Preparation method of small blood vine extract and detection method thereof

Abstract

This invention provides a method for preparing and detecting an extract of *Sargentodoxa cuneata*. The method for preparing the extract includes the following steps: (1) taking *Sargentodoxa cuneata* medicinal material, adding water and decocting, then separating the solid and liquid to obtain a filtrate; (2) concentrating and drying the filtrate from step (1), and then freeze-drying it. The freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. primary drying: the drying temperature is -45℃ to 0℃, and the vacuum degree is -0.2 to 0 mbar; c. secondary drying: the drying temperature is 5 to 25℃, and the vacuum degree is -0.1 to 0.1 mbar, to obtain the *Sargentodoxa cuneata* extract. The characteristic chromatographic detection method provided by this invention uses ultra-high performance liquid chromatography (UHPLC), which is simple, stable, highly precise, and reproducible. Furthermore, the obtained fingerprint spectrum has many peaks, good peak shapes, is easy to identify, and is accurate and reliable.

Description

Technical Field

[0001] The present invention relates to the field of modernization of traditional Chinese medicine, and particularly to a preparation method and a detection method of an extract of Rubia alata Roxb. Background Art

[0002] Rubia alata Roxb is the dried root and rhizome of Rubia alata Roxb of the family Rubiaceae. Its main effects are cooling blood for hemostasis, relieving cough and resolving phlegm, and dredging channels. It is used for hematemesis, epistaxis, metrorrhagia and metrostaxis, amenorrhea, jaundice, bronchitis, and traumatic injury. It is mainly distributed in the southwestern regions such as Yunnan, Sichuan, Guizhou, etc., and Rubia alata Roxb is widely distributed in Guizhou. Pharmacological studies have shown that it has effects such as protecting nerves, anti-inflammatory immunity, and depolymerizing platelet aggregation. A standard decoction, also known as a standard decoction liquid, is a traditional form of medication widely used clinically. The standard decoction follows the theory of traditional Chinese medicine and is standardizedly decocted according to the clinical decoction method, with solid-liquid separation, and is prepared by appropriate concentration or dried by a suitable method, serving as a standard reference object to measure whether traditional Chinese medicine formula granules are basically consistent with clinical decoctions.

[0003] Since the standard decoction is a "bridge" connecting traditional Chinese medicine decoction pieces and modern Chinese medicine preparations, providing a reference object for controlling the quality of Chinese medicine end products, standardizing different dosage forms of Chinese medicine to ensure the uniformity of quality and the consistency of efficacy, and providing a reference object for evaluating the consistency of product quality of different manufacturers, therefore, the formulation of the quality standard of Chinese medicine standard decoctions will provide a basis for the formulation of the quality standards of all end products of water decoctions derived from decoction pieces.

[0004] At present, the medicinal material standard of Rubia alata Roxb is only included in the local Chinese medicinal material standard of "Quality Standards of Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province" (2003 Edition), and there is no national medicinal material standard, and the quality control is still imperfect. Only the characters and microscopic identification are established, and there is no report on the quality control means of relevant content determination in the relevant literature reports, which is relatively crude and restricts its application and development. Summary of the Invention

[0005] Aiming at the problems in the prior art, the present invention provides a preparation method and a detection method of a standard decoction of Rubia alata Roxb to standardize the quality control and standard research of traditional Chinese medicine formula granules, realize the overall quality control and effective supervision of traditional Chinese medicine formula granules, and provide a reference for the quality control of Rubia alata Roxb formula granules.

[0006] The present invention provides a preparation method of an extract of Rubia alata Roxb, comprising the following steps:

[0007] (1) Take the medicinal material of Rubia alata Roxb, decoct with water, and perform solid-liquid separation to obtain a filtrate;

[0008] (2) The filtrate from step (1) is concentrated and dried, and then freeze-dried. The freeze-drying is divided into three stages: a. Pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. First drying: the drying temperature is -45℃ to 0℃, and the vacuum degree is -0.2 to 0mbar; c. Second drying: the drying temperature is 5 to 25℃, and the vacuum degree is -0.1 to 0.1mbar, to obtain the small blood vine extract.

[0009] Preferably, in step (1), the number of times water is added and boiled is 1-3 times;

[0010] Preferably, the boiling process is carried out twice;

[0011] Preferably, the mass ratio of the small blood vine medicinal material to water when decocting is 1:6-8.

[0012] Preferably, the boiling time is 40-60 minutes each time water is added.

[0013] Preferably, in step (2), the concentration and drying temperature is 50-65℃;

[0014] Preferably, the pre-freezing time is 150-200 minutes, and more preferably 180 minutes;

[0015] Preferably, the drying time is 2000-2500 minutes, more preferably 2340 minutes.

[0016] Preferably, the secondary drying time is 350-400 minutes, and more preferably 360 minutes.

[0017] Preferably, the yield of the *Sargentodoxa cuneata* extract is 8.1% to 21.1%.

[0018] This invention also provides a method for detecting the mass content of chlorogenic acid and / or cryptochlorogenic acid in the *Sargentodoxa cuneata* extract obtained by the above preparation method, comprising the following steps:

[0019] (1) Preparation of reference solution:

[0020] Chlorogenic acid and cryptochlorogenic acid were used as reference standards and a solution was prepared by adding methanol.

[0021] (2) Preparation of the test solution:

[0022] The *Sargentodoxa cuneata* extract obtained by the preparation method described above was extracted with a solvent.

[0023] (3) Ultra-high performance liquid chromatography analysis

[0024] The reference solution and the test solution were injected into an ultra-high performance liquid chromatograph. Octadecylsilane-bonded silica gel was used as the stationary phase, the detection wavelength was 325 nm, acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid aqueous solution was used as the mobile phase B for gradient elution to obtain the contents of chlorogenic acid 100 and cryptochlorogenic acid.

[0025] Preferably, in step (2), the solvent is selected from water, 50% ethanol, 75% ethanol, ethanol, 50% methanol, 75% methanol, and methanol. Preferably, the solvent is 50% methanol.

[0026] Preferably, in step (2), the extraction is performed using either ultrasonic extraction or reflux extraction, with ultrasonic extraction being preferred.

[0027] Preferably, the extraction time is 15-60 min, and more preferably 15 min;

[0028] Preferably, the flow rates of mobile phase A and mobile phase B are 0.20-0.30 ml / min, more preferably 0.25 ml / min.

[0029] Preferably, in step (3), the gradient elution procedure is as follows:

[0030] At 0 min, the volume percentage of mobile phase A was 1%, and the volume percentage of mobile phase B was 99%.

[0031] After 4 minutes, the volume percentage of mobile phase A was 6%, and the volume percentage of mobile phase B was 94%.

[0032] After 10 minutes, the volume percentage of mobile phase A was 9%, and the volume percentage of mobile phase B was 91%.

[0033] After 16 minutes, the volume percentage of mobile phase A was 18%, and the volume percentage of mobile phase B was 82%.

[0034] After 22 minutes, the volume percentage of mobile phase A was 20%, and the volume percentage of mobile phase B was 80%.

[0035] After 23 minutes, the volume percentage of mobile phase A was 90%, and the volume percentage of mobile phase B was 10%.

[0036] At 25.1 min, the volume percentage of mobile phase A was 1%, and the volume percentage of mobile phase B was 99%.

[0037] After 27 minutes, the volume percentage of mobile phase A was 1%, and the volume percentage of mobile phase B was 99%.

[0038] Preferably, the total mass content of chlorogenic acid and cryptochlorogenic acid in the *Sargentodoxa cuneata* extract prepared by the method is 1.01% to 6.37% (based on dried product).

[0039] Preferably, the total transfer rate of chlorogenic acid and cryptochlorogenic acid in the *Sargentodoxa cuneata* extract prepared by the method is 15.07% to 69.18%.

[0040] This invention also provides a method for detecting the characteristic spectrum of the *Sargentodoxa cuneata* extract obtained by the above preparation method, comprising the following steps:

[0041] (1) Preparation of reference solution:

[0042] Take the reference herb *Sargentodoxa cuneata* and prepare a solution with methanol;

[0043] (2) Preparation of the test solution:

[0044] The *Sargentodoxa cuneata* extract obtained by the preparation method described above was extracted with a solvent.

[0045] (3) Ultra-high performance liquid chromatography analysis

[0046] The reference solution and the test solution were injected into an ultra-high performance liquid chromatograph, and gradient elution was performed with octadecylsilane-bonded silica gel as the stationary phase to obtain the characteristic chromatogram of the small vine extract.

[0047] Preferably, in step (2), the solvent is selected from water, 95% ethanol, 75% ethanol, dilute ethanol, methanol, 75% methanol, and 50% methanol. Preferably, the solvent is 75% methanol.

[0048] Preferably, in step (2), the extraction is performed using one of reflux extraction, ultrasonic extraction, or dissolution extraction.

[0049] Preferably, the extraction is performed using ultrasonic extraction.

[0050] Preferably, the extraction time is 15-45 minutes.

[0051] Preferably, the extraction time is 15 minutes.

[0052] Preferably, in step (3), the detection wavelength is 190–400 nm, and more preferably 245 nm;

[0053] Preferably, the gradient elution procedure is as follows:

[0054] From 0 to 10 min, the volume percentage of mobile phase A changed from 6% to 8.5%, and the volume percentage of mobile phase B changed from 94% to 91.5%.

[0055] After 10-15 minutes, the volume percentage of mobile phase A changed from 8.5% to 10%, and the volume percentage of mobile phase B changed from 91.5% to 90%.

[0056] Over 15-18 minutes, the volume percentage of mobile phase A changed from 10% to 11%, and the volume percentage of mobile phase B changed from 90% to 89%.

[0057] After 18-19 minutes, the volume percentage of mobile phase A was 11%, and the volume percentage of mobile phase B was 89%.

[0058] From 19 to 31 minutes, the volume percentage of mobile phase A changed from 11% to 15%, and the volume percentage of mobile phase B changed from 89% to 85%.

[0059] Over 31-35 minutes, the volume percentage of mobile phase A changed from 15% to 18%, and the volume percentage of mobile phase B changed from 85% to 82%.

[0060] 35-35.1 min, the volume percentage of mobile phase A is 18→6%, and the volume percentage of mobile phase B is 82→94%;

[0061] From 35.1 to 37 min, the volume percentage of mobile phase A was 6%, and the volume percentage of mobile phase B was 94%.

[0062] Preferably, in step (3), the mobile phase comprises an organic phase A and an aqueous phase B.

[0063] Preferably, the organic phase A is selected from one or more combinations of acetonitrile or methanol, with acetonitrile being the preferred choice.

[0064] Preferably, the aqueous phase B is selected from one or more combinations of water, 0.05% formic acid solution, 0.1% formic acid solution, 0.1% acetic acid solution, or 0.05% phosphoric acid solution.

[0065] Preferably, the mobile phase is an acetonitrile-0.1% formic acid solution;

[0066] Preferably, the flow rate of the mobile phase is 0.20-0.30 ml / min, and more preferably 0.25 ml / min.

[0067] Preferably, the column temperature in step (3) is 30-40℃, and more preferably, the column temperature is 35℃.

[0068] Preferably, the characteristic spectrum has no less than 5 characteristic peaks, and the peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of a specified value, which includes 0.33, 0.38, 1.10, and 3.37.

[0069] The characteristic chromatographic detection method provided by this invention employs ultra-high performance liquid chromatography (UHPLC), which is simple, stable, precise, and reproducible. Furthermore, the obtained fingerprint chromatograms exhibit numerous peaks with good peak shapes, making them easy to identify and ensuring accuracy and reliability. This method can provide a quality control tool for the quality evaluation of *Sargentodoxa cuneata* extract.

[0070] In this invention, the total amount of chlorogenic acid and cryptochlorogenic acid in the extract of *Sargentodoxa cuneata* is determined by high performance liquid chromatography as an indicator, and an internal control standard for the content of the extract is established to provide a quality control method for *Sargentodoxa cuneata*. Attached Figure Description

[0071] Figure 1 The image shown is a DAD-3D diagram of the extract of *Sargentodoxa cuneata*.

[0072] Figure 2A The image shows the UPLC spectrum of the acetonitrile-0.05% phosphoric acid solution mobile phase system.

[0073] Figure 2B The figure shows the UPLC spectrum of the methanol-0.05% phosphoric acid solution mobile phase system.

[0074] Figure 3A The image shows the characteristic spectrum of *Sargentodoxa cuneata* with acetonitrile-water as the mobile phase.

[0075] Figure 3B The image shows the characteristic spectrum of *Sargentodoxa cuneata* with acetonitrile-0.05% phosphoric acid solution as the mobile phase.

[0076] Figure 3C The image shows the characteristic spectrum of *Sargentodoxa cuneata* with acetonitrile-0.1% formic acid solution as the mobile phase.

[0077] Figure 3D The image shows the characteristic spectrum of *Sargentodoxa cuneata* when the mobile phase is acetonitrile-0.1% acetic acid solution;

[0078] Figure 4A The figure shows the UPLC spectrum when water was used as the extraction solvent;

[0079] Figure 4B The figure shows the UPLC spectrum of the extraction solvent using 95% ethanol.

[0080] Figure 4C The figure shows the UPLC spectrum of the extraction solvent using 75% ethanol.

[0081] Figure 4D The figure shown is the UPLC spectrum of the extraction solvent using dilute ethanol.

[0082] Figure 4E The figure shows the UPLC spectrum of methanol as the extraction solvent.

[0083] Figure 4FThe figure shows the UPLC spectrum of the extraction solvent using 75% methanol.

[0084] Figure 4G The figure shows the UPLC spectrum of the extraction solvent using 50% methanol.

[0085] Figure 5 The image shown is a comparison of the extraction efficiency of different extraction solvents for the characteristic spectrum of *Sargentodoxa cuneata* extract.

[0086] Figure 6A The image shown is the UPLC spectrum of the ultrasonic extraction method.

[0087] Figure 6B The image shown is the UPLC spectrum examined using the reflux extraction method;

[0088] Figure 7 The image shown is a comparison of the extraction efficiency of different extraction methods for the characteristic spectra of *Sargentodoxa cuneata* extract.

[0089] Figure 8A The image shown is the UPLC spectrum after 15 minutes of ultrasonic extraction.

[0090] Figure 8B The image shown is the UPLC spectrum after 30 minutes of ultrasonic extraction.

[0091] Figure 8C The image shown is the UPLC spectrum after 45 minutes of ultrasonic extraction.

[0092] Figure 9 The figure shown is a comparison of extraction efficiency (peak area / sample weight) at different extraction times;

[0093] Figure 10A The image shown is a specificity test result of the characteristic chromatogram of *Sargentodoxa cuneata* extract using a blank solvent.

[0094] Figure 10B The figure shown is a specificity test chart of the characteristic spectrum of the extract of *Sargentodoxa cuneata* using the test sample solution;

[0095] Figure 11 The image shown is a comprehensive analysis of the characteristic spectra of *Sargentodoxa cuneata* extract.

[0096] Figure 12A The image shows the UPLC chromatogram obtained using a Dikma C18 column.

[0097] Figure 12B The image shows the UPLC chromatogram obtained using a Shim-pack GISS C18 column.

[0098] Figure 12C The image shown is of ACQUITY UPLC. UPLC chromatograms were examined using a T3 column.

[0099] Figure 13A The image shown is the UPLC spectrum observed at a column temperature of 25℃.

[0100] Figure 13B The image shown is the UPLC spectrum observed at a column temperature of 30℃.

[0101] Figure 13C The image shown is the UPLC spectrum observed at a column temperature of 35℃.

[0102] Figure 14A The UPLC chromatogram is shown at a flow rate of 0.25 ml / min.

[0103] Figure 14B The UPLC chromatogram is shown at a flow rate of 0.30 ml / min.

[0104] Figure 14C The UPLC chromatogram is shown at a flow rate of 0.35 ml / min.

[0105] Figure 15 The image shows the characteristic spectrum of the extract of *Sargentodoxa cuneata*.

[0106] Figure 16A The image shown is a spectrum obtained from a specificity study using 50% methanol.

[0107] Figure 16B The figure shown is a specificity test chromatogram using a reference solution.

[0108] Figure 16C The image shown is a chromatogram for specificity testing of the test sample solution.

[0109] Figure 17A The figure shown is a chromatogram of the peak purity of the reference solution;

[0110] Figure 17B The figure shown is a chromatogram of the peak purity of the test sample solution;

[0111] Figure 18 The graph shown is a linear regression equation for cryptochlorogenic acid.

[0112] Figure 19 Linear regression equation for cryptochlorogenic acid;

[0113] Figure 20A The figure shows the effect of the Agilent C18 column on the determination of the target component in the extract of *Sargentodoxa cuneata*.

[0114] Figure 20B The effect of the Waters C18 column on the determination of the target component in the extract of *Sargentodoxa cuneata* is shown.

[0115] Figure 20CThe effect of the Ecosil C18 column on the determination of the target component in the extract of *Sargentodoxa cuneata* is shown.

[0116] Figure 21A The image shows the effect of the Waters H-Class Plus chromatograph on the determination of the target component content in *Sargentodoxa cuneata* extract.

[0117] Figure 21B The image shows the effect of the Agilent UPLC 1290II chromatograph on the determination of the target component content in the extract of *Sargentodoxa cuneata*.

[0118] Figure 22A The figure shows the effect of column temperature at 25℃ on the determination of the target component content in *Sargentodoxa cuneata* extract;

[0119] Figure 22B The figure shows the effect of column temperature of 30℃ on the determination of the content of target components in the extract of *Sargentodoxa cuneata*.

[0120] Figure 22C The figure shows the effect of column temperature of 35℃ on the determination of the content of target components in the extract of *Sargentodoxa cuneata*.

[0121] Figure 23A The effect of a flow rate of 0.20 ml / min on the determination of the target component in the extract of *Sargentodoxa cuneata* is shown. Figure 23B The effect of a flow rate of 0.25 ml / min on the determination of the target component in the extract of *Sargentodoxa cuneata* is shown.

[0122] Figure 23C The effect of a flow rate of 0.30 ml / min on the determination of the target component in the extract of *Sargentodoxa cuneata* is shown.

[0123] Figure 24 The image shown is an overlay of the characteristic spectra of the extract of *Sargentodoxa cuneata* (S1 is the reference herb). Detailed Implementation

[0124] The term "dilute ethanol" is prepared using the test solution in Appendix XVB of the Chinese Pharmacopoeia, which involves taking 529 ml of ethanol and diluting it with water to 1000 ml. This solution should contain 49.5%–50.5% (ml / ml) of C2H5OH at 20°C.

[0125] Example 1

[0126] I. Preparation method of small bloodvine extract

[0127] (3) Preparation method

[0128] Take 100g of *Smilax china* slices (batch number YP2105-1), place them in an electric ceramic kettle, add water, and decoct twice. For the first decoction, add 8 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 60 minutes. Filter the decoction through a 300-mesh sieve while hot. For the second decoction, add 6 times the amount of water, bring to a boil over high heat, then simmer over low heat for 40 minutes. Filter the decoction through a 300-mesh sieve while hot. Combine the two decoctions.

[0129] The decoction was transferred to a 2000ml round-bottom flask and concentrated under reduced pressure using a rotary evaporator (temperature: 65℃; vacuum: -0.080~-0.090MPa) to 100ml. Under magnetic stirring, the concentrate was dispensed into 10ml amber vials, each 1ml, half-stopped, and then transferred to a vacuum freeze dryer for lyophilization. The lyophilization parameters were as follows: pre-freezing temperature of the concentrated *Sargentodoxa cuneata* extract was -50℃, pre-freezing time was 180 minutes; sublimation drying temperature was -45℃~0℃, sublimation drying time was 2340 minutes, and vacuum was -0.2mbar; desorption drying temperature was 10℃~30℃, desorption drying time was 360 minutes, and vacuum was 0mbar. The extract was then removed, capped with an aluminum cap, yielding the *Sargentodoxa cuneata* extract with batch number BT2105-1.

[0130] II. Characteristic chromatographic detection of *Sargentodoxa cuneata* extract

[0131] Establishment of Feature Map Analysis Method

[0132] 1. Instruments and reagents

[0133] Instruments: Waters ACQUITY / H-Class ultra-high performance liquid chromatograph; KQ-500DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); HH-4 digital display constant temperature water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.); 0.001% electronic balance (model: ml204).

[0134] Reagents: Acetonitrile was of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0135] Test drug: *Sargentodoxa cuneata* reference material (Zhuhai Anzhe Biotechnology Co., Ltd., XXT210426-01). Test sample: *Sargentodoxa cuneata* extract with batch number BT2105-1.

[0136] 2. Preparation of reference solution

[0137] Take about 1g of the reference herb *Sargentodoxa cuneata*, weigh it accurately, place it in a stoppered conical flask, add 25ml of 75% methanol accurately, weigh it, sonicate it (power 500W, frequency 40kHz) for 15 minutes, cool it, weigh it again, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the final product.

[0138] 3. Determination of chromatographic conditions

[0139] (1) Determination of detection wavelength

[0140] The DAD-3D diagram of the *Sargentodoxa cuneata* extract was analyzed, and the absorption spectrum in the range of 190–400 nm was recorded. The results are as follows: Figure 1 As shown.

[0141] like Figure 1 The experimental results show that the test solution of *Spatholobus suberectus* extract has the most detectable chromatographic peaks at a wavelength of 245 nm, with a stable baseline and good response of each chromatographic peak. Therefore, 245 nm was selected as the detection wavelength.

[0142] (2) Optimization of the mobile phase

[0143] ① Consider A as the organic phase and B as the aqueous phase. Acetonitrile-0.05% phosphoric acid solution and methanol-0.05% phosphoric acid solution were used as mobile phases, respectively, with gradients shown in Table 1 below.

[0144] Table 1

[0145]

[0146]

[0147] Experimental results are as follows Figure 2A and Figure 2B As shown, the elution power of acetonitrile-0.05% formic acid solution is stronger than that of methanol-0.05% phosphoric acid solution. Therefore, acetonitrile-0.05% phosphoric acid solution was chosen to explore the conditions. The various acids used in the mobile phase will be investigated later.

[0148] ② Examine the cases with and without acid.

[0149] Acetonitrile-water, acetonitrile-0.05% phosphoric acid solution, acetonitrile-0.1% formic acid solution, and acetonitrile-0.1% acetic acid solution were used as mobile phases, and elution was performed using the gradients shown in Table 2. The results are as follows: Figures 3A-3D As shown.

[0150] like Figures 3A-3D The experimental results show that the peak shape is better when acid is added than when no acid is added, and the peak shape is best when the mobile phase is acetonitrile-0.1% formic acid solution. Therefore, acetonitrile-0.1% formic acid solution is selected for elution.

[0151] (3) Determination of chromatographic conditions

[0152] The column was packed with octadecylsilane-bonded silica gel (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A and 0.1% formic acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.25 ml per minute; the column temperature was 35 °C; and the detection wavelength was 245 nm.

[0153] Table 2

[0154]

[0155] 4. Preparation of the test solution

[0156] (1) Investigation of different extraction solvents

[0157] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of *Sargentodoxa cuneata* extract. Water, ethanol, 75% ethanol, dilute ethanol, methanol, 75% methanol, and 50% methanol were selected as extraction solvents. The characteristic chromatograms of five tentatively determined chromatographic peaks were compared using the total peak area / sample weight and chromatograms. The results are as follows: Figures 4A-4G As shown in Table 3, Figure 5 The image shown is a comparison of the extraction efficiency of different extraction solvents for the characteristic spectrum of *Sargentodoxa cuneata* extract.

[0158] Table 3 Comparison of extraction efficiency of different extraction methods (peak area / sample volume)

[0159]

[0160] like Figures 4A-4G , Figure 5 As shown in Table 3, the extraction efficiency is higher when the extraction solvent is 75% methanol. Therefore, 75% methanol was selected as the extraction solvent.

[0161] (2) Examination of extraction methods

[0162] This experiment investigated the effects of different extraction methods on the characteristic chromatograms of *Sargentodoxa cuneata* extract. Ultrasonic extraction and reflux were selected as extraction methods. The characteristic chromatograms of different extraction methods were compared using the total peak area / sample weight of five tentatively determined chromatographic peaks and the chromatograms. The results are as follows: Figure 6A , Figure 6B , Figure 7 As shown in Table 4.

[0163] Table 4 Comparison of extraction efficiency of different extraction methods (peak area / sample volume)

[0164]

[0165] like Figure 6A , Figure 6B , Figure 7As shown in Table 4, the results of ultrasonic extraction and reflux extraction are not significantly different, so ultrasonic extraction was chosen.

[0166] (3) Examination of extraction time

[0167] This experiment investigated the effects of different extraction times on the characteristic chromatograms of *Sargentodoxa cuneata* extracts. Three different extraction times—15 minutes, 30 minutes, and 45 minutes—were selected. The characteristic chromatograms of the five tentatively determined peak areas / sample weights were compared using chromatograms. The results are as follows: Figures 8A-8C , Figure 9 As shown in Table 5.

[0168] Table 5 Comparison of extraction efficiency at different ultrasonic extraction times (peak area / sample volume)

[0169]

[0170] like Figures 8A-8C , Figure 9 As shown in Table 5, ultrasound for 15 minutes is optimal. The extraction efficiency is high when the extraction time is 15 minutes; therefore, 15 minutes was chosen as the extraction time.

[0171] Based on the above experimental results, the sample pretreatment method for the characteristic spectrum of *Sargentodoxa cuneata* extract can be determined as follows: Take about 1g of *Sargentodoxa cuneata* extract, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 15 minutes, cool it, weigh it again, replenish the lost weight with 75% methanol, shake it well, filter it, and take the filtrate to obtain the extract.

[0172] 5. Characteristic spectral detection method for extracts of *Sargentodoxa cuneata*

[0173] In summary, a method for detecting the characteristic spectra of *Sargentodoxa cuneata* extract is presented, as follows:

[0174] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 6; the flow rate was 0.25 ml per minute; the column temperature was 35℃; and the detection wavelength was 245 nm.

[0175] Table 6

[0176]

[0177] Preparation of reference solution: Take about 1.0g of *Sargentodoxa cuneata* reference material, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, weigh it, sonicate (power 500W) for 15 minutes, cool it, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the reference solution.

[0178] Preparation of the test solution: Weigh approximately 1.0 g of *Sargentodoxa cuneata* extract accurately, place it in a stoppered conical flask, add 25 ml of 75% methanol accurately, weigh the solution, sonicate (500 W) for 15 minutes, cool, replenish the lost weight with 75% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0179] Determination method: Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0180] Methodological validation of the 6 feature map detection method

[0181] The methodological validation of the feature map detection methods in the above 5 is carried out.

[0182] (1) Specificity examination

[0183] Accurately pipette 1 μl each of the *Sargentodoxa cuneata* extract test solution and blank solvent into the liquid chromatograph, and determine the chromatographic conditions as described above. Results are as follows: Figure 10A , Figure 10B As shown.

[0184] Depend on Figure 10A , Figure 10B The experimental results show that the solvent does not interfere with the characteristic peaks in the spectrum of the *Spatholobus suberectus* extract.

[0185] (2) Holistic Examination

[0186] The test solution of *Sargentodoxa cuneata* extract was injected into the liquid chromatograph. The elution time was doubled at the mobile phase ratio at the gradient endpoint, and the characteristic chromatograms were analyzed. The results are as follows: Figure 11 As shown.

[0187] like Figure 11 As shown, no obvious chromatographic peaks were observed after doubling the elution time under these chromatographic conditions, indicating that the chromatographic conditions basically met the principle of maximizing information content.

[0188] (3) Precision test

[0189] Take the test solution of *Sargentodoxa cuneata* extract and inject it 6 times, with an injection volume of 1 μl. Temporarily identify 5 characteristic peaks, and use peak (peak 3) as the reference peak to calculate the relative retention time. The experimental results are shown in Table 7 below.

[0190] Table 7. Precision results of the characteristic chromatograms of *Sargentodoxa cuneata* extract (relative retention time)

[0191]

[0192]

[0193] As shown in Table 7, the RSD of the relative retention time of each chromatographic peak is less than 3.0%, indicating that the instrument has good precision.

[0194] (4) Stability test

[0195] Extracts of *Sargentodoxa cuneata* were prepared using five different methods for preparing test solutions. The samples were injected at 0, 2, 4, 8, 12, and 24 hours, with an injection volume of 1 μl. Five characteristic peaks were tentatively identified, and the relative retention times were calculated using peak (peak 3) as the reference peak. The results are shown in Table 8 below.

[0196] Table 8. Stability results of the characteristic chromatograms of *Sargentodoxa cuneata* extract (relative retention time)

[0197]

[0198] The experimental results in Table 8 show that the relative retention time (RSD) of the chromatographic peak is less than 3.0%, indicating that the sample solution is relatively stable.

[0199] (5) Repeated examination

[0200] Approximately 1.0 g of the same batch of *Sargentodoxa cuneata* extract (BT2105-1) was accurately weighed and prepared in six parallel portions according to the five test solution preparation methods. 1 μl of each solution was injected. Five characteristic peaks were tentatively identified, and peak (peak 3) was used as the reference peak to calculate the relative retention time. The results are shown in Table 9.

[0201] Table 9. Repeatability results of the characteristic chromatograms of *Sargentodoxa cuneata* extract (relative retention time)

[0202]

[0203]

[0204] Table 9 shows that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, indicating that the method has good repeatability.

[0205] (6) Durability test

[0206] 1) Investigation of the chromatographic column

[0207] Three chromatographic columns were investigated: column 1 - Dikma C18 (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size); column 2 - Shim-pack GISS C18 (100 mm length, 2.1 mm inner diameter, 1.9 μm particle size); column 3 - ACQUITY UPLC. T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm). The effect of three chromatographic columns on the peak characteristics of the extract of *Sargentodoxa cuneata* was investigated. The results are as follows: Figures 12A-12CAnd as shown in Table 10

[0208] Table 10. Chromatographic column analysis results of the characteristic chromatogram of Xiaoxueteng decoction (relative retention time)

[0209]

[0210] Figures 12A-12C The experimental results in Table 10 show that different chromatographic columns have a certain impact on the peak elution. Elution with a 1-Dikma C18 column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size) yielded the best peak shape and separation effect. Therefore, it is recommended to use a 1-Dikma C18 column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size) for this method.

[0211] 2) Investigation at different column temperatures

[0212] The effects of different column temperatures (30℃, 35℃, and 40℃) on the robustness of the characteristic chromatograms of *Sargentodoxa cuneata* extract were compared. The results are as follows: Figures 13A-13C As shown in Table 11.

[0213] Table 11. Results of column temperature analysis of the characteristic chromatogram of Xiaoxueteng decoction (relative retention time)

[0214]

[0215]

[0216] Figures 13A-13C The experimental results in Table 11 show that column temperature has a certain impact on peak elution. The separation effect is best and the peak shape is better when the column temperature is 35℃. Therefore, it is recommended to use a column temperature of 35℃ for the determination.

[0217] 3) Investigation of different flow velocities

[0218] The effects of different flow rates (0.20 ml / min, 0.25 ml / min, and 0.30 ml / min) on the robustness of the characteristic chromatogram of *Sargentodoxa cuneata* extract were compared. The results are as follows: Figures 14A-14C As shown in Table 12.

[0219] Table 12. Results of flow velocity investigation (relative retention time) of Xiaoxueteng standard decoction characteristic spectrum.

[0220]

[0221] Figures 14A-14C The experimental results in Table 12 show that the flow rate has a certain impact on the peak formation, with the optimal peak shape observed at a flow rate of 0.25 ml / min. Therefore, it is recommended to use 0.25 ml / min as the measurement flow rate.

[0222] 7. Detection results of characteristic chromatograms of *Sargentodoxa cuneata* extract.

[0223] The UPLC spectrum of the *Sargentodoxa cuneata* extract sample was determined using the characteristic chromatogram detection method described in section 5. A control characteristic chromatogram of the *Sargentodoxa cuneata* extract was established using peak 3 (chlorogenic acid) as a reference peak. Figure 15 As shown.

[0224] The chromatogram of the test sample should show five characteristic peaks, corresponding to the retention times of the five characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should have the same retention time as the characteristic peak of the chlorogenic acid reference standard, and the peak corresponding to the chlorogenic acid reference standard is the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should be within ±10% of the specified values, which are 0.33 (peak 1), 0.38 (peak 2), 1.10 (peak 4), and 3.37 (peak 5).

[0225] III. Determination of the content and transfer rate of extract index components

[0226] 1. Instruments and reagents

[0227] Ultra-high performance liquid chromatograph (Waters, H-Class Plus), digital ultrasonic cleaner (model: KQ-500DB), 0.01% electronic balance (model: ml204), digital display constant temperature water bath (model: HH-4), rotary evaporator (model: YRE2000B), methanol, and acetonitrile were all chromatographically pure, with batch numbers 213476 and F21L6S113, respectively, purchased from Fisher Scientific. Water was ultrapure water, and other reagents were of analytical grade. The standard decoction was a *Sargentodoxa cuneata* extract with batch number BT2105-1.

[0228] 2. Inspection of the source and purity of the reference standard

[0229] Chlorogenic acid (batch number 110753-202119, purity 96.3%) was purchased from the China National Institutes for Food and Drug Control. Cryptochlorogenic acid (batch number ST07850120, purity 98.0%) was purchased from Shanghai Standard Technical Service Co., Ltd. Both are for content determination and require no pretreatment.

[0230] 3. Determination of chromatographic conditions

[0231] Octadecylsilane-bonded silica gel was used as the packing material (2.1 × 50 mm, inner diameter 1.9 μm); acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase for gradient elution, as shown in Table 13; the column temperature was 30 °C; the flow rate was 0.25 ml / min; and the detection wavelength was 325 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 10,000.

[0232] Table 13

[0233]

[0234] 4. Preparation of reference solution

[0235] Take appropriate amounts of chlorogenic acid and cryptochlorogenic acid reference standards, accurately weigh them, add 50% methanol to prepare a mixed reference solution containing 100 μg of chlorogenic acid and 25 μg of cryptochlorogenic acid per 1 ml, shake well, and the solution is ready.

[0236] 5. Preparation of the test solution

[0237] (1) Investigation of different extraction solvents

[0238] Take an appropriate amount of *Sargentodoxa cuneata* extract (batch number: BT2105-1), a total of 7 portions, with 2 parallel samples per portion. Place each portion in a stoppered conical flask, and accurately add 25 ml of water, 50% ethanol, 75% ethanol, ethanol, 50% methanol, 75% methanol, and methanol sequentially. Weigh the samples, sonicate for 30 min, cool, weigh again, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, accurately pipette 1 μl each of the reference solution and the test solution, inject into the ultra-high performance liquid chromatograph, and determine the content. Calculate the effect of different extraction solvents on the content of chlorogenic acid and cryptochlorogenic acid, and determine the optimal extraction solvent. The experimental results are calculated using the external standard one-point method. See Table 14 below for detailed results.

[0239] Table 14 Effects of different extraction solvents on the total amounts of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract.

[0240]

[0241] As shown in Table 14, the experimental results indicate that different solvents have a significant impact on the content of chlorogenic acid and cryptochlorogenic acid in the extract of *Spatholobus suberectus*. Considering their content, 50% methanol was selected as the optimal extraction solvent for further research.

[0242] (2) Examination of extraction methods

[0243] Take an appropriate amount of *Sargentodoxa cuneata* extract (batch number: BT2105-1), in three portions, with two parallel samples per portion. Place each portion in a stoppered conical container, and precisely add 25 ml of 50% methanol sequentially. Weigh the samples, and treat them by sonication, reflux, and shaking for 30 min each. After cooling, weigh them again, and replenish the lost weight with 50% methanol. Shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, precisely inject 1 μl each of the reference solution and the test solution into an ultra-high performance liquid chromatograph for determination. Calculate the effect of different extraction methods on the content of chlorogenic acid and cryptochlorogenic acid, respectively, to determine the optimal extraction method. The experimental results are calculated using the external standard one-point method. See Table 15 below for detailed results.

[0244] Table 15 Effects of different extraction methods on the total amounts of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract.

[0245]

[0246] As shown in Table 15, the experimental results indicate that different extraction methods do not significantly affect the content of chlorogenic acid and cryptochlorogenic acid in the extract of *Spatholobus suberectus*. Considering the differences in their content, ultrasonic extraction was selected as the extraction method for further research.

[0247] (3) Examination of different extraction times

[0248] Take an appropriate amount of *Sargentodoxa cuneata* extract (batch number: BT2105-1), in three portions, with two parallel samples per portion. Place each portion in a stoppered conical container, and accurately add 25 ml of 50% methanol sequentially. Weigh the samples, reflux for 15 min, 30 min, and 60 min respectively, cool, and replenish the lost weight with 50% methanol. Shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, accurately inject 1 μl each of the reference solution and the test solution into the ultra-high performance liquid chromatograph for determination. Calculate the effect of different extraction times on the content of chlorogenic acid and cryptochlorogenic acid, and determine the optimal extraction time. The experimental results are calculated using the external standard one-point method. See Table 16 below for detailed results.

[0249] Table 16 Effects of different extraction times on the total amounts of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract.

[0250]

[0251] As shown in Table 16, the experimental results indicate that different extraction times do not significantly affect the total amount of chlorogenic acid and cryptochlorogenic acid in the extract of *Sargentodoxa cuneata*. Considering both time cost and content differences, ultrasonic extraction for 15 minutes was selected for further research.

[0252] (4) Determination of the preparation method of the test solution

[0253] Based on the results of the sample pretreatment experiment, the preparation method for the test sample can be determined as follows:

[0254] Take about 0.15g of *Spatholobus suberectus* extract, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate for 15min (500W, 40Hz), cool it, weigh it again, replenish the lost weight with 50% methanol, shake well, filter it, and collect the filtrate to obtain the extract.

[0255] 6 Methodological Validation

[0256] (1) Specificity examination

[0257] Accurately pipette 1 μl each of the *Sargentodoxa cuneata* extract (BT 2105-1) test solution, the mixed reference solution of chlorogenic acid and cryptochlorogenic acid, and the blank solvent, and perform chromatographic analysis under the conditions described above. Record the chromatograms. Results are as follows: Figures 16A-16C As shown.

[0258] Depend on Figures 16A-16C The chromatographic results show that this analytical method is specific for the determination of chlorogenic acid and cryptochlorogenic acid in the extract of *Sargentodoxa cuneata*.

[0259] (2) Peak purity

[0260] Accurately pipette 1 μl each of the test solution and reference solution of *Sargentodoxa cuneata* extract (BT2105-1) and inject them into the ultra-high performance liquid chromatograph for determination. The peak purity of the target peak is then obtained. The results are as follows: Figure 17A \ Figure 17B As shown in Table 17.

[0261] Table 17 Matching values ​​for target peaks and peak purity

[0262]

[0263] Note: The instrument software specifies that when the purity angle is less than the purity threshold, it indicates that the purity meets the analytical requirements.

[0264] Depend on Figure 17A \ Figure 17B As shown in Table 17, the purity angles of all components in this index are less than the purity threshold, indicating that the peak purity meets the analytical requirements.

[0265] (3) Linear

[0266] Accurately weigh appropriate amounts of chlorogenic acid and cryptochlorogenic acid reference standards, place them in numbered 10ml volumetric flasks, add 50% methanol to prepare a mixed reference solution containing 1.0mg of chlorogenic acid and 0.4mg of cryptochlorogenic acid per 1ml, shake well to obtain a mixed reference stock solution of chlorogenic acid and cryptochlorogenic acid, and store it in a refrigerator for later use.

[0267] Take the mixed reference standard stock solution of chlorogenic acid and cryptochlorogenic acid and dilute it to 50, 20, 10, 5, 2.5, and 2 times to obtain mixed reference standard solutions of chlorogenic acid and cryptochlorogenic acid at different concentrations. Perform chromatographic analysis according to the conditions in section "6.2.9.3". Plot the concentration on the x-axis and the peak area on the y-axis to investigate the linear range of chlorogenic acid and cryptochlorogenic acid. The linearity test results are shown in Tables 18 and 19 below. Figure 18 and Figure 19 .

[0268] Table 18 Linearity Study of Chlorogenic Acid

[0269]

[0270] Table 19 Linearity Study of Chlorogenic Acid

[0271]

[0272] From Table 18, Table 19, Figure 18 and Figure 19 Experimental results showed that the concentration of chlorogenic acid exhibited a good linear relationship with the peak area in the range of 20.00 μg / ml to 500.00 μg / ml, with a correlation coefficient r = 0.9996. Cryptochlorogenic acid also showed a good linear relationship with the peak area in the range of 5.00 μg / ml to 100.00 μg / ml, with a correlation coefficient r = 0.9998.

[0273] (4) Precision test

[0274] 1) Instrument precision test

[0275] Accurately pipette 1 μl each of the test solution of *Sargentodoxa cuneata* extract (batch number: BT2105-1) and the mixed reference solution of chlorogenic acid and cryptochlorogenic acid, inject them into the ultra-high performance liquid chromatograph, and determine the results. Calculate the RSD (%) value of the target peak using the external standard one-point method, based on chlorogenic acid and cryptochlorogenic acid. See Table 20 below for detailed results.

[0276] Table 20 Precision test results of the method for determining the content of *Sargentodoxa cuneata* extract.

[0277]

[0278] As shown in Table 20, the RSD (%) values ​​of the target peaks chlorogenic acid and cryptochlorogenic acid are 0.70% and 0.65%, respectively, both less than 2.0%, indicating that the method has good precision.

[0279] 2) Repeatability test

[0280] Accurately weigh approximately 0.14 g of the same batch of *Sargentodoxa cuneata* extract (batch number: BT2105-1), prepare six parallel test solutions according to the above-described method, and set aside for use. Analyze the solutions under the above-described chromatographic conditions. Calculate the RSD (%) of the target peak content using the external standard single-point method, based on chlorogenic acid and cryptochlorogenic acid in the test solutions. See Table 21 below for detailed results.

[0281] Table 21 Results of repeatability experiments for the determination of the content of *Sargentodoxa cuneata* extract.

[0282]

[0283] As shown in Table 21, the RSD (%) values ​​of the target peak chlorogenic acid and cryptochlorogenic acid content were 0.48% and 0.55%, respectively, both less than 2.0%, indicating that the method has good repeatability.

[0284] 3) Intermediate precision

[0285] Other analysts in this project team operated on different dates and under different chromatographs, taking approximately 0.15g of the same batch of *Sargentodoxa cuneata* extract (batch number: BT2105-1), accurately weighed, and prepared the test solution according to the above-mentioned test solution preparation method. The test solution was then analyzed under the above-mentioned chromatographic conditions. The RSD (%) value of the target peak content was calculated using the external standard one-point method based on the chlorogenic acid and cryptochlorogenic acid content in the test solution. The specific results are detailed in Tables 22 and 23 below.

[0286] Table 22 Results of intermediate precision experiments for the determination of the content of *Sargentodoxa cuneata* extract.

[0287]

[0288] Table 23 Results of intermediate precision test for the determination of content in *Sargentodoxa cuneata* extract.

[0289]

[0290]

[0291] As shown in Tables 22 and 23, the intermediate precision RSD (%) values ​​of the target peak chlorogenic acid and cryptochlorogenic acid content are 1.29% and 1.02%, respectively, both less than 2.0%, indicating that the intermediate precision of this method is good.

[0292] 4) Accuracy test

[0293] Take 0.15g of *Sargentodoxa cuneata* extract (batch number BT2105-1, total content of chlorogenic acid and cryptochlorogenic acid 4.01%), accurately weigh 6 portions, and add 1ml of chlorogenic acid reference standard (concentration 2.5850mg / ml) and 1ml of cryptochlorogenic acid reference standard (concentration 0.4239mg / ml) prepared with 50% methanol to each portion. Then add 23ml of 50% methanol, weigh the contents, and prepare the test solution according to the test solution preparation method. Detect the solution under the above chromatographic conditions, injecting 1μl of each sample. Calculate the content of the target peak using the external standard one-point method, based on chlorogenic acid and cryptochlorogenic acid. Calculate the recovery rate and RSD (%) using the following formulas. The results are shown in Tables 24 and 25 below.

[0294]

[0295] Table 24 Results of the sample recovery experiment for the content determination method

[0296]

[0297]

[0298] Table 25 Results of the recovery experiment for the content determination method

[0299]

[0300] The experimental results in Tables 24 and 25 show that the recovery rates of chlorogenic acid and cryptochlorogenic acid in the small vine formula granules are within the range of 95%-102% and 92%-105%, respectively, and the RSD% (0.86%, 1.80%) are less than 2.0%, indicating that the accuracy of the assay method is good.

[0301] (5) Stability test

[0302] Take the test solution of *Sargentodoxa cuneata* (batch number: BT2105-1) and inject it at 0, 2, 4, 6, 8, 12 and 24 hours according to the above chromatographic conditions. The injection volume is 1 μl. The RSD (%) of the target peak content is calculated using the external standard one-point method, based on chlorogenic acid and cryptochlorogenic acid. The specific results are shown in Table 26 below.

[0303] Table 26. Determination of the content of *Sargentodoxa cuneata* granules and stability study of the test solution.

[0304]

[0305]

[0306] The experiment shown in Table 26 indicates that the RSD (%) values ​​of the target peak chlorogenic acid and cryptochlorogenic acid content within 24 hours were 0.51% and 0.86%, respectively, both less than 2.0%, indicating that the solution had good stability within 24 hours.

[0307] (6) Durability test

[0308] 1) Investigation of different chromatographic columns

[0309] The effects of three chromatographic columns—Agilent, Waters, and Ecosil—on the peak shape and resolution of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract were compared.

[0310] Take the test solution of *Sargentodoxa cuneata* extract (BT2105-1), and determine its content under the above chromatographic conditions. Calculate chlorogenic acid and cryptochlorogenic acid, and record the chromatographic data. Results are as follows: Figures 20A-20C As shown in Table 27.

[0311] Table 27 Effect of different chromatographic columns on the determination results of *Spatholobus suberectus* extract.

[0312]

[0313]

[0314] Figures 20A-20C The experimental results in Table 27 show that different columns have poor durability. The peak purity and resolution of the Ecosil UHPLC COLUMNC18 column do not meet the analytical requirements. Considering both the peak shape and resolution of the target chromatographic peak, the Agilent InfinityLab Poroshell120EC-C18 column was selected.

[0315] 2) Investigation using different chromatographs

[0316] Based on the existing equipment in the laboratory, Waters ultra-high performance liquid chromatograph and Agilent ultra-high performance liquid chromatograph were selected to compare the effects of the two chromatographs on the peak shape and resolution of chlorogenic acid and cryptochlorogenic acid in the extract of *Sargentodoxa cuneata*.

[0317] The test solution of *Sargentodoxa cuneata* extract (BT2105-1) was analyzed under the chromatographic conditions described above. Chlorogenic acid and cryptochlorogenic acid were calculated, and the chromatographic data were recorded. The experimental results are as follows: Figure 21A , Figure 21B As shown in Table 28.

[0318] Table 28 Results of Instrument Durability Test

[0319]

[0320] Figure 21A , Figure 21B The experimental results in Table 28 show that this analytical method exhibits good durability with different chromatographs. Variations in the chromatograph can meet the system adaptability requirements.

[0321] 3) Investigation at different column temperatures

[0322] The effects of column temperatures (25℃, 30℃, and 35℃) on the peak shapes of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract were compared. *Sargentodoxa cuneata* extract (BT2105-1) was used as a test solution, and the peaks were determined under the above chromatographic conditions. Chromatographic data were recorded as chlorogenic acid and cryptochlorogenic acid. The experimental results are as follows: Figures 22A-22C As shown in Table 29.

[0323] Table 29 Chromatographic results of *Sargentodoxa cuneata* extract containing the target components at different column temperatures.

[0324]

[0325] Figures 22A-22CThe results in Table 29 show that the peak shape and separation performance were good at all three column temperatures. At 30℃, the baseline of the chromatogram showed no drift, and the retention time was not significantly different compared to the other two temperatures. Considering the column's tolerance and the analysis time required, a column temperature of 30℃ was chosen.

[0326] 4) Investigation of different flow velocities

[0327] The effects of different flow rates of 0.20 ml / min, 0.25 ml / min, and 0.30 ml / min on the peak shapes of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract were compared.

[0328] Take the test solution of *Sargentodoxa cuneata* extract (BT2105-1), and determine its content under the above chromatographic conditions. Calculate chlorogenic acid and cryptochlorogenic acid, and record the chromatographic data. Experimental results are as follows: Figures 23A-23C As shown in Table 30.

[0329] Table 30 Results of the determination of *Smilax glabra* content in standard decoction at different flow rates

[0330]

[0331]

[0332] Table 30 shows that the peak shape and separation effect were good at all three flow rates. The target component showed good separation and no baseline drift at a flow rate of 0.25 ml / min; therefore, a flow rate of 0.25 ml / min was selected for this experiment.

[0333] Example 2

[0334] 1. Preparation method of small bloodvine extract

[0335] (1) Instruments

[0336] Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-5205A), ceramic health pot (Huzhou Gangdian Craft Manufacturing Factory), electronic balance (Hangzhou Youheng Weighing Equipment Co., Ltd., HLD-30002), balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd., AL104), circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd., SHZ-D(III)), low temperature coolant circulating water pump (Zhengzhou Changcheng Science and Technology Industry and Trade Co., Ltd., DLSB-5 / 20B), vacuum freeze dryer (Dalian Shuangrui Technology Co., Ltd., TRL-0.5), electric thermostatic drying oven (Tianjin Tester Instrument Co., Ltd., 202-2AB), electric blower thermostatic drying oven (Hunan Electric Furnace Drying Oven Factory, 101-4A), electric thermostatic water bath (Beijing Kewei Yongxing Instrument Co., Ltd., HH-S6A), guillotine.

[0337] (2) Materials

[0338] The following table 31 shows the sliced ​​pieces of *Sargentodoxa cuneata*:

[0339] Table 31 Information on Scutellaria baicalensis slices

[0340]

[0341]

[0342] The extract of the above-mentioned *Sargentodoxa cuneata* slices was prepared by the following method.

[0343] Take 100g of *Smilax china* slices and place them in an electric ceramic kettle. Add water and decoct twice. For the first decoction, add 8 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 60 minutes. Filter the decoction through a 300-mesh sieve while hot. For the second decoction, add 6 times the amount of water, bring to a boil over high heat, then simmer over low heat for 40 minutes. Filter the decoction through a 300-mesh sieve while hot. Combine the two decoctions.

[0344] The decoction was transferred to a 2000ml round-bottom flask and concentrated under reduced pressure using a rotary evaporator (temperature: 65℃; vacuum: -0.080~-0.090MPa) to 100ml. Under magnetic stirring, the concentrate was dispensed into 10ml amber vials, each containing 1ml. The vials were partially capped. After dispensing, the vials were transferred to a vacuum freeze dryer for lyophilization. The lyophilization parameters were as follows: pre-freezing temperature of the concentrated *Sargentodoxa cuneata* extract was -50℃, pre-freezing time was 180 minutes; sublimation drying temperature was -45℃~0℃, sublimation drying time was 2340 minutes, and vacuum was -0.2mbar; desorption drying temperature was 10℃~30℃, desorption drying time was 360 minutes, and vacuum was 0mbar. The extract was then removed, capped with an aluminum cap, and the *Sargentodoxa cuneata* extract was obtained.

[0345] 2. Method for determining the yield of ointment

[0346] The yield of the extract was determined by the amount of extract. 10g of the concentrated liquid was placed in a pre-weighed evaporating dish, dried in a water bath, and then dried in a 105℃ drying oven for 3 hours. After cooling in a desiccator for half an hour, the liquid was quickly weighed. The yield of the extract was then calculated.

[0347]

[0348] Based on the test results of 15 batches of *Spatholobus suberectus* extract, the extract yield was calculated, and the results are shown in Table 32 below.

[0349] Table 32 Determination of extract yield from 15 batches of *Sargentodoxa cuneata* extract

[0350]

[0351]

[0352] Based on the combined yield of 15 batches of *Spatholobus suberectus* extract, the fluctuation range was 10.04%–18.87%, with an average of 14.55% and a SD of 2.17%. The average yield ranged from 70% to 130%, which is 10.19%–18.92%. The mean yield range ±3SD was 8.05%–21.05%. The determined yield range for *Spatholobus suberectus* extract was the mean yield ±3SD range of 8.05%–21.05%, approximately rounded to 8.1%–21.1%.

[0353] 3. Determination of the characteristic chromatogram of *Sargentodoxa cuneata* extract

[0354] After establishing the characteristic chromatographic analysis method for *Sargentodoxa cuneata* extract and determining the characteristic chromatograms of 15 batches of *Sargentodoxa cuneata* extract, the control characteristic chromatogram of *Sargentodoxa cuneata* extract was finally determined.

[0355] Feature map detection

[0356] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 33; the flow rate was 0.25 mL / min; the column temperature was 35 °C; and the detection wavelength was 245 nm.

[0357] Chromatographic column: Dikma C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm).

[0358] Table 33

[0359]

[0360] Preparation of reference solution: Take about 1.0g of *Sargentodoxa cuneata* reference material, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, weigh it, sonicate (power 500W) for 15 minutes, cool it, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the reference solution.

[0361] Preparation of the test solution: Weigh approximately 1.0 g of *Sargentodoxa cuneata* extract accurately, place it in a stoppered conical flask, add 25 ml of 75% methanol accurately, weigh the solution, sonicate (500 W) for 15 minutes, cool, replenish the lost weight with 75% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0362] The determination method involves precisely pipetting 1 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0363] UPLC spectra of different batches of *Sargentodoxa cuneata* extract samples were determined, and the results are as follows: Figure 24And as shown in Table 34:

[0364] The results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and a total of 5 common peaks were identified.

[0365] Table 34 Sample Measurement Results

[0366]

[0367]

[0368] Figure 24 The results in Table 34 show that, with peak 3 as the reference peak, the relative retention time (RSD) values ​​of the other four characteristic peaks in the characteristic chromatograms of the 15 batches of *Sargentodoxa cuneata* extract were between 0.1% and 0.3%, all less than 3.0%, which meets the standard requirements for the characteristic chromatograms of *Sargentodoxa cuneata* extract.

[0369] The chromatogram of the test sample should show five characteristic peaks, corresponding to the retention times of the five characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should have the same retention time as the characteristic peak of the chlorogenic acid reference standard, and the peak corresponding to the chlorogenic acid reference standard is the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should be within ±10% of the specified values, which are 0.33 (peak 1), 0.38 (peak 2), 1.10 (peak 4), and 3.37 (peak 5).

[0370] 4. Determination of the content of extracts from different batches of *Spatholobus suberectus*

[0371] Determination of chlorogenic acid and cryptochlorogenic acid content in extracts of *Sargentodoxa cuneata*

[0372] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 50 mm, inner diameter 2.1 mm, particle size 1.9 μm); acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase for gradient elution, as shown in Table 35 below; the flow rate was 0.25 mL / min; the column temperature was 30 °C; and the detection wavelength was 325 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 10,000.

[0373] Table 35

[0374]

[0375] Preparation of reference solution: Take appropriate amounts of chlorogenic acid and cryptochlorogenic acid reference standards, accurately weigh them, add 50% methanol to prepare a mixed reference solution containing 100 μg of chlorogenic acid and 25 μg of cryptochlorogenic acid per 1 ml, shake well, and the solution is ready.

[0376] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.15g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate it (power 500W, 40Hz) for 15min, cool it, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0377] The assay method involves precisely pipetting 1 μl of both the reference solution and the test solution into the liquid chromatograph and measuring the concentrations. The results are shown in Table 36.

[0378] Table 36 Results of content and transfer rate of *Sargentodoxa cuneata* extract from different batches

[0379]

[0380]

[0381] Based on the determination results of the total content of chlorogenic acid and cryptochlorogenic acid in 15 batches of *Sargentodoxa cuneata* slices and extracts, the mean and range of the total content and total transfer rate of chlorogenic acid and cryptochlorogenic acid in *Sargentodoxa cuneata* extract are calculated as shown in the table below.

[0382] Table 37 Analysis of the content and transfer rate of extracts from 15 batches of *Sargentodoxa cuneata*

[0383]

[0384] Based on the determination results of the total content of chlorogenic acid and cryptochlorogenic acid in the *Sargentodoxa cuneata* extract shown in Tables 36 and 37, the average total content of chlorogenic acid and cryptochlorogenic acid in the standard decoction of *Sargentodoxa cuneata* was 3.69%. Considering that the average total content of chlorogenic acid and cryptochlorogenic acid in the standard decoction of *Sargentodoxa cuneata* is 70%–130%, the fluctuation range was calculated to be 2.58%–4.80%. Using three times the SD of the average total content of chlorogenic acid and cryptochlorogenic acid in the standard decoction of *Sargentodoxa cuneata*, the fluctuation range was calculated to be 1.01%–6.37%. The total content of chlorogenic acid and cryptochlorogenic acid in the standard decoction fluctuated between 1.01% and 6.37%.

[0385] Based on the results of the total transfer rate determination of chlorogenic acid and cryptochlorogenic acid in the *Smilax china* standard decoction in Tables 36 and 37, the mean total transfer rate of chlorogenic acid and cryptochlorogenic acid in the *Smilax china* standard decoction was 42.13%. Calculated based on a range of 70%–130% of the mean total transfer rate of chlorogenic acid and cryptochlorogenic acid in the *Smilax china* standard decoction, the fluctuation range was 29.49%–54.76%. Calculated based on three times the SD of the mean total transfer rate of chlorogenic acid and cryptochlorogenic acid in the *Smilax china* standard decoction, the fluctuation range was 15.07%–69.18%. The provisional range of fluctuation for the total transfer rate of chlorogenic acid and cryptochlorogenic acid in the standard decoction was determined to be 15.07%–69.18%.

Claims

1. A method for detecting the characteristic chromatogram of a small-flowered vine extract, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take the *Sargentodoxa cuneata* reference herb, add methanol to prepare a solution, and obtain the reference solution; (2) Preparation of the test solution: Take the small blood vine medicinal material, add water and decoct, separate the solid and liquid to obtain the filtrate, concentrate and dry the filtrate and then freeze dry it under vacuum to obtain the small blood vine extract. Then add 75% methanol solvent to the small blood vine extract and perform ultrasonic extraction to obtain the test solution. (3) Ultra-high performance liquid chromatography analysis The reference solution and the test solution were injected into the ultra-high performance liquid chromatograph. A Dikma C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm was used. Gradient elution was performed with the mobile phase to obtain the characteristic chromatogram of the *Sargentodoxa cuneata* extract. The characteristic spectrum contains at least five characteristic peaks, with the peak corresponding to the chlorogenic acid reference being the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of a specified value, which includes 0.33, 0.38, 1.10, and 3.

37. The mobile phase is acetonitrile-0.1% formic acid solution, with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B. The detection wavelength is 245 nm. In step (3), the flow rate of the mobile phase is 0.25 mL / min, and the gradient elution program is as follows: From 0 to 10 min, the volume percentage of mobile phase A changed from 6% to 8.5%, and the volume percentage of mobile phase B changed from 94% to 91.5%. Over 10-15 minutes, the volume percentage of mobile phase A changed from 8.5% to 10%, and the volume percentage of mobile phase B changed from 91.5% to 90%. Over 15-18 minutes, the volume percentage of mobile phase A changed from 10% to 11%, and the volume percentage of mobile phase B changed from 90% to 89%. At 18-19 min, the volume percentage of mobile phase A was 11%, and the volume percentage of mobile phase B was 89%. From 19 to 31 minutes, the volume percentage of mobile phase A changed from 11% to 15%, and the volume percentage of mobile phase B changed from 89% to 85%. Over 31-35 minutes, the volume percentage of mobile phase A changed from 15% to 18%, and the volume percentage of mobile phase B changed from 85% to 82%. From 35 to 35.1 min, the volume percentage of mobile phase A changed from 18% to 6%, and the volume percentage of mobile phase B changed from 82% to 94%. From 35.1 to 37 min, the volume percentage of mobile phase A was 6% and the volume percentage of mobile phase B was 94%.

2. The detection method according to claim 1, characterized in that, In step (2), the extraction time is 15-45 min.

3. The detection method according to claim 2, characterized in that, In step (2), the extraction time is 15 minutes.

4. The detection method according to claim 1, characterized in that, The column temperature in step (3) is 30-40℃.

5. The detection method according to claim 4, characterized in that, The column temperature in step (3) is 35℃.

6. The detection method according to claim 1, characterized in that, The decoction is performed 1-3 times, with the mass ratio of the small blood vine herb to water being 1:6-8 each time, and the decoction time being 40-60 minutes each time.

7. The detection method according to claim 1, characterized in that, The vacuum freeze-drying process is divided into three stages: a. Pre-freezing: The pre-freezing temperature is -50℃ to -45℃, and the pre-freezing time is 150-200 minutes; b. Single drying: The drying temperature is -45℃ to 0℃, and the vacuum degree is -0.2 to 0 mbar. The single drying time is 2000-2500 minutes. c. Secondary drying: The drying temperature is 5-25℃ and the vacuum degree is -0.1-0.1 mbar to obtain the small blood vine extract. The secondary drying time is 350-400 minutes.

8. The detection method according to claim 6, characterized in that, The boiling process involves adding water twice.

9. The detection method according to claim 1, characterized in that, The concentration and drying temperature is 50-65℃.

10. The detection method according to claim 7, characterized in that, Pre-freezing time is 180 minutes.

11. The detection method according to claim 7, characterized in that, The drying time is 2340 minutes per cycle.

12. The detection method according to claim 7, characterized in that, The secondary drying time is 360 minutes.

13. The detection method according to any one of claims 1-12, characterized in that, The yield of the extract of *Sargentodoxa cuneata* is 8.1% to 21.1%.

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