Method for determining content of vanillic acid-4-beta-d-glucoside in platycladi fructus preparation and application thereof

Abstract

The present application relates to the determination method and application of the content of vanillic acid-4-beta-D-glucoside in Platycladi Cacumen preparation, through the detection analysis of Platycladi Cacumen preparation under specific UPLC (ultra performance liquid chromatography) conditions, a content determination method for vanillic acid-4-beta-D-glucoside, which is a good water-soluble and specific effective component in Platycladi Cacumen preparation, is established, which can provide an effective evaluation method for the quality control of Platycladi Cacumen preparation including Platycladi Cacumen traditional Chinese medicine decoction pieces and formula granules; meanwhile, the method is verified to have good specificity, precision, stability, repeatability, accuracy and durability. In addition, the method can distinguish the standard decoction of Platycladi Cacumen and its common adulterants, japonica rice and linseed standard decoction, by taking the content of vanillic acid-4-beta-D-glucoside as the determination index, and the method is simple, reliable and objective.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for determining the content of vanillic acid-4-β-D-glucoside in cypress seed preparations and its application. Background Technology

[0002] Standardized decoctions of traditional Chinese medicine (TCM) are prepared using standardized processes guided by TCM theory and based on clinical application, while referencing modern extraction methods. These single-herb decoctions serve as a standard substance and system, enabling standardized clinical medication use and regulating the widely used forms of decoctions, including granules, ensuring accuracy and dosage consistency. Therefore, establishing quality standards for standardized decoctions of TCM using modern analytical techniques can provide important references for establishing quality standards for corresponding granules and other modern TCM preparations using water extraction from classic formulas.

[0003] Arborvitae seed kernel, listed in the 2020 edition of the Chinese Pharmacopoeia, is the dried, mature seed kernel of *Platycladus orientalis* (L.) Franco, a plant in the Cupressaceae family. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior-grade herb. Its flavor is sweet and neutral, entering the heart, kidney, and large intestine meridians. It has the effects of nourishing the heart and calming the mind, moistening the intestines and promoting bowel movements, and stopping sweating. As a traditional Chinese medicine, arborvitae seed kernel has a long history of medicinal use. Modern pharmacological studies have shown that it has sedative, hypnotic, antidepressant, and antioxidant effects. Clinically, it is mainly used to treat insomnia due to deficiency and restlessness, constipation due to intestinal dryness, and night sweats due to yin deficiency. Arborvitae seed kernel has a complex and diverse composition, containing both a large amount of weakly polar fatty oil and a large amount of highly polar water-soluble components. Current quality research on arborvitae seed kernel mainly focuses on the content of major components such as total saponins, polysaccharides, diterpenes, and some fat-soluble components such as β-sitosterol and carotene. Furthermore, although the amino acids in cypress seed kernels have good water solubility, these components are ubiquitous in plants and animals and lack specificity. Therefore, research reports on the water-soluble and specific components in cypress seed kernels are relatively rare, and research on the quality standards of cypress seed kernel preparations is even scarcer.

[0004] Currently, the 2020 edition of the Chinese Pharmacopoeia only includes morphological and microscopic identification items under the quality standards for Platycladus orientalis seed raw materials and processed slices, lacking indicators for the content determination of water-soluble components, indicating that the quality standards are still incomplete. Furthermore, existing literature mainly focuses on the determination of major components and fat-soluble components in Platycladus orientalis seed, with no literature found on the determination of water-soluble components. After decoction, concentration, and freeze-drying, the transfer rate of fat-soluble components in Platycladus orientalis seed preparations is low, resulting in a small amount remaining in the standard freeze-dried powder. Therefore, there is an urgent need to establish methods for determining the content of active components with better water solubility, specificity, stability, and transfer rate in Platycladus orientalis seed preparations to control their quality. Summary of the Invention

[0005] Therefore, it is necessary to provide a method for determining the content of vanillic acid-4-β-D-glucoside in cypress seed preparations and its application. This method can determine the content of vanillic acid-4-β-D-glucoside, a water-soluble and specific active ingredient in cypress seed preparations, and can provide an effective evaluation method for the quality control of cypress seed preparations.

[0006] The technical solution is as follows:

[0007] In a first aspect, the present invention provides a method for determining the content of vanillic acid-4-β-D-glucoside in a preparation of Platycladus orientalis seed, comprising the following steps:

[0008] The extract of Platycladus orientalis seed preparation was carried out using an extraction solvent to prepare a test solution;

[0009] The content of vanillic acid-4-β-D-glucoside in the cypress seed preparation was obtained by ultra-high performance liquid chromatography detection of the test sample solution.

[0010] The conditions for ultra-high performance liquid chromatography detection include: mobile phase A is acetonitrile, mobile phase B is an aqueous phosphoric acid solution, and gradient elution is used;

[0011] The gradient elution procedure includes:

[0012] From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 1%.

[0013] Between 8 and 12 minutes, the volume percentage of the mobile phase A increased from 1% to 2%.

[0014] Between 12 and 18 minutes, the volume percentage of mobile phase A increased from 2% to 3%.

[0015] The volume percentage of mobile phase A is maintained at 3% for 18-22 minutes.

[0016] Between 22 and 25 minutes, the volume percentage of the mobile phase A increased from 3% to 80%.

[0017] In one embodiment, the ultra-high performance liquid chromatography detection satisfies at least one of the following conditions:

[0018] (1) The chromatographic column is a C18 column;

[0019] (2) The column temperature is 28°C~32°C;

[0020] (3) The flow rate is 0.32 mL / min to 0.38 mL / min;

[0021] (4) The injection volume is 0.5 μL to 2 μL;

[0022] (5) The detection wavelength is 255nm~265nm.

[0023] In one embodiment, the cypress seed preparation includes at least one of the following: standard decoction of cypress seed medicinal slices, cypress seed formula granules, and water extract of cypress seed medicinal slices.

[0024] In one embodiment, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.2% to 0.4%.

[0025] In one embodiment, the extraction step satisfies at least one of the following conditions:

[0026] (1) The extraction method includes one or both of ultrasonic extraction and reflux extraction;

[0027] (2) The extraction time is 15 min to 45 min;

[0028] (3) The extraction solvent includes a methanol solution or an aqueous ethanol solution, wherein the methanol solution includes methanol or an aqueous methanol solution;

[0029] (4) The mass-volume ratio of the cypress seed preparation to the extraction solvent is 1.0 g: (15-50) mL.

[0030] In one embodiment, the methanol solution contains 30%-100% methanol by volume; and / or

[0031] The volume percentage of ethanol in the ethanol-water solution is 50%-70%.

[0032] In one embodiment, obtaining the content of vanillic acid-4-β-D-glucoside in the cypress seed preparation includes the following steps:

[0033] The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations;

[0034] The reference standard solutions of different concentrations were injected separately and detected by ultra-high performance liquid chromatography.

[0035] The peak areas corresponding to vanillic acid-4-β-D-glucoside in the ultra-high performance liquid chromatography (UHPLC) spectra of the reference solution at different concentrations were obtained. A standard curve was plotted with the concentration of the reference solution as the x-axis and the peak areas corresponding to vanillic acid-4-β-D-glucoside in the reference solution at different concentrations as the y-axis. The linear regression equation was then calculated.

[0036] The content of vanillic acid-4-β-D-glucoside in the test sample was calculated by substituting the peak area corresponding to vanillic acid-4-β-D-glucoside in the ultra-high performance liquid chromatography (UHPLC) chromatogram obtained by UHPLC detection of the test sample solution into the linear regression equation.

[0037] The reference standard includes vanillic acid-4-β-D-glucoside reference standard.

[0038] A second aspect of the present invention provides a method for identifying preparations of Platycladus orientalis seed and their adulterants, comprising the following steps:

[0039] The sample to be tested is extracted using an extraction solvent to prepare a test solution;

[0040] The reference standard was dissolved using a dissolving reagent to prepare a reference standard solution;

[0041] The test solution and the reference solution were analyzed by ultra-high performance liquid chromatography, and the presence or absence of a chromatographic peak in the chromatogram of the test solution at the position corresponding to the retention time of the chromatographic peak in the reference solution was observed.

[0042] Wherein; the reference standard includes vanillic acid-4-β-D-glucoside reference standard; the preparation method of the test solution, the preparation method of the reference solution, and the ultra-high performance liquid chromatography detection method are the same as described above.

[0043] In one embodiment, the adulterant includes one or both of the standard decoctions of japonica rice or flaxseed.

[0044] A third aspect of the present invention provides the application of the method for determining the content of vanillic acid-4-β-D-glucoside in the above-described cypress seed preparations or the method for identifying cypress seed preparations and their adulterants as described above in the quality control of cypress seed preparations.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] This application establishes a method for determining the content of vanillic acid-4-β-D-glucoside, a water-soluble and specific active ingredient in arborvitae seed preparations, using specific UPLC (ultra-high performance liquid chromatography) conditions. This method provides an effective evaluation method for the quality control of arborvitae seed preparations, including standard decoctions and granules made from arborvitae seed. Methodological validation shows that this method has good specificity, precision, stability, repeatability, accuracy, and robustness, and can provide a reference for selecting content indicators when establishing quality standards for standard decoctions, granules, and classic Chinese medicine preparations containing arborvitae seed. Furthermore, under the chromatographic conditions proposed by this method, using vanillic acid-4-β-D-glucoside content as the determination indicator can distinguish arborvitae seed preparations from their common adulterants, such as standard decoctions of japonica rice and flaxseed. The method is simple, reliable, and highly objective. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the specific embodiments of this application or 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 this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 This is a specificity test diagram for the determination of vanillic acid-4-β-D-glucoside content in the standard decoction of cypress seed slices in Example 1.

[0049] Figure 2 This is a peak purity determination chart for vanillic acid-4-β-D-glucoside in the standard decoction of cypress seed slices in Example 1.

[0050] Figure 3 This is the standard curve of vanillic acid-4-β-D-glucoside in Example 1.

[0051] Figure 4 The chromatograms are obtained from different chromatographic columns used in Example 1.

[0052] Figure 5 The chromatograms are from different column temperatures observed in Example 1.

[0053] Figure 6 The chromatograms are for different flow rates investigated in Example 1.

[0054] Figure 7 This is a chromatogram comparison of the test sample similar to the standard decoction of Platycladus orientalis seed and the vanillic acid-4-β-D-glucoside reference standard to be identified in Example 2.

[0055] Figure 8 This is a chromatogram comparison of the test sample similar to the standard decoction of Platycladus orientalis seed and the vanillic acid-4-β-D-glucoside reference standard to be identified in Example 3.

[0056] Figure 9 This is a comparison of chromatographic peaks for the determination of vanillic acid-4-β-D-glucoside in the standard decoction of Platycladus orientalis seed under different chromatographic conditions in Comparative Example 1. Detailed Implementation

[0057] The present application will be further described in detail below with reference to specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0059] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0060] In this article, "one or more" refers to any one, two or more of the listed items.

[0061] In this document, the optional range of "and / or", "or / and", and "and / or" includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0062] In this document, terms such as "further," "even further," and "particularly" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0063] In this document, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood as not constituting a closed limitation on quantity. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of those features. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified. In the description of this invention, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0064] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0065] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0066] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0067] Unless otherwise specified, the temperature parameters in this invention allow for both isothermal treatment and treatment within a certain temperature range. The isothermal treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5°C, ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C. In this invention, room temperature or ambient temperature refers to the temperature without temperature control operation, generally referring to 4°C to 35°C, preferably 20±5°C.

[0068] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0069] The composition of cypress seed kernels is complex and diverse, containing both a large amount of weakly polar fatty oils and a large number of highly polar water-soluble components. Current quality research on cypress seed kernels mainly focuses on the content of major components such as total saponins, polysaccharides, and diterpenes, as well as some fat-soluble components like β-sitosterol and carotene. Furthermore, while the amino acids in cypress seed kernels have good water solubility, these components are ubiquitous in plants and animals and lack specificity. Therefore, research reports on the water-soluble and specific components of cypress seed kernels are relatively rare, and research on the quality standards of cypress seed kernel decoctions is even scarcer. In addition, due to the shortage and rising price of cypress seed kernels, some unscrupulous merchants mix them with cheaper japonica rice. Even worse, some bleach flax seeds, which are similar in size to cypress seed kernels. Bleached flax seeds are very similar in appearance to cypress seed kernels and are difficult to distinguish without careful observation. However, japonica rice and flaxseed belong to different families and genera from cypress seed, and do not have the same functions and indications as cypress seed.

[0070] Research revealed a lack of current research on the quality standards of Platycladus orientalis seeds. These seeds primarily contain a large amount of fatty oil, along with volatile oils, terpenes, saponins, phytosterols, vitamin A, proteins, and organic acids. Currently, numerous methods are used for the quality analysis of Platycladus orientalis seeds, including microscopic identification, thin-layer chromatography (TLC), spectrophotometry, and high-performance liquid chromatography (HPLC) content determination. Reports indicate the use of colorimetric reagents and spectrophotometry to determine the total saponin content in raw and processed Platycladus orientalis seeds; ultraviolet spectrophotometry to determine the polysaccharide content; vanillin-perchloric acid colorimetric methods; ultraviolet spectrophotometry to determine the diterpenoid content; and petroleum ether to determine the fatty oil content. Additionally, a TLC method for determining β-sitosterol content in Platycladus orientalis seeds and their processed products has been established. Subsequently, high-performance liquid chromatography-evaporative light scattering (HPLC-ELISA) was used to determine the content of carotene in Platycladus orientalis seed powder. Shen Shu et al. used pre-column derivatization-HPLC to determine and compare the content of 18 amino acids in Platycladus orientalis seeds from different origins. However, the above methods lack methods for determining the content of active ingredients with better water solubility, specificity, stability, and transfer rate in Platycladus orientalis seed standard decoctions to control the quality of Platycladus orientalis seed standard decoctions and formulation granules. Studies have shown that vanillic acid-4-β-D-glucoside contained in Platycladus orientalis seeds is an organic acid compound with good water solubility and stability, and has the effects of scavenging free radicals, anti-oxidation, anti-inflammation, sedation, fatigue relief, and immune enhancement, which are somewhat related to the functional indications of Platycladus orientalis seeds. Currently, there are no reports on methods for determining the content of vanillic acid-4-β-D-glucoside in Platycladus orientalis seeds.

[0071] Based on this, the technicians of this application discovered during the experiment that the method of establishing a method for determining the content of vanillic acid-4-β-D-glucoside in cypress seed preparations from different origins by using UPLC can realize the determination of the content of vanillic acid-4-β-D-glucoside, the active ingredient in cypress seed preparations, and provide a basis for the formulation of quality standards for cypress seed preparations.

[0072] In a first aspect, the present invention provides a method for determining the content of vanillic acid-4-β-D-glucoside in a preparation of Platycladus orientalis seed, comprising the following steps:

[0073] The extract of Platycladus orientalis seed preparation was carried out using an extraction solvent to prepare a test solution;

[0074] The content of vanillic acid-4-β-D-glucoside in the cypress seed preparation was obtained by ultra-high performance liquid chromatography detection of the test sample solution.

[0075] The conditions for ultra-high performance liquid chromatography detection include: mobile phase A is acetonitrile, mobile phase B is an aqueous phosphoric acid solution, and gradient elution is used;

[0076] The gradient elution procedure includes:

[0077] From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 1%.

[0078] Between 8 and 12 minutes, the volume percentage of the mobile phase A increased from 1% to 2%.

[0079] Between 12 and 18 minutes, the volume percentage of mobile phase A increased from 2% to 3%.

[0080] The volume percentage of mobile phase A is maintained at 3% for 18-22 minutes.

[0081] Between 22 and 25 minutes, the volume percentage of the mobile phase A increased from 3% to 80%.

[0082] In some of these examples, the conditions for the ultra-high performance liquid chromatography detection include: the chromatographic column is a C18 column.

[0083] In some of these examples, the conditions for the ultra-high performance liquid chromatography detection include: the column being packed with octadecylsilane-bonded silica gel.

[0084] In some of these examples, the column type includes ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column, Thermo Hpersil Gold aQ (2.1 mm × 100 mm, 1.9 μm) column, or Agilent Zorbax SB-aq (2.1 mm × 100 mm, 1.8 μm).

[0085] In some examples, the conditions for ultra-high performance liquid chromatography (UHPLC) detection include a column temperature of 28°C to 32°C. It is understood that the column temperature for UHPLC detection includes, but is not limited to, 28°C, 29°C, 30°C, 31°C, and 32°C.

[0086] In some examples, the conditions for ultra-high performance liquid chromatography (UHPLC) detection include a flow rate of 0.32 mL / min to 0.38 mL / min. It is understood that the flow rates for UHPLC detection include, but are not limited to, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, 0.35 mL / min, 0.36 mL / min, 0.37 mL / min, and 0.38 mL / min.

[0087] In some examples, the conditions for ultra-high performance liquid chromatography (UHPLC) detection also include an injection volume of 0.5 μL to 2 μL. It is understood that the injection volume for UHPLC detection includes, but is not limited to, 0.5 μL, 1 μL, 1.5 μL, and 2 μL.

[0088] In some examples, the ultra-high performance liquid chromatography (UHPLC) detection conditions further include a detection wavelength of 255 nm to 265 nm. It is understood that the wavelengths for UHPLC detection include, but are not limited to, 255 nm, 259 nm, 261 nm, 263 nm, and 265 nm.

[0089] In some of these examples, the cypress seed preparation includes at least one of the following: standard decoction of cypress seed medicinal slices, cypress seed formula granules, and water extract of cypress seed medicinal slices.

[0090] In some of these examples, the volume percentage of phosphoric acid in the aqueous phosphoric acid solution is 0.2% to 0.4%. It is understood that the volume percentage of phosphoric acid in the aqueous phosphoric acid solution includes, but is not limited to, 0.2%, 0.25%, 0.3%, 0.35%, and 0.4%.

[0091] In some examples, the conditions for the extraction step include: the extraction method includes one or both of ultrasonic extraction and reflux extraction.

[0092] In some examples, the extraction step includes a extraction time of 15 to 45 minutes. It is understood that the extraction time includes, but is not limited to, 15, 20, 25, 30, 35, 40, and 45 minutes. Further, the extraction step includes a extraction time of 30 to 45 minutes.

[0093] In some examples, the conditions for the extraction step include: the extraction solvent is an aqueous methanol solution or an aqueous ethanol solution.

[0094] In some of these examples, the methanol solution comprises methanol or an aqueous methanol solution.

[0095] In some examples, the methanol solution contains 30%-100% methanol by volume. It is understood that the methanol aqueous solution contains, but is not limited to, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% methanol by volume. Further, the methanol solution contains 30%-70% methanol by volume.

[0096] In some of these examples, the volume percentage of ethanol in the aqueous ethanol solution is 50%-70%. It is understood that the volume percentage of ethanol in the aqueous ethanol solution includes, but is not limited to, 50%, 55%, 60%, 65%, and 70%.

[0097] In some examples, the extraction step is performed under the following conditions: the mass-to-volume ratio of the arborvitae seed preparation to the extraction solvent is 1.0 g : (15-50) mL. It is understood that the mass-to-volume ratio of the arborvitae seed preparation to the extraction solvent includes, but is not limited to, 1.0 g : 15 mL, 1.0 g : 20 mL, 1.0 g : 25 mL, 1.0 g : 30 mL, 1.0 g : 35 mL, 1.0 g : 40 mL, 1.0 g : 45 mL, and 1.0 g : 50 mL.

[0098] In some of these examples, obtaining the content of vanillic acid-4-β-D-glucoside in the said cypress seed preparation includes the following steps:

[0099] The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations;

[0100] The reference standard solutions of different concentrations were injected separately and detected by ultra-high performance liquid chromatography.

[0101] The peak areas corresponding to vanillic acid-4-β-D-glucoside in the ultra-high performance liquid chromatography (UHPLC) spectra of the reference solution at different concentrations were obtained. A standard curve was plotted with the concentration of the reference solution as the x-axis and the peak areas corresponding to vanillic acid-4-β-D-glucoside in the reference solution at different concentrations as the y-axis. The linear regression equation was then calculated.

[0102] The content of vanillic acid-4-β-D-glucoside in the test sample was calculated by substituting the peak area corresponding to vanillic acid-4-β-D-glucoside in the ultra-high performance liquid chromatography (UHPLC) chromatogram obtained by UHPLC detection of the test sample solution into the linear regression equation.

[0103] The reference standard includes vanillic acid-4-β-D-glucoside reference standard.

[0104] A second aspect of the present invention provides a method for identifying preparations of Platycladus orientalis seed and their adulterants, comprising the following steps:

[0105] The sample to be tested is extracted using an extraction solvent to prepare a test solution;

[0106] The reference standard was dissolved using a dissolving reagent to prepare a reference standard solution;

[0107] The test solution and the reference solution were analyzed by ultra-high performance liquid chromatography, and the presence or absence of a chromatographic peak in the chromatogram of the test solution at the position corresponding to the retention time of the chromatographic peak in the reference solution was observed.

[0108] The reference standard includes vanillic acid-4-β-D-glucoside reference standard; the preparation method of the test solution, the preparation method of the reference solution, and the ultra-high performance liquid chromatography detection method are the same as described above.

[0109] In some of these examples, the dissolving agent for the reference standard includes methanol.

[0110] In some of these examples, the counterfeit products include one or both of the standard decoctions of japonica rice or flaxseed.

[0111] A third aspect of the present invention provides the application of the method for determining the content of vanillic acid-4-β-D-glucoside in the above-described cypress seed preparations or the method for identifying cypress seed preparations and their adulterants as described above in the quality control of cypress seed preparations.

[0112] This application establishes for the first time a method for determining the content of vanillic acid-4-β-D-glucoside in standard decoctions of Platycladus orientalis seed slices. Methodological validation has shown that this method exhibits good specificity, precision, repeatability, accuracy, and robustness. It can provide a reference for selecting content indicators when establishing quality standards for standard decoctions of Platycladus orientalis seed slices, Platycladus orientalis seed granules, and other classic Chinese medicine preparations containing Platycladus orientalis seed. Furthermore, under the chromatographic conditions proposed in this application, it is possible to distinguish between standard decoctions of Platycladus orientalis seed and their common adulterants, such as standard decoctions of japonica rice and flaxseed.

[0113] The following detailed description is provided in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the following embodiments are commercially available; all instruments used are commercially available; and all processes involved are conventionally selected by those skilled in the art unless otherwise specified.

[0114] Example 1

[0115] This embodiment relates to the construction of a method for determining the content of vanillic acid-4-β-D-glucoside in a standard decoction of Platycladus orientalis seed, including the following steps:

[0116] 1. Preparation of Platycladus orientalis seed slices

[0117] Arborvitae seed slices were prepared according to the processing regulations under the Arborvitae Seed section of the 2020 edition of the Chinese Pharmacopoeia. The specific processing method is as follows: Take the Arborvitae Seed material and remove impurities and residual seed coat.

[0118] 2. Preparation of Standard Decoction of Cypress Seed (Platycladus orientalis)

[0119] Take 200g of cypress seed slices, crush them, place them in an electric ceramic kettle, add water and decoct twice. For the first decoction, add 8 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter while hot through a 350-mesh sieve, and quickly cool the filtrate with cold water. For the second decoction, add 6 times the amount of water, bring to a boil over high heat (500W), then simmer over low heat (200W) for 25 minutes. Filter while hot through a 350-mesh sieve, and quickly cool the filtrate with cold water. Combine the two filtrates. The decoction was transferred to a 5000mL round-bottom flask and concentrated under reduced pressure at low temperature (65℃) using a rotary evaporator to obtain 100mL of fluid extract. The extract was then dispensed into petri dishes and transferred to a vacuum freeze dryer for lyophilization. After dispensing, the extract was removed, sealed, and the standard decoction lyophilized powder was obtained. The batch numbers of the standard decoction lyophilized powder prepared from 20 batches of Chinese herbal medicine slices of Platycladus orientalis from different origins are shown in Table 1.

[0120] Table 1. Origin Information of 20 Batches of Arborvitae Seed Slices and Freeze-dried Standard Decoction Powder

[0121]

[0122] 3. Establishment of a method for determining vanillic acid-4-β-D-glucoside content

[0123] 3.1. Instruments, Reagents and Chemicals

[0124] Instruments: Waters H-Class ultra-high performance liquid chromatograph, Waters PDA detector, Empower workstation, ACQUITY UPLC® HSS T3 (2.1mm×100mm, 1.8μm) column, Thermo Hpersil Gold aQ (2.1mm×100mm, 1.9μm) column, Agilent Zorbax SB-aq (2.1mm×100mm, 1.8μm) column, analytical balance 0.001g (ME204E, Mettler Toledo), analytical balance 0.1m / million (XP26, Mettler Toledo), ultrasonic cleaner (Wuxi Ultrasonic Electronic Equipment Factory), constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., model: HWS28), ultrapure water system (Merck, Milli-Q Direct).

[0125] Reagents: Acetonitrile (Merck, chromatographic grade); Methanol (Tianjin Fuyu Chemical Reagent Co., Ltd., analytical grade); Ethanol (Tianjin Fuyu Chemical Reagent Co., Ltd., analytical grade); Phosphoric acid (Tianjin Kemio Chemical Reagent Co., Ltd., chromatographic grade); Water was ultrapure water.

[0126] Test drug: Vanillic acid-4-β-D-glucoside reference standard (PS012936, Chengdu Pusi Biotechnology Co., Ltd., content: 98.0%); the batch numbers of 20 batches of cypress seed standard decoction are shown in Table 1.

[0127] 3.2. Chromatographic conditions

[0128] An ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used; acetonitrile was used as mobile phase A and 0.3% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.35 mL per minute; the column temperature was 30 °C; the detection wavelength was 260 nm; and the injection volume was 1 μL.

[0129] Table 2 Elution gradient

[0130]

[0131] 3.3. Preparation of reference solution

[0132] Accurately weigh 2.412 mg and 2.276 mg of vanillic acid-4-β-D-glucoside reference standards, respectively, and add them to 100 mL volumetric flasks. Dilute to the mark with methanol, shake well, and prepare reference solutions containing 23.6367 μg and 22.3048 μg of vanillic acid-4-β-D-glucoside per mL, respectively.

[0133] 3.4. Investigation of pretreatment methods for test sample solutions

[0134] The extraction solvent, extraction method, extraction time, and amount of extraction solvent for the standard decoction of cypress seed were investigated to determine the optimal sample pretreatment method.

[0135] (1) Investigation of extraction solvent

[0136] Take an appropriate amount of standard decoction of cypress seed (BZR-GT-15), about 1.0 g, accurately weigh it, make 6 parallel groups, 2 portions for each group, place them in a stoppered conical flask, accurately add 25 mL each of methanol, 70% methanol, 50% methanol, 30% methanol, 70% ethanol and 50% ethanol, weigh them, sonicate (power 300W, frequency 40kHz) for 30 minutes, cool, weigh them again, add the corresponding solvent to make up the weight loss, shake well, filter, take the filtrate, inject and analyze according to the chromatographic conditions under section "3.2", record the chromatographic peak area, and calculate the content of vanillic acid-4-β-D-glucoside. The experimental results are shown in Table 3.

[0137] Table 3. Determination of vanillic acid-4-β-D-glucoside content and investigation of extraction solvents in standard decoctions of Platycladus orientalis seed.

[0138]

[0139] Experimental results showed that the content of vanillic acid-4-β-D-glucoside was highest when 70% methanol was used as the extraction solvent. Therefore, 70% methanol was selected as the extraction solvent for determining the content of vanillic acid-4-β-D-glucoside in the standard decoction of Platycladus orientalis seeds.

[0140] (2) Examination of extraction methods

[0141] Take an appropriate amount of standard decoction of cypress seed (BZR-GT-15), about 1.0 g, accurately weigh it, make 2 parallel groups, 2 portions in each group, place them in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh it, sonicate (power 300W, frequency 40kHz) for 30 minutes, heat under reflux for 30 minutes, cool, weigh it again, make up the weight loss with 70% methanol solution, shake well, filter, take the filtrate, inject and analyze it according to the chromatographic conditions under section "3.2", record the peak area, and calculate the content of vanillic acid-4-β-D-glucoside. The experimental results are shown in Table 4.

[0142] Table 4. Results of the investigation on extraction methods for determining vanillic acid-4-β-D-glucoside content in standard decoction of Platycladus orientalis seeds.

[0143]

[0144] The results showed that ultrasonic treatment and reflux extraction were equally efficient. Considering the ease of operation, ultrasonic extraction was chosen as the extraction method for determining the vanillic acid-4-β-D-glucoside content in the standard decoction of Platycladus orientalis seeds.

[0145] (3) Examination of extraction time

[0146] Take an appropriate amount of standard decoction of cypress seed (BZR-GT-15), about 1.0g, accurately weigh it, make 3 parallel groups, 2 portions in each group, place them in a stoppered conical flask, accurately add 25mL of 70% methanol solution, weigh them, and sonicate them (power 300W, frequency 40kHz) for 15 minutes, 30 minutes and 45 minutes respectively, cool them, weigh them again, make up the weight loss with 70% methanol solution, shake well, filter, take the filtrate, and inject it for analysis according to the chromatographic conditions under "3.2", record the chromatographic peak area, and calculate the content of vanillic acid-4-β-D-glucoside. The experimental results are shown in Table 5.

[0147] Table 5. Results of the Determination of Vanillic Acid-4-β-D-Glucoside Content and Extraction Time in Standard Decoction of Platycladus orientalis Seeds

[0148]

[0149] The results showed that the content of vanillic acid-4-β-D-glucoside in the standard decoction of cypress seed was comparable when the extraction time was 30 minutes and 45 minutes. To ensure the robustness of the method, 30 minutes was selected as the extraction time for determining the content of vanillic acid-4-β-D-glucoside in the standard decoction of cypress seed.

[0150] (4) Investigation on the amount of extraction solvent used

[0151] Take an appropriate amount of standard decoction of cypress seed (BZR-GT-15), about 1.0 g, accurately weigh it, make 3 parallel groups, 2 portions of each group, place them in stoppered conical flasks, accurately add 15 mL, 25 mL and 50 mL of 70% methanol solution, weigh them, sonicate them (power 300W, frequency 40kHz) for 30 minutes, cool them, weigh them again, make up the weight loss with 70% methanol solution, shake well, filter, take the filtrate, inject it for analysis according to the chromatographic conditions under section "3.2", record the peak area, and calculate the content of vanillic acid-4-β-D-glucoside. The experimental results are shown in Table 6.

[0152] Table 6. Results of the Determination of Vanillic Acid-4-β-D-Glucoside Content in Standard Decoction of Platycladus orientalis Seeds and the Amount of Extraction Solvent Used

[0153]

[0154] The results showed that different amounts of extraction solvent had no significant effect on the content of vanillic acid-4-β-D-glucoside in the standard decoction. To ensure that the extraction solvent fully wetted the sample and to save costs, 25 mL was selected as the amount of extraction solvent for determining the vanillic acid-4-β-D-glucoside content in the standard decoction of Platycladus orientalis seed.

[0155] (5) Determination of the preparation method of the test solution

[0156] Based on the above experimental results, the final preparation method of the test solution is as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 1.0 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol solution, weigh it, sonicate it (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% methanol solution, shake it well, filter it, and take the filtrate to obtain the test solution.

[0157] 3.5. Methodological Validation

[0158] (1) Specificity examination

[0159] Take an appropriate amount of the standard decoction of Platycladus orientalis seeds (BZR-GT-15), grind it into a fine powder, take about 1.0 g, and prepare the test solution according to the method for preparing the test solution specified in section "3.4". Separately, take 1 μL each of vanillic acid-4-β-D-glucoside reference solution and blank solvent, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions in section "3.2". The results are shown in the figure. Figure 1 .

[0160] The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as that of the reference sample, and the blank solvent did not interfere, indicating that the method has good specificity.

[0161] (2) Peak purity test

[0162] Take an appropriate amount of the standard decoction of cypress seed slices (BZR-GT-15), grind it into a fine powder, take about 1.0g, and prepare the test solution according to the test sample preparation method specified in section "3.4". Accurately pipette the test solution and vanillic acid-4-β-D-glucoside reference solution, inject them into the liquid chromatograph, and perform a DAD detector scan at 210~400nm under the chromatographic conditions in section "3.2". Calculate the peak purity, and the results are shown in the figure. Figure 2 .

[0163] Experimental results showed that no impurity peak was detected in the vanillic acid-4-β-D-glucoside peak in the sample, and the purity angle was less than the purity threshold. When the purity angle is less than the purity threshold, it indicates that the compound corresponding to the chromatographic peak has good purity, meaning that the purity of the vanillic acid-4-β-D-glucoside peak meets the requirements under these chromatographic conditions.

[0164] (3) Examination of linear relationships

[0165] Accurately weigh 3.12 mg of vanillic acid-4-β-D-glucoside reference standard, place it in a 20 mL volumetric flask, and add methanol solution to prepare a solution containing 154.3500 μg per mL, which is used as the reference standard stock solution.

[0166] Accurately measure 1.5 mL, 1.0 mL, 1.0 mL, 1.0 mL, 1.0 mL, 1.0 mL, and 0.5 mL of the reference standard stock solution with a concentration of 154.35 μg / mL, and place them into volumetric flasks of 2 mL, 2 mL, 5 mL, 10 mL, 20 mL, and 50 mL, respectively. Dilute to the mark with methanol solution to prepare reference standard solutions containing 115.7625 μg, 77.1750 μg, 30.8700 μg, 15.4350 μg, 7.7175 μg, and 1.5435 μg per mL, respectively. Accurately pipette the above six reference standard solutions and the stock solution of different concentrations, and inject them according to the chromatographic conditions in section "3.2". Record the peak areas. Plot a standard curve with peak area as the ordinate (y) and reference standard concentration as the abscissa (x), as shown in Table 7. Figure 3 .

[0167] Table 7. Results of linearity study of vanillic acid-4-β-D-glucoside

[0168]

[0169] The results showed that the regression equation for vanillic acid-4-β-D-glucoside was y=4878.6610x-620.8810, r=0.9997, indicating that vanillic acid-4-β-D-glucoside had a good linear relationship with peak area in the range of 1.5435μg / mL to 154.3500μg / mL.

[0170] (4) Precision test

[0171] Accurately pipette the reference solution with a concentration of 30.8700 μg / mL, and inject it 6 times repeatedly under the chromatographic conditions in section “3.2”. Calculate the RSD value based on the peak area of ​​vanillic acid-4-β-D-glucoside. The results are shown in Table 8.

[0172] Table 8. Precision test results of vanillic acid-4-β-D-glucoside content determination in standard decoction of cypress seed kernel.

[0173]

[0174] The results showed that when the same sample solution was injected six times consecutively, the RSD value of the peak area of ​​vanillic acid-4-β-D-glucoside was 0.45%, which is less than 3.0%, indicating that the instrument has good precision.

[0175] (5) Stability test

[0176] Take an appropriate amount of the standard decoction of cypress seed slices (BZR-GT-15), grind it into a fine powder, take about 1.0g, accurately weigh it, and prepare the test solution according to the test solution preparation method determined under "3.4". According to the chromatographic conditions under "3.2", inject the sample at 0, 2, 4, 8, 12 and 16 hours respectively, and determine the content of vanillic acid-4-β-D-glucoside and RSD value in the test solution. The determination results are shown in Table 9.

[0177] Table 9. Results of the stability study on the content of vanillic acid-4-β-D-glucoside in the standard decoction of cypress seed slices.

[0178]

[0179] The results showed that for the same sample solution, the RSD value of the peak area of ​​vanillic acid-4-β-D-glucoside was 1.85% when the sample was injected and measured at 0, 2, 4, 8, 12 and 16 hours, indicating that the sample solution under this method had good stability within 16 hours.

[0180] (6) Repeated examination

[0181] Take an appropriate amount of the standard decoction of cypress seed slices (BZR-GT-15), grind it into a fine powder, take about 1.0g, accurately weigh it, and prepare 6 parallel portions. Prepare 6 test solutions according to the test solution preparation method determined under "3.4", and analyze them according to the chromatographic conditions under "3.2". Calculate the content of vanillic acid-4-β-D-glucoside and the RSD value. The determination results are shown in Table 10.

[0182] Table 10. Results of Repeatability Study on Vanillic Acid-4-β-D-Glucoside Content Determination in Standard Decoction of Platycladus orientalis Seed Slices

[0183]

[0184] The results showed that when the same batch of samples was repeatedly measured 6 times, the RSD value of vanillic acid-4-β-D-glucoside was 2.86%. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.01% and 0.1%, the repeatability RSD limit is <3%, indicating that the repeatability of this analytical method is good.

[0185] (7) Intermediate precision test

[0186] Different researchers were selected to conduct the tests at different times and with different instruments. An appropriate amount of the standard decoction of Platycladus orientalis seed (BZR-GT-1) was taken, ground into a fine powder, and about 1.0 g was taken and divided into 6 parallel portions. The test solution was prepared according to the test solution preparation method determined in section "3.4". The test solution was determined according to the chromatographic conditions in section "3.2". The samples were injected separately to determine the vanillic acid-4-β-D-glucoside content in the test solution. The vanillic acid-4-β-D-glucoside content and RSD value were calculated and compared with the results of the repeatability test. See Table 11.

[0187] Table 11 Intermediate precision results for the determination of vanillic acid-4-β-D-glucoside content in standard decoctions of Platycladus orientalis seed slices.

[0188]

[0189] The results showed that when the same batch of samples was measured six times by different personnel on different instruments, the RSD value of vanillic acid-4-β-D-glucoside content was 1.49%, which was 3.46% compared with the RSD value of the six data in the repeatability test. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.01% and 0.1%, the intermediate precision RSD limit is <6%. Therefore, the intermediate precision of this method is good even when different analysts operate on different dates and under different chromatographs.

[0190] (8) Accuracy test

[0191] Reference stock solution: Accurately weigh 5.140 mg vanillic acid-4-β-D-glucoside reference standard and place it in a 200 mL volumetric flask. Add 70% methanol solution to prepare a reference stock solution containing 24.6029 μg of vanillic acid-4-β-D-glucoside per mL.

[0192] The spiking recovery method was used. The ratio of the added reference standard to the analyte in the sample was 1:1. An appropriate amount of standard decoction of Platycladus orientalis seed (BZR-GT-15) was finely ground, and approximately 0.5 g was accurately weighed. Six parallel portions were prepared and placed in stoppered conical flasks. 20 mL of the reference standard solution was accurately added to each flask, followed by 5 mL of 70% methanol solution. The sample solution was prepared according to the method specified in section "3.4". The chromatographic conditions in section "3.2" were used to determine the vanillic acid-4-β-D-glucoside content in the sample solution, and the spiking recovery rate was calculated. The results are shown in Table 12.

[0193] Table 12 Results of Vanillic Acid-4-β-D-Glucoside Content Determination in Standard Decoction of Platycladus orientalis Seeds

[0194]

[0195] The results showed that the recovery rate of vanillic acid-4-β-D-glucoside was 93.79%. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, the recovery limit is 90% to 108% when the content of the analyte in the sample is 0.01% to 0.1%. The recovery rate of vanillic acid-4-β-D-glucoside ranged from 90.72% to 95.10%, with an average recovery rate of 93.79% and an RSD of 1.73%, indicating that the method has good accuracy.

[0196] (9) Durability test

[0197] ① Investigation of different chromatographic columns

[0198] The effects of different brands of chromatographic columns—ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column, Thermo Hpersil Gold aQ (2.1 mm × 100 mm, 1.9 μm) column, and Agilent Zorbax SB-aq (2.1 mm × 100 mm, 1.8 μm) column—on the determination of vanillic acid-4-β-D-glucoside content in standard decoction of Platycladus orientalis seed were compared.

[0199] Take an appropriate amount of the standard decoction of cypress seed slices (BZR-GT-15), grind it into a fine powder, and accurately weigh about 1.0 g. Prepare the test solution according to the method specified in section "3.4". Except for the chromatographic column, all other conditions are the same as specified in section "3.2". Inject the sample for analysis, calculate the content of vanillic acid-4-β-D-glucoside and the RSD value. The experimental results are shown in Table 13. Figure 4 .

[0200] Table 13 Results of durability studies of different chromatographic columns for the determination of vanillic acid-4-β-D-glucoside content in standard decoction of Platycladus orientalis seed.

[0201]

[0202] The results showed that different brands of chromatographic columns had good separation effects, and the RSD value of vanillic acid-4-β-D-glucoside content was 0.85%, indicating that the method has good durability with different brands of chromatographic columns.

[0203] ② Investigation at different column temperatures

[0204] The effects of different column temperatures (28℃, 30℃, and 32℃) on the determination of vanillic acid-4-β-D-glucoside content in the standard decoction of Platycladus orientalis seed were compared.

[0205] Take an appropriate amount of the standard decoction of cypress seed slices (BZR-GT-15), grind it into a fine powder, and accurately weigh about 1.0g. Prepare the test solution according to the method specified in section "3.4". Except for the column temperatures of 28℃, 30℃, and 32℃, all other conditions are the same as specified in section "3.2". Inject the sample for analysis, calculate the content of vanillic acid-4-β-D-glucoside and the RSD value. The experimental results are shown in Table 14. Figure 5 .

[0206] Table 14 Results of the durability study at different column temperatures for the determination of vanillic acid-4-β-D-glucoside content in the standard decoction of cypress seed kernel.

[0207]

[0208] The results showed that the RSD value of vanillic acid-4-β-D-glucoside in the standard decoction of cypress seed slices measured at different column temperatures (±2℃) was 1.94%, indicating that the analytical method has good robustness within the column temperature range of ±2℃.

[0209] ③ Investigation of different flow velocities

[0210] The effects of different flow rates (0.32 mL / min, 0.35 mL / min, and 0.38 mL / min) on the determination of vanillic acid-4-β-D-glucoside content in the standard decoction of Platycladus orientalis seed slices were compared.

[0211] Take an appropriate amount of the standard decoction of cypress seed slices (BZR-GT-15), grind it into a fine powder, and accurately weigh about 1.0 g. Prepare the test solution according to the method specified in section "3.4". Except for the flow rates of 0.32 mL / min, 0.35 mL / min, and 0.38 mL / min, all other conditions are the same as specified in section "3.2". Inject the sample for analysis, calculate the content of vanillic acid-4-β-D-glucoside and the RSD value. The experimental results are shown in Table 15. Figure 6 .

[0212] Table 15 Results of durability study at different flow rates for the determination of vanillic acid-4-β-D-glucoside content in the standard decoction of Platycladus orientalis seed.

[0213]

[0214] The results showed that the RSD value of vanillic acid-4-β-D-glucoside content in the standard decoction of cypress seed slices was 1.72% at different flow rates (±0.03 mL / min), indicating that the analytical method has good robustness within the flow rate range of ±0.03 mL / min.

[0215] 3.6. Sample Determination

[0216] Take 20 batches of standard decoction of cypress seed slices, prepare 20 test solutions according to the test solution preparation method under section "3.4", inject and determine according to the chromatographic conditions under section "3.2", record the peak area, and calculate using the external standard method. The results are shown in Table 16.

[0217] Table 16 Results of vanillic acid-4-β-D-glucoside content determination in 20 batches of standard decoction of cypress seed slices

[0218]

[0219] The results showed that the content of vanillic acid-4-β-D-glucoside in the 20 batches of standard decoction of Platycladus orientalis seed slices ranged from 0.52 mg / g to 1.18 mg / g, with an average content of 0.76 mg / g and a standard deviation (SD) of 0.16 mg / g. The 70%–130% range of the mean was 0.53 mg / g to 0.98 mg / g, and the mean ± 3 times the SD range was 0.27 mg / g to 1.24 mg / g. Based on the data of vanillic acid-4-β-D-glucoside content in the 20 batches of standard decoction of Platycladus orientalis seed slices, the lower limit of the content was determined to be the mean - 2 SD, and the upper limit was determined to be the mean + 3 SD, i.e., 0.43 mg / g to 1.24 mg / g, rounded to 0.40 mg / g to 1.25 mg / g. The content of vanillic acid-4-β-D-glucoside in the 20 batches of standard decoction of Platycladus orientalis seed slices was within this range, which is reasonable.

[0220] Example 2

[0221] This embodiment describes a method for identifying standard decoctions of Platycladus orientalis seed slices using vanillic acid-4-β-D-glucoside as an indicator. The specific implementation steps are as follows:

[0222] 1. Chromatographic conditions

[0223] An ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used; acetonitrile was used as mobile phase A and 0.3% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.35 mL per minute; the column temperature was 30 °C; the detection wavelength was 260 nm; and the injection volume was 1 μL.

[0224] 2. Preparation of reference solution

[0225] Accurately weigh 2.412 mg and 2.276 mg of vanillic acid-4-β-D-glucoside reference standards, respectively, and add them to 100 mL volumetric flasks. Dilute to the mark with methanol, shake well, and prepare reference solutions containing 23.6367 μg and 22.3048 μg of vanillic acid-4-β-D-glucoside per mL, respectively.

[0226] 3. Preparation of the test solution

[0227] Take appropriate amounts of test samples (batch numbers: JM-GT-1, JM-GT-2) that are similar to the standard decoction of cypress seed slices, grind them into a fine powder, take about 1.0g, weigh accurately, place them in a stoppered conical flask, accurately add 25mL of 70% methanol solution, weigh, sonicate (power 300W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol solution, shake well, filter, and take the filtrate to obtain the final product.

[0228] 4. Determination method: Accurately pipette 1 μL of the reference solution and the above test solution into the liquid chromatograph and determine the result.

[0229] 5. Determination Results: The standard decoctions of *Platycladus orientalis* seed slices from batches JM-GT-1 and JM-GT-2 showed no chromatographic peak response at the position corresponding to the retention time of the vanillic acid-4-β-D-glucoside reference standard. (See attached image.) Figure 7 Therefore, the samples with batch numbers JM-GT-1 and JM-GT-2 are not the standard decoction of cypress seed. This invention can be used to identify the standard decoction of cypress seed and its common counterfeit, japonica rice standard decoction.

[0230] Example 3

[0231] Example 3 describes a method for identifying standard decoctions of Platycladus orientalis seed slices using vanillic acid-4-β-D-glucoside as an indicator. The specific implementation steps are as follows:

[0232] 1. Chromatographic conditions

[0233] An ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used; acetonitrile was used as mobile phase A and 0.3% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.35 mL per minute; the column temperature was 30 °C; the detection wavelength was 260 nm; and the injection volume was 1 μL.

[0234] 2. Preparation of reference solution

[0235] Accurately weigh 2.412 mg and 2.276 mg of vanillic acid-4-β-D-glucoside reference standards into 100 mL volumetric flasks, dilute to the mark with methanol, and shake well to prepare reference solutions containing 23.6367 μg and 22.3048 μg of vanillic acid-4-β-D-glucoside per mL, respectively.

[0236] 3. Preparation of the test solution

[0237] Take an appropriate amount of the test sample (batch number: YMZ-GT-1, YMZ-GT-2) that is similar to the standard decoction of cypress seed slices, grind it into a fine powder, take about 1.0g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% methanol solution, weigh it, sonicate it (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% methanol solution, shake it well, filter it, and take the filtrate to obtain the product.

[0238] 4. Determination method: Accurately pipette 1 μL of the reference solution and the above test solution into the liquid chromatograph and determine the result.

[0239] 5. Determination Results: The standard decoctions of *Platycladus orientalis* seed slices from batches YMZ-GT-1 and YMZ-GT-2 showed no chromatographic peak response at the position corresponding to the retention time of the vanillic acid-4-β-D-glucoside reference standard. (See attached image) Figure 8 Therefore, samples with batch numbers YMZ-GT-1 and YMZ-GT-2 are not the standard decoction of cypress seed. This invention can be used to identify the standard decoction of cypress seed and its common counterfeit, bleached flaxseed standard decoction.

[0240] Comparative Example 1

[0241] This comparative study investigated the effect of different mobile phase ratios on the determination of vanillic acid-4-β-D-glucoside content in the standard decoction of Platycladus orientalis seeds.

[0242] Take the BZR-GT-15 test solution from Example 1 and determine the vanillic acid-4-β-D-glucoside in the standard decoction of Platycladus orientalis seed using the different chromatographic conditions shown below.

[0243] (1) Ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation), ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; acetonitrile: 0.3% phosphoric acid aqueous solution (V:V=2:98) as mobile phase; flow rate of 0.35 mL per minute; column temperature of 30 °C; injection volume of 1 μL; detection wavelength of 260 nm; isocratic elution.

[0244] (2) Ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation), ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; acetonitrile as mobile phase A, 0.3% phosphoric acid solution as mobile phase B, elution according to the elution gradient in Table 17; flow rate of 0.35 mL per minute; column temperature of 30 °C; injection volume of 1 μL; detection wavelength of 260 nm.

[0245] Table 17 Elution Procedure

[0246]

[0247] (3) Ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation), ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; acetonitrile: 0.3% phosphoric acid aqueous solution (V:V=3:97) as mobile phase; flow rate of 0.35 mL per minute; column temperature of 30 °C; injection volume of 1 μL; detection wavelength of 260 nm; isocratic elution.

[0248] (4) Ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation), ACQUITY UPLC® HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; acetonitrile as mobile phase A, 0.3% phosphoric acid solution as mobile phase B, elution according to the elution gradient shown in Table 2; flow rate of 0.35 mL per minute; column temperature of 30 °C; injection volume of 1 μL; detection wavelength of 260 nm.

[0249] The results showed that, compared with the chromatographic conditions studied in this application, the resolution and peak shape of vanillic acid-4-β-D-glucoside peaks could not achieve the same effect as those in this application when using other different mobile phase elution programs and ratios. Figure 9 (4)); For example, when using isocratic acetonitrile: 0.3% phosphoric acid solution (V:V=2:98), the chromatographic peak of vanillic acid-4-β-D-glucoside cannot be separated from the adjacent chromatographic peak ( Figure 9 (1) When using isocratic acetonitrile:0.3% phosphoric acid solution (V:V=3:97), the peak shape of vanillic acid-4-β-D-glucoside was poor. Figure 9 (3)); When using other gradient elution methods, the vanillic acid-4-β-D-glucoside chromatographic peak could not be separated from adjacent chromatographic peaks ( Figure 9 (2) The results of the above comparative examples show that changing the mobile phase elution program and ratio has a significant impact on the chromatographic peak of vanillic acid-4-β-D-glucoside, and it is impossible to obtain a good resolution and peak shape. In order to more accurately determine the content of vanillic acid-4-β-D-glucoside in the standard decoction of cypress seed, the chromatographic conditions in Example 1, i.e. the conditions of Comparative Example 1 (4), were selected.

[0250] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0251] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the content of vanillic acid-4-β-D-glucoside in a preparation of Platycladus orientalis seed, characterized in that, Includes the following steps: The extract of Platycladus orientalis seed preparation is carried out using an extraction solvent to prepare a test solution; the extraction solvent includes methanol solution or aqueous ethanol solution; The content of vanillic acid-4-β-D-glucoside in the cypress seed preparation was obtained by ultra-high performance liquid chromatography detection of the test sample solution. The conditions for ultra-high performance liquid chromatography detection include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution, gradient elution is used, the detection wavelength is 255nm~265nm, and the chromatographic column is a C18 column; The gradient elution procedure includes: From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 1%. Between 8 and 12 minutes, the volume percentage of the mobile phase A increased from 1% to 2%. Between 12 and 18 minutes, the volume percentage of mobile phase A increased from 2% to 3%. The volume percentage of mobile phase A is maintained at 3% for 18-22 minutes. Between 22 and 25 minutes, the volume percentage of the mobile phase A increased from 3% to 80%.

2. The method for determining the content of vanillic acid-4-β-D-glucoside in the Platycladus orientalis seed preparation according to claim 1, characterized in that, The ultra-high performance liquid chromatography detection satisfies at least one of the following conditions: (1) The column temperature is 28°C~32°C; (2) The flow rate is 0.32 mL / min to 0.38 mL / min; (3) The injection volume is 0.5 μL to 2 μL.

3. The method for determining the content of vanillic acid-4-β-D-glucoside in the Platycladus orientalis seed preparation according to claim 1, characterized in that, The cypress seed preparation includes at least one of the following: standard decoction of cypress seed medicinal slices, cypress seed formula granules, and water extract of cypress seed medicinal slices.

4. The method for determining the content of vanillic acid-4-β-D-glucoside in the Platycladus orientalis seed preparation according to claim 1, characterized in that, The phosphoric acid aqueous solution contains phosphoric acid at a volume percentage of 0.2% to 0.4%.

5. The method for determining the content of vanillic acid-4-β-D-glucoside in the Platycladus orientalis seed preparation according to claim 1, characterized in that, The extraction step satisfies at least one of the following conditions: (1) The extraction method includes one or both of ultrasonic extraction and reflux extraction; (2) The extraction time is 15 min to 45 min; (3) The methanol solution includes methanol or an aqueous methanol solution; (4) The mass-volume ratio of the cypress seed preparation to the extraction solvent is 1.0 g: (15-50) mL.

6. The method for determining the content of vanillic acid-4-β-D-glucoside in the Platycladus orientalis seed preparation according to claim 5, characterized in that, The methanol solution contains 30%-100% methanol by volume; and / or The volume percentage of ethanol in the ethanol-water solution is 50%-70%.

7. The method for determining the content of vanillic acid-4-β-D-glucoside in the preparation of Platycladus orientalis seed according to any one of claims 1 to 6, characterized in that, Obtaining the content of vanillic acid-4-β-D-glucoside in the aforementioned Platycladus orientalis seed preparation includes the following steps: The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations; The reference standard solutions of different concentrations were injected separately and detected by ultra-high performance liquid chromatography. The peak areas corresponding to vanillic acid-4-β-D-glucoside in the ultra-high performance liquid chromatography (UHPLC) spectra of the reference solution at different concentrations were obtained. A standard curve was plotted with the concentration of the reference solution as the x-axis and the peak areas corresponding to vanillic acid-4-β-D-glucoside in the reference solution at different concentrations as the y-axis. The linear regression equation was then calculated. The content of vanillic acid-4-β-D-glucoside in the test sample was calculated by substituting the peak area corresponding to vanillic acid-4-β-D-glucoside in the ultra-high performance liquid chromatography (UHPLC) chromatogram obtained by UHPLC detection of the test sample solution into the linear regression equation. The reference standard includes vanillic acid-4-β-D-glucoside reference standard.

8. A method for identifying preparations of Platycladus orientalis seeds and their adulterants, characterized in that, Includes the following steps: The sample to be tested is extracted using an extraction solvent to prepare a test solution; The reference standard was dissolved using a dissolving reagent to prepare a reference standard solution; The test solution and the reference solution were analyzed by ultra-high performance liquid chromatography, and the presence or absence of a chromatographic peak in the chromatogram of the test solution at the position corresponding to the retention time of the chromatographic peak in the reference solution was observed. in; The reference standard includes vanillic acid-4-β-D-glucoside reference standard; the preparation method of the test solution, the preparation method of the reference solution, and the ultra-high performance liquid chromatography detection method are the same as any one of claims 1 to 7.

9. The method for identifying cypress seed preparations and their adulterants according to claim 8, characterized in that, The counterfeit products include one or both of the standard decoctions of japonica rice or flaxseed.

10. The method for determining the content of vanillic acid-4-β-D-glucoside in the cypress seed preparation according to any one of claims 1 to 7, or the method for identifying cypress seed preparations and their adulterants according to any one of claims 8 to 9, is applied to the quality control of cypress seed preparations.

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