CN103760185B - A kind of method differentiating nim oil - Google Patents
A kind of method differentiating nim oil Download PDFInfo
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- CN103760185B CN103760185B CN201410039926.1A CN201410039926A CN103760185B CN 103760185 B CN103760185 B CN 103760185B CN 201410039926 A CN201410039926 A CN 201410039926A CN 103760185 B CN103760185 B CN 103760185B
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Abstract
The present invention relates to a kind of method differentiating nim oil, comprising: 1) get nim oil or the direct feature extraction thing as becoming grouping containing fatty acid of nim oil spin-off; 2) carry out IGD carbon-13 nmr spectra finger-print to described feature extraction thing to detect, obtain several active component characteristic peak peak intensities in feature extraction thing according to finger-print; And determine the characteristic peak peak intensity of described each active component respective standard with reference to product by same way; 3) absolute content of described standard with reference to product is obtained by quantitative test means; 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 the content of each active component and the content of active component group in nim oil or nim oil spin-off.The present invention can measure in nim oil and contain which fatty acid compound and their content ratio, reaches the object to nim oil Quality Identification.
Description
Technical field
The invention belongs to the discriminating field of natural medicinal plant, particularly, relate to a kind of method differentiating nim oil.
Background technology
Nim oil is the vegetable oil squeezed from print chinaberry (AzadirachtaindicaA.Juss) fruit, having food refusal repellent, reduce insect gut vigor, suppress insect growth to be grown, suppress and stop insect molting, suppress the effects such as adult mate and oviposit, is the desirable feedstock manufacturing each biological pesticide.Have that insecticidal spectrum is wide, insect not easily develops immunity to drugs, the feature such as noresidue in environment, can with Multiple Pesticides and fungicide compounding.Excessive in India and Bangladesh etc., nim oil is the most often applied to the nursing maintenance products such as soap, hair products, body lotion and hand lotion.The scope that nim oil uses, from disease of skin, infection, inflammation disease, fever, is even applied to rheumatoid releiving.
Nim oil complicated component, each component analysis is more difficult.Usually, people carry out to nim oil the total fatty acids methyl esters that saponification obtains nim oil, then with relative content [1. sieve happiness honor of higher fatty acid contained by GC or GC-MS qualitative determination wherein, Deng. Chinese grease 2002,27 (4): 78. 2. Li Jun peaks, Deng. Yunnan Institute for nationalities's journal 2005,14 (3): 278.].Wherein main stream approach GC chromatography is owing to affecting 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.) comparatively large and need use standard items etc. reason, and repeatability and feasibility all exist many limitation; There is the problems such as MS degree of ionization and matrix interference in GC-MS analytical approach.
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, in .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.
The research and apply of nim oil IGD carbon-13 nmr spectra coupling finger-print, a new thinking is provided to the discriminating of nim oil and evaluation, also for strengthening systematization and the standardization of the inherent composition Study of nim oil, the guarantee providing science in line with international standards is realized.Along with this technology applying in other Chinese crude drugs and extract thereof, botanical pesticide, the great scientific value of this technology will be increasingly outstanding.
Summary of the invention
For solving the problem of prior art, the object of the invention is to provide a kind of method differentiating nim oil.
To achieve these goals, the present invention adopts following technical scheme:
Differentiate a method for nim oil, it comprises the following steps:
1) nim oil or the direct feature extraction thing as becoming grouping containing fatty acid of nim oil spin-off is got;
2) carry out IGD carbon-13 nmr spectra finger-print to described feature extraction thing to detect, obtain several active component characteristic peak peak intensities in feature extraction thing according to finger-print; And determine the characteristic peak peak intensity of described each active component respective standard with reference to product by same way (IGD carbon-13 nmr spectra finger-print);
3) absolute content of described standard with reference to product is obtained by quantitative test means;
4) 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 is utilized, calculate the content of each active component and the total content of this active component, the i.e. content of active component group in nim oil or nim oil spin-off.
Wherein, step 2) in, carry out IGD carbon-13 nmr spectra finger-print to feature extraction thing and detect, the solvent of dissolving characteristic extract is deuterochloroform (CDCl
3); The mass volume ratio of feature extraction thing and described solvent is 190:1-210:1(mg:ml), preferred 200:1.
Step 2) in, the active component characteristic peak in feature extraction thing is carbonyl absorption peak (i.e. C-1 absorption peak), and its chemical shift is δ
c170.0-185.0.
Step 2) in, described peak intensity can adopt peak height method, area integral method or gravimetric method to calculate.
In step 3), described quantitative test means are gas chromatography (GC) method; 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, the standard of the active component in described feature extraction thing is methyl oleate with reference to product.
Wherein, in step 4), the coupling formula calculating each active component content is:
; Wherein:
W
1in the nim oil measured for step 3) quantitative test means or nim oil spin-off, standard corresponding to a certain active component is with reference to the absolute content of product;
M
1for standard corresponding to a certain active component in described nim oil or nim oil 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 nim oil of IGD carbon-13 nmr spectra determining fingerprint pattern or nim oil spin-off with reference to product;
W
nfor the mass percentage of a certain active component in nim oil or nim oil spin-off;
M
nfor the carbon number that molecular weight/quantitatively peak is corresponding of a certain active component in nim oil or nim oil spin-off;
H
nfor the characteristic peak peak intensity by a certain active component in the nim oil of IGD carbon-13 nmr spectra determining fingerprint pattern or nim oil 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, namely refers to the summation of fatty acid active component.
In the present invention, four steps that nim oil is differentiated 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.Nim oil contains multiple fatty acid or ester constituents, and it is analyzed and detects is a difficult problem always.The qualitative and quantitative analysis aspect that currently available technology solves differential plant kind and evaluation plant source product quality also exists significant limitation.Fact proved, IGD carbon-13 nmr spectra coupling fingerprint pattern technology solves the key of existing issue just.
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.Dissimilar compound (fatty acid or ester constituents) carbon spectral difference is not very large, and 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, need have deep nuclear magnetic resonance spectrum knowledge, 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 application need solve problem.According to the feature of fatty acid ester ingredients in nim oil, what characteristic peak was selected is C-1 carbonyl absorption peak.So, need to select different specific characteristic peak, active component carbon peak according to the feature of different activities composition, one of the innovative point of this application just.
Because characteristic peak chemical shift difference is very little, in a lot of situation, only the 1st display difference after radix point, 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 fatty acid ester ingredients in nim oil, determine concrete fatty acid or ester constituents by characteristic peak, also acid or lactone component can be distinguished; This is also one of innovative point of the application.
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 composition in nim oil, what analyze quantitative means selection is gas chromatography, what standard reference product were selected is that methyl oleate is (because oleic acid boiling point is too high, gas phase is generally by after methyl oleate, measure the content of wherein methyl oleate, but in nim oil, oleic acid physical presence form is triglyceride oleate).This is one of difficult point of the application, is also one of innovative point of the application simultaneously.
The content of each active component of the present invention and the total content of this active component calculate, and are 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 following feature:
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 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 multiple 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.The present invention adopts IGD carbon-13 nmr spectra finger-print to differentiate nim oil, can reflect in nim oil and contain which fatty acid compound and the ratio between them, reach the object to nim oil Quality Identification.The range of linearity is wide, highly sensitive, repeatability and feasibility good.
Accompanying drawing explanation
Fig. 1-a is the IGD carbon-13 nmr spectra finger-print of nim oil product A in embodiment 1;
Fig. 1-b is that the local, IGD carbon-13 nmr spectra Fingerprints peak of nim oil product A in embodiment 1 widens enlarged drawing;
Fig. 2-a is the IGD carbon-13 nmr spectra finger-print of nim oil product B in embodiment 2;
Fig. 2-b is that the local, IGD carbon-13 nmr spectra Fingerprints peak of nim oil product B in embodiment 2 widens enlarged drawing;
Fig. 3-a is the IGD carbon-13 nmr spectra finger-print of nim oil products C in embodiment 3;
Fig. 3-b is that the local, IGD carbon-13 nmr spectra Fingerprints peak of nim oil products C in embodiment 3 widens enlarged drawing.
Embodiment
Below by way of specific embodiment, technical scheme of the present invention is described, but protection scope of the present invention is not limited thereto.
one, nim oil IGD carbon-13 nmr spectra finger-print research
(1) acquisition of fatty acid characteristic extract (CET)
Directly get nim oil as the feature extraction thing becoming grouping containing fatty acid.
(2) the IGD carbon-13 nmr spectra finger-print of fatty acid characteristic extract detects
Get fatty acid characteristic extract 100mg, be dissolved in 0.5mLCDCl
3in, make IGD carbon-13 nmr spectra finger-print and detect, obtain fatty acid IGD carbon-13 nmr spectra finger-print.
(3) the IGD carbon-13 nmr spectra finger-print of fatty acid characteristic extract is resolved
1. differentiate
In fatty acid characteristic extract I GD carbon-13 nmr spectra finger-print, clearly illustrate the characteristic signal of fatty acid compound.
2. each active component characteristic peak in fatty acid characteristic extract is chosen
Owing to containing a series of fitter acids and its ester compounds in fatty acid characteristic extract, carbon peak intersects more.In order to measure the ratio of each active component, respective peaks that chemical shift difference is larger must be selected as characteristic peak., investigate through reality for this reason, select δ
cone group of C-1 carbonyl carbon peak of about 170.0-185.0 is as this specific characteristic peak, active component carbon peak, and its reason is C-1 is carbonyl carbon, comparatively large, easy to identify with other carbon chemical shifts difference; Between different Compound C-1 carbonyl carbon peak, chemical shift also has certain difference.
3. standard is with reference to the selection of product
Triglyceride oleate is one of main active of liposoluble constituent in fatty acid characteristic extract, and its carbonyl carbon chemical shift is about δ
c173.2 (C-1''), 173.1 (C-1'), 172.8 (C-1), δ
c172.8 (C-1) do not have overlapping with other main fatty acid component at this.After triglyceride oleate saponification process, the overwhelming majority becomes methyl oleate, because select methyl oleate as standard with reference to product.
(4) GC method is adopted to measure the content of oleic acid in nim oil
1) chromatographic condition
With TC-FFAP quartz capillary column (thickness 0.4 μM, 600 × 0.25mm) post, column temperature scope 60 ~ 220 DEG C, 3 DEG C/min of temperature programmes, 220 DEG C of stable 30min, injection port and detector temperature are 220 DEG C.Use helium as carrier gas, flame ionization ditector detects.Test the nim oil through the direct esterification of 0.4mol/lKOH methanol solution, using methyl oleate as reference.
2) triglyceride oleate absolute content calculates
1. methyl oleate mass concentration in need testing solution is calculated by following formula:
C
x: methyl oleate mass concentration (ug/mL) in nim oil need testing solution;
C
r: standard is with reference to product solution methyl oleate mass concentration (ug/mL);
A
x: methyl oleate peak area in the nim oil need testing solution measured by GC;
A
r: the standard measured by GC is with reference to product solution methyl oleate peak area.
2. methyl oleate mass percentage in nim oil is calculated by following formula:
W'
methyl oleate(%): methyl oleate mass percentage in nim oil;
C
x: methyl oleate mass concentration (ug/mL) in nim oil need testing solution;
M
test sample: the nim oil need testing solution quality (mg) taken.
3. triglyceride oleate mass percentage calculates
The existence form of oleic acid in nim oil is triglyceride oleate, and after adding the esterification of KOH methanol solution, existence form is methyl oleate.Therefore, the content recording methyl oleate in the need testing solution after esterification is triglyceride oleate mass percentage in nim oil.
(5) by each main active content and total amount of fatty acid in coupling formulae discovery nim oil, i.e. the content of fatty acid active component group
W
1: the nim oil Plays that GC method measures is with reference to the mass percentage of product triglyceride oleate;
M
1: standard is with reference to the carbon number that molecular weight/quantitatively peak is corresponding of product triglyceride oleate;
H
1: standard is with reference to the characteristic peak peak intensity (peak height) of product triglyceride oleate;
W
n: the mass percentage of a certain fatty acid active component in the nim oil that GC method measures;
M
n: the carbon number that molecular weight/quantitatively peak is corresponding of a certain fatty acid active component in nim oil;
H
n: the characteristic peak peak intensity (peak height) of a certain fatty acid active component in nim oil.
two, instrument, reagent and material
Key instrument and equipment
Nuclear magnetic resonance spectrometer BrukerDPX400 type.
Gas chromatograph: Shimadzu GC-2010 (FID hydrogen flame ionization detector) type.
Nim oil product A (Yunnan Nanbao Biotechnology Co., Ltd.), nim oil product B (bright Neem industry exploitation incorporated company), nim oil products C (Acetar Bio-tech Inc.); Methyl oleate, chemical reference substance (Sen Beijia bio tech ltd, Nanjing); Reagent: methyl alcohol (chromatographically pure, Tianjin Siyou Fine Chemicals Co., Ltd.), methyl alcohol (analyzing pure, Tianjin Chemical Reagents Factory No.1).
three, fatty acid carbons modal data
Triglyceride oleate (Glyceroltrioleate) C
57h
104o
6
13CNMR(CDCl
3,100MHz)δ
C:173.28(C-1',1''),172.83(C-1),130.20,129.67,68.85,62.07,34.17,34.03,32.40,31.90,31.51,30.41,29.68,29.32,29.50,29.31,29.10，27.17,25.60,24.84,22.67,14.09
。
Monoolein (Glycerololeate) C
21h
40o
4
13CNMR(75MHz,CDCl
3)δ
C:173.14(C-1),33.88(C-2),24.42(C-3),29.10(C-4),29.61(C-5),29.54(C-6),29.30(C-7),27.21(C-8),130.51(C-9),128.89(C-10),24.77(C-11),29.31(C-12),29.55(C-13),26.77(C-14),29.31(C-15),31.87(C-16),22.66(C-17),14.08(C-18),68.83(C-a),62.04(C-b,C-c)
。
Linoleic acid glyceryl ester (Glyceroltrilinolet) C
57h
98o
6
13CNMR(150MHz,CDCl
3)δ
C:173.16(C-1''),173.05(C-1'),172.75(C-1),34.11(C-2'')，34.92（C-2'）,33.86(C-2),24.81(C-3''),24.41(C-3',C-3),29.29(C-4,C-4',C-4''),29.61(C-5,C-5',C-5''),29.53(C-6,C-6',C-6''),29.64(C-7,C-7',C-7''),27.20(C-8'',C-8'),27.13(C-8),130.01(C-9''),129.89(C-9',C-9),128.01(C-10''),127.82(C-10',C-10),25.56(C-11''),24.81(C-11',C-11),128.87(C-12,C-12',C-12''),130.46(C-13,C-13',C-13''),26.74(C-14,C-14',C-14''),29.53(C-15,C-15',C-15''),31.87(C-16,C-16''),31.47(C-16),22.64(C-17'',C-17'),22.52(C-17),14.05(C-18'',C-18'),14.01(C-18),68.82(C-a),62.04(C-b,C-c)
。
Monopalmitin (Glycerylmonopalmitate) C
19h
38o
4
13CNMR(100MHz,CDCl
3)δ
C:65.2(C-a),70.4(C-b),63.4(C-c),174.4(C-1),34.2(C-2),31.9(C-3),29.7-29.1(C-4toC-13),24.9(C-14),22.7(C-15),14.0(C-16).
。
Tristerin (glycerol-1-ocatadecanoate) C
21h
42o
4
13CNMR(CDCl
3,)δ
C:65.10(C-a),70.29(C-b),63.50(C-c),174.48(C-1),32.03(C-2),22.78(C-3),29.46(C-4),29.81(C-5～C-15),22.78(C-17),14.18(C-18)
。
2-stearic acid-4-hexadecylic acid-2,4-diamyl ester (2-Octadecanoicacid-4-palmiticacid-2,4-pentanediylester) C
39h
76o
4
13CNMR(CDCl
3,50MHz)δ
C:14.03(C-a),68.33(C-b),31.84(C-c),68.33(C-d),14.03(C-e),173.83(C-1,1'),64.95(C-2,2'),34.02(C-3,3'),24.81(C-4,4'),29.04-29.61(C-5,5'),22.61(C-6,6'),14.03(C-7,7')
。
18 carbonic acid-tetrahydrofuran-3,4-diester (Octadecanoicacid-tetrahydrofuran-3,4-diylester) C
40h
76o
5636
13CNMR(CDCl
3,50MHz)δ
C:65.05(C-b,e),68.45(C-c,d),173.89(C-1,1'),34.11(C-2,2'),24.90(C-3,3'),29.13(C-4,4'),31.92(C-5,5'),22.68(C-6,6'),14.09(C-7,7')
。
Ethyl palmitate (ethylpalmitate) C
18h
36o
2
13CNMR(CDCl
3,100MHz)δ
C:173.33(C-1),29.71(C-2～C-12),29.38(C-13),31.94(C-14),22.17(C-15),14.13(C-16),66.10(OCH
2)
。
Methyl hexadecanoate (methylpalmitate) C
17h
34o
2
13CNMR(CDCl
3,125MHz)δ
C：55.5(OCH
3),174.1(C-1),31.9(C-2),29.4(C-3to14),22.7(C-15),14.1(C-16)
Methyl arachidate (MethylArachidate) C
21h
42o
2
13CNMR(CDCl
3,100MHz)δ
C：174.5(C-1),51.5(OCH
3),34.0(C-2),24.9(C-3),29.0-29.7(C-4-C-17),31.9(C-18),22.7(C-19),14.1(C-20)
。
Lignoceric acid methyl esters (Lianocericacidmethylester) C
25h
50o
2
13CNMR(CDCl
3,100MHz)δ
C：174.3（C-1）,51.4(OCH
3),34.1(C-2)，31.9（C-3）,29.4(C4-C21),24.9(C-22),22.7(C-23),14.1(C-24)
。
Heptadecanoic acide (heptadecanoicacid) C
17h
34o
2
13CNMR(CDCl
3,150MHz)δ
C:179.92(C-1),34.02(C-2),24.67(C-3),29.68,29.68,29.66,29.65,29.63,29.58,29.42,29.35,29.23,29.05(C-4-C-14),31.92(C-15),22.68(C-16),14.10(C-17)
。
Stearic acid (octadecanoid acid, Stearicacid) C
18h
36o
2
13CNMR(CDCl
3,100MHz)δ
C:180.1(C-1),34.2(C-2),24.7(C-3),29.2(C-4),29.7(C-6～C-14),31.9(C-16),22.7(C-17),14.4(C-18)
。
Palmitic acid (palmitic acid, palmiticacid) C
16h
32o
2
13CNMR(CDCl
3,100MHz)δ
C:179.5(C-1),29.0-29.5(C-2～C-13)
12,31.9(C-14),22.7(C-15),14.1(C-16)
。
embodiment 1: the IGD carbon-13 nmr spectra coupling finger-print of nim oil product A
(1) feature extraction thing preparation
Select nim oil product A directly as fatty acid characteristic extract.
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get fatty acid characteristic extract 100mg, be dissolved in 0.5mLCDCl
3in, make IGD carbon-13 nmr spectra and detect, both obtain nim oil fatty acid feature extraction thing IGD carbon-13 nmr spectra finger-print.
(3) nim oil fatty acid 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 fatty acid characteristic extract of nim oil product A, clearly illustrate the characteristic signal of fatty acid compound.Fatty acid compound triglyceride oleate, linoleic acid glyceryl ester, 18 carbonic acid-tetrahydrofuran-3,4-diester, 1.2-stearic acid-4-hexadecylic acid-2,4-diamyl ester, palmitic acid, stearic acid 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) in fatty acid characteristic extract, each active component ratio measuring result is as follows:
3) nim oil product
ait is as follows that middle triglyceride oleate mass percentage measures (GC method) result:
4) nim oil product
amiddle fatty acid active component mass percentage measurement result is as follows:
embodiment 2: the IGD carbon-13 nmr spectra-GC coupling finger-print of nim oil product B
(1) feature extraction thing preparation
Select nim oil product B directly as fatty acid characteristic extract.
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get fatty acid characteristic extract 100mg, be dissolved in 0.5mLCDCl
3in, make IGD carbon-13 nmr spectra and detect, obtain fatty acid characteristic extract I GD carbon-13 nmr spectra finger-print.
(3) fatty acid characteristic extract I GD 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 fatty acid characteristic extract of nim oil product B, clearly illustrate the characteristic signal of fatty acid compound.Fatty acid compound triglyceride oleate, linoleic acid glyceryl ester, 18 carbonic acid-tetrahydrofuran-3,4-diester, 1.2-stearic acid-4-hexadecylic acid-2,4-diamyl ester, palmitic acid, stearic acid 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) in fatty acid characteristic extract, each active component ratio measuring result is as follows:
3) in nim oil product B, triglyceride oleate mass percentage mensuration (GC method) result is as follows:
4) in nim oil product B, fatty acid active component mass percentage measurement result is as follows:
embodiment 3: the IGD carbon-13 nmr spectra-GC coupling finger-print of nim oil products C
(1) feature extraction thing preparation
Select nim oil products C directly as fatty acid characteristic extract.
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get fatty acid characteristic extract 100mg, be dissolved in 0.5mLCDCl
3in, make IGD carbon-13 nmr spectra and detect, obtain fatty acid characteristic extract I GD carbon-13 nmr spectra finger-print.
(3) fatty acid characteristic extract I GD 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 fatty acid characteristic extract of nim oil products C, clearly illustrate the characteristic signal of fatty acid compound.Fatty acid compound triglyceride oleate, linoleic acid glyceryl ester, 18 carbonic acid-tetrahydrofuran-3,4-diester, 1.2-stearic acid-4-hexadecylic acid-2,4-diamyl ester, palmitic acid, stearic acid 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 fatty acid characteristic extract, each active component ratio measuring result is as follows:
3) in nim oil products C, triglyceride oleate mass percentage mensuration (GC method) result is as follows:
4) in nim oil products C, fatty acid active component mass percentage measurement result is as follows:
Claims (5)
1. differentiate a method for nim oil, it is characterized in that, comprise the following steps:
1) nim oil or the direct feature extraction thing as becoming grouping containing fatty acid of nim oil spin-off is got;
2) carry out IGD carbon-13 nmr spectra finger-print to described feature extraction thing to detect, obtain several active component characteristic peak peak intensities in feature extraction thing according to finger-print; And determine the characteristic peak peak intensity of each described active component respective standard with reference to product by same way;
3) absolute content of described standard with reference to product is obtained by quantitative test means;
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 the content of each active component and the content of active component group in nim oil or nim oil spin-off;
Step 2) in, the active component characteristic peak in feature extraction thing is carbonyl absorption peak, and its chemical shift is δ
c170.0-185.0.
2. differentiate the method for nim oil according to claim 1, it is characterized in that, step 2) in, carry out IGD carbon-13 nmr spectra finger-print to feature extraction thing and detect, the solvent of dissolving characteristic extract is deuterochloroform; The mass volume ratio of feature extraction thing and described solvent is 190mg:1ml-210mg:1ml.
3. differentiate the method for nim oil according to claim 1, it is characterized in that, in step 3), described quantitative test means are vapor-phase chromatography; 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.
4. differentiate the method for nim oil according to claim 1, it is characterized in that, the standard of the active component in feature extraction thing is methyl oleate with reference to product.
5. according to Claims 1-4 any one, differentiate the method for nim oil, it is characterized in that, in step 4), the coupling formula calculating each active component content is:
; Wherein:
W
1in the nim oil measured for step 3) quantitative test means or nim oil spin-off, standard corresponding to a certain active component is with reference to the absolute content of product;
M
1for standard corresponding to a certain active component in described nim oil or nim oil 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 nim oil of IGD carbon-13 nmr spectra determining fingerprint pattern or nim oil spin-off with reference to product;
W
nfor the mass percentage of a certain active component in nim oil or nim oil spin-off;
M
nfor the carbon number that molecular weight/quantitatively peak is corresponding of a certain active component in nim oil or nim oil spin-off;
H
nfor the characteristic peak peak intensity by a certain active component in the nim oil of IGD carbon-13 nmr spectra determining fingerprint pattern or nim oil spin-off.
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| CN101602753A (en) * | 2008-06-13 | 2009-12-16 | 四川农业大学 | Ester compound and its production and application in the nim oil |
| CN102749348A (en) * | 2012-06-18 | 2012-10-24 | 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 |
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| CN101602753A (en) * | 2008-06-13 | 2009-12-16 | 四川农业大学 | Ester compound and its production and application in the nim oil |
| CN102749348A (en) * | 2012-06-18 | 2012-10-24 | 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 |
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