Crocus sativus quality grade evaluation card based on antithrombotic titer and application

Abstract

The invention discloses a saffron quality grade evaluation card based on antithrombotic titer and application, the evaluation card comprises a color band and corresponding color grades, and the color grades comprise a first grade and a second grade; the first-grade colored tape is gradually changed from bright orange to inflammatory red, 50 < = L * < = 53, 25 < = a * < = 35, and 20 < = b * < = 30; the second-stage colored tape gradually changes from red to reddish brown, and L * lt is larger than or equal to 45; 50, 15 < = a * lt; 25, 10 < = b * lt; 20; according to the method, the color information of the stigma croci is collected, the thrombus inhibition rate of the stigma croci is measured, grading is performed according to the thrombus inhibition rate, the color information is associated with the grade of the stigma croci, and the grade quality evaluation card of the stigma croci is made, so that the quality of the stigma croci can be rapidly, accurately and conveniently measured. The method does not need a complicated sample pretreatment process, does not need to consume an organic reagent, is green and environment-friendly, and has higher practicability and clinical significance.

Description

(I) Technical field

[0001] The invention belongs to the technical field of detection of traditional Chinese medicine saffron, and relates to a saffron quality grade assessment card based on antithrombotic potency and application thereof. (II) Background technology

[0002] Saffron is the dried stigma of Crocus sativus L., a plant of the Iridaceae family. It is also known as saffron or saffron. It is a precious food and medicine product. It is not only a natural spice and condiment, but also a precious Chinese medicinal material with a wide range of uses. Its medicinal and economic value cannot be underestimated. Saffron tastes sweet, is neutral in nature, and enters the heart and liver meridians. It has the effects of promoting blood circulation and removing blood stasis, cooling blood and detoxifying, relieving depression and calming the mind. It has been officially included in the Pharmacopoeia of the People's Republic of China (Ch.P.2020).

[0003] At present, the quality standards of saffron can be roughly divided into four categories: international standards, national standards, local standards and group standards. The international standard divides it into three levels based on physical and chemical properties. Its main evaluation indicators include foreign matter (mass fraction), moisture and volatile matter content, total ash, acid-insoluble ash, cold water-soluble extracts, aroma intensity (maximum absorbance of crocin at 257nm), aromaticity (maximum absorbance of crocin aldehyde at 330nm), color value (maximum absorbance of crocin at 440nm) and artificial coloring. The national standard divides the aroma intensity, aroma and color of saffron stigma based on the absorbance of crocin, crocin aldehyde and crocin at their respective maximum absorption wavelengths determined by ultraviolet spectrophotometry. The local standard divides it into three levels only based on the color and shape of the filaments. The group standards are divided into the group standards of the China Association of Traditional Chinese Medicine and the group standards of the China Association of Traditional Chinese Medicine. The China Association of Traditional Chinese Medicine mainly focuses on the appearance phenotypic indicators of saffron stigma. Imported saffron is divided into 4 grades and domestic saffron is divided into 3 grades. However, it has no requirements for the content of medicinal ingredients in saffron. The China Association of Traditional Chinese Medicine divides saffron stigma into 3 grades (special grade, first grade and second grade) based on the content of medicinal ingredients as the indicator of quality assessment, but this standard does not focus on the appearance standards such as the shape, color, texture and smell of the stigma. The existing saffron quality standards all grade saffron based on its physical and chemical indicators, and lack the evaluation of the biological efficacy of saffron.

[0004] The quality of traditional Chinese medicine is the guarantee of its clinical efficacy. Although advanced analytical techniques and methods can accurately determine the content of chemical components in traditional Chinese medicine, it is difficult to accurately determine the clinical efficacy of traditional Chinese medicine. In addition, the current analytical methods and technologies are relatively complex and cannot meet the requirements of quickly, accurately and conveniently determining the quality of traditional Chinese medicine. (III) Summary of the invention

[0005] The purpose of the present invention is to provide a saffron quality grade assessment card based on antithrombotic potency and its application, and proposes the concept of "fuzzy" evaluation of Chinese medicine quality, that is, combining biological potency with trait evaluation, quantifying trait evaluation, and compiling a saffron quality grade assessment card based on trait analysis, which provides a research idea and method for quickly and accurately evaluating the quality of Chinese medicine.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a saffron quality grade assessment card based on antithrombotic potency. The assessment card comprises a color band and a corresponding color grade, wherein the color grade comprises a primary and a secondary grade; the primary color band is a gradient from bright orange to flaming red, with 50≤L*≤53, 25≤a*≤35, and 20≤b*≤30; the secondary color band is a gradient from blush red to reddish brown, with 45≤L*<50, 15≤a*<25, and 10≤b*<20; L* represents the brightness of the color, with a positive number representing white and a negative number representing black; a* represents a red-green value, with a positive number representing red and a negative number representing green; and the b* value represents a yellow-blue value, with a positive number representing yellow and a negative number representing blue.

[0008] Furthermore, the primary color band is a gradient from bright orange, cinnabar, vermilion to fiery red.

[0009] Furthermore, the secondary color band is a gradient from rosy red, date red, deep red, crimson to reddish brown.

[0010] The present invention also provides an application of the saffron quality grade assessment card in rapidly assessing the antithrombotic potency grade of saffron, wherein the primary color band is used to assess saffron with a thrombus inhibition rate greater than 45%, and the secondary color band is used to assess saffron with a thrombus inhibition rate less than or equal to 45%.

[0011] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0012] (1) The present invention collects the color information of saffron, measures the thrombus inhibition rate of saffron, grades saffron according to the thrombus inhibition rate, associates the color information with the grade of saffron, and produces a saffron grade quality evaluation card, which can quickly, accurately and conveniently measure the quality of saffron.

[0013] (2) Compared with the currently commonly used physical and chemical detection methods, the present invention does not require a complicated sample pretreatment process and does not require the consumption of organic reagents. The detection method is green and environmentally friendly and has greater practicality and clinical significance. (IV) Description of the drawings

[0014] Figure 1, micrographs and bar graphs of cardiac staining intensity in the zebrafish anti-thrombotic model induced by phenylhydrazine; Control represents the blank control group; PHZ only represents the model group; ASP+PHZ represents the positive control group.

[0015] Figure 2 , Bar graph of heart staining intensity (n=10) after treatment with different batches of saffron extracts.

[0016] Figure 3 , Schematic diagram of thrombosis inhibition rate and grading results after treatment with different batches of saffron extracts.

[0017] Figure 4 , micrographs of cardiac staining intensity of different grades of saffron extracts; A represents the blank control group, B represents the first-grade saffron administration group; C represents the second-grade saffron administration group, and each group has 2 parallels.

[0018] Figure 5 , PCA analysis chart of saffron color information of different grades.

[0019] Figure 6 , saffron quality grade evaluation card. (V) Specific implementation methods

[0020] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0021] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0022] Embodiment 1,

[0023] 1. Sample color information collection

[0024] 50 batches of saffron samples were crushed and passed through No. 3 sieve (50 mesh). Put them into the powder box and compact them. Set the colorimeter (model CR8, Guangdong Sanenshi Intelligent Technology Co., Ltd.) with a wavelength range of 380-780nm, a scanning speed of 600nm / min, a slit width of 1nm, an illumination source of D65, and a field of view of 10°. Black and white plate calibration. Take an appropriate amount of saffron sample powder and spread it in a cuvette. Use the colorimeter to extract the color information of each batch of samples: L* (indicates the brightness of the color, positive numbers indicate whiteness, negative numbers indicate blackness), a* (indicates red-green value, positive numbers indicate redness, negative numbers indicate greenness), b* value (indicates yellow-blue value, positive numbers indicate yellowness, negative numbers indicate blueness), and calculate the chromaticity value according to formula 1. Repeat 3 times for each sample. Take the average value.

[0025] The results of the chromaticity values ​​of 50 batches of saffron samples are shown in Table 1. The color difference values ​​of different batches of saffron ΔE*ab>6, and there are obvious color differences that can be distinguished by the naked eye.

[0026] E*ab=(L 2 +a 2 +a 2 ) 1 / 2 Formula 1

[0027] Table 1 Chromaticity results of different batches of saffron

[0028]

[0029]

[0030] 2. Determination and classification of antithrombotic activity of samples

[0031] (1) Saffron Extract

[0032] Accurately weigh 50 batches of saffron powder after color information extraction in step 1, 50 mg for each batch, place in a 50 mL brown volumetric flask, add 40 mL of 50% ethanol aqueous solution by volume, place in an ice bath for ultrasonic treatment (power 300 W, frequency 50 kHz) for 30 minutes, bring to room temperature (25-30°C), add 50% ethanol aqueous solution by volume to dilute to the scale, shake well, filter, take the filtrate, rotary evaporate to less than 10 mL, transfer to a 10 mL volumetric flask, add purified water to dilute to the scale, and obtain an extract with a saffron extract concentration of 5 mg / mL.

[0033] (2) Establishment of zebrafish antithrombotic model and drug administration: The wells of a 6-well plate were divided into 53 groups, including a blank control group (Control), a model group (PHZ), a positive control group (ASP+PHZ), and a drug administration group (50 groups). 3 mL of embryo culture medium was added to each well, and 10 randomly selected 72 hpf zebrafish larvae with normal development were added to each well.

[0034] Preparation of embryo culture medium: weigh 294 mg of anhydrous calcium chloride, 133.3 mg of magnesium sulfate heptahydrate, 63 mg of sodium bicarbonate, and 5.5 mg of potassium chloride and dissolve them in 1 L of pure water, and adjust the pH to 6.5-8.5. All chemical reagents are analytical grade.

[0035] Before modeling, 20 μg / mL aspirin (ASP) was added to each well of the positive control group; 300 μg / mL saffron extracts of different batches in step (1) were added to each well of the drug-treated group and incubated for 24 hours for pre-protection; the blank control group and the model group were incubated normally for 24 hours. When the embryos developed to 96 hpf, the culture medium of each group was replaced with 3 mL of embryo culture medium containing 1 μM phenylhydrazine (PHZ) and the model was established for 12 hours.

[0036] (3) Zebrafish hemoglobin staining: Dissolve o-dianisidine in anhydrous ethanol, shake for 10 h, prepare a storage solution with a concentration of 1.4 mg / mL, and store at 4°C in the dark. Staining solution: 2 mL of o-dianisidine storage solution, 0.5 mL of 0.1 M sodium acetate aqueous solution, 2 mL of ultrapure water, and 0.1 mL of 30% hydrogen peroxide.

[0037] Remove the culture medium in the well plate in step (2) and replace it with the staining solution. Stain for 10 minutes at room temperature in the dark. After observing that the embryo is whitish as a whole and the heart is stained red, wash it three times with PBS. Fix it with 4% paraformaldehyde overnight and photograph the stained embryo under a microscope.

[0038] Microscopic photos of the blank control group, model group and positive control group are shown in Figure 1 The microscopic images were processed with Image J, and the stained area of ​​the heart was circled. The Image J software automatically analyzed and counted the staining intensity S. Compared with the blank control group, the staining intensity of zebrafish heart red blood cells in the model group was significantly reduced (P<0.01). Due to blood flow blockage in the zebrafish, the amount of blood returning to the heart was reduced, and the staining intensity of the heart area was reduced after staining, indicating that phenylhydrazine successfully induced thrombosis in zebrafish. After the positive control group was given aspirin, the staining intensity of the zebrafish heart was significantly improved.

[0039] The intensity of heart staining in each group is shown in Figure 2 , indicating that the zebrafish heart staining intensity was significantly improved after administration of aspirin and saffron extract.

[0040] The thrombus inhibition rate was calculated according to formula 2, where S represents the staining intensity.

[0041] Formula 2:

[0042] Thrombosis inhibition rate (%) = [S (药物干预组) -S (模型组) ] / [S (空白对照组) -S (模型组) ]×100%

[0043] The thrombus inhibition rate in the drug-treated group was Figure 3 The results showed that the inhibition rate of 50 batches of saffron on zebrafish thrombosis ranged from 11.11% to 86.54%, with a large difference.

[0044] (4) Classification

[0045] According to the difference in the antithrombotic activity of saffron, 50 batches of saffron samples were divided into two levels: S1, S2, S4, S13, S41-S50 had higher activity (thrombosis inhibition rate > 45%), which was the first level; the remaining batches had lower activity (thrombosis inhibition rate ≤ 45%), which was the second level. Figure 3Among them, the typical microscopic photos of the first and second levels in the blank control group and the drug-treated group are shown in Figure 4 .

[0046] 3. Saffron color information analysis and quality grade assessment card production

[0047] Using Origin 2024 software, the color information (L*, a*, b*) of step 1 corresponding to the first and second level saffron samples in step 2 was subjected to unsupervised principal component analysis (PCA analysis). The results are shown in Figure 5 , saffron medicinal materials can be roughly divided into two categories, which are concentrated in different positions of the quadrants, indicating that these two grades of saffron can be clearly distinguished based on the difference in color information.

[0048] According to the classification in step 2, the color of each batch of saffron was extracted by Photoshop software, and the powder color was fitted. The extracted color information (L*, a*, b* values) was detected by a colorimeter to further quantify the color of saffron samples of different grades, and a saffron grade evaluation color card was made. The color card consisted of 2 color levels and 2 corresponding color bands. The primary color band was a gradient from bright orange, cinnabar, vermilion to flaming red: 50≤L*≤53, 25≤a*≤35, 20≤b*≤30; the secondary color band was a gradient from blush red, jujube red, deep red, crimson to reddish brown: 45≤L*<50, 15≤a*<25, 10≤b*<20, such as Figure 6 The saffron quality grade rating card provides a new way to quickly evaluate the grade of saffron medicinal materials.

[0049] 4. Verification of Saffron Quality Grade Card

[0050] In order to verify the accuracy of the saffron quality assessment card, this study selected 5 batches of saffron of unknown grade and used the prepared saffron quality assessment card to perform color comparison and predict the saffron grade. The results are shown in Table 2.

[0051] The antithrombotic activity of saffron of unknown grade was determined according to the experimental operation in step 2, and the grade of saffron was preliminarily determined. The results are shown in the following two tables. The accuracy of distinguishing the actual value from the predicted value is 100%, indicating that the judgment of the quality grade of saffron based on color analysis is scientific.

[0052] Table 2 Saffron quality grade card verification results

[0053]

Claims

1. A saffron quality grade assessment card based on antithrombotic potency, characterized in that: The evaluation card includes a color band and a corresponding color grade, and the color grade includes primary and secondary grades; the primary color band is a gradient from bright orange to fiery red, 50≤L*≤53, 25≤a*≤35, 20≤b*≤30; the secondary color band is a gradient from rosy red to reddish brown, 45≤L*<50, 15≤a*<25, 10≤b*<20; L* represents the brightness of the color; a* represents the red-green value; and b* represents the yellow-blue value.

2. The saffron quality grade assessment card as claimed in claim 1, characterized in that The primary color band is a gradient from bright orange, cinnabar, vermilion to fiery red.

3. The saffron quality grade assessment card as claimed in claim 1, characterized in that, The secondary color band is a gradient from rosy red, date red, deep red, crimson to reddish brown.

4. Use of the saffron quality grade assessment card according to claim 1 in rapid assessment of the antithrombotic potency grade of saffron.

5. The use according to claim 4, characterized in that The primary color band is used to evaluate saffron with a thrombus inhibition rate of more than 45%, and the secondary color band is used to evaluate saffron with a thrombus inhibition rate of ≤45%.

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