A polarized microscopic quantitative method for calcium oxalate crystals in ginseng

Abstract

The present invention discloses a polarized light microscopic quantitative method for calcium oxalate crystals in ginseng, comprising the steps of S1. crushing, sieving, accurately weighing, preparing a sample suspension, vortexing, and mounting; S2. collecting a full-field microscopic image under a polarized light microscope; S3. performing color separation, background correction, grayscale conversion, and threshold segmentation on the image to calculate the full-field calcium oxalate crystal cross-sectional area; S4. calculating the unit mass calcium oxalate crystal cross-sectional area based on a calculation formula of the calcium oxalate crystal cross-sectional area and the sample dry weight; S5. realizing the authenticity identification of ginseng and the classification and evaluation of forest ginseng and garden ginseng through the unit mass calcium oxalate crystal cross-sectional area. The present invention belongs to the field of property determination of traditional Chinese medicine, and specifically refers to a polarized light microscopic quantitative method for calcium oxalate crystals in ginseng.

Description

Technical Field

[0001] The invention belongs to the field of property determination of traditional Chinese medicines, and specifically relates to a polarized light microscopic quantitative method for calcium oxalate crystals in ginseng. Background Art

[0002] Ginseng, derived from the dried roots and rhizomes of Panax ginseng (CA Mey.), a plant of the Araliaceae family, is known as the "King of All Herbs" and boasts benefits such as tonifying vital energy, nourishing the spleen and strengthening the lungs. Its quality is closely related to factors such as its age and cultivation method (e.g., garden ginseng vs. forest ginseng). For example, forest ginseng, due to its longer growth period and more environmental stress conditions similar to those of its wild counterpart, typically contains higher saponin content than garden ginseng.

[0003] Calcium oxalate clusters are key morphological markers of plant metabolites, and their microscopic characteristics (crystal form, density, etc.) play an important role in the identification and quality evaluation of traditional Chinese medicines. The current Chinese Pharmacopoeia, under the ginseng section, only specifies a diameter range of 20 to 68 μm for calcium oxalate clusters. However, studies have shown that the density of calcium oxalate clusters in ginseng is closely related to the growth age and environment. For example, the number of calcium oxalate clusters in forest ginseng (172-383 clusters / mg) is significantly higher than that in garden ginseng (33-144 clusters / mg), and the number of calcium oxalate clusters in ginseng is much higher than that in American ginseng.

[0004] In recent years, polarized light microscopy, which exploits birefringence, has significantly enhanced the discernibility of microscopic features and has become widely used in the microscopic identification of traditional Chinese medicines. Calcium oxalate clusters exhibit bright birefringence under a polarized light microscope, providing a more reliable basis for the microscopic identification of ginseng.

[0005] Previous studies have attempted to identify ginseng by observing and counting calcium oxalate clusters using a conventional optical microscope. However, this method has significant limitations: manual counting is highly subjective, with an error rate of 15%-20%. Furthermore, it is difficult to overcome the problem of calcium oxalate crystal fragmentation during sample preparation. Small fragments with an area of ​​less than 5 μm² cannot be counted under a conventional optical microscope, resulting in an under-detection rate of over 17% (see the results). Figure 1 ), resulting in incomplete information collection and insufficiently representative data. Furthermore, the operation is time-consuming and labor-intensive, with single-sample counting and analysis taking over 30 minutes, making it difficult to meet the demands of high-throughput rapid testing. Therefore, an objective, efficient, and more accurate microscopic quantification method for calcium oxalate crystals is urgently needed to improve the scientific quality control and authenticity identification of ginseng. Summary of the Invention

[0006] The present invention mainly solves the defects of the prior art such as large manual counting errors, incomplete data collection and low detection efficiency.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows: The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng proposed by the present invention comprises the following steps:

[0008] S1. Crush, sieve, accurately weigh, prepare sample suspension, vortex, and mount;

[0009] S2. Collect full-field microscopic images under a polarizing microscope;

[0010] S3. Perform color separation, background correction, grayscale conversion, and threshold segmentation on the image to calculate the cross-sectional area of ​​calcium oxalate crystals in the entire field of view;

[0011] S4. Calculate the cross-sectional area of ​​calcium oxalate per unit mass based on the calculation formula of the cross-sectional area of ​​calcium oxalate crystals and the dry weight of the sample;

[0012] S5. The authenticity of ginseng and the classification and evaluation of forest ginseng and garden ginseng can be achieved through the cross-sectional area of ​​calcium oxalate crystals per unit mass. The authenticity of ginseng is mainly used to distinguish between American ginseng and ginseng.

[0013] Furthermore, in step S1, the sample is crushed until it passes through a No. 4 pharmacopoeia sieve, the concentration of the test sample suspension in step S1 is 20-50 mg / mL, and the optimal concentration is 30 mg / mL, wherein the device liquid used to prepare the test sample suspension is 50% glycerol: 0.5% sodium carboxymethyl cellulose: water = 9:8:3 (volume ratio), and the vortexing in step S1 means that the sample suspension must be vortexed (5000 rpm) for ≥2 seconds. If the vortex frequency changes, the processing time can be adjusted. The sampling in step S1 should be completed within 5 seconds after vortexing. The sampling volume of the test sample suspension solution is 2 μL, wherein the sampling position is the bottom of the centrifuge tube, and the loading in step S1 means distributing the sample solution within the grid lines of the cell counting plate.

[0014] Furthermore, the parameters of the polarizing microscope in step S2 are: eyepiece 10x, objective lens 10x, polarization degree 40°, and light source color temperature 5500±500K.

[0015] Furthermore, the color separation in step S3 refers to retaining only the green band (500-600nm) color of the calcium oxalate crystals in the image, the background correction in step S3 adopts the threshold automatic adaptation method to convert the image background into black, the grayscale conversion in step S3 refers to converting the image into 8-bit grayscale value, the threshold segmentation in step S3 adopts adaptive threshold segmentation, and the cross-sectional area of ​​the calcium oxalate crystals in step S3 is calculated using the software batch processing function.

[0016] Furthermore, the formula for calculating the cross-sectional area per unit mass of calcium oxalate crystals in step S4 is:

[0017] The cross-sectional area of ​​calcium oxalate crystal per unit mass (D, μm² / mg) = ΣS / [m×(1-w)×V1 / V0];

[0018] Where: ΣS is the total cross-sectional area of ​​calcium oxalate crystals in the tablet (μm²), m is the sample weight of the test sample (mg), w is the water content in the sample (%), V1 is the tablet sampling volume (mL), and V0 is the total volume of the test sample suspension (mL).

[0019] Furthermore, in step S5, the cross-sectional area of ​​calcium oxalate crystals per unit mass is used to distinguish American ginseng from Panax ginseng, and to distinguish garden ginseng from undergrowth ginseng, wherein the cross-sectional area of ​​calcium oxalate crystals per unit mass of American ginseng is significantly lower than that of Panax ginseng, and the cross-sectional area of ​​calcium oxalate crystals per unit mass of garden ginseng is significantly lower than that of undergrowth ginseng.

[0020] The beneficial effects achieved by the present invention using the above steps are as follows:

[0021] 1. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng proposed in this scheme uses a polarized light microscope to observe calcium oxalate crystals in ginseng. It can more completely and clearly capture and collect calcium oxalate crystal information, including complete calcium oxalate clusters and broken calcium oxalate crystals, and reduce the missed detection rate.

[0022] 2. This proposal proposes a polarized microscopic quantitative method for the determination of calcium oxalate crystals in ginseng. During the suspension of the sample, sodium carboxymethyl cellulose (a suspending agent) and glycerol (a humectant) are added to the device liquid. The appropriate ratio of the two can achieve uniform suspension of the sample powder, ensuring the stability and repeatability of the measurement results.

[0023] 3. The polarized microscopic quantitative method for calcium oxalate crystals in ginseng proposed in this scheme acquires full-field images of the specimen, ensuring the comprehensiveness and representativeness of the measurement results, minimizing the quantitative errors caused by manual counting using part to represent the whole, and improving the accuracy of the measurement results.

[0024] 4. This proposal proposes a polarized microscopic quantitative method for the determination of calcium oxalate crystals in ginseng. The introduction of image analysis technology determines the cross-sectional area per unit mass of calcium oxalate crystals in the sample, making the quantitative detection of calcium oxalate crystals in ginseng more accurate and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Result diagram of missed detection rate measurement using a common optical microscope;

[0026] Figure 2 Cluster analysis of the cross-sectional area of ​​calcium oxalate crystals per unit mass of American ginseng and Panax ginseng samples;

[0027] Figure 3 Flow chart for determining the cross-sectional area of ​​calcium oxalate crystals per unit mass of ginseng samples;

[0028] Figure 4 The cross-sectional area of ​​calcium oxalate crystals per unit mass of American ginseng and ginseng (garden ginseng and forest ginseng);

[0029] Figure 5 Sampling diagram;

[0030] Figure 6 Graphical representation of sample suspension;

[0031] Figure 7 Illustration of cell counting slides;

[0032] Figure 8 Illustration of the original polarized micrograph of the sample (1 / 16);

[0033] Figure 9 Color separation diagram;

[0034] Figure 10 Background correction diagram;

[0035] Figure 11 Grayscale conversion diagram;

[0036] Figure 12 Threshold adjustment icon;

[0037] Figure 13 Results of investigation on the liquid ratio of the device;

[0038] Figure 14 Sampling concentration investigation results;

[0039] Figure 15 Sampling time survey results;

[0040] Figure 16 The results of the slide inspection;

[0041] Figure 17 Results of investigation on image acquisition methods;

[0042] Figure 18 Crystal brightness measurement results;

[0043] Figure 19 Crystal cross-sectional area measurement results;

[0044] Figure 20 Linear relationship investigation;

[0045] Figure 21 Precision test results;

[0046] Figure 22 Repeatability test results;

[0047] Figure 23 Stability test results;

[0048] Figure 24Sample recovery test results.

[0049] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0051] like Figures 1 to 24 As shown, the present invention proposes a polarized light microscopic quantitative method for calcium oxalate crystals in ginseng, comprising the following steps:

[0052] S1 Sample Preparation: Ginseng sample was pulverized, passed through a No. 4 pharmacopoeia sieve, and accurately weighed before preparing a test suspension. Vortex the suspension before mounting. The test suspension concentration was 20-50 mg / mL, with an optimal concentration of 30 mg / mL. The test suspension was prepared using a 9:8:3 volume ratio of 50% glycerol: 0.5% sodium carboxymethyl cellulose: water. Vortexing was performed at 5000 rpm for ≥ 2 seconds. The vortexing time can be adjusted appropriately if the vortexing frequency is changed. Sampling was completed within 5 seconds of vortexing, with a sample volume of 2 μL from the bottom of the centrifuge tube. For mounting, a cell counting plate was used, with the sample solution distributed within the grid lines of the cell counting plate.

[0053] S2 Image Acquisition: Full-field microscopic images were collected under a polarizing microscope. The parameters of the polarizing microscope were as follows: 10x eyepiece, 10x objective, polarization degree 40°, and light source color temperature 5500 ± 500 K.

[0054] S3 Image Processing: The acquired images were subjected to color separation, background correction, grayscale conversion, and threshold segmentation to calculate the cross-sectional area of ​​calcium oxalate crystals across the entire field of view. Color separation retained only the green band (500-600 nm). Background correction used an automatic threshold adaptation method to convert the background to black. Grayscale values ​​were converted to 8-bit grayscale values. Threshold segmentation used adaptive threshold segmentation. The cross-sectional area of ​​calcium oxalate crystals was calculated using the software's batch processing function.

[0055] S4 quantitative calculation: Based on the calculation formula of calcium oxalate crystal cross-sectional area and sample dry weight, the unit mass calcium oxalate crystal cross-sectional area is calculated. The calculation formula of unit mass calcium oxalate crystal cross-sectional area is: Unit mass calcium oxalate crystal cross-sectional area (D, μm² / mg) = ΣS / [m×(1-w)×V1 / V0], where ΣS is the total cross-sectional area of ​​calcium oxalate crystals in the tablet preparation (μm²), m is the sample weight (mg), w is the water content in the sample (%), V1 is the tablet preparation sampling volume (mL), and V0 is the total volume of the test sample suspension (mL).

[0056] S5 Identification Application: Calcium oxalate crystal cross-sectional area per unit mass is used to identify the authenticity of ginseng and classify forest and garden ginseng. The calcium oxalate crystal cross-sectional area per unit mass of American ginseng is significantly lower than that of ginseng, while that of garden ginseng is significantly lower than that of forest ginseng.

[0057] Example 1: The samples selected were all American ginseng and ginseng (garden ginseng and forest ginseng) samples that had passed the identification, a total of 20 batches, and each group of samples was measured in parallel 5 times. Figure 2 、 3 、4.

[0058] Determination of cross-sectional area of ​​calcium oxalate crystals per unit mass in ginseng (forest ginseng) samples:

[0059] A 15-year-old understory ginseng sample (S12) from Jilin was taken, crushed and passed through a No. 4 pharmacopoeia sieve, 30.02 mg of powder was weighed and placed in a centrifuge tube; the device liquid was prepared according to 50% glycerol: 0.5% sodium carboxymethyl cellulose: water = 9:8:3; 1 mL of the device liquid was accurately pipetted and mixed with the sample powder to prepare a 30 mg / mL test sample suspension, and the sample was vortexed (5000 rpm) for 2 seconds to mix the sample. After vortexing (within 5 seconds), 2 μl of the sample was immediately pipetted (the sample aspiration position was fixed at the bottom of the centrifuge tube) and spread flatly on the grid lines of the cell counting plate, see attached. Figure 5 、 6 、7.

[0060] Use a 10x eyepiece and a 10x objective lens combination under a microscope, automatically expose, and capture full-field images (resolution ≥ 1280 × 720). See attached. Figure 8 .

[0061] Photoshop software (2021 version) sets up a batch processing program: (1) adjust the saturation and brightness, the saturation and brightness of the green band (500-600nm) remain unchanged, and the other colors are adjusted to -100; (2) the color level value is obtained by the threshold automatic adaptation method, and the color level is adjusted to (0.50, 2.50) to convert the background to pure black; (3) merge the color extraction layer and the background layer; (4) save; see the attached Figure 9 、 10.

[0062] ImageJ software (version 1.54d) was used for grayscale conversion and threshold segmentation, and the batch processing program was set as follows: Figure 11 、 12

[0063] run("8-bit");

[0064] resetThreshold(); / /

[0065] setThreshold(40, 255); / /

[0066] setOption("BlackBackground", true);

[0067] run("Convert to Mask");

[0068] / / run("Flatten"); / /

[0069] / / waitForUser("Verify Mask"); / /

[0070] run("Measure");

[0071] Calculation of the cross-sectional area of ​​calcium oxalate crystals per unit mass in the sample:

[0072] Cross-sectional area of ​​calcium oxalate crystal per unit mass (μm² / g) = ΣS / [m×(1-w)×V1 / V0];

[0073] The specific calculation values ​​are as follows: cross-sectional area of ​​calcium oxalate crystal per unit mass (μm² / mg) = 46.765×10 -6 μm² / [30.02mg×(1-0.11)×2×0.001mL / 1mL]=885.1μm² / mg;

[0074] in:

[0075] ΣS is the total cross-sectional area of ​​calcium oxalate crystals in the tablet preparation, ΣS=46.765μm²;

[0076] m is the sample weight of the test sample, m=30.02 mg;

[0077] w is the water content of the sample, w=11%;

[0078] V1 is the sample volume for preparation, V1=2μL=2×0.001mL;

[0079] V0 is the total volume of the test sample suspension, V0=1mL;

[0080] That is, the cross-sectional area of ​​calcium oxalate crystals per unit mass of this sample is 885.06 μm² / mg;

[0081] When used specifically:

[0082] 1. Investigation of device liquid ratio

[0083] Accurately weigh 30 mg of sample powder (S12) (passed through a No. 4 pharmacopoeia sieve) in a 2 ml EP tube. Examine the ratios of glycerol, sodium carboxymethyl cellulose, and water to prepare device solutions with different ratios. Vortex mix thoroughly and then sample 2 μL for mounting on a slide. Repeat the measurement six times to determine the cross-sectional area of ​​calcium oxalate crystals. The results are shown in the attached figure. Figure 13 When the device liquid ratio was 50% glycerol: 0.5% sodium carboxymethyl cellulose: water = 9:8:3, the measurement results were most accurate and reproducible, with an RSD of 3.01%. For other device liquid ratios, the sample powder settled too quickly or aggregated and was difficult to disperse, resulting in larger measurement results with RSD values ​​greater than 5%.

[0084] 2. Sample concentration investigation

[0085] Prepare 5, 10, 20, 30, 40, 50, 80, and 100 mg / mL sample (S12) suspensions (6 replicates for each concentration). Vortex the sample and then take 2 μL of the sample for mounting on a slide to measure the cross-sectional area of ​​calcium oxalate crystals. Investigate the repeatability of the sample suspension concentration and the measurement results, and screen the appropriate concentration range and optimal detection concentration of the sample. See the attached Figure 14 . The results showed that when the sample suspension concentration was 5 mg / mL, the RSD value of the measured calcium oxalate crystal cross-sectional area was 5.04%, and the result deviation was large; when the concentration was 10~50 mg / mL, the RSD value was less than 5%; when the sample concentration exceeded 50 mg / mL, the calcium oxalate crystal signal would be blocked by other powder features, resulting in signal loss and inability to accurately measure. In summary, 30 mg / mL is the optimal sample suspension detection concentration, and the sample suspension concentration range is 20~50 mg / mL.

[0086] 3. Sampling time inspection

[0087] Prepare a 30 mg / mL suspension of sample (S12), vortex for 2 seconds to mix, and then aspirate 2 μL of the sample from the bottom of the centrifuge tube at 2 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, and 60 seconds after vortexing to mount a slide. Measure the cross-sectional area of ​​calcium oxalate crystals. Figure 15The results showed that when samples were taken within 5 seconds of vortexing, the RSD values ​​of the cross-sectional area of ​​calcium oxalate crystals in the samples were all less than 5%. The results of samples taken 2 seconds after vortexing were 45.75±1.05μm², and the results of samples taken 5 seconds after vortexing were 47.78±1.6μm², with relatively small deviations. The RSD value of the results of samples taken 10 seconds after vortexing was 7.86%, indicating a larger deviation. Therefore, sample collection should be completed within 5 seconds after vortexing.

[0088] 4. Examination of Slides

[0089] To examine the use of ordinary glass slides and cell counting chambers, 2 μL of sample (S12) suspension was aspirated and mounted on a 25×25 mm² glass slide and a 0.0025 mm² cell counting chamber, respectively. Six samples were prepared in parallel for each group, and the cross-sectional area of ​​calcium oxalate crystals in each group was measured. Figure 16 The results showed that the results obtained with a standard glass slide were slightly higher, with an RSD of 71.64%, indicating poor reproducibility. The results obtained with a cell counting plate had an RSD of 3.01%, indicating good reproducibility. Therefore, a cell counting plate should be selected for mounting, as its grid lines can prevent duplicate or missed measurements.

[0090] 5. Investigation of image acquisition methods

[0091] Two image acquisition methods, partial field of view image acquisition and full field of view image acquisition, were investigated. A 30 mg / mL sample (S12) was prepared, vortexed for 2 seconds, and 2 μL of the sample was loaded onto a slide. 1 / 3 of the full field of view image and the full field of view image were acquired, respectively. The cross-sectional area of ​​calcium oxalate crystals per unit mass of the sample was determined. See the attached figure. Figure 17 The results showed that the RSD value of the measurement results for full-field image acquisition was 3.01%, while the RSD value of the measurement results for partial-field image acquisition was 24.63%, indicating poor repeatability. The measurement results for partial image acquisition differed by 5 to 37% from those for full-field image acquisition. Therefore, full-field image acquisition was selected as the image acquisition method.

[0092] 6. Investigation of quantitative parameters

[0093] Two quantitative indicators of calcium oxalate crystal brightness and crystal cross-sectional area were investigated. Crystal brightness determination requires manual selection of a single crystal region and calculation of the total brightness; while for crystal cross-sectional area determination, the batch processing function of ImageJ software can automatically extract the crystal region and calculate the total cross-sectional area. Sample (S12) suspensions with concentrations of 10, 20, 30, 40, and 50 mg / mL were prepared, slices were made, and images were collected. Crystal brightness and crystal cross-sectional area were used for quantification. The results showed that within the concentration range of 10~50 mg / mL, the two indicators had the same quantitative trend, but the brightness method took more time to determine. Although calcium oxalate crystals have a three-dimensional structure and brightness determination is more in line with its characteristics, the determination is time-consuming. The area determination method is more efficient and suitable for high-throughput determination. Therefore, the quantitative indicator selected was calcium oxalate crystal cross-sectional area, see Appendix. Figure 18 、 19 .

[0094] 7. Methodological investigation:

[0095] 7.1 Linear relationship investigation: 30 mg / mL sample (S7) and 30 mg / mL sample (S11) suspensions were mixed at a ratio of 100%, 80%, 60%, 40%, 20%, and 0% of sample (S11), respectively. The above experimental method and conditions were followed, and the slides were mounted in parallel and measured 6 times. The cross-sectional area of ​​calcium oxalate crystals at each ratio was determined as shown in the attached figure. Figure 20 The linear equation obtained was y=92.309x+4.9496, with γ=0.9978. This indicates a good linear relationship for the calcium oxalate crystal cross-sectional area within the range of 6.02 to 94.64 μm². For samples whose results are outside the linear range, the sample concentration can be adjusted appropriately within the range of 10 to 50 mg / mL.

[0096] 7.2 Precision Investigation: A 30 mg / mL sample (S12) suspension was prepared and operated according to the above experimental method and conditions. Parallel slides were mounted and measured 6 times. The RSD value of the cross-sectional area of ​​calcium oxalate crystals was 4.81%, indicating that the precision of the experimental instrument and method was good. Figure 21 .

[0097] 7.3 Repeatability Study: Weigh and prepare 6 portions of 30 mg / mL sample (S12) suspension, and measure the cross-sectional area of ​​calcium oxalate crystals according to the above experimental method and conditions. The RSD value of the measurement result is 3.99%, indicating that the method has good repeatability. Figure 22 .

[0098] 7.4 Stability Study: 30 mg / mL sample (S12) suspension was taken and sliced ​​at 0 h, 2 h, 4 h, 6 h, and 8 h. The RSD value of the cross-sectional area of ​​calcium oxalate crystals was 1.84%, indicating that the sample was stable. Figure 23 .

[0099] 7.5 Sample Addition Recovery Test: Accurately weigh 12 mg of sample S7 (unit mass cross-sectional area 108.81 μm² / mg) and 18 mg of sample S11 (unit mass cross-sectional area 1596.77 μm² / mg) to prepare a 30 mg / mL mixed sample suspension. Five replicates were prepared and assayed according to the above experimental method and conditions. The sample addition recovery and RSD value of the calcium oxalate crystal cross-sectional area were calculated. The results showed that the sample addition recovery of the calcium oxalate crystal cross-sectional area ranged from 98.43% to 104.99%, with an average recovery of 99.95% and an RSD of 4.12%, indicating that the experimental method was accurate. (See Appendix) Figure 24 .

[0100] In summary, the method of the present invention comprises the following steps: a ginseng sample is pulverized, passed through a No. 4 pharmacopoeia sieve, and accurately weighed to prepare a suspension. The apparatus fluid is a mixture of 50% glycerol: 0.5% sodium carboxymethyl cellulose: water (9:8:3), with an optimal sample suspension concentration of 30 mg / mL (concentration range: 20-50 mg / mL). Vortex (5000 rpm) for 2 seconds to mix thoroughly. Within 5 seconds after vortexing, 2 μl of the sample (sampling position is at the bottom of the centrifuge tube) is pipetted onto a cell counting plate. The plate is then placed under a polarizing microscope with a 40-degree polarization adjustment, observed with a 10x eyepiece and a 10x objective lens, and full-field image acquisition is performed. The cross-sectional area of ​​calcium oxalate crystals in the ginseng sample is determined by image processing, and the cross-sectional area of ​​calcium oxalate crystals per unit mass of the sample is calculated according to the formula.

[0101] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual method is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A polarized light microscopic quantitative method for calcium oxalate crystals in ginseng, characterized in that: The following steps are involved: S1. Crush, sieve, accurately weigh, prepare sample suspension, vortex, and mount; S2. Collect full-field microscopic images under a polarizing microscope; S3. Perform color separation, background correction, grayscale conversion, and threshold segmentation on the image to calculate the cross-sectional area of ​​calcium oxalate crystals in the entire field of view; S4. Calculate the cross-sectional area of ​​calcium oxalate per unit mass based on the calculation formula of the cross-sectional area of ​​calcium oxalate crystals and the dry weight of the sample; S5. The authenticity of ginseng and the classification and evaluation of forest ginseng and garden ginseng can be achieved through the cross-sectional area of ​​calcium oxalate crystals per unit mass. The authenticity of ginseng is mainly used to distinguish between American ginseng and ginseng.

2. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: In step S1, the sample is crushed until it passes through a No. 4 pharmacopoeia sieve.

3. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: The concentration of the test sample suspension in step S1 is 20-50 mg / mL, with the optimal concentration being 30 mg / mL. The volume ratio of the device liquid used to prepare the test sample suspension is 50% glycerol: 0.5% sodium carboxymethyl cellulose: water = 9:8:

3.

4. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: The vortexing in step S1 means that the sample suspension must be vortexed at a speed of 5000 rpm for ≥2 seconds. If the vortex frequency is changed, the processing time can be adjusted. The sampling in step S1 should be completed within 5 seconds after vortexing. The sampling volume of the test suspension solution is 2 μL, and the sampling position is the bottom of the centrifuge tube.

5. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: The loading in step S1 refers to distributing the sample solution within the grid lines of the cell counting plate.

6. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: The parameters of the polarizing microscope in step S2 are: eyepiece 10x, objective lens 10x, polarization degree 40°, and light source color temperature 5500±500K.

7. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: The color separation in step S3 refers to retaining only the green band of 500-600nm of the calcium oxalate crystals in the image, the background correction in step S3 uses the threshold automatic adaptation method to convert the image background into black, the grayscale conversion in step S3 refers to converting the image into 8-bit grayscale values, the threshold segmentation in step S3 uses adaptive threshold segmentation, and the cross-sectional area of ​​the calcium oxalate crystals in step S3 is calculated using the software batch processing function.

8. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: The formula for calculating the cross-sectional area per unit mass of calcium oxalate crystals in step S4 is: D = ΣS / [m×(1-w)×V1 / V0]; Where: D is the cross-sectional area of ​​calcium oxalate crystals per unit mass, in mm² / g; ΣS is the total cross-sectional area of ​​calcium oxalate crystals in the tablet preparation, in mm²; m is the sample weight of the test sample, in g; w is the water content in the sample, in %; V1 is the tablet preparation sampling volume, in mL; V0 is the total volume of the test sample suspension, in mL.

9. The polarized light microscopic quantitative method for calcium oxalate crystals in ginseng according to claim 1, characterized in that: In step S5, the unit mass cross-sectional area of ​​calcium oxalate crystals is used to distinguish American ginseng from Panax ginseng, and to distinguish garden ginseng from undergrowth ginseng. The unit mass cross-sectional area of ​​calcium oxalate crystals of American ginseng is significantly lower than that of Panax ginseng, and the unit mass cross-sectional area of ​​calcium oxalate crystals of garden ginseng is significantly lower than that of undergrowth ginseng.

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