CN115078596A - Method for detecting genistein in fermented cordyceps sinensis powder Cs-4 - Google Patents
Method for detecting genistein in fermented cordyceps sinensis powder Cs-4 Download PDFInfo
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- CN115078596A CN115078596A CN202210990118.8A CN202210990118A CN115078596A CN 115078596 A CN115078596 A CN 115078596A CN 202210990118 A CN202210990118 A CN 202210990118A CN 115078596 A CN115078596 A CN 115078596A
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- 229940045109 genistein Drugs 0.000 title claims abstract description 44
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 235000006539 genistein Nutrition 0.000 title claims abstract description 44
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 title claims abstract description 44
- 239000000843 powder Substances 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 35
- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 32
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 45
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 27
- 238000002360 preparation method Methods 0.000 claims description 21
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 19
- 238000005303 weighing Methods 0.000 claims description 17
- 239000012085 test solution Substances 0.000 claims description 15
- 239000000706 filtrate Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- 241000190633 Cordyceps Species 0.000 claims description 9
- 239000013558 reference substance Substances 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 7
- 239000012982 microporous membrane Substances 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 abstract description 4
- 239000007864 aqueous solution Substances 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 description 17
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000002137 ultrasound extraction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 3
- 229960005305 adenosine Drugs 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010003549 asthenia Diseases 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 206010006458 Bronchitis chronic Diseases 0.000 description 1
- 239000003390 Chinese drug Substances 0.000 description 1
- 208000010228 Erectile Dysfunction Diseases 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010024419 Libido decreased Diseases 0.000 description 1
- 208000019255 Menstrual disease Diseases 0.000 description 1
- 241001416980 Paecilomyces hepiali Species 0.000 description 1
- 206010041497 Spermatorrhoea Diseases 0.000 description 1
- 208000009205 Tinnitus Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 208000007451 chronic bronchitis Diseases 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 206010036596 premature ejaculation Diseases 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 231100000886 tinnitus Toxicity 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the technical field of medicines, and provides a method for detecting genistein in fermented cordyceps sinensis powder Cs-4, which comprises the steps of extracting the genistein and measuring the content of the genistein, wherein the genistein is extracted by adopting a methanol aqueous solution and passes through a microporous filter membrane, and the content measuring method is a high performance liquid chromatography. The detection method provided by the invention is more effective in quality control of products, and has higher accuracy, sensitivity and stability.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a method for detecting genistein in fermented cordyceps sinensis powder Cs-4.
Background
Fermented Cordyceps powder (Cs-4) is prepared by deep fermentation culture of Cordyceps-Paecilomyces hepiali Chen et Dai (Paecilomyces hepiali Chen et Dai) Cs-4 separated from fresh Cordyceps Cordycepsinensis (Berk) Sace, filtering the fermentation product, and drying. The fermented cordyceps sinensis powder Cs-4 has the functions of tonifying kidney and protecting lung, and replenishing essence and tonifying qi. Can be used for treating chronic bronchitis, hyperlipidemia, sexual impotence, spermatorrhea, premature ejaculation, hyposexuality, menoxenia, soreness of waist and knees, asthenia, dizziness, dim eyesight, tinnitus, senile asthenia, and late stage hemophagia, and liver cirrhosis.
The genistein is an important component in the fermented raw material soybean of the fermented cordyceps sinensis powder Cs-4, and is an important index for evaluating the fermentation degree of the fermented cordyceps sinensis powder Cs-4. At present, thin-layer chromatography, HPLC and the like are adopted for detecting genistein in cordyceps sinensis and products thereof, but the thin-layer chromatography is suitable for qualitative analysis and cannot accurately and quantitatively analyze, and the HPLC method is low in extraction rate and poor in accuracy of genistein and is not beneficial to quality control of products. At present, no relevant report about a method for measuring the content of genistein in the fermented cordyceps sinensis powder Cs-4 exists, and a scientific quality detection method is needed to be established for evaluating the quality of the fermented cordyceps sinensis powder Cs-4.
Disclosure of Invention
The invention aims to overcome at least one of the defects of the prior art and provides a method for detecting genistein in fermented cordyceps sinensis powder Cs-4. The purpose of the invention is realized based on the following technical scheme:
the invention provides a method for detecting genistein in fermented cordyceps sinensis powder Cs-4, which is determined by a high performance liquid chromatography and comprises the following steps:
preparation of a test solution: precisely weighing fermented Cordyceps powder Cs-4, adding methanol water solution for dissolving, filtering with microporous membrane, and collecting filtrate as sample solution;
preparation of control solutions: taking a genistein reference substance, precisely weighing, and dissolving with a mobile phase to obtain a reference substance solution;
the determination method comprises the following steps: precisely absorbing the reference solution and the test solution respectively, injecting into a liquid chromatograph, taking methanol-water as a mobile phase, detecting the wavelength of 260-265 nm, determining to obtain a chromatogram, and calculating the content of genistein in the fermented cordyceps sinensis powder Cs-4 according to the chromatogram.
Preferably, in the preparation of the test solution: the concentration of the aqueous methanol solution is 50 to 100 wt%.
Preferably, in the preparation of the test solution: the method for dissolving the fermented Cordyceps powder Cs-4 comprises ultrasonic treatment for 30-120min or heating and refluxing for 30-120 min.
Preferably, in the preparation of the test solution: the diameter of the microporous filter membrane is 0.3-0.6 μm.
Preferably, the preparation of the test solution specifically comprises: precisely weighing fermented Cordyceps powder Cs-4, adding 75% methanol solution, weighing, ultrasonic treating for 1 hr, cooling, weighing again, supplementing with 75% methanol solution, filtering with 0.45 μm microporous membrane, and collecting filtrate as sample solution.
Preferably, in the preparation of the control solution: the concentration of the control solution is 5-20 μ g per 1ml containing genistein.
Preferably, in the preparation of the control solution: the mobile phase is methanol-water with a volume ratio of 30-90: 70-10.
Preferably, in the assay, the chromatographic conditions are as follows: a chromatographic column C18, wherein the column temperature is 20-50 ℃, the methanol-water volume ratio is 30-90: 70-10, and the flow rate of the mobile phase is 0.5-1.5 ml/min; the sample amount is 5-50 μ l.
Preferably, in the determination method, methanol-water volume ratio of 50:50 is used as a mobile phase, and the flow rate is 1.0 ml/min; the detection wavelength is 262 nm.
Preferably, in the assay, the number of theoretical plates should not be less than 2000-5000 as calculated by adenosine peak.
The invention can obtain at least one of the following beneficial effects:
the detection method disclosed by the invention has the advantages of high extraction rate of the dye lignin, good extraction effect, more effective quality control of products, higher accuracy, sensitivity and stability, simplicity and convenience in operation, and suitability for popularization and application. The method can be used for controlling the quality of the genistein in the fermented cordyceps sinensis powder Cs-4 and can also be used for controlling the quality of the genistein in other preparations which take the fermented cordyceps sinensis powder Cs-4 as a raw material, such as tablets, granules, honeyed pills, oral liquid and the like.
Drawings
FIG. 1 is the chemical structure of genistein;
in FIG. 2, a is a liquid chromatogram of methanol-water (90:10, V: V), b is a liquid chromatogram of methanol-water (50:50, V: V), and c is a liquid chromatogram of methanol-water (30:70, V: V);
FIG. 3 is a UV spectrum of genistein;
in FIG. 4, a is a liquid chromatogram of a solvent blank, b is a liquid chromatogram of a control solution, and c is a liquid chromatogram of a test solution.
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.
The genistein reference substance is an existing product and can be purchased in the market; the chemical structure of genistein is shown in FIG. 1. The subsequent filtrate is: the filtrate filtered firstly is called as primary filtrate, the purity of the filtrate is insufficient, the primary filtrate is discarded, and the filtrate collected continuously after the primary filtrate is poured out is the subsequent filtrate.
The preferred embodiment of the invention provides a method for detecting genistein in fermented cordyceps sinensis powder Cs-4, which is measured by a high performance liquid chromatography and comprises the following steps:
preparation of a test solution: precisely weighing fermented Cordyceps powder Cs-4, adding methanol water solution for dissolving, filtering with microporous membrane, and collecting filtrate as sample solution; wherein the concentration of the methanol aqueous solution is 50-100wt%, the method for dissolving the fermented cordyceps sinensis bacteria powder Cs-4 comprises ultrasonic treatment for 60-150min or heating reflux for 60-150min, and the diameter of the microporous filter membrane is 0.3-0.6 mu m;
preparation of control solutions: taking a genistein reference substance, precisely weighing, and dissolving with a mobile phase to obtain a reference substance solution; wherein the mobile phase is methanol-water with the volume ratio of 30-90: 70-10, and the concentration of the reference solution is 5-20 mug per 1ml of genistein;
the determination method comprises the following steps: precisely absorbing a reference solution and a test solution respectively, injecting into a liquid chromatograph, taking methanol-water as a mobile phase, measuring at a wavelength of 260-265 nm to obtain a chromatogram, and calculating the content of genistein in the fermented cordyceps sinensis powder Cs-4 according to the chromatogram; the chromatographic conditions were as follows: a chromatographic column Phenomenex C18 with methanol-water (30-90: 70-10, v: v) as a mobile phase; the sample amount is 5-50 μ l; the chromatographic column has a column length of 15-25cm, an inner diameter of 2-8mm, a particle size of 2-10 μm, and a column temperature of 20-50 deg.C; the flow rate of the mobile phase is 0.5-1.5 ml/min; the number of theoretical plates should not be less than 2000-5000 calculated by adenosine peak.
Example 1
The determination method is obtained by screening, and the screening process comprises the following steps:
1. selection of extraction conditions
1.1 selection of extraction solvent
The extraction effect of ethanol and methanol as extraction solvents is investigated, and the result shows that the extraction effect of methanol is high. Further, the extraction effects of methanol of different concentrations were compared, and it was found that the extraction effect was the best when a 75% methanol solution was used as the extraction solvent.
1.2 selection of extraction method
The invention adopts a reflux extraction method and an ultrasonic extraction method to extract genistein in the fermented cordyceps sinensis powder Cs-4. The comparison shows that the extraction efficiency of the ultrasonic extraction method is higher than that of the reflux extraction method, so the ultrasonic extraction method is selected.
1.3 selection of extraction time
The ultrasonic extraction times of 30 minutes, 60 minutes, 90 minutes and 120 minutes are respectively examined, and the results show that the influence of the ultrasonic extraction time on the extraction efficiency is slightly different after 60 minutes, so that 60 minutes is selected as the ultrasonic extraction time.
2. Optimization of chromatographic conditions
2.1 selection of the Mobile phase
The mobile phase takes methanol as phase A and water as phase B, and the chromatograms of A-B (30:70, V: V), A-B (50:50, V: V) and A-B (90:10, V: V) in different proportions are shown in FIG. 2. The result shows that the main peak and the impurity peak of the mobile phase of A-B (50:50, v: v) have better separation degree, the peak shape symmetry is high, the peak emergence time is shorter, and the analysis time is shortened.
2.2 wavelength selection
The experiment was performed by UV scanning using purified water as solvent and genistein as control in a solution of appropriate concentration, as shown in FIG. 3. The result shows that the maximum absorption is between 260nm and 265nm, and the wavelength 262nm is comprehensively considered and selected as the experimental wavelength.
Example 2
Weighing 0.5g of the product, precisely weighing, adding 100ml of 75% methanol solution, weighing, performing ultrasonic treatment for 1 hr, cooling, weighing again, supplementing the lost weight with 75% methanol solution, filtering with 0.45 μm microporous membrane, and collecting the filtrate as sample solution.
Preparation of control solutions: taking a proper amount of genistein reference substance, precisely weighing, and dissolving with mobile phase to obtain a reference substance solution containing 8 μ g of genistein per 1 ml.
The chromatographic conditions were as follows: according to the experiment of high performance liquid chromatography of appendix VI D of 2015 edition of the Chinese drug dictionary, a chromatographic column Phenomenex C18 has the column length of 250mm, the inner diameter of 4.6mm and the particle size of 5 mu m; methanol-water (50:50, v: v) is used as a mobile phase, and the flow rate is 1.0 ml/min; the wavelength is 262 nm; the sample amount is 10 mul; the column temperature was 40 ℃; the number of theoretical plates should not be less than 5000 calculated from adenosine peaks.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, respectively, injecting into a liquid chromatograph, measuring to obtain chromatogram (see figure 4), and calculating genistein content according to the chromatogram.
Sample preparation: the fermented cordyceps sinensis powder Cs-4 produced by the company has the following batch numbers: 21010014, 21010015 and 21010016, and the packages are all commercial packages. The results of the measurements are shown in the following table.
TABLE 1 results of three sample batches
As can be seen from the results in Table 1, the HPLC determination method for genistein in the fermented cordyceps sinensis powder Cs-4 established by the invention has the advantages of simplicity, convenience and accuracy, and is suitable for being used as an index component for quality control of the fermented cordyceps sinensis powder Cs-4.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (9)
1. A method for detecting genistein in fermented cordyceps sinensis powder Cs-4 is characterized in that the detection is carried out by high performance liquid chromatography, and comprises the following steps:
preparation of a test solution: precisely weighing fermented Cordyceps powder Cs-4, adding methanol water solution for dissolving, filtering with microporous membrane, and collecting filtrate as sample solution;
preparation of control solutions: taking a genistein reference substance, precisely weighing, and adding methanol-water mobile phase for dissolving to obtain a reference substance solution;
the determination method comprises the following steps: precisely absorbing the reference solution and the test solution respectively, injecting into a liquid chromatograph, taking methanol-water as a mobile phase, detecting the wavelength of 260-265 nm, determining to obtain a chromatogram, and calculating the content of genistein in the fermented cordyceps sinensis powder Cs-4 according to the chromatogram.
2. The method for detecting genistein in fermented cordyceps sinensis Cs-4 according to claim 1, wherein in the preparation of the test solution: the concentration of the aqueous methanol solution is 50 to 100 wt%.
3. The method for detecting genistein in fermented cordyceps sinensis Cs-4 according to claim 1, wherein in the preparation of the test solution: the method for dissolving the fermented Cordyceps powder Cs-4 comprises ultrasonic treatment for 30-120min or heating and refluxing for 30-120 min.
4. The method for detecting genistein in fermented cordyceps sinensis powder Cs-4 according to claim 1, wherein in the preparation of the test solution: the diameter of the microporous filter membrane is 0.3-0.6 μm.
5. The method for detecting genistein in fermented cordyceps sinensis powder Cs-4 according to claim 1, wherein in the preparation of the reference solution: the concentration of the control solution is 5-20 μ g per 1ml containing genistein.
6. The method for detecting genistein in fermented cordyceps sinensis powder Cs-4 according to claim 1, wherein in the preparation of the reference solution: the volume ratio of the methanol-water mobile phase is 30-90: 70-10.
7. The method for detecting genistein in fermented cordyceps sinensis powder Cs-4 according to claim 1, wherein in the assay, the chromatographic conditions are as follows: the column temperature is 20-50 ℃, the methanol-water volume ratio is 30-90: 70-10, and the flow rate of the mobile phase is 0.5-1.5 ml/min; the sample amount is 5-50 μ l.
8. The method for detecting genistein in fermented cordyceps sinensis Cs-4 according to claim 7, wherein in the determination method, a methanol-water volume ratio of 50:50 is used as a mobile phase, and a flow rate is 1.0 ml/min; the detection wavelength is 262 nm.
9. The method for detecting genistein in fermented cordyceps sinensis powder Cs-4 according to claim 1, wherein the preparation of the test solution specifically comprises: precisely weighing fermented Cordyceps powder Cs-4, adding 75% methanol solution, weighing, ultrasonic treating for 1 hr, cooling, weighing again, supplementing with 75% methanol solution, filtering with 0.45 μm microporous membrane, and collecting filtrate as sample solution.
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