CN102735772B - 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 products determination method, be specifically related to a kind of detection method of glossy ganoderma water extract.
Background technology
Glossy ganoderma is the red sesame of On Polyporaceae
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, the effect such as relieving cough and asthma.Glossy ganoderma extraction process mainly contains water extraction and alcohol extracting, and most extraction process by water that adopts, adopt the red sesame of On Polyporaceae (
ganoderma lucidum(Leyss. ex Fr.) Karst.), purple sesame (
ganoderma sinnesezhao, Xu et Zhang) and Ganoderma tsugae (
ganodermatsugae Murr.) dry fructification get, concentrate, make after dry through water extraction.Therefore the present invention adopts the name of glossy ganoderma water extract.Research shows, mainly contains the number of chemical compositions such as polysaccharide, triterpene, nucleosides, alkaloid, flavones, amino acid and trace element in glossy ganoderma.At present, glossy ganoderma pharmacology activity research is mainly concentrated on to ganoderan and Ganoderma lucidum triterpenes components, therefore, ganoderan and triterpenes components be as the main functional component of glossy ganoderma, necessary its content measured.
The present invention is studied the assay method of the significant composition of healthy food material glossy ganoderma water extract in series of experiments process.Ganoderan is one of main active in glossy ganoderma, has immunological regulation, the multiple pharmacologically active such as antitumor, anti-oxidant, hypoglycemic.At present, some enterprise, in order to reach the dry object of even making a false report ganoderma polyoses content, adds the various auxiliary materials such as dextrin, starch in the glossy ganoderma water extract of being everlasting.If measure by the method for version Chinese Pharmacopoeia in 2010, will cause ganoderan measurement result higher.This research is extracted ganoderma lucidum crude polysaccharide by water extraction and alcohol precipitation method, wherein there is the polysaccharide of glucan structure by cupferron precipitation, adopt its content of determined by ultraviolet spectrophotometry, got rid of auxiliary material on the impact of measuring, for the needed detection technique of glossy ganoderma water extract quality control provides foundation.
Ganoderma lucidum triterpene compounds is another main pharmacodynamics composition of glossy ganoderma, anticancer owing to having, anti-inflammatory, the anti-oxidant pharmacologically active and receiving much concern of waiting.The inventor finds, contains ganoderic acid A, B, C that content is relatively high in glossy ganoderma water extract in employing high performance liquid chromatography is studied glossy ganoderma water extract triterpenes components
2.The method of the current conventional colorimetric method for determining total triterpene contents of contrast, high specificity, can reach and differentiate and effective testing product characteristic component content, finally realizes the object of quality control.
Summary of the invention
Thereby in order to assist to control product quality by testing product characteristic component content targetedly, the present invention adopts thick polyoses content and ganoderic acid A, B, C
2content sum, as the detection index of glossy ganoderma water extract, provides a kind of detection method of glossy ganoderma water extract.
The technical scheme that the present invention takes is as follows:
With ganoderic acid A, ganoderic acid B, ganoderic acid C
2content sum and thick polyoses content are as the detection index of glossy ganoderma water extract, ganoderic acid A, ganoderic acid B, ganoderic acid C
2the detection method of content and thick polyoses content.
The detection method of described ganoderic acid A, B content is as follows:
(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 to make every 1ml and contain 164 μ g ganoderic acid A and the mixed mark solution containing 52 μ g ganoderic acid Bs.
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in 25 ml volumetric flasks, add appropriate ultrasonic processing 10 min of methyl alcohol, let cool, add methyl alcohol and be diluted to scale, shake up, filter.
(3) high performance liquid chromatography detects
Take octyl bonded silica gel as filling agent, acetonitrile is mobile phase A, and 0.1 % aqueous acetic acid is 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 reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
Described ganoderic acid C
2the detection method of content is as follows:
(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 makes every 1 ml containing 33 μ g ganoderic acid Cs
2reference substance solution.
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in 25 ml volumetric flasks, add appropriate ultrasonic processing 10 min of methyl alcohol, let cool, add methyl alcohol and be diluted to scale, shake up, filter.
(3) high performance liquid chromatography detects
Take octadecyl silane as filling agent, acetonitrile is mobile phase A, and 0.1 % aqueous acetic acid is 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 reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
The detection method of described thick polyoses content is as follows:
(1) Specification Curve of Increasing:
Get that to be dried to the glucosan reference substance of constant weight appropriate, precision weighing, adds water and makes the solution of every 1 ml containing glucosan 1 mg.Accurate absorption in above-mentioned standard solution 0.1,0.2,0.4,0.6,0.8,1.0 ml to 10.0 ml measuring bottles respectively, be diluted with water to scale, draw respectively in above-mentioned solution 2.0 ml to 25 ml color comparison tubes, add 50 g/L phenol solution 1.0 ml, mix, precision adds the concentrated sulphuric acid 10.0 ml, mix, put in water-bath and boil 2 min, mix, be cooled to after room temperature and measure absorbances with spectrophotometer at 485 nm wavelength places, take corresponding reagent as blank, take glucosan concentration as horizontal ordinate, absorbance is ordinate, drawing standard curve.
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.4 g, put in 50 ml conical flasks, 25.0 ml that add water, ultrasonic 10 min, filter; Draw in filtrate 5.0 ml to 50 ml centrifuge tubes, slowly add absolute ethyl alcohol 20 ml, limit edged shakes up, and places 12 h for 4 ℃, take out centrifugal 8 min of 3000 r/min, abandoning supernatant, precipitation is dissolved in water and is transferred in 10.0 ml volumetric flasks, and the constant volume that adds water, shakes up; Draw solution 2.0 ml after above-mentioned constant volume, be placed in 15 ml centrifuge tubes, add 100 g/L sodium hydroxide solution 2.0 ml, cupferron 2.0 ml, be placed in boiling water bath and boil 3 min, be cooled to room temperature, centrifugal 8 min of 3000 r/min, abandoning supernatant; Precipitation is dissolved and is transferred in 10.0 ml volumetric flasks with 10 % sulfuric acid solution 2.0 ml, and the constant volume that adds water, shakes up, and to obtain final product.
(3) sample determination
Draw need testing solution 2.0 ml, under Specification Curve of Increasing item, from " adding 50 g/L phenol solution 1.0 ml ", measure absorbance in accordance with the law, utilize typical curve to calculate the content of glucosan.
Significant component target is: the thick polyoses content>=30.7mg/g of mensuration, in glucosan; Ganoderic acid A, B, C
2content 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 by water extraction and alcohol precipitation method, wherein there is the polysaccharide of glucan structure by cupferron precipitation, adopt its content of determined by ultraviolet spectrophotometry, get rid of auxiliary material to the impact of measuring, for characteristic component assay in glossy ganoderma water extract, provide foundation thereby reach quality control.
Red sesame, purple sesame and Ganoderma tsugae all contain characteristic component ganoderic acid A, B, the C that relative content is higher
2.The method of the current conventional colorimetric method for determining total triterpene contents of contrast, high specificity, can reach characteristic component content in discriminating and effective testing product, finally realizes the object of quality control.
Accompanying drawing explanation
Fig. 1 is the mixed mark of ganoderic acid A, B solution chromatogram, peak 1: ganoderic acid B, peak 2: ganoderic acid A.
Fig. 2 is need testing solution chromatogram, peak 1: ganoderic acid B, peak 2: ganoderic acid A.
Fig. 3 is ganoderic acid C
2reference substance solution chromatogram, peak 3: ganoderic acid C
2.
Fig. 4 is need testing solution chromatogram, peak 3: ganoderic acid C
2.
Embodiment
This product in following examples refers to the test sample of glossy ganoderma water extract.
The thick measurement of the polysaccharide content of embodiment 1
A1.1 principle
in sample, relative molecular mass is greater than 1 × 10
4polymer substance in 80% ethanolic solution, precipitate, separate with compound sugar with monose in aqueous solution, with alkaline cupric reagent selectivity from other polymer substances precipitation there is the polysaccharide of glucan structure, with the orange red compound of phenolsulfuric acid reaction generation, by its content of colorimetric estimation, its colored intensity is directly proportional to the content of glucosan in thick polysaccharide, calculates the content of thick polysaccharide 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 25 g NaOH, be dissolved in water and be diluted to 250 ml.Add solid water-free sodium sulphate to saturated, for subsequent use.
A1.2.1.4 cupferron storing solution: take 0.75 g CuS0
45H
2o, 7.5g sodium citrate, is dissolved in water and is diluted to 250 ml, mixes for subsequent use.
A1.2.1.5 cupferron solution: get cupferron storing solution 50 ml, 50 ml that add water, add solid water-free sodium sulphate 12.5 g and make its dissolving after mixing.Interim adapted.
A1.2.1.6 phenol (50 g/L): take phenol 5.0 g, be diluted with water to 100 ml, mix, its solution is placed in refrigerator and can preserves 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 appropriate, precision weighing, adds water and makes the solution of every 1 ml containing glucosan 1 mg.
A1.3 instrument
A1.3.1 ultraviolet spectrophotometer.
A1.3.2 ultrasonic cleaner.
A1.3.3 hydro-extractor (3000 r/min).
A1.4 analytical procedure
The preparation of A1.4.1 typical curve
Accurate absorption in glucosan standard reserving solution 0.1,0.2,0.4,0.6,0.8,1.0 ml to 10.0 ml measuring bottles respectively, be diluted with water to scale, draw respectively in above-mentioned solution 2.0 ml to 25 ml color comparison tubes, add 50 g/L phenol solution 1.0 ml, mix, precision adds the concentrated sulphuric acid 10.0 ml, mix, put in water-bath and boil 2 min, mix, be cooled to after room temperature and measure absorbances with spectrophotometer at 485 nm wavelength places, take corresponding reagent as blank, take glucosan concentration as horizontal ordinate, absorbance is ordinate, drawing standard curve.
The preparation of A1.4.2 sample solution
Precision takes this product 0.4 g, puts in 50 ml conical flasks, and 25.0 ml that add water, ultrasonic 10 min, filter.Draw in filtrate 5.0 ml to 50 ml centrifuge tubes, slowly add absolute ethyl alcohol 20 ml, limit edged shakes up, and places 12 h for 4 ℃, take out, with centrifugal 8 min of 3000 r/min, abandoning supernatant, precipitation is dissolved in water and is transferred in 10.0 ml volumetric flasks, is diluted with water to scale, shakes up.Draw above-mentioned solution 2.0 ml, be placed in 15 ml centrifuge tubes, add 100 g/L sodium hydroxide solution 2.0 ml, cupferron 2.0 ml, be placed in boiling water bath and boil 3 min, be cooled to room temperature, with centrifugal 8 min of 3000 r/min, abandoning supernatant.Precipitation is dissolved and is transferred in 10.0 ml volumetric flasks with 10 % sulfuric acid solution 2.0 ml, is diluted with water to scale, shakes up, and to obtain final product.Do sample blank by above-mentioned steps simultaneously.
A1.4.3 sample determination
Draw sample solution 2.0 ml and put in 25 ml color comparison tubes, under sighting target directrix curve preparation, from " adding 50 g/L phenol solution 1.0 ml ", measure absorbance in accordance with the law, the weight of reading glucosan from typical curve, calculates, and to obtain final product.
A1.4.4 result is calculated
The content (in glucosan) of thick polysaccharide in X-sample, mg/g
M
1the quality of glucosan in-sample determination liquid, mg
M
2glucosan quality in-sample blank liquid, mg
M
3-sample quality, g
V
1-sample extracting solution cumulative volume, ml
V
2-precipitate thick polysaccharide specimen in use extracting liquid volume, ml
V
3-thick polysaccharide solution volume, ml
V
4-precipitation glucosan thick polysaccharide solution volume used, ml
V
5-sample determination liquid cumulative volume, ml
V
6-test sample liquor capacity, ml
A1.4.5 tolerance
The absolute difference of the twice independent measurement result obtaining under repeated condition must not exceed 10 % of arithmetic mean.
Embodiment 2 ganoderic acid A, B, C
2the mensuration of discriminating and total amount
A2.1 principle
Sample is using methyl alcohol as solvent, ultrasonic extraction, and 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 respectively UV-detector (UV) to detect, qualitative according to the retention time of chromatographic peak, peak area external standard method is quantitative, measures respectively ganoderic acid A, B and ganoderic acid C in sample
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.
A2.3 instrument
A2.3.1 high performance liquid chromatograph (UV-detector).
A2.3.2 ultrasonic cleaner.
A2.4 analytical procedure
A2.4.1
?ganoderic acid A, B measure
A2.4.1.1 chromatographic condition
(the chromatographic column: Agilent Zorbax-SB C take octyl bonded silica gel as Bonded Phase
8150 mm × 4.6 mm, 3.5 μ m or similar specification chromatographic column), acetonitrile is mobile phase A, and 0.1 % aqueous acetic acid is Mobile phase B, and the regulation 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
A2.4.1.2 system suitability
Number of theoretical plate calculates and should be not less than 5000 by ganoderic acid A.
The preparation of A2.4.1.3 reference substance solution
Get drying under reduced pressure appropriate, accurately weighed to ganoderic acid A, the B reference substance of constant weight, add methyl alcohol and make the mixed mark solution that every 1 ml contains 52 μ g ganoderic acid Bs containing 164 μ g ganoderic acid A and every 1 ml, to obtain final product.
The preparation of A2.4.1.4 need testing solution
Get this product approximately 0.5 g, put in 25 ml volumetric flasks, add appropriate ultrasonic processing 10 min of methyl alcohol, let cool, add methyl alcohol and be diluted to scale, shake up, filter.
A2.4.1.5 determination method
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.Qualitative by main peak retention time, peak area quantification.Result as shown in Figure 1, Figure 2.
A2.4.1.6 calculates
In A2.4.1.6.1 glossy ganoderma water extract, the content of ganoderic acid A is calculated as follows:
Y
1---the content of ganoderic acid A, unit is every gram (mg/g) of milligram;
A
x---the peak area of ganoderic acid A in need testing solution chromatogram;
A
s---the peak area of ganoderic acid A in reference substance solution chromatogram;
C---the concentration of ganoderic acid A in reference substance solution, unit is every milliliter (mg/mL) of milligram;
M---sample mass, unit is gram (g);
X---sample moisture (%).
In A2.4.1.6.2 glossy ganoderma water extract, the content of ganoderic acid B is calculated as follows:
Y
2---the content of ganoderic acid B, unit is every gram (mg/g) of milligram;
A
x---the peak area of ganoderic acid B in need testing solution chromatogram;
A
s---the peak area of ganoderic acid B in reference substance solution chromatogram;
C---the concentration of ganoderic acid B in 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
(the chromatographic column: Agilent Zorbax-SB C take octadecyl silane as Bonded Phase
18250 mm × 4.6 mm, 5 μ m or similar specification chromatographic column), acetonitrile is mobile phase A, and 0.1 % aqueous acetic acid is Mobile phase B, and the regulation 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
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 appropriate, accurately weighed, adds methyl alcohol and makes every 1ml containing 33 μ g ganoderic acid Cs
2reference substance solution, to obtain final product.
The preparation of A2.4.2.4 need testing solution
Get this product approximately 0.5 g, put in 25 ml volumetric flasks, add appropriate ultrasonic processing 10 min of methyl alcohol, let cool, add methyl alcohol and be diluted to scale, shake up, filter.
A2.4.2.5 determination method
Accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.Qualitative by main peak retention time, peak area quantification.Result is as Fig. 3, Fig. 4.
A2.4.2.6 calculates
Ganoderic acid C in glossy ganoderma water extract
2content be calculated as follows:
Y
3---ganoderic acid C
2content, unit be milligram every gram (mg/g);
A
x---ganoderic acid C in need testing solution chromatogram
2peak area;
A
s---ganoderic acid C in reference substance solution chromatogram
2peak area;
C---ganoderic acid C in reference substance solution
2concentration, unit be 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 milligram every gram (mg/g);
Y
1---the content of ganoderic acid A, unit is every gram (mg/g) of milligram;
Y
2---the content of ganoderic acid B, unit is every gram (mg/g) of milligram;
Y
3---ganoderic acid C
2content, unit be milligram every gram (mg/g).
A2.4.4 tolerance
The absolute difference of the twice independent measurement result obtaining under repeated condition must not exceed 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 thick polyoses content in glossy ganoderma water extract
1 methodology checking
The method drawing standard curve that 1.1 Linear Experiments are prepared according to above-mentioned typical curve, take glucosan concentration as horizontal ordinate, absorbance is ordinate, and obtaining equation of linear regression is y=0.00514x+0.00571, related coefficient γ=0.9996, the range of linearity is 21.76 μ g～217.6 μ g.The results are shown in Table 4.
The linear result table of the thick polysaccharide of table 4
Glossy ganoderma (red sesame D9) the water extract sample of known content is got in 1.2 veracity and precision tests, adopt application of sample recovery test, precision adds a certain amount of glucosan reference substance, prepares respectively the test sample of 3 variable concentrations, each concentration is prepared the test sample of 6 parts, does sample blank simultaneously.Measure by the preparation method of above-mentioned need testing solution and condition determination, and calculate recovery rate, the results are shown in Table 5.
The thick polysaccharide veracity and precision test determination result of table 5
1.3 replica tests are got 6 parts, same glossy ganoderma (red sesame D9) water extract sample, prepare respectively need testing solution, measure by above-mentioned method, and result shows that this method has good repeatability.The results are shown in Table 6.
Table 6 replica test measurement result
1.4 method durabilities are investigated this experiment and are used different instruments to measure the thick polyoses content in same glossy ganoderma (red sesame D9) water extract, result shows, use different model ultraviolet spectrophotometer, thick polyoses content RSD<3%, shows this method good tolerance.Measurement result is in table 7.
Table 7 serviceability test result
2 conclusions
Adopt ultraviolet spectrophotometer to measure thick polyoses content in 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 cupferron separation and purification in mensuration process, and experiment shows to obtain satisfactory result.
Ganoderic acid A, ganoderic acid B, ganoderic acid C in embodiment 5 high effective liquid chromatography for measuring glossy ganoderma water extracts
2the research of the method for content
1 methodology checking
1.1 linear test precisions take ganoderic acid A, B, C
2reference substance is appropriate, adds methyl alcohol and make respectively ganoderic acid A, B, the C of variable concentrations
2reference substance solution.The 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.Result is respectively in table 8,9,10.
Table 8 ganoderic acid A Linear Experiment result table
Table 9 ganoderic acid B Linear Experiment result table
Table 10 ganoderic acid C
2linear Experiment result table
Take concentration (μ g/ml) as horizontal ordinate, respective peaks area (A) is ordinate, drawing standard curve.Obtaining 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
2within the scope of 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, be good linear relationship respectively.
Glossy ganoderma (red sesame D9) the water extract sample of known content is got in 1.2 veracity and precision tests, adopts application of sample recovery test, and precision adds a certain amount of ganoderic acid A, B, C
2reference substance, prepares respectively the test sample of 3 variable concentrations, and each concentration is prepared the test sample of 6 parts, measure by the preparation method of above-mentioned need testing solution and chromatographic condition, and calculate recovery rate, the results are shown in Table 11,12,13.
Table 11 ganoderic acid A veracity and precision test determination result
Table 12 ganoderic acid B veracity and precision test determination result
Table 13 ganoderic acid C
2veracity and precision test determination result
1.3 replica tests are got 6 parts, same glossy ganoderma (red sesame D9) water extract sample, prepare respectively need testing solution, measure by above-mentioned chromatographic condition.Result shows, this method has good repeatability.The results are shown in 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 measurement result
1.4 method durabilities
1.4.1 method stability test get same glossy ganoderma (red sesame D9) water extract sample X respectively at 0,2,4,8,16,24h sample introduction, measure ganoderic acid A, B, C by above-mentioned chromatographic condition
2content.Result shows, need testing solution is stable in 24 hours.
1.4.2 different chromatographic columns are investigated the different chromatographic columns of this experiment employing to ganoderic acid A, B, C in glossy ganoderma (red sesame D9) water extract
2content measure.Result is used different model chromatographic column, ganoderic acid A, B, C
2content RSD<2%, shows this method good tolerance, and measurement result is in table 18,19.
Table 18 serviceability test result 1
Table 19 serviceability test result 2
1.4.3 different mobile phases are investigated this experiment employing different proportion mobile phase to ganoderic acid A, B, C in glossy ganoderma (red sesame D9) water extract
2content measure.Result is used different proportion mobile phase, ganoderic acid A, B, C
2content RSD<2%, shows this method good tolerance, and measurement result is in table 20,21.
Table 20 serviceability test result 3
Table 21 serviceability test result 4
1.4.4 different instruments are investigated the different instruments of this experiment employing to ganoderic acid A, B, C in glossy ganoderma (red sesame D9) water extract
2content measure.Result is used different model high performance liquid chromatograph, ganoderic acid A, B, C
2content RSD<2%, shows this method good tolerance, and measurement result is in table 22.
Table 22 serviceability test result 5
2 conclusions
Purple its ganoderic acid of sesame water extract A, B, C
2content compared with red sesame water extract, content is lower, ganoderic acid A, the B of Ganoderma tsugae water extract, 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 foregoing is only better embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (1)
1. a detection method for glossy ganoderma water extract, is characterized in that: with ganoderic acid A, ganoderic acid B, ganoderic acid C
2content sum and thick polyoses content are as the detection index of glossy ganoderma water extract; Ganoderic acid A, ganoderic acid B, ganoderic acid C are provided
2the detection method of content and thick polyoses content;
The detection method of described ganoderic acid A, B content is as follows:
(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 to make every 1 ml and contain 164 μ g ganoderic acid A and the mixed mark solution containing 52 μ g ganoderic acid Bs;
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in 25 ml volumetric flasks, add ultrasonic processing 10 min of methyl alcohol, cooling, add methyl alcohol and be diluted to scale, shake up, filter;
(3) high performance liquid chromatography detects
Take octyl bonded silica gel as filling agent, acetonitrile is mobile phase A, and 0.1 % aqueous acetic acid is 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 reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product;
Described gradient elution is:
0th ~ 50 minutes, mobile phase A was 22% → 27%, and Mobile phase B is 78% → 73%;
50th ~ 70 minutes, mobile phase A was 27% → 28%, and Mobile phase B is 73% → 72%;
70th ~ 71 minutes, mobile phase A was 28% → 42%, and Mobile phase B is 72% → 58%;
71st ~ 76 minutes, mobile phase A was 42%, and Mobile phase B is 58%;
76th ~ 77 minutes, mobile phase A was 42% → 22%, and Mobile phase B is 58% → 78%;
77th ~ 87 minutes, mobile phase A was 22%, and Mobile phase B is 78%;
Described ganoderic acid C
2the detection method of content is as follows:
(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 makes every 1 ml containing 33 μ g ganoderic acid Cs
2reference substance solution;
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.5 g, put in 25 ml volumetric flasks, add ultrasonic processing 10 min of methyl alcohol, cooling, add methyl alcohol and be diluted to scale, shake up, filter;
(3) high performance liquid chromatography detects
Take octadecyl silane as filling agent, acetonitrile is mobile phase A, and 0.1 % aqueous acetic acid is 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 reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product;
Described gradient elution is:
0th ~ 80 minutes, mobile phase A was 20% → 24%, and Mobile phase B is 80% → 76%;
80th ~ 84 minutes, mobile phase A was 24% → 25%, and Mobile phase B is 76% → 75%;
84th ~ 90 minutes, mobile phase A was 25% → 26%, and Mobile phase B is 75% → 74%;
90th ~ 91 minutes, mobile phase A was 26% → 60%, and Mobile phase B is 74% → 40%;
91st ~ 95 minutes, mobile phase A was 60%, and Mobile phase B is 40%;
95th ~ 96 minutes, mobile phase A was 60% → 20%, and Mobile phase B is 40% → 80%;
96th ~ 106 minutes, mobile phase A was 20%, and Mobile phase B is 80%;
The detection method of described thick polyoses content is as follows:
(1) Specification Curve of Increasing
Get the glucosan reference substance that is dried to constant weight, add water and make the solution of every 1 ml containing glucosan 1 mg, the above-mentioned standard solution 0.1 of accurate absorption respectively, 0.2, 0.4, 0.6, 0.8, in 1.0 ml to 10.0 ml measuring bottles, be diluted with water to scale, draw respectively in above-mentioned solution 2.0 ml to 25 ml color comparison tubes, add 50 g/L phenol solution 1.0 ml, mix, precision adds the concentrated sulphuric acid 10.0 ml, mix, put and in water-bath, boil 2 min, mix, after being cooled to room temperature, use spectrophotometer to measure absorbances at 485 nm wavelength places, take corresponding reagent as blank, take glucosan concentration as horizontal ordinate, absorbance is ordinate, drawing standard curve,
(2) preparation of need testing solution
Get glossy ganoderma water extract 0.4 g, put in 50 ml conical flasks, 25.0 ml that add water, ultrasonic 10 min, filter; Draw in filtrate 5.0 ml to 50 ml centrifuge tubes, slowly add absolute ethyl alcohol 20 ml, limit edged shakes up, and places 12 h for 4 ℃, take out centrifugal 8 min of 3000 r/min, abandoning supernatant, precipitation is dissolved in water and is transferred in 10.0 ml volumetric flasks, and the constant volume that adds water, shakes up; Draw solution 2.0 ml after above-mentioned constant volume, be placed in 15 ml centrifuge tubes, add 100 g/L sodium hydroxide solution 2.0 ml, cupferron 2.0 ml, be placed in boiling water bath and boil 3 min, be cooled to room temperature, centrifugal 8 min of 3000 r/min, abandoning supernatant; Precipitation is dissolved and is transferred in 10.0 ml volumetric flasks with 10 % sulfuric acid solution 2.0 ml, and the constant volume that adds water, shakes up, and to obtain final product;
(3) sample determination
Draw need testing solution 2.0 ml, according to step (1), from " adding 50 g/L phenol solution 1.0 ml ", measure absorbance in accordance with the law, utilize typical curve to calculate the content of glucosan.
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| CN103604763A (en) * | 2013-12-03 | 2014-02-26 | 广东中测食品化妆品安全评价中心有限公司 | Detection method for health-care food functional components |
| CN104155250A (en) * | 2014-08-15 | 2014-11-19 | 广州衡创测试技术服务有限公司 | Crude polysaccharide determination method |
| TWI610675B (en) * | 2015-07-24 | 2018-01-11 | 東吳大學 | Ganoderma composition and uses thereof |
| CN106501439B (en) * | 2016-09-13 | 2019-03-12 | 广州神农生物技术有限公司 | A kind of red ganoderma extract reference substance and its preparation method and application |
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