CN111323513A - Method for detecting content of ginsenoside Re in pig brain extract-containing preparation - Google Patents
Method for detecting content of ginsenoside Re in pig brain extract-containing preparation Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 27
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 title claims abstract description 24
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- 239000000284 extract Substances 0.000 title claims abstract description 20
- 210000004556 brain Anatomy 0.000 title claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 93
- 239000000243 solution Substances 0.000 claims abstract description 45
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 33
- 229960001701 chloroform Drugs 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 19
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 12
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- 239000012088 reference solution Substances 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000002390 rotary evaporation Methods 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 4
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- 238000000746 purification Methods 0.000 claims description 3
- 239000003463 adsorbent Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 230000015556 catabolic process Effects 0.000 abstract description 6
- 238000006731 degradation reaction Methods 0.000 abstract description 6
- 230000002378 acidificating effect Effects 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 13
- 239000007788 liquid Substances 0.000 description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 235000008434 ginseng Nutrition 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 241000208340 Araliaceae Species 0.000 description 4
- 235000003140 Panax quinquefolius Nutrition 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
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- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 240000004371 Panax ginseng Species 0.000 description 2
- 235000002789 Panax ginseng Nutrition 0.000 description 2
- 240000005373 Panax quinquefolius Species 0.000 description 2
- 101500013104 Pelophylax ridibundus Secretoneurin Proteins 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
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- YURJSTAIMNSZAE-UHFFFAOYSA-N UNPD89172 Natural products C1CC(C2(CC(C3C(C)(C)C(O)CCC3(C)C2CC2O)OC3C(C(O)C(O)C(CO)O3)O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O YURJSTAIMNSZAE-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000002021 butanolic extract Substances 0.000 description 1
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- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
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- 229940089161 ginsenoside Drugs 0.000 description 1
- 229930182494 ginsenoside Natural products 0.000 description 1
- YURJSTAIMNSZAE-HHNZYBFYSA-N ginsenoside Rg1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YURJSTAIMNSZAE-HHNZYBFYSA-N 0.000 description 1
- CBEHEBUBNAGGKC-UHFFFAOYSA-N ginsenoside Rg1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5CC(OC6OC(CO)C(O)C(O)C6O)C34C)C CBEHEBUBNAGGKC-UHFFFAOYSA-N 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
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- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 150000003648 triterpenes Chemical class 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
- 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
-
- 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
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Library & Information Science (AREA)
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Abstract
The invention relates to the technical field of detection, and particularly relates to a method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract. The method comprises the following steps: mixing the preparation sample with trichloromethane, and extracting for 5-7 hours at the temperature of 80-90 ℃ to obtain medicine residues; in g/mL, the ratio of the preparation sample to the trichloromethane is (3-6): (80-150); mixing the dregs with methanol, and performing reflux extraction at the temperature of 80-90 ℃ for 20-25 h to obtain an extracting solution; the ratio of the preparation sample to the methanol is (3-6) in g/mL: (140-200); purifying the extracting solution to obtain a test solution; detecting by high performance liquid chromatography to obtain ginsenoside Re content. The invention increases the dosage proportion of trichloromethane and methanol, can dilute the concentration of the pig brain extract preparation extract, and reduces the pH value of the acidic extract, thereby reducing the degradation of Re content in the operation process to the maximum extent.
Description
Technical Field
The invention relates to the technical field of detection, and particularly relates to a method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract.
Background
Ginsenoside Re is a tetracyclic triterpene derivative, is colorless acicular crystal, has a structural formula shown in formula I, is present in roots, flower buds, leaves, rhizoma Phragmitis and stems of Panax ginseng (Panax ginseng C.A. Mey er) of Araliaceae, and roots of Panax quinquefolium (P.quinquefolium L.), and has effects of inhibiting central nerve, promoting DNA and RNA synthesis, increasing plasma corticosterone, and dilating blood vessel.
In the traditional technology, the product is taken and placed in a Soxhlet extractor, 30mL of trichloromethane is added, heating reflux is carried out for 1 hour, trichloromethane liquid is removed, the trichloromethane is volatilized from medicine dregs, 30mL of methanol is added, heating reflux is carried out for 3 hours, the extracting solution is evaporated to dryness at low temperature, 10mL of water is added to dissolve the chloroform, petroleum ether (30-60 ℃) is added to extract for 2 times, 10mL of the ether liquid is removed each time, the water liquid passes through a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column length is 15cm), elution is carried out by 50mL of water, and the water liquid. Eluting with 50mL of 20% ethanol, discarding 20% ethanol eluate, eluting with 80% ethanol 80mL, collecting 70mL of eluate, evaporating, dissolving the residue with methanol, transferring to 10mL measuring flask, adding methanol to the scale, shaking, filtering, and collecting the filtrate. The determination method comprises the following steps: precisely sucking 10 μ L of each of the above two reference solutions and sample solution, injecting into liquid chromatograph, and measuring. Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.05% phosphoric acid solution (20: 80) is used as a mobile phase; the detection wavelength was 203 mn. The number of theoretical plates is not less than 1500 calculated according to the peak of ginsenoside Re. For example, the content of ginseng leaves in "Chinese pharmacopoeia". However, in the traditional method, the addition amount of the trichloromethane and the methanol is small, only a small Soxhlet extractor with a small specification can be used, the preparation is generally weighed to a large amount, and the small Soxhlet extractor cannot meet the extraction requirement. And the method also has the problem of Re degradation in the extraction process of certain Re preparations.
Disclosure of Invention
In view of the above, the invention provides a method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract. The method can reduce the Re content degradation in the operation process to the maximum extent, thereby improving the accuracy and stability of detection.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract, which comprises the following steps:
step 1, mixing a preparation sample with trichloromethane, and extracting for 5-7 hours at the temperature of 80-90 ℃ to obtain medicine residues; in g/mL, the ratio of the preparation sample to the trichloromethane is (3-6): (80-150);
step 2, mixing the dregs with methanol, and performing reflux extraction for 20-25 hours at the temperature of 80-90 ℃ to obtain an extracting solution; the ratio of the preparation sample to the methanol is (3-6) in g/mL: (140-200);
step 3, purifying the extracting solution to obtain a test solution;
and 4, detecting the test solution and the reference solution by adopting high performance liquid chromatography to obtain the content of the ginsenoside Re.
The invention finds that the pH value of the pig brain extract is about 2, the acidity is strong, and the solution is acidic in the sample treatment process and can cause the decomposition of Re under the condition of heating, so that the invention increases the dosage ratio of trichloromethane and methanol, dilutes the concentration of the pig brain extract preparation extract, reduces the pH value of the acidic extract, and further reduces the degradation of Re content in the operation process to the maximum extent.
Preferably, the ratio of formulation sample to chloroform in g/mL is 1: 20.
preferably, the conditions for extraction in step 1 are: extracting at 85 deg.C for 6 h.
Preferably, the ratio of formulation sample to methanol in g/mL is 1: 30.
preferably, the reflux extraction conditions in step 2 are: extracting at 85 deg.C for 20 h.
Preferably, the step 3 purification is specifically: performing rotary evaporation on the extracting solution at 55-65 ℃ based on 1g of preparation sample, dissolving obtained residues with 25-35 mL of ammonia test solution, performing ultrasonic treatment for 4-6, and sequentially extracting with 35-45, 25-35 and 15-25 mL of water-saturated n-butyl alcohol to obtain an n-butyl alcohol extracting solution; extracting with ammonia solution for 2 times, 35-45 mL each time, discarding the ammonia solution, washing the obtained residue with 25-35 mL of n-butanol saturated water for 1 time, reserving the n-butanol solution, carrying out rotary evaporation at 55-65 ℃, adding 25-35 mL of water into the obtained residue, carrying out ultrasonic treatment for 8-12 min to dissolve the residue, and filtering; and (2) passing the filtrate through a D101 type macroporous adsorption resin column, eluting with 60-80 mL of water, 40-60 mL of 15 vt-25 vt% ethanol aqueous solution and 80-120 mL of 75 vt-85 vt% ethanol aqueous solution in sequence, collecting 75 vt-85 vt% ethanol aqueous solution eluent, evaporating at 55-65 ℃ under reduced pressure, and dissolving the obtained residue with 8-12 mL of methanol.
In a specific embodiment of the present invention, the step 3 purification specifically comprises: performing rotary evaporation on the extracting solution at 60 ℃ based on 1g of preparation sample, dissolving the obtained residue with 30mL of ammonia test solution, performing ultrasonic treatment for 5 times, and sequentially extracting with 40mL, 30mL and 20mL of water-saturated n-butanol to obtain n-butanol extracting solution; extracting with ammonia solution for 2 times (40 mL each time), discarding ammonia solution, washing the residue with 30mL n-butanol saturated water for 1 time, keeping n-butanol solution, rotary steaming at 60 deg.C, adding 30mL water into the residue, ultrasonic treating for 10min to dissolve the residue, and filtering; passing the filtrate through a D101 type macroporous adsorption resin column, eluting with 70mL of water, 50mL of 20 vt% ethanol water solution and 100mL of 80 vt% ethanol water solution in sequence, collecting 80 vt% ethanol water solution eluent, evaporating at 60 ℃ under reduced pressure, and dissolving the obtained residue with 10mL of methanol.
Preferably, the D101 type macroporous adsorbent resin column has an inner diameter of 1.5cm and a length of 15 cm.
Preferably, the packing material for high performance liquid chromatography is octadecylsilane chemically bonded silica.
Preferably, the mobile phase is acetonitrile-0.04% -0.06% phosphoric acid solution, and the volume ratio of the acetonitrile to the phosphoric acid solution is (90-100): (400-410).
Preferably, the mobile phase is acetonitrile-0.05% phosphoric acid solution, and the volume ratio of the acetonitrile to the phosphoric acid solution is 95: 405.
preferably, the detection wavelength of the high performance liquid chromatography is 200 to 205 nm.
Preferably, the detection wavelength of the high performance liquid chromatography is 203 nm.
Preferably, the sample injection amount is 8 to 12 μ L.
Preferably, the sample size is 10. mu.L.
Preferably, the reference solution is 0.4-0.6 mg/mL ginsenoside Re methanol solution.
Preferably, the control solution is 0.5mg/mL ginsenoside Re methanol solution.
The invention provides a method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract, which comprises the following steps: mixing the preparation sample with trichloromethane, and extracting for 5-7 hours at the temperature of 80-90 ℃ to obtain medicine residues; in g/mL, the ratio of the preparation sample to the trichloromethane is (3-6): (80-150); mixing the dregs with methanol, and performing reflux extraction at the temperature of 80-90 ℃ for 20-25 h to obtain an extracting solution; the ratio of the preparation sample to the methanol is (3-6) in g/mL: (140-200); purifying the extracting solution to obtain a test solution; detecting the sample solution and the reference solution by high performance liquid chromatography to obtain the content of ginsenoside Re. The technical scheme of the invention has the following beneficial effects:
according to the invention, a large Soxhlet extractor (250mL) is used instead, the dosage ratio of trichloromethane and methanol is increased, the extraction efficiency can be increased, the concentration of the pig brain extract preparation extract can be diluted on the basis of the prior art, and the pH value of the acidic extract is reduced, so that the degradation of the Re content in the operation process is reduced to the greatest extent, and the accuracy and the stability of the detection method are obviously improved.
According to the invention, the time of reflux extraction is changed into 20-25 hours, and the temperature of rotary evaporation or reduced pressure evaporation is set to be 55-65 ℃, so that the Re degradation is reduced.
Detailed Description
The invention discloses a method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The reagent or instrument used in the method for detecting the content of the ginsenoside Re in the preparation containing the pig brain extract can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
Chromatographic conditions and system applicability test: octadecylsilane bonded silica gel as filler (ZORBAX SB-C184.6 mm 250mm, 5 μm); acetonitrile-0.05% phosphoric acid solution (95: 405) as a mobile phase; the detection wavelength was 203 nm. The number of theoretical plates is not less than 2500 calculated according to the peak of ginsenoside Re.
Preparation of control solutions: accurately weighing appropriate amount of ginsenoside Re reference substance, and adding methanol to obtain solution containing 0.5mg per 1 mL.
Preparation of a test solution: taking 5g of brain peptide capsules (about 1g of crude ginseng), placing in a Soxhlet extractor, adding 100mL of chloroform, extracting at 85 ℃ for 6 hours, discarding chloroform liquid, volatilizing the chloroform from the medicine residue, placing the medicine residue in the Soxhlet extractor, adding 150mL of methanol, refluxing at 85 ℃ for 20 hours, cooling (refluxing time is recorded on the first day, refluxing time is recorded on the second day, and refluxing is completed on two days), immediately evaporating to dryness at 60 ℃ by using a rotary evaporator, adding 30mL of ammonia test solution into the residue, dissolving, ultrasonically treating for 5 minutes by using an ultrasonic cleaner, adding water-saturated n-butanol, extracting for 5 times, sequentially 40, 30 and 20mL, combining the n-butanol extract, extracting for 2 times by using the ammonia test solution, discarding 40mL each time, discarding the ammonia test solution, washing for 1 time by using 30mL of water saturated by n-butanol, discarding the water solution, preserving the n-butanol solution, evaporating to dryness at 60 ℃ by using the rotary evaporator, adding 30mL of water into the residue, carrying out ultrasonic treatment for 10 minutes to dissolve the residue, filtering, passing the filtrate through a D101 type macroporous adsorption resin column (with the inner diameter of 1.5cm and the length of 15cm), eluting with 70mL of water, discarding water solution, eluting with 50mL of 20% ethanol, discarding 20% ethanol eluent, eluting with 80% ethanol, collecting 100mL of eluent, evaporating to dryness at 60 ℃ under reduced pressure by using a rotary evaporator, dissolving the residue in methanol, transferring to a 10mL measuring flask, adding methanol to the scale, and shaking up to obtain the compound.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Comparative example 1
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica was used as a filler (ZORBAX SB-C184.6 mm 250mm, 5 μm); acetonitrile-0.05% phosphoric acid solution (20: 80) is used as a mobile phase; the detection wavelength was 203 mn. The number of theoretical plates is not less than 1500 calculated according to the peak of ginsenoside Re.
Preparation of control solutions: accurately weighing appropriate amount of ginsenoside Rg1 and ginsenoside Re, and adding methanol to obtain solutions containing ginsenoside Rg10.25mg and ginsenoside Re 0.5mg per 1mL respectively.
Preparation of a test solution: taking about 0.2g of brain peptide capsule powder, precisely weighing, placing in a Soxhlet extractor, adding 30mL of chloroform, heating and refluxing for 1 hour, discarding chloroform liquid, volatilizing chloroform from medicine residue, adding 30mL of methanol, heating and refluxing for 3 hours, evaporating the extracting solution at low temperature, adding 10mL of water for dissolving, adding petroleum ether (30-60 ℃) for extracting for 2 times, 10mL each time, discarding ether liquid, passing the water liquid through a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column length is 15cm), eluting with 50mL of water, and discarding the water liquid. Eluting with 50mL of 20% ethanol, discarding 20% ethanol eluate, eluting with 80% ethanol 80mL, collecting 70mL of eluate, evaporating, dissolving the residue with methanol, transferring to 10mL measuring flask, adding methanol to the scale, shaking, filtering, and collecting the filtrate.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the above two reference solutions and test solution, respectively, injecting into liquid chromatograph, and measuring (refer to the Chinese pharmacopoeia 2015 edition, and measuring Re content in folium Ginseng).
Test example 1 comparison of the data of the invention with those of the prior art
The prior art test results are shown in table 1:
table 1 prior art test results
The technical test results of the invention are shown in table 2:
table 2 examination results of the present invention
According to the test data, compared with the two detection methods, the detection result and the stability of the content in the same batch are obviously higher than those of the conventional method.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for detecting the content of ginsenoside Re in a preparation containing a pig brain extract is characterized by comprising the following steps:
step 1, mixing a preparation sample with trichloromethane, and extracting for 5-7 hours at the temperature of 80-90 ℃ to obtain medicine residues; in g/mL, the ratio of the preparation sample to the trichloromethane is (3-6): (80-150);
step 2, mixing the dregs with methanol, and performing reflux extraction for 20-25 hours at the temperature of 80-90 ℃ to obtain an extracting solution; the ratio of the preparation sample to the methanol is (3-6) in g/mL: (140-200);
step 3, purifying the extracting solution to obtain a test solution;
and 4, detecting the test solution and the reference solution by adopting high performance liquid chromatography to obtain the content of the ginsenoside Re.
2. The assay of claim 1, wherein the ratio of said formulation sample to said chloroform, in g/mL, is 1: 20.
3. the detection method according to claim 1, wherein the conditions extracted in step 1 are: extracting at 85 deg.C for 6 h.
4. The assay of claim 1, wherein the ratio of the formulation sample to the methanol in g/mL is 1: 30.
5. the detection method according to claim 1, wherein the conditions of the reflux extraction in the step 2 are as follows: extracting at 85 deg.C for 20 h.
6. The detection method according to claim 1, wherein the step 3 purification specifically comprises: performing rotary evaporation on the extracting solution at 55-65 ℃ based on 1g of preparation sample, dissolving obtained residues with 25-35 mL of ammonia test solution, performing ultrasonic treatment for 4-6, and sequentially extracting with 35-45, 25-35 and 15-25 mL of water-saturated n-butyl alcohol to obtain an n-butyl alcohol extracting solution; extracting with ammonia solution for 2 times, 35-45 mL each time, discarding the ammonia solution, washing the obtained residue with 25-35 mL of n-butanol saturated water for 1 time, reserving the n-butanol solution, carrying out rotary evaporation at 55-65 ℃, adding 25-35 mL of water into the obtained residue, carrying out ultrasonic treatment for 8-12 min to dissolve the residue, and filtering; and (2) passing the filtrate through a D101 type macroporous adsorption resin column, sequentially eluting with 60-80 mL of water, 40-60 mL of 15 vt-25 vt% ethanol aqueous solution and 80-120 mL of 75 vt-85 vt% ethanol aqueous solution, collecting 75 vt-85 vt% ethanol aqueous solution eluent, evaporating at 55-65 ℃ under reduced pressure, and dissolving the obtained residue with 8-12 mL of methanol.
7. The detection method according to claim 1, wherein the D101 type macroporous adsorbent resin column has an inner diameter of 1.5cm and a length of 15 cm.
8. The detection method according to claim 1, wherein the filler for high performance liquid chromatography is octadecylsilane chemically bonded silica; the mobile phase is acetonitrile-0.04% -0.06% phosphoric acid solution, and the volume ratio of the acetonitrile to the phosphoric acid solution is (90-100): (400-410).
9. The detection method according to claim 1, wherein the detection wavelength of the high performance liquid chromatography is 200 to 205nm, and the sample amount is 8 to 12 μ L.
10. The detection method according to any one of claims 1 to 9, wherein the control solution is a 0.4-0.6 mg/mL methanol solution of ginsenoside Re.
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| CN113008640A (en) * | 2021-02-26 | 2021-06-22 | 九州通医药集团股份有限公司 | Pretreatment method for rapidly detecting ginsenoside Re based on microfluidic immune chip |
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Application publication date: 20200623 |