A quality control method for wolfberries and its application

Abstract

The present invention discloses a method for quality control of wolfberries, which comprises the following steps: (1) using a statistical method of spectral-effect relationship to conduct correlation analysis on the correlation between the characteristic peaks of the oligosaccharide HPLC fingerprint of wolfberries and the anti-tumor activity; (2) obtaining quality markers that can be used for the quality control of wolfberries according to the analysis results; and (3) formulating quality control standards for the wolfberries based on the quality markers. This method further reveals the key components related to the anti-tumor activity in wolfberries, providing a new reference basis for the overall quality evaluation of wolfberries.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to a method for quality control of wolfberry fruits and its applications. Background Art

[0002] The complex chemical components and unclear active ingredients of traditional Chinese medicines are the key issues restricting the development of traditional Chinese medicine. The evaluation of the quality of a traditional Chinese medicine by chemical fingerprint has certain limitations. Some of the components in the fingerprint are not necessarily active ingredients, and different preparation methods and analysis conditions may produce different fingerprint spectra. Therefore, the fingerprint spectrum method is far from being able to comprehensively control the quality of traditional Chinese medicines. With the proposal of the concept of "spectrum-effect relationship" and the continuous development of related mathematical models, the research on the spectrum-effect relationship method has gradually received extensive attention. The spectrum-effect relationship refers to linking the peaks of the traditional Chinese medicine fingerprint spectrum with specific pharmacodynamic data, and using this relationship to find the active substances in traditional Chinese medicines, formulating quality control standards to reflect their internal quality, and providing a basis for the analysis of the main pharmacodynamic substance basis of traditional Chinese medicines.

[0003] There are many statistical methods for establishing the spectrum-effect relationship, including correlation analysis, principal component analysis, canonical correlation analysis, multiple linear regression, partial least squares regression, grey relational analysis, etc. Among them, grey relational analysis is a basic method based on grey system theory. It judges the degree of association between various factors according to the similarity of the geometric shapes of the change curves of various factors, and is often used to reveal the quantitative comparison of trends in a dynamic change system. Compared with other analysis methods such as regression analysis and canonical correlation analysis, grey relational analysis has the advantages of small sample size, small calculation amount, and good intuitiveness, and can judge the size of the correlation between the pharmacodynamic index and the chromatographic peak, providing the possibility for the prediction of active ingredients.

[0004] The Chinese Pharmacopoeia records that wolfberry fruits have the effects of tonifying the liver and kidney and improving eyesight. Among them, wolfberry polysaccharide has been reported to have various pharmacological activities such as antioxidant, immunomodulatory, and probiotic effects, and has become an index component specified in the Chinese Pharmacopoeia, stipulating that its total sugar content shall not be less than 1.8%. Due to the certain difficulty in rapidly characterizing the structure of uniform polysaccharides with definite pharmacodynamic effects, and it is relatively one-sided to evaluate the quality of wolfberry fruits only by the polysaccharide content, it is urgent to establish a rapid and effective method to supplement and improve the quality evaluation system of wolfberry fruits. In the previous research on wolfberry fruits, it was found that the content of wolfberry oligosaccharides can almost reach more than three times that of polysaccharides. As a component commonly present in traditional Chinese medicines, it is not only an important substance for life activities and biological information transmission, but also has been confirmed to have various biological activities. Compared with polysaccharides, oligosaccharides have a lower molecular weight. With the continuous development of oligosaccharide separation methods and detection means, it has become possible to rapidly analyze oligosaccharides. Summary of the Invention

[0005] Based on this, the present invention provides a quality control method for wolfberry fruits, which comprises the following steps:

[0006] (1) Use the statistical method of spectrum-effect relationship to conduct correlation analysis on the correlation between the characteristic peaks of the HPLC fingerprint of wolfberry oligose and the anti-tumor activity;

[0007] (2) According to the analysis results, obtain the quality markers that can be used for the quality control of wolfberry fruits; and

[0008] (3) Formulate the quality control standard for the wolfberry fruits according to the quality markers.

[0009] Further, the statistical method of spectrum-effect relationship is selected from one or more of the following: correlation analysis, principal component analysis, canonical correlation analysis, multiple linear regression, partial least squares regression, and grey relational analysis.

[0010] Further, the statistical method of spectrum-effect relationship is grey relational analysis.

[0011] Further, the wolfberry fruits are the dried ripe fruits of Lycium cylindricum, Lycium flexicaule, Lycium ruthenicum, Lycium truncatum, and / or Lycium barbarum.

[0012] The "wolfberry fruits" of the present invention include but are not limited to the cultivated products of the above wolfberry fruit varieties in the genus Lycium and the dried ripe fruits of the approximate species of its congeners. The wolfberry fruit varieties are similar to each other and can all be applied to the technical solution of the present invention.

[0013] Further, the place of origin of the wolfberry fruits is Ningxia, Gansu, Inner Mongolia, Xinjiang, and / or Qinghai.

[0014] Further, the wolfberry fruits are the dried ripe fruits of Lycium barbarum.

[0015] Further, the wolfberry oligose is formed by 2 to 10 monosaccharide units linked by glycosidic bonds.

[0016] In the present invention, when the number, mesh number, temperature, number of times, multiple, time, volume, rotation speed, concentration, or other values or parameters are expressed in ranges, preferred ranges, or ranges defined by a series of upper limit preferred values and lower limit preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is separately disclosed. For example, when the range "2 to 10" is disclosed, unless otherwise stated, this range is intended to include its end values and all integers within the range, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10, and the technical effects of the present invention can be achieved within the above numerical range.

[0017] Further, the wolfberry oligose is composed of 2 to 6 monosaccharide units linked by glycosidic bonds.

[0018] Further, the wolfberry oligose is composed of glucose, mannose, and / or galactose linked by glycosidic bonds.

[0019] Further, the wolfberry oligose is selected from one or more of the following: sucrose, melibiose, raffinose, kestose, nystose, fructofuranosylnystose, and 1-kestotriose.

[0020] Further, the quality marker is selected from one or more of the following: nystose, fructofuranosylnystose, and 1-kestotriose.

[0021] Further, the correlation analysis includes the following steps:

[0022] (a) Determining the analysis sequence;

[0023] (b) Nondimensionalizing the data of the analysis sequence;

[0024] (c) Calculating the correlation coefficients corresponding to each analysis sequence; and

[0025] (d) Calculating the grey correlation degree.

[0026] Further, the analysis sequence includes a reference sequence and a comparison sequence, where the inhibition rate of different batches of wolfberry oligose on HepG2 cell viability is used as the reference sequence, denoted as X0(k), and the peak areas of the common peaks in the characteristic chromatograms of different batches of wolfberry oligose are used as the comparison sequence, denoted as X i (k).

[0027] Further, the normalization method is used according to the following formula

[0028]

[0029] to nondimensionalize the data of the analysis sequence.

[0030] Further, the correlation coefficients corresponding to each comparison sequence and the reference sequence are calculated respectively according to the following formula

[0031]

[0032] where k is the batch number of the wolfberry oligose extract, X i is the inhibition rate of different batches of wolfberry oligose on HepG2 cell viability, ξ i is the correlation coefficient between the k-th batch of wolfberry oligose comparison sequence and the reference sequence, is the minimum difference between two levels, is the maximum difference between two levels, and ρ is the resolution coefficient, with a value range of 0 < ρ < 1.

[0033] Furthermore, ρ = 0.5.

[0034] Furthermore, the grey relational grade is the arithmetic mean of the correlation coefficients, and the grey relational grade γ between each chromatographic characteristic peak of wolfberry oligose and the effect of inhibiting the viability of HepG2 cells is calculated according to the following formula i ,

[0035]

[0036] where γ i is the correlation degree between the reference sequence and the comparison sequence, and N is the number of data of the comparison sequence.

[0037] According to another aspect of the present invention, a method for constructing an HPLC fingerprint of wolfberry oligose is provided, and the method includes the following steps:

[0038] Preparation of wolfberry oligose sample solution: (a) Weigh an appropriate amount of wolfberry sample and place it in a container, add water and heat under reflux, cool and filter, combine the filtrate into a 100 mL volumetric flask, add an appropriate amount of water to make up to the scale line, invert and shake well to obtain the test solution; (b) Take a graphitized carbon SPE column, rinse and activate it successively with 5 mL of water, methanol, and water each to obtain the activated graphitized carbon SPE column; and (c) Prepare an aqueous solution of 100 mg / mL based on the mass of the wolfberry medicinal material for the test solution, fully dissolve and vortex mix, then centrifuge, aspirate 1 mL of the supernatant, load it onto the activated graphitized carbon SPE column, first elute with 5 mL of distilled water, then elute with 5 mL of 50% methanol solution, blow dry the 50% methanol eluate with nitrogen, dissolve the dried sample with 0.2 mL of water and centrifuge, take the supernatant to obtain the wolfberry oligose sample solution;

[0039] Preparation of reference substance solution: Weigh sucrose, melibiose, raffinose, kestose, nystose, fructofuranosylnystose, and 1-kestotriose, and add water to prepare the reference substance solution with the concentration of each component being 0.1 - 4.2 mg / mL;

[0040] The chromatographic conditions for high performance liquid chromatography detection are as follows: Use a Prevail Carbohydrate ES (250×4.6 mm, 5 μm) chromatographic column, use methanol or acetonitrile as mobile phase A, acid aqueous solution, alkali aqueous solution, and / or buffer saline solution as mobile phase B, and the gradient elution program is: 0 - 10 min, 85% - 80% A; 10 - 30 min, 80% - 77% A; 30 - 40 min, 77% - 77% A; 40 - 65 min, 77% - 60% A; 65 - 80 min, 60% A; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 40 - 50 °C; the detector is an HPLC-CAD detector;

[0041] According to the results of high performance liquid chromatography (HPLC) detection, the HPLC fingerprint of wolfberry oligosaccharides was obtained.

[0042] In this invention, the HILIC mode is combined with a CAD detector to successfully establish the characteristic chromatogram of wolfberry oligosaccharides for 18 batches, and a total of 22 common peaks are calibrated. Similarity analysis and cluster analysis are respectively carried out. Through the investigation of the HILIC separation column in the early stage, the Prevail Carbohydrate ES column is selected as the separation liquid column for wolfberry oligosaccharides. On the basis that the oligosaccharide components can be basically well separated, the baseline of the Prevail Carbohydrate ES column is stable, less affected by the change of the mobile phase gradient, and has high precision, stability and reproducibility, becoming an ideal choice for the liquid column to establish the characteristic chromatogram of oligosaccharides.

[0043] Furthermore, the flow rate of the high performance liquid chromatography detection is about 1.0 mL / min, and the column temperature is about 45 °C.

[0044] Furthermore, the fingerprint includes peaks numbered 1 - 22. Among them, peak 1 is sucrose as the reference peak, peak 5 is melibiose, peak 10 is raffinose, peak 11 is kestotriose, peak 14 is kestotetraose, peak 20 is kestopentaose, and peak 21 is kestohexaose.

[0045] Furthermore, the acidic aqueous solution, alkaline aqueous solution and / or buffered saline solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid at different concentrations.

[0046] Furthermore, the mobile phase B is an aqueous solution of 25 - 35 mM ammonium formate (+0.2% - +0.4% formic acid), for example, an aqueous solution of about 30 mM ammonium formate (+ about 0.3% formic acid);

[0047] Further, taking chromatographic peak No. 1 as the reference peak, the relative retention times of peaks No. 2 - 22 are 1.105 - 1.115, 1.175 - 1.185, 1.26 - 1.27, 1.3 - 1.31, 1.335 - 1.345, 1.37 - 1.38, 1.49 - 1.5, 1.55 - 1.56, 1.58 - 1.59, 1.65 - 1.66, 1.84 - 1.85, 1.875 - 1.885, 2.02 - 2.03, 2.12 - 2.13, 2.13 - 2.14, 2.155 - 2.165, 2.19 - 2.20, 2.21 - 2.22, 2.235 - 2.245, 2.45 - 2.46, 2.475 - 2.485 respectively.

[0048] Further, the preparation method of the wolfberry oligose sample solution includes any one or more of the following items [1] - [8]:

[0049] [1] The wolfberry sample is the wolfberry medicinal material after being crushed and sieved through a 60 - 100 mesh sieve, such as a wolfberry sample sieved through an 80 - mesh sieve;

[0050] [2] The water is distilled water, deionized water or ultrapure water;

[0051] [3] The temperature is 50°C - 100°C, preferably 60°C - 90°C, such as about 81.3°C;

[0052] [4] The operation of heating extraction is repeated 2 - 4 times, such as 2 times;

[0053] [5] The dosage of the water is 1 - 80 times the amount (L / kg), preferably 10 - 70 times the amount, more preferably 15 - 40 times the amount, such as about 25 times the amount;

[0054] [6] The time of heating extraction is 0.5 - 10 hours, preferably 0.8 - 5 hours, more preferably 1 - 2 hours, such as about 65 min;

[0055] [7] The nitrogen blowing to dryness is carried out under nitrogen blowing in a water bath at 45°C - 55°C, such as about 50°C;

[0056] [8] The centrifugation conditions are centrifugation at a speed of 5000 rpm - 20000 rpm for 5 - 30 minutes, preferably centrifugation at a speed of 8000 rpm - 15000 rpm for 8 - 20 minutes, such as centrifugation at about 12000 rpm for about 10 minutes.

[0057] When used herein, "about" means a value within the range of ±5% of a specific value. For example, "about 81.3" includes ±5% of 81.3, or from 77.235 to 85.365, including 77.235 and 85.235.

[0058] According to another aspect of the present invention, there is provided an application of the above method in quality control and / or quality evaluation of wolfberry medicinal material or a composition comprising wolfberry medicinal material.

[0059] Beneficial effects of the present invention:

[0060] The present invention mainly uses the inhibition rate of wolfberry oligosaccharides on HepG2 cell viability as an in vitro activity index to investigate the difference in anti-liver cancer activity of wolfberry oligosaccharides of different batches. The viability of HepG2 cells acted on wolfberry oligosaccharides of the same concentration from different batches was detected using CCK-8 reagent to obtain the inhibition rate of HepG2 cell viability of wolfberry oligosaccharides of different batches. Based on the established characteristic spectra of wolfberry oligosaccharides of different batches, the grayscale correlation analysis method was used to analyze the correlation between the characteristic peaks of the characteristic spectra of wolfberry oligosaccharides of each batch and the anti-liver cancer activity, explore the main pharmacodynamic material basis of wolfberry oligosaccharides and in vitro inhibition of HepG2 cell viability, further reveal the key components in wolfberry related to anti-tumor activity, and provide a new reference for the overall quality evaluation of wolfberry. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments 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 exceeding the scope of protection required by the present invention.

[0062] Figure 1 Schematic diagram of the yield of wolfberry oligosaccharides in each batch (n=3).

[0063] Figure 2 Schematic diagram of HPLC results of precision experiment.

[0064] Figure 3 Schematic diagram of HPLC results of stability experiment.

[0065] Figure 4 Schematic diagram of HPLC results of reproducibility experiment.

[0066] Figure 5 The HPLC characteristic graph of wolfberry oligosaccharide. (a) is the characteristic graph of 18 batches of wolfberry oligosaccharide by HPLC; (b) is the common pattern of the characteristic graph of wolfberry oligosaccharide.

[0067] Figure 6 This is a schematic diagram of the cluster analysis results of characteristic profiles of 18 batches of wolfberry oligosaccharides.

[0068] Figure 7Schematic diagram of the inhibitory rate results of different batches of LBOS on HepG2 cells. The error bars represent SD, n = 3 independent experiments.

[0069] Figure 8 Non - dimensionalized result graph of the peak areas of each characteristic peak of Lycium barbarum oligosaccharides and the inhibitory rate on HepG2 cells.

[0070] Figure 9 Correlation coefficient graph of each characteristic peak of Lycium barbarum oligosaccharides and the inhibitory rate on HepG2 cells.

[0071] Figure 10 Schematic diagram of the results of the effects of different concentration gradients (400, 200, 100, 50, 25 μg / mL) of pure products of Lycium barbarum oligosaccharides (LBOS - 20 - 1, sucrose; LBOS - 20 - 2, melibiose; LBOS - 20 - 3, kestotriose; LBOS - 20 - 4, raffinose; LBOS - 20 - 5, kestotetraose; LBOS - 20 - 6, kestopentaose; LBOS - 20 - 7, kestohexaose) on the viability of HepG2 cells. *** p < 0.001; ** p < 0.005; * p < 0.01 indicates significant difference from the 0 μg / mL group. (a) 24 h; (b) 48 h. The error bars represent SD, n = 5 independent experiments. Detailed implementation manners

[0072] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0073] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the present invention or the field to which the term is applied. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the implementation process of the present invention, the preferred methods, conditions, substances or materials are described herein.

[0074] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. Next, the present invention will be described in detail in conjunction with the embodiments.

[0075] The following further describes the present invention in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed in the present application.

[0076] Embodiment

[0077] Establishment of the Characteristic Chromatogram of Lycium barbarum Polysaccharides

[0078] The fingerprint of traditional Chinese medicine focuses on reflecting the information characteristics of the overall chemical components in traditional Chinese medicine, and it is a quality standard evaluation method with the characteristics of traditional Chinese medicine that is widely accepted at home and abroad. The content of this section is mainly based on the theory of hydrophilic interaction chromatography. The oligosaccharide components of Lycium barbarum herbs from 18 different producing areas and batches are analyzed by high performance liquid chromatography, the characteristic chromatogram of Lycium barbarum oligosaccharides is established, and 22 common peaks are initially determined. The similarity analysis and hierarchical clustering analysis of the obtained oligosaccharide characteristic chromatogram are carried out by using the similarity calculation software of traditional Chinese medicine fingerprint and SPSS 22 software, and the overall quality of Lycium barbarum herbs is comprehensively evaluated.

[0079] 1.1 Experimental Instruments and Materials

[0080] 1.1.1 Reagents and Materials

[0081] Absolute ethanol (Sinopharm Chemical Reagent Co., Ltd., China)

[0082] Purified water (Shanghai Wahaha Drinking Water Co., Ltd., China)

[0083] SUGAR KS-802 sugar analysis column (Showa, Shodex, Japan)

[0084] Graphitized carbon black SPE small column (CNWBOND Carbon-GCB Cartridge) (Shanghai Anpu Experimental Technology Co., Ltd., China)

[0085] Acetonitrile (Fisher, USA)

[0086] Ammonium formate (Aldrich, USA)

[0087] Formic acid (J.T.Baker, USA)

[0088] Methanol (Sinopharm Chemical Reagent Co., Ltd., China)

[0089] Prevail Carbohydrate ES 5u (GRACE, USA)

[0090] 1.1.2 Instruments and Equipment

[0091] Electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., China)

[0092] Tabletop centrifuge (Shanghai Feiqiaer Analytical Instrument Co., Ltd., China)

[0093] HWS24 type constant temperature water bath (Shanghai Chengxian Instrument Equipment Co., Ltd., China)

[0094] MILLI-Q ultrapure water preparation instrument (Merck Millipore, MERCK MILLIPORE, USA)

[0095] Multi-sample automatic nitrogen blowing concentrator (ATR Technology Company, USA)

[0096] Differential high performance liquid chromatography (Agilent, Aglient, USA)

[0097] Pipette (Eppendorf, Eppendorf, USA)

[0098] HPLC U3000 - Electrospray detector (Thermo Fisher Scientific, Thermo, Germany)

[0099] Standard test sieve (80 mesh) (Huafeng Hardware Instruments Co., Ltd., Shangyu City, Zhejiang Province, China)

[0100] High - speed crusher (Yili Industry and Trade Co., Ltd., Zhejiang Province, China)

[0101] Vortex oscillator (Qilinbeier Instrument Manufacturing Co., Ltd., China)

[0102] 1.1.3 Medicinal materials

[0103] Source of medicinal materials: The wolfberry fruits of each production area and batch were identified by Researcher WU Lihong of the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine. The specimens are stored in the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine. The sample information is shown in Table 1.

[0104] Table 1 Information of wolfberry fruit batches

[0105]

[0106]

[0107] 1.2 Experimental methods

[0108] 1.2.1 Preparation of test sample solution

[0109] Precisely weigh 1.0 g of wolfberry fruit samples of each batch (passed through 80 - mesh sieve), place them in a round - bottom flask, add 25 mL of pure water, heat under reflux in a water bath at 81.3 °C for 65 min, cool and filter. Add 25 mL of pure water to the residue, heat under reflux in a water bath at 81.3 °C for 65 min, cool and filter. Combine the secondary filtrates and transfer them to a 100 - mL volumetric flask, add pure water to the scale line, shake well, and obtain the solution.

[0110] 1.2.2 Determination of the content of wolfberry oligosaccharides

[0111] Accurately pipette 2mL of the sample solution and blow dry it in a 50℃ water bath with nitrogen. Add 1mL of the prepared 70% ethanol solution to the dried sample, vortex thoroughly, centrifuge at 12000rpm for 10min, carefully pipette 800μL of the supernatant, blow dry it in a 50℃ water bath with nitrogen, reconstitute the sample with 500μL of ultrapure water, vortex mix, and perform liquid phase detection. The detection conditions are as follows: Agilent 1100 high performance liquid chromatograph, RID differential detector, SUGAR KS-802 chromatographic column, column oven temperature of 40±0.1℃, ultrapure water as mobile phase, flow rate of 0.8mL / min, injection volume of 10 μL.

[0112] 1.2.3 Preparation of Lycium barbarum oligosaccharide samples

[0113] CNWBOND Carbon-GCB Cartridge SPE cartridge activation: Take a graphitized carbon SPE cartridge and rinse and activate it with 5 mL of water, 5 mL of methanol, and 5 mL of water in sequence.

[0114] Prepare the test solution for each batch of wolfberry according to the method under 1.2.1. Prepare a 100 mg / mL aqueous solution based on the weight of the raw wolfberry medicinal material, fully dissolve, vortex mix, centrifuge at 12000rpm for 10min, carefully draw 1mL of the supernatant, and load it on the activated graphitized carbon SPE column. Elute with 5mL of distilled water to remove most of the monosaccharide components and impurities such as inorganic salts, and then elute the oligosaccharide components with 5mL of 50% methanol solution. The methanol eluent is blown dry in a 50℃ water bath with nitrogen, and the residue is dissolved in 0.2mL of ultrapure water, centrifuged at 12000rpm for 10min, and the supernatant is taken for use.

[0115] 1.2.4 Establishment of HPLC analysis method

[0116] Chromatographic column: Prevail Carbohydrate ES (250×4.6 mm, 5 μm); mobile phase: acetonitrile-30 mM ammonium formate (+0.3% formic acid) aqueous solution; flow rate: 1 mL / min; column temperature: 45° C., gradient elution program as shown in Table 2.

[0117] Table 2 Gradient elution program

[0118]

[0119] 1.2.5 Methodological investigation of oligosaccharide characteristic profiles

[0120] (1) Precision inspection

[0121] Precisely weigh 1.0 g of wolfberry fruit powder from Jiuquan City, Gansu Province (S5) (passed through 80-mesh sieve), prepare the supernatant of wolfberry fruit oligosaccharide sample according to the method under item 1.2.3, repeat the injection 6 times according to the above chromatographic conditions, record the HPLC chromatogram, and calculate the RSD values of the relative retention time (RRT) and relative peak area (RPA) of each chromatographic peak respectively.

[0122] (2) Stability investigation

[0123] Precisely weigh 1.0 g of wolfberry fruit powder from Jiuquan City, Gansu Province (S5) (passed through 80-mesh sieve), prepare the supernatant of wolfberry fruit oligosaccharide sample according to the method under item 1.2.3, and conduct the determination at 0, 2, 4, 8, 16, and 24 h respectively according to the above chromatographic conditions, record the HPLC chromatogram, and calculate the RSD values of the RRT and RPA of each chromatographic peak respectively.

[0124] (3) Reproducibility investigation

[0125] Precisely weigh 1.0 g of wolfberry fruit powder from Jiuquan City, Gansu Province (S5) (passed through 80-mesh sieve), take 6 parallel portions, prepare the supernatant of wolfberry fruit oligosaccharide sample according to the method under item 1.2.3, conduct the determination respectively according to the above chromatographic conditions, record the HPLC chromatogram, and calculate the RSD values of the RRT and RPA of each chromatographic peak respectively.

[0126] 1.2.6 Establishment of the characteristic chromatogram of wolfberry fruit oligosaccharide and similarity analysis

[0127] Take 18 batches of wolfberry fruit medicinal materials, prepare the supernatant of wolfberry fruit oligosaccharide samples of each batch according to the method under item 1.2.3, and conduct the analysis by the high performance liquid chromatography method under item 1.2.4 to obtain the characteristic chromatograms of wolfberry fruit oligosaccharides of each batch. Use the similarity calculation software for traditional Chinese medicine fingerprints (version A in 2004) to process the characteristic chromatograms of 18 batches of wolfberry fruit oligosaccharides according to the following conditions: take sample S1 as the reference chromatogram, select the median method, the time window width is 0.2 min, perform multi-point calibration and automatic matching, generate the reference chromatogram by the software and calculate the similarity.

[0128] 1.2.7 Cluster analysis of the characteristic chromatogram of wolfberry fruit oligosaccharide

[0129] Apply the between-group average linkage method of SPSS 22 software, take the chromatographic peak areas of 18 batches of wolfberry fruit oligosaccharides as the source data, combine with the origin batch information for cluster analysis, and obtain the dendrogram of cluster analysis of wolfberry fruit oligosaccharide samples of different batches.

[0130] 1.3 Experimental results

[0131] 1.3.1 Determination of the content of wolfberry fruit oligosaccharide

[0132] Extract the wolfberry samples of each batch by the method shown in 1.2.1, in triplicate. Determine the peak area of oligosaccharides by HPLC-RID, and calculate the oligosaccharide yield of wolfberries in each batch as follows Figure 1 shown. The average oligosaccharide yield of wolfberries in each batch was 7.40 ± 0.96%, and except for the oligosaccharide yield of S1 (Wuzhong City, Ningxia) exceeding 10%, the oligosaccharide yields of the other batches were all between 6% and 8.5%, with small differences between batches. Considering the wolfberry specification information, the fewer the number of grains per unit weight of wolfberries, that is, the greater the weight of each wolfberry, the higher the oligosaccharide yield of wolfberries.

[0133] 1.3.2 Methodology investigation of the oligosaccharide characteristic fingerprint

[0134] (1) Precision investigation

[0135] Inject the prepared wolfberry oligosaccharide sample into the instrument 6 times repeatedly according to the above chromatographic conditions. The HPLC results are as follows Figure 2 shown. Taking chromatographic peak No. 1 as the reference peak, calculate the RRT and RPA of each chromatographic peak. The results show that the RSD value of RRT of each chromatographic peak ≤ 0.23%, and the RSD value of RPA of each chromatographic peak ≤ 1.99%, indicating good instrument precision.

[0136] (2) Stability investigation

[0137] Measure the prepared wolfberry oligosaccharide sample at 0, 2, 4, 8, 16, and 24 h respectively according to the above chromatographic conditions. The HPLC results are as follows Figure 3 shown. Taking chromatographic peak No. 1 as the reference peak, calculate the RRT and RPA of each chromatographic peak. The results show that the RSD value of RRT of each chromatographic peak ≤ 0.28%, and the RSD value of RPA of each chromatographic peak ≤ 2.39%, indicating that the test solution is stable within 24 h at room temperature.

[0138] (3) Reproducibility investigation

[0139] Measure the 6 parallelly prepared wolfberry oligosaccharide samples respectively according to the above chromatographic conditions. The HPLC results are as follows Figure 4 shown. Taking chromatographic peak No. 1 as the reference peak, calculate the RRT and RPA of each chromatographic peak. The results show that the RSD value of RRT of each chromatographic peak ≤ 0.16%, and the RSD value of RPA of each chromatographic peak ≤ 2.60%, indicating good reproducibility of this method.

[0140] 1.3.3 Establishment of the oligosaccharide characteristic fingerprint of wolfberries and similarity analysis

[0141] Perform high-performance liquid chromatography analysis on 18 batches of wolfberry medicinal materials collected from various places, and process them with the similarity calculation software for traditional Chinese medicine fingerprints (version A in 2004) to obtain the oligosaccharide characteristic fingerprints of wolfberries in each batch ( Figure 5a), The common pattern of the characteristic fingerprint of wolfberry oligosaccharides ( Figure 5 b) and the similarity results. It can be seen from the above results that the similarity results among different batches of wolfberries are all above 0.98, indicating that the differences in the chemical components of wolfberry oligosaccharides among different batches are relatively small, showing good similarity.

[0142] 1.3.4 Cluster analysis of the characteristic fingerprint of wolfberry oligosaccharides

[0143] To verify the similarity analysis results and further clarify the similarity relationship among different batches of wolfberry samples, hierarchical cluster analysis was performed using the peak areas of each chromatographic characteristic peak as the source data, and the cluster analysis results are as Figure 6 shown. The results show that when the threshold is taken as 25, the wolfberry samples can be divided into two major categories. Group 2 includes S12 and S13, and the rest can be classified into Group 1. When the threshold is taken as 15, Group 1 can be further divided into two subgroups. Among them, S10, S11, S14, S15, and S18 can be classified into Group 1-2, and the rest are classified into Group 1-1. From the classification results, it can be seen that the wolfberries from two batches in Tianjingshan are quite different from those from other regions, which may be related to the large geographical location difference between Tianjingshan and other production areas. The cluster analysis results are relatively consistent with the similarity results and can be mutually verified. Moreover, the cluster analysis results can more intuitively show the differences among different batches from different production areas when the similarity differences are not significant.

[0144] 2 Spectrum-effect relationship of wolfberry oligosaccharides against tumors

[0145] 2.1 Experimental instruments and materials

[0146] 2.1.1 Reagents and materials

[0147] DMEM medium (GIBCO, USA)

[0148] 0.25% Trypsion-EDTA (GIBCO, USA)

[0149] Fetal bovine serum (GIBCO, USA)

[0150] DPBS buffer (GIBCO, USA)

[0151] Cell culture plates (CORNING, USA)

[0152] Cell culture dishes (Thermo Fisher Scientific, USA)

[0153] Dimethyl sulfoxide (Amresco, USA)

[0154] Double antibody (Penicillin-Streptomycin, P / S) (GIBCO, USA)

[0155] Cell Counting Kit-8 (CCK-8) (Shanghai Yisheng Biotechnology Co., Ltd., China)

[0156] Other substances of the present invention can also be obtained through commercial purchase.

[0157] 2.1.2 Instruments and Equipment

[0158] Electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., China)

[0159] Desktop centrifuge (Shanghai Feiqiaer Analytical Instrument Co., Ltd., China)

[0160] Freeze dryer (Labconco, USA)

[0161] Constant temperature water bath (Shanghai Zhicheng Analytical Instrument Co., Ltd., China)

[0162] CO2 cell incubator (Eppendorf, USA)

[0163] Laminar flow hood (Suzhou Antai Air Technology Co., Ltd., China)

[0164] Low-temperature high-speed centrifuge (Eppendorf, USA)

[0165] Microplate reader (Molecular Devices, USA)

[0166] Pipette (Eppendorf, USA)

[0167] Vortex oscillator (Qilinbeier Instrument Manufacturing Co., Ltd., China)

[0168] Liquid nitrogen tank (Shanghai Longtuo Instrument Equipment, China)

[0169] MLS-3780SANYO autoclave (SANYO, Japan)

[0170] 2.2 Experimental Methods

[0171] 2.2.1 Preparation of Lycium barbarum polysaccharide samples of different batches

[0172] Precisely weigh 2 g of wolfberry samples of each batch (passed through 80-mesh sieve), in triplicate. Prepare the test solution according to the method under 1.2.1, and use the CNWBOND Carbon-GCB Cartridge SPE column to remove most of the monosaccharide components and inorganic salts and other impurities in the sample according to the method under 1.2.3 to obtain the oligosaccharides of wolfberry of each batch. After drying, add an appropriate amount of DPBS buffer to prepare a stock solution of 500 mg / mL (the concentration is calculated based on the quality of the corresponding crude drug), and store it at -20 °C for later use. Before adding the drug, dilute it to different concentrations with DMEM complete medium and filter and sterilize it with a 0.22 μm microporous filter membrane.

[0173] 2.2.2 Effects of wolfberry oligosaccharides of different batches on the viability of HepG2 cells

[0174] Configure HepG2 cells in the logarithmic growth phase into 5×10 5 cells / mL, add 100 μL of cell suspension to each well of a 96-well plate, and culture it overnight in a cell incubator. Carefully aspirate and discard the medium. Add the LBOS solution with a concentration of 8 mg / mL of different batches to the drug treatment group (the LBOS concentration is calculated based on the quality of the corresponding crude drug). Add the same volume of DMEM complete medium to the blank control group, and continue to culture it in the cell incubator for 48 h. Add 10 μL of CCK-8 solution to each well, incubate it in the incubator for 30 min, take it out and detect the absorbance at 450 nm with a multifunctional microplate reader. Calculate the cell survival rate of HepG2 cells using the formula: cell survival rate % = [(OD of the drug treatment group - OD of the blank group) / (OD of the control group - OD of the blank group)] × 100%.

[0175] 2.2.3 Grey relational analysis of the inhibition of the viability of HepG2 cells by wolfberry oligosaccharides of different batches

[0176] (1) Determine the analysis sequences

[0177] First, determine the reference sequence and the comparison sequences. In this study, the inhibition rate of the viability of HepG2 cells by wolfberry oligosaccharides of different batches is used as the reference sequence, denoted as X0(k), and the peak areas of the common peaks in the characteristic chromatograms of wolfberry oligosaccharides of each batch are used as the comparison sequences, denoted as X i (k).

[0178] (2) Nondimensionalization of data

[0179] Since the dimensions of each sequence are different, it is necessary to perform nondimensionalization processing on each sequence to make the dimensions of each sequence consistent. In this study, the normalization method is used to perform nondimensionalization processing on each sequence. The calculation formula is as follows:

[0180]

[0181] (3) Calculate the correlation coefficient

[0182] Calculate the correlation coefficient corresponding to each comparison sequence and the reference sequence respectively according to the following formula:

[0183]

[0184] where k is the 18 batches of wolfberry oligose extracts, X i is the inhibition rate of the 18 batches of wolfberry oligose on the viability of HepG2 cells, and ξ i is the correlation coefficient between the k-th batch of wolfberry oligose comparison sequence and the reference sequence. is the minimum difference between two levels, is the maximum difference between two levels. ρ is the resolution coefficient, and its value range is 0 < ρ < 1. In this invention, ρ takes the value of 0.5 for calculating the correlation coefficient.

[0185] (4) Calculate the correlation degree

[0186] The correlation degree is the arithmetic mean of the correlation coefficients, and is calculated according to the following formula to obtain the grey correlation degree γ between each chromatographic characteristic peak of wolfberry oligose and the effect of inhibiting the viability of HepG2 cells. i . Where γ i is the correlation degree between the reference sequence and the comparison sequence, and N is the number of data in the comparison sequence.

[0187]

[0188] 2.2.4 Influence of the common peaks of wolfberry oligose on the viability of HepG2 cells

[0189] Precisely weigh about 10 mg of pure wolfberry oligose products (LBOS-20-1, sucrose; LBOS-20-2, melibiose; LBOS-20-3, kestotriose; LBOS-20-4, raffinose; LBOS-20-5, kestotetraose; LBOS-20-6, kestopentaose; LBOS-20-7, kestohexaose), add a certain amount of DPBS buffer solution to prepare a 20 mg / mL stock solution, and store it at -20 °C for later use. Before adding the drug, dilute it to different concentrations with DMEM complete medium and filter and sterilize it with a 0.22 μm microporous filter membrane.

[0190] HepG2 cells growing to the logarithmic phase are configured into 5×10 4Cell suspension at a concentration of [[[number of cells]]] cells / mL. Add 100 μL of cell suspension to each well of a 96-well plate and incubate overnight in a cell culture incubator. Carefully aspirate and discard the culture medium. Add purified samples of LBOS-20 at concentrations of 400, 200, 100, 50, and 25 μg / mL to the treatment groups. Add the same volume of complete DMEM medium to the blank control group and continue to incubate in the cell culture incubator for 24 and 48 h respectively. Add 10 μL of CCK-8 solution to each well, incubate in the incubator for 30 min, measure the absorbance at 450 nm using a multifunctional microplate reader, and calculate the cell viability of HepG2 cells.

[0191] 2.3 Experimental results

[0192] 2.3.1 Effects of wolfberry oligosaccharides from different batches on the viability of HepG2 cells

[0193] Wolfberry oligosaccharides from each batch extracted under the same conditions were purified by CNWBOND Carbon-GCB Cartridge SPE columns and then used to treat HepG2 cells at a concentration of 8 mg / mL (the concentration of LBOS was calculated based on the mass of the corresponding crude drug). After 48 h, the effects of wolfberry oligosaccharides from different batches on the viability of HepG2 cells were detected by CCK-8, and the inhibition rate results were calculated as Figure 7 shown: The average inhibition rate of wolfberry oligosaccharides from each batch on HepG2 cells was 45.1%. Under the same conditions, there were significant differences in the inhibitory activities of wolfberry oligosaccharides from different batches on HepG2 cells, and the maximum difference in the inhibition rate could reach more than 40%. Among them, S1 (Wuzhong City, Ningxia), S11 (Base of Zhongning Company), and S16 (Yumen, Gansu) showed weak inhibitory activities on HepG2 cells, while S5 (Jiuquan City, Gansu) and S13 (Tianjingshan) showed strong inhibitory activities on HepG2 cells. Thus, it can be seen that there are significant differences in the contents of active ingredients that inhibit the viability of HepG2 cells in wolfberries from different batches.

[0194] 2.3.2 Grey relational analysis of the inhibition of HepG2 cell viability by wolfberry oligosaccharides from different batches

[0195] Grey relational analysis is a statistical method for judging the degree of correlation between factors based on the similarity of the change curves of various factors, and it is a simple and effective method for comprehensively evaluating the spectrum-effect relationship. Based on the grey relational analysis method, the present invention analyzed the correlation between the characteristic peaks of wolfberry oligosaccharides and the inhibition of HepG2 cell viability.

[0196] (1) Nondimensionalization of data

[0197] The nondimensionalization treatment of each sequence was carried out according to the above formula, and the results were as Figure 8 shown.

[0198] (2) Calculate the correlation coefficient

[0199] Calculate the correlation coefficient corresponding to each comparison sequence and the reference sequence for the dimensionless processed sequences according to the above formula, and represent the correlation coefficient results as a heat map. The results are as Figure 9 shown. It can be seen from the figure that the correlation coefficient between the 1st peak and the inhibitory activity of HepG2 cells is the lowest, both lower than 0.4. The correlation coefficients between the 3rd peak, the 12th peak and the inhibitory activity of HepG2 cells are relatively low compared with other characteristic peaks, and the correlation coefficients are between 0.7 and 0.8. The correlation coefficients of the remaining characteristic peaks are all higher than 0.8.

[0200] (3) Calculate the correlation degree

[0201] Calculate the grey correlation degree γ between each chromatographic characteristic peak of Lycium barbarum polysaccharide oligosaccharide and the effect of inhibiting the viability of HepG2 cells according to the above formula i Parallel correlation order. The results are shown in Table 3-21. Except for the 1st characteristic peak, the correlation degrees between the remaining characteristic peaks and the effect of inhibiting the viability of HepG2 cells are all greater than 0.7, indicating that these components are related to the inhibitory activity of HepG2 cells. In particular, the correlation degrees of the 8th peak, the 9th peak, the 15th peak, the 18th peak, the 19th peak, and the 20th peak are higher than 0.98, indicating that these components are highly correlated with the inhibitory activity of HepG2 cells. According to the size of the correlation degree, the correlation order is arranged, and the contribution of each characteristic peak to the inhibitory activity of HepG2 cells is 9>8>18>19>20>15>21>6>14>7>2> 4>13>16>22>10>17>5>11>12>3>1 in turn.

[0202] Table 3 Correlation degree and correlation order between each characteristic peak of Lycium barbarum polysaccharide oligosaccharide and the inhibition rate of HepG2 cells

[0203]

[0204]

[0205]

[0206] 2.3.3 Effect of common peaks of Lycium barbarum polysaccharide oligosaccharide on the viability of HepG2 cells

[0207] Each purified part of oligosaccharide of LBOS-20 (LBOS-20-1, sucrose; LBOS-20-2, melibiose; LBOS-20-3, fructooligosaccharide trisaccharide; LBOS-20-4, raffinose; LBOS-20-5, fructooligosaccharide tetrasaccharide; LBOS-20-6, fructooligosaccharide pentasaccharide; LBOS-20-7, fructooligosaccharide hexasaccharide) was used to act on HepG2 cells at the same concentration gradients (400, 200, 100, 50, 25, 0 μg / mL) for 24 h and 48 h respectively. The effect on the viability of HepG2 cells is asFigure 10 As shown: There was no statistically significant difference between LBOS-20-1 (peak 1, sucrose) and the blank group at 24 h and 48 h. Except for LBOS-20-1, the remaining purified fractions all showed good inhibitory effects on the viability of HepG2 cells at 400 μg / mL, and the inhibitory effect was more obvious at 48 h than at 24 h. However, under the condition of 25 μg / mL, the inhibitory effect on the viability of HepG2 cells at 48 h was relatively weaker than that at 24 h. Among them, LBOS-20-5 (peak 14, fructosylnystose), LBOS-20-6 (peak 20, fructosylfructosylnystose), and LBOS-20-7 (peak 21, fructosylfructosylfructosylnystose) had strong inhibitory effects on the viability of HepG2 cells at 24 h and 48 h. LBOS-20-2 (peak 5, melibiose), LBOS-20-3 (peak 10, fructosylnystose), and LBOS-20-4 (peak 11, raffinose) had weak inhibitory effects on the viability of HepG2 cells, which was consistent with the correlation degree results predicted by grey relational analysis, indicating that grey relational analysis could better predict the correlation between the fingerprint and in vitro activity, and determine the effective component group. Among them, LBOS-20-5 (peak 14, fructosylnystose), LBOS-20-6 (peak 20, fructosylfructosylnystose), and LBOS-20-7 (peak 21, fructosylfructosylfructosylnystose) can be used as quality indicators for the quality control of Lycium barbarum L. medicinal materials.

[0208] The above has introduced the embodiments of the present invention in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. At the same time, changes or deformations made by those skilled in the art based on the idea of the present invention, within the specific implementation manner and application scope of the present invention, all belong to the protection scope of the present invention. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A quality control method for wolfberries, characterized in that, The method comprises the following steps: (1) Using the statistical method of spectrum-effect relationship, the grey correlation analysis was performed on the correlation between the characteristic peaks of the HPLC fingerprint of wolfberry oligosaccharides and the anti-tumor activity; (2) obtaining, based on the analysis results, a quality marker that can be used for quality control of wolfberry; and (3) formulating the quality control standard of the wolfberry according to the quality marker; The wolfberry oligosaccharide HPLC fingerprint is constructed by the following steps: Preparation of wolfberry oligosaccharide sample solution: (a) weigh an appropriate amount of wolfberry sample and place it in a container, add water and heat to reflux, filter after cooling, combine the filtrate into a 100mL volumetric flask, add an appropriate amount of water to the scale line, shake upside down to obtain a test solution; (b) take a graphitized carbon SPE column, rinse and activate it with 5mL of water, methanol, and water in turn, to obtain an activated graphitized carbon SPE column; and (c) prepare the test solution into a 100mg / mL aqueous solution based on the mass of wolfberry medicinal material, fully dissolve and vortex mix, then centrifuge, take 1mL of the supernatant, load it on the activated graphitized carbon SPE column, first elute with 5mL of distilled water, then elute with 5mL of 50% methanol solution, blow dry the 50% methanol eluate with nitrogen, dissolve the dried sample in 0.2mL of water, and then centrifuge, take the supernatant, and obtain the wolfberry oligosaccharide sample solution; Preparation of reference solution: weigh sucrose, melibiose, raffinose, sucrose triose, sucrose tetraose, sucrose pentose and sucrose hexaose, add water to prepare the reference solution containing each component at a concentration of 0.1 to 4.2 mg / mL; The chromatographic conditions for high performance liquid phase detection are as follows: using a Prevail Carbohydrate ES chromatographic column, acetonitrile as mobile phase A, 30 mM ammonium formate aqueous solution as mobile phase B, and a gradient elution program of: 0-10 min, 85%-80% A; 10-30 min, 80%-77% A; 30-40 min, 77%-77% A; 40-65 min, 77%-60% A; 65-80 min, 60% A; the flow rate is 1.0 mL / min; the column temperature is 45° C.; the detector is an HPLC-CAD detector; wherein the specification of the chromatographic column is 250×4.6 mm, 5 μm; the concentration of formic acid in the ammonium formate aqueous solution is 0.3%; According to the results of HPLC detection, the HPLC fingerprint of wolfberry oligosaccharide was obtained; The fingerprint spectrum includes peaks 1-22, wherein peak 1 is sucrose as a reference peak, peak 5 is melibiose, peak 10 is raffinose, peak 11 is kestose triose, peak 14 is kestose tetraose, peak 20 is kestose pentasaccharide, and peak 21 is kestose hexaose; Among them, taking chromatographic peak No. 1 as the reference peak, the relative retention times of peaks No. 2 - 22 are 1.105 - 1.115, 1.175 - 1.185, 1.26 - 1.27, 1.3 - 1.31, 1.335 - 1.345, 1.37 - 1.38, 1.49 - 1.5, 1.55 - 1.56, 1.58 - 1.59, 1.65 - 1.66, 1.84 - 1.85, 1.875 - 1.885, 2.02 - 2.03, 2.12 - 2.13, 2.13 - 2.14, 2.155 - 2.165, 2.19 - 2.20, 2.21 - 2.22, 2.235 - 2.245, 2.45 - 2.46, 2.475 - 2.485 respectively; Among them, the wolfberry oligosaccharides are sucrose, melibiose, raffinose, kestose, nystose, fructofuranosylnystose and 1 - kestotriose; Among them, the quality markers are nystose, fructofuranosylnystose and 1 - kestotriose.

2. The method according to claim 1, characterized in that, The wolfberries are the dry and mature fruits of Lycium cylindricum, Lycium flexicaule, Lycium ruthenicum, Lycium truncatum and / or Lycium barbarum.

3. The method according to claim 1, wherein The producing areas of the wolfberries are Ningxia, Gansu, Inner Mongolia, Xinjiang and / or Qinghai.

4. The method according to claim 1, wherein The wolfberries are the dry and mature fruits of Lycium barbarum.

5. The method according to claim 1, wherein The correlation analysis includes the following steps: (a) Determine the analysis sequence; (b) Perform dimensionless processing on the data of the analysis sequence; (c) Calculate the correlation coefficients corresponding to each of the analysis sequences; and (d) Calculate the grey correlation degree.

6. The method according to claim 5, wherein The analysis sequence includes a reference sequence and a comparison sequence, where the inhibition rate of the oligosaccharides from wolfberries of different batches on the viability of HepG2 cells is used as the reference sequence, denoted as X0(k), and the peak areas of the common peaks in the characteristic chromatograms of the oligosaccharides from wolfberries of different batches are used as the comparison sequence, denoted as X i (k).

7. The method according to claim 6, characterized in that, Calculate the correlation coefficients corresponding to each of the comparison sequences and the reference sequence respectively according to the following formula, where k is the batch number of the wolfberry oligosaccharide extract, and X i is the inhibition rate of wolfberry oligosaccharides from different batches on the viability of HepG2 cells, and ξ i is the correlation coefficient between the comparison sequence and the reference sequence of wolfberry oligosaccharides in the k-th batch, is the two-level minimum difference, is the two-level maximum difference, and ρ is the resolution coefficient, with the value range 0 < ρ < 1.

8. The method according to claim 7, characterized in that, ρ = 0.

5.

9. The method according to claim 6, wherein The grey relational grade is the arithmetic mean of the correlation coefficients, and the grey relational grade γ between each chromatographic characteristic peak of wolfberry oligose and the effect of inhibiting the viability of HepG2 cells is calculated according to the following formula i , where γ i is the degree of association between the reference sequence and the comparison sequence, and N is the number of data in the comparison sequence.

10. A method for constructing an HPLC fingerprint of wolfberry oligosaccharides, characterized in that, The method includes the following steps: Preparation of wolfberry oligosaccharide sample solution: (a) Weigh an appropriate amount of wolfberry sample and place it in a container, add water and heat under reflux, cool and then filter, combine the filtrate into a 100 mL volumetric flask, add an appropriate amount of water to make up to the scale line, invert and shake well to obtain the test solution; (b) Take a graphitized carbon SPE column, rinse and activate it successively with 5 mL of water, 5 mL of methanol and 5 mL of water to obtain the activated graphitized carbon SPE column; and (c) Prepare an aqueous solution of 100 mg / mL based on the mass of the wolfberry medicinal material for the test solution, fully dissolve and vortex - mix, then centrifuge, pipette 1 mL of the supernatant, load it onto the activated graphitized carbon SPE column, first elute with 5 mL of distilled water, then elute with 5 mL of 50% methanol solution, blow - dry the 50% methanol eluate with nitrogen, dissolve the dried sample with 0.2 mL of water and centrifuge, take the supernatant to obtain the wolfberry oligosaccharide sample solution; Preparation of reference solution: Weigh sucrose, melibiose, raffinose, kestose, nystose, fructofuranosylnystose and 1 - kestotriose, add water to prepare the reference solution with the concentration of each component being 0.1 - 4.2 mg / mL; The chromatographic conditions for high-performance liquid chromatography detection are as follows: Prevail Carbohydrate ES chromatographic column is used, acetonitrile is used as mobile phase A, and 30 mM ammonium formate aqueous solution is used as mobile phase B. The gradient elution program is: 0 - 10 min, 85% - 80% A; 10 - 30 min, 80% - 77% A; 30 - 40 min, 77% - 77% A; 40 - 65 min, 77% - 60% A; 65 - 80 min, 60% A; the flow rate is 1.0 mL / min; the column temperature is 45 °C; the detector is an HPLC-CAD detector; among them, the specifications of the chromatographic column are: 250×4.6 mm, 5 μm; the concentration of formic acid in the ammonium formate aqueous solution is 0.3%; According to the high-performance liquid chromatography detection results, the HPLC fingerprint of wolfberry oligosaccharides is obtained; Among them, the fingerprint includes peaks numbered 1 - 22. Among them, peak 1 is sucrose as the reference peak, peak 5 is melibiose, peak 10 is raffinose, peak 11 is kestotriose, peak 14 is kestotetraose, peak 20 is kestopentaose, and peak 21 is kestohexaose; Among them, with chromatographic peak 1 as the reference peak, the relative retention times of peaks 2 - 22 are 1.105 - 1.115, 1.175 - 1.185, 1.26 - 1.27, 1.3 - 1.31, 1.335 - 1.345, 1.37 - 1.38, 1.49 - 1.5, 1.55 - 1.56, 1.58 - 1.59, 1.65 - 1.66, 1.84 - 1.85, 1.875 - 1.885, 2.02 - 2.03, 2.12 - 2.13, 2.13 - 2.14, 2.155 - 2.165, 2.19 - 2.20, 2.21 - 2.22, 2.235 - 2.245, 2.45 - 2.46, 2.475 - 2.485 respectively.

11. The method according to claim 10, wherein The preparation method of the wolfberry oligosaccharide sample solution includes any one or more of the following [1] - [8]: [1] The wolfberry sample is a wolfberry sample obtained by pulverizing wolfberry medicinal materials and passing through a 60 - 100 mesh sieve; [2] The water is distilled water, deionized water or ultrapure water; [3] The temperature of the heating under reflux is 50 °C - 100 °C; [4] The operation of adding water, heating under reflux and filtering after cooling is repeated 2 - 4 times; [5] The dosage of water is 1 - 80 times the amount, with the unit of L / kg; [6] The time of heating under reflux is 0.5 - 10 hours; [7] The nitrogen blowing to dryness is carried out by nitrogen blowing under a water bath at 45 °C - 55 °C; [8] The centrifugation conditions are centrifugation at 5000 rpm - 20000 rpm for 5 - 30 minutes.

12. The method according to claim 11, wherein In item [1] of the method, the wolfberry sample is a wolfberry sample obtained by pulverizing wolfberry medicinal materials and passing through an 80 - mesh sieve.

13. The method according to claim 11, characterized in that, In item [3] of the method, the temperature is 60 °C - 90 °C.

14. The method according to claim 13, characterized in that The temperature is 77.235 - 85.365 °C.

15. The method according to claim 11, wherein In item [4] of the method, the operation of adding water, heating under reflux and filtering after cooling is repeated 2 times.

16. The method according to claim 11, wherein In item [5] of the said method, the amount of water used is 10 to 70 times the amount, with the unit of L / kg.

17. The method according to claim 16, wherein The amount of water used is 15 to 40 times the amount, with the unit of L / kg.

18. The method according to claim 17, wherein The amount of water used is 25 times the amount, with the unit of L / kg.

19. The method according to claim 11, wherein In item [6] of the said method, the time of heating under reflux is 0.8 to 5 hours.

20. The method according to claim 19, wherein The time of heating under reflux is 1 to 2 hours.

21. The method according to claim 20, characterized in that, The time of heating under reflux is 65 min.

22. The method according to claim 11, wherein In item [7] of the said method, the nitrogen blowing to dryness is carried out under nitrogen blowing in a water bath at 50 °C.

23. The method according to claim 11, wherein In item [8] of the said method, the conditions for centrifugation are centrifugation at a rotation speed of 8000 rpm to 15000 rpm for 8 to 20 minutes.

24. The method according to claim 23, wherein The conditions for centrifugation are centrifugation at a rotation speed of 12000 rpm for 10 minutes.

25. The method according to claim 10, characterized in that, Hierarchical cluster analysis was performed using the peak areas of each chromatographic characteristic peak as the source data. When the thresholds were taken as 25 and 15, the cluster analysis results were obtained to distinguish wolfberries from different producing areas and batches.

26. Use of the method according to any one of claims 1 to 25 in the quality control and / or quality evaluation of wolfberry medicinal materials or compositions containing wolfberry medicinal materials.

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