A preparation method of wolfberry oligosaccharide

Abstract

The invention discloses a method for preparing total wolfberry oligosaccharides and / or wolfberry oligosaccharides, the method comprising the following steps: (1) weighing an appropriate amount of wolfberry medicinal material, adding water and heating and refluxing at a certain temperature for extraction, merging the filtered extracts, concentrating under reduced pressure to a certain volume and centrifuging, discarding the medicinal residues, adding ethanol precipitation to the supernatant, stirring evenly and standing overnight, centrifuging, collecting the supernatant and concentrating under reduced pressure to ethanol-free, freeze-drying to obtain total wolfberry oligosaccharides; and optionally (2) subjecting the total wolfberry oligosaccharides to column chromatography by an activated carbon-diatomaceous earth column and column chromatography by an ODS-AQ preparative column to separate and purify to obtain the wolfberry oligosaccharides. The process is simple, convenient, pollution-free, and the total wolfberry oligosaccharides and / or wolfberry oligosaccharides have high yield and purity.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for preparing wolfberry oligosaccharides. Background Art

[0002] So far, there is no report on the extraction method of wolfberry oligosaccharides. In addition, compared with Dendrobium officinale, wolfberry oligosaccharides have the characteristics of fast dissolution and low viscosity of the extract, so the extraction method of Dendrobium officinale oligosaccharides cannot be directly applied. In addition, previous studies have shown that there are large differences in the experimental results of the two, and different extraction conditions may produce different material components with large differences in content. Therefore, it is urgent to establish a standardized and efficient extraction method to maximize the utilization of wolfberry oligosaccharides. Summary of the invention

[0003] Based on this, the present invention provides a method for preparing wolfberry total oligosaccharides and / or wolfberry oligosaccharides, the method comprising the following steps:

[0004] (1) weighing an appropriate amount of wolfberry medicinal material, adding water, heating and refluxing at a certain temperature for extraction, combining the filtered extracts, concentrating under reduced pressure to a certain volume and centrifuging, discarding the drug residue, adding ethanol to the supernatant for precipitation, stirring well and then standing overnight, centrifuging, collecting the supernatants, combining them, concentrating under reduced pressure until there is no ethanol, and freeze-drying to obtain wolfberry total oligosaccharides; and

[0005] Optionally (2) the total wolfberry oligosaccharide is subjected to column chromatography through an activated carbon-diatomaceous earth column and a preparative ODS-AQ column to separate and purify the wolfberry oligosaccharide.

[0006] Furthermore, the wolfberry is the dried mature fruit of cylindrical wolfberry, soft-stem wolfberry, black-fruited wolfberry, calyx-truncate wolfberry or Ningxia wolfberry.

[0007] Furthermore, the wolfberry is produced in Ningxia, Gansu, Inner Mongolia, Xinjiang and / or Qinghai.

[0008] Furthermore, the wolfberry is the dried mature fruit of Ningxia wolfberry.

[0009] Further, the wolfberry oligosaccharide is composed of 2 to 10 monosaccharide units bonded by glycosidic bonds. Further, the wolfberry oligosaccharide is composed of 2 to 6 monosaccharide units bonded by glycosidic bonds. Further, the wolfberry oligosaccharide is composed of glucose, mannose and / or galactose bonded by glycosidic bonds. Further, the wolfberry oligosaccharide is selected from one or more of the following: sucrose, melibiose, raffinose, kestose, kestose, pentose and kestose.

[0010] Furthermore, step (1) of the method includes any one or more of the following items [1] to

[11] :

[0011] [1] crushing the wolfberry medicinal material;

[0012] [2] Passing the crushed wolfberry medicinal material through a 60-100 mesh sieve, such as an 80 mesh sieve;

[0013] [3] The water is distilled water or deionized water;

[0014] [4] The temperature is 50°C to 100°C, preferably 70°C to 90°C, more preferably 77°C to 85°C, for example about 81.3°C;

[0015] [5] The heating extraction operation is repeated 2 to 4 times, for example 2 times;

[0016] [6] The amount of water is 1 to 80 times (L / kg), preferably 10 to 60 times, more preferably 15 to 60 times, particularly preferably 20 to 40 times, particularly preferably 23 to 27 times, for example about 25 times;

[0017] [7] The heating extraction time is 0.5 to 10 hours, preferably 1 to 5 hours, more preferably 1 to 1.15 hours, for example about 65 minutes;

[0018] [8] The certain volume is 1 / 10 to 1 / 2 of the volume of the added water, preferably 2 / 5;

[0019] [9] The centrifugation conditions are 2000 rpm to 6000 rpm for 5 to 30 minutes, preferably 3000 rpm to 5000 rpm for 8 to 20 minutes, for example, about 3600 rpm for about 10 minutes;

[0020]

[10] The amount of anhydrous ethanol is 1 to 5 times the volume of the supernatant, for example about 2.4 times the volume;

[0021]

[11] The concentration of ethanol used in the ethanol precipitation is 60% to 100%, preferably about 100%.

[0022] Furthermore, the activated carbon-diatomaceous earth column used in step (2) of the method is prepared by the following method:

[0023] Weigh an appropriate amount of granular activated carbon, boil it with 10% to 30% acetic acid for 10 to 60 minutes, then wash it with water until it is neutral, and then mix it with an equal weight of diatomaceous earth with water to form a slurry, pour it into a chromatography column, wait for it to settle naturally, cover the carbon surface with a piece of filter paper, rinse with distilled water until the carbon surface no longer drops, and when the water on the carbon surface is reduced to 1 μm to 10 μm, the activated carbon-diatomaceous earth column is obtained.

[0024] Further, a proper amount of granular activated carbon was weighed and boiled with about 20% acetic acid for about 30 minutes.

[0025] Furthermore, step (2) of the method further comprises the following steps:

[0026] (a) weighing an appropriate amount of the total oligosaccharides of wolfberry, adding water to fully dissolve and centrifuging, adding the supernatant to the activated carbon-diatomaceous earth column, and after natural sedimentation, sequentially using different mobile phases for gradient elution, after the phenol-sulfuric acid method detects that no sugar fraction flows out, switching to the next gradient, and concentrating the eluate of each part under reduced pressure in a water bath and then freeze-drying to obtain crude oligosaccharides of wolfberry; and

[0027] (b) Using a CAPCELL PAK C18-AQ (250×10 mm, 5 μm) preparative column and ultrapure water as the mobile phase, the crude wolfberry oligosaccharide was purified and detected online, and the wolfberry oligosaccharide was collected and freeze-dried according to the peak results to obtain the wolfberry oligosaccharide.

[0028] Furthermore, steps (a) and (b) of the method respectively include any one or more of the following items [1] to [8]:

[0029] [1] The water is distilled water or deionized water;

[0030] [2] The amount of water used is 1 to 80 times (L / kg), preferably 10 to 50 times, for example about 20 times;

[0031] [3] The centrifugation conditions are 6000 rpm to 20000 rpm for 5 to 30 minutes, preferably 8000 rpm to 15000 rpm for 8 to 20 minutes, for example, about 10000 rpm for about 15 minutes;

[0032] [4] The different mobile phases are distilled water, 15% to 25% ethanol solution and 45% to 55% ethanol solution, preferably distilled water, about 20% ethanol solution and about 50% ethanol solution;

[0033] [5] The temperature of the water bath is 40°C to 100°C, preferably 50°C to 80°C, for example about 60°C;

[0034] [6] The crude wolfberry oligosaccharide is eluted with about 20% ethanol solution;

[0035] [7] The flow rate of the CAPCELL PAK C18-AQ preparative column is 1-3 mL / min, the column oven temperature is 25±0.1°C to 35±0.1°C, the injection volume is 45-55 μL, the sample concentration is 15-25 mg / mL, preferably about 2 mL / min, the column oven temperature is 30±0.1°C, the injection volume is about 50 μL, and the sample concentration is about 20 mg / mL;

[0036] [8] The instrument used for the online detection is an Agilent 1100 high performance liquid chromatograph and a RID differential detector.

[0037] According to another aspect of the present invention, there is provided wolfberry total oligosaccharides and / or wolfberry oligosaccharides prepared by the above method.

[0038] Beneficial effects of the present invention:

[0039] The response surface methodology (RSM) adopted in the present invention is a mathematical statistical model that can be used to improve and optimize multi-factor processes and determine the influence of a single factor on the result. The main advantage of the response surface methodology is that it reduces the number of experiments required to evaluate a single parameter and its interaction. Box-Behnken design (BBD) is a response surface model. Compared with other methods, BBD is more effective and easier to organize. Therefore, based on the single factor experiment, the present invention uses the response surface methodology combined with the Box-Behnken design to optimize the extraction process of wolfberry oligosaccharides. The process is simple, convenient, pollution-free, and the yield and purity of wolfberry total oligosaccharides and / or wolfberry oligosaccharides are high. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 This is a schematic diagram of the effect of ethanol precipitation concentration on the yield of wolfberry oligosaccharides.

[0042] Figure 2 Schematic diagram of the effect of extraction times on the yield of wolfberry oligosaccharides.

[0043] Figure 3 Schematic diagram of the effect of extraction temperature on the yield of wolfberry oligosaccharides.

[0044] Figure 4 Schematic diagram of the effect of extraction time on the yield of wolfberry oligosaccharides.

[0045] Figure 5 Schematic diagram of the effect of the solid-liquid ratio on the yield of wolfberry oligosaccharides.

[0046] Figure 6 Schematic diagram of the results of statistical and variance analysis of experimental data using Design-Expert 8.0.6 software, (a) normal probability distribution of residuals; (b) correspondence between residuals and equation predicted values; (c) correspondence between predicted values ​​and experimental values.

[0047] Figure 7 Schematic diagram of the effect of interaction on the extraction rate of wolfberry oligosaccharides.

[0048] Figure 8 This is a schematic diagram of the elution curve results of CAPCELL PAK C18-AQ at the LBOS-20 site.

[0049] Fig. 9 Schematic diagram of HPLC-CAD chromatographic results of purified parts of LBOS-20 and mixed standards (glucose, sucrose, fructo-oligosaccharides DP 3-6, fructose, melibiose and raffinose). DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0051] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0052] As described in the background technology section, there is no report on the separation and purification method of wolfberry oligosaccharides in the prior art, and there is a problem that the separation and purification conditions are difficult to master. In order to solve the above problems, the present invention provides a method for preparing wolfberry total oligosaccharides and / or wolfberry oligosaccharides, which comprises the following steps:

[0053] (1) weighing an appropriate amount of wolfberry medicinal material, adding water, heating and refluxing at a certain temperature for extraction, combining the filtered extracts, concentrating under reduced pressure to a certain volume and centrifuging, discarding the drug residue, adding ethanol to the supernatant for precipitation, stirring well and then standing overnight, centrifuging, collecting the supernatants, combining them, concentrating under reduced pressure until there is no ethanol, and freeze-drying to obtain wolfberry total oligosaccharides; and

[0054] Optionally (2) the total wolfberry oligosaccharide is subjected to column chromatography through an activated carbon-diatomaceous earth column and a preparative ODS-AQ column to separate and purify the wolfberry oligosaccharide.

[0055] In a preferred embodiment, the wolfberry is the dried mature fruit of Lycium barbarum, Lycium barbarum with soft stems, Lycium barbarum with black fruit, Lycium barbarum with calyx or Lycium barbarum with Ningxia.

[0056] The "wolfberry" of the present invention includes but is not limited to the cultivated products of the above-mentioned wolfberry varieties in the genus Lycium and the dried mature fruits of similar species of plants of the same genus. The wolfberry varieties are similar to each other and can all be applied to the technical solution of the present invention.

[0057] In a preferred embodiment, the wolfberry is produced in Ningxia, Gansu, Inner Mongolia, Xinjiang and / or Qinghai.

[0058] In a preferred embodiment, the wolfberry is the dried mature fruit of Ningxia wolfberry.

[0059] In a preferred embodiment, the wolfberry oligosaccharide is composed of 2 to 10 monosaccharide units linked by glycosidic bonds.

[0060] In the present invention, when the number, mesh number, temperature, number of times, multiples, time, volume, rotation speed, concentration or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, it should be understood that all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "2 to 10" is disclosed, unless otherwise specified, the range is intended to include its end value 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.

[0061] In a preferred embodiment, the wolfberry oligosaccharide is composed of 2 to 6 monosaccharide units linked by glycosidic bonds.

[0062] In a preferred embodiment, the wolfberry oligosaccharide is formed by combining glucose, mannose and / or galactose via glycosidic bonds.

[0063] In a preferred embodiment, the wolfberry oligosaccharide is selected from one or more of the following: sucrose, melibiose, raffinose, kestose, kestetetrose, kestose, pentose and kestose.

[0064] In a preferred embodiment, step (1) of the method includes any one or more of the following items [1] to

[11] :

[0065] [1] crushing the wolfberry medicinal material;

[0066] [2] Passing the crushed wolfberry medicinal material through a 60-100 mesh sieve, such as an 80 mesh sieve;

[0067] [3] The water is distilled water or deionized water;

[0068] [4] The temperature is 50°C to 100°C, preferably 70°C to 90°C, more preferably 77°C to 85°C, for example about 81.3°C (about 81.3°C can improve the diffusion and solubility of oligosaccharides and enhance fluidity);

[0069] [5] The heating extraction operation is repeated 2 to 4 times, for example 2 times;

[0070] [6] The amount of water used is 1 to 80 times (L / kg), preferably 10 to 60 times, more preferably 15 to 60 times, particularly preferably 20 to 40 times, and particularly preferably 23 to 27 times, for example about 25 times (about 25 times can not only facilitate the dissolution of the oligosaccharide component and obtain a higher oligosaccharide yield, but also reduce the additional pressure and material and manpower investment caused by the subsequent concentration process due to an excessively high solid-liquid ratio);

[0071] [7] The heating extraction time is 0.5 to 10 hours, preferably 1 to 5 hours, more preferably 1 to 1.15 hours, for example about 65 minutes;

[0072] [8] The certain volume is 1 / 10 to 1 / 2 of the volume of the added water, preferably 2 / 5;

[0073] [9] The centrifugation conditions are 2000 rpm to 6000 rpm for 5 to 30 minutes, preferably 3000 rpm to 5000 rpm for 8 to 20 minutes, for example, about 3600 rpm for about 10 minutes;

[0074]

[10] The amount of anhydrous ethanol is 1 to 5 times the volume of the supernatant, for example about 2.4 times the volume;

[0075]

[11] The concentration of ethanol used in the ethanol precipitation is 60% to 100%, preferably about 100%.

[0076] As used herein, "about" refers to a value within a range of ±5% of a particular value. For example, "about 81.3" includes ±5% of 81.3, or from 77.235 to 85.365, including 77.235 and 85.365.

[0077] In a preferred embodiment, the activated carbon-diatomaceous earth column used in step (2) of the method is prepared by the following method:

[0078] Weigh an appropriate amount of granular activated carbon, boil it with 10% to 30% acetic acid for 10 to 60 minutes, then wash it with water until it is neutral, and then mix it with an equal weight of diatomaceous earth with water to form a slurry, pour it into a chromatography column, wait for it to settle naturally, cover the carbon surface with a piece of filter paper, rinse with distilled water until the carbon surface no longer drops, and when the water on the carbon surface is reduced to 1 μm to 10 μm, the activated carbon-diatomaceous earth column is obtained.

[0079] In a preferred embodiment, a proper amount of granular activated carbon is weighed and boiled with about 20% acetic acid for about 30 minutes.

[0080] In a preferred embodiment, step (2) of the method further comprises the following steps:

[0081] (a) weighing an appropriate amount of the total oligosaccharides of wolfberry, adding water to fully dissolve and centrifuging, adding the supernatant to the activated carbon-diatomaceous earth column, and after natural sedimentation, sequentially using different mobile phases for gradient elution, after the phenol-sulfuric acid method detects that no sugar fraction flows out, switching to the next gradient, and concentrating the eluate of each part under reduced pressure in a water bath and then freeze-drying to obtain crude oligosaccharides of wolfberry; and

[0082] (b) Using a CAPCELL PAK C18-AQ (250×10 mm, 5 μm) preparative column and ultrapure water as the mobile phase, the crude wolfberry oligosaccharide was purified and detected online, and the wolfberry oligosaccharide was collected and freeze-dried according to the peak results to obtain the wolfberry oligosaccharide.

[0083] In a preferred embodiment, steps (a) and (b) of the method respectively include any one or more of the following items [1] to [8]:

[0084] [1] The water is distilled water or deionized water;

[0085] [2] The amount of water used is 1 to 80 times (L / kg), preferably 10 to 50 times, for example about 20 times;

[0086] [3] The centrifugation conditions are 6000 rpm to 20000 rpm for 5 to 30 minutes, preferably 8000 rpm to 15000 rpm for 8 to 20 minutes, for example, about 10000 rpm for about 15 minutes;

[0087] [4] The different mobile phases are distilled water, 15% to 25% ethanol solution and 45% to 55% ethanol solution, preferably distilled water, about 20% ethanol solution and about 50% ethanol solution;

[0088] [5] The temperature of the water bath is 40°C to 100°C, preferably 50°C to 80°C, for example about 60°C;

[0089] [6] The crude wolfberry oligosaccharide is eluted with about 20% ethanol solution;

[0090] [7] The flow rate of the CAPCELL PAK C18-AQ preparative column is 1-3 mL / min, the column oven temperature is 25±0.1°C to 35±0.1°C, the injection volume is 45-55 μL, the sample concentration is 15-25 mg / mL, preferably about 2 mL / min, the column oven temperature is 30±0.1°C, the injection volume is about 50 μL, and the sample concentration is about 20 mg / mL;

[0091] [8] The instrument used for the online detection is an Agilent 1100 high performance liquid chromatograph and a RID differential detector.

[0092] According to another aspect of the present invention, there is provided wolfberry total oligosaccharides and / or wolfberry oligosaccharides prepared by the above method.

[0093] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0094] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0095] Example

[0096] 1 Optimization of Extraction Technology of Lycium barbarum Oligosaccharides by Response Surface Methodology

[0097] The present invention mainly optimizes the extraction process of wolfberry oligosaccharides by combining response surface methodology and Box-Behnken design. By optimizing the three main influencing factors of hot water extraction method: extraction temperature, extraction time, and solid-liquid ratio, the maximum enrichment of wolfberry oligosaccharides is achieved, and the best extraction scheme is established, providing a standard and feasible scheme for the establishment of wolfberry oligosaccharide characteristic map and subsequent research.

[0098] 1.1 Experimental instruments and materials

[0099] 1.1.1 Reagents and materials

[0100] Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., China); purified water (Shanghai Wahaha Drinking Water Co., Ltd., China); SUGAR KS-802 sugar analysis column (Showa, Shodex, Japan); D-glucose (Sigma-Aldrich, USA); sucrose (Aladdin Reagent (Shanghai) Co., Ltd., China); wolfberry (Wuzhong City, Ningxia); heavy water (Sigma-Aldrich, USA); D-arabinose (Sigma-Aldrich, USA); L-fucose (Sigma-Aldrich, USA); L-rhamnose (Sigma-Aldrich, USA); D-xylose (Sigma-Aldrich, USA); D-mannose (Sigma-Aldrich, USA); D-galactose (Sigma-Aldrich, USA); melibiose (Shanghai Yuanye Biotechnology Co., Ltd., China); raffinose (Shanghai Yuanye Biotechnology Co., Ltd., China); fructo-oligosaccharide (DP3-6) (Chengdu Pusi Biotechnology Co., Ltd. , China); acetonitrile (Fisher, USA); ammonium formate (Aldrich, USA); formic acid (JTBaker, USA); 1-methylimidazole (Sigma-Aldrich, USA); dimethyl sulfoxide (Sigma-Aldrich, USA); sodium borohydride (Sinopharm Chemical Reagent Co., Ltd., China); glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd., China); trifluoroacetic acid (Sinopharm Chemical Reagent Co., Ltd., China); acetic anhydride (Sinopharm Chemical Reagent Co., Ltd., China); chloroform (Sinopharm Chemical Reagent Co., Ltd., China); anhydrous sodium sulfate (Sinopharm Chemical Reagent Co., Ltd., China); granular activated carbon (Sinopharm Chemical Reagent Co., Ltd., China); diatomaceous earth (Sinopharm Chemical Reagent Co., Ltd., China) Prevail Carbohydrate ES 5u (GRACE, USA); CAPCELL PAK C18-AQ (Shiseido, Japan).

[0101] 1.1.2 Instruments and equipment

[0102] Electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., China); desktop centrifuge (Shanghai Feichaer Analytical Instrument Co., Ltd., China); HWS24 constant temperature water bath (Shanghai Chengxian Instrument Equipment Co., Ltd., China); MILLI-Q ultrapure water preparation instrument (MERCK MILLIPORE, USA); multi-sample automatic nitrogen blowing concentrator (ATR Technology Inc, USA); HPLC (Agilent, USA); pipette (Eppendorf, USA); standard test sieve (80 mesh) (Zhejiang Shangyu Huafeng Hardware Instrument Co., Ltd., China); high-speed pulverizer (Zhejiang Yili Industry and Trade Co., Ltd., China); vortex oscillator (Qilin Bell Instrument Manufacturing Co., Ltd., China); freeze dryer (Labconco, USA); LC-6M centrifuge (Shanghai Centrifugal Machinery Research Institute, China); N-1100 rotary evaporator (Ruihui Electronics Co., Ltd., China); SHB-Ⅲ circulating water multi-purpose vacuum pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd., China); Thermo Trace DSQ gas chromatograph (Agilent, USA); AM-600 and AM-400 nuclear magnetic resonance (Bruker, USA); Agilent 1290 Infinity LC / 6546Q-TOF (Agilent, USA); HPLC Electrospray detector (Thermo, Germany).

[0103] 1.2 Experimental methods

[0104] 1.2.1 Establishment of oligosaccharide content determination method

[0105] (1) HPLC conditions

[0106] The oligosaccharide content in the sample was determined by high performance liquid gel chromatography (HPGPC). The detection conditions were as follows: Agilent 1100 high performance liquid chromatograph, RID differential detector for detection, 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 10μL, isocratic elution for 20min, and the sum of peak areas between 8.10min-10.40min was counted.

[0107] (2) Determination of standard curve

[0108] Preparation of standard solution: Accurately weigh 84.0 mg of sucrose standard, add appropriate amount of ultrapure water to fully dissolve, transfer to a 5 mL volumetric flask, add ultrapure water to dilute to the scale, shake up and down to obtain a 16.8 mg / mL standard solution.

[0109] Preparation of sample solution: 1600μL, 800μL, 400μL, 200μL, 100μL, 50μL, 25μL, 12.5μL, 6.25μL, 3.13μL of standard solution were taken respectively, and diluted to 1600μL with ultrapure water, and the solution was prepared in triplicate to form a series of sucrose solutions of different concentrations. After thorough mixing, the solution was centrifuged at 12000rpm, and the supernatant was taken and added to the liquid phase vial for liquid phase detection under the above conditions. The standard curve of sucrose concentration and peak area was established with the concentration of sucrose standard solution as the horizontal axis and the peak area as the vertical axis, and the regression equation was established.

[0110] (3) Preparation of test solution

[0111] Weigh 1.0g of wolfberry sample accurately, place it in a round-bottom flask, add 15mL of pure water, heat and reflux in a 70℃ water bath for 1.5h, filter after cooling, add 15mL of pure water to the residue for secondary extraction, combine the secondary filtrates and transfer them to a 50mL volumetric flask, add pure water to the scale, and shake upside down. Accurately transfer 2mL of filtrate and blow dry in a 50℃ water bath with nitrogen. Add 1mL of the prepared 70% ethanol solution to the dried sample, vortex thoroughly to mix, centrifuge at 12000rpm for 10min, carefully aspirate 800μL of the supernatant, blow dry in a 50℃ water bath with nitrogen, add 500μL of ultrapure water to the sample, vortex to mix, and obtain.

[0112] 1.2.2 Methodological investigation

[0113] (1) Precision inspection

[0114] Prepare the test solution according to the test solution preparation method in 1.2.1. Inject the sample 6 times continuously according to the HPLC conditions in 1.2.1 to examine the precision and calculate the RSD.

[0115] (2) Repeatability study

[0116] Prepare the test solution according to the test solution preparation method in 1.2.1, in 6 replicates, and perform the determination according to the HPLC conditions in 1.2.1. Investigate the repeatability of the sample and calculate the RSD.

[0117] (3) Stability investigation

[0118] Prepare the test solution according to the test solution preparation method in 1.2.1, and measure it at 0, 3, 6, 9, 12, and 24 h according to the HPLC conditions in 1.2.1 to examine the stability of the sample and calculate the RSD.

[0119] (4) Investigation of sample recovery rate

[0120] Sample content determination: Prepare the test solution according to the test solution preparation method under 1.2.1, in triplicate, and determine according to the HPLC conditions under 1.2.1.

[0121] Recovery rate investigation of sample addition: accurately weigh 1.0 g of wolfberry powder, add 50%, 100%, and 150% sucrose reference substances respectively in 9 parallel portions, with 3 parallel portions in each group. Prepare the test solution according to the test solution preparation method under 1.2.1, determine the content according to the HPLC conditions under 1.2.1, and calculate the recovery rate and RSD.

[0122] 1.2.3 Single factor experimental investigation

[0123] 1.0 g of wolfberry powder was accurately weighed and placed in a round-bottom flask. Distilled water was used as the extraction solvent and reflux extraction was performed in a water bath. The effects of ethanol precipitation concentration, extraction times, extraction temperature, extraction time, and solid-liquid ratio on the yield of wolfberry oligosaccharides were investigated.

[0124] (1) Effect of ethanol precipitation concentration on the yield of wolfberry oligosaccharides

[0125] Accurately weigh 1.0 g of wolfberry sample, place it in a round-bottom flask, extract twice at a solid-liquid ratio of 15 mL / g, heat under reflux at 70°C in a water bath for 1.5 h, treat the sample according to the method under 1.2.1-1.2.3, select 60%, 65%, 70%, 75%, 80%, and 85% ethanol concentration for alcohol precipitation, and perform HPLC-RID liquid phase detection to analyze the effect of ethanol precipitation concentration on the yield of wolfberry oligosaccharides.

[0126] (2) Effect of extraction times on the yield of wolfberry oligosaccharides

[0127] Accurately weigh 1.0 g of wolfberry sample and place it in a round-bottom flask. At a solid-liquid ratio of 15 mL / g and reflux at 70°C in a water bath for 1.5 h, extract it once, twice, and three times respectively. Treat the sample according to the methods under 1.2.1-1.2.3, and perform HPLC-RID liquid phase detection to analyze the effect of extraction times on the yield of wolfberry oligosaccharides.

[0128] (3) Effect of extraction temperature on the yield of wolfberry oligosaccharides

[0129] Accurately weigh 1.0 g of wolfberry sample and place it in a round-bottom flask. At a solid-liquid ratio of 15 mL / g, heat under reflux in a water bath at 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 1.5 h, respectively, and extract twice. Treat the sample according to the methods under 1.2.1-1.2.3, and perform HPLC-RID liquid phase detection to analyze the effect of extraction temperature on the yield of wolfberry oligosaccharides.

[0130] (4) Effect of extraction time on the yield of wolfberry oligosaccharides

[0131] Accurately weigh 1.0 g of wolfberry sample and place it in a round-bottom flask with a solid-liquid ratio of 15 mL / g. Heat and reflux in a 70°C water bath for 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, and 3 h. Extract twice. Treat the sample according to the method under 1.2.1-1.2.3, and perform HPLC-RID liquid phase detection to analyze the effect of extraction time on the yield of wolfberry oligosaccharides.

[0132] (5) Effect of solid-liquid ratio on the yield of wolfberry oligosaccharides

[0133] Accurately weigh 1.0 g of wolfberry sample and place it in a round-bottom flask. Heat under reflux in a 70°C water bath at a solid-liquid ratio of 5 mL / g, 10 mL / g, 15 mL / g, 20 mL / g, 25 mL / g, and 30 mL / g, respectively. Extract twice. Treat the sample according to the method under 1.2.1-1.2.3, and perform HPLC-RID liquid phase detection to analyze the effect of the solid-liquid ratio on the yield of wolfberry oligosaccharides.

[0134] 1.2.4 Optimization of extraction of wolfberry oligosaccharides by response surface analysis

[0135] According to the results of the single factor experiment, three factors, namely, extraction temperature, extraction time and solid-liquid ratio, were selected, and the three levels with the highest yield of wolfberry oligosaccharides were selected. The three-factor and three-level response surface experiment was designed using Design-Expert 8.0.6 software to investigate the effects of the three factors on the yield of wolfberry oligosaccharides. The response surface factors and level design are shown in Table 1. The corresponding predictions by the quadratic polynomial are as follows:

[0136]

[0137] Where Y is the response value (i.e., the extraction rate of wolfberry oligosaccharides), a0 is the coefficient constant, and a i , a ii and a ij are the coefficients of the linear term, quadratic term and interaction term, respectively, X i and X j Represent two different variables (i≠j).

[0138] Table 1 Factor level table of response surface design for extracting wolfberry oligosaccharides

[0139]

[0140]

[0141] 1.3 Experimental Results

[0142] 1.3.1 Establishment of standard curve

[0143] By performing HPLC-RID liquid phase analysis on sucrose standard solutions of different concentrations, a standard curve of the relationship between the concentration of sucrose standard solution and the peak area was established. The linear regression equation between the concentration of sucrose standard and the peak area was: y = 124133x + 17134, R 2 =0.9995. The linear range is 0.03-16.80 mg / mL.

[0144] 1.3.2 Methodological investigation

[0145] (1) Precision inspection

[0146] The prepared wolfberry oligosaccharide sample was injected 6 times continuously, and the sum of the peak areas from 8.10min to 10.40min was calculated. The results are shown in Table 2. The RSD value was 0.35% < 3%, indicating that the instrument had good precision.

[0147] Table 2 Precision test

[0148]

[0149] (2) Repeatability study

[0150] Six samples of wolfberry oligosaccharide were prepared in parallel, and the sum of the peak areas from 8.10min to 10.40min was calculated by liquid phase analysis. The results are shown in Table 3. The RSD value was 2.03% < 3%, indicating that the method had good reproducibility.

[0151] Table 3 Repeatability test

[0152]

[0153]

[0154] (3) Stability investigation

[0155] The prepared wolfberry oligosaccharide samples were measured at 0, 3, 6, 9, 12, and 24 hours, and the liquid phase analysis was performed to calculate the sum of the peak areas from 8.10 min to 10.40 min. The results are shown in Table 4. The RSD value was 2.59% < 3%, indicating that the sample extracted by this method was stable within 24 hours.

[0156] Table 4 Stability test

[0157]

[0158] (4) Investigation of sample recovery rate

[0159] Three samples of wolfberry oligosaccharide were prepared in parallel, and the sum of the peak areas of 8.10min-10.40min was calculated by liquid phase analysis, and the sample content was calculated to be about 3.73mg. 1.0g of wolfberry powder was accurately weighed, and 9 parallel samples were added with 50%, 100%, and 150% sucrose reference substances, respectively, and 3 parallel samples were added in each group. The sum of the peak areas of 8.10min-10.40min was calculated, and the recovery rate and RSD were calculated. The results are shown in Table 5. The sample recovery rates were all between 95% and 105%, and RSD = 1.75% < 3%, indicating that the method had good accuracy.

[0160] Table 5 Sample recovery test

[0161]

[0162]

[0163] 1.3.3 Single factor experimental investigation

[0164] (1) Effect of ethanol precipitation concentration on the yield of wolfberry oligosaccharides

[0165] In order to enrich oligosaccharides as much as possible and eliminate the influence of polysaccharides in the water extract on liquid phase detection, the alcohol precipitation method was considered to remove the polysaccharide components in the water extract as much as possible and enrich the oligosaccharide components to facilitate subsequent liquid phase detection. This experiment selected 6 ethanol concentration gradients from 60% to 85% for alcohol precipitation of wolfberry water extract. The results are as follows Figure 1 As shown in the figure, when the concentration of ethanol was 60%-70%, the yield of wolfberry oligosaccharides only decreased by about 0.12%, and when the concentration of ethanol was 70%-85%, the yield of wolfberry oligosaccharides decreased significantly. Therefore, 70% ethanol concentration was selected to precipitate the water extract.

[0166] (2) Effect of extraction times on the yield of wolfberry oligosaccharides

[0167] Increasing the number of extractions is a common method to increase the yield of medicinal materials, but it will also increase the time, manpower and other costs. This experiment investigated the yield of oligosaccharides by extracting wolfberry powder once, twice and three times under the same conditions. The results are as follows: Figure 2 As shown in the figure, the yield of wolfberry oligosaccharides extracted twice was 5.26%, which was 4.02% higher than that extracted once, and the yield of 5.76% extracted three times was not significantly increased compared with that extracted twice. After comprehensive consideration, extraction twice was selected as the condition for subsequent investigation.

[0168] (3) Effect of extraction temperature on the yield of wolfberry oligosaccharides

[0169] The extraction temperature has a potential impact on the transport and solubility of the solute. The appropriate extraction temperature will promote the release of the solute and increase the solubility of the solute in the solvent, while too high a temperature may destroy the structure of the target compound. This experiment investigated the effects of six temperature gradients between 50℃ and 100℃ on the yield of wolfberry oligosaccharides. The results are as follows: Figure 3 As shown in the figure, the yield of wolfberry oligosaccharides increases at 50℃-80℃, reaching the highest at 80℃, and the yield of wolfberry oligosaccharides reaches 5.89% at 80℃. As the temperature continues to rise, the yield of wolfberry oligosaccharides begins to decline, indicating that wolfberry oligosaccharides may degrade with the increase of temperature, causing the yield of wolfberry oligosaccharides to decrease. Therefore, 70℃-90℃ was selected as the factor level for subsequent response surface investigation.

[0170] (4) Effect of extraction time on the yield of wolfberry oligosaccharides

[0171] Appropriate extraction time can promote sufficient contact between medicinal materials and solvents. This experiment investigated the effects of six time gradients between 0.5h and 3h on the yield of wolfberry oligosaccharides. Figure 4 As shown in the figure, as the extraction time increases, the yield of wolfberry oligosaccharides increases rapidly. After reaching the highest point at 1h of extraction time, it decreases slightly and then decreases rapidly as time continues to increase. This may be because wolfberry oligosaccharides can diffuse quickly into the extraction solvent, and too long extraction time will lead to a decrease in the yield of wolfberry oligosaccharides. Therefore, 0.5h-1.5h was selected as the factor level for subsequent response surface investigation.

[0172] (5) Effect of solid-liquid ratio on the yield of wolfberry oligosaccharides

[0173] Within a certain range, increasing the solid-liquid ratio is beneficial for the extraction solvent to completely infiltrate the medicinal materials, allowing the medicinal materials to fully swell, which is beneficial to increase the contact area between the medicinal materials and the extraction solvent, thereby improving the yield. This experiment investigated the effects of six solid-liquid ratio gradients between 5mL / g and 30mL / g on the yield of wolfberry oligosaccharides. The results are as follows Figure 5 As shown in the figure, as the solid-liquid ratio increases, the yield of wolfberry oligosaccharides gradually increases. It is speculated that increasing the solid-liquid ratio may help to completely infiltrate the medicinal materials, reduce the viscosity of the water extract, and thus promote the dissolution of the target product. Since the solid-liquid ratio of 30mL / g is only increased by 0.7% compared with 25mL / g, and a higher solid-liquid ratio will bring unnecessary investment to subsequent steps such as concentration, 15mL / g-25mL / g is selected as the subsequent response surface investigation factor level.

[0174] 1.3.4 Optimization of extraction of wolfberry oligosaccharides by response surface analysis

[0175] (1) Establishment and analysis of regression equation

[0176] Based on the results of the single-factor experiment, the three factors of extraction temperature, extraction time, and solid-liquid ratio were used as independent variables, and the yield of wolfberry oligosaccharides was used as the response value. The Box-Behnken factorial design (BBD) was used, and the three-factor three-level experimental design was performed using Design-Expert8.0.6 software, including 17 groups of experimental schemes. According to the experimental scheme, wolfberry powder was extracted, liquid phase detection was performed, and the yield of wolfberry oligosaccharides was calculated. The experimental results are shown in Table 6.

[0177] The experimental results were analyzed using Design-Expert 8.0.6 software, and a quadratic multinomial regression model of the yield of wolfberry oligosaccharides (Y) on extraction temperature (X1), extraction time (X2), and solid-liquid ratio (X3) was obtained:

[0178] Y=7.29+0.22X1+0.46X2+0.32X3+0.047X1X2-0.053X1X3-0.053X2X3-0.67X1 2 -1.12X2 2 -0.10X3 2

[0179] Table 6 Experimental design of independent variable response

[0180]

[0181] (2) Response surface variance analysis

[0182] The experimental data were statistically analyzed and analyzed by variance analysis using Design-Expert 8.0.6 software. The results are shown in Table 7 and Figure 6 As shown. Figure 6 (a) It can be seen that the residual normal probability distribution is close to a straight line, which meets the normal hypothesis, indicating that the regression model has a good fit. Figure 6 (b) It can be seen that the residual is randomly distributed, given by Figure 6 (c) It can be seen that the predicted value is well correlated with the experimental value. As can be seen from the table, the model F value is 147.46, P < 0.01, and the difference is significant, indicating that the regression model has a high significance, and only less than 0.01% of the probability is caused by noise. The F value of the lack of fit term of the regression model is 0.016, P = 0.6800 > 0.05, and the lack of fit term is not significant, indicating that the lack of fit is not significant relative to the pure error, and the regression model is sufficient for predicting variation. Model correlation coefficient R 2 =0.9948, corrected correlation coefficient R adj 2 =0.9880, indicating that there is a good correlation between the experimental value and the model prediction value. The coefficient of variation CV value is 1.40%, indicating that the regression model has high precision and good reliability.

[0183] The significance of the influence between different variables and response values ​​in the regression equation is related to the size of the F value. As shown in Table 7, the influence of the three factors on the yield of wolfberry oligosaccharides is in the following order: X2>X3>X1, that is, extraction time>solid-liquid ratio>extraction temperature.

[0184] Table 7 Results of variance analysis of quadratic polynomial fitting for wolfberry oligosaccharide yield

[0185]

[0186]

[0187] Note: ** indicates a highly significant difference (P<0.01), * indicates significant difference (P<0.05)

[0188] (3) Effects of interactive factors on the yield of wolfberry oligosaccharides

[0189] In order to study the effects of extraction temperature, extraction time, solid-liquid ratio and their interaction on the yield of wolfberry oligosaccharides, Design-Expert 8.0.6 software was used to draw 3D response surface diagrams and contour diagrams to show the relationship between the yield of wolfberry oligosaccharides and any two independent variables. The three-dimensional response surface diagram and two-dimensional contour diagram were drawn under the condition of controlling the third factor to 0 level. Figure 7 The interaction between variables can be observed more intuitively.

[0190] (4) Response surface experimental model verification

[0191] Through comprehensive analysis of regression equation and response surface, the optimal extraction conditions of wolfberry oligosaccharide were obtained as follows: extraction temperature 81.31℃, extraction time 1.09h, solid-liquid ratio 25mL / g. In order to facilitate operation, the optimal extraction conditions were adjusted to: extraction temperature 81.3℃, extraction time 65min, solid-liquid ratio 25mL / g. The extraction process of wolfberry oligosaccharide was verified under these conditions. The experiment was repeated three times, and the extraction yield of wolfberry oligosaccharide was 7.62±0.08%, which was close to the predicted value of 7.55%. This shows that the model prediction is relatively accurate.

[0192] 2 Extraction, separation, purification and structural identification of wolfberry oligosaccharides

[0193] Clarifying the structural information of the oligosaccharide components contained in wolfberry is the basis for the quality control of medicinal materials and the study of pharmacological activity. This section mainly uses the extraction method optimized by response surface methodology to extract wolfberry oligosaccharides, and further separates and purifies the oligosaccharides through activated carbon column chromatography and CAPCELL PAK C18-AQ preparative column to obtain pure oligosaccharides. The oligosaccharide structure is determined by HPLC-QTOF-MS, monosaccharide composition analysis and NMR analysis. And compare it with the liquid phase results of the standard to determine the ownership of the common peaks of the oligosaccharide characteristic spectrum.

[0194] 2.1 Experimental methods

[0195] 2.1.1 Extraction of total oligosaccharides from wolfberry

[0196] Weigh 500 g (passed through a 80-mesh sieve) of wolfberry medicinal materials from batch S5 (Jiuquan, Gansu), and extract the wolfberry medicinal materials using the extraction method optimized by response surface, i.e., add 12.5 L of water, reflux extraction at 81.3 °C for 65 min, repeat the extraction twice, combine the filtered extracts, concentrate under reduced pressure to about 5 L, centrifuge at 3600 rpm for 10 min in a large centrifuge, discard the residue, add about 12 L of anhydrous ethanol to the supernatant, stir well and let stand overnight, centrifuge at 3600 rpm for 10 min in a large centrifuge, collect the supernatants, combine them, concentrate under reduced pressure to free of ethanol, and freeze-dry to obtain total wolfberry oligosaccharides.

[0197] 2.1.2 Isolation and purification of wolfberry oligosaccharides

[0198] Boil 200g of granular activated carbon with 20% acetic acid for half an hour, then wash with water until neutral, and then mix it with an equal weight of diatomaceous earth with water to form a slurry. Pour it into a chromatography column and wait for it to settle naturally. Cover the carbon surface with a piece of filter paper and rinse with distilled water until the carbon surface no longer sinks. When the water on the carbon surface is reduced to a thin layer, you can load the sample.

[0199] Weigh 5g of total oligosaccharide sample of wolfberry, add 100mL of distilled water to fully dissolve, centrifuge at 10000rpm for 15min, and gently add the supernatant to the column. After natural sedimentation, use distilled water, 20% ethanol solution, and 50% ethanol solution as mobile phases for gradient elution. After the phenol-sulfuric acid method detects that there is no sugar effluent, switch to the next gradient, and concentrate the eluate of each part in a 60℃ water bath under reduced pressure and freeze-dry. The oligosaccharide component is mainly concentrated in the 20% ethanol elution part, named LBOS-20.

[0200] LBOS-20 was further purified using a CAPCELL PAK C18-AQ (250×10 mm, 5 μm) preparative column, ultrapure water as the mobile phase, a flow rate of 2 mL / min, a column oven temperature of 30±0.1°C, an injection volume of 50 μL, and a sample concentration of 20 mg / mL. Agilent 1100 high performance liquid chromatograph was used for online detection with a RID differential detector, and the peaks were collected and freeze-dried according to the elution results.

[0201] 2.1.3 Analysis of monosaccharide composition of wolfberry oligosaccharides

[0202] After the oligosaccharides are completely hydrolyzed into monosaccharides by trifluoroacetic acid, hydrochloric acid or sulfuric acid, they can be reduced to sugar alcohols by reducing agents such as sodium borohydride, and then acetylated to sugar alcohol acetate by acetic anhydride. After chloroform extraction, they are analyzed by GC-MS and compared with the chromatographic peaks of monosaccharide standards to determine the monosaccharide composition of the oligosaccharides.

[0203] Take about 1 mg of sample and place it in a 5 mL ampoule, add 1 mL of 2 M trifluoroacetic acid (TFA) solution to dissolve; seal it and place it in a 120 ° C oven for hydrolysis for 2 h; take it out, cool it to room temperature, absorb 200 μL of the hydrolyzate into a 15 mL test tube, add 200 μL of 2 M NaOH solution to neutralize it, and then add 1 mL of newly prepared 0.5 M NaBH4-DMSO solution, vortex mix, and reduce in a 40°C water bath for 90 minutes; take out, add 100 μL of glacial acetic acid dropwise, shaking while adding until no bubbles appear, add 200 μL of 1-methylimidazole and 1 mL of acetic anhydride, vortex mix, and acetylate in a 40°C water bath for 10 minutes; add 2 mL of distilled water and vortex mix to terminate the reaction; take out, after cooling to room temperature, add 1.5 mL of chloroform, vortex mix to extract the acetylated product, after stratification, carefully aspirate the upper aqueous layer, add 2 to 3 mL of distilled water to the lower solution and continue washing for 3 times; aspirate the lower solution, dry it over anhydrous sodium sulfate, place it in a sample bottle, and perform GC-MS detection and analysis.

[0204] GC-MS analysis conditions:

[0205] Gas phase part: chromatographic column Agilent DM-5MS (60m×0.25mm×0.25μm); carrier gas is helium, flow rate is 1mL / min; injection volume is 1μL; injection port ion source temperature is 250℃; column oven program temperature rise: starting temperature 140℃, increase to 192℃ at 4℃ / min, increase to 204℃ at 1℃ / min, keep warm for 4min, increase to 215℃ at 1℃ / min, keep warm for 4min, increase to 250℃ at 5℃ / min, keep temperature for 9min, total 60min.

[0206] Mass spectrometry: EI source, 70 eV; ion source temperature 230°C; quadrupole 150°C; scanning range: m / z = 40 to 500; mass spectrometry on time: 8 to 60 min.

[0207] 2.1.4 HPLC-QTOF-MS analysis of wolfberry oligosaccharides

[0208] HPLC-QTOF-MS analysis of each purified part of LBOS-20 can obtain the molecular weight of each purified part, and thus calculate the degree of polymerization of each purified part. Chromatographic column: Prevail Carbohydrate ES (250×4.6mm, 5μm); mobile phase: acetonitrile-30mM ammonium formate (+0.3% formic acid) aqueous solution; flow rate: 0.8mL / min; column temperature: 45℃, gradient elution program is shown in Table 8. In negative ion mode, capillary voltage: 3.5kV, nozzle voltage 1kV, drying gas temperature 320℃, drying gas flow rate 8L / min, nebulizer pressure: 35psi, fragmentation voltage 175V, scanning range m / z 50-1700Da, reference ion m / z: 112.9855, 1033.9881.

[0209] Table 8 Gradient elution program

[0210]

[0211] 2.1.5 Determination of Lycium barbarum Oligosaccharide by Nuclear Magnetic Resonance Spectroscopy

[0212] Take about 20 mg of each purified part of LBOS-20 sample, add 500 μL of heavy water to dissolve, centrifuge at 10000 rpm for 10 min, and draw the supernatant into a 5 mm NMR tube for 1 H. 13 C. 13 C DEPT 135 NMR detection.

[0213] 2.2 Experimental Results

[0214] 2.2.1 Extraction of total oligosaccharides from wolfberry

[0215] 500g of Chinese wolfberry fruit was extracted by water, precipitated by alcohol, concentrated and freeze-dried by response surface optimization, and 53.6g of total wolfberry oligosaccharides were obtained with a yield of 10.7%. The total wolfberry oligosaccharides were dark red-brown and easily absorbed water.

[0216] 2.2.2 Isolation and purification of wolfberry oligosaccharides

[0217] The total oligosaccharides of wolfberry were first separated by activated carbon-diatomite column chromatography, and then eluted by ethanol solutions of different concentrations to obtain three groups of fractions: LBOS-W, LBOS-20 and LBOS-50. The yields were 3.17%, 0.98% and 0.42%, respectively. CAPCELL PAK C18-AQ (250×10mm, 5μm) preparative column was used with ultrapure water as the mobile phase to further purify LBOS-20. The elution curve obtained is as follows: Figure 8 As shown, they were collected and freeze-dried respectively, and marked with serial numbers. The detection was carried out under the established wolfberry oligosaccharide characteristic spectrum HPLC-CAD liquid phase conditions and compared with the standard. STD-1 contained glucose, sucrose, and sugar cane oligosaccharide DP3-6, and STD-2 contained fructose, melibiose, and raffinose. The results are shown in Fig. 9 shown.

[0218] 2.2.3 Analysis of monosaccharide composition of wolfberry oligosaccharides

[0219] The purified oligosaccharide products were completely acid-hydrolyzed and then analyzed for monosaccharide composition. By comparing the peak time with that of the standard, it was found that LBOS-20-1, LBOS-20-3, LBOS-20-5, LBOS-20-6, and LBOS-20-7 were composed of mannose and glucose, LBOS-20-2 was composed of glucose and galactose, and LBOS-20-4 was composed of mannose, glucose, and galactose.

[0220] 2.2.4 Lycium barbarum oligosaccharide HPLC-QTOF-MS analysis + NMR spectroscopy analysis

[0221] Each purified part of LBOS-20 was subjected to HPLC-QTOF-MS detection and analysis to obtain the quasi-molecular ion peak [MH]- of each part and to calculate the degree of polymerization of each purified part by molecular formula. 1 H. 13 C. 13 C DEPT135NMR analysis, combined with the monosaccharide composition analysis results and the molecular weight and degree of polymerization information obtained by QTOF-MS analysis, can be used to perform structural analysis and common peak attribution for each purified part. The results are shown in Table 9.

[0222] Table 9 HPLC-QTOF-MS results of each purified part of LBOS-20

[0223]

[0224]

[0225] According to the prior art literature, LBOS-20-1 is sucrose; LBOS-20-2 is melibiose; LBOS-20-3 is kestose; LBOS-20-4 is raffinose; LBOS-20-5 is kestose; LBOS-20-6 is kestose pentose; and LBOS-20-7 is kestose hexaose.

[0226] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing wolfberry oligosaccharide, characterized in that: The method comprises the following steps: (1) Weighing an appropriate amount of wolfberry medicinal material, adding 25 times the amount of water, heating and refluxing at 81.3° C. for 65 minutes, combining the filtered extracts, concentrating to a certain volume under reduced pressure and centrifuging, discarding the drug residue, adding ethanol to the supernatant for precipitation, stirring well and standing overnight, centrifuging, collecting the supernatants, combining them, concentrating under reduced pressure until there is no ethanol, and freeze-drying to obtain wolfberry total oligosaccharides; and (2) Weighing an appropriate amount of the total wolfberry oligosaccharides, adding water to fully dissolve and centrifuging, adding the supernatant to an activated carbon-diatomaceous earth column, and after natural sedimentation, sequentially performing gradient elution with different mobile phases, wherein the different mobile phases are distilled water, 15% to 25% ethanol solution and 45% to 55% ethanol solution, and after the phenol-sulfuric acid method detects that no sugar is eluted, switching to the next gradient, and concentrating the eluate of the 15% to 25% ethanol solution under reduced pressure in a water bath and then freeze-drying to obtain crude wolfberry oligosaccharides, and performing column chromatography on the crude wolfberry oligosaccharides through an ODS-AQ preparative column to separate and purify the wolfberry oligosaccharides; The wolfberry oligosaccharides are sucrose, melibiose, raffinose, kestose, kestetetrose, kestetetetrose, kestetetrose and kestetetrose.

2. The method according to claim 1, characterized in that The wolfberry is the dried mature fruit of cylindrical wolfberry, soft-stem wolfberry, black-fruited wolfberry, calyx-truncate wolfberry or Ningxia wolfberry.

3. The method according to claim 2, characterized in that The wolfberry is produced in Ningxia, Gansu, Inner Mongolia, Xinjiang and / or Qinghai.

4. The method according to claim 3, characterized in that The wolfberry is the dried mature fruit of Ningxia wolfberry.

5. The method according to claim 1, characterized in that Step (1) of the method includes any one or more of the following items [1] to [8]: [1] crushing the wolfberry medicinal material; [2] Passing the crushed wolfberry medicinal material through a 60-100 mesh sieve; [3] The water is distilled water or deionized water; [4] The heating reflux extraction operation is repeated 2 to 4 times; [5] The certain volume is 1 / 10 to 1 / 2 of the volume of the water added; [6] The centrifugation condition is 2000 rpm to 6000 rpm for 5 to 30 minutes; [7] The amount of ethanol is 1 to 5 times the volume of the supernatant; [8] The concentration of the ethanol used in the ethanol precipitation is 60% to 100%.

6. The method according to claim 5, characterized in that In item [2] of the method, the ground wolfberry medicinal material is passed through an 80-mesh sieve.

7. The method according to claim 5, characterized in that In item [4] of the method, the heating reflux extraction operation is repeated twice.

8. The method according to claim 5, characterized in that In item [5] of the method, the certain volume is 2 / 5 of the volume of the added water.

9. The method according to claim 5, characterized in that In item [6] of the method, the centrifugation condition is centrifugation at a rotation speed of 3000 rpm to 5000 rpm for 8 to 20 minutes.

10. The method according to claim 9, characterized in that The centrifugation condition is centrifugation at 3600 rpm for 10 minutes.

11. The method according to claim 5, characterized in that In item [7] of the method, the amount of ethanol is 2.4 times the volume of the supernatant.

12. The method according to claim 5, characterized in that In item [8] of the method, the concentration of the ethanol used in the ethanol precipitation is 100%.

13. The method according to claim 1, characterized in that The activated carbon-diatomaceous earth column used in step (2) of the method is prepared by the following method: Weigh an appropriate amount of granular activated carbon, boil it with 10% to 30% acetic acid for 10 to 60 minutes, then wash it with water until it is neutral, and then mix it with an equal weight of diatomaceous earth with water to form a slurry, pour it into a chromatography column, wait for it to settle naturally, cover the carbon surface with a piece of filter paper, rinse with distilled water until the carbon surface no longer drops, and when the water on the carbon surface is reduced to 1 μm to 10 μm, the activated carbon-diatomaceous earth column is obtained.

14. The method according to claim 13, characterized in that Weigh an appropriate amount of granular activated carbon and boil it with 20% acetic acid for 30 minutes.

15. The method according to claim 1, characterized in that Step (2) of the method further comprises the following steps: A 250×10 mm, 5 μm CAPCELL PAK C18-AQ preparative column was used and ultrapure water was used as the mobile phase to purify the crude wolfberry oligosaccharide and detect it online. The crude wolfberry oligosaccharide was collected and freeze-dried according to the peak results to obtain the wolfberry oligosaccharide.

16. The method according to claim 15, characterized in that Step (2) of the method includes any one or more of the following items [1] to [6]: [1] The water is distilled water or deionized water; [2] The amount of water is 1 to 80 times the amount, in L / kg; [3] The centrifugation condition is 6000 rpm to 20000 rpm for 5 to 30 minutes; [4] The different mobile phases are distilled water, 20% ethanol solution and 50% ethanol solution; [5] The temperature of the water bath is 40°C to 100°C; [6] The wolfberry crude oligosaccharide is the elution portion of 20% ethanol solution.

17. The method according to claim 16, characterized in that In item [2] of the method, the amount of water used is 10 to 50 times the amount, in units of L / kg.

18. The method according to claim 17, characterized in that The amount of water used is 20 times the amount, and the unit is L / kg.

19. The method according to claim 16, characterized in that In item [3] of the method, the centrifugation condition is centrifugation at a rotation speed of 8000 rpm to 15000 rpm for 8 to 20 minutes.

20. The method according to claim 19, characterized in that The centrifugal condition is centrifugation at 10000 rpm for 15 minutes.

21. The method according to claim 16, characterized in that In item [5] of the method, the temperature of the water bath is 50°C to 80°C.

22. The method according to claim 21, characterized in that The temperature of the water bath was 60°C.

23. The method according to claim 15, characterized in that The flow rate of the CAPCELL PAK C18-AQ preparative column is 1-3 mL / min, the column oven temperature is 25±0.1° C. to 35±0.1° C., the injection volume is 45-55 μL, and the sample concentration is 15-25 mg / mL.

24. The method according to claim 23, characterized in that The flow rate of the CAPCELL PAK C18-AQ preparative column was 2 mL / min, the column oven temperature was 30±0.1° C., the injection volume was 50 μL, and the sample concentration was 20 mg / mL.

25. The method according to claim 15, characterized in that The instruments used for the online detection are Agilent 1100 high performance liquid chromatograph and RID differential detector.

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