A method for constructing a characteristic chromatogram and thin-layer chromatogram of a North Ledum drug preparation

Abstract

This invention belongs to the field of traditional Chinese medicine quality testing technology, specifically relating to a method for constructing characteristic chromatograms and thin-layer chromatography for *Artemisia annua* (Northern Liu Artemisia) preparations. The test sample solution is analyzed using high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material. The mobile phase includes an acidic aqueous solution and acetonitrile. Through continuous optimization of the gradient elution program, effective separation of 11 common characteristic peaks is achieved. Furthermore, the obtained characteristic chromatograms exhibit stable baselines, good peak shapes, and short detection times, providing a scientific basis for comprehensively establishing quality control standards for *Artemisia annua* preparations. The chromatograms fully reflect the integrity and characteristics of *Artemisia annua* preparations (e.g., standard decoction lyophilized powder, formula granules, etc.). The method for constructing the characteristic chromatograms of this invention can also be used for the identification of *Artemisia annua* preparations from easily confused products.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine quality detection, and specifically relates to a method for constructing a characteristic chromatogram and thin-layer chromatography of a Siphonostegia chinensis Benth. drug preparation Background Art

[0002] Siphonostegia chinensis Benth. is a traditional Chinese medicine name. It is the dried whole herb of the plant Siphonostegia chinensis Benth. of the family Scrophulariaceae. The root is short and curved, with slightly branched. The stem is cylindrical, with a grayish-brown or yellowish-brown surface, 30-80 cm long, densely covered with rust-colored short hairs, with edges; it is brittle and easy to break, the cross-section is yellowish-white, the edge is fibrous, and the center is white and loose pith. The leaves are opposite, and the upper leaves are mostly alternate, mostly broken and fallen off. The complete ones are pinnately deeply lobed and dark green. The raceme is terminal, the flowers have short pedicels, with a tubular calyx, about 1.5 cm long, with 10 raised longitudinal ribs on the surface, and 5-lobed at the top. The yellowish-brown labiate corolla remains. The capsule is oblong, brownish-black, with many longitudinal lines, brittle and easy to break. The seeds are small, numerous, and the surface is wrinkled. It has a slight smell and a light taste. It is cold in nature and bitter in taste. It belongs to the spleen meridian, stomach meridian, liver meridian, and gallbladder meridian. Its functions and indications are: promoting blood circulation to remove blood stasis, dredging meridians to stop pain, cooling blood, stopping bleeding, clearing heat and promoting diuresis. It belongs to the category of drugs for activating blood circulation and regulating menstruation under the classification of drugs for promoting blood circulation and removing blood stasis

[0003] The 2020 edition of the Chinese Pharmacopoeia uses luteolin and acteoside as the quality evaluation indicators for Siphonostegia chinensis Benth. medicinal materials, but it cannot comprehensively reflect its quality. The standard decoction of traditional Chinese medicine is the material benchmark for measuring whether the traditional Chinese medicine formula granules are basically consistent with the decoction. However, drug preparations such as freeze-dried powder of the standard decoction have lost the microscopic and morphological identification characteristics of the original medicinal materials and cut pieces, that is, it is impossible to check and identify from the shape, size, color, surface, texture, etc. of the medicinal materials Summary of the Invention

[0004] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic chromatogram and thin-layer chromatography of a Siphonostegia chinensis Benth. drug preparation. Among them, the construction method of the characteristic chromatogram and the identification method of thin-layer chromatography can respectively establish the characteristic chromatogram and thin-layer chromatography of this variety according to the characteristics of the Siphonostegia chinensis Benth. drug preparation (freeze-dried powder of the standard decoction), achieve effective separation of each characteristic peak, increase the number of characteristic peaks or spots, shorten the detection and analysis time, improve the separation effect, and can quickly and effectively identify the genuine and easily confused products of the Siphonostegia chinensis Benth. drug preparation, providing a scientific basis for comprehensively establishing the quality control standard of the Siphonostegia chinensis Benth. drug preparation

[0005] For this reason, the present invention provides a method for constructing a characteristic chromatogram of a Siphonostegia chinensis Benth. drug preparation, including the following steps

[0006] Preparation of the test solution

[0007] Determination: The test solution was analyzed by high performance liquid chromatography (HPLC). Octadecylsilane-bonded silica gel was used as the stationary phase, and the mobile phase consisted of an acidic aqueous solution and acetonitrile, with gradient elution. The column temperature was 25-35℃, and the detection wavelength was 250-350nm. The gradient elution program included: 0→10-17min→15-37min→23-45min→38-50min, and the volume percentage of acetonitrile in the mobile phase was: 15%→15%→16-36%→15-36%→15-40%. The characteristic chromatogram of the *Ligusticum striatum* preparation was obtained.

[0008] Furthermore, the high-performance liquid chromatography detection process also satisfies at least one of the following (1)-(4):

[0009] (1) The detection wavelength is 280-350 nm; and / or the flow rate is 0.25-0.8 mL / min; and / or the injection volume is 5-20 μl; and / or the column temperature is 25-30 °C;

[0010] (2) The acid in the acid-containing aqueous solution is selected from one or more of glacial acetic acid, formic acid, and phosphoric acid; and / or, the volume percentage of acid in the acid-containing aqueous solution is 0.05-0.15%;

[0011] (3) A chromatographic column with a specification of 4.6×150mm and 2.7μm is used in the high performance liquid chromatography detection; and / or, an Agilent InfinityLab Poroshell 120 SB-C18 column or an Agilent InfinityLab Poroshell 120 EC-C18 column is used in the high performance liquid chromatography detection;

[0012] (4) The gradient elution program includes: 0→15min→30min→45min→50min, and the volume percentage of acetonitrile in the mobile phase is: 15%→15%→23%→36%→40%; or, the gradient elution program includes: 0→17min→37min→37.1min→45min, and the volume percentage of acetonitrile in the mobile phase is: 15%→15%→36%→15%→15%; the gradient elution program includes: 0→10min→15min→23min→38min→60min→60.1min→70min, and the volume percentage of acetonitrile in the mobile phase is: 15%→15%→16%→16%→18%→36%→15%→15%.

[0013] Furthermore, the chromatographic column used in the high performance liquid chromatography detection is selected from the Agilent InfinityLab Poroshell 120 EC-C18 column, the Agilent InfinityLab Poroshell 120 SB-C18 column, or the Agilent InfinityLab Poroshell HPH-C18 column.

[0014] Further, the preparation of the test sample solution includes the following steps: weighing the test sample, extracting with solvent to obtain the extract, separating the solid and liquid phases, and taking the liquid, which is the test sample solution; preferably, the preparation of the test sample solution also satisfies any one or more of the following A:

[0015] A. The ratio of the mass of the test sample to the volume of the solvent is 0.1-0.8:10-100; the relationship between mass and volume is g / mL;

[0016] B. The extraction method is either reflux extraction or ultrasonic extraction, preferably with an ultrasonic power of 210-300W;

[0017] C. The extraction time is ≥15 min, preferably 15-60 min;

[0018] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0019] E. The solvent is selected from water, pure methanol, pure ethanol, methanol aqueous solution or ethanol aqueous solution; preferably, it is methanol aqueous solution with a volume concentration of 70% or more, ethanol aqueous solution with a volume concentration of 70% or more, pure methanol or pure ethanol.

[0020] Further, the preparation method of the test solution includes: weighing 0.1-0.8g of the test sample accurately, placing it in an Erlenmeyer flask, accurately adding 10-100ml of methanol aqueous solution with a volume concentration of 70% or higher, ethanol aqueous solution with a volume concentration of 70% or higher, pure methanol or pure ethanol, weighing the sample, sonicating or refluxing for 15-60min, weighing the sample again, cooling the sample, weighing the sample again, shaking the sample well, filtering the sample, and collecting the filtrate to obtain the test solution.

[0021] Further, the construction method further includes the step of preparing a reference solution by dissolving luteolin, verbascoside, luteolin-7-O-β-D-glucuronide, caffeic acid, apigenin, and isocitric acid in a solvent, respectively, and detecting them using the high-performance liquid chromatography method described in claim 1 or 2; preferably, each 1 mL of the reference solution contains 10-100 µg of luteolin, 10-100 µg of verbascoside, 10-100 µg of luteolin-7-O-β-D-glucuronide, 10-100 µg of caffeic acid, 10-100 µg of apigenin, or 10-100 µg of isocitric acid; preferably, the solvent is methanol or an aqueous methanol solution.

[0022] Further, the construction method further includes the step of preparing a reference solution by dissolving luteolin, verbascoside, apigenin-7-O-β-D-glucuronide, caffeic acid, apigenin, and isocitric acid in a solvent, respectively, and detecting them using the high-performance liquid chromatography method described in claim 1 or 2; preferably, each 1 mL of the reference solution contains 10-100 µg of luteolin, 10-100 µg of verbascoside, 10-100 µg of apigenin-7-O-β-D-glucuronide, 10-100 µg of caffeic acid, 10-100 µg of apigenin, or 10-100 µg of isocitric acid; preferably, the solvent is methanol or an aqueous methanol solution.

[0023] Furthermore, the *Artemisia annua* pharmaceutical preparation is selected from *Artemisia annua* standard decoction freeze-dried powder or *Artemisia annua* formula granules.

[0024] Furthermore, it also includes importing the characteristic chromatograms of different batches of Artemisia annua drug preparations into the similarity evaluation system of chromatographic fingerprint chromatograms of traditional Chinese medicine promulgated by the National Pharmacopoeia Commission to generate a reference characteristic chromatogram of Artemisia annua drug preparations. The reference characteristic chromatogram of Artemisia annua drug preparations has 11 common characteristic peaks. Peaks 1, 3, 4, 7, 10, and 11 are, in order, caffeic acid, luteolin-7-O-β-D-glucuronide, verbascoside, iso-citric acid, luteolin, and apigenin.

[0025] In some embodiments, the method further includes constructing a reference characteristic chromatogram for the *Artemisia annua* drug preparation. The characteristic chromatograms obtained from multiple batches of *Artemisia annua* drug preparation samples are used to generate a reference characteristic chromatogram for the *Artemisia annua* drug preparation using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission. At least two batches of *Artemisia annua* formula granules or standard decoction lyophilized powder are used, for example, 2 batches, 6 batches, 15 batches, or 18 batches of *Artemisia annua* formula granules or standard decoction lyophilized powder.

[0026] In some embodiments, after generating the control characteristic spectrum of the *Ligusticum striatum* drug preparation using the fingerprint spectrum similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, the step of marking common characteristic peaks is also included.

[0027] Furthermore, the characteristic chromatogram of the *Artemisia annua* preparation satisfies at least one of the following (1)-(2):

[0028] (1) It has 11 common characteristic peaks, with peaks 1, 3, 4, 7, 10, and 11 being caffeic acid, luteolin-7-O-β-D-glucuronide, verbascoside, iso-citric acid, luteolin, and apigenin, respectively; and / or,

[0029] (2) Peaks 3, 4, and 10 correspond to the retention times of luteolin-7-O-β-D-glucuronide, verbascoside, and luteolin reference standards. The peak corresponding to the verbascoside reference standard peak is peak S1. The relative retention times of peaks 1-2 and 5-8 with peak S1 are within ±10% of the specified values. The specified values ​​for peaks 1-2 and 5-8 are 0.29, 0.52, 1.06, 1.12, 1.19, and 1.21, respectively. The peak corresponding to the luteolin reference standard peak is peak S2. The relative retention times of peaks 9 and 11 with peak S2 are within ±10% of the specified values. The specified values ​​for peaks 9 and 11 with peak S2 are 0.84 and 1.14, respectively.

[0030] In some preferred embodiments, the method for constructing the characteristic spectrum of the *Artemisia annua* drug formulation includes the following steps:

[0031] Preparation of the test solution: Weigh 0.1-0.8g of the test sample accurately, place it in an Erlenmeyer flask, accurately add 10-100ml of methanol aqueous solution with a volume concentration of 70% or higher, weigh the sample, sonicate or reflux for 15-60min, weigh the sample again, cool the sample, weigh the sample again, shake well, filter the sample, and collect the filtrate to obtain the test solution.

[0032] Preparation of the reference solution: Accurately weigh appropriate amounts of luteolin, verbascoside, luteolin-7-O-β-D-glucuronide, caffeic acid, apigenin, and isocitric acid, and add methanol to prepare a solution containing 10-100 µg of luteolin, 10-100 µg of verbascoside, 10-100 µg of luteolin-7-O-β-D-glucuronide, 10-100 µg of caffeic acid, 10-100 µg of apigenin, and 10-100 µg of isocitric acid per mL of reference solution.

[0033] High-performance liquid chromatography (HPLC) was used to detect the test solution and reference solution. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase (150 mm × 4.6 mm, 2.7 µm); acetonitrile was used as mobile phase A; and 0.05-0.15% glacial acetic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below. The detection wavelength was 280-350 nm; the column temperature was 25-35 °C; the flow rate was 0.25-0.8 mL / min; and the injection volume was 5-20 μl.

[0034]

[0035] This invention also provides a method for constructing a thin-layer chromatography method for a drug preparation of Artemisia capillaris, comprising the following steps:

[0036] Preparation of the test solution;

[0037] Thin-layer chromatography was performed on the test solution using methanol-formic acid-water with a volume ratio of 2:1:3-5 as the developing solvent to obtain the thin-layer chromatogram of the *Liu Ji Nu* drug preparation.

[0038] Furthermore, in the thin-layer chromatography detection, any one or more of the following AC conditions are met:

[0039] A. Spot the test solution onto a thin-layer plate. Preferably, the spotting volume is 1-3 μl.

[0040] B, with methanol-formic acid-water in a volume ratio of 2:1:3 as the developing solvent;

[0041] C. After unfolding and drying, spray with aluminum trichloride solution, heat and then examine under ultraviolet light.

[0042] Further, the preparation of the test sample solution includes taking the test sample, extracting with methanol, filtering, and obtaining the solution; preferably, the mass ratio of the test sample to the volume of methanol is 0.1-0.3:5; the mass-to-volume relationship is g / mL; preferably, the extraction method is ultrasonic extraction.

[0043] The present invention also provides a method for identifying a *Artemisia annua* pharmaceutical preparation from adulterants, comprising the steps of constructing a characteristic chromatogram of the product to be identified using the product to be identified according to any of the above-described construction methods and comparing it with the characteristic chromatogram of the *Artemisia annua* pharmaceutical preparation; or, the steps of constructing a thin-layer chromatogram of the product to be identified using the product to be identified according to the above-described construction methods and comparing it with the thin-layer chromatogram of the *Artemisia annua* pharmaceutical preparation.

[0044] For thin-layer chromatography, if the thin-layer chromatogram of the product to be identified shows fluorescent spots of the same color at the corresponding positions as those of the reference medicinal material *Artemisia capillaris*, verbascoside, and luteolin-7-O-β-D-glucuronide, then it is a genuine *Artemisia capillaris* pharmaceutical preparation. Otherwise, it is a counterfeit product.

[0045] The present invention also provides a quality testing method for a *Artemisia annua* pharmaceutical preparation, comprising the steps of constructing a characteristic chromatogram of the product to be tested using any of the above-described construction methods and comparing it with the characteristic chromatogram of the *Artemisia annua* pharmaceutical preparation; or, constructing a thin-layer chromatography (TLC) chromatogram of the product to be tested using the above-described construction methods and comparing it with the TLC chromatogram of the *Artemisia annua* pharmaceutical preparation.

[0046] For thin-layer chromatography, if the thin-layer chromatogram of the product to be tested shows fluorescent spots of the same color at the corresponding positions as those of the reference medicinal material *Artemisia capillaris*, verbascoside, and luteolin-7-O-β-D-glucuronide, then the product is qualified. Otherwise, the product is unqualified.

[0047] The technical solution of this invention has the following advantages:

[0048] 1. The method for constructing the characteristic chromatogram of the *Artemisia annua* pharmaceutical preparation of the present invention uses octadecylsilane-bonded silica gel as the packing material, and employs an acid-containing aqueous solution and acetonitrile as the mobile phase for gradient elution. The column temperature is 25-35℃, and the detection wavelength is 250-350nm. The gradient elution program includes: 0→10-17min→15-37min→23-45min→38-50min, and the volume percentage of acetonitrile in the mobile phase is: 15%→15%→16-36%→15-36%→15-40%. The resulting characteristic chromatogram of the *Artemisia annua* pharmaceutical preparation yields multiple characteristic peaks, achieving effective separation of 11 active ingredients, including luteolin, verbascoside, luteolin-7-O-β-D-glucuronide, caffeic acid, apigenin, and iso-citric acid. Furthermore, the obtained characteristic chromatogram has a stable baseline, good peak shape, and short detection time, providing a scientific basis for comprehensively establishing quality control standards for *Artemisia annua* formulation granules. Furthermore, it can accurately locate the peak position of the aforementioned reference standard, fully reflecting the integrity and characteristics of *Artemisia capillaris* drug preparations (such as formulation granules).

[0049] The above-mentioned construction method is not only applicable to the quality testing of *Artemisia annua* drug preparations, but also to the identification of genuine and adulterated *Artemisia annua* drug preparations, enabling more comprehensive quality monitoring of *Artemisia annua* formula granules or standard decoction freeze-dried powder.

[0050] By optimizing chromatographic conditions, extraction solvents, and solvent dosage, the optimal extraction process and chromatographic conditions were determined, resulting in higher peak areas and better separation effects. This allows for more comprehensive quality monitoring of *Artemisia annua* formula granules or standard decoction freeze-dried powder.

[0051] 2. The method for constructing the thin-layer chromatography of the *Artemisia annua* drug preparation of the present invention involves using methanol-formic acid-water in a volume ratio of 2:1:3-5 as the developing solvent to perform thin-layer chromatography detection on the test solution, thereby obtaining the thin-layer chromatogram of the *Artemisia annua* drug preparation. The developing solvent at this specific volume ratio can simultaneously detect nine spots (including verbascoside, luteolin-7-O-) in the test solution of the *Artemisia annua* drug preparation. β The chromatographic information (-D-glucuronide) develops well, has high resolution, produces numerous clear spots that do not aggregate, provides comprehensive chromatographic information, and is not affected by negative interference, making the identification process fast and convenient, and the identification results accurate and reliable. Attached Figure Description

[0052] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0053] Figure 1 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method one in Experiment Example 1 is shown below.

[0054] Figure 2 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method 2 in Experimental Example 1 is shown below.

[0055] Figure 3 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method 3 in Experiment Example 1 is shown below.

[0056] Figure 4 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method four in Experiment Example 1 is shown below.

[0057] Figure 5 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method five in Experiment Example 1 is shown below.

[0058] Figure 6 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method six in Experiment Example 1 is shown below.

[0059] Figure 7 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method seven in Experiment Example 1 is shown below.

[0060] Figure 8 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method eight in Experiment Example 1 is shown below.

[0061] Figure 9 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method nine in Experimental Example 1 is shown below.

[0062] Figure 10 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method ten in Experimental Example 1 is shown below.

[0063] Figure 11 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method eleven in Experiment Example 1;

[0064] Figure 12 The chromatogram of the standard decoction (lyophilized powder) of *Artemisia annua* obtained by method 12 in Experimental Example 1 is shown below.

[0065] Figure 13 The chromatogram at wavelength 230 nm is from Experiment Example 1;

[0066] Figure 14 The chromatogram at wavelength 254 nm is from Experiment Example 1;

[0067] Figure 15 The chromatogram at wavelength 270 nm is from Experiment Example 1;

[0068] Figure 16 The chromatogram at wavelength 290 nm is from Experiment Example 1;

[0069] Figure 17 The chromatogram at wavelength 310 nm is shown in Experiment Example 1.

[0070] Figure 18 The chromatogram at wavelength 350 nm is from Experiment Example 1;

[0071] Figure 19 The chromatogram for the acetonitrile-0.1% glacial acetic acid system in Experimental Example 1 is shown.

[0072] Figure 20 The chromatogram for the methanol-0.1% glacial acetic acid system in Experimental Example 1 is shown.

[0073] Figure 21The chromatogram for the acetonitrile-0.1% glacial acetic acid system in Experimental Example 1 is shown below.

[0074] Figure 22 The chromatogram for the acetonitrile-0.1% phosphoric acid system in Experimental Example 1 is shown below.

[0075] Figure 23 The chromatogram for the acetonitrile-0.1% formic acid system in Experimental Example 1 is shown below.

[0076] Figure 24 The chromatogram for the acetonitrile-0.05% glacial acetic acid system in Experimental Example 1 is shown below.

[0077] Figure 25 The chromatogram for the acetonitrile-0.15% glacial acetic acid system in Experimental Example 1 is shown below.

[0078] Figure 26 The chromatogram for Experiment 1 at a column temperature of 25℃ is shown below.

[0079] Figure 27 The chromatogram for Experiment 1 at a column temperature of 35℃ is shown below.

[0080] Figure 28 The chromatogram for a flow rate of 0.6 ml / min in Experiment Example 1;

[0081] Figure 29 The chromatogram for a flow rate of 0.8 ml / min in Experiment Example 1;

[0082] Figure 30 The chromatogram of the test sample under the conditions of Poroshell 120 SB-C18 column in Experiment Example 1;

[0083] Figure 31 The chromatogram of the test sample under the conditions of the Poroshell HPH-C18 column in Experiment Example 1;

[0084] Figure 32 The chromatogram of methanol extraction in Experiment Example 1;

[0085] Figure 33 The chromatogram of ethanol extraction in Experiment Example 1;

[0086] Figure 34 The chromatogram of 85% methanol extraction in Experiment Example 1;

[0087] Figure 35 The chromatogram of 70% methanol extraction in Experiment Example 1;

[0088] Figure 36 The chromatogram of 50% methanol extraction in Experiment Example 1;

[0089] Figure 37 The chromatogram of the 30% methanol extraction in Experiment Example 1;

[0090] Figure 38 This is the characteristic spectrum of the control in Experiment Example 1; where peak 1: caffeic acid; peak 3: luteolin-7-O- β -D-glucuronide; Peak 4: verbascoside; Peak 7: heterologous cimicifugin; Peak 10: luteolin; Peak 11: apigenin;

[0091] Figure 39 This is a comparison chromatogram of the test sample and the reference sample in Experiment Example 1;

[0092] Figure 40 The results show the comparison of the characteristic spectra of the freeze-dried powder of the standard decoction of Artemisia annua and the freeze-dried powder of Artemisia annua in Example 2.

[0093] Figure 41 The results of the comparison of the characteristic spectra of the freeze-dried powder of the standard decoction of Artemisia annua and the freeze-dried powder of Clematis chinensis in Example 2 are shown.

[0094] Figure 42 The results of the comparison of the characteristic spectra of the freeze-dried powder of the standard decoction of Artemisia annua and the freeze-dried powder of Gynostemma pentaphyllum in Example 2 are shown.

[0095] Figure 43 The thin-layer identification chromatogram obtained in Example 3; wherein, 1: verbascoside; 2: luteolin-7-O- β 3: D-glucuronide; 4: Test sample; 5: Reference herb.

[0096] Figure 44 The thin-layer identification chromatogram obtained in Example 4; wherein, 1: verbascoside; 2: luteolin-7-O- β 3: D-glucuronide; 4: Test sample; 5: Reference herb.

[0097] Figure 45 Thin-layer chromatography comparison results between the freeze-dried powder of the standard decoction of Artemisia capillaris and the freeze-dried powder of Artemisia capillaris; where 1: verbascoside; 2: luteolin-7-O-β-D-glucuronide; 3: Artemisia capillaris reference material; 4-6: freeze-dried powder of the standard decoction of Artemisia capillaris; 7-9: freeze-dried powder of the standard decoction of Artemisia capillaris.

[0098] Figure 46 Thin-layer chromatography comparison results of freeze-dried powder of *Artemisia annua* standard decoction and freeze-dried powder of *Gnaphalium affine*; where 1: verbascoside; 2: luteolin-7-O-β-D-glucuronide; 3: *Artemisia annua* reference material; 4-6: freeze-dried powder of *Artemisia annua* standard decoction; 7-9: freeze-dried powder of *Gnaphalium affine* standard decoction;

[0099] Figure 47Thin-layer chromatography comparison results of freeze-dried powder of *Artemisia annua* standard decoction and freeze-dried powder of *Gnaphalium affine*. Among them, 1: verbascoside; 2: luteolin-7-O-β-D-glucuronide; 3: *Artemisia annua* reference material; 4-6: freeze-dried powder of *Artemisia annua* standard decoction; 7-9: freeze-dried powder of *Gnaphalium affine* standard decoction.

[0100] Figure 48 The thin-layer identification chromatogram obtained in Comparative Example 1; where 1: verbascoside; 2: luteolin-7-O- β 3: D-glucuronide; 4: Test sample; 5: Reference herb.

[0101] Figure 49 The thin-layer identification chromatogram obtained in Comparative Example 2; where 1: verbascoside; 2: luteolin-7-O- β 3: D-glucuronide; 4: Test sample; 5: Reference herb.

[0102] Figure 50 The thin-layer identification chromatogram obtained in Experimental Example 3; where, 1: negative control; 2: verbascoside (1µl); 3: verbascoside (2µl); 4: verbascoside (4µl); 5: luteolin-7-O- β -D-glucuronide (1µl); 6: Luteolin-7-O- β -D-glucuronide (2µl); 7: Luteolin-7-O- β 8: D-glucuronide (3µl); 9: Control material (1µl); 10: Control material (2µl); 11: Test sample (1µl); 12: Test sample (2µl); 13: Test sample (3µl);

[0103] Figure 51 The image shows the thin-layer identification chromatogram obtained in Experimental Example 3; where 1: verbascoside (3µl); 2: luteolin-7-O-β-D-glucuronide (3µl); 3: reference medicinal material (1µl); 4: reference medicinal material (2µl); 5: test sample (2µl). Figure 1-42 The horizontal and vertical columns represent elution time, in minutes. Detailed Implementation

[0104] The following embodiments are provided to better understand the present invention and are not intended to limit the preferred embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0105] Preparation of standard decoction freeze-dried powders: Freeze-dried powders of *Artemisia capillaris*, *Artemisia capillaris*, *Gynostemma pentaphyllum*, and *Gynostemma pentaphyllum* were prepared using *Artemisia capillaris* slices, *Artemisia capillaris* slices, *Gynostemma pentaphyllum* slices, and *Gynostemma pentaphyllum* slices, respectively, as raw materials, employing conventional methods in this field. ， For example, the present invention is prepared according to the following steps: Take an appropriate amount of medicinal slices, soak for 30 minutes, decoct twice. For the first decoction, add 12 times the amount of water to the medicinal slices (the mass ratio of medicinal slices to water is 1:12), bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. For the second decoction, add 10 times the amount of water to the medicinal slices (the mass ratio of medicinal slices to water is 1:10), bring to a boil over high heat (500W), then simmer over low heat (200W) for 25 minutes. Combine the filtrates, concentrate (65℃) until the mass ratio of material to liquid is about 1:1 (relative density is 1.02-1.12 (60℃)), freeze dry, and the product is obtained.

[0106] Experiment Example 1: Examination of Construction Methods

[0107] 1. Instruments and reagents

[0108] 1.1 Instruments and Equipment

[0109] Table 1. Statistics on Instruments and Equipment

[0110]

[0111] 1.2 Reagents and Test Chemicals

[0112] Table 2. Statistics of Reagents and Test Items

[0113]

[0114] 2. Preparation of the test solution

[0115] Using the freeze-dried powder of the standard decoction of Artemisia annua as the test sample, take about 0.3g of the test sample powder, accurately weigh it, place it in an Erlenmeyer flask, accurately add 25ml of 85% methanol, weigh it, sonicate (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, shake it well, filter it, and take the filtrate to obtain the test sample.

[0116] 3. Optimization of chromatographic conditions

[0117] (1) Optimization experiment of mobile phase gradient

[0118] The test solution prepared according to the method described in section 2 of this embodiment was injected into a high-performance liquid chromatograph and detected under the following chromatographic conditions:

[0119] Method 1: A CORTECS® UPLC® T3 (2.1 × 100 mm, 1.6 μm) column was used with methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, following the gradient elution specified in the table below; the flow rate was 0.25 mL / min; the column temperature was 25 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. Results are shown below. Figure 1 As shown, the chromatographic peak tailed and without baseline separation occurred at 25-35 minutes.

[0120] Method 2: Using a CORTECS® UPLC® T3 (2.1 × 100 mm, 1.6 μm) column, with methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.25 mL / min; the column temperature was 25 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. Results are as follows: Figure 2 As shown, the chromatographic peak splits at 6-8 minutes, and the chromatographic peak tails and does not show baseline separation at 25-32 minutes.

[0121] Method 3: Using a CORTECS® UPLC® T3 (2.1 × 100 mm, 1.6 μm) column, with methanol as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.25 mL / min; the column temperature was 25 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. Results are as follows: Figure 3 As shown, the chromatographic peak splits at 6-8 minutes, and the chromatographic peak does not show baseline separation at 25-32 minutes. After further adjustment of the elution gradient, a chromatogram with good separation effect was still not obtained.

[0122] Method 4: Using an Agilent Poroshell 120 EC-C18 (2.1 × 100 mm, 1.9 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 mL / min; the column temperature was 30 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. Results are as follows: Figure 4 As shown, there are basically no chromatographic peaks from 0 to 5 minutes, while the chromatographic peaks are dense from 15 to 25 minutes, indicating poor separation.

[0123] Method 5: Using an Agilent Poroshell 120 EC-C18 (2.1×100mm, 1.9μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 mL / min; the column temperature was 30℃; the detection wavelength was 310 nm; and the injection volume was 2 µL. The results are as follows: Figure 5 As shown, the chromatographic peak separation was poor after 30 minutes. After further adjustments to the elution gradient, a chromatogram with good separation was still not obtained.

[0124] Method Six: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.4 mL / min; the column temperature was 30 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. Results are as follows: Figure 6 As shown, the chromatogram contains rich chromatographic information and the resolution of each chromatographic peak is significantly improved, but the baseline is unstable and the resolution of characteristic peaks is poor due to the excessively long acquisition time.

[0125] Method 7: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.4 mL / min; the column temperature was 40 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. Results are as follows: Figure 7 As shown, the chromatogram contains rich chromatographic information, but the characteristic peaks exhibit overlapping peaks, indicating a long acquisition time.

[0126] Method 8: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.5 mL / min; the column temperature was 40 °C; the detection wavelength was 310 nm; and the injection volume was 2 µL. The results are as follows: Figure 8 As shown, the chromatogram contains abundant chromatographic information, but the number of characteristic peaks is relatively small, and there are cases of overlapping peaks among the characteristic peaks.

[0127] Method 9: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.8 mL / min; the column temperature was 40 °C; the detection wavelength was 310 nm; and the injection volume was 5 µL. The results are as follows: Figure 9As shown, when the flow rate is increased to 0.8 ml per minute, no chromatographic peaks appear after 20-30 minutes, the number of characteristic peaks is small, and there are cases of characteristic peaks overlapping.

[0128] Method 10: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.8 mL / min; the column temperature was 40 °C; the detection wavelength was 310 nm; and the injection volume was 5 µL. The results are as follows: Figure 10 As shown, there may be more than one chromatographic peak in the 21-minute chromatographic peak, indicating poor separation.

[0129] Method 11: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.8 mL / min; the column temperature was 30 °C; the detection wavelength was 310 nm; and the injection volume was 5 µL. Results are as follows: Figure 11 As shown, an additional chromatographic peak appears at 24-25 minutes, but the separation of the chromatographic peaks at 13 minutes and 33 minutes is poor.

[0130] Method 12: Using an Agilent Poroshell 120 EC-C18 (4.6 × 150 mm, 2.7 μm) column, with acetonitrile mobile phase A and 0.1% glacial acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.7 mL / min; the column temperature was 30 °C; the detection wavelength was 310 nm; and the injection volume was 5 µL. Results are as follows: Figure 12 As shown, the chromatogram contains rich chromatographic information, a large number of characteristic peaks, good separation of each characteristic peak, short detection time, stable baseline, and good peak shape.

[0131] Table 3 Gradient conditions for methods one through three

[0132]

[0133] Table 4. Gradient conditions for methods four through six

[0134]

[0135] Table 5 Gradient conditions for methods 7 to 9.

[0136]

[0137] Table 6 Gradient conditions for methods 10 to 12

[0138]

[0139] like Figure 1-12 As shown, compared with other methods, the number of characteristic peaks in methods six, eleven, and twelve is significantly increased, and the overall separation effect is significantly improved, especially method twelve.

[0140] (2) Experiments on different detection wavelengths

[0141] The lyophilized powder of *Artemisia annua* standard decoction was prepared according to the method described in section 2 of this experimental example to obtain a test solution of the lyophilized powder of *Artemisia annua* standard decoction. The chromatographic conditions of method 12 in section 3(1) of this experimental example were used to detect the lyophilized powder test solution of *Artemisia annua* standard decoction, with the only difference being the detection wavelength. The effects of different wavelengths (230 nm, 254 nm, 270 nm, 290 nm, 310 nm (reference wavelength), 350 nm) on the robustness of the characteristic chromatogram of the lyophilized powder of *Artemisia annua* standard decoction were compared. The results are shown in […]. Figure 13-18 As shown.

[0142] The results showed that the number of chromatographic peaks did not differ significantly within the detection wavelength range of 210–230 nm, but the baseline fluctuations were substantial. Compared to 280–350 nm, the number of chromatographic peaks did not differ significantly between the 250–270 nm and 280–350 nm ranges, but the latter was more representative of the chemical components of *Artemisia capillaris*. The chromatographic peak at 310 nm showed stronger absorption intensity than those at 290 nm and 350 nm; therefore, 310 nm was the preferred detection wavelength for the characteristic chromatogram of the *Artemisia capillaris* standard decoction.

[0143] (3) Investigation experiment of mobile phase A

[0144] The test solution prepared according to the method in section 2 of this experimental example was injected into a high-performance liquid chromatograph. Methanol and acetonitrile were used as mobile phase A, respectively, and the remaining samples were detected under the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results are shown in […]. Figure 19-20 As shown.

[0145] The results showed that both mobile phase systems had a significant impact on each chromatographic peak. The chromatogram eluted by the acetonitrile-0.1% glacial acetic acid system had richer chromatographic information, more major chromatographic peaks, better resolution, and a more stable baseline. Therefore, the acetonitrile-0.1% glacial acetic acid mobile phase system with relatively better separation performance was selected for subsequent condition screening and investigation in order to obtain better separation results.

[0146] (4) Experiment to investigate the types of acids in the mobile phase

[0147] The test solution prepared according to the method in section 2 of this experimental example was injected into a high-performance liquid chromatograph. Mobile phase B was prepared using 0.1% phosphoric acid, 0.1% glacial acetic acid, and 0.1% formic acid, respectively. The remaining samples were detected under the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results are shown in the table below. Figure 21-23 As shown.

[0148] Table 7 Applicability parameters for different acid selection systems

[0149]

[0150] By comparing the chromatograms and system suitability parameters, compared with 0.1% glacial acetic acid, 0.1% phosphoric acid has one less chromatographic peak after 22 minutes, which is suspected to be two chromatographic peaks that are not separated and are completely enclosed. 0.1% formic acid has two chromatographic peaks that are not separated between 17 and 19 minutes, and the chromatographic peak (peak 8) at 17.878 minutes is the content determination chromatographic peak. Therefore, 0.1% glacial acetic acid is preferred.

[0151] (5) Experiment to investigate the concentration of glacial acetic acid

[0152] The test solution prepared according to the method in section 2 of this experimental example was injected into a high-performance liquid chromatograph. 0.05% glacial acetic acid and 0.15% glacial acetic acid were used as mobile phase B, respectively. The remaining chromatographic conditions were determined according to method 12 in section 3(1) of this experimental example. The results are shown in [Figure 1]. Figure 24-25 And as shown in the table below, and compared with the characteristic spectrum obtained using 0.1% glacial acetic acid (see...). Figure 21 The parameters were compared with the system adaptability parameters (see Table 7).

[0153] Table 8. Applicability parameters of the system for different glacial acetic acid concentrations.

[0154]

[0155] The results show that, compared to other concentrations, the chromatogram produced by elution with acetonitrile-0.1% glacial acetic acid exhibits better system suitability parameters for the main chromatographic peaks and a more stable baseline. Therefore, acetonitrile-0.1% glacial acetic acid, with its relatively better separation effect, is the preferred choice for subsequent condition screening and evaluation.

[0156] (6) Experiment on column temperature

[0157] The test solution prepared according to the method in section 2 of this experimental example was injected into a high-performance liquid chromatograph, and the column temperatures were 25°C and 35°C, respectively. The other chromatographic conditions were determined according to the chromatographic conditions in section 3(1) of this experimental example. The results are shown in [Figure 1]. Figure 26-27 As shown, and compared with the characteristic spectrum obtained at 30℃ (see...). Figure 21 The parameters were compared with the system adaptability parameters (see Table 7).

[0158] Table 9. Applicability parameters of the system for different column temperatures.

[0159]

[0160] The results showed that the separation of chromatographic peaks and system suitability parameters (including resolution and peak area) at a column temperature of 30℃ were not significantly different from those at 25℃, and were even better than those at 35℃. Therefore, a column temperature of 25-30℃ is preferred. Furthermore, the peak shape of chromatographic peak 12 was optimal at a column temperature of 30℃. Therefore, a column temperature of 30℃ was selected as the optimal temperature for subsequent screening and evaluation of the standard decoction (lyophilized powder).

[0161] (7) Experiment on flow velocity

[0162] The test solution prepared according to the method in section 2 of this experimental example was injected into the high-performance liquid chromatograph at flow rates of 0.6 ml / min and 0.8 ml / min, respectively. The remaining samples were detected under the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results are shown in [Figure 1]. Figures 28-29 As shown, and compared with the characteristic spectrum obtained using 0.7 ml / min (see...). Figure 21 The parameters were compared with the system adaptability parameters (see Table 7).

[0163] Table 10 Applicability parameters of the system for different flow velocities

[0164]

[0165] The results showed that different flow rates had no significant effect on the suitability parameters of the chromatogram system obtained by this method (including resolution, peak area, tailing factor, etc.). 0.6-0.8 ml / min can be selected, and 0.7 ml / min is tentatively set for subsequent condition investigation.

[0166] (8) Investigation of different chromatographic columns

[0167] The test solution prepared according to the method in section 2 of this experimental example was injected into a high-performance liquid chromatograph, using the following columns: Agilent InfinityLab Poroshell 120 SB-C18 2.7μm, 150×4.6mm and Agilent InfinityLab Poroshell HPH-C18 2.7μm, 100×3.0mm. The other chromatographic conditions were determined according to method 12 in section 3(1) of this experimental example. The results are shown in [Figure 1]. Figure 30 and 31 The characteristic maps obtained were compared with those obtained using the Agilent Infinity LabPoroshell 120 EC-C18 2.7μm laser (see...). Figure 21 The parameters were compared with the system adaptability parameters (see Table 7).

[0168] Table 11 System suitability parameters for chromatographic peaks obtained from different chromatographic columns

[0169]

[0170] The results showed that the number of peaks on the Agilent InfinityLab Poroshell 120 SB-C18 and Agilent InfinityLab Poroshell 120 EC-C18 columns was significantly better than that on the Agilent InfinityLab Poroshell HPH-C18 column. The peak shape and other characteristics of the chromatograms obtained from the Agilent InfinityLab Poroshell 120 EC-C18 column were also superior to those obtained from the Agilent InfinityLab Poroshell 120 SB-C18 column. Therefore, the Agilent InfinityLab Poroshell 120 EC-C18 column is preferred.

[0171] (9) Investigation using different instruments

[0172] The test solution prepared according to the method under section 2 of this experimental example was injected into a high-performance liquid chromatograph using an Agilent chromatograph. The remaining samples were detected under the chromatographic conditions of method 12 under section 3(1) of this experimental example, and compared with the characteristic chromatogram obtained using a Water chromatograph (see...). Figure 21 The parameters were compared with the system adaptability parameters (see Table 7).

[0173] The results show that different instruments have no significant impact on the suitability parameters of the chromatogram system obtained by this method (including resolution, peak area, tailing factor, etc.), indicating good robustness.

[0174] 4. Preparation of the test solution

[0175] (1) Investigation of extraction solvent

[0176] Methanol and ethanol were used as extraction solvents, respectively. All other solutions were prepared according to the method described in section 2 of this experimental example, and the chromatographic conditions were determined according to method 12 in section 3(1) of this experimental example. The results are as follows: Figure 32 and 33 As shown, when methanol is used as the extraction solvent, the peak areas of each characteristic peak are significantly higher than those of ethanol. Apart from this, there are no significant differences in the system suitability parameters (including resolution, tailing factor, etc.) of each chromatographic peak between the two extraction solvent systems; therefore, methanol is the preferred extraction solvent.

[0177] (2) Investigation of methanol concentration

[0178] Extraction solvents were 85% methanol, 70% methanol, 50% methanol, and 30% methanol, respectively. All other solvents were prepared according to the method described in section 2 of this experimental example. Chromatographic analysis was performed under the conditions of method 12 in section 3(1) of this experimental example. The results are shown in 34-37 and compared with the characteristic chromatograms obtained using methanol (see [reference]). Figure 32 The results showed that when extracted with 100% methanol, 85% methanol, and 70% methanol, the peak area and resolution of each chromatographic peak did not change significantly. However, when the methanol concentration was reduced to 50%, the peak areas of peaks 15 and 16 decreased significantly or even disappeared. Based on the results of the content determination, methanol with a concentration of 70% or higher was selected as the preferred extraction solvent.

[0179] (3) Investigation of extraction method, extraction time and sample concentration

[0180] The effects of different extraction methods on the standard decoction of Artemisia annua (lyophilized powder) were investigated: ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes and reflux for 30 minutes on the extraction effect of the standard decoction of Artemisia annua (lyophilized powder). The test solutions prepared according to the method in item 2 of this experimental example were determined according to the chromatographic conditions of method 12 in item 3(1) of this experimental example. The results showed that the extraction method had no significant effect on the information content of the chromatographic peaks and the system suitability parameters (including resolution, peak area, tailing factor, etc.). Both ultrasonic extraction and reflux extraction were feasible. For ease of operation, ultrasonic extraction was selected as the extraction method for subsequent investigation.

[0181] (4) Examination of extraction time

[0182] The ultrasonic time was examined for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively. For the rest, the test solution was prepared according to the method in item 2 of this experimental example and determined according to the chromatographic conditions of method 12 in item 3(1) of this experimental example.

[0183] The results showed that the extraction time had no significant effect on the information content of the chromatographic peaks and the system suitability parameters (including resolution, peak area, tailing factor, etc.). Ultrasonic time of 15-60 minutes was feasible. In order to ensure complete extraction, ultrasonic time of 30 minutes was selected as the extraction time for subsequent investigation.

[0184] (5) Examination of ultrasonic power

[0185] The effects of ultrasonic treatment power (① 210W, 40kHz; ② 240W, 40kHz; ③ 300W, 40kHz) on the extraction effect of *Artemisia capillaris* standard decoction (lyophilized powder) were investigated. All other test solutions were prepared according to the method described in section 2 of this experimental example and determined under the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results showed that at ultrasonic power of 210W, peak 6 had a smaller peak area (not integrated); ultrasonic power of 240W and 300W did not significantly affect the information content of the chromatographic peaks or the system suitability parameters (including resolution, peak area, tailing factor, etc.). For complete extraction, an extraction power of 240-300W was preferred for subsequent investigation.

[0186] (5) Investigation of the amount of solvent added

[0187] The effects of solvent addition volumes of 10 ml, 25 ml, 50 ml, and 100 ml on the extraction efficiency of *Artemisia annua* standard decoction (lyophilized powder) were investigated. All other test solutions were prepared according to the method described in section 2 of this experimental example and analyzed under the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results showed that different solvent volumes effectively separated all characteristic peaks in *Artemisia annua* standard decoction (lyophilized powder), significantly affecting the peak response. Solvent addition volumes of 10-100 mL were feasible. Considering that a solvent volume of 25 ml resulted in a relatively moderate peak response, 25 ml of 70% methanol solution was chosen as the solvent volume for this experiment.

[0188] (6) Examination of sampling size

[0189] The effects of sample amounts of 0.1 g, 0.2 g, 0.4 g, and 0.8 g on the extraction efficiency of *Artemisia annua* standard decoction (lyophilized powder) were investigated during the preparation of the test solution. All other test solutions were prepared according to the method described in section 2 of this experimental example and determined under the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results showed that all characteristic peaks in *Artemisia annua* standard decoction (lyophilized powder) could be effectively separated under different sample amounts. With the increase of sample amount, the peak area of ​​the chromatographic peaks increased proportionally, indicating that when the sample amount was between 0.1 and 0.8 g, the components in *Artemisia annua* standard decoction (lyophilized powder) could be basically completely extracted. Based on the overall response values ​​of the characteristic peaks, 0.2 g was selected as the optimal sample amount for this experiment.

[0190] (7) Investigation of injection volume

[0191] The test solution prepared according to the method in section 2 of this experimental example was examined for the separation effect of different injection volumes of the test solution: 5 μl, 10 μl, 15 μl, and 20 μl. The remaining samples were determined according to the chromatographic conditions of method 12 in section 3(1) of this experimental example. The results showed that there was no significant difference in the system suitability parameters (including resolution, peak area, tailing factor, etc.) of the chromatographic peaks obtained with different injection volumes of the test solution. However, with the increase of injection volume, the peak area of ​​the chromatographic peaks in the test sample increased relatively, and the non-characteristic peaks became more obvious, resulting in a slight accumulation of chromatographic peaks in the chromatogram. Among them, the 10 μl peak had the best peak shape, the best resolution of the chromatographic peak, and the peak height and peak width were relatively moderate. Therefore, the preferred injection volume was 10 μl.

[0192] 5. Determination of characteristic peaks and establishment of reference spectra

[0193] (1) Construction method

[0194] Preparation method of test solution: Test solution and reference solution were prepared from 18 batches of *Artemisia annua* standard decoction lyophilized powder and reference medicinal material according to the method in Example 1. The test solution and reference solution were then subjected to high-performance liquid chromatography (HPLC) for detection, under the same chromatographic conditions as in Example 1.

[0195] Characteristic chromatograms of 18 batches of *Artemisia annua* standard decoction lyophilized powder were obtained. Using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, control characteristic chromatograms were generated from the 18 batches of *Artemisia annua* standard decoction lyophilized powder. (See attached image.) Figure 38 As shown. The characteristic chromatograms of 18 batches of *Artemisia annua* standard decoction (lyophilized powder) showed a total of 11 chromatographic peaks. Peaks 3, 4, and 10 are respectively associated with luteolin-7-O- β The retention times of the reference peaks for -D-glucuronide, verbascoside, and luteolin correspond to those of the standard references. The peak corresponding to the verbascoside reference peak is peak S1. The relative retention times of peaks 1-2 and 5-8 with peak S1 are calculated to be 0.29 (peak 1), 0.52 (peak 2), 1.06 (peak 5), 1.12 (peak 6), 1.19 (peak 7), and 1.21 (peak 8). The peak corresponding to the luteolin reference peak is peak S2. The relative retention times of peaks 9 and 11 with peak S2 are calculated to be 0.84 (peak 9) and 1.14 (peak 11). The characteristic chromatogram results of 18 batches of *Artemisia annua* standard decoction lyophilized powder show that the 11 common peaks have good transferability, and the relative retention times of each characteristic peak in the 18 batches of *Artemisia annua* standard decoction lyophilized powder are all within ±10% of the specified values.

[0196] Table 12. Results of relative retention time determination of characteristic spectra of 18 batches of Bei Liu Ji Nu standard decoction (lyophilized powder)

[0197]

[0198] The results are as follows Figure 4-8 As shown, where Figure 4 In the figure, S1(11)~S18(11) are the characteristic chromatograms of freeze-dried powder of 18 batches of Bei Liu Ji Nu standard decoction, and R(11) is the control characteristic chromatogram.

[0199] (2) Identification of characteristic peaks

[0200] Preparation of the reference solution: Accurately weigh appropriate amounts of luteolin, verbascoside, luteolin-7-O-β-D-glucuronide, caffeic acid, apigenin-7-O-β-D-glucuronide, apigenin, and iso-citricascoside, and add methanol to prepare a solution containing 10 µg luteolin, 40 µg verbascoside, 20 µg luteolin-7-O-β-D-glucuronide, 10 µg caffeic acid, 20 µg apigenin-7-O-β-D-glucuronide, 10 µg apigenin, or 20 µg iso-citricascoside per mL of reference solution.

[0201] The lyophilized powder test solution of the *Artemisia annua* standard decoction was prepared according to the preparation method of Example 1. The reference solutions and the lyophilized powder test solution of the *Artemisia annua* standard decoction were then analyzed by high-performance liquid chromatography (HPLC) according to the method of Example 1 and compared. The results are as follows: Figure 39 .

[0202] Summary: In the chromatogram of the freeze-dried powder of the standard decoction of Artemisia annua, peak 1 is caffeic acid, peak 3 is luteolin-7-O-β-D-glucuronide, peak 4 is verbascoside, peak 6 is apigenin-7-O-β-D-glucuronide, peak 7 is iso-citric acid, peak 10 is luteolin, and peak 11 is apigenin.

[0203] The characteristic peaks were identified and assigned using LC / MS / MS. The results showed that peak 2 was (S)-Suspensaside or its isomer, peak 5 was Bacopaside B or its isomer, peak 8 was Crenatoside, and peak 9 was Isocrenatoside.

[0204] Table 13 LC / MS / MS Analysis Results of *Liu Jinu* (a type of *Pleurotus ostreatus*)

[0205]

[0206] Experiment Example 2: Methodological Validation

[0207] 1. Precision

[0208] (1) Precision

[0209] Take the same sample solution of the standard decoction of *Ligusticum striatum* (lyophilized powder) prepared according to the method of Example 1, and inject it 6 times repeatedly under the chromatographic conditions of Example 1. Measure the relative retention time and relative peak area of ​​the above 11 characteristic peaks, and calculate the RSD%. The RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area of ​​each characteristic peak is less than 3%, indicating that the instrument has good precision.

[0210] (2) Repeatability experiment

[0211] Six test solutions were prepared by repeating the method of Example 1 using the same batch of Bei Liu Ji Nu standard decoction (lyophilized powder). The solutions were injected and analyzed under the chromatographic conditions of Example 1. The relative retention time and relative peak area of ​​the above 11 characteristic peaks were determined and the RSD% was calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2% and the RSD of the relative peak area of ​​each characteristic peak was less than 3%, indicating that the method has good repeatability.

[0212] (3) Different personnel (intermediate precision)

[0213] Three individuals, A, B, and C, each took two portions of the same batch of *Artemisia annua* standard decoction (lyophilized powder) and prepared test solutions according to the method in Example 1. The solutions were then analyzed on the same instrument under the chromatographic conditions of Example 1. The relative retention times and relative peak areas of the 11 characteristic peaks were measured, and the RSD% was calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, and the RSD of the relative peak area of ​​each characteristic peak was less than 2%, indicating that the method has good repeatability.

[0214] (4) Intermediate precision of different instruments

[0215] Six portions of the same batch of Bei Liu Ji Nu standard decoction (lyophilized powder) were taken by different inspectors at different times. Six test solutions were prepared repeatedly according to the method in Example 1. The samples were injected and analyzed according to the chromatographic conditions in Example 1. The relative retention time and relative peak area of ​​the above 11 characteristic peaks were determined and the RSD% was calculated. The results showed that the RSD value of the relative retention time of each characteristic peak was less than 2%, indicating good intermediate precision of different instruments.

[0216] 2. Stability

[0217] The same sample solution of *Artemisia annua* standard decoction (lyophilized powder) prepared according to the method of Example 1 was injected at 0, 2, 4, 8, 12, 18, 24, 36, and 48 hours, respectively, and the relative retention time and relative peak area of ​​the above 11 characteristic peaks were determined according to the chromatographic conditions of Example 1. The RSD% was calculated. The results showed that the RSD value of the relative retention time of each characteristic peak was less than 2%, indicating that the sample solution was stable within 48 hours and met the determination requirements.

[0218] 3. Exclusivity

[0219] Accurately weigh the standard decoction of Artemisia annua (lyophilized powder) and prepare the test solution according to the method in Example 1. Accurately pipette 10 μL each of the test solution and the blank solvent (70% methanol) and inject them into the high-performance liquid chromatograph. Perform the test according to the method in Example 1. The results show that the blank solvent has no interference.

[0220] Example 1

[0221] This embodiment provides a method for constructing the characteristic spectrum of freeze-dried powder of Bei Liu Ji Nu standard decoction, including the following steps:

[0222] Preparation of the test solution: Take about 0.2g of the test sample powder, accurately weigh it, place it in an Erlenmeyer flask, accurately add 25ml of 70% methanol, weigh it, sonicate (power 300W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0223] Preparation of the reference solution: Take appropriate amounts of luteolin, verbascoside, and luteolin-7-O-β-D-glucuronide, accurately weigh them, and add methanol to prepare a solution containing 20 µg luteolin, 40 µg verbascoside, and 10 µg luteolin-7-O-β-D-glucuronide per mL of reference solution.

[0224] Reference solution for the reference herb: Take 1g of Artemisia capillaris reference herb, accurately weigh it, place it in an Erlenmeyer flask, add 50ml of water, heat under reflux for 30 minutes, remove it, centrifuge, and evaporate the supernatant under reduced pressure at 50~65℃. Accurately add 25ml of 70% methanol, weigh it, and sonicate it (300W power, 40 kHz frequency) for 30 minutes. Cool it, weigh it again, and replenish the lost weight with 70% methanol. Shake well, filter it, and collect the filtrate to obtain the solution.

[0225] High-performance liquid chromatography (HPLC) detection: Test solution, reference solution, and reference herb solution were analyzed separately. Chromatographic conditions were as follows: Octadecylsilane-bonded silica gel (Poroshell 120 EC-C18 (150 mm × 4.6 mm, 2.7 µm)) was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% glacial acetic acid solution was used as mobile phase B, with gradient elution as specified in the table below; flow rate was 0.7 mL / min; column temperature was 30 °C; detection wavelength was 310 nm; the theoretical plate number, calculated based on the verbascoside peak, should not be less than 3000.

[0226]

[0227] Table 14 Suitability Parameters of Reference Standard System

[0228]

[0229] Table 15. Systemic Suitability Parameters for Comparative Medicinal Materials

[0230]

[0231] Table 16 System Suitability Parameters for Test Samples

[0232]

[0233] The results showed that the chromatogram of the test sample contained 11 characteristic peaks, which should correspond to the 11 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 4, and 10 should correspond to the retention times of the reference peaks of luteolin-7-O-β-D-glucuronide, verbascoside, and luteolin, respectively. The peak corresponding to the verbascoside reference peak is designated as peak S1. The relative retention times of peaks 1-2 and 5-8 with peak S1 were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are 0.29 (peak 1), 0.52 (peak 2), 1.06 (peak 5), 1.12 (peak 6), 1.19 (peak 7), and 1.21 (peak 8). The peak corresponding to the luteolin reference peak is designated as peak S2. The relative retention times of peaks 9 and 11 with peak S2 were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are 0.84 (peak 9) and 1.14 (peak 11).

[0234] Example 2

[0235] Common adulterants of *Artemisia anomala* (Northern Artemisia) include *Arfemisia anomala* S. Moore (Asteraceae family), *Hypericum sampsonii* Hance (Hypericaceae family), and *Hypericum japonicum* Thunb. (Dried whole herb). The medicinal parts and effects of *Artemisia anomala* differ from these adulterants, and they should not be used interchangeably.

[0236] This embodiment provides a method for identifying pharmaceutical preparations containing Artemisia capillaris, comprising the following steps:

[0237] Preparation of test solutions: Test solutions were prepared according to the method in Example 1 using three batches of freeze-dried powder of *Artemisia annua* standard decoction from the north, three batches of freeze-dried powder of *Artemisia annua* standard decoction from the south, three batches of freeze-dried powder of *Gnaphalium affine* standard decoction, and three batches of freeze-dried powder of *Gnaphalium affine* standard decoction.

[0238] Detection by high performance liquid chromatography: Take the test solution separately for detection, and the chromatographic conditions are the same as in Example 1.

[0239] See results Figures 40-42 As shown, the chromatogram of the lyophilized powder of *Artemisia annua* standard decoction exhibits 11 characteristic peaks. The chromatograms of *Artemisia annua* and its lyophilized powder show only one peak corresponding to the lyophilized powder of *Artemisia annua* standard decoction (peak 1: caffeic acid). The chromatogram of *Artemisia annua* lyophilized powder with a retention time similar to characteristic peak 4 is determined by spectral analysis to be different compounds. The chromatogram of *Gnaphalium affine* and its lyophilized powder shows only one peak corresponding to the lyophilized powder of *Artemisia annua* standard decoction (peak 1: caffeic acid). The chromatogram of *Gnaphalium affine* lyophilized powder with a retention time similar to characteristic peaks 2, 8, and 10 is determined by spectral analysis to be different compounds. The chromatogram of *Gnaphalium affine* and its lyophilized powder shows only one peak corresponding to the lyophilized powder of *Artemisia annua* standard decoction (peak 1: caffeic acid). The chromatogram of *Gnaphalium affine* and its lyophilized powder with a retention time similar to characteristic peak 10 is determined by spectral analysis to be different compounds. Therefore, the identification method of the present invention can distinguish the freeze-dried powder of the standard decoction of Artemisia annua from its common adulterants (Artemisia annua, Diercao, Yuanbaocao).

[0240] Example 3

[0241] This embodiment provides a method for constructing a thin-layer chromatography (TLC) of a *Ligustrum lucidum* (a type of medicinal preparation), comprising the following steps:

[0242] Preparation of test solution: Take 0.2g of freeze-dried powder of Bei Liu Ji Nu standard decoction, add 5ml of methanol, sonicate for 30 minutes, filter, and use as test solution.

[0243] Preparation of control herb solution: Take 1g of Artemisia capillaris control herb, add 50ml of water, heat under reflux for 1 hour, centrifuge, evaporate to dryness under reduced pressure, add 5ml of methanol to the residue, sonicate for 30 minutes, filter, and use as control herb solution.

[0244] Luteolin-7-O- β -D-glucuronide reference solution: Take luteolin-7-O- β -D-glucuronide reference standard, taken in appropriate amount, is added to methanol to prepare a solution containing luteolin-7-O- per ml. β A solution of 50 µg of D-glucuronide was used as a reference solution.

[0245] Verbascoside reference solution: Take an appropriate amount of verbascoside reference standard, add methanol to prepare a solution containing 50 µg of verbascoside per ml, and use it as the reference solution.

[0246] Take 1 μl each of the test solution, the reference herb solution, the reference solution verbascoside, and the luteolin-7-O-β-D-glucuronide reference solution, and spot them separately on the same polyamide thin-layer plate. Develop the plate using methanol-formic acid-water (volume ratio 2:1:5), remove the plate, air dry it, spray it with 1% aluminum trichloride solution, heat it at 105℃ for several minutes, and examine it under ultraviolet light (365nm).

[0247] See results Figure 43 As shown in the results, the chromatogram of the test sample shows that, compared with the reference herb *Artemisia capillaris*, it contains verbascoside, luteolin-7-O- β -D-glucuronide reference standard chromatogram shows the same color spots at the corresponding positions, and the spots are abundant.

[0248] Example 4

[0249] This embodiment provides a method for constructing a thin-layer chromatography (TLC) method for a *Artemisia capillaris* (Northern Liu Artemisia) pharmaceutical preparation, which is basically the same as that in Example 3, except that the volume ratio of the developing solvent, methanol-formic acid-water, is 2:1:3. The results are shown in [Figure 3]. Figure 44 As shown, in the chromatogram of the test sample, spots of the same color are present at the corresponding positions as those of the reference medicinal material Artemisia capillaris, verbascoside, and luteolin-7-O-β-D-glucuronide. The spots are abundant and well separated, and the Rf values ​​of verbascoside and luteolin-7-O-β-D-glucuronide are appropriate.

[0250] Example 5: Identification of Common Adulterants

[0251] This embodiment provides a method for identifying pharmaceutical preparations containing Artemisia capillaris, comprising the following steps:

[0252] Preparation of test solutions: Test solutions were prepared according to the method in Example 4 using three batches of *Artemisia annua* standard decoction lyophilized powder, three batches of *Artemisia annua* (Southern) standard decoction lyophilized powder, three batches of *Gnaphalium affine* standard decoction lyophilized powder, and three batches of *Gnaphalium affine* standard decoction lyophilized powder as test samples. 3 µl of verbascoside reference solution and luteolin-7-O- were respectively taken. β Thin-layer chromatography was constructed by spotting 3 µl of D-glucuronide reference solution, 1 µl of *Artemisia annua* reference medicinal material solution, 2 µl of *Artemisia annua* standard decoction lyophilized powder test solution, and 2 µl of mixed standard decoction lyophilized powder test solution according to the method in Example 4.

[0253] See results Figures 45-47As shown, the results indicate that the lyophilized powder of the *Artemisia annua* standard decoction exhibits fluorescent spots of the same color at the corresponding positions as the reference medicinal material and reference substance. However, the lyophilized powders of *Artemisia annua* standard decoction, *Gnaphalium affine* standard decoction, and *Gnaphalium affine* standard decoction show no fluorescent spots of the same color at the corresponding positions of verbascoside and luteolin-7-O-β-D-glucuronide, and are significantly different from the chromatograms of the *Artemisia annua* reference medicinal material and the lyophilized powder of the *Artemisia annua* standard decoction. Therefore, the identification method of this invention can distinguish the lyophilized powder of the *Artemisia annua* standard decoction from its common adulterants (*Artemisia annua*, *Gnaphalium affine*, and *Gnaphalium affine*).

[0254] Comparative Example 1

[0255] This comparative example provides a method for constructing a thin-layer chromatography of a drug preparation of *Artemisia capillaris*, which is basically the same as that in Example 3, except that the developing solvent is replaced with toluene-ethyl formate-formic acid (volume ratio 5:4:1).

[0256] See results Figure 48 As shown, in the chromatogram of the test sample, spots of the same color are present at the corresponding positions as in the chromatogram of the reference herb, but the chromatogram contains less chromatographic information, with more spots accumulating at the origin and not developing.

[0257] Comparative Example 2

[0258] This embodiment provides a method for constructing a thin-layer chromatography (TLC) method for a *Artemisia capillaris* (Northern Liu Artemisia) pharmaceutical preparation, which is basically the same as that in Example 3, except that the volume ratio of the developing solvent, methanol-formic acid-water, is 2:1:7. The results are shown in [Figure 3]. Figure 49 As shown, in the chromatogram of the test sample, the same color spots appear at the corresponding positions as in the chromatograms of Artemisia capillaris, verbascoside, and luteolin-7-O-β-D-glucuronide reference materials. However, in this chromatogram, luteolin-7-O-β-D-glucuronide did not develop.

[0259] Experimental Example 3: Sample Size and Specificity Investigation

[0260] Preparation of the test solution: Same as in Example 4.

[0261] Preparation of the control herbal solution: Same as in Example 4.

[0262] Luteolin-7-O- β -D-glucuronide reference solution: Same as in Example 4.

[0263] Verbascoside reference solution: Take an appropriate amount of verbascoside reference standard and add methanol to prepare a solution containing 0.15 mg of verbascoside per ml, which is used as the reference solution.

[0264] Preparation of negative control solution: Take 0.2g of maltodextrin, add 5ml of methanol, sonicate for 30 minutes, filter, and use as negative control solution.

[0265] Development and color development: Take 1µl, 2µl, and 3µl of the test solution of *Artemisia annua* standard decoction (lyophilized powder) (batch number: 2108001Y-BT), 1µl, 2µl, and 3µl of the *Artemisia annua* reference material solution, 1µl, 2µl, and 3µl of the verbascoside reference solution, and luteolin-7-O- β 1 µl, 2 µl, and 3 µl of D-glucuronide reference solution and 3 µl of negative control solution were spotted onto the same polyamide thin-layer plate. The plate was developed using methanol-formic acid-water (2:1:3) as the developing solvent. After development, the plate was removed, dried, sprayed with 1% aluminum trichloride solution, heated at 105 °C for several minutes, and examined under ultraviolet light (365 nm).

[0266] See results Figure 50 As shown in the results, the chromatogram of the test sample shows that, compared with the reference medicinal material, verbascoside, and luteolin-7-O- β -D-glucuronide reference standard showed the same color spots at the corresponding positions on the chromatogram, and there was no negative interference, indicating good specificity of the method. Verbascoside 3µl, luteolin-7-O- β When 3 µl of -D-glucuronide and 2 µl of the test sample were used, the spots were distinct. Therefore, verbascoside and luteolin-7-O- were tentatively identified. β The spotting volumes of -D-glucuronide and the test sample were 3 µl, 3 µl, and 2 µl, respectively. When the spotting volume of the control herb was 1 µl, its spot was significantly darker than that of the test sample; therefore, the control herb solution was diluted by half and spotted again. The results are shown below. Figure 51 As shown in the results, the chromatogram of the test sample shows that, compared with the reference medicinal material, verbascoside, and luteolin-7-O- β The D-glucuronide reference standard showed the same color spots at the corresponding positions on the chromatogram, and when the amount of the reference medicinal material was 1µl, each spot was equivalent to that of the test sample. Therefore, the amount of the reference medicinal material was tentatively set at 1µl.

[0267] Experiment Example 4: Methodological Validation

[0268] 1. Investigation at different temperatures

[0269] Take 2 µl of the test solution of the standard decoction (lyophilized powder) of *Artemisia annua* prepared according to Example 3 (batch numbers: 2108001Y-BT, 2108004Y-BT, 2108005Y-BT), 1 µl of *Artemisia annua* reference material solution, 3 µl of verbascoside reference solution, and luteolin-7-O- β3 µl of the -D-glucuronide reference solution was spotted onto the same polyamide thin-layer plate. Methanol-formic acid-water (2:1:3) was used as the developing solvent. The plate was developed at room temperature, low temperature (2-8℃), and high temperature (40℃), respectively. After development, the plate was removed, dried, sprayed with 1% aluminum trichloride solution, heated at 105℃ for several minutes, and examined under ultraviolet light (365nm). The results showed that, under room temperature, low temperature, and high temperature conditions, the chromatogram of the *Artemisia annua* standard decoction (lyophilized powder) was similar to that of the *Artemisia annua* reference material in terms of verbascoside, luteolin-7-O-... β -D-glucuronide reference standard showed spots of the same color at the corresponding positions in the chromatogram, and the chromatographic spots were well separated. The experimental results showed that temperature had no significant effect on the thin-layer identification of Bei Liu Ji Nu standard decoction (lyophilized powder), and the thin-layer identification method had good temperature resistance.

[0270] 2. Investigation of different humidity levels

[0271] Take 2 µl of the test solution of the standard decoction (lyophilized powder) of *Artemisia annua* prepared according to Example 3 (batch numbers: 2108001Y-BT, 2108004Y-BT, 2108005Y-BT), 1 µl of *Artemisia annua* reference material solution, 3 µl of verbascoside reference solution, and luteolin-7-O- β 3 µl of the -D-glucuronide reference solution was spotted onto the same polyamide film layer. Methanol-formic acid-water (2:1:3) was used as the developing solvent, and the mixture was developed under normal humidity and high humidity (75%) conditions. The film was then removed, air-dried, sprayed with 1% aluminum trichloride solution, heated at 105℃ for several minutes, and examined under ultraviolet light (365 nm). The results showed that, under normal humidity and high humidity conditions, the chromatogram of the *Artemisia annua* standard decoction (lyophilized powder) was significantly different from that of the *Artemisia annua* reference material in terms of verbascoside and luteolin-7-O- β -D-glucuronide reference standard showed spots of the same color at the corresponding positions in the chromatogram, and the chromatographic spots were well separated. The experimental results showed that humidity had no significant effect on the thin-layer identification of Bei Liu Ji Nu standard decoction (lyophilized powder), and the thin-layer identification method had good durability against humidity.

[0272] 3. Examination of thin-layer boards from different manufacturers

[0273] Take 2 µl of the test solution of the standard decoction (lyophilized powder) of *Artemisia annua* prepared according to Example 3 (batch numbers: 2108001Y-BT, 2108004Y-BT, 2108005Y-BT), 1 µl of *Artemisia annua* reference material solution, 3 µl of verbascoside reference solution, and luteolin-7-O- β3 µl of the -D-glucuronide reference solution was spotted onto the same polyamide thin-layer plate from different manufacturers. Methanol-formic acid-water (2:1:3) was used as the developing solvent. The plates were removed, dried, sprayed with 1% aluminum trichloride solution, heated at 105℃ for several minutes, and examined under ultraviolet light (365 nm). The results showed that, on polyamide thin-layer plates from different manufacturers, the chromatogram of the *Artemisia annua* standard decoction (lyophilized powder) showed spots of the same color at the corresponding positions as those of the *Artemisia annua* reference medicinal material, verbascoside, and luteolin-7-O-β-D-glucuronide reference standard. The chromatographic spot separation was also good. The experimental results indicate that the polyamide thin-layer plate manufacturer has no significant impact on the thin-layer identification of the *Artemisia annua* standard decoction (lyophilized powder), and this thin-layer identification method has good durability for polyamide thin-layer plate manufacturers.

[0274] 4. Examination of reproducibility

[0275] The test solutions were prepared by different personnel (A and B) according to the method in Example 3. 2 µl of the test solution of *Artemisia annua* standard decoction (lyophilized powder) (batch numbers: 2108001Y-BT, 2108004Y-BT, 2108005Y-BT), 1 µl of *Artemisia annua* reference herb solution, 3 µl of verbascoside reference solution, and luteolin-7-O- β 3 µl of the -D-glucuronide reference solution was spotted onto the same polyamide thin-layer plate from different companies. Methanol-formic acid-water (2:1:3) was used as the developing solvent. The plates were removed, dried, sprayed with 1% aluminum trichloride solution, heated at 105 °C for several minutes, and examined under ultraviolet light (365 nm). The results showed that the chromatograms of the *Artemisia capillaris* standard decoction (lyophilized powder) prepared by different personnel (A and B) differed from those of the *Artemisia capillaris* reference material in terms of verbascoside, luteolin-7-O-... β -D-glucuronide reference standard showed spots of the same color at the corresponding positions in the chromatogram, and the chromatographic spots were well separated. The experimental results show that the thin-layer identification method of Bei Liu Ji Nu standard decoction (lyophilized powder) has good reproducibility.

[0276] Experimental Example 5: Identification of Multiple Batches

[0277] Eighteen batches of *Artemisia annua* standard decoction (lyophilized powder) were prepared according to Example 3, and the test solutions were developed under the thin-layer chromatography conditions of Example 4. The solutions were then removed, dried, and examined under ultraviolet light (365 nm). The results are shown in the figure. The results show that the chromatograms of the 18 batches of *Artemisia annua* standard decoction (lyophilized powder) showed spots of the same color at the corresponding positions as the chromatograms of *Artemisia annua* reference medicinal material, verbascoside, and luteolin-7-O-β-D-glucuronide reference standards, and the chromatographic spot separation was good.

[0278] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of a *Artemisia annua* pharmaceutical preparation, characterized in that, Includes the following steps, Preparation of the test solution; the extraction solvent in the preparation of the test solution is selected from pure methanol, pure ethanol, methanol aqueous solution with a volume concentration of 70% or more, or ethanol aqueous solution with a volume concentration of 70% or more. Determination: The test solution and reference solution were analyzed by high performance liquid chromatography (HPLC). The mobile phase consisted of 0.1% glacial acetic acid aqueous solution and acetonitrile, with gradient elution. The column temperature was 25-35℃, and the detection wavelength was 250-350nm. An Agilent InfinityLab Poroshell 120 EC-C18 column with specifications of 4.6×150mm and 2.7μm was used. The gradient elution program included: 0→15min→30min→45min→50min, and the volume percentage of acetonitrile in the mobile phase was: 15%→15%→23%→36%→40%. The characteristic chromatogram of the *Artemisia annua* preparation was obtained. The construction method further includes dissolving luteolin, verbascoside, luteolin-7-O-β-D-glucuronide, caffeic acid, apigenin, and iso-citric acid in solvents to prepare reference solutions.

2. The construction method according to claim 1, characterized in that, The high-performance liquid chromatography detection process also satisfies: The detection wavelength is 280-350 nm; and / or the flow rate is 0.25-0.8 mL / min; and / or the injection volume is 5-20 μl; and / or the column temperature is 25-30 °C.

3. The construction method according to claim 1, characterized in that, The preparation of the test solution includes the following steps: weigh the test sample, add solvent to extract, obtain the extract, separate the solid and liquid, and take the liquid, which is the test solution.

4. The construction method according to claim 3, characterized in that, The preparation of the test solution also satisfies any one or more of the following AD: A. The ratio of the mass of the test sample to the volume of the solvent is 0.1-0.8:10-100; the relationship between mass and volume is g / mL. B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥15 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

5. The construction method according to claim 4, characterized in that, The ultrasonic power extracted by ultrasound is 210-300W.

6. The construction method according to claim 4, characterized in that, In the preparation of the test solution, the extraction time is 15-60 min.

7. The construction method according to claim 1, characterized in that, Each 1 mL of the reference solution contains 10-100 µg of luteolin, 10-100 µg of verbascoside, 10-100 µg of luteolin-7-O-β-D-glucuronide, 10-100 µg of caffeic acid, 10-100 µg of apigenin, or 10-100 µg of iso-citric acid.

8. The construction method according to claim 1, characterized in that, The solvent used in the preparation of the reference solution is methanol or an aqueous methanol solution.

9. The construction method according to any one of claims 1-7, characterized in that, The characteristic chromatogram of the *Liu Ji Nu* pharmaceutical preparation satisfies at least one of the following (1)-(2): (1) It has 11 common characteristic peaks, with peaks 1, 3, 4, 7, 10, and 11 being caffeic acid, luteolin-7-O-β-D-glucuronide, verbascoside, iso-citric acid, luteolin, and apigenin, respectively; and / or, (2) It has 11 common characteristic peaks. Peaks 3, 4, and 10 correspond to the retention times of the reference peaks of luteolin-7-O-β-D-glucuronide, verbascoside, and luteolin. The peak corresponding to the reference peak of verbascoside is peak S1. The relative retention times of peaks 1-2 and 5-8 with peak S1 are within ±10% of the specified values. The specified values ​​of peaks 1-2 and 5-8 are 0.29, 0.52, 1.06, 1.12, 1.19, and 1.21, respectively. The peak corresponding to the reference peak of luteolin is peak S2. The relative retention times of peaks 9 and 11 with peak S2 are within ±10% of the specified values. The specified values ​​of peaks 9 and 11 with peak S2 are 0.84 and 1.14, respectively.

10. A method for constructing a thin-layer chromatography formula of *Artemisia annua*, characterized in that, Includes the following steps: Preparation of the test solution; the preparation of the test solution includes taking the test sample, extracting it with methanol, filtering it, and obtaining the solution. Thin-layer chromatography was performed on the test solution and the reference solution using methanol-formic acid-water with a volume ratio of 2:1:3-5 as the developing solvent to obtain the thin-layer chromatogram of the *Ligusticum striatum* preparation; polyamide thin-layer plates were used for the detection. It also includes the preparation of reference solutions using verbascoside and luteolin-7-O-β-D-glucuronide reference standards.

11. The construction method according to claim 10, characterized in that, In the thin-layer chromatography detection, any one or more of the following AC conditions are met: A. Spot the test solution onto a thin-layer plate; B, with methanol-formic acid-water in a volume ratio of 2:1:3 as the developing solvent; C. After unfolding and drying, spray with aluminum trichloride solution, heat and then examine under ultraviolet light.

12. The construction method according to claim 11, characterized in that, The sample volume is 1-3 μl.

13. The construction method according to claim 10, characterized in that, The mass ratio of the test sample to the volume of methanol is 0.1-0.3:5; the mass-volume relationship is g / mL.

14. The construction method according to claim 10, characterized in that, The extraction method is ultrasonic extraction.

15. A method for identifying a pharmaceutical preparation of *Artemisia capillaris* from adulterants, characterized in that, The method includes the steps of constructing a characteristic chromatogram of the product to be identified using the construction method according to any one of claims 1-9 and comparing it with the characteristic chromatogram of the *Artemisia annua* drug preparation; or, the steps of constructing a thin-layer chromatography (TLC) chromatogram of the product to be identified using the construction method according to any one of claims 10-14 and comparing it with the TLC chromatogram of the *Artemisia annua* drug preparation, wherein the adulterants are *Artemisia nanmu*, *Gnaphalium affine*, and *Gnaphalium affine*.

16. A quality testing method for a preparation of *Artemisia capillaris*, characterized in that, This includes the steps of constructing a characteristic chromatogram of the product to be tested using the construction method according to any one of claims 1-9 and comparing it with the characteristic chromatogram of the *Artemisia annua* drug preparation; or, the steps of constructing a thin-layer chromatography (TLC) chromatogram of the product to be tested using the construction method according to any one of claims 10-14 and comparing it with the TLC chromatogram of the *Artemisia annua* drug preparation.

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