Method for detecting glycosides in cinnamon and application thereof

Abstract

The present application relates to a kind of detection method of glycosides component in cassia and its application, it is related to the technical field of quality control of traditional Chinese medicine.The detection method includes the following steps: pretreatment: the measured product is mixed with polyphenol agent, remove polyphenolic compounds, obtain polyphenol product, mixed with impurity removing agent, remove impurities, obtain sample to be measured;Detection: using liquid chromatography-mass spectrometry to detect the sample to be measured, glycosides component is analyzed;Glycosides component includes benzyl alcohol apiofuranosyl glucoside and / or dihydrocoumaric acid apiofuranosyl glucoside.The detection method can detect glycosides component in cassia, and based on the detection method, the characteristics of chemical components in cassia can be reflected, which is beneficial to the overall control of the quality of cassia and its products, and ensures product quality.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality monitoring technology, and in particular to a method for detecting glycosides in cinnamon and its application. Background Technology

[0002] The current edition of the Chinese Pharmacopoeia only lists three chemical component analysis items for the quality control of cinnamon: thin-layer chromatography identification of cinnamaldehyde (or cinnamaldehyde), and determination of volatile oil and cinnamaldehyde content. There are no quality control methods for glycosides in cinnamon. The chemical component indicators reported in the literature for cinnamon quality control mainly include volatile oil and phenylpropanoids (cinnamaldehyde, cinnamyl alcohol, cinnamic acid, coumarins, etc.). To date, no quality control methods for glycosides in cinnamon and its products have been reported.

[0003] Existing quality control standards and research on cinnamon and its products lack reports on quality control methods for glycoside components, failing to fully reflect the characteristics of the chemical components in cinnamon. Therefore, current quality control methods for cinnamon and its products still need further improvement. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for detecting glycosides in cinnamon. This method can detect glycosides in cinnamon and, based on this method, can reflect the characteristics of chemical components in cinnamon, which is beneficial for comprehensive control of the quality of cinnamon and its products and ensures product quality.

[0005] To achieve the above objectives, the present invention provides a method for detecting glycosides in cinnamon, comprising the following steps:

[0006] Pretreatment: The sample to be tested is mixed with a polyphenol removal agent to remove polyphenolic compounds, resulting in a polyphenol-removed product. This product is then mixed with a purification agent to remove impurities, yielding the sample to be tested.

[0007] Detection: The test samples were analyzed using liquid chromatography-mass spectrometry to detect glycoside components; the glycoside components included benzyl alcohol apirudate glucoside and / or dihydrocoumaric acid apirudate glucoside.

[0008] Benzyl alcohol, produced by the metabolic hydrolysis of benzyl alcohol apiracerulic acid glucoside, is a component with analgesic, local anesthetic, and antibacterial effects. The glycosides of aromatic compounds in cinnamon also have analgesic effects, suggesting they could serve as indicators of pharmacological efficacy. However, existing quality control standards and research on cinnamon and its products do not comprehensively reflect the quality control of the pharmacologically active components in cinnamon. Therefore, the inventors propose the aforementioned detection method for detecting glycosides in cinnamon. Based on this method, the characteristics of the chemical components in cinnamon can be reflected, which is beneficial for comprehensive quality control of cinnamon and its products, ensuring product quality.

[0009] In one embodiment, the polyphenol removal agent in the pretreatment step includes polyamide and / or aluminum oxide;

[0010] The impurity removal agent includes macroporous resin and / or activated carbon.

[0011] The above-mentioned polyphenol remover can adsorb polyphenolic impurities, and the above-mentioned impurity remover can adsorb impurities of medium to small polarity.

[0012] In one embodiment, the mass ratio of the test sample to the polyphenol remover is 1:(0.5-1.5);

[0013] The mass ratio of the test sample to the impurity removal agent is 1:(0.5-1.5).

[0014] In one embodiment, the pretreatment step, the removal of polyphenolic compounds includes: removing polyphenolic compounds by passing a mixture of the test sample and a polyphenol removal agent through column chromatography; or removing polyphenolic compounds by adsorption of the polyphenol removal agent.

[0015] The removal of impurities includes: removing impurities by passing a mixture of polyphenol removal product and impurity removal agent through column chromatography; or removing impurities by adsorption of the impurity removal agent.

[0016] In one embodiment, the analysis in the detection step includes: analyzing the ion chromatographic peaks of the glycoside components; the m / z of benzyl alcohol apirudate glucoside is 415.12, and the m / z of dihydrocoumaric acid apirudate glucoside is 473.13.

[0017] In one embodiment, the detection includes: filtering the sample to be tested, taking the filtrate, and injecting the filtrate into a liquid chromatography-mass spectrometry instrument to determine under predetermined conditions;

[0018] The predetermined liquid phase conditions include:

[0019] Stationary phase: A chromatographic column packed with octadecylsilane-bonded silica gel;

[0020] Mobile phase: Acetonitrile was used as mobile phase A, and formic acid aqueous solution with a volume percentage concentration of 0.1% was used as mobile phase B. Elution method was isocratic elution or gradient elution.

[0021] In one embodiment, the filtrate used in the detection is a continuous filtrate.

[0022] In one embodiment, when the elution method employs gradient elution, the gradient elution conditions are as follows:

[0023] From 0 min to 5 min, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0024] From 5 min to 35 min, the volume ratio of mobile phase A to mobile phase B changed from 5:95 to 45:55.

[0025] In one embodiment, the liquid phase conditions further include:

[0026] Injection volume: 2 ± 0.2 μL;

[0027] Flow rate: 0.2 ± 0.05 mL / min;

[0028] Column temperature: 35±3℃.

[0029] In one embodiment, the mass spectrometry conditions of the predetermined conditions include: electrospray ionization using negative ion mode, interface voltage -3.0kV, and ion source temperature 400°C.

[0030] This invention also provides the application of the detection method in the description, evaluation and quality monitoring of cinnamon quality.

[0031] This invention also provides a quality control method for a product, wherein the product is cinnamon or a cinnamon-containing product. The quality control method includes the following steps: detecting the product using the aforementioned detection method to obtain a total ion chromatogram; comparing the m / z values ​​of the chromatographic peaks with the m / z values ​​of benzyl apirulentinuron glucoside and / or hydrocoumaric acid apirulentinuron glucoside to evaluate the quality of the product.

[0032] In one embodiment, the evaluation includes: if the product detects at least one chromatographic peak, and the difference between the m / z value of the chromatographic peak and the m / z value of benzyl alcohol apirulentinuron glucoside and / or hydrocoumaric acid apirulentinuron glucoside is within ±5% of the mass, then the product is judged to be qualified.

[0033] The above methods can be used for quality control of glycosides in cinnamon and its products, which is conducive to comprehensive quality control of cinnamon and its products, ensuring product quality, and protecting the rights and interests of producers and consumers.

[0034] In one embodiment, during the evaluation, if the product detects at least two chromatographic peaks, and the difference between the m / z value of the chromatographic peak and the m / z value of benzyl alcohol apirulentinurate glucoside and hydrocoumaric acid apirulentinurate glucoside is within ±5% of the mass, then the product is judged to be qualified.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention discloses a method for detecting glycosides in cinnamon and its application. This method can detect glycosides in cinnamon and, based on this method, can reflect the characteristics of chemical components in cinnamon, which is beneficial for comprehensive control of the quality of cinnamon and its products and ensures product quality. Attached Figure Description

[0037] Figure 1 The total ion chromatogram of Guigang cinnamon from Guangxi Zhuang Autonomous Region in Example 2;

[0038] Figure 2 This is the mass spectrum of compound A: benzyl alcohol apirulic acid glucoside from Guigang cinnamon in Guangxi Zhuang Autonomous Region in Example 2;

[0039] Figure 3 This is the mass spectrum of compound B: dihydrocoumaric acid apigenate glucoside from Guigang cinnamon in Guangxi Zhuang Autonomous Region, as shown in Example 2.

[0040] Figure 4 The total ion chromatogram of cinnamon from Luoding, Guangdong Province, in Example 2;

[0041] Figure 5 The mass spectrum of compound A: benzyl alcohol apigenate glucoside from Luoding cinnamon in Example 2;

[0042] Figure 6 This is the mass spectrum of compound B from Luoding cinnamon in Example 2: dihydrocoumaric acid apigenate glucoside;

[0043] Figure 7 The total ion chromatogram of cinnamon from Gaoyao, Guangdong Province, in Example 2;

[0044] Figure 8 The mass spectrum of compound A: benzyl alcohol apigenate glucoside from Gaoyao cinnamon in Guangdong Province in Example 2;

[0045] Figure 9 This is the mass spectrum of compound B from Gaoyao cinnamon in Guangdong Province, namely benzyl alcohol apirulic acid glucoside, in Example 2.

[0046] Figure 10 The total ion chromatogram of cinnamon from Guigang, Guangxi Zhuang Autonomous Region, in Comparative Example 1. Detailed Implementation

[0047] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] definition:

[0050] Benzyl apirudate glucoside:

[0051] Its English name is benzyl alcohol 7-O-β-D-apiuronyl(1→6)-β-D-glucoside.

[0052] Dihydrocoumaric acid apiuron glucoside:

[0053] Its English name is dihydrocoumaric acid 2-O-β-D-apiuronyl(1→6)-β-D-glucoside.

[0054] source:

[0055] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0056] Example 1

[0057] A method for detecting glycosides in cinnamon.

[0058] I. Preprocessing.

[0059] 1. Extract cinnamon or cinnamon-containing products with water (if the product itself is an aqueous extract, use it directly for processing). Following the ratio of 1g of crude drug to 1g of polyamide (polyphenol remover) (in this example, the mass ratio of the test sample to the polyphenol remover is 1:1), soak the purified polyamide in ethanol for 24 hours, filter, wash several times with pure water, then suspend in pure water, pack into a glass chromatography column, allow the polyamide to settle, open the stopcock to release solvent until the liquid surface submerges the polyamide packing, immediately add the aqueous extract into the column, allowing all the aqueous extract to pass through the polyamide, and collect the eluent after passing through the column.

[0060] 2. According to the ratio of 1g of crude drug to 1g of macroporous resin (impurity remover) (in this embodiment, the mass ratio of the test sample to the impurity remover is 1:1), the purified macroporous resin is soaked in ethanol for 24 hours, filtered, washed multiple times with pure water, then suspended in pure water, packed into a glass chromatography column, and allowed to stand until the macroporous resin settles. The stopcock is opened to release the solvent to the liquid surface to submerge the macroporous resin packing. The effluent collected after step 1 of this embodiment is immediately added to the column, allowing it to pass entirely through the macroporous resin. The effluent after passing through the column is collected.

[0061] II. Testing.

[0062] The treated water extract was filtered through a 0.45 μm nylon microporous membrane, and 2 μL of the filtrate was injected into a liquid chromatography-mass spectrometry system.

[0063] Liquid chromatography conditions: An octadecylsilanized bonded silica gel column was used as the stationary phase;

[0064] Elution was performed using an isocratic elution with a mobile phase of acetonitrile:0.1% formic acid aqueous solution (15:85), or a gradient elution method (mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, 0 to 5 minutes, ratio A:B = 5:95; 5 to 35 minutes, the ratio changes linearly from A:B = 5:95 to 45:55) at a flow rate of 0.2 mL / min and a column temperature of 35 °C. In this embodiment, a gradient elution method was used for the liquid phase.

[0065] Mass spectrometry conditions: The mass spectrometer used parameters optimized by automatic instrument tuning; electrospray ionization was performed in negative ion mode with an interface voltage of -3.0 kV and an ion source temperature of 400℃. The measured total ion chromatogram showed ion chromatographic peaks at m / z 415.12 (benzyl alcohol apiuron glucoside) and m / z 473.13 (dihydrocoumaric acid apiuron glucoside) (m / z values ​​not exceeding ±5% of the mass number).

[0066] Example 2

[0067] The detection method of Example 1 was used to test cinnamon from three different origins.

[0068] 1. Guigang cinnamon from Guangxi Zhuang Autonomous Region (commercially available).

[0069] Test results as follows Figure 1 , 2 As shown in Figure 3, there are ion chromatographic peaks with m / z of 415.12 (compound A: benzyl alcohol apiuron glucoside) and m / z of 473.13 (compound B: dihydrocoumaric acid apiuron glucoside) (m / z values ​​not exceeding 0.05% of the mass).

[0070] 2. Cinnamon from Luoding, Guangdong Province (commercially available).

[0071] Test results as follows Figure 4 , 5 As shown in Figure 6, there are ion chromatographic peaks with m / z of 415.12 (compound A: benzyl alcohol apirudate glucoside) and m / z of 473.13 (compound B: dihydrocoumaric acid apirudate glucoside) (m / z values ​​not exceeding 0.05% of the mass).

[0072] 3. Cinnamon from Gaoyao, Guangdong Province (commercially available).

[0073] Test results as follows Figure 7 , 8 As shown in Figure 9, there are ion chromatographic peaks with m / z of 415.12 (compound A: benzyl alcohol apiuron glucoside) and m / z of 473.13 (compound B: dihydrocoumaric acid apiuron glucoside) (m / z values ​​not exceeding 0.05% of the mass).

[0074] Comparative Example 1

[0075] The cinnamon from Guigang, Guangxi Zhuang Autonomous Region, in Example 2 was analyzed using liquid chromatography-mass spectrometry.

[0076] The difference between the detection method of Comparative Example 1 and Example 1 is that the sample of the comparative example was not treated with polyphenol removal agent and impurity removal agent after being prepared into an aqueous extract.

[0077] The test results of this comparative example are as follows: Figure 10 As shown, it is evident that the ion chromatographic peaks (B) of benzyl alcohol apirudate glucoside (A) and dihydrocoumaric acid apirudate glucoside cannot be effectively detected.

[0078] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting glycosides in cinnamon, characterized in that, Includes the following steps: Pretreatment: The sample to be tested is mixed with a polyphenol remover to remove polyphenolic compounds, resulting in a polyphenol-removed product. This product is then mixed with a purifying agent to remove impurities, yielding the sample to be tested. The polyphenol remover includes polyamide, and the purifying agent includes macroporous resin. Detection: The sample was analyzed using liquid chromatography-mass spectrometry (LC-MS) to identify glycosides. The glycosides included benzyl alcohol apeguronate glucoside and dihydrocoumaric acid apeguronate glucoside. The detection process involved filtering the sample, collecting the filtrate, and injecting the filtrate into the LC-MS instrument for determination under predetermined conditions. These predetermined LC conditions included: stationary phase: an octadecylsilane-bonded silica gel column; mobile phase: acetonitrile as mobile phase A and 0.1% (v / v) formic acid aqueous solution as mobile phase B, with isocratic elution or gradient elution. When gradient elution was used, the gradient elution conditions were: from 0 min to 5 min, the volume ratio of mobile phase A to mobile phase B was 5:95; from 5 min to 35 min, the volume ratio of mobile phase A to mobile phase B changed from 5:95 to 45:

55.

2. The detection method according to claim 1, characterized in that, The mass ratio of the test sample to the polyphenol remover is 1:(0.5-1.5). The mass ratio of the test sample to the impurity removal agent is 1:(0.5-1.5).

3. The detection method according to claim 1, characterized in that, In the pretreatment step, the removal of polyphenolic compounds includes: removing polyphenolic compounds by passing a mixture of the sample and the polyphenol removal agent through column chromatography; or removing polyphenolic compounds by adsorption of the polyphenol removal agent. The removal of impurities includes: removing impurities by passing a mixture of polyphenol removal product and impurity removal agent through column chromatography; or removing impurities by adsorption of the impurity removal agent.

4. The detection method according to claim 1, characterized in that, In the detection step, the analysis includes: analyzing the ion chromatographic peaks of the glycoside components; the m / z of benzyl alcohol apirudate glucoside is 415.12, and the m / z of dihydrocoumaric acid apirudate glucoside is 473.

13.

5. The detection method according to claim 1, characterized in that, The predetermined mass spectrometry conditions include: electrospray ionization in negative ion mode, interface voltage -3.0 kV, and ion source temperature 400℃.

6. The application of the detection method according to any one of claims 1-5 in the description, evaluation and quality monitoring of cinnamon quality.

7. A quality control method for a product, characterized in that, The product is cinnamon or a cinnamon-containing product. The quality control method includes the following steps: testing the product using the detection method described in any one of claims 1-5 to obtain the total ion chromatogram of the product; comparing the m / z value of the chromatographic peak with the m / z value of benzyl alcohol apirulentinuron glucoside and / or hydrocoumaric acid apirulentinuron glucoside to evaluate the quality of the product.

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