CN101648870A - Method for separating and preparing monomer with milk increasing activity from cowherb seeds - Google Patents

Abstract

The invention aims at providing a method for separating and preparing a monomer with milk increasing activity from cowherb seeds, which has a simple preparation method, high purity and low cost. The preparation method comprises the following steps: crudely extracting cowherb seeds; sieving an active site of a cowherb seed extract; preparing a cowherb seed milk increasing monomer; and identifying the structure of a milk increasing monomer. An identifying result ensures dibutyl phthalate as a target compound; and the prepared dibutyl phthalate can promote a secretion level of lactose and beta-casein of in-vitro cultured milk cow thymic epithelial cells. In the method for separating and preparing a monomer with milk increasing activity from cowherb seeds, organic solvents, such as alcohol, and the like which are adopted in an extracting process have low cost and no toxicity and can be recycled; and the adopted polyamide column chromatography and the silica gel column chromatography can enlarge large-scale production, thereby obtaining a large number of milk increasing components. The method for separating and preparing a monomer with milk increasing activity from cowherb seeds is important for the displacement of a Chinese medicine resource of the cowherb seeds to some extents and must bring some economic benefits.

Description

A kind ofly from Semen Vaccariae, separate preparation and have the method that increases newborn reactive monomer

(1) technical field

The present invention relates to Natural Medicine Chemistry and biocytology, is exactly a kind ofly to separate preparation have the method that increases newborn reactive monomer from Semen Vaccariae specifically.

(2) background technology

China's Chinese herbal medicine resource is abundant, and Chinese medicine is used to increase milk, has unique advantages such as toxic side effect is little, price is low, no resistance, noresidue.Herbal medicine is come from China the earliest as the research that increases milk agent and fodder additives, had many milk prescriptions that increase to be used for fodder additives and to improve the milk cow milk production at present, some increase the natural traditional Chinese medicine and the preparation also widespread use clinically thereof of breast simultaneously.Dugoua etc. (2006,2009) have systematically discussed the pharmacological property that increases the Ruzhong medicine, estimate increasing the exactness and the security of Ruzhong medicine in gestation and lactation clinical application.

Semen Vaccariae (Semen Vaccariae) is the seed of pinkwort cow-fat, is traditional conventional Chinese medicine.Semen Vaccariae is as the important Ruzhong medicine that increases, and its ingredient kind is many, complicated component.The basic substance of natural drug validity is its effective constituent.Therefore just need search out wherein to have and increase newborn active monomer component if want to verify the newborn mechanism of increasing of Semen Vaccariae, could pass through the method control natural drug of assay and the quality of preparation thereof, thereby better study its pharmacodynamics and pharmacokinetics, the development and use that Semen Vaccariae increased milk active substance have important theory and actual value.Screening the composition screening from the flavour of a drug of Chinese medicine compound prescription is the main flow of herbal medicine efficacy research.Utilizing cell culture technology screening herbal medicine efficacy composition is ten minutes method efficiently and effectively.Along with Natural Medicine Chemistry Study on Technology progress, dibutyl phthalate separates acquisition from some natural active matters.Wei Song etc. (1997) separate preparation from Amaranthaceae Achyranthes plant Radix Achyranthis Bidentatae and identify 7 kinds of compounds, comprising dibutyl phthalate.Zhou Zhongbo etc. (2007) find to contain the dibutyl phthalate composition in the red building.Han Zhihui etc. (2006) find to contain the dibutyl phthalate composition in the skunk bush.Have in addition and also contain dibutyl phthalate in the Radix Rhapontici seu Radix Echinopsis that increases newborn effect.Because ecotope and terrestrial life are totally different, containing the active substance of a large amount of structure uniquenesses in the marine organisms, marine natural product becomes one of most active fields in the present Natural Medicine Chemistry research.History waits (2005) that the chemical ingredients of basic root bristle algae is carried out separation and purification greatly forever, identifies and wherein contains dibutyl phthalate.Sun Yi (2008) separates from sea lettuce from green alga and obtains 8 compounds, comprising dibutyl phthalate.Qu Xinyan etc. (2004) prepare dibutyl phthalate from the actinomycetes meta-bolites separation of marine source, and it has anti-tumor activity.Dibutyl phthalate is a kind of environmental estrogens, has the biological activity of the biological sample of oestrogenic hormon.In recent years some studies show that the p-Hydroxybenzoate experiment in vitro has weak estrogen activity, can combine with estrogen receptor (ER).Less about the bioactive research report of dibutyl phthalate, the result of some in vivo test shows that the dibutyl phthalate biology can be to promoting that uterus weight has certain effect (Yang Bo etc., 2002).Dibutyl phthalate can promote the propagation of estrogen-dependent mammary cancer MCF-7 cell, may have quasi-estrin effect (Jin Qiumei etc., 2005).

(3) summary of the invention

The object of the present invention is to provide the preparation that from Semen Vaccariae, separates that a kind of preparation method is easy, purity is high, cost is low to have the method that increases newborn reactive monomer.

The object of the present invention is achieved like this: it is as follows that the separation preparation has the method steps that increases newborn reactive monomer from Semen Vaccariae:

Step 1: Semen Vaccariae slightly extracts: gets Semen Vaccariae meal 10kg, uses the alcohol heating reflux of volume fraction 70% to extract 3 times, and each 2h, decompression and solvent recovery, the dilution mother liquor successively with ether, ethyl acetate extraction, obtains the ethyl acetate extraction position.

Step 2: the screening of Semen Vaccariae extract reactive site: ethyl acetate extract is carried out polyamide column chromatography, sample on the dry method, the difference water, 10%, 30%, 50%, 70%, 95% ethanol system carries out gradient elution, collect different gradient eluents, obtaining the traditional Chinese medicine ingredients at different wash-outs position behind the rotary evaporation, is that Semen Vaccariae increases newborn reactive site by its ethanol elution position to influence establishment 50% of lactation model of cow mammary gland epithelial cells cell viability, lactose and milk-protein secretion capacity.

Step 3: Semen Vaccariae increases newborn monomeric preparation: the efficient part to 50% carries out further separation and purification, polyamide column chromatography is separated the 50% ethanol elution position that obtains carry out silica gel thin-layer chromatography, Rf value situation according to the thin layer result of silica gel G plate selects to grope different solvent systemss, finally establishing the petroleum ether-ethyl acetate system is effective separation system, adopt the petroleum ether-ethyl acetate system that silica gel column chromatography is carried out at 50% ethanol elution position, isolated compound is screened, wherein sherwood oil: contain one in 20: 1 the wash-out flow point of ethyl acetate and have and increase newborn active monomer component; Adopt analysis mode and preparation HPLC method to carry out to this preparation that increases newborn monomeric compound, adopt 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% methyl alcohol that the crude product of monomeric compound is carried out the ultraviolet full wavelength scanner and examine knowledge, the uv-absorbing wavelength of establishing this compound is 275nm, adopt half preparation HPLC to the compound continuous sample introduction with purifying and prepare highly purified purpose compound.

Step 4: increase newborn monomeric structure and identify: adopt UV spectrum, proton nmr spectra, carbon-13 nmr spectra, high resolution electrospray ionization mass spectrum (HR-ESI-MS) that the purpose compound is carried out structure and identify, to determine molecular weight, molecular formula and the specific constructive form thereof of purpose compound, qualification result determines that the purpose compound is a dibutyl phthalate; The dibutyl phthalate of preparing can promote the cow mammary gland epithelial cells lactose of vitro culture and the secretion level of beta-casein; And the animal test results of the traditional Chinese medicine monomer injection liquid of dibutyl phthalate preparation shows the raising that it can promote mouse and milk production of cow.

The present invention is a kind of to be separated preparation and has the method that increases newborn reactive monomer from Semen Vaccariae, organic solvents such as the ethanol that in leaching process, adopts with low cost, nontoxic, can reclaim.The polyamide column chromatography and the silica gel column chromatography that adopt can enlarge large-scale production, thereby can obtain a large amount of milk-contents that increases.Simultaneously can obtain monomer component by preparation HPLC, its preparation method is easy and purity is high.Semen Vaccariae is increased milk-content---the definite of o-benzoic acid dibutylester can provide important thinking for the exploitation that increases newborn new drug.Exploitation for the Semen Vaccariae natural resources of Chinese medicinal materials has certain meaning, also will inevitably bring certain economic benefits.

(4) description of drawings

Fig. 1 is sample chromatogram figure before the separation of the present invention;

Fig. 2 is the silica gel thin-layer chromatography figure that increases milk active substance of the present invention;

Fig. 3 for of the present invention increase newborn reactive monomer and separate before;

Fig. 4 for of the present invention increase newborn reactive monomer and separate after;

Fig. 5 is the newborn monomeric structure iron that increases of the present invention.

(5) embodiment

The invention will be further described for example below in conjunction with accompanying drawing.

Embodiment 1, and in conjunction with Fig. 1-Fig. 5, the present invention is a kind of to be separated preparation and have the method that increases newborn reactive monomer from Semen Vaccariae, and method steps is as follows:

Step 1: Semen Vaccariae slightly extracts: gets Semen Vaccariae meal 10kg, uses the alcohol heating reflux of volume fraction 70% to extract 3 times, and each 2h, decompression and solvent recovery, the dilution mother liquor successively with ether, ethyl acetate extraction, obtains the ethyl acetate extraction position;

Step 2: the screening of Semen Vaccariae extract reactive site: ethyl acetate extract is carried out polyamide column chromatography, sample on the dry method, the difference water, 10%, 30%, 50%, 70%, 95% ethanol system carries out gradient elution, collect different gradient eluents, obtaining the traditional Chinese medicine ingredients at different wash-outs position behind the rotary evaporation, is that Semen Vaccariae increases newborn reactive site by its ethanol elution position to influence establishment 50% of lactation model of cow mammary gland epithelial cells cell viability, lactose and milk-protein secretion capacity;

Step 3: Semen Vaccariae increases newborn monomeric preparation: the efficient part to 50% carries out further separation and purification, polyamide column chromatography is separated the 50% ethanol elution position that obtains carry out silica gel thin-layer chromatography, Rf value situation according to the thin layer result of silica gel G plate selects to grope different solvent systemss, finally establishing the petroleum ether-ethyl acetate system is effective separation system, adopt the petroleum ether-ethyl acetate system that silica gel column chromatography is carried out at 50% ethanol elution position, isolated compound is screened, wherein sherwood oil: contain one in 20: 1 the wash-out flow point of ethyl acetate and have and increase newborn active monomer component; Adopt analysis mode and preparation HPLC method to carry out to this preparation that increases newborn monomeric compound, adopt 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% methyl alcohol that the crude product of monomeric compound is carried out the ultraviolet full wavelength scanner and examine knowledge, the uv-absorbing wavelength of establishing this compound is 275nm, adopt half preparation HPLC to the compound continuous sample introduction with purifying and prepare highly purified purpose compound;

Step 4: increase newborn monomeric structure and identify: adopt UV spectrum, proton nmr spectra, carbon-13 nmr spectra, high resolution electrospray ionization mass spectrum (HR-ESI-MS) that the purpose compound is carried out structure and identify, to determine molecular weight, molecular formula and the specific constructive form thereof of purpose compound, qualification result determines that the purpose compound is a dibutyl phthalate; The dibutyl phthalate of preparing can promote the cow mammary gland epithelial cells lactose of vitro culture and the secretion level of beta-casein; And the animal test results of the traditional Chinese medicine monomer injection liquid of dibutyl phthalate preparation shows the raising that it can promote mouse and milk production of cow.

Embodiment 2: in conjunction with Fig. 1-Fig. 5, the present invention is a kind of to be separated preparation and has the method that increases newborn reactive monomer from Semen Vaccariae, and experimental technique is as follows:

Experiment reagent:

The ether that chemical analysis is pure, ethyl acetate, sherwood oil, ethanol; Hplc grade methanol; Chromatography polymeric amide (80～100 order); Silica gel for chromatography (200～300 order).

Experimental installation:

R-205 type Rotary Evaporators, win the Shen, Shanghai; ZHJH-1112 vertical current Bechtop, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.; CO-150 type CO ₂Incubator, Japan; The automatic dual pure water distiller of SZ-93, Shanghai Yarong Biochemical Instrument Plant; The LC-10AT high performance liquid chromatograph, day island proper Tianjin, SPD-10A UV-detector; 2695-2996 type HPLC chromatographic instrument, U.S. Waters; Delta-600-2487 preparation HPLC chromatographic instrument, U.S. Waters; Bruker-400 type superconduction nuclear magnetic resonance spectrometer; SHIMADZU UV-1601 type ultraviolet spectrometer; IonSpec Ultima 7.0T FTICR high resolution electrospray mass spectrometer (HR-ESI-MS); Hypersil-ODS II (5 μ m, 10 * 250mm) half preparative scale chromatography posts, Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.; (5 μ m, 4.6 * 200mm) analysis mode chromatographic columns, Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for Hypersil ODS II.

Experimental technique:

(1) Semen Vaccariae slightly extracts:

Get Semen Vaccariae meal 10kg, use the alcohol heating reflux of volume fraction 70% to extract 3 times, each 2h, decompression and solvent recovery.The dilution mother liquor is successively with ether, ethyl acetate extraction.Obtain the ethyl acetate extraction position.

(2) screening of Semen Vaccariae extract reactive site:

Ethyl acetate extract is carried out polyamide column chromatography, sample on the dry method, the difference water, 10%, 30%, 50%, 70%, 95% ethanol system carries out gradient elution.Collect different gradient eluents, obtain the traditional Chinese medicine ingredients at different wash-outs position behind the rotary evaporation.By its ethanol elution position to influence establishment 50% of lactation model of cow mammary gland epithelial cells cell viability, lactose and milk-protein secretion capacity is that Semen Vaccariae increases newborn reactive site.

(3) Semen Vaccariae increases newborn monomeric preparation:

Efficient part to 50% carries out further separation and purification.Silica gel thin-layer chromatography is carried out at the 50% ethanol elution position that polyamide column chromatography separate to obtain, select to grope different solvent systemss according to the thin layer result's of silica gel G plate Rf value situation, finally establishing the petroleum ether-ethyl acetate system is effective separation system.Adopt the petroleum ether-ethyl acetate system that silica gel column chromatography is carried out at 50% ethanol elution position.Isolated compound is screened, and wherein from sherwood oil: the isolating compound in ethyl acetate (20: 1) position has that to increase breast active.

The preparation that increases newborn monomeric compound adopts the HPLC method of analysis mode and preparation type to carry out.Adopt 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% methyl alcohol that the crude product of monomeric compound is carried out the ultraviolet full wavelength scanner and examine knowledge.With the uv-absorbing wavelength of establishing this compound is 275nm.Adopt half preparation HPLC to the compound continuous sample introduction with purifying and prepare highly purified purpose compound.

(4) increasing newborn monomeric structure identifies:

Adopting UV spectrum, proton nmr spectra, carbon-13 nmr spectra, high resolution electrospray ionization mass spectrum (HR-ESI-MS) that this compound is carried out structure identifies.To determine molecular weight, molecular formula and the specific constructive form thereof of this compound.The dibutyl phthalate of preparing can promote the cow mammary gland epithelial cells lactose of vitro culture and the secretion level of beta-casein.And the animal test results of the traditional Chinese medicine monomer injection liquid of dibutyl phthalate preparation shows the raising that it can promote mouse and milk production of cow.

Experimental result:

(1) Semen Vaccariae increases the separation preparation of newborn reactive monomer

Adopt the HPLC method of analysis mode and preparation type that the point of destination of thin-layer chromatography is analyzed and separate preparation, wherein Fig. 1 is sample chromatogram figure before separating, the ultraviolet full wavelength scanner result of analysis mode HPLC shows that this compound has maximum absorption under 275nm, the HPLC analytical results shows impurity peaks, its purity low (Fig. 3).Adopt the Waters preparation HPLC this compound is carried out purifying, obtained the monomeric compound (Fig. 4) that 30mg has single HPLC chromatographic peak.

(2) Semen Vaccariae increases the structure qualification result of newborn reactive monomer

This research separates having obtained a kind of monomer component that increases newborn effect that has by the research of Chinese medicine to the lactation model of cow mammary gland epithelial cells lactation function effect of vitro culture from Semen Vaccariae.Be the clearly structural performance of this activeconstituents and the basic substance of pharmacodynamics thereof, this traditional Chinese medicine ingredients that the preparation HPLC purifying obtains carried out UV spectrum, mass spectrum, carbon spectrum, hydrogen spectrum and superconduction NMR (Nuclear Magnetic Resonance) spectrum, to determine its structure.The structure qualification result shows that this compound is dibutyl phthalate (Dibutyl phthalate).Its concrete structure is seen shown in Figure 5.

Increasing newborn monomer is colourless oil liquid, is soluble in methyl alcohol.Can be miscible with alcohol, ether, ketone etc., be slightly soluble in water.Relative density: 1.047～1.049, zero pour :-35 ℃, boiling point: 340 ℃.In HR-ESI-MS (pos.) spectrum, at the m/z301.1413 place visible [M+Na] ⁺Molecular ion peak, the molecular weight that shows compound is 278.In conjunction with ¹H-NMR and ¹³The C-NMR spectrum infers that its molecular formula is C ₁₆H ₂₂O ₄(calculated value: 301.1416), calculating degree of unsaturation is 6.

Table 4-1 is the data and the ownership of the nuclear magnetic resonance spectrum of this compound.According to this experimental result at compound ¹In the H-NMR spectrum, because of the symmetry of its structure, so only show the proton signal feature of half structure.Low place δ 7.71 (2H, dd, J=6.0,3.2Hz) and 7.61 (J=6.0 3.2Hz) locates as seen to belong to two groups of hydrogen signals on the adjacent disubstituted benzenes for 2H, dd; (4H, t J=6.4Hz) locate visible 2 oxo methylene radical hydrogen signals to δ 4.28; 1.71 (4H, m) and 1.45 (4H m) locates visible 4 methylene radical hydrogen signals; (6H, t J=3.6Hz) locate visible 2 methyl hydrogen signals to δ 0.97.

Compound ¹³Provide 16 carbon signals in the C-NMR spectrum,, also only show the carbon signal feature of half structure because of the symmetry of its structure.Comprising 6 carbon signals that belong to phenyl ring (δ 132.4 * 2,129.9 * 2 and 133.6 * 2); At the visible ester carbonyl group carbon signal in low place δ 169.3 places; In addition, at δ 66.6 places visible oxo mesomethylene carbon signal; The visible mesomethylene carbon signal in δ 31.7 and 20.3 places; This visible methyl carbon signal in external δ 14.1 places.

With compound ¹Whole hydrogen signals of H-NMR spectrum and ¹³All carbon signals of C-NMR spectrum have carried out resolving accurately and belonging to.And the Wave Spectrum data and the known compound dibutyl phthalate of compound contrasted both basically identicals.Comprehensive above the analysis, authenticating compound is a dibutyl phthalate.

Table 4-1THL compound ¹H-NMR and ¹³The statistic data of C-NMR

(3) Semen Vaccariae increases the newborn effect of increasing of newborn reactive monomer

1. dibutyl phthalate can significantly promote the raising of cow mammary gland epithelial cells lactose and beta-casein secretion capacity.2. dibutyl phthalate improve lactation period mouse and the milk produced by animals test of milk cow show that this monomer has that to increase breast active.

Claims (1)

1. one kind is separated preparation and has the method that increases newborn reactive monomer from Semen Vaccariae, and it is characterized in that: method steps is as follows:

Step 1: Semen Vaccariae slightly extracts: gets Semen Vaccariae meal 10kg, uses the alcohol heating reflux of volume fraction 70% to extract 3 times, and each 2h, decompression and solvent recovery, the dilution mother liquor successively with ether, ethyl acetate extraction, obtains the ethyl acetate extraction position;

Step 2: the screening of Semen Vaccariae extract reactive site: ethyl acetate extract is carried out polyamide column chromatography, sample on the dry method, the difference water, 10%, 30%, 50%, 70%, 95% ethanol system carries out gradient elution, collect different gradient eluents, obtaining the traditional Chinese medicine ingredients at different wash-outs position behind the rotary evaporation, is that Semen Vaccariae increases newborn reactive site by its ethanol elution position to influence establishment 50% of lactation model of cow mammary gland epithelial cells cell viability, lactose and milk-protein secretion capacity;

Step 3: Semen Vaccariae increases newborn monomeric preparation: the efficient part to 50% carries out further separation and purification, polyamide column chromatography is separated the 50% ethanol elution position that obtains carry out silica gel thin-layer chromatography, Rf value situation according to the thin layer result of silica gel G plate selects to grope different solvent systemss, finally establishing the petroleum ether-ethyl acetate system is effective separation system, adopt the petroleum ether-ethyl acetate system that silica gel column chromatography is carried out at 50% ethanol elution position, isolated compound is screened, wherein sherwood oil: contain one in 20: 1 the wash-out flow point of ethyl acetate and have and increase newborn active monomer component; Adopt analysis mode and preparation HPLC method to carry out to this preparation that increases newborn monomeric compound, adopt 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% methyl alcohol that the crude product of monomeric compound is carried out the ultraviolet full wavelength scanner and examine knowledge, the uv-absorbing wavelength of establishing this compound is 275nm, adopt half preparation HPLC to the compound continuous sample introduction with purifying and prepare highly purified purpose compound;

Step 4: increase newborn monomeric structure and identify: adopt UV spectrum, proton nmr spectra, carbon-13 nmr spectra, high resolution electrospray ionization mass spectrum (HR-ESI-MS) that the purpose compound is carried out structure and identify, to determine molecular weight, molecular formula and the specific constructive form thereof of purpose compound, qualification result determines that the purpose compound is a dibutyl phthalate; The dibutyl phthalate of preparing can promote the cow mammary gland epithelial cells lactose of vitro culture and the secretion level of beta-casein; And the animal test results of the traditional Chinese medicine monomer injection liquid of dibutyl phthalate preparation shows the raising that it can promote mouse and milk production of cow.

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