A method for quality control of ingredients in *Smilax glabra*

Abstract

This invention discloses a method for quality control of *Gynostemma pentaphyllum* components, comprising the following steps: Step 1: Screening for flavonoids and phenolic acids in *Gynostemma pentaphyllum* using liquid chromatography-high-resolution mass spectrometry (LC-MS / MS); Step 2: Determining characteristic flavonoids and phenolic acids in *Gynostemma pentaphyllum* using LC-MS / MS (a); Step 3: Determining characteristic amino acids in *Gynostemma pentaphyllum* using LC-MS / MS (b); Step 4: Performing ionomics analysis to screen for seven mineral elements in *Gynostemma pentaphyllum*, and further determining characteristic mineral elements in *Gynostemma pentaphyllum* based on principal component analysis results; Step 5: Using the characteristic components obtained in steps 1-4 as evaluation indicators for assessing the quality of *Gynostemma pentaphyllum* and for quality control. This method is rapid and highly accurate, enabling objective, comprehensive, and accurate evaluation of the quality of *Gynostemma pentaphyllum*, which is of great significance for controlling the quality of *Gynostemma pentaphyllum* and ensuring clinical efficacy.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis and relates to a quality control method for traditional Chinese medicinal materials, specifically a method for quality control of components in *Smilax glabra*. Background Technology

[0002] White-backed Panax notoginseng, also known as white-leaved Panax notoginseng or white-seed vegetable, is a dicotyledonous plant belonging to the genus Panax notoginseng in the family Asteraceae. It has tuberous rhizomes, generally with a broad, oblong-ovate base or oblong-obovate leaves, the widest part of which has irregular notches and distinct serrations. It has a faint odor. Because it prefers warmth and dislikes cold, it grows faster in sunny environments and is distributed in Yunnan, Guangdong, and Guangxi provinces of my country. The entire plant of White-backed Panax notoginseng can be used medicinally. Its main chemical components include flavonoids, phenolic acids, terpenes, sterols, volatile oils, and other compounds of various structural types. It also contains trace elements and amino acids. Studies have shown that White-backed Panax notoginseng has certain therapeutic effects on various diseases such as obesity, bronchial asthma, kidney stones, cardiovascular disease, and tumors. Besides being used as a traditional Chinese medicine, it has also been used in recent years as a functional health vegetable, or processed into White-backed Panax notoginseng tea, powders, and biscuits, possessing high nutritional and therapeutic value.

[0003] Studies have shown that flavonoids are the main active components in antiarrhythmic, vasodilatory, microcirculation-improving, and blood lipid-lowering effects; polyphenols have antioxidant properties and can lower blood pressure, blood lipids, and cholesterol; amino acids, as essential substances for the human body, regulate physiological functions and maintain normal metabolism, and the amino acid content in *Smilax glabra* is closely related to its medicinal effects; mineral elements constitute human tissues and maintain normal physiological functions, which is the main reason why this herb has the effects of lowering blood pressure, blood sugar, and cholesterol, anti-cancer, and anti-aging. Therefore, the content of bioactive components, amino acids, and mineral elements is closely related to the medicinal effects of *Smilax glabra*.

[0004] Given the potential medicinal value of *Gynostemma pentaphyllum*, it is necessary to find indicative components that can more comprehensively highlight the basic quality characteristics of *Gynostemma pentaphyllum* and are related to its in vivo efficacy. Establishing scientific evaluation methods and unified evaluation criteria is of great significance for the screening of high-quality *Gynostemma pentaphyllum*.

[0005] Chinese Patent (Patent No.: ZL202110313629.1) discloses a cultivation method and its analysis method for improving the effective components of *Lysimachia christinae*, including the following steps: (1) spraying abscisic acid during the cultivation of *Lysimachia christinae*; (2) sampling: selecting *Lysimachia christinae* leaves, washing the soil off the surface of the harvested leaves with water, and then drying them; (3) sample pretreatment: taking out the dried *Lysimachia christinae* leaf samples, grinding them into powder with a grinder, weighing the powder and placing it in a headspace vial, and tightening the vial opening; (4) headspace extraction: placing the headspace vial containing the sample on a headspace sample tray for equilibration, and after equilibration, the quantitative looping gas in the headspace extraction enters the injection port of the gas chromatograph-mass spectrometer; (5) using gas chromatography-mass spectrometry to perform qualitative analysis on the volatile components of *Lysimachia christinae* leaves. The analytical method of this invention has the advantages of simple operation, low cost, and no need for solvents, and can complete the entire process of sampling, extraction and analysis of the volatile components of *Lysimachia christinae* in a short time.

[0006] Chinese patent application (application number: 201711046763.X) discloses a method for extracting flavonoids from *Gynostemma pentaphyllum*. The method includes tissue culture, hydroponics, greenhouse cultivation, and flavonoid extraction. The flavonoid extraction process is divided into reflux extraction and reflux combined with microwave extraction. Ethanol (50%-60% volume fraction) is added at a material-to-liquid ratio of 1:30-1:40, and the mixture is soaked for 90-120 minutes at 60°C to obtain the flavonoid extract. After obtaining the flavonoid extract, reflux extraction is performed at 60-70°C for 60 minutes. Following reflux extraction, microwave extraction is performed for 1.5-2.5 minutes at an intensity of 360-480W. This invention addresses the issues of low efficiency and low purity in current flavonoid extraction processes by improving the growth methods and optimizing the processing and extraction of *Gynostemma pentaphyllum*.

[0007] Chinese patent application (application number: 201710615115.5) discloses a mineral water containing the efficacy of *Lysimachia christinae* and its preparation method. The invention discloses a mineral water containing the efficacy of *Lysimachia christinae* and its preparation method, including the following steps: (1) Take mineral water, add *Lysimachia christinae* powder and licorice powder to the mineral water in proportion, wherein, by weight, the mineral water is 80-100 parts, *Lysimachia christinae* powder is 0.7-1 parts, and licorice is 0.2-0.5 parts; (2) Heat the solution to 30-60℃ and maintain it for 1-3 hours. When the powder gradually dissolves until there are no obvious particles, stir it with a stirrer until it is completely dissolved; (3) First, pass the solution through an 80-200 mesh coarse sieve, and then remove bacteria with a hollow fiber filter membrane. After standing, take the clear liquid from the top to obtain the product. The invention makes full use of the nutritional and health care functions of *Lysimachia christinae*, which can improve the human immune function, improve the body function, and has certain disease prevention and anti-aging effects.

[0008] Chinese Patent (Patent No.: ZL201510408907.6) discloses a *Gynostemma pentaphyllum* polysaccharide and its application in the preparation of drugs and functional foods for immunomodulation and anti-tumor purposes. This invention provides a *Gynostemma pentaphyllum* polysaccharide and its application in the preparation of drugs and functional foods for immunomodulation and anti-tumor purposes. The *Gynostemma pentaphyllum* polysaccharide prepared by this invention can promote the phagocytosis of neutral red and the release of nitric oxide by mononuclear macrophages; it can promote the proliferation of B cells and T cells; it can significantly increase the spleen and thymus indices of immunosuppressed mice, increase the transformation and proliferation of T cells and B cells, and increase the white blood cell level in immunosuppressed mice; it can significantly inhibit the growth of tumor tissue; and it can also protect the body, inhibit weight loss, and increase body weight. Moreover, polysaccharides are the main active ingredient of *Gynostemma pentaphyllum*, possessing various health benefits. They are derived from pure natural sources, have no side effects, and can be developed into immunomodulatory, anti-tumor, and anti-angiogenic drugs and health products, thus having good market application prospects.

[0009] The germplasm of *Gynostemma pentaphyllum* is quite diverse, with significant differences in its phenotypic characteristics across different regions, resulting in substantial variations in the effective components it contains. A review of the current research status on the chemical composition and quality evaluation of *Gynostemma pentaphyllum* reveals the following shortcomings: First, some literature reports that the quality evaluation indicators for *Gynostemma pentaphyllum* are singular and lack specificity. For example, some scholars have used ultraviolet spectrophotometry to determine the total flavonoid and total polyphenol content in *Gynostemma pentaphyllum*. It is well known that flavonoids and polyphenols are widely present in medicinal plants; using their total amount as an indicator lacks specificity, and analysis with a single indicator is insufficient to reflect the multi-component characteristics of the medicinal material. Second, the quality evaluation indicators reported in the literature lack specificity. For specific diseases, not all chemical components are effective; comprehensive analysis using unrelated indicators fails to reflect the unique characteristics of the medicinal material.

[0010] In addition, different planting conditions can affect the chemical composition and mineral elements of *Gynostemma pentaphyllum*. It is necessary to find differential secondary metabolites and marker elements, and then conduct quantitative analysis on them to provide new ideas for the quality control of *Gynostemma pentaphyllum*. Summary of the Invention

[0011] The technical problems to be solved by this disclosure include: providing a method for quality control of the components of *Gynostemma pentaphyllum*, with the aim of screening out four categories of physiologically related characteristic components through reasonable testing methods, using them as quality control indicators for *Gynostemma pentaphyllum*, providing technical support for objectively evaluating the quality of *Gynostemma pentaphyllum*, which is of great significance for controlling the quality of *Gynostemma pentaphyllum*-related products and ensuring efficacy, and at the same time providing a reference method for the material control of traditional Chinese medicine.

[0012] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0013] A method for quality control of *Lysimachia christinae* components includes the following steps:

[0014] Step 1: Metabolomics analysis of methanol extracts of *Smilax glabra* under different abscisic acid treatments was performed using liquid chromatography-high resolution mass spectrometry to screen out flavonoids and phenolic acids contained in *Smilax glabra*.

[0015] Step 2: The contents of known flavonoids and phenolic acids in the methanol extract of *Smilax china* were determined by liquid chromatography-mass spectrometry (a), and the characteristic flavonoids and phenolic acids in *Smilax china* were determined by combining the results of principal component analysis.

[0016] Step 3: The contents of 17 amino acids in the medicinal materials of *Smilax glabra* under different treatments were determined by liquid chromatography-mass spectrometry (b), and the characteristic amino acid components in *Smilax glabra* were determined by combining the results of principal component analysis.

[0017] Step 4: Determine 22 mineral elements in *Sedum morganianum* under different treatments according to GB 5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food". At the same time, perform ionomics analysis to screen out 7 mineral element-like substances in *Sedum morganianum*. Combine with the principal component analysis results, further determine the characteristic mineral element components in *Sedum morganianum*.

[0018] Step 5: Use the various characteristic components obtained in Steps 1 to 4 as evaluation indicators to assess the quality of *Euphorbia hirta* and as a reference indicator for quality control.

[0019] Preferably, the flavonoids mentioned in step 2 are rutin, isoquercitrin, safflower glycoside, astragaloside, quercetin, and kaempferol.

[0020] Preferably, the phenolic acid components mentioned in step 2 are chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isovanillic acid, p-coumaric acid, scopolamine lactone, ferulic acid, isochlorogenic acid B, and isochlorogenic acid C.

[0021] Preferably, the characteristic flavonoid components mentioned in step 2 are rutin and flavin.

[0022] Preferably, the characteristic phenolic acid components in step 2 are chlorogenic acid, neochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C.

[0023] Preferably, the amino acid components mentioned in step 3 are proline, valine, threonine, leucine, isoleucine, cystine, aspartic acid, lysine, glutamic acid, histidine, phenylalanine, arginine, tyrosine, methionine, glycine, alanine, and serine.

[0024] Preferably, the characteristic amino acid components mentioned in step 3 are isoleucine, glutamic acid, serine, and proline.

[0025] Preferably, the 22 mineral elements in step 4 are phosphorus, potassium, calcium, magnesium, sodium, boron, lithium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, strontium, molybdenum, cadmium, lead, aluminum, and iron.

[0026] Preferably, the seven mineral elements mentioned in step 4 are phosphorus, potassium, calcium, magnesium, sodium, aluminum, and iron.

[0027] Preferably, the characteristic mineral element components mentioned in step 4 are potassium and iron.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) This invention selects four major categories of components that are closely related to the functions of *Smilax glabra* in lowering blood sugar, lowering blood lipids, dilating blood vessels, fighting cancer, inhibiting gastric acid secretion, and enhancing human immunity, and have relatively high content and representativeness, such as flavonoids, phenolic acids, amino acids, and mineral elements, as content determination indicators, which can effectively control the quality of *Smilax glabra*.

[0030] (2) This invention establishes four categories of chemical component indicators, namely flavonoids, phenolic acids, amino acids and mineral elements, for the quality evaluation and quality control of *Sedum morganianum*. Compared with the current quality standard of *Sedum morganianum* which only uses the total amount of flavonoids as the content determination indicator, this invention can more comprehensively control the quality of *Sedum morganianum*.

[0031] (3) The liquid chromatography-mass spectrometry and inductively coupled plasma method used in this invention to determine the four major characteristic components of Gynostemma pentaphyllum are rapid, simple and accurate. They can objectively, comprehensively and accurately evaluate the quality of Gynostemma pentaphyllum, which is of great significance for controlling the quality of Gynostemma pentaphyllum and ensuring clinical efficacy. Attached Figure Description

[0032] Figure 1 This is an HPLC-MS chromatogram of flavonoids and phenolic acids.

[0033] Figure 2 The graph shows the changes in the content of flavonoids in *Lysimachia christinae* under different abscisic acid treatments.

[0034] Figure 3 The graph shows the changes in the content of phenolic acids in *Lysimachia christinae* under different abscisic acid treatments.

[0035] Figure 4 This is a diagram showing the composition of phenolic acids.

[0036] Figure 5 This is the HPLC-MS chromatogram of amino acid substances.

[0037] Figure 6 The graph shows the changes in amino acid content in *Lysimachia christinae* under different abscisic acid treatments.

[0038] Figure 7 This is a diagram of the amino acid composition.

[0039] Figure 8 The graph shows the changes in aluminum and iron content in *Lysimachia christinae* under different abscisic acid treatments.

[0040] Figure 9 The graph shows the changes in potassium and calcium content in *Lysimachia christinae* under different abscisic acid treatments.

[0041] Figure 10 The graph shows the changes in phosphorus, magnesium, and sodium content in *Lysimachia christinae* under different abscisic acid treatments.

[0042] Figure 11 This is a diagram showing the elemental composition of minerals. Detailed Implementation

[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention by those skilled in the art in various equivalent forms will fall short of the scope defined by the appended claims.

[0044] The herb *Gynostemma pentaphyllum* contains numerous chemical components. The quality control indicators involved in this invention prioritize marker components with clearly defined effects, relatively high content, and significant variability due to environmental factors. To overcome the shortcomings of existing testing technologies, this invention aims to conduct quality evaluation research on the bioactive components, essential amino acids, and mineral elements for maintaining physiological functions in *Gynostemma pentaphyllum*, which are associated with medicinal efficacy.

[0045] 1. Confirmation of the bioactive components of *Smilax china*

[0046] This invention first performs metabolomics analysis on extracts of *Lysimachia christinae* under different abscisic acid treatments, screening out 6 flavonoids and 9 phenolic acids in *Lysimachia christinae*. Then, the content of these substances is further confirmed through targeted metabolomics. Combined with principal component analysis results, the characteristic active ingredients in *Lysimachia christinae* are identified as one of the quality control indicators.

[0047] 2. The amino acid composition of *Smilax glabra* has been confirmed.

[0048] This invention determines the content of 17 amino acids in *Symplocos rubra* herb under different abscisic acid treatments, and uses the principal component analysis results to identify the characteristic amino acid components in *Symplocos rubra* as one of the quality control indicators.

[0049] 3. Confirmation of the mineral element composition of *Smilax glabra*

[0050] This invention first performs ionomics analysis on extracts of *Symplocos rubra* under different abscisic acid treatments to screen out 7 mineral elements in *Symplocos rubra*, and then further confirms their content. Combined with the principal component analysis results, the characteristic mineral elements in *Symplocos rubra* are determined as one of the quality control indicators.

[0051] To make the present invention fully disclosed, the present invention will be described in the following manner.

[0052] A method for quality control of *Lysimachia christinae* components includes the following steps:

[0053] Step 1: Metabolomics analysis of methanol extracts of *Sedum morganianum* under different treatments was performed using liquid chromatography-high resolution mass spectrometry to screen out flavonoids and phenolic acids contained in *Sedum morganianum*.

[0054] Step 2: The contents of known flavonoids and phenolic acids in the methanol extract of *Smilax china* were determined by liquid chromatography-mass spectrometry (a), and the characteristic flavonoids and phenolic acids in *Smilax china* were determined by combining the results of principal component analysis.

[0055] Step 3: The contents of 17 amino acids in the medicinal materials of *Smilax glabra* under different treatments were determined by liquid chromatography-mass spectrometry (b), and the characteristic amino acid components in *Smilax glabra* were determined by combining the results of principal component analysis.

[0056] Step 4: Determine 22 mineral elements in *Sedum morganianum* under different treatments according to GB 5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food". At the same time, perform ionomics analysis to screen out 7 mineral element-like substances in *Sedum morganianum*. Combine with the principal component analysis results, further determine the characteristic mineral element components in *Sedum morganianum*.

[0057] Step 5: Use the various characteristic components obtained in Steps 1 to 4 as evaluation indicators to assess the quality of *Euphorbia hirta* and as a reference indicator for quality control.

[0058] Preferably, the flavonoids mentioned in step 2 are rutin, isoquercitrin, safflower glycoside, astragaloside, quercetin, and kaempferol.

[0059] Preferably, the phenolic acid components mentioned in step 2 are chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isovanillic acid, p-coumaric acid, scopolamine lactone, ferulic acid, isochlorogenic acid B, and isochlorogenic acid C.

[0060] Preferably, the characteristic flavonoid components mentioned in step 2 are rutin and flavin.

[0061] Preferably, the characteristic phenolic acid components in step 2 are chlorogenic acid, neochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C.

[0062] Preferably, the amino acid components mentioned in step 3 are proline, valine, threonine, leucine, isoleucine, cystine, aspartic acid, lysine, glutamic acid, histidine, phenylalanine, arginine, tyrosine, methionine, glycine, alanine, and serine.

[0063] Preferably, the characteristic amino acid components mentioned in step 3 are isoleucine, glutamic acid, serine, and proline.

[0064] Preferably, the 22 mineral elements in step 4 are phosphorus, potassium, calcium, magnesium, sodium, boron, lithium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, strontium, molybdenum, cadmium, lead, aluminum, and iron.

[0065] Preferably, the seven mineral elements mentioned in step 4 are phosphorus, potassium, calcium, magnesium, sodium, aluminum, and iron.

[0066] Preferably, the characteristic mineral element components mentioned in step 4 are potassium and iron.

[0067] Preferably, the different treatments of *Lysimachia christinae* mentioned in step 1 refer to the same variety of *Lysimachia christinae* treated with different concentrations of abscisic acid.

[0068] Preferably, the relevant information of the *Lysimachia christinae* samples under different abscisic acid treatments described in step 1 is as follows:

[0069] On the 15th day of the growing season of *Gynostemma pentaphyllum*, abscisic acid (ABA) was sprayed twice, with a 7-day interval. Other management measures were the same as conventional management methods. Seven comparative experiments were set up, with ABA concentrations of 10 mg / L (treatment 1), 25 mg / L (treatment 2), 50 mg / L (treatment 3), 100 mg / L (treatment 4), 250 mg / L (treatment 5), 500 mg / L (treatment 6), and a water control (CK). The effects of different ABA concentrations on *Gynostemma pentaphyllum* were tested.

[0070] Preferably, the metabolomics analysis of the methanol extracts of *Lysimachia christinae* under different treatments using liquid chromatography-high-resolution mass spectrometry (LC-HPLC-MS / MS) in step 1 is as follows:

[0071] Step 1.1 Preparation of the test solution:

[0072] Accurately weigh 0.1 g of *Lysimachia christinae* (accurate to 0.0001 g) into a 10 mL centrifuge tube, and add 5 mL of 80% methanol-water solution containing 0.2% formic acid. Vortex to mix, and sonicate for 20 min at a controlled temperature (below 25℃). Allow to settle, and take 1 mL of the supernatant. Place this supernatant into a centrifuge tube pre-filled with 0.1 mg of C18 adsorbent powder, vortex to mix, and filter the supernatant through a 0.22 μm microporous membrane. The filtrate is the test solution.

[0073] Step 1.2 The conditions for the liquid chromatography-high resolution mass spectrometry (LC-MS / MS) are as follows:

[0074] Instrument Model: Q-Exactive High-Resolution Liquid Chromatography-Mass Spectrometer

[0075] Chromatographic column: Waters ACQUITY UPLC HSS T3 1.8μm 2.1*100mm

[0076] Mobile phases: Mobile phase A is 0.1% formic acid in water, and mobile phase B is acetonitrile.

[0077] The gradient elution program was as follows: 0 min-3 min: 5%-15% acetonitrile; 3 min-5 min: 15% acetonitrile; 5 min-11 min: 15%-50% acetonitrile; 11 min-14 min: 50%-95% acetonitrile; 14 min-15 min: 95%-95% acetonitrile; 15 min-15.1 min: 95%-5% acetonitrile; 15.1 min-18 min: 5% acetonitrile.

[0078] Column temperature: 40℃;

[0079] Column flow rate: 0.3 ml / min;

[0080] Injection volume: 5 μL;

[0081] Scanning method: Two-stage MS / MS data scanning was performed using both positive and negative ion scanning modes;

[0082] Scan range: m / z 100~1500.

[0083] Step 1.3 Data Analysis:

[0084] Raw mass spectrometry data were imported into CompoundDiscoverer 3.0 software for preprocessing, including peak extraction, retention time correction, peak alignment, and noise removal. The preprocessed mass spectrometry data was then matched with the mzClond and mzVault databases. Compounds were assigned and identified using precise relative molecular masses and fragment ions from the headphone mass spectrometry data.

[0085] Preferably, the step 2, which involves determining flavonoids and phenolic acids using liquid chromatography-mass spectrometry (a), is as follows:

[0086] Step 2.1 Preparation of the test solution:

[0087] Accurately weigh 0.1 g of *Lysimachia christinae* (accurate to 0.0001 g) into a 10 mL centrifuge tube, and add 5 mL of 80% methanol-water solution containing 0.2% formic acid. Vortex to mix, and sonicate for 20 min at a controlled temperature (below 25℃). Allow to settle, and take 1 mL of the supernatant. Place this supernatant into a centrifuge tube pre-filled with 0.1 mg of C18 adsorbent powder, vortex to mix, and filter the supernatant through a 0.22 μm microporous membrane. The filtrate is the test solution.

[0088] Step 2.2 Preparation of the reference solution:

[0089] Weigh appropriate amounts of rutin, isoquercitrin, safflowerin, astragalin, quercetin, kaempferol, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isovanillic acid, p-coumaric acid, scopolamine, ferulic acid, isochlorogenic acid B, and isochlorogenic acid C standards to prepare a mixed reference solution. Prepare a series of gradient flavonoid and phenolic acid standard solutions with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 ng / mL using 0.2% formic acid and 80% methanol to create a flavonoid and phenolic acid standard solution curve.

[0090] Step 2.3 Liquid chromatography coupled with mass spectrometry (a) Instrument conditions are as follows:

[0091] Chromatographic column: Waters ACQUITY UPLC HSS T3 1.8μm 2.1*100mm

[0092] Mobile phases: Mobile phase A is 0.1% formic acid in water, and mobile phase B is acetonitrile.

[0093] The gradient elution program was as follows: 0 min-3 min: 5%-15% acetonitrile; 3 min-5 min: 15% acetonitrile; 5 min-11 min: 15%-50% acetonitrile; 11 min-14 min: 50%-95% acetonitrile; 14 min-15 min: 95%-95% acetonitrile; 15 min-15.1 min: 95%-5% acetonitrile; 15.1 min-18 min: 5% acetonitrile.

[0094] Column temperature: 40℃;

[0095] Column flow rate: 0.3 ml / min;

[0096] Injection volume: 5 μL;

[0097] Scanning mode: MRM positive ion;

[0098] Scanning ion pairs: See Appendix 1 for details.

[0099] Table 1. Scanning Ions and Collision Energy of Flavonoids and Phenolic Acids

[0100]

[0101]

[0102] Step 2.4 Determination of flavonoids and phenolic acids in the sample:

[0103] The test solution prepared in step 2.1 and the mixed reference solution prepared in step 2.2 were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for analysis to obtain chromatograms, as shown in the figure. Figure 1 The contents of rutin, isoquercitrin, safflowerin, astragaloside, quercetin, kaempferol, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isovanillic acid, p-coumaric acid, scopolamine, ferulic acid, isochlorogenic acid B, and isochlorogenic acid C were calculated using the external standard method. The results are shown in Tables 2 and 5. Figure 2 , 3 .

[0104] Table 2. Flavonoid content in *Lysimachia christinae* (unit: mg / kg)

[0105]

[0106]

[0107] Table 3. Main component analysis of flavonoids

[0108]

[0109] Table 4 Flavonoid Loading

[0110] Components First principal component loading Rutin 0.963 Isoquercitrin 0.933 Astragaloside 0.919 Fireworks Glycoside 0.881 Kaempferol 0.822 Quercetin 0.818

[0111] Principal component analysis (PCA) of six flavonoid components was performed using SPSS software (see Table 3). Factors with an eigenvalue (λ) greater than 1 were selected as the extraction criterion. The results showed that the first extracted factor was the principal factor, contributing a cumulative value of 79.423%. Therefore, one principal component can represent the information on the content of most flavonoid chemical components in *Smilax glabra*. The loading of each eigenvector was calculated (see Table 4). In the first principal component eigenvector, the components with higher loadings were rutin, isoquercitrin, astragaloside, and safflower glycoside. Since rutin and safflower glycoside have high content and are greatly affected by environmental factors, only two components (rutin and safflower glycoside) were selected as the characteristic flavonoid components of *Smilax glabra*.

[0112] Table 5. Content of phenolic acids in *Lysimachia christinae* (unit: mg / kg)

[0113]

[0114] Table 6. Main Component Analysis of Phenolic Acids

[0115]

[0116] Table 7. Loading capacity of phenolic acids

[0117]

[0118]

[0119] Principal component analysis of the nine phenolic acid components was performed using SPSS software, as shown in Table 6 and 2017. Figure 4 The extraction principle was to select factor eigenvalues ​​(λ) greater than 1. The results showed that the first two factors were extracted as principal factors, with a cumulative contribution of 78.986%. Therefore, the two principal components can represent the information on the content of phenolic acid chemical components in most of the *Sedum aizoon*. The loading of each eigenvector was calculated, as shown in Table 7. In the first principal component eigenvector, the five components with higher loadings were neochlorogenic acid, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, and cryptochlorogenic acid. Since the content of cryptochlorogenic acid was low in different *Sedum aizoon* species, only four components (chlorogenic acid, neochlorogenic acid, isochlorogenic acid C, and isochlorogenic acid B) were selected as the representative components of the first principal component. In the second principal component eigenvector, the component with higher loading was ferulic acid. As can be seen from the principal component analysis table, the contribution value of the first principal component accounted for 53.446%, while the content of the second principal component components was relatively low. Therefore, only four components (chlorogenic acid, neochlorogenic acid, isochlorogenic acid C, and isochlorogenic acid B) were selected as the characteristic phenolic acid components of *Lysimachia christinae*.

[0120] Preferably, step 3 of the liquid chromatography-mass spectrometry (b) method is as follows:

[0121] Step 3.1 Preparation of the test solution:

[0122] Accurately weigh 0.1 g of *Lysimachia christinae* (accurate to 0.0001 g), place it in a 15 mL centrifuge tube, add 10 mL of 25% ethanol-water solution containing 0.2% formic acid, vortex for 45 s, and sonicate at ≤25℃ for 30 min. Take an appropriate amount of supernatant into a 2 mL centrifuge tube, centrifuge at 12000 r / min for 10 min in a low-temperature high-speed centrifuge, filter through a 0.22 μm needle filter membrane, and derivatize using the AccQ.Tag method to obtain the test solution.

[0123] Step 3.2 Preparation of the reference solution:

[0124] Weigh out glutamic acid, alanine, and serine standards and prepare a mixed reference solution. Prepare a series of gradient amino acid compound standard solution curves using 0.2% formic acid and 80% methanol, and derivatize them using the AccQ.Tag method to obtain the reference solution.

[0125] The instrument conditions for determining amino acid components using liquid chromatography-mass spectrometry as described in step 3.3 are as follows:

[0126] Chromatographic column: ACQUITYUPLC HSS T3 column (2.1*100mm, 1.8μm);

[0127] Mobile phase: A is a 0.1% formic acid aqueous solution; mobile phase B is acetonitrile containing 0.1% formic acid;

[0128] The gradient elution program was as follows: 0–1.0 min, 96% A; 1.0–1.5 min, 96% A–90% A; 1.5–10.0 min, 90% A–72% A; 10.0–11.0 min, 72% A–5% A; 11.0–13.0 min, 5% A–5% A; 13.0–13.1 min, 5% A–96% A; 13.1–18.0 min, 96% A–96% A.

[0129] Column temperature: 40℃;

[0130] Column flow rate: 0.3 ml / min;

[0131] Injection volume: 2.0 μL;

[0132] Scanning mode: MRM positive ion;

[0133] Scanning ion pairs: See attached Table 8 for details.

[0134] Table 8. Scanning Ions and Collision Energy of Amino Acids

[0135]

[0136] Step 3.4 Determination of amino acid components in the sample:

[0137] The test solution prepared in step 3.1 and the mixed reference solution prepared in step 3.2 were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for analysis to obtain chromatograms, as shown in the figure. Figure 5 The contents of proline, valine, threonine, leucine, isoleucine, cystine, aspartic acid, lysine, glutamic acid, histidine, phenylalanine, arginine, tyrosine, methionine, glycine, alanine, and serine were calculated using the external standard method. The results are shown in Table 9. Figure 6 .

[0138] Table 9. Amino acid content in *Lysimachia christinae* (unit: mg / kg)

[0139] name CK(n=3) Process 1 (n=3) Process 2 (n=3) Process 3 (n=3) Process 4 (n=3) Process 5 (n=3) Process 6 (n=3) proline 88.71±3.33 128.23±4.04 130.09±2.25 159.71±6.52 162.52±11.50 94.08±3.81 91.74±1.10 Valine 49.13±5.51 47.72±7.69 50.98±2.11 53.11±3.26 90.77±12.51 50.59±7.57 45.30±4.75 threonine 132.39±4.08 138.68±10.10 127.34±14.47 122.46±7.53 106.26±2.25 80.39±4.25 73.41±5.14 Leucine 29.11±2.81 26.65±2.50 36.39±0.42 40.80±6.50 83.84±16.66 45.79±2.84 46.33±2.13 Isoleucine 88.95±3.56 113.20±5.63 124.34±2.92 121.72±13.67 144.14±3.58 58.76±1.44 53.02±1.92 Aspartic acid 770.06±48.96 1320.06±87.57 835.63±1.08 828.61±37.93 680.96±124.78 498.60±78.52 498.21±31.89 Lysine 27.90±3.07 23.82±4.62 20.09±1.97 20.30±0.33 36.51±3.65 20.58±4.08 17.47±0.93 glutamic acid 1501.84±85.34 1476.92±40.67 1317.77±174.58 1190.38±100.85 1114.43±53.39 1174.37±17.11 1037.65±19.76 Histidine 34.33±1.99 34.34±6.55 24.49±4.74 27.62±3.45 28.53±3.93 13.21±2.09 15.24±0.41 Phenylalanine 55.04±6.21 55.61±10.98 54.65±5.24 57.51±5.29 73.95±9.95 44.98±8.36 43.17±1.51 Arginine 41.44±4.71 51.07±8.64 49.83±9.69 50.25±7.36 79.27±12.00 32.38±4.15 34.62±2.51 Tyrosine 24.86±0.87 31.63±3.27 30.77±2.08 35.25±4.07 44.92±8.98 19.73±1.12 23.59±1.57 Methionine 2.59±0.16 2.94±0.16 1.64±0.16 2.42±0.24 4.56±0.83 1.62±0.16 2.92±0.35 glycine 25.13±3.21 22.37±3.56 17.31±3.31 22.95±1.63 21.54±3.09 14.26±1.90 16.81±1.88 alanine 252.50±8.32 266.38±7.78 246.50±2.72 193.09±1.41 199.08±8.45 183.55±8.22 189.99±6.78 Serine 273.08±5.73 263.40±263.40 197.23±5.25 168.12±7.51 158.05±3.67 125.77±1.79 127.96±4.27

[0140] Table 10. Main component analysis of amino acid substances

[0141]

[0142] Table 11 Amino Acid Loading Capacity

[0143]

[0144]

[0145] Since cystine was not detected in *Lysimachia christinae*, principal component analysis of the 16 amino acid components was performed using SPSS software, as shown in Table 10. Figure 7 The extraction principle was to select factor eigenvalues ​​(λ) greater than 1. The results showed that the first three factors were extracted as principal factors, with a cumulative contribution of 85.942%. Therefore, the three principal components can represent the information on the content of most amino acid chemical components in *Smilax glabra*. The loading of each eigenvector was calculated, as shown in Table 11. In the first principal component eigenvector, the four components with higher loading were isoleucine, phenylalanine, histidine, and arginine. Since the content of phenylalanine, histidine, and arginine was low, one component (isoleucine) was selected as the representative component of the first principal component. In the second principal component eigenvector, the components with higher loading were leucine, glutamic acid, and serine. Since the content of leucine was low, two components (glutamic acid and serine) were selected as the representative components of the second principal component. In the third principal component eigenvector, the component with higher loading was proline. Therefore, only four components (isoleucine, glutamic acid, serine, and proline) were selected as the characteristic amino acid components of *Smilax glabra*.

[0146] Preferably, the inductively coupled plasma mass spectrometry determination step 4 is as follows:

[0147] Step 4.1 Preparation of the test solution:

[0148] Weigh 0.2g–0.5g (accurate to 0.001g) of solid sample (0.5g–1.5g for fresh sample) into the microwave digestion vessel. Add 5mL of nitric acid, cover and let stand for 1 hour. Tighten the lid and digest according to the standard operating procedure of the microwave digester (refer to Table B.1 of GB5009.268-2016 for digestion reference conditions). After cooling, remove the vessel, slowly open the lid to release air, rinse the inner lid with a small amount of water, place the digestion vessel on a temperature-controlled heating plate, heat at 100℃ for 30 minutes, cool to room temperature, and dilute to 25mL or 50mL with water. Mix well and set aside. Perform a blank test simultaneously.

[0149] Step 4.2 Preparation of the reference solution:

[0150] Take appropriate amounts of calcium, potassium, magnesium, sodium, and iron reference standard stock solutions, and dilute them stepwise with nitric acid solution (5+95) to prepare a series of mixed standard working solutions (adjust the mass concentration range of each element in the standard series appropriately according to the mass concentration level of the elements in the sample).

[0151] The instrument conditions for the inductively coupled plasma instrumentation (ICP-IPV) measurement in step 4.3 are shown in Table 12.

[0152] Table 12 Reference Conditions for Inductively Coupled Plasma Analyzers

[0153] Parameter name parameter RF power (W) 1500 Atomizing gas flow rate / (SLM) 0.6 Auxiliary gas flow rate (SLM) 1 Cooling airflow rate (SLM) 12 Number of repetitions 2 Rinsing time (s) 25 Flushing pump speed (rmp) 30 Analyze pump speed (rmp) 30

[0154] Step 4.4 Determination of representative mineral element components in the sample:

[0155] The test solution prepared in step 4.1 and the mixed reference solution prepared in step 4.2 were injected into an inductively coupled plasma mass spectrometer for determination, and the content of each element was calculated by the external standard method.

[0156] The contents of seven mineral elements in *Lysimachia christinae* under different treatments are shown in Table 13 and 14. Figure 8-10 .

[0157] Table 13 Content of 7 mineral elements in *Lysimachia christinae* (g / kg)

[0158]

[0159] Table 14 Main Component Analysis of Mineral Elements

[0160]

[0161] Table 15 Mineral Element Loading

[0162] Components First principal component loading Second principal component loading sodium 0.876 0.226 iron -0.846 0.490 phosphorus 0.818 0.201 aluminum -0.766 0.423 Potassium -0.494 0.794 magnesium 0.434 0.793 calcium 0.592 0.719

[0163] Principal component analysis of the seven mineral elements was performed using SPSS software, as shown in Table 14 and 15. Figure 11 The extraction principle was to select factor eigenvalues ​​(λ) greater than 1. The results showed that the first two factors were extracted as principal factors, with a cumulative contribution of 82.996%. Therefore, the two principal components can represent the information on the content of most mineral elements in *Sedum aizoon*. The loading of each eigenvector was calculated, as shown in Table 15. In the first principal component eigenvector, the three components with higher loadings were sodium, iron, and phosphorus; in the second principal component eigenvector, the components with higher loadings were potassium, magnesium, and calcium. Since the potassium content was much higher than that of other elements, and the iron content varied greatly under different planting conditions, two components (potassium and iron) were selected as the characteristic representative components of mineral elements in *Sedum aizoon*.

[0164] Step 5: Establishment of quality evaluation standards for *Clerodendrum trichotomum* (a type of herb):

[0165] Based on the content of various characteristic components measured in steps 1, 2, 3, and 4, representative components of each type were analyzed, compared, and screened. These representative components were then used as evaluation indicators, as shown in Table 16, as reference standards for assessing the quality and controlling the quality of *Euphorbia hirta*.

[0166] Table 16 Representative components of various characteristics of *Lysimachia christinae*

[0167] category Representative ingredients Flavonoids Rutin, pyroside Phenolic acids Chlorogenic acid, neochlorogenic acid, isochlorogenic acid C, isochlorogenic acid B amino acids Isoleucine, glutamic acid, serine, proline Mineral elements potassium and iron

[0168] This invention discloses the establishment of four major categories of chemical component indicators: flavonoids, phenolic acids, amino acids, and mineral elements. Twelve representative components are selected for the quality evaluation and control of *Gynostemma pentaphyllum*, covering different categories of components. Compared with the current *Gynostemma pentaphyllum* quality standard, which only uses total flavonoids as the content determination indicator, this method provides a more comprehensive control over the quality of *Gynostemma pentaphyllum*, enabling objective, comprehensive, and accurate quality evaluation. This is of great significance for quality control of *Gynostemma pentaphyllum*. Simultaneously, it provides a multi-indicator screening reference method for multi-indicator quality evaluation of Chinese medicinal materials, promoting the precision and collaborative research of Chinese medicine quality standards.

[0169] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the present invention, should fall within the protection scope of the present invention.

Claims

1. A method for quality control of a component of Eupatorium odoratum L., characterized by, Includes the following steps: Step 1: Metabolomics analysis of methanol extracts of *Smilax glabra* under different abscisic acid treatments was performed using liquid chromatography-high resolution mass spectrometry to screen out flavonoids and phenolic acids contained in *Smilax glabra*. Step 2: The contents of known flavonoids and phenolic acids in the methanol extract of *Smilax glabra* were determined by liquid chromatography-mass spectrometry (a), and the characteristic flavonoids and phenolic acids in *Smilax glabra* were determined by combining the results of principal component analysis. Step 3: The contents of 17 amino acids in the *Smilax china* herb under different treatments were determined by liquid chromatography-mass spectrometry (b). The 17 amino acid components were proline, valine, threonine, leucine, isoleucine, cystine, aspartic acid, lysine, glutamic acid, histidine, phenylalanine, arginine, tyrosine, methionine, glycine, alanine, and serine. The characteristic amino acid components in *Smilax china* were determined by combining the principal component analysis results. Step 4: Following GB 5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food", 22 mineral elements in *Euphorbia hirta* under different treatments were determined, namely phosphorus, potassium, calcium, magnesium, sodium, boron, lithium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, strontium, molybdenum, cadmium, lead, aluminum, and iron. Simultaneously, ionomics analysis was performed to screen out 7 mineral elements in *Euphorbia hirta*, namely phosphorus, potassium, calcium, magnesium, sodium, aluminum, and iron. The characteristic mineral element components in *Euphorbia hirta* were further determined based on the principal component analysis results. Step 5: Use the various characteristic components obtained in steps 1 to 4 as evaluation indicators to assess the quality of *Euphorbia hirta* and as reference indicators for quality control. The characteristic flavonoid components mentioned in step 2 are rutin and flavin glycoside; The characteristic phenolic acid components mentioned in step 2 are chlorogenic acid, neochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C; The characteristic amino acid components mentioned in step 3 are isoleucine, glutamic acid, serine, and proline; The characteristic mineral element components mentioned in step 4 are potassium and iron.

2. The method for quality control of *Lysimachia christinae* components according to claim 1, characterized in that, The flavonoids mentioned in step 2 are rutin, isoquercitrin, safflower glycoside, astragaloside, quercetin, quercetin, and kaempferol.

3. The method for quality control of *Lysimachia christinae* components according to claim 1, characterized in that, The phenolic acid components mentioned in step 2 are chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isovanillic acid, p-coumaric acid, scopolamine lactone, ferulic acid, isochlorogenic acid B, and isochlorogenic acid C.

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