CN104000857A - Method for extracting common traditional Chinese medicine from traditional Chinese medicinal materials and authenticating common traditional Chinese medicine through MALDI-TOF-MS - Google Patents
Method for extracting common traditional Chinese medicine from traditional Chinese medicinal materials and authenticating common traditional Chinese medicine through MALDI-TOF-MS Download PDFInfo
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- CN104000857A CN104000857A CN201410260787.5A CN201410260787A CN104000857A CN 104000857 A CN104000857 A CN 104000857A CN 201410260787 A CN201410260787 A CN 201410260787A CN 104000857 A CN104000857 A CN 104000857A
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Abstract
The invention discloses a method for extracting a common traditional Chinese medicine from traditional Chinese medicinal materials and authenticating the common traditional Chinese medicine through MALDI-TOF-MS, and belongs to the technical field of biology. According to the method, the traditional Chinese medicinal materials are smashed at high speed, total protein and polypeptide are extracted from the traditional Chinese medicinal materials, dialysis desalination is conducted, then the MALDI-TOF-MS is adopted for detection, and a traditional Chinese medicinal material protein map is obtained. The simple protein extracting technology and Malditof are combined for authenticating the protein maps of the common traditional Chinese medicinal materials such as astragalus membranaceus, ginseng and liquorice. The method has the advantages of being easy and reliable to implement, and provides selections and references for authenticating the common traditional Chinese medicinal materials.
Description
Technical field
The present invention relates to a kind of conventional Chinese medicine method with MALDI-TOF-MS qualification extracted from Chinese crude drug, be specifically related to a kind of method of the Malditof of application qualification parts of generic medicinal plants Radix Astragali, Radix Ginseng, Radix Glycyrrhizae, belong to biological technical field.
Background technology
Chinese crude drug is the raw material of the prepared slices of Chinese crude drugs, Chinese medicine extract and Chinese patent medicine, and its front end is medicinal plants, medicinal animal, mineral drug and specific medicinal part thereof, or it is former to be called the base of Chinese crude drug.The precise Identification of Chinese crude drug is the basic link of Chinese medicine operation and development, receives much concern always.The species confirmation of originate of the relevant medical material standard Main Basis means of taxonomic research of country, defines medicinal part, has substantially clarified confusion.But medical material market is in dynamic change, there is fake and forged phenomenon in some important drugs resources, and the simultaneously former variation of base and new medical material type constantly occur, the Chinese crude drug in the different places of production there are differences, and identification research work still can not be relaxed.
Along with going deep into and scientific and technical development of Chinese medicine research, existing several classes are for the identification of the new technique of Chinese crude drug at present.The first kind is chemical identification technology.The material base of Chemistry for Chinese Traditional Medicine qualification is its contained chemical composition group.Chinese crude drug ingredient complexity is various, and chemical identification is selected the chemical composition of minority tool feature conventionally, or forms feature as appraisal basis using the entirety of chemical composition.Along with the development of chromatographic isolation, analytical technology, HPLC, GC, the methods such as HPCE and with the development of spectrum multiple techniques, can reflect meticulousr and exactly the features such as Chinese crude drug chemical group mark and amount, become the effective ways of Materia Medica Identification and quality control.Equations of The Second Kind is Molecular Identification technology.Mainly to identify for DNA molecular and protein.DNA bar codes technique based on sequence analysis is that impact is at present large, application DNA authenticate technology more widely.Compared with other method for identifying molecules, the qualification of DNA bar code has three advantages: qualification result is repeatable good; Method highly versatile; Can build unified database and qualification platform, be easy to promote and standardization.
Mainly contain protein electrophorese technology, ELISA detection technique, albumen flight time mass spectrum authenticate technology for the authenticate technology of albumen.Substance assistant laser desorpted ionized flight time mass spectrum (Matrix-assisted laser desorption ionization-time of flight massspectrometry, MALDI-TOF-MS) technology is the novel organic mass spectrometry of a kind of soft ionization that development in recent years is got up, by introducing substrate molecule, make testing molecule not produce fragment, solved non-volatile and problem thermal instability biomacromolecule desorption ionization.The features such as that MALDI-TOF-MS has is highly sensitive, accuracy is high, resolution is high, collection of illustrative plates is simple and clear, mass range is wide and speed is fast, have special superiority measuring polypeptide, protein and synthetic high polymer application aspect.
At present MALDI-TOF-MS has obtained comparatively successfully application aspect microbial identification, but application aspect Materia Medica Identification is also relatively less.Chinese crude drug also has more protein component, and the composition of the Chinese crude drug of same breed more existing conservatives on albumen composition also has the heterogeneity of the impact such as the factor that is subject to the place of production.Therefore applying the mass spectrum that MALDI-TOF-MS collects the Chinese crude drug in the different places of production of different Chinese crude drugs, same breed not only can evaluate but also can be for the identification of Chinese crude drug.The present invention's application Protein Extraction technology has been collected the mass spectrum of the common Chinese medicine material Radix Astragali, Radix Glycyrrhizae, Radix Ginseng in conjunction with sample treatment application MALDI-TOF-MS, for its qualification provides selection and reference.
Summary of the invention
The object of the invention is to set up a kind of method of identifying the parts of generic medicinal plants Radix Astragali, Radix Ginseng, Radix Glycyrrhizae, for identifying that conventional Chinese crude drug provides selection and reference.
Technical scheme of the present invention, the present invention carries out high speed pulverization and has extracted Chinese crude drug total protein and polypeptide Chinese crude drug, after dialysis desalination, detects and has obtained Chinese crude drug protein profiling with MALDI-TOF-MS.
A kind of method of extracting conventional Chinese medicine from Chinese crude drug, step is: get traditional Chinese medicinal material samples and pulverize through vertical and high-speed pulverizer, then getting 4g powder sample uses containing 50mM NaCl, pH 8.0 25mM Tris-HCl buffer and dissolves according to the mass volume ratio example of 1:10,50 DEG C of water-bath lixiviate 4h; The centrifugal 30min of 8000 rpm, gets supernatant subsequently, with 15mL centrifugal ultrafiltration pipe concentrating sample be 1mL to volume, it blocks molecular weight is 3KD, pure water dialysis 2 days for sample obtains total protein and the polypeptide of conventional Chinese medicine in Chinese crude drug.
Described conventional Chinese medicine is the Radix Astragali, Radix Ginseng or Radix Glycyrrhizae.
The method of Malditof qualification for the described conventional Chinese medicine extracting from Chinese crude drug, step is: will in Chinese crude drug, extract the total protein of conventional Chinese medicine and polypeptide according to the upper quadrat method loading of conventional albumen, get 1 μ L point on Malditof target, cover on sample Deng getting 1 μ L TA substrate liquid after its natural drying, sample detection after natural drying, obtains the collection of illustrative plates of conventional Chinese medicine.
Beneficial effect of the present invention: the method for the application Malditof qualification parts of generic medicinal plants Radix Astragali provided by the invention, Radix Ginseng, Radix Glycyrrhizae, adopt high speed pulverization method Chinese crude drug to be processed to and extracted in conjunction with simple solution leach extraction method total protein and the polypeptide of Chinese crude drug, after dialysis, be the collection of illustrative plates that available Malditof obtains the proteins and peptides of sample, and then realize the qualification to Chinese crude drug.Have compared with other method simple to operate, analysis time is short, the feature of method highly versatile; With similar unified database and the qualification platform of building of DNA bar codes technique, be easy to promote and standardization.
Brief description of the drawings
The Malditof mass spectrum of the Radix Astragali of Fig. 1-a Inner Mongol.
The Malditof mass spectrum of the Radix Astragali in Fig. 1-b Suzhou.
The Malditof mass spectrum of the Radix Astragali in Fig. 1-c Gansu.
The Malditof mass spectrum of the Radix Glycyrrhizae of Fig. 2-a Inner Mongol.
The Malditof mass spectrum of the west of Gansu Province, Fig. 2-b Gansu Radix Glycyrrhizae.
The Malditof mass spectrum of the Radix Glycyrrhizae in Fig. 2-c Xinjiang.
The Malditof mass spectrum of Fig. 3-a northeast Radix Ginseng.
The Malditof mass spectrum of Fig. 3-b American Ginseng.
Detailed description of the invention
Further illustrate by the following examples the present invention.
Embodiment 1
Instrument:
Substance assistant laser desorpted ionized flight time mass spectrum (ultrafle Xtreme MALDI-TOF-MS), German Brooker
;
YS-04 miniature high-speed pulverizer, the Yanshan Mountain, Beijing Zheng De plant equipment company limited;
70mm (MD77) bag filter, Cutoff is 3KDa, USA;
TGL-40B table-type low-speed centrifuge, Anting Scientific Instrument Factory, Shanghai;
KFLOW water purification machine, Kai Folong company;
The horizontal shaking table of ZD-9556, granary science and education equipment factory;
Adjustable pipettor, Thermo Labsystems company;
Turbine mixer, Shanghai Hu Xi Instrumental Analysis factory.
Two, reagent: Tris base, Sigma-Aldrich; Other reagent are analytical reagent.
Three, step, taking Chinese medicine astragalus as example
1, traditional Chinese medicinal material samples is processed and Protein Extraction
Get the dry Radix Astragali after 15g section, pulverize through vertical and high-speed pulverizer.Getting 4g powder sample uses containing 50mM NaCl, pH 8.0 25mM Tris-HCl buffer and dissolves according to the mass volume ratio example of 1:10,50 DEG C of water-bath lixiviate 4h.The centrifugal 30min of 8000 rpm, gets supernatant, and using 15mL centrifugal ultrafiltration pipe (millipore, blocking molecular weight is 3KDa) concentrating sample is 1mL to volume.Pure water dialysis (blocking molecular weight is 3KD) dialysis 2 days for sample, removes salt ion, obtains total protein and the polypeptide of the Radix Astragali.
2, Malditof qualification parts of generic medicinal plants
By the total protein of the conventional Chinese medicine extracting in Chinese crude drug and polypeptide according to the upper quadrat method loading of conventional albumen, get 1 μ L point on Malditof target, cover on sample Deng getting 1 μ L TA substrate liquid after its natural drying, sample detection after natural drying, obtains the collection of illustrative plates of conventional Chinese medicine.
Put forward after total protein and polypeptide the Malditof mass spectrum of the Radix Astragali of identifying the Inner Mongol, Suzhou and the Gansu that obtain with Malditof respectively as shown in Fig. 1-a, Fig. 1-b and Fig. 1-c according to above-mentioned steps; The Malditof mass spectrum of the Radix Glycyrrhizae in Inner Mongol, the west of Gansu Province, Gansu and Xinjiang is as shown in Fig. 2-a, Fig. 2-b and Fig. 2-c; The Malditof mass spectrum of the Radix Ginseng of northeast and the U.S. is as shown in Fig. 3-a and Fig. 3-b.
Claims (3)
1. one kind is extracted the method for conventional Chinese medicine from Chinese crude drug, it is characterized in that step is: get traditional Chinese medicinal material samples and pulverize through vertical and high-speed pulverizer, then getting 4g powder sample uses containing 50mM NaCl, pH 8.0 25mM Tris-HCl buffer and dissolves according to the mass volume ratio example of 1:10,50 DEG C of water-bath lixiviate 4h; The centrifugal 30min of 8000 rpm, gets supernatant subsequently, with 15mL centrifugal ultrafiltration pipe concentrating sample be 1mL to volume, it blocks molecular weight is 3KD, pure water dialysis 2 days for sample obtains total protein and the polypeptide of conventional Chinese medicine in Chinese crude drug.
2. the method for extracting according to claim 1 conventional Chinese medicine from Chinese crude drug, is characterized in that: described conventional Chinese medicine is the Radix Astragali, Radix Ginseng or Radix Glycyrrhizae.
3. the method for Malditof qualification for the conventional Chinese medicine that described in claim 1, method is extracted from Chinese crude drug, it is characterized in that step is: will in Chinese crude drug, extract the total protein of conventional Chinese medicine and polypeptide according to the upper quadrat method loading of conventional albumen, get 1 μ L point on Malditof target, cover on sample Deng getting 1 μ L TA substrate liquid after its natural drying, sample detection after natural drying, obtains the collection of illustrative plates of conventional Chinese medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105574474A (en) * | 2014-10-14 | 2016-05-11 | 中国科学院大连化学物理研究所 | Mass spectrometry information-based biological characteristic image identification method |
| CN105572212A (en) * | 2014-10-14 | 2016-05-11 | 中国科学院大连化学物理研究所 | Visual mass spectrometry information-based sun-dried ginseng and red ginseng rapid identification method |
| CN110596230A (en) * | 2019-09-18 | 2019-12-20 | 山东省分析测试中心 | MALDI-TOF-MS-based high-throughput analysis method for capsule single-particle medicine |
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| CN1687115A (en) * | 2005-03-25 | 2005-10-26 | 中国农业大学 | Agglutinin protein of astragalus root and preparation method thereof |
| CN102260340A (en) * | 2011-06-20 | 2011-11-30 | 长春中医药大学 | Chinese magnoliavine protein, and preparation method and medicinal application thereof |
| CN103627699A (en) * | 2012-08-21 | 2014-03-12 | 中国中医科学院中药研究所 | Traditional Chinese medicinal material genome DNA extraction method suitable for DNA amplification technology |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105574474A (en) * | 2014-10-14 | 2016-05-11 | 中国科学院大连化学物理研究所 | Mass spectrometry information-based biological characteristic image identification method |
| CN105572212A (en) * | 2014-10-14 | 2016-05-11 | 中国科学院大连化学物理研究所 | Visual mass spectrometry information-based sun-dried ginseng and red ginseng rapid identification method |
| CN105574474B (en) * | 2014-10-14 | 2019-03-12 | 中国科学院大连化学物理研究所 | A kind of biometric image recognition methods based on Information in Mass Spectra |
| CN110596230A (en) * | 2019-09-18 | 2019-12-20 | 山东省分析测试中心 | MALDI-TOF-MS-based high-throughput analysis method for capsule single-particle medicine |
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