A quality evaluation method and application for processed Polygonatum multiflorum

Abstract

This invention relates to the field of analytical testing technology, and more specifically, to a method and application for quality evaluation of processed Polygonatum cyrtonema. The method for quality evaluation of processed Polygonatum cyrtonema includes the following steps: S1, analyzing the molecular weight of polysaccharides in processed Polygonatum cyrtonema using MEC-RI technology to construct a MEC-RI fingerprint; S2, determining the contents of free D-glucose, D-fructose, and 12 free amino acids in processed Polygonatum cyrtonema using LEC-RI and HILIC-MS / MS technologies; S3, establishing a quality evaluation method for processed Polygonatum cyrtonema based on the similarity of the MEC-RI fingerprint and the results of the contents of free D-glucose, D-fructose, and free amino acids. The analytical method of this invention has high specificity and accuracy, and has strong industry and market application value.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and more specifically, to a method and application for quality evaluation of processed Polygonatum odoratum. Background Technology

[0002] Polygonatum sibiricum Red. is a herbaceous plant belonging to the genus Polygonatum in the family Liliaceae. It is mainly distributed in Northeast China and is divided into three types: Polygonatum sibiricum, Polygonatum yunnanense, and Polygonatum multiflorum. It has a sweet taste and neutral properties, and enters the spleen, lung, and kidney meridians. It has the effects of tonifying qi and nourishing yin, strengthening the spleen, benefiting the kidneys, and moistening the lungs. However, taking raw Polygonatum can cause numbness and irritation of the mouth and tongue, so it needs to be processed before use to enhance its efficacy. Steaming is the most common processing method for Polygonatum. As a traditional Chinese medicine that is both food and medicine, its chemical components mainly include polysaccharides, saponins, flavonoids, lignans, amino acids, trace elements, and volatile oils. Among them, Polygonatum polysaccharides are its main components, possessing pharmacological effects such as lowering blood sugar and blood lipids. Polysaccharides are the only quality control component of Polygonatum specified in the 2020 edition of the Chinese Pharmacopoeia. Currently, domestic and international literature reports, as well as the 2020 edition of the Chinese Pharmacopoeia, primarily employ the sulfuric acid-anthrone-UV-Vis spectroscopy method for analyzing the polysaccharide components of Polygonatum cyrtonema. This method uses glucose as a reference standard to determine the polysaccharide content, exhibiting poor specificity. Furthermore, the sample preparation process is cumbersome, involving the use of potentially explosive sulfuric acid and potentially toxic anthrone reagents, raising safety concerns. In addition, existing quality evaluation methods for processed Polygonatum cyrtonema mainly focus on controlling the content of polysaccharides, flavonoids, Polygonatine A, fructose, glucose, total saponins, EBC, and 5-hydroxymethylfurfural. However, these evaluation indicators and methods are still somewhat one-sided and lack sufficient accuracy.

[0003] High-performance gel permeation chromatography (GPC) is a type of size exclusion chromatography (MEC) used for molecular weight fractionation and molecular weight distribution determination of polymers. In this study, the Shodex SUGAR KS-804 column from Showa Denko Scientific Instruments Co., Ltd. (Japan) was used, employing high-performance gel permeation chromatography (GPC) as the packing material, thus possessing size exclusion capabilities. Size exclusion chromatography-refractive index detection (MEC-RI) can be used for qualitative analysis of carbohydrate components.

[0004] Ligand exchange chromatography (LEC) is a separation technique developed based on polymeric resins that utilize ligand exchange interactions. Ligand exchange resins are highly sulfonated cation exchange resins containing group 1, 2, or transition metals. The sulfonic acid groups on the resin can tightly adsorb metal ions onto the column through ionic attraction, preventing elution. For example, the Xtimate Sugar-Ca column used in this study by Shanghai Yuexu Co., Ltd. is a calcium-type sugar column, while the Shodex SUGAR KS-804 column from Showa Denko Scientific Instruments Co., Ltd. in Japan also functions as a sodium-type sugar column due to the presence of sodium ions on its packing material surface. For sugar molecules, each hydroxyl group carries a very weak negative charge, while the hydroxyl groups on the terminal isomeric carbons can be deprotonated, thus acquiring a strong negative charge. The interaction between these negative charges on the sugar molecules and the positive charges of the metal ions on the resin surface allows the sugar to be retained, achieving separation. A weak ionic attraction also exists between water and the metal ions on the column; therefore, water is used as the mobile phase to competitively elute the adsorbed sugars. Ligand exchange chromatography-refractive index detection (LEC-RI) can be used for the quantitative analysis of carbohydrate components.

[0005] Hydrophilic chromatography is a method for separating highly polar compounds. The mobile phase in hydrophilic chromatography consists of an aqueous buffer (<40%) and an organic solvent, while the stationary phase is a strongly hydrophilic polar adsorbent, such as silica-bonded phases, polar polymer packing materials, or ion exchange adsorbents. These stationary phases share the common characteristic of strong interaction with water, thus exhibiting "hydrophilicity," which increases the retention time for highly polar compounds, thereby achieving separation. Hydrophilic chromatography-triple quadrupole mass spectrometry (HILIC-MS / MS) can enable the quantitative analysis of highly polar compounds.

[0006] Traditional Chinese medicine (TCM) fingerprinting technology is currently recognized both domestically and internationally as one of the most effective, accurate, and direct methods for quality control of TCM. It provides a wealth of information, comprehensively reflecting the types and contents of chemical components, and demonstrating the overall effect of TCM components to evaluate quality. It is not only applied to quality control of TCM but also widely used in research on the pharmacodynamic material basis of TCM. Summary of the Invention

[0007] This invention aims to provide a quality evaluation method for processed Polygonatum odoratum based on qualitative analysis using MEC-RI molecular weight and fingerprint spectroscopy, and quantitative analysis using LEC-RI free monosaccharides and HILIC-MS / MS free amino acids. This method is highly specific, more complete, and has strong application value.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for quality evaluation of processed Polygonatum multiflorum includes the following steps:

[0010] S1. Collect MEC-RI chromatograms of processed Polygonatum multiflorum, analyze the molecular weight of free polysaccharides in the sample based on the relationship between retention time and molecular weight function, and construct MEC-RI fingerprint chromatograms using the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines".

[0011] S2. Determine the contents of free D-glucose, D-fructose, and free amino acids in processed Polygonatum odoratum; among which, free amino acids include: L-alanine, L-arginine, L-glutamic acid, L-glycine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, and L-valine.

[0012] S3. Based on the similarity of the fingerprint spectrum of processed Polygonatum multiflorum and the results of the content of free D-glucose, D-fructose and free amino acids, a quality evaluation method for processed Polygonatum multiflorum is established.

[0013] There is no specific order between steps S1 and S2.

[0014] In one preferred embodiment, the method for preparing processed Polygonatum multiflorum includes: adding rice wine to Polygonatum multiflorum for moistening, steaming, and then drying in stages by blowing air; repeating the moistening-steaming-stage drying process 9 times until the Polygonatum turns black, thus obtaining processed Polygonatum multiflorum.

[0015] In one preferred embodiment, the processed Polygonatum odoratum needs to be pulverized and passed through a No. 3 sieve to obtain processed Polygonatum odoratum fine powder.

[0016] In one preferred embodiment, the molecular weight of the processed Polygonatum multiflorum polysaccharide was analyzed by MEC-RI method, and a MEC-RI fingerprint was constructed using the software of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System".

[0017] In one preferred embodiment, the molecular weight analysis of processed Polygonatum multiflorum polysaccharide and the construction of its MEC-RI fingerprint include the following steps:

[0018] (1) Preparation of MEC-RI test sample: Grind the processed Polygonatum odoratum into fine powder, add water, heat under reflux for 120 min, cool, add weight, filter, and the test sample is obtained.

[0019] (2) Preparation of dextran reference standards: Dextran T100 (molecular weight approximately 100,000 Da), dextran T50 (molecular weight approximately 50,000 Da), dextran T20 (molecular weight approximately 20,000 Da), dextran T10 (molecular weight approximately 10,000 Da), dextran T5 (molecular weight approximately 5,000 Da), dextran T2 (molecular weight approximately 2,000 Da), and dextran T1 (molecular weight approximately 1,000 Da) with a concentration of 10 mg / ml were prepared as reference standards using ultrapure water, and ultrapure water was used as a blank reference standard;

[0020] (3) MEC-RI chromatographic conditions: porous gel and sodium-modified polystyrene / divinylbenzene were used as the packing material; ultrapure water was used as the mobile phase; column temperature was 50℃; flow rate was 0.5 ml / min; differential refractive index detector was used at 35℃ and detection time was 30 min.

[0021] (4) MEC-RI analysis: Accurately pipette 10 μl each of blank reference, reference and test sample and inject them into the liquid chromatograph. ① Fit the equation of retention time and molecular weight of the main chromatographic peaks according to the MEC-RI chromatogram of dextran reference and calculate the relative molecular mass of each polysaccharide in the test sample; ② Construct the MEC-RI fingerprint of the test sample.

[0022] In one preferred embodiment, a standard fingerprint is generated based on the MEC-RI fingerprint of a qualified test sample.

[0023] In one preferred embodiment, the contents of free D-glucose and D-fructose in the processed Polygonatum odoratum were determined by LEC-RI method.

[0024] In one preferred embodiment, the method for determining the content of free D-glucose and D-fructose in processed Polygonatum cyrtonema includes the following steps:

[0025] (1) Preparation of LEC-RI test sample: Grind the processed Polygonatum odoratum into fine powder, add water, heat under reflux for 120 min, cool, add weight, filter, and the test sample is obtained; take 1 ml of the test sample, dilute with water to 10 ml, filter, and the test sample is obtained.

[0026] (2) Preparation of D-glucose and D-fructose reference standards: D-fructose and D-glucose at a concentration of 1 mg / ml were prepared using ultrapure water as reference standards, and ultrapure water was used as blank reference standards.

[0027] (3) LEC-RI chromatographic conditions: Calcified polystyrene / divinylbenzene was used as the packing material, and ultrapure aqueous solution was used as the mobile phase; column temperature was 80℃; flow rate was 0.5 ml / min; differential refractive index detector was used, temperature was 35℃, and detection time was 30 min;

[0028] (4) LEC-RI analysis: Accurately pipette blank reference standard, D-glucose, D-fructose reference standard, test solution, and 10 μl of test solution into the liquid chromatograph, collect the LEC-RI chromatogram, and calculate the content of D-glucose or D-fructose based on the peak area information.

[0029] In one preferred embodiment, the content of free amino acids in processed Polygonatum multiflorum was determined by HILIC-MS / MS.

[0030] In one preferred embodiment, the method for determining the content of free amino acids in processed Polygonatum multiflorum includes the following steps:

[0031] (1) Preparation of HILIC-MS / MS test sample: Grind the processed Polygonatum odoratum into fine powder, add water, heat under reflux for 120 min, cool, add weight, filter, and the test sample is obtained.

[0032] (2) Preparation of amino acid reference standards: amino acids with a concentration of 1 μg / ml were prepared using ultrapure water as reference standards, and ultrapure water was used as blank reference standards.

[0033] (3) HILIC-MS / MS chromatographic conditions: Triple-bonded amide-bonded silica gel was used as the stationary phase, and acetonitrile (A)-0.1% formic acid (B) was used as the mobile phase. Gradient elution was performed (0-5 min, 90% A; 5-15 min, 90% A-70% A; 15-20 min, 70% A; 20-21 min, 70% A-90% A; 21-30 min, 90% A), with a flow rate of 0.3 mL / min and a column temperature of 35℃. Triple quadrupole mass spectrometry was used for detection: electrospray ionization (ESI) source, and multiple ion detection (MRM) mode was used for multiple ion scanning of each compound. In positive ion mode, the injection voltage (EP) and collision chamber exit voltage (CXP) were 10.

[0034] (4) HILIC-MS / MS mass spectrometry conditions: In one preferred embodiment, the parent ion (m / z), daughter ion (m / z), debulking voltage (DP), and collision voltage (CE) of each amino acid are shown in Table 1;

[0035] Table 1. MRM conditions for 12 amino acid components in HILIC-MS / MS

[0036]

[0037]

[0038] (5) HILIC-MS / MS analysis: Accurately pipette 1 μl of blank reference standard, amino acid reference standard and test sample into the high performance liquid chromatograph-mass spectrometer, collect the MRM chromatogram of HILIC-MS / MS, and calculate the content based on the peak area.

[0039] In one preferred embodiment, evaluating the quality of processed Polygonatum multiflorum includes the following steps:

[0040] (1) Whether the similarity of the MEC-RI fingerprint spectrum of the processed Polygonatum multiflorum sample is above 0.9;

[0041] (2) Whether the D-glucose content of the test sample is between 50 and 150 mg / g, and whether the D-fructose content of the test sample is between 100 and 200 mg / g;

[0042] (3) Whether the contents of L-alanine, L-arginine, L-glutamic acid, L-glycine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-tyrosine, and L-valine in the test sample are within the range of 0-300 μg / g;

[0043] If all conditions are met, the processed Polygonatum odoratum raw material is deemed qualified; if any one condition is not met, the processed Polygonatum odoratum raw material is deemed unqualified.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] This invention innovatively establishes a quality evaluation method for processed Polygonatum cyrtonema based on MEC-RI molecular weight and fingerprint spectroscopy for qualitative analysis, LEC-RI for free monosaccharides, and HILIC-MS / MS for quantitative analysis of free amino acids. The analytical method of this invention has high specificity and accuracy, uses mainly water and acetonitrile as reagents, requires no sample derivatization, and is simple, reasonable, and feasible. This quality evaluation method for processed Polygonatum cyrtonema is more complete and has strong industry and market application value. Attached Figure Description

[0046] Figure 1 This is the MEC-RI chromatogram of each dextran reference standard obtained in Example 1 of the present invention.

[0047] Figure 2 This is a graph showing the retention time-molecular weight fitting equations of the various dextran reference standards obtained in Example 1 of this invention.

[0048] Figure 3 This is the MEC-RI standard fingerprint spectrum of processed Polygonatum multiflorum obtained in Example 1 of this invention.

[0049] Figure 4 This is the MEC-RI fingerprint of the processed Polygonatum multiflorum sample obtained in Example 1 of this invention.

[0050] Figure 5This is the LEC-RI chromatogram of D-glucose and D-fructose obtained in Example 1 of the present invention.

[0051] Figure 6 This is the LEC-RI chromatogram of the processed Polygonatum multiflorum sample obtained in Example 1 of this invention.

[0052] Figure 7 This is a HILIC-MS / MS-MRM chromatogram of each amino acid reference standard obtained in Example 1 of this invention.

[0053] Figure 8 This is the HILIC-MS / MS-MRM chromatogram of the processed Polygonatum multiflorum sample obtained in Example 1 of this invention. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are illustrative rather than limiting, and will be understood by those skilled in the art. In the description of the present invention, it should be noted that unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Instruments or reagents whose manufacturers are not specified are all commercially available conventional products.

[0055] The main instruments, equipment, materials, and reagents used in the examples are briefly described below:

[0056] The following instruments were used: AB Sciex Exion LC AD ultra-high performance liquid chromatograph (USA); AB Sciex 3500 triple quadrupole mass spectrometer (USA); AB Sciex X-500R time-of-flight quadrupole mass spectrometer (USA); Thermo Fisher Scientific Ultimate liquid chromatograph (equipped with UV detector and differential refractive index detector); and LabTech UV900B UV-Vis spectrophotometer (Beijing).

[0057] Water was ultrapure water (18.2 MΩ); acetonitrile (mass spectrometry grade, 18100306LM01) was purchased from OCEAN PAK; methanol (mass spectrometry grade, 17111204LM01) was purchased from OCEAN PAK; formic acid (88.0%, 176131) was purchased from Fisher Chemical; rice wine (11% ethanol concentration, 20220315) was purchased from Wangzhihe Co., Ltd.; sulfuric acid (95-98%, 20170726) was purchased from Zhuzhou Xingkong Chemical Glass Co., Ltd.; anhydrous ethanol (95%, 20180413) and anthrone (98%, 20200312) were purchased from Sinopharm Chemical Reagent Co., Ltd.; D-fructose (99%, LOT: J31M9R62568) and D(+)-anhydrous glucose (98%, LOT: Y19F1) were also purchased. 1J108781), Dextran T100 (lot number: H29M11B114192), Dextran T50 (LOT: H23S11B125400), Dextran T20 (LOT: M01GS142715), Dextran T10 (LOT: A19GS145781), Dextran T5 (LOT: N08GS166773), Dextran T2 (LOT: H21S8B44410), Dextran T1 (LOT: A14GS15773) 4), L-alanine (98%, LOT: J04GB153374), L-arginine (98%, LOT: H11M10Y82633), L-glutamic acid (99%, LOT: S12A10I85582), glycine (99%, LOT: S29A10I96410), L-histidine (98%, LOT: A13GB144942), L-lysine (98%, LOT: J16GB155185), L-phenylalanine (98%) The following substances were purchased from Shanghai Yuanye Biotechnology Co., Ltd.: L-proline (99%, LOT: Z12O11H127142), L-serine (98%, LOT: J01GB150454), L-threonine (98%, LOT: O10GB162607), L-tyrosine (99%, LOT: H22O11Y128127), and L-valine (98%, LOT: S05J12I135887).

[0058] All samples of Polygonatum cyrtonema were provided by Hunan Yinshuang Biotechnology Co., Ltd., and identified by Professor Wang Zhi of the Department of Medicinal Plant Identification at Hunan University of Traditional Chinese Medicine as dried rhizomes of Polygonatum cyrtonema Hua, a plant of the Liliaceae family. Sample information is detailed in Table 2.

[0059] Table 2 Information on Processed Polygonatum multiflorum Samples

[0060] Place of origin Abbreviation abbreviation Purchase batch number Wild species from Xinhua County, Loudi City, Hunan Province Xinye DHJHJ-XY 20220315 Nanping, Fujian Nanping DHJHJ-NP 20220315 Chenzhou, Hunan Chenzhou DHJHJ-CZ 20220315 Shaoyang, Hunan Shaoyang DHJHJ-SY 20220315 Xiangxi, Hunan Xiangxi DHJHJ-XX 20220315 Kaili, Guizhou Kerry DHJHJ-KL 20220315 Xinhua County, Loudi City, Hunan Province (artificial) Xinlin DHJHJ-XL 20220315 Yueyang, Hunan Yueyang DHJHJ-YY 20220315 Meishan, Sichuan Meishan DHJHJ-MS 20220315 Yuxi, Yunnan Yuxi DHJHJ-YX 20220315 Tongren, Guizhou Tongren DHJHJ-TR 20220315

[0061] The preparation of processed Polygonatum multiflorum is as follows: Weigh approximately 400g of Polygonatum multiflorum, spread it evenly in a 4.0L earthenware pot, add 80g of rice wine, and let it soak for at least 12 hours. Place the soaked Polygonatum multiflorum in a steamer (26cm, three layers) and steam for 4 hours. Then place the Polygonatum multiflorum on a white square iron plate and place it in a forced-air drying oven at 60℃ for at least 12 hours. The water in the steamer is not poured out but used as a soaking solvent. Repeat the "steaming" and "drying" process until the total number of times is 9 (nine steaming and nine processing), until the Polygonatum multiflorum turns black. The processed Polygonatum multiflorum is then obtained.

[0062] The preparation of processed Polygonatum odoratum fine powder is generally as follows: Pulverize processed Polygonatum odoratum and pass it through a No. 3 sieve to obtain processed Polygonatum odoratum fine powder.

[0063] All Polygonatum multiflorum and processed Polygonatum multiflorum were tested according to the Polygonatum multiflorum entry in Part I of the 2020 edition of the Chinese Pharmacopoeia, using the sulfuric acid-anthrone-ultraviolet-visible spectroscopy method (hereinafter referred to as the "Pharmacopoeia Method") to determine the content of Polygonatum multiflorum polysaccharides in processed Polygonatum multiflorum.

[0064] Example 1

[0065] I. A qualitative method for analyzing the molecular weight of processed Polygonatum multiflorum polysaccharides and constructing fingerprint spectra based on MEC-RI, including the following steps:

[0066] (1) Preparation of test solution: Weigh 2.0g of processed polysaccharide powder from 11 origins that have passed the pharmacopoeia test, add 200mL of water, seal tightly, weigh, heat (80℃) under reflux for 120min, cool, weigh again, replenish the lost mass with water, shake well, filter through a 0.22μm microporous membrane to obtain the test sample.

[0067] (2) Preparation of dextran reference solutions: Dextran T100 (molecular weight 100,000), dextran T50 (molecular weight 50,000), T20 (molecular weight 20,000), dextran T10 (molecular weight 100,000), dextran T5 (molecular weight 5,000), dextran T2 (molecular weight 2000), and dextran T1 (molecular weight 1000) with concentrations of 10.1100, 10.3500, 10.2100, 10.3200, 10.0400, 11.3200, and 10.0100 mg / ml were prepared using ultrapure water. Blank reference solutions were prepared simultaneously using the same method.

[0068] (3) MEC-RI chromatographic conditions: porous gel and sodium-modified polystyrene / divinylbenzene were used as the packing material (Shodex SUGAR KS-804, 8.0*300m, Showa Denko Scientific Instruments Co., Ltd., Japan); ultrapure water was used as the mobile phase; column temperature was 50℃; flow rate was 0.5ml / min; differential refractive index detector was used at 35℃ and detection time was 30min.

[0069] (4) MEC-RI analysis: Accurately measure 10 μl of various dextran, blank reference standards, and test sample solutions from different origins, inject them into the liquid chromatograph, and collect the chromatograms of the test samples under the MEC-RI analysis conditions. ① Plot the retention time (min) of the main chromatographic peak of each reference standard as the abscissa (X) and the molecular weight (Da) as the ordinate (Y), and fit the retention time-molecular weight equation according to DPS version 9.05: Molecular weight = 164628014.4796e (-0.552888×保留时间) ① Calculate the molecular weight of the main MEC-RI chromatographic peaks in the test solution according to the equation; ② Using the MEC-RI chromatogram of processed Polygonatum multiflorum from Xinye County, which passed the pharmacopoeia test, as a reference chromatogram, perform full-spectrum peak matching using the average method, and construct the standard MEC-RI fingerprint chromatogram of processed Polygonatum multiflorum using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". The MEC-RI chromatograms of each dextran reference standard are as follows: Figure 1 As shown, the retention time-molecular weight equation for dextran is as follows: Figure 2 As shown, the MEC-RI standard fingerprint spectrum of processed Polygonatum multiflorum is as follows: Figure 3 As shown, the MEC-RI fingerprint spectra of the test samples from various origins are as follows: Figure 4 As shown.

[0070] (5) Precision test: The sample solution from Xinye was injected 6 times consecutively, and the RSD% of the similarity of the MEC-RI fingerprint spectrum was analyzed to test the precision. The result was 0.183%, indicating that the method has good precision.

[0071] (6) Repeatability test: Six sample solutions from Xinye were prepared simultaneously in parallel according to the method in (2). The RSD% of the similarity of the MEC-RI fingerprint spectrum was analyzed to test the repeatability results. The result was 0.293%, indicating that the method has good repeatability.

[0072] (7) Stability test: The test solution from Xinye was injected at 0.0, 1.5, 3.0, 4.5, 6.0, 12, 18 and 24 h after preparation. The RSD% of the MEC-RI fingerprint similarity was analyzed to test the stability. The result was 0.275%, indicating that the method has good stability.

[0073] (8) The MEC-RI fingerprint spectra of processed Polygonatum cyrtonema from 11 production areas were randomly determined according to the above method. The MEC-RI fingerprint spectra of the fine powder of processed Polygonatum cyrtonema from various regions that passed the pharmacopoeia verification are shown in Table 3.

[0074] Table 3 shows the MEC-RI fingerprint similarity of the processed Polygonatum odoratum samples.

[0075]

[0076] The results showed that the similarity of the test samples from the 11 production areas that passed the pharmacopoeia verification was all above 90%.

[0077] For some processed Polygonatum multiflorum that failed the pharmacopoeia verification, the MEC-RI fingerprint spectrum similarity drawn according to this method ranged from 60% to 85%. Therefore, a fingerprint spectrum similarity of 90% or more is one of the standards for passing the test.

[0078] II. Establishment of quantitative analytical methods for determining free monosaccharides in processed Polygonatum cyrtonema using LEC-RI and free amino acids using HILIC-MS / MS, including the following steps:

[0079] (1) Preparation of test solution: Weigh 2.0g of processed polysaccharide powder that has passed the pharmacopoeia test, add 200mL of water, stopper tightly, weigh, heat under reflux for 120min, cool, weigh again, replenish the lost mass with water, shake well, filter through a 0.22μm microporous membrane to obtain the test sample (glucose / amino acid), accurately measure 1ml of the test sample filtrate, place it in a 10ml volumetric flask, add water to the mark, shake well, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the test sample (fructose).

[0080] (2) Preparation of reference solutions: D-glucose, D-fructose, and various amino acids with concentrations of approximately 90–1000 μg / ml and 0–7 μg / ml were prepared using ultrapure water. Blank reference solutions were prepared simultaneously using the same method. See Table 4.

[0081] Table 4 Concentrations of 14 Components

[0082]

[0083] (3) The LEC-RI chromatographic conditions were as follows: calcified polystyrene / divinylbenzene was used as the packing material (Xtimate Sugar-Ca, 5um, 7.8*300mm, Shanghai Yuexu Technology Co., Ltd.), and ultrapure aqueous solution was used as the mobile phase; the column temperature was 80℃; the flow rate was 0.5ml / min; a differential refractive index detector was used, the temperature was 35℃, and the detection time was 30min.

[0084] (4) LEC-RI analysis: Accurately pipette 10 μl of the above series of D-glucose, D-fructose and blank reference standards, inject into the liquid chromatograph, acquire the LEC-RI chromatogram, and determine the corresponding peak area, such as... Figure 5 As shown. A standard curve was plotted with peak area on the ordinate and injection concentration on the abscissa. Separately, 10 μl of the test solution was precisely pipetted into the liquid chromatograph, and a LEC-RI chromatogram was acquired, as shown. Figure 6 As shown. The concentrations of D-glucose and D-fructose in the test solution (glucose, fructose) are read from the standard curve based on the peak area of ​​the chromatogram. The concentration ranges of D-glucose and D-fructose in the qualified sample, as determined by the pharmacopoeia method, are then calculated.

[0085] (5) The chromatographic conditions for HILIC-MS / MS were as follows: triple bonded amide-bonded silica gel was used as the stationary phase (WATERS ACQUITY UPLC@BEH Amid type column, 1.7 μm, 2.1 × 150 mm column), acetonitrile (A)-water (B) was used as the mobile phase, gradient elution (0–5 min, 90% A; 5–15 min, 90% A–70% A; 15–20 min, 70% A; 20–21 min, 70% A–90% A; 21–30 min, 90% A), flow rate 0.3 mL / min, column temperature 35 °C. The mass spectrometry conditions for HILIC-MS / MS were as follows: triple quadrupole mass spectrometer was used for detection: electrospray ionization (ESI) source, multiple ion detection (MRM) mode was used for multiple ion scanning of each compound, positive ion mode: injection voltage (EP) and collision chamber exit voltage (CXP) were 10. The parent ion (m / z), daughter ion (m / z), debulking voltage (DP), and collision voltage (CE) of each analyte are shown in Table 5.

[0086] Table 5. MRM conditions for 12 amino acid components in HILIC-MS / MS

[0087]

[0088]

[0089] (6) HILIC-MS / MS Analysis: Accurately pipette 1 μl of each of the above-mentioned series of amino acid reference solutions and inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument. Acquire the MRM chromatogram of the HILIC-MS / MS instrument and determine the peak area. Figure 7 As shown. A standard curve was plotted with peak area on the ordinate and injection concentration on the abscissa. Separately, 1 μl of the above test solution was precisely pipetted into the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) instrument, and the MRM chromatogram was acquired. The peak area was measured, as shown. Figure 8As shown. Based on the peak area of ​​the chromatographic peak, the concentrations of L-alanine, L-arginine, L-glutamic acid, L-glycine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, and L-valine in the liquid chromatography-mass spectrometry sample were read from the standard curve. The concentration range of each amino acid in the qualified sample that passed the pharmacopoeia test was then calculated. (7) Linearity investigation: Each reference solution was injected, and the linear regression equation and linear range were calculated with the concentration of each component as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 6.

[0090] Table 6 Results of linear relationship investigation of 14 components

[0091]

[0092] (8) Precision test: The test solution (glucose and fructose) from Xinye, which passed the pharmacopoeia test, was injected 6 times consecutively. The RSD% of each component was calculated to test the precision. The results are shown in Table 7.

[0093] (9) Repeatability test: Six samples of test solution and test solution (glucose and fructose) from Xinye County that passed the pharmacopoeia test were prepared simultaneously in parallel according to the method in (2). The RSD% of each component was calculated after injection to test the repeatability results. The results are shown in Table 7.

[0094] (10) Stability study: The test solution and test solution (glucose and fructose) from Xinye, which were tested and qualified according to the pharmacopoeia, were injected at 0.0, 1.5, 3.0, 4.5, 6.0, 12, 18 and 24 h after preparation. The RSD% of each component was calculated to study the stability results, which are shown in Table 7.

[0095] (11) Accurately weigh 1.0 g of the test solution and test solution (glucose, fructose) from Xinye, which are qualified according to the pharmacopoeia method and have known content of each component. Add 200 mL of the reference solution and aqueous solution of known concentration. Prepare the test solution and test solution (glucose, fructose) solution for spiking recovery according to the method in (2). Calculate the spiking recovery rate and RSD% of each component. Examine the spiking recovery rate. The results are shown in Table 7.

[0096] Table 7 Results of precision, repeatability, stability, and (spiking) recovery of 14 components.

[0097] Element Precision RSD% Repeatability RSD% Stability RSD% Recovery rate % Recovery rate RSD% D-glucose 0.836 1.367 1.288 98.038 1.341 D-fructose 0.581 0.982 0.862 98.935 0.907 L-alanine 0.931 1.896 1.770 94.617 1.915 L-arginine 0.852 1.313 1.384 96.837 1.403 L-glutamic acid 1.381 2.659 2.724 93.573 2.819 L-glycine 1.271 2.583 2.612 94.272 2.691 L-histidine 1.312 2.601 2.729 95.176 2.715 L-Lysine 1.296 2.653 2.538 95.783 2.657 L-phenylalanine 1.101 2.536 2.588 95.317 2.646 L-proline 1.196 2.495 2.692 95.015 2.586 L-serine 1.097 2.385 2.386 95.373 2.628 L-threonine 0.573 1.106 1.083 97.318 1.195 L-tyrosine 1.173 2.486 2.520 96.011 2.475 L-valine 1.057 2.116 2.063 95.957 2.105

[0098] The determination of 14 components in processed Polygonatum cyrtonema from Xinye, which passed the pharmacopoeia test, by LEC-RI and HILIC-MS / MS methods, showed linearity, precision, repeatability, stability, and recovery rate that met the relevant requirements for quantitative analytical method validation.

[0099] Based on the analysis results of qualified processed Polygonatum multiflorum from 11 production areas, the qualified range of the test samples was determined as follows: whether the MEC-RI fingerprint similarity of the test samples was above 0.9; whether the D-glucose content was 50–150 mg / g; whether the fructose content was 100–200 mg / g; and whether the L-alanine content, L-arginine content, L-glutamic acid content, and L-glycine content were 0–300 μg / g, 0–2000 μg / g, 0–100 μg / g, and 0–100 μg / g, respectively. The following parameters are used to determine whether the processed Polygonatum odoratum raw material is qualified: whether the L-histidine content is within the range of 0-100 μg / g, the L-lysine content is within the range of 0-200 μg / g, the L-phenylalanine content is within the range of 0-200 μg / g, the L-proline content is within the range of 0-500 μg / g, the L-serine content is within the range of 0-200 μg / g, the L-threonine content is within the range of 0-3000 μg / g, the L-tyrosine content is within the range of 0-200 μg / g, and the L-valine content is within the range of 0-300 μg / g.

[0100] Example 2

[0101] Optimization of parameters for MEC-RI-based polysaccharide molecular weight and fingerprint spectroscopy qualitative analysis, LEC-RI-based free monosaccharide analysis, and HILIC-MS / MS-based free amino acid quantification methods.

[0102] (1) Investigation of the extraction solvent for the test sample

[0103] In the HILIC-MS / MS method, the extraction effects of water, methanol, and ethanol as extraction solvents on 12 free amino acid components in processed polyanthraquinone powder were investigated. The results showed that the sum of extraction rates of the 12 free amino acid components was highest when water was used as the extraction solvent. In the MEC-RI and LEC-RI methods, if the test sample contains methanol or ethanol, interference peaks will be generated on the tested MEC-RI and LEC-RI chromatograms; therefore, water is chosen as the solvent for the test sample.

[0104] (2) Investigation of the sample extraction method

[0105] Referring to the 2020 edition of the Chinese Pharmacopoeia, the preparation of test samples for content determination of components in traditional Chinese medicine usually adopts extraction methods such as ultrasound and reflux. This invention investigated the extraction effects of ultrasound (250W, 50K Hz, 60min) and reflux (80℃, 120min) on 14 components in processed Polygonum multiflorum powder. The results showed that there was no statistically significant difference in extraction rate between ultrasound and reflux. Since ultrasound requires less time and is simpler to operate, it was selected.

[0106] (3) Optimization of sample size

[0107] This invention investigated the extraction effect of 14 components from processed polyanthemum extract powder on samples of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g with 200 ml of water. The results showed that the RSD% of the repeatability test results was too high when the sample size was 0.5, 1.0, and 1.5 g, while the RSD% of the repeatability test results met the relevant requirements for quantitative analysis when the sample size was 2.0 g. When the sample size was 2.5 and 3.0 g, the extraction rates of the three components were lower than those when the sample size was 2.0 g, and the difference was significant. Therefore, the preferred material-to-liquid ratio is: 2.0 g sample size, 200 ml water for extraction.

[0108] (4) Investigation of acid addition to the mobile phase

[0109] In the HILIC-MS / MS method, acetonitrile was used as the mobile phase (A) and pure water as the mobile phase (B). The separation effect of mobile phase (B) on the chromatographic peaks of 12 free amino acid components was investigated under conditions of no formic acid, addition of 0.1% and 0.2% formic acid. It was found that the chromatographic peaks separated by the column were finer after the addition of 0.1% and 0.2% formic acid, and the resolution was >2.0. However, 0.2% formic acid caused some unidentified peaks to appear. Therefore, this method selected 0.1% formic acid aqueous solution as the mobile phase (B) for analysis and measurement. In the MEC-RI and LEC-RI methods, the Xtimate Sugar-Ca column used by Shanghai Yuexu Company is a calcium-type sugar column. The Shodex SUGAR KS-804 column from Showa Denko Scientific Instruments Co., Ltd. of Japan uses high-efficiency gel as the chromatographic packing material, and the packing material surface has sodium ions, which makes it have both molecular exclusion and sodium-type sugar column functions. For sugar molecules, each hydroxyl group carries a very weak negative charge, while the hydroxyl groups on the terminal isomeric carbons can be deprotonated, thus acquiring a strong negative charge. The interaction between these negative charges on the sugar molecules and the positive charges of metal ions on the resin surface allows the sugar to be retained, achieving separation. A weak ionic attraction also exists between water and the metal ions on the column; therefore, water is chosen as the mobile phase to competitively elute the adsorbed sugar, thereby achieving separation and determination.

[0110] (5) Analysis of injection volume

[0111] In the HILIC-MS / MS method, the separation effect of chromatographic peaks of 12 free amino acid components was investigated with injection volumes of 1, 2, 5, and 10 μL. It was found that when the injection volume exceeded 2 μL, the chromatographic peaks of components such as L-glycine were tailed, and when the injection volume exceeded 5 μL, the chromatographic peaks of components such as L-glutamic acid were broken. Therefore, this method selected an injection volume of 1 μL for analysis and measurement.

[0112] (6) Optimization of mass spectrometry MRM conditions

[0113] This invention optimized the MRM mass spectrometry determination conditions of UPLC-MS / MS for 12 free amino acid components. The results showed that L-alanine exhibited the highest signal response value under the following conditions: parent ion 90.3 (m / z), daughter ion 44.2 (m / z), decolumning voltage -48.0 e V, and collision voltage -16.0 e V. L-arginine showed the highest signal response value under the following conditions: parent ion 175.2 (m / z), daughter ion 70.2 (m / z), decolumning voltage -70.0 e V, and collision voltage -20.0 e V. L-glutamic acid showed the highest signal response value under the following conditions: parent ion 148.1 (m / z), daughter ion 84.1 (m / z), decolumning voltage -50.7 e V, and collision voltage -21.0 e V. The highest signal response values ​​were observed for L-glycine under the following conditions: parent ion 76.0 (m / z), daughter ion 30.2 (m / z), declustering voltage -54.0 e V, and collision voltage -17.0 e V. For L-histidine, the highest signal response values ​​were observed for 156.1 (m / z) parent ion, 110.2 (m / z) daughter ion, -61.0 e V, and -20.0 e V. For L-lysine, the highest signal response values ​​were observed for 147.2 (m / z) parent ion, 84.2 (m / z) daughter ion, -116.60 e V, and -13.0 e V. For L-phenylalanine, the highest signal response values ​​were observed for 166.1 (m / z) parent ion, 120.2 (m / z) daughter ion, and -68.0 e V. The highest signal response values ​​were obtained for L-proline at a precursor ion of 116.1 eV, a daughter ion of 70.1 eV, a decolumning voltage of -76.0 eV, and a collision voltage of -19.0 eV. For L-serine, the highest signal response values ​​were obtained at a precursor ion of 106.1 eV, a daughter ion of 60.1 eV, a decolumning voltage of -48.0 eV, and a collision voltage of -15.0 eV. For L-threonine, the highest signal response values ​​were obtained at a precursor ion of 120.1 eV, a daughter ion of 74.0 eV, a decolumning voltage of -53.0 eV, and a collision voltage of -13.0 eV. For L-tyrosine, the highest signal response values ​​were obtained at a precursor ion of 182.1 eV, a daughter ion of 136.2 eV, and a decolumning voltage of -75.0 eV. The highest signal response value was obtained under the conditions of a parent ion of 118.2 (m / z), a daughter ion of 72.1 (m / z), a decoupling voltage of -69.0 e V, and a collision voltage of -15.0 e V. Therefore, the above MRM conditions of UPLC-MS / MS were selected for analysis and measurement.

[0114] Example 3

[0115] According to the 2020 edition of the Chinese Pharmacopoeia, the polysaccharides in processed Polygonatum cyrtonema were quantified using the sulfuric acid-anthrone-UV-Vis spectrophotometry method (hereinafter referred to as the "Pharmacopoeia Method") under the Polygonatum cyrtonema entry, and the qualification of the samples was determined. The specific process and results are as follows:

[0116] (1) Preparation of the test solution: Accurately weigh approximately 0.25 g of the processed polysaccharide powder dried to constant weight at 60℃, place it in a round-bottom flask, add 150 ml of 80% ethanol, heat under reflux in a water bath for 1 h, filter while hot, wash the residue three times with 10 ml of 80% hot ethanol each time, place the residue and filter paper in a flask, add 150 ml of water, heat under reflux in a boiling water bath for 1 h, filter while hot, wash the residue and flask four times with hot water each time. Combine 10 ml of the filtrate and washings, cool, transfer to a 250 ml volumetric flask, add water to the mark, shake well, then accurately measure 1 ml of this solution into a 10 ml stoppered dry test tube, add water to 2.0 ml, shake well, slowly add 0.2% anthrone-sulfuric acid solution to the mark in an ice-water bath, mix well, cool, and incubate in a water bath for 10 min, remove, and immediately cool in an ice-water bath for 10 min, remove, and use the corresponding reagent as a blank to obtain the final product.

[0117] (2) Preparation of D-glucose reference solution: Weigh 33 mg of D-glucose reference standard dried to constant weight at 105℃, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, then accurately measure 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml of the solution and place them in 10 ml stoppered graduated test tubes respectively, add water to each to 2.0 ml, shake well, slowly add 0.2% anthrone-sulfuric acid solution to the mark in an ice-water bath, mix well, cool and incubate in a water bath for 10 min, remove and immediately cool in an ice-water bath for 10 min, remove, and use the corresponding reagent as a blank to obtain the solution.

[0118] (3) Determination of Polygonatum polysaccharides: Measure the absorbance at a wavelength of 582 nm using ultraviolet-visible spectrophotometry (General Rule 0401). Plot a standard curve with absorbance (Y) as the ordinate and concentration (X) as the abscissa. Read the weight (mg) of D-glucose in the test solution from the standard curve and calculate the concentration.

[0119] (4) Linearity test: The D-glucose standard solution was measured, and the linear regression equation was calculated as Y = 0.027X + 0.054, R = 0.996, with a linear range of 0–20.400 μg·mL. -1 .

[0120] (6) Precision test: The test solution from Xinye was measured 6 times consecutively, and the RSD% of the content of Polygonatum polysaccharide was calculated to test the precision. The result was 0.3364%.

[0121] (7) Repeatability test: Six samples of the test product from Xinye were prepared simultaneously in parallel according to the method in (2), and the RSD% of the content of Polygonatum polysaccharide was determined and calculated to test repeatability. The result was 2.632%.

[0122] (8) Stability test: The test solution from Xinye was injected at 0.0, 1.5, 3.0, 4.5, 6.0, 12, 18 and 24 h after preparation. The RSD% of the content of Polygonatum polysaccharide was calculated to test the stability. The result was 5.946%.

[0123] (9) Recovery rate test: 1.0 g of the test sample solution with known polysaccharide content from Xinye was accurately weighed, and a total of 6 portions were prepared. 200 mL of D-glucose reference solution and aqueous solution of known concentration were added. The test sample solution was prepared according to the method in (2). The recovery rate of polysaccharide component of Polysaccharide was determined to be 88.50%, and the RSD% was 12.99%.

[0124] Comparative analysis showed that the repeatability, stability, and recovery rate of the sulfuric acid-anthrone-UV-Vis spectroscopy method for determining Polygonatum polysaccharides in processed Polygonatum odoratum were all inferior to those of this invention.

[0125] Example 4

[0126] Quality Evaluation Methods and Optimization of Processed Polygonatum multiflorum

[0127] (1) Following the detection methods for each indicator in Examples 1, 2, and 3, the unqualified processed Polygonatum odoratum powder from 11 production areas was analyzed. Each production area had multiple batches of processed Polygonatum odoratum. The MEC-RI fingerprint similarity and D-glucose and D-fructose data of some qualified samples are shown in Table 8. The samples that met the requirements for MEC-RI fingerprint similarity and D-glucose and D-fructose content were then subjected to subsequent amino acid content testing.

[0128] Table 8. Mean values ​​of three indicators for processed Polygonum multiflorum powder (n=3)

[0129] Place of origin Similarity <![CDATA[D-glucose / mg·g -1 > <![CDATA[D-fructose / mg·g -1 > Xinye 0.999 75.76±0.84 158.32±0.20 Nanping 0.944 116.68±1.33 160.95±0.44 Chenzhou 0.987 65.81±0.81 149.85±0.42 Shaoyang 0.989 72.68±1.23 155.71±0.33 Xiangxi 0.984 71.12±3.00 154.35±0.42 Kerry 0.993 58.77±2.80 146.63±0.42 Xinlin 0.998 71.49±1.82 158.65±0.45 Yueyang 0.997 59.31±2.54 143.08±0.50 Meishan 0.998 70.34±1.48 162.05±0.51 Yuxi 0.989 66.38±2.01 157.06±0.48 Tongren 0.991 61.06±1.79 150.35±0.42

[0130] Samples that passed the MEC-RI fingerprint similarity test were tested using the pharmacopoeia method, and the pass rate for Polygonatum polysaccharides was 90.90%. However, for samples that passed the D-glucose and D-fructose content test, the pass rate for Polygonatum polysaccharides was only 81.81%. This indicates that methods that only test MEC-RI fingerprint similarity or only test D-glucose and D-fructose are insufficient to meet the requirements for screening qualified samples.

[0131] Samples that passed the MEC-RI fingerprint similarity test and had qualified D-glucose and D-fructose content tests were tested using the pharmacopoeia method. The pass rate of Polygonatum polysaccharide was 100%, indicating that the method of detecting MEC-RI fingerprint similarity combined with D-glucose and D-fructose content is as accurate as the pharmacopoeia method in screening qualified samples.

[0132] For samples that passed the MEC-RI fingerprint similarity test and had qualified D-glucose and D-fructose content tests, the content of 12 amino acids was further determined. Some qualified data are shown in Table 9.

[0133] Table 9. Average content of 12 amino acids in processed Polygonum multiflorum powder (n=3)

[0134] Place of origin <![CDATA[L-alanine / μg·g -1 > <![CDATA[L-arginine / μg·g -1 > <![CDATA[L-glutamic acid / μg·g -1 > <![CDATA[L-glycine / μg·g -1 > <![CDATA[L-Histidine / μg·g -1 > <![CDATA[L-lysine / μg·g -1 > Xinye 258.2±4.65 1291±16.78 55.94±1.45 47.73±1.19 39.74±1.03 95.97±2.50 Nanping 152.5±2.75 331.8±4.31 38.03±0.99 27.73±0.69 25.96±0.67 43.89±1.14 Chenzhou 168.6±3.03 157.1±2.04 36.09±0.94 37.68±0.94 17.98±0.47 36.68±0.95 Shaoyang 256.5±4.62 1022±13.29 79.08±2.06 44.28±1.11 37.11±0.96 150.8±3.92 Xiangxi 165.2±2.97 761.8±9.90 20.79±0.54 12.24±0.31 23.85±0.62 37.81±0.98 Kerry 146.6±2.64 371±4.82 57.48±1.49 20.32±0.51 20.72±0.54 32.94±0.86 Xinlin 197.3±3.55 857.3±11.14 34.39±0.89 29.83±0.75 28.59±0.74 48.32±1.26 Yueyang 34.68±0.62 87.89±1.14 4.22±0.11 3.26±0.08 14.27±0.37 3.4±0.09 Meishan 121.8±2.19 508.4±6.61 16.57±0.43 15.18±0.38 21.68±0.56 51.12±1.33 Yuxi 203.4±3.66 1793±23.31 38.1±0.99 20.01±0.50 27.32±0.71 81.97±2.13 Tongren 159.3±2.87 721.7±9.38 16.64±0.43 13.53±0.34 22.49±0.58 46.32±1.20 Place of origin <![CDATA[L-Phenylalanine / μg·g -1 > <![CDATA[L-proline / μg·g -1 > <![CDATA[L-serine / μg·g -1 > <![CDATA[L-Threonine / μg·g -1 > <![CDATA[L-tyrosine / μg·g -1 > <![CDATA[L-Valine / μg·g -1 > Xinye 47.51±1.19 394.4±9.47 138.6±3.19 1478±16.26 137.5±3.84 159.8±3.36 Nanping 29.85±0.75 111.9±2.69 118.8±2.73 1421±15.63 83.8±2.85 118.9±2.50 Chenzhou 26.5±0.66 43.54±1.04 38.64±0.89 1318±14.50 50.59±2.32 96.47±2.03 Shaoyang 110.8±2.77 180.0±4.32 143.6±3.30 1225±13.48 121±4.79 199.6±4.19 Xiangxi 69.39±1.73 56.89±1.37 27.27±0.63 1452±15.97 105.9±3.81 158.7±3.33 Kerry 40.09±1.00 179±4.30 59.58±1.37 2759±30.35 57.36±3.04 126.8±2.66 Xinlin 53.3±1.33 261.9±6.29 79.91±1.84 748.8±8.24 91.73±1.85 77.2±1.62 Yueyang 9.38±0.23 94.66±2.27 4.38±0.10 307.2±3.38 11.95±0.73 30.54±0.64 Meishan 66.95±1.67 84.98±2.04 54.34±1.25 982.1±10.80 67.98±1.89 78.61±1.65 Yuxi 62.71±1.57 149.7±3.59 132±3.04 1992±21.91 97.68±4.80 200.1±4.20 Tongren 68.09±1.70 87.00±2.09 81.66±1.88 1414±15.55 97.95±3.66 152.6±3.20

[0135] The results showed that even among processed Polygonatum cyrtonema samples that passed MEC-RI fingerprint similarity, D-glucose and D-fructose content tests, and pharmacopoeia-based tests, some amino acid contents were still substandard. For example, the L-glycine, L-glutamic acid, and L-histidine contents of three samples from Yueyang were not within the range of 0–100 μg / g, but were much higher. However, these processed Polygonatum cyrtonema samples passed pharmacopoeia-based tests, indicating that the quality evaluation method for processed Polygonatum cyrtonema of this invention is as accurate as, or even more comprehensive than, the pharmacopoeia method, and can meet the requirements for screening qualified processed Polygonatum cyrtonema samples.

[0136] Furthermore, the results showed that 6.06% of the processed Polygonatum multiflorum samples had amino acid content within the range, but failed the pharmacopoeia test. Therefore, relying solely on MEC-RI fingerprint similarity, monosaccharide content, or amino acid content alone is insufficient to meet the requirements for screening qualified processed Polygonatum multiflorum samples.

[0137] In summary, this invention provides a quality analysis method for processed Polygonatum cyrtonema based on MEC-RI molecular weight and fingerprint chromatogram for qualitative analysis, and LEC-RI for free monosaccharides and HILIC-MS / MS for quantification of free amino acids. The MEC-RI method for constructing the MEC-RI fingerprint chromatogram of processed Polygonatum cyrtonema exhibits precision, repeatability, and stability that meet the relevant regulations for fingerprint chromatogram research of traditional Chinese medicine. The LEC-RI and HPLC-MS / MS methods for determining the content of 14 components in processed Polygonatum cyrtonema exhibit linearity, precision, repeatability, stability, and recovery rate that meet the relevant regulations for content determination method validation in the 2020 edition of the Chinese Pharmacopoeia.

[0138] This invention application also provides a quality evaluation method for processed Polygonatum multiflorum, based on: whether the MEC-RI fingerprint similarity of the test sample is above 0.9, whether the D-glucose content is 50-150 mg / g, whether the fructose content in the test sample is 100-200 mg / g; and whether the L-alanine content, L-arginine content, L-glutamic acid content, and L-glycine content in the test sample are 0-300 μg / g, 0-2000 μg / g, 0-100 μg / g, and 0-100 μg / g, respectively. The quality of the processed Polygonatum odoratum is determined by whether the contents of L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, and L-valine are within the ranges of 0–100 μg / g, 0–200 μg / g, 0–200 μg / g, 0–3000 μg / g, 0–200 μg / g, and 0–300 μg / g.

[0139] This invention innovatively establishes a quality evaluation method for processed Polygonatum cyrtonema based on MEC-RI molecular weight and fingerprint spectroscopy for qualitative analysis, LEC-RI for free monosaccharides, and HILIC-MS / MS for quantitative analysis of free amino acids. The analytical method of this invention has high specificity and accuracy, uses mainly water and acetonitrile as reagents, requires no sample derivatization, and is simple, reasonable, and feasible. This quality evaluation method for processed Polygonatum cyrtonema is more complete and has strong industry and market application value.

[0140] The above embodiments should be understood as being used only to illustrate the invention more clearly, and not to limit the scope of the invention. After reading this invention, any modifications of the embodiments by those skilled in the art in various equivalent forms fall within the scope defined by the appended claims. For example, any of the claimed embodiments in the above claims can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of the invention, and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for quality evaluation of processed Polygonatum multiflorum, characterized in that, Includes the following steps: S1. Collect MEC-RI spectra of processed Polygonatum multiflorum, analyze the molecular weight of free polysaccharides in the sample based on the relationship between retention time and molecular weight function, and construct MEC-RI fingerprint spectrum; S2. Determine the contents of free D-glucose, D-fructose, and free amino acids in processed Polygonatum odoratum; among which, free amino acids include: L-alanine, L-arginine, L-glutamic acid, L-glycine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, and L-valine. S3. Based on the similarity of the fingerprint spectrum of processed Polygonatum multiflorum and the results of the contents of free D-glucose, D-fructose and free amino acids, establish a quality evaluation method for processed Polygonatum multiflorum. The molecular weight of processed Polygonatum multiflorum polysaccharides was analyzed by MEC-RI, and a MEC-RI fingerprint was constructed, including the following steps: (1) Preparation of MEC-RI test sample: Grind the processed Polygonatum odoratum into fine powder, add water, heat under reflux for 120 min, cool, add weight, filter, and the test sample is obtained. (2) Preparation of dextran reference standards: Dextran T100, T50, T20, T10, T5, T2 and T1 with a concentration of 10 mg / ml were prepared using ultrapure water as reference standards, and ultrapure water was used as blank reference standard. (3) MEC-RI chromatographic conditions: porous gel and sodium-modified polystyrene / divinylbenzene were used as the packing material; ultrapure water was used as the mobile phase; column temperature was 50℃; flow rate was 0.5 ml / min; differential refractive index detector was used, temperature was 35℃, and detection time was 30 min; (4) MEC-RI analysis: Accurately pipette 10 μl each of blank reference, reference and test sample and inject them into the liquid chromatograph. Fit the equation of retention time and molecular weight of the main chromatographic peaks according to the MEC-RI chromatogram of dextran reference and calculate the relative molecular mass of each polysaccharide in the test sample; construct the MEC-RI fingerprint of the test sample according to the MEC-RI chromatogram. The content of free D-glucose and D-fructose in processed Polygonatum cyrtonema was determined by LEC-RI method, including the following steps: 1) Preparation of LEC-RI test sample: Grind the processed Polygonatum odoratum into a fine powder, add water, heat under reflux for 120 min, cool, add weight, filter, and the test sample is obtained; take 1 ml of the test sample, dilute it with water to 10 ml, filter, and the test sample is obtained. 2) Preparation of D-glucose and D-fructose reference standards: D-fructose and D-glucose at a concentration of 1 mg / ml were prepared using ultrapure water as reference standards, and ultrapure water was used as a blank reference standard. 3) LEC-RI chromatographic conditions: Calcified polystyrene / divinylbenzene was used as the stationary phase, and ultrapure aqueous solution was used as the mobile phase; column temperature was 80℃; flow rate was 0.5 ml / min; differential refractive index detector was used, temperature was 35℃, and detection time was 30 min; 4) LEC-RI analysis method: Accurately pipette blank reference standard, D-glucose, D-fructose reference standard, test solution, and 10 μl of test solution into the liquid chromatograph, collect the LEC-RI chromatogram, and calculate the content of D-glucose or D-fructose based on the peak area information.

2. The quality evaluation method according to claim 1, characterized in that, The preparation method of processed Polygonatum multiflorum includes: adding rice wine to Polygonatum multiflorum for moistening and steaming, and then drying in stages by blowing air; repeating the moistening-steaming-stage drying process 9 times until Polygonatum turns black, thus obtaining processed Polygonatum multiflorum.

3. The quality evaluation method according to claim 1, characterized in that, The content of free amino acids in processed Polygonatum multiflorum was determined by HILIC-MS / MS.

4. The quality evaluation method according to claim 3, characterized in that, The method for determining the content of free amino acids in processed Polygonatum multiflorum includes the following steps: (1) Preparation of HILIC-MS / MS test sample: The processed Polygonatum odoratum was ground into fine powder, water was added, and the mixture was heated under reflux for 120 min. After cooling, the weight was added, and the mixture was filtered to obtain the test sample. (2) Preparation of amino acid reference standards: amino acids with a concentration of 1 µg / ml were prepared using ultrapure water as reference standards, and ultrapure water was used as blank reference standards; (3) HILIC-MS / MS chromatographic conditions: Triple-bonded amide-bonded silica gel was used as the stationary phase, and acetonitrile (A)-0.1% formic acid (B) was used as the mobile phase. Gradient elution was performed, and the gradient elution process was as follows: 0~5 min, 90% A; 5~15 min, 90% A~70% A; 15~20 min, 70% A; 20~21 min, 70% A~90% A; 21~30 min, 90% A, flow rate 0.3 mL / min, column temperature 35℃; Triple quadrupole mass spectrometer was used for detection: electrospray ionization source, multiple ion detection mode was used for each compound, positive ion mode: injection voltage and collision chamber outlet voltage were 10; (4) HILIC-MS / MS mass spectrometry conditions: The parent ion, daughter ion, debulking voltage, and collision voltage of each amino acid are shown below; ； (5) HILIC-MS / MS analysis: Accurately pipette 1 μl of blank reference standard, amino acid reference standard and test sample into the high performance liquid chromatograph-mass spectrometer, collect the MRM chromatogram of HILIC-MS / MS, and calculate the content based on the peak area.

5. The quality evaluation method according to claim 1, characterized in that, Evaluating the quality of processed Polygonatum multiflorum includes the following steps: (1) Whether the similarity of the MEC-RI fingerprint spectrum of the processed Polygonatum multiflorum sample is above 0.9; (2) Whether the D-glucose content of the test sample is within 50~150 mg / g, and whether the D-fructose content of the test sample is within 100~200 mg / g; (3) Whether the content of L-alanine in the test sample is within the range of 0~300 µg / g, L-arginine within the range of 0~2000 µg / g, L-glutamic acid within the range of 0~100 µg / g, L-glycine within the range of 0~100 µg / g, L-histidine within the range of 0~100 µg / g, L-lysine within the range of 0~200 µg / g, L-phenylalanine within the range of 0~200 µg / g, L-proline within the range of 0~500 µg / g, L-serine within the range of 0~200 µg / g, L-threonine within the range of 0~3000 µg / g, L-tyrosine within the range of 0~200 µg / g, and L-valine within the range of 0~300 µg / g; If all conditions are met, the raw material for processing Polygonatum odoratum is deemed qualified; if any one condition is not met, the raw material for processing Polygonatum odoratum is deemed unqualified.

Images