CN103808749A - Method for identifying forsythia suspensa derivatives - Google Patents

Method for identifying forsythia suspensa derivatives Download PDF

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CN103808749A
CN103808749A CN201410044108.0A CN201410044108A CN103808749A CN 103808749 A CN103808749 A CN 103808749A CN 201410044108 A CN201410044108 A CN 201410044108A CN 103808749 A CN103808749 A CN 103808749A
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capsule
active component
weeping forsythia
benzyl carbinol
lignanoid
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CN103808749B (en
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董建军
张海艳
赵天增
郭唯
李晓
陈玲
范毅
常霞
***
魏悦
李倩
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Wang Shuncai
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Henan Kegao Vegetable Natural Product Development Engineering Technology Co ltd
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Abstract

The invention relates to a method for identifying forsythia suspensa derivatives. The method comprises the following steps: (1) directly obtaining the forsythia suspensa derivatives as a forsythia suspensa lignans and phenylethanoid glycoside characteristic extract containing an active component group; (2) carrying out IGD (Integrated Graphics Driver) nuclear magnetic resonance spectroscopy fingerprint spectrum detection on the forsythia suspensa lignans and phenylethanoid glycoside characteristic extract so as to obtain peak intensities of characteristic peaks of a plurality of active components in the characteristic extract according to a fingerprint spectrum; and detecting the peak intensity of the characteristic peak of a standard reference corresponding to each active component in the same manner; (3) obtaining the absolute content of the standard reference by a quantitative analysis means; (4) calculating the content of each active component and the content of the active component group in the forsythia suspensa derivatives in a ratio of the peak intensities of the characteristic peaks and the absolute content. The method provided by the invention can be used for reflecting the lignans and phenylethanoid glycoside compounds contained in the forsythia suspensa derivatives and a ratio of the lignans compounds to the phenylethanoid glycoside compounds, so that the purpose of identifying the quality of the forsythia suspensa derivatives can be realized.

Description

A kind of method of differentiating capsule of weeping forsythia spin-off
Technical field
The invention belongs to the discriminating field of natural medicinal plant, particularly, relate to a kind of method of differentiating capsule of weeping forsythia spin-off.
Background technology
The capsule of weeping forsythia is one of conventional traditional Chinese medicine of Chinese Clinical, for Oleaceae (Oleaceae) Forsythia Vahl (Forsythia) the plant capsule of weeping forsythia (Forsythia suspensa(Thunb.) Vahl) dry fruit, fruit just ripe still gathering when band is green is called green grass or young crops and sticks up, and gathers to be called always and stick up when the well-done color jaundice of fruit.The capsule of weeping forsythia is returned lung, the heart, small intestinl channel, and the property of medicine is slightly cold, bitter, have clearing heat and detoxicating, dispersing swelling and dissipating binds, dispelling wind and heat from the body effect [Chinese Pharmacopoeia Commission. one of Pharmacopoeia of People's Republic of China version in 2010. Chinese Medicine science and technology publishing house, 159.].Its active component is mainly lignanoids and benzyl carbinol glycosides compounds, have antiviral, anti-inflammatory, antibacterial, anti-oxidant, fall blood ester, the effect such as protect the liver [Wang Jinmei, etc. research and development of natural products 2007,19:153.].
Above-mentioned certain constituents of qualitative discriminating or its certain active component content of quantitative measurement, become the important method (for example Chinese Pharmacopoeia 2010 editions is that lignan component forsythin in capsule of weeping forsythia spin-off and phenylethanoid glycoside Forsythoside are carried out to qualitative and quantitative analysis) of evaluating capsule of weeping forsythia spin-off quality; This quality testing pattern of carrying out qualitative and quantitative analysis for single component can not effectively be controlled the inherent quality of Chinese medical extract, cannot meet current for the objective an urgent demand of effectively evaluating and control the capsule of weeping forsythia and the quality of the pharmaceutical preparations thereof.
Finger-print is current internationally recognized control Chinese medicine or natural drug quality one of the most effective mode.People rely on practical finger-print not only can confirm the true and false of this product, can judge its stability simultaneously.At present; the finger-print research work of the lignanoid of capsule of weeping forsythia spin-off and benzyl carbinol glycosides compounds; concentrate on HPLC finger-print [1. [and Chinese Pharmacopoeia Commission. one of Pharmacopoeia of People's Republic of China version in 2010. Chinese Medicine science and technology publishing house; 381. ②Sun states are auspicious; Deng. Chinese patent drug 2007; 29 (2): 161.], capillary electrophoresis fingerprint (HPCE method) [Sun Guoxiang; Deng. chromatogram 2006; 24 (2): 196.], tablets by HPLC-MS (LC-MS) [Qu HH; et al.MICROCHEMICAL JOURNAL, 2008; 89 (2): 159] etc.Wherein main stream approach HPLC finger-print is owing to being subject to non-chromatographic condition (as chromatographic column internal diameter, length, the fixing phase trade mark, carrier granularity, flow rate of mobile phase, the mutually each component ratio of mixed flow, column temperature, sample size, detector sensitivity etc.) impact greatly and needing by standard items etc. reason, and repeatability and feasibility all exist many limitation; Though HPCE method is not vulnerable to pollution and be better than HPLC method of efficient, quick, easy and post with it, but still have that reappearance is poor, the range of linearity is narrow and the deficiency such as sensitivity is lower; Single LC-MS analytical approach is due to the complicacy of Chinese medicine compound prescription and lack the present situation of standard items, and MS also has the problem such as degree of ionization and matrix interference, is conventionally not enough to Multiple components to carry out that structure is qualitative accurately.
IGD carbon-13 nmr spectra coupling (IGD 13c NMR coupling) fingerprint pattern technology, be also inverted gated decoupling carbon-13 nmr spectra coupling fingerprint pattern technology, this technology be study proton nmr spectra for many years ( 1h NMR) fingerprint pattern technology [Zhao Tianzeng, etc. 1hNMR fingerprint technique plant identification Chinese medicine, Chinese herbal medicine 2000,31 (11): 868-870] on basis, combine other technologies (such as current most widely used high efficiency liquid phase (HPLC) fingerprint pattern technology [Xie Peishan etc., chromatographic fingerprints of Chinese materia medica, People's Health Publisher, 2005] a kind of new comprehensive fingerprint pattern technology of non-single means) proposing.So far, for capsule of weeping forsythia spin-off and Related product, and have no the report differentiated with IGD carbon-13 nmr spectra coupling fingerprint pattern technology and the disclosure of technology contents.
The quality of capsule of weeping forsythia spin-off does not lie in certain single component, and its curative effect is the result of the collaborative proportioning of multiple compositions.Therefore, foundation can not only be controlled capsule of weeping forsythia spin-off active component, and it is imperative to control the IGD carbon-13 nmr spectra coupling finger-print of ratio of these active components.Research and the application of capsule of weeping forsythia spin-off carbon-13 nmr spectra coupling finger-print, not only can solve the difficult problem that China's capsule of weeping forsythia spin-off is evaluated, also for strengthening systematization and the standardization of the inherent composition Study of capsule of weeping forsythia spin-off, realize the assurance that science is provided in line with international standards.Along with this technology applying in other Chinese medical extracts, the great scientific value of this technology will be increasingly outstanding.
Summary of the invention
In order to solve the problem of prior art, the object of this invention is to provide a kind of method of differentiating capsule of weeping forsythia spin-off.
To achieve these goals, the method for discriminating capsule of weeping forsythia spin-off provided by the invention, comprises the following steps:
1) get capsule of weeping forsythia spin-off directly as the capsule of weeping forsythia lignanoid and the benzyl carbinol glycosides feature extraction thing that contain active component group (capsule of weeping forsythia lignanoid and benzyl carbinol glycosides);
2) described capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing are carried out to the detection of IGD carbon-13 nmr spectra finger-print, obtain several active component characteristic peak peak intensities in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing according to finger-print; And determine the characteristic peak peak intensity of described each active component respective standard with reference to product by same way (IGD carbon-13 nmr spectra finger-print);
3) obtain the absolute content of described standard with reference to product by quantitative test means;
4) utilize the ratio of described characteristic peak peak intensity (each active component characteristic peak peak intensity and respective standard are with reference to the characteristic peak peak intensity of product) and the described standard absolute content with reference to product, calculate the content of each active component and the total content of this active component, the i.e. content of active component group in capsule of weeping forsythia spin-off.
Wherein, adopt and there is the extraction process that obtains clear IGD carbon-13 nmr spectra finger-print as the extracting mode of described capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing.
Wherein, step 2) in, capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing are carried out to the detection of IGD carbon-13 nmr spectra finger-print, the solvent that dissolves capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing is deuterated methanol (CD 3or deuterated dimethyl sulfoxide (DMSO-d OD) 6), preferably deuterated dimethyl sulfoxide; The mass volume ratio of capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing and coordinative solvent is 55:1～65:1(mg:mL), preferably 60:1.
Wherein, step 2) in, in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing, the characteristic peak of lignanoids active component is C-7 absorption peak, its chemical shift is δ c83.0～88.0.
Preferably, select C-7 ' further to distinguish forsythin and the table rosin element-4-β-D-Glucose glycosides in lignanoids active component as characteristic peak, their chemical shift is δ c81.0～82.0.
In capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing, the characteristic peak of benzyl carbinol glycosides active component is C-9 ' absorption peak, and its chemical shift is δ c165.0～170.0.
Preferably, select C-1 or C-6 further to distinguish forsythiaside A and the Forsythoside H in benzyl carbinol glycosides active component as characteristic peak, their chemical shift is respectively δ c119.0～120.0 and δ c129.0～130.0.
Wherein, step 2) in, according to the size of characteristic peak peak intensity and position, several active components in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing are sorted.
Further, step 2) in, according to the size of characteristic peak peak intensity and position, the active component in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing: forsythin, table rosin element-4-β-D-Glucose glycosides, loose ester element-β-D-Glucose glycosides, Forsythingenin, " O-glucoside, forsythiaside A, Forsythoside H, Forsythoside I, Forsythoside F's 1-hydroxyl-rosin element 4 sort.
Wherein, step 2) in, described peak intensity can adopt peak height method, area integral method or gravimetric method to calculate.
Wherein, in step 3), described quantitative test means are: high efficiency liquid phase (HPLC) method.
Further, in the time differentiating capsule of weeping forsythia lignanoids active component, the condition of described high-efficient liquid phase technique comprises:
Mobile phase is acetonitrile: water=(20～30): (70～80), preferably 25:75; Detection wavelength is 277nm.
Further, in the time differentiating benzyl carbinol glycosides active component, the condition of described high-efficient liquid phase technique comprises:
Mobile phase is acetonitrile: glacial acetic acid solution=(10～20): (80～90), preferably 15:85; Detection wavelength is 330nm.
Wherein, in step 3), described standard refers to reference to the absolute content of product: the standard of measuring by quantitative test means is with reference to the quality percentage composition of product.
Further, in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing, the standard of lignanoids active component and benzyl carbinol glycosides active component is forsythin or forsythiaside A with reference to product.
Wherein, in step 4), the coupling formula that calculates the content of each active component is:
W n = W 1 M n h n M 1 h 1 ; Wherein:
W 1the standard that in the capsule of weeping forsythia spin-off of measuring by quantitative test means for step 3), a certain active component is corresponding is with reference to the absolute content of product;
M 1for standard that in described capsule of weeping forsythia spin-off, a certain active component is corresponding is with reference to the molecular weight/quantitatively carbon number corresponding to peak of product;
H 1for standard that in the capsule of weeping forsythia spin-off by IGD carbon-13 nmr spectra determining fingerprint pattern, a certain active component is corresponding is with reference to the characteristic peak peak intensity of product;
W nfor the quality percentage composition of a certain active component in capsule of weeping forsythia spin-off;
M nfor the molecular weight/quantitatively carbon number corresponding to peak of a certain active component in capsule of weeping forsythia spin-off;
H nfor the characteristic peak peak intensity of a certain active component in the capsule of weeping forsythia spin-off by IGD carbon-13 nmr spectra determining fingerprint pattern; The total content of this active component is exactly the W of similar each active component nsum, the i.e. content of active component group.
The derivation of above-mentioned formula is:
Figure BDA0000463966010000052
Active component group described in the inventive method is the active component group in capsule of weeping forsythia spin-off.Described capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing refer to contain capsule of weeping forsythia lignanoid and benzyl carbinol glycosides active component in this extract, and active component group refers to the summation of corresponding same active component.
Wherein, described capsule of weeping forsythia spin-off comprises: forsythia suspense extraction or capsule of weeping forsythia natural drug.
Forsythia suspense extraction of the present invention or spin-off, comprise each position of capsule of weeping forsythia plant, as extract or the spin-off of root, skin, stem, leaf, flower and fruit etc.
Further, described forsythia suspense extraction can comprise: capsule of weeping forsythia berry extract, and capsule of weeping forsythia lignanoid or benzyl carbinol glycosides extract, chromocor extract, triterpenic acid extract, or Folium Forsythiae extract etc.
The calculating of the content of the each active component of the present invention and the total content of this active component is by IGD carbon-13 nmr spectra and the coupling of analysis quantitative means by coupling formula.Compared to the prior art, the present invention adopts IGD 13c NMR coupling finger-print has several features below:
1. stability (repeatability): IGD 13the chemical shift data that C NMR obtains is second after radix point, and explanation property is good, reproducible; Non-chromatographic condition (as chromatographic column internal diameter, length, the fixing phase trade mark, carrier granularity, flow rate of mobile phase, the mutually each component ratio of mixed flow, column temperature, sample size, the detector sensitivity etc.) change of HPLC, GC etc., the retention time data variation obtaining is very large, mean the variation of monolithic chromatogram figure, repeatability is bad.
2. globality (comprehensive): IGD 13in C NMR finger-print, comprise the corresponding spectrum peak of each the active component carbon in sample; There is not this relation in HPLC, GC, UV, IR, MS.
3. reliability (unicity): IGD 13c NMR spectrum peak and the carbon on different activities composition in sample and different group thereof are strict one-to-one relationships; There is not this relation in HPLC, GC, UV, IR, MS.
4. feasibility (the easily property distinguished): IGD 13c NMR finger-print regularity is very strong, generally, can belong to each the carbon peak in collection of illustrative plates; HPLC, GC need reference substance; IR is difficult for resolving; UV quantity of information is few; MS has the problem such as degree of ionization and matrix interference.
IGD carbon-13 nmr spectra finger-print can only show in feature extraction thing, there is which active component, and quantitative ratio between these active components, and the absolute content of these active components must be analyzed quantitative means with reference to product and other by standard, then obtain by coupling formula.
The advantage that the present invention has is as follows:
1, four steps of the present invention are an entirety, indispensable; Four steps have distinctive feature separately; If separately or simple combination can not detect ratio and the content of complicated ingredient in medicinal plant.
As everyone knows, although Chinese medicine, botanical pesticide are time-honored medicines, its analyzing and testing is a difficult problem always.Therefore, need especially a kind of method can differential plant kind and extract in various active components, and it is quantitative, then select best extract component or its composition, to give full play to its effect.In addition, one of obstacle that Chinese medicine, botanical pesticide are gone abroad is there is no constituent and quantitative identifying thereof, and the qualitative and quantitative analysis aspect that reason is currently available technology solution differential plant kind and evaluation plant source product quality exists significant limitation (specifically seeing the content that the application's background technology is described).Fact proved, the gordian technique that IGD carbon-13 nmr spectra coupling fingerprint pattern technology addresses this problem just, this is seeing in present specification existing explanation.
Although 2 conventional extraction processes are known by those of ordinary skill, never there is people to obtain clear collection of illustrative plates and representative active component group selects technological parameter to obtain feature extraction thing as standard.Utilize the method, can make final better for the identification result of extract, this is one of difficult point of the present invention, is also one of innovative point of the present invention.
3, IGD 13c NMR coupling finger-print is the mixed spectrum of multiple active components, unavoidably can cause the crowded, even overlapping of excellent peak one by one.In order to make result of calculation accurate, it is necessary selecting specific characteristic peak, each active component carbon peak in active component group that chemical shift difference is larger.Because dissimilar compound carbon spectral difference is very not large, the selection of characteristic peak can be ever-changing; Same type compound carbon spectral difference is not very little, and a lot of peak overlaps are serious, need have deep nuclear magnetic resonance spectrum knowledge to instruct, and through in many ways comparing, turning over, could select good characteristic peak; And characteristic peak selection is bad, have no idea active component group to carry out accurate quantitative analysis analysis this problem that also the present invention need to solve just.According to the feature of lignanoids in the capsule of weeping forsythia and phenylethanoid glycoside, select C-7 to distinguish lignan component as characteristic peak, and select C-7 ' as further this overlapping constituents of difference of characteristic peak; Select C-9 ' as characteristic peak difference phenylethanoid glycoside, and select C-1 or C-6 as further this overlapping constituents of difference of characteristic peak.So need to select different specific characteristic peaks, active component carbon peak according to the feature of different activities composition is also the application's another innovative point.
4, because characteristic peak chemical shift difference is very little, in a lot of situations, only after radix point, the 1st potential difference is other, so the sequence of characteristic peak is the key of determining main active and ratio thereof, there is no deep nuclear magnetic resonance spectrum knowledge and separate basis, be difficult to determine active component and the ratio thereof of characteristic peak representative, also just cannot carry out accurate qualitative and quantitative analysis to active component group, according to the feature of the carbon spectrum nuclear magnetic data of lignanoids in the capsule of weeping forsythia and phenylethanoid glycoside, the characteristic peak of two constituents is carried out to accurate sequence; This is one of difficult point of the present invention, is also one of innovative point of the present invention.
5, coupling calculating key is the selection of choice criteria product and quantitative analysis tech.Analyze quantitative means can select high efficiency liquid phase, gas chromatography, thin-layered chromatography and weighing method etc., standard with reference to product can be a certain active component as interior mark, can be also additional with reference to product as external standard.According to the feature of lignanoids in the capsule of weeping forsythia and phenylethanoid glycoside, what analyze quantitative means selection is liquid phase, and what standard was selected with reference to product is forsythin or Forsythoside (can have different choice according to different situations).This is one of difficult point of the present invention, is also one of innovative point of the present invention.
The present invention adopts IGD carbon-13 nmr spectra finger-print to differentiate capsule of weeping forsythia spin-off, can reflect in capsule of weeping forsythia spin-off and contain which capsule of weeping forsythia lignanoids and phenylethyl alcohol glycoside and the ratio between them, reaches the object to capsule of weeping forsythia spin-off Quality Identification.The range of linearity is wide, highly sensitive, and repeatability and feasibility are good.Not only can effectively control the inherent quality of capsule of weeping forsythia spin-off, also can meet current for the objective an urgent demand of effectively evaluating and controlling capsule of weeping forsythia quality.All in all, not only can solve the difficult problem that China's capsule of weeping forsythia spin-off is evaluated, also, for strengthening systematization and the standardization of the inherent composition Study of capsule of weeping forsythia spin-off, realize the assurance that science is provided in line with international standards.
Accompanying drawing explanation
Fig. 1-a is the IGD carbon-13 nmr spectra finger-print that embodiment 1 makes Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract by oneself.
Fig. 1-b, 1-c and 1-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak that embodiment 1 makes Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract by oneself widens enlarged drawing.
Fig. 2-a is the IGD carbon-13 nmr spectra finger-print of embodiment 2 commercially available forsythia suspense extractions.
Fig. 2-b and 2-c are that the part, IGD carbon-13 nmr spectra Fingerprints peak of embodiment 2 commercially available forsythia suspense extractions widens enlarged drawing.
Fig. 3-a is the IGD carbon-13 nmr spectra finger-print that embodiment 3 makes Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract first (precipitation method) by oneself.
Fig. 3-b, 3-c and 3-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak that embodiment 3 makes Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract first (precipitation method) by oneself widens enlarged drawing.
Fig. 4-a is that embodiment 4 makes Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract (resin method) IGD carbon-13 nmr spectra finger-print by oneself.
Fig. 4-b, 4-c and 4-d are that embodiment 4 makes Luanchuan, Yuzhou Folium Forsythia lignanoid by oneself and part, benzyl carbinol glycosides extract (resin method) IGD carbon-13 nmr spectra Fingerprints peak widens enlarged drawing.
Fig. 5-a is the IGD carbon-13 nmr spectra finger-print that embodiment 5 makes Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second (resin method) by oneself.
Fig. 5-b, 5-c and 5-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak that embodiment 5 makes Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second (resin method) by oneself widens enlarged drawing.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
If without particularly pointing out, the material that the present invention uses is the conventional material of this area, and NM method of operating is also the method for this area routine.
In the present invention, the concentration of ethanol is percent by volume.
One, capsule of weeping forsythia spin-off IGD carbon-13 nmr spectra finger-print research
(1) acquisition of capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing (CET)
Get capsule of weeping forsythia spin-off directly as capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing (CET)
(2) the IGD carbon-13 nmr spectra finger-print of capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing detects
Get capsule of weeping forsythia lignanoid and/or benzyl carbinol glycosides feature extraction thing 30mg, be dissolved in 0.5mL deuterated dimethyl sulfoxide (DMSO-d 6) or deuterated methanol in, do IGD carbon-13 nmr spectra finger-print detect, Ji get capsule of weeping forsythia lignanoid and benzyl carbinol glycosides IGD carbon-13 nmr spectra finger-print.
(3) the IGD carbon-13 nmr spectra finger-print of capsule of weeping forsythia lignanoids and benzyl carbinol glycosides feature extraction thing is resolved
1. differentiate
In capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing (CET) IGD carbon-13 nmr spectra finger-print, clearly illustrate the characteristic signal of capsule of weeping forsythia lignanoids and benzyl carbinol glycosides compounds, forsythin, loose ester element-β-D-Glucose glycosides, forsythiaside A, Forsythoside H, Forsythoside I etc. all have corresponding NMR signal.
2. the each active component characteristic peak in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing is chosen
Need to select different specific characteristic peaks, active component carbon peak according to the feature of different activities composition.Selection principle is as follows: 1. the specific characteristic peak of similar compound is preferably identical carbon carbon peak, each compound position; 2. between the specific characteristic peak of each compound and other carbon peaks, chemical shift difference is larger; 3. between the specific characteristic peak of each compound, chemical shift difference is larger; 4. the chemical shift effect difference at specific characteristic peak itself that affects each compound is larger.
Owing to containing a series of lignanoids and benzyl carbinol glycosides compounds (active component) in capsule of weeping forsythia lignanoids and benzyl carbinol glycosides feature extraction thing, carbon peak intersects morely, in order to measure the ratio of each active component, must select the larger respective peaks of chemical shift difference as characteristic peak., investigate through reality for this reason, select δ cδ, as specific characteristic peak, lignanoids active component carbon peak, is selected in 83.0～88.0 C-7 tertiary carbon carbon peak, one group of left and right c165.0～170.0 C-9 ' carbonyl carbon carbon peak, one group of left and right are as specific characteristic peak, benzyl carbinol glycosides active component carbon peak; It is former because Lignanoids compounds C-7 tertiary carbon and benzyl carbinol glycosides compounds C-9 ' carbonyl carbon and other carbon geochemistry displacement difference are larger, easy to identify; And between dissimilar compound and different Compound C-9 ' carbonyl carbon carbon of the same type peak, chemical shift also has certain difference.Preferably, can select C-7 ' further to distinguish overlapping forsythin and the table rosin element-4-β-D-Glucose glycosides of C-7 characteristic peak in lignanoids active component as characteristic peak, their chemical shift is δ c81.0～82.0; Can select C-1 or C-6 further to distinguish overlapping forsythiaside A and the Forsythoside H of C-9 ' characteristic peak in benzyl carbinol glycosides active component as characteristic peak, their chemical shift is respectively δ c119.0～120.0 and δ c129.0～130.0.
3. standard is with reference to the selection of product
Forsythin is capsule of weeping forsythia medicinal material and extract thereof, and main lignanoids active component in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing, and its C-7 chemical shift is about δ c86.7(DMSO-d 6) do not have overlapping at this with other main lignanoids and phenylethanoid glycoside; Forsythiaside A is main benzyl carbinol glycosides active component in capsule of weeping forsythia medicinal material and extract thereof, and the chemical shift of its C-1 or C-6 is respectively δ c119.0～120.0 and δ c129.0～130.0, do not have overlapping at this with other main lignanoids and phenylethanoid glycoside; Therefore, select forsythin or Forsythoside as the standard of 2 active components with reference to product.
(4) content of forsythin and Forsythoside in employing HPLC method mensuration capsule of weeping forsythia spin-off
1. forsythin
I) chromatographic condition (with reference to 2010 editions pharmacopeia)
Mobile phase is acetonitrile: water=25:75;
Chromatographic column: C18(250*4.6mm, 5um);
Detect wavelength: 277nm.
Ii) forsythin standard is with reference to the preparation of product solution
Accurately take forsythin 5mg, put in 50mL volumetric flask, to scale, after shaking up, obtain forsythin standard with reference to product solution with methyl alcohol dissolved dilution.
Iii) contain the preparation of the need testing solution of capsule of weeping forsythia lignanoids active component
Accurately take test sample: capsule of weeping forsythia spin-off (as forsythia suspense extraction) 200mg is in 100mL volumetric flask, adding appropriate methyl alcohol (30-40mL) dissolves, after sonic oscillation, be diluted to scale, i.e. capsule of weeping forsythia spin-off need testing solution (as forsythia suspense extraction need testing solution).
Iv) determination method
The accurate forsythin standard of drawing is with reference to product solution and each 20 μ 1 of need testing solution (being above-mentioned capsule of weeping forsythia spin-off need testing solution) that contain capsule of weeping forsythia lignanoids active component respectively, and injection liquid chromatography, measures, and obtains Determination of forsythin.
V) in capsule of weeping forsythia spin-off, forsythin absolute content calculates
Calculated forsythin mass concentration in the need testing solution that contains lignanoids active component by following formula:
C X = C R × A X A R
C x: forsythin mass concentration (ug/mL) in the need testing solution that contains lignanoids active component;
C r: forsythin standard is with reference to product concentration of polymer solution (ug/mL);
A x: the peak area of the need testing solution that contains lignanoids active component;
A r: forsythin standard is with reference to the peak area of product solution.
Vi) calculate forsythin quality percentage composition in capsule of weeping forsythia spin-off by following formula
Figure BDA0000463966010000122
W forsythin(%): forsythin quality percentage composition in capsule of weeping forsythia spin-off;
C x: the forsythin mass concentration (ug/mL) in the need testing solution that contains lignanoids active component;
M: the quality (mg) of the capsule of weeping forsythia spin-off taking.
2. Forsythoside assay
I) chromatographic condition (with reference to 2010 editions pharmacopeia)
Mobile phase is acetonitrile: glacial acetic acid solution=15:85;
Chromatographic column: C18(250*4.6mm, 5um);
Detect wavelength: 330nm.
Ii) Forsythoside standard is with reference to the preparation of product solution
Accurately take Forsythoside 5mg, put in 50mL volumetric flask, to scale, after shaking up, obtain Forsythoside standard with reference to product solution with methyl alcohol dissolved dilution.
Iii) contain the preparation of the need testing solution of capsule of weeping forsythia benzyl carbinol glycosides active component
Accurately take test sample: capsule of weeping forsythia spin-off (as forsythia suspense extraction) 200mg is in 100mL volumetric flask, adding appropriate methyl alcohol (30-40mL) dissolves, after sonic oscillation, be diluted to scale, i.e. capsule of weeping forsythia spin-off need testing solution (as forsythia suspense extraction need testing solution).
Iv) determination method
The accurate Forsythoside standard of drawing is with reference to product solution and each 20 μ 1 of need testing solution (being above-mentioned capsule of weeping forsythia spin-off need testing solution) that contain capsule of weeping forsythia benzyl carbinol glycosides active component respectively, and injection liquid chromatography, measures, and obtains Determination of forsythin.
V) in capsule of weeping forsythia spin-off, Forsythoside absolute content calculates
Calculated forsythin mass concentration in the need testing solution that contains benzyl carbinol glycosides active component by following formula:
C X = C R × A X A R
C x: Forsythoside mass concentration (ug/mL) in the need testing solution that contains benzyl carbinol glycosides active component;
C r: Forsythoside standard is with reference to product concentration of polymer solution (ug/mL);
A x: the peak area of the need testing solution that contains benzyl carbinol glycosides active component;
A r: Forsythoside standard is with reference to the peak area of product solution.
Vi) calculate Forsythoside quality percentage composition in capsule of weeping forsythia spin-off by following formula
Figure BDA0000463966010000141
W forsythoside(%): Forsythoside quality percentage composition in capsule of weeping forsythia spin-off;
C x: the Forsythoside mass concentration (ug/mL) in the need testing solution that contains benzyl carbinol glycosides active component;
M: the quality (mg) of the capsule of weeping forsythia spin-off taking.
(5) by content and the total amount of lignanoids or each main active of phenylethyl alcohol glycoside in coupling formula calculating capsule of weeping forsythia spin-off, the i.e. content of lignanoids or benzyl carbinol glycosides active component group
W n = W 1 M n h n M 1 h 1
W 1: capsule of weeping forsythia spin-off Plays is with reference to the quality percentage composition of product forsythin or Forsythoside;
M 1: standard is with reference to the molecular weight/quantitatively carbon number corresponding to peak of product forsythin or Forsythoside;
H 1: the characteristic peak peak intensity (peak height) by the standard of IGD carbon-13 nmr spectra determining fingerprint pattern with reference to product forsythin or Forsythoside;
W n: the quality percentage composition of a certain lignanoids or benzyl carbinol glycosides active component in capsule of weeping forsythia spin-off;
M n: the molecular weight of a certain lignanoids or benzyl carbinol glycosides active component in capsule of weeping forsythia spin-off/quantitatively carbon number corresponding to peak;
H n: by a certain lignanoids in the capsule of weeping forsythia spin-off of IGD carbon-13 nmr spectra determining fingerprint pattern or benzyl carbinol glycosides active component characteristic peak peak intensity (peak height).
Two, instrument, reagent and material
Key instrument and equipment
Nuclear magnetic resonance spectrometer Bruker DPX400 type.
Mass spectrometer: the Q-Tof MicroTM of Waters Micromass company type.
Half preparative high-performance liquid chromatographic instrument: Waters600 type.
High performance liquid chromatograph: Agilent1200 type.
2000mL distilling flask, 5000mL distilling flask, spherical condensating tube, 2000mL separating funnel.
DE-52AA Rotary Evaporators: Shanghai Yarong Biochemical Instrument Plant.
DEF-6020 type vacuum drying chamber: the above grand experimental facilities of Nereid company limited.
Green grass or young crops sticks up, always sticks up (pluck from local for 2012 Luanchuan In Henan, Yuzhou), commercially available forsythia suspense extraction (Kang Peiji bio tech ltd, Xi'an), and medicinal material is identified through professor Zhu Changshan of In Henan Agriculture university; Forsythin, chemical reference substance, laboratory self-control (identifying through spectroscopic data).
Reagent: chromatographically pure (methyl alcohol, Tianjin Siyou Fine Chemicals Co., Ltd.) and analyze pure (Tianjin Chemical Reagents Factory No.1).
Three, fundamental research
(1) structure of main active and carbon-13 nmr spectra data in capsule of weeping forsythia spin-off
Figure BDA0000463966010000151
Forsythin: R 1=Me, R 2=Glc
Forsythingenin: R 1=Me, R 2=H
Table rosin element-4-β-D-Glucose glycosides: R 1=H, R 2=Glc
Show loose ester element-4 '-O-glucoside: R 1=Glc, R 2=H
Pine ester element-β-D-Glucose glycosides: R 1=H, R 2=Glc
Figure BDA0000463966010000162
1-hydroxyl-rosin element 4 " O-glucosides
Figure BDA0000463966010000163
The blue A:R=H of the capsule of weeping forsythia
The blue B:R=Me of the capsule of weeping forsythia
Figure BDA0000463966010000164
Forsythiaside A
Figure BDA0000463966010000171
Forsythiaside B
Forsythoside H
Figure BDA0000463966010000173
Forsythoside F
Figure BDA0000463966010000174
Forsythoside I
Figure BDA0000463966010000181
Forsythoside J
Figure BDA0000463966010000182
N-butoxy Forsythoside
Forsythin (Phillyrin)
13C?NMR(100MHz,DMSO-d 6)δ C:135.2(C-1),110.3(C-2),148.8
(C-3),145.8(C-4),115.1(C-5),118.0(C-6),86.5(C-7),53.9
(C-8),70.2(C-9),131.2(C-1′),109.3(C-2′),148.5(C-3′),146.0
(C-4′),111.4(C-5′),117.4(C-6′),81.1(C-7′),49.2(C-8′),68.9
(C-9′),55.4,55.4,55.6(3OMe)，100.0(Glc-1),73.1(Glc-2),76.7
(Glc-3),69.6(Glc-4),76.9(Glc-5),60.6(Glc-6)。
Forsythingenin (Phillygenin)
13C?NMR(100MHz,DMSO-d 6)δ C:132.2(C-1),110.2(C-2),147.4*
(C-3),145.9(C-4),115.1(C-5),118.5(C-6),86.9(C-7),53.8
(C-8),70.2(C-9),131.1(C-1′),109.4(C-2′),148.4(C-3′),147.6*
(C-4′),111.5(C-5′),117.5(C-6′),81.3(C-7′),49.3(C-8′),68.7
(C-9′),55.4,55.4,55.5(3OMe)。(* ownership is interchangeable)
Table rosin element-4-β-D-Glucose glycosides (epipinoresinol-4-β-D-glucoside)
13C?NMR(100MHz,DMSO-d 6)δ C:135.3(C-1),110.4(C-2),148.9(C-3),145.9(C-4),115.1(C-5),118.1*(C-6),86.6(C-7),54.0(C-8),70.2(C-9),129.6(C-1′),109.7(C-2′),147.3(C-3′),145.2(C-4′),115.1(C-5′),117.9*(C-6′),81.4(C-7′),49.3(C-8′),69.0(C-9′),55.5,55.6(2OMe)，100.1(Glc-1),73.2(Glc-2),76.8**(Glc-3),69.6(Glc-4),77.0*(Glc-5),60.6(Glc-6)。(* ownership is interchangeable)
Pine ester element-β-D-Glucose glycosides [(+)-pinoresinol-β-D-glucoside]
13C?NMR(100MHz,DMSO-d 6)δ C:135.2(C-1),115.1(C-2),148.9(C-3),145.9(C-4),114.8(C-5),118.6(C-6),85.1(C-7),53.5(C-8),70.9(C-9),132.1(C-1′),110.4(C-2′),148.9(C-3′),148.9(C-4′),115.1(C-5′),118.6(C-6′),84.8(C-7′),53.5(C-8′),70.9(C-9′),55.6(2OMe)，100.0(Glc-1),73.1(Glc-2),76.9(Glc-3),69.6(Glc-4),76.9(Glc-5),60.6(Glc-6)。(* ownership is interchangeable)
1-hydroxyl-rosin element 4 " O-glucoside (Hydroxypinoresinol-4 "-O-β-D-glucoside)
13C?NMR(100MHz,DMSO-d 6)δ C:135.3(C-1),110.9(C-2),148.9(C-3),145.9(C-4),115.2(C-5),118.4(C-6),85.1(C-7),60.8(C-8),70.3(C-9),128.0(C-1′),112.4(C-2′),146.9(C-3′),146.0(C-4′),114.6(C-5′),120.2(C-6′),87.2(C-7′),91.0(C-8′),74.8(C-9′),55.6,55.8(2OMe)，100.1(Glc-1),73.2(Glc-2),76.8(Glc-3),69.6(Glc-4),77.0(Glc-5),60.6(Glc-6)。(* ownership is interchangeable)
The blue A(forsythialan A of the capsule of weeping forsythia)
13C?NMR(75MHz,CDCL 3)δ:132.30(C-1),110.04(C-2),146.84(C-3),114.06(C-4),145.57(C-5),120.08(C-6),83.85(C-7),52.22(C-8),61.35(C-9),129.42(C-1′),110.39(C-2′),146.85(C-3′),150.84(C-4′),114.07(C-5′),123.81(C-6′),197.99(C-7′),49.62(C-8′),70.78(C-9′)，56.07(OCH 3-3)，55.96(OCH 3-3′)。
The blue B(forsythialan B of the capsule of weeping forsythia)
13C?NMR(75MHz,CDCL 3)δ:132.32(C-1),108.97(C-2),146.85(C-3),114.04(C-4),145.62(C-5),120.11(C-6),83.88(C-7),52.25(C-8),61.42(C-9),129.79(C-1′),110.67(C-2′),149.27(C-3′),153.71(C-4′),10.15(C-5′),123.16(C-6′),197.96(C-7′),49.66(C-8′),70.80(C-9′)，56.01(OCH 3-3)，56.01(OCH 3-3′)。
Show loose ester element-4 '-O-glucoside (epipinoresinol-4 '-O-glucoside)
13C?NMR(100MHz,DMSO-d 6)δ C:132.2*(C-1),110.2(C-2),147.4(C-3),145.9(C-4),114.8(C-5),118.6(C-6),86.9(C-7),53.8(C-8),68.8(C-9),132.3*(C-1′),109.9(C-2′),148.5(C-3′),145.4(C-4′),115.1(C-5′),117.5(C-6′),81.1(C-7′),49.2(C-8′),68.8(C-9′),55.5,55.6(2OMe)，100.0(Glc-1),73.2(Glc-2),76.8(Glc-3),69.6(Glc-4),76.9(Glc-5),60.6(Glc-6)。(* ownership is interchangeable)
Forsythiaside A [Forsythoside A (forsythiaside)]
13C?NMR(100Mz,DMSO)δ:129.2(C-1),115.5(C-2),144.9(C-3),143.5(C-4),116.3(C-5),119.5(C-6),35.1(C-7),70.3(C-8),125.4(C-1′),114.8(C-2′),145.6(C-3′),148.6(C-4′),115.8(C-5′),121.4(C-6′),145.6(C-7′),113.6(C-8′),165.9(C-9′),102.9(Glc-1),73.0(Glc-2),73.5(Glc-3),71.0(Glc-4),73.9(Glc-5),66.1(Glc-6),100.5(Rha-1),70.6(Rha-2),70.6(Rha-3),71.9(Rha-4),68.4(Rha-5),17.8(Rha-6)
13C?NMR(100Mz,CD 3OD)δ:131.4(C-1),116.4(C-2),146.1(C-3),144.7(C-4),117.2(C-5),121.3(C-6),36.7(C-7),72.4(C-8),127.7(C-1′),115.3(C-2′),146.9(C-3′),149.8(C-4′),116.6(C-5′),123.2(C-6′),147.7(C-7′),114.7(C-8′),168.3(C-9′),104.5(Glc-1),75.2(Glc-2),75.8(Glc-3),72.4(Glc-4),74.8(Glc-5),67.7(Glc-6),102.3(Rha-1),72.3(Rha-2),
72.1(Rha-3),74.0(Rha-4),69.9(Rha-5),18.0(Rha-6)。
Forsythiaside B (Forsythoside B)
13C?NMR(125Mz,CD 3OD)δ:131.4(C-1),115.3(C-2),146.0(C-3),144.5(C-4),116.3(C-5),121.3(C-6),36.5(C-7),72.2(C-8),127.6(C-1′),114.7(C-2′),146.7(C-3′),149.6(C-4′),116.5(C-5′),123.2(C-6′),148.0(C-7′),117.1(C-8′),168.2(C-9′),104.1(Glc-1),81.6(Glc-2),76.0(Glc-3),70.4(Glc-4),74.4(Glc-5),68.3(Glc-6),103.0(Rha-1),72.2(Rha-2),72.0(Rha-3),73.7(Rha-4),70.8(Rha-5),18.4(Rha-6)，110.9(APi-1),78.0(APi-2),80.6(APi-3),75.0(APi-4),65.6(APi-5)。
Forsythoside H(Forsythoside H)
13C?NMR(100Mz,DMSO)δ:129.1(C-1),115.4(C-2),144.9(C-3),143.5(C-4),116.2(C-5),119.6(C-6),35.1(C-7),69.8(C-8),125.5(C-1′),114.8(C-2′),145.6(C-3′),148.5(C-4′),115.8(C-5′),121.3(C-6′),145.2(C-7′),114.2(C-8′),165.7(C-9′),100.2(Glc-1),73.4(Glc-2),74.1(Glc-3),70.7(Glc-4),75.5(Glc-5),66.6(Glc-6),100.7(Rha-1),70.5(Rha-2),70.3(Rha-3),72.0(Rha-4),68.4(Rha-5),18.0(Rha-6)。
Forsythoside F(Forsythoside F)
13C?NMR(100Mz,DMSO)δ:129.2(C-1),115.5(C-2),144.9(C-3),143.5(C-4),116.4(C-5),119.6(C-6),35.0(C-7),69.4(C-8),125.4(C-1′),114.7(C-2′),145.9(C-3′),148.6(C-4′),115.8(C-5′),121.5(C-6′),145.6(C-7′),113.3(C-8′),166.0(C-9′),102.2(Glc-1),73.0(Glc-2),78.9(Glc-3),70.3(Glc-4),73.2(Glc-5),68.8(Glc-6),101.3(Rha-1),71.7(Rha-2),71.7(Rha-3),76.3(Rha-4),70.4(Rha-5),18.2(Rha-6),103.8(Xyl-1),74.4(Xyl-2)，78.8(Xyl-3)，70.5(Xyl-4)，65.6(Xyl-5)。
Forsythoside I(Forsythoside I)
13C?NMR(100Mz,DMSO)δ:129.5(C-1),115.5(C-2),144.7(C-3),143.5(C-4),116.3(C-5),119.5(C-6),35.1(C-7),70.2(C-8),125.6(C-1′),114.9(C-2′),145.3(C-3′),148.2(C-4′),115.7(C-5′),121.3(C-6′),144.9(C-7′),114.7(C-8′),166.1(C-9′),102.7(Glc-1),71.4(Glc-2),77.5(Glc-3),68.1(Glc-4),75.1(Glc-5),66.5(Glc-6),100.7(Rha-1),70.6(Rha-2),70.4(Rha-3),71.9(Rha-4),68.4(Rha-5),17.9(Rha-6)。
Forsythoside J(Forsythoside J)
13C?NMR(100Mz,DMSO)δ:129.2(C-1),115.4(C-2),144.9(C-3),143.5(C-4),116.2(C-5),119.6(C-6),35.0(C-7),69.8(C-8),125.5(C-1′),114.8(C-2′),145.6(C-3′),148.5(C-4′),115.8(C-5′),121.3(C-6′),145.1(C-7′),114.2(C-8′),165.7(C-9′),100.1(Glc-1),73.3(Glc-2),74.1(Glc-3),70.0(Glc-4),75.7(Glc-5),65.7(Glc-6),104.0(Xyl-1),73.3(Xyl-2),76.6(Xyl-3),69.6(Xyl-4),68.2(Xyl-5)。
N-butoxy Forsythoside [(-)-Suspensaside B)
13C?NMR(100Mz,DMSO)δ:131.8(C-1),115.0(C-2),146.5(C-3),146.3(C-4),116.5(C-5),119.7(C-6),81.9(C-7),75.4(C-8),127.7(C-1′),115.2(C-2′),145.6(C-3′),148.5(C-4′),115.8(C-5′),121.3(C-6′),145.1(C-7′),114.2(C-8′),167.5(C-9′),100.1(Glc-1),73.3(Glc-2),74.1(Glc-3),70.0(Glc-4),75.7(Glc-5),65.7(Glc-6),104.1(Xyl-1),73.3(Xyl-2),76.6(Xyl-3),69.4(Xyl-4),68.2(Xyl-5)。
Embodiment 1: self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract I GD carbon-13 nmr spectra coupling finger-print
(1) feature extraction thing preparation
Self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract, adopt the method preparation of Macroporous Adsorption Resin purifying after alcohol extracting: take Luanchuan green grass or young crops and stick up 20g, add and be equivalent to 20% ethanol that medicinal material 8 volumes doubly measure (160mL) refluxing extraction 3 times at 85 ℃, each 1h, filter, merging filtrate, reduced pressure concentration is approximately extremely without alcohol taste, filter, D101 resin column on filtrate (its quality is 50g), successively with water, 40% ethanol rush post (rush the water of post and the volume of ethanol be respectively concentrated after 25 and 40 times of filtrate volume), collect 40% ethanol eluate, evaporated under reduced pressure, obtain homemade Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract 0.78g, set it as feature extraction thing.
(2) self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract I GD carbon-13 nmr spectra finger-print detect
Get Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract 30mg that step makes, be dissolved in 0.5mL DMSO-d 6in, make IGD carbon-13 nmr spectra and detect, obtain Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract I GD carbon-13 nmr spectra finger-print.
(3) self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides feature extraction thing IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
In the IGD carbon-13 nmr spectra finger-print of Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract, clearly illustrate the characteristic signal of capsule of weeping forsythia Lignanoids compounds: δ c132.0-136.0,108.0-116.5,144.5-149.0,143.0-146.0,114.0-116.0,118.0-121.0,83.0-88.0,53.0-62.0,68.0-71.0,129.0-133.0,108.0-113.0,146.0-150.0,145.0-154.0,111.0-116.0,117.0-124.0,81.0-82.0 or 84.5-88.0 or 197.0-199.0,49.0-51.0 or 90.0-92.0,68.0-75.0 or 61.0-62.0 are respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,9,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal; In the IGD carbon-13 nmr spectra finger-print of capsule of weeping forsythia lignanoid and benzyl carbinol glycosides extract, clearly illustrate the characteristic signal of capsule of weeping forsythia benzyl carbinol glycosides compounds: δ c129.0-132.0,115.0-117.0,144.0-147.0,143.0-147.0,116.0-118.0,119.0-122.0,34.5-37.0 or 81.0-82.0,69.0-76.0,125.0-128.0,104.0-116.0,145.0-146.0,148.0-150.0,115.0-116.0,121.0-124.0,144.0-148.0,113.0-115.0,165.0-170.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal.Capsule of weeping forsythia lignanoid and benzyl carbinol glycosides compounds: forsythin, table rosin element-4-β-D-Glucose glycosides, loose ester element-β-D-Glucose glycosides, forsythiaside A, Forsythoside H, Forsythoside I etc. all have corresponding NMR signal in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 1-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 1-b, 1-c and 1-d.
2) in self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract, each active component ratio measuring result is as follows:
Figure BDA0000463966010000241
(4) HPLC method is measured forsythin and Forsythoside quality percentage composition in self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract, and measurement result is as follows:
Figure BDA0000463966010000242
(5) in self-control Luanchuan Qing Qiao lignanoid and benzyl carbinol glycosides extract, lignanoid and benzyl carbinol glycosides active component quality percentage composition measurement result are as follows:
Figure BDA0000463966010000243
Figure BDA0000463966010000251
As can be seen from the table, the forsythiaside A content that IGD carbon-13 nmr spectra finger-print calculates is that the forsythiaside A content that 18.22%, HPLC measures is 17.44%, and the two error is in 5%.
Embodiment 2: commercially available forsythia suspense extraction IGD carbon-13 nmr spectra coupling finger-print
(1) feature extraction thing is selected
Directly select commercially available forsythia suspense extraction (Kang Peiji bio tech ltd, Xi'an) as feature extraction thing.
(2) commercially available forsythia suspense extraction IGD carbon-13 nmr spectra finger-print detects
Get commercially available forsythia suspense extraction 30mg, be dissolved in 0.5mL DMSO-d 6in, make IGD carbon-13 nmr spectra and detect, obtain commercially available forsythia suspense extraction IGD carbon-13 nmr spectra finger-print.
(3) commercially available forsythia suspense extraction IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
In the IGD carbon-13 nmr spectra finger-print of commercially available forsythia suspense extraction, clearly illustrate the characteristic signal of capsule of weeping forsythia Lignanoids compounds: δ c132.0-136.0,108.0-116.5,144.5-149.0,143.0-146.0,114.0-116.0,118.0-121.0,83.0-88.0,53.0-62.0,68.0-71.0,129.0-133.0,108.0-113.0,146.0-150.0,145.0-154.0,111.0-116.0,117.0-124.0,81.0-82.0 or 84.5-88.0 or 197.0-199.0,49.0-51.0 or 90.0-92.0,68.0-75.0 or 61.0-62.0 are respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,9,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal; In the IGD carbon-13 nmr spectra finger-print of commercially available forsythia suspense extraction, clearly illustrate the characteristic signal of capsule of weeping forsythia benzyl carbinol glycosides compounds: δ c129.0-132.0,115.0-117.0,144.0-147.0,143.0-147.0,116.0-118.0,119.0-122.0,34.5-37.0 or 81.0-82.0,69.0-76.0,125.0-128.0,104.0-116.0,145.0-146.0,148.0-150.0,115.0-116.0,121.0-124.0,144.0-148.0,113.0-115.0,165.0-170.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal.Capsule of weeping forsythia lignanoid and benzyl carbinol glycosides compounds: forsythin, loose ester element-β-D-Glucose glycosides, forsythiaside A, Forsythoside H, Forsythoside F, Forsythoside I etc. all have corresponding NMR signal in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 2-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 2-b and 2-c.
2) in commercially available forsythia suspense extraction, each active component ratio measuring result is as follows:
Figure BDA0000463966010000261
(4) HPLC method is measured forsythin and Forsythoside quality percentage composition in commercially available forsythia suspense extraction, and measurement result is as follows:
Forsythin quality percentage composition W in commercially available forsythia suspense extraction Forsythin（%） 0.67%
Forsythoside quality percentage composition W in commercially available forsythia suspense extraction Forsythoside（%） 10.34%
(5) in commercially available forsythia suspense extraction, lignanoid and benzyl carbinol glycosides active component quality percentage composition measurement result are as follows:
Figure BDA0000463966010000262
Figure BDA0000463966010000271
As can be seen from the table, the forsythiaside A content that IGD carbon-13 nmr spectra finger-print calculates is that the forsythiaside A content that 10.63%, HPLC measures is 10.34%, and the two error is in 5%.
Embodiment 3: lignanoid and benzyl carbinol glycosides extract first (precipitation method) the IGD carbon-13 nmr spectra coupling finger-print of self-control Luanchuan Folium Forsythia
(1) feature extraction thing preparation
Self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract first, adopt the method preparation of the refining purifying of poach postprecipitation: take Luanchuan Folium Forsythia 20g, add and be equivalent to water that medicinal material 8 volumes doubly measure (160mL) refluxing extraction 2 times at 95 ℃, each 1h, filters, and after precipitation oven dry, dissolves with MeOH, filter, filtrate dries, and obtains homemade Luanchuan leaf lignanoid and benzyl carbinol glycosides extract 1.56g, sets it as feature extraction thing.
(2) self-control Folium Forsythia lignanoid and benzyl carbinol glycosides extract first IGD carbon-13 nmr spectra finger-print detect
Get Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract first 30mg that step makes, be dissolved in 0.5mL DMSO-d 6in, make IGD carbon-13 nmr spectra and detect, must make Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract I GD carbon-13 nmr spectra finger-print by oneself.
(3) self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides feature extraction thing first IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
In the IGD carbon-13 nmr spectra finger-print of Folium Forsythia lignanoid and benzyl carbinol glycosides extract first, clearly illustrate the characteristic signal of capsule of weeping forsythia Lignanoids compounds: δ c132.0-136.0, 108.0-116.5, 146.0-149.0, 145.0-146.0, 114.0-116.0, 118.0-121.0, 83.0-88.0, 53.0-62.0, 68.0-71.0 or 60.0-62.0, 128.0-133.0, 108.0-113.0, 146.0-150.0, 145.0-154.0, 111.0-116.0, 117.0-124.0, 81.0-82.0 or 84.5-88.0 or 197.0-199.0, 49.0-51.0 or 90.0-92.0, 68.0-75.0 or 61.0-62.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal, in the IGD carbon-13 nmr spectra finger-print of self-control capsule of weeping forsythia lignanoid and benzyl carbinol glycosides extract, clearly illustrate the characteristic signal of capsule of weeping forsythia benzyl carbinol glycosides compounds: δ c129.0-132.0,115.0-117.0,144.0-147.0,143.0-147.0,116.0-118.0,119.0-122.0,34.5-37.0 or 81.0-82.0,69.0-76.0,125.0-128.0,104.0-116.0,145.0-146.0,148.0-150.0,115.0-116.0,121.0-124.0,144.0-148.0,113.0-115.0,165.0-170.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal.Capsule of weeping forsythia lignanoid and benzyl carbinol glycosides compounds: forsythin, Forsythingenin, loose ester element-β-D-Glucose glycosides, " O-glucoside, forsythiaside A, Forsythoside H, Forsythoside I etc. all have corresponding NMR signal to 1-hydroxyl-rosin element 4 in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 3-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 3-b, 3-c and 3-d.
2) in self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract first, each active component ratio measuring result is as follows:
Figure BDA0000463966010000281
(4) HPLC method is measured forsythin and Forsythoside quality percentage composition in self-control Folium Forsythia lignanoid and benzyl carbinol glycosides extract first, and measurement result is as follows:
(5) self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract Jia Zhong capsule of weeping forsythia lignanoid and benzyl carbinol glycosides active component quality percentage composition measurement result are as follows:
As can be seen from the table, the forsythiaside A content that IGD carbon-13 nmr spectra finger-print calculates is that the forsythiaside A content that 7.87%, HPLC measures is 7.49%, and the two error is in 5%.
Embodiment 4: lignanoid and benzyl carbinol glycosides extract (resin method) the IGD carbon-13 nmr spectra coupling finger-print of self-control Yuzhou Folium Forsythia
(1) feature extraction thing preparation
Self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract, adopt the method preparation of Macroporous Adsorption Resin purifying after alcohol extracting: take Yuzhou Folium Forsythia 20g, add and be equivalent to water that medicinal material 8 volumes doubly measure (160mL) refluxing extraction 3 times at 85 ℃, each 1h, filter, merging filtrate, reduced pressure concentration is approximately extremely without alcohol taste, filter, D101 resin column on filtrate (its quality is 50g), successively with water, 40% ethanol rush post (rush the water of post and the volume of ethanol be respectively concentrated after 25 and 40 times of filtrate volume), collect 40% ethanol eluate, evaporated under reduced pressure, must make Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract 1.79g by oneself, set it as feature extraction thing.
(2) self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract I GD carbon-13 nmr spectra finger-print detect
Get lignanoid and benzyl carbinol glycosides extract second 30mg that step makes, be dissolved in 0.5mLDMSO-d 6in, make IGD carbon-13 nmr spectra and detect, must make Folium Forsythia lignanoid and benzyl carbinol glycosides extract second IGD carbon-13 nmr spectra finger-print by oneself.
(3) self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides feature extraction thing IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
In the IGD carbon-13 nmr spectra finger-print of self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract, clearly illustrate the characteristic signal of capsule of weeping forsythia Lignanoids compounds: δ c132.0-136.0, 108.0-116.5, 144.5-149.0, 143.0-146.0, 114.0-116.0, 118.0-121.0, 83.0-88.0, 53.0-62.0, 68.0-71.0 or 60.0-62.0, 128.0-133.0, 108.0-113.0, 146.0-150.0, 145.0-154.0, 111.0-116.0, 117.0-124.0, 81.0-82.0 or 84.5-88.0 or 197.0-199.0, 49.0-51.0 or 90.0-92.0, 68.0-75.0 or 61.0-62.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal, in the IGD carbon-13 nmr spectra finger-print of self-control Folium Forsythia lignanoid and benzyl carbinol glycosides extract second, clearly illustrate the characteristic signal of capsule of weeping forsythia benzyl carbinol glycosides compounds: δ c129.0-132.0,115.0-117.0,144.0-147.0,143.0-147.0,116.0-118.0,119.0-122.0,34.5-37.0 or 81.0-82.0,69.0-76.0,125.0-128.0,104.0-116.0,145.0-146.0,148.0-150.0,115.0-116.0,121.0-124.0,144.0-148.0,113.0-115.0,165.0-170.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal.Capsule of weeping forsythia lignanoid and benzyl carbinol glycosides compounds: forsythin, loose ester element-β-D-Glucose glycosides, " O-glucoside, forsythiaside A, Forsythoside H, Forsythoside I etc. all have corresponding NMR signal to 1-hydroxyl-rosin element 4 in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 4-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 4-b, 4-c and 4-d.
2) in self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract, each active component ratio measuring result is as follows:
Figure BDA0000463966010000311
(4) HPLC method is measured forsythin and Forsythoside quality percentage composition in self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract, and measurement result is as follows:
Figure BDA0000463966010000312
(5) in self-control Yuzhou Folium Forsythia lignanoid and benzyl carbinol glycosides extract, capsule of weeping forsythia lignanoid and benzyl carbinol glycosides active component quality percentage composition measurement result are as follows:
Figure BDA0000463966010000321
As can be seen from the table, the Determination of forsythin that IGD carbon-13 nmr spectra finger-print calculates is that the forsythiaside A content that 6.99%, HPLC measures is 6.70%, and the two error is in 5%.
Embodiment 5: self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second (resin method) IGD carbon-13 nmr spectra coupling finger-print
(1) feature extraction thing preparation
Self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second, adopt the method preparation of Macroporous Adsorption Resin purifying after alcohol extracting: take Luanchuan Folium Forsythia 20g, add and be equivalent to water that medicinal material 8 volumes doubly measure (160mL) refluxing extraction 3 times at 85 ℃, each 1h, filter, merging filtrate, reduced pressure concentration is approximately extremely without alcohol taste, filter, D101 resin column on filtrate (its quality is 50g), successively with water, 40% ethanol rush post (rush the water of post and the volume of ethanol be respectively concentrated after 30 and 40 times of filtrate volume), collect 40% ethanol eluate, evaporated under reduced pressure, must make Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second 1.56g by oneself, set it as feature extraction thing.
(2) self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second IGD carbon-13 nmr spectra finger-print detect
Get lignanoid and benzyl carbinol glycosides extract second 30mg that step makes, be dissolved in 0.5mLDMSO-d 6in, make IGD carbon-13 nmr spectra and detect, must make Folium Forsythia lignanoid and benzyl carbinol glycosides extract second IGD carbon-13 nmr spectra finger-print by oneself.
(3) self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides feature extraction thing second IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
In the IGD carbon-13 nmr spectra finger-print of self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second, clearly illustrate the characteristic signal of capsule of weeping forsythia Lignanoids compounds: δ c132.0-136.0, 108.0-116.5, 144.5-149.0, 143.0-146.0, 114.0-116.0, 118.0-121.0, 83.0-88.0, 53.0-62.0, 68.0-71.0 or 60.0-62.0, 128.0-133.0, 108.0-113.0, 146.0-150.0, 145.0-154.0, 111.0-116.0, 117.0-124.0, 81.0-82.0 or 84.5-88.0 or 197.0-199.0, 49.0-51.0 or 90.0-92.0, 68.0-75.0 or 61.0-62.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal, in the IGD carbon-13 nmr spectra finger-print of self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second, clearly illustrate the characteristic signal of capsule of weeping forsythia benzyl carbinol glycosides compounds: δ c129.0-132.0,115.0-117.0,144.0-147.0,143.0-147.0,116.0-118.0,119.0-122.0,34.5-37.0 or 81.0-82.0,69.0-76.0,125.0-128.0,104.0-116.0,145.0-146.0,148.0-150.0,115.0-116.0,121.0-124.0,144.0-148.0,113.0-115.0,165.0-170.0 is respectively on lignanoid and benzyl carbinol glycosides skeleton 1,2,3,4,5,6,7,8,1 ', 2 ', 3 ', 4 ', 5 ', 6 ', 7 ', 8 ', 9 ' position carbon signal.Capsule of weeping forsythia lignanoid and benzyl carbinol glycosides compounds: forsythin, loose ester element-β-D-Glucose glycosides, forsythiaside A, Forsythoside H, Forsythoside I etc. all have corresponding NMR signal in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 5-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 5-b, 5-c and 5-d.
2) in self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second, each active component ratio measuring result is as follows:
Figure BDA0000463966010000341
(4) HPLC method is measured forsythin and Forsythoside quality percentage composition in self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract second, and measurement result is as follows:
Figure BDA0000463966010000342
(5) self-control Luanchuan Folium Forsythia lignanoid and benzyl carbinol glycosides extract Yi Zhong capsule of weeping forsythia lignanoid and benzyl carbinol glycosides active component quality percentage composition measurement result are as follows:
Figure BDA0000463966010000343
As can be seen from the table, the Determination of forsythin that IGD carbon-13 nmr spectra finger-print calculates is that the forsythiaside A content that 7.84%, HPLC measures is 7.69%, and the two error is in 5%.

Claims (11)

1. a method of differentiating capsule of weeping forsythia spin-off, comprises the following steps:
1) get capsule of weeping forsythia spin-off directly as the capsule of weeping forsythia lignanoid and the benzyl carbinol glycosides feature extraction thing that contain active component group;
2) described capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing are carried out to the detection of IGD carbon-13 nmr spectra finger-print, obtain several active component characteristic peak peak intensities in feature extraction thing according to finger-print; And determine the characteristic peak peak intensity of described each active component respective standard with reference to product by same way;
3) obtain the absolute content of described standard with reference to product by quantitative test means;
4) utilize each active component characteristic peak peak intensity and respective standard with reference to the ratio of the characteristic peak peak intensity of product and described standard with reference to the absolute content of product, calculate the content of each active component in capsule of weeping forsythia spin-off and the content of active component group.
2. method according to claim 1, is characterized in that, adopts and has the extraction process that obtains clear IGD carbon-13 nmr spectra finger-print as the extracting mode of described capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing.
3. method according to claim 1, it is characterized in that, step 2) in, capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing are carried out to the detection of IGD carbon-13 nmr spectra finger-print, the solvent that dissolves capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing is deuterated dimethyl sulfoxide or deuterated methanol.
4. according to the method described in claim 1～3 any one, it is characterized in that step 2) in, in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing, the characteristic peak of lignanoids active component is C-7 absorption peak, its chemical shift is δ c83.0～88.0; Preferably, select C-7 ' further to distinguish forsythin and the table rosin element-4-β-D-Glucose glycosides in lignanoids active component as characteristic peak, their chemical shift is δ c81.0～82.0;
In capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing, the characteristic peak of benzyl carbinol glycosides active component is C-9 ' absorption peak, and its chemical shift is δ c165.0～170.0; Preferably, select C-1 or C-6 further to distinguish forsythiaside A and the Forsythoside H in benzyl carbinol glycosides active component as characteristic peak, their chemical shift is respectively δ c119.0～120.0 and δ c129.0～130.0.
5. method according to claim 4, is characterized in that step 2) in, according to the size of characteristic peak peak intensity and position, several active components in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing are sorted.
6. according to the method described in claim 1～5 any one, it is characterized in that, in step 3), described quantitative test means are high-efficient liquid phase technique.
7. method according to claim 6, is characterized in that, in the time differentiating capsule of weeping forsythia lignanoids active component, the condition of described high-efficient liquid phase technique comprises that mobile phase is acetonitrile: water=(20～30): (70～80), preferably 25:75; Detection wavelength is 277nm;
In the time differentiating benzyl carbinol glycosides active component, the condition of described high-efficient liquid phase technique comprises that mobile phase is acetonitrile: glacial acetic acid solution=(10～20): (80～90), preferably 15:85; Detection wavelength is 330nm.
8. according to the method described in claim 1～7 any one, it is characterized in that, in step 3), described standard refers to that with reference to the absolute content of product the standard of using quantitative test means mensuration is with reference to the quality percentage composition of product.
9. according to the method described in claim 1～8 any one, it is characterized in that, the standard of the active component in capsule of weeping forsythia lignanoid and benzyl carbinol glycosides feature extraction thing is forsythin or forsythiaside A with reference to product.
10. according to the method described in claim 1～9 any one, it is characterized in that, in step 4), the coupling formula that calculates the content of each active component is:
W n = W 1 M n h n M 1 h 1 ; Wherein:
W 1the standard that in the capsule of weeping forsythia spin-off of measuring by quantitative test means for step 3), a certain active component is corresponding is with reference to the absolute content of product;
M 1for standard that in described capsule of weeping forsythia spin-off, a certain active component is corresponding is with reference to the molecular weight/quantitatively carbon number corresponding to peak of product;
H 1for standard that in the capsule of weeping forsythia spin-off by IGD carbon-13 nmr spectra determining fingerprint pattern, a certain active component is corresponding is with reference to the characteristic peak peak intensity of product;
W nfor the quality percentage composition of a certain active component in capsule of weeping forsythia spin-off;
M nfor the molecular weight/quantitatively carbon number corresponding to peak of a certain active component in capsule of weeping forsythia spin-off;
H nfor the characteristic peak peak intensity of a certain active component in the capsule of weeping forsythia spin-off by IGD carbon-13 nmr spectra determining fingerprint pattern.
11. according to the method described in claim 1～10 any one, it is characterized in that, described capsule of weeping forsythia spin-off comprises: forsythia suspense extraction or capsule of weeping forsythia natural drug.
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Patentee before: HENAN BRANCH OF PLANT NATURAL PRODUCTS OF HIGH ENGINEERING TECHNOLOGY DEVELOPMENT CO.,LTD.