Method for detecting contents of five components in roundbell flower and application of method for detecting contents of five components in roundbell flower

Abstract

The invention provides a method for detecting the content of five components in melastoma dodecandrum and application of the method. Specifically, the invention provides a method for detecting the contents of chlorogenic acid, codonopsis pilosula glycoside I, luteoloside, apigenin 7-O-beta-D-glucoside and lobetyolin in the centella asiatica. The detection method disclosed by the invention is accurate, high in sensitivity, good in repeatability and reliable in result, provides a basis for quality control and evaluation of the round-clock flowers, and has a good application prospect in formulating a round-clock flower quality standard, reasonably developing and utilizing resources and the like.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology of traditional Chinese medicinal materials, specifically, it relates to a method and application for detecting the content of five components in *Cyclocarya paliurus*. Background Technology

[0002] Cyclocodon lancifolius (Roxburgh) Kurz., belonging to the genus Cyclocodon in the family Campanulaceae, is an erect or creeping perennial herb. First recorded in the 1872 edition of *Flora of China*, it is commonly known as "meat abacus," "mountain water chestnut," and "long-leaved lancifolius," and is a traditional medicinal and edible plant resource characteristic of Guizhou Province. Every part of the Cyclocodon lancifolius is valuable; the fruit is edible, and the roots, stems, and leaves can be used medicinally. In southern and southeastern Guizhou, the Miao, Buyi, and western Hunan ethnic minorities also use its roots as a tonic. Currently, the root of Cyclocodon lancifolius is listed under the medicinal name "Spider Fruit" in the 2019 edition of *Guizhou Province Standards for the Quality of Traditional Chinese and Ethnic Medicines*. It is classified as entering the lung and large intestine meridians, possessing the effects of tonifying qi, resolving phlegm, and relieving pain. It is mainly used to treat qi deficiency and fatigue, traumatic injuries, abdominal pain, and pulmonary tuberculosis cough, and is a commonly used medicinal material among ethnic minorities in Guizhou Province.

[0003] Modern research shows that *Flammulina velutipes* contains various chemical components, including flavonoids, polysaccharides, organic acids, alkynes, and glycosides, which possess antioxidant properties and inhibit α-glucosidase and xanthine oxidase. In recent years, the medicinal value of *Flammulina velutipes* has received increasing attention and recognition. However, current research on quality control of different parts of *Flammulina velutipes* (roots, stems, leaves, and fruits) is limited, with only reports on the determination of chlorogenic acid, luteolin, and apigenin content in the roots. Therefore, developing quality control research on roots, stems, leaves, and fruits is of great significance for the comprehensive development and utilization of *Flammulina velutipes* resources. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies where quality control research on *Flammulina velutipes* is limited to measuring individual non-characteristic components in the roots, while lacking systematic quality control methods for other parts, making it difficult to comprehensively evaluate its quality. This invention provides a method and application for detecting the content of five components in *Flammulina velutipes*. The detection method of this invention is accurate, highly sensitive, has good repeatability, and reliable results, providing a basis for the quality control and evaluation of *Flammulina velutipes*, and has good application prospects in the formulation of *Flammulina velutipes* quality standards and the rational development and utilization of resources.

[0005] The present invention solves the above-mentioned technical problems through the following technical solution:

[0006] This invention discloses a method for detecting the content of chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside, and codonopsis glycoside in *Cyclocarya paliurus*, comprising the following steps: detecting the *Cyclocarya paliurus* sample using high-performance liquid chromatography (HPLC).

[0007] In chromatographic conditions:

[0008] The chromatographic column is C10. 18 Chromatographic column;

[0009] Mobile phase: Gradient elution of 0.2% phosphoric acid aqueous solution-methanol-acetonitrile:

[0010] .

[0011] In one aspect of the present invention, the sample to be tested is the root, stem, leaf or fruit of the bellflower; for example, the leaf, stem or root of the bellflower; or for example, the leaf or stem of the bellflower.

[0012] In one aspect of the present invention, when the test sample of *Cyclocarya paliurus* is the root of *Cyclocarya paliurus*, the detected components are chlorogenic acid, codonopsis glycoside I, luteolin and codonopsis glycoside or chlorogenic acid, luteolin and codonopsis glycoside.

[0013] In one aspect of this invention, when the test sample of *Cyclocarya paliurus* is the fruit of *Cyclocarya paliurus*, the detected components are chlorogenic acid, codonopsis glycoside I, luteolin glycoside, and codonopsis glycoside.

[0014] In this invention, the chromatographic column can be selected from various C66 columns according to conventional methods in the art. 18 Chromatographic columns; for example, Shiseido MGII BP-C18 columns, Shiseido CAPCELL PAK C18 columns, Welch Xtimate® C18 columns, or Waters HSS T3® C18 columns; for example, Shiseido MG II BP-C18 columns.

[0015] In one embodiment of the present invention, the length of the chromatographic column is 200-300 mm; for example, 250 mm.

[0016] In one embodiment of the present invention, the inner diameter of the chromatographic column is 4.6 mm.

[0017] In one embodiment of the present invention, the particle size of the packing material in the chromatographic column is 4-6µm; for example, 5µm.

[0018] In one embodiment of the present invention, the chromatographic column has the following specifications: length 250 mm, inner diameter 4.6 mm, and packing particle size 5 µm.

[0019] In this invention, the column temperature of the chromatographic column can be selected in accordance with conventional methods in the art, for example, 20-35°C, or 25-35°C, or even 25°C or 27°C.

[0020] In this invention, the detection wavelength in the high-performance liquid chromatography method can be selected according to conventional methods in the art, for example, 200-360 nm, or even 245-360 nm, or more specifically, 267 nm or 340 nm. The detector can be conventional equipment in the art, such as a DAD or UV detector.

[0021] In one aspect of the present invention, the injection volume in the high-performance liquid chromatography method is 3-8 μL, for example, 5 μL.

[0022] In this invention, the flow rate of the mobile phase can be as conventional in the art, for example 0.6-1.0 mL / min, or for example 0.8 mL / min.

[0023] In one aspect of the present invention, before detecting the sample of *Callicarpa spp.* using high performance liquid chromatography, the sample of *Callicarpa spp.* is pretreated, which includes the following steps: extracting the sample of *Callicarpa spp.* in a solvent.

[0024] The extraction method is a conventional method for extracting this type of substance in the art; for example, reflux extraction or ultrasonic extraction, and ultrasonic extraction is another example.

[0025] In this invention, the solvent can be selected according to conventional methods in the art, for example, an alcohol solvent and / or water; or, for example, an alcohol solvent and water; the alcohol solvent can be methanol and / or ethanol.

[0026] Preferably, the solvent is a 25% methanol aqueous solution, a 25% ethanol aqueous solution, a 50% methanol aqueous solution, a 50% ethanol aqueous solution, a 75% methanol aqueous solution, a 75% ethanol aqueous solution, methanol, or ethanol, for example, a 75% methanol aqueous solution.

[0027] In one aspect of the present invention, the sample of *Callicarpa japonica* to be tested needs to be made into powder before extraction to obtain *Callicarpa japonica* sample powder.

[0028] In this invention, the preparation method of the *Cymbidium goeringii* test sample powder can be selected according to conventional methods in the art, such as drying, grinding, and sieving.

[0029] In this invention, the drying temperature is selected according to conventional methods in the art, for example, 40-60°C, or for example, 50°C.

[0030] In this invention, the grinding process can be selected according to conventional operations in the art, such as using a grinding machine.

[0031] In this invention, the mesh size of the sieve is 10-50 mesh; for example, 24 mesh; or for example, a No. 2 sieve for traditional Chinese medicine.

[0032] In this invention, the temperature of the reflux method can be selected according to conventional methods in the art, for example, 60-100℃; for example, 80℃.

[0033] In this invention, the reflux time can be selected according to conventional methods in the art, for example, 20-120 min; or for example, 60 min.

[0034] In this invention, the temperature of the ultrasonic extraction method can be selected according to conventional methods in the art, for example, 20-50℃, or for example, 30℃.

[0035] In this invention, the time for the ultrasonic extraction method can be selected according to conventional methods in the art, for example, 20-120 min; or for example, 30 min, 60 min or 90 min.

[0036] In this invention, the mass-to-volume ratio of *Callicarpa arvensis* to solvent in the ultrasonic extraction method can be selected according to conventional methods in the art, for example, 1:(5-60) g / mL, or for example, 1:10 g / mL, 1:20 g / mL, 1:30 g / mL or 1:50 g / mL; further for example, 1:30 g / mL.

[0037] In one aspect of the present invention, the pretreatment further includes the following post-treatment steps: cooling and filtration.

[0038] In one aspect of the present invention, the filtration is performed using a 0.15-0.30 μm microporous membrane; for example, a 0.22 μm microporous membrane.

[0039] In one aspect of the present invention, the cooling is to cool to 15-30°C; for example, 20-25°C.

[0040] In one aspect of the present invention, the cooling process further includes adding a solvent to replenish the weight lost during the extraction process; the solvent is the solvent used in the extraction method described above.

[0041] In one embodiment of the present invention, the detection method further includes the preparation of a reference solution: chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside, and codonopsis glycoside are mixed in a solvent to prepare a reference solution.

[0042] In one aspect of the present invention, the solvent used in the preparation of the reference solution is methanol.

[0043] In one embodiment of the present invention, the high-performance liquid chromatograph used in the high-performance liquid chromatography method is a Thermo U3000 high-performance liquid chromatograph or an Aglient 1260 Infinity high-performance liquid chromatograph.

[0044] In one embodiment of the present invention, the place of origin of the bellflower is Guizhou Province, Yunnan Province, Sichuan Province or Guangxi Zhuang Autonomous Region; for example, Wengyan Village, Taiping Township, Wanshan District, Tongren City, Guizhou Province; Wenshan City, Yunnan Province; Jinghe Village, Dewang District, Jiangkou County, Tongren City, Guizhou Province; Miaowan Village, Huangban Town, Songtao Miao Autonomous County, Tongren City, Guizhou Province; Xiema, Jiajiang County, Leshan City, Sichuan Province; Hongya County, Meishan City, Sichuan Province; Lüchun County, Honghe City, Yunnan Province; Malipo County, Wenshan City, Yunnan Province; Jinxiu Yao Autonomous County, Laibin City, Guangxi Zhuang Autonomous Region; Wanshan District, Tongren City, Guizhou Province or Maguan County, Wenshan City, Yunnan Province.

[0045] In one embodiment of the present invention, the detection method is to use high performance liquid chromatography to detect the reference standard and the test sample of *Cyclocarya paliurus* as described above; then, the contents of chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside and codonopsis glycoside in *Cyclocarya paliurus* as described above are calculated by the external standard method.

[0046] This invention discloses a method for detecting the content of chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside and codonopsis glycoside in *Cyclocarya paliurus*, and its application in the quality control of *Cyclocarya paliurus* or its preparations; the detection method is as described in any embodiment of this invention.

[0047] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0048] All reagents used in this invention are commercially available.

[0049] The significant advantages of this invention are as follows: It provides an HPLC method for determining the content of five components in the roots, stems, leaves, and fruits of *Flammulina velutipes*. By optimizing the extraction method and chromatographic conditions, the content of these five chemical components in the roots, stems, leaves, and fruits of *Flammulina velutipes* is determined, providing a research basis for the formulation of quality standards for this medicinal material and the rational development and utilization of its resources. The method is accurate, highly sensitive, reproducible, and reliable, providing a basis for the quality control and evaluation of *Flammulina velutipes*. Detailed Implementation

[0050] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0051] 1. Experimental Materials and Instruments

[0052] 1.1 Experimental Materials

[0053] Chlorogenic acid (batch number CFS202401), luteolin (batch number 12937), codonopsis glycoside I (batch number CFS202202), and codonopsis glycoside (batch number 16457, ≥95%) were all purchased from Wuhan Zhongbiao Technology Co., Ltd.; apigenin-7-O-β-D glucoside (cosmos glycoside) (batch number MUST23050611, 99.35%) was purchased from Chengdu Dester Technology Co., Ltd. Acetonitrile and methanol were chromatographic grade; water was ultrapure water; all other reagents were analytical grade.

[0054] A total of 15 batches of whole *Cymbidium goeringii* plants were collected. S1-S10 were artificially cultivated and provided by Guizhou Chuangxing Agricultural Development Co., Ltd.; S11-S15 were wild-grown and purchased from local pharmaceutical merchants; and 5 batches of fruits were also provided by Guizhou Chuangxing Agricultural Development Co., Ltd. The identification numbers, varieties, and origin information of the medicinal materials are shown in Table 1.

[0055] Table 1. Information on various parts of the 15th batch of cycads

[0056]

[0057]

[0058] 1.2 Experimental Apparatus

[0059] Mettler MS 204TS electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Mettler MS105 (DU) electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); SB-5200DTD ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); Aglient 1260 Infinity high performance liquid chromatograph (Agilent Technologies Inc.); Thermo U3000 high performance liquid chromatograph (Thermo Fisher Scientific (China) Co., Ltd.); Mili-Q50 SP pure water system (Milipore, USA).

[0060] 2. Experimental methods and results

[0061] 2.1 Chromatographic conditions

[0062] Column: Shiseido MG II BP-C18 (4.6 mm × 250 mm, 5 μm); Column temperature: 25℃; Flow rate: 0.8 mL / min; Detection wavelength: 267 nm, 340 nm (chlorogenic acid); Injection volume: 5 μL; Mobile phase conditions (volume ratio): 0.2% phosphoric acid aqueous solution (A) - methanol (B) - acetonitrile (C); Gradient elution, elution program is shown in Table 2.

[0063] Table 2 Mobile phase elution conditions

[0064]

[0065] 2.2 Preparation of reference solution

[0066] Accurately weigh appropriate amounts of chlorogenic acid, codonopsis pilosula glycoside I, luteolin, apigenin 7-O-β-D-glucoside, and codonopsis pilosula glycoside reference standards, and prepare reference stock solutions with concentrations of 1346.0, 263.2, 636.0, 1087.0, and 987.0 μg / mL respectively by dissolving them in methanol. Accurately pipette appropriate amounts of the reference stock solutions and mix them thoroughly to prepare mixed reference solutions with concentrations of 67.30, 21.06, 63.60, 43.48, and 39.48 μg / mL. Prepare two parallel aliquots of these solutions.

[0067] 2.3 Investigation of the preparation method of the test solution

[0068] 2.3.1 Examination of Extraction Methods

[0069] The leaves and stems of *Lysimachia christinae* (Y-S8) were dried at 50℃, pulverized using a pulverizer, and passed through a No. 2 sieve for traditional Chinese medicine to obtain powder. Four portions, each 1.0 g, were accurately weighed and placed in stoppered conical flasks. 20 mL of 75% methanol aqueous solution was accurately added, the flasks were sealed, and the weights were measured. The flasks were then subjected to reflux for 60 minutes (finally maintained at 80℃) and ultrasonic treatment (power 250W, frequency 40kHz, initial temperature controlled at 30℃) for 60 minutes, respectively. After cooling, the weights were measured again, and the lost weight was replenished with 75% methanol aqueous solution. The mixture was shaken well and filtered through a 0.22 μm microporous membrane. The chromatographic analysis was performed according to the conditions in "2.1," with two parallel determinations. The content of each chemical component was calculated, and the results are shown in Table 3.

[0070] Table 3. Results of different extraction methods for *Flammulina velutipes* (n=2)

[0071]

[0072] As shown in Table 3, reflux yielded better results for the five components obtained by both extraction methods. However, considering that the final results were relatively similar and that ultrasonic extraction was more convenient and safer, ultrasonic extraction was chosen as the subsequent extraction method.

[0073] Content (%) is a percentage by mass.

[0074] 2.3.2 Investigation of Extraction Solvents

[0075] The leaves and stems of *Lysimachia christinae* (Y-S8) were dried at 50℃, pulverized using a pulverizer, and passed through a No. 2 sieve for traditional Chinese medicine to obtain powder. Twelve portions, each 1.0 g, were accurately weighed and placed in stoppered conical flasks. 20 mL each of 25% methanol aqueous solution, 25% ethanol aqueous solution, 50% methanol aqueous solution, 50% ethanol aqueous solution, 75% methanol aqueous solution, 75% ethanol aqueous solution, methanol, and ethanol were accurately added to each flask. The flasks were sealed tightly, weighed, and soaked for 10 min. They were then sonicated (250 W power, 40 kHz frequency, initial temperature controlled at 30℃) for 60 min, cooled, and weighed again. The lost weight was replenished with the appropriate solvent, and the mixture was shaken well. The flasks were filtered through a 0.22 μm microporous membrane. The samples were analyzed under the chromatographic conditions described in "2.1," with two parallel determinations. The content of each chemical component was calculated, and the results are shown in Table 4.

[0076] Table 4 Results of the investigation of different extraction solvents for *Flammulina velutipes* (n=2)

[0077]

[0078] As shown in Table 4, using methanol and ethanol of different concentrations as extraction solvents significantly affected the extraction rates of chemical components with different polarities from *Centella asiatica*. Chlorogenic acid showed the highest extraction yield in a 50%-75% ethanol system, while codonopsis glycoside I, luteolin, and apigenin 7-O-β-D-glucoside all showed the highest extraction yields in a 75% ethanol system. Codonopsis glycoside showed the lowest extraction yield in a 100% ethanol system. Considering all factors, 75% methanol was chosen as the subsequent extraction solvent.

[0079] 2.3.3 Investigation into the amount of extraction solvent used

[0080] The leaves and stems of *Lysimachia christinae* (Y-S8) were dried at 50℃, ground into powder using a pulverizer, and then passed through a No. 2 sieve for traditional Chinese medicine to obtain powder. Eight portions, each 1.0 g, were accurately weighed and placed in stoppered conical flasks. 10 mL, 20 mL, 30 mL, and 50 mL of 75% methanol were accurately added to each flask, respectively. The flasks were then sealed tightly, weighed, and sonicated (250 W, 40 kHz) for 60 minutes. After cooling, the flasks were weighed again. The lost weight was replenished with methanol of the corresponding concentration, and the mixture was shaken well. The flasks were then filtered through a 0.22 μm microporous membrane. The chromatographic analysis was performed under the conditions described in "2.1," with two parallel determinations. The content of each chemical component was calculated, and the results are shown in Table 5.

[0081] Table 5 Results of the investigation of different extraction solvent dosages for *Flammulina velutipes* (n=2)

[0082]

[0083] As shown in Table 5, except for two components with higher content that increased by about 3% after increasing the solvent, the other components showed little difference. Considering factors such as saving solvent, 30 mL of 75% methanol was selected as the subsequent extraction solvent.

[0084] 2.3.4 Examination of extraction time

[0085] The leaves and stems of *Lysimachia christinae* (Y-S8) were dried at 50℃, ground into powder using a pulverizer, and then passed through a No. 2 sieve for traditional Chinese medicine to obtain powder. Six portions, each weighing 1.0 g, were accurately weighed and placed in stoppered conical flasks. 30 mL of 75% methanol was accurately added, the flasks were sealed, and the weights were verified. The flasks were then ultrasonically treated (250 W power, 40 kHz frequency, initial temperature controlled at 30℃) for 30 minutes, 60 minutes, and 90 minutes, respectively. After cooling, the weights were verified again, and the lost weight was replenished with methanol. The mixture was shaken well and filtered through a 0.22 μm microporous membrane. The chromatographic analysis was performed under the conditions described in "2.1," with two parallel determinations. The content of each chemical component was calculated, and the results are shown in Table 6.

[0086] Table 6. Results of the investigation of different extraction times of *Cymbidium goeringii* (n=2)

[0087]

[0088] As shown in Table 6, the content of the five components did not differ significantly at different ultrasonic extraction times. Therefore, considering the timeliness, 30 min was ultimately selected as the subsequent extraction time.

[0089] 2.3.5 Determined method for preparing test solution

[0090] Take samples of *Lysimachia christinae*: dry at 50℃, grind into powder using a pulverizer, and pass through a No. 2 sieve for traditional Chinese medicine to obtain powder. Accurately weigh 1.0g per portion, place in a stoppered conical flask, accurately add 30mL of 75% methanol aqueous solution, weigh, seal tightly, and sonicate (power 250W, frequency 40kHz, initial temperature controlled at 30℃) for 30 minutes. Cool to room temperature (20-25℃), weigh again, replenish the lost weight with 75% methanol aqueous solution, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0091] 2.4 Determination Method

[0092] Samples of leaves, stems, and roots of *Lysimachia christinae* were collected; batch number S14; fruit samples were collected (batch number 20). Test solutions were prepared according to the method described in section "2.3.5". 5 μL of both the reference solution and the test solution were accurately injected into the liquid chromatograph, and the chromatographic conditions described in section "2.1" were applied. The chromatographic peaks and peak areas were recorded, and the contents of the five components were calculated using the external standard method. The results are shown in Table 7.

[0093] Table 7. Chromatogram Information of Mixed Standards and Five Components from Roots, Stems, Leaves, and Fruits

[0094]

[0095] 2.5 Methodological Examination

[0096] 2.5.1 Examination of linearity, limit of quantitation, and limit of detection.

[0097] Accurately pipette the reference solution from section "2.2" and dilute it with methanol to obtain reference solutions of different concentrations. The concentrations of chlorogenic acid were 1.900, 19.000, 190.000, 380.000, 760.000, and 1520.000 μg / mL; the concentrations of codonopsis glycoside I were 0.616, 6.156, 61.563, 123.125, 246.250, and 492.500 μg / mL; and the concentrations of luteolin were 1.650, 16.500, and 82 μg / mL. The concentrations of apigenin 7-O-β-D-glucoside were 1.902, 19.023, 38.045, 76.090, and 152.180 μg / mL, respectively; the concentrations of codonopsis glycoside were 0.410, 8.200, 41.000, 102.500, 164.000, and 410.000 μg / mL, respectively. The samples were injected according to the chromatographic conditions described in section “2.1”. A standard curve was plotted with the concentration of the reference standard (μg / mL) as the abscissa (X) and the peak area of ​​the reference standard (mAu) as the ordinate (Y). The linear regression equation was calculated. The limit of detection (LOD) was the concentration of the compound when the signal-to-noise ratio (S / N) was equal to 3, and the limit of quantitation (LOQ) was the concentration of the compound when the signal-to-noise ratio (S / N) was equal to 10. The results are shown in Table 8. It can be seen that the five chemical components showed good linearity within their respective concentration ranges, with correlation coefficients r ≥ 0.9995. The limits of detection for chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside, and codonopsis glycoside were 0.570, 0.103, 0.050, 0.095, and 0.370 μg / mL, respectively, and the limits of quantitation were 0.950, 0.308, 0.066, 0.171, and 0.518 μg / mL, respectively.

[0098] Table 8. Linearity, Limit of Detection, and Limit of Quantification of Five Chemical Components

[0099]

[0100] 2.5.2 Precision Test

[0101] The mixed reference solution prepared under section "2.2" was injected six times consecutively under the chromatographic conditions under section "2.1", and the RSD of the peak area and retention time of each reference standard was calculated. The results of the precision test are shown in Tables 9 and 10. It can be seen that the RSD of the peak area of ​​the five components is less than 0.41%, and the RSD of the retention time is less than 0.18%, indicating that the method has good precision.

[0102] Table 9. Peak area results of precision tests (n=6)

[0103]

[0104] Table 10 Retention time results of precision test (n=6)

[0105]

[0106] 2.5.3 Stability Test

[0107] A sample of the leaf part of *Lysimachia christinae* (Y-S7) was taken, and a test solution was prepared according to the method described in section "2.3.5". The sample was injected under the chromatographic conditions described in section "2.1", and detected at time points of 0.0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 15.0, 18.0, and 24.0 h. The content of each component and the RSD of the retention time were calculated. The results are shown in Tables 11 and 12. It can be seen that the RSD of the content of all five components is less than 2.80%, and the RSD of the retention time is less than 0.29%, indicating that the test solution prepared by this method has good stability within 24 hours.

[0108] Table 11 Content results from stability tests

[0109]

[0110] Table 12 Retention time results of stability tests

[0111]

[0112] 2.5.4 Repeatability Test

[0113] Six sample solutions (Y-S7) from the leaf parts of *Lysimachia christinae* were prepared in parallel according to the method described in section "2.3.5". The solutions were injected under the chromatographic conditions described in section "2.1", and the RSD of the content and retention time of each component was calculated. The results are shown in Tables 13 and 14. The RSDs of the contents of all five components were less than 2.61%, and the RSDs of the retention times were all less than 0.52%, indicating that the method has good repeatability.

[0114] Table 13 Content results from repeatability tests (n=6)

[0115]

[0116] Table 14 Retention time results of repeatability tests (n=6)

[0117]

[0118] 2.5.5 Recovery Test

[0119] Take about 0.5g of a sample of *Lysimachia christinae* leaf (Y-S7) with known content, and add appropriate amounts of reference standards to each of the five components in the herb according to 100% of their content. Prepare six test solutions in parallel according to the method in section “2.3.5”, and inject them according to the chromatographic conditions in section “2.1”. Determine the analytes according to the method, and calculate the recovery rate and RSD of each analyte. The results are shown in Table 15. The average recovery rate of chlorogenic acid was 99.56%, with an RSD of 0.250%; the average recovery rate of codonopin I was 95.08%, with an RSD of 0.993%; the average recovery rate of luteolin was 98.42%, with an RSD of 0.369%; the average recovery rate of apigenin 7-O-β-D-glucoside was 99.81%, with an RSD of 0.342%; and the average recovery rate of codonopinin was 92.81%, with an RSD of 1.168%. The average recovery rates ranged from 92.81% to 99.81%, and the RSDs ranged from 0.250% to 1.168%, which meets the relevant requirements of the "9101 Analytical Method Validation Guidelines" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, indicating that the method is accurate and feasible.

[0120] Table 15. Experimental results of the recovery rate of five chemical components (n=6)

[0121]

[0122]

[0123] 2.5.6 Durability Test

[0124] (1) Investigation of different chromatographic columns

[0125] Samples of *Lysimachia christinae* leaves (Y-S7) were collected, and test solutions were prepared according to the method described in section "2.3.5". Chromatographic columns were used at different speeds: a Shiseido CAPCELL PAK C18 column (4.6*250mm, 5μm), a Welch Xtimate® C18 column (4.6*250mm, 5μm), and a Waters HSS T3® C18 column (4.6*250mm, 5μm). The chromatographic conditions described in section "2.1" were followed, and the content and RSD of each component were calculated. The results are shown in Table 16. Table 16 shows that the RSD values ​​of each component were less than 4.06% when using different types of columns, indicating that this method has good column robustness.

[0126] Table 16. Content results of *Lysimachia christinae* medicinal material under different chromatographic columns.

[0127]

[0128] (2) Investigation of different column temperatures

[0129] The sample of *Lysimachia christinae* leaves (Y-S7) was prepared according to the method described in section "2.3.5". The column temperature was adjusted to 23, 25, and 27℃ respectively, and the sample was injected according to the chromatographic conditions described in section "2.1". The content and RSD of each component were calculated respectively, and the results are shown in Table 17. As can be seen from Table 17, the RSD values ​​of each component were less than 3.76% when using different column temperatures, indicating that the method has good robustness to column temperature.

[0130] Table 17. Content of *Flammulina velutipes* medicinal material at different column temperatures.

[0131]

[0132] (3) Investigation of different flow velocities

[0133] A sample of *Lysimachia christinae* leaves (Y-S7) was taken, and a test solution was prepared according to the method described in section "2.3.5". The flow rates were adjusted to 0.8, 1.0, and 1.2 mL / min, respectively, and the sample was injected according to the chromatographic conditions described in section "2.1". The content and RSD of each component were calculated, and the results are shown in Table 18. As can be seen from Table 18, the RSD values ​​of each component were less than 1.50% at different flow rates, indicating that the method has good robustness.

[0134] Table 18. Content of *Flammulina velutipes* medicinal material at different flow rates

[0135]

[0136] (4) Investigation using different instruments

[0137] Samples of *Lysimachia christinae* leaves (Y-S7) were taken, and test solutions were prepared according to the method described in section "2.3.5". The samples were injected using a Thermo U3000 high-performance liquid chromatograph and an Aglient 1260 Infinity high-performance liquid chromatograph, respectively, under the chromatographic conditions described in section "2.1". The content and RSD of each component were calculated, and the results are shown in Table 19. Table 19 shows that the RSD values ​​of each component were less than 0.60% when using different instruments, indicating that the method has good robustness.

[0138] Table 19 Content results of medicinal materials under different instruments

[0139]

[0140] 2.6 Content Determination

[0141] Fifteen different batches of *Lysimachia christinae* medicinal materials were grouped according to Table 1. Test solutions were prepared according to the method in section “2.3.5”, and the samples were tested under the chromatographic conditions in section “2.1”. The tests were performed in parallel twice, and the contents of five chemical components were calculated using the external standard method. The results are shown in Table 20.

[0142] Table 20. Results of content determination of various parts of 15 batches of medicinal materials.

[0143]

[0144]

[0145] "-" indicates that the content is below the detection limit; it also indicates that the corresponding part does not contain the component.

[0146] Comparative Example 1

[0147] Take the R-S2 sample of *Fuchsia argentea*: Prepare the test solution according to the method in section “2.3.5”, and perform detection according to the following chromatographic conditions and the aforementioned chromatographic conditions in section “2.1”:

[0148] Chromatographic conditions: Pntulips BP-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile-methanol-0.1% phosphoric acid, gradient elution, elution table below; flow rate: 1.0 mg / mL; detection wavelength: 340 nm; column temperature: 25 ℃; injection volume: 10 μL. Elution program is shown in Table 21.

[0149] Table 21

[0150]

[0151] The results showed that, compared with the chromatographic conditions described in “2.1” of this application, the above chromatographic conditions, using 340 nm, could not detect the peaks of codonopsis glycoside I, codonopsis glycoside, and apigenin-7-O-β-D-glucoside. Furthermore, luteolin and the unidentifiable peaks overlapped and could not be separated.

[0152] Comparative Example 2

[0153] Take leaves of *Lysimachia christinae* Y-S10 and prepare a test solution according to the method in section “2.3.5”. Detect the solution under the following chromatographic conditions and the chromatographic conditions in section “2.1”, and perform two parallel determinations.

[0154] Chromatographic column: Shiseido MG II BP-C18 (4.6 mm × 250 mm, 5 μm); column temperature: 25℃; flow rate: 0.8 mL / min; detection wavelength: 267 nm, 340 nm (chlorogenic acid); injection volume: 5 μL; mobile phase conditions: 0.2% phosphoric acid aqueous solution (A) - methanol (B) - acetonitrile (C); gradient elution, the elution program is shown in Table 22.

[0155] Table 22

[0156]

[0157] The results showed that, compared with the chromatographic conditions described in "2.1" of this application, the structures obtained under the above chromatographic conditions differed in the detection results.

[0158] The peak of codonopsis glycoside could not be detected at 267 nm; at 340 nm, there was a significant interference peak after the chlorogenic acid peak at 8.075 minutes, and the peaks were similar in UV light and could not be separated.

[0159] Comparative Example 3

[0160] Take the stem of *Callicarpa japonica* J-S10 and prepare the test solution according to the method in section “2.3.5”. Detect the solution according to the chromatographic conditions shown below and the chromatographic conditions in section “2.1”, and perform two parallel determinations.

[0161] Chromatographic column: Shiseido MG II BP-C18 (4.6 mm × 250 mm, 5 μm); column temperature: 25℃; flow rate: 0.8 mL / min; detection wavelength: 267 nm, 340 nm (chlorogenic acid); injection volume: 5 μL; mobile phase conditions: 0.2% phosphoric acid aqueous solution (A) - methanol (B) - acetonitrile (C); gradient elution, the elution program is shown in Table 23.

[0162] Table 23

[0163]

[0164] The results showed that, compared with the chromatographic conditions described in "2.1" of this application, the above chromatographic conditions resulted in the following: a significant interfering peak preceded the ginsenoside peak at 267 nm for 25.655 minutes, with similar UV absorption, making separation impossible; a significant interfering peak followed the chlorogenic acid peak at 340 nm for 8.075 minutes, with similar UV absorption, also making separation impossible; and the luteolin peak showed low UV purity and interfering substances at the same time.

[0165] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A method for detecting the content of chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside and codonopsis glycoside in *Cyclocarya paliurus*, characterized in that, It includes the following steps: High-performance liquid chromatography (HPLC) is used to detect the *Flammulina* sample: In chromatographic conditions: The chromatographic column is C10. 18 Chromatographic column; Mobile phase: Gradient elution of 0.2% phosphoric acid aqueous solution-methanol-acetonitrile: 。 2. The detection method according to claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The sample of *Cyclocarya paliurus* to be tested is the root, stem, leaf or fruit of *Cyclocarya paliurus*; (2) The chromatographic column is a Shiseido MG II BP-C18 column, a Shiseido CAPCELL PAK C18 column, a Welch Xtimate® C18 column or a Waters HSS T3® C18 column; (3) The length of the chromatographic column is 200-300 mm; (4) The inner diameter of the chromatographic column is 4.6 mm; (5) The particle size of the packing material in the chromatographic column is 4-6 µm; (6) The column temperature of the chromatographic column is 20-35℃; (7) In the high performance liquid chromatography method, the detection wavelength is 200-360 nm; (8) In the high performance liquid chromatography method described above, the injection volume is 3-8 μL; (9) The flow rate of the mobile phase is 0.6-1.0 mL / min; (10) Before using high performance liquid chromatography to detect the sample of *Callicarpa spp.*, the sample of *Callicarpa spp.* is pretreated, which includes the following steps: extracting the sample of *Callicarpa spp.* in a solvent.

3. The detection method according to claim 2, characterized in that, It satisfies one or more of the following conditions: (1) The sample of *Cyclocarya paliurus* to be tested is the leaf, stem or root of *Cyclocarya paliurus*; (2) The chromatographic column is a Shiseido MG II BP-C18 column; (3) The length of the chromatographic column is 250 mm; (4) The particle size of the packing material in the chromatographic column is 5µm; (5) The column temperature of the chromatographic column is 25-35℃; (6) In the high performance liquid chromatography method, the detection wavelength is 245-360 nm; (7) In the high performance liquid chromatography method, the injection volume is 5 μL; (8) The flow rate of the mobile phase is 0.8 mL / min; (9) The extraction method is either reflux extraction or ultrasonic extraction. (10) The solvent is an alcohol solvent and / or water; Before extraction as described in (11), the sample of *Cyclocarya paliurus* to be tested needs to be made into powder to obtain *Cyclocarya paliurus* sample powder.

4. The detection method according to claim 3, characterized in that, It satisfies one or more of the following conditions: (1) The sample of *Flammulina* to be tested is the leaf or stem of *Flammulina*; (2) The column temperature of the chromatographic column is 25℃ or 27℃; (3) In the high performance liquid chromatography method, the detection wavelength is 267 nm or 340 nm; (4) When the test sample of the flower is the root of the flower, the components detected are chlorogenic acid, codonopsis glycoside I, luteolin and codonopsis glycoside or chlorogenic acid, luteolin and codonopsis glycoside. (5) The specifications of the chromatographic column are: length 250 mm, inner diameter 4.6 mm, and packing particle size 5 µm; (6) The extraction method is ultrasonic extraction; The solvents mentioned in (7) are alcohol solvents and water.

5. The detection method according to claim 4, characterized in that, It satisfies one or more of the following conditions: (1) The alcohol solvent is methanol and / or ethanol; (2) When the test sample of the flower is the fruit of the flower, the components detected are chlorogenic acid, codonopsis glycoside I, luteolin and codonopsis glycoside. The preparation method of the test sample powder of *Cymbidium goeringii* described in (3) is to dry, grind, and then sieve.

6. The detection method according to claim 5, characterized in that, It satisfies one or more of the following conditions: (1) The solvent is 25% methanol aqueous solution, 25% ethanol aqueous solution, 50% methanol aqueous solution, 50% ethanol aqueous solution, 75% methanol aqueous solution, 75% ethanol aqueous solution, methanol or ethanol; (2) The drying temperature is 40-60℃; (3) The grinding is done using a grinding machine; (4) The mesh size of the sieve is 10-50 mesh; (5) The temperature of the reflux method is 60-100℃; (6) The reflux time is 20-120 min; (7) The temperature of the ultrasonic extraction method is 20-50℃; (8) The time for the ultrasonic extraction method is 20-120 min; (9) In the ultrasonic extraction method, the mass-to-volume ratio of *Cymbidium goeringii* to solvent is 1:(5-60) g / mL. (10) The pretreatment also includes the following post-treatment steps: cooling and filtration.

7. The detection method according to claim 6, characterized in that, It satisfies one or more of the following conditions: (1) The solvent is a 75% methanol aqueous solution; (2) The drying temperature is 50℃; (3) The sieve mesh size is 24 mesh; (4) The temperature of the reflux method is 80℃; (5) The reflux time is 60 min; (6) The temperature of the ultrasonic extraction method is 30℃; (7) The time for the ultrasonic extraction method is 30 min, 60 min or 90 min; (8) In the ultrasonic extraction method, the mass-to-volume ratio of *Cymbidium goeringii* to solvent is 1:10 g / mL, 1:20 g / mL, 1:30 g / mL or 1:50 g / mL; (9) The filtration is performed using a 0.15-0.30 μm microporous membrane; (10) The cooling is to cool to 15-30℃; (11) The detection method further includes the preparation of a reference solution: chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside, and codonopsis glycoside are mixed in a solvent to prepare a reference solution; (12) The high performance liquid chromatograph used in the high performance liquid chromatography method is a Thermo U3000 high performance liquid chromatograph or an Aglient 1260 Infinity high performance liquid chromatograph; (13) The place of origin of the bellflower is Guizhou Province, Yunnan Province, Sichuan Province or Guangxi Zhuang Autonomous Region.

8. The detection method according to claim 7, characterized in that, It satisfies one or more of the following conditions: (1) The cooling process further includes adding solvent to replenish the weight lost during extraction; the solvent is any one of claims 2-7; (2) The sieving process is the No. 2 sieve for Chinese medicine; (3) In the ultrasonic extraction method, the mass-to-volume ratio of *Cymbidium goeringii* to solvent is 1:30 g / mL; (4) The filtration is performed using a 0.22μm microporous membrane; (5) The cooling refers to cooling to 20-25℃; (5) In the preparation of the reference solution, the solvent is methanol; The places of origin of the bellflower mentioned in (6) are Wengyan Village, Taiping Township, Wanshan District, Tongren City, Guizhou Province; Wenshan City, Yunnan Province; Jinghe Village, Dewang District, Jiangkou County, Tongren City, Guizhou Province; Miaowan Village, Huangban Town, Songtao Miao Autonomous County, Tongren City, Guizhou Province; Xiema, Jiajiang County, Leshan City, Sichuan Province; Hongya County, Meishan City, Sichuan Province; Lvchun County, Honghe City, Yunnan Province; Malipo County, Wenshan City, Yunnan Province; Jinxiu Yao Autonomous County, Laibin City, Guangxi Zhuang Autonomous Region; Wanshan District, Tongren City, Guizhou Province; or Maguan County, Wenshan City, Yunnan Province.

9. The detection method according to claim 8, characterized in that, The detection method involves using high performance liquid chromatography to detect the reference standard as described in claim 7 or 8 and the sample of *Cyclocarya paliurus* as described in any one of claims 1-8; then, the contents of chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside and codonopsis glycoside in *Cyclocarya paliurus* as described in any one of claims 1-8 are calculated by the external standard method.

10. The application of a method for detecting the content of chlorogenic acid, codonopsis glycoside I, luteolin, apigenin 7-O-β-D-glucoside and codonopsis glycoside in *Cyclocarya paliurus* in the quality control of *Cyclocarya paliurus* or its preparations; the detection method is as described in any one of claims 1-9.

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