A method for simultaneously determining the contents of 6 saccharide components in Polygonatum sibiricum

Abstract

The present invention relates to the field of traditional Chinese medicine analysis, and particularly to a method for simultaneously determining the contents of 6 saccharide components in Polygonatum sibiricum. The present invention discloses a method for simultaneously determining the contents of 6 saccharide components in Polygonatum sibiricum. This method uses inexpensive and easily obtainable sucrose as an internal reference substance, establishes the relative correction factors and relative intercepts of 5 saccharide components including fructose, glucose, kestose, nystose, and fructofuranosylnystose in Polygonatum sibiricum with respect to the internal reference substance, and realizes the calculation of the contents of sucrose and the other 5 saccharide components in Polygonatum sibiricum by only using one reference substance, sucrose, and utilizing the relative correction factors, relative intercepts, and relative retention values. Thus, it can simply, quickly, comprehensively, and accurately conduct multi-index quality evaluation on Polygonatum sibiricum (raw product and processed product), which helps to ensure the quality controllability and stable efficacy of this product, and at the same time saves detection costs and time.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine analysis, and particularly to a method for simultaneously determining the contents of 6 saccharide components in Polygonati Rhizoma. Background Art

[0002] Polygonati Rhizoma is also known as fairy's surplus food, tiger ginger, chicken head ginseng, etc. The traditional Chinese medicine Polygonati Rhizoma included in the Chinese Pharmacopoeia (2020 Edition) is the dried rhizome of the liliaceous plants Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red., or Polygonatum cyrtonema Hua., which are mainly distributed in Hebei, Gansu, Inner Mongolia, Shaanxi and southwestern regions. It is usually harvested in autumn and used fresh or after drying. Polygonati Rhizoma has the effects of invigorating the spleen and replenishing qi, nourishing yin and moistening the lungs, and tonifying the kidney and filling essence. There are special discussions on this in the earliest existing diet therapy monograph in the world, Materia Dietetica, and it is a typical traditional Chinese medicinal material with both medicine and food homology in China.

[0003] Polygonati Rhizoma was first recorded in Miscellaneous Records of Famous Physicians. "Polygonati Rhizoma tastes sweet, is flat, and is non-toxic. It mainly replenishes the middle qi, dispels wind-dampness, and tranquilizes the five internal organs. Taking it for a long time can make the body light, prolong life, and not feel hungry." Polygonati Rhizoma was listed as the top of the herb category in the Illustrated Classic of Materia Medica. Zhang Hua's Bowu Zhi in the Jin Dynasty said: "The herb of the sun is called Polygonati Rhizoma. Eating it can lead to immortality." Wufujing recorded: "Polygonati Rhizoma obtains the pure essence of heaven and earth, so it is named Wuji Zhi." Rihua Ziben Cao said: "It supplements the five kinds of overstrain and seven kinds of injuries, helps the muscles and bones, stops hunger, withstands cold and heat, benefits the spleen and stomach, and moistens the heart and lungs." Compendium of Materia Medica, a great work on Chinese materia medica before the 16th century written by Li Shizhen in the Ming Dynasty, recorded: "Because it obtains the essence of Kun earth, it is called Polygonati Rhizoma." "Polygonati Rhizoma supplements various deficiencies, fills the marrow, and tonifies qi and blood evenly and moistly."

[0004] The Chinese Pharmacopoeia (2020 Edition) records that Polygonati Rhizoma is "sweet in nature, flat in taste; attributive to the spleen, lung, and kidney meridians; has the effects of invigorating qi and nourishing yin, strengthening the spleen, moistening the lungs, and tonifying the kidneys." Traditional medicine believes that clinically, Polygonati Rhizoma is mostly used to treat symptoms such as spleen-stomach weakness, abdominal pain and diarrhea, fatigue, lung deficiency cough, internal heat and polydipsia, tidal fever and night sweats, etc.

[0005] Modern research shows that Polygonatum sibiricum mainly contains active substances such as sugars, flavonoids, steroidal saponins, terpenes, alkaloids, cardiac glycosides, lignans, etc., which have biological activities such as anti-oxidation, anti-fatigue, anti-tumor, improving immunity, protecting nerves, lowering blood lipids, lowering blood pressure, lowering blood sugar, anti-virus, anti-inflammatory and bactericidal. The sugar components in Polygonatum sibiricum mainly include Polygonatum sibiricum polysaccharides, Polygonatum sibiricum oligosaccharides, Polygonatum sibiricum monosaccharides and starch, among which Polygonatum sibiricum polysaccharides are the most important components, with about 37 kinds. The 2020 edition of the Chinese Pharmacopoeia stipulates that the total polysaccharide content in raw Polygonatum sibiricum shall not be less than 7.0% by the phenol-sulfuric acid method measured with glucose, and the total polysaccharide content in wine Polygonatum sibiricum shall not be less than 4.0%. However, raw Polygonatum sibiricum is irritating and can "spike people in the throat". It can only be used as medicine after steaming and drying. The processing methods of successive dynasties are mainly steaming and boiling, and most of the herbal medicines record the nine-steaming and nine-drying processing methods. Clinically, Polygonatum sibiricum is mostly used as medicine in processed products.

[0006] In the process of steaming and drying, not only the appearance and taste of Polygonatum odoratum change greatly, but also its material basis and medicinal efficacy change significantly. Studies have shown that during the processing of Polygonatum odoratum, polysaccharides will be hydrolyzed into oligosaccharides in large quantities, and oligosaccharides will be further hydrolyzed into monosaccharides. Some people have determined the 6 monosaccharide and low sugar components in Polygonatum odoratum. The results show that the content of raw products is 29.23%, the content of wine steamed products is 45.93%, and the content of stewed products is 41.86%. Among them, D-fructose has the highest content in all processed products. Fructose is called "healthy sugar" because of its low glycemic index. It is the sweetest sugar among natural sugars, with excellent flavor and taste, and has the functions of nourishing the stomach, protecting the liver, moisturizing the intestines, and smoothing the intestines. Glucose is a physiological sugar with the functions of nutrition, detoxification, cardiotonic, and diuresis. It is the main source of energy required by the body. It has a protective and detoxifying function on the liver and can promote the excretion of toxins. In addition, glucose is very important for brain function, which can enhance memory, stimulate calcium absorption and increase communication between cells. Sucrose provides energy for brain tissue function, human muscle activity, etc. and maintains body temperature. It can also increase the synthesis of ATP in the body, which is beneficial to the activity of amino acids and the synthesis of proteins. Other studies have reported that Polygonatum oligosaccharides have antiviral effects. Therefore, people are increasingly aware that Polygonatum monosaccharides and oligosaccharides should be used as the core indicators for controlling the quality of Polygonatum and its medicinal materials.

[0007] Monosaccharides and oligosaccharide components are ubiquitous in natural medicines. However, due to the lack of ultraviolet absorption, their accurate determination has always been a difficult problem. It was not until the emergence of mass detectors (such as ELSD, CAD, and MS) that this problem was effectively solved. Among them, the evaporative light-scattering detector (ELSD) is the most popular and relatively low-cost mass detector, so it is the most widely used in the determination of saccharide components. However, the detection cost of saccharide components remains high, and there are two reasons for this: First, the reference substances of oligosaccharide components are expensive in themselves, and because the ELSD detector is not as sensitive as the PDA detector / ultraviolet detector, the usage amount of the reference substance is much larger than that of the reference substance used in ultraviolet detection; Second, the separation of saccharide components usually uses a normal-phase column (such as an amino column, an Amide column, a HIlIC column, etc.) as the stationary phase, and the mobile phase uses a large proportion of organic phase, resulting in a huge consumption of solvents and high detection costs.

[0008] The "Quantitative Analysis of Multi-components by Single Marker" (QAMS) method draws on the comprehensive advantages of the classical internal standard method, correction factor method, ultraviolet percentage absorption coefficient method, and self-control method of the main component. Using the relative correction factor (RCF) between components, the determination of components can be carried out under the condition of lacking reference substances, so as to realize the quality control of traditional Chinese medicines. This method greatly reduces the detection cost and time, and is easy to operate, effectively improving the practicability and detection efficiency of the method.

[0009] The basic principle of the "Quantitative Analysis of Multi-components by Single Marker" method is that within a certain linear range, the component amount (mass or concentration) is proportional to the response value of the detector. In the multi-index quality evaluation, a stable, cheap, and easily available reference substance is set as the internal reference substance, and at the same time, the relative correction factor (f i / s ) between other components to be measured (i) and the internal reference substance (s) is established. Through f i / s , the content of other components can be calculated. Suppose there are n components to be measured. In the first step, select 1-2 components as the internal reference substance, and use the formula f i / s = (Ai / Ci) / (As / Cs) to calculate the relative correction factor f i / s between other components and the internal reference substance; where As is the peak area of the internal reference substance, Cs is the concentration of the internal reference substance, Ai is the peak area of the reference substance of the analyte, and Ci is the concentration of the analyte; in the second step, according to the peak areas of each component measured in the test sample, use the following formula to obtain the concentration of the component to be measured:

[0010] Cx = Ax × Cs' / As'f i / s;

[0011] In the formula, f i / s is the obtained relative correction factor, Ax is the peak area of the component to be measured in the test sample, As' is the peak area of the internal reference substance in the test sample, Cs' is the concentration of the internal reference substance measured in the test sample by the conventional method, and Cx is the concentration of the component to be measured.

[0012] Since the proposal of QAMS, many scholars in the domestic and foreign pharmaceutical fields have continuously strengthened the research on the mechanism and process of its quality control mode, providing good theoretical and practical references for expanding the application scope and applicable fields of QAMS. The application scope of QAMS has also been extended from the determination of the same type of components to the determination of different types of components. The components determined by the QAMS method in the 2020 edition of the Chinese Pharmacopoeia include alkaloids, quinones, phenols, flavonoids and saponins, and the dosage forms determined include traditional Chinese medicine pieces, extracts, tablets, pills, capsules. However, so far, almost all the components determined by the "one assay for multiple evaluations" method are detected by ultraviolet detectors or diode array detectors. Although there are literatures that have tried to establish the "one assay for multiple evaluations" method for 4 kinds of saccharide components in traditional Chinese medicine by ELSD detection, the research results have not been successful. Summary of the Invention

[0013] The purpose of the present invention is to provide a method for simultaneously determining the contents of 6 saccharide components in Polygonatum sibiricum Redoute to solve the problems existing in the above-mentioned prior art.

[0014] Evaporative Light-scattering Detector (ELSD) is a general detector, and the detection result is related to the sample concentration. It can detect any sample with a volatility lower than that of the mobile phase. The working principle of the evaporative light-scattering detector is as follows: After the sample solution separated by the chromatographic system enters the detector, it is first atomized by a high-pressure gas stream. The tiny droplets formed by atomization are mixed in the carrier gas and enter the drift tube through shunting. The mobile phase and low-boiling components are evaporated, and the aerosol of the high-boiling sample enters the light-scattering cell. When the light beam passes through the scattering cell, it is scattered by the sample aerosol, and the scattered light is amplified by the phototube and processed into a digital signal of the chromatographic workstation - a chromatogram.

[0015] The content of six saccharide components (D-fructose, glucose, kestotriose, kestotetraose, and kestopentaose) in Polygonatum sibiricum Red. was determined by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method in this invention. Using sucrose as the internal reference substance, the relative correction factors and relative intercepts of the other five saccharide components relative to sucrose were calculated, and the contents of the other five saccharide components were calculated based on the relative correction factors and relative intercepts. The relative standard deviations (RSDs) of the QAMS determination results and the external standard method calculation results of the six sugars were within 5%, which proved the accuracy of the method provided by this invention. This invention not only invented the "one measure for multiple evaluations" method for simultaneously determining the contents of six saccharide components in traditional Chinese medicine Polygonatum sibiricum Red., but also successfully realized the application of the "one measure for multiple evaluations" method on ELSD.

[0016] To achieve the above object, this invention provides the following solutions:

[0017] This invention provides a method for simultaneously determining the contents of six saccharide components in Polygonatum sibiricum Red., including the following steps:

[0018] Using the high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method to determine the peak areas of D-fructose, glucose, sucrose, kestotriose, kestotetraose, and kestopentaose in the test solution of Polygonatum sibiricum Red.; then using sucrose as the internal reference substance, and calculating the contents of D-fructose, glucose, kestotriose, kestotetraose, and kestopentaose in the test solution of Polygonatum sibiricum Red. according to the "one measure for multiple evaluations" method.

[0019] Preferably, in the high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method, the high performance liquid chromatography separation uses 0.1% ammonia water·acetonitrile as mobile phase A and 0.1% ammonia water as mobile phase B for gradient elution.

[0020] Preferably, in the high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method, the detection conditions for high performance liquid chromatography detection are: the chromatographic column is BEH Amide chromatographic column, the column temperature is 40 °C, and the injection volume is 10 μL - 20 μL.

[0021] Preferably, in the high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method, the detection conditions for evaporative light scattering detection are: the drift tube temperature is 80 °C; the nebulizer mode; heating, and the power level is 59%; the gas pressure is 30 psi.

[0022] This invention selects an amino chromatographic column, mobile phase: 0.1% ammonia water·acetonitrile (A) - 0.1% ammonia water (B), gradient elution, and uses an evaporative light scattering detector for detection, successfully realizing the determination of the contents of six saccharide components in Polygonatum sibiricum Red. with only one reference substance sucrose, as well as the application of the "one measure for multiple evaluations" method on the evaporative light scattering detector.

[0023] Preferably, the Polygonatum sibiricum Red. includes raw Polygonatum sibiricum Red. and processed Polygonatum sibiricum Red.

[0024] Preferably, the processed polygonatum sibiricum includes polygonatum sibiricum prepared with wine and polygonatum sibiricum prepared by nine steaming and nine sunning.

[0025] Preferably, the method for preparing the polygonatum sibiricum test solution includes the steps of ultrasonic extraction and filtration of polygonatum sibiricum to obtain the polygonatum sibiricum test solution.

[0026] Preferably, the condition parameters of the ultrasonic extraction are: power 300 Kw, frequency 40 Hz, and time 30 min.

[0027] The present invention provides the application of the above method in evaluating the quality of polygonatum sibiricum.

[0028] The present invention provides the application of the above method in identifying the authenticity of polygonatum sibiricum.

[0029] The present invention discloses the following technical effects:

[0030] The present invention discloses a method for simultaneously determining the contents of 6 saccharide components in polygonatum sibiricum. The method uses inexpensive and easily available sucrose as an internal reference substance, establishes the relative correction factors and relative intercepts of 5 saccharide components, namely D-fructose, glucose, 1-kestose, nystose, and fructofuranosylnystose, in polygonatum sibiricum with respect to the internal reference substance, and realizes the determination of the contents of sucrose and the other 5 saccharide components in polygonatum sibiricum by using only one reference substance of sucrose, calculating with the relative correction factors, relative intercepts, and relative retention values. Thus, it can simply, quickly, comprehensively, and accurately conduct multi-index quality evaluation on polygonatum sibiricum (raw and processed products), which helps to ensure the quality of polygonatum sibiricum and its related products, and at the same time saves detection costs and time.

[0031] The results of the specific embodiments of the present invention show that the RSD between the QAMS determination results and the external standard method calculation results of 6 sugars is within 5%, which proves the accuracy of the method provided by the present invention. Thus, it can be seen that the present invention not only invents the "one assay for multiple evaluations" method for simultaneously determining the contents of 6 saccharide components in traditional Chinese medicine polygonatum sibiricum, but also successfully realizes the application of the "one assay for multiple evaluations" method on ELSD. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 For the standard curves (regression equations) and correlation coefficients of 6 saccharide components;

[0034] Figure 2It is the HPLC-ELSD chromatogram of 6 kinds of saccharide components in freeze-dried fresh polygonatum sibiricum redoute; among them, 1-6 in the figure are D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose in sequence;

[0035] Figure 3 It is the HPLC-ELSD chromatogram of 6 kinds of saccharide components in raw polygonatum sibiricum redoute; among them, 1-6 in the figure are D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose in sequence;

[0036] Figure 4 It is the HPLC-ELSD chromatogram of 6 kinds of saccharide components in polygonatum sibiricum redoute processed by nine steaming and nine sunning; among them, 1-3 in the figure are D-fructose, glucose and sucrose in sequence;

[0037] Figure 5 It is the HPLC-ELSD chromatogram of the mixed reference substance; among them, 1-6 in the figure are D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose in sequence. Detailed implementation mode

[0038] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing special implementation modes and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0041] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the specification of the present invention, which are obvious to those skilled in the art. Other implementation modes obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The polygonatum sibiricum medicinal material used in the present invention is purchased from medicinal material markets and decoction piece factories in various places, and is identified as the dried rhizome of polygonatum yunnanensis, polygonatum sibiricum or polygonatum cyrtonema of the lily family.

[0044] Example 1 HPLC chromatograms of six sugar components in different polygonatum samples

[0045] 1. High performance liquid chromatography conditions (HPLC conditions)

[0046] Waters Alliance HPLC, 2695 Quaternary Solvent Manager, 2424 Evaporative Light Scattering Detector, Empower 3 Chromatography workstation; BEH Amide chromatographic column, 5μm, 4.6×250mm Column.

[0047] The mobile phase was 0.1% aqueous ammonia·acetonitrile (A)-0.1% aqueous ammonia (B), gradient elution, and the gradient elution program was: 0-15 min, 85%→80%, A; 15-40 min, 85%→75%, A; 40-60 min, 75%→70%, A; 60-90 min, 70%, A; "%" in the gradient elution program represents the meaning of "volume percentage".

[0048] The column temperature is 40° C.; the flow rate is 1 mL / min; the injection volume is 10 μL to 20 μL, and the injection volume in this embodiment is 20 μL.

[0049] 2. Preparation of reference solution

[0050] Accurately weigh 100.03 mg of D-fructose, 50.03 mg of glucose, 100.10 mg of sucrose, 100.60 mg of konjac triose, 50.60 mg of konjac tetraose and 50.60 mg of konjac pentasaccharide, place them in 25 mL volumetric flasks respectively, add 70% methanol to dissolve and dilute to the scale, shake well, and use them as the stock solutions of each monosaccharide reference substance.

[0051] Accurately pipette 0.7 mL of the above 6 monosaccharide stock solutions into the same 5 mL volumetric flask, add 70% methanol to dissolve and dilute to the mark, shake well, and use as mixed reference solution.

[0052] 3. Preparation of test solution

[0053] Take 0.25 g of Lichuan nine-steamed and nine-sunned polygonatum sibiricum, 0.25 g of fresh Lichuan polygonatum sibiricum, and 0.25 g of freeze-dried fresh Lichuan polygonatum sibiricum. Weigh accurately, add 25 mL of 70% methanol solution respectively, weigh again, extract by ultrasonic wave (power 300 Kw, frequency 40 Hz) for 30 min, take out, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the continuous filtrate to obtain.

[0054] 4. HPLC-ELSD Chromatograms of 6 Kinds of Carbohydrate Components in Different Polygonatum Sibiricum Samples

[0055] The HPLC-ELSD chromatograms of 6 kinds of carbohydrate components in different polygonatum sibiricum samples are as Figures 2 to 5 shown.

[0056] Example 2

[0057] A method for simultaneously determining the contents of 6 kinds of carbohydrate components (D-fructose, glucose, sucrose, 1-kestose, nystose, and fructofuranosylnystose) in polygonatum sibiricum with sucrose as the internal reference substance, the steps are as follows:

[0058] 1. High Performance Liquid Chromatography Conditions (HPLC Conditions) and ELSD Detection Conditions

[0059] 1.1. HPLC Conditions:

[0060] Waters Alliance high performance liquid chromatograph, 2695 quaternary solvent manager, 2424 evaporative light scattering detector, Empower 3 chromatography workstation; BEH Amide chromatographic column, 5 μm, 4.6×250 mm Column.

[0061] The mobile phase is 0.1% ammonia water·acetonitrile (A)-0.1% ammonia water (B), gradient elution, gradient elution program: 0 - 15 min, 85% → 80%, A; 15 - 40 min, 85% → 75%, A; 40 - 60 min, 75% → 70%, A; 60 - 90 min, 70%, A.

[0062] The column temperature is 40°C; the flow rate is 1 mL / min; the injection volume is 10 μL - 20 μL, and the injection volume in this example is 20 μL.

[0063] 1.2. ELSD Detection Conditions:

[0064] Drift tube temperature: 80°C; nebulizer mode; heating, power level is 59%, gas pressure: 30 psi (2.07 slpm).

[0065] 2. Preparation of Reference Solution

[0066] Accurately weigh 100.03 mg of D-fructose, 50.03 mg of glucose, 100.10 mg of sucrose, 100.60 mg of 1-kestose, 50.60 mg of nystose, and 50.60 mg of fructofuranosylnystose, respectively, place them in 25-mL volumetric flasks, dissolve with 70% methanol and dilute to the mark, shake well, and use as the stock solutions (C0) of each monosaccharide reference substance.

[0067] Accurately pipette 0.7 mL of the above 6 kinds of monosaccharide stock solutions into the same 5-mL volumetric flask, dissolve with 70% methanol and dilute to the mark, shake well, and use as the mixed reference substance solution (C1).

[0068] 3. Preparation of test solution

[0069] Take about 0.5 g of crude powder of raw Polygonati Rhizoma (take 0.25 g of Polygonati Rhizoma Praeparatum cum Vinum, 0.25 g of Polygonati Rhizoma Praeparatum by Repeated Steaming and Drying, 0.25 g of fresh Polygonati Rhizoma), accurately weigh, accurately add 25 mL of 70% methanol solution respectively, weigh, extract by ultrasonic wave (power 300 Kw, frequency 40 Hz) for 30 min, take out, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate to obtain.

[0070] 4. Plotting of standard curve and determination of detection limit and quantification limit

[0071] Take the mixed reference substance solution C1, inject 1, 2, 4, 10, 20, 30, 40, and 50 μL respectively, use the natural logarithm (Ln μg) of the injection volume of the reference substance as the abscissa, and the natural logarithm (Ln Area) of the peak area as the ordinate to plot the standard curve, conduct linear regression, calculate the correlation coefficient and determine the detection limit and quantification limit. The regression equations and correlation coefficients of the 6 kinds of saccharide components are shown in Table 1 and Figure 1 as follows. The results show that fructose has a good linear relationship in the range of 1.12 - 28.01 μg, glucose in the range of 0.560 - 14.01 μg, sucrose in the range of 1.12 - 28.03 μg, 1-kestose in the range of 1.13 - 28.17 μg, nystose in the range of 0.570 - 14.25 μg, and fructofuranosylnystose in the range of 0.567 - 14.17 μg.

[0072] Table 1 Results of investigation on linear relationship of 6 kinds of saccharide components

[0073]

[0074] 5. Methodology investigation

[0075] 5.1 Precision test:

[0076] Take about 0.5 g of the crude powder of Polygonatum sibiricum (No. SHJ_HN_10), accurately weigh it, prepare the test solution according to the method under "3. Preparation of the test solution", and continuously inject samples 6 times under the chromatographic conditions in "1. High-performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions" to determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose. The results are shown in Table 2. The results show that the RSDs of the natural logarithms of the peak areas of the six saccharide components of D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose in Polygonatum sibiricum are 0.086%, 0.28%, 0.056%, 0.56%, 0.49% and 0.36% respectively, indicating that the precision of the instrument is good.

[0077] Table 2 Results of precision test

[0078]

[0079]

[0080] 5.2. Repeatability test:

[0081] Take about 0.5 g of the crude powder of Polygonatum sibiricum (No. SHJ_HN_10), accurately weigh it, prepare the test solution according to the method under "3. Preparation of the test solution", and inject samples successively under the chromatographic conditions in "1. High-performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions" to determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose. The results are shown in Table 3. The results show that the average mass fractions of the six saccharide components of D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose in Polygonatum sibiricum are 2.79%, 0.847%, 2.60%, 0.852%, 0.286% and 0.441% respectively, and the RSDs are 1.5%, 0.47%, 1.8%, 0.78%, 0.51% and 0.27% respectively, indicating that the repeatability of this method is good.

[0082] Table 3 Results of repeatability test

[0083]

[0084] 5.3. Stability test:

[0085] Take about 0.5 g of the crude powder of Polygonatum sibiricum Red. (No. SHJ_HN_10), accurately weighed, and prepare the test solution according to the method under "3. Preparation of the test solution". At 0 min, 100 min, 200 min, 300 min, 400 min, 500 min, 1400 min, and 1500 min after the preparation of the test solution, inject the sample according to the chromatographic conditions under "1. HPLC conditions and ELSD detection conditions", and determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose, and fructofuranosylnystose. The results are shown in Table 4. The results show that the RSDs of the natural logarithms of the peak areas of the six saccharide components of D-fructose, glucose, sucrose, 1-kestose, nystose, and fructofuranosylnystose in Polygonatum sibiricum Red. are 0.078%, 0.24%, 0.058%, 0.49%, 0.048%, and 0.39% respectively, indicating that the test solution has good stability within 25 h after preparation.

[0086] Table 4 Results of the stability test

[0087]

[0088] 5.4 Spike recovery test:

[0089] Take about 0.25 g of the crude powder of Polygonatum sibiricum Red. (No. SHJ_HN_10), accurately weighed, place it in a 50 mL stoppered conical flask, accurately add the reference substance according to the ratio of the sample content to the added amount of the reference substance of approximately 1:1, prepare the test solution according to the method under "3. Preparation of the test solution", in parallel for 6 portions, inject 20 μL according to the chromatographic conditions under "1. HPLC conditions and ELSD detection conditions", determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose, and fructofuranosylnystose, and calculate the average spike recovery of the above six saccharide components in the test solution. The results are shown in Table 5. The results show that the average spike recoveries of D-fructose, glucose, sucrose, 1-kestose, nystose, and fructofuranosylnystose are 103.52%, 93.66%, 99.37%, 95.59%, 94.22%, and 97.55% respectively, and the RSDs are 3.2%, 1.7%, 3.7%, 3.6%, 3.0%, and 0.95% respectively, indicating that the accuracy of this method is good.

[0090] Table 5 Spike recovery test of six components in Polygonatum sibiricum Red.

[0091]

[0092] 6. Sample determination (standard curve method)

[0093] Take the polygonatum samples of different batches, prepare the test solution according to the method under the step "3. Preparation of the test solution", inject the sample according to the chromatographic conditions under the step "1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions", determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose, and calculate the contents. The results are shown in Table 6.

[0094] Table 6 Determination results of 6 kinds of saccharide components in polygonatum samples of multiple batches (standard curve method) Unit: / %

[0095]

[0096]

[0097] Note: "-" indicates not detected.

[0098] 7. Calculation of relative correction factor and relative intercept

[0099] There are two calculation methods for the relative correction factor. One is the slope method, and the other is the multi-point correction method. Previous research results have shown that for components with a standard curve approximately passing through the origin (y = ax + b, a / b > 100), there is no significant difference in the results obtained by the two calculation methods. However, for components with a standard curve not passing through the origin, only the slope method can be used to calculate the relative correction factor. The specific steps are as follows:

[0100] Slope method calculation formula: f i / s = a i / a s ; In the formula: f is the relative correction factor, a is the slope; s is the internal reference substance reference, and i is other control components.

[0101] Taking sucrose as the internal reference substance, the relative correction factors of the other 5 kinds of saccharide components to sucrose were calculated by the slope method. The results are shown in Table 7.

[0102] Calculation of relative intercept (Relative Intercept, RI): Since in the standard curve y = ax + b of the 6 kinds of saccharide components, the value of a / b is close to 0.1 and the intercept cannot be ignored far, therefore, based on the intercept of the internal reference substance sucrose, the relative intercepts of the other 5 kinds of sugars were calculated. The calculation formula is: RI i / s = I i / I s ; In the formula, I is the intercept; s is the internal reference substance reference, and i is other control components.

[0103] Taking sucrose as the internal reference substance, the relative intercepts of the other 5 kinds of saccharide components to sucrose were calculated according to the above formula. The results are shown in Table 7.

[0104] Table 7 Results of linear relationship investigation (not passing through the origin)

[0105]

[0106] Note: The regression equation here is the standard curve of the second experiment.

[0107] 8. Location of the chromatographic peaks of the components to be measured

[0108] Adopt BEH Amide chromatographic column. According to the test method under the step "4. Drawing of the standard curve and determination of the detection limit and quantification limit", inject 2, 6, 15, 25, 35, and 45 μL of the reference solution C1 respectively. Through the formula: r i / s = t Ri / t Rs Calculate the relative retention values (r i / s ) between D-fructose, glucose, 1-kestose, nystose, and fructofuranosylnystose and the internal reference substance sucrose. In the formula, t R is the retention time; s is the internal reference substance reference, and i is other control components. The results are shown in Table 8.

[0109] Table 8 Relative retention values of 6 saccharide components

[0110]

[0111]

[0112] 9. Sample determination ("one determination for multiple evaluations" method, QAMS method)

[0113] Take Polygonatum sibiricum samples of different batches, prepare the test solution according to the method under the step "3. Preparation of the test solution", inject the sample according to the chromatographic conditions under the step "1. High-performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions", and determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose, and fructofuranosylnystose; then, using sucrose as the internal reference substance, calculate the contents of each component to be measured through the relative correction factor (RCF), relative intercept (RI), and relative retention value (r i / s ) of the other 5 saccharide components established for sucrose. The results are shown in Table 9 - Table 10.

[0114] Table 9 Results of the content determination of 6 saccharide components in Polygonatum sibiricum samples of multiple batches (QAMS method)

[0115]

[0116]

[0117] Table 10 Location results of 6 saccharide components in Polygonatum sibiricum samples of multiple batches (QAMS method) Unit: / min

[0118]

[0119] 10. Calculation of the deviation of the determination results by two methods

[0120] Take polygonatum samples of different batches, prepare the test solution according to the method under the step "3. Preparation of the test solution", inject the sample according to the chromatographic conditions under the step "1. HPLC conditions and ELSD detection conditions", and determine the peak areas of D-fructose, glucose, sucrose, 1-kestose, nystose and fructofuranosylnystose; then, using sucrose as the internal reference substance, position each component to be measured through the relative retention values (r i / s ), relative correction factors (RCF) and relative intercepts (RI) of the other five saccharide components established for sucrose, and calculate the contents of each component to be measured; at the same time, use the external standard method for detection; then compare the results of the external standard method and QAMS, and use the RSD values of the two determination results to represent the difference between the two methods. The results are shown in Tables 11 - 14. The results show that: the positioning results of QAMS are basically the same, RSD < 1%, there is no obvious difference in the content determination results of the two methods, RSD < 3%, and furthermore, the results show that the r i , RCF and RI of the determined saccharide components are reliable, and the calculation results of QAMS are relatively accurate.

[0121] Table 11 Comparison of the results of D-fructose, glucose and sucrose by two determination methods

[0122]

[0123]

[0124] Table 12 Comparison of the results of 1-kestose, nystose and fructofuranosylnystose by two determination methods

[0125]

[0126]

[0127] Note "-" indicates not detected, the same as the following tables.

[0128] Table 13 Comparison of the positioning results of D-fructose, glucose and sucrose by two determination methods

[0129]

[0130]

[0131] Table 14 Comparison of the positioning results of 1-kestose, nystose and fructofuranosylnystose by two determination methods

[0132]

[0133]

[0134] 11. Durability Investigation of Relative Correction Factor, Relative Intercept and Relative Retention Value

[0135] 11.1 Investigation of Different Chromatographic Columns

[0136] The high performance liquid chromatography conditions (HPLC conditions), ELSD detection conditions, preparation of reference solution and preparation of test solution were the same as those in Example 1, with the only difference being the use of different chromatographic columns. The chromatographic column information and relative correction factors are shown in Table 15, the relative intercepts of different chromatographic columns are shown in Table 16, and the relative retention values of 6 saccharide components on different chromatographic columns are shown in Table 17.

[0137] Table 15 Relative Correction Factors of 6 Saccharide Components Measured on Different Chromatographic Columns

[0138]

[0139] The results showed that different chromatographic columns had different effects on the relative correction factor, with the greatest effect on fructooligosaccharide, followed by glucose and D-fructose, and the RSD were 15.5%, 13.4% and 5.3% respectively, all >5%, and the effects on fructooligosaccharide and 1-kestose were smaller, with RSD of 2.8% and 4.9% respectively, both <5%, indicating that the relative correction factor of saccharide components had poor adaptability on different chromatographic columns. Therefore, the "one test for multiple evaluations" detection method for saccharide components was not suitable for use on different chromatographic columns (Table 15).

[0140] Table 16 Relative Intercepts of 6 Saccharide Components Measured on Different Chromatographic Columns

[0141]

[0142] Table 17 Relative Retention Values of 6 Saccharide Components on Different Chromatographic Columns

[0143]

[0144] The results showed that different chromatographic columns had different effects on the relative intercept, with the greatest effect on glucose, followed by fructooligosaccharide and fructooligosaccharide, and the RSD were 20.4%, 9.6% and 5.8% respectively, all >5%, and the effects on D-fructose and 1-kestose were smaller, with RSD of 1.3% and 2.7% respectively, both <5%, indicating that the relative intercept of saccharide components had poor adaptability on different chromatographic columns (Table 16). Therefore, the "one test for multiple evaluations" method should be used with a fixed chromatographic column for the detection of saccharide components. However, different chromatographic columns had little effect on the relative retention values of each component, and the RSD were all less than 5% (Table 17).

[0145] 11.2 Investigation of Different Evaporative Light Scattering Detectors

[0146] The high performance liquid chromatography conditions (HPLC conditions), ELSD detection conditions, preparation of reference substance solution and preparation of test sample solution were the same as those in Example 1, except that different chromatographs and evaporative light scattering detectors were used. The chromatograph and detector information and relative correction factors are shown in Table 18, the relative intercepts of different chromatographs and detectors are shown in Table 19, and the relative retention values of 6 saccharide components on different chromatographs and detectors are shown in Table 20.

[0147] Table 18 Relative Correction Factors Measured on Different Chromatographs and Different Detectors

[0148]

[0149] Table 19 Relative Intercepts Measured on Different Chromatographs and Different Detectors

[0150]

[0151] Table 20 Relative Retention Values Measured on Different Chromatographs and Different Detectors

[0152]

[0153] The results showed that the relative correction factors, relative intercepts and relative retention values measured on the same chromatographic column with different brands of liquid phases and different types of evaporative light scattering (low temperature type / high temperature type, split / non-split) detectors were relatively stable, and the RSDs were all <5%, indicating that different instruments had good system adaptability to this method (Tables 18 - 20). Therefore, the established relative correction factors, relative intercepts and relative retention values can be used for different instruments.

[0154] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for simultaneously determining the contents of six sugar components in polygonatum, characterized in that: The following steps are involved: The peak areas of D-fructose, glucose, sucrose, kestose, kestetetrose and kestose in the test solution of Polygonatum sibiricum were determined by HPLC-ELSD. Then, the contents of D-fructose, glucose, sucrose, kestose, kestetetrose and kestose in the test solution of Polygonatum sibiricum were calculated by the "one measurement, multiple evaluations" method with sucrose as the internal reference. The method for preparing the polygonatum test solution comprises the steps of ultrasonically extracting and filtering polygonatum, using 70% methanol as the solvent, to obtain the polygonatum test solution; The detection conditions of the high performance liquid chromatography detection in the high performance liquid chromatography-evaporative light scattering detector coupling method are as follows: the chromatographic column is BEH Amide column, with 0.1% ammonia acetonitrile solution as mobile phase A, 0.1% ammonia as mobile phase B, gradient elution, gradient elution program: 0-15 min, 85%→80%, A; 15-40 min, 85%→75%, A; 40-60 min, 75%→70%, A; 60-90 min, 70%, A; "%" in the gradient elution program means "volume percentage".

2. The method according to claim 1, characterized in that The method according to claim 1 is characterized in that the detection conditions of the high performance liquid chromatography detection in the high performance liquid chromatography-evaporative light scattering detector combination method are: column temperature 40°C, injection volume 10μL~20μL.

3. The method according to claim 1, characterized in that The detection conditions of the evaporative light scattering detector in the high performance liquid chromatography-evaporative light scattering detector coupling method are: drift tube temperature 80° C.; nebulizer mode; heating, power level: 59%; gas pressure 30 psi.

4. The method according to claim 1, characterized in that: The polygonatum sibiricum includes raw polygonatum sibiricum and processed polygonatum sibiricum.

5. The method according to claim 4, characterized in that The processed polygonatum includes wine polygonatum and nine-steamed and nine-dried polygonatum.

6. The method according to claim 1, characterized in that The ultrasonic extraction conditions are as follows: power 300Kw, frequency 40Hz, time 30min.

7. Use of the method according to any one of claims 1 to 6 in evaluating the quality of Polygonatum sibiricum.

8. Use of the method according to any one of claims 1 to 6 in identifying the authenticity of polygonatum.

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