A method for constructing characteristic spectrum of Piper longum samples and a method for detecting the same

Abstract

The present invention relates to a method for constructing a characteristic spectrum of a Piper longum sample and a detection method. The present invention adopts ultra-high performance liquid chromatography and reasonably controls the chromatographic conditions to find out ultra-high performance liquid chromatography conditions that are simultaneously applicable to Piper longum medicinal materials, decoction pieces, water extracts, and standard decoctions and formula granules thereof, thereby constructing a characteristic spectrum of the Piper longum sample. The characteristic spectrum of the present invention contains 10 characteristic peaks, which enables the quality control of Piper longum to rise from the original content determination of a single component or the determination of a few common peaks to the overall control of the intrinsic quality of the Piper longum sample, providing an effective method for the quality detection of Piper longum and its modern preparations.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine analysis and quality control, and in particular to a method for constructing a characteristic spectrum of a Piper longum sample and a method for detecting the same. Background Art

[0002] Piper longum L. is the dried nearly mature or mature fruit spike of Piper longum L., a plant of the Piperaceae family. It is pungent and warm in nature. It enters the spleen, stomach, kidney, and bladder meridians. It has the effects of warming the middle and dispelling cold, promoting qi and relieving pain. It is used for stomach cold vomiting, cold pain in the abdomen, abdominal pain due to cold hernia, cold dampness stagnation, and turbid urine. Modern pharmacological studies have found that Piper longum L. also has antibacterial, anti-inflammatory, antiviral, anti-tumor, antioxidant, and regulates metabolism and immune regulation in the body.

[0003] The clinical use of Piper longum is mainly in the form of decoctions. The content of active ingredients in decoctions directly affects the clinical effectiveness of traditional Chinese medicine. The traditional Chinese medicine granules produced by modern equipment are produced based on standard decoctions. In order to ensure the consistency of chemical composition in the process from medicinal materials to standard decoctions to formula granules, a detection method is urgently needed. This detection method can not only detect Piper longum medicinal materials, but also be suitable for the detection of Piper longum standard decoctions and its traditional Chinese medicine formula granules, etc., which is crucial for the quality detection and control of Piper longum modern preparations. The 2020 edition of the "Chinese Pharmacopoeia" uses the content of piperine as a quality evaluation indicator for Piper longum. However, due to the diversity and complexity of the active ingredients of traditional Chinese medicine, the content determination of a single ingredient is not enough to explain the intrinsic quality of the medicinal material and lacks specificity. The UPLC characteristic spectrum can provide more comprehensive and rich information. Pseudocanthine, which belongs to the same genus of Piperaceae on the market, is similar in appearance to Piper longum and is often mixed. Literature reports (Cui Guojing, Liu Fang. Piper longum and its counterfeit pseudo-conch [J]. Capital Food and Medicine, 2015, 0(9); Xu Yaling, Tian Jing, Shu Juan. Identification of Piper longum and its counterfeit pseudo-conch [J]. Journal of Guiyang College of Traditional Chinese Medicine, 2005, 27(4). etc.) mainly use the properties, physicochemical, microscopic, thin layer identification and piperine content of the original plant and medicinal pieces. The property identification relies on experienced Chinese medicine identification personnel. Similar appearance is prone to errors. Moreover, for standard decoctions and formula granules that lose their medicinal properties after water extraction, it is difficult to identify the authenticity of Piper longum by properties, physicochemical, and thin layer. Using the content of piperine as the identification point, identification based on only one characteristic component is prone to misdetection and is not comprehensive. There is currently no report on the identification of Piper longum and its counterfeit products through UPLC fingerprint / characteristic spectrum. Therefore, it is necessary to establish a comprehensive, accurate, rapid and specific detection method, which can not only detect Piper longum medicinal materials, decoction pieces and water extracts, but is also suitable for the detection of Piper longum standard decoctions and their formula granules, etc., which is crucial for the quality inspection and authenticity identification of Piper longum modern preparations. Summary of the invention

[0004] Problem that the invention aims to solve

[0005] In order to solve the above problems in the prior art, the purpose of the present invention is to provide a method for constructing a characteristic spectrum of a Piper longum sample, wherein the characteristic spectrum constructed by this method can comprehensively control the quality of Piper longum medicinal materials, decoction pieces, water extracts, standard decoctions and / or formula granules.

[0006] Furthermore, the purpose of the present invention is to provide a method for detecting a Piper longum sample, which method is simple and efficient, has objective results, is precise and reliable, has strong specificity, high sensitivity, good stability and tolerance, and can distinguish the authenticity of Piper longum, and further, can distinguish Piper longum from Pseudo Piper longum.

[0007] Solutions for solving problems

[0008] Through long-term research, the inventors found that the above technical problems can be solved by implementing the following technical solutions:

[0009] [1] A method for constructing a characteristic spectrum of a Piper longum sample, wherein the construction method comprises the following steps:

[0010] (a) Preparation of reference solution: comprising the steps of taking a piperlongumin reference substance, a piperine reference substance, and a pseudopiperlongumamide B reference substance to prepare a reference solution;

[0011] (b) Preparation of test solution: extracting the Piper longum sample with an extraction solvent, collecting the extract, and preparing the test solution, wherein the Piper longum sample is a Piper longum medicinal material, a Piper longum decoction piece, a Piper longum water extract, a Piper longum standard decoction, and / or a Piper longum formula granule;

[0012] (c) Establishment of characteristic spectrum: The reference solution and the test solution are tested by ultra-high performance liquid chromatography to obtain a reference spectrum and a test spectrum respectively, and a Chinese medicine chromatographic fingerprint similarity evaluation system is used to analyze and establish a characteristic spectrum;

[0013] The chromatographic conditions of the ultra-high performance liquid chromatography include:

[0014] Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.6-1.8 μm;

[0015] Mobile phase: acetonitrile as mobile phase A, glacial acetic acid or 0.1-0.5wt% glacial acetic acid aqueous solution as mobile phase B;

[0016] Gradient elution program:

[0017] From 0min to 5min, the volume percentage of the mobile phase A increases from 45% to 62%; from 5min to 7min, the volume percentage of the mobile phase A increases from 62% to 70%; from 7min to 10min, the volume percentage of the mobile phase A increases from 70% to 85%; from 10min to 15min, the volume percentage of the mobile phase A increases from 85% to 90%; from 15min to 23min, the volume percentage of the mobile phase A remains at 90%.

[0018] [2] The construction method according to [1], wherein the characteristic spectrum contains 10 peaks, wherein peak 1 is piperine, peak 2 is piperine, and peak 5 is pseudopiperilamide B.

[0019] [3] The construction method according to [1] or [2], wherein peak 5 is used as the reference peak to calculate the relative retention time of peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 5, and the relative retention time should be within ±10% of the specified value, and the specified values ​​corresponding to peak 3, peak 4, peak 6, peak 7, peak 8, peak 9 and peak 10 are 0.52, 0.92, 1.24, 1.69, 1.92, 1.98 and 2.04 respectively.

[0020] [4] The method according to any one of the technical solutions [1] to [3], wherein the preparation of the test solution comprises:

[0021] Ultrasonic extraction of Piper longum L. medicinal materials was performed using methanol or a methanol aqueous solution containing 70% to 90% methanol by volume, and the extract was collected to prepare a test solution;

[0022] Alternatively, ultrasonic extraction is performed on the standard decoction of Piper longum using methanol or a methanol aqueous solution containing 50% to 90% methanol by volume, and the extract is collected to prepare a test solution;

[0023] Alternatively, the Longan granules are ultrasonically extracted with methanol or a methanol aqueous solution containing 50% to 90% methanol by volume, and the extract is collected to prepare a test solution;

[0024] [5] The construction method according to [4], wherein the duration of the ultrasonic extraction is 15 min to 60 min, the power is 250 W to 300 W, and the frequency is 35 kHz to 45 kHz.

[0025] [6] The construction method according to any one of the technical solutions [1] to [5], wherein the preparation of the reference solution comprises adding methanol or a methanol aqueous solution containing 70% to 90% methanol by volume to prepare a reference solution containing 5 μg to 20 μg of the piperine reference, 10 μg to 50 μg of the piperine reference, and 2 μg to 25 μg of the pseudopiperilamide reference B per 1 ml.

[0026] [7] The construction method according to any one of the technical solutions [1] to [6], wherein the preparation of the reference solution further comprises taking an appropriate amount of Piper longum reference medicinal material, adding methanol or a methanol aqueous solution containing 50% to 90% methanol by volume, ultrasonically treating for 30 minutes, shaking well, and filtering to obtain the reference solution.

[0027] [8] The construction method according to any one of the technical solutions [1] to [7], wherein the column length of the chromatographic column is 100 mm and the inner diameter is 2.1 mm; the chromatographic conditions also include: 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 250 nm to 280 nm.

[0028] [9] The present invention also provides a method for detecting a Piper longum sample, wherein the detection method comprises the following steps:

[0029] Extracting the Piper longum sample to be tested with an extraction solvent, collecting the extract, and preparing a test solution; the Piper longum sample is a Piper longum medicinal material, a Piper longum decoction piece, a Piper longum water extract, a Piper longum standard decoction, and / or a Piper longum formula granule;

[0030] The test solution is tested by ultra-high performance liquid chromatography, and the test spectrum obtained by the test is compared with the characteristic spectrum constructed by the construction method according to any one of claims 1 to 8 to detect the quality and / or authenticity of the Piper longum sample;

[0031] Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.6-1.8 μm;

[0032] Mobile phase: acetonitrile as mobile phase A, glacial acetic acid or 0.1-0.5wt% glacial acetic acid aqueous solution as mobile phase B;

[0033] Gradient elution program:

[0034] From 0min to 5min, the volume percentage of the mobile phase A increases from 45% to 62%; from 5min to 7min, the volume percentage of the mobile phase A increases from 62% to 70%; from 7min to 10min, the volume percentage of the mobile phase A increases from 70% to 85%; from 10min to 15min, the volume percentage of the mobile phase A increases from 85% to 90%; from 15min to 23min, the volume percentage of the mobile phase A remains at 90%.

[0035]

[10] The present invention also provides an application of the detection method described in [9] in distinguishing Piper longum from Piper pseudocone.

[0036] Effects of the Invention

[0037] The present invention selects ultra-high performance liquid chromatography conditions that are simultaneously applicable to Piper longum medicinal materials, Piper longum decoction pieces, Piper longum water extracts and their standard decoctions and / or Chinese medicine formula granules, and uses the medicinal materials, standard decoctions and / or Chinese medicine formula granules as test samples for detection, thereby constructing a characteristic spectrum. The characteristic spectrum contains 10 characteristic peaks, which can not only reflect the quality of Piper longum medicinal materials, but also reflect the quality of Piper longum decoction pieces, water extracts, standard decoctions and Chinese medicine formula granules, and is particularly applicable to the quality detection of Piper longum medicinal materials, their standard decoctions and their Chinese medicine formula granules, thereby providing an effective method for the quality detection of Piper longum and its modern preparations, and also providing a scientific experimental basis for the comprehensive quality evaluation of Piper longum and its modern preparations.

[0038] The characteristic spectrum is used to detect Piper longum medicinal materials, Piper longum decoction pieces, Piper longum water extract, standard decoctions and Chinese medicine formula granules, which is simple, rapid and efficient, with objective results, precise and reliable, strong specificity, high sensitivity, good stability and tolerance, and is particularly suitable for Piper longum medicinal materials, standard decoctions and / or formula granules, and can also distinguish genuine Piper longum from counterfeit Piper longum. The characteristic spectrum of the present invention contains 10 characteristic peaks, which enables the quality control of Piper longum to rise 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 Piper longum and specificity identification.

[0039] The present invention applies ultra-high performance liquid chromatography to the quality inspection of Piper longum medicinal materials and their modern preparations for the first time, filling the gap in the quality inspection of Piper longum medicinal materials and their modern preparations, and realizing the quality control and specificity identification of Piper longum from medicinal materials to Chinese medicine formula granules in terms of intrinsic quality, which can not only solve the problem of difficulty in identifying decoction pieces, water extracts, standard decoctions and Chinese medicine formula granules due to the lack of raw material appearance, but also reduce the adverse effects of counterfeit and adulterated products on the Chinese medicine market from the source to the terminal product, thereby ensuring the quality of Piper longum medicinal materials, decoction pieces, water extracts, standard decoctions and formula granules, and facilitating the safety and effectiveness of clinical drug use.

[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 23 minutes, the solvent consumption is small, and the environmental pollution is small. The separation of the characteristic peaks of the spectrum of the present invention is good, and the chemical information of all characteristic peaks is confirmed by the reference substance and mass spectrum information. The specificity is strong, the analysis efficiency is high, the stability is high, the repeatability is good, the application range is wide, and it is suitable for the detection of products such as standard decoctions and formula granules of Piper longum medicinal materials, and is particularly suitable for the 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 It is the characteristic spectrum of the investigation result of the mobile phase in the present invention.

[0043] Figure 2 This is the investigation result of the elution time gradient 1 of the present invention.

[0044] Figure 3 This is the investigation result of elution time gradient 2 of the present invention.

[0045] Figure 4 This is the investigation result of the elution time gradient 3 of the present invention.

[0046] Figure 5 It is the characteristic spectrum of the reference substance in Example 1 of the present invention.

[0047] Figure 6 It is the superimposed characteristic spectrum of 15 batches of Piper longum in Example 1 of the present invention.

[0048] Figure 7 It is the superimposed characteristic spectrum of 15 batches of standard decoction of Piper longum in Example 1 of the present invention.

[0049] Figure 8 It is the superimposed characteristic spectrum of three batches of granules of Piper longum in Example 1 of the present invention.

[0050] Fig. 9 This is the reference characteristic spectrum of the Piper longum medicinal material in Example 1 of the present invention.

[0051] Fig.10 It is the reference characteristic spectrum of the standard decoction of Piper longum in Example 1 of the present invention.

[0052] Fig.11 This is a comparative characteristic spectrum of the Piper longum formula granules in Example 1 of the present invention.

[0053] Fig.12 This is an investigation of different column temperatures of the characteristic spectrum of Piper longum in Example 1 of the present invention.

[0054] Fig.13 This is an investigation of different flow rates of the characteristic spectrum of Piper longum in Example 1 of the present invention.

[0055] Fig.14 The characteristic spectrum of Piper longum in Example 1 of the present invention is investigated using different chromatographic columns.

[0056] Fig.15 It is a comparison chart of the characteristic spectra of the standard decoctions of Piper longum and Pseudocong konjac in Example 6 of the present invention.

[0057] Fig.16 This is a comparison of the characteristic spectra of Piper longum and Pseudocong in Example 6 of the present invention. DETAILED DESCRIPTION

[0058] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and embodiments obtained by appropriately combining the technical means disclosed in different embodiments and embodiments are also included in the technical scope of the present invention. In addition, all the documents recorded in this specification are cited in this specification as references.

[0059] Unless otherwise defined, 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 the invention belongs.

[0060] 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.

[0061] In this specification, the 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.

[0062] In this specification, the meaning 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 the situation where the event occurs and the situation where the event does not occur.

[0063] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "some specific / preferred technical solutions", "other specific / preferred technical solutions", etc. refer to the specific elements (e.g., features, structures, properties and / or characteristics) related to the embodiments described herein being included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0064] The term "comprising" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0065] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0066] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0067] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0068] 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, which 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.

[0069] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0070] 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.

[0071] Unless otherwise specified, the term "subsequent filtrate" used herein refers to the filtrate collected after the primary filtrate is discarded during filtration. Compared with the primary filtrate, the subsequent filtrate is closer to the actual concentration of the sample, because the filter medium (such as filter membrane, filter paper, etc.) may adsorb solutes, resulting in a lower sample concentration in the primary filtrate; in addition, the subsequent filtrate is cleaner, because the solutes adsorbed by the filter medium can form a filter cake, which reduces the filter pore size and can thus intercept smaller particles.

[0072] Unless otherwise specified, the term "ultra-high 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.

[0073] Unless otherwise specified, the term "retention time" used in this article refers to the time from the start of injection to the appearance of the maximum concentration of the separated component after the column, that is, the time from the start of injection to the appearance of the apex of the chromatographic peak of the separated component; the term "relative retention time" refers to the ratio of the calibrated retention time of the separated component to the calibrated retention time of the standard sample.

[0074] Unless otherwise specified, the term "reference peak" (or "control peak") used herein refers to the chromatographic peak corresponding to the standard sample when calculating the relative retention time.

[0075] Unless otherwise specified, the percentage "%" in this article refers to the volume percentage. For example, 0.1% phosphoric acid aqueous solution means adding 1mL of phosphoric acid to 1000mL of pure water. Since the measuring cylinder is not recommended as a container for mixed solutions, 0.1% phosphoric acid aqueous solution is also approximately equal to mixing 1000mL of pure water with 1mL of phosphoric acid.

[0076] <Method for constructing characteristic spectrum of Piper longum samples>

[0077] The method for constructing the characteristic spectrum of the Piper longum sample of the present invention mainly comprises the following steps:

[0078] (a) Preparation of reference solution: Take the reference substance of Piperglinide, piperine reference substance, and pseudopiperglinamide B reference substance respectively to prepare the reference substance solution; further, dissolve the reference substance in water, alcohol or alcohol-water solution. In some specific embodiments of the present invention, the preparation method of the above-mentioned reference substance solution is as follows: take an appropriate amount of the reference substance, accurately weigh it, add the above-mentioned solvent, mix until it is dissolved, and obtain it. Further, the extraction solvent is alcohol or alcohol-water solution, such as methanol, methanol-water solution, and further, the volume fraction of methanol in the methanol-water solution is not less than 20%, and the concentration of the extraction solvent is preferably 70% to 90% in terms of the volume fraction of methanol. More specifically, the concentration of the Piperglinide reference substance is 5μg to 20μg / mL, the concentration of the piperine reference substance is 10μg to 50μg / mL, and the concentration of the pseudopiperglinamide B reference substance is 2μg to 25μg / mL.

[0079] (b) Preparation of test solution: extracting the Piper longum sample with an extraction solvent, collecting the extract, and preparing the test solution. The Piper longum sample can be Piper longum medicinal material, Piper longum standard decoction, water extract, decoction piece, and / or Piper longum formula granule. In some specific embodiments of the present invention, the Piper longum sample can be Piper longum medicinal material, Piper longum standard decoction and / or Piper longum formula granule; wherein, "medicinal material" or "raw drug" refers to Chinese medicine raw materials that have not been processed or made into finished products; "standard decoction" refers to a single Chinese medicine decoction piece water decoction prepared by standardized process under the guidance of traditional Chinese medicine theory and based on clinical application, with reference to modern extraction methods; "formulated granule" is a granule prepared by extracting and concentrating a single Chinese medicine decoction piece according to traditional standards for use in Chinese medicine clinical prescriptions. In some specific embodiments of the present invention, the extraction solvent is selected from methanol or a methanol aqueous solution containing 20% ​​to 90% methanol by volume. In order to obtain a better peak shape (or peak shape) and comprehensively consider the total extraction efficiency, the extraction reagent is preferably a methanol aqueous solution or methanol with a methanol volume percentage of 50% to 90%; more preferably, a methanol aqueous solution or methanol with a methanol volume percentage of 70% to 90%. Regarding the extraction method, for the Piper longum medicinal material, it is preferred to sieve the medicinal material powder first, preferably using a No. 4 sieve. In order to obtain richer chromatographic peak information, the extraction can be carried out by ultrasound and heating reflux. Preferably, the ultrasonic treatment extraction method with simpler operation is adopted. In some preferred embodiments of the present invention, a methanol aqueous solution with a methanol volume percentage of 50% to 90% or methanol is used to extract the sieved Piper longum medicinal material powder. More preferably, a methanol aqueous solution with a methanol volume percentage of 70% to 90% or methanol is used to extract the sieved Piper longum medicinal material powder; the extraction method adopted is ultrasonic treatment, with a power of 250W to 300W and a frequency of 35kHz to 45kHz; further, considering the extraction efficiency of each chromatographic peak comprehensively, the duration of ultrasonic extraction is 15min to 60min. For the standard decoction of Piper longum, the medicinal material is first subjected to pretreatment, decoction, low-temperature concentration, freeze-drying and other processes to obtain a freeze-dried powder, and the freeze-dried powder is preferably ground into powder, and then preferably extracted with a methanol aqueous solution or methanol with a methanol volume percentage of 50% to 90%. The extraction can be carried out by ultrasound, heating reflux or shaking. In order to simplify the extraction operation, ultrasonic treatment is preferably used. The power and frequency of ultrasound have little effect on the present invention. Furthermore, the duration of ultrasonic extraction is 15min to 60min, the power is 250W to 300W, and the frequency is 35kHz to 45kHz.In some specific embodiments of the present invention, the amount of the extraction solvent added relative to the Piper longum sample is 300-600 mL / g, further 350-550 mL / g, and further, for Piper longum powder, the amount of the extraction solvent added relative to the Piper longum powder is 400-500 mL / g; for Piper longum standard decoction or Piper longum formula granules, the relative amount of the extraction solvent added is 450-520 mL / g. If the concentration of the test sample is too small, it is likely to be unfavorable for the detection of the chromatographic peak, and if the concentration is too large, the chromatographic column is overloaded and the chromatographic peak shape is easily poor. In some specific embodiments of the present invention, the preparation method of the test solution is as follows: take an appropriate amount of Piper longum medicinal material, crush it, sieve it (No. 4 sieve), accurately weigh it, put it in a stoppered conical bottle, accurately add a methanol aqueous solution with a volume fraction of 50-90%, seal it, weigh it, ultrasonically treat it for 15min-60min, let it cool, weigh it again, make up the lost weight with a methanol aqueous solution with a volume fraction of 60%-80%, shake it well, filter it, and take the filtrate to obtain; or, take an appropriate amount of Piper longum standard decoction or Piper longum formula granules The amount is measured, ground into powder, accurately weighed, placed in a stoppered conical flask, accurately added with 50-90% by volume methanol aqueous solution, plugged, weighed, ultrasonically treated for 15-60 min, cooled, weighed again, supplemented with 60-80% by volume methanol aqueous solution to make up for the lost weight, shaken, filtered, and the filtrate is obtained; wherein, the mass volume ratio of Piper longum medicinal materials, Piper longum standard decoction or Piper longum formula granules to 50-90% by volume methanol aqueous solution is 1g:(200-600mL).

[0080] (c) Establishment of characteristic spectrum: The reference solution and the test solution are tested by ultra-high performance liquid chromatography to obtain a reference spectrum and a test spectrum respectively, and a Chinese medicine chromatographic fingerprint similarity evaluation system is used to analyze and establish a characteristic spectrum;

[0081] The present invention has determined, through in-depth research, the ultra-high performance liquid chromatography conditions that are applicable to Piper longum medicinal materials, decoction pieces, water extracts and their standard decoctions and / or formula granules, which include:

[0082] Stationary phase: a chromatographic column with octadecylsilane bonded silica as filler and a particle size (filler particle size) of 1.5 to 2 μm; further, in order to obtain better column efficiency, the particle size is 1.6 to 1.8 μm; further, the column length of the chromatographic column is 100 to 150 mm and the inner diameter is 2.1 mm. In some specific embodiments of the present invention, the chromatographic column is selected from any one of Eclipse Plus C18RRHD (Agilent, 2.1 mm × 100 mm, 1.8 μm), CORTECS T3 (Waters, 2.1 × 100 mm, 1.6 μm), and BEH C18 (Waters, 2.1 × 100 mm, 1.7 μm); further, the chromatographic column preferably uses an Eclipse Plus C18 RRHD (Agilent, 2.1 mm × 100 mm, 1.8 μm) chromatographic column, and the separation using this chromatographic column has better separation of characteristic peaks and a more beautiful overall spectrum.

[0083] Mobile phase: acetonitrile is used as mobile phase A, and glacial acetic acid or a 0.05-0.7 wt % glacial acetic acid aqueous solution is used as mobile phase B; in some preferred embodiments of the present invention, glacial acetic acid or a 0.1-0.5 wt % glacial acetic acid aqueous solution is used as mobile phase B, which helps to obtain a good peak shape.

[0084] The gradient elution procedure of the present invention is as follows:

[0085] 0min~5min, the volume percentage of the mobile phase A increases from 45% to 62%; 5min~7min, the volume percentage of the mobile phase A increases from 62% to 70%; 7min~10min, the volume percentage of the mobile phase A increases from 70% to 85%; 10min~15min, the volume percentage of the mobile phase A increases from 85% to 90%; 15min~23min, the volume percentage of the mobile phase A remains at 90%. The use of this condition can achieve that no matter for samples of Piper longum medicinal materials, standard decoctions or formula granules, each chromatographic peak can be fully separated and has a good peak shape, and the analysis cycle is short, and the separation efficiency and separation degree are higher.

[0086] The detector used in the present invention can be an ultraviolet detector. Furthermore, the chromatographic conditions of the present invention also include: the detection wavelength is 250nm~280nm. In some preferred embodiments of the present invention, the detection wavelength is 240nm~260nm. The chromatographic peak information is relatively rich under the detection wavelength in this range, 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: the flow rate is 0.25mL / min~0.35mL / min; and / or the column temperature is 25℃~35℃. In the construction method of the present invention, within the range of column temperature or flow rate, smaller column temperature changes or flow rate changes can meet the system applicability requirements. In some preferred embodiments of the present invention, the flow rate is 0.28mL / min~0.32mL / min; and / or the column temperature is 28℃~33℃.

[0087] Furthermore, the present invention detects at least 15 batches of test solutions and reference solutions of Piper longum medicinal materials from different origins, analyzes and compares them, selects common peaks to establish characteristic spectra, and identifies common peaks by referring to the positioning of the chromatographic peaks of the reference products. Finally, the chromatograms of the obtained medicinal materials, standard decoctions, and formula granules 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 characteristic spectra. The characteristic spectrum of the Piper longum sample obtained by the aforementioned construction method of the present invention contains at least 10 peaks. Further, peak 1 is the peak of the Piper longum reference substance, peak 2 is the peak of the piperine reference substance, and peak 5 is the peak of the pseudopiper longum amide B reference substance. Since peak 5 has good separation and moderate retention time, peak 5 is used as the reference peak to calculate the retention time of other peaks. The relative retention times of peak 3, peak 4, peak 6, peak 7, peak 8, peak 9 and peak 10 are 0.52 (±10%), 0.92 (±10%), 1.24 (±10%), 1.69 (±10%), 1.92 (±10%), 1.98 (±10%), and 2.04 (±10%), respectively. The elution time of peak 1 and peak 2 is located by the reference substance. The 10 common peaks can stably appear in various medicinal materials, standard decoctions, and formula granules, and the degree of consistency is good.

[0088] The present invention shortens the analysis time and improves the analysis efficiency by optimizing the test sample preparation and chromatographic analysis conditions, establishes ultra-high performance liquid chromatography conditions that are simultaneously applicable to the Piper longum medicinal material and its standard decoction and formula granules, and truly reflects the original chemical composition of the Piper longum medicinal material, the standard decoction and the formula granules.

[0089] <Testing Method of Piper longum Samples>

[0090] On the other hand, the characteristic spectrum of the present invention can be used for a detection method for quality control of Piper longum samples (such as Piper longum medicinal materials, water extracts, decoction pieces, standard decoctions, and / or formula granules), and the detection method comprises the following steps:

[0091] The Piper longum sample to be tested is extracted with an extraction solvent, and the extract is collected to prepare a test solution; the Piper longum sample can be a Piper longum medicinal material, a decoction piece, an aqueous extract, a Piper longum standard decoction, and / or a Piper longum formula granule. In some specific embodiments of the present invention, the Piper longum sample can be a Piper longum medicinal material, a Piper longum standard decoction, and / or a Piper longum formula granule;

[0092] The test solution is tested by ultra-high performance liquid chromatography, and the test spectrum obtained by the test is compared with the characteristic spectrum constructed by the construction method of the present invention to detect the quality and / or authenticity of the Piper longum sample; further, if the UPLC characteristic spectra of the two are consistent, it is a qualified product; if the UPLC characteristic spectra of the two are inconsistent, it is an unqualified product. In some specific embodiments of the present invention, the test solution preparation method and chromatographic conditions of the present invention are used for detection.

[0093] In some specific embodiments of the present invention, the characteristic spectrum of Piper longum L. medicinal materials, standard decoctions and / or formula granules is used to detect the sample to be tested. The construction and detection method of the present invention provides a new analytical means for the intrinsic quality control of Piper longum L. from raw materials to finished products. The method is simple to operate, has high precision, good stability, good repeatability, high accuracy, maintains the consistency of the quality of Piper longum medicinal materials, decoction pieces, water extracts, standard decoctions and formula granules, and provides an important multi-index parameter basis for evaluating process stability.

[0094] Example

[0095] The present invention will be further described below through specific embodiments:

[0096] Experimental instruments, reagents and medicines

[0097] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters Corporation); Agilent 12920 ultra-high performance liquid chromatograph (Agilent Corporation); METTLER TOLEDO XP6 one-millionth balance (Mettler-Toledo (Shanghai) Instrument Co., Ltd.); ME204E electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.).

[0098] 15 batches of Piper longum medicinal materials were collected and identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd. 15 batches of Piper longum standard decoctions were prepared from raw medicinal materials by Jiangyin Tianjiang Pharmaceutical Co., Ltd. according to the guiding principles of "Preparation of decoctions for research and characterization of standard decoctions" in the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules". 3 batches of Piper longum formula granule samples were produced and prepared by the company. Pseudoconchoa was identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd. and prepared as Pseudoconchoa standard decoction. Piper longum control medicinal materials, batch number 121023-201103, were purchased from the China Food and Drug Inspection Institute.

[0099] Piperine (batch number: 110775-201706) was purchased from the China Food and Drug Administration. Piperine (batch number: B23090) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Pseudopiperine B (batch number: 10445) was purchased from Shanghai Shidande Biotechnology Co., Ltd.

[0100] Methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); glacial acetic acid (chromatographic grade, Aladdin); water is ultrapure water.

[0101] Example 1: Construction of characteristic profiles of Piper longum medicinal materials, standard decoctions and formulated granules

[0102] 1. Preparation of reference solution

[0103] Take 0.1 g of Piper longum reference medicinal material, place it in a stoppered conical flask, add 50 ml of 70% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0104] Take the piperlongin reference substance, piperine reference substance, and pseudopiperlongamide B reference substance separately, add methanol to prepare a solution containing 5 μg of piperlongin, 25 μg of piperine, and 5 μg of pseudopiperlongamide B per 1 ml, which is used as the reference substance solution.

[0105] 2. Preparation of test solution

[0106] (1) Piper longum drug test solution: Take an appropriate amount of Piper longum drug, crush it, pass it through a No. 4 sieve, take out about 0.1 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0107] (2) Test solution of standard decoction of Piper longum: Grind an appropriate amount of freeze-dried powder of standard decoction of Piper longum prepared by pretreatment, decoction, low-temperature concentration and freeze drying of Piper longum medicinal materials, take about 0.1 g, weigh accurately, put it in a stoppered conical flask, accurately add 50 ml of 70% methanol, stopper, weigh accurately, treat with ultrasound (power 250 W, frequency 40 kHz) for 30 min, let cool, weigh accurately again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the product.

[0108] (3) Test solution of Piper longum formula granules: Take an appropriate amount of the Chinese medicine formula granules obtained by pre-treatment, extraction, concentration, spray drying, granulation and packaging of Piper longum medicinal materials, grind them into powder, take about 0.1 g, weigh accurately, put it in a stoppered conical flask, accurately add 50 ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0109] 3. Chromatographic conditions

[0110] (1) Investigation of mobile phase

[0111] The chromatographic column used was Eclipse Plus C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm), mobile phase A was methanol, mobile phase B (0.1% phosphoric acid, 0.1% formic acid, 0.5% glacial acetic acid, water, respectively), gradient elution was performed as specified in Table 1, the flow rate was 0.3 ml per minute, the column temperature was 30°C, and the detection wavelength was 320 nm.

[0112] Table 1: Gradient elution table for mobile phase investigation

[0113] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～4 11→14 89→86 4～6 14 86 6～9 14→17 86→83 9～13 17 83 13～16 17→27 83→73 16～19 27 73 19～21 27→45 73→55 21～25 45 55

[0114] The separation effect of each characteristic peak was investigated by setting different mobile phases B. The results are shown in Figure 1 From the separation results, it can be seen that the use of 0.5% glacial acetic acid has a better separation effect on the characteristic peaks.

[0115] (2) Investigation of elution time gradient

[0116] (2-1)

[0117] The chromatographic column used was Eclipse Plus C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); mobile phase A was methanol, mobile phase B was 0.5% glacial acetic acid solution, and gradient elution was performed according to the provisions in Table 2; the flow rate was 0.3 ml per minute; the column temperature was 30° C.; and the detection wavelength was 320 nm.

[0118] Table 2: Investigation of elution time gradient 1

[0119] Time (minutes) Methanol(%) Mobile phase B (%) 0～3 11→14 89→86 3～5 14 86 5～20 14→17 86→83 20～24 17 83 24～30 17→27 83→73 30～38 27 73

[0120] Elute according to the time settings in Table 2. See the results. Figure 2 It was found that there was no chromatographic peak with a high response value from 0 to 15 min.

[0121] (2-2)

[0122] The chromatographic column used was Eclipse Plus C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm), mobile phase A was methanol, mobile phase B was 0.5% glacial acetic acid solution, gradient elution was performed according to the provisions in Table 3, the flow rate was 0.3 ml per minute, the column temperature was 30° C., and the detection wavelength was 320 nm.

[0123] Table 3: Investigation of elution time gradient 2

[0124]

[0125]

[0126] Elute according to the time settings in Table 3. See the results. Figure 3 It was found that although the separation effect of each characteristic peak was good under this time setting condition, the analysis time was long.

[0127] (2-3)

[0128] The chromatographic column used was Eclipse Plus C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm), mobile phase A was methanol, mobile phase B was 0.5% glacial acetic acid solution, gradient elution was performed according to the provisions in Table 4, the flow rate was 0.3 ml per minute, the column temperature was 30° C., and the detection wavelength was 320 nm.

[0129] Table 4: Investigation of elution time gradient 3

[0130] Time (minutes) Methanol(%) Mobile phase B (%) 0～5 45→62 55→38 5～7 62→70 38→30 7～10 70→85 30→15 10～15 85→90 15→10 15～19 90 10

[0131] Elute according to the time settings in Table 4. See the results. Figure 4 ,It was found that under this chromatographic condition, the chromatographic peak information was rich, the various characteristic peaks were well separated, and the analysis time was moderate.

[0132] According to the above investigation of the mobile phase and the elution time gradient, the chromatographic conditions of the present invention are: using an EclipsePlus C18 RRHD (column length of 100 mm, inner diameter of 2.1 mm, particle size of 1.8 μm) chromatographic column; acetonitrile is used as mobile phase A, 0.1% glacial acetic acid solution is used as mobile phase B, and gradient elution is performed according to Table 5; the column temperature is 30°C; the flow rate is 0.30 mL / min; and the detection wavelength is 254 nm.

[0133] Table 5: Mobile phase gradient elution table

[0134] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 45→62 55→38 5～7 62→70 38→30 7～10 70→85 30→15 10～15 85→90 15→10 15～23 90 10

[0135] 4. Establishment of feature maps

[0136] 4.1 Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, record the chromatogram, and obtain the characteristic spectrum of Piper longum medicinal material, standard decoction, and Chinese medicine formula granules.

[0137] 4.2 Identification of chromatographic peaks

[0138] The results of positioning using the reference substances of piperlongumine, piperine and pseudopiperlongumamide B showed that peak 1 was piperlongumine, peak 2 was piperine and peak 5 was pseudopiperlongumamide B. Figure 5 .

[0139] Selection of reference peak and common peak

[0140] The chromatograms obtained from 15 batches of Piper longum L. from different origins revealed 10 common peaks. Figure 6 . Figure 6 Overlay characteristic maps of 15 batches of Piper longum medicinal materials.

[0141] The chromatograms of 15 batches of self-produced standard decoction of Piper longum were determined, and 10 common peaks were identified. Figure 7 . Figure 7 The characteristic spectra of 15 batches of standard decoctions of Piper longum were superimposed.

[0142] The chromatograms obtained from the three batches of pilot-scale production of Piper longum formula granules revealed 10 common peaks. Figure 8 . Figure 8 Superimpose characteristic spectra of three batches of Chinese medicine granules of Piper longum.

[0143] Construction of control profiles and similarity evaluation

[0144] The chromatograms of 15 batches of Piper longum L. medicinal materials, 15 batches of Piper longum standard decoctions, and 3 batches of Piper longum formula granules were imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" and the median method was used to generate their respective superimposed characteristic spectra (see Figure 6 , Figure 7 , Figure 8 ) and control characteristic spectrum (see Fig. 9 , Fig.10 , Fig.11 ).

[0145] 5. Methodological investigation

[0146] 5.1 Precision test

[0147] Take 1 sample each of Piper longum medicinal material (YC122008003), standard decoction (DG122008003), and Chinese medicine formula granules (20010049), prepare them according to the method described in "2. Preparation of test solution", accurately pipette 1 μL of the same test solution, inject it continuously 6 times, determine according to the method under "3. Chromatographic conditions", and record the chromatogram. The three peaks of Piper longum, piperine and pseudopiper longum amide B should correspond to the retention time of the corresponding reference substance peaks; the peak corresponding to the pseudopiper longum amide B reference substance peak is the S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak.

[0148] The results of the precision test showed that the relative retention time RSD values ​​of each characteristic peak of Piper longum medicinal material were all less than 0.04%, and the relative peak area RSD values ​​were all less than 1.55%; the relative retention time RSD values ​​of each characteristic peak of Piper longum standard decoction were all less than 0.13%, and the relative peak area RSD values ​​were all less than 1.43%; the relative retention time RSD values ​​of each characteristic peak of Piper longum Chinese medicine formula granules were all less than 0.25%, and the relative peak area RSD values ​​were all less than 2.39%, indicating that the instrument has good precision.

[0149] 5.2 Repeatability test

[0150] Take 6 samples of Piper longum medicinal materials (YC122008003), standard decoction (DG122008003), and Chinese medicine formula granules (20010049), prepare them according to the method described in "2. Preparation of test solution", accurately pipette 1 μL of the test solution respectively, measure according to the method under "3. Chromatographic conditions", and record the chromatogram, among which the three peaks of Piper longum, piperine and pseudopiper longum amide B should correspond to the retention time of the corresponding reference substance peaks; the peak corresponding to the pseudopiper longum amide B reference substance peak is the S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak.

[0151] The results of the repeatability test showed that the relative retention time RSD values ​​of each characteristic peak of Piper longum medicinal material were all less than 0.09%, and the relative peak area RSD values ​​were all less than 1.67%; the relative retention time RSD values ​​of each characteristic peak of Piper longum standard decoction were all less than 0.11%, and the relative peak area RSD values ​​were all less than 1.84%; the relative retention time RSD values ​​of each characteristic peak of Piper longum formula granules were all less than 0.16%, and the relative peak surface RSD values ​​were all less than 1.92%, indicating that the method has good repeatability.

[0152] 5.3 Stability test

[0153] Take 1 portion each of Piper longum medicinal material (YC122008003), standard decoction (DG122008003), and Chinese medicine formula granules (20010049), prepare them according to the method described in "2. Preparation of test solution", and measure them according to the method under "3. Chromatographic conditions" at 0h, 2h, 4h, 8h, 12h, 18h, and 24h, and record the chromatogram. The three peaks of piperine, piperine, and pseudopiper longum amide B should correspond to the retention time of the corresponding reference substance peaks; the peak corresponding to the pseudopiper longum amide B reference substance peak is the S peak. Calculate the relative retention time and relative peak area of ​​each characteristic peak.

[0154] The results of the stability test showed that the relative retention time RSD values ​​of each characteristic peak of Piper longum medicinal material were all less than 0.09%, and the relative peak area RSD values ​​were all less than 1.65%; the relative retention time RSD values ​​of each characteristic peak of Piper longum standard decoction were all less than 0.12%, and the relative peak area RSD values ​​were all less than 2.71%; the relative retention time RSD values ​​of each characteristic peak of Piper longum formula granules were all less than 0.08%, and the relative peak surface RSD values ​​were all less than 2.97%, indicating that the method has good stability.

[0155] 5.4 Durability test

[0156] Column temperature investigation: The chromatograms at 25℃, 30℃, and 35℃ were compared. The results showed that the column temperature variation within the range of 25℃ to 35℃ could meet the system suitability requirements, and the preferred column temperature was 30℃. Fig.12 .

[0157] Flow rate investigation: The chromatograms of 0.25mL / min, 0.30mL / min, and 0.35mL / min were compared. The results showed that the flow rate ranged from 0.25 to 0.35ml / min, and the flow rate change could meet the system suitability requirements. The preferred flow rate was 0.30mL / min, see Fig.13 .

[0158] Chromatographic column investigation: Three different types of chromatographic columns were compared, namely Eclipse Plus c18 (Agilent, 2.1×100mm, 1.8μm), CORTECS T3 (Waters, 2.1×100mm, 1.6μm), and BEH C18 (Waters, 2.1×100mm, 1.7μm). The peak shapes of the three columns were good and the separation was good, as shown in Figure 2. Fig.14 .

[0159] Example 2: Detection method of Piper longum medicinal material

[0160] 1. Preparation of sample solution

[0161] Take an appropriate amount of Piper longum medicinal material, crush it, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0162] 2. Detection

[0163] The sample solution to be tested is accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph are the same as those in the item "3. Chromatographic conditions" in Example 1.

[0164] By comparing the sample to be tested with Fig. 9 From the characteristic spectrum of the reference of Piper longum medicinal material, it can be seen that the similarity between the detection spectrum of the sample to be tested and the characteristic spectrum of the reference of Piper longum medicinal material is 0.96, and 10 characteristic peaks can be detected, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material reference material correspond to each other, among which peak 1, peak 2, and peak 5 should correspond to the retention times of the reference peaks of Piper longum, piperine, and pseudopiper longum amide B, respectively. The peak corresponding to the pseudopiper longum amide B reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention times of peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, and peak 10 are 0.52, 0.92, 1.23, 1.70, 1.95, 2.02, and 2.09, respectively, which are in line with the ±10% range of the specified value. The specified values ​​are: 0.52 (peak 3), 0.92 (peak 4), 1.24 (peak 6), 1.69 (peak 7), 1.92 (peak 8), 1.98 (peak 9), 2.04 (peak 10). It can be considered that the quality of the sample to be tested is stable, meets the quality requirements, and is a qualified medicinal material.

[0165] Embodiment 3: Detection method of standard decoction of Piper longum

[0166] 1. Preparation of sample solution

[0167] Take an appropriate amount of freeze-dried powder of standard decoction of Piper longum prepared by pre-treatment, decoction, low-temperature concentration and freeze-drying of Piper longum medicinal materials, grind it into powder, take about 0.1g, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0168] 2. Detection

[0169] The sample solution to be tested is accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph are the same as those in the item "3. Chromatographic conditions" in Example 1.

[0170] By comparing the sample to be tested with Fig.10 The characteristic spectrum of the standard decoction of Piper longum is known to have a similarity of 0.92 between the detection spectrum of the sample to be tested and the characteristic spectrum of the standard decoction of Piper longum. Ten characteristic peaks can be detected, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material reference material correspond to each other, wherein peak 1, peak 2, and peak 5 should correspond to the retention times of the reference material peaks of Piper longum, piperine, and pseudopiper longum amide B, respectively. The peak corresponding to the pseudopiper longum amide B reference material peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention times of peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, and peak 10 are 0.52, 0.92, 1.23, 1.70, 1.93, 2.00, and 2.05, respectively, which are in line with the ±10% range of the specified value. The specified values ​​are: 0.52 (peak 3), 0.92 (peak 4), 1.24 (peak 6), 1.69 (peak 7), 1.92 (peak 8), 1.98 (peak 9), 2.04 (peak 10). It can be considered that the quality of the sample to be tested is stable, meets the quality requirements, and is a qualified standard decoction.

[0171] Example 4: Detection method of Piper longum formula granules

[0172] 1. Preparation of sample solution

[0173] Take an appropriate amount of Chinese medicine formula granules obtained by pre-treatment, extraction, concentration, spray drying, granulation and packaging of Piper longum medicinal materials, grind them into powder, take about 0.1g, accurately weigh, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0174] 2. Detection

[0175] The sample solution to be tested is accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph are the same as those in the item "3. Chromatographic conditions" in Example 1.

[0176] By comparing the sample to be tested with Fig.11 The characteristic spectrum of the Piper longum formula granules shows that the similarity between the test spectrum of the sample to be tested and the characteristic spectrum of the Piper longum formula granules is 0.96, and 10 characteristic peaks can be detected, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material reference material correspond to each other, among which peak 1, peak 2, and peak 5 should correspond to the retention times of the reference material peaks of Piper longum, piperine, and pseudopiper longum amide B, respectively. The peak corresponding to the pseudopiper longum amide B reference material peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention times of peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, and peak 10 are 0.52, 0.92, 1.23, 1.70, 1.93, 2.00, and 2.05, respectively, which are in line with the ±10% range of the specified value. The specified values ​​are: 0.52 (peak 3), 0.92 (peak 4), 1.24 (peak 6), 1.69 (peak 7), 1.92 (peak 8), 1.98 (peak 9), 2.04 (peak 10). It can be considered that the quality of the sample to be tested is stable, meets the quality requirements, and is a qualified Piper longum formula granule.

[0177] Example 5: Consistency evaluation of Piper longum medicinal materials, standard decoctions, and Chinese medicine granules

[0178] In this embodiment, the constructed characteristic spectrum method of Piper longum medicinal materials, standard decoctions and formula granules is applied to sample detection and quality evaluation at various stages of the production process of Piper longum formula granules. One batch of medicinal materials, standard decoctions and formula granules in the pilot production process are tested.

[0179] 1. Preparation of test solution

[0180] Medicinal materials: Take an appropriate amount of Piper longum medicinal material, crush it, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 60 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0181] Standard decoction: Take an appropriate amount of freeze-dried powder of standard decoction of Piper longum prepared by pre-treatment, decoction, low-temperature concentration and freeze-drying of Piper longum medicinal materials, grind it into powder, take about 0.1g, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 45 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0182] Chinese herbal formula granules: Take an appropriate amount of Chinese herbal formula granules obtained by pre-treatment, extraction, concentration, spray drying, granulation and packaging of Piper longum medicinal materials, grind them into powder, take about 0.1g, accurately weigh, put it in a stoppered conical bottle, accurately add 50ml of methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0183] 2. Detection

[0184] The sample solution to be tested is accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph are the same as those in the item "3. Chromatographic conditions" in Example 1.

[0185] The chromatograms of a batch of pilot-produced Piper longum medicinal materials, the corresponding standard decoction and Chinese medicine formula granules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition". The median method was used to generate their respective superimposed feature maps. The pilot-produced medicinal materials were used as the reference map to perform similarity comparison. The comparison results are shown in Table 6 below.

[0186] Table 6: Similarity and consistency analysis of pilot-scale medicinal materials, standard decoctions, and formula granules from the same batch of raw materials

[0187] No. 1 Similarity Medicinal Materials 1 1.00 Standard decoction 1 0.93 Chinese medicine formula granules 1 0.92

[0188] According to Table 6, the spectra of Piper longum L. and its standard decoction and TCM granules are extremely similar, which means that the characteristic spectrum constructed with the medicinal material as the test sample can not only reflect the quality of the medicinal material, but also reflect the quality of the standard decoction and TCM granules. Therefore, it can be used for the detection of medicinal materials, standard decoctions and / or TCM granules, and can be used for the quality evaluation of each link of the production of granules, so as to ensure that the chemical components corresponding to each chromatographic peak can be well transferred from the raw materials to the final product during the production process.

[0189] Example 6: Identification of counterfeit products of Piper longum medicinal materials and standard decoctions

[0190] Piper longum is the dried nearly mature or mature fruit cluster of Piper longum L., a plant of the Piperaceae family. Pseudoconiferum, which is also of the Piperaceae family, is similar in appearance to Piper longum in the market and is often used interchangeably.

[0191] Take the standard decoction of Pseudocong and Pseudocong medicinal materials, prepare according to the method under "2. Preparation of test solution", accurately pipette 1 μL of the test solution, measure according to the method under "3. Chromatographic conditions", record the chromatogram, and the results are shown in Fig.15 , Fig.16 .

[0192] The test results show that compared with the chromatogram of the genuine Piper longum, the pseudocong has fewer chromatographic peaks, lacking characteristic peaks 1, peak 3, peak 4, peak 7, peak 8, peak 9, and peak 10. The intrinsic chemical components of Piper longum and pseudocong are significantly different. Peaks 1, peak 3, peak 4, peak 7, peak 8, peak 9, and peak 10 can all be used as identification and analysis points for Piper longum adulterated products, and the similarity between Piper longum standard decoction, Chinese medicine formula granules and their corresponding medicinal materials is extremely high, and the consistency of the chromatograms of the three is strong. Therefore, the identification points of the characteristic spectra of the above-mentioned medicinal materials and standard decoctions can also be used as identification points for the formula granules.

[0193] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0194] The above-mentioned embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a Piper longum sample, characterized in that: The construction method comprises the following steps: (a) Preparation of reference solution: comprising the steps of preparing reference solution by taking piperlongumin reference substance, piperine reference substance and pseudopiperlongumamide B reference substance respectively; (b) Preparation of test solution: extracting the Piper longum sample with an extraction solvent, collecting the extract, and preparing the test solution, wherein the Piper longum sample is Piper longum medicinal material, Piper longum decoction slices, Piper longum water extract, Piper longum standard decoction and / or Piper longum formula granules, and the extraction solvent is methanol or a methanol aqueous solution containing 20% ​​to 90% methanol by volume; (c) Establishment of characteristic spectrum: the reference solution and the test solution are tested by ultra-high performance liquid chromatography to obtain a reference spectrum and a test spectrum respectively, and a Chinese medicine chromatographic fingerprint similarity evaluation system is used to analyze and establish a characteristic spectrum; The chromatographic conditions of the ultra-high performance liquid chromatography include: Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.6-1.8 μm; Mobile phase: acetonitrile as mobile phase A, 0.1-0.5wt% glacial acetic acid aqueous solution as mobile phase B; Gradient elution program: 0min~5min, the volume percentage of the mobile phase A increases from 45% to 62%; 5min~7min, the volume percentage of the mobile phase A increases from 62% to 70%; 7min~10min, the volume percentage of the mobile phase A increases from 70% to 85%; 10min~15min, the volume percentage of the mobile phase A increases from 85% to 90%; 15min~23min, the volume percentage of the mobile phase A remains at 90%, The detection wavelength is 250nm~280nm.

2. The construction method according to claim 1, characterized in that: The characteristic spectrum contains 10 peaks, wherein peak 1 is piperine, peak 2 is piperine, and peak 5 is pseudopiperilamide B.

3. The construction method according to claim 2, characterized in that: Taking Peak 5 as the reference peak, calculate the relative retention time of Peak 3, Peak 4, Peak 6, Peak 7, Peak 8, Peak 9, Peak 10 and Peak 5, and the relative retention time should be within ±10% of the specified value. The specified values ​​corresponding to Peak 3, Peak 4, Peak 6, Peak 7, Peak 8, Peak 9 and Peak 10 are 0.52, 0.92, 1.24, 1.69, 1.92, 1.98 and 2.04, respectively.

4. The construction method according to any one of claims 1 to 3, characterized in that: The preparation of the test solution includes: Ultrasonic extraction of Piper longum L. medicinal materials was performed using methanol or a methanol aqueous solution containing 70% to 90% methanol by volume, and the extract was collected to prepare a test solution; Alternatively, ultrasonic extraction is performed on the standard decoction of Piper longum using methanol or a methanol aqueous solution containing 50% to 90% methanol by volume, and the extract is collected to prepare a test solution; Alternatively, the Longan granules are ultrasonically extracted with methanol or a methanol aqueous solution containing 50% to 90% methanol by volume, and the extract is collected to prepare a test solution.

5. The construction method according to claim 4, characterized in that: The ultrasonic extraction lasts for 15 minutes to 60 minutes, with a power of 250W to 300W and a frequency of 35kHz to 45kHz.

6. The construction method according to any one of claims 1 to 3, characterized in that: The preparation of the reference solution includes adding methanol or a methanol aqueous solution containing 70% to 90% methanol by volume to prepare a reference solution containing 5 μg to 20 μg of piperine reference, 10 μg to 50 μg of piperine reference, and 2 μg to 25 μg of pseudopiperilamide B reference per 1 ml.

7. The construction method according to any one of claims 1 to 3, characterized in that: The preparation of the reference solution also includes taking an appropriate amount of Piper longum reference medicinal material, adding methanol or a methanol aqueous solution containing 50% to 90% methanol by volume, ultrasonically treating for 30 minutes, shaking well, and filtering to obtain the reference solution.

8. The construction method according to any one of claims 1 to 3, characterized in that: The column length of the chromatographic column is 100 mm and the inner diameter is 2.1 mm; the chromatographic conditions also include: a flow rate of 0.25 mL / min to 0.35 mL / min; and / or a column temperature of 25° C. to 35° C.

9. A method for detecting a Piper longum sample, characterized in that: The detection method comprises the following steps: The Piper longum sample to be tested is extracted with an extraction solvent, and the extract is collected to prepare a test solution; the Piper longum sample is a Piper longum medicinal material, a Piper longum decoction piece, a Piper longum water extract, a Piper longum standard decoction and / or a Piper longum formula granule, and the extraction solvent is methanol or a methanol aqueous solution containing 20% ​​to 90% methanol by volume; The test solution is tested by ultra-high performance liquid chromatography, and the test spectrum obtained by the test is compared with the characteristic spectrum constructed by the construction method according to any one of claims 1 to 8 to detect the quality and / or authenticity of the Piper longum sample; Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.6-1.8 μm; Mobile phase: acetonitrile as mobile phase A, 0.1-0.5wt% glacial acetic acid aqueous solution as mobile phase B; Gradient elution program: 0min~5min, the volume percentage of the mobile phase A increases from 45% to 62%; 5min~7min, the volume percentage of the mobile phase A increases from 62% to 70%; 7min~10min, the volume percentage of the mobile phase A increases from 70% to 85%; 10min~15min, the volume percentage of the mobile phase A increases from 85% to 90%; 15min~23min, the volume percentage of the mobile phase A remains at 90%, The detection wavelength is 250nm~280nm.

10. Use of the detection method according to claim 9 in distinguishing Piper longum and Piper pseudocone.

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