CN102735772A - Quality controlling method of ganoderma aqueous extract - Google Patents
Quality controlling method of ganoderma aqueous extract Download PDFInfo
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Abstract
The invention relates to a quality analysis analysis method of medicines or health care products, and specifically relates to a quality controlling method of a ganoderma aqueous extract. For controlling the product quality with high specificity, according to the invention, crude polysaccharide contents and a sum of ganoderma acid A, B, and C2 contents are adopted as quality controlling indexes of the ganoderma aqueous extract. According to the invention, ganoderma crude polysaccharide is extracted with a water extraction and alcohol precipitation method; polysaccharide with a glucan structure is precipitated by using a copper reagent; and the content of the polysaccharide is determined by using an ultraviolet spectrophotometry method. Therefore, the influences of auxiliary materials on determination are eliminated, and a basis is provided for quality controlling of the ganoderma aqueous extract. The contents of the characteristic components ganoderma acid A, B, and C2 in ganoderma lucidum, ganoderma sinnese, and ganoderma tsugae are relatively high. Compared to a currently commonly-used colorimetric method for determining total triterpenoid contents, the specificity of the method provided by the invention is high. With the method, purposes of authentication and quality control can be achieved.
Description
Technical field
The present invention relates to a kind of medicine or health quality analysis method, be specifically related to a kind of method of quality control of glossy ganoderma water extract.
Background technology
Glossy ganoderma is the red sesame of Polyporaceae fungi
Ganoderma lucidum(Leyss.ex Fr.) Karst. or purple sesame
Ganoderma sinenseZhao, Xu et Zhang or Ganoderma tsugae
GanodermaThe dry fructification of tsugae Murr. has invigorating qi for tranquilization, effect such as relieving cough and asthma.The glossy ganoderma extraction process mainly contains that water is carried and alcohol extracting, and most extraction process by water that adopts, promptly adopt the red sesame of Polyporaceae fungi (
Ganoderma lucidum(Leyss. ex Fr.) Karst.), purple sesame (
Ganoderma sinneseZhao, Xu et Zhang) and Ganoderma tsugae (
GanodermaTsugae Murr.) dry fructification is extracted, is concentrated, makes after the drying through water.So the present invention adopts the name of glossy ganoderma water extract.Research shows, mainly contains number of chemical compositions such as polysaccharide, triterpene, nucleosides, alkaloid, flavones, amino acid and trace element in the glossy ganoderma.At present, the glossy ganoderma pharmacology activity research is mainly concentrated on GL-B and ganodenic acid constituents, therefore, GL-B and triterpenes components be as the main functional component of glossy ganoderma, necessary its content measured.
The present invention studies the assay method of the significant composition of healthy food material glossy ganoderma water extract in a series of experimentations.GL-B is one of main active in the glossy ganoderma, has immunological regulation, multiple pharmacologically active such as antitumor, anti-oxidant, hypoglycemic.At present, some enterprise adds various auxiliary materials such as dextrin, starch in order to reach dry even to make a false report the purpose of ganoderma polyoses content in the glossy ganoderma water extract of being everlasting.If the method by version Chinese Pharmacopoeia in 2010 is measured, it is higher to cause GL-B to measure the result.This research is extracted ganoderma lucidum crude polysaccharide through water extraction and alcohol precipitation method, precipitates the polysaccharide that wherein has glucan structure with DDTC, adopts its content of determined by ultraviolet spectrophotometry, has got rid of the influence of auxiliary material to measuring, for the quality control of glossy ganoderma water extract provides foundation.
Ganoderma lucidum triterpene compounds is another main pharmacodynamics composition of glossy ganoderma, owing to have anticancerly, anti-inflammatory anti-oxidantly waits pharmacologically active and receives much concern.The inventor finds in the employing high performance liquid chromatography is studied glossy ganoderma water extract triterpenes components, contains content higher relatively ganoderic acid A, B, C in the glossy ganoderma water extract
2Contrast is the method for colorimetric method for determining total triterpene contents commonly used at present, and high specificity can reach and differentiate and the purpose of quality control.
Summary of the invention
In order to control product quality targetedly, the present invention adopts crude polysaccharides content and ganoderic acid A, B, C
2The content sum provides a kind of method of quality control of glossy ganoderma water extract as the quality control index of glossy ganoderma water extract.
The technical scheme that the present invention takes is following:
With ganoderic acid A, ganoderic acid B, ganoderic acid C
2Content sum and crude polysaccharides content are as the quality control index of glossy ganoderma water extract.
Said ganoderic acid A, B content detecting method are following:
(1) preparation of reference substance solution
Get ganoderic acid A, the B reference substance of drying under reduced pressure to constant weight, add methyl alcohol and process the mixed mark solution that every 1ml contains 164 μ g ganoderic acid A and contains 52 μ g ganoderic acid B.
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in the 25 ml volumetric flasks, add an amount of sonicated 10 min of methyl alcohol, put coldly, add methyl alcohol and be diluted to scale, shake up, filter.
(3) high performance liquid chromatography detects
With the octyl bonded silica gel is filling agent, and acetonitrile is a mobile phase A, and 0.1 % aqueous acetic acid is a Mobile phase B, gradient elution; Detecting wavelength is 257 nm; Column temperature is 22 ℃; Flow velocity is 1.0 ml/min; Number of theoretical plate is pressed ganoderic acid A and is calculated, and should be not less than 5000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.
Said ganoderic acid C
2Content detecting method is following:
(1) preparation of reference substance solution
Get the ganoderic acid C of drying under reduced pressure to constant weight
2Reference substance adds methyl alcohol and processes per 1 ml and contain 33 μ g ganoderic acid C
2Reference substance solution.
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in the 25 ml volumetric flasks, add an amount of sonicated 10 min of methyl alcohol, put coldly, add methyl alcohol and be diluted to scale, shake up, filter.
(3) high performance liquid chromatography detects
With the octadecyl silane is filling agent, and acetonitrile is a mobile phase A, and 0.1 % aqueous acetic acid is a Mobile phase B, gradient elution; Detecting wavelength is 257 nm; Column temperature is 20 ℃; Flow velocity is 1.0 ml/min; Number of theoretical plate is pressed ganoderic acid C
2Calculate, should be not less than 5000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.
Said crude polysaccharides content detecting method is following:
(1) typical curve is drawn:
It is an amount of to get the glucosan reference substance that is dried to constant weight, and precision weighing adds water and processes the solution that per 1 ml contains glucosan 1 mg.Accurate respectively the absorption in above-mentioned standard solution 0.1,0.2,0.4,0.6,0.8,1.0 ml to the 10.0 ml measuring bottles, thin up is drawn respectively in above-mentioned solution 2.0 ml to the 25 ml color comparison tubes to scale; Add 50 g/L phenol solution, 1.0 ml, mixing, the accurate concentrated sulphuric acid 10.0 ml that add; Mixing is put and is boiled 2 min, mixing in the water-bath; Measuring absorbance with spectrophotometer in 485 nm wavelength after being cooled to room temperature, is blank with the reagent corresponding, is horizontal ordinate with glucosan concentration; Absorbance is an ordinate, the drawing standard curve.
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.4 g, put in the 50 ml conical flasks, add water 25.0 ml, ultrasonic 10 min filter; Draw in filtrating 5.0 ml to the 50 ml centrifuge tubes, slowly add absolute ethyl alcohol 20 ml, the limit edged shakes up, and places 12 h for 4 ℃; Take out centrifugal 8 min of 3000 r/min, abandoning supernatant; Deposition is dissolved in water and is transferred in the 10.0 ml volumetric flasks, adds the water constant volume, shakes up; Draw solution 2.0 ml behind the above-mentioned constant volume, place 15 ml centrifuge tubes, add 100 g/L sodium hydroxide solutions, 2.0 ml, DDTC 2.0 ml, place boiling water bath to boil 3 min, be cooled to room temperature, centrifugal 8 min of 3000 r/min, abandoning supernatant; Deposition is dissolved with 10 % sulfuric acid solutions, 2.0 ml and is transferred in the 10.0 ml volumetric flasks, adds the water constant volume, shakes up, and promptly gets.
(3) sample determination
Draw need testing solution 2.0 ml, the sighting target directrix curve is drawn item down, from " adding 50 g/L phenol solution, 1.0 ml ", measures absorbance in accordance with the law, utilizes typical curve to calculate the content of glucosan.
Significant component target is: the crude polysaccharides content>=30.7mg/g of mensuration, in glucosan; Ganoderic acid A, B, C
2The content sum, in dry product, red sesame, Ganoderma tsugae water extract>=13.3mg/g, purple sesame water extract>=1.8mg/g.
This research is extracted ganoderma lucidum crude polysaccharide through water extraction and alcohol precipitation method, precipitates the polysaccharide that wherein has glucan structure with DDTC, adopts its content of determined by ultraviolet spectrophotometry, has got rid of the influence of auxiliary material to measuring, for the quality control of glossy ganoderma water extract provides foundation.
Red sesame, purple sesame and Ganoderma tsugae all contain relative content higher characteristic component ganoderic acid A, B, C
2Contrast is the method for colorimetric method for determining total triterpene contents commonly used at present, and high specificity can reach and differentiate and the purpose of quality control.
Description of drawings
Fig. 1 is that ganoderic acid A, B mix mark solution chromatogram, peak 1: ganoderic acid B, peak 2: ganoderic acid A.
Fig. 2 is the need testing solution chromatogram, peak 1: ganoderic acid B, peak 2: ganoderic acid A.
Fig. 3 is ganoderic acid C
2The reference substance solution chromatogram, peak 3: ganoderic acid C
2
Fig. 4 is the need testing solution chromatogram, peak 3: ganoderic acid C
2
Embodiment
These article in following examples refer to the confession test agent of glossy ganoderma water extract.
The A1.1 principle
Relative molecular mass is greater than 1 * 10 in the sample
4Polymer substance in 80% ethanolic solution, precipitate; Separate with compound sugar with monose in the WS; From other polymer substances, precipitate polysaccharide with alkaline cupric reagent selectivity ground, generate orange red compound with the phenolsulfuric acid reaction, through its content of colorimetric estimation with glucan structure; Its colored intensity is directly proportional with the content of glucosan in the crude polysaccharides, calculates the content of crude polysaccharides in this product with this.
A1.2 reagent
A1.2.1 reagent and material
A1.2.1.1 ethanol: analyze pure.
The A1.2.1.2 concentrated sulphuric acid: analyze pure.
A1.2.1.3 sodium hydroxide solution (100 g/L): take by weighing 25 g NaOH, be dissolved in water and be diluted to 250 ml.Add solid water-free sodium sulphate to saturated, subsequent use.
A1.2.1.4 DDTC storing solution: take by weighing 0.75 g CuS0
45H
2O, the 7.5g sodium citrate is dissolved in water and is diluted to 250 ml, and mixing is subsequent use.
A1.2.1.5 DDTC solution: get DDTC storing solution 50 ml, add water 50 ml, add solid water-free sodium sulphate 12.5 g behind the mixing and make its dissolving.Interim adapted.
A1.2.1.6 phenol (50 g/L): take by weighing phenol 5.0 g, thin up to 100 ml, mixing, its solution place refrigerator can preserve one month.
A1.2.1.7 glucosan standard reserving solution: the glucosan reference substance that get molecular weight 500000, is dried to constant weight is an amount of, and precision weighing adds water and processes the solution that per 1 ml contains glucosan 1 mg.
The A1.3 instrument
The A1.3.1 ultraviolet spectrophotometer.
The A1.3.2 ultrasonic cleaner.
A1.3.3 hydro-extractor (3000 r/min).
The A1.4 analytical procedure
The preparation of A1.4.1 typical curve
Accurate respectively the absorption in glucosan standard reserving solution 0.1,0.2,0.4,0.6,0.8,1.0 ml to the 10.0 ml measuring bottles, thin up is drawn respectively in above-mentioned solution 2.0 ml to the 25 ml color comparison tubes to scale; Add 50 g/L phenol solution, 1.0 ml, mixing, the accurate concentrated sulphuric acid 10.0 ml that add; Mixing is put and is boiled 2 min, mixing in the water-bath; Measuring absorbance with spectrophotometer in 485 nm wavelength after being cooled to room temperature, is blank with the reagent corresponding, is horizontal ordinate with glucosan concentration; Absorbance is an ordinate, the drawing standard curve.
The preparation of A1.4.2 sample solution
Precision takes by weighing these article 0.4 g, puts in the 50 ml conical flasks, adds water 25.0 ml, and ultrasonic 10 min filter.Draw in filtrating 5.0 ml to the 50 ml centrifuge tubes, slowly add absolute ethyl alcohol 20 ml, the limit edged shakes up, and places 12 h for 4 ℃; Take out, with centrifugal 8 min of 3000 r/min, abandoning supernatant; Deposition is dissolved in water and is transferred in the 10.0 ml volumetric flasks, and thin up shakes up to scale.Draw above-mentioned solution 2.0 ml, place 15 ml centrifuge tubes, add 100 g/L sodium hydroxide solutions, 2.0 ml, DDTC 2.0 ml, place boiling water bath to boil 3 min, be cooled to room temperature, with centrifugal 8 min of 3000 r/min, abandoning supernatant.Deposition is dissolved with 10 % sulfuric acid solutions, 2.0 ml and is transferred in the 10.0 ml volumetric flasks, and thin up shakes up to scale, promptly gets.Do sample blank by above-mentioned steps simultaneously.
The A1.4.3 sample determination
Draw sample solution 2.0 ml and put in the 25 ml color comparison tubes, sighting target directrix curve preparation item from " adding 50 g/L phenol solution, 1.0 ml ", is measured absorbance down in accordance with the law, from the weight that typical curve is read glucosan, calculates, and promptly gets.
A1.4.4 result calculates
The content of crude polysaccharides (in glucosan) in X-sample, mg/g
m
1The quality of glucosan in the-sample determination liquid, mg
m
2Glucosan quality in the-sample blank liquid, mg
m
3-sample quality, g
V
1-sample extracting solution cumulative volume, ml
V
2-deposition crude polysaccharides specimen in use extracting liquid volume, ml
V
3-crude polysaccharides liquor capacity, ml
V
4The used crude polysaccharides liquor capacity of-deposition glucosan, ml
V
5-sample determination liquid cumulative volume, ml
V
6-test sample liquor capacity, ml
The A1.4.5 tolerance
The twice independent result's of mensuration who under repeated condition, obtains absolute difference must not surpass 10 % of arithmetic mean.
The A2.1 principle
Sample with methyl alcohol as solvent, ultrasonic Extraction, ganoderic acid A, ganoderic acid B are through performance liquid chromatographic column (C
8) separate; Ganoderic acid C
2Through performance liquid chromatographic column (C
18) separate, use UV-detector (UV) to detect respectively, qualitative according to the retention time of chromatographic peak, the peak area external standard method is quantitative, measures ganoderic acid A in the sample, B and ganoderic acid C respectively
2Content, and calculate its total amount.
A2.2 reagent
A2.2.1
Water: purified water.
A2.2.2
Acetonitrile: chromatographically pure.
A2.2.3
Glacial acetic acid: analyze pure.
A2.2.4 methyl alcohol: analyze pure.
A2.2.5
Ganoderic acid A, B, C
2Reference substance: purity>=98 % is provided by institute of Materia Medica,Chinese Academy of Medical Sciences.
The A2.3 instrument
A2.3.1 high performance liquid chromatograph (UV-detector).
The A2.3.2 ultrasonic cleaner.
The A2.4 analytical procedure
A2.4.1
Ganoderic acid A, B measure
The A2.4.1.1 chromatographic condition
With the octyl bonded silica gel is bonding phase (chromatographic column: Agilent Zorbax-SB C
8150 mm * 4.6 mm, 3.5 μ m or similar specification chromatographic column), acetonitrile is a mobile phase A, and 0.1 % aqueous acetic acid is a Mobile phase B, and the regulation in the according to the form below is carried out gradient elution; Detecting wavelength is 257 nm; Column temperature is 22 ℃; Flow velocity is 1.0 ml/min.
Table 1 eluent gradient type of elution
The test of A2.4.1.2 system suitability
Number of theoretical plate calculates by ganoderic acid A should be not less than 5000.
The preparation of A2.4.1.3 reference substance solution
Ganoderic acid A, the B reference substance of getting drying under reduced pressure to constant weight are an amount of, accurate claim surely, add methyl alcohol and process per 1 ml and contain the mixed mark solution that 164 μ g ganoderic acid A and per 1 ml contain 52 μ g ganoderic acid B, promptly get.
The preparation of A2.4.1.4 need testing solution
Get about 0.5 g of these article, put in the 25 ml volumetric flasks, add an amount of sonicated 10 min of methyl alcohol, put coldly, add methyl alcohol and be diluted to scale, shake up, filter.
The A2.4.1.5 determination method
Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.Qualitative with the main peak retention time, peak area quantification.Result such as Fig. 1, Fig. 2.
A2.4.1.6 calculates
The content of ganoderic acid A is calculated as follows in the A2.4.1.6.1 glossy ganoderma water extract:
Y
1---the content of ganoderic acid A, unit are milligram every gram (mg/g);
A
x---the peak area of ganoderic acid A in the need testing solution chromatogram;
A
s---the peak area of ganoderic acid A in the reference substance solution chromatogram;
C---the concentration of ganoderic acid A in the reference substance solution, unit is every milliliter (mg/mL) of milligram;
M---sample mass, unit is gram (g);
X---sample moisture (%).
The content of ganoderic acid B is calculated as follows in the A2.4.1.6.2 glossy ganoderma water extract:
Y
2---the content of ganoderic acid B, unit are milligram every gram (mg/g);
A
x---the peak area of ganoderic acid B in the need testing solution chromatogram;
A
s---the peak area of ganoderic acid B in the reference substance solution chromatogram;
C---the concentration of ganoderic acid B in the reference substance solution, unit is every milliliter (mg/mL) of milligram;
M---sample mass, unit is gram (g);
X---sample moisture (%).
A2.4.2 ganoderic acid C
2Measure
A2.4.2.1 chromatographic condition and system suitability test
With the octadecyl silane is bonding phase (chromatographic column: Agilent Zorbax-SB C
18250 mm * 4.6 mm, 5 μ m or similar specification chromatographic column), acetonitrile is a mobile phase A, and 0.1 % aqueous acetic acid is a Mobile phase B, and the regulation in the according to the form below is carried out gradient elution; Detecting wavelength is 257 nm; Column temperature is 20 ℃; Flow velocity is 1.0 ml/min.
Table 2 eluent gradient type of elution
The test of A2.4.2.2 system suitability
Number of theoretical plate is pressed ganoderic acid C
2Calculating should be not less than 5000.
The preparation of A2.4.2.3 reference substance solution
Get the ganoderic acid C of drying under reduced pressure to constant weight
2Reference substance is an amount of, accurate claims surely, adds methyl alcohol and processes every 1ml and contain 33 μ g ganoderic acid C
2Reference substance solution, promptly get.
The preparation of A2.4.2.4 need testing solution
Get about 0.5 g of these article, put in the 25 ml volumetric flasks, add an amount of sonicated 10 min of methyl alcohol, put coldly, add methyl alcohol and be diluted to scale, shake up, filter.
The A2.4.2.5 determination method
Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.Qualitative with the main peak retention time, peak area quantification.Result such as Fig. 3, Fig. 4.
A2.4.2.6 calculates
Ganoderic acid C in the glossy ganoderma water extract
2Content be calculated as follows:
Y
3---ganoderic acid C
2Content, unit be the milligram every gram (mg/g);
A
x---ganoderic acid C in the need testing solution chromatogram
2Peak area;
A
s---ganoderic acid C in the reference substance solution chromatogram
2Peak area;
C---ganoderic acid C in the reference substance solution
2Concentration, unit be the milligram every milliliter (mg/mL);
M---sample mass, unit is gram (g);
X---sample moisture (%).
A2.4.3 ganoderic acid A, B, C
2Total amount (Y) is calculated
Y=Y
1+Y
2+Y
3
Y---ganoderic acid A, B, C
2Total amount, unit be the milligram every gram (mg/g);
Y
1---the content of ganoderic acid A, unit are milligram every gram (mg/g);
Y
2---the content of ganoderic acid B, unit are milligram every gram (mg/g);
Y
3---ganoderic acid C
2Content, unit be the milligram every gram (mg/g).
The A2.4.4 tolerance
The twice independent result's of mensuration who under repeated condition, obtains absolute difference must not surpass 10% of arithmetic mean.
Significant component target should meet the regulation of table 3.
The significant component target of table 3
Embodiment 4 measures the research of the method for crude polysaccharides content in the glossy ganoderma water extract
The checking of 1 methodology
1.1 linear experiment is according to the method drawing standard curve of above-mentioned typical curve preparation; With glucosan concentration is horizontal ordinate, and absorbance is an ordinate, and getting equation of linear regression is y=0.00514x+0.00571; Related coefficient γ=0.9996, the range of linearity are 21.76 μ g～217.6 μ g.The result sees table 4.
The linear table as a result of table 4 crude polysaccharides
1.2 glossy ganoderma (red sesame D9) the water extract sample of known content is got in accuracy and precision test; Adopt the application of sample recovery test, precision adds a certain amount of glucosan reference substance, prepares the test sample of 3 variable concentrations respectively; The test sample that each prepared at concentrations is 6 parts is done sample blank simultaneously.Preparation method and condition determination by above-mentioned need testing solution are measured, and calculate recovery rate, and the result sees table 5.
Accuracy of table 5 crude polysaccharides and precision test determination result
1.3 replica test is got 6 parts in same glossy ganoderma (red sesame D9) water extract sample, prepares need testing solution respectively, measures by above-mentioned method, the result shows that this method has good repeatability.The result sees table 6.
Table 6 replica test is measured the result
1.4 investigating this experiment, the method durability use different instruments that the crude polysaccharides content in same glossy ganoderma (the red sesame D9) water extract is measured; The result shows; Use the different model ultraviolet spectrophotometer, crude polysaccharides content RSD 3%, show this method good tolerance.Measure the result and see table 7.
Table 7 serviceability test result
2 conclusions
Adopt ultraviolet spectrophotometer to measure crude polysaccharides content in the glossy ganoderma water extract, its disturbing factor is starch, dextrin and other carbohydrates.In order to get rid of above-mentioned interference, this method adopts the DDTC separation and purification in the mensuration process, and experiment shows can obtain satisfactory result.
Ganoderic acid A, ganoderic acid B, ganoderic acid C in the embodiment 5 high effective liquid chromatography for measuring glossy ganoderma water extracts
2The research of the method for content
The checking of 1 methodology
1.1 the linear test precision takes by weighing ganoderic acid A, B, C
2Reference substance is an amount of, adds ganoderic acid A, B, C that methyl alcohol is processed variable concentrations respectively
2Reference substance solution.Accurate 10 μ l of absorption or 20 μ l reference substance solution are injected high performance liquid chromatograph, measure by above-mentioned chromatographic condition.The result sees Table 8,9,10 respectively.
The linear experimental result table of table 8 ganoderic acid A
The linear experimental result table of table 9 ganoderic acid B
Table 10 ganoderic acid C
2Linear experimental result table
(μ g/ml) is horizontal ordinate with concentration, and respective peaks area (A) is an ordinate, the drawing standard curve.Getting ganoderic acid A equation of linear regression is y=8677.49x+2832.86, correlation coefficient r=1.0000; Ganoderic acid B equation of linear regression is y=8520.66x+4165.17, correlation coefficient r=1.0000; Ganoderic acid C
2Equation of linear regression be y=17504.5x-15909.5, correlation coefficient r=0.9999.Test shows ganoderic acid A, B, C
2In 3.504 μ g/ml～525.60 μ g/ml, 3.232 μ g/ml～484.80 μ g/ml, 2.036～203.600 μ g/ml scopes, be good linear relationship respectively.
1.2 glossy ganoderma (red sesame D9) the water extract sample of known content is got in accuracy and precision test, adopts the application of sample recovery test, accurate a certain amount of ganoderic acid A, B, the C of adding
2Reference substance prepares the test sample of 3 variable concentrations respectively, the test sample that each prepared at concentrations is 6 parts, and by the preparation method and the chromatographic condition mensuration of above-mentioned need testing solution, and calculate recovery rate, the result sees table 11,12,13.
A accuracy of table 11 ganoderic acid and precision test determination result
B accuracy of table 12 ganoderic acid and precision test determination result
Table 13 ganoderic acid C
2Accuracy and precision test determination result
1.3 replica test is got 6 parts in same glossy ganoderma (red sesame D9) water extract sample, prepares need testing solution respectively, measures by above-mentioned chromatographic condition.The result shows that this method has good repeatability.The result sees table 14,15,16.
Table 14 ganoderic acid A replica test measurement result
Table 15 ganoderic acid B replica test measurement result
Table 16 ganoderic acid C
2Replica test is measured the result
1.4 method durability
1.4.1 the method stability test get same glossy ganoderma (red sesame D9) water extract sample X respectively at 0,2,4,8,16, the 24h sample introduction, measure ganoderic acid A, B, C by above-mentioned chromatographic condition
2Content.The result shows that need testing solution is stable in 24 hours.
1.4.2 investigating this experiment, different chromatographic columns adopt different chromatographic columns to ganoderic acid A, B, C in glossy ganoderma (the red sesame D9) water extract
2Content measure.The result uses the different model chromatographic column, ganoderic acid A, B, C
2Content RSD<2%, show this method good tolerance, measure the result and see table 18,19.
Table 18 serviceability test result 1
Table 19 serviceability test result 2
1.4.3 investigating this experiment, different moving phases adopt different proportion moving phase to ganoderic acid A, B, C in glossy ganoderma (the red sesame D9) water extract
2Content measure.The result uses different proportion moving phase, ganoderic acid A, B, C
2Content RSD<2%, show this method good tolerance, measure the result and see table 20,21.
Table 20 serviceability test result 3
Table 21 serviceability test result 4
1.4.4 investigating this experiment, different instruments adopt different instruments to ganoderic acid A, B, C in glossy ganoderma (the red sesame D9) water extract
2Content measure.The result uses the different model high performance liquid chromatograph, ganoderic acid A, B, C
2Content RSD<2%, show this method good tolerance, measure the result and see table 22.
Table 22 serviceability test result 5
2 conclusions
Purple its ganoderic acid of sesame water extract A, B, C
2Content compare with red sesame water extract, content is lower, the ganoderic acid A of Ganoderma tsugae water extract, B, C
2The content of content and red sesame water extract is suitable, therefore purple sesame water extract and red sesame, its ganoderic acid of Ganoderma tsugae water extract A, B, C
2The limit of total amount should separately be formulated.
The above is merely preferable embodiment of the present invention, and all equalizations of doing according to claim of the present invention change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. the method for quality control of a glossy ganoderma water extract is characterized in that: with ganoderic acid A, ganoderic acid B, ganoderic acid C
2Content sum and crude polysaccharides content are as the quality control index of glossy ganoderma water extract.
2. the method for quality control of glossy ganoderma water extract according to claim 1 is characterized in that:
Said ganoderic acid A, B content detecting method are following:
(1) preparation of reference substance solution
Get ganoderic acid A, the B reference substance of drying under reduced pressure to constant weight, add methyl alcohol and process the mixed mark solution that per 1 ml contains 164 μ g ganoderic acid A and contains 52 μ g ganoderic acid B;
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in the 25 ml volumetric flasks, add methyl alcohol sonicated 10 min, cooling adds methyl alcohol and is diluted to scale, shakes up, and filters;
(3) high performance liquid chromatography detects
With the octyl bonded silica gel is filling agent, and acetonitrile is a mobile phase A, and 0.1 % aqueous acetic acid is a Mobile phase B, gradient elution; Detecting wavelength is 257 nm; Column temperature is 22 ℃; Flow velocity is 1.0 ml/min; Number of theoretical plate is pressed ganoderic acid A and is calculated, and should be not less than 5000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get;
Said ganoderic acid C
2Content detecting method is following:
(1) preparation of reference substance solution
Get the ganoderic acid C of drying under reduced pressure to constant weight
2Reference substance adds methyl alcohol and processes per 1 ml and contain 33 μ g ganoderic acid C
2Reference substance solution;
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in the 25 ml volumetric flasks, add methyl alcohol sonicated 10 min, cooling adds methyl alcohol and is diluted to scale, shakes up, and filters;
(3) high performance liquid chromatography detects
With the octadecyl silane is filling agent, and acetonitrile is a mobile phase A, and 0.1 % aqueous acetic acid is a Mobile phase B, gradient elution; Detecting wavelength is 257 nm; Column temperature is 20 ℃; Flow velocity is 1.0 ml/min; Number of theoretical plate is pressed ganoderic acid C
2Calculate, should be not less than 5000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.
3. the method for quality control of glossy ganoderma water extract according to claim 1 is characterized in that: said crude polysaccharides content detecting method is following:
(1) typical curve is drawn
Get the glucosan reference substance that is dried to constant weight, add water and process the solution that per 1 ml contains glucosan 1 mg, accurate respectively the absorption in above-mentioned standard solution 0.1,0.2,0.4,0.6,0.8,1.0 ml to the 10.0 ml measuring bottles, thin up is to scale; Draw respectively in above-mentioned solution 2.0 ml to the 25 ml color comparison tubes, add 50 g/L phenol solution, 1.0 ml, mixing; The accurate concentrated sulphuric acid 10.0 ml that add, mixing is put and is boiled 2 min in the water-bath; Mixing is cooled to after the room temperature and measures absorbance with spectrophotometer in 485 nm wavelength, is blank with the reagent corresponding; With glucosan concentration is horizontal ordinate, and absorbance is an ordinate, the drawing standard curve;
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.4 g, put in the 50 ml conical flasks, add water 25.0 ml, ultrasonic 10 min filter; Draw in filtrating 5.0 ml to the 50 ml centrifuge tubes, slowly add absolute ethyl alcohol 20 ml, the limit edged shakes up, and places 12 h for 4 ℃; Take out centrifugal 8 min of 3000 r/min, abandoning supernatant; Deposition is dissolved in water and is transferred in the 10.0 ml volumetric flasks, adds the water constant volume, shakes up; Draw solution 2.0 ml behind the above-mentioned constant volume, place 15 ml centrifuge tubes, add 100 g/L sodium hydroxide solutions, 2.0 ml, DDTC 2.0 ml, place boiling water bath to boil 3 min, be cooled to room temperature, centrifugal 8 min of 3000 r/min, abandoning supernatant; Deposition is dissolved with 10 % sulfuric acid solutions, 2.0 ml and is transferred in the 10.0 ml volumetric flasks, adds the water constant volume, shakes up, and promptly gets;
(3) sample determination
Draw need testing solution 2.0 ml,,, measure absorbance in accordance with the law, utilize typical curve to calculate the content of glucosan from " adding 50 g/L phenol solution, 1.0 ml " according to step (1).
4. the method for quality control of glossy ganoderma water extract according to claim 1 is characterized in that: the crude polysaccharides content>=30.7mg/g of mensuration, in glucosan; Ganoderic acid A, B, C
2The content sum, in dry product, red sesame, Ganoderma tsugae water extract>=13.3mg/g, purple sesame water extract>=1.8mg/g.
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