CN111855860A - Preparation of detection standard substance for sasanquasaponin and quantitative detection method for sasanquasaponin - Google Patents
Preparation of detection standard substance for sasanquasaponin and quantitative detection method for sasanquasaponin Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 229930192760 sasanquasaponin Natural products 0.000 title claims abstract description 28
- 239000000126 substance Substances 0.000 title claims abstract description 19
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 claims abstract description 51
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 claims abstract description 51
- 235000018597 common camellia Nutrition 0.000 claims abstract description 40
- 229930182490 saponin Natural products 0.000 claims abstract description 40
- 150000007949 saponins Chemical class 0.000 claims abstract description 40
- 240000001548 Camellia japonica Species 0.000 claims abstract description 39
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims abstract description 36
- 239000003208 petroleum Substances 0.000 claims abstract description 26
- 238000001914 filtration Methods 0.000 claims abstract description 24
- 239000000178 monomer Substances 0.000 claims abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 6
- 238000007873 sieving Methods 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 54
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 54
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 54
- 235000017709 saponins Nutrition 0.000 claims description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 241001122767 Theaceae Species 0.000 claims description 24
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000012043 crude product Substances 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
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- 239000007788 liquid Substances 0.000 claims description 11
- 230000007935 neutral effect Effects 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 241000526900 Camellia oleifera Species 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 6
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- 238000006460 hydrolysis reaction Methods 0.000 claims description 6
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- 238000000926 separation method Methods 0.000 claims description 6
- 238000000149 argon plasma sintering Methods 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 230000008020 evaporation Effects 0.000 claims description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical group [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 claims description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 4
- 238000005481 NMR spectroscopy Methods 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 238000009835 boiling Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 238000000825 ultraviolet detection Methods 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 230000005540 biological transmission Effects 0.000 claims description 2
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- 239000012159 carrier gas Substances 0.000 claims description 2
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- 239000003595 mist Substances 0.000 claims description 2
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- 238000012856 packing Methods 0.000 claims description 2
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 238000010567 reverse phase preparative liquid chromatography Methods 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims 1
- -1 saponin compound Chemical class 0.000 abstract description 2
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 16
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- 238000011161 development Methods 0.000 description 3
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- 239000003921 oil Substances 0.000 description 3
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 3
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 3
- 235000012141 vanillin Nutrition 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000006356 dehydrogenation reaction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 241000209507 Camellia Species 0.000 description 1
- ZMUQGGLSCIDPLS-UHFFFAOYSA-N Saponin 5 Natural products COC(=O)C1(C)CCC2(CCC3(C)C(=CCC4C5(C)CCC(OC6OC(CO)C(O)C(O)C6OC7OC(CO)C(O)C(O)C7OC8OC(C)C(O)C(O)C8O)C(C)(C)C5CCC34C)C2C1)C(=O)O ZMUQGGLSCIDPLS-UHFFFAOYSA-N 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
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- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004566 building material Substances 0.000 description 1
- 239000010495 camellia oil Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- TWHXWYVOWJCXSI-UHFFFAOYSA-N phosphoric acid;hydrate Chemical compound O.OP(O)(O)=O TWHXWYVOWJCXSI-UHFFFAOYSA-N 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 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
- G01N30/02—Column chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention discloses a preparation method of a detection standard substance of sasanquasaponin and a quantitative detection method of sasanquasaponin, wherein S1: firstly, crushing and sieving oil-tea camellia seeds or oil-tea camellia cakes, then ultrasonically extracting the sieved oil-tea camellia seeds or oil-tea camellia cakes for 2 times by using petroleum ether, combining extracting solutions, filtering the combined petroleum ether extracting solutions, concentrating under reduced pressure to a degree of no petroleum ether, and recovering the petroleum ether, and relates to the technical field of sapogenin preparation and detection. According to the preparation method of the camellia saponin detection standard product and the quantitative detection method of the camellia saponin, the camellia saponin compound monomer can be directly and rapidly prepared and separated through the preparation method of the camellia saponin detection standard product, the structure is accurately analyzed, the camellia saponin monomer is detected through a high performance liquid chromatography, the purity is over 98.0%, the feasibility of the quantitative detection method of the sapogenin prepared by the preparation method of the camellia saponin detection standard product is improved, and the preparation method of the camellia saponin detection standard product is convenient to implement.
Description
Technical Field
The invention relates to the technical field of sapogenin preparation and detection, in particular to a preparation method of a detection standard substance of sasanquasaponin and a quantitative detection method of sasanquasaponin.
Background
Camellia oleifera belongs to the family Theaceae and the genus Camellia, and the seeds of Camellia oleifera contain abundant saponins. The oil tea saponin is a pentacyclic triterpenoid compound, and the basic structure of the oil tea saponin is composed of saponin mother nucleus, glycosyl and organic acid. As a green environment-friendly nonionic surfactant, the sasanquasaponin has wide application in industries such as building materials, aquatic products, pesticides, washing and the like. Meanwhile, the sasanquasaponin is an excellent bioactive substance, has wide biological activities of sterilization, antibiosis, antivirus, anticancer and the like, is increasingly applied in the pharmaceutical and cosmetic industries, and the conventional vanillin concentrated sulfuric acid color development method which is widely applied is to ensure that the saponin is subjected to dehydrogenation reaction under the condition of strong oxidizing acid.
According to the existing preparation of the camellia saponin standard substance and the quantitative detection method of the camellia saponin, due to the characteristics of complexity and amphipathy of a molecular structure of the camellia saponin, formation of a mixed aggregate in a solution and the like, detection work of the content of the camellia saponin is very challenging, in addition, saponin molecules lack strong chromophores, difficulty is brought to given amount detection, the applicability to camellia cake with low mass fraction or a saponin crude extract is not strong, and the existing vanillin concentrated sulfuric acid color development method which is widely applied is to perform dehydrogenation reaction on the saponin under the condition of strong oxidizing acid, and the accuracy of the vanillin color development method is interfered because a detection sample also contains a large amount of compounds such as flavone and phenols which are similar to the saponin structure.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a preparation method of a camellia saponin detection standard substance and a quantitative detection method of camellia saponin, and solves the problems that the detection work of the content of camellia saponin is difficult and the quantitative detection of the camellia saponin is difficult in the existing preparation method of the camellia saponin detection standard substance and the quantitative detection method of the camellia saponin.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: the preparation method of the detection standard substance of the sasanquasaponin and the quantitative detection method of the sasanquasaponin specifically comprise the following steps:
s1: firstly, crushing and sieving camellia seeds or camellia cake, then ultrasonically extracting the sieved camellia seeds or camellia cake for 2 times by using petroleum ether, combining extracting solutions, filtering the combined petroleum ether extracting solution, concentrating under reduced pressure until the petroleum ether is absent, and recovering the petroleum ether;
s2: drying the filter residue after filtering the petroleum ether extracting solution, adding 75% methanol-water solution into the dried filter residue for three times of ultrasonic extraction, combining the extracting solutions, filtering the combined alcohol extracting solution, removing the filter residue, concentrating under reduced pressure until no alcohol exists, and recovering methanol;
s3: purifying the obtained solution after filtering the alcohol extract by an AB-8 macroporous adsorption resin chromatographic column, performing gradient elution by using sodium hydroxide and methanol, collecting eluent containing saponin, and performing reduced pressure concentration until no alcohol degree exists to obtain a crude product of the total saponins of camellia oleifera;
s4: putting the obtained crude product of the tea seed total saponin into a reaction kettle, adding 20 times of 50% methanol-water solution of 3mol/L hydrochloric acid, adjusting the pH value of the solution to 1-2, heating in a water bath at 100 ℃, stirring and refluxing, monitoring the pH change of the solution in real time during the refluxing process, supplementing hydrochloric acid in a proper amount, maintaining the pH of the solution for hydrolysis, naturally cooling the hydrolyzed crude product of the tea seed total saponin, adjusting the pH of the solution to be neutral by using 10% sodium hydroxide, extracting for 3 times by using ethyl acetate, and combining the extract liquor;
s5: filtering the extract, recovering ethyl acetate under reduced pressure to obtain extract, suspending the extract with sodium hydroxide, heating and stirring in boiling water bath, cooling the heated and stirred extract, filtering, and washing the extract with pure water to neutrality to obtain total sapogenin of Camellia oleifera.
Preferably, the raw material in S1 can be one or combination of fresh or dried oil tea seeds and tea cake pulp, and the size of the screen is 20-100 meshes.
Preferably, the alcohol in S2 is one or a combination of methanol, ethanol and n-butanol, the proportion of the alcohol is 50-100%, the dosage of the alcohol is 3-10 times of the raw materials, and the ultrasonic time is 30-120 min.
Preferably, the alcohol content in S3 is 10-100%, and the sodium hydroxide and methanol gradient elution is performed by washing with 1% sodium hydroxide solution until the effluent is substantially colorless, washing with purified water until the effluent is neutral, washing with 5 column volumes of 10% methanol-water and 30% methanol-water sequentially, and finally washing with 80% methanol-water until no saponin is present.
Preferably, the hydrolysis time in S4 is 1-10hrs, and the hydrolysis pH is maintained between 1-2.
Preferably, the proportion of the sodium hydroxide in the S5 is 1-10%, and the stirring reflux time of the boiling water bath is 0.5-5 hrs.
The invention also discloses a quantitative detection method of the sapogenin prepared by the preparation method of the detection standard substance of the oil-tea camellia sapogenin, which specifically comprises the following steps:
l1: firstly, mixing a tea seed total sapogenin crude product with silica gel, performing silica gel column chromatography, performing gradient elution in the sequence of petroleum ether to ethyl acetate, then to ethyl acetate and then to methanol, performing reversed-phase preparative liquid chromatography, eluting with a methanol-acid solution, detecting by TLC, and performing reduced pressure recovery of a solvent to obtain a sapogenin crude product;
l2: dissolving the obtained sapogenin crude product with methanol, filtering, repeatedly performing high performance preparative liquid chromatography for multiple times for preparative separation until single component HPLC monomer with purity of more than 98% is obtained, and detecting by MS and NMR to obtain tea oil sapogenin;
l3: simultaneously separating the other half of the crude sapogenin solution after being dissolved in methanol and filtered by a chromatographic column, adding the separated crude sapogenin solution into an evaporative light scattering detector, introducing the separated crude sapogenin solution into an atomizer along with a mobile phase, and atomizing the crude sapogenin solution into fine droplets by high-speed carrier gas flow;
l4: and then the atomized fine droplets enter an evaporator with controllable temperature, so that the mobile phase and the solvent in the evaporator are vaporized and evaporated, the non-volatile components are changed into fine mist particles and pass through an optical path of the evaporator and a light source at a high speed, the laser beam irradiates on the solute particles to generate light scattering, and a light collector collects the scattered light and converts the scattered light into an electric signal through a photomultiplier tube to obtain a detection result of the sasanquagenin.
Preferably, the sample-mixing silica gel in the L1 is 100-mesh 300-mesh, the amount of the silica gel is 1-5 times of that of the sample, the ethyl acetate is methanol 100:0-0:100, the ultraviolet detection chromatography conditions include a chromatographic column, a flow rate, an isocratic elution of acetonitrile and a detection wavelength, the chromatographic column is nano-micro UniSil5-120C18Ultra (SS4.6 × 250mm, i.d.5 μm), the flow rate is 1.0ml/min, the isocratic elution of acetonitrile is 0.2% phosphoric acid water 90:10, the detection wavelength is 203nm, the sample introduction amount is 10 μ L, and the column temperature is 30 ℃.
Preferably, the reversed-phase preparative chromatography packing in the L2 is C18, the particle size is 5-12 μm, the MS analysis condition is electrospray ESI negative ion mode, the capillary voltage is negative 2000V, and the temperature of the ion transmission tube is 300 ℃. The evaporation temperature is 338 ℃, the sheath gas pressure is 56.9psi, the auxiliary gas pressure is 6.5psi, the back-blowing gas pressure is 0.5psi, the scanning range is 200-1250u, and the solvent used in nuclear magnetic resonance is DMSO-d6 or CDCl 3.
(III) advantageous effects
The invention provides a preparation method of a detection standard substance of sasanquasaponin and a quantitative detection method of sasanquasaponin. Compared with the prior art, the method has the following beneficial effects: the preparation method of the detection standard substance for the sasanquasaponin comprises the following steps of S1: firstly, crushing and sieving camellia seeds or camellia cake, then ultrasonically extracting the sieved camellia seeds or camellia cake for 2 times by using petroleum ether, combining extracting solutions, filtering the combined petroleum ether extracting solution, concentrating under reduced pressure until the petroleum ether is absent, and recovering the petroleum ether; s2: at the moment, filter residue obtained after the petroleum ether extracting solution is filtered is dried, 75% methanol-water solution is added into the dried filter residue for three-time ultrasonic extraction, the extracting solutions are combined, the combined alcohol extract is filtered, the filter residue is discarded, the pressure reduction concentration is carried out until the alcohol content is zero, the methanol is recovered, the oil-tea sapogenin compound monomer can be directly and rapidly prepared and separated by the preparation method of the oil-tea sapogenin detection standard product, the structure is accurately analyzed without a derivative presumption structure, the high performance liquid chromatography is used for detecting the oil-tea sapogenin monomer, the purity is more than 98.0%, the feasibility of the quantitative detection method of the sapogenin prepared by the preparation method of the oil-tea sapogenin detection standard product is improved, the preparation method of the oil-tea sapogenin detection standard product is convenient to implement and wide in applicability, and is suitable for the preparation and separation of sapogenin monomer compounds in other plants, and the baseline drift caused by temperature change, the method can provide a stable base line during gradient elution, simultaneously eliminate solvent interference during UV detection by the evaporative light scattering detector, greatly improve the separation effect, has low noise of the base line of the evaporative light scattering detector, is not influenced by the gradient, can obtain better peak shape, and only responds to nonvolatile analytes, so that the impurity peaks are fewer, and compared with a differential refraction detector, the base line drift of the differential refraction detector is not influenced by temperature.
Drawings
FIG. 1 is a molecular structure diagram of sasanquasaponin of the present invention;
FIG. 2 is a HPLC structure diagram of the sasanquasaponin monomer product of the invention;
FIG. 3 is a diagram of a mass spectrum structure of the sasanquasaponin monomer product of the invention;
FIG. 4 is a nuclear magnetic carbon spectrum and hydrogen spectrum structural diagram of a first sasanquasaponin monomer product of the invention;
fig. 5 is a nuclear magnetic carbon spectrum and hydrogen spectrum structural diagram of a second sasanquasaponin monomer product of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-5, the present invention provides two technical solutions: the preparation method of the detection standard substance of the sasanquasaponin and the quantitative detection method of the sasanquasaponin specifically comprise the following steps:
example 1
Weighing 20kg of tea seed cake, crushing and sieving with a 80-mesh sieve. Adding 160L petroleum ether, ultrasonic defatting twice for 2 hr and 1 hr, filtering, mixing filtrates, and recovering petroleum ether at 50 deg.C under reduced pressure. Adding 75% methanol-water solution into the dried residue, and ultrasonic extracting for 3 times (2 hr, 1 hr, and 1 hr each time), wherein the amount of solution used is 8 times, 5 times, and 5 times; mixing extractive solutions, filtering, removing residue, and recovering alcohol from the filtrate at 45 deg.C under reduced pressure until no alcohol exists. Suspending the obtained extract with 1 time of purified water, purifying with AB-8 macroporous adsorbent resin chromatographic column at flow rate of 4BV/h, washing with 1% sodium hydroxide solution until effluent liquid is almost colorless, washing with purified water until effluent liquid is neutral, washing with 5 times column volume of 10% methanol-water and 30% methanol-water sequentially, washing with 80% methanol-water until saponin is absent, collecting 80% methanol-water effluent liquid, and concentrating under reduced pressure at 45 deg.C until alcohol degree is absent to obtain crude product 1005g of total saponins of Camellia oleifera.
Putting the crude product of the tea seed total saponin into a reaction kettle, adding 20 times of 50% methanol-water solution of 3mol/L hydrochloric acid, adjusting the pH value of the solution to 1-2, heating in a water bath at 100 ℃, stirring and refluxing for 5 hours, monitoring the pH change of the solution in real time during the refluxing process, adding hydrochloric acid in a proper amount, and maintaining the pH of the solution between 1-2. Cooling to room temperature after the acidolysis is finished, adjusting the pH of the solution to be neutral by using 10% sodium hydroxide, extracting for 3 times by using 3 times of ethyl acetate, combining ethyl acetate layers, filtering, recovering an ethyl acetate reagent at 60 ℃ under reduced pressure, diluting residues by using 10 times of 2% sodium hydroxide, heating in a water bath at 100 ℃, stirring and refluxing for 1h, cooling the solution to the room temperature, filtering, and washing solids to be neutral by using pure water to obtain about 130g of a crude product of the tea seed total sapogenin.
Taking the crude tea seed total sapogenin, mixing the crude tea seed total sapogenin with 3 times of 200-mesh silica gel with 300 meshes, performing silica gel column chromatography, performing gradient elution by using petroleum ether with 3 times of column volume, ethyl acetate (30:1-1:1) → ethyl acetate → methanol respectively, collecting fractions with the concentration of 1/500 ml, detecting by TLC, using 10% sulfuric acid-ethanol solution as a color developing agent, combining the fractions with the same Rf value, recovering the solvent under reduced pressure at 60 ℃ to obtain a plurality of different tea seed sapogenin components, and drying to obtain 23g of crude tea seed sapogenin.
Example 2
Weighing 0.1kg of tea seed cake, crushing and sieving with a 80-mesh sieve. Adding 160L petroleum ether, ultrasonic defatting twice for 2 hr and 1 hr, filtering, mixing filtrates, and recovering petroleum ether at 50 deg.C under reduced pressure. Adding 75% methanol-water solution into the dried residue, and ultrasonic extracting for 3 times (2 hr, 1 hr, and 1 hr each time), wherein the amount of solution used is 8 times, 5 times, and 5 times; mixing extractive solutions, filtering, removing residue, and recovering alcohol from the filtrate at 45 deg.C under reduced pressure until no alcohol exists. Suspending the obtained extract with 1 time of purified water, purifying with AB-8 macroporous adsorbent resin chromatographic column at flow rate of 4BV/h, washing with 1% sodium hydroxide water solution until effluent liquid is almost colorless, washing with purified water until effluent liquid is neutral, washing with 5 times column volume of 10% methanol-water and 30% methanol-water sequentially, washing with 80% methanol-water until saponin is absent, collecting 80% methanol-water effluent liquid, and concentrating under reduced pressure at 45 deg.C until alcohol degree is absent to obtain crude product of Camellia oleifera total saponin 5 g.
Putting the crude product of the tea seed total saponin into a reaction kettle, adding 20 times of 50% methanol-water solution of 3mol/L hydrochloric acid, adjusting the pH value of the solution to 1-2, heating in a water bath at 100 ℃, stirring and refluxing for 5 hours, monitoring the pH change of the solution in real time during the refluxing process, adding hydrochloric acid in a proper amount, and keeping the pH of the solution between 1 and 2 all the time. Cooling to room temperature after the acidolysis is finished, adjusting the pH of the solution to be neutral by using 10% sodium hydroxide, extracting for 3 times by using 3 times of ethyl acetate, combining ethyl acetate layers, filtering, recovering an ethyl acetate reagent at 60 ℃ under reduced pressure, diluting residues by using 10 times of 2% sodium hydroxide, heating in a water bath at 100 ℃, stirring and refluxing for 1h, cooling the solution to the room temperature, filtering, and washing solids to be neutral by using pure water to obtain about 0.65 g of crude tea seed total sapogenin.
Detecting and analyzing the liquid phase conditions of the sapogenin content: a chromatographic column: UniSil5-120C18Ultra SS4.6 x 250 x 5; flow rate: 1.0 ml/min; mobile phase A: acetonitrile; mobile phase B: 0.2% phosphoric acid-water; detection wavelength: 203 nm; column temperature: 30 ℃; the content of sapogenin in 100g of oil-tea camellia cake is as follows: 0.65 g (0.038+0.0273+0.0956+0.1041+0.0405+0.1273) ═ 0.28g, the percentage content is 0.28%.
And those not described in detail in this specification are well within the skill of those in the art.
In conclusion, the preparation method of the standard product for detecting the sasanqua sapogenin can directly and quickly prepare and separate sasanqua sapogenin compound monomers, accurately resolve the structure, does not need to derive a presumed structure, detects the sasanqua sapogenin monomers by the high performance liquid chromatography, has the purity of over 98.0 percent, improves the feasibility of the quantitative detection method of the sapogenin prepared by the preparation method of the standard product for detecting the sasanqua sapogenin, is convenient to implement, has wide applicability, can be suitable for the preparation and separation of the sapogenin monomer compounds in other plants, can eliminate the baseline drift caused by the temperature change by the evaporation light scattering detector, can provide a stable baseline during gradient elution, simultaneously eliminates the solvent interference when using a UV detector by the evaporation light scattering detector, greatly improves the separation effect, has small baseline noise of the evaporation light scattering detector, is not influenced by the gradient, better peak shape can be obtained and only response to non-volatile analytes, so that the impurity peaks are fewer and the baseline drift is not affected by temperature compared to a differential refractometer detector.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. The preparation method of the detection standard substance for the sasanquasaponin is characterized by comprising the following steps of: the method specifically comprises the following steps:
s1: firstly, crushing and sieving camellia seeds or camellia cake, then ultrasonically extracting the sieved camellia seeds or camellia cake for 2 times by using petroleum ether, combining extracting solutions, filtering the combined petroleum ether extracting solution, concentrating under reduced pressure until the petroleum ether is absent, and recovering the petroleum ether;
s2: drying the filter residue after filtering the petroleum ether extracting solution, adding 75% methanol-water solution into the dried filter residue for three times of ultrasonic extraction, combining the extracting solutions, filtering the combined alcohol extracting solution, removing the filter residue, concentrating under reduced pressure until no alcohol exists, and recovering methanol;
s3: purifying the obtained solution after filtering the alcohol extract by an AB-8 macroporous adsorption resin chromatographic column, performing gradient elution by using sodium hydroxide and methanol, collecting eluent containing saponin, and performing reduced pressure concentration until no alcohol degree exists to obtain a crude product of the total saponins of camellia oleifera;
s4: putting the obtained crude product of the tea seed total saponin into a reaction kettle, adding 20 times of 50% methanol-water solution of 3mol/L hydrochloric acid, adjusting the pH value of the solution to 1-2, heating in a water bath at 100 ℃, stirring and refluxing, monitoring the pH change of the solution in real time during the refluxing process, supplementing hydrochloric acid in a proper amount, maintaining the pH of the solution for hydrolysis, naturally cooling the hydrolyzed crude product of the tea seed total saponin, adjusting the pH of the solution to be neutral by using 10% sodium hydroxide, extracting for 3 times by using ethyl acetate, and combining the extract liquor;
s5: filtering the extract, recovering ethyl acetate under reduced pressure to obtain extract, suspending the extract with sodium hydroxide, heating and stirring in boiling water bath, cooling the heated and stirred extract, filtering, and washing the extract with pure water to neutrality to obtain total sapogenin of Camellia oleifera.
2. The preparation method of the sasanquasaponin detection standard substance according to claim 1, which is characterized in that: the raw materials in the S1 can be one or a combination of fresh or dried oil tea seeds and tea cake dregs, and the size of the screen is 20-100 meshes.
3. The preparation method of the sasanquasaponin detection standard substance according to claim 1, which is characterized in that: the alcohol in the S2 is one or combination of methanol, ethanol and n-butanol, the proportion of the alcohol is 50-100%, the dosage of the alcohol is 3-10 times of the raw materials, and the ultrasonic time is 30-120 min.
4. The preparation method of the sasanquasaponin detection standard substance according to claim 1, which is characterized in that: the proportion of alcohol in S3 is 10-100%, sodium hydroxide and methanol are eluted in a gradient mode by using 1% sodium hydroxide solution until effluent liquid is basically colorless, then the effluent liquid is washed by using purified water until the effluent liquid is neutral, and finally the effluent liquid is washed by using 10% methanol-water and 30% methanol-water which are 5 times of column volume in sequence, and finally the effluent liquid is washed by using 80% methanol-water until saponin is absent.
5. The preparation method of the sasanquasaponin detection standard substance according to claim 1, which is characterized in that: the hydrolysis time in S4 is 1-10hrs, and the hydrolysis pH is maintained at 1-2.
6. The preparation method of the sasanquasaponin detection standard substance according to claim 1, which is characterized in that: the proportion of sodium hydroxide in the S5 is 1-10%, and the stirring reflux time in a boiling water bath is 0.5-5 hrs.
7. The quantitative detection method for the sapogenin prepared by the preparation method of the camellia sapogenin detection standard according to any one of claims 1 to 6, is characterized by comprising the following steps:
l1: firstly, mixing a tea seed total sapogenin crude product with silica gel, performing silica gel column chromatography, performing gradient elution in the sequence of petroleum ether to ethyl acetate, then to ethyl acetate and then to methanol, performing reversed-phase preparative liquid chromatography, eluting with a methanol-acid solution, detecting by TLC, and performing reduced pressure recovery of a solvent to obtain a sapogenin crude product;
l2: dissolving the obtained sapogenin crude product with methanol, filtering, repeatedly loading one half of the filtered sapogenin crude product solution for multiple times by high performance preparative liquid chromatography for preparative separation until a single component HPLC monomer with purity of more than 98% is obtained, and determining the single component as the oil tea sapogenin by MS and NMR detection;
l3: simultaneously separating the other half of the crude sapogenin solution after being dissolved in methanol and filtered by a chromatographic column, adding the separated crude sapogenin solution into an evaporative light scattering detector, introducing the separated crude sapogenin solution into an atomizer along with a mobile phase, and atomizing the crude sapogenin solution into fine droplets by high-speed carrier gas flow;
l4: and then the atomized fine droplets enter an evaporator with controllable temperature, so that the mobile phase and the solvent in the evaporator are vaporized and evaporated, the non-volatile components are changed into fine mist particles and pass through an optical path of the evaporator and a light source at a high speed, the laser beam irradiates on the solute particles to generate light scattering, and a light collector collects the scattered light and converts the scattered light into an electric signal through a photomultiplier tube to obtain a detection result of the sasanquagenin.
8. The quantitative detection method for the sapogenin prepared by the preparation method of the camellia sapogenin detection standard according to claim 7, is characterized by comprising the following steps of: the sample mixing silica gel in the L1 is 100-300 meshes, the silica gel amount is 1-5 times of that of a sample, ethyl acetate is methanol 100:0-0:100, the ultraviolet detection chromatographic conditions comprise a chromatographic column, flow rate, isocratic elution and detection wavelength, the chromatographic column is nano UniSil5-120C18Ultra (SS4.6 x 250mm, i.d.5 mu m), the flow rate is 1.0ml/min, the isocratic elution is acetonitrile 0.2% phosphoric acid water 90:10, the detection wavelength is 203nm, the sample introduction amount is 10 mu L, and the column temperature is 30 ℃.
9. The quantitative detection method for the sapogenin prepared by the preparation method of the camellia sapogenin detection standard according to claim 7, is characterized by comprising the following steps of: the reversed-phase preparative chromatographic packing in the L2 is C18, the particle size is 5-12 μm, the MS analysis condition is an electrospray ESI negative ion mode, the capillary voltage is negative 2000V, and the temperature of an ion transmission tube is 300 ℃. The evaporation temperature is 338 ℃, the sheath gas pressure is 56.9psi, the auxiliary gas pressure is 6.5psi, the back-blowing gas pressure is 0.5psi, the scanning range is 200-1250u, and the solvent used in nuclear magnetic resonance is DMSO-d6 or CDCl 3.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114950480A (en) * | 2022-07-06 | 2022-08-30 | 西北大学 | Carbon-based solid acid catalyst and method for preparing tea sapogenin by catalyzing tea saponin through carbon-based solid acid catalyst |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838302A (en) * | 2010-05-25 | 2010-09-22 | 南昌大学 | Method for extracting sasanquasaponin |
| CN102504007A (en) * | 2011-12-15 | 2012-06-20 | 成都普思生物科技有限公司 | Method for separation and purification of ruscogenin monomer |
| CN102659904A (en) * | 2012-05-21 | 2012-09-12 | 上海砝码斯医药生物科技有限公司 | Preparation method for hederagenin and salts thereof |
| CN102911240A (en) * | 2011-08-02 | 2013-02-06 | 苏州宝泽堂医药科技有限公司 | Method for preparing high-purity bayogenin |
| CN109265494A (en) * | 2018-11-30 | 2019-01-25 | 中南林业科技大学 | The method of Kaempferol glucoside compounds is extracted from the camellia of Yunnan |
| CN110794075A (en) * | 2019-11-21 | 2020-02-14 | 华侨大学 | Analysis method of oil-tea camellia seed oil saponin compounds |
-
2020
- 2020-07-31 CN CN202010762482.XA patent/CN111855860A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838302A (en) * | 2010-05-25 | 2010-09-22 | 南昌大学 | Method for extracting sasanquasaponin |
| CN102911240A (en) * | 2011-08-02 | 2013-02-06 | 苏州宝泽堂医药科技有限公司 | Method for preparing high-purity bayogenin |
| CN102504007A (en) * | 2011-12-15 | 2012-06-20 | 成都普思生物科技有限公司 | Method for separation and purification of ruscogenin monomer |
| CN102659904A (en) * | 2012-05-21 | 2012-09-12 | 上海砝码斯医药生物科技有限公司 | Preparation method for hederagenin and salts thereof |
| CN109265494A (en) * | 2018-11-30 | 2019-01-25 | 中南林业科技大学 | The method of Kaempferol glucoside compounds is extracted from the camellia of Yunnan |
| CN110794075A (en) * | 2019-11-21 | 2020-02-14 | 华侨大学 | Analysis method of oil-tea camellia seed oil saponin compounds |
Non-Patent Citations (2)
| Title |
|---|
| 孙劲毅: "茶皂素的分离纯化及性能研究", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
| 干丽: "油茶饼粕中茶皂素苷元的制备与分析研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114950480A (en) * | 2022-07-06 | 2022-08-30 | 西北大学 | Carbon-based solid acid catalyst and method for preparing tea sapogenin by catalyzing tea saponin through carbon-based solid acid catalyst |
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