CN102175707A - New method for identifying and evaluating bulbus fritillariae cirrhosae - Google Patents
New method for identifying and evaluating bulbus fritillariae cirrhosae Download PDFInfo
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- CN102175707A CN102175707A CN2011100486105A CN201110048610A CN102175707A CN 102175707 A CN102175707 A CN 102175707A CN 2011100486105 A CN2011100486105 A CN 2011100486105A CN 201110048610 A CN201110048610 A CN 201110048610A CN 102175707 A CN102175707 A CN 102175707A
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Abstract
The invention relates to a method for identifying and evaluating bulbus fritillariae cirrhosae, wherein four types of pharmaceutically acceptable bulbus fritillariae cirrhosae are used as objects; the total extractives of the four types of bulbus fritillariae cirrhosae are used as analysis objects; 1H-NMR (Nuclear Magnetic Resonance) is used as a detection means; principal component analysis or minimum partial least square is used as a data processing method; and the 1H-NMR data of the total extractives of the four types of bulbus fritillariae cirrhosae are used as a base for judging whether the unknown bulbus fritillariae cirrhosae sample belongs to the pharmaceutically acceptable bulbus fritillariae cirrhosae sample or not.
Description
Technical field
The present invention relates to the discriminating and the evaluation method of Chinese crude drug Bulbus Fritillariae Cirrhosae.
Background technology
Bulbus Fritillariae Cirrhosae flavor is sweet, little hardship is arranged, and the effect of clearing heat and moistening lung, preventing phlegm from forming and stopping coughing is arranged, and cures mainly lung-heat type cough, coughs up sputum streaked with blood, disease such as excessive sputum and oppression feeling over the chest
[1]At present the Fritillaria plant of finding has 60 kinds, 45 mutation and 5 modification.Four Ji Yuan kinds of the Bulbus Fritillariae Cirrhosae that 2005 editions pharmacopeia are recorded are the liliaceous plant leaf roll bulb of fritillary (Bulbus Fritillariae Cirrhosae)
Fritillaria cirrhosa, the dark violet bulb of fritillary
F. unibratata, the Gansu bulb of fritillary
F. przewalskiiAnd Bulbus Fritillariae cirrhosae
F. delavayiDry bulb
[2]Former three is practised title " loose shellfish " and " blue or green shellfish " respectively by the proterties difference, and the latter practises title " lubei ".Investigate from plant origin
[3], provinces and regions such as leaf roll bulb of fritillary main product Sichuan, Tibet, Yunnan; Aba Tibetan Autonomous Prefecture, dark violet bulb of fritillary main product Sichuan, Qinghai; There is a small amount of cultivation in counties such as Sichuan Ruoergai, Xiao Jin, Nanchuan; Provinces such as the Gansu bulb of fritillary (claiming the Min shellfish again) main product Gansu, Qinghai, Yunnan, Sichuan; Provinces and regions such as Bulbus Fritillariae cirrhosae main product Yunnan, Sichuan, Qinghai, Tibet are born in the rock crevice on the quicksand beach of height above sea level 3000m-4700m.From the medicinal material specification, loose shellfish plant provenance is mainly Bulbus Fritillariae Cirrhosae, and is best in quality; Blue or green shellfish plant provenance is mainly the dark violet bulb of fritillary, and quality is also excellent; The lubei plant of generally originating is a Bulbus Fritillariae cirrhosae, and quality is inferior to loose shellfish, blue or green shellfish.
Because the effect widespread use clinically of the significant clearing heat and moistening lung of Bulbus Fritillariae Cirrhosae, preventing phlegm from forming and stopping coughing.But output is little, and consumption is big, and supply falls short of demand in market, causes the market price to rise steadily, thereby some pseudo-product have appearred in various places in succession.What Bulbus Fritillariae Cirrhosae itself existed simultaneously uses with mutually, disorderly serious for abusing phenomenon.According to investigations
[4], except that pharmacopeia is recorded kind, still have the bulb of many these other plants of genus to do " Bulbus Fritillariae Cirrhosae " use in different regions.Their sections belong to approaching, and profile has similarity, and the common trait of Fritillaria plant metabolites is all to have different steroid alkaloid, and chemical constitution roughly the same.Therefore, how to differentiate that different types of bulb of fritillary class medicinal material commodity have become one of difficult point in the Chinese traditional medicine identification.
At present a lot of to the distinguishing method between true and false of Bulbus Fritillariae Cirrhosae, as Chen Junhua etc.
[5]Outward appearance shape property and powder characteristics to certified products tendril-leaved fritillary bulb and pseudo-product are discussed.Cheng Cungui etc.
[6]Adopt fourier transform infrared spectroscopy directly to measure the infrared spectrum of botanical herbs material, Bulbus Fritillariae Cirrhosae, fritillaria thunbergii and Bulbus Fritillariae thunbergii are distinguished evaluation, find that its infrared spectrum differs greatly.Meng Yan etc.
[7]The easily mixed little flat shellfish of product to Bulbus Fritillariae Cirrhosae
F ritillaria ussuriensisBulb carried out medicinal material proterties, microscopic features and thin-layer chromatography Study on Identification, and compare with loose shellfish, flat shellfish.Zhang Ping etc.
[8]With atomic absorption spectrophotometry (FAAS method) 8 kinds of inorganic element contents in Hupeh Fritillary Bulb, fritillaria thunbergii and three kinds of bulbs of fritillary of Bulbus Fritillariae Cirrhosae have been carried out measuring relatively.Yu Hua etc.
[9]Utilize high performance liquid chromatography, the total effective constituent bulb of fritillary suffering of the Bulbus Fritillariae Cirrhosae of four Ji Yuan kinds is carried out quantitative test for index, to reach the quantivative approach of setting up control Bulbus Fritillariae Cirrhosae quality.Ma Liqiong etc.
[10]Adopt the double phase titration method to measure the Bulbus Fritillariae Cirrhosae alkaloids content in 17 different places of production of four Ji Yuan kinds that Chinese Pharmacopoeia records, it is higher with Bulbus Fritillariae Cirrhosae to record in the Bulbus Fritillariae Cirrhosae in 17 different places of production total alkaloid contents, the dark violet bulb of fritillary is on the low side, and (content of Bulbus Fritillariae Cirrhosae is 0.08 %, the dark violet bulb of fritillary is 0.04 %, Bulbus Fritillariae cirrhosae is 0.07 %, and the Gansu bulb of fritillary is 0.05 %).Also have much about Bulbus Fritillariae Cirrhosae and differentiate related articles with quality controling research, but these research methods are nothing more than being exactly that crude drug character identification, microstructure discriminating, physicochemical identification, thin-layer chromatography are differentiated, high-efficient liquid phase technique is measured its alkaloid or the like.These methods can not reach the purpose of difference separate sources medicinal material fully.The present invention has gathered the Bulbus Fritillariae Cirrhosae of four the Ji Yuan kinds in main producing region, adopts
1H-NMR binding pattern identification (PR) method is studied this four Ji Yuan kind Bulbus Fritillariae Cirrhosae sample, and attempts setting up the forecast model of Bulbus Fritillariae Cirrhosae, forecast model is verified as the test set sample with the Bulbus Fritillariae Cirrhosae sample commodity that circulate on the market then.The result shows
1The H-NMR-PR method can be differentiated four Ji Yuan kind Bulbus Fritillariae Cirrhosaes effectively.The forecast model of being set up also can carry out Fast Classification to the Bulbus Fritillariae Cirrhosae sample that circulates on the market well and differentiate.
Summary of the invention
Technical matters to be solved by this invention provides a kind of discriminating and evaluation method of new Chinese crude drug Bulbus Fritillariae Cirrhosae.
The Chinese crude drug Bulbus Fritillariae Cirrhosae of indication of the present invention is the Bulbus Fritillariae Cirrhosae of four kinds of Ji Yuan stipulating in the version in 2005 of the Pharmacopoeia of the People's Republic of China.
The evaluation index of indication of the present invention is the Changing Pattern of the overall chemical composition of Bulbus Fritillariae Cirrhosae.
The used determination techniques of the present invention is the hydrogen nuclear magnetic resonance technology.
The data processing method of indication of the present invention is principal component analysis (PCA) and minimum square law partially.
The present invention relates to Chinese crude drug Bulbus Fritillariae Cirrhosae sample collecting, processing, hydrogen nuclear magnetic resonance mensuration, data processing and analysis and as a result judgment mode finish by following manner.
1. Bulbus Fritillariae Cirrhosae sample collecting:
The Bulbus Fritillariae Cirrhosae sample of four kinds of Ji Yuan is collection in the open air, and identifies by pharmacopeia and traditional plant evaluation mode, is respectively:
The leaf roll bulb of fritillary: Fritillaria cirrhosa
The Gansu bulb of fritillary: Fritillaria przewalskii
Bulbus Fritillariae cirrhosae: Fritillaria delavayi
The dark violet bulb of fritillary: Fritillaria unibratata
2. The pretreatment
Take by weighing the about 1.2g of Bulbus Fritillariae Cirrhosae sample (crossing 80 mesh sieves) of grinding, add methyl alcohol 15ml, weigh, in 55 ℃ of water-bath backflow 90min; Weigh again, and complement to weight before the water-bath, filter, discard filtrate just, get subsequent filtrate 10ml, be evaporated to dried, vacuum drying 30 minutes; Residue 0.05ml DMSO-d
6Dissolving changes f5mm nuclear-magnetism pipe over to, promptly gets to supply
1The sample solution that the H-NMR spectrum is measured.
3. hydrogen nuclear magnetic resonance is measured
1The condition determination of H-NMR: constant temperature is at 296K, with DMSO-d
6As internal lock, to scan 128 times, the sampling time territory is 64 k, spectrum width is 12335.526 Hz, and the sampling time is 2.66 s, and recurrent interval D1 is 3.00 s, presaturation pulse train (zgpr) the compacting water peak-to-peak signal of employing standard adopts CPMG pulse train to suppress big molecular signal.Deamplification (FID signal) gains freedom.The FID signal transfers the one-dimensional NMR spectrogram to through Fourier transform.With the tms signal peak is that 0 of interior mark is adjusted chemical displacement value, and it is corresponding promptly to obtain each sample
1The H-NMR collection of illustrative plates.With per 0.05 chemical displacement value unit to each sample
1The H-NMR collection of illustrative plates is divided into 200 sections, and carries out integration respectively, promptly obtains each the chemical displacement value section and the corresponding signal peak area integral value of each sample.With the sample is horizontal ordinate, and integrated value is an ordinate, the composition data matrix.
4. data processing and analysis
To respectively organize data in the data matrix, carry out normalized, import statistics software then, start the principal component analysis (PCA) pattern and analyze by unified mode.
5. judgment mode as a result
Each major component that obtains is respectively horizontal ordinate carries out the X-Y scheme making.The distributed points regional assignment of Bulbus Fritillariae Cirrhosae sample in X-Y scheme of clearly identifying is the discriminating fundamental region.Unknown Bulbus Fritillariae Cirrhosae sample and above-mentioned and fundamental region sample spot are compared, be in the fundamental region, judge that then this unknown Bulbus Fritillariae Cirrhosae sample is that pharmacopeia allows the Bulbus Fritillariae Cirrhosae that uses as unknown Bulbus Fritillariae Cirrhosae sample; As being in outside the fundamental region, judge that then this unknown Bulbus Fritillariae Cirrhosae sample is the Bulbus Fritillariae Cirrhosae that the non-permission of pharmacopeia is used as unknown Bulbus Fritillariae Cirrhosae sample.
Description of drawings
Fig. 1 is the leaf roll bulb of fritillary among the embodiment 1
1The H-NMR diagram.
Fig. 2 is the dark violet bulb of fritillary among the embodiment 1
1The H-NMR diagram.
Fig. 3 is the Gansu bulb of fritillary among the embodiment 1
1The H-NMR diagram.
Fig. 4 is a Bulbus Fritillariae cirrhosae among the embodiment 1
1The H-NMR diagram.
Fig. 5 is 1 four kinds of basic sources of embodiment Bulbus Fritillariae Cirrhosae
1H-NMR-PCA analysis result diagram.
Fig. 6 is embodiment 1 unknown Bulbus Fritillariae Cirrhosae sample and four kinds of basic source Bulbus Fritillariae Cirrhosaes
1H-NMR-PCA analysis result diagram.
Embodiment
Embodiment 1:
With the dark violet bulb of fritillary that derives from Xining, Qinghai, Maqin, the Gansu bulb of fritillary of Guide, Qinghai, Xining, Qinghai, Luhuo, state, Ganzi, the leaf roll bulb of fritillary in kangding county and each 6 each sample of Bulbus Fritillariae cirrhosae that derive from Ding County, Shanghai, Sichuan are object, take by weighing the about 1.2g of Bulbus Fritillariae Cirrhosae sample (crossing 80 mesh sieves) of grinding respectively, add methyl alcohol 15ml, weigh, in 55 ℃ of water-bath backflow 90min; Weigh again, and complement to weight before the water-bath, filter, discard filtrate just, get subsequent filtrate 10ml, be evaporated to dried, vacuum drying 30 minutes; Residue 0.05ml DMSO-d
6Dissolving changes f5mm nuclear-magnetism pipe over to, promptly gets to supply
1The sample solution that the H-NMR spectrum is measured.
At constant temperature at 296K, with DMSO-d
6As internal lock, scan 128 times, the sampling time territory is 64 k, spectrum width is 12335.526 Hz, and the sampling time is 2.66 s, and recurrent interval D1 is 3.00 s, presaturation pulse train (zgpr) the compacting water peak-to-peak signal of employing standard, adopt CPMG pulse train to suppress under the condition of big molecular signal, measure the deamplification that gains freedom (FID signal) in Bruker AV II-600 nuclear magnetic resonance analyser.The FID signal is that 0 of interior mark is adjusted chemical displacement value through the Fourier transform conversion with the tms signal peak, and it is corresponding promptly to obtain each sample
1The H-NMR collection of illustrative plates (Fig. 1-Fig. 4).With per 0.05 chemical displacement value unit to each sample
1The H-NMR collection of illustrative plates is divided into 200 sections, and carries out integration respectively, promptly obtains each the chemical displacement value section and the corresponding signal peak area integral value of each sample.With the sample is horizontal ordinate, and integrated value is an ordinate, the composition data matrix.
Data matrix is imported SIMCA-P software, starts the principal component analysis (PCA) program, extract major component, with major component 2(PC2) and major component 1(PC1) score value be the score scatter diagram (Fig. 5) of horizontal ordinate drafting.With the four kinds of residing zone location of Bulbus Fritillariae Cirrhosae sample fundamental regions.
17 Bulbus Fritillariae Cirrhosae samples (wz1-17) in Chinese medicine market and each pharmacy will be derived from, by same way as extract, after mensuration, collection of illustrative plates and the data processing, with the data importing SIMCA-P software that obtains, with major component 2(PC2) and major component 1(PC1) score value is the score scatter diagram of horizontal ordinate drafting and compares (Fig. 6) with the four kinds of residing zone location of Bulbus Fritillariae Cirrhosae sample fundamental regions.
Wz2, wz9, wz11, wz13, wz14, wz15, wz16, wz17 sample are in the fundamental region in the unknown Bulbus Fritillariae Cirrhosae sample, judge that then these samples are the Bulbus Fritillariae Cirrhosae that pharmacopeia allows use; Wz1, wz3, wz5, wz6, wz7, wz8, wz10 and wz12 then are in outside the fundamental region, then are judged as the medicine SARS and allow to use kind.
Claims (6)
1. new method of differentiating and estimating Bulbus Fritillariae Cirrhosae, its content comprises (1) Bulbus Fritillariae Cirrhosae; (2) Bulbus Fritillariae Cirrhosae sample pretreatment; (3) Bulbus Fritillariae Cirrhosae sample determination method; (4) data processing and analysis; (5) database is set up; (6) parts such as discriminating and evaluation.
2. (1) Bulbus Fritillariae Cirrhosae described in claim 1, Bulbus Fritillariae Cirrhosae refer to the legal four kinds of medicinal Bulbus Fritillariae Cirrhosaes of defined (the leaf roll bulb of fritillary, the dark violet bulb of fritillary, the Gansu bulb of fritillary and Bulbus Fritillariae cirrhosae) under the Bulbus Fritillariae Cirrhosae item in the Pharmacopoeia of the People's Republic of China version in 2005.
3. (2) Bulbus Fritillariae Cirrhosae sample pretreatment described in claim 1, the Bulbus Fritillariae Cirrhosae sample pretreatment refers to the physical behavior according to (1) Bulbus Fritillariae Cirrhosae described in the claim 1, correspondingly take, the pulverizing to adapt to the assay method needs, extraction, concentrate, methods such as drying, freeze-drying, dissolving.
4. (3) assay method described in claim 1 refers to the hydrogen nuclear magnetic resonance method among the present invention, and the megacycle number of used instrument generally should be more than 400 megacycles (400MHz).
5. (4) data processing and the analysis described in claim 1 can be the principal component analysis (PCA) among the present invention, also can be the PLS method, the PLS-DA method, the neural network method or adopt various data filters after analytic approach.
6. (5) described in claim 1 are differentiated and evaluation method, mainly be the data that the Bulbus Fritillariae Cirrhosae sample based on some obtains after above-mentioned pre-service, mensuration, data processing, constitute product database, with this database data is standard, and the Bulbus Fritillariae Cirrhosae sample is differentiated and estimated; Data as new Bulbus Fritillariae Cirrhosae obtains by identical method compare with database data, both unanimities, and then declaring this Bulbus Fritillariae Cirrhosae sample is that pharmacopeia allows to use kind; Inconsistent as both, then declaring this Bulbus Fritillariae Cirrhosae sample is that non-pharmacopeia allows to use kind.
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Application publication date: 20110907 |