Method for simultaneously determining contents of six carbohydrate components in rhizoma polygonati

Abstract

The invention relates to the field of traditional Chinese medicine analysis, in particular to a method for simultaneously determining the content of six carbohydrate components in rhizoma polygonati. The invention discloses a method for simultaneously determining the contents of six carbohydrate components in rhizoma polygonati, which comprises the following steps of: establishing a relative correction factor and a relative intercept between the five carbohydrate components of fructose, glucose, kestose, nystose and nystose in the rhizoma polygonati and an internal reference substance by taking cane sugar which is low in cost and easy to obtain as the internal reference substance; according to the method, the contents of sucrose and other five carbohydrate components in the rhizoma polygonati are calculated by only adopting sucrose as a reference substance and utilizing a relative correction factor, a relative intercept and a relative retention value, so that multi-index quality evaluation on the rhizoma polygonati (crude products and processed products) is simple, convenient, rapid, comprehensive and accurate, and the quality controllability and the stable curative effect of the product are ensured; meanwhile, the detection cost and time are saved.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine analysis, and in particular to a method for simultaneously determining the contents of six sugar components in polygonatum. Background Art

[0002] Huangjing is also known as Xianren Yuliang, Tiger Ginger, and Jitoushen. The 2020 edition of the Chinese Pharmacopoeia lists Huangjing as the dried rhizome of Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red., or Polygonatum cyrtonema Hua. of the Liliaceae family. It is mainly distributed in Hebei, Gansu, Inner Mongolia, Shaanxi, and the southwest region. It is usually harvested in autumn and used fresh or dried. Huangjing has the effects of strengthening the spleen and replenishing qi, nourishing yin and moistening the lungs, and replenishing the kidneys and essence. There is a special discussion on this in the world's earliest existing food therapy monograph "Food Therapy Materia Medica", which is a typical traditional Chinese medicine with the same origin of medicine and food.

[0003] Polygonatum sibiricum was first recorded in Ming Yi Bie Lu, "Polygonatum sibiricum is sweet, mild and non-toxic. It mainly nourishes the middle and replenishes qi, eliminates rheumatism, and soothes the five internal organs. Long-term use can make the body light, prolong life, and prevent hunger"; "Zheng Lei Ben Cao" lists Polygonatum sibiricum as the first of the grass section; Zhang Hua's "Bo Wu Zhi" in the Jin Dynasty said: "The grass of the sun is called Polygonatum sibiricum. Eating it can make you live forever"; "Wu Fu Jing" records: "Polygonatum sibiricum obtains the pure essence of heaven and earth, so it is named Wu Ji Zhi"; "Rihuazi Ben Cao" says: "It replenishes the five labors and seven injuries, helps the muscles and bones, stops hunger, withstands cold and heat, benefits the spleen and stomach, and moisturizes the heart and lungs"; "Compendium of Materia Medica", a great work of Chinese herbal medicine before the 16th century written by Li Shizhen in the Ming Dynasty, records: "Because it obtains the essence of Kun soil, it is called Polygonatum sibiricum" "Polygonatum sibiricum replenishes all deficiencies, fills the essence, and nourishes qi and blood and moisturizes".

[0004] The 2020 edition of the Chinese Pharmacopoeia records that Polygonatum sibiricum is "sweet in nature, mild in taste; it enters the spleen, lung, and kidney meridians; it has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys." Traditional medicine believes that Polygonatum sibiricum is clinically used to treat spleen and stomach weakness, abdominal pain and diarrhea, fatigue, lung deficiency cough, internal heat and thirst, hot flashes 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 oligosaccharides are common in natural medicines, but their accurate determination has always been a problem due to the lack of ultraviolet absorption. This problem was not effectively solved until the emergence of mass detectors (such as ELSD, CAD and MS); among them, the evaporative light-scattering detector (ESLD) is the most popular and relatively low-cost mass detector, so it is the most widely used in the determination of sugar components. However, the cost of sugar component detection is still high, for two reasons: first, the reference substance of oligosaccharide components is expensive, and because the ELSD detector is not as sensitive as the PDA detector / ultraviolet detector, the amount of reference substance used is much greater than the reference substance for ultraviolet detection; second, the separation of sugar components usually uses a normal phase column (such as an amino column, an Amide column, a HIIC column, etc.) as the stationary phase, and the mobile phase uses a large proportion of organic phase, which consumes a huge amount of solvent, resulting in high detection costs.

[0008] The "Quantitative Analysis of Multi-components by Single Marker, QAMS" method draws on the comprehensive advantages of the classic internal standard method, correction factor method, ultraviolet absorption coefficient method and main component self-control method, and uses the relative correction factors (RCF) between components to determine the components in the absence of reference substances, thereby achieving quality control of traditional Chinese medicine. This method significantly reduces the cost and time of testing, and is easy to operate, which effectively improves the practicality and detection efficiency of the method.

[0009] The basic principle of the "one test, multiple evaluations" method is that within a certain linear range, the amount (mass or concentration) of the component 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 a relative correction factor (f) is established between the other components to be tested (i) and the internal reference substance (s). i / s ), through f i / s The content of other components can be calculated. Assuming there are n components to be tested, in step 1, select 1 to 2 components as internal references and use formula f i / s =(Ai / Ci) / (As / Cs) Calculate the relative correction factor f between other components and internal reference i / s ; Where As is the peak area of ​​the internal reference, Cs is the concentration of the internal reference, Ai is the peak area of ​​the reference substance, and Ci is the concentration of the substance being tested; Step 2, according to the peak area of ​​each component measured in the test sample, the concentration of the component to be tested is calculated using the following formula:

[0010] Cx=Ax×Cs' / As'f i / s;

[0011] In the formula, f i / s To obtain the relative correction factor, Ax is the peak area of ​​the component to be tested 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 in the test sample measured by the conventional method, and Cx is the concentration of the component to be tested.

[0012] Since the proposal of QAMS, many domestic and foreign scholars in the medical field have continuously strengthened the research on the mechanism and process of its quality control model, providing a good theoretical and practical reference for expanding the application scope and applicable fields of QAMS. The application scope of QAMS has also expanded 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 Chinese herbal medicines, extracts, tablets, pills, and capsules. However, to date, almost all components determined by the "one test, multiple evaluations" method are detected by ultraviolet detectors or diode array detectors. Although some literature has attempted to use ELSD detection to establish a "one test, multiple evaluations" method for 4 sugar components in traditional Chinese medicine, the research results were unsuccessful. Summary of the invention

[0013] The purpose of the present invention is to provide a method for simultaneously determining the contents of six sugar components in Polygonatum sibiricum, so as to solve the problems existing in the above-mentioned prior art.

[0014] The evaporative light-scattering detector (ESLD) is a universal detector. The detection result is related to the sample concentration. It can detect any sample with lower volatility than 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 the high-pressure airflow. The tiny droplets formed by the atomization are mixed in the carrier gas and enter the drift tube through the diversion. The mobile phase and low-boiling point components are evaporated, and the remaining high-boiling point sample aerosol enters the light scattering cell. When the light beam passes through the scattering cell, it is scattered by the sample aerosol. The scattered light is amplified by the photoelectric tube and processed into a digital signal of the chromatographic workstation - the chromatogram.

[0015] The present invention adopts a high performance liquid chromatography-evaporative light scattering detector coupling method to determine the contents of six sugar components (D-fructose, glucose, saccharide, saccharide tetraose and saccharide pentasaccharide) in polygonatum, and takes sucrose as an internal reference, calculates the relative correction factors and relative intercepts of the other five sugar components relative to sucrose, and calculates the contents of the other five sugar components based on the relative correction factors and relative intercepts. As a result, the RSD of the QAMS determination results of the six sugars and the calculation results of the external standard method are within 5%, which proves the accuracy of the method provided by the present invention. The present invention not only invents a "one-measurement-multiple-evaluation" method for synchronously determining the contents of six sugar components in the traditional Chinese medicine polygonatum, but also successfully realizes the application of the "one-measurement-multiple-evaluation" method on ELSD.

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

[0017] The present invention provides a method for simultaneously determining the contents of six sugar components in polygonatum, comprising the following steps:

[0018] The peak areas of D-fructose, glucose, sucrose, ketose triose, ketose tetraose and ketose pentasaccharide in the Polygonatum sibiricum test solution were determined by HPLC-ELSD. Then, sucrose was used as the internal reference and the contents of D-fructose, glucose, ketose triose, ketose tetraose and ketose pentasaccharide in the Polygonatum sibiricum test solution were calculated according to the "one measurement, multiple evaluations" method.

[0019] Preferably, in the HPLC-ELSD coupling method, 0.1% ammonia water·acetonitrile is used as mobile phase A, 0.1% ammonia water is used as mobile phase B, and gradient elution is performed.

[0020] Preferably, the detection conditions of the high performance liquid chromatography detection in the high performance liquid chromatography-evaporative light scattering detector coupling method are: the chromatographic column is BEH Amide column, column temperature 40℃, injection volume 10μL~20μL.

[0021] Preferably, the detection conditions of evaporative light scattering detection in the HPLC-ELSD coupling method are: drift tube temperature 80° C.; nebulizer mode; heating, power level 59%; gas pressure 30 psi.

[0022] The present invention selects an amino chromatographic column, a mobile phase of 0.1% ammonia water·acetonitrile (A)-0.1% ammonia water (B), gradient elution, and an evaporative light scattering detector for detection, and successfully achieves the determination of the contents of six sugar components in polygonatum by using only one reference substance, sucrose, and the application of a "one-measurement-multiple-evaluations" method on an evaporative light scattering detector.

[0023] Preferably, the polygonatum includes raw polygonatum and processed polygonatum.

[0024] Preferably, the processed polygonatum includes wine polygonatum and nine-steamed and nine-dried polygonatum.

[0025] Preferably, the method for preparing the polygonatum test solution comprises the steps of ultrasonically extracting and filtering polygonatum to obtain the polygonatum test solution.

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

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

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

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

[0030] The invention discloses a method for simultaneously determining the contents of six sugar components in polygonatum. The method uses sucrose, which is cheap and easily available, as an internal reference, establishes relative correction factors and relative intercepts between five sugar components, namely, D-fructose, glucose, saccharide, saccharide tetraose and saccharide pentasaccharide, in polygonatum and the internal reference, and uses only one reference substance, namely, sucrose, to calculate the contents of sucrose and other five sugar components in polygonatum by using relative correction factors, relative intercepts and relative retention values, thereby simply, quickly, comprehensively and accurately performing multi-index quality evaluation on polygonatum (raw product and processed product), which is helpful to ensure the quality of polygonatum and related products thereof, and saves detection cost and time at the same time.

[0031] The results of the specific embodiment of the present invention show that the RSD of the QAMS determination results of the six sugars and the calculation results of the external standard method are within 5%, which proves the accuracy of the method provided by the present invention. It can be seen that the present invention not only invented a "one-measurement-multiple-evaluation" method for synchronously determining the contents of six sugar components in the traditional Chinese medicine Polygonatum odoratum, but also successfully realized the application of the "one-measurement-multiple-evaluation" method on ELSD. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 The standard curves (regression equations) and correlation coefficients of the six sugar components;

[0034] Figure 2The HPLC-ELSD spectrum of six sugar components in freeze-dried fresh polygonatum; wherein 1-6 in the figure are D-fructose, glucose, sucrose, ketose triose, ketose tetraose and ketose pentose;

[0035] Figure 3 This is the HPLC-ELSD spectrum of 6 sugar components in raw polygonatum; 1-6 in the figure are D-fructose, glucose, sucrose, ketose triose, ketose tetraose and ketose pentose, respectively;

[0036] Figure 4 This is the HPLC-ELSD spectrum of six sugar components in nine-steamed and nine-dried polygonatum; 1-3 in the figure are D-fructose, glucose and sucrose respectively;

[0037] Figure 5 It is the HPLC-ELSD spectrum of the mixed reference substance; wherein, 1-6 in the figure are D-fructose, glucose, sucrose, kestose, kestose and pentose, respectively. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[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 0.25g of Lichuan nine-steamed and nine-dried polygonatum, 0.25g of Lichuan fresh polygonatum, and 0.25g of Lichuan freeze-dried fresh polygonatum, accurately weigh, accurately add 25mL of 70% methanol solution to each of them, weigh the weight, extract with ultrasound (power 300Kw, frequency 40Hz) for 30min, take out, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0054] 4. HPLC-ELSD spectra of six sugar components in different Polygonatum samples

[0055] HPLC-ELSD spectra of six sugar components in different Polygonatum samples are shown in Figure 2. Figure 2 to Figure 5 shown.

[0056] Example 2

[0057] A method for simultaneously determining the contents of six sugar components (D-fructose, glucose, sucrose, nitrosaccharide, nitrosaccharide tetraose and nitrosaccharide pentasaccharide) in polygonatum using sucrose as an internal reference, 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 HPLC, 2695 Quaternary Solvent Manager, 2424 Evaporative Light Scattering Detector, Empower 3 Chromatography workstation; BEH Amide chromatographic column, 5μm, 4.6×250mm Column.

[0061] 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.

[0062] 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.

[0063] 1.2. ELSD detection conditions:

[0064] Drift tube temperature: 80°C; Nebulizer mode; heating, power level 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 konjac triose, 50.60 mg of konjac tetraose and 50.60 mg of konjac pentasaccharide, respectively, and place them in 25 mL volumetric flasks, add 70% methanol to dissolve and dilute to the scale, shake well, and use them as the reference stock solutions (C0) of each monosaccharide.

[0067] 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 this as the mixed reference solution (C1).

[0068] 3. Preparation of test solution

[0069] Take about 0.5g of raw polygonatum powder (0.25g of wine polygonatum, 0.25g of nine-steamed and nine-dried polygonatum, and 0.25g of fresh polygonatum), accurately weigh, accurately add 25mL of 70% methanol solution to each, weigh, extract with ultrasound (power 300Kw, frequency 40Hz) for 30min, take out, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

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

[0071] Take the mixed reference solution C1 and inject 1, 2, 4, 10, 20, 30, 40 and 50 μL respectively. The natural logarithm of the reference sample injection amount (Ln μg) is used as the abscissa and the natural logarithm of the peak area (Ln Area) is used as the ordinate. The standard curve is drawn, and linear regression is performed to calculate the correlation coefficient and determine the detection limit and quantification limit. The regression equations and correlation coefficients of the six sugar components are shown in Tables 1 and Figure 1 The results showed that the linear relationship was good in the range of fructose 1.12-28.01μg, glucose 0.560-14.01μg, sucrose 1.12-28.03μg, nitrosaccharide 1.13-28.17μg, nitrosaccharide 0.570-14.25μg, and nitrosaccharide 0.567-14.17μg.

[0072] Table 1 Results of linear relationship investigation of 6 sugar components

[0073]

[0074] 5. Methodological investigation

[0075] 5.1. Precision test:

[0076] About 0.5 g of coarse powder of Polygonatum sibiricum (No. SHJ_HN_10) was taken and accurately weighed. The test solution was prepared according to the method in step "3. Preparation of test solution". According to the chromatographic conditions in step "1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions", the peak areas of D-fructose, glucose, sucrose, nitrosaccharide, nitrosaccharide and nitrosaccharide were measured for 6 times. The results are shown in Table 2. The results show that the RSDs of the natural logarithms of the peak areas of the six sugar components D-fructose, glucose, sucrose, nitrosaccharide, nitrosaccharide and nitrosaccharide 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 Precision test results

[0078]

[0079]

[0080] 5.2. Repeatability test:

[0081] About 0.5 g of coarse powder of Polygonatum sibiricum (No. SHJ_HN_10) was taken and accurately weighed. The test solution was prepared according to the method in step "3. Preparation of test solution". The samples were injected in sequence according to the chromatographic conditions in step "1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions". The peak areas of D-fructose, glucose, sucrose, nitrosaccharide, nitrosaccharide and nitrosaccharide were determined. The results are shown in Table 3. The results showed that the average mass fractions of the six sugar components of D-fructose, glucose, sucrose, nitrosaccharide, nitrosaccharide and nitrosaccharide in Polygonatum sibiricum were 2.79%, 0.847%, 2.60%, 0.852%, 0.286% and 0.441%, respectively, and the RSDs were 1.5%, 0.47%, 1.8%, 0.78%, 0.51% and 0.27%, respectively, indicating that the method had good repeatability.

[0082] Table 3 Repeatability test results

[0083]

[0084] 5.3 Stability test:

[0085] Take about 0.5 g of coarse powder of Polygonatum sibiricum (No. SHJ_HN_10), accurately weigh it, prepare the test solution according to the method in step “3. Preparation of test solution”, and inject the sample according to the chromatographic conditions in “1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions” 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, and determine the peak areas of D-fructose, glucose, sucrose, sucrose triose, sucrose tetraose and sucrose pentasaccharide. The results are shown in Table 4. The results showed that the RSDs of the natural logarithms of the peak areas of the six sugar components in Polygonatum sibiricum, including D-fructose, glucose, sucrose, ketose triose, ketose tetraose and ketose pentasaccharide, were 0.078%, 0.24%, 0.058%, 0.49%, 0.048% and 0.39%, respectively, indicating that the test solution had good stability within 25 hours after preparation.

[0086] Table 4 Stability test results

[0087]

[0088] 5.4. Sample recovery test:

[0089] Take about 0.25g of coarse powder of Polygonatum sibiricum (No. SHJ_HN_10), accurately weigh it, put it in a 50mL stoppered conical flask, accurately add the reference substance at a ratio of sample content / reference substance addition of approximately 1:1, prepare the test solution according to the method in step "3. Preparation of test solution", parallel 6 parts, inject 20μL according to the chromatographic conditions in "1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions", determine the peak areas of D-fructose, glucose, sucrose, sucrose triose, sucrose tetraose and sucrose pentasaccharide, calculate the average sample recovery rate of the above 6 sugar components in the test solution, and the results are shown in Table 5. The results showed that the average recoveries of D-fructose, glucose, sucrose, ketose triose, ketose tetraose and ketose pentose were 103.52%, 93.66%, 99.37%, 95.59%, 94.22% and 97.55%, respectively, and the RSDs were 3.2%, 1.7%, 3.7%, 3.6%, 3.0% and 0.95%, respectively, indicating that the method has good accuracy.

[0090] Table 5 Sample recovery test of 6 components in Polygonatum sibiricum

[0091]

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

[0093] Take different batches of Polygonatum sibiricum samples, prepare the test solution according to the method in step “3. Preparation of test solution”, inject the samples according to the chromatographic conditions in step “1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions”, determine the peak areas of D-fructose, glucose, sucrose, saccharide, saccharide tetraose and saccharide pentasaccharide, and calculate the contents. The results are shown in Table 6.

[0094] Table 6 Determination results of the contents of 6 sugar components in multiple batches of Polygonatum cyrtonema samples (standard curve method) Unit: / %

[0095]

[0096]

[0097] Note: “-” means not detected.

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

[0099] There are two methods for calculating the relative correction factor, one is the slope method, and the other is the multi-point correction method. Previous research results show that for components whose standard curves approximately pass 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 whose standard curves do not pass 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, and i is other control components.

[0101] With sucrose as the internal reference, the slope method was used to calculate the relative correction factors of the other five sugar components to sucrose. The results are shown in Table 7.

[0102] Calculation of Relative Intercept (RI): Because the a / b value of the standard curve y=ax+b of the six sugar components is close to 0.1, the intercept is far from being negligible. Therefore, we used the intercept of the internal reference sucrose as a benchmark to calculate the relative intercepts of the other five sugars. The calculation formula is: RI i / s =I i / I s ; In the formula, I is the intercept; s is the internal reference substance, and i is other control components.

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

[0104] Table 7 Linear relationship investigation results (but not the origin)

[0105]

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

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

[0108] use For the BEH Amide column, according to the test method in step "4, drawing of standard curve and determination of detection limit and quantitation limit", 2, 6, 15, 25, 35, and 45 μL of the reference solution C1 were injected respectively. According to the formula: i / s =t Ri / t Rs The relative retention values ​​(r) between D-fructose, glucose, ketose triose, ketose tetraose and ketose pentose and the internal reference sucrose were calculated. i / s ), where t R is the retention time; s is the internal reference substance, and i is other control components. The results are shown in Table 8.

[0109] Table 8 Relative retention values ​​of 6 sugar components

[0110]

[0111]

[0112] 9. Sample determination (“one test, multiple evaluations” method, QAMS method)

[0113] Different batches of Polygonatum sibiricum samples were taken, and the test solutions were prepared according to the method in step "3. Preparation of test solution". The samples were injected according to the chromatographic conditions in step "1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions", and the peak areas of D-fructose, glucose, sucrose, sucrose triose, sucrose tetraose and sucrose pentasaccharide were determined; then, sucrose was used as the internal reference, and the relative correction factor (RCF), relative intercept (RI) and relative retention value (R) of the other five sugar components to sucrose were established. i / s ) Calculate the content of each component to be tested. The results are shown in Tables 9 and 10.

[0114] Table 9 Determination results of the contents of 6 sugar components in multiple batches of Polygonatum sibiricum samples (QAMS method)

[0115]

[0116]

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

[0118]

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

[0120] Different batches of Polygonatum sibiricum samples were taken, and the test solutions were prepared according to the method in step "3. Preparation of test solution". The samples were injected according to the chromatographic conditions in step "1. High performance liquid chromatography conditions (HPLC conditions) and ELSD detection conditions", and the peak areas of D-fructose, glucose, sucrose, sucrose triose, sucrose tetraose and sucrose pentasaccharide were determined; then, sucrose was used as the internal reference, and the relative retention values ​​(r i / s ), relative correction factor (RCF) and relative intercept (RI) were used to locate each component to be tested and calculate the content of each component to be tested; the external standard method was used for detection at the same time; the results of the external standard method and QAMS were then compared, and the RSD value of the two determination results was used to represent the difference between the two methods. The results are shown in Tables 11-14. The results showed that the QAMS positioning results were basically consistent, with RSD < 1%, and there was no significant difference in the content determination results of the two methods, with RSD < 3%. In addition, the results showed that the r value of the determined carbohydrate components was i , RCF and RI are reliable, and the calculation results of QAMS are more 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 two determination methods for ketose triose, ketose tetraose and ketose pentose

[0125]

[0126]

[0127] Note: “-” indicates not detected, the same as in the following table.

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

[0129]

[0130]

[0131] Table 14 Comparison of the localization results of ketose triose, ketose tetraose and ketose pentose by two determination methods

[0132]

[0133]

[0134] 11. Durability study 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 the reference solution and preparation of the test solution were the same as those in Example 1, with the only difference being that different chromatographic columns were used. 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 the six sugar components on different chromatographic columns are shown in Table 17.

[0137] Table 15 Relative correction factors of 6 sugar components measured on different chromatographic columns

[0138]

[0139] The results showed that different chromatographic columns had different effects on the relative correction factor. The greatest impact was on pentasaccharide, followed by glucose and D-fructose, with RSDs of 15.5%, 13.4% and 5.3%, respectively, all >5%. The impact on tetrasaccharide and trisaccharide was smaller, with RSDs of 2.8% and 4.9%, respectively, both <5%, indicating that the relative correction factor of carbohydrate components had poor adaptability on different chromatographic columns. Therefore, the "one test, multiple evaluations" detection method for carbohydrate components should not be carried out on different chromatographic columns (Table 15).

[0140] Table 16 Relative intercepts of 6 sugar components measured on different chromatographic columns

[0141]

[0142] Table 17 Relative retention values ​​of 6 sugar components on different chromatographic columns

[0143]

[0144] The results showed that different chromatographic columns had different effects on relative intercepts, with the greatest impact on glucose, followed by sucrose pentose and sucrose tetraose, with RSDs of 20.4%, 9.6% and 5.8%, respectively, all >5%, and less impact on D-fructose and sucrose triose, with RSDs of 1.3% and 2.7%, respectively, all <5%, indicating that the relative intercept of sugar components had poor adaptability on different chromatographic columns (Table 16). Therefore, the "one test, multiple evaluations" method for detecting sugar components should be carried out with a fixed chromatographic column. However, different chromatographic columns had little effect on the relative retention values ​​of each component, with RSDs of 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 the reference solution and preparation of the test solution are the same as those in Example 1, except that different chromatographs and evaporative light scattering detectors are used. The chromatograph and detector information and the 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 the six sugar 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 factor, relative intercept and relative retention value measured by the same chromatographic column on different brands of liquid phase and different types of evaporative light (low temperature type / high temperature type, split / splitless) detectors were relatively stable, and the RSD was <5%, indicating that different instruments had good system adaptability to the method (Table 18-Table 20). Therefore, the established relative correction factor, relative intercept and relative retention value can be used for different instruments.

[0154] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field 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 pentasaccharide in the test solution of Polygonatum sibiricum were determined by HPLC-ELSD. Then, sucrose was used as the internal reference and the contents of D-fructose, glucose, sucrose, kestose, kestetetrose and pentasaccharide in the test solution of Polygonatum sibiricum were calculated according to the "one measurement and multiple evaluations" method.

2. The method according to claim 1, characterized in that: In the high performance liquid chromatography-evaporative light scattering detector coupling method, the high performance liquid chromatography separation uses 0.1% ammonia acetonitrile solution as mobile phase A and 0.1% ammonia water as mobile phase B, and gradient elution.

3. The method according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography detection in the high performance liquid chromatography-evaporative light scattering detector coupling method are: the chromatographic column is BEH Amide column, column temperature 40℃, injection volume 10μL~20μL.

4. 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.

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

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

7. The method according to claim 1, characterized in that The method for preparing the polygonatum test solution comprises the steps of ultrasonically extracting and filtering polygonatum to obtain the polygonatum test solution.

8. The method according to claim 7, characterized in that The ultrasonic extraction conditions are as follows: power 300Kw, frequency 40Hz, time 30min.

9. Use of the method according to any one of claims 1 to 8 in evaluating the quality of Polygonatum sibiricum.

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

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