CN103808751B - A kind of method differentiating traditional Chinese medicine honeysuckle or spin-off - Google Patents
A kind of method differentiating traditional Chinese medicine honeysuckle or spin-off Download PDFInfo
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- CN103808751B CN103808751B CN201410044444.5A CN201410044444A CN103808751B CN 103808751 B CN103808751 B CN 103808751B CN 201410044444 A CN201410044444 A CN 201410044444A CN 103808751 B CN103808751 B CN 103808751B
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- honeysuckle
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- organic acid
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Abstract
The present invention relates to a kind of method differentiating traditional Chinese medicine honeysuckle or spin-off, comprising: traditional Chinese medicine honeysuckle or spin-off are extracted, obtain honeysuckle organic acid containing active component group or/and iridoid glycoside feature extraction thing; Carry out IGD carbon-13 nmr spectra finger-print to feature extraction thing to detect, obtain organic acid according to finger-print or/and several active component characteristic peak peak intensities in iridoid glycoside feature extraction thing; And determine each active component respective standard with reference to product characteristic peak peak intensity by same way; The absolute content of the standard that obtains with reference to product is measured by quantitative test means; Utilize the ratio of characteristic peak peak intensity and the absolute content of standard reference product, calculate the content of each active component in traditional Chinese medicine honeysuckle or spin-off and the content of active component group.The present invention can reflect in honeysuckle containing which honeysuckle organic acid or/and iridoid glycoside compounds and the ratio between them, reach the object to traditional Chinese medicine honeysuckle kind and Quality Identification.
Description
Technical field
The invention belongs to the discriminating field of natural medicinal plant, particularly, relate to a kind of method differentiating traditional Chinese medicine honeysuckle or spin-off.
Background technology
Honeysuckle (Loniceraejaponicaeflos) is dry flower or the first flower opened of band of caprifoliaceae plant honeysuckle (looonicerajaponicaThunb.), is the Chinese crude drug that China is famous.Honeysuckle returns lung, the heart, stomach warp, and the property of medicine is trembled with fear, and taste is sweet, have clearing heat and detoxicating, dispelling wind and heat from the body effect [Chinese Pharmacopoeia Commission. Pharmacopoeia of People's Republic of China version in 2010. China Medical Science Press, 205].Its active component is mainly organic acid, iridoid glycoside and flavone compound, there is effect [the 1. Zhang Shoupings such as inhibitory anti-virus, antipyretic anti-inflammatory, hepatic cholagogic, reducing blood lipid, hemostasis, antioxidation, Deng. Chinese traditional Chinese medicine information magazine 2007,14 (3): 84. 2. Wang Li rivers. Agriculture of Anhui science 2009,37 (5): 2036.].
Qualitive test certain active component above-mentioned or certain active component content of quantitative measurement, become the important method evaluating honeysuckle and the quality of the pharmaceutical preparations thereof.But this quality testing pattern of carrying out qualitative and quantitative analysis for single component effectively can not control the inherent quality of Chinese crude drug, cannot meet current for the objective an urgent demand effectively evaluating and control honeysuckle and the quality of the pharmaceutical preparations thereof.
Fingerprint pattern technology is the most 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, the finger-print research work of traditional Chinese medicine honeysuckle and preparation thereof, concentrate on HPLC finger-print [1. Bai Xuemei, Deng. Chinese patent drug 2004,26 (7): 521. 2. bear is gorgeous, Deng. CHINA JOURNAL OF CHINESE MATERIA MEDICA 2009,34 (8): 1015.], capillary electrophoresis fingerprint (HPCE method) [Sun Guoxiang, Deng. chromatogram 2007,25 (1): 96.], tablets by HPLC-MS (LC-MS) [Zhang Qian, Deng. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2012,37 (23): 3564.] etc.Wherein main stream approach HPLC finger-print due to and polycomponent comparatively large by non-chromatographic condition (as chromatographic column internal diameter, length, the Stationary liquid trade mark, carrier fractions, flow rate of mobile phase, the mutually each component ratio of mixed flow, column temperature, sample size, detector sensitivity etc.) impact quantitatively need by multiple standard items etc. reason, all there is many limitation in repeated and feasibility; Though efficient with it, quick, the easy and post of HPCE method not vulnerable to pollution and be better than HPLC method, 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 due to Chinese medicine compound prescription complicacy and lack the present situation of standard items, MS also has the problem such as degree of ionization and matrix interference, be typically not enough to Multiple components is carried out structure-characterized accurately.
IGD carbon-13 nmr spectra coupling (IGD
13cNMRcoupling) fingerprint pattern technology, is also inverted gated decoupling carbon-13 nmr spectra coupling fingerprint pattern technology, this technology be study proton nmr spectra for many years (
1hNMR) fingerprint pattern technology [Zhao Tianzeng, etc.
1hNMR fingerprint technique plant identification Chinese medicine, Chinese herbal medicine 2000,31 (11): 868-870] basis is combined other technologies (such as current most widely used efficient liquid phase (HPLC) fingerprint pattern technology [Xie Peishan etc., chromatographic fingerprints of Chinese materia medica, People's Health Publisher, 2005] a kind of comprehensive fingerprint pattern technology of non-single means newly) proposed.So far, differentiate traditional Chinese medicine honeysuckle or spin-off, utilize IGD carbon-13 nmr spectra coupling fingerprint pattern technology and have no the disclosure of relevant report and technology contents.
The quality of honeysuckle does not lie in certain single component, and its curative effect is the result that multiple composition works in coordination with proportioning.Therefore, foundation can not only control honey suckle active composition, and the IGD carbon-13 nmr spectra coupling finger-print of the ratio that can control these active components is imperative.The research and apply of traditional Chinese medicine honeysuckle or spin-off carbon-13 nmr spectra coupling finger-print, not only can solve a difficult problem for China's traditional Chinese medicine honeysuckle or spin-off discriminating and evaluation, also for strengthening systematization and the standardization of traditional Chinese medicine honeysuckle or the inherent composition Study of spin-off, the guarantee providing science in line with international standards is realized.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 differentiating traditional Chinese medicine honeysuckle or spin-off, this process employs IGD carbon-13 nmr spectra coupling fingerprint pattern technology.
To achieve these goals, the method for discriminating traditional Chinese medicine honeysuckle provided by the invention or spin-off, comprises the following steps:
1) traditional Chinese medicine honeysuckle or spin-off are extracted, obtain honeysuckle organic acid containing active component group (organic acid is or/and iridoid glycoside) or/and iridoid glycoside feature extraction thing;
2) to described honeysuckle organic acid or/and iridoid glycoside feature extraction thing carries out IGD carbon-13 nmr spectra finger-print detects, obtain described honeysuckle organic acid according to finger-print or/and several active component characteristic peak peak intensities in iridoid glycoside feature extraction thing; 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) absolute content obtaining described standard reference product is measured by quantitative test means;
4) ratio of described characteristic peak peak intensity (the characteristic peak peak intensities of each active component characteristic peak peak intensity and respective standard reference product) and the absolute content of described standard reference product is utilized, calculate each organic acid in traditional Chinese medicine honeysuckle or spin-off or/and the content of iridoid glycoside active component and the total content of main active, the i.e. content of active component group.
Wherein, adopt have obtain clear IGD carbon-13 nmr spectra finger-print extraction process as described honeysuckle organic acid or/and the extracting mode of iridoid glycoside feature extraction thing.
Wherein, in step 1), honeysuckle organic acid is or/and the preparation method of iridoid glycoside feature extraction thing, comprise: take traditional Chinese medicine honeysuckle or medicine materical crude slice is pulverized, add 20 ~ 25% alcohol refluxs to extract or ultrasonic extraction 2 ~ 3 times, each extraction 1 ~ 2 hour, merging filtrate after filtering, reduced pressure concentration; Get the filtrate after concentrating, fill post with macroporous absorbent resin, rush post with water-ethanol system, collect different elution fraction, evaporated under reduced pressure, obtain honeysuckle organic acid or/and iridoid glycoside feature extraction thing.
Further, the mass volume ratio of described traditional Chinese medicine honeysuckle or medicine materical crude slice and 20 ~ 25% ethanol is 1:(6 ~ 10) (g:mL).
Further, the weight of macroporous absorbent resin is 1 ~ 2 times of the filtrate weight after concentrating.
Further, the temperature of described refluxing extraction is 90 ~ 95 DEG C, and the temperature of ultrasonic extraction is 50 ~ 60 DEG C.
Further, traditional Chinese medicine honeysuckle or medicine materical crude slice cross 24 ~ 65 mesh sieves after pulverizing.
Further, the model of described macroporous absorbent resin is HP-20, D-101, D-201, SP-825 or SP-70.
Further, the blade diameter length ratio of described macroporous absorbent resin is 8 ~ 15:1, preferably 10 ~ 12:1.
Wherein, step 2) in, to honeysuckle organic acid or/and iridoid glycoside feature extraction thing carries out the detection of IGD carbon-13 nmr spectra finger-print, dissolve honeysuckle organic acid or/and the solvent of iridoid glycoside feature extraction thing is deuterochloroform (CDCl
3), deuterated methanol (CD
3cOCD
3) or deuterated dimethyl sulfoxide (DMSO-d
6), preferred deuterated methanol.Honeysuckle organic acid is or/and the mass volume ratio of iridoid glycoside feature extraction thing and coordinative solvent is 55:1 ~ 65:1(mg:mL), preferred 60:1.
Except honeysuckle medicine materical crude slice, other honeysuckle spin-offs, as honeysuckle organic acid or/and iridoid glucoside extract can directly as honeysuckle organic acid or/and iridoid glycoside feature extraction thing, differentiate with said method.
Wherein, step 2) in, honeysuckle organic acid is or/and the active component characteristic peak in iridoid glycoside feature extraction thing is ester carbonyl group absorption peak, and its chemical shift is δ
cthe deuterated DMSO-d of 164.0 ~ 167.0(
6dissolve honeysuckle organic acid or/and iridoid glycoside feature extraction thing time) or δ
c168.0 ~ 170.0(with deuterated methanol dissolve honeysuckle organic acid or/and iridoid glycoside feature extraction thing time).
Wherein, step 2) in, according to size and the position of characteristic peak peak intensity, to described honeysuckle organic acid or/and in iridoid glycoside feature extraction thing several active components sort.
Further; step 2) in; according to size and the position of characteristic peak peak intensity; to described honeysuckle organic acid or/and active component in iridoid glycoside feature extraction thing: chlorogenic acid, chlorogenic acid butyl ester, Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside), the two caffeoyl quinic acid ethyl ester of chlorogenic acid butyl ester+3,4-O-, forulic acid, NSC 619661, α-morroniside+Secologanindimethylacetal, β-morroniside, sweroside, kingisides 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: efficient liquid phase (HPLC) method.
Further, when detect honeysuckle organic acid or/and iridoid glycoside feature extraction thing time, the condition of described efficient liquid phase comprises: mobile phase is acetonitrile: 0.4% phosphoric acid solution=(10 ~ 20): (80 ~ 95), preferred 13:87; Determined wavelength is 240nm.
Wherein, in step 3), described standard refers to reference to the absolute content of product: the standard measured by quantitative test means is with reference to the mass percentage of product.
Further, honeysuckle organic acid is or/and the standard of active component in iridoid glycoside feature extraction thing is chlorogenic acid with reference to product.
Wherein, in step 4), the coupling formula calculating the content of each active component is:
W
1in the traditional Chinese medicine honeysuckle measured for step 3) quantitative test means or spin-off, standard corresponding to a certain active component is with reference to the absolute content of product;
M
1for standard corresponding to active component a certain in described traditional Chinese medicine honeysuckle or spin-off is with reference to the carbon number that molecular weight/quantitatively peak is corresponding of product;
H
1for by the characteristic peak peak intensity of standard corresponding to a certain active component in the traditional Chinese medicine honeysuckle of IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off with reference to product;
W
nfor the mass percentage of active component a certain in traditional Chinese medicine honeysuckle or spin-off;
M
nfor the carbon number that molecular weight/quantitatively peak is corresponding of active component a certain in traditional Chinese medicine honeysuckle or spin-off;
H
nfor the characteristic peak peak intensity by a certain active component in the traditional Chinese medicine honeysuckle of 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 both can be the active component group in honeysuckle single medicinal material, may also be the active component group in honeysuckle spin-off.Described honeysuckle organic acid is or/and iridoid glycoside feature extraction thing refers to contain honeysuckle organic acid class in this extract or/and iridoid glycoside active component, and namely active component group refers to the summation of corresponding same active component.
Wherein, described honeysuckle spin-off comprises: honeysuckle medicine materical crude slice, Honegsukle flower P.E (as honeysuckle organic acid extract, honeysuckle iridoid glucoside extract or honeysuckle chromocor extract, or two or more combination in them) or honeysuckle natural drug.
Traditional Chinese medicine honeysuckle of the present invention, comprises each position of honeysuckle plant, as root, skin, stem, leaf, flower and fruit etc.
The calculating of the content of 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
13cNMR coupling finger-print has several feature below:
1. stability (repeatability): IGD
13the chemical shift data that CNMR obtains is second after radix point, and explanation property is good, reproducible; The non-chromatographic condition (as chromatographic column internal diameter, length, the Stationary liquid trade mark, carrier fractions, flow rate of mobile phase, the mutually each component ratio of mixed flow, column temperature, sample size, detector sensitivity etc.) of HPLC, GC changes, the retention time data variation obtained is very large, mean the variation of monolithic chromatogram figure, repeatability is bad.
2. globality (comprehensive): IGD
13the corresponding spectrum peak of each the active component carbon in sample is comprised in CNMR finger-print; There is not this relation in HPLC, GC, UV, IR, MS.
3. reliability (unicity): IGD
13it is strict one-to-one relationship from the carbon on different activities composition in sample and different group thereof that CNMR composes peak; There is not this relation in HPLC, GC, UV, IR, MS.
4. feasibility (easily the property distinguished): IGD
13cNMR 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 not easily resolves; UV quantity of information is few; MS then has the problem such as degree of ionization and matrix interference.
IGD carbon-13 nmr spectra finger-print can only show there is which active component in feature extraction thing, and the quantitative ratio between these active components, and the absolute content of these active components must analyze quantitative means by standard with reference to product and other, then obtained by coupling formula.
Advantage of the present invention is:
1, four steps in the present invention are an entirety, indispensable; Four steps have respective distinctive feature; If separately or simple combination then 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, it is analyzed and detects is a difficult problem always.Therefore, special need a kind of method can various active components in differential plant kind and extract thereof, 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 does not have 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 also exists significant limitation (specifically seeing the content that the application's background technology describes).Fact proved, the gordian technique that IGD carbon-13 nmr spectra coupling fingerprint pattern technology addresses this problem just, this embodies in the instructions of the application.
Although 2 traditional extraction techniques are known by those of ordinary skill, people is never had to obtain clear collection of illustrative plates and representative active component group obtains feature extraction thing as standard to select technological parameter.Utilize the method, the identification result finally for extract can be made better, and this is one of difficult point of the present invention, is also one of innovative point of the present invention.
3, IGD
13cNMR coupling finger-print is the mixed spectrum of multiple active component, inevitably causes the crowded of excellent peak one by one, even overlapping.In order to make result of calculation accurate, specific characteristic peak, each active component carbon peak in the active component group that chemical shift difference is larger is selected to be necessary.Because dissimilar compound carbon spectral difference is not very large, the selection of characteristic peak can be ever-changing; Identical type compound carbon spectral difference is not very little, and a lot of peak overlap is serious, deep nuclear magnetic resonance spectrum knowledge need be had to instruct, through in many ways comparing, turning over, could select good characteristic peak; And characteristic peak select bad, have no idea to carry out accurate quantitative analysis to active component group, this also just the present invention need solve problem.According to the feature of Chlorogenic Acid of Flos Lonicerae class and iridoid glycoside constituents, ester carbonyl group peak is selected to distinguish 2 constituents as characteristic peak.So, need to select different specific characteristic peaks, active component carbon peak to be also the another innovative point of the application according to the feature of different activities composition.
4, because characteristic peak chemical shift difference is very little, in a lot of situation, only after radix point, the 1st potential difference is other, so the sequence of characteristic peak is the key determining main active and ratio thereof, there is no deep nuclear magnetic resonance spectrum knowledge and be separated basis, the active component being difficult to determine that characteristic peak represents and ratio thereof, also just accurate qualitative and quantitative analysis cannot be carried out to active component group, according to the feature of the carbon spectrum nuclear magnetic data of Chlorogenic Acid of Flos Lonicerae class and iridoid glycoside constituents, accurate sequence is carried out to the characteristic peak of two constituents; This is one of difficult point of the present invention, is also one of innovative point of the present invention.
5, coupling calculates the selection that key is choice criteria product and quantitative analysis tech.Analyze quantitative means can select efficient 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, also can be additional with reference to product as external standard.According to the feature of Chlorogenic Acid of Flos Lonicerae class and iridoid glycoside constituents, what analyze that quantitative means selects be liquid phase, and standard is chlorogenic acid with reference to product selection.This is one of difficult point of invention, is also one of innovative point of the present invention.
Research project involved in the present invention is by quality inspection public welfare industry scientific research specific project expenditure Funded Projects (bullets: 2012104019-1).
The present invention adopts IGD carbon-13 nmr spectra finger-print to differentiate traditional Chinese medicine honeysuckle or spin-off, can reflect in traditional Chinese medicine honeysuckle or spin-off and contain which honey suckle active composition and the ratio between them, reach the object to traditional Chinese medicine honeysuckle or spin-off kind and Quality Identification.The range of linearity is wide, highly sensitive, repeatability and feasibility good.Effectively can not only control the inherent quality of traditional Chinese medicine honeysuckle or spin-off, also can meet current for the objective an urgent demand effectively evaluated and control quality of Flos Lonicerae.All in all, not only can solving a difficult problem for China's traditional Chinese medicine honeysuckle or spin-off discriminating and evaluation, also for strengthening systematization and the standardization of traditional Chinese medicine honeysuckle or the inherent composition Study of spin-off, realizing the guarantee providing science in line with international standards.
Accompanying drawing explanation
Fig. 1-a is that embodiment 1 commercially available traditional Chinese medicine honeysuckle A organic acid is or/and the IGD carbon-13 nmr spectra finger-print of iridoid glycoside feature extraction thing.
Fig. 1-b is that embodiment 1 commercially available traditional Chinese medicine honeysuckle A organic acid is or/and the local, IGD carbon-13 nmr spectra Fingerprints peak of iridoid glycoside feature extraction thing widens enlarged drawing.
Fig. 2-a is that embodiment 2 commercially available traditional Chinese medicine honeysuckle B organic acid is or/and the IGD carbon-13 nmr spectra finger-print of iridoid glycoside feature extraction thing.
Fig. 2-b is that embodiment 2 commercially available traditional Chinese medicine honeysuckle B organic acid is or/and the local, IGD carbon-13 nmr spectra Fingerprints peak of iridoid glycoside feature extraction thing widens enlarged drawing.
Fig. 3-a is the IGD carbon-13 nmr spectra finger-print of embodiment 4 commercially available chlorogenic acid 20% Honegsukle flower P.E (upper standing grain).
Fig. 3-b is that the local, IGD carbon-13 nmr spectra Fingerprints peak of embodiment 4 commercially available chlorogenic acid 20% Honegsukle flower P.E (upper standing grain) widens enlarged drawing.
Fig. 4-a is the IGD carbon-13 nmr spectra finger-print that embodiment 5 makes Honegsukle flower P.E by oneself.
Fig. 4-b be embodiment 5 make by oneself Honegsukle flower P.E IGD carbon-13 nmr spectra Fingerprints peak local widen enlarged drawing.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
If not specified, the material used in the present invention is the conventional material of this area, and NM method of operating is also the conventional method of this area.
In the present invention, the concentration of methyl alcohol and ethanol is percent by volume.
One, traditional Chinese medicine honeysuckle or the research of spin-off IGD carbon-13 nmr spectra finger-print
(1) honeysuckle organic acid that obtains is extracted or/and the IGD carbon-13 nmr spectra finger-print of iridoid glycoside feature extraction thing by traditional Chinese medicine honeysuckle or spin-off
1) honeysuckle organic acid is or/and the acquisition of iridoid glycoside feature extraction thing
1. obtain from traditional Chinese medicine honeysuckle or medicine materical crude slice
Take traditional Chinese medicine honeysuckle or medicine materical crude slice pulverizing (crossing 24 ~ 65 mesh sieves), adding volume is 6 ~ 10 times amount, 20% ~ 25%(volume ratio) ethanol reflux at 90 ~ 95 DEG C or ultrasonic extraction 2 ~ 3 times at 50 ~ 60 DEG C, each extraction 1 ~ 2 hour, merging filtrate after filtering, reduced pressure concentration; Get the filtrate after concentrating, be the macroporous absorbent resin dress post of 1 ~ 2 times amount by its weight, rush post with water-ethanol system, collect different elution fraction, obtain honeysuckle organic acid or/and iridoid glycoside feature extraction thing (CET).
Described macroreticular resin is HP-20, D-101, D-201, SP-825 or SP-70; The blade diameter length ratio of described macroporous absorbent resin is 8 ~ 15:1, preferably 10 ~ 12:1.
2. obtain from other honeysuckle spin-offs
Directly get other honeysuckle spin-offs (the honeysuckle spin-off except honeysuckle medicine materical crude slice, as honeysuckle organic acid or/and iridoid glucoside extract) as honeysuckle organic acid or/and iridoid glycoside feature extraction thing.
2) honeysuckle organic acid is or/and the IGD carbon-13 nmr spectra finger-print of iridoid glycoside feature extraction thing detects
Extracting honeysuckle organic acid, or/and iridoid glycoside feature extraction thing, is dissolved in deuterochloroform, DMSO-d
6or in deuterated methanol, make IGD carbon-13 nmr spectra finger-print and detect, obtain honeysuckle organic acid or/and iridoid glycoside IGD carbon-13 nmr spectra finger-print.
Honeysuckle organic acid is or/and the mass volume ratio of iridoid glycoside feature extraction thing and coordinative solvent is 55:1 ~ 65:1(mg:mL), preferred 60:1.
3) honeysuckle organic acid is or/and the IGD carbon-13 nmr spectra finger-print of iridoid glycoside feature extraction thing is resolved
1. differentiate
Honeysuckle organic acid is or/and in iridoid glycoside feature extraction thing (CET) IGD carbon-13 nmr spectra finger-print, clearly illustrate honeysuckle organic acid or/and the characteristic signal of iridoid glycoside compounds.
2. honeysuckle organic acid is or/and each active component characteristic peak in 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; Between the specific characteristic peak of 3. each compound, chemical shift difference is larger; 4. the chemical shift effect difference affecting the specific characteristic peak of each compound itself is larger.
Due to honeysuckle organic acid or/and containing this compounds a series of in iridoid glycoside feature extraction thing, carbon peak intersects more, in order to measure the ratio of each active component, the larger respective peaks of chemical shift difference must be selected as characteristic peak., investigate through reality for this reason, all select ester carbonyl group carbon carbon peak as two specific characteristic peaks, active component carbon peak, its reason is that ester carbonyl group carbon and other carbon chemical shifts difference are comparatively large, easy to identify; And chemical shift also has certain difference between different compound carbonyl carbon carbon peak.
3. standard is with reference to the selection of product
Chlorogenic acid is honeysuckle organic acid or/and one of main active in iridoid glycoside feature extraction thing, and its carbonyl chemical shift does not have overlapping with other principal ingredients at this; Therefore, chlorogenic acid is selected as standard with reference to product.
4) HPLC method is adopted to measure the content of traditional Chinese medicine honeysuckle or spin-off Content of Chlorogenic Acid
I) chromatographic condition (with reference to 2010 editions pharmacopeia)
Mobile phase: acetonitrile: 0.4% phosphoric acid solution=13:87;
Chromatographic column: C18(250*4.6mm, 5um);
Determined wavelength: 240nm.
Ii) chlorogenic acid standard is with reference to the preparation of product solution
Accurately take chlorogenic acid 5mg, put in 50mL volumetric flask, with mass concentration be the methyl alcohol dissolved dilution of 50% to scale, namely obtain chlorogenic acid standard after shaking up with reference to product solution.
Iii) the preparation of the need testing solution of honeysuckle organic acid and/or iridoid glycoside active component is contained
Accurately take test sample: the powder 0.5g of traditional Chinese medicine honeysuckle/medicine materical crude slice, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 50mL, weighed weight, adds hot reflux 1 hour, lets cool, supply weight with 50% methyl alcohol, filter, get subsequent filtrate, as traditional Chinese medicine honeysuckle/medicine materical crude slice need testing solution.Other honeysuckle spin-offs 30-50mg, puts in 50mL volumetric flask, with mass concentration be the methyl alcohol dissolved dilution of 50% to scale, namely obtain other honeysuckle spin-off need testing solutions after shaking up.
Iv) determination method
Respectively accurate chlorogenic acid standard of drawing is with reference to product solution and traditional Chinese medicine honeysuckle/medicine materical crude slice need testing solution or each 20 μ 1 of spin-off need testing solution, and injection liquid chromatography, mensuration, obtains chlorogenic acid content.
2. chlorogenic acid absolute content calculates (traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off Content of Chlorogenic Acid absolute content calculate)
I) traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off need testing solution Content of Chlorogenic Acid mass concentration is calculated by following formula:
C
x: traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off need testing solution Content of Chlorogenic Acid mass concentration (ug/mL);
C
r: chlorogenic acid standard is with reference to product concentration of polymer solution (ug/mL);
A
x: the peak area of traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off need testing solution;
A
r: chlorogenic acid standard is with reference to the peak area of product solution.
Ii) traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off Content of Chlorogenic Acid mass percentage is calculated by following formula
W
1(%): traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off Content of Chlorogenic Acid mass percentage;
C
x: the chlorogenic acid mass concentration (ug/mL) in traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off need testing solution;
M: the quality (mg) of the traditional Chinese medicine honeysuckle taken or spin-off.
5) by organic acid in coupling formulae discovery traditional Chinese medicine honeysuckle or spin-off or/and the content of each main active of iridoid glycosides and total amount, namely organic acid is or/and the content of iridoid glycoside active component group
W
1: the mass percentage of traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off Plays reference product chlorogenic acid;
M
1: standard is with reference to the carbon number that molecular weight/quantitatively peak is corresponding of product chlorogenic acid;
H
1: standard is with reference to the characteristic peak peak intensity (peak height) of product chlorogenic acid;
W
n: in traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off, a certain organic acid is or/and the mass percentage of iridoid glycoside active component;
M
n: a certain organic acid in traditional Chinese medicine honeysuckle/medicine materical crude slice or spin-off is or/and the carbon number that molecular weight/quantitatively peak is corresponding of iridoid glycoside active component;
H
n: in traditional Chinese medicine honeysuckle or spin-off, a certain organic acid is or/and the characteristic peak peak intensity (peak height) of iridoid glycoside active component.
Two, instrument, reagent and material
Key instrument and equipment
Nuclear magnetic resonance spectrometer BrukerDPX400 type.
Mass spectrometer: WatersMicromass company Q-TofMicroTM 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 upper grand experimental facilities company limited of Nereid.
Column chromatography silica gel G and tlc silica gel H: Haiyang Chemical Plant, Qingdao.
Silica gel column chromatography 6cm × 70cm(diameter × highly).
Commercially available traditional Chinese medicine honeysuckle A(Central Plains Zheng Xin company, in June, 2013 purchased from medicinal material market, Hui nationality, Henan, the place of production), honeysuckle (Central Plains Zheng Xin company, in July, 2013 purchased from medicinal material market, Hui nationality, Henan, the place of production), all identify through In Henan Agriculture university professor Zhu Changshan; Chlorogenic acid, chemical reference substance, purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Reagent: chromatographically pure (methyl alcohol, Tianjin Siyou Fine Chemicals Co., Ltd.) and analyzing pure (Tianjin Chemical Reagents Factory No.1).
Three, the structure of main active and carbon-13 nmr spectra data in traditional Chinese medicine honeysuckle or spin-off
Chlorogenic acid chlorogenic acid butyl ester
Protocatechuic acid caffeic acid
NSC 619661's forulic acid
Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside)
Methylchlorogenate(3-caffeoylquinicacidmethylester)
The two caffeoyl quinic acid ethyl ester of 3,4-O-
Loganin 7-dehydrogenation loganin
Morroniside sweroside
7-O-methyl-morroniside Secologanindimethylacetal
Kingiside (Kingiside) 8-epi-KINGISIDE
Chlorogenic acid (Chlorogenicacid, C
16h
18o
9)
13CNMR(75MHz,DMSO-d
6)δ:73.6(C-1),36.4(C-2),70.5(C-3),70.9(C-4),68.3(C-5),37.3(C-6),175.1(C-7)，125.6(C-1′),114.3(C-2′),145.0(C-3′),146.0(C-4′),115.8(C-5′),121.4(C-6′),148.4(C-7′),114.8(C-8′),165.8(C-9′)。
13CNMR(125MHz,CD
3OD)δ:127.8(C-1),115.2(C-2),146.8(C-3),149.6(C-4),116.0(C-5),123.0(C-6),147.1(C-7),115.3(C-8),168.7(C-9);B:76.1(C-1),38.8(C-2),72.0(C-3),73.4(C-4),71.3(C-5),38.2(C-6),177.0(C-7)
Chlorogenic acid butyl ester
13C-NMR(MHz,DMSO-d6)δ:73.2(C-1),37.3(C-2),69.4(C-3),71.1(C-4),66.9(C-5),35.2(C-6),173.2(C-7),64.2(C-8),30.1(C-9),18.6(C-10),13.6(C-11),125.4(C-1′),114.6(C-2′),145.2(C-3′),148.8(C-4′),115.9(C-5′),121.4(C-6′),145.7(C-7′),113.9(C-8′),165.5(C-9′)。
The two caffeoyl quinic acid ethyl ester of 3,4-O-
13C-NMR(MHz,CD
3OD)δ:75.1(C-1),36.8(C-2),75.1(C-3),69.8(C-4),66.0(C-5),41.3(C-6),175.7(C-7),62.5,14.2(-7-OCH
2CH
3),127.7,127.6(C-1′,1′′),115.2,115.1(C-2′,2′′),146.8,146.8(C-3′,3′′),149.0,149.0(C-4′,4′′),116.5,116.5(C-5′,5′′),123.2,123.1(C-6′,6′′),147.4,147.4(C-7′,7′′),115.0,115.0(C-8′),168.5,168.4(C-9′)。
Caffeic acid (C
9h
8o
4)
13CNMR(CD
3OD,75MHz)δ:127.8(C-1),115.1(C-2),147.0(C-3),149.4(C-4),115.5(C-5),122.8(C-6),146.8(C-7),116.8(C-8),170.9(C-9)
NSC 619661 (C
11h
12o
4)
13CNMR(CD
3OD,75MHz)δ:127.7(C-1),115.0(C-2),146.8(C-3),149.5(C-4),115.2(C-5),122.9(C-6),146.7(C-7),116.5(C-8),169.3(C-9)，61.4,14.6(-OCH
2CH
3)
Forulic acid (C
10h
10o
4)
13CNMR(125MHz,CD
3OD):δ
C51.9(q,OCH
3-3),114.8(d,C-8),115.1(d,C-2),116.4(d,C-5),122.7(d,C-6),127.6(s,C-1),146.6(s,C-4),146.7(s,C-7),149.3(s,C-3),169.5(s,C-9).
Protocatechuic acid
13CNMR(CD
3OD,125MHz)δ
C:123.2(C-1)，115.8(C-2)，146.1(C-3)，151.5(C-4)，117.8(C-5)，123.9(C-6)，170.3(C-7);
13CNMR(DMSO-d
6,100MHz)δ
C:121.7(C-1),116.7(C-2),144.8(C-3),149.8(C-4),115.1(C-5),122.5(s,C-6),167.7(C-7)。
Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside) (vanillic acid 7-o-β-D – (6-o-phenylpyran glucose)
13C-NMR(MHz,CD
3OD)δ:126.1(C-1),114.3(C-2),150.4(C-3),151.6(C-4),116.5(C-5),124.6(C-6),169.5(C-7),56.5(-OMe),131.2(C-1′),130.6(C-2′),129.6(C-3′),134.4(C-4′),129.6(C-5′),130.6(C-6′),167.7(C-7′)。
Methylchlorogenate (3-caffeoylquinicacidmethylester) [methyl chlorogenate (
3-caffeoyl guinic acid methyl esters)]
13C-NMR(MHz,MeOD)δ:75.8(C-1),38.0(C-2),70.3(C-3),72.1(C-4),72.1(C-5),37.7(C-6),175.4(C-7),53.0(OMe),127.6(C-1′),115.1(C-2′),146.9(C-3′),149.7(C-4′),116.5(C-5′),123.0(C-6′),145.7(C-7′),115.0(C-8′),168.2(C-9′)。
Loganin
13CNMR(100MHz,MeOD)δ
C:97.71(C-1),152.10(C-3),114.03(C-4),32.15(C-5),42.70(C-6),75.04(C-7),42.16(C-8),46.50(C-9),13.41(C-10),169.52(C-11),51.62(C-12),100.05(C-1′),74.72(C-2′),78.02(C-3′),71.59(C-4′),78.35(C-5′),62.76(C-6′)
13CNMR(100MHz,DMSO-d
6)δ
C:96.15(C-1),150.71(C-3),112.21(C-4),30.88(C-5),41.74(C-6),73.11(C-7),40.58(C-8),44.80(C-9),13.62(C-10),167.14(C-11),51.13(C-12),98.63(C-1′),72.18(C-2′),76.64(C-3′),70.05(C-4′),77.31(C-5′),61.12(C-6′)
7-dehydrogenation loganin
13CNMR(100MHz,D
2O)δ
C:95.04(C-1),152.60(C-3),110.35(C-4),26.74(C-5),42.52(C-6),225.04(C-7),44.08(C-8),44.87(C-9),12.61(C-10),169.78(C-11),52.26(C-12),99.07(C-1′),73.00(C-2′),75.95(C-3′),69.94(C-4′),76.72(C-5′),61.07(C-6′)
Sweroside
13CNMR(100MHz,DMSO-d
6)δ
C:95.75(C-1),151.60(C-3),105.01(C-4),26.94(C-5),24.45(C-6),67.86(C-7),132.54(C-8),41.69(C-9),120.48(C-10),164.83(C-11),98.27(Glc-1),73.30(Glc-2),76.57(Glc-3),70.22(Glc-4),77.52(Glc-5),61.21(Glc-6)
13CNMR(100MHz,MeOD)δ
C:98.2(C-1),153.9(C-3),106.0(C-4),28.5(C-5),26.0(C-6),69.7(C-7),133.5(C-8),43.8(C-9),120.1(C-10),168.5(C-11),99.7(C-1′),74.7(C-2′),77.8(C-3′),71.5(C-4′),78.4(C-5′),62.7(C-6′)
α-morroniside
13CNMR(100MHz,DMSO-d
6)δ
C:95.38(C-1),152.95(C-3),109.92(C-4),30.30(C-5),36.39(C-6),94.37(C-7),73.74(C-8),39.99(C-9),19.56(C-10),166.51(C-11),51.34(C-12),98.41(C-1′),71.60(C-2′),76.82(C-3′),70.35(C-4′),77.65(C-5′),61.24(C-6′)
13CNMR(CD
3OD)97.2(C-1),154.6(C-3),111.0(C-4),34.7(C-5),37.4(C-6),96.1(C-7).75.1(C-8),40.0(C-9),20.0(C-10),168.8(C-11),100.2(C-1′).74.2(C-2′),78.0(C-3′),71.7(C-4′),78.5(C-5′),62.9(C-6′).
β-morroniside
13CNMR(100MHz,DMSO-d
6)δ
C:93.98(C-1),153.00(C-3),110.70(C-4),25.74(C-5),33.64(C-6),90.01(C-7),40.12(C-8),40.43(C-9),19.52(C-10),166.47(C-11),51.28(C-12),98.37(C-1′),73.74(C-2′),76.82(C-3′),70.35(C-4′),77.70(C-5′),78.46.(C-5′),61.39(C-6′)
13CNMR(CD
3OD)δ
C:95.8(C-1),154.6(C-3),111.8(C-4),27.6(C-5),34.7(C-6).92.5(C-7),66.0(C-8).40.7(C-9),20.0(C-IO),168.8(C-11),100.2(C-1′),74.2(C-2’),78.0(C-3’),71.7(C-4’),78.5(C-5’),62.9(C-6’).
7-O-methyl-morroniside
13CNMR(100MHz,CD
3Cl
3)δ
C:95.1(C-1),152.0(C-3),110.7(C-4),29.9(C-5),33.8(C-6),102.4(C-7),71.6(C-8),38.9(C-9),18.4(C-10),166.1(C-11),50.9(C-12),96.7(C-1′),70.7(C-2′),72.0(C-3′),68.6(C-4′),72.5(C-5′),61.6(C-6′)
13CNMR(100MHzCD
3Cl
3,)δ
C:94.7(C-1),152.1(C-3),111.7(C-4),26.1(C-5),32.6(C-6),97.7(C-7),64.1(C-8),39.4(C-9),18.6(C-10),166.5(C-11),51.0(C-12),96.7(C-1′),71.0(C-2′),72.0(C-3′),68.6(C-4′),72.5(C-5′),61.7(C-6′)
Secologanindimethylacetal(driffractive ring loganin dimethyl-acetal)
13CNMR(100MHz,DMSO-d
6)δ
C:95.7(C-1),151.5(C-3),109.7(C-4),25.2(C-5),31.7(C-6),102.1(C-7),134.5(C-8),43.1(C-9),119.3(C-10),166.6(C-11),98.7(C-1′),73.0(C-2′),77.4(C-3′),69.9(C-4′),76.7(C-5′),61.0(C-6′)
Kingiside
13CNMR(MeOD)δ
C:94.4(C-1),154.3(C-3),111.4(C-4),28.0(C-5),34.3(C-6),174.3(C-7),76.8(C-8),39.9(C-9),18.4(C-10),168.2(C-11),100.1(C-1′),74.8(C-2′),78.0(C-3′),71.6(C-4′),78.5(C-5′),62.8(C-6′)
8-epi-KINGISIDE(8-shows kingiside)
13CNMR(MeOD)δ
C:96.3(C-l),154.4(C-3),109.6(C-4),28.1(C-5),34.6(C-6),174.7(C-7),75.8(C-8),41.9(C-9),21.7(C-lo),168.3(C-11).52.0(OMe),100.7(C-l'),74.7(C-2'),78.5(C-3'),71.7(C-4'),77.9(C-5'),62.9(C-6').
Embodiment 1: commercially available traditional Chinese medicine honeysuckle A(Central Plains is just believed) organic acid and iridoid glycoside feature extraction thing IGD carbon-13 nmr spectra coupling finger-print
(1) feature extraction thing preparation
Take the commercially available traditional Chinese medicine honeysuckle A25 gram after pulverizing (crossing 65 mesh sieves), adding volume is 6,6,10 times amount (150mL, 150mL, 250mL), 20%(volume ratio) ethanol refluxing extraction 3 times at 90 DEG C, each extraction 1 hour, merging filtrate after filtering, be evaporated to without alcohol taste, about 500mL, adjusts pH=1 with concentrated hydrochloric acid; Get the filtrate after acid adjustment, fill post with the macroporous absorbent resin (model: HP-20, blade diameter length ratio 12:1) that its weight is 2 times amount, rush post with water-ethanol system, collect 30% elution fraction, obtain honeysuckle A organic acid and iridoid glycoside feature extraction thing (CET).
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get commercially available honeysuckle A(Central Plains just to believe) organic acid and iridoid glycoside feature extraction thing 30mg, be dissolved in 0.5mLDMSO-d
6in, make IGD carbon-13 nmr spectra finger-print and detect, obtain honeysuckle A organic acid and iridoid glycoside IGD carbon-13 nmr spectra finger-print.
(3) commercially available honeysuckle A(Central Plains is just believed) organic acid and iridoid glycoside feature extraction thing IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
Commercially available honeysuckle A(Central Plains is just believed) in the honeysuckle organic acid of medicinal material and the IGD carbon-13 nmr spectra finger-print of iridoid glycoside feature extraction thing (CET), clearly illustrate the characteristic signal of honeysuckle organic acid and iridoid glycoside compounds.Honeysuckle organic acid and iridoid glycosides: chlorogenic acid, chlorogenic acid butyl ester, NSC 619661, Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside), α-morroniside, Secologanindimethylacetal, β-morroniside, sweroside 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 local widens enlarged drawing and sees accompanying drawing 1-b.
2) commercially available honeysuckle A(Central Plains is just believed) each active component ratio measuring result is as follows in the organic acid of medicinal material and iridoid glycoside feature extraction thing:
(4) commercially available honeysuckle A(Central Plains is just believed) medicinal material Content of Chlorogenic Acid mass percentage measurement result is as follows:
(5) commercially available honeysuckle A(Central Plains is just believed) in medicinal material organic acid and iridoid glycoside active component mass percentage measurement result as follows:
The molecular formula of α in following table-morroniside+Secologanindimethylacetal is with α-morroniside molecular formula representatively (following examples are all with embodiment 1).
Embodiment 2: commercially available traditional Chinese medicine honeysuckle B(Central Plains is just believed) organic acid and iridoid glycoside feature extraction thing IGD carbon-13 nmr spectra coupling finger-print
(1) feature extraction thing preparation
The commercially available honeysuckle B(Central Plains taken after pulverizing (crossing 24 mesh sieves) is just believed) medicine materical crude slice 25g, adding volume is 10,10 times amount (250mL, 250mL), 25%(volume ratio) ethanol ultrasonic extraction 2 times at 50 DEG C, each extraction 2 hours, merging filtrate after filtering, be evaporated to without alcohol taste, about 500mL, adjusts pH=2 with concentrated hydrochloric acid; Get the filtrate after acid adjustment; Get the filtrate after concentrating, fill post with the macroporous absorbent resin (model: D-101, blade diameter length ratio 12:1) that its weight is 1 times amount, rush post with water-ethanol system, collect 35% elution fraction, obtain honeysuckle B organic acid and iridoid glycoside feature extraction thing (CET).
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get commercially available honeysuckle B(Central Plains just to believe) organic acid and iridoid glycoside feature extraction thing 30mg, be dissolved in the deuterated DMSO-d of 0.5mL
6in, make IGD carbon-13 nmr spectra finger-print and detect, obtain honeysuckle B organic acid and iridoid glycoside IGD carbon-13 nmr spectra finger-print.
(3) commercially available honeysuckle B(Central Plains is just believed) organic acid and iridoid glycoside feature extraction thing IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
Commercially available honeysuckle B(Central Plains is just believed) in the organic acid of medicinal material and the IGD carbon-13 nmr spectra finger-print of honeysuckle iridoid glycoside feature extraction thing (CET), clearly illustrate the characteristic signal of honeysuckle organic acid and iridoid glycoside compounds.Honeysuckle organic acid and iridoid glycosides: chlorogenic acid, chlorogenic acid butyl ester, NSC 619661, Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside), α-morroniside, Secologanindimethylacetal, β-morroniside, sweroside 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 local widens enlarged drawing and sees accompanying drawing 2-b.
2) commercially available honeysuckle B(Central Plains is just believed) each active component ratio measuring result is as follows in the organic acid of medicinal material and iridoid glycoside feature extraction thing:
(4) commercially available honeysuckle B(Central Plains is just believed) medicinal material Content of Chlorogenic Acid mass percentage measurement result is as follows:
(5) commercially available honeysuckle B(Central Plains is just believed) in medicinal material organic acid and iridoid glycoside active component mass percentage measurement result as follows:
Embodiment 3: commercially available Honegsukle flower P.E IGD carbon-13 nmr spectra coupling finger-print
(1) feature extraction thing is selected
Direct selection commercially available chlorogenic acid 20% Honegsukle flower P.E (Shang He bio tech ltd, Changsha) is as feature extraction thing.
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get standing grain chlorogenic acid 20% Honegsukle flower P.E 30mg, be dissolved in the deuterated DMSO-d of 0.5mL
6in, make IGD carbon-13 nmr spectra finger-print and detect, obtain standing grain chlorogenic acid 20% Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print.
(3) upper standing grain chlorogenic acid 20% Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print
1) IGD carbon-13 nmr spectra finger-print is differentiated
Chlorogenic acid 20% Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print in, clearly illustrate the characteristic signal of honeysuckle organic acid compounds.Honeysuckle organic acid class: chlorogenic acid, chlorogenic acid butyl ester, 3,4-O-two caffeoyl quinic acid ethyl ester, NSC 619661, forulic acids 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 local widens enlarged drawing and sees accompanying drawing 3-b.
2) in upper standing grain chlorogenic acid 20% Honegsukle flower P.E, each active component ratio measuring result is as follows:
(4) upper standing grain chlorogenic acid 20% Honegsukle flower P.E Content of Chlorogenic Acid mass percentage measurement result is as follows:
(5) in upper standing grain chlorogenic acid 20% Honegsukle flower P.E, active component mass percentage measurement result is as follows:
Embodiment 4: self-control Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print
(1) feature extraction thing is selected
Direct self-control Honegsukle flower P.E of selecting [takes commercially available traditional Chinese medicine honeysuckle (the Central Plains Zheng Xin company after pulverizing (crossing 65 mesh sieves), source is medicinal material market, Hui nationality, Henan, the place of production) 50 grams, adding volume is 6,6,10 times amount, 20%(volume ratio) ethanol refluxing extraction 3 times at 90 DEG C, each extraction 1 hour, merging filtrate after filtering, is evaporated to without alcohol taste, about 500mL, adjusts pH=2 with concentrated hydrochloric acid; Be extracted with ethyl acetate 4 times, combined ethyl acetate liquid, evaporate to dryness, derive from prepared honeysuckle extract 2.22g] as feature extraction thing.
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Take from prepared honeysuckle extract 30mg, be dissolved in 0.5mL deuterated methanol, make IGD carbon-13 nmr spectra finger-print and detect, obtain standing grain chlorogenic acid 25% Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print.
(3) Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print is made by oneself
1) IGD carbon-13 nmr spectra finger-print is differentiated
Self-control Honegsukle flower P.E IGD carbon-13 nmr spectra finger-print in, clearly illustrate the characteristic signal of honeysuckle organic acid and iridoid glycoside compounds.Honeysuckle organic acid and iridoid glycosides: chlorogenic acid, chlorogenic acid butyl ester, NSC 619661, morroniside, sweroside, kingiside 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 local widens enlarged drawing and sees accompanying drawing 4-b.
2) each active component ratio measuring result in Honegsukle flower P.E is made by oneself as follows:
(4) Honegsukle flower P.E Content of Chlorogenic Acid mass percentage measurement result is made by oneself as follows:
(5) active component mass percentage measurement result in Honegsukle flower P.E is made by oneself as follows:
Claims (9)
1. differentiate a method for traditional Chinese medicine honeysuckle or spin-off, comprise the following steps:
1) traditional Chinese medicine honeysuckle or spin-off are extracted, obtain honeysuckle organic acid containing active component group or/and iridoid glycoside feature extraction thing;
Honeysuckle organic acid is or/and the preparation method of iridoid glycoside feature extraction thing, comprise: take traditional Chinese medicine honeysuckle or medicine materical crude slice is pulverized, add 20 ~ 25% alcohol reflux and extract or ultrasonic extraction 2 ~ 3 times, extract 1 ~ 2 hour at every turn, merging filtrate after filtering, reduced pressure concentration; Get the filtrate after concentrating, fill post with macroporous absorbent resin, rush post with water-ethanol system, collect different elution fraction, evaporated under reduced pressure, obtain honeysuckle organic acid or/and iridoid glycoside feature extraction thing;
The mass volume ratio of described traditional Chinese medicine honeysuckle or medicine materical crude slice and 20 ~ 25% ethanol is 1:(6 ~ 10);
The weight of described macroporous absorbent resin is 1 ~ 2 times of the filtrate weight after concentrating;
The temperature of described refluxing extraction is 90 ~ 95 DEG C, and the temperature of ultrasonic extraction is 50 ~ 60 DEG C;
2) to described honeysuckle organic acid or/and iridoid glycoside feature extraction thing carries out IGD carbon-13 nmr spectra finger-print respectively detects, obtain described honeysuckle organic acid according to finger-print or/and several active component characteristic peak peak intensities in iridoid glycoside feature extraction thing; And determine the characteristic peak peak intensity of described each active component respective standard with reference to product by same way;
Wherein, honeysuckle organic acid is or/and the active component characteristic peak in iridoid glycoside feature extraction thing is ester carbonyl group absorption peak, and its chemical shift is δ
c164.0 ~ 167.0 or δ
c168.0 ~ 170.0;
Honeysuckle organic acid is or/and the standard of active component in iridoid glycoside feature extraction thing is chlorogenic acid with reference to product;
3) absolute content obtaining described standard reference product is measured by efficient liquid phase means;
The condition of described efficient liquid phase comprises: mobile phase is acetonitrile: 0.4% phosphoric acid solution=(10 ~ 20): (80 ~ 95), and determined wavelength is 240nm;
4) utilize the ratio of the characteristic peak peak intensity of each active component characteristic peak peak intensity and respective standard reference product and the absolute content of described standard reference product, calculate each organic acid in traditional Chinese medicine honeysuckle or spin-off or/and the content of iridoid glycoside active component and the content of two active component groups.
2. method according to claim 1, is characterized in that, adopt have obtain clear IGD carbon-13 nmr spectra finger-print extraction process as described honeysuckle organic acid or/and the extracting mode of iridoid glycoside feature extraction thing.
3. the method according to claim 1 ~ 2 any one, is characterized in that, the model of described macroporous absorbent resin is HP-20, D-101, D-201, SP-825 or SP-70; The blade diameter length ratio of described macroporous absorbent resin is 8 ~ 15:1.
4. method according to claim 3, it is characterized in that, step 2) in, to honeysuckle organic acid or/and iridoid glycoside feature extraction thing carries out the detection of IGD carbon-13 nmr spectra finger-print, dissolve honeysuckle organic acid or/and the solvent of iridoid glycoside feature extraction thing is deuterochloroform, deuterated methanol or deuterated dimethyl sulfoxide.
5. the method according to claim 1 or 2 or 4, is characterized in that, step 2) in, according to size and the position of characteristic peak peak intensity, to described honeysuckle organic acid or/and in iridoid glycoside feature extraction thing several active components sort.
6. method according to claim 5, is characterized in that, the condition of described efficient liquid phase comprises: mobile phase is acetonitrile: 0.4% phosphoric acid solution=13:87.
7. the method according to claim 1 or 2 or 4 or 6, is characterized in that, step 3) in, described standard refers to reference to the absolute content of product: the standard measured by quantitative test means is with reference to the mass percentage of product.
8. method according to claim 7, is characterized in that, step 4) in, the coupling formula calculating the content of each active component is:
W
1for step 3) a certain active component is corresponding in the traditional Chinese medicine honeysuckle that measures by quantitative test means or spin-off standard is with reference to the absolute content of product;
M
1for standard corresponding to active component a certain in described traditional Chinese medicine honeysuckle or spin-off is with reference to the carbon number that molecular weight/quantitatively peak is corresponding of product;
H
1for by the characteristic peak peak intensity of standard corresponding to a certain active component in the traditional Chinese medicine honeysuckle of IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off with reference to product;
W
nfor the mass percentage of active component a certain in traditional Chinese medicine honeysuckle or spin-off;
M
nfor the carbon number that molecular weight/quantitatively peak is corresponding of active component a certain in traditional Chinese medicine honeysuckle or spin-off;
H
nfor the characteristic peak peak intensity by a certain active component in the traditional Chinese medicine honeysuckle of IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off.
9. the method according to claim 1 or 2 or 4 or 6 or 8, it is characterized in that, honeysuckle spin-off comprises: honeysuckle medicine materical crude slice, Honegsukle flower P.E or honeysuckle natural drug.
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| CN109030666B (en) * | 2018-10-09 | 2022-01-11 | 山东中医药大学 | Method for identifying honeysuckle and lonicera confusa by utilizing high performance liquid chromatography |
| CN112730677B (en) * | 2020-01-17 | 2022-06-21 | 成都中医药大学 | UPLC-Q-TOF/MS method for detecting multiple chemical components in paederia scandens |
| CN117310050B (en) * | 2023-11-28 | 2024-02-09 | 中国中医科学院中医药健康产业研究所 | Screening method of honeysuckle antioxidation quality markers |
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| CN100409864C (en) * | 2005-05-18 | 2008-08-13 | 中国人民解放军第三军医大学第一附属医院 | Method for sieving and separating active substances from Chinese herbs, composition containing said active substances and application thereof |
| CN1793105A (en) * | 2005-12-16 | 2006-06-28 | 抚州苍源药业开发有限公司 | Tech. for extracting high purity chlorogenic acid from honeysuckle |
| CN100575358C (en) * | 2006-06-07 | 2009-12-30 | 石家庄汉康生化药品有限公司 | Flos Lonicerae extract, its preparation method and application |
| CN101985421B (en) * | 2010-10-26 | 2013-01-09 | 西北农林科技大学 | Method for simultaneously preparing chlorogenic acid and luteoloside from honeysuckle flower |
| CN102692460A (en) * | 2012-05-22 | 2012-09-26 | 辽宁中医药大学 | Process for determining content of four components of honeysuckle during full-time-interval three-wavelength fusion |
| CN102749348B (en) * | 2012-06-18 | 2014-06-11 | 河南省科高植物天然产物开发工程技术有限公司 | Method for identifying active components in medicinal plant |
| CN102818869A (en) * | 2012-09-10 | 2012-12-12 | 山东汉方制药有限公司 | High performance liquid chromatograph |
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