CN100491999C - Method for detecting glossy ganoderma spore oil content in glossy ganoderma oil preparation - Google Patents
Method for detecting glossy ganoderma spore oil content in glossy ganoderma oil preparation Download PDFInfo
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Abstract
The measuring method measures contents of general ganoderma lucidum acid, ganoderma lucidum acid E in spore oil preparation of ganoderma lucidum. The testing standard is as following: in each 1.0g of spore oil preparation of ganoderma lucidum (equivalent to 5g of crude drugs of ganoderma lucidum spore), general ganoderma lucidum acid contained is not allowed less than 1.0mg counted by ganoderma lucidum acid E (C30H54O7); content of ganoderma lucidum acid E is between 0.001mg-0.050mg, i.e. content of ganoderma lucidum acid E is between 0.010%-0.50%. The invention makes stable testing and controlling quality of spore oil preparation of ganoderma lucidum.
Description
Technical field
The present invention relates to medicine detection method field, particularly relate to ganoderma lucidum spore oil content detecting method in a kind of Reishi sporule oil formulation.
Background technology
Reishi sporule (Ganoderma lucidiumspore) is the glossy ganoderma growth and maturity phase to eject ultrafine spore from cap, is the reproduction cell of glossy ganoderma, has the whole hereditary active substance of glossy ganoderma.That the pharmacological action of Reishi sporule mainly contains is antitumor, immunological regulation, adjusting blood fat and neural, cardiovascular and respiratory system had the improvement of adjusting effect.Ganoderma lucidum spore oil is that Reishi sporule passes through the oily shape extract that adopts methods such as supercritical extract or organic solvent extraction to obtain behind the broken wall.The complex chemical composition of ganoderma lucidum spore oil, compositions such as main oil-containing lipid, fatty acid, sterols and triterpenes.Modern pharmacological research shows that ganoderma lucidum spore oil is that Reishi sporule is antitumor, immunological regulation, adjusting blood fat the main force.And triterpenes components particularly the ganoderic acid constituents be the strongest active composition in glossy ganoderma, the Reishi sporule.
The method of Reishi sporule oil content is to adopt the content of total triterpene (in ursolic acid) in the visible spectrophotometry detection of drugs in the existing detection Reishi sporule oil formulation, and its standard is that total triterpene contents is more than 15～24.9%.Because of this method in the mensuration process of total triterpenoid, ursolic acid product have in contrast been adopted, but in the result of study of three terpene components of glossy ganoderma, lucidum spore powder, ganoderma lucidum spore oil, find no ursolic acid and exist, product are more appropriate in contrast should to adopt representative composition in its triterpene in the mensuration process; And this method is in the mensuration process of total triterpenoid, adopted with perchloric acid oxidation's colour developing, used the visible spectrophotometry colorimetric estimation, in the mensuration process, measurement result differs greatly along with difference such as the time, the temperature of colour developing, the time of mensuration of colour developing, the poor reproducibility of measurement result, the measurement result instability; Again because of this method in the mensuration process of total triterpenoid, total triterpene is not separated, directly adopted ganoderma lucidum spore oil to develop the color with the perchloric acid oxidation, and a large amount of long-chain fat hydro carbons composition that exists in the ganoderma lucidum spore oil also can be developed the color by the perchloric acid oxidation, thereby make to measure and false positive results occurs, using this method measures other edible vegetable oil and also can measure high level, can't form relation one to one with the content of three terpene components of ganoderma lucidum spore oil in the medicine, therefore, directly develop the color with ganoderma lucidum spore oil with the perchloric acid oxidation, the content that detects ganoderma lucidum spore oil three terpene components in the Reishi sporule oil formulation is subjected to the interference of other composition of ganoderma lucidum spore oil easily, is unfavorable for controlling aborning the quality with monitor drug.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists on the existing ganoderma lucidum spore oil detection method of content, provide a kind of detection steady quality, easily the ganoderma lucidum spore oil content detecting method of control.
To achieve these goals, the present invention adopts following technical scheme:
1, the assay of ganoderic acid E in the Reishi sporule oil formulation:
The preparation of reference substance solution: precision takes by weighing the reference substance through the ganoderic acid E of phosphorus pentoxide dried overnight, adds methyl alcohol and makes the solution that every 1ml contains 0.001～0.100mg, promptly gets reference substance solution;
The preparation of need testing solution: Reishi sporule oil formulation 5.0～15.0g decided in accurate title, add sherwood oil 50～150ml dissolving, add the silicagel column (10g that has handled well, on the 18mm * 400mm), add 100～200ml ethyl acetate: the mixed solution wash-out of sherwood oil (60～90 ℃)=65～75: 35～25, discard eluent, silicagel column continues to add 100～200ml ethyl acetate: methyl alcohol=80～90: 20～10 mixed solution wash-out, collect eluent, reclaim solvent to doing, residue adds dissolve with methanol, be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, promptly get need testing solution;
Use high effective liquid chromatography for measuring: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile-0.1% formic acid solution or acetonitrile-0.1% acetic acid solution 28～45:72～55 are moving phase; The detection wavelength is 252nm; Number of theoretical plate is pressed ganoderic acid E peak and is calculated, and is not less than 1500;
Measurement result: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject high performance liquid chromatograph, measure, calculate; Examination criteria be the content of ganoderic acid E in every 1.0g Reishi sporule oil formulation between 0.001mg～0.050mg, promptly the content of ganoderic acid E should be between 0.010%～0.50%.
Above-mentioned Reishi sporule oil formulation is clinical acceptable forms such as capsule (comprising Reishi sporule fat capsule, self-emulsifying Reishi sporule fat capsule, ganoderma lucidum spore oil liquid-filling capsule), emulsion, granule, injection, dripping pill, film or aerosol.
When the Reishi sporule oil formulation was Reishi sporule fat capsule, self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule, the content assaying method of ganoderic acid E also can adopt following method:
Chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile-0.1% formic acid solution 31:69 is a moving phase; The detection wavelength is 252nm; Number of theoretical plate is pressed ganoderic acid E peak and is calculated, and is not less than 1500;
The preparation of reference substance solution: precision takes by weighing the reference substance through the ganoderic acid E of phosphorus pentoxide dried overnight, adds methyl alcohol and makes the solution that every 1ml contains 0.010mg, promptly gets reference substance solution;
The preparation of need testing solution: get 30 in Reishi sporule fat capsule or self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule sample, cut off, incline and content, mixing, precision takes by weighing 10.0g, adds sherwood oil 100ml dissolving, add the silicagel column (10g that has handled well, on the 18mm * 400mm), add 150ml ethyl acetate: the mixed solution wash-out of sherwood oil (60～90 ℃)=70:30 discards eluent, silicagel column continues to add 150ml ethyl acetate: the mixed solution wash-out of methyl alcohol=85:15, collect eluent, reclaim solvent to doing, residue adds dissolve with methanol, be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, promptly get need testing solution;
Measurement result: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject high performance liquid chromatograph, measure, calculate; Examination criteria be the content of ganoderic acid E in every 1.0g Reishi sporule oil formulation between 0.001mg～0.050mg, promptly the content of ganoderic acid E should be between 0.010%～0.50%.
2, total ganoderic acid content is measured in the Reishi sporule oil formulation:
The preparation of reference substance solution: precision takes by weighing through the ganoderic acid E of phosphorus pentoxide dried overnight reference substance 2.5～7.5mg, places the 25ml measuring bottle, adds saturated NaHCO
3The solution dissolving, and be diluted to scale, shaking up, containing ganoderic acid E among every 1ml is 0.1～0.3mg;
The preparation of typical curve: precision is measured reference substance solution 0.5ml, 2.5ml, 5.0ml, 7.5ml, 10.0ml, puts respectively in the 10ml measuring bottle, adds saturated NaHCO
3Solution shakes up to scale; With corresponding saturated NaHCO
3Solution is blank, according to ultraviolet-spectrophotometric method, measures absorbance log at 262nm wavelength place, is that ordinate, concentration are horizontal ordinate with the absorbance log, the drawing standard curve;
Determination method: get Reishi sporule oil formulation 1～5g, the accurate title, decide, add sherwood oil 10～50ml dissolving, add the silicagel column (8g that has handled well, on the 18mm * 400mm), add 100～150ml ethyl acetate: the mixed solution wash-out of sherwood oil (60～90 ℃)=10～25:75～90, discard eluent, silicagel column continues to add 100～150ml ethyl acetate: the mixed solution wash-out of methyl alcohol=80～95:5～20, collect eluent, and reclaim solvent to doing, residue adds chloroform 20ml, ultrasonic 2min dissolving is shifted and is put in the separating funnel, uses saturated NaHCO
3Extract 4 times, each 20ml, combining extraction liquid is put in the 100ml measuring bottle, adds saturated NaHCO
3Solution dilution shakes up to scale, as need testing solution.According to spectrophotometric method, measure absorbance log at 262nm wavelength place, from the weight that typical curve is read ganoderic acid E the need testing solution, calculate, promptly; Examination criteria is to contain total ganoderic acid in ganoderic acid E in every 1.0g Reishi sporule oil formulation, should be no less than 1.0mg.
Above-mentioned Reishi sporule oil formulation is clinical acceptable forms such as capsule (comprising Reishi sporule fat capsule, self-emulsifying Reishi sporule fat capsule, ganoderma lucidum spore oil liquid-filling capsule), emulsion, granule, injection, dripping pill, film or aerosol.
When the Reishi sporule oil formulation was Reishi sporule fat capsule, self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule, the content assaying method of total ganoderic acid also can adopt following method:
The preparation of reference substance solution: precision takes by weighing through the ganoderic acid E of phosphorus pentoxide dried overnight reference substance 5.0mg, places the 25ml measuring bottle, adds saturated NaHCO
3The solution dissolving, and be diluted to scale, shaking up, containing ganoderic acid E among every 1ml is 0.2mg;
The preparation of typical curve: precision is measured reference substance solution 0.5ml, 2.5ml, 5.0ml, 7.5ml, 10.0ml, puts respectively in the 10ml measuring bottle, adds saturated NaHCO
3Solution shakes up to scale; With corresponding saturated NaHCO
3Solution is blank, according to ultraviolet-spectrophotometric method, measures absorbance log at 262nm wavelength place, is that ordinate, concentration are horizontal ordinate with the absorbance log, the drawing standard curve;
Determination method: 20 in Reishi sporule fat capsule or self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule sample, cut off, incline and content, mixing, precision takes by weighing 3.0g, adds sherwood oil 50ml dissolving, adds the silicagel column (8g that has handled well, on the 18mm * 400mm), add 120ml ethyl acetate: the mixed solution wash-out of sherwood oil (60～90 ℃)=25:75, discard eluent, silicagel column continues to add 120ml ethyl acetate: the mixed solution wash-out of methyl alcohol=85:15, collect eluent, reclaim solvent to doing, add chloroform 20ml, ultrasonic 2min dissolving, transfer is put in the separating funnel, uses saturated NaHCO
3Extract 4 times, each 20ml, combining extraction liquid is put in the 100ml measuring bottle, adds saturated NaHCO
3Solution dilution shakes up to scale, as need testing solution.According to spectrophotometric method, measure absorbance log at 262nm wavelength place, from the weight that typical curve is read ganoderic acid E the need testing solution, calculate, promptly; Examination criteria is to contain total ganoderic acid in ganoderic acid E in every 1.0g Reishi sporule oil formulation, should be no less than 1.0mg.
Compared with prior art, the present invention has following beneficial effect: the triterpenes chemical constitution is an active very high composition in the ganoderma lucidum spore oil in the ganoderma lucidum spore oil, and the triterpenic acid constituents is the major component of triterpenes components in the ganoderma lucidum spore oil, and content and Reishi sporule oil content are linear under certain condition.So on basis with the total ganoderic acid content of colorimetric method for determining, the content of one of ganoderic acid constituents ganoderic acid E in the ganoderma lucidum spore oil is measured, detect the content of ganoderma lucidum spore oil in the Reishi sporule oil formulation with this, Reishi sporule oil content quality standard is able to perfect, raising in the Reishi sporule oil formulation thereby make, and accomplishes the stable, controlled of the ganoderma lucidum spore oil quality of the pharmaceutical preparations conscientiously.
The present invention carries out first separation to the total ganoderic acid in the Reishi sporule oil formulation, is standard with the ganoderic acid E in total ganoderic acid, with the assay that ultraviolet spectrophotometry is carried out, and this method maturation, accurately and reliably.In order to control the quality of Reishi sporule oil formulation further, the present invention measures with high performance liquid chromatography first to the content of ganoderic acid E in the Reishi sporule oil formulation, this method accurately, stable, favorable reproducibility, recovery height, measure noiseless.
Description of drawings
Fig. 1 is a ganoderic acid E reference substance uv absorption spectra;
Fig. 2 is a ganoderma lucidum spore oil test sample uv absorption spectra.
Embodiment
Ganoderic acid E Determination on content in embodiment 1 ganoderma lucidum spore oil
(1) instrument and reagent
High performance liquid chromatograph comprises Waters 1525 pumps, 2487 binary channels UV-detector, Waters column oven, Breeze liquid chromatography workstation, Brasson ultrasonic cleaning instrument.Ganoderic acid E reference substance (extract to separate obtain in the Reishi sporule, through mp, IR, UV, MS,
1H-NMR etc. are accredited as ganoderic acid E, and it is 99.6% that the HPLC normalization method records purity), acetonitrile is a U.S. Tedia chromatographically pure, and water is ultrapure water, and it is pure that other reagent is analysis.Drug sample to be measured is a Reishi sporule fat capsule (lot number: 051114,051217,060123).
(2) chromatographic condition
Chromatographic column: Kromasil C18,4.6mm * 250mm, 5 μ m; Column temperature: 30 ℃; Moving phase: acetonitrile-0.1% formic acid solution (31:69); Detect wavelength 252nm; Flow velocity: 1.0ml/min.
(3) selection of detection wavelength
Get ganoderic acid E reference substance solution, in the interscan of 200～400nm scope, the result has absorption maximum at the 252nm place, so select 252nm as detecting wavelength.
(4) preparation of reference substance solution
Precision takes by weighing through the ganoderic acid E of phosphorus pentoxide dried overnight reference substance 2.675mg, puts in the measuring bottle of 25ml, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets reference substance solution.Its concentration is that every 1ml contains 0.107mg.
(5) preparation of need testing solution
Get 30 of Reishi sporule fat capsules, cut off, incline and content, mixing, precision takes by weighing 10.0g, adds sherwood oil 100ml dissolving, add the silicagel column (10g that has handled well, on the 18mm * 400mm), add 150ml ethyl acetate: the mixed solution wash-out of sherwood oil (60～90 ℃)=70:30 discards eluent, silicagel column continues to add 150ml ethyl acetate: the mixed solution wash-out of methyl alcohol=85:15, collect eluent, reclaim solvent to doing, residue adds dissolve with methanol, be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, promptly get need testing solution; (6) selection of eluting solvent in the need testing solution preparation
Respectively with sherwood oil → sherwood oil: ethyl acetate (90:10) → sherwood oil: ethyl acetate (80:20) → sherwood oil: ethyl acetate (70:30) → sherwood oil: ethyl acetate (60:40) → sherwood oil: ethyl acetate (50:50) → sherwood oil: ethyl acetate (40:60) → sherwood oil: ethyl acetate (35:65) → sherwood oil: ethyl acetate (25:75) → sherwood oil: ethyl acetate (10:90) → ethyl acetate → ethyl acetate: methyl alcohol (90:10) → ethyl acetate: the order of methyl alcohol (80:20) is carried out wash-out, collect the eluent of each section, carry out the assay analysis after concentrating, found that, at sherwood oil: wash-out section and ethyl acetate before the ethyl acetate (25:75): the wash-out Duan Zhongjun of methyl alcohol (8:2) does not find ganoderic acid E, the best eluting order that shows ganoderic acid E be with sherwood oil: the wash-out section wash-out impurity that ethyl acetate (25:75) was former, and with ethyl acetate: the wash-out section wash-out ganoderic acid E that methyl alcohol (90:10) is later.
(7) selection of eluting solvent consumption in the need testing solution preparation
Respectively with sherwood oil: ethyl acetate (40:60) 150ml → sherwood oil: ethyl acetate (30:70) 150ml → ethyl acetate: methyl alcohol (85:15) 150ml → ethyl acetate: methyl alcohol (80:20) 150ml is wash-out successively, collect the each several part eluent, the difference evaporate to dryness, and add methanol constant volume to 2ml.Sample introduction 10 μ l carry out assay respectively then, the result is at sherwood oil: ethyl acetate (40:60) wash-out partly contains significant quantities of fat oil, at sherwood oil: ethyl acetate (30:70) and ethyl acetate: methyl alcohol (80:20) part is not all found the chromatographic peak of ganoderic acid E, at ethyl acetate: methyl alcohol (85:15) part is found the chromatographic peak of ganoderic acid E, the consumption that eluting solvent is described is that 150ml both can effectively remove impurity, ganoderic acid E can be eluted fully again.
(8) high performance liquid chromatography moving phase: investigate ganoderic acid E at Kromasil C18 post, each parameter in the different moving phases, the result is good with the peak shape of ganoderic acid E under acetonitrile-1% formic acid or acetic acid aqueous solution (31:69), methyl alcohol-1% formic acid or acetic acid aqueous solution (48:52) condition, degree of separation all can be used as moving phase greater than 1.5.
(9) linear relationship is investigated
Precision is measured the solution of ganoderic acid E reference substance solution (0.107mg/ml) 5 μ l, 10 μ l, 15 μ l, 20 μ l, 25 μ l respectively, injects liquid chromatograph respectively, and (μ g) is horizontal ordinate with sample size, is ordinate with the peak area, the drawing standard curve.The results are shown in Table 1.
Table 1, linear relationship experimental data (n=5)
Y＝631929.1X-23648.2，R＝0.9999。
Above result shows: ganoderic acid E is good linear relationship in 0.535 μ g～2.675 μ g scopes.
(10) precision test: accurate ganoderic acid E reference substance solution (0.107mg/ml) the 10 μ l that draw, repeat sample introduction 5 times, RSD＜2% of ganoderic acid E peak area value the results are shown in Table 2.
Table 2 precision test figure (n=5)
Evidence, precision is good.
(10) stability test: sample thief test liquid (lot number 051114) is respectively at 0 hour, and 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, sample introduction 10 μ l respectively recorded RSD＜2% of ganoderic acid E peak area in the sample, the results are shown in Table 3.
Table 3 stability test data
Test findings shows that the sample test liquid is good at 24 hours internal stabilities.
(11) reappearance test: according to the operation down of assay item, same batch sample (lot number 051114) is measured for 5 parts, trying to achieve relative standard deviation RSD is 1.077%.
Table 4 reappearance test figure
Table 4 test findings shows that it is good that ganoderic acid E measures reappearance.
(12) recovery test: reclaim with application of sample, precision takes by weighing the sample (lot number: 051114 of known content, 0.01416mg/g) sample 10.0g, accurate respectively ganoderic acid E reference substance solution (0.04276mg/ml) 5ml that adds, according to the operation down of assay item, be calculated as follows the recovery, the results are shown in Table 5.
Table 5 ganoderic acid E recovery test result
Test findings shows that average recovery is good.
According to the operation of the method for above-mentioned assay, measured the content of ganoderic acid E in 6 batches of Reishi sporule oil formulations, the results are shown in Table 6.
The assay result (n=2) of ganoderic acid E in table 6 sample
The content that records ganoderic acid E in every 1g ganoderma lucidum spore oil is between 0.00252～0.04618mg.
Record every 1.0g Reishi sporule oil formulation (being equivalent to crude drug 5g) ganoderic acid E content between 0.00252～0.04618mg, consider the loss error in the industry amplification, on this basis, row fluctuates, and therefore the examination criteria that obtains is that every 1.0g Reishi sporule oil formulation (being equivalent to crude drug 5g) ganoderic acid E content is (being that ganoderic acid E content is between 0.01%～0.50%) between 0.001～0.050mg.
The mensuration of total ganoderic acid content in embodiment 2 ganoderma lucidum spore oils
(1) instrument and reagent
UV Agilent8453 ultraviolet-visible spectrophotometer (U.S. Anjelen Sci. ﹠ Tech. Inc).Drug sample to be measured be the Reishi sporule fat capsule (lot number: 051114,051217,060123), ganoderic acid E reference substance (self-control, purity is 99.6%); It is pure that agents useful for same is analysis.
(2) preparation of reference substance solution
Precision takes by weighing ganoderic acid E reference substance 5.36mg, puts in the 25ml measuring bottle, adds saturated NaHCO
3Solution dissolves, and is diluted to scale, in contrast product solution.
(3) need testing solution preparation
Get test sample content 3g, the accurate title, decide, and adds sherwood oil 50ml dissolving, add the silicagel column handled well and (on the 8g, 18mm * 400mm), add 150ml ethyl acetate: the mixed solution wash-out of sherwood oil (60～90 ℃)=25:75, discard eluent, silicagel column continues to add 150ml ethyl acetate: the mixed solution wash-out of methyl alcohol=90:10, collect eluent, and reclaim solvent to doing, residue adds chloroform 20ml, ultrasonic 2min dissolving is shifted and is put in the separating funnel, uses saturated NaHCO
3Extract 4 times, each 20ml, combining extraction liquid is put in the 100ml measuring bottle, adds saturated NaHCO
3Solution dilution is to scale, as the ganoderma lucidum spore oil need testing solution.
(4) the method feasibility is investigated
Get above-mentioned ganoderic acid E reference substance solution, need testing solution respectively, with saturated NaHCO
3One times of solution dilution carries out UV scanning in 200～400nm wavelength coverage, record UV scanning figure sees Fig. 1, the ultraviolet spectrogram basically identical of ganoderic acid E reference substance, ganoderma lucidum spore oil as a result, and all absorption maximum is arranged at 262nm wavelength place.So determine with saturated NaHCO3 solution to be solvent, measure wavelength and be defined as 262nm.
(5) typical curve preparation
Accurate respectively ganoderic acid E reference substance solution (0.2144mgml-1) 0.5,2.5,5.0,7.5, the 10.0ml of drawing puts in the 10ml measuring bottle, adds saturated NaHCO
3Solution dilution is measured absorption value to scale at 262nm wavelength place, be horizontal ordinate with concentration, and absorbance log is an ordinate, basis of calculation curve, and ganoderic acid E is good linear relationship in 0.01072～0.2144mgml-1 scope as a result; Regression equation is Y=9.671X+0.0866, r=0.9995.
(6) stability test
Get test sample (lot number 051114) content, be equipped with the ganoderma lucidum spore oil need testing solution according to (3) below legal system, respectively 0,0.5,1,2,4,8,12,24h, measure absorption value in 262nm wavelength place, the result shows that absorption value does not have significant change in the 24h, and RSD is 1.57%.
(7) replica test
Get 5 parts of test sample (lot number 051114) contents, be equipped with need testing solution according to (3) below legal system respectively, measure absorption value at 262nm wavelength place, calculate the content (in ganoderic acid E) of total ganoderic acid in each bottle sample.RSD is 1.84% (n=5) as a result.
(8) determination of recovery rates
Precision takes by weighing test sample (lot number 051114) content 2.0g, totally 5 parts, accurate respectively ganoderic acid E reference substance chloroformic solution (0.502mgml-1) 3.0ml that adds, be equipped with need testing solution according to (3) below legal system, measure absorption value at 262nm wavelength place, calculate, average recovery rate is 99.42% as a result, RSD is 0.57%, the results are shown in Table 7.
Table 7 recovery test result
Test findings shows that average recovery is good.
According to the method operation of the assay of total ganoderic acid in the above-mentioned Reishi sporule oil formulation, measured the content of total ganoderic acid in 6 batches of Reishi sporule oil formulations, the results are shown in Table 8.
The assay result (n=2) of total ganoderic acid in table 8 sample
Claims (9)
1, ganoderma lucidum spore oil content detecting method in a kind of Reishi sporule oil formulation is characterized in that comprising the steps: by measuring the content of ganoderic acid E in the Reishi sporule oil formulation
(1) preparation of reference substance solution: the dry ganoderic acid E reference substance of learning from else's experience, add methyl alcohol and make the solution that every 1ml contains 0.001～0.100mg, promptly get reference substance solution;
(2) preparation of need testing solution: get the Reishi sporule oil formulation, add petroleum ether dissolution, add on the silicagel column, add 60～90 ℃ ethyl acetate: the mixed solution wash-out of sherwood oil 65～75:35～25 discards eluent, silicagel column continues to add ethyl acetate: the mixed solution wash-out of methyl alcohol 80～90:20～10, collect eluent, reclaim solvent to doing, residue adds dissolve with methanol, shake up, promptly get need testing solution;
(3) use high effective liquid chromatography for measuring: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile-0.1% formic acid solution or acetonitrile-0.1% acetic acid solution 28～45:72～55 are moving phase; The detection wavelength is 252nm; Draw reference substance solution and need testing solution respectively, inject high performance liquid chromatograph, measure, calculate the content of ganoderic acid E; The content of ganoderic acid E should be between 0.001～0.050 milligram in every kairine Ganoderma lucidum spore oil formulation.
2, detection method according to claim 1, it is characterized in that being prepared as of step (2) described need testing solution: get Reishi sporule oil formulation 5.0～15.0g, add sherwood oil 50～150ml dissolving, add on the silicagel column, add 100～200ml, 60～90 ℃ ethyl acetate: the mixed solution wash-out of sherwood oil 65～75:35～25, discard eluent, silicagel column continues to add 100～200ml ethyl acetate: the mixed solution wash-out of methyl alcohol 80～90:20～10, collect eluent, reclaim solvent to doing, residue adds dissolve with methanol, be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, promptly get need testing solution;
3, detection method according to claim 1 is characterized in that comprising the steps:
(1) preparation of reference substance solution: the dry ganoderic acid E reference substance of learning from else's experience, add methyl alcohol and make the solution that every 1ml contains 0.010mg, promptly get reference substance solution;
(2) preparation of need testing solution: get Reishi sporule fat capsule or self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule sample, cut off, incline and content, mixing, sample thief, add petroleum ether dissolution, add on the silicagel column, add 60～90 ℃ ethyl acetate: the mixed solution wash-out of sherwood oil 70:30 discards eluent, silicagel column continues to add ethyl acetate: the mixed solution wash-out of methyl alcohol 85:15, collect eluent, reclaim solvent to doing, residue adds dissolve with methanol, shake up, promptly get need testing solution;
(3) use high effective liquid chromatography for measuring: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile-0.1% formic acid solution 31:69 is a moving phase; The detection wavelength is 252nm; Draw reference substance solution and need testing solution respectively, inject high performance liquid chromatograph, measure, calculate the content of ganoderic acid E.
4, ganoderma lucidum spore oil content detecting method in a kind of Reishi sporule oil formulation is characterized in that comprising the steps: by measuring total ganoderic acid content in the Reishi sporule oil formulation
(1) preparation of reference substance solution: the dry ganoderic acid E reference substance of learning from else's experience adds saturated NaHCO
3The solution dissolving shakes up, and containing ganoderic acid E among every 1ml is 0.1～0.3mg;
(2) preparation of typical curve: measure reference substance solution 0.5ml, 2.5ml, 5.0ml, 7.5ml, 10.0ml, put respectively in the 10ml measuring bottle, add saturated NaHCO
3Solution shakes up to scale; With corresponding saturated NaHCO
3Solution is blank, according to ultraviolet-spectrophotometric method, measures absorbance log at 262nm wavelength place, is that ordinate, concentration are horizontal ordinate with the absorbance log, the drawing standard curve; (3) preparation of need testing solution: get the Reishi sporule oil formulation, add petroleum ether dissolution, add on the silicagel column, add 60～90 ℃ ethyl acetate: the mixed solution wash-out of sherwood oil 10～25:75～90, discard eluent, silicagel column continues to add ethyl acetate: the mixed solution wash-out of methyl alcohol 80～95:5～20, collect eluent, and reclaim solvent to doing, residue adds chloroform, ultrasonic dissolution shifts and puts in the separating funnel, uses saturated NaHCO
3Extraction, combining extraction liquid adds saturated NaHCO
3Solution dilution shakes up, as need testing solution;
(4) according to spectrophotometric method, measure absorbance log at 262nm wavelength place, from the weight that typical curve is read ganoderic acid E the need testing solution, calculate total ganoderic acid content in ganoderic acid E; Contain total ganoderic acid in ganoderic acid E in every kairine Ganoderma lucidum spore oil formulation, should be less than 1 milligram.
5, detection method according to claim 4, it is characterized in that being prepared as of step (3) described need testing solution: get Reishi sporule oil formulation 1～5g, add sherwood oil 10～50ml dissolving, add on the silicagel column, add 100～150ml, 60～90 ℃ ethyl acetate: the mixed solution wash-out of sherwood oil 10～25:75～90, discard eluent, silicagel column continues to add 100～150ml ethyl acetate: the mixed solution wash-out of methyl alcohol 80～95:5～20, collect eluent, reclaim solvent to doing, residue adds chloroform 20ml, ultrasonic 2min dissolving, transfer is put in the separating funnel, uses saturated NaHCO
3Extract 4 times, each 20ml, combining extraction liquid to the 100ml measuring bottle, adds saturated NaHCO
3Solution dilution shakes up to scale, as need testing solution.
6, detection method according to claim 4 is characterized in that comprising the steps:
(1) preparation of reference substance solution: the dry ganoderic acid E reference substance of learning from else's experience adds saturated NaHCO
3The solution dissolving shakes up, and containing ganoderic acid E among every 1ml is 0.2mg;
(2) preparation of typical curve: measure reference substance solution 0.5ml, 2.5ml, 5.0ml, 7.5ml, 10.0ml, put respectively in the 10ml measuring bottle, add saturated NaHCO
3Solution shakes up to scale; With corresponding saturated NaHCO
3Solution is blank, according to ultraviolet-spectrophotometric method, measures absorbance log at 262nm wavelength place, is that ordinate, concentration are horizontal ordinate with the absorbance log, the drawing standard curve;
(3) preparation of need testing solution: get Reishi sporule fat capsule or self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule sample, cut off, incline and content, mixing, sample thief adds petroleum ether dissolution, add on the silicagel column, add 60～90 ℃ of ethyl acetate: the mixed solution wash-out of sherwood oil 25:75, discard eluent, silicagel column continues to add ethyl acetate: the mixed solution wash-out of methyl alcohol 85:15, collect eluent, reclaim solvent to doing, add chloroform, ultrasonic dissolution, transfer is put in the separating funnel, uses saturated NaHCO
3Extraction, combining extraction liquid adds saturated NaHCO
3Solution dilution shakes up, as need testing solution;
(4) according to spectrophotometric method, measure absorbance log at 262nm wavelength place, from the weight that typical curve is read ganoderic acid E the need testing solution, calculate total ganoderic acid content in ganoderic acid E.
7,, it is characterized in that adopting the dry ganoderic acid E of phosphorus pentoxide reference substance according to claim 1 or 4 described detection methods.
8,, it is characterized in that described Reishi sporule oil formulation is capsule, emulsion, granule, injection, dripping pill, film or aerosol according to each described detection method of claim 1～6.
9, according to claim 3 or 6 described detection methods, it is characterized in that described Reishi sporule oil formulation is a capsule preparations, this capsule preparations is Reishi sporule fat capsule, self-emulsifying Reishi sporule fat capsule or ganoderma lucidum spore oil liquid-filling capsule.
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| CN109212085B (en) * | 2018-10-22 | 2021-05-25 | 广州白云山汉方现代药业有限公司 | Fingerprint of ganoderma lucidum spore oil and construction method and application thereof |
| CN111562250A (en) * | 2020-05-26 | 2020-08-21 | 河北省食品检验研究院 | Method for rapidly detecting ganoderic acid G in ganoderma lucidum spore oil |
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