A method for constructing a fingerprint of Pseudoconch and its application

Abstract

The present invention belongs to the field of quality evaluation of pseudocong, and specifically relates to a method for constructing a pseudocong fingerprint and its application. The method identifies the pseudocong characteristic spectrum by using vitexin-2-O-rhamnoside, vitexin, pellitorine reference substances and pseudocong reference medicinal materials, constructs a fingerprint of pseudocong medicinal materials, can be used for the identification of true and false pseudocong medicinal materials, can distinguish pseudocong from similar medicinal materials on the market, provides an accurate and reliable identification method for the formulation of quality standards for pseudocong medicinal materials, and provides a basis for further improving the quality standards of pseudocong medicinal materials.

Description

Technical Field

[0001] The invention belongs to the field of quality evaluation of Pseudocong, and particularly relates to a construction method and application of a Pseudocong fingerprint. Background Art

[0002] Pseudocong is a plant of the Piperaceae family. Piper sarmentosum Roxb.) can be harvested throughout the year and sun-dried or low-temperature dried. However, the quality standards for Pseudocong medicinal materials are still imperfect, and it is difficult to quickly and accurately determine the content of active ingredients and chemical composition characteristics. Therefore, it is not possible to quickly and effectively distinguish Pseudocong medicinal materials from other similar medicinal materials, which brings difficulties to the authenticity identification of medicinal materials. Therefore, this study established the HPLC fingerprint method of Pseudocong medicinal materials, constructed the fingerprint and characteristic spectrum of Pseudocong medicinal materials, and established a standardized process for the distinction and quality evaluation of Pseudocong medicinal materials. Summary of the invention

[0003] The invention provides a method for constructing a fingerprint spectrum of Pseudocong, and an application thereof, which can quickly identify Pseudocong medicinal materials and realize quality evaluation thereof.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A method for constructing a fingerprint of Pseudokonjac comprises the following steps:

[0006] (1) Preparation of reference solution and test sample solution

[0007] Take the reference Pseudoconium konjac medicinal material, weigh it accurately, put it in a stoppered conical bottle, add methanol, weigh it, treat it with ultrasound for 15 minutes, cool it, make up the weight loss with methanol, filter it through a 0.22 µm microporous filter membrane, and take the filtrate to obtain the reference solution;

[0008] Take a sample of Pseudoconium konjac and weigh it accurately, put it in a stoppered conical bottle, add methanol, weigh it, treat it with ultrasound for 15 min, cool it, make up the weight loss with methanol, filter it through a 0.22 µm microporous filter membrane, and take the filtrate to obtain the sample solution;

[0009] (2) HPLC conditions

[0010] The HPLC was performed using a Welch Ultimate® AQ-C18 column; the mobile phase was acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), the flow rate was 1 mL / min, the detection wavelength was 320 nm, the column temperature was 30°C, and the injection volume was 10 μL.

[0011] The HPLC gradient elution program was:

[0012] Time / min A / % B / % 0-20 5 to 20 95 to 80 20-25 Keep 20 Maintain 80 25-35 20 to 30 80 to 70 35-45 30 to 40 70 to 60 45-50 40 to 45 60 to 55 50-60 45 to 70 55 down to 30 60-70 70 to 72 30 down to 28 70-75 72 down to 5 28 to 95 75-75 Keep 5 Maintain 95

[0013] (3) Fingerprint map construction

[0014] The reference solution and the sample solution were injected into the high performance liquid chromatograph for elution to obtain the reference spectrum and the sample spectrum respectively. The sample spectrum was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" software, and the reference spectrum was used as the reference spectrum. Through the multi-point correction method, the control spectrum was generated as the average, and the fingerprint spectrum of Pseudoconch was obtained.

[0015] Furthermore, in the step (1), 15 samples of Pseudocongmus medicinal material are used.

[0016] Furthermore, in the step (1), the ultrasonic treatment has an ultrasonic power of 300 W and a frequency of 40 kHz.

[0017] Furthermore, in step (2), the chromatographic column C18 column has a specification of (4.6×250 mm, 5 µm).

[0018] Furthermore, the constructed fingerprint of Pseudoconazole has 8 characteristic peaks, among which peak 2 is vitexin-2-O-rhamnoside, peak 3 is vitexin, and peak 8 is pellitorine.

[0019] The fingerprint of Pseudocong is constructed by the above-mentioned method for constructing the fingerprint of Pseudocong.

[0020] The application of the above-mentioned Pseudocong fingerprint spectrum in identifying Pseudocong medicinal materials.

[0021] Furthermore, the fingerprint of Pseudocong can distinguish Pseudocong from other medicinal materials.

[0022] Furthermore, the other medicinal material is Betel nut leaf.

[0023] A method for identifying a Pseudocongium konjac medicinal material comprises the following steps:

[0024] (1) Preparation of sample solution

[0025] Take the sample to be tested, add 25 mL of 60% methanol, ultrasonically treat for 30 min, cool, shake well, filter, and take the filtrate as the test solution;

[0026] (2) HPLC conditions

[0027] The HPLC was performed using a Welch Ultimate® AQ-C18 column; the mobile phase was acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), the flow rate was 1 mL / min, the detection wavelength was 320 nm, the column temperature was 30°C, and the injection volume was 10 μL.

[0028] The HPLC gradient elution program was:

[0029] Time / min A / % B / % 0-20 5 to 20 95 to 80 20-25 Keep 20 Maintain 80 25-35 20 to 30 80 to 70 35-45 30 to 40 70 to 60 45-50 40 to 45 60 to 55 50-60 45 to 70 55 down to 30 60-70 70 to 72 30 down to 28 70-75 72 down to 5 28 to 95 75-75 Keep 5 Maintain 95

[0030] (3) Map construction and comparison

[0031] The test sample solution is injected into a high performance liquid chromatograph for gradient elution to obtain a test sample spectrum, and the test sample spectrum is compared with the Pseudocong fingerprint spectrum. If there are peaks in the test sample corresponding to the 8 peaks in the Pseudocong fingerprint spectrum, the test sample is the Pseudocong medicinal material; otherwise, the test sample is not the Pseudocong medicinal material.

[0032] Beneficial effects of the present invention:

[0033] The fingerprint construction method of the present invention uses vitexin-2-O-rhamnoside, vitexin, pellitorine reference substances and pseudocone control medicinal materials to identify the pseudocone characteristic spectrum, the sample is extracted with 60% methanol, acetonitrile-0.05% phosphoric acid aqueous solution is used as the mobile phase system, and a chromatographic column and an ultraviolet detector with a C18 bonded stationary phase as the filler are used to separate and identify the characteristic peaks in the sample, and a fingerprint of the pseudocone medicinal material is constructed, which has good repeatability and strong characteristics. It can be used to identify the true and false medicinal materials of pseudocone, and to distinguish pseudocone from similar medicinal materials on the market. Even for the betel leaves (plants of the same family) that are very similar in appearance to the pseudocone medicinal materials, the fingerprint of the present invention can also be accurately distinguished. The pseudocone fingerprint constructed by the construction method of the pseudocone characteristic spectrum of the present invention provides an accurate and reliable identification method for the formulation of the quality standards of pseudocone medicinal materials, and provides a basis for further improving the quality standards of pseudocone medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0035] Figure 1 This is a diode array scanning 3D image (200-400 nm) of the methanol extract of the Pseudoconazole sample described in the present invention.

[0036] Figure 2 HPLC graphs of Pseudocong samples under different UV wavelengths.

[0037] Figure 3 The effect of different extraction solvents on the chromatographic peaks (methanol).

[0038] Figure 4 The effect of different extraction solvents on the chromatographic peaks (water).

[0039] Figure 5 The effect of different extraction solvents on the chromatographic peaks (ethanol).

[0040] Figure 6 The effect of different extraction times on the chromatographic peaks.

[0041] Figure 7 The influence of different material-liquid ratios on the chromatographic peaks.

[0042] Figure 8 These are the results of the precision investigation. The different numbers on the right represent the numbers of the 6 precision injections.

[0043] Fig. 9 To investigate the repeatability of the results, the different numbers on the right represent the numbers of the 6 parallel injections for repeatability.

[0044] Fig.10 These are the results of the stability study. The different numbers on the right represent injections at 0, 2, 4, 8, 12, and 24 hours, respectively.

[0045] Fig.11 HPLC chromatograms of 16 batches of Pseudocongol samples.

[0046] Fig.12 This is the HPLC reference spectrum of Pseudocong.

[0047] Fig.13 This is a HPLC comparison chart of different medicinal parts of Pseudocong.

[0048] Fig.14 HPLC spectra and UV absorption wavelengths of different samples, including: (A) HPLC spectrum of mixed reference; (B) HPLC spectrum of Pseudocong, (C) UV absorption wavelengths of peaks 3, 4, and 8 in the reference; (D) UV absorption wavelengths of peaks 3, 4, and 8 in the Pseudocong test sample. Fig.15 This is the fingerprint of Pseudokonjac.

[0049] Fig.16 The HPLC characteristic spectrum of Pseudocong (A) is compared with the HPLC spectrum of Betel leaves (B) and the HPLC spectrum of Pseudocong (C). Peaks 1 to 8 are characteristic peaks of Pseudocong, among which peak 2 is vitexin-2-O-rhamnoside, peak 3 is vitexin, and peak 8 is pellitorine.

[0050] Fig.17 The corresponding comparison of characteristic peaks of HPLC characteristic spectrum of Pseudocong (A), HPLC spectrum of Betel leaf (B), and HPLC spectrum of Pseudocong (C), among which 1~8 are characteristic peaks of Pseudocong, among which peak 2 is vitexin-2-O-rhamnoside, peak 3 is vitexin, and peak 8 is pellitorine. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Example 1

[0053] 1 sample

[0054] We collected 16 batches of medicinal materials from different sources in Hainan Island, including 1 batch of control medicinal materials (JJ-16) and 15 batches of medicinal materials. All samples were identified as Pseudocongolonia truncatula of the Piperaceae family. Piper sarmentosum Roxb. Among them, 12 batches were above-ground parts and 3 batches were whole plants (JJ-9, JJ-11, and JJ-15). The samples were stored in the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences. Specific information is shown in Table 1. The samples were shade-dried or sun-dried after collection.

[0055] Table 1 List of Pseudocongium konjac sample information

[0056] serial number Collection location Latitude and longitude Collection time JJ-1 Ruixi Town, Chengmai County, Hainan Province <![CDATA[E:110 o 7’ 10’’;N:19 o 43’ 51’’]]> 2023.5.11 JJ-2 Xida Farm, Chengmai County, Hainan Province <![CDATA[E:109 o 53’ 13’’;N:19 o 32’ 20’’]]> 2023.5.11 JJ-3 Xilian Farm, Danzhou City, Hainan Province <![CDATA[E:109 o 32’ 36’’;N:19 o 34’ 36’’]]> 2023.5.11 JJ-4 Hainan Province, Danzhou City, Lanyang <![CDATA[E:109 o 39’ 58’’;N:19 o 27’ 6’’]]> 2023.5.11 JJ-5 Dacheng Town, Danzhou City, Hainan Province <![CDATA[E:109 o 24’ 41’’;N:19 o 28’ 36’’]]> 2023.5.11 JJ-6 Bangxi Town, Baisha County, Hainan Province <![CDATA[E:109 o 8’ 1’’;N:19 o 21’ 34’’]]> 2023.5.11 JJ-7 Da'an Town, Baisha County, Hainan Province <![CDATA[E:109 o 19’ 41’’;N:19 o 17’ 5’’]]> 2023.5.11 JJ-8 Xishui Township, Baisha County, Hainan Province <![CDATA[E:109 o 32’ 57’’ ;N:19 o 12’ 21’’]]> 2023.5.11 JJ-9 Yinggeling, Qiongzhong County, Hainan Province <![CDATA[E:109 o 33’ 56’’;N:19 o 3’ 16’’]]> 2023.5.11 JJ-10 Maoyang Town, Wuzhishan City, Hainan Province <![CDATA[E:109 o 32’ 38’’;N:18 o 55’ 5’’]]> 2023.5.11 JJ-11 Yunlong Town, Haikou City, Hainan Province <![CDATA[E:110 o 27’ 11’’;N:19 o 54’ 7’’]]> 2023.5.11 JJ-12 Xiangou Town, Ding'an County, Hainan Province <![CDATA[E:110 o 22’ 44’’;N:19 o 41’ 5’’]]> 2023.5.11 JJ-13 Jiaji Town, Qionghai City, Hainan Province <![CDATA[E:110 o 26’ 32’’;N:19 o 16’ 13’’]]> 2023.5.11 JJ-14 Longgun Town, Wanning City, Hainan Province <![CDATA[E:110 o 29’ 17’’;N:19 o 2’ 34’’]]> 2023.5.11 JJ-15 Timeng Township, Lingshui County, Hainan Province <![CDATA[E:109 o 58’ 45’’;N:18 o 35’ 7’’]]> 2023.5.11 JJ-16 Hainan Tropical Botanical Garden, Danzhou City, Hainan Province <![CDATA[E:109 o 29' 40'';N: 19o 30' 46'']]> 2023.5.11

[0057] 2 Chromatographic conditions

[0058] Welch Ultimate® AQ-C18 chromatographic column, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm (4.6×250 mm, 5 μm). Mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), flow rate 1 mL / min, detection wavelength 320 nm, column temperature 30°C, injection volume 10 μL, gradient elution program see Table 2.

[0059] Table 2 Gradient elution program

[0060] Time / min A / % B / % 0 5 95 20 20 80 25 20 80 35 30 70 45 40 60 50 45 55 60 70 30 70 72 28 75 5 95 85 5 95

[0061] Take the methanol extract of the reference medicinal material of Pseudoconch (JJ-16) with a volume concentration of 60% and scan it in the wavelength range of 200~400 nm. The results are shown in Figure 1 The results show that there are more strong absorption peaks at 320 nm than at 260 nm ( Figure 2 ), so 320nm is selected as the detection wavelength.

[0062] 3 Investigation of the preparation method of test solution

[0063] (1) Investigation of extraction solvent

[0064] Take 11 portions of Pseudoconium powder, about 0.5 g each, accurately weigh them, put them in a stoppered conical flask, add 25 mL of methanol, 80% methanol, 60% methanol, 40% methanol, 20% methanol, water, anhydrous ethanol, 80% ethanol, 60% ethanol, 40% ethanol, 20% ethanol (volume concentration), weigh the mass, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 min, cool, make up the weight loss with methanol, filter through a 0.22 µm microporous filter membrane, take 10 μL of the filtrate and detect it according to the chromatographic conditions under item "2", record the chromatogram, and the results are shown in the table. Figure 3-5 The results showed that when 80% methanol, 60% methanol, 80% ethanol and 60% ethanol (volume concentration) were used as extraction solvents, the number and peak height of the chromatographic peaks in the chromatogram of the test sample were the best overall. In order to reduce the use of organic solvents, it was more appropriate to choose 60% methanol as the extraction solvent.

[0065] (2) Extraction time investigation

[0066] Take 3 portions of Pseudoconium powder, about 0.5 g each, accurately weigh, place in a stoppered conical flask, add 25 mL of 60% methanol, weigh the mass, ultrasonically treat (power 300 W, frequency 40 kHz) for 15, 30, and 45 min, respectively, cool, make up the weight loss with 60% methanol, filter through a 0.22 µm microporous filter membrane, take 10 μL of the filtrate and test according to the chromatographic conditions under item "2", record the chromatogram, and the results are shown in Figure 6 The results showed that there was no significant difference in the chromatographic peaks of the test samples when ultrasonic extraction was performed for 15 to 45 min, so ultrasonic extraction for 15 min was selected.

[0067] (3) Investigation of material-liquid ratio

[0068] Take 3 portions of Pseudoconium powder, about 0.5 g each, accurately weigh, place in a stoppered conical flask, add 60% methanol at a solid-liquid ratio of 1:20, 1:50, and 1:100, respectively, and the solvent volume is 10, 25, and 50 mL, respectively. Weigh the mass, ultrasonically treat (power 300 W, frequency 40 kHz) for 15 min, cool, make up the weight loss with 60% methanol, filter through a 0.22 µm microporous filter membrane, take 10 μL of the filtrate and detect it according to the chromatographic conditions under item "2", record the chromatogram, and the results are shown in Figure 7 The results showed that when the solid-liquid ratio was 1:20, the concentration of the test solution was too high and the chromatographic peak response value was large. When the solid-liquid ratio was 1:100, the concentration of the test solution was too low and the chromatographic peak response of the components with lower content was low, which was not conducive to observing the characteristics. When the solid-liquid ratio was 1:50, the overall characteristics of the chromatogram were better, so a solid-liquid ratio of 1:50 was selected for extraction.

[0069] In summary, the preparation method of the test solution is determined as follows: take about 0.5 g of Pseudoconium konjac powder, weigh it accurately, put it in a stoppered conical flask, add 25 mL of 60% methanol, weigh the mass, ultrasonically treat (power 300 W, frequency 40 kHz) for 15 min, cool, make up the weight loss with 60% methanol, filter through a 0.22 µm microporous filter membrane, and take the filtrate (i.e. the filtrate after filtering with the microporous filter membrane).

[0070] 4 Methodological considerations

[0071] (1) Precision test

[0072] Take the same test solution (JJ-1), inject and test 6 times continuously, and record the chromatogram. The obtained HPLC chromatogram is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" software for peak matching. The results are shown in Figure 8 ; Taking peak S as the reference peak, the relative retention time and relative peak area of ​​other common peaks were calculated. The relative retention time RSD of each characteristic peak was <2%, and the relative peak area RSD was <5%, indicating that the instrument had good precision.

[0073] (2) Repeatability test

[0074] Take the same source of Pseudocones japonicus (JJ-1) sample, prepare 6 test sample solutions in parallel according to the method under "3", inject the samples separately, and record the chromatograms. The obtained HPLC chromatograms were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" software for peak matching. The results are shown in Fig. 9 ; Taking peak S as the reference peak, the relative retention time and relative peak area of ​​other common peaks were calculated. The relative retention time RSD of each characteristic peak was <2%, and the relative peak area RSD was <5%, indicating that the sample repeatability was good.

[0075] (3) Stability test

[0076] Take the same test sample JJ-1 sample solution, inject and test at 0, 2, 4, 8, 12, and 24 h, and record the chromatogram. The obtained HPLC chromatogram was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" software for peak matching. The results are shown in Fig.10 ; Taking peak S as the reference peak, the relative retention time and relative peak area of ​​other common peaks were calculated. The relative retention time RSD of each characteristic peak was <2%, and the relative peak area RSD was <5%, indicating that the sample had good stability.

[0077] 5 Fingerprint

[0078] Take 15 batches of Pseudocong samples, prepare samples according to the method under "3", and determine the HPLC fingerprint of Pseudocong according to the chromatographic conditions under "2". Import the HPLC spectra of the 16 batches of Pseudocong samples into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" software, use the reference herbal medicine (JJ-16) spectrum as the reference spectrum, and use the multi-point correction method to generate the reference spectrum with the average number for fingerprint matching and similarity evaluation. The matching results and reference spectrum are shown in Fig.11 and Fig.12 , a total of 17 common peaks were successfully matched. The similarity results of each batch of samples are shown in Table 3. Except for JJ-12 and 14, the similarities between other batches and the reference spectrum are all greater than 0.85, indicating that the generated reference spectrum is highly representative.

[0079] Table 3 Similarity between the spectra of the Pseudocongium samples and the reference

[0080] Sample No. Similarity Sample No. Similarity JJ-1 0.974 JJ-9 0.930 JJ-2 0.839 JJ-10 0.887 JJ-3 0.960 JJ-11 0.909 JJ-4 0.961 JJ-12 0.753 JJ-5 0.972 JJ-13 0.869 JJ-6 0.909 JJ-14 0.708 JJ-7 0.912 JJ-15 0.889 JJ-8 0.969 JJ-16 0.918

[0081] 6 Comparative study on HPLC spectra of different medicinal parts of Pseudocong

[0082] Take different medicinal parts (aerial parts, roots, stems, leaves) of the same batch of Pseudoconium konjac samples (JJ-11), prepare samples according to the method under "3", and inject and test according to the chromatographic conditions under "2", and record the chromatogram, see Fig.13 . The results showed that there were fewer chromatographic peaks of chemical components in the roots of Pseudocongeria, mainly common peaks 15 and 16, no obvious peak 7 (wallwortine), and the response value of peak 4 (vitexin-2-O-rhamnoside) was extremely low; the number of chromatographic peaks in the stems of Pseudocongeria was not significantly increased compared with the roots, and the response value of peak 4 (vitexin-2-O-rhamnoside) was slightly increased; the number and response intensity of peaks in the leaves of Pseudocongeria increased significantly, and obvious peak 17 (wallwortine) was seen. Compared with the chromatogram of the aboveground parts of Pseudocongeria, there was no significant difference in quantity, and the peak response value was stronger. The above results showed that under this condition, the chemical components in the leaves of Pseudocongeria were richer in variety and higher in content, followed by the stems, and the least in the roots. Considering the factors of sustainable utilization of resources and biomass, combined with local usage habits and the ease of actual harvesting, it is more appropriate to choose the aboveground parts as the medicinal part.

[0083] 7 Characteristic map research

[0084] 7.1 Peak identification

[0085] The reference substances of 8 compounds, including 8-hydroxyquinoline, sinomenine hydrochloride, apigenin, piperine, norharman, vitexin, vitexin-2-O-rhamnoside and pellitorine, were initially selected for peak identification. Appropriate amounts of the above reference substances were taken, dissolved in 60% methanol, fixed to volume, and shaken to obtain the reference substance solutions.

[0086] Take the Pseudoconium test solution and the above-mentioned reference solution respectively, filter them with a 0.22 µm microporous filter membrane, inject the filtrate into the high performance liquid chromatograph, use the full wavelength (200-800 nm) scanning mode, detect according to the "2" chromatographic conditions, record the chromatogram, and mark the peaks in the HPLC chromatogram of the test sample according to the retention time and ultraviolet absorption wavelength. The results are shown in Fig.14 By comparing with the reference substance, three characteristic peaks were identified, namely, vitexin-2-O-rhamnoside (peak 4), vitexin (peak 5) and pellitoryrine (peak 8) ( Fig.15 ).

[0087] 7.2 Characteristic spectrum of Pseudoconch

[0088] (1) Preparation of reference solution

[0089] Take appropriate amounts of vitexin-2-O-rhamnoside, vitexin and pellitorycin reference substances, weigh them accurately, and add 60% methanol to prepare a mixed solution containing 100 μg of vitexin-2-O-rhamnoside, 50 μg of vitexin and 50 μg of pellitorycin per 1 mL.

[0090] (2) Preparation of reference solution

[0091] Take about 0.5 g of Pseudoconium konjac reference medicinal material, place it in a stoppered conical flask, add 25 mL of 60% methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 15 min, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0092] (3) Preparation of test solution

[0093] Take about 0.5 g of Pseudoconium konjac powder, weigh it accurately, put it in a stoppered conical flask, add 25 mL of 60% methanol, weigh the mass, treat it with ultrasound (power 300 W, frequency 40 kHz) for 15 min, cool it, make up the weight loss with 60% ethanol, filter it through a 0.22 µm microporous filter membrane, and take the filtrate to obtain the product.

[0094] (4) Determination method

[0095] Based on the fingerprint of Pseudocong obtained under item "5" and the peak identification results in "7.1", the characteristic spectrum analysis conditions of Pseudocong are obtained as follows:

[0096] Determined according to the high performance liquid chromatography method (General Chapter 0512) in the 2020 edition of the "Chinese Pharmacopoeia".

[0097] Chromatographic conditions and system suitability test: Octadecyl bonded silica gel (non-hydrophilic) was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as the mobile phase A, 0.05% phosphoric acid aqueous solution was used as B, and gradient elution was performed according to the provisions in the following table; column temperature was 30°C; detection wavelength was 320 nm. The theoretical plate number calculated based on vitexin-2-O-rhamnoside should be no less than 10,000.

[0098] The HPLC gradient elution program was:

[0099] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 5 95 20 20 80 25 20 80 35 30 70 45 40 60 50 45 55 60 70 30 70 72 28 75 5 95

[0100] Accurately pipette 10 μL of reference solution, reference substance solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0101] The test sample should show 8 characteristic peaks in the chromatogram, and should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Fig.15 Among them, peak 2, peak 3, and peak 8 should be consistent with the retention time of the reference peaks of vitexin-2-O-rhamnoside, vitexin, and pellitorycin.

[0102] Fingerprint identification of Pseudocong and Betel leaves

[0103] Fake Konjac Piper sarmentosum Roxb. and Piper betel Piper betle L. are all plants of the genus Piper in the family Piperaceae, widely distributed in southern my country, and the two have very different efficacy and use areas. Pseudocong is often used as a medicine, with warming the middle and dispelling cold, dispelling wind and dampness, and reducing swelling and relieving pain. It is used for stomach and abdominal cold pain, wind-cold cough, edema, dysentery, toothache, rheumatic bone pain, and traumatic injuries. Betel leaves are often used as food by local residents in Hainan, often wrapped in betel nut and chewed. Because the two are derived from the same family, the plant morphology is very similar, and it is more difficult to identify after drying, which increases the difficulty of identifying the authenticity of Pseudocong medicinal materials. The Pseudocong fingerprint map constructed in the present invention has good repeatability and strong characteristic, and can be used to accurately distinguish Pseudocong and Betel leaves.

[0104] Take the leaves of Pseudocong and Betel leaves, randomly number them as sample 1 and sample 2, prepare the test solutions of the two samples according to "(3) Preparation of test solution" in "7.2 Characteristic spectrum of Pseudocong", and obtain HPLC chromatograms according to the characteristic spectrum analysis conditions provided in "7.2 Characteristic spectrum of Pseudocong", which are compared with the fingerprint spectrum of Pseudocong, that is, Fig.15 The results are shown in Fig.16 and Fig.17 .

[0105] HPLC characteristic spectrum of Pseudocong Fig.16 A. Fig.17A), sample 2 can show 8 characteristic peaks (numbered 1 to 8) under this condition ( Fig.16 C), the retention time of each peak corresponds to the HPLC characteristic spectrum of Pseudocong ( Fig.17 C), so sample 2 is Pseudo-cong medicinal material. And sample 1 ( Fig.16 B. Fig.17 B) does not have the above characteristic peaks, so sample 1 is Piper betel. The above identification results are consistent with the samples taken. Therefore, the fingerprint of Pseudocongruentia serrata described in the present invention can accurately identify Pseudocongruentia serrata and Piper betel.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of Pseudokonjac, characterized in that: The following steps are involved: (1) Preparation of reference solution and test sample solution Take the reference Pseudoconium konjac medicinal material, weigh it accurately, put it in a stoppered conical bottle, add methanol, weigh it, treat it with ultrasound for 15 min, cool it, make up the weight loss with methanol, filter it through a 0.22 µm microporous filter membrane, and take the filtrate to obtain the reference solution; Take a sample of Pseudoconium konjac and weigh it accurately, put it in a stoppered conical bottle, add methanol, weigh it, treat it with ultrasound for 15 min, cool it, make up the weight loss with methanol, filter it through a 0.22 µm microporous filter membrane, and take the filtrate to obtain the sample solution; (2) HPLC conditions The HPLC was performed using a Welch Ultimate® AQ-C18 column; the mobile phase was acetonitrile A-0.05% phosphoric acid aqueous solution B, the flow rate was 1 mL / min, the detection wavelength was 320 nm, the column temperature was 30°C, and the injection volume was 10 μL; The HPLC gradient elution program was: (3) Fingerprint map construction The reference solution and the sample solution were injected into a high performance liquid chromatograph for gradient elution to obtain a reference spectrum and a sample spectrum, respectively. The sample spectrum was imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" software, and the reference spectrum was used as a reference spectrum. Through the multi-point correction method, a control spectrum was generated with the average number to obtain the fingerprint of Pseudoconazole. The constructed fingerprint of Pseudoconazole has 8 characteristic peaks, of which peak 2 is vitexin-2-O-rhamnoside, peak 3 is vitexin, and peak 8 is pellitorine.

2. The method for constructing a fingerprint of Pseudokonjac as claimed in claim 1, characterized in that: In the step (1), the ultrasonic treatment has an ultrasonic power of 300 W and a frequency of 40 kHz.

3. The method for constructing a fingerprint of Pseudokonjac as claimed in claim 1, characterized in that: In the step (2), 15 samples of Pseudocongmus medicinal material are used.

4. The method for constructing a fingerprint of Pseudokonjac as claimed in claim 1, characterized in that: In the step (2), the chromatographic column has a specification of 4.6×250 mm and 5 µm.

5. Application of the method for constructing the fingerprint of Pseudocongol as claimed in any one of claims 1 to 4 in identifying Pseudocongol medicinal materials.

6. The use according to claim 5, characterized in that: The method for constructing the fingerprint spectrum of Pseudocong can distinguish Pseudocong from other medicinal materials.

7. The use according to claim 6, characterized in that: The other medicinal material is Betel leaf.

8. A method for identifying a medicinal material of Pseudocongmus, characterized in that: The following steps are involved: (1) Preparation of sample solution Take the sample to be tested, add 25 mL of 60% methanol, ultrasonically treat for 30 min, cool, shake well, filter, and take the filtrate as the test solution; (2) HPLC conditions The HPLC was performed using a Welch Ultimate® AQ-C18 column; the mobile phase was acetonitrile A-0.05% phosphoric acid aqueous solution B, the flow rate was 1 mL / min, the detection wavelength was 320 nm, the column temperature was 30°C, and the injection volume was 10 μL; The HPLC gradient elution program was: (3) Map construction and comparison The test sample solution is injected into a high performance liquid chromatograph for gradient elution to obtain a test sample spectrum, and the test sample spectrum is compared with the Pseudocong fingerprint spectrum. If there are peaks in the test sample corresponding to the 8 characteristic peaks in the Pseudocong fingerprint spectrum, the test sample is the Pseudocong medicinal material; otherwise, the test sample is not the Pseudocong medicinal material; wherein peak 2 is vitexin-2-O-rhamnoside, peak 3 is vitexin, and peak 8 is pellitorine.

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