Detection method of glycoside components in cinnamon and application of detection method

Abstract

The invention relates to a method for detecting glycoside components in cinnamon and application of the method, and relates to the technical field of quality monitoring of traditional Chinese medicines. The detection method comprises the following steps: pretreatment: mixing a to-be-detected product with a polyphenol removal agent, removing polyphenol compounds to obtain a polyphenol-removed product, mixing the polyphenol-removed product with an impurity removal agent, and removing impurities to obtain a to-be-detected sample; detecting: detecting the to-be-detected sample by adopting a liquid chromatography-mass spectrometry method, and analyzing glycoside components; and the glycoside component comprises benzyl alcohol apipuronic acid glucoside and / or dihydrocoumaric acid apipuronic acid glucoside. The detection method can be used for detecting glycoside components in the cinnamon, and on the basis of the detection method, the characteristics of chemical components in the cinnamon can be reflected, the quality of the cinnamon and the quality of products of the cinnamon can be comprehensively controlled, and the product quality is guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of quality monitoring of traditional Chinese medicines, and in particular to a method for detecting glycoside components in cinnamon and an application thereof. Background Art

[0002] In the current edition of the Chinese Pharmacopoeia, the quality control items for cinnamon only include three chemical component analysis items: thin layer identification of cinnamaldehyde (or cinnamaldehyde) and determination of volatile oil and cinnamaldehyde content. There is no quality control method for glycoside components in cinnamon. The chemical component indicators used for cinnamon quality control reported in the literature are mainly volatile oil and phenylpropanoid components (cinnamaldehyde, cinnamyl alcohol, cinnamic acid, coumarin, etc.). So far, there is no report on the quality control method of glycoside components in cinnamon and its products.

[0003] The existing quality control standards and research on cinnamon and its products do not report the quality control methods of glycoside components, which cannot fully reflect the characteristics of the chemical components in cinnamon. Therefore, the quality control methods of cinnamon and its products still need to be further improved. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for detecting glycoside components in cinnamon, which can detect the glycoside components in cinnamon, and based on the detection method, can reflect the characteristics of chemical components in cinnamon, which is conducive to comprehensively controlling the quality of cinnamon and its products and ensuring product quality.

[0005] In order to achieve the above object, 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 polyphenol compounds, thereby obtaining a polyphenol removal product, which is then mixed with an impurity removal agent to remove impurities, thereby obtaining a sample to be tested;

[0007] Detection: Liquid chromatography-mass spectrometry is used to detect the sample to be tested and the glycoside components are analyzed; the glycoside components include benzyl alcohol apigenin glucoside and / or dihydrocoumaric acid apigenin glucoside.

[0008] Benzyl alcohol apigenin glucoside can be metabolized and hydrolyzed to produce benzyl alcohol, which is a component with analgesic, local anesthesia and antibacterial effects. The glycoside components of aromatic compounds in cinnamon have analgesic effects, suggesting that they can be used as index components of medicinal ingredients. However, the existing quality control standards and research on cinnamon and its products do not fully reflect the quality control of medicinal ingredients in cinnamon. Therefore, the inventors propose the above-mentioned detection method to detect the glycoside components in cinnamon, and based on the detection method, it can reflect the characteristics of the chemical components in cinnamon, which is conducive to comprehensively controlling the quality of cinnamon and its products and ensuring product quality.

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

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

[0011] The above-mentioned polyphenol removing agent can adsorb polyphenol impurities, and the above-mentioned impurity removing agent can adsorb impurities with medium to small polarity.

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

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

[0014] In one embodiment, in the pretreatment step, the removal of polyphenolic compounds comprises: removing polyphenolic compounds from a mixture of the sample and the polyphenolic removal agent by column chromatography; or removing polyphenolic compounds by adsorption of the polyphenolic removal agent;

[0015] The impurity removal comprises: removing impurities from the mixture of the polyphenol-removed product and the impurity-removing agent through column chromatography; or removing impurities through adsorption of the impurity-removing agent.

[0016] In one embodiment, in the detection step, the analysis includes: analyzing the ion chromatographic peaks of the glycoside components; the m / z of the benzyl alcohol apigenin glucoside is 415.12, and the m / z of the dihydrocoumaric acid apigenin 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 spectrometer for determination under predetermined conditions;

[0018] The liquid phase conditions of the predetermined conditions include:

[0019] Stationary phase: chromatographic column with octadecylsilane bonded silica as filler;

[0020] Mobile phase: acetonitrile is used as mobile phase A, 0.1% by volume formic acid aqueous solution is used as mobile phase B, and the elution method is isocratic elution or gradient elution.

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

[0022] In one embodiment, when the elution method adopts gradient elution, the gradient elution conditions are:

[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±0.2μL;

[0027] Flow rate: 0.2±0.05mL / min;

[0028] Column temperature: 35±3℃.

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

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

[0031] The present invention also provides a product quality control method, the product is cinnamon or a product containing cinnamon, the quality control method comprises the following steps: using the detection method to detect the product, obtaining a total ion flow chart of the product, comparing the m / z value of the chromatographic peak with the m / z value of benzyl alcohol apigenin glucoside and / or hydrocoumaric acid apigenin glucoside, and evaluating the quality of the product

[0032] In one embodiment, the evaluation includes: if at least one chromatographic peak is detected in the product, and the difference between the m / z value of the chromatographic peak and the m / z value of benzyl alcohol apigenin and / or hydrocoumaric acid apigenin is no more than 5 parts per 100,000 by mass, the product is judged to be qualified.

[0033] The method can be used to control the quality of the glycoside components in cinnamon and its products, which is beneficial to comprehensively control the quality of cinnamon and its products, ensure product quality, and protect the rights and interests of producers and consumers.

[0034] In one of the embodiments, in the evaluation, if at least two chromatographic peaks are detected in the product, and the difference between the m / z values ​​of the chromatographic peaks and the m / z values ​​of benzyl alcohol apigenin and hydrocoumaric acid apigenin is no more than 0.5 parts per 100,000 by mass, the product is judged to be qualified.

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

[0036] The invention discloses a method for detecting glycoside components in cinnamon and application thereof. The method can detect the glycoside components in cinnamon, and based on the method, the characteristics of chemical components in cinnamon can be reflected, which is beneficial to comprehensively control the quality of cinnamon and its products and ensure product quality. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] Figure 2 The mass spectrum of compound A: benzyl alcohol apigenin glucoside from Guigang cinnamon bark in Guangxi Zhuang Autonomous Region in Example 2;

[0039] Figure 3 The mass spectrum of compound B: dihydrocoumaric acid apigenin glucoside in Guigang, Guangxi Zhuang Autonomous Region in Example 2;

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

[0041] Figure 5 The mass spectrum of compound A: benzyl alcohol apigenin glucoside from cinnamon bark from Luoding, Guangdong Province in Example 2;

[0042] Figure 6 The mass spectrum of compound B of cinnamon bark from Luoding, Guangdong Province in Example 2: dihydrocoumaric acid apigenin glucoside;

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

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

[0045] Fig. 9 The mass spectrum of compound B: benzyl alcohol apigenin glucoside from cinnamon bark from Gaoyao, Guangdong Province in Example 2;

[0046] Fig.10 This is the total ion current diagram of Guigang cinnamon in Guangxi Zhuang Autonomous Region in Comparative Example 1. DETAILED DESCRIPTION

[0047] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0049] definition:

[0050] Benzyl alcohol apigenin glucoside:

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

[0052] Dihydrocoumaric acid apigenin glucoside:

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

[0054] source:

[0055] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0056] Example 1

[0057] A method for detecting glycoside components in cinnamon.

[0058] 1. Pre-processing

[0059] 1. Extract cinnamon or a product containing cinnamon with water (if the product itself is a water extract, it is directly used for treatment), and the amount of polyamide (excluding polyphenols) corresponding to 1g of crude drug (in this embodiment, the mass ratio of the test product to the polyphenols removing agent is 1:1) is used. The clean polyamide is soaked in ethanol for 24 hours, filtered, washed with pure water for multiple times, and then suspended with pure water. It is loaded into a glass chromatography column, allowed to stand for the polyamide to settle, the piston is opened to release the solvent until the liquid surface submerges the polyamide filler, and the water extract is immediately added to the column, allowing the water extract to pass through the polyamide completely, and the effluent after passing through the column is collected.

[0060] 2. According to the amount of 1g of crude drug corresponding to 1g of macroporous resin (impurity remover) (in this embodiment, the mass ratio of the test product to the impurity remover is 1:1), the clean macroporous resin is soaked in ethanol for 24 hours, filtered, washed with pure water for many times, and then suspended with pure water, loaded into a glass chromatography column, and allowed to stand until the macroporous resin settles, the piston is opened to release the solvent to the liquid surface to immerse the macroporous resin filler, and the effluent collected after the treatment in step 1 of this embodiment is immediately added to the column, and it is allowed to pass through the macroporous resin, and the effluent after the column is collected.

[0061] 2. Testing.

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

[0063] Liquid phase conditions: chromatographic column using octadecyl silane bonded silica gel as stationary phase;

[0064] Acetonitrile: 0.1% formic acid aqueous solution (15:85) was used as the mobile phase for isocratic elution, or gradient elution (mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid aqueous solution, 0 to 5 minutes, the ratio was A:B = 5:95; 5 to 35 minutes, the ratio changed linearly from A:B = 5:95 to 45:55), flow rate: 0.2 mL / min; column temperature: 35° C. In this embodiment, the liquid phase conditions adopted a gradient elution mode.

[0065] Mass spectrometry conditions: The mass spectrometer used the parameters optimized by instrument automatic tuning; the electrospray ionization used the negative ion mode, the interface voltage was -3.0 kV, and the ion source temperature was 400°C. On the measured total ion current, there were ion chromatographic peaks with m / z of 415.12 (benzyl alcohol apigenin glucoside) and m / z of 473.13 (dihydrocoumaric acid apigenin glucoside) (the m / z value was not more than 5 mass numbers of 100,000).

[0066] Example 2

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

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

[0069] Test results such as Figure 1 , 2 As shown in 3, there are ion chromatographic peaks with m / z of 415.12 (Compound A: benzyl alcohol apigenin glucoside) and m / z of 473.13 (Compound B: dihydrocoumaric acid apigenin glucoside) (the mass number of m / z value is not more than 5 parts per 100,000).

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

[0071] Test results such as Figure 4 , 5 As shown in 6, there are ion chromatogram peaks with m / z of 415.12 (Compound A: Benzyl alcohol apigenin glucoside) and m / z of 473.13 (Compound B: Dihydrocoumaric acid apigenin glucoside) (the mass number of m / z value is not more than 5 parts per 100,000).

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

[0073] Test results such as Figure 7 , 8 As shown in Figure 9, there are ion chromatographic peaks with m / z of 415.12 (Compound A: Benzyl alcohol apigenin glucoside) and m / z of 473.13 (Compound B: Dihydrocoumaric acid apigenin glucoside) (the m / z value does not exceed 5 mass numbers of one hundred thousandths).

[0074] Comparative Example 1

[0075] The Guigang cinnamon of Guangxi Zhuang Autonomous Region in Example 2 was measured by liquid chromatography-mass spectrometry.

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

[0077] The test results of this comparative example are as follows Fig.10 As shown, it can be seen that the ion chromatogram peaks of benzyl alcohol apigenin glucoside (A) and dihydrocoumaric acid apigenin glucoside (B) cannot be effectively detected.

[0078] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting glycosides in cinnamon, characterized in that: The following steps are involved: Pretreatment: the sample to be tested is mixed with a polyphenol removal agent to remove polyphenol compounds, thereby obtaining a polyphenol removal product, which is then mixed with an impurity removal agent to remove impurities, thereby obtaining a sample to be tested; Detection: Liquid chromatography-mass spectrometry is used to detect the sample to be tested and the glycoside components are analyzed; the glycoside components include benzyl alcohol apigenin glucoside and / or dihydrocoumaric acid apigenin glucoside.

2. The detection method according to claim 1, characterized in that: In the pre-treatment step, the polyphenol removal agent includes polyamide and / or aluminum oxide; The impurity remover includes macroporous resin and / or activated carbon.

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

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

5. 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 the benzyl alcohol apigenin glucoside is 415.12, and the m / z of the dihydrocoumaric acid apigenin glucoside is 473.

13.

6. The detection method according to any one of claims 1 to 5, characterized in that: The detection comprises: filtering the sample to be tested, taking the filtrate, and injecting the filtrate into a liquid chromatography-mass spectrometer for determination under predetermined conditions; The liquid phase conditions of the predetermined conditions include: Stationary phase: chromatographic column with octadecylsilane bonded silica as filler; Mobile phase: acetonitrile is used as mobile phase A, 0.1% by volume formic acid aqueous solution is used as mobile phase B, and the elution method is isocratic elution or gradient elution.

7. The detection method according to claim 6, characterized in that: When the elution method adopts gradient elution, the gradient elution conditions are: 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.

8. The detection method according to claim 6, characterized in that: The predetermined mass spectrometry conditions include: electrospray ionization in negative ion mode, interface voltage -3.0 kV, and ion source temperature of 400°C.

9. Application of the detection method according to any one of claims 1 to 8 in quality description, evaluation and quality monitoring of cinnamon.

10. A method for controlling product quality, characterized in that: The product is cinnamon or a product containing cinnamon, and the quality control method comprises the following steps: using the detection method described in any one of claims 1 to 8 to detect the product, obtaining a total ion flow chart of the product, comparing the m / z value of the chromatographic peak with the m / z value of benzyl alcohol apigenin glucoside and / or hydrocoumaric acid apigenin glucoside, and evaluating the quality of the product.

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