CN113514595A - Rapid detection method for semi-quantitatively determining hydroxysafflor yellow A - Google Patents
Rapid detection method for semi-quantitatively determining hydroxysafflor yellow A Download PDFInfo
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- IAVUBSCVWHLRGE-UXEKTNMQSA-N (6e)-2,5-dihydroxy-6-[(e)-1-hydroxy-3-(4-hydroxyphenyl)prop-2-enylidene]-2,4-bis[(2s,3r,4r,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]cyclohex-4-ene-1,3-dione Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C(C(C(O)([C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C1=O)=O)=C(O)\C1=C(/O)\C=C\C1=CC=C(O)C=C1 IAVUBSCVWHLRGE-UXEKTNMQSA-N 0.000 title claims abstract description 38
- ZZMASNSDVDSYKO-UHFFFAOYSA-N hydroxysafflor yellow A Natural products OCC1OC(C(O)C(O)C1O)C2=C(O)C(O)(C3OC(CO)C(O)C(O)C3O)C(=O)C(=C2O)C(=O)C=Cc4ccc(O)cc4 ZZMASNSDVDSYKO-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 244000020518 Carthamus tinctorius Species 0.000 claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 36
- 239000012085 test solution Substances 0.000 claims abstract description 26
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- WIYUJTDPESFNJV-UHFFFAOYSA-N chloroform formic acid methanol hydrate Chemical compound O.OC.OC=O.ClC(Cl)Cl WIYUJTDPESFNJV-UHFFFAOYSA-N 0.000 claims abstract description 7
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 claims abstract description 4
- 235000003255 Carthamus tinctorius Nutrition 0.000 claims description 17
- 238000005303 weighing Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 5
- 210000001672 ovary Anatomy 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000007781 pre-processing Methods 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims description 2
- 238000005375 photometry Methods 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 230000004580 weight loss Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 4
- 241000208838 Asteraceae Species 0.000 abstract description 3
- 238000002137 ultrasound extraction Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- DYQVDISPPLTLLR-HJQYTNQXSA-N Carthamin Natural products CC[C@H]1O[C@H]([C@H](O)[C@@H](O)[C@@H]1O)[C@]2(O)C(=C(C=C/3C(=O)C(=C(O)[C@](O)([C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)C3=O)C(=O)C=Cc5ccc(O)cc5)C(=O)C(=C2O)C(=O)C=Cc6ccc(O)cc6)O DYQVDISPPLTLLR-HJQYTNQXSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- -1 hydroxyl carthamin Chemical compound 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- WLYGSPLCNKYESI-RSUQVHIMSA-N Carthamin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1[C@@]1(O)C(O)=C(C(=O)\C=C\C=2C=CC(O)=CC=2)C(=O)C(\C=C\2C([C@](O)([C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(O)=C(C(=O)\C=C\C=3C=CC(O)=CC=3)C/2=O)=O)=C1O WLYGSPLCNKYESI-RSUQVHIMSA-N 0.000 description 1
- 241000208809 Carthamus Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- NAYRZLVSOLIQSH-UHFFFAOYSA-N acetonitrile;methanol;phosphoric acid Chemical compound OC.CC#N.OP(O)(O)=O NAYRZLVSOLIQSH-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- KINGXFAMZNIVNL-SXQDSXCISA-N safflor yellow A Natural products OC[C@@H]1O[C@H]2[C@H](OC3=C2C(=O)C(=C(O)C=Cc4ccc(O)cc4)C(=O)[C@]3(O)[C@@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@@H](O)[C@H]1O KINGXFAMZNIVNL-SXQDSXCISA-N 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- Life Sciences & Earth Sciences (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a rapid detection method for semi-quantitatively determining hydroxysafflor yellow A, and relates to the technical field of detection of hydroxysafflor yellow A components of Compositae safflor. The method is characterized in that a safflower plant sample is dried and then crushed into fine powder, acetone ultrasonic extraction is carried out to prepare a test solution, the test solution and a contrast solution are subjected to sample application, chloroform-formic acid-water-methanol is used as a developing agent for development, a sulfuric acid ethanol solution with the concentration of 10% is used as a color developing agent for color development, and whether the content of hydroxysafflor yellow A in the safflower plant sample corresponding to the test solution meets the medicinal standard or not is rapidly judged by observing the color difference between the test solution and the contrast solution.
Description
Technical Field
The invention relates to the technical field of detection of a component A of hydroxysafflor yellow A of Compositae safflower, in particular to a rapid detection method for semi-quantitatively determining hydroxysafflor yellow A.
Background
Safflower (Carthamus tinctorius L.) is an annual herbaceous plant of Carthamus of Compositae (Compositae), is a drug collected in the 'Chinese pharmacopoeia' 2020 edition, belongs to a common traditional Chinese medicine, and has the effects of promoting blood circulation to remove blood stasis, and stimulating the menstrual flow to relieve pain. One of the major pharmaceutical active ingredients of safflower is Hydroxysafflor yellow a (HSYA), which is widely used for treating blood vessel occlusion, angina pectoris, coronary heart disease, etc. The safflower germplasm resource is a precious material for researching and utilizing medicinal safflower and is a material basis for cultivating new varieties of high-yield and high-quality safflower. A certain unit in Yunnan has already preserved more than 4700 safflower germplasm resources all over the world, and the quantity is still increasing. The medicinal standard of the safflower germplasm resources is that the content of hydroxysafflor yellow A (C27H32O16) is not less than 1.0 percent, so that the hydroxysafflor yellow A in various safflower germplasm resources is required to be measured. At present, the content of the hydroxyl carthamin yellow A (HSYA) is determined according to the high performance liquid chromatography (general rule 0512) of Chinese pharmacopoeia, concretely, safflower is ground, sieved and precisely weighed, methanol is added for weighing, ultrasonic treatment and filtration are carried out, filtrate is taken and put on a computer, the high performance liquid chromatography is adopted, octadecylsilane chemically bonded silica is used as a stationary phase, methanol-acetonitrile-phosphoric acid is used as a mobile phase, the peak area is determined according to the detection wavelength, and the weight of the hydroxyl carthamin yellow A is obtained by calculation. The detection method is not satisfactory when used for screening medical resources in large quantities, firstly, a special instrument needs to be purchased, the instrument is expensive and can be used after being trained, and secondly, the analysis operation is complicated, so that the detection time is long, the cost is high, and the popularization and the application in basic units are difficult. Therefore, a method for rapidly detecting whether the content of the medicinal component hydroxy safflower yellow A in the safflower germplasm resources is more than 1.0 percent is urgently needed to be developed so as to rapidly screen out germplasm resources meeting medicinal standards in laboratories and basic units.
Disclosure of Invention
The invention aims to provide a rapid detection method for semi-quantitatively determining hydroxysafflor yellow A, which solves the problems of expensive instruments and long detection time and high cost caused by complicated operation in the existing determination method.
In order to solve the technical problems, the invention adopts the following technical scheme: a rapid detection method for semi-quantitatively determining hydroxysafflor yellow A is characterized by comprising the following steps:
s1, preprocessing a safflower plant, drying for 4-5 hours at 100-105 ℃, crushing and screening to obtain powder;
s2, placing the powder in a centrifugal tube, adding an acetone solution, weighing, performing ultrasonic treatment, centrifuging, taking supernatant as a test solution, and taking a hydroxysafflor yellow A standard solution with a constant volume and a concentration of 1.0% by taking an 80% acetone solution as a solvent as a control solution;
s3, spotting a sample on the same thin-layer plate by using the test solution and the control solution, then using chloroform-formic acid-water-methanol as a developing agent, and taking out and airing after developing, wherein the developing height is 4-5 cm;
s4, spraying a sulfuric acid ethanol solution with the concentration of 10% as a color developing agent on the thin-layer plate, and heating until spots are clear;
s5, comparing R in the hydroxysafflor yellow A in the test solution and the reference solutionfAnd (3) when spots with the same value appear at the positions corresponding to the chromatograms of the control solutions and show the same color, judging according to the color depth of the spots: when the color of the spot is obviously darker than that of the control solution, judging that the content of hydroxysafflor yellow A in the safflower plant corresponding to the test solution is higher than 1.0 percent; when the color of the spot is obviously lighter than that of the contrast solution, judging that the content of hydroxysafflor yellow A in the safflower plant corresponding to the test solution is lower than 1.0 percent; when the color of the spot is obviously close to that of the control solution, the content of hydroxysafflor yellow A in the safflower plant corresponding to the test solution is judged to be approximate to 1.0 percent.
The further technical scheme is that the pretreatment in the step S1 is specifically to select a dry tubular flower of the safflower plant without an ovary, remove leaves, the ovary, bracts, seeds and worm eggs in safflower petals, ensure that the content of impurities is not more than 2.0 percent, and ensure that the water content is less than 13 percent after drying.
The further technical scheme is that the safflower plants dried in the step S1 are cooled for 30min, the total gray scale of the safflower plants is required to be not more than 15.0%, the acid insoluble ash content is required to be not more than 5.0%, the absorbance of the ultraviolet-visible light spectrophotometry at the wavelength of 518nm is required to be not less than 0.2, and the safflower plants are crushed and sieved by 0.425mm to obtain powder.
The further technical scheme is that the specific steps of preparing the test solution in the step S2 are that about 0.1g of the powder is taken firstly, precisely weighed and placed in a 2ml centrifuge tube with a cover, 1ml of 80% acetone solution is precisely added, the weighed weight is subjected to ultrasonic treatment at 30 ℃ for 30 minutes, the mixture is cooled and weighed again, the 80% acetone solution is used for complementing the lost weight, the centrifugation is carried out for 5-10 minutes at 16000r/min at room temperature, and after the standing is carried out for 1 hour, the supernatant is taken as the test solution.
The further technical scheme is that 1ul of test solution and 1ul of control solution are respectively taken in the step S3, and the mass ratio of chloroform-formic acid-water-methanol in the developing agent is 7:2:3: 0.4.
The further technical proposal is that the heating temperature in the step S4 is 105 ℃.
Compared with the prior art, the invention has the beneficial effects that: the method has the characteristics of quickness, simplicity, easiness in operation and low detection cost, can be used for rapidly screening safflower germplasm resources in laboratories and basic units, is easy to master and popularize, and has a good application prospect.
Drawings
FIG. 1 is a flow chart of the present invention.
FIG. 2 is a graph comparing the results of detection in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
S1, selecting dry tubular flowers of a safflower plant (Yunhuaqi) without ovaries, removing leaves, ovaries, bracts, seeds, ova and the like in safflower petals, and ensuring that the content of impurities is not more than 2.0%. Drying the treated safflower plant for 4 hours at 100-105 ℃, sealing, cooling for 30 minutes, weighing until the water content is not more than 13%, requiring that the total gray scale is not more than 15.0%, the acid insoluble ash content is not more than 5.0%, and the absorbance of the ultraviolet-visible light spectrophotometry at the wavelength of 518nm is not less than 0.2, and sieving by 0.425mm after crushing to obtain powder.
S2, firstly taking 0.1g of the powder, precisely weighing, placing the powder in a 2ml centrifuge tube with a cover, precisely adding 1ml of 80% acetone solution, weighing the powder, carrying out ultrasonic treatment at 30 ℃ for 30 minutes, cooling the powder, weighing the powder again, complementing the lost weight with 80% acetone solution, centrifuging the powder for 5-10 min at 16000r/min at room temperature, standing the powder for 1h, and taking supernatant as a test solution. And taking a hydroxysafflor yellow A standard solution with the volume fixed and the concentration of 1.0 percent by taking an 80 percent acetone solution as a solvent as a control solution.
S3, taking 1ul of each of the test solution and the control solution, spotting a sample on the same thin-layer plate, taking chloroform-formic acid-water-methanol as a developing agent, taking the chloroform-formic acid-water-methanol as a developing agent according to the mass ratio of 7:2:3:0.4, and taking out and airing the developing agent after developing, wherein the developing height is 4-5 cm.
S4, spraying a sulfuric acid ethanol solution with the concentration of 10% as a color developing agent on the thin-layer plate, and heating at 105 ℃ until spots are clear.
S5, comparing R in the hydroxysafflor yellow A in the test solution and the reference solutionfWhen spots having the same color appear at positions corresponding to the color spectrum of the control solution, the color of the spots is judged according to the shade of the color. The detection result is shown in fig. 1, and if the color of the spot in the test solution is obviously darker than that of the control solution, the content of hydroxysafflor yellow A in the safflower plant is judged to be higher than 1.0%.
38 parts of plant samples of different safflower varieties are taken, the HSYA rapid semi-quantitative detection method is adopted for operation and judgment, and the judgment result is compared with the quantitative analysis result of an HPLC instrument, so that the judgment result accuracy is 94.73% (shown in Table 1).
TABLE 1 semi-definite determination results of HSYA content of plant samples of different safflower species
28 parts of different plant samples of the same safflower variety are taken, the HSYA rapid semi-quantitative detection method is adopted for operation and judgment, and the judgment result is compared with the quantitative analysis result of an HPLC instrument, so that the judgment result accuracy is 92.86% (shown in Table 2).
TABLE 2 semi-definite results of HSYA content determination of different plant samples of the same safflower species
As shown in tables 1 and 2, the HSYA rapid semi-quantitative detection method disclosed by the invention is high in accuracy.
Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts or arrangements of the subject combination arrangement within the scope of the disclosure, the drawings and the appended claims. In addition to variations and modifications in the component parts or arrangements, other uses will also be apparent to those skilled in the art.
Claims (6)
1. A rapid detection method for semi-quantitatively determining hydroxysafflor yellow A is characterized by comprising the following steps:
s1, preprocessing a safflower plant, drying for 4-5 hours at 100-105 ℃, crushing and screening to obtain powder;
s2, placing the powder into a centrifugal tube, adding 80% acetone solution, weighing, performing ultrasonic treatment, centrifuging, taking supernatant as a test solution, and taking a hydroxysafflor yellow A standard solution with a constant volume and a concentration of 1.0% by taking the 80% acetone solution as a solvent as a control solution;
s3, spotting a sample on the same thin-layer plate by using the test solution and the control solution, then using chloroform-formic acid-water-methanol as a developing agent, and taking out and airing after developing, wherein the developing height is 4-5 cm;
s4, spraying a sulfuric acid ethanol solution with the concentration of 10% as a color developing agent on the thin-layer plate, and heating until spots are clear;
s5, comparing R in the hydroxysafflor yellow A in the test solution and the reference solutionfAnd (3) when spots with the same value appear at the positions corresponding to the chromatograms of the control solutions and show the same color, judging according to the color depth of the spots: when the color of the spot is obviously darker than that of the control solution, judging that the content of hydroxysafflor yellow A in the safflower plant corresponding to the test solution is higher than 1.0 percent; when the color of the spot is obviously lighter than that of the contrast solution, judging that the content of hydroxysafflor yellow A in the safflower plant corresponding to the test solution is lower than 1.0 percent; when the color of the spot is obviously close to that of the control solution, the content of hydroxysafflor yellow A in the safflower plant corresponding to the test solution is judged to be approximate to 1.0 percent.
2. The rapid detection method for semi-quantitatively determining hydroxysafflor yellow A according to claim 1, wherein the rapid detection method comprises the following steps: the pretreatment in the step S1 is to select dry tubular flowers of the safflower plants without ovaries, remove leaves, bracts, seeds and ova in safflower petals, ensure that the content of impurities is not more than 2.0 percent, and dry the flowers until the water content is less than 13 percent.
3. The rapid detection method for semi-quantitatively determining hydroxysafflor yellow A according to claim 1, wherein the rapid detection method comprises the following steps: cooling the dried safflower plant in the step S1 for 30min, wherein the total gray scale is required to be not more than 15.0%, the acid insoluble ash content is not more than 5.0%, the absorbance of the safflower plant at 518nm by using an ultraviolet-visible light photometry is not less than 0.2, and sieving the safflower plant by using a 0.425mm sieve after crushing to obtain powder.
4. The rapid detection method for semi-quantitatively determining hydroxysafflor yellow A according to claim 1, wherein the rapid detection method comprises the following steps: the specific steps of preparing the test solution in the step S2 are that about 0.1g of the powder of the product is taken firstly, precisely weighed, placed in a 2ml centrifuge tube with a cover, precisely added with 1ml of 80% acetone solution, weighed, ultrasonically treated for 30 minutes at 30 ℃, cooled and weighed again, the weight loss is compensated by 80% acetone solution, centrifuged for 5-10 min at 16000r/min at room temperature, and after standing for 1h, the supernatant is taken as the test solution.
5. The rapid detection method for semi-quantitatively determining hydroxysafflor yellow A according to claim 1, wherein the rapid detection method comprises the following steps: in the step S3, 1ul of test solution and 1ul of control solution are respectively taken, and the mass ratio of chloroform-formic acid-water-methanol in the developing solvent is 7:2:3: 0.4.
6. The rapid detection method for semi-quantitatively determining hydroxysafflor yellow A according to claim 1, wherein the rapid detection method comprises the following steps: the heating temperature in the step S4 was 105 ℃.
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