Establishment method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of chinaroot greenbrier, fingerprint

Abstract

The invention provides an establishment method of an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of chinaroot greenbrier, which comprises the following steps: S1, preparation of a test solution: weighing a chinaroot greenbrier sample, adding a methanol solution, extracting, cooling, complementing the lost weight with the methanol solution, and filtering to obtain the test solution; s2, preparation of reference substance solutions: weighing six reference substances, namely chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, engeletin and resveratrol, and respectively adding the reference substances into the methanol solution for dissolving to obtain the reference substance solutions; and S3, establishing a fingerprint spectrum, namely respectively measuring the prepared test solution and the reference solution by using HPLC, recording chromatograms, comparing, and identifying common peaks, thereby obtaining the fingerprint spectrum of the chinaroot greenbrier produced from the Hubei Province, i.e., the chinaroot greenbrier produced from the Hubei Province and the chinaroot greenbrier produced from the Hubei Province. According to the method, an extraction method and chromatographic conditions of a chinaroot greenbrier test sample are optimized, a chinaroot greenbrier HPLC fingerprint spectrum research method is established, common peaks of chinaroot greenbrier are identified, and fingerprint spectrum similarity analysis and chemometrics analysis are performed, so that the overall characteristics of chinaroot greenbrier are well reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine fingerprint analysis, and particularly relates to a method for establishing an HPLC fingerprint of Smilax china L. produced in Hubei, its fingerprint and application. Background Art

[0002] Smilax china L. is the dried rhizome of the plant Smilax china L. of the Liliaceae family, and has the effects of promoting diuresis to remove turbidity, expelling wind and removing obstruction in the meridians, and detoxifying and dissipating stasis. The chemical components in Smilax china L. mainly include flavonoids, phenolic acids, stilbenes, and steroidal saponins, etc. Pharmacological studies have revealed that Smilax china L. has pharmacological effects such as anti-inflammatory, anti-gout, lipid-lowering, and anti-tumor. The traditional Chinese medicine fingerprint technology can mark the characteristic and common components of medicinal materials, realize the identification and quality consistency evaluation of traditional Chinese medicinal materials, and provide a reference for the quality control and comprehensive evaluation of traditional Chinese medicinal materials.

[0003] There are rich resources of Smilax china L. in Hubei, and there is no relevant research on fingerprint for Smilax china L. produced in Hubei at present. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing an HPLC fingerprint of Smilax china L. produced in Hubei, which can at least solve some defects existing in the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical scheme:

[0006] A method for establishing an HPLC fingerprint of Smilax china L. produced in Hubei includes the following steps:

[0007] S1. Preparation of test solution: Weigh the Smilax china L. sample produced in Hubei, add methanol solution, weigh, extract, cool, make up the lost weight with methanol solution, shake well, filter, and obtain the test solution;

[0008] S2. Preparation of reference solution: Weigh 6 reference substances including chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, engeletin, and resveratrol, and dissolve them in methanol solution respectively to obtain the reference solution;

[0009] S3. Establishment of fingerprint: The above-prepared test solution and reference solution are respectively determined by HPLC, the chromatograms are recorded and compared, and the common peaks are identified to obtain the fingerprint of Smilax china L. produced in Hubei.

[0010] Further, in the step S1, the concentration of the methanol solution is 50-90%, the extraction method is ultrasonic extraction or water bath reflux extraction, and the extraction time is 30 min - 1.5 h.

[0011] Furthermore, in the reference substance solution obtained in step S2, the concentration of chlorogenic acid is 45 μg / mL, the concentration of cryptochlorogenic acid is 17 μg / mL, the concentration of polydatin is 16 μg / mL, the concentration of astilbin is 20 μg / mL, the concentration of engeletin is 40 μg / mL, and the concentration of resveratrol is 16 μg / mL.

[0012] Furthermore, in step S3, the chromatographic conditions of HPLC are as follows: the chromatographic column is CAPCELL PAK C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is acetonitrile - 0.1% phosphoric acid solution, and gradient elution is performed; the detection wavelength is 303 nm; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 35°C; the injection volume is 10 μL; the number of theoretical plates calculated by the engeletin peak should be not less than 3000.

[0013] Furthermore, the gradient elution program is as follows by volume percentage: 0 - 5 min, 8% acetonitrile, 92% 0.1% phosphoric acid solution; 5 - 15 min, 8% - 12% acetonitrile, 92% - 88% 0.1% phosphoric acid solution; 15 - 20 min, 12% - 15% acetonitrile, 88% - 85% 0.1% phosphoric acid solution; 20 - 30 min, 15% acetonitrile, 85% 0.1% phosphoric acid solution; 30 - 60 min, 15% - 29% acetonitrile, 85% - 71% 0.1% phosphoric acid solution; 60 - 65 min, 29% - 8% acetonitrile, 71% - 92% 0.1% phosphoric acid solution; 65 - 70 min, 8% acetonitrile, 92% 0.1% phosphoric acid solution.

[0014] Furthermore, in step S3, the chromatogram of the test sample solution is imported into the "Similarity Evaluation System Software for Traditional Chinese Medicine Chromatographic Fingerprints" to perform similarity evaluation with the chromatogram of the reference substance solution, determine the similarity, and determine the common peaks.

[0015] Furthermore, 12 common peaks are determined, and 6 of the common peaks are identified, namely chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, engeletin, and resveratrol, as the fingerprint of Smilax china L. produced in Hubei.

[0016] Furthermore, the Smilax china L. produced in Hubei is Smilax china L. medicinal material or Smilax china L. cut crude drug.

[0017] In addition, the present invention also provides a fingerprint obtained by the method for establishing the HPLC fingerprint of Smilax china L. produced in Hubei as described above.

[0018] The present invention also provides the application of the method for establishing the HPLC fingerprint of Smilax china L. produced in Hubei as described above in the determination of the component content of Smilax china L. produced in Hubei and the quality detection of Smilax china L. produced in Hubei.

[0019] Compared with the prior art, the beneficial effects of the present invention:

[0020] (1) By optimizing the extraction method of the test sample of Smilax china L. produced in Hubei and the chromatographic conditions, the invention establishes a research method for HPLC fingerprint of Smilax china L. produced in Hubei, identifies its common peaks, conducts fingerprint similarity analysis and chemometric analysis, so as to better reflect the overall characteristics of Smilax china L. produced in Hubei, scientifically evaluate the stability and uniformity of its quality, and is conducive to overall evaluating the scientificity and rationality of the quality of Smilax china L. produced in Hubei.

[0021] (2) The invention has the characteristics of simple and easy operation, good method precision and repeatability, and provides a technical reference for the establishment of fingerprint and quality evaluation of Smilax china L. produced in Hubei.

[0022] The following will further elaborate on the present invention in conjunction with the attached drawings. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the HPLC superposition chromatogram of 13 batches of Smilax china L. produced in Hubei and the control chromatogram R in Example 1;

[0024] Figure 2 It is the HPLC chromatogram of the chlorogenic acid reference substance solution in Example 1;

[0025] Figure 3 It is the HPLC chromatogram of the cryptochlorogenic acid reference substance solution in Example 1;

[0026] Figure 4 It is the HPLC chromatogram of the polydatin reference substance solution in Example 1;

[0027] Figure 5 It is the HPLC chromatogram of the astilbin reference substance solution in Example 1;

[0028] Figure 6 It is the HPLC chromatogram of the engeletin reference substance solution in Example 1;

[0029] Figure 7 It is the HPLC chromatogram of the resveratrol reference substance solution in Example 1;

[0030] Figure 8 It is the HPLC chromatogram of the mixed reference substance solution in Example 1;

[0031] Figure 9 It is the HPLC chromatogram of Smilax china L. produced in Hubei in Example 1;

[0032] Figure 10 It is the dendrogram of cluster analysis (CA) in Example 2;

[0033] Figure 11 It is the score plot of principal component analysis (PCA) in Example 2;

[0034] Figure 12It is the score plot of orthogonal partial least squares-discriminant analysis (OPLS-DA) in Example 2;

[0035] Figure 13 It is the OPLS-DA VIP value plot in Example 2;

[0036] Figure 14 It is the chromatogram of the Smilax china sample extracted by ultrasonic with methanol for 45 min in Example 3;

[0037] Figure 15 It is the chromatogram of the Smilax china sample extracted by ultrasonic with 50% methanol for 45 min in Example 3;

[0038] Figure 16 It is the chromatogram of the Smilax china sample extracted by ultrasonic with 70% methanol for 45 min in Example 3;

[0039] Figure 17 It is the chromatogram of the Smilax china sample extracted by reflux with methanol for 1 h in Example 3;

[0040] Figure 18 It is the chromatogram of the Smilax china sample extracted by reflux with 50% methanol for 1 h in Example 3;

[0041] Figure 19 It is the chromatogram of the Smilax china sample extracted by reflux with 70% methanol for 1 h in Example 3;

[0042] Figure 20 It is the chromatogram of the Smilax china sample extracted by ultrasonic with ethanol for 45 min in Example 3;

[0043] Figure 21 It is the chromatogram of the Smilax china sample extracted by ultrasonic with 50% ethanol for 45 min in Example 3;

[0044] Figure 22 It is the chromatogram of the Smilax china sample extracted by ultrasonic with 70% ethanol for 45 min in Example 3;

[0045] Figure 23 It is the chromatogram of the Smilax china sample extracted by reflux with ethanol for 1 h in Example 3;

[0046] Figure 24 It is the chromatogram of the Smilax china sample extracted by reflux with 50% ethanol for 1 h in Example 3;

[0047] Figure 25 It is the chromatogram of the Smilax china sample extracted by reflux with 70% ethanol for 1 h in Example 3;

[0048] Figure 26 It is the chromatogram of the Smilax china sample extracted by reflux with water for 1 h in Example 3;

[0049] Figure 27 It is the chromatogram of the Smilax china sample extracted by reflux with water for 2 h in Example 3. Detailed implementation mode

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0051] Experimental instruments and reagents:

[0052] 1. Instruments

[0053] UltiMate3000 high performance liquid chromatograph (Thermo Fisher Scientific (China) Co., Ltd.); XS105DU electronic balance (Mettler Toledo Technology (China) Co., Ltd.); KQ-500DE digital control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); WHL-45B electrothermal constant temperature forced air drying oven (Tianjin Test Instrument Co., Ltd.); PGJ-20-YS ultrapure water instrument (Wuhan Pinguang Instrument Equipment Co., Ltd.); YF1000 traditional Chinese medicine grinder (Ruian Yongli Pharmaceutical Machinery Co., Ltd.).

[0054] 2. Test drugs and reagents

[0055] Chlorogenic acid (batch number: 110753-202119, mass fraction: 96.3%); astilbin reference substance (batch number: 111798-201805, mass fraction: 93.6%); resveratrol reference substance (batch number: 111535-201703, purchased from the National Institutes for Food and Drug Control, mass fraction: 99.4%); engelitin reference substance (batch number: 111906-201103, purchased from the National Institutes for Food and Drug Control, mass fraction: 93.7%); polydatin reference substance (batch number: 111538-201603, purchased from the National Institutes for Food and Drug Control, mass fraction: 87.3%); cryptochlorogenic acid reference substance (CAS number: 905-99-7, purchased from Shanghai Taopu Biotechnology Co., Ltd.; mass fraction: 98%); Smilax china L. reference crude drug (batch number: 121466-201804, purchased from the National Institutes for Food and Drug Control).

[0056] A total of 13 batches of Smilax china L. samples (numbered S1-S13 in Table 1) were all collected in Hubei Province and identified as the rhizomes of Smilax china L. of the Liliaceae family by Associate Professor Tian Liwen of Southern Medical University; the rhizomes of Smilax china L. were washed with sediment, the fibrous roots were removed, sliced while fresh, dried in an oven at 60°C, pulverized, and passed through a No. 3 sieve for standby.

[0057] Acetonitrile and methanol are both chromatographically pure, purchased from Sallou Company of Spain; phosphoric acid is of superior grade purity, purchased from Sinopharm Chemical Reagent Co., Ltd.; water is self-made ultrapure water.

[0058] Table 1: Source Information of Smilax china L. Samples

[0059]

[0060]

[0061] Example 1: Establishment of HPLC Fingerprint of Smilax china L. Produced in Hubei

[0062] 1. Preparation of Solutions

[0063] 1.1 Reference Substance Solution: Appropriate amounts of chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, engeletin and resveratrol reference substances were accurately weighed, dissolved in 50% methanol and prepared into reference substance solutions with concentrations of 45 μg / mL, 17 μg / mL, 16 μg / mL, 20 μg / mL, 40 μg / mL and 16 μg / mL respectively. That is how to obtain it.

[0064] 1.2 Test Solution: About 1 g of the powder of Smilax china L. sample was accurately weighed, placed in a 100 mL stoppered conical flask, accurately added 50 mL of 50% methanol, extracted by ultrasonic wave (400 W, 40 kHz) for 45 min, cooled, weighed again, made up the weight with 50% methanol, shaken well, and filtered through a 0.45 μm microporous filter membrane. That is how to obtain it.

[0065] 2. Chromatographic Conditions

[0066] Chromatographic column: CAPCALPAK C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.1% phosphoric acid solution (B), eluted according to the gradient elution program shown in Table 2, column temperature 35 °C, flow rate 1.0 mL / min, detection wavelength 303 nm, injection volume 10 μL.

[0067] Table 2: Gradient Elution Program

[0068] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0～5 8％ 92％ 5～15 8％→12％ 92％→88％ 15～20 12％→15％ 88％→85％ 20～30 15％ 85％ 30～60 15％→29％ 85％→71％ 60～65 29％→8％ 71％→92％ 65～70 8％ 92％

[0069] 3. Methodology Investigation of Fingerprint

[0070] 3.1 Precision Test: Take the Smilax china L. sample (S3), prepare the test solution according to the method of "1.2 Test Solution", inject samples continuously for 6 times according to the "2. Chromatographic Conditions", take the chromatographic peak of engeletin as the reference peak, calculate the relative retention time RSD of each common peak < 0.17%, and the relative peak area RSD < 2.68%. It shows that the precision of the instrument is good.

[0071] 3.2 Repeatability test: Take the same batch of Smilax china L. sample powder (S3), and prepare 6 portions of test solution in parallel according to the method of "1.2 Test solution". Inject and determine according to the "2. Chromatographic conditions". Using the chromatographic peak of engelitin as the reference peak, the relative retention time RSD of each common peak is calculated to be <0.02%, and the relative peak area RSD is <2.84%, indicating that this method has good repeatability.

[0072] 3.3 Stability test: Take the Smilax china L. sample (S3), and prepare the test solution according to the method of "1.2 Test solution". Inject and determine at 0, 2, 4, 8, 12, and 24 h at room temperature under the "2. Chromatographic conditions". Using the chromatographic peak of engelitin as the reference peak, the relative retention time RSD of each common peak is calculated to be <0.04%, and the relative peak area RSD is <1.27%, indicating that the test solution has good stability within 24 h.

[0073] 3.4 Establishment of fingerprint and identification of chromatographic peaks: Take 13 batches of Smilax china L. (S1 - S13), and prepare the test solution according to the method of "1.2 Test solution". Inject and determine under the "2. Chromatographic conditions". Import the relevant data into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to establish the HPLC fingerprint. Using S1 as the reference fingerprint, adopt the average method, set the time width to 0.1 min, perform multi-point calibration and Mark peak matching to generate the superimposed map and control map (R, common pattern map) of 13 batches of samples. As Figure 1 shown, 12 common peaks are calibrated from 13 batches of samples, and the proportion of the peak area of non-common peaks is less than 10%. Compare the retention time and ultraviolet absorption spectrum of the chromatogram of Smilax china L. sample with those of the chromatograms of each reference substance solution (as Figures 2 - 8 shown), and 6 compounds are identified, which are: chlorogenic acid at peak 1, cryptochlorogenic acid at peak 2, polydatin at peak 7, astilbin at peak 8, engelitin at peak 9, and resveratrol at peak 11. Among them, the peak shape of engelitin at peak 9 is good, the peak area is large, and the retention time is moderate, so it is selected as the reference peak (S). Thus, the HPLC fingerprint of Smilax china L. produced in Hubei is obtained as Figure 9 shown, in which 12 common peaks are marked, and 6 common peaks are identified, which are chlorogenic acid at peak 1, cryptochlorogenic acid at peak 2, polydatin at peak 7, astilbin at peak 8, engelitin at peak 9, and resveratrol at peak 11.

[0074] Example 2: Quality analysis of Smilax china L. produced in Hubei

[0075] 1. Similarity analysis: Use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to compare and analyze the chromatograms of 13 batches of Smilax china L. samples with the control chromatogram (R), and calculate the similarity. The results are shown in Table 3. The results show that the similarity of the fingerprint chromatograms of 13 batches of Smilax china L. is above 0.9, indicating that the similarity of Smilax china L. from different origins is good.

[0076] Table 3 Similarity of Smilax china Samples

[0077] Number Similarity Number Similarity S1 0.979 S8 0.990 S2 0.989 S9 0.990 S3 0.978 S10 0.991 S4 0.901 S11 0.997 S5 0.972 S12 0.983 S6 0.976 S13 0.994 S7 0.973

[0078] 2. Cluster analysis (CA): The peak areas of 12 common peaks of 13 batches of Smilax china were imported into the SPSS 26.0 software system for variable standardization. Through the between-group linkage clustering method, CA clustering analysis was carried out with the squared Euclidean distance as the measurement scale. When the distance between classes is less than 20, as Figure 10 shown, the samples of each batch can be divided into 3 categories: S5-S7 are clustered into the first category, S1-S3 are clustered into the second category, and S4, S8-S13 are clustered into the third category.

[0079] 3. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA):

[0080] The peak areas of the common peaks of 13 batches of Smilax china samples were imported into the SPSS 26.0 software system for variable standardization. Through the PCA method, dimensionality reduction factor analysis was carried out to calculate the eigenvalues of the principal components of Smilax china and the contribution rate of their cumulative variance. The results of the PCA analysis are as Figure 11 shown. The cumulative variance contribution rate is 97.051% (calculated based on the principal component factors with eigenvalues greater than 1). The results show that the selected principal component factors contain 97.051% of the information of 12 components and have good representativeness.

[0081] The PCA and OPLS-DA score plots of Smilax china samples were drawn using SIMCA 14.1 software, and the results are as Figure 12 shown. It can be seen from the figure that the 13 batches of samples are divided into 3 regions, which is consistent with the results of CA and PCA.

[0082] 4. Variable importance in projection (VIP) is a variable screening method based on partial least squares regression, which can be used to evaluate the explanatory ability of the corresponding principal components of each common peak for sample classification and discrimination. Taking the VIP value > 1 as the standard to screen the main marker components, it is found that 4 common peaks meet the requirements, as Figure 13 shown, namely peak 9 (engeletin), peak 1 (chlorogenic acid), peak 5 (unknown), and peak 10 (unknown).

[0083] Example 3: Preparation method of test solution and optimization of chromatographic conditions

[0084] 1. Selection of extraction method and extraction solvent

[0085] Take the powder of the same batch of Smilax china samples (S3), weigh 1 g accurately, place it in a 100 mL stoppered conical flask, accurately add 50 mL of extraction solution, extract, let it cool, weigh again, make up the weight, shake well, filter through a membrane, and obtain; among them, the extraction solvent and extraction method are shown in Table 4.

[0086] Table 4: Extraction Solvent and Extraction Method

[0087]

[0088]

[0089] The extracts obtained by using the above different extraction solvents and extraction methods were detected by HPLC, and the results are as Figures 14 - 27 shown. Taking the peak areas of the main peak chlorogenic acid and engelitin as the investigation objects, the results showed that for the same extraction solvent, in terms of ultrasonic extraction time, the peak area obtained by extracting for 45 min was slightly larger than that for 30 min, and the extraction method of ultrasonic extraction with 50% methanol for 45 min was superior to the ultrasonic extraction methods with other solvents; in the reflux method, with the extension of the reflux time, some components were degraded, and the extraction method of reflux extraction with 70% ethanol for 1 h was superior to the reflux extraction methods with other solvents; in the water reflux extraction method, components such as engelitin and astilbin were significantly degraded with the extension of time; the ultrasonic extraction method was relatively simpler than the water bath reflux extraction method. Therefore, in this invention, the extraction method of ultrasonic extraction with 50% methanol (400 W, 40 kHz) was selected as the extraction method for the test solution.

[0090] 2. Selection of Mobile Phase

[0091] An appropriate amount of powder of the same batch of Smilax china L. samples (S3) was taken and prepared according to the preparation method of the test solution in Example 1. The following mobile phases were compared using a C18 chromatographic column: (1) methanol - 0.2% acetic acid solution, (2) methanol - 0.1% phosphoric acid solution, (3) acetonitrile - 0.2% acetic acid solution, (4) acetonitrile - 0.1% phosphoric acid solution, (5) acetonitrile - 0.2% phosphoric acid solution, and gradient elution was carried out according to the gradient elution program in Example 1.

[0092] The results showed that on the same chromatographic column, the separation effect of the Smilax china L. samples with acetonitrile as the mobile phase was better than that with methanol, and the peak shape of acetonitrile was better. Therefore, acetonitrile was selected as the organic phase; compared with 0.1% phosphoric acid and 0.2% acetic acid, the former was superior to the latter in improving the peak tailing of the sample; both 0.1% phosphoric acid and 0.2% phosphoric acid could better improve the peak tailing phenomenon. Considering the protection of the chromatographic column, finally 0.1% phosphoric acid solution was used as the aqueous phase of the mobile phase.

[0093] 3. Selection of Column Temperature of Chromatographic Column

[0094] Four portions of the same batch of Smilax china L. samples (S3) were taken and prepared according to the preparation method of the test solution in Example 1, and were injected into a high performance liquid chromatograph for analysis, with the column temperatures being room temperature, 30 °C, 35 °C, and 40 °C respectively.

[0095] The results show that at room temperature, the peak emergence time of the chromatographic peaks fluctuates before and after with the change of the external temperature. Under the condition of a column temperature of 30 °C compared with 35 °C, the peak emergence time of the chromatographic peaks is delayed, resulting in an extended analysis time. Under the condition of a column temperature of 40 °C compared with 35 °C, the peaks emerge earlier, but some peaks of the sample cannot be separated. Therefore, the column temperature condition of 35 °C was finally considered.

[0096] 4. Selection of chromatographic column

[0097] An appropriate amount of the same batch of Smilax china samples (S3) was taken and prepared according to the preparation method of the test solution in Example 1, and then injected into a high-performance liquid chromatograph for analysis. Columns of different brands, namely Thermo, Waters, Agilent ZORBAX, Shiseido CAPCELLPAK, Shimadzu WondaSil, and Chinese Nano Chrom, all with C18 (5um, 4.6*250mm), were used for analysis.

[0098] The results show that the chromatographic columns of four brands, Thermo, Waters, Nano Chrom, and Agilent ZORBAX, can separate the chromatographic peaks of the mixed control solution well, but the separation effect on the Smilax china samples is not good (some peaks are wrapped, and the resolution of some adjacent peaks is not good); the separation effect of the Shimadzu WondaSil model chromatographic column on the peaks of the mixed control solution is also not good. Compared with other model chromatographic columns, the Shiseido CAPCELL PAK model column has a better separation effect on both the mixed control solution and the Smilax china samples. Therefore, the Shiseido CAPCELL PAK C18 (5um, 4.6*250mm) column was finally selected as the chromatographic column for the analysis of Smilax china samples.

[0099] In summary, by optimizing the extraction method of Smilax china samples and the HPLC chromatographic conditions, a research method for the HPLC fingerprint of Hubei-produced Smilax china and the HPLC fingerprint of Hubei-produced Smilax china were established, and the common peaks were identified. A total of 6 main common peaks were identified and attributed, and 6 active ingredients could be determined simultaneously; the fingerprint similarity analysis and chemometric analysis were carried out on the chromatograms of 13 batches of Hubei-produced Smilax china samples and the reference fingerprint chromatogram, so as to better reflect the overall characteristics of Hubei-produced Smilax china. The types of chemical components contained in each batch of samples are basically the same, and there is good homogeneity among the samples; this detection method is accurate, reliable, and easy to operate, and can provide a basis for the quality control of Hubei-produced Smilax china.

[0100] The above examples are only illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. Any design identical or similar to the present invention falls within the protection scope of the present invention.

Claims

1. A method for establishing the HPLC fingerprint of Smilax china produced in Hubei, characterized in that: The steps include: S1. Preparation of the test solution: Weigh the sample of Smilax china produced in Hubei, add methanol solution, weigh it, extract it, cool it, make up the lost weight with methanol solution, shake it well, filter it, and obtain the test solution; S2. Preparation of reference solution: weigh 6 reference substances including chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, cyperitoside and resveratrol, and add methanol solution to dissolve them respectively to obtain reference solution; S3. Establishment of fingerprint: The test solution and reference solution prepared above were measured by HPLC respectively, the chromatograms were recorded and compared, the common peaks were identified, and the fingerprint of Smilax china produced in Hubei was obtained.

2. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 1, characterized in that: In the step S1, the concentration of the methanol solution is 50-90%, the extraction method is ultrasonic extraction or water bath reflux extraction, and the extraction time is 30 minutes to 1.5 hours.

3. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 1, characterized in that: In the reference solution obtained in step S2, the concentration of chlorogenic acid is 45 μg / mL, the concentration of cryptochlorogenic acid is 17 μg / mL, the concentration of polydatin is 16 μg / mL, the concentration of astilbin is 20 μg / mL, the concentration of cyperidin is 40 μg / mL, and the concentration of resveratrol is 16 μg / mL.

4. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 1, characterized in that: In step S3, the chromatographic conditions of HPLC are as follows: the chromatographic column is CAPCELL PAK C18, 250 mm×4.6 mm, 5 μm; the mobile phase is acetonitrile-0.1% phosphoric acid solution, and gradient elution is performed; the detection wavelength is 303 nm; the flow rate is 0.8-1.2 mL / min; the column temperature is 35° C.; the injection volume is 10 μL; and the theoretical plate number calculated based on the flavonoids glycoside peak should be no less than 3000.

5. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 4, characterized in that: The gradient elution program is as follows by volume percentage: 0-5 min, 8% acetonitrile, 92% 0.1% phosphoric acid solution; 5-15 min, 8%-12% acetonitrile, 92%-88% 0.1% phosphoric acid solution; 15-20 min, 12%-15% acetonitrile, 88%-85% 0.1% phosphoric acid solution; 20-30 min, 15% acetonitrile, 85% 0.1% phosphoric acid solution; 30-60 min, 15%-29% acetonitrile, 85%-71% 0.1% phosphoric acid solution; 60-65 min, 29%-8% acetonitrile, 71%-92% 0.1% phosphoric acid solution; 65-70 min, 8% acetonitrile, 92% 0.1% phosphoric acid solution.

6. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 1, characterized in that: In step S3, the chromatogram of the test solution is imported into the "Chinese medicine chromatographic fingerprint similarity evaluation system software" to perform similarity evaluation with the chromatogram of the reference solution, determine the similarity, and determine the common peaks.

7. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 6, characterized in that: Twelve common peaks were determined, and six of them were identified as chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, cyperitoside and resveratrol, which were used as the fingerprint of Smilax china produced in Hubei.

8. The method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in claim 1, characterized in that: The Smilax china produced in Hubei is Smilax china medicinal material or Smilax china decoction pieces.

9. A fingerprint obtained by the method for establishing the HPLC fingerprint of Smilax china produced in Hubei according to any one of claims 1 to 8.

10. Application of the method for establishing the HPLC fingerprint of Smilax china produced in Hubei as claimed in any one of claims 1 to 8 in the determination of component contents of Smilax china produced in Hubei and the quality inspection of Smilax china produced in Hubei.

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