CN101435801A - Fingerprint pattern identifying method of ganoderma lucidum spore oil - Google Patents
Fingerprint pattern identifying method of ganoderma lucidum spore oil Download PDFInfo
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- CN101435801A CN101435801A CNA2007101352008A CN200710135200A CN101435801A CN 101435801 A CN101435801 A CN 101435801A CN A2007101352008 A CNA2007101352008 A CN A2007101352008A CN 200710135200 A CN200710135200 A CN 200710135200A CN 101435801 A CN101435801 A CN 101435801A
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- ganoderma lucidum
- lucidum spore
- spore oil
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Abstract
The invention discloses a fingerprint chromatogram identification method for ganoderma lucidum spore oil. Firstly, a standard high-performance liquid phase fingerprint chromatogram of a triterpene component of the ganoderma lucidum spore oil is established, then a high-performance liquid phase fingerprint chromatogram of a triterpene component of a ganoderma lucidum spore oil sample to be tested is determined by the same method, and finally the high-performance liquid phase fingerprint chromatogram of the ganoderma lucidum spore oil sample to be tested is compared with the standard high-performance liquid phase fingerprint chromatogram and is identified. The method establishes the high-performance liquid phase fingerprint chromatogram of the ganoderma lucidum spore oil, thereby contributing to ensuring that the quality of triterpene compounds in the ganoderma lucidum spore oil is steady and controllable. The ganoderma lucidum spore oil is identified through the high-performance liquid phase fingerprint chromatogram of the ganoderma lucidum spore oil, so as to comprehensively reflect the variety and quantity of triterpenes contained in the ganoderma lucidum spore oil, effectively embody the complexity of traditional Chinese medicine components, and be used for identifying the true or false of the ganoderma lucidum spore oil and whether the adulteration is doped. At the same time, the method can also provide pharmaceutical research information for the research and development of novel ganoderma lucidum triterpene medicines.
Description
Technical field
The invention belongs to the analytical chemistry field, be specifically related to a kind of method and differentiate ganoderma lucidum spore oil with high-efficiency liquid-phase fingerprint.
Background technology
The English Reishi mushroom by name of red sesame Ganoderma lucidum (Curtis:Fr.) Karst., claim auspicious grass, polyporus lucidus again, Japan is called 10000 years young pilose antlers, belong to Polyporaceae (Polyporaceae) Ganoderma (Ganoderma) medicinal fungi, be traditional rare Chinese medicine of China, formally recorded in the Pharmacopoeia of the People's Republic of China in 2000.Modern science proves that red sesame has pharmacological action widely.Wherein triterpenes is one of main effective constituent of red sesame, from red sesame fructification, separate to obtain triterpene compound up to the present from people such as Kubota.T in nineteen eighty-two first, successively from red sesame fructification and conidia powder, be separated to 103 kinds of new triterpenes chemical constitutions.Triterpene compound has multiple physiologically active and pharmacological action, as ganoderic acid A, B, C, D can suppress the release of mouse muscle cell histamine, the aminotransferase in mice that compatriot's spore acid A then causes phenixin and galactosamine and Propionibacterium raises the reduction effect is arranged, and other ganoderma lucidum triterpene compounds also all have physiologically active widely, as suppress tumour, antiviral, anti-ageing, regulate effects such as immune system and cardiovascular system, antibacterial, analgesia, liver protecting.
Traditional Chinese medicine fingerprint is meant that Chinese medicine is after suitably handling, adopt certain analysis means, obtain to indicate the total peak collection of illustrative plates of this Chinese medicine feature, by holding the feature of Chinese medicine fingerprint, differentiate the quality of Chinese medicine effectively, guarantee that Chinese crude drug and Chinese patent drug quality are relatively stable, become a kind of effective method of control traditional Chinese medicine quality now.But because of the ganoderma lucidum spore oil complicated component, especially the triterpenes chemical constitution is of a great variety, has brought very big trouble for the quality control of ganoderma lucidum spore oil.
Chinese patent application 200510035987.1 discloses the method for building up of a kind of Reishi sporule and ganoderma lucidum spore oil finger-print, attempt to provide a kind of quality control and discrimination method to Reishi sporule and ganoderma lucidum spore oil, but adopt the total basic ingredient olein of all plant oils in its method as object of reference, thereby make resulting collection of illustrative plates sizable defective be arranged to ganoderma lucidum spore oil selectivity and selectivity, and the olein peak area in its collection of illustrative plates greatly and in the ganoderma lucidum spore oil peak of effective ingredient less, be difficult to guarantee accurately distinguish other plant oil and ganoderma lucidum spore oil, say nothing of the discriminating other plant oil condition of in ganoderma lucidum spore oil, having mixed.Simultaneously the moving phase in its chromatographic condition to the selectivity of ganoderma lucidum spore oil effective ingredient a little less than and cost an arm and a leg, detecting device is rare, these have all limited the application of the method.
Summary of the invention
The objective of the invention is control problem for the difficult quality that overcomes ganoderma lucidum spore oil, utilize high performance liquid chromatography, set up effective ingredient triterpene component to ganoderma lucidum spore oil and had strong specific aim and high-efficiency liquid-phase fingerprint optionally, thereby ganoderma lucidum spore oil has been carried out quality control.
This method has overcome the conventional thought that needs to use object of reference in the step that finger-print is set up, accurately measure earlier the ganoderma lucidum spore oil certified products, then do not have object of reference condition under accurately select chromatographic condition, thereby by a large amount of experiments select to ganoderma lucidum spore oil triterpene component have strong specific aim and optionally high-efficient liquid phase chromatogram condition also further obtained ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint; By the content that this is pointed to the triterpene component and the selectivity finger-print is identified ganoderma lucidum spore oil and detected ganoderma lucidum spore oil.
Purpose of the present invention can reach by following measure:
A kind of fingerprint discrimination method of ganoderma lucidum spore oil comprises the steps:
The first step is set up ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint, and method is as follows:
The preparation of a, need testing solution: get Reishi sporule oil samples 300mg, with the mixed solution dissolving of 50% methyl alcohol-50% ethyl acetate, constant volume is to 25ml;
B, efficient liquid phase chromatographic analysis: get 20ul need testing solution sample introduction, continuous sample introduction 5 times; Chromatographic condition: chromatographic column is a luna C18 post, moving phase be second fine-phosphoric acid (preferred volume ratio is 40: 60), column temperature is 30 ℃, flow velocity is 0.8ml/min, the detection wavelength is 234 ± 1nm; Obtain ganoderma lucidum spore oil triterpene component high-efficiency liquid-phase fingerprint;
The high-efficiency liquid-phase fingerprint of c, 10 batches of ganoderma lucidum spore oils of mensuration also compares, and obtains the ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint that is made of its common characteristic peak;
In the ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint, have 1,2,3,4,5,6,7,8,9,100 main chromatographic peak, wherein the retention time at the first eight peak is respectively 32.22min, 33.52min, 34.60min, 35.36min, 38.95min, 39.77min, 40.08min, 41.22min, the peak area of ten main chromatographic peaks all accounts for more than 5% of total peak area, and its area sum accounts for more than 80% of total peak area; The Duplication of 10 batches of samples of the present invention is better, all more than 90%.
In second step, measure Reishi sporule oil samples triterpene component high-efficiency liquid-phase fingerprint to be measured with above-mentioned same procedure.
The 3rd step, testing sample high-efficiency liquid-phase fingerprint and above-mentioned standard high-efficiency liquid-phase fingerprint are compared, differentiate: when sample has retention time sequence consensus on 10 main chromatographic peaks and retention time and the standard finger-print, peak 1,2,5,6,7, the number percent that 8 peak area sum accounts for total peak area can assert that 80% when above the testing sample ganoderma lucidum spore oil is a certified products.
The present invention sets up the ganoderma lucidum spore oil high-efficiency liquid-phase fingerprint, helps guaranteeing the steady quality of triterpene compound in the ganoderma lucidum spore oil and controlled.Differentiate ganoderma lucidum spore oil by the ganoderma lucidum spore oil high-efficiency liquid-phase fingerprint, the kind and the quantity that can reflect contained triterpene in the ganoderma lucidum spore oil comprehensively, embody the complicacy of traditional Chinese medicine ingredients effectively, and can be used for differentiating the true and false of ganoderma lucidum spore oil and whether mixing.Simultaneously, the research and development that also can be the ganodenic acid kind new medicine provide the study of pharmacy data.In the ganoderma lucidum spore oil high-efficiency liquid-phase fingerprint of the present invention the triterpene component is had good selectivity and selectivity, thus the quality of reflection ganoderma lucidum spore oil that can be sensitiveer.
Description of drawings
Fig. 1 is a ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint.
Embodiment
The foundation of embodiment 1 ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint
1 instrument and material
1.1 instrument
The Agilentl100 high performance liquid chromatograph, quaternary pump, diode array detector, vacuum degassing machine, column oven, automatic sampler, U.S. Agilent company product, process software: Agilent chem workstation.
Chromatographic condition: chromatographic column: (250mm * 4.6mm), moving phase: second is fine: phosphoric acid (40:60), flow velocity: 0.8ml/min detects wavelength: 234nm to U.S. luna C18 post.Solvent: 50% methyl alcohol, 50% ethyl acetate.Get 20ul need testing solution sample introduction, continuous sample introduction 5 times.
1.2 material
CO
2Supercritical extract ganoderma lucidum spore oil (being provided by NanJing ZhongKe Pharmacy Co., Ltd), methyl alcohol, ethyl acetate are chromatographically pure.Get Reishi sporule oil samples 300mg, with the mixed solution dissolving of 50% methyl alcohol-50% ethyl acetate, constant volume is to 25ml.
2 methods
2.1 determining of relative retention value finger-print
Select the placed in the middle and chromatographic peak that in each sample, all exist of a retention time as object of reference, specify its α=1, obtain the α (α=t of each component in each sample respectively
R(i)/t
R(s), t wherein
R(i) be the appearance time of each component, t
R(s) be appearance time) with reference to the peak.
The chromatographic peak total area with a certain sample is a denominator, and each chromatographic peak area is a molecule in each sample, tries to achieve peak area ratio (ratio of peak area, the A of this chromatographic peak
r), it is marked on its α value item down, the α with each sample is arranged in rows by ascending order at last, obtains the relative retention value (α/A of 10 batches of Reishi sporule oil samples
r) finger-print, wherein there is not corresponding A
rThe time represent that this sample does not have the chromatographic peak of this α.
2.2 the Duplication of chromatographic peak (overlap ratio, R
o)
Choosing with reference to sample and be the contrast benchmark, is denominator with its peak number sum that goes out that goes out the peak number order and desire comparative sample, and the peak number sum that both have identical α value is a molecule, obtains the Duplication of sample respectively with this.
2.3 stability, instrument precision and reappearance test
Get same Reishi sporule oil samples,, respectively at 0h, 4h, 8h, 12h and 24h detect finger-print, investigate the α and the A of main chromatographic peak
rConsistance.
Get same Reishi sporule oil samples, after treatment, continuous sample introduction 5 times is investigated the α and the A of main chromatographic peak
rConsistance.Relative standard deviation (RSD) is a kind of investigation standard of test confidence level, according to detecting data, calculates RSD separately.
5 parts of same spore oil samples,, investigate the α and the A of main chromatographic peak
rConsistance, and calculate separately RSD.
3 results
3.1 main chromatographic peak area A
pWith the sample Duplication
Fig. 1 provides ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint, and as can be seen, essentially no chromatographic peak occurs after separating 40min in the record spectrogram, illustrates that active principle separates in 40min basically.
In 10 batches of samples being measured, because No. 1 sample chromatogram peak total area is bigger, peak number is more and representative, so the standard of being assigned therein as, the peak work that with the retention time is 26.94min calculates the α and the A at each peak to be measured with reference to the peak
rThe relative peak area of these 10 main chromatographic peaks all accounts for total peak area more than 5%.Result in the table 1 shows that peak area accounts for the number percent A that 10 the main chromatographic peak area sums of total peak area more than 5% account for total peak area
pAll more than 80%, and the Duplication of 10 batches of samples is better, all more than 90%.
The area ratio and the Duplication of the main chromatographic peak of table 1 10 batches of ganoderma lucidum spore oil test samples HPLC
3.2 stability, accuracy of instrument and reproducible test results
As shown in table 2, get the spore oil samples No. 1, detect through different time, the relative retention time of 10 main chromatographic peaks of investigation and the consistance of peak area ratio are better, illustrate that ganoderma lucidum spore oil test sample performance is more stable.
The relative retention time of table 2 stability test and peak area ratio
As shown in table 3, get the spore oil samples No. 1, continuous sample introduction 5 times (accuracy of instrument test), and 5 parts of the spore oil samples (reappearance test) of getting No. 1 lot number, the α of 10 main chromatographic peaks and A in the efficient liquid-phase chromatograph finger print atlas
rRDS all less than 3%.
The experimental result of test of table 3 accuracy of instrument and reappearance test
Claims (3)
1, a kind of fingerprint discrimination method of ganoderma lucidum spore oil comprises the steps:
The first step is set up ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint, and method is as follows:
The preparation of a, need testing solution: get Reishi sporule oil samples 300mg, with the mixed solution dissolving of 50% methyl alcohol-50% ethyl acetate, constant volume is to 25ml;
B, efficient liquid phase chromatographic analysis: get 20ul need testing solution sample introduction, continuous sample introduction 5 times; Chromatographic condition: chromatographic column is a luna C18 post, moving phase be second fine-phosphoric acid, column temperature is 30 ℃, flow velocity is 0.8ml/min, the detection wavelength is 234 ± 1nm; Obtain ganoderma lucidum spore oil triterpene component high-efficiency liquid-phase fingerprint;
The high-efficiency liquid-phase fingerprint of c, 10 batches of ganoderma lucidum spore oils of mensuration also compares, and obtains the ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint that is made of its common characteristic peak;
In second step, measure Reishi sporule oil samples triterpene component high-efficiency liquid-phase fingerprint to be measured with above-mentioned same procedure;
In the 3rd step, testing sample high-efficiency liquid-phase fingerprint and above-mentioned standard high-efficiency liquid-phase fingerprint are compared, differentiate.
2, discrimination method according to claim 1, wherein moving phase second fine with volume ratio phosphoric acid be 40:60.
3, discrimination method according to claim 1, in the described ganoderma lucidum spore oil triterpene component standard high-efficiency liquid-phase fingerprint, have 1,2,3,4,5,6,7,8,9,100 main chromatographic peak, wherein the retention time at the first eight peak is respectively 32.22min, 33.52min, 34.60min, 35.36min, 38.95min, 39.77min, 40.08min, 41.22min, the peak area of ten main chromatographic peaks all accounts for more than 5% of total peak area, and its area sum accounts for more than 80% of total peak area.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101352008A CN101435801A (en) | 2007-11-13 | 2007-11-13 | Fingerprint pattern identifying method of ganoderma lucidum spore oil |
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|---|---|---|---|
| CNA2007101352008A CN101435801A (en) | 2007-11-13 | 2007-11-13 | Fingerprint pattern identifying method of ganoderma lucidum spore oil |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732364B (en) * | 2009-12-07 | 2012-05-23 | 浙江工业大学 | Method for identifying lucid ganoderma alkaloids substance finger-print |
| CN103837622A (en) * | 2014-03-24 | 2014-06-04 | 中南林业科技大学 | Method for rapidly identifying types of tea oil |
| CN110749673A (en) * | 2019-10-25 | 2020-02-04 | 陇南市祥宇油橄榄开发有限责任公司 | Primary olive oil comparison fingerprint spectrum and construction method and application thereof |
-
2007
- 2007-11-13 CN CNA2007101352008A patent/CN101435801A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732364B (en) * | 2009-12-07 | 2012-05-23 | 浙江工业大学 | Method for identifying lucid ganoderma alkaloids substance finger-print |
| CN103837622A (en) * | 2014-03-24 | 2014-06-04 | 中南林业科技大学 | Method for rapidly identifying types of tea oil |
| CN110749673A (en) * | 2019-10-25 | 2020-02-04 | 陇南市祥宇油橄榄开发有限责任公司 | Primary olive oil comparison fingerprint spectrum and construction method and application thereof |
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Open date: 20090520 |