A microscopic detection method for detecting quality of raw material of ramie

Abstract

A kind of microscopic detection method for detecting quality of moxa, comprising the following steps: S1, photographic calculation;S2, sieving;S4, dyeing;S5, making slice ’ ;S6, obtaining stem retention: dividing average area S ’ By average width W0 in step S1, the average length of stem of sieved moxa is obtained, and slice is made three times for each batch of sample, measurement and calculation are carried out, and the final average length L ’ Is obtained by three results, stem retention Y=L ’ / L 0 ×100%, stem retention Y is maximum 60%, the smaller stem retention Y is, the worse quality is indicated.The method is simple, convenient to operate, low in detection cost, small in workload, high in efficiency, good in detection effect, effectively used for microscopic detection method for detecting quality of moxa, the purpose of detecting moxa processing quality is achieved, and there are significant social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of moxa detection, and particularly to a microscopic detection method for detecting the quality of moxa. BACKGROUND

[0002] Moxa is the raw material for making moxa sticks and the main material for moxibustion. The traditional method of making moxa is to repeatedly sun, pound, crush, and sieve impurities and dust from dried moxa leaves to obtain a soft and fine moxa-like substance. The quality of moxa directly affects the efficacy of moxibustion. Ancient physicians believed that moxa for moxibustion should be processed to the extent of "soft and fine as cotton" and "soft, fine, and yellow". Modern moxa processing often uses large machines to crush and sieve moxa leaves or stems and leaves connected to each other, removing stems, leaf flesh, leaf veins, and leaf stems to obtain moxa.

[0003] The degree of moxa processing is called fineness, and the processing ratio is commonly used to represent the fineness of commercially available moxa, i.e., the output ratio of raw moxa leaves to moxa, for example, 5 kg of moxa leaves processed into 1 kg of moxa, with a fineness ratio of 5:1. Some documents clearly state that "the higher the moxa ratio (fineness), the better, and the fewer the impurities, the better". Machine-processed moxa can continuously remove leaf tissue, leaf veins, and other impurities through repeated crushing and sieving. In this case, it is concluded that "the more times the crushing and sieving are repeated, the higher the quality of the obtained moxa". However, due to various uncertain factors such as different production manufacturers and different processing techniques, there has been no objective identification method and standard to objectively evaluate whether commercially available moxa meets the processing ratio indicated on the factory label. Currently, the national standard (Moxa for Moxibustion GB / T 40976-2021) uses microscopic fineness to reflect the fineness of moxa in microscopic detection of moxa. Microscopic fineness focuses on the fineness of leaf tissue and other impurities. If the fineness is too low or the impurities are not completely removed, it will affect the fineness of moxa, resulting in reduced quality. This method can only roughly classify moxa into moxa for moxa stick moxibustion and moxa for direct moxibustion.

[0004] At present, the widely recognized mechanism of moxibustion is the combined effect of physical effect, chemical effect and comprehensive effect. The back (lower surface) of the leaf is covered with gray-white fluff, which is the main source of fluff moxa. According to literature, fine moxa is mainly composed of T-shaped non-glandular hairs derived from the lower surface of the leaf. Recent studies have shown that the T-shaped non-glandular hair stalk can spontaneously fluoresce, and the stalk cells store complex secondary metabolites (polyphenols, flavonoids, volatile oils, etc.), and the stalk cell wall is keratinized. These components are directly related to the burning value of moxa, and the products after burning may be closely related to the quality of moxa and the effect of moxibustion. Therefore, if the crushing is excessive and the screening times are too many, the length of the T-shaped non-glandular hair top cell and the stalk will be continuously reduced, and the secondary metabolites stored in the stalk cells will also be excessively lost, which will affect the quality of moxa and moxibustion. On the other hand, the length of the T-shaped non-glandular hair top cell and the stalk is closely related to the fineness of the moxa. Within a certain processing ratio range, as the processing ratio increases, the micro-crushing degree increases, and the average length of the T-shaped non-glandular hair top cell and the stalk also decreases, and the fineness of the moxa also increases. However, when the fineness is too high, the moxa will be processed into ash, which will affect the processing and production of moxa products and the actual therapeutic effect. Therefore, the average length or the degree of preservation of the T-shaped non-glandular hair top cell and the stalk in the moxa can be used as an index for detecting the quality of moxa.

[0005] In actual operation, it is very inconvenient to directly measure the length of the T-shaped non-glandular hair top cell because it is extremely long and twisted, and different T-shaped non-glandular hair top cells are intertwined with each other. Actual measurement shows that the length variation characteristics of the T-shaped non-glandular hair top cell are very consistent with those of the stalk. If the length of the T-shaped non-glandular hair stalk in the moxa can be counted and measured, it can become a best microscopic detection index for detecting the processing quality of moxa. However, if the length of the T-shaped non-glandular hair stalk is still manually observed and measured one by one to complete the detection of moxa, it will be a large amount of work, consume a lot of manpower, and have low efficiency and poor practical operability. Therefore, how to use the T-shaped non-glandular hair stalk to detect the quality of moxa has not been reported so far. SUMMARY

[0006] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present application is to provide a microscopic detection method for detecting the quality of moxa, which can effectively solve the problem that there is no use of T-shaped non-glandular hair stalk to detect the processing quality of moxa, resulting in unstable quality of moxa.

[0007] To achieve the above purpose, the technical solution solved by the present application is a microscopic detection method for detecting the quality of moxa, comprising the following steps:

[0008] S1, photographic calculation: the back of the leaf of Artemisia argyi T-shaped non-glandular hairs is photographed under ultraviolet light by a fluorescence microscope, and the average length and average width of the T-shaped non-glandular hair stalk are calculated to obtain the average length L0 and the average width W0, then the leaf of Artemisia argyi is repeatedly crushed and sieved to obtain the leaf of Artemisia argyi;

[0009] S2, sieving: 1-2 g of crushed leaf of Artemisia argyi is taken out with tweezers, spread under a dissection microscope, and made fluffy, the fluffy leaf of Artemisia argyi is sieved with a 20-100 mesh sieve, and the large granular substances in the leaf of Artemisia argyi are removed to obtain the sieved leaf of Artemisia argyi;

[0010] S3, methanol treatment: 10-20 mg of sieved leaf of Artemisia argyi is taken out with tweezers and placed in 3-5 ml of methanol, and treated for 5-10 min to obtain the methanol-treated leaf of Artemisia argyi;

[0011] S4, dyeing: the methanol-treated leaf of Artemisia argyi is taken out and transferred into another culture dish, and dyed with 0.1-0.5% 2-aminoethyl biphenyl borate for 1-3 min to obtain dyed leaf of Artemisia argyi;

[0012] S5, tablet preparation: 1-2 mg of dyed leaf of Artemisia argyi is taken out with tweezers, spread on a glass slide, and 0.02 ml of 20%-40% dilute glycerol is added dropwise, mixed, covered with a cover glass, observed and photographed under ultraviolet light by a fluorescence microscope, and the image is processed by image J, the small fluorescent background spots are removed by setting the threshold value, and the total number and total area of the T-shaped non-glandular hair stalk are counted and measured, and the average area S ’ ;

[0013] S6, stalk retention degree calculation: the average area S ’ is divided by the average width W0 in step S1 to obtain the average length of the stalk of the sieved leaf of Artemisia argyi, and the measurement and calculation are performed three times for each batch of sample tablets, and the final average length L ’ is obtained through the three results, and the stalk retention degree Y is calculated as Y=L ’ / L0x100%, the maximum stalk retention degree Y is 60%, and the smaller the stalk retention degree Y, the worse the quality.

[0014] The method is simple, convenient to operate, low in detection cost, small in workload, high in efficiency, good in detection effect, effectively used for the microscopic detection method for detecting the quality of leaf of Artemisia argyi, achieves the purpose of detecting the processing quality of leaf of Artemisia argyi, and has remarkable social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the dense hair diagram of the back of the leaf of Artemisia argyi of the application;

[0016] Figure 2 is the T-shaped non-glandular hair morphology diagram (the stalk part is a short rod, emitting blue fluorescence) of the application.

[0017] Figure 3 is a T-shaped non-glandular hair morphology chart of the present application (the handle emits bright fluorescence);

[0018] Figure 4 is an artemisia floss chart containing more mesophyll powder, etc. of the present application;

[0019] Figure 5 is an artemisia floss chart observed by fluorescence microscope (bright field) of the present application;

[0020] Figure 6 is a T-shaped non-glandular hair chart of artemisia floss observed by fluorescence microscope after fluorescence staining (and Figure 2 is the same field of view, excited by ultraviolet light) of the present application;

[0021] Figure 7 is a processed chart after processing Figure 3 of the present application by image J;

[0022] Figure 8 is a chart counted and measured by image J of the present application. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application will be described in detail below in combination with the drawings and specific cases.

[0024] The present application is embodied in the following examples.

[0025] A microscopic detection method for detecting the quality of artemisia floss, comprising the following steps:

[0026] S1, photographic calculation: photograph the T-shaped non-glandular hair on the back of the artemisia leaf under ultraviolet light by fluorescence microscope, and calculate the average length and average width of the handle of the T-shaped non-glandular hair, to obtain the average length L0 and the average width W0, then repeatedly crush and sieve the artemisia leaf to obtain artemisia floss;

[0027] S2, sieving: take 1-2g of crushed artemisia floss with tweezers, spread it under a dissecting microscope to make it fluffy, sieve the fluffy artemisia floss with a 20-100 mesh ultrasonic vibration sieve to remove small mesophyll tissue blocks, stomata and glandular hairs and other fine powders existing in the artemisia floss, and pick out the remaining granular substances such as large mesophyll tissues and leaf stems with tweezers to obtain sieved artemisia floss;

[0028] S3, methanol treatment: take 10-20mg of sieved artemisia floss with tweezers, place it in a container containing 3-5ml of methanol, and treat it for 5-10min to obtain methanol-treated artemisia floss;

[0029] S4, dyeing: the processed mugwort silk was taken out and transferred into another culture dish, and then dyed with 0.1-0.5% 2-aminoethyl diphenylboronic acid (DPBA) (95% ethanol as solvent) for 1-3 min;

[0030] S5, tabletting: 1-2 mg of the dyed and processed mugwort silk was taken with tweezers, spread on a glass slide, and 0.02 ml of 20%-40% dilute glycerol was added dropwise, mixed well, covered with a cover glass, and observed and photographed under ultraviolet light using a fluorescence microscope. The image was processed using image J, the threshold value was set to remove small fluorescent background points, and the total number and total area of T-shaped non-glandular hair stalks were counted and measured. The average area S was obtained by dividing the total area by the total number. ’ ;

[0031] S6, stalk retention rate: the average area S ’ was divided by the average width W0 in step S1 to obtain the average length of the stalk of the sieved mugwort silk. The tabletting was performed three times for each batch of samples, and the measurement and calculation were performed. The final average length L ’ was obtained by the three results, and the stalk retention rate Y = L ’ / L0 x 100% was calculated. The maximum stalk retention rate Y was 60%, and the smaller the stalk retention rate Y, the better the quality.

[0032] As shown in Figure 1 , the back of the mugwort leaf is covered with fluff. The T-shaped non-glandular hairs on the back of the mugwort leaf were photographed under ultraviolet light using a fluorescence microscope, as shown in Figure 2 , 3 , and the average length and average width of the T-shaped non-glandular hair stalks were calculated to obtain the average length L0 and the average width W0, as shown in Table 1 below.

[0033] Table 1 Average length L0 and average width W0

[0034] average width (W0) of the shank 12.53 μm average length of the shank (L0) 35.07009224 μm

[0035] The mugwort leaf was crushed continuously for 3 times and sieved with a 20-mesh sieve. The obtained mugwort silk still contained a lot of leaf pulp and other powders, as shown in Figure 4 . 1-2 g of the mugwort silk was taken with tweezers, spread under a dissecting microscope, and puffed up. The puffed-up mugwort silk was sieved with a 20-100 mesh sieve, and the large granular substances in the mugwort silk were removed to obtain the sieved mugwort silk, as shown in Figure 5 . The sieved mugwort silk was subjected to dyeing treatment, and the dyed mugwort silk was observed and photographed under ultraviolet light using a fluorescence microscope, as shown in Figure 6 . Then, the image was processed using image J, and the stalk part of the mugwort silk was highlighted (black part in the figure), as shown in Figure 7 . After setting the threshold value to remove small fluorescent background points, the T-shaped non-glandular hair stalks were obtainedFigure 8 , the total number and total area of T-shaped non-glandular hair stalks are counted and measured, and the results are shown in Table 2 below. Figure 8

[0036] Table 2 Measurement results of stalk length

[0037] Handle numbering Area (pm 2 ) Handle numbering Area (pm 2 ) <!-- 3 -->]]> 1 138.197 15 449.918 2 52.018 16 302.404 3 244.175 17 333.459 4 110.247 18 102.872 5 177.793 19 138.197 6 237.964 20 126.552 7 461.952 21 81.909 8 703.021 22 284.159 9 266.302 23 339.282 10 148.29 24 835.007 11 257.761 25 252.715 12 56.288 26 148.679 13 171.582 27 146.738 14 69.875 28 161.489

[0038] According to the total number and total area of T-shaped non-glandular hair stalks, the average area S ’ is 242.816 μm 2 , the average area S ’ is divided by the average width W0 in step S1, and the measurement and calculation are performed three times for each batch of sample slices, and the final average length L is obtained by the three results ’ is 19.3788 μm.

[0039] The stalk retention Y = L ’ / L0 x 100% = 19.3788 / 35.07009224 = 55.3%, which is close to the upper limit of 60%, indicating that the moxa processed in the embodiment has good quality.

[0040] Table 3 Calculation of average area, length and retention of stalk

[0041]

[0042] As can be seen from the above, the present application is quite different from the practice of focusing on the fineness of mesophyll tissue impurities in the current national standard (Moxa for moxibustion GB / T 40976-2021). The detection method of the present application not only can detect the fineness of moxa processing, but also can reflect the retention degree of T-shaped non-glandular stalks in moxa processing, thereby regulating the current widespread problem of excessive processing of moxa. Since the stalks store polyphenols, flavonoids and other medicinal ingredients, the number characteristics of the stalks are directly related to the quality of moxa, and the present application focuses on the T-shaped non-glandular stalks of moxa, which can more directly and accurately reflect the processing quality of moxa, and has advantages over the indicators used in the current national standard (Moxa for moxibustion GB / T 40976-2021).

[0043] ​The method is repeatedly tried and experimented, and the T-shaped non-glandular stalk of the processed moxa is clear and prominent in the fluorescence image, the background is dark, and it is very suitable for image J to process and analyze the image, which is convenient for detection, short in experimental time, small in workload and important in value. According to the structural characteristics of the T-shaped non-glandular stalk, counting and measuring the stalk are the core of the method, and the method has three key points, one is that the ultrasonic vibration sieve 20-100 mesh sieve can sieve out the small powder in the leaf pulp tissue block, stoma and glandular hair in the moxa, and even the small powder particles adhered to the T-shaped non-glandular stalk can be removed, so as to avoid the interference in the later microscopic observation; the tweezers is used to remove the large leaf pulp tissue, leaf stem and other granular substances, so as to avoid the fluorescence interference of the non-observation target objects, the methanol is used for treatment, so as to wash away the fluorescence substances of the non-glandular stalk, and avoid the fluorescence interference; the 2-aminoethyl diphenyl borate (DPBA) is used for fluorescence staining of the stalk, so as to enhance the fluorescence imaging; the image J is used for image analysis and detection, so as to improve the efficiency. The operation instrument is simple, the operation is convenient, the cost is low, the workload is small, the efficiency is high, the effect is good, it is a kind of effective microscopic detection method for detecting the quality of moxa, the method can achieve the purpose of detecting the processing quality of moxa, and has remarkable social and economic benefits.

Claims

1. A microscopic detection method for detecting the quality of moxa wool, characterized in that, Includes the following steps: S1. Photographic calculation: The T-shaped non-glandular hairs on the back of the mugwort leaf were photographed under ultraviolet light using a fluorescence microscope, and the average length and average width of the stalk of the T-shaped non-glandular hairs were calculated to obtain the average length L0 and average width W0. Then the mugwort leaf was repeatedly crushed and sieved to obtain mugwort floss. S2. Sieving: Take 1-2g of crushed moxa wool with tweezers, spread it out under a dissecting microscope to make it fluffy, and sieve the fluffy moxa wool through a 20-100 mesh sieve to remove large particles in the moxa wool, and obtain sieved moxa wool. S3. Methanol treatment: Take 10-20mg of sieved moxa wool with tweezers and place it in 3-5ml of methanol. Treat for 5-10 minutes to obtain methanol-treated moxa wool. S4. Staining: Take out the methanol-treated mugwort floss, transfer it to another petri dish, and then stain it with 0.1~0.5% 2-aminoethyl biphenyl borate ester for 1~3 min to obtain stained mugwort floss; S5. Slide Preparation: Take 1-2 mg of stained Artemisia floss with tweezers, spread it on a glass slide, and add 0.02 ml of 20%-40% dilute glycerol (by volume). Mix well, cover with a coverslip, and observe and photograph the Artemisia floss under ultraviolet light using a fluorescence microscope. Process the images with ImageJ, remove small fluorescent background noise by setting a threshold, and count and measure the total number and total area of ​​T-shaped non-glandular trichomes. Divide the total area by the total number to obtain the average area S. ’ ; S6. Calculate the handle retention rate: Calculate the average area S... ’ Divide by the average width W0 from step S1 to obtain the average length of the stem of the sieved moxa wool. Prepare sheets three times for each batch of samples, measure and calculate, and obtain the final average length L from the three measurements. ’ The handle retention rate Y=L was calculated. ’ / L0×100%, the maximum handle retention Y is 60%. The smaller the handle retention Y, the worse the quality.

2. The microscopic detection method for detecting the quality of moxa wool according to claim 1, characterized in that, The sieving process is performed using a 20-100 mesh ultrasonic vibrating screen.

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