Construction method of characteristic spectrum of bamboo shavings and identification method

Abstract

The invention discloses a method for constructing a characteristic spectrum of bamboo shavings and an identification method. The method comprises: extracting bamboo shavings with 50 vol% to 90 vol% methanol, separating solid and liquid, dissolving the solid phase with water, extracting with ethyl acetate, separating solid and liquid, and dissolving the solid phase with 50 vol% to 100 vol% methanol to obtain a test solution; dissolving a p-hydroxybenzoic acid reference substance and a 4-coumaric acid reference substance with 30 vol% to 100 vol% methanol to obtain a reference substance solution; measuring the test solution and the reference substance solution with a liquid chromatograph to obtain a characteristic spectrum of bamboo shavings; and implementing the invention can effectively identify bamboo shavings medicinal materials and counterfeits thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and in particular to a method for constructing a characteristic spectrum of bamboo shavings and an identification method. Background Art

[0002] Bamboo shavings were first recorded in the Synopsis of the Golden Chamber, named "bamboo skin". The 2020 edition of the Chinese Pharmacopoeia stipulates that bamboo shavings are the dried middle layer of the stems of the Poaceae plants Bambusa tuldoides Munro, Sinocalamus beecheyanus (Munro) McClure var.pubescens PFLi or Phyllostachys nigra (Lodd.) Munro var.Henonis (Mitf.) Stapf ex Rendle. Bamboo shavings are commonly used in Chinese medicine prescriptions and patent medicines, with the effects of clearing heat and resolving phlegm, eliminating restlessness and stopping vomiting. It is used for cough with phlegm heat, phlegm with gallbladder fire, palpitations and restlessness, insomnia, phlegm due to stroke, tongue stiffness and speechlessness, vomiting due to stomach heat, nausea during pregnancy, and restless fetuses. There are many types of bamboo, with more than 1,200 species in the world. Common bamboos include moso bamboo, water bamboo, purple bamboo, Buddha belly bamboo, tea pole bamboo, etc. At present, bamboo shavings substitutes that often appear on the market are mostly moso bamboo, but moso bamboo is a medicine for treating hematuria, and its skin does not have the effect of clearing away heat, resolving phlegm and stopping vomiting, so it cannot be mixed with it. Other closely related plants such as pink single bamboo, small-leaved dragon bamboo, ma bamboo, cilia chinensis, and bamboo are difficult to distinguish from each other in terms of morphology, especially the silk strips scraped off by bamboo craftsmen during the process of weaving bamboo utensils are also mixed in and used as bamboo shavings for medicinal purposes. In order to ensure the safety of clinical drug use, it is necessary to distinguish bamboo shavings (green stalk bamboo, big head bamboo, light bamboo) from counterfeits.

[0003] So far, the quality control standards under the bamboo shavings medicinal materials in the 2020 edition of the Chinese Pharmacopoeia only include water and extract items. At present, there are only a few literature reports on the characteristics / characteristic spectra of bamboo shavings, and the number of chromatographic peaks that can be identified is small, and the applicability remains to be verified. For example, Zhou Bingqian et al. established the characteristic spectrum of bamboo shavings medicinal materials by HPLC gradient elution, and selected 13 chromatographic common peaks, including uridine, adenosine, guanosine, p-hydroxybenzoic acid and p-coumaric acid, to provide a reference for the comprehensive evaluation of the quality of bamboo shavings medicinal materials from different origins, but failed to distinguish the origin of bamboo shavings and compare different counterfeits. The rest of the quality analysis focused on the standard research of formula granules. For example, Zhao Shushu et al. used high-performance liquid chromatography to establish the characteristic spectrum of ginger bamboo shavings formula granules, and Wang Xiaoya et al. established the characteristic spectrum of bamboo shavings formula granules. The research results showed that there were 4 common peaks, and only 4-coumaric acid was identified. Among the provincial local standards for bamboo shavings formula granules, there is currently only one standard for green bamboo, one of the three basic ingredients. Except for the Shanghai standard, the characteristic spectrum of the other standards has only four chromatographic peaks, two of which are identified, namely p-hydroxycinnamic acid and (+)-candlewood resin phenol-9'-O-glucoside. The characteristic spectrum of the Shanghai standard has seven common peaks, and only 4-coumaric acid is identified. Similarly, these standards do not have information on relevant identification items for counterfeit products. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of bamboo shavings, which has strong specificity and can be used to identify common counterfeits. It also has many identification components and rich information.

[0005] In order to solve the above technical problems, the present invention provides a method for constructing a characteristic spectrum of bamboo shavings, which comprises:

[0006] The bamboo shavings are extracted with 50 vol% to 90 vol% methanol, the solid-liquid separation is performed, the obtained solid phase is dissolved in water, extracted with ethyl acetate, the solid-liquid separation is performed, and the obtained solid phase is dissolved with 50 vol% to 100 vol% methanol to obtain a test solution; wherein the concentration of the methanol-water solution for the first extraction is exemplarily 54 vol%, 60 vol%, 66 vol%, 72 vol%, 78 vol%, 84 vol% or 88 vol%, but is not limited thereto; the concentration of the methanol-water solution used for dissolution can be exemplarily 54 vol%, 60 vol%, 66 vol%, 72 vol%, 78 vol%, 84 vol%, 88 vol%, 92 vol% or 96 vol%, but is not limited thereto.

[0007] The p-hydroxybenzoic acid reference substance and the 4-coumaric acid reference substance are dissolved in 30 vol% to 100 vol% methanol to obtain a reference substance solution; wherein the concentration of the methanol aqueous solution used for dissolution is exemplarily 35 vol%, 40 vol%, 45 vol%, 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70 vol%, 75 vol%, 80 vol%, 85 vol% or 90 vol%, but is not limited thereto.

[0008] The test solution and the reference solution are measured by liquid chromatography to obtain a characteristic spectrum of bamboo shavings;

[0009] The chromatographic column of the liquid chromatograph uses octadecylsilane bonded silica gel as the stationary phase, acetonitrile as the mobile phase A, and 0.02 vol% to 0.1 vol% acetic acid aqueous solution as the mobile phase B for gradient elution, and the gradient elution curve is:

[0010] 0min~15min, mobile phase A is 5%, mobile phase B is 95%;

[0011] 15min~35min, mobile phase A from 5%→10%, mobile phase B from 95%→90%;

[0012] 35min~50min, mobile phase A from 10%→12%, mobile phase B from 90%→88%;

[0013] 50min-60min, mobile phase A from 12% to 15%, mobile phase B from 88% to 85%;

[0014] 60min~65min, mobile phase A is 15%, mobile phase B is 85%;

[0015] The concentration of acetic acid in the acetic acid aqueous solution is exemplified by 0.03 vol%, 0.04 vol%, 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol% or 0.09 vol%, but is not limited thereto.

[0016] As an improvement of the above technical solution, the mobile phase B is a 0.05 vol% acetic acid aqueous solution.

[0017] As an improvement of the above technical solution, the column length of the chromatographic column is 150mm to 300mm, the column diameter is 3mm to 5mm, and the particle size of the stationary phase is 3μm to 10μm. Exemplarily, the column length of the chromatographic column is 150mm, 200mm, 250mm or 300mm, but not limited thereto. The column diameter of the chromatographic column is 3mm, 4mm or 4.6mm, but not limited thereto. The particle size of the stationary phase in the chromatographic column is 3μm, 5μm, 9μm or 10μm, but not limited thereto.

[0018] As an improvement of the above technical solution, the chromatographic column is an Agilent ZORBAX SB-AQ chromatographic column, with a column length of 250 mm and a column diameter of 4.6 mm, and the particle size of the stationary phase is 5 μm.

[0019] As an improvement of the above technical solution, the column temperature of the chromatographic column is 30°C to 40°C; exemplary temperatures are 32°C, 34°C, 36°C or 38°C, but not limited thereto.

[0020] The flow rate of the liquid chromatograph is 0.9 mL / min to 1.1 mL / min, and exemplarily is 0.92 mL / min, 0.95 mL / min, 0.98 mL / min, 1.04 mL / min or 1.08 mL / min, but not limited thereto.

[0021] The detection wavelength is 250 nm to 270 nm, exemplified by 252 nm, 256 nm, 260 nm, 264 nm or 268 nm, but not limited thereto.

[0022] The injection volume of the test solution is 5 μL to 15 μL, exemplified by 7.5 μL, 9 μL, 11.5 μL, 13 μL or 14.5 μL, but not limited thereto.

[0023] The injection volume of the reference solution is 5 μL to 15 μL, exemplified by 7.5 μL, 9 μL, 11.5 μL, 13 μL or 14.5 μL, but not limited thereto.

[0024] As an improvement of the above technical solution, the column temperature of the chromatographic column is 35°C;

[0025] The flow rate of the liquid chromatograph was 1.0 mL / min, and the detection wavelength was 260 nm; the injection volume of the test solution was 10 μL, and the injection volume of the reference solution was 10 μL.

[0026] As an improvement of the above technical solution, the preparation method of the test solution is:

[0027] (1) Extracting bamboo shavings with 50 vol% to 90 vol% methanol under heating reflux for 20 min to 40 min, separating the obtained extract into solid and liquid, and dissolving the obtained solid in water to obtain an extract; wherein the amount ratio of bamboo shavings to 50 vol% to 90 vol% methanol is 1.5 g to 3 g: 30 mL to 80 mL; and the volume ratio of water to 50 vol% to 90 vol% methanol is 1:2 to 1:5;

[0028] Exemplarily, the usage ratio of bamboo shavings to 50 vol% to 90 vol% methanol is 1.8 g: 35 mL, 2 g: 40 mL, 2.4 g: 45 mL, 2.5 g: 51 mL, 2.7 g: 60 mL or 2.9 g: 68 mL, but is not limited thereto. Preferably, the usage ratio of bamboo shavings to 50 vol% to 90 vol% methanol is 1.8 g to 2.2 g: 45 mL to 55 mL.

[0029] Exemplarily, the volume ratio of water to 50 vol% to 90 vol% methanol is 1:2.5, 1:2.9, 1:3.4, 1:3.9, 1:4.3 or 1:4.7, but not limited thereto, preferably 1:3 to 1:4.5.

[0030] (2) extracting the extract with ethyl acetate for 1 to 5 times, combining the ethyl acetate layers to obtain an extract; wherein the volume ratio of ethyl acetate to water is 1:0.8 to 1:1.5; illustratively, the volume ratio of ethyl acetate to water used in each extraction is 1:0.9, 1:1.1, 1:1.3 or 1:1.4, but not limited thereto. Preferably, it is 1:0.8 to 1:1.1.

[0031] (3) The extract is separated into solid and liquid, and the obtained solid is dissolved in 50 vol% to 100 vol% methanol to obtain a test solution.

[0032] As an improvement of the above technical solution, the preparation method of the test solution is: take 2.0 g of bamboo shavings, add 50 mL of 70% methanol, heat and reflux for 30 minutes, take out, cool, centrifuge at 4000 rpm for 5 minutes, take the supernatant and evaporate it to dryness, add 15 mL of water to the residue to dissolve it, extract it with ethyl acetate for 3 times, 15 mL each time, combine the ethyl acetate layers, evaporate to dryness, add methanol to the residue to dissolve it and make it up to 5 mL, shake it well, filter it, and take the filtrate to obtain it.

[0033] As an improvement of the above technical solution, in the reference solution, the concentration of the parahydroxybenzoic acid reference substance is 5 μg / mL to 20 μg / mL, exemplified by 6 μg / mL, 8.5 μg / mL, 11 μg / mL, 13.5 μg / mL, 16 μg / mL or 18.5 μg / mL, but not limited thereto; preferably, it is 8 μg / mL to 15 μg / mL, and more preferably, it is 10 μg / mL.

[0034] The concentration of the 4-coumaric acid reference substance is 150 μg / mL to 300 μg / mL, exemplified by 175 μg / mL, 190 μg / mL, 215 μg / mL, 230 μg / mL, 250 μg / mL or 275 μg / mL, but not limited thereto; preferably, it is 180 μg / mL to 220 μg / mL, and more preferably, it is 200 μg / mL.

[0035] As an improvement of the above technical solution, the characteristic spectrum includes 7 common peaks, wherein peak 1 is p-hydroxybenzoic acid, peak 2 is p-hydroxybenzaldehyde, peak 3 is vanillic acid, peak 4 is syringic acid, peak 5 is coniferyl alcohol, and peak 6 is 4-coumaric acid;

[0036] Taking peak 1 as S1 peak, calculate the relative retention time of peak 2 and peak 3. The relative retention time of each peak is within ±10% of the specified value, where the specified values ​​of peak 2 and peak 3 are 1.36 and 1.46 respectively.

[0037] Taking Peak 6 as the S2 peak, the relative retention times of Peak 4, Peak 5, and Peak 7 were calculated. The relative retention time of each peak was within ±10% of the specified value, wherein the specified values ​​of Peak 4, Peak 5, and Peak 7 were 0.83, 0.90, and 1.07, respectively.

[0038] As an improvement of the above technical solution, taking Peak 6 as the S2 peak, the relative peak areas of Peak 1 and Peak 3 are calculated, then the relative peak area of ​​Peak 1 is ≥1.5, and the relative peak area of ​​Peak 3 is ≥0.5.

[0039] Correspondingly, the present invention also discloses a method for identifying bamboo shavings, which is used to identify bamboo shavings and their counterfeits, and comprises:

[0040] Provide the substance to be identified;

[0041] Constructing the characteristic spectrum of the substance to be identified by using the above-mentioned method for constructing the characteristic spectrum of bamboo shavings;

[0042] If the characteristic spectrum shows peaks 1 to 7, and the relative peak area of ​​peak 1 is ≥1.5, and the relative peak area of ​​peak 3 is ≥0.5, then the substance to be identified includes or is bamboo shavings, otherwise it is a counterfeit; the relative peak area is the relative peak area calculated with peak 7 as the S peak;

[0043] Among them, the counterfeits include pointed green bamboo, water bamboo, sweet bamboo, purple bamboo, pole bamboo, gold-inlaid jade bamboo, tortoise shell bamboo, yellow-striped diamond bamboo, golden bamboo, square bamboo, Buddha belly bamboo, tea pole bamboo, spicy leek bamboo, flower bamboo, filial bamboo, Chengdu bamboo, green-skinned bamboo, golden bamboo and moso bamboo.

[0044] The implementation of the present invention has the following beneficial effects:

[0045] The characteristic spectrum of bamboo shavings constructed by the present invention can effectively identify bamboo shavings medicinal materials (green bamboo, large-headed bamboo, light bamboo) that have lost their property characteristics and other counterfeits through their own chemical composition differences, and the identification components are relatively large and the characteristic peak information is rich. In addition, the construction method of the characteristic spectrum of the present invention has good specificity, high accuracy, and is simple and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1It is a superposition of characteristic spectra of 16 batches of bamboo shavings in Example 1;

[0047] Figure 2 is the reference characteristic spectrum of the bamboo shavings reference medicinal material in Example 1;

[0048] Figure 3 It is a comparison diagram of the chromatographic peaks of the characteristic spectrum of bamboo shavings in Example 2 and the retention time of the reference substance;

[0049] Figure 4 It is a comparison diagram of the characteristic spectrum chromatographic peak of bamboo shavings in Example 2 and the ultraviolet absorption curve of the reference substance;

[0050] Figure 5 It is a superposition diagram of the characteristic spectra of 4 batches of moso bamboo and bamboo ruts in Example 3;

[0051] Figure 6 It is a superposition of the characteristic spectra of 18 batches of counterfeit products and bamboo shavings in Example 4;

[0052] Figure 7 This is a comparison of the characteristic spectrum results of bamboo shavings under different chromatographic conditions in Comparative Example 1;

[0053] Figure 8 This is a comparison of the characteristic spectrum results of bamboo shavings when different chromatographic columns are used in Comparative Example 2;

[0054] Fig. 9 This is a comparison of the characteristic spectrum results of bamboo shavings when different pre-treatment methods are used in Example 3;

[0055] Fig.10 This is a comparison diagram of the characteristic spectrum results of bamboo shavings using different characteristic spectrum determination methods in Comparative Example 4;

[0056] Among them: Peak 1 is p-hydroxybenzoic acid; Peak 2 is p-hydroxybenzaldehyde; Peak 3 is vanillic acid; Peak 4 is syringic acid; Peak 5 is coniferyl alcohol; Peak 6 is 4-coumaric acid. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0058] Example 1

[0059] The method for establishing a characteristic spectrum of a bamboo shavings medicinal material in this embodiment is as follows:

[0060] 1. Instruments, reagents and test drugs

[0061] Instruments: Waters high performance liquid chromatograph (Arc, Waters Corporation), Agilent ZORBAX SB-AQ chromatographic column (4.6 mm × 250 mm, 5 μm), one ten-thousandth balance (Mettler-Toledo, ME204E), one millionth balance (Mettler-Toledo, XP26), electric constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., HWS-28), ultrapure water machine (Milli-Q Direct, Merck & Co., Ltd.).

[0062] Reagents: ethanol, methanol, and ethyl acetate (Xilong Scientific Co., Ltd.) were of analytical grade; glacial acetic acid (Tianjin Komiou Chemical Reagent Co., Ltd.) and acetonitrile (Merck Co., Ltd.) were of HPLC grade for liquid phase; and water was ultrapure water (self-made in the laboratory).

[0063] Reagents: 4-coumaric acid (batch number: 112037-202102, content: 99.7%, China Food and Drug Inspection Institute); p-hydroxybenzoic acid (batch number: wkq2102204, content: 99.8%, Sichuan Weikeqi Company); p-hydroxybenzaldehyde (batch number: wkq20120204, content: 98.0%, Sichuan Weikeqi Company); vanillic acid (batch number: 20041703, content: 99.3%, Chengdu Pufeide Biotechnology Co., Ltd.); syringic acid (batch number: ST03780120, content: 98.0%, Shanghai Shidande Biotechnology Co., Ltd.); coniferyl alcohol (batch number: 10979, content: 95.0%, Shanghai Shidande Standard Technology Service Co., Ltd.).

[0064] The detailed information of 16 batches of bamboo shavings is shown in Table 1.

[0065] Table 1 Information on the source of medicinal materials

[0066]

[0067]

[0068] 2. Preparation of test solution

[0069] Take an appropriate amount of bamboo shavings powder (passed through No. 3 sieve), take about 2.0g, place it in a stoppered conical flask, add 50mL of 70% methanol, heat and reflux for 30min, take it out, cool, centrifuge (4000rpm) for 5min, take the supernatant and evaporate it to dryness, add 15mL of water to dissolve the residue, extract with ethyl acetate 3 times, 15mL each time, combine the ethyl acetate layers, evaporate to dryness, add methanol to dissolve the residue, transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain the product.

[0070] 3. Preparation of reference solution

[0071] Take appropriate amount of p-hydroxybenzoic acid reference substance and 4-coumaric acid reference substance, weigh accurately, add methanol to make mixed solutions containing 10 μg and 200 μg per mL respectively, as reference substance solutions.

[0072] 4. Chromatographic conditions

[0073] Chromatographic column: Agilent ZORBAX SB-AQ (4.6 mm × 250 mm, 5 μm); acetonitrile as mobile phase A, 0.05% glacial acetic acid as mobile phase B, gradient elution as specified in Table 2; flow rate 1.0 mL / min; column temperature 35°C; detection wavelength 260 nm. The theoretical plate number calculated based on the 4-coumaric acid peak should not be less than 3000.

[0074] Table 2 Elution program

[0075]

[0076] 5. Determination

[0077] Accurately pipette 10 μL of reference solution and 10 μL of test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0078] 6. Establishment of the characteristic map of bamboo shavings

[0079] The “Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)” was used to process the data of 16 batches of bamboo shavings medicinal materials and generate superimposed spectra. Figure 1 , selected 16 batches of green stalk bamboo, big head bamboo, light bamboo three different original medicinal materials characteristic spectrum common peaks, and the relative retention time and relative peak area RSD relatively stable 7 common peaks as characteristic peaks, generate control characteristic spectrum, see Figure 2 .

[0080] 7. Determination of the characteristic spectrum of bamboo shavings

[0081] The characteristic spectra of 16 batches of bamboo shavings were analyzed. Taking p-hydroxybenzoic acid as the reference peak S1, the relative retention time of peaks 2 to 3 and S1 was calculated. Taking the chromatographic peak of 4-coumaric acid as the reference peak S2, the relative retention time of peaks 4, 5, 7 and S2 was calculated, and the RSD value was calculated. The relative peak area of ​​each characteristic peak and peak 7 was calculated, and the RSD value was calculated. The experimental results are shown in Tables 3 and 4:

[0082] Table 3 Characteristic spectra of 16 batches of bamboo shavings (relative retention time)

[0083]

[0084] Table 4 Characteristic spectrum results of 16 batches of bamboo shavings (relative peak area)

[0085]

[0086]

[0087] With the reference characteristic spectrum of bamboo shavings as a reference, Mark peak matching was used to calculate the similarity between each batch of samples and the reference characteristic spectrum of medicinal materials, and the results are shown in Table 5. The experimental results showed that the similarity between the characteristic spectrum of 16 batches of bamboo shavings of different origins and the reference characteristic spectrum was above 0.95, which met the requirements of the characteristic spectrum, indicating that the reference characteristic spectrum of bamboo shavings established by this method is reliable, highly credible, and highly representative.

[0088] Table 5 Similarity calculation results of characteristic spectra of 16 batches of bamboo shavings

[0089]

[0090] Furthermore, the characteristic spectra of 16 batches of bamboo shavings have 7 common peaks, and the similarity is greater than 0.95, indicating that the 7 common peaks established by this method are all components of green bamboo, large-headed bamboo, and light bamboo. Taking p-hydroxybenzoic acid as reference peak S1 and 4-coumaric acid chromatographic peak as reference peak S2, the relative retention time RSD values ​​of the other 5 characteristic peaks of the characteristic spectra of 16 batches of bamboo shavings are between 0.2% and 1.4%, which meets the standard requirements of the characteristic spectra of bamboo shavings; the RSD of the relative peak area of ​​each characteristic peak of the characteristic spectra of 16 batches of bamboo shavings and peak 7 is between 104.8% and 252.4%. The results show that there are certain differences in the content of the corresponding components of the characteristic peaks of bamboo shavings from different origins.

[0091] Therefore, the characteristic spectrum standard of bamboo shavings is stipulated as follows: 7 characteristic peaks should be present in the chromatogram of the test sample, of which peak 1 and peak 6 should be consistent with the retention time of the reference peaks of p-hydroxybenzoic acid and 4-coumaric acid reference substances, respectively. The peak corresponding to the reference peak of p-hydroxybenzoic acid is the S1 peak, and the relative retention time of peaks 2 to 3 and S1 is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.36 (peak 2), 1.46 (peak 3). The peak corresponding to the reference peak of 4-coumaric acid is the S2 peak, and the relative retention time of peaks 4, 5, and 7 and S2 is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.83 (peak 4), 0.90 (peak 5), and 1.07 (peak 7).

[0092] Example 2 Confirmation of Bamboo Shavings Characteristic Spectrum Chromatographic Peak Component Reference Substance

[0093] In this example, each chromatographic peak in the characteristic spectrum of the bamboo shavings medicinal material in Example 1 is identified. Specifically, the ZR5 test solution in Example 1 is taken for reference substance identification. Among them, the reference substances used include: p-hydroxybenzoic acid reference substance, p-hydroxybenzaldehyde reference substance, vanillic acid reference substance, syringic acid reference substance, coniferyl alcohol reference substance and 4-coumaric acid reference substance. Methanol was added to each reference substance to prepare a reference substance solution containing 10μg, 10μg, 10μg, 5μg, 10μg, and 200μg per 1mL. Samples were injected and analyzed according to the chromatographic conditions in Example 1, and the retention time and 3D ultraviolet absorption curve were compared (see Figure 3 , Figure 4 ).

[0094] The results showed that peaks 1, 2, 3, 4, 5 and 6 in the characteristic spectrum of bamboo shavings were consistent with the retention time and 3D ultraviolet absorption curve of p-hydroxybenzoic acid reference, p-hydroxybenzaldehyde reference, vanillic acid reference, syringic acid reference, coniferyl alcohol reference and 4-coumaric acid reference, respectively.

[0095] Example 3 Identification of Bamboo Shavings and Moso Bamboo

[0096] This embodiment provides a method for identifying bamboo shavings and moso bamboo. Specifically, the characteristic spectrum of moso bamboo is first established by the method of embodiment 1. The moso bamboo information used in the establishment process is shown in Table 6.

[0097] Table 6 Source information of 4 batches of bamboo

[0098]

[0099] The characteristic spectra of 4 batches of bamboo were analyzed. Taking p-hydroxybenzoic acid as the reference peak S1, the relative retention time of peaks 2 to 3 and S1 was calculated. Taking the chromatographic peak of 4-coumaric acid as the reference peak S2, the relative retention time of peaks 4, 5, 7 and S2 was calculated, and the RSD value was calculated; the relative peak area of ​​each characteristic peak and peak 7 was calculated, and the RSD value was calculated. The experimental results are shown in Tables 7 and 8:

[0100] Table 7 Characteristic spectra of four batches of bamboo (relative retention time)

[0101]

[0102] Table 8 Characteristic spectrum results of 4 batches of bamboo (relative peak area)

[0103]

[0104] The results showed that the characteristic spectra of the four batches of bamboo had the same main common peaks as the characteristic spectra of bamboo shavings (see Figure 5), a few chromatographic peaks are missing, and the types of components are highly similar. The reason may be that green bamboo, large-headed bamboo, light bamboo, and moso bamboo all belong to the bamboo trees of the Poaceae family, and the composition of the components is relatively small. However, the size of some chromatographic peaks is quite different. For example, peaks 1, 3, and 4 of moso bamboo are relatively small, while peak 7 is relatively large. Therefore, they can be distinguished by the size of the relative peak area. In order to maximize the above differences, this study used peak 7 in 16 batches of bamboo shavings as a reference, calculated the relative peak area of ​​different chromatographic peaks (see Table 9), and formulated the relative peak area range exclusive to bamboo shavings (green bamboo, large-headed bamboo, and light bamboo).

[0105] Table 9 Comparison of relative peak area fluctuation ranges of 16 batches of bamboo shavings and 4 batches of moso bamboo

[0106]

[0107] Taking peak 7 as the reference peak, the relative peak area fluctuation ranges of the other 6 characteristic peaks of the characteristic spectra of 16 batches of bamboo shavings are all higher than those of the 4 batches of bamboo, but there is an intersection in peak 5. Among the other 4 chromatographic peaks, the fluctuation ranges of peak 1 (p-hydroxybenzoic acid) and peak 3 (vanillic acid) in the two types of samples differ the most, which can be used as a basis for distinguishing between authentic bamboo shavings and bamboo. Therefore, the relative peak area regulations are added to the characteristic spectra standards of bamboo shavings. That is, compared with peak 7, the relative peak areas of peaks 1 and 3 shall not be less than 1.6 and 0.6, and shall be revised to 1.5 and 0.5. Therefore, preferably, the standard of the characteristic spectra of the modified bamboo shavings is as follows:

[0108] There should be 7 characteristic peaks in the chromatogram of the test sample, of which peak 1 and peak 6 should be consistent with the retention time of the reference peaks of p-hydroxybenzoic acid and 4-coumaric acid reference substances, respectively. The peak corresponding to the reference peak of p-hydroxybenzoic acid is the S1 peak. The relative retention time of peaks 2 to 3 and S1 is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.36 (peak 2) and 1.46 (peak 3). The peak corresponding to the reference peak of 4-coumaric acid is the S2 peak. The relative retention time of peaks 4, 5, and 7 and S2 is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.83 (peak 4), 0.90 (peak 5), and 1.07 (peak 7). Calculate the relative peak areas of peaks 1, 3, and 7, and their relative peak areas should be within the specified range: not less than 1.5 (peak 1) and 0.5 (peak 3).

[0109] Example 4 Identification of Bamboo Shavings and Counterfeit Products

[0110] This embodiment provides a method for distinguishing bamboo shavings from common counterfeits. Specifically, the method of Embodiment 1 is first used to establish a characteristic spectrum of counterfeits. The counterfeit information used in the establishment process is detailed in Table 10.

[0111] Table 10 Source information of 18 batches of counterfeit products

[0112]

[0113] The relative retention time determination results of the characteristic spectra of 18 batches of counterfeit products are shown in Table 11, the relative peak area determination results are shown in Table 12, and the spectrum results are shown in Figure 6 .

[0114] Table 11 Characteristic spectrum results of 18 batches of counterfeit products (relative retention time)

[0115]

[0116]

[0117] Table 12 Characteristic spectrum results of 18 batches of counterfeit products (relative peak area)

[0118]

[0119] Table 13 Analysis of characteristic spectra of 18 batches of counterfeit products

[0120]

[0121] The results showed that among the 18 batches of Zhu Ru counterfeit characteristic spectra, 9 batches of data did not present 7 characteristic peaks consistent with the Zhu Ru medicinal material control, and only one batch of peak 1 of the 18 batches of samples was not less than 1.5, and the other batches were far below the prescribed value of 1.5; and the relative peak area of ​​peak 3 was 0.0-0.2, which did not reach the prescribed value of 0.5. As a result, these 18 batches of samples can be judged as non-Zhu Ru authentic medicinal materials. Therefore, the results obtained in Example 1 can be used to distinguish the number of chromatographic peaks and the size of the two relative peak areas as the distinguishing conditions to identify the authentic and counterfeit Zhu Ru medicinal materials.

[0122] In summary, since different original bamboo trees all come from the Gramineae family and have similar chemical composition, it is difficult to identify and distinguish a single variety through specific components. The characteristic spectrum of bamboo shavings established by the method of this study can effectively characterize the content of its known chemical components through the number of characteristic peaks and the size of the relative peak area, and can effectively distinguish it from other counterfeits, and has a certain degree of specificity.

[0123] Comparative Example 1

[0124] This experiment investigated the effects of different chromatographic conditions (mobile phase, elution procedure, etc.) and the elution conditions of the reference "HPLC fingerprint and chemometric analysis of bamboo shavings from different sources" on the characteristic spectra of bamboo shavings.

[0125] The ZR7 test solution in Example 1 was taken and the characteristic spectrum of the bamboo shavings was determined using different chromatographic conditions.

[0126] Conditions (1): High performance liquid chromatograph (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm); acetonitrile as mobile phase A, 0.05% glacial acetic acid as mobile phase B, gradient elution as specified in Table 14; flow rate 1.0 mL / min; column temperature 35° C.; detection wavelength 260 nm. Same as Example 1.

[0127] Table 14 Gradient elution table

[0128]

[0129] Condition (2): High performance liquid chromatograph (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm); elution conditions according to the reference “HPLC fingerprint and chemometric analysis of bamboo shavings from different sources”, with methanol as mobile phase A and 0.1% formic acid as mobile phase B, and gradient elution as specified in Table 15; flow rate of 1.0 mL / min; column temperature of 30°C; detection wavelength of 260 nm.

[0130] Table 15 Gradient elution table

[0131]

[0132] Condition (3): high performance liquid chromatograph (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm); methanol as mobile phase A, 0.05% glacial acetic acid as mobile phase B, gradient elution as specified in Table 16; flow rate of 1.0 mL / min; column temperature of 35°C; detection wavelength of 260 nm.

[0133] Table 16 Gradient elution table

[0134]

[0135] Condition (4): high performance liquid chromatograph (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm); acetonitrile as mobile phase A, 0.05% glacial acetic acid as mobile phase B, gradient elution as specified in Table 17; flow rate of 1.0 mL / min; column temperature of 35°C; detection wavelength of 260 nm.

[0136] Table 17 Gradient elution table

[0137]

[0138] (5) High performance liquid chromatography (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm); acetonitrile as mobile phase A, 0.05% glacial acetic acid as mobile phase B, gradient elution as specified in Table 18; flow rate of 1.0 mL / min; column temperature of 35°C; detection wavelength of 260 nm.

[0139] Table 18 Gradient elution table

[0140]

[0141] The results show that, compared with the chromatographic conditions of the present invention, the chromatograms obtained by using other mobile phases and elution procedures cannot achieve the effect of the present invention in terms of the number of chromatographic peaks or the separation of the main chromatographic peaks (see Figure 7 -Condition (1)). For example, referring to the mobile phase and elution gradient in the literature, it can be seen from the characteristic spectrum results that the overall elution effect is shifted to the back, the analysis time is longer, the baseline impurities in the second half of the chromatogram are more, and the separation is poor (see Figure 7 -Condition (2)). The organic phase was changed to methanol, and a methanol-0.05% glacial acetic acid elution system was used. Since the elution capacity of methanol was lower than that of acetonitrile, the chromatographic peak took a long time to appear, resulting in poor peak shape and separation (see Figure 7 -Condition (3)). Changing the elution procedure greatly affected the number, shape and separation of the chromatographic peaks, and it was not possible to present the seven characteristic peaks well. Figure 7 -Condition (4), Figure 7 -Condition (5). In order to fully present the seven characteristic peaks of bamboo shavings, the chromatographic conditions in Example 1 were selected.

[0142] Comparative Example 2

[0143] This experiment investigated the effects of different types of chromatographic columns on the characteristic spectrum of bamboo shavings under the same mobile phase and elution procedure.

[0144] Take the ZR7 test solution in Example 1, use different chromatographic columns, and determine the characteristic spectrum of the bamboo shavings medicinal material according to the chromatographic conditions of this study in Example 1.

[0145] High performance liquid chromatograph (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm), Waters Xselect HSS T3 chromatographic column (4.6 mm×250 mm, 5 μm), Phenomenex SynergiPolar-RP chromatographic column (4.6 mm×250 mm, 5 μm), YMC-Triart C18 chromatographic column (4.6 mm×250 mm, 5 μm), Thermo Acclaim C18 chromatographic column (4.6 mm×250 mm, 5 μm); Using the same acetonitrile-0.05% glacial acetic acid system and elution procedure, column temperature, flow rate, detection wavelength and other chromatographic conditions as in Example 1, the separation effects of different chromatographic columns were compared, see Figure 8 .

[0146] The results showed that there were significant differences in the elution time of each characteristic peak, the separation degree and peak shape of peak 6 when using different brands of chromatographic columns. Only the Agilent column and the Phenomenex column could achieve baseline separation of peak 6, but the peaks 2 and 3 of the Phenomenex column were not separated. Therefore, it is recommended to choose the Agilent ZORBAX SB-AQ (4.6mm×250mm, 5μm) chromatographic column.

[0147] Comparative Example 3

[0148] This experiment investigated the effects of different pretreatment methods on the characteristic spectrum of bamboo shavings. First, the two extraction methods of 70% methanol extraction and 70% methanol extraction followed by ethyl acetate extraction in Example 1 of this study were investigated. Then, the sample preparation method of the reference "HPLC fingerprint and chemometric analysis of bamboo shavings from different sources" was referred to. The effects of different extraction methods on the characteristic spectrum of bamboo shavings were investigated by comparing the peak shape, separation and baseline of the seven characteristic peaks. The results are shown in Fig. 9 .

[0149] Pretreatment (1): Take an appropriate amount of powder (batch number: ZR7; passed through a No. 3 sieve), take about 2.0 g, place in a stoppered conical flask, add 50 mL of 70% methanol, heat and reflux for 30 min, take out, cool, centrifuge (4000 rpm) for 5 min, take the supernatant and evaporate to dryness, add 15 mL of water to dissolve the residue, extract with ethyl acetate 3 times, 15 mL each time, combine the ethyl acetate layers, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5 mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain.

[0150] Pretreatment (2): Take an appropriate amount of powder (batch number: ZR7; passed through a No. 3 sieve), take about 2.0 g, place in a stoppered conical flask, add 50 mL of 70% methanol, heat and reflux for 30 min, take out, cool, centrifuge (4000 rpm) for 5 min, take the supernatant and evaporate to dryness, add methanol to dissolve the residue, transfer to a 5 mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0151] Pretreatment (3): Take an appropriate amount of powder (batch number: ZR7; sieve No. 2), take about 2.0g, put it in a 150mL stoppered bottle, add 100mL of water, weigh the mass, extract by ultrasonic for 45min, take it out and let it cool to make up for the weight loss, centrifuge at 10000r / min for 20min. Take the supernatant, evaporate to dryness in a water bath, re-dissolve it in 50% methanol to 2mL, shake well, centrifuge at high speed and take the supernatant.

[0152] The chromatographic conditions were the same as in Example 1.

[0153] The results showed that the peak shape, separation effect and baseline level of each characteristic peak were significantly different when different extraction methods were used. The method for preparing the sample in the literature, i.e., the extraction of aqueous solution by reflux, contained more impurities, had poor chromatographic peak separation, uneven baseline and lacked peak 5 (coniferyl alcohol), which was not suitable for the requirements of the present invention for identifying the authenticity of bamboo shavings (see Fig. 9 -Pretreatment (3)). The peak shape and separation effect of the characteristic spectrum obtained by 70% methanol extraction and reflux basically meet the requirements, but there are many background impurities and further purification is required (see Fig. 9 - pretreatment (2)). Therefore, the step of ethyl acetate extraction and impurity removal was added on the basis of 70% methanol extraction and reflux. After impurity removal, the characteristic peaks had good separation, the baseline was stable and the impurities were few (see Fig. 9 - pretreatment (1)), and therefore selected as the sample preparation method of the present invention.

[0154] Comparative Example 4

[0155] This experiment investigates the influence of the characteristic spectrum of the present invention and the characteristic spectrum obtained by the literature method (HPLC fingerprint spectrum and chemometric analysis of bamboo shavings medicinal materials from different sources) on the characteristic spectrum of bamboo shavings.

[0156] ① Take the ZR7 test solution in Example 1 and determine the characteristic spectrum of the bamboo shavings medicinal material according to the chromatographic conditions of Example 1.

[0157] ②Methods provided by references:

[0158] High performance liquid chromatography (Arc, Waters Corporation); Agilent ZORBAX SB-AQ chromatographic column (4.6 mm×250 mm, 5 μm); methanol as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution as specified in Table 15; flow rate of 1.0 mL / min; column temperature of 30°C; detection wavelength of 260 nm.

[0159] Take an appropriate amount of powder (batch number: ZR7; pass through No. 2 sieve), take about 2.0g, put it in a 150mL stoppered bottle, add 100mL of water, weigh the mass, extract by ultrasonic for 45min, take it out and let it cool to make up for the weight loss, centrifuge at 10000r / min for 20min. Take the supernatant, evaporate it in a water bath, dissolve it in 50% methanol to 2mL, shake it well, centrifuge it at high speed and take the supernatant.

[0160] The characteristic spectrum of bamboo shavings was determined under the above chromatographic conditions. The results are shown in Fig.10 .

[0161] The results showed that the characteristic spectrum obtained in the literature was quite different from the results of this study, the analysis time was long, and the number of chromatographic peaks and separation degree did not meet the judgment criteria of this study for distinguishing the authenticity of bamboo shavings. Therefore, the method in this literature has no reference value for distinguishing the authentic bamboo shavings.

[0162] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of bamboo shavings, characterized in that: include: The bamboo shavings are extracted with 50 vol% to 90 vol% methanol under heating reflux for 20 min to 40 min, the obtained extract is centrifuged, the supernatant obtained by centrifugation is evaporated to dryness, and the obtained residue is dissolved in water to obtain an extract; wherein the dosage ratio of the bamboo shavings to 50 vol% to 90 vol% methanol is 1.5 g to 3 g: 30 mL to 80 mL; the volume ratio of water to 50 vol% to 90 vol% methanol is 1:2 to 1:5; Extracting the extract with ethyl acetate for 1 to 5 times, combining the ethyl acetate layers to obtain an extract; wherein the volume ratio of ethyl acetate to water is 1:0.8 to 1:1.5; The extract is evaporated to dryness, and the residue obtained by evaporation is dissolved in 50 vol% to 100 vol% methanol to obtain a test solution; Dissolve the p-hydroxybenzoic acid reference substance and the 4-coumaric acid reference substance in 30 vol% to 100 vol% methanol to obtain a reference substance solution; The test solution and the reference solution are measured by liquid chromatography to obtain a characteristic spectrum of bamboo shavings; The chromatographic column of the liquid chromatograph is an Agilent ZORBAX SB-AQ chromatographic column, with a column length of 250 mm, a column diameter of 4.6 mm, and a particle size of the stationary phase of 5 μm. The liquid chromatograph uses acetonitrile as mobile phase A and 0.02 vol% to 0.1 vol% acetic acid aqueous solution as mobile phase B for gradient elution, and the gradient elution curve is: 0min~15min, mobile phase A is 5%, mobile phase B is 95%; 15min~35min, mobile phase A from 5% to 10%, mobile phase B from 95% to 90%; 35min~50min, mobile phase A from 10% to 12%, mobile phase B from 90% to 88%; 50min~60min, mobile phase A from 12% to 15%, mobile phase B from 88% to 85%; 60min~65min, mobile phase A is 15%, mobile phase B is 85%; The detection wavelength of the liquid chromatograph is 250nm~270nm; The characteristic spectrum includes 7 common peaks, wherein peak 1 is p-hydroxybenzoic acid, peak 2 is p-hydroxybenzaldehyde, peak 3 is vanillic acid, peak 4 is syringic acid, peak 5 is coniferyl alcohol, and peak 6 is 4-coumaric acid; Taking peak 1 as S1 peak, calculate the relative retention time of peak 2 and peak 3. The relative retention time of each peak is within ±10% of the specified value. The specified values ​​of peak 2 and peak 3 are 1.36 and 1.46 respectively. Taking Peak 6 as the S2 peak, the relative retention times of Peak 4, Peak 5, and Peak 7 were calculated. The relative retention time of each peak was within ±10% of the specified value, among which the specified values ​​of Peak 4, Peak 5, and Peak 7 were 0.83, 0.90, and 1.07, respectively.

2. The method for constructing the characteristic spectrum of bamboo shavings according to claim 1, characterized in that: The mobile phase B is a 0.05 vol% acetic acid aqueous solution.

3. The method for constructing the characteristic spectrum of bamboo shavings according to claim 1, characterized in that: The column temperature of the chromatographic column is 30°C to 40°C; The flow rate of the liquid chromatograph is 0.9 mL / min~1.1 mL / min; the injection volume of the test solution is 5 μL~15 μL, and the injection volume of the reference solution is 5 μL~15 μL.

4. The method for constructing the characteristic spectrum of bamboo shavings according to claim 1 or 3, characterized in that: The column temperature of the chromatographic column is 35°C; The flow rate of the liquid chromatograph was 1.0 mL / min, and the detection wavelength was 260 nm; the injection volume of the test solution was 10 μL, and the injection volume of the reference solution was 10 μL.

5. The method for constructing the characteristic spectrum of bamboo shavings according to claim 1, characterized in that: The preparation method of the test solution is as follows: take 2.0 g of bamboo shavings, add 50 mL of 70% methanol, heat and reflux for 30 minutes, take out, cool, centrifuge at 4000 rpm for 5 minutes, evaporate the supernatant, add 15 mL of water to the residue to dissolve it, extract with ethyl acetate for 3 times, 15 mL each time, combine the ethyl acetate layers, evaporate to dryness, add methanol to the residue to dissolve it and make it up to 5 mL, shake well, filter, and take the filtrate to obtain the solution.

6. The method for constructing the characteristic spectrum of bamboo shavings according to claim 1, characterized in that: In the reference substance solution, the concentration of the p-hydroxybenzoic acid reference substance is 5 μg / mL to 20 μg / mL, and the concentration of the 4-coumaric acid reference substance is 150 μg / mL to 300 μg / mL.

7. The method for constructing the characteristic spectrum of bamboo shavings according to claim 1, characterized in that: Taking Peak 6 as the S2 peak, the relative peak areas of Peak 1 and Peak 3 are calculated, then the relative peak area of ​​Peak 1 is ≥1.5, and the relative peak area of ​​Peak 3 is ≥0.

5.

8. A method for identifying bamboo shavings, used for distinguishing bamboo shavings from their counterfeits, characterized in that: include: Provide the substance to be identified; Constructing the characteristic spectrum of the substance to be identified by the method for constructing the characteristic spectrum of bamboo shavings according to any one of claims 1 to 7; If the characteristic spectrum shows peaks 1 to 7, and the relative peak area of ​​peak 1 is ≥1.5, and the relative peak area of ​​peak 3 is ≥0.5, then the substance to be identified is bamboo shavings, otherwise it is a counterfeit; the relative peak area is the relative peak area calculated with peak 7 as the S peak; Among them, the counterfeits include pointed green bamboo, water bamboo, sweet bamboo, purple bamboo, pole bamboo, gold-inlaid jade bamboo, tortoise shell bamboo, yellow-striped diamond bamboo, golden bamboo, square bamboo, Buddha belly bamboo, tea pole bamboo, spicy leek bamboo, flower bamboo, filial bamboo, Chengdu bamboo, green-skinned bamboo, golden bamboo and moso bamboo.

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