Summary of the invention
The object of the invention is to: a kind of Chinese traditional medicine Ganoderma lucidum granules and preparation method thereof and detection method are provided.The present invention is directed to the deficiencies in the prior art, provide a kind of dissolve fast, bioavailability is high, the ganoderma particles preparation of good stability, has also developed scientific and reasonable preparation technology and detection method, effectively to control and to improve the quality of products.
Technical scheme of the present invention: a kind of Chinese traditional medicine Ganoderma lucidum granules, it is to take Ganoderma 1500g as crude drug, adds 950g lactose to make as adjuvant.
The preparation method of aforementioned Chinese traditional medicine Ganoderma lucidum granules is: get Ganoderma, decoct with water three times, add for the first time 10 times of water gagings and extract 4 hours, second and third time respectively adds 8 times of water gagings and extract 3 hours, collecting decoction, filters, and filtrate is concentrated into thick paste shape, adds lactose, mix, granulation, dry, obtain.
The detection method of aforementioned Chinese traditional medicine Ganoderma lucidum granules comprises character, inspection, assay project; Wherein assay is the assay to adenosine in preparation, and the content assaying method of adenosine is to take adenosine reference substance as contrast, take acetonitrile: 0.04mol/L potassium dihydrogen phosphate=5: 95 high performance liquid chromatography that are mobile phase.
Concrete detection method comprises following items:
Character: product is that light brown is to brown granule, sweet, the micro-hardship of taste;
Check: should meet relevant every regulation under appendix IC granule item of Chinese Pharmacopoeia version in 2005;
Assay: shine appendix VID high effective liquid chromatography for measuring of Chinese Pharmacopoeia version in 2005:
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler, acetonitrile: 0.04mol/L potassium dihydrogen phosphate=5: 95 is mobile phase, and detection wavelength is 260nm, and number of theoretical plate calculates and should be not less than 4000 by adenosine peak;
It is that the desiccant drying under reduced pressure adenosine reference substance of 24 hours is appropriate that the preparation precision of reference substance solution takes with phosphorus pentoxide, adds methanol and makes the solution that contains 16ug in every 1ml, obtains;
Ganoderma particles 2.5g is got in the preparation of need testing solution, accurately weighed, puts in conical flask, and precision adds 15% methanol solution 25ml, weigh, supersound process 30 minutes, cooling, weigh, with 15% methanol, supply the weight of loss, centrifugal or filter through microporous filter membrane, get supernatant or filtrate, obtain;
Algoscopy is accurate reference substance solution and each 10ul of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains; The every 1g of this granule contains Ganoderma with adenosine C
10h
13n
5o
4meter, must not be less than 0.10mg.
The applicant has carried out preparation technology and the detection method that series of experiments is selected preparation of the present invention, to guarantee its science, reasonable, feasible, and has good curative effect.
The research of Determination of Adenosine method:
In Ganoderma, contain various active composition, mainly comprise polysaccharide, triterpenoid compound, adenosine etc., according to the medicinal document mycology > > report of documents and materials < < China, adenosine is one of main effective ingredient of Ganoderma, determine that adenosine is the quality detecting index of ganoderma particles, adopt the content of adenosine in high effective liquid chromatography for measuring ganoderma particles.
1, instrument and reagent
HP1100 series of high efficiency chromatograph of liquid, comprises 1311A quaternary pump, 1313 automatic samplers, 1316A column oven, 1314A purple light visible detection device, 1315B DAD; Agilent chem workstation (A.06.03 version).
Acetonitrile is chromatographically pure, and methanol, potassium dihydrogen phosphate are analytical pure, double distilled water.
Adenosine reference substance (Adenosine, lot number: 3 batches, Ganoderma sample (lot number 011203,020101,020103) 879-200001) is provided by Nat'l Pharmaceutical & Biological Products Control Institute.
2, chromatographic condition
Chromatographic column: DiamonsilC18 post (250 * 4.6mm, 5um); Mobile phase: acetonitrile-0.04mol/L potassium dihydrogen phosphate (5: 95); Flow velocity: 1.0ml/min; Column temperature: 30 ℃, detect wavelength: 260nm.
Once adopted 1. methanol-0.1mol/L potassium dihydrogen phosphate (75: 25), 2. acetonitrile-methanol-0.05mol/L potassium dihydrogen phosphate (42: 23: 35), 3. acetonitrile-0.04mol/L potassium dihydrogen phosphate (5: 95) is tested respectively for mobile phase, result take acetonitrile-0.04mol/L potassium dihydrogen phosphate (5: 95) as mobile phase separating degree best.
In test sample, adenosine composition can reach better separated with other compositions with this understanding.Experiment records adenosine reference substance solution has absorption maximum at 260nm wavelength place, selects 260nm for measuring wavelength.
3, the preparation of reference substance solution
It is that the desiccant drying under reduced pressure adenosine reference substance of 24 hours is appropriate that precision takes with phosphorus pentoxide, adds 15% methanol and makes the reference substance stock solution that contains 0.1585mg in every 1ml, by 10 times of this solution dilutions (0.01585mg/ml).
4, the preparation of need testing solution
A, the comparison of extracting solvent: sample is selected different extraction solvents, and all the other,, with the extracting method of test sample described in technical solution of the present invention, make need testing solution separately, sample introduction is measured, and content results is in Table 1.Select to adopt 15% methanol to make to extract solvent.
Table 1 extracts solvent comparison
Extract solvent |
Water |
15% methanol |
50% methanol |
Sample size (mg/g) |
0.165 |
0.165 |
0.165 |
The comparison of b, supersound extraction time: select the different supersound extraction time, all the other make need testing solution separately with the extracting method of test sample described in technical solution of the present invention, sample introduction is measured, assay the results are shown in Table 2, can find out, 30,45,60 minutes gained content results of supersound extraction are all in range of error, and selecting the supersound extraction time is 30 minutes.
The comparison of table 2 ultrasonic time
The supersound extraction time |
30 minutes |
45 minutes |
60 minutes |
Sample size (mg/g) |
0.165 |
0.170 |
0.168 |
5, linear relationship is investigated
Accurate adenosine reference substance solution (0.01585mg/ml) 4,8,10,12,16, the 20ul of drawing, injection liquid chromatography, by above-mentioned condition, measure peak area, take peak area integrated value as vertical coordinate (Y), adenosine sample size is that abscissa (X) is made regression equation, obtain Y=3.2475X-3.7209, r
2=1.0000, show that adenosine sample size has good linear relationship within the scope of 63.4~317ng.
6, precision test
The above-mentioned reference substance solution of accurate absorption, repeats sample introduction 5 times (10ul), measures peak area, and the relative standard deviation of result integrated value (RSD) is 0.3% (n=5), in Table 3.
Table 3 Precision test result
Tested number |
1 |
2 |
3 |
4 |
5 |
Meansigma methods |
RSD% |
Peak area |
510.01 |
510.47 |
510.54 |
513.01 |
513.52 |
511.29 |
0.3 |
7, stability test
Get need testing solution, by above chromatographic condition, sample introduction is 1 time at regular intervals, measures peak area, result RSD=0.48% (n=8), and show sample is at least stable in 16 hours, in Table 4.
Table 4 stability test
Time (hr) |
0 |
2 |
4 |
6 |
8 |
12 |
16 |
Meansigma methods |
RSD |
Peak area |
510.01 |
510.18 |
513.01 |
513.52 |
514.71 |
516.24 |
515.82 |
513.67 |
0.48% |
8, repeatability test
Get 6 parts of same batch samples (lot number: 011203), difference is accurately weighed, extracts and measure peak area by method described in technical solution of the present invention, calculate adenosine content, in this batch sample of result, adenosine average content is 0.164mg/g, and RSD=1.08% (n=6), the results are shown in Table 5.
Table 5 sample replica test result
Numbering |
1 |
2 |
3 |
4 |
5 |
6 |
Meansigma methods |
RSD% |
Content (mg/g) |
0.165 |
0.162 |
0.162 |
0.163 |
0.164 |
0.166 |
0.164 |
1.08 |
9, recovery test
Adopt application of sample absorption method, (lot number: it is appropriate that 011203) precision adds adenosine reference substance, records peak area and calculates content through following formula by method described in technical solution of the present invention, the results are shown in Table 6 to get 6 parts, the sample of known content.Result shows that this law has the good response rate.
The response rate=(measure adenosine amount-sample suitable adenosine amount)/add adenosine reference substance amount
Table 6 recovery test result
10, blank test
Experiment makes blank solution to lacking Ganoderma blank sample equally by need testing solution preparation method, and sample introduction is measured, and result shows, lacks Ganoderma blank noiseless to adenosine peak, proves that the adenosine composition of measuring by this method has specificity.
11, sample determination
By content assaying method described in technical solution of the present invention, measure 3 batches of ganoderma particles, the results are shown in Table 7.According to 3 batches of determination datas, adenosine content limit in sample is fixed tentatively as " the every 1g of this granule is containing adenosine (C
10h
13n
5o
4) must not be less than 0.10mg ".
Table 7 ganoderma particles sample determination result
Lot number |
Measure content 1 (mg/g) |
Measure content 2 (mg/g) |
Average content (mg/g) |
020103 |
0.114 |
0.113 |
0.114 |
020102 |
0.228 |
0.225 |
0.228 |
011203 |
0.163 |
0.165 |
0.164 |
Compared with prior art, it is fast that ganoderma particles of the present invention has dissolving, and bioavailability is high, the advantage of good stability; Detection method precision of the present invention is high simultaneously, favorable reproducibility, and measurement result is accurate, can effectively guarantee the clinical efficacy of said preparation.
The specific embodiment
Embodiment 1: the preparation of ganoderma particles: get Ganoderma 1500g, decoct with water three times, add for the first time 10 times of water gagings and extract 4 hours, second and third time respectively adds 8 times of water gagings and extract 3 hours, collecting decoction, filters, and filtrate is concentrated into thick paste shape, adds lactose 950g, mix, granulation, dry, obtain.
Embodiment 2: the detection method of ganoderma particles: comprise following items:
Character: product is that light brown is to brown granule, sweet, the micro-hardship of taste;
Check: should meet relevant every regulation under appendix IC granule item of Chinese Pharmacopoeia version in 2005;
Assay: shine appendix VID high effective liquid chromatography for measuring of Chinese Pharmacopoeia version in 2005:
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler, acetonitrile: 0.04mol/L potassium dihydrogen phosphate=5: 95 is mobile phase, and detection wavelength is 260nm, and number of theoretical plate calculates and should be not less than 4000 by adenosine peak;
It is that the desiccant drying under reduced pressure adenosine reference substance of 24 hours is appropriate that the preparation precision of reference substance solution takes with phosphorus pentoxide, adds methanol and makes the solution that contains 16ug in every 1ml, obtains;
Ganoderma particles 2.5g is got in the preparation of need testing solution, accurately weighed, puts in conical flask, and precision adds 15% methanol solution 25ml, weigh, supersound process 30 minutes, cooling, weigh, with 15% methanol, supply the weight of loss, centrifugal or filter through microporous filter membrane, get supernatant or filtrate, obtain;
Algoscopy is accurate reference substance solution and each 10ul of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains; The every 1g of this granule contains Ganoderma with adenosine C
10h
13n
5o
4meter, must not be less than 0.10mg.