CN103868943A - Method for identifying glossy privet fruit medicinal material or derivative - Google Patents
Method for identifying glossy privet fruit medicinal material or derivative Download PDFInfo
- Publication number
- CN103868943A CN103868943A CN201410044033.6A CN201410044033A CN103868943A CN 103868943 A CN103868943 A CN 103868943A CN 201410044033 A CN201410044033 A CN 201410044033A CN 103868943 A CN103868943 A CN 103868943A
- Authority
- CN
- China
- Prior art keywords
- glossy privet
- fruit
- active component
- iridoid glycoside
- ligustri lucidi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000830535 Ligustrum lucidum Species 0.000 title claims abstract description 135
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- 239000000284 extract Substances 0.000 claims abstract description 49
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- STKUCSFEBXPTAY-GSUVRYNNSA-N methyl (4s,5z,6s)-5-ethylidene-4-[2-oxo-2-[[(2r,3s,4s,5r,6r)-3,4,5-trihydroxy-6-[2-(4-hydroxyphenyl)ethoxy]oxan-2-yl]methoxy]ethyl]-6-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4h-pyran-3-carboxylate Chemical compound O([C@@H]\1OC=C([C@H](C/1=C/C)CC(=O)OC[C@@H]1[C@H]([C@H](O)[C@@H](O)[C@H](OCCC=2C=CC(O)=CC=2)O1)O)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O STKUCSFEBXPTAY-GSUVRYNNSA-N 0.000 claims description 49
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- QZCJFXSHMSZCLH-QAPDQXECSA-N methyl (1S,4aS,8S,8aS)-1-methyl-3-oxo-8-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,4a,8,8a-tetrahydro-1H-pyrano[3,4-c]pyran-5-carboxylate Chemical compound O([C@@H]1OC=C([C@H]2CC(=O)O[C@@H](C)[C@H]21)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QZCJFXSHMSZCLH-QAPDQXECSA-N 0.000 description 9
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Abstract
The invention relates to a method for identifying a glossy privet fruit medicinal material or a derivative. The method comprises the following steps: extracting the glossy privet fruit medicinal material or the derivative to obtain a glossy privet fruit iridoid glycoside characteristic extract containing an active ingredient group; performing IGD (Inverted Gated Decoupling) nuclear magnetic resonance carbon spectrum fingerprint detection on the glossy privet fruit iridoid glycoside characteristic extract, and obtaining characteristic peak intensities of a plurality of active ingredients in the glossy privet fruit iridoid glycoside characteristic extract according to the fingerprint; measuring the characteristic peak intensity of a standard reference object corresponding to each active ingredient in the same manner; measuring the absolute content of the standard reference object through a quantitative analysis method; calculating the content of each active ingredient and the content of the active ingredient group in the glossy privet fruit medicinal material or a derivative by using the ratio of the characteristic peak intensities and the absolute content of the standard reference object. According to the method, the glossy privet fruit iridoid glycoside compounds contained in the glossy privet fruit and the ratio of the compounds can be reflected so as to achieve the aims of variety identification and quality determination of the glossy privet fruit medicinal material.
Description
Technical field
The invention belongs to the discriminating field of natural medicinal plant, particularly, relate to a kind of method of differentiating Fructus Ligustri Lucidi or spin-off.
Background technology
The fruit of glossy privet is the dry mature fruit of Oleaceae plants glossy privet (Ligustrum lucidum Ait.), is the famous Chinese crude drug of China.The fruit of glossy privet is returned liver, kidney channel, and the property of medicine is cool, and taste is sweet, bitter, have black of nourishing liver and kidney, improving eyesight effect [Chinese Pharmacopoeia Commission. one of Pharmacopoeia of People's Republic of China version in 2010. Chinese Medicine science and technology publishing house, 43].Its active component is mainly iridoid glycoside compounds, has hypoglycemic, anti-hemorrhagic shock, inhibition platelet aggregation, regulates immunity, antimicrobial antiphlogistic etc. to act on [Liu Hong, etc. Nanjing University of Traditional Chinese Medicine's journal 2003,19 (4): 254.].
Above-mentioned certain active component of qualitative discriminating or its certain active component content of quantitative measurement, become the important method of evaluating the fruit of glossy privet and the quality of the pharmaceutical preparations thereof.For example Chinese Pharmacopoeia 2010 editions is, by HPLC method, iridoid glycoside constituents Specnuezhenide in Fructus Ligustri Lucidi is carried out to qualitative and quantitative analysis.But this quality testing pattern of carrying out qualitative and quantitative analysis for single component can not effectively be controlled the inherent quality of Chinese crude drug, cannot meet current for the objective an urgent demand of effectively evaluating and control the fruit of glossy privet and the quality of the pharmaceutical preparations thereof.
Fingerprint pattern technology is the effective method of current internationally recognized control Chinese medicine or natural drug quality.People rely on practical finger-print not only can confirm the true and false of this product, can evaluate its quality simultaneously.At present, Fructus Ligustri Lucidi and preparation thereof finger-print research work, concentrate on triterpenoids HPLC finger-print [Xu Changgen, Deng. new Chinese medicine and pharmacology clinical 2005,16 (5): 1922.] and X-ray diffraction Fourier finger-print [Shi Kunbo, Deng. Chinese crude drug 2007,30 (6) :].Iridoid glycoside compounds finger-print has no report.And 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 is large and needing by multiple standard items etc. reason, all there are many limitation in repeated and feasibility; X ray finger-print is because of the stack of the each component diffraction effect of Chinese medicine seem comparatively complexity and abundant information, between dactylogram and chemical composition, lack correlativity, sample pre-treatments and test condition directly affect measurement result, qualitative and quantitative analysis to specific sample gained spectrogram need to participate in [Sun Guoxiang by highly qualified specialist, Deng. Central-South pharmacy 2009,10 (6) :].。
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, will not differentiate glossy privet medicinal material or spin-off, utilize the report of IGD carbon-13 nmr spectra coupling fingerprint pattern technology.
The quality of the fruit of glossy privet 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 fruit of glossy privet 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 Fructus Ligustri Lucidi or spin-off IGD carbon-13 nmr spectra coupling finger-print, not only can solve the difficult problem that China's Fructus Ligustri Lucidi or spin-off are differentiated and evaluated, also for strengthening systematization and the standardization of the inherent composition Study of Fructus Ligustri Lucidi or spin-off, realize the assurance that science is provided in line with international standards.Along with this technology applying in other Chinese crude drugs or spin-off, 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 the present invention is to provide a kind of method of differentiating Fructus Ligustri Lucidi or spin-off, the method has been utilized IGD carbon-13 nmr spectra coupling fingerprint pattern technology.
To achieve these goals, the method for discriminating Fructus Ligustri Lucidi provided by the invention or spin-off, comprises the following steps:
1) Fructus Ligustri Lucidi or spin-off are extracted, obtain the fruit of glossy privet iridoid glycoside feature extraction thing that contains active component group (iridoid glycoside);
2) described fruit of glossy privet iridoid glycoside feature extraction thing is carried out to IGD carbon-13 nmr spectra finger-print and detect, obtain several active component characteristic peak peak intensities in described fruit of glossy privet iridoid glycoside 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) measure and 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 Fructus Ligustri Lucidi or 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 fruit of glossy privet iridoid glycoside feature extraction thing.
Wherein, in step 1), the preparation method of fruit of glossy privet iridoid glycoside feature extraction thing, comprise: take Fructus Ligustri Lucidi or medicine materical crude slice and pulverize, add 90～95% alcohol refluxs extractions or ultrasonic extraction 2～3 times, extract 1～2 hour at every turn, merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add water to disperse, then fill post with macroporous absorbent resin, successively rush post with water, 50～70% ethanol and 90～95% ethanol, collect 50～70% ethanol eluates, evaporated under reduced pressure, obtains fruit of glossy privet iridoid glycoside feature extraction thing.
Further, the mass volume ratio (g:mL) of described Fructus Ligustri Lucidi or medicine materical crude slice and 90～95% ethanol is 1:(6～10).
Further, be 20 times of medicinal material weight for disperseing the weight of water of medicinal extract.
Further, the weight of described macroporous absorbent resin is 0.8～2 times of medicinal material weight.
Further, the temperature of refluxing extraction is 90～95 ℃, and the temperature of ultrasonic extraction is 55～65 ℃.
Further, Fructus Ligustri Lucidi or medicine materical crude slice are crossed 24～50 mesh sieves after pulverizing.
Described macroporous absorbent resin is D101, AB-8, HP-20 etc.
Wherein, step 2) in, fruit of glossy privet iridoid glycoside feature extraction thing being carried out to IGD carbon-13 nmr spectra finger-print and detect, the solvent that dissolves fruit of glossy privet iridoid glycoside feature extraction thing is deuterated methanol (CD
3cOCD
3) or deuterated dimethyl sulfoxide (DMSO-d
6), preferably deuterated methanol.The mass volume ratio of fruit of glossy privet iridoid glycoside feature extraction thing and coordinative solvent is 35:1～65:1(mg:mL), preferably 60:1.
Except fruit of glossy privet medicine materical crude slice, other fruit of glossy privet spin-offs, as fruit of glossy privet iridoid glucoside extract can be directly as fruit of glossy privet iridoid glycoside feature extraction thing, differentiate with said method.
Wherein, step 2) in, the active component characteristic peak in fruit of glossy privet iridoid glycoside feature extraction thing is carbonyl absorption peak.
Further, step 2) in, the active component characteristic peak in fruit of glossy privet iridoid glycoside feature extraction thing is C-4 " or C-7 carbonyl absorption peak, their chemical shift is for being respectively δ
c144.0～158.0 and δ
c171.0～176.0.Using deuterated methanol to dissolve fruit of glossy privet iridoid glycoside feature extraction thing obtains.
Wherein, step 2) in, according to the size of characteristic peak peak intensity and position, several active components in fruit of glossy privet iridoid glycoside feature extraction thing are sorted.
Further, step 2) in, according to the size of characteristic peak peak intensity and position, the active component in fruit of glossy privet iridoid glycoside feature extraction thing: Specnuezhenide, 8E-Nuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Lucidumoside C, Kingiside, 8-epikingiside etc. 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, the condition of described high-efficient liquid phase technique comprises: mobile phase is methyl alcohol: water=30～50:50～70, preferably 40:60; Detection wavelength is 224nm.
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, the standard of the active component in fruit of glossy privet iridoid glycoside feature extraction thing is Specnuezhenide with reference to product.
Wherein, in step 4), the coupling formula that calculates the content of each active component is:
W
1the standard that in the Fructus Ligustri Lucidi of measuring by quantitative test means for step 3) or spin-off, a certain active component is corresponding is with reference to the absolute content of product;
M
1for standard that in described Fructus Ligustri Lucidi or 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 Fructus Ligustri Lucidi by IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off, 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 Fructus Ligustri Lucidi or spin-off;
M
nfor the molecular weight/quantitatively carbon number corresponding to peak of a certain active component in Fructus Ligustri Lucidi or spin-off;
H
nfor the characteristic peak peak intensity of a certain active component in the Fructus Ligustri Lucidi by IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off; 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:
Active component group described in the inventive method, can be both the active component group in fruit of glossy privet single medicinal material, also the active component group in fruit of glossy privet spin-off.Described fruit of glossy privet iridoid glycoside feature extraction thing refers to contain fruit of glossy privet iridoid glycoside active component in this extract, and active component group refers to the summation of corresponding same active component.
Wherein, described fruit of glossy privet spin-off comprises: fruit of glossy privet medicine materical crude slice, glossy privet fruit extract (as fruit of glossy privet iridoid glucoside extract) or fruit of glossy privet natural drug.
Fructus Ligustri Lucidi of the present invention, comprises each position of fruit of glossy privet plant, as root, skin, stem, leaf, flower and fruit 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.
Advantage of the present invention is:
1, four steps in 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, these embody in present specification.
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, there is no deep nuclear magnetic resonance spectrum knowledge, is difficult to select 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." and C-7 carbonyl peak is distinguished different iridoid methods of glycosides as characteristic peak according to the feature of iridoid glycoside constituents in the fruit of glossy privet and actual collection of illustrative plates, to select respectively C-4.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, 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 iridoid glycoside constituents in the fruit of glossy privet and actual collection of illustrative plates, to iridoid glycoside constituents (Specnuezhenide in the fruit of glossy privet, 8E-Nuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Lucidumoside C, Kingiside, 8-epikingiside etc.) characteristic peak carried out accurate sequence and calculated its ratio, 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 iridoid glycoside constituents in the fruit of glossy privet, what analyze quantitative means selection is liquid phase, what standard was selected with reference to product is that (Specnuezhenide is one of main active in fruit of glossy privet iridoid glycoside feature extraction thing to Specnuezhenide, " carbonylation displacement and other principal ingredients do not have overlapping its C-4 at this, δ
c156.7～156.8 left and right; Therefore, select Specnuezhenide as standard with reference to product).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 Fructus Ligustri Lucidi or spin-off, can reflect in Fructus Ligustri Lucidi or spin-off and contain which fruit of glossy privet iridoid glycosides and the ratio between them, reach the object to Fructus Ligustri Lucidi or spin-off kind and quality 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 Fructus Ligustri Lucidi or spin-off, also can meet current for the objective an urgent demand of effectively evaluating and controlling fruit of glossy privet quality.All in all, not only can solve the difficult problem that China's Fructus Ligustri Lucidi or spin-off are differentiated and evaluated, also, for strengthening systematization and the standardization of the inherent composition Study of Fructus Ligustri Lucidi or 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 of the special extract of fruit of glossy privet iridoid glycoside of embodiment 1 Fructus Ligustri Lucidi A.
Fig. 1-b, 1-c and 1-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak of the special extract of fruit of glossy privet iridoid glycoside of embodiment 1 Fructus Ligustri Lucidi A widens enlarged drawing.
Fig. 2-a is the IGD carbon-13 nmr spectra finger-print of the special extract of fruit of glossy privet iridoid glycoside of embodiment 2 Fructus Ligustri Lucidi B.
Fig. 2-b, 2-c and 2-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak of the special extract of fruit of glossy privet iridoid glycoside of embodiment 2 Fructus Ligustri Lucidi B widens enlarged drawing.
Fig. 3-a is the IGD carbon-13 nmr spectra finger-print of the special extract of fruit of glossy privet iridoid glycoside of embodiment 3 Fructus Ligustri Lucidi C.
Fig. 3-b, 3-c and 3-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak of the special extract of fruit of glossy privet iridoid glycoside of embodiment 3 Fructus Ligustri Lucidi C widens enlarged drawing.
Fig. 4-a is the IGD carbon-13 nmr spectra finger-print of the special extract of fruit of glossy privet iridoid glycoside of embodiment 4 Fructus Ligustri Lucidi D.
Fig. 4-b, 4-c and 4-d are that the part, IGD carbon-13 nmr spectra Fingerprints peak of the special extract of fruit of glossy privet iridoid glycoside of embodiment 4 Fructus Ligustri Lucidi D 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 do not specialize, the material using in the present invention is the conventional material of this area, and NM operation is the routine operation of this area.
In the present invention, the percentage concentration of ethanol is percent by volume.
One, Fructus Ligustri Lucidi or the research of spin-off IGD carbon-13 nmr spectra finger-print
(1) extracted the IGD carbon-13 nmr spectra finger-print of the fruit of glossy privet iridoid glycoside feature extraction thing obtaining by Fructus Ligustri Lucidi or spin-off
1) acquisition of fruit of glossy privet iridoid glycoside feature extraction thing
1. from Fructus Ligustri Lucidi or medicine materical crude slice, obtain
Take Fructus Ligustri Lucidi or medicine materical crude slice and pulverize (crossing 24～50 mesh sieves), adding volume is the ultrasonic extraction 2～3 times at refluxing extraction or 55～65 ℃ at 90～95 ℃ of 6～10 times of amounts, 90%～95% ethanol, each extraction 1～2 hour, merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add water to disperse, then fill post with macroporous absorbent resin, successively rush post with water, 50～70% ethanol and 90～95% ethanol, collect 50～70% ethanol eluates, evaporated under reduced pressure, obtains fruit of glossy privet iridoid glycoside feature extraction thing (CET).
The mass volume ratio (g:mL) of described Fructus Ligustri Lucidi or medicine materical crude slice and 90～95% ethanol is 1:(6～10); The weight that is used for the water that disperses medicinal extract is 20 times of medicinal material weight; The weight of described macroporous absorbent resin is 0.8～2 times of medicinal material weight; Described macroporous absorbent resin is AF-8, HP20, D101 etc.
2. from other fruit of glossy privet spin-offs, obtain
Directly get other fruit of glossy privet spin-offs (fruit of glossy privet spin-off except fruit of glossy privet medicine materical crude slice, as fruit of glossy privet iridoid glucoside extract) as fruit of glossy privet iridoid glycoside feature extraction thing.
2) the IGD carbon-13 nmr spectra finger-print of fruit of glossy privet iridoid glycoside feature extraction thing detects
Get fruit of glossy privet iridoid glycoside feature extraction thing 30mg, be dissolved in the deuterated DMSO-d of 0.5mL
6or CD
3cOCD
3in, make IGD carbon-13 nmr spectra finger-print and detect, obtain fruit of glossy privet iridoid glycoside IGD carbon-13 nmr spectra finger-print.
3) the IGD carbon-13 nmr spectra finger-print of fruit of glossy privet iridoid glycoside feature extraction thing is resolved
1. differentiate
In fruit of glossy privet iridoid glycoside feature extraction thing (CET) IGD carbon-13 nmr spectra finger-print, clearly illustrate the characteristic signal of fruit of glossy privet iridoid glycoside compounds, Specnuezhenide, 8E-Nuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Lucidumoside C, Kingiside, 8-epikingiside etc. all have corresponding NMR signal.
2. the each active component characteristic peak in fruit of glossy privet iridoid glycoside 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 iridoid glycoside compounds in fruit of glossy privet iridoid glycoside 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
c164.0～220.0 left and right C-4 " or C-7 carbonyl carbon carbon peak is as this specific characteristic peak, active component carbon peak, it is former because C-4 " or C-7 carbonyl carbon and other carbon geochemistry displacement difference are larger, easy to identify; " or between C-7 carbonyl carbon carbon peak, chemical shift also has certain difference in and different Compound C-4.
3. standard is with reference to the selection of product
Specnuezhenide is one of main active in fruit of glossy privet iridoid glycoside feature extraction thing, and " carbonylation displacement and other principal ingredients do not have overlapping its C-4 at this, δ
c156.7～156.8 left and right; Therefore, select Specnuezhenide as standard with reference to product.
4) adopt HPLC method to measure the content of Specnuezhenide in Fructus Ligustri Lucidi or spin-off
1. Specnuezhenide assay
I) chromatographic condition (with reference to 2010 editions pharmacopeia)
Mobile phase: methyl alcohol: water=40:60;
Chromatographic column: C18(250*4.6mm, 5um);
Detect wavelength: 224nm.
Ii) Specnuezhenide standard is with reference to the preparation of product solution
Accurately take Specnuezhenide 5mg, put in 50mL volumetric flask, the methyl alcohol dissolved dilution that is 80% by mass concentration, to scale, obtains Specnuezhenide standard with reference to product solution after shaking up.
Iii) contain the preparation of the need testing solution of fruit of glossy privet iridoid glycoside active component
Accurately take test sample: the powder of Fructus Ligustri Lucidi/medicine materical crude slice or other fruit of glossy privet spin-offs 0.5g, accurately weighed, put in tool plug conical flask, precision adds Diluted Alcohol 50mL, weighed weight, add hot reflux 1 hour, let cool, supply weight with Diluted Alcohol, filter, get subsequent filtrate, as Fructus Ligustri Lucidi, medicine materical crude slice or other fruit of glossy privet spin-off need testing solutions (as fruit of glossy privet iridoid glucoside extract need testing solution).
Iv) determination method
The accurate Specnuezhenide standard of drawing is with reference to product solution and each 20 μ 1 of need testing solution that contain fruit of glossy privet iridoid glycoside active component respectively, and injection liquid chromatography, measures, and obtains Specnuezhenide content.
2. Specnuezhenide absolute content calculates (in Fructus Ligustri Lucidi or spin-off, Specnuezhenide absolute content calculates)
I) calculate Specnuezhenide mass concentration in the need testing solution that contains iridoid glycoside active component by following formula:
C
x: Specnuezhenide mass concentration (ug/mL) in the need testing solution that contains iridoid glycoside active component;
C
r: Specnuezhenide standard is with reference to product concentration of polymer solution (ug/mL);
A
x: the peak area of the need testing solution that contains iridoid glycoside active component;
A
r: Specnuezhenide standard is with reference to the peak area of product solution.
Ii) calculate Specnuezhenide quality percentage composition in Fructus Ligustri Lucidi or spin-off by following formula
W
1(%): Specnuezhenide quality percentage composition in Fructus Ligustri Lucidi or spin-off;
C
x: the Specnuezhenide mass concentration (ug/mL) in the need testing solution that contains iridoid glycoside active component;
M: the Fructus Ligustri Lucidi taking or the quality of spin-off (mg).
5) by content and the total amount of each main active of iridoid glycosides in coupling formula calculating Fructus Ligustri Lucidi or spin-off, the i.e. content of iridoid glycoside active component group
W
1: Fructus Ligustri Lucidi or spin-off Plays are with reference to the quality percentage composition of product Specnuezhenide;
M
1: standard is with reference to the molecular weight/quantitatively carbon number corresponding to peak of product Specnuezhenide;
H
1: standard is with reference to the characteristic peak peak intensity (peak height) of product Specnuezhenide;
W
n: the quality percentage composition of a certain iridoid glycoside active component in Fructus Ligustri Lucidi or spin-off;
M
n: a certain iridoid glycoside active component molecular weight in Fructus Ligustri Lucidi or spin-off/quantitatively carbon number corresponding to peak;
H
n: a certain iridoid glycoside active component characteristic peak peak intensity (peak height) in Fructus Ligustri Lucidi or spin-off.
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.
Column chromatography silica gel G and tlc silica gel H: Haiyang Chemical Plant, Qingdao.
Silica gel column chromatography 6cm × 70cm(diameter × highly).
Fructus Ligustri Lucidi A(Central Plains Zheng Xin medicinal material company, Henan, the place of production), Fructus Ligustri Lucidi B(Central Plains Zheng Xin medicinal material company, Jiangsu, the place of production), Fructus Ligustri Lucidi C(Central Plains Zheng Xin medicinal material company, market, Hui nationality), Fructus Ligustri Lucidi D(Central Plains Zheng Xin medicinal material company, Zhejiang, the place of production) all identify through professor Zhu Changshan of In Henan Agriculture university; Specnuezhenide, 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).
D101, AB-8 macroreticular resin (technical grade, Nankai chemical plant); HP20 macroreticular resin (technical grade, Mitsubishi chemical company product, the green hundred careless development in science and technology company limiteds in Beijing).
Three, structure and the nuclear magnetic data of main active in glossy privet medicinal material
Specnuezhenide
13C-NMR(DMSO)δ：92.92(C-1)，153.33(C-3)，129.24(C-4)，30.02(C-5)，39.49(C-6)，170.60(C-7)，123.10(C-8)，107.74(C-9)，12.99(C-10)，166.14(C-11)，51.18(C-OCH
3)，70.06(C-α)，34.83(C-β)，98.95(C-1')，73.23(C-2')，73.50(C-3')，69.92(C-4')，76.41(C-5')，61.05(C-6')，128.61(C-1")，114.97(C-2")，129.66(C-3")，155.54(C-4")，129.66(C-5")，114.97(C-6")，102.78(C-1"')，73.23(C-2"')，77.30(C-3"')，70.06(C-4"')，76.47(C-5"')，64.03(C-6"')。
Oleuropein
13C-NMR?(CD
3OD)δ：95.3(C-1)，155.1(C-3)，109.4(C-4)，31.6(C-5),42.9(C-6)，173.1(C-7)，124.9(C-8)，130.4C-9)，13.5(C-10)，168.8(C-11)，51.8(-OCH3)，100.9(C-1')，74.5(C-2')，77.8(C-3')，71.6(C-4')，78.6(C-5')，62.5(C-6')，66.8(C-α)，?36.2(C-β)，131.2(C-1")，117.0(C-2")，146.7(C-3")，144.6(C-4")，116.3(C-5")，121.2(C-6")。
lucidumosides?B
13C-NMR(CD
3OD)δ：98.8(C-1)，154.1(C-3)，110.7(C-4)，30.9(C-5)，38.4(C-6)，174.2(C-7)，35.1(C-8)，37.8(C-9)，14.3(C-10)，168.9(C-11)，51.7(-OCH3)，100.9(C-1')，74.6(C-2')，78.1(C-3')，71.8(C-4')，78.3(C-5')，62.7(C-6')，67.0(C-α)，36.0(C-β)，131.4(C-1")，117.0(C-2")，146.8(C-3")，144.8(C-4")，116.4(C-5")，121.8(C-6")。
lucidumosides?C
13C-NMR(CD
3OD)δ：93.7(C-1)，153.8(C-3)，107.9(C-4)，30.4(C-5)，39.7(C-6)，171.7(C-7)，123.6(C-8)，128.9(C-9)，12.4(C-10)，167.3(C-11)，50.7(-OCH3)，99.3(C-1')，73.3(C-2')，76.4(C-3')，70.0(C-4')，76.9(C-5')，61.3(C-6')，67.9(C-α)，79.2(C-β)，129.8(C-1")，114.9(C-2")，145.0(C-3")，144.9(C-4")，113.4(C-5")，118.3(C-6")，14.3,63.9(C-OCH
2CH
3)。
lucidumosides?D
13C-NMR(CD
3OD)δ：95.1(C-1)，154.8(C-3)，109.6(C-4)，31.6(C-5),42.5(C-6)，173.4(C-7)，124.6(C-8)，130.3(C-9)，14.1(C-10)，168.7(C-11)，51.3，56.7,56.8(-OCH3)，99.5(C-1')，74.1(C-2')，78.4(C-3')，71.8(C-4')，78.5(C-5')，62.4(C-6')，66.5(C-α)，36.1(C-β)，130.5(C-1")，112.1(C-2")，149.6(C-3")，147.6(C-4")，113.1(C-5")，121.8(C-6")。
lucidumosides?A
13C-NMR(CD
3OD)δ：98.4(C-1)，154.0(C-3)，110.4(C-4)，30.8(C-5)，36.6(C-6)，174.1(C-7)，35.0(C-8)，37.4(C-9)，14.3(C-10)，168.6(C-11)，51.8(-OCH
3)，100.6(C-1')，74.7(C-2')，77.9(C-3')，71.5(C-4')，78.3(C-5')，62.8(C-6')，66.9(C-α)，35.1(C-β)，129.9(C-1")，130.8(C-2")，116.2(C-3")，156.9(C-4")，116.2(C-5")，130.8(C-6")。
8-epikingiside(8-shows kingiside)
13C-NMR(CD
3OD)δ：96.3(C-1)，154.4(C-3)，109.6(C-4)，28.2(C-5)，34.6(C-6)，?174.8(C-7)，75.8(C-8)，41.9(C-9)，21.7(C-10)，168.3(C-11)，52.0(-OCH3)，100.6(C-1')，74.7(C-2')，78.5(C-3')，71.7(C-4')，77.9(C-5')，62.9(C-6')。
Iso?8-epikingiside
1
3C-NMR(CD
3OD)δ：98.2(C-1)，156.2(C-3)，105.4(C-4)，32.5(C-5)，69.3(C-6)，177.0(C-7)，36.0(C-8)，40.6(C-9)，17.6(C-10)，168.4(C-11)，51.9(-OCH3)，100.8(C-1')，74.7(C-2')，78.5(C-3')，71.7(C-4')，77.9(C-5')，62.9(C-6')。
6'-Elenolylnicotiflorine
13C-NMR(CD
3OD)δ：202.1/95.8(C-1)，155.7/158.4(C-3)，108.5/109.8(C-4)，29.2/32.2(C-5)，40/41.5(C-6)，173.2/172.1(C-7/EtO)，72/125.6(C-8)，52.4/131.3(C-9)，18.5/14.3(C-10)，169.9/169.1(C-11)，52.4/52.8(C-OCH3)，104.9/101.5(C-1')，75.6/75.4(C-2')，78.3/78.3(C-3')，71.9/71.6(C-4')，75.2/78.8(C-5')，65/63.1(C-6')，104.9(C-1")，75.6(C-2")，78.3(C-3")，71.9(C-4")，75.2(C-5")，65.0(C-6")，122.9(C-1"')，131.4(C-2"')，138.3(C-3"')，131.4(C-4"')，122.9(C-5"')，151.0(C-6"')，72.1(C-7"')，37.0(C-8"')。
ligustaloside?A
13C-NMR(CD
3OD)δ：97.9(C-1)，154.1(C-3)，110.6(C-4)，30.8(C-5)，36.6(C-6)，174.3(C-7)，42.0(C-8)，37.3(C-9)，202.8(C-10)，168.9(C-11)，51.8(-OCH3)，100.8(C-1')，74.8(C-2')，78.4(C-3')，71.7(C-4')，78.0(C-5')，62.8(C-6')，66.9(C-α)，35.4(C-β)，130.9(C-1")，116.5(C-2")，146.2(C-3")144.9(C-4")，117.1(C-5")，121.3(C-6")。
ligustaloside?B
13C-NMR(CD
3OD)δ：97.8(C-1)，154.1(C-3)，110.6(C-4)，30.8(C-5)，36.5(C-6)，174.3(C-7)，42.0(C-8)，37.4(C-9)，202.7(C-10)，168.9(C-11)，51.8(-OCH
3)，100.8(C-1')，74.8(C-2')，78.4(C-3')，71.7(C-4')，78.0(C-5')，62.8(C-6')，66.9(C-α)，35.2(C-β)，130.1(C-1")，130.9(C-2")，116.4(C-3")，157.0(C-4")，116.4(C-5")，130.9(C-6")。
10-Hydroxyoleuropein(10-hydroxyl-oleuropein)
13C-NMR(CD
3OD)δ：94.9(C-1)，155.1(C-3)，109.4(C-4)，32.4(C-5),41.4(C-6)，173.1(C-7)，129.5(C-8)，131.2(C-9)，59.3(C-10)，168.5(C-11)，52.0(-OCH
3)，100.1(C-1')，74.8(C-2')，78.5(C-3')，71.6(C-4')，78.0(C-5')，62.9(C-6')，66.9(C-α)，35.4(C-β)，130.8(C-1")，116.6(C-2")，146.3(C-3")，145.0(C-4"),117.2(C-5")，121.4(C-6")。
L0-acetoxyoleuropein(10-acetyl group Cleupin)
13C-NMR(CD
3OD)δ：94.3(C-1)，154.9(C-3)，109.1(C-4)，32.4(C-5)，41.2(C-6)，172.8(C-7)，124.4(C-8)，133.9(C-9)，61.8(C-10)，168.3(C-11)，52.0(-OCH
3)，100.9(C-1')，74.7(C-2')，78.4(C-3')，71.4(C-4')，77.9(C-5')，62.7(C-6')，66.9(C-α)，35.4(C-β)，130.7(C-1")，116.4(C-2")，146.2(C-3")，146.9(C-4")，117.1(C-5")，121.3(C-6")。
dihydroligustaloside?A
13C-NMR(CD
3OD)δ：98.5(C-1)，154.1(C-3)，110.7(C-4)，30.7(C-5),36.5(C-6)，174.4(C-7)，30.6(C-8)，37.6(C-9)，60.7(C-10)，168.9(C-11)，51.7(-OCH
3)，100.6(C-1')，74.8(C-2')，78.4(C-3')，71.7(C-4')，78.0(C-5')，62.9(C-6')，66.8(C-α)，35.2(C-β)，130.1(C-1")，130.9(C-2")，116.4(C-3")，157.1(C-4"),116.4(C-5")，130.9(C-6")。
dihydroligustaloside?B
13C-NMR(CD
3OD)δ：98.4(C-1)，154.0(C-3)，110.5(C-4)，30.6(C-5),36.4(C-6)，174.3(C-7)，30.5(C-8)，37.4(C-9)，60.6(C-10)，168.9(C-11)，51.7(-OCH
3)，100.4(C-1')，74.7(C-2')，78.2(C-3')，71.5(C-4')，77.9(C-5')，62.8(C-6')，66.8(C-α)，35.3(C-β)，130.8(C-1")，116.4(C-2")，146.1(C-3")，144.8(C-4"),117.0(C-5")，121.2(C-6")。
(8Z)-Nuezhenide((8Z)-Ligustrum lucidum Ait)
13C-NMR(CD
3OD)δ：93.7(C-1)，154.2(C-3)，112.2(C-4)，33.6(C-5)，37.7(C-6)，173.6(C-7)，126.2(C-8)，132.1(C-9)，13.5(C-10)，168.6(C-11)，51.8(C-OCH3)，100.1(C-1')，74.7(C-2')，78.0(C-3')，71.6(C-4')，78.4(C-5')，62.8(C-6')，72.3(C-α)，36.5(C-β)，130.8(C-1")，131.0(C-2")，116.2(C-3")，156.9(C-4")，116.2(C-5")，131.0(C-6")，104.5(C-1"')，75.0(C-2"')，78.1(C-3"')，71.6(C-4"')，75.4(C-5"')，64.8(C-6"')。
Specnuezhenide
13C-NMR(CD
3OD)δ：92.9(C-1)，153.3(C-3)，129.2(C-4)，30.0(C-5)，39.5(C-6)，170.6(C-7/EtO)，123.1(C-8)，107.7(C-9)，13.0(C-10)，166.1(C-11)，99.0(C-1')，73.2(C-2')，73.5(C-3')，69.9(C-4')，76.4(C-5')，61.1(C-6')，70.1(C-α)，34.8(C-β)，128.6(C-1")，115.0(C-2")，130.0(C-3")，155.5(C-4")，130.0(C-5")，115.0(C-6")，102.8(C-1"')，73.2(C-2"')，77.3(C-3"')，70.1(C-4"')，76.5(C-5"')，64.0(C-6"')。
Nuezhenoside
13C-NMR(CD
3OD)δ：92.9(C-1)，153.3(C-3)，129.6(C-4)，30.0(C-5)，39.5(C-6)，170.6(C-7/EtO)，123.0(C-8)，107.7(C-9)，13.0(C-10)，166.1(C-11)，51.1(C-OCH
3)，98.9(C-1')，73.2(C-2')，73.5(C-3')，69.9(C-4')，76.4(C-5')，61.0(C-6')，70.1(C-α)，34.8(C-β)，128.6(C-1")，114.9(C-2")，129.6(C-3")，155.5(C-4")，129.6(C-5")，115.0(C-6")，102.8(C-1"')，73.5(C-2"')，77.3(C-3"')，70.1(C-4"')，76.5(C-5"')，64.0(C-6"')。
Ligstroside
13C-NMR(CD
3OD)δ：95.1(C-1)，155.1(C-3)，109.4(C-4)，31.8(C-5)，41.3(C-6)，173.2(C-7)，124.9(C-8)，130.5(C-9)，13.5(C-10)，168.7(C-11)，51.9(C-OCH
3)，100.9(C-1')，74.7(C-2')，77.9(C-3')，71.4(C-4')，78.4(C-5')，62.7(C-6')，66.9(C-α)，35.4(C-β)，130.7(C-1")，117.1(C-2")，146.2(C-3")，144.9(C-4")，116.4(C-5")，121.3(C-6")。
Ligustroside
13C-NMR(CD
3OD)δ：94.9(C-1)，155.0(C-3)，109.1(C-4)，31.5(C-5)，41.1(C-6)，173.0(C-7)，124.8(C-8)，130.0(C-9)，13.6(C-10)，168.4(C-11)，51.9(C-OCH
3)，100.5(C-1')，74.4(C-2')，77.6(C-3')，71.2(C-4')，78.5(C-5')，62.5(C-6')，66.7(C-α)，34.9(C-β)，129.7(C-1")，130.8(C-2")，116.1(C-3")，156.7(C-4")，116.1(C-5")，130.8(C-6")。
Oleonuezhenide
13C-NMR(D
2O)δ：94.7/94.8(C-1)，154.4/154.4(C-3)，108.2/108.2(C-4)，29.8/30.2(C-5)，39.4/40(C-6)，171/173(C-7/EtO)，124.6/124.8(C-8)，129.8/129.8(C-9)，12.7/12.8(C-10)，168.4/168.4(C-11)，51.7/51.7(C-OCH
3)，99.6/99.6(C-1')，72.8/72.8(C-2')，76.4/76.4(C-3')，69.6/69.6(C-4')，75.8/75.8(C-5')，60.8/60.8(C-6')，70.2(C-α)，34.3(C-β)，130.1(C-1")，129.8(C-2")，115.2(C-3")，154.4(C-4")，115.2(C-5")，129.8(C-6")，100.2(C-1"')，73.9(C-2"')，73.9(C-3"')，70.0(C-4"')，73.4(C-5"')，63.9(C-6"')。
(8E)-Nuezhenide
13C-NMR(CD
3OD)δ：95.2(C-1)，155.2(C-3)，109.5(C-4)，31.8(C-5)，41.4(C-6)，173.1(C-7)，125.0(C-8)，130.6(C-9)，13.8(C-10)，168.7(C-11)，52.0(C-OCH
3)，100.9(C-1')，74.8(C-2')，78.0(C-3')，71.6(C-4')，78.5(C-5')，62.8(C-6')，72.3(C-α)，36.5(C-β)，130.8(C-1")，131.0(C-2")，116.2(C-3")，156.9(C-4")，116.2(C-5")，131.0(C-6")，102.2(C-1"')，72.6(C-2"')，75.6(C-3"')，69.7(C-4"')，73.1(C-5"')，63.8(C-6"')。
Ligustaloside?B
13C-NMR(D
2O)δ：96.4(C-1)，152.2(C-3)，108.8(C-4)，29.2(C-5)，34.9(C-6)，171.7(C-7)，29.3(C-8)，35.1(C-9)，102.5(C-10)，52.5/53.1(C-OCH
3)，166.3(C-11)，50.9(C-OCH
3)，99.1(C-1')，73.2(C-2')，77.2(C-3')，70.0(C-4')，76.6(C-5')，61.2(C-6')，64.9(C-α)，33.4(C-β)，127.8(C-1")，129.6(C-2")，115.1(C-3")，155.8(C-4")，115.1(C-5")，129.6(C-6")。
Ligustroside?acid
13C-NMR(CDCl
3)δ：93.3(C-1)，152.8(C-3)，108.2(C-4)，31.3(C-5)，39.4(C-6)，171.7(C-7)，119.0(C-8)，147.1(C-9)，166.3(C-10)，166.6(C-11)，51.7(C-OCH
3)，?97.4(C-1')，71.0(C-2')，72.7(C-3')，68.4(C-4')，72.5(C-5')，61.9(C-6')，65.1(C-α)，34.4(C-β)，135.7(C-1")，130.1(C-2")，121.9(C-3")，149.8(C-4")，121.9(C-5")，130.1(C-6")。
Oleoside11-methyl?ester
13C-NMR(CDCl
3)δ：94.1(C-1)，153.4(C-3)，109.0(C-4)，30.4(C-5)，39.9(C-6)，125.0(C-8)，128.5(C-9)，13.5(C-OCH
3)，167.1(C-11)，51.5(C-OCH
3)，97.4(C-1')，71.0(C-2')，72.8(C-3')，68.5(C-4')，72.4(C-5')，62.0(C-6')。
Kingiside
13C-NMR(CD
3OD)δ：95.1(C-1)，152.4(C-3)，110.1(C-4)，35.2(C-5)，35.8(C-6)，175.2(C-7)，38.8(C-8)，82.1(C-9)，172.3(C-10)，166.4(C-11)，51.4(C-OCH
3)，102.1(C-1')，74.6(C-2')，78.4(C-3')，71.5(C-4')，78.7(C-5')，63.0(C-6')
Isooleonuezhenide
13C-NMR(CD
3OD)δ：95.09/95.03(C-1)，156.35/156.35(C-3)，109.2/109.09(C-4)，31.78/31.56(C-5)，41.42/41.42(C-6)，172.73/172.48(C-7/EtO)，124.99/124.87(C-8)，130.73/130.46(C-9)，13.93/13.8(C-10)，168.36/168.36(C-11)，52.1/51.05(C-OCH
3)，100.7(C-1')，74.9(C-2')，77.7(C-3')，71.3(C-4')，74.5(C-5')，64.7(C-6')，72.0(C-α)，36.3(C-β)，130.1(C-1")，130.0(C-2")，116.0(C-3")，154.9(C-4")，116.0(C-5")，130.0(C-6")，100.7(C-1"')，74.8(C-2"')，77.6(C-3"')，71.1(C-4"')，77.6(C-5"')，62.6(C-6"')。
Glossy privet G13
13C-NMR(D
2O)δ：94.8/95.0(C-1)，154.4/154.6(C-3)，108.1/108.1(C-4)30.3/30.3(C-5)，39.8/40.1(C-6)，170.2/173.2(C-7)，124.1/125.1(C-8)，128.5/128.5(C-9)，12.8/12.9(C-10)，168.5/168.5(C-11)，51.8/51.8(-OCH
3)，99.7/99.8(C-1')，72.7/72.7(C-2')，76.3/76.3(C-3')，69.5/69.5(C-4')，75.7/75.7(C-5')，60.7/60.7(C-6')，70.4(C-α)，34.7(C-β)，136.8(C-1'')，130.0(C-2'')，121.2(C-3'')，148.6(C-4'')，121.1(C-5'')，130.6(C-6'')，102.3(C-1''')，73.0(C-2''')，75.7(C-3''')，69.8(C-4''')，73.2(C-5''')，63.9(C-6''')。
Ligusides?A
13C-NMR(CD
3OD)δ：95.5/95.4(C-1)，155.3/155.4(C-3)，109.3/109.3(C-4)，31.6/?31.8(C-5)，40.7/41(C-6)，172.4/171.7(C-7/EtO)，125.4/125.1(C-8)，130.4/130.6(C-9)，13.8/13.8(C-10)，168.7/168.7(C-11)，52/52(C-OCH3)，101.2/101(C-1')，74.6/74.7(C-2')，78.3/78.3(C-3')，71.2/71.4(C-4')，77.9/77.9(C-5')，62.4/62.5(C-6')，71.6(C-α)，36.5(C-β)，137.9(C-1")，131.0(C-2")，122.5(C-3")，150.5(C-4")，122.5(C-5")，131.0(C-6")，104.2(C-1"')，75.1(C-2"')，75.8(C-3"')，72.9(C-4"')，75.6(C-5"')，62.6(C-6"')。
Ligusides?B
13C-NMR(CD
3OD)δ：202.1/95.8(C-1)，155.7/158.4(C-3)，108.5/109.8(C-4)，29.2/32.2(C-5)，40/41.5(C-6)，173.2/172.1(C-7/EtO)，72/125.6(C-8)，52.4/131.3(C-9)，18.5/14.3(C-10)，169.9/169.1(C-11)，52.4/52.8(C-OCH
3)，104.9/101.5(C-1')，75.6/75.4(C-2')，78.3/78.3(C-3')，71.9/71.6(C-4')，75.2/78.8(C-5')，65/63.1(C-6')，72.1(C-α)，37.0(C-β)，122.9(C-1")，131.4(C-2")，138.3(C-3")，131.4(C-4")，122.9(C-5")，151.0(C-6")，104.9(C-1"')，75.6(C-2"')，78.3(C-3"')，71.9(C-4"')，75.2(C-5"')，65.0(C-6"')。
Embodiment 1: the special extract I GD of the fruit of glossy privet iridoid glycoside carbon-13 nmr spectra coupling finger-print of Fructus Ligustri Lucidi A
(1) feature extraction thing preparation
The Fructus Ligustri Lucidi A(taking after pulverizing crosses 24 mesh sieves) 50g, adding volume is 6/6/10 times of amount (300/300/500mL), 95%(volume ratio) ethanol refluxing extraction 3 times at 95 ℃, each extraction 1 hour, merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add after water (1000g) dispersion of 20 times of medicinal material weight, centrifugal; AB-8 macroporous absorbent resin (50g) the dress post of using again 1 times of medicinal material weight, successively rushes post with water, 60% ethanol and 90% ethanol, collects 60% ethanol eluate, and evaporated under reduced pressure, obtains Fructus Ligustri Lucidi A iridoid glycoside feature extraction thing (CET).
(2) the IGD carbon-13 nmr spectra finger-print of Fructus Ligustri Lucidi A iridoid glycoside feature extraction thing detects
Get fruit of glossy privet iridoid glycoside feature extraction thing 30mg, be dissolved in 0.5mL deuterated methanol, make IGD carbon-13 nmr spectra finger-print and detect, obtain fruit of glossy privet iridoid glycoside IGD carbon-13 nmr spectra finger-print.
(3) Fructus Ligustri Lucidi A iridoid glycoside 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 the iridoid glycoside feature extraction thing (CET) of Fructus Ligustri Lucidi A, clearly illustrate the characteristic signal of fruit of glossy privet iridoid glycoside compounds.Fruit of glossy privet iridoid glycosides: Specnuezhenide, 8E-Nuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Lucidumoside C, Kingiside, 8-epikingiside 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 the special extract of the fruit of glossy privet iridoid glycoside of Fructus Ligustri Lucidi A, each active component ratio measuring result is as follows:
(4) in Fructus Ligustri Lucidi A, Specnuezhenide quality percentage composition measurement result is as follows:
| Fruit of glossy privet Specnuezhenide quality percentage composition W in medicinal material A Specnuezhenide（%） | 0.93% |
(5) in Fructus Ligustri Lucidi A, fruit of glossy privet iridoid glycoside active component quality percentage composition measurement result is as follows:
Embodiment 2: the special extract I GD of the fruit of glossy privet iridoid glycoside carbon-13 nmr spectra coupling finger-print of Fructus Ligustri Lucidi B
(1) feature extraction thing preparation
The Fructus Ligustri Lucidi B(taking after pulverizing crosses 50 mesh sieves) 50g, adding volume is 6/8 times of amount (300/400mL), 90% ethanol refluxing extraction 2 times at 90 ℃, extracts 2 hours at every turn, merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add after water (1000g) dispersion of 20 times of medicinal material weight, centrifugal; The HP-20 macroporous absorbent resin (75g) of 1.5 times of medicinal material weight dress post, successively rushes post with water, 70% ethanol and 95% ethanol again, collects 70% ethanol eluate, and evaporated under reduced pressure, obtains Fructus Ligustri Lucidi B iridoid glycoside feature extraction thing (CET).
(2) the IGD carbon-13 nmr spectra finger-print of Fructus Ligustri Lucidi B iridoid glycoside feature extraction thing detects
With embodiment 1.
(3) Fructus Ligustri Lucidi B iridoid glycoside feature extraction thing IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated (891/ZHTZ)
In the IGD carbon-13 nmr spectra finger-print of the fruit of glossy privet iridoid glycoside feature extraction thing (CET) of Fructus Ligustri Lucidi B, clearly illustrate the characteristic signal of fruit of glossy privet iridoid glycoside compounds.Fruit of glossy privet iridoid glycosides: Specnuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Kingiside, 8-epikingiside 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, 2-c and 2-d.
2) in the special extract of the fruit of glossy privet iridoid glycoside of Fructus Ligustri Lucidi B, each active component ratio measuring result is as follows:
(4) in Fructus Ligustri Lucidi B, Specnuezhenide quality percentage composition measurement result is as follows:
| Specnuezhenide quality percentage composition W in Fructus Ligustri Lucidi B Specnuezhenide（%） | 0.82% |
(5) in Fructus Ligustri Lucidi B, fruit of glossy privet iridoid glycoside active component quality percentage composition measurement result is as follows:
Embodiment 3: the special extract I GD of the fruit of glossy privet iridoid glycoside carbon-13 nmr spectra coupling finger-print of Fructus Ligustri Lucidi C
Take Fructus Ligustri Lucidi C(100g) pulverize (crossing 30 mesh sieves), ultrasonic extraction 2 times is extracted 1.5 hours at every turn, and each temperature of extracting is 55 ℃; Merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add after water (2000g) dispersion of 20 times of medicinal material weight, centrifugal; HP-20 macroporous absorbent resin (150g) the dress post of using again 1.5 medicinal material weight, successively rushes post with water, 50% ethanol and 90% ethanol, collects 50% ethanol eluate, and evaporated under reduced pressure, obtains fruit of glossy privet iridoid glycoside feature extraction thing (CET).
(2) the IGD carbon-13 nmr spectra finger-print of Fructus Ligustri Lucidi C iridoid glycoside feature extraction thing detects
With embodiment 1.
(3) fruit of glossy privet iridoid glycoside 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 the fruit of glossy privet iridoid glycoside feature extraction thing (CET) of Fructus Ligustri Lucidi C, clearly illustrate the characteristic signal of fruit of glossy privet iridoid glycoside compounds.Fruit of glossy privet iridoid glycosides: Specnuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Lucidumoside C, Kingiside, 8-epikingiside etc. all have corresponding NMR signal 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 the special extract of the fruit of glossy privet iridoid glycoside of Fructus Ligustri Lucidi C, each active component ratio measuring result is as follows:
(4) in Fructus Ligustri Lucidi C, Specnuezhenide quality percentage composition measurement result is as follows:
| Fruit of glossy privet Specnuezhenide quality percentage composition W in medicinal material C Specnuezhenide（%） | 0.87% |
(5) in Fructus Ligustri Lucidi C, fruit of glossy privet iridoid glycoside active component quality percentage composition measurement result is as follows:
Embodiment 4: the special extract I GD of the fruit of glossy privet iridoid glycoside carbon-13 nmr spectra coupling finger-print of Fructus Ligustri Lucidi D
Take Fructus Ligustri Lucidi D(50g) pulverize (crossing 30 mesh sieves), ultrasonic extraction 2 times is extracted 1.5 hours at every turn, and each temperature of extracting is 65 ℃; Merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add after water (1000g) dispersion of 20 times of medicinal material weight, centrifugal; D101 macroporous absorbent resin (200g) the dress post of using again 2 times of medicinal material weight, successively rushes post with water, 60% ethanol and 90% ethanol, collects 60% ethanol eluate, and evaporated under reduced pressure, obtains fruit of glossy privet iridoid glycoside feature extraction thing (CET).
(2) the IGD carbon-13 nmr spectra finger-print of fruit of glossy privet iridoid glycoside feature extraction thing detects
With embodiment 1.
(3) fruit of glossy privet iridoid glycoside 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 the fruit of glossy privet iridoid glycoside feature extraction thing (CET) of Fructus Ligustri Lucidi D, clearly illustrate the characteristic signal of fruit of glossy privet iridoid glycoside compounds:.Fruit of glossy privet iridoid glycosides: Specnuezhenide, Ligustroside, Oleuropein, Lucidumoside B, Lucidumoside D, 6'-Elenolylnicotiflorine, Lucidumoside C, Kingiside, 8-epikingiside etc. all have corresponding NMR signal 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 the special extract of the fruit of glossy privet iridoid glycoside of Fructus Ligustri Lucidi D, each active component ratio measuring result is as follows:
(4) in Fructus Ligustri Lucidi D, Specnuezhenide quality percentage composition measurement result is as follows:
| Specnuezhenide quality percentage composition W in Fructus Ligustri Lucidi D Specnuezhenide（%） | 0.83% |
(5) in Fructus Ligustri Lucidi D, fruit of glossy privet iridoid glycoside active component quality percentage composition measurement result is as follows:
Claims (12)
1. a method of differentiating Fructus Ligustri Lucidi or spin-off, comprises the following steps:
1) Fructus Ligustri Lucidi or spin-off are extracted, obtain the fruit of glossy privet iridoid glycoside feature extraction thing that contains active component group;
2) described fruit of glossy privet iridoid glycoside feature extraction thing is carried out to IGD carbon-13 nmr spectra finger-print and detect, obtain several active component characteristic peak peak intensities in described fruit of glossy privet iridoid glycoside 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) measure and 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 Fructus Ligustri Lucidi or 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 fruit of glossy privet iridoid glycoside feature extraction thing.
3. method according to claim 2, it is characterized in that, in step 1), the preparation method of fruit of glossy privet iridoid glycoside feature extraction thing, comprise: take Fructus Ligustri Lucidi or medicine materical crude slice and pulverize, add 90～95% alcohol refluxs extractions or ultrasonic extraction 2～3 times, extract 1～2 hour at every turn, merging filtrate after filtering, is evaporated to medicinal extract; In this medicinal extract, add water to disperse, then fill post with macroporous absorbent resin, successively rush post with water, 50～70% ethanol and 90～95% ethanol, collect 50～70% ethanol eluates, evaporated under reduced pressure, obtains fruit of glossy privet iridoid glycoside feature extraction thing;
The mass volume ratio of described Fructus Ligustri Lucidi or medicine materical crude slice and 90～95% ethanol is 1:(6～10); The weight that is used for the water that disperses medicinal extract is 20 times of medicinal material weight; The weight of described macroporous absorbent resin is 0.8～2 times of medicinal material weight; The temperature of refluxing extraction is 90～95 ℃, and the temperature of ultrasonic extraction is 55～65 ℃; Described macroporous absorbent resin is D101, AB-8 or HP-20.
4. according to the method described in claim 1～3 any one, it is characterized in that, step 2) in, fruit of glossy privet iridoid glycoside feature extraction thing is carried out to IGD carbon-13 nmr spectra finger-print and detect, the solvent that dissolves fruit of glossy privet iridoid glycoside feature extraction thing is deuterated methanol or deuterated dimethyl sulfoxide.
5. according to the method described in claim 1～4 any one, it is characterized in that step 2) in, the active component characteristic peak in fruit of glossy privet iridoid glycoside feature extraction thing is carbonyl absorption peak; Preferably C-4 " or C-7 carbonyl absorption peak, their chemical shift is respectively δ
c144.0～158.0 and δ
c171.0～176.0.
6. method according to claim 5, is characterized in that step 2) in, according to the size of characteristic peak peak intensity and position, several active components in fruit of glossy privet iridoid glycoside feature extraction thing are sorted.
7. according to the method described in claim 1～6 any one, it is characterized in that, in step 3), described quantitative test means are high-efficient liquid phase technique.
8. method according to claim 7, is characterized in that, the condition of described high-efficient liquid phase technique comprises that mobile phase is methyl alcohol: water=30～50:50～70, preferably 40:60; Detection wavelength is 224nm.
9. according to the method described in claim 1～8 any one, it is characterized in that, 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.
10. according to the method described in claim 1～9 any one, it is characterized in that, the standard of the active component in fruit of glossy privet iridoid glycoside feature extraction thing is Specnuezhenide with reference to product.
11. according to the method described in claim 1～10 any one, it is characterized in that, in step 4), the coupling formula that calculates the content of each active component is:
W
1the standard that in the Fructus Ligustri Lucidi of measuring by quantitative test means for step 3) or spin-off, a certain active component is corresponding is with reference to the absolute content of product;
M
1for standard that in described Fructus Ligustri Lucidi or 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 Fructus Ligustri Lucidi by IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off, 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 Fructus Ligustri Lucidi or spin-off;
M
nfor the molecular weight/quantitatively carbon number corresponding to peak of a certain active component in Fructus Ligustri Lucidi or spin-off;
H
nfor the characteristic peak peak intensity of a certain active component in the Fructus Ligustri Lucidi by IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off.
12. according to the method described in claim 1～11 any one, it is characterized in that, described fruit of glossy privet spin-off comprises: fruit of glossy privet medicine materical crude slice, glossy privet fruit extract or fruit of glossy privet natural drug.
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