A method for constructing and detecting an UPLC characteristic spectrum of pollen typhae

Abstract

The present application relates to a kind of UPLC characteristic atlas of Typhae Pollen medicinal material construction method and detection method.The present application is by using ultra-high performance liquid chromatography, and chromatographic condition is reasonably controlled, find out while applicable to Typhae Pollen medicinal material different base (water candle cattail and oriental cattail) Ultra-high performance liquid chromatography condition, to construct the UPLC characteristic atlas of Typhae Pollen medicinal material.The water candle cattail characteristic atlas of the present application contains 9 characteristic peaks, and the oriental cattail characteristic atlas contains 10 characteristic peaks, this method is strong in special property, high in stability, good in repeatability, can be used to distinguish water candle cattail and oriental cattail these two base, also can be used for the identification of Typhae Pollen sample, realizes the purpose of effective identification Typhae Pollen multi-base, simultaneously helps to perfect the quality control standard of Typhae Pollen medicinal material.

Description

Technical Field

[0001] The present invention relates to the fields of traditional Chinese medicine analysis and quality control, and in particular to a method for constructing a UPLC characteristic spectrum of Pollen Typhae and a method for detecting the same. Background Art

[0002] The use of cattail pollen as a medicine was first recorded in Shennong's Herbal Classic and was listed as a top-grade product. It is the dried pollen of Typhaangustifolia L., Typha orientalis Presl., or plants of the same genus, and has the effects of stopping bleeding, removing blood stasis, and relieving stranguria. It is used to treat hematemesis, epistaxis, hemoptysis, metrorrhagia, traumatic bleeding, amenorrhea, dysmenorrhea, chest and abdominal stabbing pain, swelling and pain caused by falls, hematuria, and astringent pain (National Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China: Volume 1 [S]. 2020 Edition. Beijing: China Medical Science and Technology Press, 2020: 368-369). Studies have shown that the chemical components of cattail pollen mainly include flavonoids, sterols, alkanes, organic acids, polysaccharides, and tannins (Kong Xiangpeng, Chen Peidong, et al. Study on the chemical components of cattail pollen [J]. Jilin Traditional Chinese Medicine, 2011, 31(1): 66-68). It is mainly used clinically to treat cardiovascular diseases, digestive tract diseases, fundus hemorrhage and other diseases (Hu Lihong, Fang Shiming, et al. Research progress on the chemical composition and pharmacological activity of cattail pollen [J]. Journal of Tianjin University of Traditional Chinese Medicine, 2016, 35(2):136-138). It has high medicinal value, wide clinical application, and large market demand. Plants of the genus Typha in the family Typhaceae are widely distributed in shallow water wetlands in North China, Northeast China, Northwest China, and Southwest China (Editorial Committee of Flora of China, Chinese Academy of Sciences. Flora of China [M]. Beijing: Science Press, 1992.2). According to research, Typha truncatum is widely distributed and is also the mainstream variety in the current medicinal market. Although Typha orientalis is sporadically distributed in most provinces and regions, the resource quantity is relatively small.

[0003] Typhae pollen is a multi-origin medicinal material. Due to its small pollen grains, species identification is difficult based on traditional medicinal properties. It has been reported that the chemical composition of flavonoids varies significantly among different species of Typha (Zhang Zehong, Zhang Nairong, Wang Shiqiang et al. Comparative study on the contents of quercetin and isorhamnetin in five species of Typhae pollen (Typhae pollen) [J]. Hunan Journal of Traditional Chinese Medicine, 1999, 5(2):36). To ensure the stable and controllable quality of Typhae pollen, the origin should be clarified. Currently, the identification of Typhae styracifolia and Typha orientalis is mostly done using microscopic identification and molecular identification methods. Cai Shaoqing et al. studied the microscopic characteristics of different origins of Typha palustris. The results showed that the pollen of Typha serrata and Typha orientalis are single grains, and their size, surface patterns, the ratio of mesh to ridge, and the ratio of the outer layer to the inner layer of the pollen wall are all different, which can achieve the purpose of identification (Cai Shaoqing, Li Shenghua. Variety and quality research of commonly used Chinese medicinal materials (Northern Edition Volume 4) [M]. Beijing Medical University Press, 2001). Ma Xiaoxi et al. studied the DNA barcode identification of pollen-based medicinal materials such as Typha palustris and pine pollen and their mixed products. They can clearly separate Typha palustris and its mixed products, but they cannot make a good distinction between different species within the genus Typha (Ma Xiaoxi, Sun Wei, Ren Weichao et al. DNA barcode identification of pollen-based medicinal materials such as Typha palustris and pine pollen and their mixed products [J]. China Journal of Traditional Chinese Medicine, 2014, 39(12): 2189-2193).

[0004] At present, the legal standards have set standards for the source, properties, microscopy, thin layer, inspection items and content determination of Typha laevigata medicinal materials, but the existing quality evaluation methods are difficult to distinguish the two origins of Typha laevigata and Typha orientalis. According to the above literature reports, there are still some defects in the identification research of Typha laevigata and Typha orientalis: (1) Microscopic identification has the disadvantage of low resolution, especially for medicinal materials such as Typha laevigata with multiple origins and mixed varieties, its identification is more difficult and has a lot of subjectivity; (2) DNA barcode identification can distinguish Typha laevigata and its mixed products, but it cannot distinguish Typha laevigata and Typha orientalis, which are closely related. Therefore, it is necessary to establish a comprehensive, accurate, rapid and specific detection method that can distinguish the two different origins of Typha laevigata medicinal materials. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] In order to solve the above problems in the prior art, the present invention aims to provide a method for constructing a UPLC characteristic spectrum of Typhae Pollen medicinal material, wherein the characteristic spectrum constructed by this method can be used for quality evaluation of Typhae Pollen medicinal material.

[0007] Furthermore, the purpose of the present invention is to provide a detection method for Typhae Pollen medicinal material (or a method for identifying the origin of Typhae Pollen medicinal material), which has strong specificity, high stability and good repeatability, and can be used to distinguish the two origins of Typhae Pollen and Typhae Orientale, thereby achieving the purpose of effectively identifying the multiple origins of Typhae Pollen, and at the same time helping to improve the quality control standards of Typhae Pollen medicinal material.

[0008] Solutions for solving problems

[0009] Through long-term research, the inventors found that the above technical problems can be solved by implementing the following technical solutions:

[0010] [1] The present invention provides a method for constructing a UPLC characteristic spectrum of the medicinal material Typhae Pollen, wherein the construction method comprises the following steps:

[0011] (a) Preparation of control medicinal material solution: Prepare control medicinal material solutions by taking Typha aculeatus and Typha orientalis as control medicinal materials respectively;

[0012] (b) Preparation of reference solution: prepare reference solution by taking uridine, p-hydroxybenzoic acid, vanillic acid, quercetin-3-O-neohesperidin, typhaneoside, kaempferol-3-O-neohesperidin, isorhamnetin-3-O-neohesperidin and rutin reference substances respectively;

[0013] (c) Establishing a characteristic spectrum: Using ultra-performance liquid chromatography, the control medicinal material solution prepared in step (a) and the reference substance solution prepared in step (b) are tested. Based on the retention times of the eight reference substances in their respective chromatograms, it is determined whether the two control medicinal materials have chromatographic peaks corresponding to the eight reference substances in their respective chromatograms. The two control medicinal materials are analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to construct characteristic spectra of the two control medicinal materials.

[0014] The chromatographic conditions of the ultra-high performance liquid chromatography method include:

[0015] Stationary phase: Octadecylsilane bonded silica gel as filler;

[0016] Mobile phase: acetonitrile as mobile phase A, water or aqueous solution of phosphoric acid, acetic acid or formic acid as mobile phase B;

[0017] The gradient elution method was adopted, and the gradient elution procedure was as follows:

[0018]

[0019] [2] The construction method according to [1], wherein the preparation of the control medicinal material solution comprises extracting the control medicinal material with a methanol aqueous solution having a methanol volume concentration of 10% or more; the extraction is performed by any one of ultrasonic treatment, heating reflux and shaking extraction, preferably, ultrasonic treatment is used for extraction; the ultrasonic treatment lasts for 10 minutes to 70 minutes, the power is 200W to 300W, and the frequency is 35kHz to 45kHz;

[0020] The preparation of the reference substance solution comprises adding a methanol aqueous solution with a methanol volume concentration of more than 10% to prepare reference substance solutions containing 10 μg to 30 μg of a uridine reference substance, 30 μg to 50 μg of a p-hydroxybenzoic acid reference substance, 30 μg to 50 μg of a vanillic acid reference substance, 30 μg to 50 μg of a quercetin-3-O-neohesperidin reference substance, 40 μg to 60 μg of a typhaneoglycan reference substance, 10 μg to 30 μg of a kaempferol-3-O-neohesperidin reference substance, 40 μg to 60 μg of an isorhamnetin-3-O-neohesperidin reference substance, and 40 μg to 60 μg of a rutin reference substance per ml.

[0021] [3] The construction method according to [1] or [2], wherein the chromatographic conditions of the ultra-high performance liquid chromatography method include: a chromatographic column having a length of 100 mm to 150 mm and an inner diameter of 2.1 mm; a mobile phase B of 0.05% v / v to 0.2% v / v formic acid aqueous solution; a flow rate of 0.25 mL / min to 0.35 mL / min; and / or a column temperature of 25°C to 35°C; and / or a detection wavelength of 210 nm to 400 nm; and an injection speed of 0.5 to 1.5 μl / min.

[0022] [4] The construction method according to any one of the technical solutions [1] to [3], wherein the characteristic spectrum of the Typha rapa control medicinal material contains 9 common peaks, and the characteristic spectrum of the Typha orientalis control medicinal material contains 10 common peaks.

[0023] [5]. The construction method according to [4], wherein the characteristic spectrum of the Typha truncatum reference medicinal material contains 7 common peaks corresponding to the reference substance, wherein: peak 1 corresponds to uridine, peak 2 corresponds to p-hydroxybenzoic acid, peak 3 corresponds to vanillic acid, peak 5 corresponds to quercetin-3-O-neohesperidin, peak 7 corresponds to typhaneoglycan, peak 8 corresponds to kaempferol-3-O-neohesperidin, and peak 9 corresponds to isorhamnetin-3-O-neohesperidin;

[0024] The characteristic spectrum of the Typha orientalis reference medicinal material includes 8 common peaks corresponding to the reference substance, wherein: in addition to the 7 common peaks, the remaining 1 common peak corresponds to rutin.

[0025] [6]. In addition, the present invention also provides a method for detecting the medicinal material Typhae Pollen, wherein the detection method comprises the following steps:

[0026] (a) Preparation of test solution: Prepare a test solution using a test medicinal material, wherein the test medicinal material is one or more of the counterfeit products of Typha australis, Typha orientalis, or Pollen Typhae.

[0027] (b) Chromatographic detection: The test solution prepared in step (a) is detected by ultra-performance liquid chromatography, and the chromatogram of the test solution is compared with the characteristic spectrum of the control medicinal materials of Typha australis and / or Typha orientalis constructed by the construction method according to any one of claims 1 to 5 to identify the origin and / or authenticity of the medicinal material Typhae pollen;

[0028] The chromatographic conditions of the ultra-high performance liquid chromatography method include:

[0029] Stationary phase: Octadecylsilane bonded silica gel as filler;

[0030] Mobile phase: acetonitrile as mobile phase A, water or aqueous solution of phosphoric acid, acetic acid or formic acid as mobile phase B;

[0031] The gradient elution method was adopted, and the gradient elution procedure was as follows:

[0032]

[0033] [7]. The detection method according to [6], wherein the preparation of the test solution comprises extracting the medicinal material to be tested using a methanol aqueous solution with a methanol volume concentration of more than 10%, preferably a methanol aqueous solution with a methanol volume concentration of 30%; the extraction is performed by any one of ultrasonic treatment, heating reflux and shaking extraction, preferably, ultrasonic treatment is used for extraction; the duration of the ultrasonic treatment is 10 min to 70 min, the power is 200 W to 300 W, and the frequency is 35 kHz to 45 kHz.

[0034] [8] The detection method according to [6] or [7], wherein the preparation of the test solution further comprises collecting the extract and then weighing the weight, supplementing the lost weight with a methanol aqueous solution with a methanol volume concentration of more than 10%, shaking, filtering, and taking the filtrate.

[0035] [9]. The detection method according to any one of the technical solutions [6] to [8], wherein the identification criteria are as follows: if the chromatogram of the medicinal material to be tested corresponds to the characteristic spectrum of the control medicinal material Typha aquatica, it is identified as Typha aquatica-based; if the chromatogram of the medicinal material to be tested corresponds to the characteristic spectrum of the control medicinal material Typha orientalis, it is identified as Typha aquatica-based; if neither corresponds, it is identified as a counterfeit Typha aquatica.

[0036]

[10] . Application of the detection method according to any one of the technical solutions [6] to [9] in identifying the origin and / or authenticity of the medicinal material Typhae Pollen.

[0037] Effects of the Invention

[0038] The present invention selects ultra-high performance liquid chromatography conditions suitable for Typha palinurus samples (Typha serrata medicinal materials and Typha orientalis medicinal materials), uses Typha serrata medicinal materials and Typha orientalis medicinal materials as test samples for detection, and thus constructs characteristic spectra. The characteristic spectra of Typha serrata contain 9 characteristic peaks, and the characteristic spectra of Typha orientalis contain 10 characteristic peaks. These spectra can not only reflect the quality of the Typha palinurus medicinal materials, but also distinguish between two different origins of Typha palinurus, thereby providing an effective method for quality detection of Typha palinurus and a scientific experimental basis for comprehensive quality evaluation of Typha palinurus.

[0039] The characteristic spectrum is used to detect the Puhuang medicinal material, and the method is simple, rapid, and efficient, with objective results, precise and reliable results, strong specificity, high sensitivity, and good stability and tolerance. The characteristic spectrum constructed by the method of the present invention can comprehensively reflect the characteristic peak information of the sample, fully demonstrate the differences in chemical components of Puhuang medicinal materials of different origins, establish a determination method for the specificity of Puhuang, improve the scientific nature of quality monitoring and quality evaluation, provide a new analytical means for distinguishing Puhuang medicinal materials of different origins, and enable the quality control of Puhuang to be upgraded from the original content determination of a single component or the determination of a few common peaks to the control of the intrinsic quality of the entire Puhuang and specificity identification.

[0040] The method of the present invention is simple, reproducible, accurate, reliable, easy to operate, and saves time compared to HPLC. By optimizing the chromatographic conditions, the detection time is shortened to 43 minutes, the solvent consumption is low, and the environmental pollution is small. The characteristic peaks of the spectrum of the present invention are well separated, and the chemical information of all characteristic peaks is confirmed by reference substances and mass spectrum information. The method has strong specificity, high analysis efficiency, high stability, good repeatability, and a wide range of applications. It is suitable for distinguishing Typha australis and Typha orientalis medicinal materials and identifying mixed and counterfeit products, etc., and is particularly suitable for detection and monitoring of large-scale production.

[0041] It should be noted that the above description does not disclose all embodiments of the present invention and all advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Plant morphology of Typha truncatum and Typha orientalis

[0043] Figure 2 :Chromatograms of different extraction solvents

[0044] Figure 3 :Chromatograms of different extraction methods

[0045] Figure 4 :Chromatograms of different extraction times

[0046] Figure 5 :Investigation results of different mobile phase elution systems for Typha aquatica

[0047] Figure 6 :Investigation results of different mobile phase elution systems for oriental cattail medicinal materials

[0048] Figure 7 : Elution time gradient of Typhae Pollen medicinal material 1

[0049] Figure 8 : Elution time gradient of Typhae Pollen medicinal material 2

[0050] Figure 9 : Characteristic spectrum of Typha aquatica medicinal material

[0051] Figure 10 : Comparative Atlas of Medicinal Materials of Typha aquatica

[0052] Figure 11 :Characteristics of Oriental Typha Medicinal Materials

[0053] Figure 12 : Comparative Atlas of Oriental Typha Medicinal Materials

[0054] Figure 13 :Reference material identification spectrum

[0055] Figure 14 :Investigation charts of different chromatographic columns

[0056] Figure 15 :Investigation chart of different column temperatures

[0057] Figure 16 :Investigation graphs of different flow rates

[0058] Figure 17 : Comparison of the characteristic spectra of Typha aquatica and Typha orientalis

[0059] Figure 18 : Characteristic spectrum detection diagram of water candle cattail

[0060] Figure 19 :Characteristic map of Oriental Cattail

[0061] Figure 20 :Pine pollen characteristic spectrum detection diagram DETAILED DESCRIPTION

[0062] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below; various modifications may be made within the scope of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included within the technical scope of the present invention. In addition, all documents listed in this specification are cited as references in this specification.

[0063] Unless defined otherwise, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] In the context of describing this specification (especially in the context of the following claims), the terms "a," "an," and "the" and similar language are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0065] In this specification, a numerical range expressed using "numerical value A to numerical value B" or "numerical value A - numerical value B" means a range including the endpoints numerical values ​​A and B.

[0066] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both cases where the event occurs and cases where the event does not occur.

[0067] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "some specific / preferred technical solutions," "other specific / preferred technical solutions," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiment are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in various embodiments in any appropriate manner.

[0068] The term "comprises" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0069] Unless otherwise specified, the percentage contents mentioned in the present invention refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0070] The percentage concentrations mentioned in the present invention, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0071] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0072] Unless otherwise specified, the term "reference substance" used in this article refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance. It is usually reviewed and approved by the national drug testing agency, and its standard should not be lower than the quality standard of the product.

[0073] Unless otherwise specified, the term "test article" used herein refers to an experimental sample used for detection or identification.

[0074] Unless otherwise specified, the terms "precision weighing or precision weighing" used in this article mean that the weight should be weighed accurately to one thousandth of the weight taken, the term "weighing" means that the weight should be weighed accurately to one hundredth of the weight taken, the term "precision addition" means that the volume should be measured accurately to one thousandth of the volume taken, and the term "precision aspiration" refers to the operation method of accurately measuring samples through a microinjector.

[0075] Unless otherwise specified, the term "subsequent filtrate" as used herein refers to the filtrate collected after the primary filtrate is discarded during filtration. Compared to the primary filtrate, the subsequent filtrate is closer to the true concentration of the sample because the filter medium (such as the membrane or filter paper) may adsorb solutes, resulting in a lower sample concentration in the primary filtrate. Furthermore, the subsequent filtrate is cleaner because solutes adsorbed by the filter medium form a filter cake, reducing the filter pore size and thus retaining smaller particles.

[0076] Unless otherwise specified, the term "ultra-performance liquid chromatography" (or "UPLC") used in this article refers to a new technology developed based on high-performance liquid chromatography (HPLC) that has the characteristics of small filler particles, fast detection speed, large analytical throughput, and high sensitivity.

[0077] Unless otherwise noted, percentages (%) in solution concentrations are by volume. For example, a 0.1% formic acid solution is equivalent to adding 1 mL of formic acid to 1000 mL of pure water. Since graduated cylinders are not recommended for mixing solutions, a 0.1% formic acid solution is also equivalent to mixing 1 mL of formic acid with 1000 mL of pure water.

[0078] <Construction Method of UPLC Characteristic Chromatogram of Pollen Typhae>

[0079] The method for constructing the UPLC characteristic spectrum of the medicinal material Typhae Pollen of the present invention mainly comprises the following steps:

[0080] (a) Preparation of control medicinal material solution: Prepare control medicinal material solutions by taking Typha aculeatus and Typha orientalis as control medicinal materials respectively;

[0081] In some specific embodiments of the present invention, the step of preparing the control medicinal material solution includes extracting the control medicinal materials of Typha aquatica and Typha orientalis respectively with a methanol aqueous solution having a methanol volume concentration of 10% or more, preferably, using a methanol aqueous solution having a methanol volume concentration of 30% or more for extraction, more preferably, using a methanol aqueous solution having a volume concentration of 30% or more and 70% or less, and most preferably using a methanol aqueous solution having a methanol volume concentration of 30% or more for extraction of the control medicinal materials of Typha aquatica and Typha orientalis respectively;

[0082] In order to obtain richer chromatographic peak information, extraction can be carried out by any one of ultrasonic treatment, heating reflux and shaking extraction. However, the inventors found through experiments that the total peak area / sample weight value of heating reflux was slightly higher, but the difference in peak area ratio between different extraction methods was relatively small. Taking into account the simplicity of the extraction method and the response of each peak, ultrasonic treatment is preferably used; the ultrasonic treatment time is 10 min to 70 min, the power is 200 W to 300 W, and the frequency is 35 kHz to 45 kHz; preferably, the power is 250 W and the frequency is 40 kHz; further, taking into account the extraction efficiency of each chromatographic peak, the ultrasonic treatment time is 15 min to 60 min, and more preferably 30 min.

[0083] In some specific embodiments of the present invention, the preparation method of the above-mentioned control medicinal material solution is as follows: take Typha australis and Typha orientalis control medicinal materials, place them in stoppered conical flasks respectively, add methanol aqueous solution with a methanol volume concentration of 30%, and extract by ultrasonic treatment (time is 30 minutes, power is 250W, frequency is 40kHz), cool, shake well, filter, and take the filtrate as the control medicinal material solution.

[0084] The method further includes (b) preparing a reference solution: preparing a reference solution by taking uridine, p-hydroxybenzoic acid, vanillic acid, quercetin-3-O-neohesperidin, typhaneoside, kaempferol-3-O-neohesperidin, isorhamnetin-3-O-neohesperidin and rutin reference substances respectively;

[0085] In some specific embodiments of the present invention, a methanol aqueous solution with a methanol volume concentration of 10% or more is added to prepare a reference solution containing 10 μg to 30 μg of uridine reference substance, 30 μg to 50 μg of p-hydroxybenzoic acid reference substance, 30 μg to 50 μg of vanillic acid reference substance, 30 μg to 50 μg of quercetin-3-O-neohesperidin reference substance, 40 μg to 60 μg of typhaneoglycan reference substance, 10 μg to 30 μg of kaempferol-3-O-neohesperidin reference substance, 40 μg to 60 μg of isorhamnetin-3-O-neohesperidin reference substance and 40 μg to 60 μg of rutin reference substance per 1 ml; preferably, a methanol aqueous solution with a methanol volume concentration of 30% or more is used to extract the reference substance, more preferably, a methanol aqueous solution with a methanol volume concentration of 30% or more and less than 70% is used, and most preferably, a methanol aqueous solution with a methanol volume concentration of 30% is used to extract the reference substance; preferably, each 1 ml is prepared respectively. ml methanol aqueous solution contains 15μg~25μg of uridine reference substance, 35μg~45μg of p-hydroxybenzoic acid reference substance, 35μg~45μg of vanillic acid reference substance, 35μg~45μg of quercetin-3-O-neohesperidin reference substance, 45μg~55μg of typhalenol reference substance, 15μg~25μg of kaempferol-3-O-neohesperidin reference substance, 45μg~55μg of isorhamnetin-3-O-neohesperidin reference substance and rutin reference substance More preferably, a reference solution containing 20 μg of uridine reference substance, 40 μg of p-hydroxybenzoic acid reference substance, 40 μg of vanillic acid reference substance, 40 μg of quercetin-3-O-neohesperidin reference substance, 50 μg of typhalenol reference substance, 20 μg of kaempferol-3-O-neohesperidin reference substance, 50 μg of isorhamnetin-3-O-neohesperidin reference substance and 50 μg of rutin reference substance is prepared per 1 ml of methanol aqueous solution.

[0086] In some specific embodiments of the present invention, the preparation method of the above-mentioned reference solution is as follows: uridine, p-hydroxybenzoic acid, vanillic acid, quercetin-3-O-neohesperidin, typhaneoside, kaempferol-3-O-neohesperidin, isorhamnetin-3-O-neohesperidin and rutin reference substances are taken respectively, accurately weighed, and methanol aqueous solution with a methanol volume concentration of 30% is added to prepare reference solution containing 20 μg, 40 μg, 40 μg, 40 μg, 50 μg, 20 μg, 50 μg and 50 μg per 1 ml, respectively. (c) Establishing a characteristic spectrum: Using ultra-performance liquid chromatography, the control medicinal material solution prepared in step (a) and the reference substance solution prepared in step (b) are tested. Based on the retention times of the eight reference substances in their respective chromatograms, it is determined whether the two control medicinal materials have chromatographic peaks corresponding to the eight reference substances in their respective chromatograms. The two control medicinal materials are analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to construct characteristic spectra of the two control medicinal materials.

[0087] The inventors of the present invention have determined through in-depth research that the ultra-high performance liquid chromatography conditions suitable for the control medicinal materials of Typha australis and Typha orientalis include:

[0088] Stationary phase: A chromatographic column with octadecylsilane bonded silica as a filler and a particle size (filler particle size) of 1.6 to 1.8 μm; the column length is 100 mm to 150 mm, and the inner diameter is 2.1 mm. In some embodiments of the present invention, the chromatographic column is selected from any one of ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm), ZORBAX SB-Aq RRHD (2.1 mm × 100 mm, 1.8 μm), and CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm). Furthermore, the chromatographic column is preferably an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm). Using this chromatographic column for separation provides better resolution of characteristic peaks and a more aesthetically pleasing overall chromatogram.

[0089] Mobile phase: acetonitrile is used as mobile phase A, and water or an aqueous solution of phosphoric acid, acetic acid or formic acid is used as mobile phase B. In some specific embodiments of the present invention, 0.05% v / v to 0.2% v / v formic acid aqueous solution is preferably used as mobile phase B, and 0.1% v / v formic acid aqueous solution is more preferably used as mobile phase B.

[0090] The present invention adopts gradient elution mode, and the gradient elution procedure is as follows:

[0091]

[0092] Using this condition, whether it is the medicinal material Typha aquatica or Typha orientalis, each chromatographic peak can be fully separated and the peak shape is good, and the analysis cycle is short, and the separation efficiency and resolution are higher.

[0093] The detector used in the present invention can be an ultraviolet detector. Furthermore, the chromatographic conditions of the present invention also include: a detection wavelength of 210nm to 400nm. In some preferred embodiments of the present invention, the detection wavelength is 240nm to 350nm. Under the detection wavelength in this range, the chromatographic peak information is relatively rich, the separation effect of each chromatographic peak is good, and the baseline is relatively stable. Furthermore, the chromatographic conditions of the present invention also include: a flow rate of 0.25mL / min to 0.35mL / min; and / or, a column temperature of 25℃ to 35℃. Within the column temperature or flow rate range, the construction method of the present invention can meet the system applicability requirements with smaller column temperature changes or flow rate changes. In some preferred embodiments of the present invention, the flow rate is 0.28mL / min to 0.32mL / min; and / or, the column temperature is 28℃ to 33℃; and / or, the injection speed is 0.5 to 1.5μl / min.

[0094] Furthermore, the present invention detects at least 19 batches of Typha aquatica medicinal materials and at least 5 batches of Typha orientalis medicinal materials and reference medicinal materials and reference solution solutions, and performs analysis and comparison, selects common peaks to establish the characteristic spectrum of Typha aquatica and the characteristic spectrum of Typha orientalis, and identifies the common peaks with reference to the positioning of the chromatographic peaks of the reference products. Finally, the chromatograms of the medicinal materials obtained are each imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", and the median method is used to generate respective superimposed characteristic spectra and reference spectra. The characteristic spectrum of the Typha aquatica reference medicinal material obtained by the aforementioned construction method of the present invention contains 9 common peaks, and the 9 common peaks can all appear stably in each medicinal material, and the degree of agreement is good; the characteristic spectrum of the Typha orientalis reference medicinal material contains 10 common peaks, and the 10 common peaks can all appear stably in each medicinal material, and the degree of agreement is good. Among them, the characteristic spectrum of the Typha orientalis reference medicinal material includes 7 common peaks corresponding to the reference substance, among which: peak 1 corresponds to uridine, peak 2 corresponds to p-hydroxybenzoic acid, peak 3 corresponds to vanillic acid, peak 5 corresponds to quercetin-3-O-neohesperidin, peak 7 corresponds to typhaneoglycans, peak 8 corresponds to kaempferol-3-O-neohesperidin, and peak 9 corresponds to isorhamnetin-3-O-neohesperidin; the characteristic spectrum of the Typha orientalis reference medicinal material includes 8 common peaks corresponding to the reference substance, among which: in addition to the 7 common peaks, the remaining 1 common peak corresponds to rutin.

[0095] The present invention shortens the analysis time and improves the analysis efficiency by optimizing the preparation of reference medicinal materials and reference substances and the chromatographic analysis conditions, establishes ultra-high performance liquid chromatography conditions that are applicable to both Typha aquatica and Typha orientalis medicinal materials, and truly reflects the original chemical composition of Typhae pollen medicinal materials.

[0096] <Testing Methods for Pollen Typhae>

[0097] On the other hand, the characteristic spectrum constructed by the present invention can be used for the detection of Typhae Pollen medicinal materials, such as the identification of Typhae Rhizoma and Typhae Orientalis, and the distinction between counterfeit and similar products of Typhae Pollen medicinal materials. The detection method comprises the following steps:

[0098] (a) Preparation of a test solution: preparing a test solution from a test medicinal material; the preparation of the test solution refers to extracting the test medicinal material with an extraction solvent, collecting the extract, and preparing the test solution, wherein the test medicinal material is one or more of the counterfeit products of Typha australis, Typha orientalis, or Pollen Typhae;

[0099] In some specific embodiments of the present invention, the extraction solvent is selected from a methanol aqueous solution with a methanol volume concentration of more than 10%. In order to obtain a better peak type (or peak shape) and comprehensively consider the total extraction efficiency, the extraction solvent is preferably a methanol aqueous solution with a methanol volume concentration of more than 10% and less than 70%; more preferably, a methanol aqueous solution with a methanol volume concentration of 30%. The present invention has no special requirements for the pretreatment of the medicinal materials to be tested. It is preferred that the medicinal material powder be sieved to remove impurities, and preferably a No. 7 sieve can be used (the mass of the impurities sieved out cannot exceed 10% of the total mass of the medicinal materials). Regarding the extraction method, in order to obtain richer chromatographic peak information, the extraction can be carried out by any one of ultrasonic treatment, heating reflux and shaking extraction. However, the inventors found through experiments that the total peak area / sample weight value of heating reflux is slightly higher, but the difference in peak area ratio between different extraction methods is relatively small. Considering the simplicity of the extraction method and the response of each peak, ultrasonic treatment is preferably used; the ultrasonic treatment time is 10min~70min, the power is 200W~300W, and the frequency is 35kHz~45kHz; preferably, the power is 250W and the frequency is 40kHz; further, considering the extraction efficiency of each chromatographic peak, the ultrasonic treatment time is 15min~60min, more preferably 30min.

[0100] In some specific embodiments of the present invention, the amount of the extraction solvent added relative to the medicinal material to be tested is 30 to 60 mL / g, preferably 35 to 55 mL / g, and more preferably 40 to 45 mL / g. If the test sample concentration is too small, it is likely to be detrimental to the detection of the chromatographic peak. If the concentration is too large, the chromatographic column will be overloaded and easily lead to poor chromatographic peak shape. In some specific embodiments of the present invention, the preparation method of the test solution is as follows: take an appropriate amount of the medicinal material to be tested, crush it, sieve it (No. 7 sieve), accurately weigh it, put it in a stoppered conical flask, accurately add a methanol aqueous solution with a methanol volume concentration of more than 10%, seal it, weigh it, ultrasonically treat it (time is 30 min, power is 250 W, frequency is 40 kHz), let it cool, weigh it again, make up the lost weight with a methanol aqueous solution with a methanol volume concentration of more than 10%, shake it well, filter it, and take the filtrate to obtain it.

[0101] (b) Chromatographic detection: The test solution prepared in step (a) is detected by ultra-performance liquid chromatography, and the chromatogram of the test solution is compared with the characteristic spectrum of the control medicinal materials of Typha australis and / or Typha orientalis constructed by the above-mentioned construction method to identify the origin and / or authenticity of the Typhae pollen medicinal material; in some specific embodiments of the present invention, the detection is performed using the above-mentioned chromatographic conditions of the present invention.

[0102] The identification criteria are as follows: if the chromatogram of the medicinal material to be tested corresponds to the characteristic spectrum of the reference medicinal material Typha aquatica, it is identified as Typha aquatica-based; if the chromatogram of the medicinal material to be tested corresponds to the characteristic spectrum of the reference medicinal material Typha orientalis, it is identified as Typha aquatica-based; if neither corresponds, it is identified as a counterfeit Typha aquatica.

[0103] The construction and detection method of the present invention provides a new analytical means for the intrinsic quality control of Typhae Pollen medicinal material. The method is simple to operate, has high precision, good stability, good repeatability, and high accuracy. It can not only be used to identify similar and counterfeit products of Typhae Pollen medicinal material, but also can distinguish the different origins of Typhae Pollen samples (Typha australis and Typha orientalis), providing an important multi-index parameter basis for evaluating the stability of the quality of Typhae Pollen medicinal material.

[0104] Example

[0105] The present invention will be further described below through specific embodiments:

[0106] Laboratory instruments, reagents and medicines

[0107] A Waters Acquity UPLC ultra-high-performance liquid chromatograph (Waters); an Empower 3 workstation (Waters); an Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); an electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); a KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); a temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); a pure water system (Sartorius); and an AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.) were used. Acetonitrile (chromatographic grade, Thermo Fisher); ultrapure water; and acetic acid (chromatographic grade, Aladdin). All other reagents were of analytical grade.

[0108] Vanillic acid (batch number: 110776-201503), typhanoside (batch number: 111573-201405), isorhamnetin-3-O-neohesperidin (batch number: 111571-201205), uridine (batch number: 110887-202104), and rutin (batch number: 100080-202012) were purchased from the China Food and Drug Administration. Quercetin-3-O-neohesperidin and kaempferol-3-O-neohesperidin reference standards were purchased from Yuanye Biotechnology Co., Ltd., batch numbers P25M10S89184 and P25M10S89185, respectively. Para-hydroxybenzoic acid (batch number: ST05300120-2169) was purchased from Shanghai Standard Standard Technology Service Co., Ltd. Pollen Typhae control medicinal material was purchased from China Food and Drug Inspection Institute with batch number 121225-201003.

[0109] 19 batches of experimental samples came from major production areas and authentic production areas across the country, all of which met the requirements of the 2020 edition of the Chinese Pharmacopoeia. The medicinal material information is shown in Table 1, and the plant characteristics are shown in Table 2. Figure 1 ; Typha latifolia (water candle cattail) reference medicinal material (batch number: 121225-2010000); Typha latifolia (oriental cattail) reference medicinal material was obtained after being identified as Typha latifolia (oriental cattail) by Tianjiang Research Institute.

[0110] Table 1: Origin information of different origins of Pollen Typhae

[0111]

[0112]

[0113] Example 1: Construction of the Characteristic Spectrum of Pollen Typhae

[0114] 1. Preparation of control medicinal material solution

[0115] 1.1 Investigation of extraction solvent

[0116] Take about 0.6 g of Typha Pollen (Typha rapa) control medicinal material powder, accurately weigh it, place it in a stoppered conical flask, accurately add water, methanol solution with volume concentrations of 10%, 30%, 50%, and 70%, and 25 ml of methanol, weigh the weight, ultrasonically treat for 30 minutes, let cool, weigh the weight again, make up the lost weight with methanol solution with a volume concentration of 30%, shake well, filter, and take the filtrate to obtain.

[0117] By comparing the characteristic spectra of 6 different extraction solvents Figure 2 It can be found that when methanol solutions with a methanol volume concentration of 10%, 30% and 70% are used as extraction solvents, the total peak area / sample weight is larger. Considering that the methanol solution with a methanol volume concentration of 30% has a good chromatographic peak shape and high extraction efficiency when used as the extraction solvent, it is selected as the final extraction solvent.

[0118] 1.2 Investigation of extraction methods

[0119] Take about 0.6 g of Typha Pollen (Typha rapa) control medicinal material powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of methanol solution with a methanol volume concentration of 30%, weigh the weight, ultrasonically treat, shake to extract, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with methanol solution with a methanol volume concentration of 30%, shake well, filter, and take the filtrate to obtain.

[0120] Comparison of chromatograms obtained by three extraction methods: ultrasonic treatment, shaking extraction, and heating reflux Figure 3 It was found that the total peak area / sample weight value of heating reflux was slightly higher, but considering the simplicity of the extraction method and the response of each peak, ultrasonic treatment was finally selected as the extraction method for the test solution.

[0121] 1.3 Investigation of extraction time Take about 0.6 g of Typha pollen (Typha rapa) control medicinal material powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of methanol solution with a methanol volume concentration of 30%, weigh the weight, and ultrasonically treat it for 15 min, 30 min, 45 min, and 60 min respectively. Let it cool, weigh it again, make up the lost weight with methanol solution with a methanol volume concentration of 30%, shake it evenly, filter it, and take the filtrate.

[0122] By comparing different extraction times, the chromatograms Figure 4 ,according to Figure 4 The results showed that the characteristic peak areas obtained at different extraction times were not much different, indicating that the extraction was complete at 15 minutes. To ensure sufficient extraction, the extraction time was selected as 30 minutes.

[0123] Based on the above investigations on the extraction solvent, extraction method and extraction time, the reference solution preparation method of the present invention is as follows: about 0.6 g of each of Typha australis and Typha orientalis reference medicinal materials are taken, accurately weighed, placed in a stoppered conical flask, accurately added with 25 ml of a methanol aqueous solution with a methanol volume concentration of 30%, weighed, ultrasonically treated for 30 minutes, cooled, weighed again, supplemented with a methanol solution with a methanol volume concentration of 30%, shaken, filtered, and a filtrate is obtained.

[0124] 2. Preparation of Reference Solution

[0125] Uridine, p-hydroxybenzoic acid, vanillic acid, quercetin-3-O-neohesperidin, typhaneoside, kaempferol-3-O-neohesperidin, isorhamnetin-3-O-neohesperidin and rutin reference substances were accurately weighed and added with a methanol aqueous solution with a methanol volume concentration of 30% to prepare reference substance solutions containing 20 μg, 40 μg, 40 μg, 40 μg, 50 μg, 20 μg, 50 μg and 50 μg per ml respectively.

[0126] 3. Chromatographic conditions

[0127] 3.1 Mobile phase investigation

[0128] The control medicinal material solutions of Typha aculeatus and Typha orientalis were taken and chromatographically analyzed on an ACQUITY UPLC HSS T3 (2.1mm×100mm,1.8μm) column; the mobile phases were acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% acetic acid, acetonitrile-0.1% formic acid, and acetonitrile-water for gradient elution, with a flow rate of 0.30ml / min, a column temperature of 30℃, a detection wavelength of 254nm, and an injection volume of 1μl.

[0129] The results of the investigation can be found in Figure 5 、 Figure 6 ,in Figure 5 This is the result of investigation on different mobile phase elution systems for Typha aquatica. Figure 6 The results of an investigation of different mobile phase elution systems for Typha orientalis were presented. The results showed that the chromatographic peak information was poor when acetonitrile-water was used as the elution system, while the resolution at Peak 1 was poor when acetonitrile-0.1% phosphoric acid and acetonitrile-0.1% acetic acid were used as the elution systems. Therefore, the chromatographic peak shape, separation effect, and rich chromatographic peak information were better when the mobile phase was acetonitrile-0.1% formic acid.

[0130] 3.2 Elution time gradient investigation

[0131] Chromatographic column: ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution as specified in the table below; flow rate, 0.3 ml / min; column temperature, 30°C, detection wavelength, 254 nm.

[0132] Table 2: Elution time gradient 1

[0133]

[0134] The results of the investigation can be found in Figure 7 As can be seen from the figure, elution according to this elution gradient results in greater noise in the chromatographic peaks and poor separation of some peaks.

[0135] After further optimization of the elution gradient, the chromatographic column was ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B. Gradient elution was performed as specified in the table below; the flow rate was 0.3 ml / min; the column temperature was 30° C., and the detection wavelength was 254 nm.

[0136] Table 3: Elution time gradient 2

[0137]

[0138] The results of the investigation can be found in Figure 8 As can be seen from the figure, under the elution conditions, the separation of each chromatographic peak is good, the chromatographic peak information is rich, and the analysis time is moderate.

[0139] Based on the above investigation of the mobile phase and elution time gradient, the chromatographic conditions of the present invention are as follows: using an ACQUITY UPLC HSS T3 column (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution as specified in Table 3; flow rate 0.3 ml / min; column temperature 30° C., and detection wavelength 254 nm.

[0140] 4. Establishment of feature maps

[0141] 19 batches of Typhae pollicis (Typhae truncatum) medicinal material samples and 5 batches of Typhae pollicis (Typhae orientalis) medicinal material samples were taken, each sample 0.6 g, and the sample characteristic spectra were determined according to the above preparation method and chromatographic conditions. The characteristic spectra of the 19 batches of Typhae pollicis (Typhae truncatum) medicinal material samples were superimposed using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" promulgated by the Chinese Pharmacopoeia Commission. Figure 9 ; The reference atlas of Typha pueraria (Typha truncatum) is shown in Figure 10 ; The characteristic maps of 5 batches of Typha palinurus (Oriental Typha) medicinal material samples were superimposed. Figure 11 ; The reference atlas of Typha rapa (Oriental Typha) is shown in Figure 12 .

[0142] 4.1 Identification of chromatographic peaks

[0143] The results showed that peak 1 was uridine, peak 2 was p-hydroxybenzoic acid, peak 3 was vanillic acid, peak 5 was quercetin-3-O-neohesperidin, peak 7 was typhaneoglycan, peak 8 was kaempferol-3-O-neohesperidin, peak 9 was isorhamnetin-3-O-neohesperidin, and peak a was rutin. Figure 13 .

[0144] 5. Durability inspection

[0145] 5.1 Column inspection

[0146] The chromatograms of different columns, ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm), ZORBAX SB-Aq RRHD (2.1 mm × 100 mm, 1.8 μm), and CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm), were compared. Figure 14 The results showed that the ZORBAX SB-Aq RRHD (2.1 mm × 100 mm, 1.8 μm) and CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm) had poor resolution for peak 2, while the remaining peaks were well resolved. This application used the ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) for subsequent studies.

[0147] 5.2 Column temperature investigation

[0148] The chromatograms at column temperatures of 25°C, 30°C, and 35°C are compared. Figure 15 The results showed that the method had good separation when the column temperature was 25℃~35℃, and the relative retention time of each characteristic peak was within the specified range. The system suitability requirements could be met with a small change in column temperature, and the optimal column temperature was 30℃.

[0149] 5.3 Flow rate investigation

[0150] The chromatograms at flow rates of 0.25 mL / min, 0.30 mL / min, and 0.35 mL / min are compared. Figure 16 The results showed that the peak shapes were good within the flow rate range of 0.25 ml / min to 0.35 ml / min, and the relative retention times of the characteristic peaks were within the specified range, indicating that the method had good durability for different flow rates.

[0151] Example 2: Detection of Pollen Typhae

[0152] 1. Preparation of test solution

[0153] Take about 0.6 g of the medicinal material powder (A, B, C) to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of a methanol aqueous solution with a methanol volume concentration of 30%, weigh the weight, ultrasonicate for 30 minutes, let cool, weigh the weight again, make up the lost weight with a methanol aqueous solution with a methanol volume concentration of 30%, shake well, filter, and take the filtrate to obtain.

[0154] 2. Detection

[0155] The sample solution to be tested was accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph were the same as those in "3. Chromatographic conditions" in Example 1.

[0156] The detection spectrum obtained by the test was compared with the characteristic spectrum of Typha aquatica Figure 10 oriental cattail characteristic map Figure 12 The comparison is performed to detect the quality of the sample to be tested and / or to distinguish between Typha aquatica and Typha orientalis; wherein, if the chromatogram of the medicinal material to be tested corresponds to the characteristic spectrum of the Typha aquatica reference medicinal material, it is identified as Typha aquatica-based; if the chromatogram of the medicinal material to be tested corresponds to the characteristic spectrum of the Typha orientalis reference medicinal material, it is identified as Typha aquatica-based; if neither corresponds, it is identified as a counterfeit Typha aquatica.

[0157] Comparison of the characteristic patterns of Typha aquatica and Typha orientalis medicinal materials can be seen in Figure 17 . It can be seen that peak a can be used as an identification and analysis point for the medicinal materials Typha aquatica and Typha orientalis. The results show that the medicinal material A to be tested has peaks 1 to 9, but no peak a, and is identified as Typha aquatica; the medicinal material B to be tested has peaks 1 to 9 and peak a, and is identified as Typha orientalis; the medicinal material C to be tested does not correspond to the characteristic spectra of Typha aquatica and Typha orientalis, and is identified as a counterfeit. After further identification, it was concluded that it is a counterfeit pine pollen medicinal material of Typha aquatica. The characteristic spectrum detection results of Typha aquatica (Typha aquatica), Typha aquatica (Typha orientalis) and its counterfeit pine pollen medicinal material are shown in Figure 2. Figure 18 、 Figure 19 and Figure 20 .

[0158] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0159] The above-described embodiments merely illustrate several implementations of the present invention, facilitating a specific and detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A method for constructing a UPLC characteristic spectrum of Pollen Typhae, characterized in that: The construction method comprises the following steps: (a) Preparation of a control medicinal material solution: preparing control medicinal material solutions from Typha aculeatus and Typha orientalis control medicinal materials, respectively, wherein the control medicinal material solutions are extracted using a methanol aqueous solution having a methanol concentration of 10% or more by volume; (b) Preparation of reference solution: prepare reference solution by taking uridine, p-hydroxybenzoic acid, vanillic acid, quercetin-3-O-neohesperidin, typhaneoside, kaempferol-3-O-neohesperidin, isorhamnetin-3-O-neohesperidin and rutin reference substances respectively; (c) Establishing a characteristic spectrum: Using ultra-high performance liquid chromatography, the control medicinal material solution prepared in step (a) and the reference substance solution prepared in step (b) are tested. Based on the retention times of the eight reference substances in their respective chromatograms, it is determined whether the two control medicinal materials have chromatographic peaks corresponding to the eight reference substances in their respective chromatograms. The two control medicinal materials are analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to construct characteristic spectra of the two control medicinal materials. The chromatographic conditions of the ultra-high performance liquid chromatography method include: Column: ACQUITY UPLC HSS T3, 2.1 mm inner diameter, 100 mm length, 1.8 µm particle size; Mobile phase: acetonitrile as mobile phase A, 0.05% v / v~0.2% v / v formic acid in water as mobile phase B; The detection wavelength is 254nm, The gradient elution method was adopted, and the gradient elution procedure was as follows: 。 2. The construction method according to claim 1, characterized in that The extraction adopts any one of ultrasonic treatment, heating reflux and shaking extraction.

3. The construction method according to claim 2, characterized in that The extraction is performed by ultrasonic treatment; the duration of the ultrasonic treatment is 10 minutes to 70 minutes, the power is 200W to 300W, and the frequency is 35kHz to 45kHz.

4. The construction method according to any one of claims 1 to 3, characterized in that The preparation of the reference substance solution comprises adding a methanol aqueous solution with a methanol volume concentration of 10% or more to prepare reference substance solutions containing 10 μg to 30 μg of a uridine reference substance, 30 μg to 50 μg of a p-hydroxybenzoic acid reference substance, 30 μg to 50 μg of a vanillic acid reference substance, 30 μg to 50 μg of a quercetin-3-O-neohesperidin reference substance, 40 μg to 60 μg of a typhaneoglycan reference substance, 10 μg to 30 μg of a kaempferol-3-O-neohesperidin reference substance, 40 μg to 60 μg of an isorhamnetin-3-O-neohesperidin reference substance, and 40 μg to 60 μg of a rutin reference substance per 1 ml.

5. The construction method according to any one of claims 1 to 3, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography method include: a flow rate of 0.25 mL / min to 0.35 mL / min; a column temperature of 25° C. to 35° C.; and an injection speed of 0.5 to 1.5 μl / min.

6. The construction method according to any one of claims 1 to 3, characterized in that The characteristic spectrum of the Typha aquatica control medicinal material includes 9 common peaks, and the characteristic spectrum of the Typha orientalis control medicinal material includes 10 common peaks.

7. The construction method according to claim 6, characterized in that: The characteristic spectrum of the Typha truncatum reference medicinal material contains 7 common peaks corresponding to the reference substance, wherein: peak 1 corresponds to uridine, peak 2 corresponds to p-hydroxybenzoic acid, peak 3 corresponds to vanillic acid, peak 5 corresponds to quercetin-3-O-neohesperidin, peak 7 corresponds to typhaneoglycan, peak 8 corresponds to kaempferol-3-O-neohesperidin, and peak 9 corresponds to isorhamnetin-3-O-neohesperidin; The characteristic spectrum of the Typha orientalis reference medicinal material includes 8 common peaks corresponding to the reference substance, wherein: in addition to the 7 common peaks, the remaining 1 common peak corresponds to rutin.

8. A method for detecting Typhae Pollen medicinal material, characterized in that: The detection method comprises the following steps: (a) Preparation of a test solution: A test solution is prepared from a test medicinal material, wherein the test medicinal material is Typha truncatum or Typha orientalis. The test solution preparation comprises extracting the test medicinal material with a methanol aqueous solution having a methanol concentration of 10% or more by volume; (b) Chromatographic detection: The test solution prepared in step (a) is detected by ultra-performance liquid chromatography, and the chromatogram of the test solution is compared with the characteristic spectrum of the control medicinal material Typha aquatica or Typha orientalis constructed by the construction method according to any one of claims 1 to 7 to identify the origin of the medicinal material Typhae pollen; The chromatographic conditions of the ultra-high performance liquid chromatography method include: Column: ACQUITY UPLC HSS T3, 2.1 mm inner diameter, 100 mm length, 1.8 µm particle size; Mobile phase: acetonitrile as mobile phase A, 0.05% v / v~0.2% v / v formic acid in water as mobile phase B; The detection wavelength is 254nm, The gradient elution method was adopted, and the gradient elution procedure was as follows: 。 9. The detection method according to claim 8, characterized in that The preparation of the test solution includes extracting the medicinal material to be tested using a methanol aqueous solution with a methanol volume concentration of 30%.

10. The detection method according to claim 8 or 9, characterized in that The extraction adopts any one of ultrasonic treatment, heating reflux and shaking extraction.

11. The detection method according to claim 10, characterized in that: Ultrasonic treatment is used for extraction; the duration of the ultrasonic treatment is 10 minutes to 70 minutes, the power is 200W to 300W, and the frequency is 35kHz to 45kHz.

12. The detection method according to claim 8, characterized in that The preparation of the test solution further includes collecting the extract and then weighing it, supplementing the lost weight with a methanol aqueous solution with a methanol volume concentration of more than 10%, shaking it well, filtering it, and taking the filtrate to obtain the product.

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