CN117007584A - Method for detecting content of triterpene compounds in cactus - Google Patents
Method for detecting content of triterpene compounds in cactus Download PDFInfo
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- -1 triterpene compounds Chemical class 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 68
- 241000219357 Cactaceae Species 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 239000012488 sample solution Substances 0.000 claims abstract description 38
- 238000012360 testing method Methods 0.000 claims abstract description 31
- 239000000523 sample Substances 0.000 claims abstract description 29
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000010438 heat treatment Methods 0.000 claims abstract description 23
- 238000002360 preparation method Methods 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 239000007800 oxidant agent Substances 0.000 claims abstract description 20
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000013558 reference substance Substances 0.000 claims abstract description 15
- 238000010992 reflux Methods 0.000 claims abstract description 15
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 claims abstract description 15
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000012141 vanillin Nutrition 0.000 claims abstract description 15
- 229960000583 acetic acid Drugs 0.000 claims abstract description 13
- 238000001816 cooling Methods 0.000 claims abstract description 13
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 13
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000002798 spectrophotometry method Methods 0.000 claims abstract description 8
- 150000003839 salts Chemical class 0.000 claims abstract description 4
- 238000000605 extraction Methods 0.000 claims description 36
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 claims description 10
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 claims description 10
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 claims description 10
- 229940100243 oleanolic acid Drugs 0.000 claims description 10
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 6
- 230000001590 oxidative effect Effects 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- 125000000955 oleanolic acid group Chemical group 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 27
- 230000000052 comparative effect Effects 0.000 description 18
- 238000002835 absorbance Methods 0.000 description 17
- 235000013399 edible fruits Nutrition 0.000 description 17
- 239000000843 powder Substances 0.000 description 15
- 239000000203 mixture Substances 0.000 description 14
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Substances OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 239000012086 standard solution Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 238000009833 condensation Methods 0.000 description 5
- 230000005494 condensation Effects 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 238000012417 linear regression Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000000874 microwave-assisted extraction Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 240000001155 Opuntia dillenii Species 0.000 description 3
- 235000006544 Opuntia dillenii Nutrition 0.000 description 3
- 238000004880 explosion Methods 0.000 description 3
- 239000012088 reference solution Substances 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 238000011056 performance test Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 150000003648 triterpenes Chemical class 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 description 1
- 235000009434 Actinidia chinensis Nutrition 0.000 description 1
- 244000298697 Actinidia deliciosa Species 0.000 description 1
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000985694 Polypodiopsida Species 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000002249 anxiolytic agent Substances 0.000 description 1
- 230000000949 anxiolytic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013572 fruit purees Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000005839 oxidative dehydrogenation reaction Methods 0.000 description 1
- VKJKEPKFPUWCAS-UHFFFAOYSA-M potassium chlorate Chemical compound [K+].[O-]Cl(=O)=O VKJKEPKFPUWCAS-UHFFFAOYSA-M 0.000 description 1
- SATVIFGJTRRDQU-UHFFFAOYSA-N potassium hypochlorite Chemical compound [K+].Cl[O-] SATVIFGJTRRDQU-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 description 1
- 229940096998 ursolic acid Drugs 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/44—Sample treatment involving radiation, e.g. heat
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N2021/786—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour with auxiliary heating for reaction
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- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The application relates to the technical field of compound detection, and particularly discloses a method for detecting the content of triterpene compounds in cactus. The method for detecting the triterpene compounds in the cactus comprises the steps of preparing a reference substance solution, preparing a test sample solution and detecting by an ultraviolet spectrophotometry; sample solution preparation: dissolving a sample to be detected, heating and refluxing for 20-40min, and directly radiating and extracting for 10-30s under 500-900W microwave power without cooling to obtain an extracting solution; centrifuging and separating the extracting solution, taking supernatant, adding 5-7 mass% vanillin glacial acetic acid solution and strong oxidizing agent into the supernatant, heating, cooling and fixing the volume to obtain a sample solution; the strong oxidizing agent is concentrated sulfuric acid, hypochlorous acid and salts thereof. The method for detecting the content of the triterpene compounds in the cactus has high accuracy and good safety, and can be popularized and used in a large scale.
Description
Technical Field
The application relates to the technical field of compound detection, in particular to a method for detecting the content of triterpene compounds in cactus.
Background
Triterpenes are important active natural products widely existing in fungi, ferns, single-leaf and double-leaf plants, and have tranquilizing, hypnotizing, anxiolytic, analgesic and antiinflammatory effects. Thus, triterpenes have been the subject of hot research in medicine.
At present, the method for extracting triterpene compounds from plants mainly comprises the following steps: organic solvent extraction method and supercritical fluid extractionMethods and ultrasonic extraction methods. The organic solvent extraction method is the earliest extraction method, and the method needs to consume a large amount of organic solvent during extraction, has poor extraction selectivity, and obtains an extract with more impurities and residual organic solvent. Supercritical fluid extraction is carried out by supercritical fluid, e.g. CO 2 The method has high extraction efficiency and no pollution to the environment, but the required equipment is expensive, and the equipment is difficult to clean, so that the method is difficult to popularize and use on a large scale; the ultrasonic extraction method has high extraction speed and high extraction efficiency, but if the extraction time is too long, the structure of the sample is affected.
At present, most of the triterpene compounds are detected by using ursolic acid or oleanolic acid as a reference substance, developing by using a vanillin-perchloric acid color development method, and then measuring the content of the triterpene compounds by using an ultraviolet spectrophotometry. However, perchloric acid is used in the detection method, and is a dangerous chemical easy to explode, and after being dissolved in water, the perchloric acid reacts with the water to release a large amount of hydrogen ions, so that explosion is easy to occur, and the danger coefficient is high; and perchloric acid is expensive and purchasing is severely controlled, so that the detection method of the vanillin-perchloric acid color development method is difficult to realize large-scale popularization and use.
Disclosure of Invention
In order to overcome the defects in the prior art, the application provides the method for detecting the content of the triterpene compounds in the cactus, which has high accuracy, good safety and large-scale use.
The application provides a method for detecting the content of triterpene compounds in cactus, which adopts the following technical scheme:
a method for detecting the content of triterpene compounds in cactus comprises the steps of preparing a reference substance solution, preparing a test substance solution and detecting by an ultraviolet spectrophotometry;
sample solution preparation: dissolving a cactus sample to be measured, heating and refluxing for 20-40min, then directly placing the cactus sample into microwave equipment without cooling, and performing irradiation extraction for 10-30s under the microwave power of 500-900W to obtain an extracting solution; centrifuging and separating the extracting solution, taking supernatant, adding 5-7 mass% vanillin glacial acetic acid solution and strong oxidizing agent into the supernatant, heating, cooling and fixing the volume to obtain a sample solution;
the strong oxidizing agent is concentrated sulfuric acid, hypochlorous acid and salts thereof.
In the preparation step of the sample of the method for detecting the content of the triterpene compounds, the method for extracting the triterpene compounds by heating reflux-microwave synergy is adopted, the extraction method can effectively extract the triterpene compounds in the sample to be detected of the cactus, and then the content detection of the triterpene compounds in the cactus can be completed by detecting the triterpene compounds by an ultraviolet spectrophotometer. Compared with the extraction method in the related technology, the heating reflux-microwave synergistic method has the advantages of high extraction efficiency, less solvent use, good selectivity and simple equipment, thereby being capable of realizing large-scale popularization and use. In addition, in the preparation step of the sample solution, concentrated sulfuric acid, hypochlorous acid or hypochlorite (such as potassium hypochlorite) is used as a strong oxidizing agent, so that the explosion risk of the prior art adopting perchloric acid as the strong oxidizing agent is effectively avoided, and the sources of the concentrated sulfuric acid, potassium chlorate or hypochlorous acid are relatively easy and low in price. Therefore, the method for detecting the content of the triterpene compound provided by the application can be popularized and used on a large scale.
In the application, most of the triterpene compounds in the cactus are separated from impurities by heating and refluxing, and then the triterpene compounds remained in the cactus are further dissolved out by treatment under 500-900W for 10-30 s. The microwave power and the treatment time can influence the structure and dissolution rate of the triterpene compounds, so the application controls the microwave treatment conditions within the range, not only can extract the triterpene compounds of the cactus to the maximum extent, but also can ensure that the structure of the triterpene compounds is not influenced. Therefore, the preparation steps of the sample solution provided by the application lay a foundation for the accurate detection of the content of the triterpene compounds in the cactus.
In some embodiments, the heating reflux time may be 15-30min or 30-45min.
In a specific embodiment, the heating reflux time may also be 15min, 30min or 45min.
In some embodiments, the microwave power may be 500-600W, 500-700W, 500-850W, 600-700W, 600-850W, 600-900W, 700-850W, 700-900W, or 850-900W.
In a specific embodiment, the microwave power may also be 500W, 600W, 700W, 850W or 900s.
In some embodiments, the irradiation extraction time may be 10-15s, 10-20s, 15-30s, or 20-30s.
In a specific embodiment, the irradiation extraction time may be 10s, 15s, 20s or 30s.
Preferably, the microwave power is 600-850W, and the irradiation extraction time is 10-20s.
According to the application, the microwave power and the irradiation extraction time are further controlled within the ranges, the extraction efficiency of the triterpene compounds of the sample to be detected is higher, the structure of the triterpene compounds is more complete, and the obtained detection result of the triterpene compound content in the sample to be detected is more accurate.
Preferably, the solvent used for dissolving the cactus sample to be measured is 80-95 mass% ethanol.
Preferably, the strong oxidizing agent is an agent having a concentration of not less than 85 mass%.
In the application, in the presence of a strong oxidizing reagent, the triterpene compound is subjected to oxidative dehydrogenation to form a double bond, the double bond compound is subjected to condensation color development with vanillin in a reaction system, and the content of the color development product is detected by an ultraviolet spectrophotometer, so that the content of the triterpene compound can be obtained. The experiment researches show that the concentration of the strong oxidizing reagent can influence the success rate of the condensation reaction and further influence the accuracy of the detection result, so that the concentration of the strong oxidizing reagent is controlled to be more than 85 mass percent, the triterpene compound can be fully oxidized, and the obtained detection result has higher accuracy.
In a specific embodiment, the strong oxidizing agent may also be 75 mass% concentrated sulfuric acid, 85 mass% concentrated sulfuric acid, 98 mass% concentrated sulfuric acid, or 90 mass% hypochlorous acid.
Preferably, after 5-7 mass% vanillin glacial acetic acid solution and strong oxidizing agent are added into the supernatant, the heating temperature is 65-75 ℃ and the heating time is 10-45min.
The application controls the temperature and time of the condensation color reaction within the above range, and can ensure that the condensation color reaction is smoothly carried out.
In a specific embodiment, the volumetric ratio of vanillin glacial acetic acid solution to supernatant is 2:1, a step of; the volume ratio of the strong oxidizing agent to the supernatant is 8:1.
preferably, the solvent used for the constant volume is ethyl acetate, n-heptane, n-hexane or diethyl ether.
Preferably, in the ultraviolet spectrophotometry detection step, the detection wavelength is 530-555nm.
Preferably, the reference substance is oleanolic acid reference substance.
Preferably, the quantitative limit of the detection method is 0.00385mg/mL.
Cactus, also called radix et caulis Opuntiae Dillenii, kiwi berry, etc., is a succulent plant of genus Cactus of family Cactaceae, the cactus contains various active ingredients, and the triterpene compound is one of the active ingredients. The method for detecting the content of the triterpene compounds is suitable for extracting the triterpene compounds in the cactus.
In the application, when the triterpene compounds in the cactus are detected, the cactus is required to be pretreated to obtain a sample to be detected of the cactus fruit paste, the cactus fruit residue or the cactus fruit powder.
The cactus fruit puree is a muddy substance obtained by cutting, crushing and stirring cactus pulp; the cactus pomace is residues obtained after juice extraction of cactus pulp. The cactus fruit powder is powdery material obtained by squeezing pulp of peeled cactus and drying juice;
in summary, the application has the following beneficial effects:
1. the application provides a method for detecting the content of triterpene compounds in cactus, which comprises the steps of firstly extracting the triterpene compounds in the cactus by adopting a heating reflux-microwave synergistic method, controlling the heating reflux time to be 20-40min, and controlling the microwave power and the irradiation extraction time to be in the following ranges: the triterpene compounds in the sample are fully extracted by irradiation extraction for 10-30s under the microwave power of 500-900W. The extraction method has simple operation and high extraction efficiency, and does not need a large amount of organic solvents and expensive equipment, so that the method can be popularized and used on a large scale.
2. The application further controls the microwave power and the irradiation extraction time in the preparation step of the sample solution within the following range: the method for detecting the content of the triterpene compound is high in accuracy and better in repeatability, and the radiation extraction is carried out for 10-20s under the microwave power of 600-850W.
3. The detection method provided by the application uses concentrated sulfuric acid, hypochlorous acid and salts thereof as strong oxidizing agents to carry out condensation color reaction, so that the safety of the detection method is improved and the detection cost is reduced while the reaction efficiency is ensured.
4. The concentration of the strong oxidizing reagent is controlled to be more than 85 mass percent, so that the triterpene compound can be fully oxidized, and the accuracy of the obtained detection result is higher.
Detailed Description
The application provides a method for detecting the content of triterpene compounds in cactus, which specifically comprises the following steps:
(1) Preparing a reference substance solution: precisely weighing oleanolic acid reference substance 5mg, dissolving with methanol, and fixing volume in 25mL volumetric flask to obtain reference substance solution with concentration of 0.1834 mg/mL.
(2) Drawing a standard curve: precisely sucking 0mL, 0.1mL, 0.3mL, 0.5mL, 0.7mL and 0.9mL of reference substance solution, respectively placing into 6 15mL test tubes with plugs, volatilizing, and cooling; then, 0.2mL of each 5 mass% vanillin glacial acetic acid solution and 0.8mL of each 98 mass% concentrated sulfuric acid are respectively added into the test tube with the plug, the mixture is uniformly shaken, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, 4mL of ethyl acetate is precisely added, and the mixture is uniformly shaken to obtain a series of standard solutions. The absorbance was measured at a wavelength of 530-555nm. A solution in a volumetric flask containing 0mL of standard solution was used as a reference solution. And drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate.
(3) Sample solution preparation: 1.0g of a cactus sample to be measured is taken, precisely weighed, placed into a conical flask, added with 25mL of 80-95 mass% ethanol for dissolution, and heated and refluxed for 20-40min by a heating plate; then pouring the reaction liquid into a test tube with a plug immediately, placing the test tube into a microwave extraction instrument, and performing irradiation extraction for 10-30s under the microwave power of 500-900W to obtain an extraction liquid; centrifuging and separating the extracting solution to obtain 25mL of supernatant; taking 0.1mL of supernatant, placing the supernatant into a 15mL test tube with a plug, volatilizing, and cooling; then 0.2mL of 5 mass percent vanillin glacial acetic acid solution and 0.8mL of 98 mass percent concentrated sulfuric acid are added into the test tube with the plug, the mixture is shaken uniformly, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, and 4mL of ethyl acetate is precisely added, and the mixture is shaken uniformly to obtain a sample solution.
(4) Ultraviolet spectrophotometry detection: the sample solution was transferred to an ultraviolet-visible spectrophotometer and absorbance was measured at a wavelength of 530-555nm. And (3) according to the absorbance of the sample solution, detecting the corresponding concentration on a standard curve, and calculating the content of the triterpene compound in the sample to be detected.
The method for calculating the content of the triterpene compounds comprises the following steps:
wherein X is the content of triterpene compounds in the cactus sample to be detected, and g/100g; a is the absorbance value of the sample solution; b is the intercept of the standard curve regression equation; n is dilution multiple; a is the slope of a standard curve regression equation; m is the sample weighing amount and g; v is the volume of the sample solution, mL; 100. 1000 is a conversion coefficient.
In the application, the lot number of the oleanolic acid reference substance is 110709-202109, and the purity is: 95.8%; vanillin was purchased from Shanghai Yi En chemical technologies limited; the cactus sample to be measured is cactus fruit powder; the model of the microwave extraction instrument is AL-IMC1; the remaining materials, reagents, solvents, and the like are commercially available.
The present application will be described in further detail with reference to examples and performance test.
Example 1
Embodiment 1 provides a method for detecting the content of triterpene compounds, which specifically comprises the following steps:
(1) Preparing a reference substance solution: precisely weighing oleanolic acid reference substance 5mg, dissolving with methanol, and fixing volume in 25mL volumetric flask to obtain reference substance solution with concentration of 0.1834 mg/mL.
(2) Drawing a standard curve: precisely sucking 0mL, 0.1mL, 0.3mL, 0.5mL, 0.7mL and 0.9mL of reference substance solution, respectively placing into 6 15mL test tubes with plugs, volatilizing, and cooling; then, 0.2mL of each 5 mass% vanillin glacial acetic acid solution and 0.8mL of each 98 mass% concentrated sulfuric acid are respectively added into the test tube with the plug, the mixture is uniformly shaken, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, 4mL of ethyl acetate is precisely added, and the mixture is uniformly shaken to obtain a series of standard solutions. Absorbance was measured at a wavelength of 546 nm. A solution in a volumetric flask containing 0mL of standard solution was used as a reference solution. And drawing a standard curve by taking absorbance as an ordinate and concentration as an abscissa.
(3) Sample solution preparation: 1.0g of cactus fruit powder is taken, precisely weighed, placed into a conical flask, added with 25mL of 95 mass percent ethanol for dissolution, and heated and refluxed for 30min by a heating plate; then the reaction liquid is immediately poured into a test tube with a plug and placed into a microwave extraction instrument to be irradiated and extracted for 15s under the microwave power of 750W, so as to obtain an extracting liquid; centrifuging and separating the extracting solution to obtain 25mL of supernatant; taking 0.1mL of supernatant, placing in a 15mL test tube with a plug, volatilizing, and cooling; then 0.2mL of 5 mass percent vanillin glacial acetic acid solution and 0.8mL of 98 mass percent concentrated sulfuric acid are added into the test tube with the plug, the mixture is shaken uniformly, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, and 4mL of ethyl acetate is precisely added, and the mixture is shaken uniformly to obtain a sample solution.
(4) Ultraviolet spectrophotometry detection: the sample solution was transferred to an ultraviolet-visible spectrophotometer, and absorbance was measured at a wavelength of 546 nm. And (3) according to the absorbance of the sample solution, detecting the corresponding concentration on a standard curve, and calculating the content of the triterpene compound in the sample to be detected.
Linear relationship investigation
6 gradient standard solutions (each concentration is shown in table 1) were prepared according to the method of step (1) -step (2) in example 1, absorbance of each standard solution at 546nm wavelength was detected, and then linear regression was performed with the concentration of the reference solution as abscissa and absorbance (y) as ordinate to obtain a linear regression equation, and specific results are shown in table 1.
TABLE 1 Linear relationship detection results and Linear regression equations
From the results in Table 1, it can be seen that the fitting degree R of the linear regression equation 2 The ratio of the oleanolic acid to the water is 0.9975, which shows that the oleanolic acid has good linear relationship in the range of 0.0038519 mg/mL-0.034667 mg/mL.
Precision test
Taking a standard solution with the concentration of 0.01100mg/L, detecting the absorbance at the wavelength of 546nm according to the method of the step (4), detecting for 6 times in parallel, and recording the absorbance. The results of the precision test are shown in Table 2.
TABLE 2 results of precision test
From the above results, the RSD value of the 6 detection results was only 1.88%. Therefore, the instrument precision is good.
Stability test
Taking 1.0g of a sample to be measured, precisely weighing, and preparing a sample solution according to the method of the step (3) in the example 1; and detecting the absorbance of the sample solution at 546nm wavelength for 0min, 5min, 10min, 15min, 20min, 25min and 30min according to the method of the step (4). The stability test results are shown in Table 3.
TABLE 3 stability test results
From the above results, the absorbance of the sample solution was substantially the same at 0min, 5min, 10min, 15min, 20min, 25min, and 30min, and the RSD value was only 1.1%, indicating that the stability of the sample solution was good.
Repeatability test
Precisely weighing 6 parts (1.0 g each) of a sample to be measured, and preparing a sample solution according to the method of the step (3) in the example 1; and detecting the absorbance at 546nm according to the method of the step (4). The repeatability test results are shown in Table 4
TABLE 4 repeatability test results
According to the results, the average value of the content of the triterpene compounds in the detection sample is 0.85g/100g, and the RSD is 0.88% (< 2%), which shows that the repeatability of the method for detecting the content of the triterpene compounds provided by the application is good.
Label recovery test
9 parts (0.5 g each) of the sample to be measured are precisely weighed and divided into 3 groups (3 parts each). Oleanolic acid control (the addition amounts are shown in table 5) was added at 3 levels of 80%, 100% and 120% of the known triterpene compound content, respectively, and then a test solution was prepared in the same manner as in step (3) of example 1; and (3) respectively detecting the absorbance at 546nm wavelength according to the method of the step (4), calculating the content of the determined triterpene compound according to a standard curve, and calculating the standard recovery rate, wherein the standard recovery rate is calculated according to the following method. The specific results are shown in Table 5.
Standard recovery = (measurement value-triterpene compound content in sample)/(oleanolic acid control addition amount) ×100% of standard recovery = (measurement value-triterpene compound content in sample)
TABLE 5 labeling recovery test results
According to the detection results, the average standard adding recovery rate of the triterpene compounds at the content limit of about 80%, 100% and 120% is 100.1%, and the RSD is 1.45 (< 2.0%), so that the accuracy of the method for detecting the triterpene compound content provided by the application is good.
Examples 2 to 3
Examples 2 to 3 respectively provide a method for detecting the content of triterpene compounds, which is different from example 1 in that: in the test solution preparation step, heating and refluxing are carried out for a time. Specifically, the results are shown in Table 6.
TABLE 6 heating reflux time in sample solution preparation step of examples 1-3
| Examples | Adding reflux time (min) |
| 1 | 30 |
| 2 | 15 |
| 3 | 45 |
Examples 4 to 10
Examples 4 to 10 respectively provide a method for detecting the content of triterpene compounds, which is different from example 1 in that: in the preparation step of the sample solution, microwave power and irradiation extraction time. Specifically, the results are shown in Table 7.
TABLE 7 microwave Power and irradiation extraction time in sample solution preparation steps of example 1, examples 4-10
Examples 11 to 13
Examples 11 to 13 respectively provide a method for detecting the content of triterpene compounds, which is different from example 1 in that: type and concentration of strong oxidizing agent. As shown in table 8.
TABLE 8 concentration of concentrated sulfuric acid in example 1, examples 11-13
Comparative example 1
Comparative example 1 provides a method for detecting the content of triterpene compounds, which is different from example 1 in that: the sample extraction method in the test solution preparation step is ultrasonic treatment. The method comprises the following steps:
(3) 1.0g of cactus fruit powder is taken, precisely weighed, placed into a conical flask, added with 25mL of 95 mass percent ethanol, subjected to ultrasonic treatment for 30min at the power of 100W and the frequency of 40kHz, and centrifuged and separated to obtain 25mL of supernatant; taking 0.1mL of supernatant, placing in a 15mL test tube with a plug, volatilizing, and cooling; then 0.2mL of 5 mass percent vanillin glacial acetic acid solution and 0.8mL of 98 mass percent concentrated sulfuric acid are added into the test tube with the plug, the mixture is shaken uniformly, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, and 4mL of ethyl acetate is precisely added, and the mixture is shaken uniformly to obtain a sample solution.
Comparative example 2
Comparative example 2 provides a method for detecting the content of triterpene compounds, which is different from example 1 in that: the sample extraction method in the sample solution preparation step is heating reflux treatment. The method comprises the following steps:
(3) Sample solution preparation: 1.0g of cactus fruit powder is taken, precisely weighed, placed into a conical flask, added with 25mL of 95 mass percent ethanol for dissolution, and heated and refluxed for 30min by a heating plate to obtain an extracting solution; centrifuging and separating the extracting solution to obtain 25mL of supernatant; taking 0.1mL of supernatant, placing in a 15mL test tube with a plug, volatilizing, and cooling; then 0.2mL of 5 mass percent vanillin glacial acetic acid solution and 0.8mL of 98 mass percent concentrated sulfuric acid are added into the test tube with the plug, the mixture is shaken uniformly, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, and 4mL of ethyl acetate is precisely added, and the mixture is shaken uniformly to obtain a sample solution.
Comparative example 3
Comparative example 3 provides a method for detecting the content of triterpene compounds, which is different from example 1 in that: the sample extraction method in the test sample solution preparation step is microwave irradiation treatment. The method comprises the following steps:
(3) Sample solution preparation: 1.0g of cactus fruit powder is taken, precisely weighed, placed in a test tube with a plug, added with 25mL of 95 mass percent ethanol for dissolution, placed in a microwave extraction instrument, and subjected to irradiation extraction for 15s under the microwave power of 750W to obtain an extract; centrifuging and separating the extracting solution to obtain 25mL of supernatant; taking 0.1mL of supernatant, placing in a 15mL test tube with a plug, volatilizing, and cooling; then 0.2mL of 5 mass percent vanillin glacial acetic acid solution and 0.8mL of 98 mass percent concentrated sulfuric acid are added into the test tube with the plug, the mixture is shaken uniformly, heated in a water bath at 70 ℃ for 15min, immediately cooled in the ice bath for 5min, taken out, and 4mL of ethyl acetate is precisely added, and the mixture is shaken uniformly to obtain a sample solution.
Comparative example 4
Comparative example 4 provides a method for detecting the content of triterpene compounds, which is different from example 1 in that: in the sample solution preparation step, the microwave power is 400W.
Comparative example 5
Comparative example 5 provides a method for detecting the content of triterpene compounds, which is different from example 1 in that: the strong oxidizing agent is perchloric acid.
Performance test
Precisely weighing 54 parts (1 g each part) of cactus fruit powder, and dividing the cactus fruit powder into 18 groups (3 parts each group); then, the triterpene compound content in the above-mentioned Opuntia Dillenii fruit powder was measured according to the measurements provided in examples 1 to 13 and comparative examples 1 to 5, respectively, and 1 group was measured by each method, and the measurement results are shown in Table 9. (actual content of triterpene compounds in Opuntia Dillenii fruit powder is 0.83-0.86g/100 g).
TABLE 9 detection results obtained by the detection methods of examples 1 to 13 and comparative examples 1 to 5
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According to the detection results of Table 9, the triterpene compound content in the Opuntia Dillenii fruit powder obtained by the detection methods provided in examples 1-13 was 0.830-0.853g/100g, and the RSD values of the detection results were all < 1.5%. The method for detecting the content of the triterpene compounds can accurately detect the content of the triterpene compounds in the cactus, and has high detection accuracy and good detection repeatability.
The triterpene compound content in cactus obtained by the detection method provided in comparative example 1 is only 0.775g/100g; the ultrasonic treatment of the cactus fruit powder cannot completely extract the triterpene compounds in the cactus, so that the accuracy of the detection result is poor.
The content of the triterpene compounds in the cactus obtained by the detection method provided in comparative example 2 is 0.810g/100g, and the triterpene compounds in the cactus cannot be completely extracted even if the cactus fruit powder is subjected to heating reflux treatment, so that the accuracy of the detection result is poor.
The content of the triterpene compounds in the cactus obtained by the detection method provided in comparative example 3 is 0.783g/100g, and the cactus fruit powder is subjected to microwave irradiation treatment only, so that the triterpene compounds in the cactus cannot be completely extracted, and further the accuracy of the detection result is poor.
In the detection method provided in comparative example 4, when the microwave power is controlled to be 400W, the content of the triterpene compounds in the cactus obtained by detection is 0.825g/100g, which indicates that when the microwave power is too low, the triterpene compounds in the cactus are difficult to thoroughly extract, so that the accuracy of the detection result is poor.
In the detection method provided in comparative example 4, perchloric acid is adopted as a strong oxidizing reagent to carry out condensation color reaction, and then the content of the triterpene compounds in the cactus is detected, so that the detection accuracy is good, but the repeatability is poor; in addition, the method has explosion risk and high risk in the detection process, so that the method is not suitable for large-scale use.
According to the detection results of the examples 1-3, when the reflux time is controlled to be 15-45min in the examples 1-3, the content of the triterpene compounds in the cactus obtained by detection is 0.832-0.852g/100g, and the RSD values are all less than 1.5%; in the preparation step of the sample solution, the heating reflux time of the sample to be detected has influence on the content of the extracted triterpene compounds, and the adding reflux time is controlled within the range of 15-45min, so that the obtained detection result has higher accuracy and better repeatability.
According to the detection results of the embodiment 1, the embodiment 4-10 and the comparative example 4, along with the increase of the microwave power and the extension of the irradiation extraction time, the content of the triterpene compound in the cactus obtained by detection shows the trend of increasing and then decreasing, the microwave power is controlled to be 500-900W, the irradiation extraction time is controlled to be 10-30s, the content of the triterpene compound is 0.834-0.853g/100g, and the RSD value is 0.49-1.131%; further comparison shows that the triterpene compounds obtained in examples 1, 5-6 and 8-9 have a content of 0.843-0.853g/100g and an RSD value of < 1%. Therefore, the application further controls the microwave power in the preparation step of the sample solution to be 600-850W, and controls the irradiation extraction time to be 10-20s, so that the accuracy and repeatability of the obtained detection method are better.
As is clear from the results of the tests in examples 1 and 11 to 13, in examples 1 and 12 to 13, when concentrated sulfuric acid and hypochlorous acid were used as the strong oxidizing agent and the concentration of the strong oxidizing agent was controlled to 85 mass% or more, the content of the triterpene compound obtained was 0.844 to 0.852g/100g and the RSD value was < 1%. Example 11A triterpene compound was obtained in an amount of 0.830g/100g and an RSD value of 1.33% by using 75% by mass of concentrated sulfuric acid as a strong oxidizing agent. Therefore, the method for detecting the content of the triterpene compounds has the advantages that the concentration of the strong oxidizing agent is controlled to be more than or equal to 85 mass percent, the repeatability of the detection method is better, and the obtained detection result is more accurate.
While the application has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the application as claimed.
Claims (9)
1. The method for detecting the content of the triterpene compounds in the cactus is characterized by comprising the steps of preparing a reference substance solution, preparing a test substance solution and detecting by an ultraviolet spectrophotometry;
sample solution preparation: dissolving a cactus sample to be measured, heating and refluxing for 20-40min, and directly radiating and extracting for 10-30s under 500-900W microwave power without cooling to obtain an extracting solution; centrifuging and separating the extracting solution, taking supernatant, adding 5-7 mass% vanillin glacial acetic acid solution and strong oxidizing agent into the supernatant, heating, cooling and fixing the volume to obtain a sample solution;
the strong oxidizing agent is concentrated sulfuric acid, hypochlorous acid and salts thereof.
2. The method according to claim 1, wherein the microwave power is 600-850W and the irradiation extraction time is 10-20s.
3. The method according to claim 1, wherein the solvent used for dissolving the cactus sample to be measured is 80-95 mass% ethanol.
4. The method according to claim 1, wherein the strongly oxidizing reagent is a reagent having a concentration of not less than 85 mass%.
5. The method according to claim 2, wherein the supernatant is heated at 65 to 75 ℃ for 10 to 45 minutes after adding 5 to 7 mass% vanillin glacial acetic acid solution and a strong oxidizing agent.
6. The method according to claim 1, wherein the solvent used for the constant volume is ethyl acetate, n-heptane, n-hexane or diethyl ether.
7. The method according to claim 1, wherein in the ultraviolet spectrophotometry detection step, the detection wavelength is 530-555nm.
8. The method of claim 1, wherein the reference is oleanolic acid reference.
9. The method according to claim 1, wherein the quantitative limit of the method is 0.00385mg/mL.
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