Application of difructose anhydride I and difructose anhydride III as markers in rapid detection of polygonatum kingianum and wine-processed polygonatum kingianum and detection method of difructose anhydride I and difructose anhydride III in polygonatum kingianum

Abstract

The invention relates to the technical field of analysis and detection, in particular to application of difructose anhydride I and difructose anhydride III as markers in rapid detection of rhizoma polygonati and wine rhizoma polygonati and a detection method of difructose anhydride I and difructose anhydride III in rhizoma polygonati. The invention provides application of difructose anhydride I and difructose anhydride III as markers in rapid detection of polygonatum kingianum and wine-processed polygonatum kingianum. The total content of the difructose anhydride I and the difructose anhydride III can effectively distinguish the raw rhizoma polygonati from the wine rhizoma polygonati.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to the application of difructose anhydride I and difructose anhydride III as markers in the rapid detection of Polygonatum sibiricum and Polygonatum sibiricum, and a method for detecting difructose anhydride I and difructose anhydride III in Polygonatum sibiricum. Background Technology

[0002] Polygonatum (PR) is a perennial herb belonging to the genus Polygonatum in the family Liliaceae. It is mainly distributed in East and Southeast Asia. The raw product has a slight odor, a sweet taste, and is sticky when chewed. It is neutral in nature and enters the spleen, lung, and kidney meridians. It has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It can be used for spleen and stomach qi deficiency, fatigue, and internal heat with thirst. PR is a traditional and classic medicinal and edible plant, widely consumed as a valuable commodity in the functional food, dietary supplement, and health product industries. It has significant health benefits and is considered to have nourishing, strengthening, and longevity-promoting effects. However, the raw product contains irritating components, which can cause a numbing sensation on the tongue when consumed. After processing, the irritation is eliminated, and the tonic effects are enhanced.

[0003] In the theoretical system of Traditional Chinese Medicine (TCM), the concepts of "judging quality by form" and "interrelationship between exterior and interior" provide a unique perspective on the processing of Polygonatum odoratum. Traditionally, it is believed that processed Polygonatum odoratum is best when it is "black in color." Meanwhile, polysaccharides are the only quality control component of Polygonatum odoratum as specified in the 2020 edition of the Chinese Pharmacopoeia. Currently, domestic and international literature reports and the analysis of the sugar components of Polygonatum odoratum in Part I of the 2020 edition of the Chinese Pharmacopoeia mainly use the sulfuric acid-anthrone-UV-Vis spectroscopy method. This method uses glucose as a reference to determine the polysaccharide content of Polygonatum odoratum, resulting in poor specificity. Furthermore, the preparation process of the test sample is cumbersome, involving the use of potentially explosive sulfuric acid and potentially toxic anthrone reagents, raising safety concerns. In addition, existing quality evaluation methods for processed Polygonatum odoratum mainly focus on the determination of polysaccharide, flavonoid, Polygonatine A, fructose, glucose, total saponins, EBC, and 5-hydroxymethylfurfural content. However, these evaluation indicators and methods are still relatively one-sided and lack accuracy. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an application of difructose anhydride I (DFA I) and difructose anhydride III (DFA III) as markers for rapid detection of Polygonatum sibiricum and Polygonatum sibiricum, and a method for detecting difructose anhydride I and difructose anhydride III in Polygonatum sibiricum. The total content of difructose anhydride I and difructose anhydride III can effectively distinguish between raw Polygonatum sibiricum and Polygonatum sibiricum.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of difructose anhydride I and difructose anhydride III as markers in the rapid detection of Polygonatum sibiricum and Polygonatum sibiricum.

[0006] This invention also provides a method for detecting difructose anhydride I and difructose anhydride III in Polygonatum sibiricum, comprising the following steps: The Polygonatum sibiricum to be tested was mixed with the extractant and extracted to obtain the extract; The extract was subjected to high performance liquid chromatography-electrospray detection to obtain the peak areas of difructose anhydride I and difructose anhydride III, respectively. Based on the peak areas of difructose anhydride I and difructose anhydride III and their respective predetermined standard curves, the mass concentrations of difructose anhydride I and difructose anhydride III are obtained respectively. The predetermined standard curve is a linear relationship between the peak area and mass concentration of difructose anhydride I or difructose anhydride III. The separation conditions for the high performance liquid chromatography include: mobile phase A is acetonitrile, and mobile phase B is an aqueous ammonia solution.

[0007] Preferably, the elution method of the mobile phase in the high-performance liquid chromatography is isocratic elution; The volume ratio of mobile phase A to mobile phase B is 87:13; The ammonia solution is an ammonia solution with a volume concentration of 0.1%.

[0008] Preferably, the flow rate of the mobile phase is 1.0 mL / min and the column temperature is 30 °C.

[0009] Preferably, the sampling frequency of the electro-fogging detection is 5Hz, the filtering time is 2.0s, and the evaporation temperature is 60℃.

[0010] Preferably, the linear relationship between the peak area and mass concentration of the difructose anhydride I is: Y = 60.473X + 2.2758; Where Y is the peak area and X is the mass concentration.

[0011] Preferably, the linear relationship between the peak area and mass concentration of the difructose anhydride III is: Y = 53.697X + 2.1644; Where Y is the peak area and X is the mass concentration.

[0012] Preferably, the extractant is ethanol and water in a volume ratio of 80:20.

[0013] Preferably, the ratio of the amount of Polygonatum sibiricum to the extractant is 0.5g:40mL.

[0014] Preferably, the extraction is performed under ultrasonic conditions; The extraction process also includes filtration using a 0.22 μm filter membrane.

[0015] This invention provides an application of difructose anhydride I and difructose anhydride III as biomarkers in the rapid detection of Polygonatum sibiricum and Polygonatum sibiricum processed with wine. The total content of difructose anhydride I and difructose anhydride III can effectively distinguish between raw Polygonatum sibiricum and Polygonatum sibiricum processed with wine, and has the potential to serve as a key indicator for evaluating the processing technology and quality of Polygonatum sibiricum processed with wine.

[0016] This invention also provides a method for detecting difructose anhydride I and difructose anhydride III in Polygonatum sibiricum, comprising the following steps: mixing the Polygonatum sibiricum to be tested with an extractant for extraction to obtain an extract; performing high-performance liquid chromatography-electrospray spectroscopy on the extract to obtain the peak areas of difructose anhydride I and difructose anhydride III, respectively; obtaining the mass concentrations of difructose anhydride I and difructose anhydride III based on the peak areas of difructose anhydride I and difructose anhydride III and their respective predetermined standard curves; the predetermined standard curves are linear relationships between the peak area and mass concentration of difructose anhydride I or difructose anhydride III; the separation conditions of the high-performance liquid chromatography include: mobile phase A being acetonitrile and mobile phase B being an ammonia solution. This detection method features high sensitivity, convenient pretreatment, and rapid and accurate determination results. Attached Figure Description

[0017] Figure 1 The mass concentrations of difructose anhydride I, difructose anhydride III, and total mass concentration are given for the test objects numbered 2, 5, 8-23. Figure 2 The total mass concentration of difructose anhydride I and difructose anhydride III in the analytes numbered K0~K9, C0~C9, and S0~S9. Detailed Implementation

[0018] This invention provides the application of difructose anhydride I and difructose anhydride III as markers in the rapid detection of Polygonatum sibiricum and Polygonatum sibiricum.

[0019] In this invention, the structural formula of the difructose anhydride I is: .

[0020] In this invention, the structural formula of the difructose anhydride III is: .

[0021] This invention also provides a method for detecting difructose anhydride I and difructose anhydride III in Polygonatum sibiricum, comprising the following steps: The Polygonatum sibiricum to be tested was mixed with the extractant and extracted to obtain the extract; The extract was subjected to high performance liquid chromatography-electrospray detection to obtain the peak areas of difructose anhydride I and difructose anhydride III, respectively. Based on the peak areas of difructose anhydride I and difructose anhydride III and their respective predetermined standard curves, the mass concentrations of difructose anhydride I and difructose anhydride III are obtained respectively. The predetermined standard curve is a linear relationship between the peak area and mass concentration of difructose anhydride I or difructose anhydride III. The separation conditions for the high performance liquid chromatography include: mobile phase A is acetonitrile, and mobile phase B is an aqueous ammonia solution.

[0022] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0023] In this invention, the Polygonatum sibiricum to be tested is mixed with an extractant for extraction to obtain an extract.

[0024] In this invention, before mixing the Polygonatum sibiricum to be tested with the extractant, it is preferable to sieve the fine powder of the Polygonatum sibiricum to be tested. Preferably, a No. 5 sieve is used for sieving.

[0025] In this invention, the extractant is preferably an ethanol and water mixture with a volume ratio of 80:20 (i.e., an 80% ethanol solution).

[0026] In this invention, the preferred ratio of the amount of the test Polygonatum to the extractant is 0.5g:40mL.

[0027] In this invention, the extraction is preferably performed under ultrasonic conditions, with the ultrasonic time preferably being 30 minutes, the temperature being room temperature, and the power being 250W. In this invention, room temperature can be understood as no additional heating or cooling being performed.

[0028] After the extraction is completed, cooling is also preferably included. The present invention does not have any special limitation on the cooling method, and any method known to those skilled in the art can be used.

[0029] After cooling, it is preferable to replenish the lost weight with an extractant of the same type as the extractant, then shake and filter in sequence; the filtration is preferably performed using a 0.22 μm filter membrane.

[0030] After obtaining the extract, the present invention performs high performance liquid chromatography-electrospray detection on the extract to obtain the peak areas of difructose anhydride I and difructose anhydride III, respectively; based on the peak areas of difructose anhydride I and difructose anhydride III and their respective predetermined standard curves, the mass concentrations of difructose anhydride I and difructose anhydride III are obtained, respectively.

[0031] In this invention, the elution method of the mobile phase in the high-performance liquid chromatography is preferably isocratic elution; the volume ratio of mobile phase A to mobile phase B is preferably 87:13. The ammonia solution is preferably a 0.1% (v / v) ammonia solution.

[0032] In this invention, the flow rate of the mobile phase is preferably 1.0 mL / min, and the column temperature is preferably 30 °C.

[0033] In this invention, the preferred acquisition frequency of the electro-fogging detection is 5Hz, the preferred filtering is 2.0s, and the preferred evaporation temperature is 60℃.

[0034] In this invention, the preferred linear relationship between the peak area and mass concentration of the difructose anhydride I is: Y = 60.473X + 2.2758; where Y is the peak area and X is the mass concentration.

[0035] In this invention, the preferred linear relationship between the peak area and mass concentration of the difructose anhydride III is: Y = 53.697X + 2.1644; where Y is the peak area and X is the mass concentration.

[0036] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] Example 1 The linear relationship between the peak area and mass concentration of the difructose anhydride I was plotted as follows: The difructose anhydride I was mixed with an 80% ethanol solution and diluted to a volumetric flask of 10 mL to obtain a difructose anhydride I reference solution with a concentration of 0.522 mg / mL. Using 80% ethanol solution as a solvent, the 0.522 mg / mL difructose anhydride I reference solution was serially diluted to obtain a series of difructose anhydride I solutions with mass concentrations of 0.522 mg / mL, 0.261 mg / mL, 0.131 mg / mL, 0.065 mg / mL, 0.033 mg / mL and 0.016 mg / mL, respectively. The series of difructose anhydride I solutions of the above-mentioned mass concentrations were subjected to high performance liquid chromatography-electrospray detection (elution mode: isocratic elution, mobile phase A is acetonitrile, mobile phase B is 0.1% ammonia solution; the volume ratio of mobile phase A to mobile phase B is 87:13, the flow rate of the mobile phase is 1.0 mL / min, and the column temperature is 30℃). The electro-fogging detection was performed at a sampling frequency of 5 Hz, a filtering time of 2.0 s, and an evaporation temperature of 60 ℃. Peak areas were recorded. Linear regression was performed with peak area as the ordinate (Y) and mass concentration as the abscissa (X). The linear relationship curves are shown in Table 1. The linear relationship between the peak area and mass concentration of the difructose anhydride III was plotted as follows: The difructose anhydride III was mixed with an 80% ethanol solution and diluted to a volumetric flask of 10 mL to obtain a difructose anhydride III reference solution with a concentration of 0.518 mg / mL. Using 80% ethanol solution as a solvent, the 0.518 mg / mL difructose anhydride III reference solution was serially diluted to obtain a series of difructose anhydride III solutions with mass concentrations of 0.518 mg / mL, 0.259 mg / mL, 0.130 mg / mL, 0.065 mg / mL, 0.032 mg / mL and 0.016 mg / mL, respectively. The series of difructose anhydride III solutions of the above-mentioned mass concentrations were subjected to high performance liquid chromatography-electrospray detection (elution mode: isocratic elution, mobile phase A is acetonitrile, mobile phase B is 0.1% ammonia solution; the volume ratio of mobile phase A to mobile phase B is 87:13, the flow rate of the mobile phase is 1.0 mL / min, and the column temperature is 30℃). The electro-fogging detection was performed at a sampling frequency of 5 Hz, a filtering time of 2.0 s, and an evaporation temperature of 60 ℃. Peak areas were recorded. Linear regression was performed with peak area as the ordinate (Y) and mass concentration as the abscissa (X). The linear relationship curves are shown in Table 1. Table 1. Linear relationship between peak area and mass concentration of difructose anhydride I and difructose anhydride III

[0038] Test object: K0: Batch number YNDH202401, collected from Yunnan Dehong Polygonatum; Yellow wine dosage: 20kg of yellow wine for every 100kg of Polygonatum.

[0039] K1: Add 1 kg of rice wine to 5 kg of K0 and let it soak until the Polygonatum is absorbed and softens. Place it in a steamer, add water, and steam for 3 hours. Mix the juice into the steamed Polygonatum and let it air dry. Take 500g, slice it, and sun-dry it for 48 hours (i.e., after one steaming and one sun-drying process) to obtain K1; K2: Following the above procedure, the remaining unsliced ​​Polygonatum samples were subjected to two steaming and two sun-drying processes (during the subsequent steaming and sun-drying processes, the steamed Polygonatum was mixed with the juice and dried). Then, 500g was taken, sliced, and sun-dried for 48 hours (i.e., the second steaming and sun-drying process) to obtain K2; K3, K4, K5, K6, K7 and K8 follow the same pattern, K9: follow the above steaming and drying process, and perform nine steaming and nine drying cycles; C0: Batch number AHBZ202401, collected from Polygonatum cyrtonema in Bozhou, Anhui; Yellow wine dosage: 20 kg of yellow wine for every 100 kg of Polygonatum cyrtonema.

[0040] C1: Add 1 kg of rice wine to 5 kg of the above C0 mixture, let it soak until the Polygonatum is absorbed and softens, then place it in a steamer, add water and steam for 3 hours. Mix the juice into the steamed Polygonatum and air dry. Take 500g, slice it, and sun-dry for 48 hours (i.e., after one steaming and one sun-drying process) to obtain C1; C2: Following the above procedure, the remaining unsliced ​​Polygonatum samples were subjected to two steaming and two sun-drying processes (during the subsequent steaming and sun-drying processes, the steamed Polygonatum was mixed with the juice and dried). Then, 500g was taken, sliced, and sun-dried for 48 hours (i.e., the second steaming and sun-drying process) to obtain C2; C3, C4, C5, C6, C7 and C8 follow the same pattern, C9: follow the above steaming and drying process, and perform nine steaming and nine drying cycles; S0: Batch number HNLY202401, collected from Polygonatum sibiricum (chicken head Polygonatum) in Luoyang, Henan; Yellow wine dosage: 20 kg of yellow wine for every 100 kg of Polygonatum sibiricum.

[0041] S1: Add 1 kg of rice wine to 5 kg of the mixture described in S0, let it soak until the Polygonatum is absorbed and softens, then place it in a steamer, add water and steam for 3 hours. Mix the juice into the steamed Polygonatum, and let it air dry. Take 500g, slice it, and sun-dry it for 48 hours (i.e., after one steaming and one sun-drying process) to obtain S1; S2: Following the above procedure, the remaining unsliced ​​Polygonatum samples were subjected to two steaming and two sun-drying processes (during the subsequent steaming and sun-drying processes, the steamed Polygonatum was mixed with the juice and dried). Then, 500g was taken, sliced, and sun-dried for 48 hours (this is the second steaming and sun-drying process) to obtain S2; S3, S4, S5, S6, S7 and S8 follow the same pattern, S9: follow the above steaming and drying process, and perform nine steaming and nine drying cycles; Other market samples are shown in Table 2: Table 2. Relevant Sources of Samples Collected from Other Markets

[0042] After passing the fine powder of the above-mentioned test objects through a No. 5 sieve, 0.5g of each powder was weighed into a stoppered conical flask, 40mL of 80% ethanol solution was added, the powder was weighed, and the mixture was sonicated at room temperature for 30min (power of 250W). The mixture was then removed, cooled, and the lost weight was replenished with 80% ethanol solution. The mixture was shaken well and filtered through a 0.22μm filter membrane to obtain the extract. The extract was subjected to high performance liquid chromatography-electrospray detection (elution mode: isocratic elution, mobile phase A was acetonitrile, mobile phase B was 0.1% ammonia solution; the volume ratio of mobile phase A to mobile phase B was 87:13, the flow rate of the mobile phase was 1.0 mL / min, the column temperature was 30℃; the acquisition frequency of electrospray detection was 5 Hz, the filtering time was 2.0 s, and the evaporation temperature was 60℃), and the peak areas of difructose anhydride I and difructose anhydride III were obtained respectively. Based on the peak areas of difructose anhydride I and difructose anhydride III and their respective predetermined standard curves, the mass concentrations of difructose anhydride I and difructose anhydride III were obtained respectively (results are shown in Table 3 and...). Figures 1-2 (as shown) Table 3. Mass concentrations of fructosine I and fructosine III in the above-mentioned test subjects.

[0043] Figure 1 The mass concentrations of difructose anhydride I, difructose anhydride III, and total mass concentration are given for the test objects numbered 1 to 18. Figure 2 The total mass concentrations of difructose anhydride I and difructose anhydride III in the analytes numbered K0~K9, C0~C9, and S0~S9 (where PK is K0~K9, PC is C0~C9, and PS is S0~S9); from Figures 1-2 As shown in Table 3, the total mass concentrations of difructose anhydride and difructose anhydride III in all raw Polygonatum sibiricum were significantly lower than those in processed Polygonatum sibiricum. Furthermore, during processing, their content initially increased and then stabilized, reaching a stable level after "three steamings and three sun-dryings" to "four steamings and four sun-dryings." Analysis of market samples further indicated that the total mass concentrations of DFAIII and DFAI in the three original Polygonatum sibiricum varieties listed in the Pharmacopoeia and other raw Polygonatum sibiricum products were also significantly lower than those in processed products, exhibiting the same trend. These results demonstrate that the total content of DFAIII and DFAI can effectively distinguish between raw and processed Polygonatum sibiricum, possessing the potential to serve as a key indicator for evaluating the processing technology and quality of processed Polygonatum sibiricum. In addition, the CAD detection method established in this study is significantly superior to the UV-Vis spectrophotometry method in terms of sensitivity and ease of pretreatment, making it suitable for rapid and accurate determination of this indicator.

[0044] Precision test: The above-mentioned 0.522 mg / mL difructose anhydride I reference solution and 0.518 mg / mL difructose anhydride III reference solution were mixed to obtain a mixed reference solution; 10 μL of the mixed reference solution was detected under the above-mentioned high performance liquid chromatography-electrospray detection conditions, and the sample was injected 6 times consecutively. After 6 measurements, the RSDs of the peak areas of difructose anhydride III and difructose anhydride I were 0.60% and 0.86%, respectively, indicating that the instrument precision was good. Stability test: 10 μL of extract K3 was accurately pipetted and injected at 0, 2, 4, 8, 16 and 24 h. After 6 measurements, the RSDs of the peak areas of difructose anhydride III and difructose anhydride I were 1.11% and 0.76%, respectively. Repeatability test: Take 0.5g of the fine powder of the test sample numbered K3 into 6 portions, prepare the extract according to the above process, and then detect it according to the above high performance liquid chromatography-electrospray detection conditions. Record the peak area. The average contents of difructose anhydride III and difructose anhydride I are 1.12% and 1.68%, respectively, and the RSDs are 1.17% and 1.18%, respectively, indicating that the method has good repeatability. Recovery test: Accurately weigh 0.25 g of the fine powder of 6 test samples (K3) with known content, and accurately add 0.522 mg / mL difructose anhydride I reference solution and 0.518 mg / mL difructose anhydride III reference solution. Prepare the extract according to the above procedure, and then detect it according to the above high performance liquid chromatography-electrospray detection conditions. Record the peak area. The average recoveries of difructose anhydride III and difructose anhydride I were 98.86% and 98.14%, respectively, with RSDs of 1.29% and 1.14%, respectively.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of difructose anhydride I and difructose anhydride III as biomarkers in the rapid detection of Polygonatum sibiricum and Polygonatum sibiricum.

2. A method for detecting difructose anhydride I and difructose anhydride III in Polygonatum sibiricum, characterized in that, Includes the following steps: The Polygonatum sibiricum to be tested was mixed with the extractant and extracted to obtain the extract; The extract was subjected to high performance liquid chromatography-electrospray detection to obtain the peak areas of difructose anhydride I and difructose anhydride III, respectively. Based on the peak areas of difructose anhydride I and difructose anhydride III and their respective predetermined standard curves, the mass concentrations of difructose anhydride I and difructose anhydride III are obtained respectively. The predetermined standard curve is a linear relationship between the peak area and mass concentration of difructose anhydride I or difructose anhydride III. The separation conditions for the high performance liquid chromatography include: mobile phase A is acetonitrile, and mobile phase B is an aqueous ammonia solution.

3. The detection method as described in claim 2, characterized in that, The elution method of the mobile phase in the high performance liquid chromatography is isocratic elution; The volume ratio of mobile phase A to mobile phase B is 87:13; The ammonia solution is an ammonia solution with a volume concentration of 0.1%.

4. The detection method as described in claim 3, characterized in that, The flow rate of the mobile phase is 1.0 mL / min, and the column temperature is 30 °C.

5. The detection method as described in claim 2, characterized in that, The electro-fogging detection has a sampling frequency of 5Hz, a filtering time of 2.0s, and an evaporation temperature of 60℃.

6. The detection method as described in claim 2, characterized in that, The linear relationship between the peak area and mass concentration of the difructose anhydride I is: Y = 60.473X + 2.2758; Where Y is the peak area and X is the mass concentration.

7. The detection method as described in claim 2, characterized in that, The linear relationship between the peak area and mass concentration of the difructose anhydride III is: Y = 53.697X + 2.1644; Where Y is the peak area and X is the mass concentration.

8. The detection method as described in claim 2, characterized in that, The extractant is ethanol and water in a volume ratio of 80:

20.

9. The detection method as described in claim 2 or 8, characterized in that, The ratio of the amount of Polygonatum sibiricum to the extractant was 0.5g:40mL.

10. The detection method as described in claim 9, characterized in that, The extraction was performed under ultrasonic conditions; The extraction process also includes filtration using a 0.22 μm filter membrane.

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