A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs

Abstract

A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, comprising the following steps: (1) weighing the sample; (2) treating with formaldehyde solution; (3) rinsing; (4) treating with 2-phenylboronic acid 2-aminoethyl ester reagent; (5) microscopic observation: dripping glycerol on the moxa, observing under ultraviolet light excitation of a fluorescence microscope, judging the authenticity of the moxa according to the microscopic characteristics, if the T-shaped non-glandular hairs are observed to have handles or shedding marks of varying lengths, it is true moxa, otherwise it is false moxa. The method of the present invention is simple and easy to operate. Under the action of formaldehyde and 2-phenylboronic acid 2-aminoethyl ester, the top cells of the real moxa after mechanical processing regain fluorescence, and the stem cells are also strengthened. It can be clearly seen that the T-shaped non-glandular hairs in the moxa have handles or shedding marks of varying lengths, while the fake moxa does not have the handles or shedding marks unique to the T-shaped non-glandular hairs. The authenticity of the moxa is effectively solved, the treatment and efficacy are ensured, and the economic and social benefits are huge.

Description

Technical Field

[0001] The invention relates to the field of moxa identification, in particular to a moxa authenticity identification method based on non-glandular hair fluorescence microscopic characteristics. Background Art

[0002] Moxibustion therapy has a long history and is widely used. The main material of moxibustion therapy is moxa, which is processed from moxa leaves. In recent years, with the popularization of moxibustion health care, the moxa industry has flourished, but the understanding of the properties of qualified moxa is still at the level of description by the ancients. The identification of properties requires high professional skills and experience of the identifier, and even ordinary practitioners find it difficult to master and accurately identify them. Microscopic identification is simple and accurate. However, the microscopic identification characteristics of moxa leaves in the Chinese Pharmacopoeia from the 1977 edition to the 2020 edition have not been improved and improved. The biggest problem is that there are two types of non-glandular hairs in moxa: T-shaped non-glandular hairs and (extremely long apical cells) uniseriate non-glandular hairs. In this case, inferior cotton, various plant fibers and even synthetic fibers are processed and mixed into moxa, which affects the quality of moxa and directly affects the efficacy. The Chinese Pharmacopoeia has classified the national standards for moxa for moxibustion, but there are certain problems with the microscopic identification of moxa, which undoubtedly brings difficulties to the microscopic identification of moxa.

[0003] According to research, the non-glandular hairs of moxa leaves are all T-shaped non-glandular hairs, and there are no uniseriate non-glandular hairs as described in the current 2020 edition of the Chinese Pharmacopoeia; the main component of high-precision moxa is the slender and curved T-shaped non-glandular hairs of the apical cells. In the process of processing into moxa, with repeated crushing and sieving, the integrity of the T-shaped non-glandular hair stalks is continuously reduced or even falls off. However, in the current microscopic identification of moxa, the method usually used is to permeabilize the moxa with chloral hydrate and then conduct microscopic observation. Most of the T-shaped non-glandular hairs in moxa are tangled together into a mess, and it is difficult to see the existence of the T-shaped non-glandular hair stalks under an ordinary optical microscope ( Figure 1 ).

[0004] The study found that the stalks or shedding marks of T-shaped non-glandular hairs scraped off from the leaves with a blade are keratinized, and some secondary metabolites exist in the stalk cells, which can spontaneously fluoresce ( Figure 2 ), the intensity is much higher than the autofluorescence of the apical cells, which is of great value for identifying the authenticity of moxa. However, the current microscopic identification of moxa is carried out after treatment with chloral hydrate. Due to the influence of chloral hydrate, this fluorescence feature cannot be seen even with a fluorescence microscope; on the other hand, since the current moxa is generally processed mechanically, during the repeated crushing process, the fluorescence of the apical cells and stalks of the T-shaped non-glandular hairs fades one by one due to the influence of high temperature, and the structural characteristics of the T-shaped non-glandular hairs cannot be clearly seen under a fluorescence microscope ( Figure 3). Therefore, how to restore and enhance the fluorescence characteristics of moxa after processing it in a certain way, so that the fluorescence microscopic characteristics of the unique T-shaped non-glandular hairs of moxa can be clearly seen under a fluorescence microscope, and thereby identify the authenticity of moxa, has very important practical value. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, which can effectively solve the problem of identifying the authenticity of moxa.

[0006] To achieve the above object, the technical solution provided by the present invention is a method for authenticating moxa based on the fluorescence microscopic characteristics of non-glandular hairs, comprising the following steps:

[0007] (1) Sample weighing: weigh 5-10 mg of moxa as a sample;

[0008] (2) Formaldehyde solution treatment: Add 3-5 ml of formaldehyde solution with a volume concentration of 10%-20% to the sample and immerse for 1-2 minutes at room temperature;

[0009] (3) Rinse: Take out the moxa from the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 10-20 seconds;

[0010] (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 3-5 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.5%-1% into the weighing bottle, and immerse for 1-5 minutes at room temperature;

[0011] (5) Microscopic observation: Take 3-5 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.02-0.05 ml of 20% glycerol by volume on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation of a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic characteristics. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

[0012] The method of the present invention is simple and easy to operate. Under the action of formaldehyde and diphenyl boric acid-2-aminoethyl ester, the top cells of the real moxa after mechanical processing regain fluorescence and the stem cells are also strengthened. It can be clearly seen that the T-shaped non-glandular hairs in the moxa have stems or shedding marks of varying lengths, while the fake moxa does not have the stems or shedding marks unique to the T-shaped non-glandular hairs. The method effectively solves the identification of the authenticity of the moxa, ensures the treatment and efficacy, and has huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a micrograph of moxa under a bright field of an ordinary microscope of the present invention.

[0014] Figure 2 This is a spontaneous fluorescence micrograph (ultraviolet light excitation) of T-shaped non-glandular hairs scraped off by a wormwood blade of the present invention.

[0015] Figure 3 This is a micrograph of the T-shaped non-glandular hairs of moxa after mechanical processing according to the present invention (fluorescence fades).

[0016] Figure 4 This is a fluorescence micrograph of the T-shaped non-glandular hairs recovered after being treated with real moxa in the experimental group of the present invention.

[0017] Figure 5 This is a micrograph of the fake moxa sample 1 in the control group of the present invention after being treated under bright field observation.

[0018] Figure 6 This is a micrograph of the fake moxa sample 1 in the control group of the present invention after being treated under ultraviolet light (with Figure 5 same field of view).

[0019] Figure 7 This is a micrograph of the fake moxa sample 2 in the control group of the present invention after being treated under bright field observation.

[0020] Figure 8 This is a micrograph of the fake moxa sample 2 in the control group of the present invention after being treated under ultraviolet light (with Figure 7 same field of view). DETAILED DESCRIPTION

[0021] The specific implementation of the present invention is described in detail below with reference to the accompanying drawings and examples. Example 1

[0022] A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, comprising the following steps:

[0023] (1) Sample weighing: weigh 5 mg of moxa as sample;

[0024] (2) Formaldehyde solution treatment: Add 3 ml of 20% formaldehyde solution to the sample and immerse for 2 min at room temperature;

[0025] (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 10 seconds;

[0026] (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 3 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 1% into the weighing bottle, and immerse for 5 minutes at room temperature;

[0027] (5) Microscopic observation: Take 3 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.02 ml of 20% glycerol on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa. Example 2

[0028] A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, comprising the following steps:

[0029] (1) Sample weighing: weigh 10 mg of moxa as the sample;

[0030] (2) Formaldehyde solution treatment: Add 5 ml of 10% formaldehyde solution to the sample and immerse for 1 min at room temperature;

[0031] (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 20 seconds;

[0032] (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 5 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.5% into the weighing bottle, and immerse for 1 min at room temperature;

[0033] (5) Microscopic observation: Take 5 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.05 ml of 20% glycerol by volume on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa. Example 3

[0034] A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, comprising the following steps:

[0035] (1) Sample weighing: weigh 7 mg of moxa as sample;

[0036] (2) Formaldehyde solution treatment: Add 4 ml of 15% formaldehyde solution to the sample and immerse for 2 min at room temperature;

[0037] (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 15 seconds;

[0038] (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 4 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.8% into the weighing bottle, and immerse for 3 minutes at room temperature;

[0039] (5) Microscopic observation: Take 4 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.03 ml of 20% glycerol on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa. Example 4

[0040] A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, comprising the following steps:

[0041] (1) Sample weighing: weigh 9 mg of moxa as sample;

[0042] (2) Formaldehyde solution treatment: Add 5 ml of 12% formaldehyde solution to the sample and immerse for 2 min at room temperature;

[0043] (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 18 seconds;

[0044] (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 3 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.6% into the weighing bottle, and immerse for 2 minutes at room temperature;

[0045] (5) Microscopic observation: Take 5 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.05 ml of 20% glycerol by volume on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

[0046] The authenticity identification method of moxa floss based on the fluorescence microscopic characteristics of non-glandular hairs of the present invention has the advantage of accurate identification, and the experimental identification effect is good. The real moxa floss is used as the experimental group, and two groups of false moxa floss samples are set as the control group. The real moxa floss in the experimental group and the false moxa floss in the control group are treated in the same way as the treatment method in Example 3, and observed under ultraviolet light with a fluorescence microscope. Figure 4 As shown in Figure 1, the T-shaped non-glandular hairs in the experimental group recovered fluorescence after being treated with the real moxa reagent, and it can be clearly seen that the T-shaped non-glandular hairs in the moxa have handles of varying lengths or shedding marks. Figure 5 , 6 ) and sample 2 (such as Figure 7 , 8 ) the stalk or shedding marks characteristic of T-shaped non-glandular hairs are absent.

[0047] While the present invention was experimented with the treatment method of Example 3, the real moxa and the fake moxa were also experimentally treated with the treatment methods of Examples 1, 2, and 4, and the same and similar results were obtained, which are not listed one by one here, indicating that the present invention has good effect and stable and reliable efficacy.

[0048] From the above, we can see that after mechanical processing, under the action of formaldehyde and diphenyl boric acid-2-aminoethyl ester, the top cells of the real moxa have regained fluorescence, and the stem cells have also been strengthened. It can be clearly seen that the T-shaped non-glandular hairs in the moxa have stems of varying lengths or shedding marks ( Figure 4 ), while fake moxa, such as inferior cotton, various plant fibers and synthetic fibers, do not have the unique handle or shedding marks of T-shaped non-glandular hairs. This method has a good effect on identifying inferior moxa mixed with moxa residues and fake moxa mixed with various plant fibers, and has important practical value for identifying the authenticity of moxa. After experiments and field applications, the accuracy rate reached more than 99%, which effectively solved the identification of the authenticity of moxa, ensured the treatment and efficacy, and had huge economic and social benefits.

Claims

1. A method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs, characterized in that: The following steps are involved: (1) Sample weighing: weigh 5-10 mg of moxa as a sample; (2) Formaldehyde solution treatment: Add 3-5 ml of formaldehyde solution with a volume concentration of 10%-20% to the sample and immerse for 1-2 minutes at room temperature; (3) Rinse: Take out the moxa from the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 10-20 seconds; (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 3-5 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.5%-1% into the weighing bottle, and immerse for 1-5 minutes at room temperature; (5) Microscopic observation: Take 3-5 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.02-0.05 ml of 20% glycerol by volume on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation of a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic characteristics. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

2. The method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs according to claim 1, characterized in that: The following steps are involved: (1) Sample weighing: weigh 5 mg of moxa as sample; (2) Formaldehyde solution treatment: Add 3 ml of 20% formaldehyde solution to the sample and immerse for 2 min at room temperature; (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 10 seconds; (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 3 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 1% into the weighing bottle, and immerse for 5 minutes at room temperature; (5) Microscopic observation: Take 3 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.02 ml of 20% glycerol on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

3. The method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs according to claim 1, characterized in that: The following steps are involved: (1) Sample weighing: weigh 10 mg of moxa as the sample; (2) Formaldehyde solution treatment: Add 5 ml of 10% formaldehyde solution to the sample and immerse for 1 min at room temperature; (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 20 seconds; (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 5 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.5% into the weighing bottle, and immerse for 1 min at room temperature; (5) Microscopic observation: Take 5 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.05 ml of 20% glycerol by volume on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

4. The method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs according to claim 1, characterized in that: The following steps are involved: (1) Sample weighing: weigh 7 mg of moxa as sample; (2) Formaldehyde solution treatment: Add 4 ml of 15% formaldehyde solution to the sample and immerse for 2 min at room temperature; (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 15 seconds; (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 4 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.8% into the weighing bottle, and immerse for 3 minutes at room temperature; (5) Microscopic observation: Take 4 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.03 ml of 20% glycerol on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

5. The method for identifying the authenticity of moxa based on the fluorescence microscopic characteristics of non-glandular hairs according to claim 1, characterized in that: The following steps are involved: (1) Sample weighing: weigh 9 mg of moxa as sample; (2) Formaldehyde solution treatment: Add 5 ml of 12% formaldehyde solution to the sample and immerse for 2 min at room temperature; (3) Rinsing: Take the moxa out of the formaldehyde solution, place it in a petri dish, and rinse it with distilled water for 18 seconds; (4) Treatment with diphenylboronic acid-2-aminoethyl ester reagent: transfer the rinsed moxa from the culture dish into a weighing bottle, add 3 ml of diphenylboronic acid-2-aminoethyl ester reagent with a mass concentration of 0.6% into the weighing bottle, and immerse for 2 minutes at room temperature; (5) Microscopic observation: Take 5 mg of moxa treated with diphenylboronic acid-2-aminoethyl ester reagent, spread it on a glass slide, add 0.05 ml of 20% glycerol by volume on the moxa, cover it with a coverslip, and observe it under ultraviolet light excitation under a fluorescence microscope. The authenticity of the moxa can be determined based on the microscopic features. If T-shaped non-glandular hairs with stalks of varying lengths or shedding marks are observed, it is real moxa, otherwise it is fake moxa.

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