A quality control method for Dendrobium and its application

Abstract

The present invention discloses a quality control method for Dendrobium officinale, and the method comprises the following steps: (1) performing correlation analysis on the correlation degree between the characteristic peaks of the HPLC fingerprint of Dendrobium officinale oligosaccharides and the anti-inflammatory activity by using the statistical method of spectrum-effect relationship; (2) obtaining quality markers that can be used for the quality control of Dendrobium officinale according to the analysis results; and (3) formulating a quality control standard for Dendrobium officinale according to the quality markers. The method further reveals the key components related to the anti-inflammatory activity in Dendrobium officinale, and provides a new reference basis for the overall quality evaluation of Dendrobium officinale.

Description

Technical Field

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

[0002] The complex chemical components and unknown 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 material 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] As a precious medicinal material and health care product, Dendrobium officinale has always been highly favored. However, the wild resources of Dendrobium officinale have rapidly decreased due to overexploitation. Although the success of artificial cultivation has alleviated the resource tension to a certain extent, due to the high cultivation input and long growth cycle, it still cannot meet the huge market demand. In addition to traditional methods such as morphology and "sticky teeth when chewed" for the identification of Dendrobium officinale, only the total sugar content and the proportion of monosaccharide composition of Dendrobium officinale polysaccharide are specified in the Chinese Pharmacopoeia for its chemical components. Since polysaccharides are high molecular polymers, it is very difficult to rapidly characterize the homogeneous polysaccharides with definite pharmacodynamic effects, which also provides an opportunity for adulteration and counterfeiting of Dendrobium officinale. Compared with polysaccharides, oligosaccharides have lower molecular weights and better water solubility. As a component commonly present in traditional Chinese medicines, they are not only important substances for life activities and biological information transmission, but also have been proven to have a variety of biological activities. Moreover, 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 method for quality control of Dendrobium, which comprises the following steps:

[0006] (1) Using statistical methods of spectrum-effect relationship to conduct correlation analysis on the correlation between characteristic peaks of the HPLC fingerprint of Dendrobium oligosaccharides and anti-inflammatory activity;

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

[0008] (3) Formulating quality control standards for this Dendrobium 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 Dendrobium is selected from one or more of the following: fresh or dried stems of Dendrobium nobile Lindl., Dendrobium huoshanense C. Z. Tang & S. J. Cheng, Dendrobium chrysotoxum Lindl., Dendrobium fimbriatum Hook., Dendrobium wardianum Rchb. f., Dendrobium officinale Kimura & Migo, Dendrobium chrysanthum Wall., and Dendrobium flagelliforme (Lindl.) Bateman ex Lindl.

[0012] The "Dendrobium" in the present invention includes but is not limited to cultivated products of the above Dendrobium varieties in Orchidaceae plants and fresh or dried stems of approximate species of its congeners. Dendrobium varieties are similar to each other and can all be applicable to the technical solutions of the present invention.

[0013] Further, the Dendrobium is fresh or dried stems of Dendrobium officinale Kimura & Migo.

[0014] Further, the Dendrobium oligosaccharides are composed of 2 to 10 monosaccharide units bonded by glycosidic bonds.

[0015] In the present invention, when the number, mesh number, temperature, number of times, multiple, time, volume, rotational speed, concentration, or other values or parameters are expressed in ranges, preferred ranges, or ranges defined by a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, 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 this 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.

[0016] Further, the Dendrobium oligosaccharides are composed of 2 to 7 monosaccharide units bonded by glycosidic bonds.

[0017] Furthermore, the Dendrobium oligosaccharide is formed by the combination of glucose, fructose, and / or mannose through glycosidic bonds.

[0018] Furthermore, the Dendrobium oligosaccharide is selected from one or more of the following: sucrose, maltotriose, maltotetraose, maltopentaose, maltohexaose, mannohexaose, and maltoheptaose.

[0019] Furthermore, the quality marker is selected from one or more of the following: maltotetraose, maltopentaose, maltohexaose, and mannohexaose.

[0020] Furthermore, the correlation analysis includes the following steps:

[0021] (a) Determine the analysis sequence;

[0022] (b) Perform dimensionless processing on the data of the analysis sequence;

[0023] (c) Calculate the correlation coefficient corresponding to each analysis sequence; and

[0024] (d) Calculate the grey correlation degree.

[0025] Furthermore, the analysis sequence includes a reference sequence and a comparison sequence, where the inhibition rate of different batches of Dendrobium oligosaccharide on the NO secretion of LPS-induced RAW 264.7 macrophages 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 Dendrobium oligosaccharide are used as the comparison sequence, denoted as X i (k).

[0026] Furthermore, the normalization method is used according to the following formula

[0027]

[0028] to perform dimensionless processing on the data of the analysis sequence.

[0029] Furthermore, the correlation coefficient corresponding to each comparison sequence and the reference sequence is calculated respectively according to the following formula

[0030]

[0031] where k is the batch number of the Dendrobium oligosaccharide extract, X i is the inhibition rate of different batches of Dendrobium oligosaccharide on the NO secretion of LPS-induced RAW264.7 macrophages, ξ i is the correlation coefficient between the k-th batch of Dendrobium oligosaccharide 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.

[0032] Furthermore, ρ = 0.5.

[0033] 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 dendrobium oligosaccharide and the anti-inflammatory effect of inhibiting the NO secretion of RAW 264.7 macrophages induced by LPS is calculated according to the following formula i ,

[0034]

[0035] 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.

[0036] According to another aspect of the present invention, a method for constructing an HPLC fingerprint of dendrobium oligosaccharide is provided, and the method includes the following steps:

[0037] Preparation of dendrobium oligosaccharide sample solution: (a) Weigh an appropriate amount of dendrobium 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, absorb 2 mL of the dendrobium water extract, blow dry with nitrogen, add 1 mL of ethanol solution to the dried sample for ethanol precipitation, vortex thoroughly and centrifuge, carefully absorb 800 μL of the supernatant, blow dry with nitrogen, add 500 μL of water to the dried sample for reconstitution, vortex to mix well to obtain the test solution; (b) Take a graphitized carbon SPE column, rinse and activate it with 5 mL of water, methanol, and water in sequence to obtain the graphitized carbon SPE column after activation treatment; and (c) Prepare an aqueous solution of 100 mg / mL based on the mass of the dendrobium medicinal material for the test solution, dissolve it fully and vortex to mix well, centrifuge, absorb 1 mL of the supernatant, load it onto the graphitized carbon SPE column after activation treatment, 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 dendrobium oligosaccharide sample solution;

[0038] Preparation of reference substance solution: Weigh sucrose, maltotriose, maltotetraose, maltopentaose, maltohexaose, mannohexaose, and maltoheptaose, add water to make the reference substance solution with the concentration of each component being 0.1 - 4.2 mg / mL;

[0039] The chromatographic conditions for high performance liquid chromatography (HPLC) are as follows: A Prevail Carbohydrate ES column (250×4.6 mm, 5 μm) is used. Methanol or acetonitrile is used as mobile phase A, and an aqueous acid solution, an aqueous base solution, and / or a buffered saline solution is used as mobile phase B. The gradient elution program is as follows: from 0 to 60 min, 80% - 50% A; from 60 to 80 min, 50% 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.

[0040] Based on the results of the high performance liquid chromatography (HPLC) detection, the HPLC fingerprint of dendrobium oligosaccharides is obtained.

[0041] In this invention, the HILIC mode is combined with a CAD detector, and the characteristic chromatogram of 48 batches of dendrobium oligosaccharides is successfully established. Ten common peaks are determined, and 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 dendrobium oligosaccharides. On the basis that the oligosaccharide components can be basically well separated, the Prevail Carbohydrate ES column has a stable baseline, is less affected by the gradient change of the mobile phase, and has high precision, stability and reproducibility, making it an ideal choice for establishing the liquid column of the oligosaccharide characteristic chromatogram.

[0042] Furthermore, the flow rate of this high performance liquid chromatography (HPLC) detection is about 1.0 mL / min, and the column temperature is about 45 °C.

[0043] Furthermore, this fingerprint includes peaks No. 1 - 10. Among them, peak No. 1 is sucrose as the reference peak, peak No. 3 is maltotriose, peak No. 4 is maltotetraose, peak No. 5 is maltopentaose, peak No. 6 is maltohexaose, peak No. 7 is mannohexaose, and peak No. 8 is maltoheptaose.

[0044] Furthermore, the aqueous acid solution, the aqueous base solution, and / or the 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.

[0045] Furthermore, mobile phase B is an aqueous solution of 25 - 35 mM ammonium formate (+0.2% - +0.4% formic acid), such as an aqueous solution of about 30 mM ammonium formate (+ about 0.3% formic acid);

[0046] Further, taking chromatographic peak No. 1 as the reference peak, the relative retention times of peaks No. 2 - 10 are 1.155 - 1.165, 1.315 - 1.325, 1.40 - 1.41, 1.545 - 1.555, 1.90 - 1.91, 2.19 - 2.21, 2.42 - 2.43, and 2.62 - 2.63, respectively.

[0047] Further, the method for preparing the oligosaccharide sample solution of Dendrobium includes any one or more of the following items [1] - [9]:

[0048] [1] The Dendrobium sample is the Dendrobium medicinal material after being pulverized and sieved through a 60 - 100 mesh sieve, such as the Dendrobium sample sieved through an 80 - mesh sieve;

[0049] [2] The water is distilled water, deionized water or ultrapure water;

[0050] [3] The temperature is 50°C - 100°C, preferably 60°C - 80°C, such as about 70°C;

[0051] [4] The operation of heating extraction is repeated 2 - 4 times, such as 2 times;

[0052] [5] The dosage of the water is 1 - 80 times the amount (L / kg), preferably 20 - 70 times the amount, more preferably 40 - 60 times the amount, such as about 60 times the amount;

[0053] [6] The time of heating extraction is 1 - 10 hours, preferably 2 - 8 hours, more preferably 2 - 5 hours, such as about 2 hours;

[0054] [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;

[0055] [8] The conditions for centrifugation 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 a speed of about 12000 rpm for about 10 minutes;

[0056] [9] The concentration of the ethanol used for ethanol precipitation is 60% - 100%, preferably 70% - 90%, such as about 80%.

[0057] When used herein, "about" means a value within the range of ±5% of a specific value. For example, "about 70" includes ±5% of 70, or from 66.5 to 73.5, including 66.5 and 73.5.

[0058] According to another aspect of the present invention, there is provided an application of the above - mentioned method in the quality control and / or quality evaluation of Dendrobium medicinal materials or compositions containing Dendrobium medicinal materials.

[0059] Advantages of the present invention:

[0060] This section mainly uses the LPS-induced RAW 264.7 macrophage inflammation model to compare the differences in anti-inflammatory activities of different batches of Dendrobium oligosaccharides. The Griess reagent was used to detect the NO content in the cell supernatant treated with different batches of Dendrobium oligosaccharides at the same concentration, and the NO inhibition rates of different batches of Dendrobium oligosaccharides were obtained. Based on the established characteristic chromatograms of different batches of Dendrobium oligosaccharides, the grey relational analysis method was used to analyze the correlation between the characteristic peaks of the characteristic chromatograms of each batch of Dendrobium oligosaccharides and the anti-inflammatory activity, explore the main pharmacodynamic material basis of the components of Dendrobium oligosaccharides in inhibiting the secretion of NO by macrophages in vitro, further reveal the key components related to anti-inflammatory activity in Dendrobium, and provide a new reference basis for the overall quality evaluation of Dendrobium. Brief Description of the Drawings

[0061] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, 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 results of Dendrobium officinale oligosaccharides for each batch (n = 3).

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

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

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

[0066] Figure 5 HPLC characteristic chromatogram of Dendrobium officinale oligosaccharides. Where a is the HPLC oligosaccharide characteristic chromatogram of 48 batches of Dendrobium officinale; b is the common pattern of the Dendrobium officinale oligosaccharide characteristic chromatogram.

[0067] Figure 6 Cluster analysis result diagram of the characteristic chromatograms of 48 batches of Dendrobium officinale oligosaccharides

[0068] Figure 7 NO inhibition rates of Dendrobium officinale oligosaccharides (DOOS) from different batches on LPS-induced RAW 264.7 macrophages. The error bars represent SD, and n = 3 independent experiments.

[0069] Figure 8 Dimensionless result diagram of the peak areas of each characteristic peak of Dendrobium officinale oligosaccharides and the NO inhibition rate

[0070] Figure 9 It is the correlation coefficient diagram between the characteristic peaks of Dendrobium officinale oligosaccharides and the NO inhibition rate.

[0071] Figure 10 It is a schematic diagram of the inhibitory effect of different concentration gradients (400, 200, 100, 50, 25 μg / mL) of pure Dendrobium officinale oligosaccharides (DOOS-20-2, sucrose; DOOS-20-3, maltotriose; DOOS-20-4, maltotetraose; DOOS-20-5, maltopentaose; DOOS-20-6, maltohexaose; DOOS-20-7, mannohexaose; DOOS-20-8, maltoheptaose) on the NO secretion of LPS-induced RAW 264.7 macrophages. *** p < 0.001 indicates significant difference from the LPS group. Among them, AH is aminoguanidine hydrochloride (50 μg / mL). 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 some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0073] Unless otherwise specified, 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 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 may be combined with each other. Next, the present invention will be described in detail in conjunction with the embodiments.

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

[0076] Embodiment

[0077] 1 Establishment of the characteristic fingerprint of Dendrobium officinale oligosaccharides

[0078] The fingerprint of traditional Chinese medicine (TCM) emphasizes the information characteristics of the overall chemical components in TCM, and it is a quality standard evaluation method with the characteristics of TCM that is widely accepted at home and abroad. This invention is mainly based on the theory of hydrophilic interaction chromatography, and high-performance liquid chromatography analysis is carried out on the oligosaccharide components of Dendrobium officinale Kimura et Migo from 48 batches of medicinal materials from different producing areas, and a characteristic fingerprint of Dendrobium officinale oligosaccharides is established, and 10 common peaks are initially determined. The similarity calculation software for TCM fingerprints and SPSS 22 software are used to analyze the similarity and hierarchical clustering of the obtained oligosaccharide characteristic fingerprints, and comprehensively evaluate the overall quality of Dendrobium officinale medicinal materials.

[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 Feiqia'er 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 Technologies, USA)

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

[0097] Pipette (Eppendorf, Eppendorf, USA)

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

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

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

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

[0102] 1.1.3 Medicinal materials

[0103] Source of medicinal materials: Dendrobium officinale from various production areas and batches was identified by Researcher Xu Hong 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. The reference medicinal materials of Dendrobium officinale were purchased from the National Institutes for Food and Drug Control.

[0104] Table 1 Batch information of Dendrobium officinale

[0105]

[0106]

[0107]

[0108]

[0109] 1.2 Experimental methods

[0110] 1.2.1 Preparation of test sample solution

[0111] Precisely weigh 0.5 g of Dendrobium officinale samples of each batch (passed through 80-mesh sieve), place them in a round-bottom flask, add 30 mL of pure water, heat under reflux in a water bath at 70 °C for 2 h, filter after cooling, add another 30 mL of pure water to the residue, repeat the extraction twice, then combine the two filtrates into a 100-mL volumetric flask, add appropriate pure water to volume to the calibration line, invert and shake well. Precisely pipette 2 mL of the water extract of Dendrobium officinale, and blow-dry under nitrogen in a water bath at 50 °C. Add 1 mL of the prepared 80% ethanol solution to the dried sample, vortex thoroughly, centrifuge at 12,000 rpm for 10 min, carefully aspirate 800 μL of the supernatant, blow-dry under nitrogen in a water bath at 50 °C, and then re-dissolve the sample with 500 μL of ultrapure water, vortex to mix well, and it is ready.

[0112] 1.2.2 Determination of the content of oligosaccharides in Dendrobium officinale

[0113] Prepare the test sample solutions of each batch in triplicate. After vortexing and mixing, centrifuge at 12,000 rpm for 10 min, and carefully aspirate the supernatant for HPLC-RID liquid phase detection. The chromatographic conditions are as follows: Agilent 1100 high-performance liquid chromatograph, using an RID differential refractive index detector, selecting a SUGAR KS-802 chromatographic column, the column oven temperature is 40 ± 0.1 °C, ultrapure water is used as the mobile phase, the flow rate is 0.8 mL / min, and the injection volume is 10 μL. Calculate the peak area of oligosaccharides from 8.10 min to 10.40 min, and obtain the average yield of oligosaccharides in each batch of samples.

[0114] 1.2.3 Preparation of Dendrobium officinale oligosaccharide samples

[0115] Activation of CNWBOND Carbon-GCB Cartridge SPE column: Take a graphitized carbon SPE column and rinse and activate it successively with 5 mL of water, 5 mL of methanol, and 5 mL of water each.

[0116] Prepare the test solutions of Dendrobium officinale of each batch according to the method under 1.2.1. Based on the Dendrobium officinale medicinal materials, prepare an aqueous solution of 100 mg / mL, dissolve it completely, vortex and mix well, centrifuge at 12,000 rpm for 10 min, carefully aspirate 1 mL of the supernatant, and load it onto the activated graphitized carbon SPE column. Elute with 5 mL of distilled water to remove most of the monosaccharide components and impurities such as inorganic salts, and then elute the oligosaccharide components with 5 mL of 50% methanol solution. Blow-dry the methanol eluate under nitrogen in a water bath at 50 °C, dissolve the residue with 0.2 mL of ultrapure water, centrifuge at 12,000 rpm for 10 min, and take the supernatant for use.

[0117] 1.2.4 Establishment of a high-performance liquid chromatography analysis method

[0118] 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, and the gradient elution program is shown in Table 2.

[0119] Table 2 Gradient elution program

[0120]

[0121] 1.2.5 Methodology investigation of the characteristic chromatogram of oligosaccharides

[0122] (1) Precision investigation

[0123] Accurately weigh 0.5 g of Dendrobium officinale powder from Yandang Mountain in Zhejiang (S13) (passed through 80 - mesh sieve), prepare the supernatant of Dendrobium officinale oligosaccharide sample according to the method under 1.2.3. According to the above chromatographic conditions, inject samples repeatedly for 6 times, record the HPLC chromatogram for 80 min, and calculate the RSD values of the relative retention time (RRT) and relative peak area (RPA) of each chromatographic peak respectively.

[0124] (2) Stability investigation

[0125] Accurately weigh 0.5 g of Dendrobium officinale powder from Yandang Mountain in Zhejiang (S13) (passed through 80 - mesh sieve), prepare the supernatant of Dendrobium officinale oligosaccharide sample according to the method under 1.2.3. According to the above chromatographic conditions, conduct determinations at 0, 2, 4, 8, 16, and 24 h respectively, record the HPLC chromatogram for 80 min, and calculate the RSD values of the RRT and RPA of each chromatographic peak respectively.

[0126] (3) Reproducibility investigation

[0127] Accurately weigh 0.5 g of Dendrobium officinale powder from Yandang Mountain in Zhejiang (S13) (passed through 80 - mesh sieve), take 6 parallel portions, prepare the supernatant of Dendrobium officinale oligosaccharide sample according to the method under 1.2.3. According to the above chromatographic conditions, conduct determinations respectively, record the HPLC chromatogram for 80 min, and calculate the RSD values of the RRT and RPA of each chromatographic peak respectively.

[0128] 1.2.6 Establishment of the characteristic chromatogram of Dendrobium officinale oligosaccharides and similarity analysis

[0129] Take 48 batches of Dendrobium officinale medicinal materials, prepare the supernatant of each batch of Dendrobium officinale oligosaccharide samples according to the method under 1.2.3, analyze them by the high performance liquid chromatography method under 1.2.4, and obtain the characteristic spectrum of each batch of Dendrobium officinale medicinal materials oligosaccharides. The characteristic spectrum of 48 batches of Dendrobium officinale oligosaccharides was processed according to the following conditions using the similarity calculation software of traditional Chinese medicine fingerprint spectrum (2004 version A): use sample S48 as the reference spectrum, select the median method, the time window width is 0.2min, multi-point correction and automatic matching, and the software generates a reference spectrum and calculates the similarity.

[0130] 1.2.7 Cluster analysis of characteristic profiles of oligosaccharides from Dendrobium officinale

[0131] SPSS 22 software was used to perform cluster analysis on the chromatographic peak areas of 48 batches of Dendrobium officinale oligosaccharides as source data, combined with the origin batch information, and the cluster analysis dendrogram of different batches of Dendrobium officinale oligosaccharide samples was obtained.

[0132] Similarity analysis and cluster analysis of the characteristic spectrum of Dendrobium officinale oligosaccharides showed that the similarity of the oligosaccharide components of different batches of Dendrobium officinale was quite different, indicating that Dendrobium officinale oligosaccharides were easily affected by soil conditions, harvesting season, altitude and cultivation environment. Analysis of the characteristic spectrum of Dendrobium officinale oligosaccharides at different harvesting times showed that storage time also had a great influence on the chemical composition of Dendrobium officinale oligosaccharides. In the batches of Dendrobium officinale with a longer storage time, the content of oligosaccharides with a higher degree of polymerization decreased significantly.

[0133] 1.3 Experimental Results

[0134] 1.3.1 Determination of oligosaccharide content in Dendrobium officinale

[0135] Extract each batch of Dendrobium officinale samples using the method shown in 1.2.1 in triplicate, measure the oligosaccharide peak area at 8.10min-10.40min, and calculate the oligosaccharide yield of each batch of Dendrobium officinale samples as follows: Figure 1 As shown. The average yield of oligosaccharides in each batch of Dendrobium officinale was 7.65±2.23%, and the average yield of the control medicinal materials was 7.59±0.01%. Combined with the batch information of the origin of Dendrobium officinale, the earlier the batch was collected, the lower the oligosaccharide yield, indicating that the length of storage time has a greater impact on the oligosaccharide content. From the perspective of origin, the oligosaccharide content of Dendrobium officinale batches in Fujian is higher, and the oligosaccharide content of Dendrobium officinale batches in Yunnan fluctuates greatly, which may be related to the large differences in geographical environment, climate and humidity in different regions of Yunnan.

[0136] 1.3.2 Methodological investigation of oligosaccharide characteristic profiles

[0137] (1) Precision inspection

[0138] The prepared Dendrobium officinale oligosaccharide sample was injected repeatedly 6 times under the above chromatographic conditions. The HPLC results are as follows Figure 2 shown. Taking chromatographic peak No. 1 as the reference peak, the RRT and RPA of each chromatographic peak were calculated. The results showed that the RSD value of the RRT of each chromatographic peak ≤ 0.19%, and the RSD value of the RPA of each chromatographic peak ≤ 1.94%, indicating good instrument precision.

[0139] (2) Stability investigation

[0140] The prepared Dendrobium officinale oligosaccharide sample was determined at 0, 2, 4, 8, 16, and 24 h respectively under the above chromatographic conditions. The HPLC results are as follows Figure 3 shown. Taking chromatographic peak No. 1 as the reference peak, the RRT and RPA of each chromatographic peak were calculated. The results showed that the RSD value of the RRT of each chromatographic peak ≤ 0.21%, and the RSD value of the RPA of each chromatographic peak ≤ 1.80%, indicating that the test solution was stable within 24 h at room temperature.

[0141] (3) Reproducibility investigation

[0142] Six parallelly prepared Dendrobium officinale oligosaccharide samples were determined respectively under the above chromatographic conditions. The HPLC results are as follows Figure 4 shown. Taking chromatographic peak No. 1 as the reference peak, the RRT and RPA of each chromatographic peak were calculated. The results showed that the RSD value of the RRT of each chromatographic peak ≤ 0.23%, and the RSD value of the RPA of each chromatographic peak ≤ 2.85%, indicating good reproducibility of this method.

[0143] 1.3.3 Establishment of the characteristic chromatogram of Dendrobium officinale oligosaccharide and similarity analysis

[0144] Forty-eight batches of Dendrobium officinale medicinal materials were analyzed by high performance liquid chromatography and processed with the similarity calculation software for traditional Chinese medicine fingerprints (version A in 2004) to obtain the characteristic chromatograms of Dendrobium officinale oligosaccharide Figure 5 a), the common pattern of the characteristic chromatogram of Dendrobium officinale oligosaccharide Figure 5 b) and the similarity results. It can be seen from the above results that there are great overall differences among different batches of Dendrobium officinale oligosaccharide, and the similarity results are between 0.222 - 0.995, indicating that there are significant differences in chemical components among different batches. In particular, S27 (Yunnan Yishi), S45 (Anguo, Hebei) and S9 (Yandang Mountain, Zhejiang) have the greatest differences compared with other batches, and the similarities are all lower than 0.4. Among them, the overall similarity of the Yandang Mountain batch in Zhejiang is relatively low compared with other origin batches. In order to better reveal the origin differences among Dendrobium officinale, cluster analysis was combined to comprehensively analyze Dendrobium officinale from different origin batches.

[0145] 1.3.4 Cluster analysis of the characteristic chromatogram of Dendrobium officinale oligosaccharide

[0146] To verify the results of similarity analysis and further clarify the similarity relationship among Dendrobium officinale samples, hierarchical clustering analysis was performed. The clustering analysis was based on the between-group linkage method as the merging criterion and the square of the Euclidean distance as the metric standard. The clustering analysis results are as Figure 6 shown. The results show that when the threshold is set at 15, the Dendrobium officinale samples can be divided into two major groups. Group 1 includes S4, S9, S10, S11, S12, S13, S14, S27, S45, and S47, and the rest are classified into Group 2. When the threshold is set at 11, each group can be further divided into two subgroups. S45 and S47 can be classified into Group 1-2, and the rest are classified into Group 1-1. S18, S19, S26, S33, S34, S35, S36, S37, S38, and S39 are classified into Group 2-1, and the rest can be classified into Group 2-2. From the clustering analysis results, it is found that the correlation between Dendrobium officinale samples from different origins is relatively consistent with the results of similarity analysis. Compared with Group 2, Group 1 has a higher similarity with the reference medicinal material, and the similarity among the origin batches within Group 2 is higher. The Dendrobium officinale produced in Yandang Mountain, Zhejiang is basically classified into Group 2, indicating that the growth environment, climate humidity, and altitude will all affect the chemical composition of Dendrobium officinale.

[0147] 2 Spectrum-effect relationship of Dendrobium officinale oligosaccharides

[0148] 2.1 Experimental instruments and materials

[0149] 2.1.1 Reagents and materials

[0150] DMEM medium (GIBCO, USA)

[0151] 0.25% Trypsion-EDTA (GIBCO, USA)

[0152] Fetal bovine serum (GIBCO, USA)

[0153] DPBS buffer (GIBCO, USA)

[0154] Cell culture plates (CORNING, USA)

[0155] Cell culture dishes (Thermo Fisher Scientific, USA)

[0156] Griess kit (Promega, USA)

[0157] Dimethyl sulfoxide (Amresco, USA)

[0158] Dual antibiotics (Penicillin-Streptomycin, P / S) (GIBCO, USA)

[0159] Lipopolysaccharide (LPS) (Sigma, USA)

[0160] Aminoguanidine hydrochloride (AH) (Shanghai Yisheng Biotechnology Co., Ltd., China)

[0161] Absolute ethanol (Sinopharm Chemical Reagent Co., Ltd., China)

[0162] Methanol (Sinopharm Chemical Reagent Co., Ltd., China)

[0163] Purified water (Shanghai Wahaha Drinking Water Co., Ltd., China)

[0164] CNWBOND Carbon - GCB Cartridge (Shanghai Anpu Experimental Technology Co., Ltd., China)

[0165] Other substances of the present invention can also be sourced from commercial purchases.

[0166] 2.1.2 Instruments and equipment

[0167] Electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., China)

[0168] Desktop centrifuge (Shanghai Feiqiaer Analytical Instrument Co., Ltd., China)

[0169] Freeze dryer (Labconco, USA)

[0170] Constant temperature water bath (Shanghai Zhicheng Analytical Instrument Co., Ltd., China)

[0171] CO2 cell incubator (Eppendorf, USA)

[0172] Laminar flow hood (Suzhou Antai Air Technology Co., Ltd., China)

[0173] Low - temperature high - speed centrifuge (Eppendorf, USA)

[0174] Microplate reader (Molecular Devices, USA)

[0175] Pipette (Eppendorf, USA)

[0176] Inverted fluorescence microscope (Olympus Corporation, Japan)

[0177] Vortex oscillator (Qilinbeier Instrument Manufacturing Co., Ltd., China)

[0178] Liquid nitrogen tank (Shanghai Longtuo Instrument Equipment, China)

[0179] MLS - 3780SANYO autoclave (SANYO, Japan)

[0180] 2.2 Experimental methods

[0181] 2.2.1 Preparation of oligosaccharide samples from Dendrobium officinale Kimura et Migo of different batches

[0182] Precisely weigh 0.5 g of Dendrobium officinale Kimura et Migo 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 CNWBOND Carbon-GCB Cartridge SPE column to remove most of the monosaccharide components and impurities such as inorganic salts in the samples according to the method under 1.2.3 to obtain oligosaccharides from Dendrobium officinale Kimura et Migo 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.

[0183] 2.2.2 Effects of oligosaccharides from Dendrobium officinale Kimura et Migo of different batches on NO secretion in RAW 264.7 macrophage inflammation model

[0184] Configure RAW 264.7 macrophages 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 place it in a cell culture incubator overnight. The drug administration group is added with oligosaccharide solutions from Dendrobium officinale Kimura et Migo of different batches (DOOS) with a final concentration of 5 mg / mL (the DOOS concentration is calculated based on the quality of the corresponding crude drug) and LPS with a final concentration of 1 μg / mL. The positive drug group is added with aminoguanidine hydrochloride with a final concentration of 50 μg / mL and LPS with a final concentration of 1 μg / mL. The model group is added with LPS with a final concentration of 1 μg / mL, and the blank control group is added with the same volume of complete medium, and then continue to culture in a cell culture incubator for 24 h.

[0185] Take 100 μL of cell supernatant, add 50 μL of sulfanilamide solution, incubate at room temperature in the dark for 10 min, then add 50 μL of NED solution, incubate at room temperature in the dark for 10 min, and measure the absorbance at 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader. At the same time, use a NaNO2 standard solution to make a standard curve between absorbance and NO concentration, substitute the absorbance value into the standard curve to obtain the NO secretion amount of each group, and calculate the NO inhibition rate of oligosaccharides from Dendrobium officinale Kimura et Migo of each batch. The calculation formula is as follows:

[0186]

[0187] 2.2.3 Grey relational analysis of anti-inflammatory activities of oligosaccharides from Dendrobium officinale Kimura et Migo of different batches

[0188] (1) Determine the analysis sequence

[0189] First, determine the reference sequence and the comparison sequence. Among them, the reference sequence reflects the system behavior characteristics, and the comparison sequence is the data sequence composed of the factors affecting the system. In the present invention, the Dendrobium officinale oligosaccharides are regarded as a whole, that is, a grey system. Among them, the inhibition rate of different batches of Dendrobium officinale oligosaccharides on the NO secretion of LPS-induced RAW 264.7 macrophages is used as the reference sequence, denoted as X0(k), and the peak areas of the common peaks in the characteristic chromatograms of each batch of Dendrobium officinale oligosaccharides are used as the comparison sequence, denoted as X i (k).

[0190] (2) Nondimensionalization of data

[0191] 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 the present invention, the normalization method is adopted to perform nondimensionalization processing on each sequence, and the calculation formula is as follows:

[0192]

[0193] (3) Calculate the correlation coefficient

[0194] Calculate the correlation coefficient corresponding to each comparison sequence and the reference sequence respectively according to the following formula,

[0195]

[0196] where k is the Dendrobium officinale oligosaccharide extract of 48 batches, X i is the inhibition rate of 48 batches of Dendrobium officinale oligosaccharides on the NO secretion of LPS-induced RAW264.7 macrophages, ξ i is the correlation coefficient between the kth batch of Dendrobium officinale oligosaccharide 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 the value range is 0 < ρ < 1. In this invention, ρ = 0.5 is taken for the calculation of the correlation coefficient.

[0197] (4) Calculate the correlation degree

[0198] 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 Dendrobium officinale oligosaccharides and the anti-inflammatory effect of inhibiting the NO secretion of LPS-induced RAW 264.7 macrophages i . Among them, 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.

[0199]

[0200] 2.2.4 Verification of the activity of the common peaks of Dendrobium officinale oligosaccharides in inhibiting the NO secretion of RAW 264.7 macrophages

[0201] Accurately weigh about 10 mg of pure Dendrobium officinale oligosaccharides (DOOS-20-2, sucrose; DOOS-20-3, maltotriose; DOOS-20-4, maltotetraose; DOOS-20-5, maltopentaose; DOOS-20-6, maltohexaose; DOOS-20-7, mannohexaose; DOOS-20-8, maltoheptaose), add a certain amount of DPBS buffer, prepare a stock solution of 20 mg / mL, and store it at -20 °C for later use. Before adding the drug, dilute it to different concentrations with DMEM complete medium and filter sterilize it with a 0.22 μm microporous filter membrane.

[0202] Configure RAW 264.7 macrophages 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 place it in a cell culture incubator to culture overnight. The drug treatment group was added with pure Dendrobium officinale oligosaccharide samples with final concentrations of 400, 200, 100, 50, 25 μg / mL and LPS with a final concentration of 1 μg / mL. The positive drug group was added with aminoguanidine hydrochloride with a final concentration of 50 μg / mL and LPS with a final concentration of 1 μg / mL. The model group was added with LPS with a final concentration of 1 μg / mL, and the blank control group was added with the same volume of complete medium, and then placed in a cell culture incubator to continue culturing for 24 h.

[0203] Take 100 μL of cell supernatant, add 50 μL of sulfanilamide solution, incubate at room temperature in the dark for 10 min, then add 50 μL of NED solution, incubate at room temperature in the dark for 10 min, and measure the absorbance value at 540 nm with an enzyme-labeled instrument.

[0204] 2.3 Experimental results

[0205] 2.3.1 Effects of different batches of Dendrobium officinale oligosaccharides on the secretion of NO in the RAW 264.7 macrophage inflammation model

[0206] Extract oligosaccharides from Dendrobium officinale of each batch under the same conditions. After purification with a CNWBOND Carbon-GCB Cartridge SPE column, they were applied to LPS-induced RAW 264.7 macrophages at the same concentration. The effects of different batches of DOOS on the release of NO by LPS-induced RAW 264.7 macrophages were detected by a Griess kit, and the NO inhibition rate of each batch of DOOS was calculated. The results are as Figure 7As shown in the figure, under the condition of the same dose, the inhibitory rates of oligosaccharides from different batches of Dendrobium officinale on the NO secretion of LPS-induced RAW 264.7 macrophages vary greatly. Among them, S8 (Wuyi, Zhejiang), S13 (Yandang Mountain, Zhejiang), and S37 (Gengma, Yunnan) show the strongest inhibitory effect on NO secretion. It can be seen that there are significant differences in the inhibitory effect on NO secretion among different batches from different origins, which may be related to the differences in the content of anti-inflammatory active ingredients in different batches.

[0207] 2.3.2 Grey relational analysis of the anti-inflammatory activity of oligosaccharides from different batches of Dendrobium officinale

[0208] Grey relational analysis is a statistical method for judging the degree of association 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 analyzes the correlation between the characteristic peaks of oligosaccharides from Dendrobium officinale and the anti-inflammatory activity.

[0209] (1) Nondimensionalization of data

[0210] According to the above formula, each sequence is nondimensionalized, and the results are as Figure 8 shown.

[0211] (2) Calculation of correlation coefficients

[0212] According to the above formula, the correlation coefficients corresponding to each comparison sequence and the reference sequence are calculated respectively for the sequences after nondimensionalization, and the correlation coefficients are represented in the form of a heat map. The results are as Figure 9 shown. It can be seen from Figure 9 that the correlation between Peak 1, Peak 3, and Peak 8 and the activity of inhibiting the secretion of NO by LPS-induced macrophages is not significant. The remaining characteristic peaks have a greater correlation with the activity of inhibiting the secretion of NO by macrophages.

[0213] (3) Calculation of correlation degree

[0214] According to the above formula, the grey correlation degree γi between each chromatographic characteristic peak of oligosaccharides from Dendrobium officinale and the anti-inflammatory effect of inhibiting the secretion of NO by LPS-induced RAW 264.7 macrophages is calculated and the correlation order is arranged. The results are shown in Table 3. Except for Characteristic Peak 1, the correlation degrees of the remaining characteristic peaks with the activity of inhibiting the secretion of NO by macrophages are all greater than 0.6, indicating that these components are related to the NO inhibition rate. According to the correlation degree, the correlation order is arranged, and the contribution of each characteristic peak to the NO inhibition effect is 7>4>2>9>5>10>6>3>8>1 in turn.

[0215] Table 3 Correlation degree and correlation order between each characteristic peak of oligosaccharides from Dendrobium officinale and NO inhibition rate

[0216]

[0217]

[0218] 2.3.3 Verification of the Activity of Inhibiting NO Secretion by RAW 264.7 Macrophages with the Common Peaks of Oligosaccharides from Dendrobium officinale

[0219] The pure products of Dendrobium officinale oligosaccharides (DOOS-20-2, sucrose; DOOS-20-3, maltotriose; DOOS-20-4, maltotetraose; DOOS-20-5, maltopentaose; DOOS-20-6, maltohexaose; DOOS-20-7, mannohexaose; DOOS-20-8, maltoheptaose, which can be obtained from the extraction, separation and purification of Dendrobium officinale oligosaccharides or purchased commercially) were used to act on LPS-induced RAW 264.7 macrophages at the same concentration gradients (400, 200, 100, 50, 25 μg / mL). The effects of each purified part of DOOS-20 on the release of NO by LPS-induced RAW 264.7 macrophages were detected by Griess kit. The results are as Figure 10 shown: Except that at the concentration of 25 μg / mL of DOOS-20-2 (peak 1, sucrose) and DOOS-20-3 (peak 3, maltotriose), the inhibition of NO secretion by LPS-induced RAW 264.7 macrophages had no statistical significance compared with the LPS group, the inhibition of NO secretion by macrophages at each concentration gradient of the other parts had statistical significance compared with the LPS group (p < 0.001). Among them, DOOS-20-4 (peak 4, maltotetraose), DOOS-20-5 (peak 5, maltopentaose), DOOS-20-6 (peak 6, maltohexaose), and DOOS-20-7 (peak 7, mannohexaose) had a relatively significant inhibitory effect on NO secretion, and DOOS-20-8 (peak 8, maltoheptaose) had a weak inhibitory effect on NO, which was consistent with the correlation results predicted by grey relational analysis, indicating that grey relational analysis could better predict the correlation between fingerprint and in vitro activity and determine the effective ingredient group. Among them, maltotetraose, maltopentaose, maltohexaose and mannohexaose can be used as quality indicators for the quality control of Dendrobium officinale medicinal materials.

[0220] 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, those skilled in the art, based on the idea of the present invention, the changes or deformations made in 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 Dendrobium, characterized in that, The method includes the following steps: (1) Using the statistical method of spectrum-effect relationship to conduct grey relational analysis on the correlation between the characteristic peaks of the HPLC fingerprint of dendrobium oligosaccharides and the anti-inflammatory activity; (2) Obtaining quality markers that can be used for the quality control of dendrobium according to the analysis results; and (3) Formulating the quality control standard of the dendrobium according to the quality markers; Among them, the HPLC fingerprint of dendrobium oligosaccharides is constructed through the following steps: Preparation of the dendrobium oligosaccharide sample solution: (a) Weigh an appropriate amount of dendrobium 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 volume to the calibration line, invert and shake well, absorb 2 mL of the dendrobium water extract, blow dry with nitrogen, add 1 mL of ethanol solution to the dried sample for ethanol precipitation, vortex thoroughly and centrifuge, carefully absorb 800 μL of the supernatant, blow dry with nitrogen, add 500 μL of water to the dried sample for reconstitution, and vortex to obtain the test solution; (b) Take a graphitized carbon SPE column, rinse and activate it with 5 mL of water, 5 mL of methanol, and 5 mL of water in sequence to obtain the activated graphitized carbon SPE column; and (c) Prepare an aqueous solution of 100 mg / mL based on the mass of the dendrobium medicinal material with the test solution, fully dissolve and vortex, centrifuge, absorb 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 in 0.2 mL of water and centrifuge, take the supernatant to obtain the dendrobium oligosaccharide sample solution; Preparation of the reference solution: Weigh sucrose, maltotriose, maltotetraose, maltopentaose, maltohexaose, mannohexaose, and maltoheptaose, and 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: Using a Prevail Carbohydrate ES chromatographic column, with acetonitrile as mobile phase A and 30 mM ammonium formate aqueous solution as mobile phase B, the gradient elution program is: 0 - 60 min, 80% - 50% A; 60 - 80 min, 50% 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, obtain the HPLC fingerprint of dendrobium oligosaccharides; Among them, the fingerprint includes peaks No. 1 - 10, among which, peak No. 1 is sucrose as the reference peak, peak No. 3 is maltotriose, peak No. 4 is maltotetraose, peak No. 5 is maltopentaose, peak No. 6 is maltohexaose, peak No. 7 is mannohexaose, and peak No. 8 is maltoheptaose; Among them, taking chromatographic peak No. 1 as the reference peak, the relative retention times of peaks No. 2-10 are 1.155-1.165, 1.315-1.325, 1.40-1.41, 1.545-1.555, 1.90-1.91, 2.19-2.21, 2.42-2.43, 2.62-2.63 respectively; Among them, the Dendrobium oligosaccharides are sucrose, maltotriose, maltotetraose, maltopentaose, maltohexaose, mannohexaose and maltoheptaose; Among them, the quality markers are maltotetraose, maltopentaose, maltohexaose and mannohexaose.

2. The method according to claim 1, wherein The Dendrobium is selected from one or more of the following: fresh or dried stems of Dendrobium nobile, Dendrobium huoshanense, Dendrobium chrysotoxum, Dendrobium fimbriatum, Dendrobium wardianum, Dendrobium officinale, Dendrobium chrysanthum and Dendrobium flagelliforme.

3. The method according to claim 2, wherein The Dendrobium is fresh or dried stems of Dendrobium officinale.

4. The method according to claim 1, characterized in that, 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.

5. The method according to claim 4, wherein The analysis sequence includes a reference sequence and a comparison sequence, where the inhibition rate of different batches of dendrobium oligosaccharides on the NO secretion of LPS-induced RAW 264.7 macrophages 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 dendrobium oligosaccharides are used as the comparison sequence, denoted as X i (k).

6. The method according to claim 5, wherein 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 dendrobium oligosaccharide extract, X i is the inhibition rate of different batches of dendrobium oligosaccharides on the NO secretion of LPS-induced RAW 264.7 macrophages, ξ i is the correlation coefficient between the comparison sequence and the reference sequence of the dendrobium oligosaccharide of the kth batch, is the two-stage minimum difference, is the two-stage maximum difference, and ρ is the resolution coefficient with a value range of 0 < ρ < 1.

7. The method according to claim 6, wherein ρ = 0.

5.

8. The method according to claim 5, characterized in that, The grey relational grade is the arithmetic mean of the correlation coefficients, and the grey relational grade γ between each chromatographic characteristic peak of dendrobium oligosaccharide and the anti-inflammatory effect of inhibiting NO secretion in LPS-induced RAW 264.7 macrophages is calculated according to the following formula 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.

9. A method for constructing an HPLC fingerprint of dendrobium oligosaccharides, characterized in that, The method includes the following steps: Preparation of Dendrobium oligosaccharide sample solution: (a) Weigh an appropriate amount of Dendrobium 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 volume to the scale line, invert and shake well, pipette 2 mL of the aqueous Dendrobium extract, blow dry with nitrogen, add 1 mL of ethanol solution to the dried sample for ethanol precipitation, vortex thoroughly and centrifuge, carefully pipette 800 μL of the supernatant, blow dry with nitrogen, add 500 μL of water to the dried sample for reconstitution, vortex to mix evenly to obtain the test solution; (b) Take a graphitized carbon SPE column, rinse and activate it with 5 mL of water, 5 mL of methanol and 5 mL of water in sequence to obtain the graphitized carbon SPE column after activation treatment; and (c) Prepare the test solution into an aqueous solution of 100 mg / mL based on the mass of the Dendrobium medicinal material, fully dissolve and vortex to mix evenly and then centrifuge, pipette 1 mL of the supernatant, load it onto the graphitized carbon SPE column after activation treatment, 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 Dendrobium oligosaccharide sample solution; Preparation of reference solution: Weigh sucrose, maltotriose, maltotetraose, maltopentaose, maltohexaose, mannohexaose and maltoheptaose, 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 - 60 min, 80% - 50% A; 60 - 80 min, 50% A; the flow rate is 1.0 mL / min; the column temperature is 45 °C; the detector is 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 dendrobium oligosaccharides is obtained; Among them, the fingerprint includes peaks No. 1 - 10. Among them, peak No. 1 is sucrose as the reference peak, peak No. 3 is maltotriose, peak No. 4 is maltotetraose, peak No. 5 is maltopentaose, peak No. 6 is maltohexaose, peak No. 7 is mannohexaose, and peak No. 8 is maltoheptaose; Among them, taking chromatographic peak No. 1 as the reference peak, the relative retention times of peaks No. 2 - 10 are 1.155 - 1.165, 1.315 - 1.325, 1.40 - 1.41, 1.545 - 1.555, 1.90 - 1.91, 2.19 - 2.21, 2.42 - 2.43, 2.62 - 2.63 respectively.

10. The method according to claim 9, wherein The preparation method of the dendrobium oligosaccharide sample solution includes any one or more of the following [1] - [9]: [1] The dendrobium sample is a dendrobium sample obtained by pulverizing dendrobium 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 for 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 heating time under reflux is 1 - 10 hours; [7] The nitrogen blowing to dryness is carried out under nitrogen blowing in a water bath at 45 °C - 55 °C; [8] The centrifugation conditions are centrifugation at 5000 rpm - 20000 rpm for 5 - 30 minutes; [9] The concentration of the ethanol used for ethanol precipitation is 60% - 100%.

11. The method according to claim 10, wherein In item [1] of the method, the dendrobium sample is a dendrobium sample obtained by pulverizing dendrobium medicinal materials and passing through an 80 - mesh sieve.

12. The method according to claim 10, wherein In item [3] of the method, the temperature is 60 °C - 80 °C.

13. The method according to claim 12, wherein The temperature is 66.5 - 73.5 °C.

14. The method according to claim 10, characterized in that, In item [4] of the method, the operation of adding water for heating under reflux and filtering after cooling is repeated 2 times.

15. The method according to claim 10, wherein In item [5] of the method, the dosage of water is 20 - 70 times the amount, with the unit of L / kg.

16. The method according to claim 15, characterized in that The dosage of water is 40 - 60 times the amount, with the unit of L / kg.

17. The method according to claim 16, wherein The dosage of water is 60 times the amount, with the unit of L / kg.

18. The method according to claim 10, wherein In item [6] of the method, the heating time under reflux is 2 - 8 hours.

19. The method according to claim 18, characterized in that, The heating time under reflux is 2 - 5 hours.

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

21. The method according to claim 10, wherein In item [7] of the method, the nitrogen blowing to dryness is carried out under nitrogen blowing in a water bath at 50 °C.

22. The method according to claim 10, wherein In item [8] of the said method, the conditions for centrifugation are centrifuging at a rotational speed of 8000 rpm to 15000 rpm for 8 to 20 minutes.

23. The method according to claim 22, wherein The conditions for centrifugation are centrifuging at a rotational speed of 12000 rpm for 10 minutes.

24. The method according to claim 10, wherein In item [9] of the said method, the concentration of the ethanol used for ethanol precipitation is 70% to 90%.

25. The method according to claim 24, wherein The concentration of the ethanol used for ethanol precipitation is 80%.

26. The method according to claim 9, wherein Based on the group average linkage method as the merging criterion and the square of the Euclidean distance as the metric, when the thresholds are taken as 15 and 11, the cluster analysis results are obtained to distinguish Dendrobium between different production area batches.

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

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