CN102907426B - Original celastrus angulatus medicine as well as preparation method and mass detection method thereof - Google Patents
Original celastrus angulatus medicine as well as preparation method and mass detection method thereof Download PDFInfo
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- CN102907426B CN102907426B CN201210376286.4A CN201210376286A CN102907426B CN 102907426 B CN102907426 B CN 102907426B CN 201210376286 A CN201210376286 A CN 201210376286A CN 102907426 B CN102907426 B CN 102907426B
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- celastrus angulatus
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Abstract
The invention relates to original celastrus angulatus medicine, a water suspension agent of the original celastrus angulatus medicine, as well as a preparation method and a mass detection method of the original celastrus angulatus medicine and the water suspension agent. The original celastrus angulatus medicine comprises the following active ingredients by mass percentage: 2.5%-4.0% of angulatus A, 3.0%-5.0% of angulatus B, 1.5%-3.0% of angulatus C, angulatus F and angulatus P, 1.5%-3.0% of angulatus G, 0.05%-1.00% of celastrus angulatus IV, 3.5%-5.5% of angulatus A, angulatus E and celastrus angulatus XIX, and 1.0%-2.0% of angulatus H. The original celastrus angulatus medicine is prepared by a precipitant precipitating method. The mass detection method adopts an IGD (inverted gated decoupling) carbon nuclear magnetic resonance coupling fingerprint technology. The content of the original celastrus angulatus medicine is increased greatly, and the mass detection method is higher in accuracy, stability, repeatability and practicability.
Description
Technical field
The invention belongs to botanical pesticide technical field, particularly, related to the former medicine of a kind of Celastrus angulatus and aqueous suspension agent thereof, and its preparation method and quality determining method.
Background technology
Celastrus angulatus (the Celastrus angulatus MaXim) bark of also grieving, dish medicine, belong to Celastraceae celastraceae plants.In China the Changjiang river, district of each province, the Huanghe valley distribute very wide.Its root skin and leaf are used as insecticide by among the people always, also can be used as medicine and treat the swollen pain of sore for treatment, there is effects for removing toxic heat, wherein the insecticidal action of root skin best [Chinese Academy of Sciences's northwest Institute of Zoology. Qinling Mountains flora 1981, Beijing: Science Press].The insecticidal active ingredient of Celastrus angulatus is to separate a series of 4-OH-β-dihydroagarofuran polyol ester compounds that obtain, insect is had to poisoning, food refusal and anesthetic effect, can be used to prevent and treat cabbage caterpillar on vegetables, rice plant skipper, paddy rice mythimna separata, blister beetle sawfly larva, pest of stored grain corn weevil etc. [Wu Wenjun. the research of vegetable insecticide Celastrus angulatus and application [M]. Chemical Industry Press, 2010].
The extracting method of the Celastrus angulatus of bibliographical information is solvent reflux extraction at present, extracts solvent and comprises benzinum, benzene, acetone, ether, toluene, methyl alcohol, ethanol etc.; Wherein suitability for industrialized production mostly adopt benzene [red legend is beautiful, etc. the chemical composition of root bark of celastrus angulatus. Chinese experimental pharmacology of traditional Chinese medical formulae magazine, 2011,17 (13), 271-276.].Although ethanol is environmentally friendly solvent, but compared with other solvent extractable matters or former medicine, Celastrus angulatus ethanol extract (or the former medicine obtaining with alcohol extract) chemical composition is extremely complicated, various, contain other compositions such as a large amount of flavones, dulcin, triterpene, 4-OH-β-dihydroagarofuran polyol ester kind compound content very low (as Celangulin A content 0.5% left and right), dissolubility is poor, is difficult to make corresponding formulation.Therefore, Celastrus angulatus does not adopt alcohol extract substantially.
The quality of Celastrus angulatus alcohol extract does not lie in certain single-activity composition, and its effect is the result of the collaborative proportioning of multiple active components.Studies have shown that in Celastrus angulatus, main active insecticidal components is polyol ester compound and the alkaloid thereof with 4-OH-β-dihydroagarofuran sequiterpene skeleton, be referred to as Celastrus angulatus.Celastrus angulatus chemical composition is very complicated, contains taking Celangulin A, Celangulin B as main multiple insecticidal constituent.At present, the detection method of Celastrus angulatus component in Celastrus angulatus extract or former medicine: the one, measure the benzoic acid producing after Celastrus angulatus extract or former liquid medicine solution, operating procedure is extremely complicated, and result is unreliable, and feasibility is poor; The 2nd, only one of them component Celangulin A (or Celangulin V) is carried out to quantitative analysis [Wu Wenjun, Deng. the research and development .Pesticides (agricultural chemicals) of vegetable insecticide 0.2% Celastrus angulatus missible oil, 2001,40 (3): 17; 2. Lu Gui honor, measure the content of Celangulin A in Celastrus angulatus Deng .RP-HPLC method. Henan science, 2007,25(3): 395.], this quality testing pattern of carrying out quantitative analysis for single component can not effectively be controlled the inherent quality of Celastrus angulatus extract, cannot meet current for the objective an urgent demand of effectively evaluating and controlling Celastrus angulatus product quality.Fingerprint pattern technology oneself become internationally recognized difference evaluate the most effective means of natural plant product and raw material thereof [Luo Guoan, etc. the classification of traditional Chinese medicine fingerprint and development. Chinese Journal of New Drugs, 2002,11 (1): 46.].Have no report about product and the finger-print detection thereof of Celastrus angulatus extract or former medicine at present.
IGD carbon-13 nmr spectra coupling (IGD
13c NMR coupling) fingerprint pattern technology, be also inverted gated decoupling carbon-13 nmr spectra coupling fingerprint pattern technology, be study proton nmr spectra for many years (
1h NMR) fingerprint pattern technology [Zhao Tianzeng, etc.
1hNMR fingerprint technique plant identification Chinese medicine. Chinese herbal medicine, 2000,31 (11): 868-870.] on basis, combine a kind of new comprehensive fingerprint pattern technology of non-single means that other technologies (for example current most widely used high efficiency liquid phase (HPLC) fingerprint pattern technology) propose.
Therefore, if can develop the former medicine product of the Celastrus angulatus making new advances, and develop the method for utilizing IGD carbon-13 nmr spectra coupling fingerprint pattern technology to detect the relevant former medicine product of Celastrus angulatus, not only can solve the wherein qualitative question of active component, also for strengthening systematization and the standardization of its inherent composition Study, accelerate the modern development of botanical pesticide Celastrus angulatus, realize the guarantee that science is provided in line with international standards.
Summary of the invention
The problem existing in order to solve the aspects such as botanical pesticide prior art, quality, the object of the invention is to, and the former medicine of a kind of alcohol extracting Celastrus angulatus and aqueous suspension agent thereof are provided.
Another object of the present invention is to, the preparation method of the former medicine of a kind of described Celastrus angulatus and aqueous suspension agent thereof is provided.
Another object of the present invention is to, the quality determining method of the former medicine of a kind of described Celastrus angulatus and aqueous suspension agent thereof is provided.
For achieving the above object, the former medicine of a kind of Celastrus angulatus provided by the invention, wherein contain the active component of following quality percentage composition: Celangulin A2.5 ~ 4.0%, Celangulin B3.0 ~ 5.0%, Celangulin C, Celangulin F and Celangulin P1.5 ~ 3.0%, Celangulin G1.5 ~ 3.0%, Celastrus angulatus IV0.05 ~ 1.00%, Celangulin A, Celangulin E and Celastrus angulatus XIX3.5 ~ 5.5%, Celangulin H1.0 ~ 2.0%.
Described active component in the former medicine of Celastrus angulatus of the present invention is 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound.
Wherein, the quality percentage composition of Celangulin C is 0.5 ~ 2.0%, the quality percentage composition of Celangulin F is 0.5 ~ 1.0%, the quality percentage composition of Celangulin P is 0.05 ~ 0.75%, the quality percentage composition of Celangulin E is 0.4 ~ 1.0%, the quality percentage composition of Celastrus angulatus XIX is 0.2 ~ 0.7%, and the quality percentage composition of Celastrus angulatus III is 0.5 ~ 1.0%, and the quality percentage composition of Celangulin J is 0.3 ~ 2.5%.
The total quality percentage composition of each active component in the former medicine of Celastrus angulatus of the present invention (4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound) is not less than 13%.Preferably 15%.
Further, in the former medicine of Celastrus angulatus, the total quality percentage composition of each active component is 15.25 ~ 17.05%.
The former medicine of Celastrus angulatus of the present invention is solid shape.
The preparation method of the former medicine of Celastrus angulatus provided by the invention comprises: take Celastrus angulatus root or/and skin, pulverize, be that the ethanol of 2 ~ 6 times refluxes or ultrasonic extraction 2 ~ 3 times at 60 ~ 80 DEG C with volume, extract merging filtrate after filtering at every turn 1 ~ 2 hour, reduced pressure concentration becomes filtrate, get this filtrate, add volume and be the precipitating reagent of 1 ~ 2 times, precipitate 12 ~ 24 hours, after centrifugal, be precipitated, i.e. the former medicine of described Celastrus angulatus.
Further, Celastrus angulatus root is or/and skin is crossed 10 ~ 24 mesh sieves after pulverizing
Wherein, the mass ratio of described ethanol is 90 ~ 95%.
Further, the density of concentrated filtrate is 1.0 ~ 1.2, preferably 1.08 ~ 1.13.
Wherein, described precipitating reagent is: water or be 5 ~ 30% ethanol, preferably 10 ~ 30% ethanol.The percentage " % " is here mass ratio.
Celastrus angulatus root is or/and the active component in skin, except the composition described in the former medicine of Celastrus angulatus of the present invention, also comprises Celangulin U, X, Celastrus angulatus II, Celangulin V II; The 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compounds such as celangulatinC and flavones, alkaloid, triterpene isoreactivity composition.
The former medicine of Celastrus angulatus provided by the invention, can make the various formulations such as aqueous suspension agent, wetting powder, aqueous emulsion for botanical pesticide insecticide.
A kind of Celastrus angulatus aqueous suspension agent provided by the invention, comprises the former medicine of described Celastrus angulatus.
Described Celastrus angulatus aqueous suspension agent provided by the invention, comprises the composition of following weight portion: 10 ~ 20 parts of the former medicines of described Celastrus angulatus, 3 ~ 7 parts of wetting dispersing agents, 2 ~ 3 parts of thickeners, 3 ~ 5 parts of antifreezing agents, 0.2 ~ 0.5 part of defoamer.
Described Celastrus angulatus aqueous suspension agent provided by the invention, preferably, comprises the composition of following weight portion: 16 ~ 17 parts of the former medicines of described Celastrus angulatus, 5.5 ~ 6 parts of wetting dispersing agents, 2.5 ~ 3.0 parts of thickeners, 4 ~ 5 parts of antifreezing agents, 0.3 ~ 0.5 part of defoamer.
Wherein, described wetting dispersing agent is selected from: sodium metnylene bis-naphthalene sulfonate (NNO), the sodium salt (MorwetD425) of alkyl naphthalene sulfonic acid condensation polymer, sodium lignin sulfonate (wooden sodium), dispersing agent MF (MF), polycarboxylic acid salt (GYD), alkylphenol polyoxyethylene sulfosuccinates (agriculture breast 2000#), calcium dodecyl benzene sulfonate (agriculture breast 500#), phenethyl phenol APEO (agriculture breast 601#), alkylaryl polyoxyethylene polyoxypropylene ether (agriculture breast 33#), one or more in phenethyl phenol polyoxyethylene poly-oxygen propylene aether (agriculture breast 1601#) and castor oil polyoxyethylene ether (BY125) etc.Preferred: sodium metnylene bis-naphthalene sulfonate, one or more in the sodium salt of alkyl naphthalene sulfonic acid condensation polymer and alkylaryl polyoxyethylene polyoxypropylene ether.
Wherein, described thickener is selected from: gum Arabic, xanthans, CMC, polyvinyl alcohol, one or more in polyvinyl pyrrolidone, aluminium-magnesium silicate and bentonite.Preferred: bentonite, one or more in xanthans and CMC.
Wherein, described antifreezing agent is selected from: ethylene glycol, propane diols, glycerine, diethylene glycol (DEG), triethylene glycol, polyethylene glycol, one or more in urea and inorganic salts.Preferred: ethylene glycol.
Wherein, described defoamer is: organosilicon ketone, C
8~ C
10aliphatic alcohols, C
10~ C
20aliphatic carboxylic acid and lipid thereof, ester-ether type compound or cocoa butter EO-PO polymer etc.Preferred: organosilicon.
A kind of Celastrus angulatus aqueous suspension agent provided by the invention, also comprises solvent.
Wherein, solvent is water, adds water to 100%.
Celastrus angulatus aqueous suspension agent of the present invention, auxiliary material wherein, i.e. wetting dispersing agent, thickener, antifreezing agent, defoamer and solvent, can be also conventional kind and the consumption of this area.
Total quality percentage composition of each active component in Celastrus angulatus aqueous suspension agent of the present invention (4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound) is not less than 2%.
Further, in Celastrus angulatus aqueous suspension agent, the total quality percentage composition of each active component is 2.3 ~ 3.0%.
Further, in the former medicine of Celastrus angulatus that kind is Baoji, the total quality percentage composition of active component is 2.5 ~ 2.9%, and kind is that in the former medicine of the Celastrus angulatus of Hua County, Shaanxi, the total quality percentage composition of active component is 2.6 ~ 3.0%.
The preparation method of described Celastrus angulatus aqueous suspension agent provided by the invention, comprising: former described Celastrus angulatus medicine is mixed in described ratio with wetting dispersing agent, thickener, antifreezing agent, defoamer and solvent, to obtain final product.
The quality determining method of the former medicine of described Celastrus angulatus provided by the invention, comprises the following steps:
1) the former medicine of described Celastrus angulatus is processed, obtained the former medicine feature extraction of the Celastrus angulatus thing that contains active component group;
2) the former medicine feature extraction of described Celastrus angulatus thing is carried out to IGD carbon-13 nmr spectra finger-print and detect, obtain several active component characteristic peak peak intensities in the former medicine feature extraction of described Celastrus angulatus thing according to finger-print; And determine the characteristic peak peak intensity of the corresponding standard of described each active component with reference to product by same way (IGD carbon-13 nmr spectra finger-print);
3) measure and obtain standard described in the former medicine of Celastrus angulatus with reference to the absolute content of product by quantitative analysis means;
4) utilize ratio and the described absolute content of described characteristic peak peak intensity (each active component characteristic peak peak intensity and corresponding standard are with reference to the characteristic peak peak intensity of product), calculate the content of each active component and the total content of this active component, the i.e. content of active component group in the former medicine of Celastrus angulatus.
Wherein, in step 1), the processing method of the former medicine feature extraction of Celastrus angulatus thing, comprise: take the former medicine of described Celastrus angulatus, adding volume is the chloroform of 6 ~ 12 times of amounts, reflux or ultrasonic extraction 20 ~ 40min at 60 ~ 80 DEG C, after filtration at 60 ~ 70 DEG C reduced pressure concentration, reclaim solvent to dry, obtain the former medicine feature extraction of Celastrus angulatus thing.
The former medicine feature extraction of Celastrus angulatus object detecting method is as follows: get the former medicine feature extraction of Celastrus angulatus thing, be dissolved in CDCl
3in, make IGD carbon-13 nmr spectra finger-print and detect.
Wherein, step 2) in, the active component characteristic peak in the former medicine feature extraction of described Celastrus angulatus thing is: C-15, its chemical shift is δ
c60.0 ~ 66.0.
Further, select C-9 and/or C-1 further to distinguish Celangulin C, Celangulin F and Celangulin P as characteristic peak, its (C-9 or C-1) chemical shift is 70.6 ~ 72.6.
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 standard refers to reference to the absolute content of product: the former medicine Plays of Celastrus angulatus of measuring by quantitative analysis means is with reference to the quality percentage composition of product.
Wherein, described quantitative analysis means are high performance liquid chromatography (HPLC) method.
Further, the condition of HPLC method is: chromatographic column is taking octadecyl silane as filler, mobile phase is methyl alcohol: acetonitrile: water=(35 ~ 45): (15 ~ 25): the mixed solvent of (35 ~ 45), and flow velocity is 1mL/min, detection wavelength is 242nm.Methyl alcohol: acetonitrile: the preferred 40:20:40 of water.
Wherein, described standard is Celangulin B with reference to product.
What the present invention mainly measured is the content of 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound in the former medicine of Celastrus angulatus.
Wherein, in the former medicine of Celastrus angulatus, the content of each active component and the calculating of the total content of this active component are by IGD carbon-13 nmr spectra and the coupling of quantitative analysis means by coupling computing formula, be in step 4), the coupling formula that calculates the content of each active component is:
W
1the standard that in the former medicine of Celastrus angulatus of measuring by quantitative analysis means for step 3), a certain active component is corresponding is with reference to the absolute content (quality percentage composition) of product;
M
1for standard corresponding to described a certain active component is with reference to the molecular weight of product;
H
1for standard corresponding to a certain active component described in the former medicine feature extraction of the Celastrus angulatus thing by IGD carbon-13 nmr spectra determining fingerprint pattern is with reference to the characteristic peak peak intensity of product;
W
nfor the quality percentage composition of a certain active component in the former medicine of Celastrus angulatus;
M
nfor the molecular weight of a certain active component;
H
nfor the characteristic peak peak intensity of a certain active component in the former medicine feature extraction of the Celastrus angulatus thing by IGD carbon-13 nmr spectra determining fingerprint pattern.
Also can calculate coefficient and the overall coefficient of main active in the former medicine of Celastrus angulatus by coupling formula.
Further, the computing formula of described coefficient is:
Wherein Fn is standard that in the former medicine of Celastrus angulatus, a certain active component is corresponding with it ratio coefficient with reference to product quality percentage composition.M
1, h
1, M
nand h
nimplication with calculating the corresponding definition in each active component content coupling formula in Celastrus angulatus former medicine.
This coefficient Fn is applicable to calculate active component and the active component group in Celastrus angulatus aqueous suspension agent too.
The quality determining method of Celastrus angulatus aqueous suspension agent provided by the invention, utilizes said method to detect and obtains the content of certain each active component in the former medicine of Celastrus angulatus, then extrapolate the quality percentage composition of each active component in Celastrus angulatus aqueous suspension agent.The Celastrus angulatus aqueous suspension agent is here to be prepared by the former medicine of Celastrus angulatus.
Wherein, in Celastrus angulatus aqueous suspension agent, the cubage formula of each active component is:
X
n=X
1fn; Wherein:
X
nfor a certain active component quality percentage composition in Celastrus angulatus aqueous suspension agent;
X
1for standard that in the Celastrus angulatus aqueous suspension agent of measuring by quantitative analysis means, a certain active component is corresponding is with reference to the absolute content (quality percentage composition) of product;
Wherein, described quantitative analysis means are high performance liquid chromatography.
In the former medicine of Celastrus angulatus or Celastrus angulatus aqueous suspension agent, the total content of this active component is exactly the X of similar each active component
nsum, i.e. the content of active component group, single reactive compound coefficient sum is Celastrus angulatus overall coefficient.
Active component group described in the inventive method, is after Celastrus angulatus medicinal material is extracted, the active component group in the former medicine of Celastrus angulatus obtaining, and in fact the former medicine of described Celastrus angulatus is exactly Celastrus angulatus extract.
Quality determining method of the present invention can be used for detecting 13%, 15% or the former medicine of Celastrus angulatus of other concentration, and the present invention 2% Celastrus angulatus aqueous suspension agent or the Celastrus angulatus aqueous suspension agent of other concentration.
Active component group described in the inventive method, after especially Celastrus angulatus medicinal material being extracted, the active component group in the former medicine of Celastrus angulatus and the Celastrus angulatus aqueous suspension agent obtaining.
Celastrus angulatus medicinal material of the present invention, refers to that the root of Celastrus angulatus plant is or/and the position of skin.
The calculating of the content of the each active component of the present invention and the total content of this active component is by IGD carbon-13 nmr spectra and the coupling of analysis quantitative means by coupling formula.Compared to the prior art, the present invention adopts IGD
13c NMR coupling finger-print has several features below:
1. stability (repeatability): IGD
13the chemical shift data that C NMR obtains is second after decimal point, and explanation property is good, reproducible; Non-chromatographic condition (as chromatographic column internal diameter, length, the fixing phase trade mark, carrier granularity, flow rate of mobile phase, the mutually each component ratio of mixed flow, column temperature, sample size, the detector sensitivity etc.) change of HPLC, GC etc., the retention time data variation obtaining is very large, mean the variation of monolithic chromatogram figure, repeatability is bad.
2. globality (comprehensive): IGD
13in C NMR finger-print, comprise the corresponding spectrum peak of each the active component carbon in sample; There is not this relation in HPLC, GC, UV, IR, MS.
3. reliability (unicity): IGD
13c NMR spectrum peak and the carbon on different activities composition in sample and different group thereof are strict one-to-one relationships; There is not this relation in HPLC, GC, UV, IR, MS.
4. feasibility (the easily property distinguished): IGD
13c NMR finger-print regularity is very strong, generally, can belong to each the carbon peak in collection of illustrative plates; HPLC, GC need reference substance; IR is difficult for resolving; UV information content is few; MS has the problem such as degree of ionization and matrix interference.
The present invention gos deep into Celastrus angulatus alcohol extract, systematic research, find a series of active insecticidal components and determined its chemical constitution, and adopt the prior art active insecticidal components that all there is no the enrichment of the used precipitating reagent precipitation method, compare with the former medicine of current existing Celastrus angulatus, the Celastrus angulatus ethanol extract obtaining or former medicine Celastrus angulatus content are higher, and (wherein effective active composition is 12 kinds, its total amount is not less than 13%, especially in 15%(the present invention, represent with 15%T12), Celangulin A content is greater than 2.5%, Celangulin B content is greater than 3.0%, Celangulin G content is greater than 1.5%, and impurity content is few.Greatly reduce with respect to toxicity such as Celastrus angulatus benzene extractiveses, be easy to be prepared to follow-up formulation products.And technique of the present invention has the features such as equipment is simple, index is controlled, easy to operate, cost is low, and yield is high, non-environmental-pollution, be applicable to suitability for industrialized production.
Aqueous suspension agent of the present invention advantage is compared with prior art: aqueous suspension agent is as the representative of the water base property formulations of pesticide, compared with existing formulation, reduce consumption of organic solvent, use and store and transport safe ready, Environmental compatibility is good, and drug effect is high, and cost is low, having vast potential for future development, is the focus of formulations of pesticide research both at home and abroad.
The present invention is directed to the former medicine of Celastrus angulatus (being Celastrus angulatus extract), especially ethanol extract active ingredient is few, impurity is many, and the limitation of efficient liquid-phase chromatograph finger print atlas and proton nmr spectra (1HNMR) finger-print, IGD carbon-13 nmr spectra coupling finger-print is studied in great detail, the technical scheme of determining can qualitatively reflect in the former medicine of Celastrus angulatus, mainly to contain which 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound, and can reflect their content separately, proportionate relationship and total content, reach the object to the former medicine quality testing of Celastrus angulatus and control.Stability and veracity, repeatability and feasibility compared with prior art improve a lot.
In a word, the invention enables that the former medicine definite ingredients of Celastrus angulatus, content are clear, quality controllable, stable performance, can effectively control the inherent quality of the former medicine of Celastrus angulatus, meet currently for the objective an urgent demand of effectively evaluating and controlling Celastrus angulatus product quality, strengthened systematization and the standardization of the inherent composition Study of the former medicine of Celastrus angulatus.
Brief description of the drawings
Fig. 1-a is the former medicine first of the 15%T12 Celastrus angulatus feature extraction thing IGD carbon-13 nmr spectra finger-print of embodiment 1.
Fig. 1-b is that the part, the former medicine first of 15%T12 Celastrus angulatus feature extraction thing IGD carbon-13 nmr spectra Fingerprints peak of embodiment 1 widens enlarged drawing.
Fig. 2-a is the former medicine first of the 15%T12 Celastrus angulatus feature extraction thing IGD carbon-13 nmr spectra finger-print of embodiment 2.
Fig. 2-b is that the part, the former medicine first of 15%T12 Celastrus angulatus feature extraction thing IGD carbon-13 nmr spectra Fingerprints peak of embodiment 2 widens enlarged drawing.
Fig. 3-a is the former medicine second of the 15%T12 Celastrus angulatus feature extraction thing IGD carbon-13 nmr spectra finger-print of embodiment 3.
Fig. 3-b is that the part, the former medicine second of 15%T12 Celastrus angulatus feature extraction thing IGD carbon-13 nmr spectra Fingerprints peak of embodiment 3 widens enlarged drawing.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in further detail, but protection scope of the present invention is not limited to this.
1, the preparation method of the former medicine of 15%T12 Celastrus angulatus, aqueous suspension agent
(1) the former medicine of 15%T12 Celastrus angulatus
Take Celastrus angulatus root or/and skin, pulverize (10 ~ 24 mesh sieve), at 60 ~ 80 DEG C, reflux or ultrasonic extraction 2 ~ 3 times with the ethanol (mass ratio 90 ~ 95%) that volume is 2 ~ 6 times, extract 1 ~ 2 hour at every turn, merging filtrate after filtering, reduced pressure concentration becomes filtrate (density is 1.08 ~ 1.13), gets this filtrate, adds its volume and be the precipitating reagent (water or be 5 ~ 30% ethanol (mass ratioes) of 1 ~ 2 times, preferably 10 ~ 30% ethanol), precipitate 12 ~ 24 hours, be precipitated the i.e. former medicine of described Celastrus angulatus after centrifugal.
(2) 15%T12 Celastrus angulatus aqueous suspension agent
Comprise the composition of following weight portion: 10 ~ 20 parts of the former medicines of described Celastrus angulatus, 3 ~ 7 parts of wetting dispersing agents, 2 ~ 3 parts of thickeners, 3 ~ 5 parts of antifreezing agents, 0.2 ~ 0.5 part of defoamer.Mix in proportion, to obtain final product.
2, the former medicine IGD of 15%T12 Celastrus angulatus carbon-13 nmr spectra finger print quality detecting method research step
(1) feature extraction thing obtains program research
Take the former medicine 1 ~ 2g of Celastrus angulatus, adding volume is the chloroform of 6 ~ 12 times of amounts (mL), reflux or ultrasonic extraction 20 ~ 40min at 60 ~ 80 DEG C, after filtration at 60 ~ 70 DEG C reduced pressure concentration, reclaim solvent to dry, obtain the former medicine feature extraction of 15%T12 Celastrus angulatus thing.
(2) feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get the former medicine feature extraction of above-mentioned 15% Celastrus angulatus thing, be dissolved in 0.5mL CDCl
3in, make IGD carbon-13 nmr spectra finger-print and detect, obtain IGD carbon-13 nmr spectra finger-print.
(3) feature extraction thing IGD carbon-13 nmr spectra finger-print is resolved
1) differentiate
In feature extraction thing IGD carbon-13 nmr spectra finger-print, should clearly illustrate the characteristic signal of 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound, and all or major part contain Celangulin A, E, H, Celastrus angulatus XIX; Celangulin B, C, F, G, P, Celastrus angulatus IV; Celangulin J, Celastrus angulatus III signal.
Concrete data are as follows:
δ
c74.9-75.5 or 70.5-70.9 or 75.8-76.0,66.9-68.1,41.1-42.1,72.1-72.2 or 69.7-69.9,91.3-91.7 is respectively A ring 1,2,3,4,5 alicyclic ring carbon signals, 76.2-77.0 or 75.0-75.6, 53.0-53.8, 73.3-74.7 or 75.6-76.5 or 69.7-69.8, 74.9-75.5 or 70.6-72.6, 50.1-50.8 or 53.9-54.5 or 52.9-53.0 are respectively B ring 6, 7, 8, 9, 10 alicyclic ring carbon signals, 82.5-84.6 be 4-OH-beta-dihydroagarofuran sesquiterpene polyolester Compound C-11 carbon signals, 29.3-30.1, 26.2-26.4 or 25.4-25.7 or 24.3-24.4, 24.1-24.6 is 4-OH-beta-dihydroagarofuran sesquiterpene polyolester Compound C-12, C-13, C-14 methyl carbon signal, 61.4-61.8 or 65.0-65.7 or 60.2-60.7 are 4-OH-beta-dihydroagarofuran sesquiterpene polyolester Compound C-15 mesomethylene carbon signals.
2) in the former medicine feature extraction of 15%T12 Celastrus angulatus thing, each active component characteristic peak is chosen owing to containing a series of active component 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compounds in feature extraction thing, carbon peak intersects morely, in order to measure the ratio of each active component, must select respective peaks that chemical shift difference is larger as characteristic peak., investigate through reality for this reason, selected δ
c60.0-66.0 C-15 peak, one group of left and right.Its former because: generally, C-15 peak, as connecting oxygen carbon, is easy to identification; C-15 position is subject to the γ effect of the upper α of C-9 and β bit substituent different, and its chemical shift difference is larger.If the chemical shift of Celangulin A, E, H, Celastrus angulatus XIX is δ
c61.0 left and right; The chemical shift of Celangulin B, C, F, G, P, Celastrus angulatus IV is δ
c65.0 left and right; The chemical shift of Celangulin J, Celastrus angulatus III is δ
c60.0 left and right.Alternative selects C-9 and/or C-1 further distinguishes Celangulin C, Celangulin F, Celangulin P as characteristic peak, and the chemical shift of C-9 or C-1 is δ
c70.6 ~ 72.6 left and right.
3) standard is with reference to the selection of product
Celangulin B is one of main active insecticidal components of Insecticidal Plant Celastrus Angulatus, and the chemical shift of its characteristic peak is δ
c65.4, do not have overlapping at this with other main active characteristic peaks.Therefore, select Celangulin B as standard with reference to product.
(4) adopt HPLC to measure the content of Celangulin B in the former medicine of 15%T12 Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent
1) HPLC detects
1. chromatographic condition
Instrument: Shimadzu LC-20AT
Mobile phase: (methyl alcohol: acetonitrile: water)=40:20:40
Flow velocity: 1mL/min
Chromatographic column: Sunfire C18 (4.6mm × 150mm, 5 μ are m);
Detector: ultraviolet
Wavelength: 242nm
Sample size: 20 μ L;
2. standard is with reference to the preparation of product solution
Accurately take Celangulin B5mg, put in 50mL volumetric flask, with methyl alcohol dissolved dilution, to scale, the standard that obtains after shaking up is with reference to product solution (Celangulin B100 μ g/mL).
3. calibration curve and detection limit
Concentration range: 1 ~ 100 μ g/mL(ppm); Standard is respectively with reference to product concentration: 1 μ g/mL, 5 μ g/mL, 50 μ g/mL, 100 μ g/mL.
Under above-mentioned chromatographic condition, carry out HPLC analysis, Celangulin B total peak area Y to the equation of linear regression of concentration C is: Y=12472.59*C+396.23(n=5, R=0.9999).
Detect and be limited to: 0.5ug/mL (S/N=3).
According to canonical plotting, in selected concentration range, the working curve linear relation of the standard liquid of Celangulin B is good.
4. the preparation of need testing solution
Accurately take the former medicine of 15%T12 Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent 200mg in 100mL volumetric flask, adding appropriate methyl alcohol dissolves, after sonic oscillation, be diluted to scale, after shaking up, obtain the former medicine of 15% Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent need testing solution.
5. precision is measured
Need testing solution repeats sample introduction 5 times, peak area relative standard deviation RSD=1.02%, retention time relative standard deviation RSD=0.45%.
6. the mensuration of test sample
Draw each need testing solution, sample introduction, surveys its peak area, tries to achieve Celangulin B content.
7. determination of recovery rates
Employing standard adds method, mark-on 100 μ g/mL in the former medicine test sample of Celastrus angulatus, and average recovery rate is 103.5%.
2) Celangulin B absolute content calculates
1. calculate Celangulin B mass concentration in need testing solution by following formula
C
x: Celangulin B mass concentration (ug/mL) in the former medicine of 15%T12 Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent need testing solution;
C
r: standard is with reference to product (Celangulin B) concentration of polymer solutions (ug/mL);
A
x: the peak area of Celangulin B in the former medicine need testing solution of 15%T12 Celastrus angulatus of being measured by HPLC or 2% Celastrus angulatus aqueous suspension agent;
A
r: the standard of being measured by HPLC is with reference to the peak area of Celangulin B in product solution.
2. calculate Celangulin B quality percentage composition in the former medicine of 15%T12 Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent by following formula
W
celangulin B(%): in the former medicine of 15%T12 Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent Celangulin B quality percentage composition, standard is with reference to the absolute content (quality percentage composition) of product Celangulin B;
C
x: Celangulin B mass concentration (ug/mL) in the former medicine test sample of 15% Celastrus angulatus or 2% Celastrus angulatus aqueous suspension agent solution;
M
test sample: the former medicine of 15%T12 Celastrus angulatus taking or 2% Celastrus angulatus aqueous suspension agent quality (mg).
(5) calculate main active content and total amount in the former medicine of 15%T12 Celastrus angulatus by coupling formula
W
n(%): a certain active component quality percentage composition % in the former medicine of 15%T12 Celastrus angulatus;
W
1(%): Celangulin B quality percentage composition % in the former medicine of 15%T12 Celastrus angulatus, standard is with reference to the absolute content (quality percentage composition) of product Celangulin B, i.e. W
celangulin B(%);
M
1: Celangulin B(standard is with reference to product) molecular weight;
H
1: by Celangulin B(standard in the former medicine feature extraction of the 15%T12 Celastrus angulatus thing of IGD carbon-13 nmr spectra determining fingerprint pattern with reference to product) characteristic peak peak intensity (peak height);
M
n: a certain active component molecular weight in the former medicine feature extraction of 15%T12 Celastrus angulatus thing;
H
n: by a certain active component characteristic peak peak intensity (peak height) in the former medicine feature extraction of the 15%T12 Celastrus angulatus thing of IGD carbon-13 nmr spectra determining fingerprint pattern.
Total amount is W
nsum.
(6) calculate main single active component coefficient and overall coefficient in the former medicine of 15% Celastrus angulatus by coupling formula
1. coefficient formulas
F
n: in the former medicine of 15% Celastrus angulatus, a certain active component and Celangulin B(standard are with reference to product) ratio coefficient of quality percentage composition;
M
1: Celangulin B(standard is with reference to product) molecular weight;
H
1: by Celangulin B(standard in the former medicine feature extraction of the 15% Celastrus angulatus thing of IGD carbon-13 nmr spectra determining fingerprint pattern with reference to product) characteristic peak peak intensity (peak height);
M
n: a certain active component molecular weight in the former medicine feature extraction of 15% Celastrus angulatus thing;
H
n: by a certain active component characteristic peak peak intensity (peak height) in the former medicine feature extraction of the 15% Celastrus angulatus thing of IGD carbon-13 nmr spectra determining fingerprint pattern.
This coefficient F
nbe applicable to too calculate active component and active component group in the agent of Celastrus angulatus aqueous suspension agent.
Main active content and calculation of total in (7) 2% Celastrus angulatus aqueous suspension agents
X
n（%）=X
1（%）Fn
X
n: a certain active component quality percentage composition % in 2% Celastrus angulatus aqueous suspension agent;
X
1: Celangulin B quality percentage composition % in 2% Celastrus angulatus aqueous suspension agent, standard is with reference to the absolute content of product Celangulin B.
3, instrument, reagent and material
Nuclear magnetic resonance chemical analyser Bruker DPX 400 types.
Mass spectrograph: Waters Micromass Q-Tof MicroTM type.
Half preparative high-performance liquid chromatographic instrument: Waters 600 types.
High performance liquid chromatograph: Agilent 1200 types.
2000mL distilling flask, 5000mL distilling flask, spherical condensating tube, 2000mL separatory funnel.
DE-52AA Rotary Evaporators: Shanghai Yarong Biochemical Instrument Plant.
DEF-6020 type vacuum drying chamber: the above grand experimental facilities of Nereid Co., Ltd.
Column chromatography silica gel G and tlc silica gel H: Haiyang Chemical Plant, Qingdao.
Silica gel column chromatography 6cm × 70cm(diameter × highly).
Celastrus angulatus medicinal material (Baoji, in November, 2009, a large amount of purchases were from local), Celastrus angulatus medicinal material (Hua County, Shaanxi, in November, 2010, a large amount of purchases were from local), Celastrus angulatus medicinal material (Xichuan County, henan Province, in November, 2009, a large amount of purchases were from local), all identifies through professor Zhu Changshan of In Henan Agriculture university.Celangulin B, standard is with reference to product, and (through spectroscopic data qualification) made in laboratory by oneself.
Reagent: chromatographically pure (methyl alcohol, Tianjin Siyou Fine Chemicals Co., Ltd.) and analyze pure (Tianjin Chemical Reagents Factory No.1).
4, basic research
(1) separation and Extraction flow process 1
Take the Xichuan County, henan Province drying in the shade and produce Celastrus angulatus root, skin 1kg, pulverize, by 6 times of amount volume benzene refluxing extraction 3 times, filtrate merges rear 60 DEG C of reduced pressure concentrations, reclaim solvent to medicinal extract shape, after medicinal extract dissolves with the methyl alcohol of 6 times of amount bulk purity 80%, the petroleum ether extraction of 60mL 1 time, reduced pressure concentration after methanol layer filters, reclaims solvent and obtains medicinal extract (27.5g).Get this medicinal extract silica gel column chromatography and separate, carry out gradient elution with petroleum ether-ethyl acetate (10:1 ~ 4:6) dicyandiamide solution, every 250mL collects 1 part, merges identical cut.The 38th part through preparative chromatography purifying, obtains Celangulin E(18mg); 42nd ~ 43 parts obtain Celangulin A sterling (60mg); The 54th part through preparative chromatography purifying, obtains Celangulin B(25mg), Celangulin H(40mg); The 61st part obtains Celangulin G(95mg); 69th ~ 70 parts through preparative chromatography purifying, obtains Celangulin F(45mg); The 76th part obtains Celangulin J(45mg through preparative chromatography purifying).
(2) separation and Extraction flow process 2
Take 1kg Baoji and produce root bark of celastrus angulatus powder, add successively refluxing extraction 2h at 80 DEG C of 2 times of amount 95% ethanol of 3 ︰ 2 ︰, filter, merge three times filtrate, be evaporated to medicinal extract.According to said method extract altogether root bark of celastrus angulatus powder 7kg, altogether obtain medicinal extract 1120g.Take above-mentioned alcohol extract medicinal extract, add six times of amount chloroforms, refluxing extraction 40min at 80 DEG C, filters, and is evaporated to medicinal extract, obtains medicinal extract 147g.Upper prop after this medicinal extract silica gel mixed sample, adopt petrol ether/ethyl acetate (10:1～4:6) carry out gradient elution, through high efficiency liquid phase prepare purifying [Sunfire C18 chromatographic column (and 150mm × 10mm, 10 μ m); Mobile phase: methanol-water; Column temperature: 25 DEG C; Flow velocity: 10mL/min; Detect wavelength: 232nm; Sample size: 200 μ l] obtain following 4-OH-β-dihydroagarofuran polyol ester compounds.381-385 part obtains Celangulin E (27mg) through preparing purifying [methanol-water 65:35]; The 420th part obtains Celangulin A (80mg) through preparing purifying [methanol-water 62:38]; 546-554 part obtains Celastrus angulatus XIX (43mg) through preparing purifying [methanol-water 60:40]; 590-594 part obtains Celangulin C (28mg) and Celangulin P (16mg) through preparing purifying [methanol-water 68:32]; 613-614 part obtains Celangulin B (95mg), Celangulin H (60mg) through preparing purifying [methanol-water 62:38]; 679-681 part obtains Celastrus angulatus III (26mg) through preparing purifying [methanol-water 60:40]; The 820th part obtains Celangulin J (29mg) through preparing purifying [methanol-water 68:32].
Celastrus angulatus IV data see document [Wu Wenjun. vegetable insecticide Celastrus angulatus research and application [M]. Chemical Industry Press, 2010].
(3) structure and the carbon-13 nmr spectra data of main active in the former medicine of 15%T12 Celastrus angulatus
Celangulin A:R
1=R
4=OiBu, R
2=R
5=H, R
3=OBz, R
6=OH, R
7=OAc
Celangulin B:R
1=R
5=OiBu, R
2=OFu, R
3=R
4=H, R
6=R
7=OAc
Celangulin C:R
1=R
5=OiBu, R
2=OBu, R
3=R
4=H, R
6=R
7=OAc
Celangulin E:R
1=OiPet, R
2=R
5=H, R
3=OBz, R
4=OiBu, R
6=OH, R
7=OAc
Celangulin F:R
1=R
6=R
7=OAc, R
2=R
5=OFu, R
3=R
4=H
Celangulin G:R
1=R
6=R
7=OAc, R
2=OFu, R
3=R
4=H, R
5=OiBu
Celangulin H:R
1=R
7=OAc, R
2=R
5=H, R
3=OBz, R
4=OiBu, R
6=OH
Celangulin J:R
1=R
5=R
6=R
7=OAc, R
2=R
4=H, R
3=OBz
Celangulin P:R
1=OiPet, R
2=OBu, R
3=R
4=H, R
5=OiBu, R
6=R
7=OAc
Celastrus angulatus III: R
1=OiBu, R
2=R
4=H, R
3=OBz, R
5=R
6=R
7=OAc
Celastrus angulatus IV:R
1=OiBu, R
2=R
5=OFu, R
3=R
4=H, R
6=R
7=OAc
Celastrus angulatus XIX:R
1=OiBu, R
2=R
5=H, R
3=OBz, R
4=OFu, R
6=OH, R
7=OAc
Celangulin A
13C NMR(100MHz,CDCl
3)δ
C:75.01(C-1)，67.26(C-2),41.15(C-3),72.13(C-4)，91.41(C-5)，76.89(C-6)，53.51(C-7)，73.69(C-8)，75.28(C-9)，50.52(C-10),84.54(C-11),30.05(C-12)，26.35(C-13)，24.14(C-14)，61.69(C-15)
OAC:169.49,169.60，20.50，21.13
OiBu:175.82,176.77,34.09,34.31,18.46,18.66,19.06,19.14
OBz:165.67,129.26,129.44,128.63,133.45
Celangulin B
13C NMR(100MHz,CDCl
3)δ
C:70.63(C-1)，67.96(C-2),42.00(C-3),69.84(C-4)，91.33(C-5)，75.39(C-6)，52.97(C-7)，76.05(C-8)，71.44(C-9)，53.87(C-10),83.44(C-11),29.59(C-12)，25.45(C-13)，24.47(C-14)，65.45(C-15)
OAC:169.54,169.66,169.79,20.55,21.12,21.48
OiBu:175.74,176.90,33.95,34.10,18.73,18.99,19.00,19.06
OFu:160.91,148.99,117.81,109.69,144.00
Celangulin C
13C NMR(100MHz,CDCl
3)δ
C:70.86(C-1),67.98(C-2),42.04(C-3),69.88(C-4)，91.42(C-5),75.48(C-6),53.04(C-7),76.19(C-8),72.03(C-9),54.10(C-10),83.54(C-11),29.61(C-12),25.66(C-13),24.50(C-14),65.55(C-15);
OA
c: 169.44,169.57,169.26,20.38,21.13*, 21.50* (CH
3) (* ownership is interchangeable)
OiBu:175.77,176.95,33.98,34.12,18.78,18.91,19.04,19.08
OBz:164.52,128.31,130.18,128.47,133.87
Celangulin E
13C NMR(100MHz,CDCl
3)δ
C:75.09(C-1)，67.31(C-2),41.21(C-3),72.15(C-4)，91.49(C-5)，76.92(C-6)，53.53(C-7)，73.80(C-8)，75.23(C-9)，50.49(C-10),84.54(C-11),30.08(C-12)，26.31(C-13)，24.06(C-14)，61.68(C-15)
OAC:169.55,169.46,20.48,21.13
OiBu:175.72,34.11,18.48,18.64
OiPet:176.19,41.56,26.59,11.69,16.92
OBz:165.68,129.36,129.49,128.61,133.42
Celangulin F
13C NMR(100MHz,CDCl
3)δ
C:70.53(C-1)，67.80(C-2),41.93(C-3),69.90(C-4)，91.31(C-5)，75.34(C-6)，53.80(C-7)，76.54(C-8)，71.65(C-9)，54.33(C-10),83.12(C-11),25.55(C-12)，29.51(C-13)，24.32(C-14)，65.56(C-15)
OAC:169.51,169.75,169.86,170.52,20.47,21.13,21.52,21.11
OFu:161.61,160.53,148.69,148.87,118.84,118.05,109.96,109.78,144.01,144.01
Celangulin G
13C NMR(100MHz,CDCl
3)δ
C:70.49(C-1)，67.82(C-2),41.92(C-3),69.81(C-4)，91.51(C-5)，75.00(C-6)，53.34(C-7)，76.10(C-8)，72.65(C-9)，53.97(C-10),83.27(C-11),29.45(C-123)，25.48(C-13)，24.35(C-14)，65.67(C-15)
OAC:169.47,169.69,169.74,170.53,20.45,21.14,21.52,21.04
OiBu:175.86,33.92,18.81,18.89
OFu:160.91,148.99,117.83,109.71,144.02
Celangulin H
13C NMR(100MHz,CDCl
3)δ
C:74.89(C-1)，67.31(C-2),41.10(C-3),72.11(C-4)，91.44(C-5)，76.87(C-6)，53.59(C-7)，73.36(C-8)，75.40(C-9)，50.64(C-10),84.52(C-11),30.03(C-123)，26.24(C-132)，24.23(C-14)，61.38(C-15)
OAC:169.39,169.55,170.28,20.49,21.10,21.47
OiBu:175.88,34.02,18.41,18.58
OBz:165.72,129.23,129.40,128.64,133.40
Celangulin J
13C NMR(100MHz,CDCl
3)δ
C:75.96(C-1),67.85(C-2),41.92(C-3),69.68(C-4),91.70(C-5),75.00(C-6),53.25(C-7),69.75(C-8),72.01(C-9),52.90(C-10),82.62(C-11),29.28(C-12),24.34(C-13),24.11(C-14),60.23(C-15)
OAC:169.52,169.71,169.52,169.92,170.47,20.27,20.99*, 21.22*, 21.46*, 21.55 (* ownership is interchangeable)
OBz:164.69,129.00,129.54,128.74,133.59
Celangulin P
13C NMR(100MHz,CDCl
3)：δ
C：70.94（C-1），68.07（C-2），42.09（C-3），69.92（C-4），91.42（C-5），76.22（C-6），52.98（C-7），75.58（C-8），72.01（C-9），54.08（C-10），83.55（C-11），29.62（C-12），25.63.（C-13），24.55（C-14），65.56（C-15）
OAc：169.52，169.75，169.52，20.40,21.10,21.50
OiBu-8：175.77，33.99，18.84，18.84
OiPet-15：176.55，41.21，26.50，11.67，16.57
OBz：164.52，128.47，130.19,128.34,133.85
Celastrus angulatus III
13C NMR(100MHz,CDCl
3)δ
C:75.84(C-1),67.83(C-2),42.02(C-3),69.73(C-4),91.57(C-5),75.39(C-6),53.27(C-7),69.84(C-8),71.95(C-9),53.05(C-10),82.47(C-11),29.27(C-12),24.45(C-13),24.27(C-14),60.74(C-15)
OAC:169.59*, 169.59*, 169.59*, 170.07*, 20.30,20.93**, 21.23**, 21.50** (*, * * ownership is interchangeable)
OiBu-15:177.29,33.92,19.12,19.32
OBz-9:164.58,129.07,129.50,128.73,133.59
Celastrus angulatus IV
13c NMR (100MHz, CDCl
3) δ
c: 70.3* (C-1,), 70.6* (C-2), 42.0 (C-3), 69.9 (C-4), 91.4 (C-5), 75.6* (C-6), 53.6 (C-7), 76.1* (C-8), 68.0* (C-9), 54.4 (C-10), 83.4 (C-11), 29.6 (C-12), 25.5 (C-13), 24.4 (C-14), 65.0 (C-15) (* ownership is interchangeable)
OAC:169.6×3,20.5,21.0,21.4
OiBu:176.7,34.0,18.8,18.9
OFu:161.3,160.6,148.9,148.7,118.8,118.1,109.8,109.7,143.9×2
Celastrus angulatus XIX
13C NMR(100MHz,CDCl
3)δ
C:75.08(C-1),67.37(C-2),41.28(C-3),72.17(C-4),91.51(C-5),76.84(C-6),53.39(C-7),74.71(C-8),75.08(C-9),50.06(C-10),84.57(C-11),30.05(C-12),26.31(C-13),24.23(C-14),61.76(C-15)
OAC:169.47*, 169.59*, 20.43,21.15 (* ownership is interchangeable)
OiBu-15:176.74,34.37,19.15,19.15
OBz-9:165.74,129.27,129.46,128.62,133.44
OFu-8:161.86,148.25,118.65,109.59,143.87
Embodiment 1
(1) the former medicine first of 15%T12 Celastrus angulatus preparation method
By the Baoji Celastrus angulatus root that conforms with the regulations after screening, or/and skin (24 order), adding successively volume is 2 times of amount 95%(mass ratioes of 3 ︰ 2 ︰) ethanol, at 80 DEG C, refluxing extraction 2h(extracts 3 times, each all 2h), filter, merge three times filtrate, be evaporated to the filtrate of density 1.08.Get this filtrate, add the precipitating reagent that its volume is 1 times of amount (ethanol that mass ratio is 10%), precipitation 12h, must precipitate through centrifugal, i.e. the former medicine first of 15%T12 Celastrus angulatus.
(2) the former medicine first of 15%T12 Celastrus angulatus quality determining method
1. feature extraction thing preparation
Get the former medicine first of 15%T12 Celastrus angulatus 1g, adding volume is the chloroform of 6 times of amounts (6mL), refluxing extraction 40min at 80 DEG C, after filtration at 60 DEG C reduced pressure concentration, reclaim solvent to dry, obtain the former medicine first of 15%T12 Celastrus angulatus feature extraction thing.
2. feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get above-mentioned feature extraction thing, be dissolved in 0.5mL CDCl
3in, make IGD carbon-13 nmr spectra finger-print and detect, obtain IGD carbon-13 nmr spectra finger-print.
3. feature extraction thing IGD carbon-13 nmr spectra finger-print is resolved
1) differentiate
In the IGD carbon-13 nmr spectra finger-print of the former medicine first of 15%T12 Celastrus angulatus feature extraction thing, clearly illustrate the characteristic signal of 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound.According to the chemical shift of its parent nucleus signal and C-15 characteristic peak signal thereof, confirm 12 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound Celangulin A, B, C, E, F, G, H, J, P, Celastrus angulatus III, IV, XIX all has corresponding NMR signal in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 1-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 1-b.
2) the each active component ratio measuring of the former medicine first of 15%T12 Celastrus angulatus result is as follows:
3) in the former medicine first of 15%T12 Celastrus angulatus, Celangulin B concentration assay result is as follows:
| Take former medicine quality | 200mg |
| Celangulin B mass concentration in the former medicine first of 15%T12 Celastrus angulatus | 67.62ug/mL |
| 15%T12 Celastrus angulatus Yuan Yao B-grade in the first class quality percentage composition | 3.34% |
4) the former medicine first of 15%T12 Celastrus angulatus assay result is as follows:
In following table, the molecular formula of Celangulin C+ Celangulin F+ Celangulin P is the molecular formula using Celangulin C as representative, and the molecular formula of Celangulin A+ Celangulin E+ Celastrus angulatus XIX is the molecular formula (following examples are all with embodiment 1) using Celangulin A as representative.
Embodiment 2
(1) the former medicine first of 15%T12 Celastrus angulatus preparation method
Other conditions are with embodiment 1, and difference is to use the water of filtrate doubling dose volume to do precipitating reagent, and effect is similar to Example 1.
(2) the former medicine first of 15%T12 Celastrus angulatus quality determining method
1. feature extraction thing preparation-with embodiment 1.
2. feature extraction thing IGD carbon-13 nmr spectra finger-print detects
With embodiment 1.
3. feature extraction thing IGD carbon-13 nmr spectra finger-print is resolved
1) differentiate
In the IGD carbon-13 nmr spectra finger-print of the former medicine first of 15%T12 Celastrus angulatus feature extraction thing, clearly illustrate the characteristic signal of 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound.According to the chemical shift of its parent nucleus signal and C-15 characteristic peak signal thereof, confirm 12 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound Celangulin A, B, C, E, F, G, H, J, P, Celastrus angulatus III, IV, XIX all has corresponding NMR signal in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 2-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 2-b.
2) the each active component ratio measuring of the former medicine first of 15%T12 Celastrus angulatus result is as follows:
3) in the former medicine first of 15%T12 Celastrus angulatus, Celangulin B concentration assay result is as follows:
| Take former medicine quality | 200mg |
| Celangulin B mass concentration in the former medicine first of 15%T12 Celastrus angulatus | 68.00ug/mL |
| Celangulin B quality percentage composition in the former medicine first of 15%T12 Celastrus angulatus | 3.40% |
4) the former medicine first of 15%T12 Celastrus angulatus assay result is as follows:
Embodiment 3
(1) the former medicine second of 15%T12 Celastrus angulatus preparation method
By Hua County, the Shaanxi Celastrus angulatus root that conforms with the regulations after screening, or/and skin (10 order), adding successively volume is 6 times of amount 90%(mass ratioes of 6 ︰) ethanol, ultrasonic extractions 1h(extraction 2 times at 60 DEG C, all extract 1h) at every turn, filter, merge twice filtrate, be evaporated to the filtrate of density 1.13.Get this filtrate, add the precipitating reagent that its volume is 2 times of amounts (ethanol that mass ratio is 30%), precipitation 24h, must precipitate through centrifugal, i.e. the former medicine second of 15%T12 Celastrus angulatus.
(2) the former medicine second of 15%T12 Celastrus angulatus quality determining method
1. feature extraction thing preparation
Get 15%T12 Celastrus angulatus second 1g, adding volume is the chloroform of 12 times of amounts (12mL), ultrasonic extraction 20min at 60 DEG C, after filtration at 70 DEG C reduced pressure concentration, reclaim solvent to dry, obtain the former medicine second of 15%T12 Celastrus angulatus feature extraction thing.
2. feature extraction thing IGD carbon-13 nmr spectra finger-print detects
Get above-mentioned feature extraction thing, be dissolved in 0.5mL CDCl
3in, make IGD carbon-13 nmr spectra finger-print and detect, obtain IGD carbon-13 nmr spectra finger-print.
3. feature extraction thing IGD carbon-13 nmr spectra finger-print is resolved
1) differentiate
In the IGD carbon-13 nmr spectra finger-print of the former medicine second of 15%T12 Celastrus angulatus feature extraction thing, clearly illustrate the characteristic signal of 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound.According to the chemical shift of its parent nucleus signal and C-15 characteristic peak signal thereof, confirm 12 4-OH-beta-dihydroagarofuran sesquiterpene polyolester compound Celangulin A, B, C, E, F, G, H, J, P, Celastrus angulatus III, IV, XIX all has corresponding NMR signal in IGD carbon-13 nmr spectra finger-print.Accompanying drawing 3-a is shown in by IGD carbon-13 nmr spectra finger-print, and its characteristic peak part widens enlarged drawing and sees accompanying drawing 3-b.
2) the each active component ratio measuring of the former medicine second of 15%T12 Celastrus angulatus result is as follows:
3) in the former medicine second of 15%T12 Celastrus angulatus, Celangulin B concentration assay result is as follows:
| Take former medicine quality | 200mg |
| Celangulin B mass concentration in the former medicine second of 15%T12 Celastrus angulatus | 63.74ug/mL |
| Celangulin B quality percentage composition in the former medicine second of 15%T12 Celastrus angulatus | 3.19% |
4) the former medicine second of 15%T12 Celastrus angulatus assay result is as follows:
Above-described embodiment 1 ~ 3 explanation: the former medicine of Celangulin obtaining by preparation method of the present invention, the wherein stable content of effective active composition, and detection method of the present invention, repeatability and stability are very high.
Embodiment 4
(1) 2%T12 Celastrus angulatus aqueous suspension agent preparation
Be placed in reactor by detecting the qualified former medicine of 15%T12 Celastrus angulatus, add the auxiliary material (wetting dispersing agent, thickener, antifreezing agent and defoamer) in following table according to proportional quantity, stir, then solubilizer is to specifying content, be stirred to completely and dissolve, obtain 2%T12 Celastrus angulatus aqueous suspension agent.Configuration proportion sees the following form:
(2) field trial-carry out with reference to the raw pesticide field efficacy medicine test criterion of formulating chamber of surveying of the Institute for the Control of Agrochemicals of the Ministry of Agriculture,PRC.
1) materials and methods
A. reagent agent
2% Celastrus angulatus aqueous suspension agent (seeing the aqueous suspension agent of the first row in the present embodiment above table), 10% imidacloprid suspending agent (commercially available).
B. controlling object and trial crops
Controlling object is wheat aphid: English grain aphid, the connected valleys aphid of hanging, greenbug.Trial crops is wheat.
C. experimental field and crop
Experimental field be located at experimental field, Tai’an, Shandong Province, this Orchard Soil is fertile, fertility is even, managerial skills are consistent, and wheat aphid all has generation over the years.
D. chemicals treatment
If 3 processing of 2% 800,1000,1200 times of Celastrus angulatus aqueous suspension agents liquid, taking 40% flolimat, 20% sumicidin to 2000 times of liquid of water dilution as contrast medicament, spray clear water is blank.Community area 20m2, order is arranged, and repeats 3 times.Random district group is arranged.
E. spraying time and method
Be administered to for the first time and carry out on May 12nd, 2011, cause harm the Sheng phase on the occasion of wheat aphid.Test weather gas on the same day is fine is partly cloudy weather, 20.1 DEG C of mean temperature of air.With the dispenser of bodyguard's board WS-16P knapsack hand sprayer.
F. control time and method
In dispenser Qian Mei community, press 5 samplings of diagonal, list and fix 5 points, choose for every and have the wheat of aphid 5 strains, aphid number on strain wheat flag leaf and fringe is determined in investigation, using this as dispenser before insect population radix.After dispenser 1,3,7d investigates remaining borer population alive on the wheat of mark, calculate each treatment region control efficiency and carry out significance of difference test.
Preventive effect (%)=[borer population of living before borer population × check plot medicine of living after 1 one treatment region medicines/(borer population of living before borer population × treatment region medicine of living after the medicine of check plot)] × 100
2) statistic analysis result
Wheat aphid field efficacy experiment analysis results sees the following form.As can be seen from the table, it is better that 2% Celastrus angulatus aqueous suspension agent is diluted to 800 ~ 1000 times of preventive effect effects, and after its medicine, the control efficiency of 1 day is 81.93% ~ 92.16%; Best to 800 times for the treatment of region preventive effects of water dilution, after its medicine, the control efficiency of 3 days is 91.90% ~ 92.28%; Be better than contrasting medicament 10% imidacloprid suspending agent doubly to 1000 times of water dilutions.
Similar with the above-mentioned aqueous suspension agent effect that provides field trial according to other aqueous suspension agents of the present embodiment preparation, taking the above-mentioned aqueous suspension agent that provides field trial as optimum.
Embodiment 5
(1) 2%T12 Celastrus angulatus aqueous suspension agent Celastrus angulatus coefficient calculations
Get the mean value of the former medicine first of 15%T12 Celastrus angulatus (Baoji) Celastrus angulatus overall coefficient of embodiment 1-2 as the former medicine first of Celastrus angulatus (Baoji) Celastrus angulatus overall coefficient.
Get the former medicine second of 15%T12 Celastrus angulatus (Hua County, Shaanxi) the Celastrus angulatus overall coefficient real value of embodiment 3 as the former medicine second of Celastrus angulatus (Hua County, Shaanxi) Celastrus angulatus overall coefficient.
Result of calculation is as follows:
| The medicinal material place of production | The former medicine Celastrus angulatus of Celastrus angulatus overall coefficient |
| Baoji | 4.68 |
| Hua County, Shaanxi | 5.40 |
(2) 2%T12 Celastrus angulatus aqueous suspension agent Celangulin B concentration assay result is as follows:
(3) 2%T12 Celastrus angulatus aqueous suspension agent Celastrus angulatus cubage
Claims (4)
1. a quality determining method for the former medicine of Celastrus angulatus, comprises the following steps:
1) the former medicine of Celastrus angulatus is extracted, obtain the former medicine feature extraction of the Celastrus angulatus thing that contains active component group;
The preparation method of the former medicine of described Celastrus angulatus, comprise: taking Celastrus angulatus root or/and skin, pulverize, is that the ethanol of 2～6 times refluxes or ultrasonic extraction 2～3 times at 60～80 DEG C with volume, each extraction 1～2 hour, merging filtrate after filtering, reduced pressure concentration becomes filtrate, gets this filtrate, add volume and be the precipitating reagent of 1～2 times, precipitate 12～24 hours, be precipitated after centrifugal, obtain the former medicine of Celastrus angulatus; Described precipitating reagent is: water or be 5～30% ethanol;
The preparation method of the former medicine feature extraction of described Celastrus angulatus thing, comprise: take the former medicine of Celastrus angulatus, adding volume is the chloroform of 6～12 times of amounts, at 60～80 DEG C, reflux or ultrasonic extraction 20～40min, after filtration at 60～70 DEG C reduced pressure concentration, reclaim solvent to dry, obtain the former medicine feature extraction of Celastrus angulatus thing;
2) the former medicine feature extraction of Celastrus angulatus thing is carried out to IGD carbon-13 nmr spectra finger-print and detect, obtain several active component characteristic peak peak intensities in the former medicine feature extraction of described Celastrus angulatus thing according to finger-print; And determine the characteristic peak peak intensity of the corresponding standard of described each active component with reference to product by same way;
Active component characteristic peak in the former medicine feature extraction of described Celastrus angulatus thing is: C-15, its chemical shift is δ
c60.0～66.0, select C-9 and/or C-1 further to distinguish Celangulin C, Celangulin F and Celangulin P as characteristic peak, its chemical shift is 70.6～72.6;
Described standard is Celangulin B with reference to product;
3) measure and obtain standard described in the former medicine of Celastrus angulatus with reference to the absolute content of product by quantitative analysis means;
4) utilize each active component characteristic peak peak intensity and corresponding standard with reference to ratio and the described absolute content of the characteristic peak peak intensity of product, calculate the content of each active component and the content of active component group in the former medicine of Celastrus angulatus.
2. quality determining method according to claim 1, is characterized in that step 3) in, described standard refers to reference to the absolute content of product: the former medicine Plays of Celastrus angulatus of measuring by quantitative analysis means is with reference to the quality percentage composition of product; Described quantitative analysis means are high performance liquid chromatography.
3. quality determining method according to claim 1 and 2, is characterized in that step 4) in, the coupling formula that calculates the content of each active component is:
W
1for step 3) a certain active component is corresponding in the former medicine of Celastrus angulatus measured by quantitative analysis means standard is with reference to the absolute content of product;
M
1for standard corresponding to described a certain active component is with reference to the molecular weight of product;
H
1for standard corresponding to a certain active component described in the former medicine feature extraction of the Celastrus angulatus thing by IGD carbon-13 nmr spectra determining fingerprint pattern is with reference to the characteristic peak peak intensity of product;
W
nfor the quality percentage composition of a certain active component in the former medicine of Celastrus angulatus;
M
nfor the molecular weight of a certain active component;
H
nfor the characteristic peak peak intensity of a certain active component in the former medicine feature extraction of the Celastrus angulatus thing by IGD carbon-13 nmr spectra determining fingerprint pattern.
4. quality determining method according to claim 3, is characterized in that, in the former medicine of calculating Celastrus angulatus, the coefficient of each active component and the formula of overall coefficient are:
Wherein F
nfor standard that in the former medicine of Celastrus angulatus, a certain active component is corresponding with it is with reference to the ratio coefficient of product quality percentage composition; M
1, h
1, M
nand h
nimplication with the corresponding definition in claim 3, overall coefficient refers to the coefficient F of each active component
nsum.
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| Title |
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| 新型植物源农药苦皮藤素的研究综述;李广领等;《安徽农业科学》;20060730;第34卷(第21期);第5594-5595页 * |
| 杀虫植物苦皮藤的研究新进展;李衍方等;《农药》;20060310;第45卷(第3期);第148-150，154页 * |
| 李广领等.新型植物源农药苦皮藤素的研究综述.《安徽农业科学》.2006,第34卷(第21期),第5594-5595页. * |
| 李衍方等.杀虫植物苦皮藤的研究新进展.《农药》.2006,第45卷(第3期),第148-150，154页. * |
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