese herbal medicine Luanxiong;

[0037] Fig.10 This is a schematic diagram of the results of content determination of samples No. 1-13 of Luanxiong leaves in Example 4 in the quality standard detection method of the Chinese herbal medicine Luanxiong; DETAILED DESCRIPTION

[0038] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0039] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0040] In addition, in order to better illustrate the present application, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, and elements well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0041] Reference Figure 1-Figure 10 , the present invention provides 5 technical solutions:

[0042] Embodiment 1: A quality standard detection method for the Chinese herbal medicine Luanqiu. Currently, most of the research on Luanqiu is the extraction and separation of chemical components and the research on pharmacological activity. There is no comprehensive method for evaluating its quality. Therefore, this embodiment uses the content of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Luanqiu leaves as the main standard for quality evaluation, and uses the moisture, total ash and extract of Luanqiu leaves as auxiliary standards for quality evaluation to complete the quality standard detection of the Chinese herbal medicine Luanqiu, including the following specific steps:

[0043] S1: Collection of Koelreuteria serrata leaves; multiple groups of Koelreuteria serrata leaves were collected, each group of Koelreuteria serrata leaves were collected from different locations, and the collection time of the collected Koelreuteria serrata leaves covered the whole year;

[0044] S2: Establish a high performance liquid chromatography method for determining the content of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Luanxiong leaves and perform content determination; according to the original characteristics of Luanxiong leaves collected in S1, complete the establishment of the test sample preparation method and chromatographic conditions, prepare the test sample solution and the reference solution, and finally complete the determination and record the chromatogram;

[0045] S3: Verification: Methodological verification of S2 was performed, and the content determination results of Luanxiong leaf samples were calculated;

[0046] S4: Determination of the content of the collected Koelreuteria osmanthus leaves; moisture determination, total ash determination and extract determination are performed based on the collected Koelreuteria osmanthus leaves.

[0047] S5: Determine whether the quality is excellent and establish the standard for the collection area; determine the quality of the Luan osmanthus leaves and establish the standard for the sampling area based on the content of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, moisture content, total ash content and extract content in the collected Luan osmanthus leaves.

[0048] The establishment of the test sample preparation method in S2, the selection of the extraction method is completed based on the original characteristics of Luanqiu leaves, and the ideal extraction time and extraction solvent are selected, and finally the preparation of the test solution is established. For the content detection of Luanqiu leaves isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C, there are multiple extraction methods, multiple extraction solvents, and multiple extraction times. After extraction, the test solution for quality inspection can be prepared, and the selection of the ideal extraction time, extraction solvent, and extraction method needs to be determined in combination with the original characteristics of Luanqiu leaves. Therefore, the following embodiment will discuss in detail how to select;

[0049] Selection of extraction method; determined by the spectral separation effect in the chromatogram of different extraction methods, the peak height of the chromatogram of the measured component, and the peak area of ​​the chromatogram of the measured component;

[0050] The extraction methods mainly include ultrasonic extraction, heating reflux and cold soaking. The above methods are used to treat the Luanxiong leaf samples, and the chromatograms are compared to judge the spectral separation effect in the chromatograms, determine the peak height of the component chromatograms, and determine the peak area of ​​the component chromatograms to select a suitable extraction method;

[0051] Selection of extraction solvent: It is determined by the number of highly polar components in the chromatograms of different extraction solvents, the shape of the chromatogram peaks, the peak height of the chromatogram of the measured components, the peak area of ​​the chromatogram of the measured components, and whether the components are completely extracted;

[0052] The extraction solvents are mainly water, ethanol, 50% methanol, and methanol. The above extraction solvents are used to treat the Luanxiong leaf samples, and the chromatograms are compared to determine the number of components with high polarity in the chromatograms, the peak shape of the chromatograms, the peak height of the chromatograms of the measured components, the peak area of ​​the chromatograms of the measured components, and whether the components are completely extracted to determine the extraction solvent;

[0053] Selection of extraction time: The peak height and peak area of ​​the chromatogram of the index component in the chromatogram at different extraction times are determined by comprehensively considering economic and environmental protection, experimental operation and required time;

[0054] The extraction time is mainly 30 minutes, 60 minutes and 120 minutes of heating reflux extraction. After processing the Luanxiong leaf sample, the chromatograms are compared to judge the peak height and peak area of ​​the chromatogram of the index component in the chromatogram. The extraction time is selected by comprehensively considering the economy, environmental protection, experimental operation and required time.

[0055] The preparation of the test solution also includes the following specific steps:

[0056] Q1: Accurately weigh 0.5 g of Luanxiong leaf powder and pass it through a No. 2 sieve, then place it in a stoppered conical flask;

[0057] Q2: Accurately pipette the extraction solvent selected in S2, seal it, weigh it, and carry out the treatment method selected in S2 at 87°C for the extraction time selected in S2;

[0058] Q3: Remove the conical flask, quickly seal it, let it cool, weigh it again, accurately pipette the extraction solvent selected in S2 to make up the weight, shake it well, filter it with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0059] The establishment of chromatographic conditions in S2 includes the optimization of mobile phase conditions and the selection of column temperature and flow rate;

[0060] Optimization of mobile phase conditions; determined by the retention time of the component to be determined and other components in the chromatogram of different mobile phase systems, whether the component to be determined is eluted too early or too late, spectral information, separation effect, separation effect of the chromatographic peaks of the component to be determined, and retention time;

[0061] The mobile phase systems are mainly acetonitrile-water, methanol-water, acetonitrile-phosphoric acid aqueous solution, methanol-phosphoric acid aqueous solution;

[0062] The selection of column temperature is determined by the chromatographic peak separation of the components to be determined in the chromatogram under different column temperature conditions and the chromatographic separation effect;

[0063] The column temperature is mainly 25℃, 30℃ and 35℃. The appropriate column temperature is determined according to the chromatographic peak separation of the components to be determined and the chromatographic separation effect under different column temperature conditions.

[0064] The selection of flow rate is determined by the retention time of the chromatographic peaks in the chromatogram at different flow rates.

[0065] The flow rates are mainly 0.8mL / min, 0.9mL / min, and 1.0mL / min. The appropriate flow rate is selected considering the retention time of the chromatographic peak.

[0066] The preparation of the reference solution also includes the following specific steps: accurately weighing appropriate amounts of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances, adding the extraction solution selected in S2, sonicating for 3 minutes to dissolve, and fixing the volume to prepare mixed reference solutions of different concentrations.

[0067] The determination of the test solution and the reference solution in S2 is carried out based on the established chromatographic conditions, and the chromatograms are recorded. After the determination results are obtained, methodological validation is carried out. The methodological validation includes linear range validation, precision test, stability test, repeatability test, and sample recovery test, which aims to evaluate the precision of the instrument used in the method, the stability of the test solution, the repeatability of the content measurement of the same sample, and the recovery rate.

[0068] The specific steps taken for the moisture determination in S4 are: accurately weigh 2 g of Luanxiong leaf powder, pass it through a No. 2 sieve and spread it flat in a flat weighing bottle that has been dried to constant weight, with a thickness not exceeding 10 mm, accurately weigh it, open the bottle cap and dry it at 100-105°C for 5 hours, cover the bottle cap and quickly transfer it to a dryer, cool it to room temperature, accurately weigh it, then dry it at the above temperature for 1 hour, cool it for 30 minutes, and weigh it until the difference between two consecutive weighings does not exceed 5 mg; calculate the water content in the Luanxiong leaf sample based on the lost weight.

[0069] The specific steps taken for the determination of total ash in S4 are: accurately weigh 2-3g of Koelreuteria osmanthus leaf powder, pass it through a No. 2 sieve, place it in a crucible that has been incinerated to a constant weight, accurately weigh the weight, slowly heat it on an electric furnace to avoid burning, and after complete carbonization, gradually increase the temperature to 550°C to achieve complete ashization and constant weight. After cooling, accurately weigh the crucible and total ash weight; calculate the total ash content in the sample based on the weight of the residue.

[0070] The specific steps taken for the determination of extracts in S4 are as follows: 4 g of Luanxiong leaf powder was accurately weighed, passed through a No. 2 sieve, placed in a 150 ml stoppered conical flask, 50 ml of 50% ethanol was accurately added, the flask was sealed, and the weight was determined. After standing for 1 hour, a reflux condenser was connected, and the flask was heated to boiling and kept at a slight boiling temperature for 1 hour.

[0071] After cooling, remove the conical flask, quickly seal it, weigh it again, add 50% ethanol to make it heavier, shake it well, filter it with a drying filter, accurately measure 10 ml of the filtrate, place it in an evaporating dish that has been dried to constant weight, evaporate it to dryness on a water bath, dry it at 105°C for 3h, cool it in a desiccator for 30min, and quickly and accurately weigh it; calculate the content of the extract in the test sample based on the dry product.

[0072] Example 2: In view of the description of Example 1, this example takes 13 groups of Luanxiong leaves from different regions as samples for quality standard testing. The samples are numbered 1-13 in sequence, and when taken from a single region, multiple samples are taken as alternatives;

[0073] In the process of preparing samples 1-13 for testing:

[0074] Firstly, the samples of Luanxiong leaf were treated by ultrasonic extraction, heating reflux and cold soaking respectively, and the chromatograms were compared. Figure 3 As shown in the attached figure, A: ultrasonic extraction method; B: cold soaking method; C: heating reflux method, the spectrum separation effect obtained by ultrasonic extraction method is not good, the chromatograms of cold soaking method and heating reflux method are basically the same, but the peak heights of the three measured components in the spectrum of heating reflux method are higher, the peak area is larger, and the extraction is more complete, so the heating reflux method is selected as the treatment method for the test sample;

[0075] Then, samples 1-13 of Luanxiong leaves were treated with water, ethanol, 50% methanol, and methanol as extraction solvents, and the chromatograms were compared. Figure 4 As shown, A: water; B: ethanol; C: 50% methanol; D: methanol. When water is used as the solvent, there are too many components with high polarity, which have a greater impact on the peaks of the three components to be determined; when ethanol is used as the solvent, the peak shape is not good; when methanol is used as the solvent, the peak heights of the three components to be determined are lower than those of 50% methanol, the peak area is smaller, and the three components are not completely extracted; when the solvent is 50% methanol, the peak heights and peak areas of the three components to be determined are the largest among all solvents, which is suitable for content determination, so 50% methanol is selected as the extraction solvent for the test sample;

[0076] Finally, the samples of Luanxiong leaf were extracted by heating under reflux for 30 minutes, 60 minutes and 120 minutes respectively, and the chromatograms were compared. Figure 5 As shown, A: 30 minutes; B: 60 minutes; C: 120 minutes, the chromatogram obtained by heating reflux extraction for 60 minutes has no obvious difference with that of 30 minutes, but the peak height of the index component is higher and the peak area is larger, while the spectrum obtained by heating reflux extraction for 120 minutes has a component to be tested with a smaller peak height and peak area than that of the extraction for 60 minutes. Considering the economic and environmental protection and the simplicity of experimental operation and time saving, the extraction time is selected as 60 minutes;

[0077] Therefore, in this embodiment, a high performance liquid chromatography method for determining the contents of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in Luanxiong leaves was established, and the extraction method, extraction time and extraction solvent involved were heating reflux method, 50% methanol and 60 minutes, respectively.

[0078] Therefore, based on the above selection, perform the following steps:

[0079] Q1: Accurately weigh 0.5 g of Luanxiong leaf powder and pass it through a No. 2 sieve, then place it in a stoppered conical flask;

[0080] Q2: Accurately pipette 50% methanol, seal, weigh, and heat under reflux at 87°C for 60 min;

[0081] Q3: Remove the conical flask, quickly seal it, let it cool, weigh it again, accurately pipette 50% methanol to make up the weight again, shake it well, filter it with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0082] After the final test solution is prepared, the corresponding HPLC conditions are established;

[0083] First, the effects of four mobile phase systems, acetonitrile-water, methanol-water, acetonitrile-phosphoric acid aqueous solution, and methanol-phosphoric acid aqueous solution, on the chromatogram were investigated. Figure 6 As shown, A: acetonitrile-phosphoric acid; B: methanol-phosphoric acid; C: acetonitrile-water; D: methanol-water. When the mobile phase is methanol-phosphoric acid aqueous solution, the retention time of the three components to be determined and other components is too long and they are eluted by the chromatographic column later. When acetonitrile-water or methanol-water is used as the mobile phase, the three components to be determined are eluted too early, the spectrum information is less, and it cannot be determined. When acetonitrile-phosphoric acid aqueous solution is used as the mobile phase, the spectrum is complete and the separation effect is good.

[0084] When acetonitrile-phosphoric acid aqueous solution is used as the mobile phase, different concentrations of phosphoric acid aqueous solution should be considered. Figure 7 As shown, A: 0.05% phosphoric acid; B: 0.1% phosphoric acid; C: 0.4% phosphoric acid. When the concentration is 0.1%, the chromatographic peaks of the three components to be determined have a good separation effect and a suitable retention time. Therefore, the mobile phase system is selected as acetonitrile-phosphoric acid aqueous solution with a concentration of 0.1%;

[0085] Then select three column temperatures of 25℃, 30℃, and 35℃ respectively. According to the chromatograms under different column temperatures, as shown in the attached Figure 8 As shown, A: 25℃; B: 30℃; C: 35℃. Under the column temperature condition of 25℃, the chromatographic peaks of the three components to be determined have good separation and excellent chromatographic separation effect, so the column temperature is selected to be 25℃.

[0086] Finally, three flow rate conditions of 0.8mL / min, 0.9mL / min, and 1.0mL / min were selected, as shown in the attached Fig. 9 As shown, A: 0.8mL / min; B: 0.9mL / min; C: 1.0mL / min, the three flow rates have little effect on the chromatogram. Considering the retention time of the chromatographic peak, the retention time of the chromatographic peak is the shortest when the flow rate is 1.0mL / min, so the flow rate is selected as 1.0mL / min;

[0087] It should be noted that regardless of the choice of any condition such as the mobile phase system, extraction method, extraction time, etc., the control experiments used samples of Luanxiong leaves from the same region;

[0088] Then, in this embodiment, the chromatographic conditions established for the quality inspection of the collected Luan osmanthus leaf samples are specifically as follows: the chromatographic column is a Phenomenex Luna Omega PS C18 chromatographic column; the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, and gradient elution is performed according to Table 2-4; the detection wavelength is 340nm; the column temperature is 25°C; the flow rate is 1.0mL / min; and the injection volume is 10μL.

[0089] Embodiment three:

[0090] On the basis of Example 2: accurately weigh appropriate amounts of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances, add 50% methanol and ultrasonically dissolve for 3 minutes, and adjust the volume to prepare mixed reference substance solutions with concentrations of 216.40 μg / ml, 76.80 μg / ml, and 202.40 μg, respectively;

[0091] Accurately pipette the test solution and the reference solution, measure according to the chromatographic conditions of Example 2, and record the chromatogram, as shown in the attached figure. Fig. 9 As shown, A: reference substance; B: test substance; 1: isochlorogenic acid B; 2: isochlorogenic acid A; 3: isochlorogenic acid C. Based on this content comparison result, it is necessary to conduct methodological verification and calculate the content determination results of the Luanxiong leaf sample.

[0092] The specific operation is: repeatedly measure the same test solution 6 times using the chromatographic conditions of Example 2, record the peak area, and calculate the RSD of the peak area value of each component.

[0093] The RSDs of the peak area values ​​of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C were 0.42%, 0.67% and 0.74% respectively, indicating that the instrument used in this method has excellent precision. Excluding the influence of external factors on the results, the precision test results are shown in the following table:

[0094]

[0095]

[0096] The stability test is as follows: the peak area of ​​the same test solution is measured using the chromatographic conditions of Example 2 at 0, 2, 4, 6, 8, 10, 12, and 24 hours respectively. The RSD of the peak area value of each component is calculated, and the RSD of the peak area values ​​of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C are 0.70%, 1.16%, and 0.92%, respectively, that is, the stability of the test solution remains good within 24 hours. The stability test results are shown in the following table:

[0097]

[0098] The repeatability test is as follows: prepare parallel test solutions by the method of Example 2, repeat the injection of each sample twice, and record the peak area. Calculate the RSD of the content of each component, and the RSD of the content of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C are 0.71%, 0.73% and 0.92%, respectively, that is, this method has excellent repeatability, and the stability test results are shown in the following table:

[0099]

[0100] The recovery test was as follows: 11.80 mg, 2.69 mg and 10.42 mg of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C reference substances were accurately weighed in a 500 ml volumetric flask, 50% methanol was added for 3 min of ultrasonic dissolution, and the volume was fixed to prepare mixed reference substance solutions with concentrations of 23.60, 5.38 and 20.84 μg / ml, respectively. 6 portions of Luanxiong leaf powder (S7) with known contents were accurately weighed in parallel, and 50 ml of the mixed reference substance solution was accurately pipetted respectively. The test sample was prepared using the steps of Example 2, and each portion was injected twice, and the peak area was recorded. The recovery was calculated. The average recoveries of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C were 98.95%, 98.81% and 99.03%, respectively, and the RSDs were 0.36%, 0.70% and 0.91%, respectively. That is, the recovery rate of this method is relatively good. The results of the sample recovery test are shown in the following table:

[0101]

[0102]

[0103]

[0104] Therefore, this example verifies that the determination of the content of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in the Luanwujing leaves in the standard detection method used in Example 2 has good stability, repeatability, and recovery rate according to multiple groups of control experiments;

[0105] Example 4: Based on the feasibility of Example 2 verified in Example 3: it is necessary to determine the content of the samples No. 1-13 of Luanxiong leaves, prepare 13 batches of Luanxiong leaf test solution by the method mentioned in Example 2, determine under the chromatographic conditions of Example 2, record the chromatogram, and calculate the content of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C of 13 batches of Luanxiong leaves with the dried product;

[0106] As attached Fig.10As shown, according to the content determination results of samples No. 1-13, the average content of isochlorogenic acid A was calculated to be 1.11%, the average content of isochlorogenic acid B was 0.22%, and the average content of isochlorogenic acid C was 1.07%. Then, the minimum standards for the contents of the three components in the thirteen regions collected were as follows: the average contents of the three components were lowered by 30% as the minimum limits to obtain 0.78%, 0.16% and 0.75% respectively, and the minimum quality standards for isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C of Luanwujing leaves in the sampled regions were obtained;

[0107] The contents of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in the leaves of Luan osmanthus collected from different origins and harvesting times were accurately and efficiently determined, and the minimum and maximum contents of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C were obtained. The average content of each origin was reasonably lowered as the minimum limit, providing a basis for rapid quality evaluation for subsequent collection;

[0108] And on the basis of condition investigation and methodological verification, the results of this quality standard detection method are further made reliable, providing a reasonable and accurate basis and detection method for the subsequent development and utilization of Luanxiong resources and quality evaluation in the collection area.

[0109] Example 5: Based on Example 1 and Example 2: The test standards for moisture determination, total ash determination and extract determination are:

[0110] 1. The moisture content of 13 batches of Luanxiong leaves from different origins was measured, and the results showed that the moisture content was between 10.27% and 14.09%, with an average moisture content of 12.53%. The average value was adjusted up by 20%, rounded to 15.0%, and referring to the moisture content limit of leaf medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, it is stipulated that the moisture content of Luanxiong leaves shall not exceed 15.0%;

[0111] 2. By measuring the total ash content of 13 batches of Luanxiong leaves from different origins and harvesting times, the total ash content of Luanxiong leaves in this experiment ranged from 12.38% to 17.46%, and the average total ash content was 14.05%. The average value was adjusted up by 20%, rounded to 17.0%. Referring to the total ash content limit of leaf medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, it is stipulated that the total ash content of Luanxiong leaves shall not exceed 17.0%;

[0112] 3. The results of the 50% ethanol hot-infusion method of 13 batches of Luanxiong leaves from different origins showed that the content of the 50% ethanol hot-infusion method was between 25.25% and 35.41%, with an average content of 31.37%. The average value was adjusted down by 20% and rounded to 25%, and it was stipulated that the alcohol-soluble extract of Luanxiong leaves should not be less than 25%.

[0113] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0114] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A quality standard detection method for Chinese herbal medicine Luanxiong, characterized in that: The specific steps include: S1: Collection of Koelreuteria serrata leaves; multiple groups of Koelreuteria serrata leaves were collected, each group of Koelreuteria serrata leaves were collected from different locations, and the collection time of the collected Koelreuteria serrata leaves covered the whole year; S2: Establish a high performance liquid chromatography content determination method for isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Luanqiu leaves and perform content determination; according to the original characteristics of Luanqiu leaves collected in S1, complete the establishment of the test sample preparation method and chromatographic conditions, prepare the test sample solution and the reference solution, and finally complete the determination and record the chromatogram; S3: Verification: Methodological verification of S2 was performed, and the content determination results of Luanxiong leaf samples were calculated; S4: Determination of the content of the collected Koelreuteria osmanthus leaves; moisture determination, total ash determination and extract determination are performed based on the collected Koelreuteria osmanthus leaves. S5: Determine whether the quality is excellent and establish the standard for the collection area; determine the quality of the Luan osmanthus leaves and establish the standard for the sampling area based on the content of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, moisture content, total ash content and extract content in the collected Luan osmanthus leaves.

2. The quality standard detection method of a Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The establishment of the test sample preparation method in S2 is to complete the selection of the extraction method based on the original characteristics of the Koelreuteria osmanthus leaves, select the ideal extraction time and extraction solvent, and finally establish the preparation of the test sample solution.

3. The quality standard detection method of a Chinese herbal medicine Luanxiong according to claim 2, characterized in that: Selection of extraction method; determined by the spectral separation effect in the chromatogram of different extraction methods, the peak height of the chromatogram of the measured component, and the peak area of ​​the chromatogram of the measured component; Selection of extraction solvent: It is determined by the number of highly polar components in the chromatograms of different extraction solvents, the shape of the chromatogram peaks, the peak height of the chromatogram of the measured components, the peak area of ​​the chromatogram of the measured components, and whether the components are completely extracted; Selection of extraction time; based on the peak height and peak area of ​​the chromatogram of the index component in the chromatogram at different extraction times, and comprehensive consideration of economic and environmental protection, experimental operation and required time.

4. The quality standard detection method of a Chinese herbal medicine Luanxiong according to claim 2, characterized in that: The preparation of the test solution also includes the following specific steps: Q1: Accurately weigh 0.5 g of Luanxiong leaf powder and pass it through a No. 2 sieve, then place it in a stoppered conical flask; Q2: Accurately pipette the extraction solvent selected in S2, seal it, weigh it, and carry out the treatment method selected in S2 at 87°C for the extraction time selected in S2; Q3: Remove the conical flask, quickly seal it, let it cool, weigh it again, accurately pipette the extraction solvent selected in S2 to make up the weight, shake it well, filter it with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution.

5. The quality standard detection method of a Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The establishment of the chromatographic conditions in S2 includes the optimization of mobile phase conditions and the selection of column temperature and flow rate; Optimization of mobile phase conditions; determined by the retention time of the component to be determined and other components in the chromatogram of different mobile phase systems, whether the component to be determined is eluted too early or too late, spectral information, separation effect, separation effect of the chromatographic peaks of the component to be determined, and retention time; The selection of column temperature is determined by the chromatographic peak separation of the components to be determined in the chromatogram under different column temperature conditions and the chromatographic separation effect; The selection of flow rate is determined by the retention time of the chromatographic peaks in the chromatogram at different flow rates.

6. The quality standard detection method of a Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The preparation of the reference solution also includes the following specific steps: accurately weighing appropriate amounts of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances, adding the extraction solution selected in S2, sonicating for 3 minutes to dissolve, and fixing the volume to prepare mixed reference solutions of different concentrations.

7. The quality standard detection method of a Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The determination of the test solution and the reference solution in S2 is carried out based on the established chromatographic conditions, and the chromatograms are recorded. After the determination results are obtained, methodological validation is carried out. The methodological validation includes linear range validation, precision test, stability test, repeatability test, and sample recovery test, which aims to judge the precision of the instrument used in the method, the stability of the test solution, the repeatability of the content measurement of the same sample, and the recovery rate.

8. The quality standard detection method of the Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The specific steps taken for the moisture determination in S4 are: accurately weigh 2 g of Luanxiong leaf powder, pass it through a No. 2 sieve and spread it flat in a flat weighing bottle that has been dried to constant weight, with a thickness not exceeding 10 mm, accurately weigh it, open the bottle cap and dry it at 100-105°C for 5 hours, cover the bottle cap and quickly transfer it to a dryer, cool it to room temperature, accurately weigh it, then dry it at the above temperature for 1 hour, cool it for 30 minutes, and weigh it until the difference between two consecutive weighings does not exceed 5 mg; calculate the water content in the Luanxiong leaf sample based on the lost weight.

9. The quality standard detection method of the Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The specific steps taken for the determination of the total ash content in S4 are as follows: accurately weigh 2-3 g of Koelreuteria osmanthus leaf powder, pass it through a No. 2 sieve, place it in a crucible that has been incinerated to a constant weight, accurately weigh the weight, slowly heat it on an electric furnace to avoid burning, and after complete carbonization, gradually increase the temperature to 550°C to achieve complete ash conversion and constant weight. After cooling, accurately weigh the crucible and the total ash weight; calculate the total ash content in the sample based on the weight of the residue.

10. The quality standard detection method of the Chinese herbal medicine Luanxiong according to claim 1, characterized in that: The specific steps taken for the determination of the extract in S4 are as follows: 4 g of Luanxiong leaf powder is accurately weighed, passed through a No. 2 sieve, placed in a 150 ml stoppered conical flask, 50 ml of 50% ethanol is accurately added, the flask is tightly stoppered, the weight is determined, and after standing for 1 hour, a reflux condenser is connected, and the flask is heated to boiling and kept at a slight boiling temperature for 1 hour; After cooling, remove the conical flask, quickly seal it, weigh it again, add 50% ethanol to make it heavier, shake it well, filter it with a drying filter, accurately measure 10 ml of the filtrate, place it in an evaporating dish that has been dried to constant weight, evaporate it to dryness on a water bath, dry it at 105°C for 3h, cool it in a desiccator for 30min, and quickly and accurately weigh it; calculate the content of the extract in the test sample based on the dry product.

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