CN102579540B - Preparation method and application of sowthistle-leaf ixeris seedling flavonoid aglycone - Google Patents
Preparation method and application of sowthistle-leaf ixeris seedling flavonoid aglycone Download PDFInfo
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Abstract
The invention discloses a preparation method of sowthistle-leaf ixeris seedling flavonoid aglycone. The preparation method is characterized by comprising the following steps of: smashing sowthistle-leaf ixeris seedling; performing enzymolysis by using an inherent hydrolase; extracting a medicinal material which is subjected to enzymolysis to obtain over 20 percent by weight of a crude extract of sowthistle-leaf ixeris seedling flavonoid aglycone; and purifying the sowthistle-leaf ixeris seedling flavonoid aglycone crude extract through macroporous resin column chromatography and a solvent recrystallizing method to obtain a sowthistle-leaf ixeris seedling flavonoid aglycone extract, wherein the content of flavonoid aglycone is more than 80 percent; and the flavonoid aglycone is taken as a bulk pharmaceutical for application to a medicament for treating various relevant diseases. After a novel enzymolysis technology is adopted, a high-purity sowthistle-leaf ixeris seedling flavonoid aglycone extract is obtained, belongs to a novel effective fraction in ixeris sonchifolia, mainly comprises luteolin and apigenin of which the total content surpasses over 50 percent of the total solid content, has stable quality and a definite curative effect, is safe and controllable, and is consistent with the requirement of preparation of a Chinese medical injection.
Description
Technical field
The present invention relates to a kind of preparation method of medicine material medicine, be specifically related to the novel preparation method of sowthistle-leaf ixeris seedling flavonoid aglycone.Method of the present invention refers to that the Herba Ixeritis Sonchifoliae flavonoid glycoside enzymic hydrolysis in medicinal material is become sowthistle-leaf ixeris seedling flavonoid aglycone by the lytic enzyme existing by Herba Ixeritis Sonchifoliae self, then in conjunction with the method for chromatographic separation, from Ixeris sonchifolia Hance direct production sowthistle-leaf ixeris seedling flavonoid aglycone.
Sowthistle-leaf ixeris seedling flavonoid aglycone preparation method's of the present invention advance and practical value are, present method does not need to adopt inflammable and explosive organic solvent to carry out degreasing to medicinal material, utilizes the intrinsic lytic enzyme of Herba Ixeritis Sonchifoliae itself directly to prepare sowthistle-leaf ixeris seedling flavonoid aglycone from medicinal material simultaneously.The method is simple and practical, is more suitable for suitability for industrialized production.The conversion preparation that adopts at present the own lytic enzyme of medicinal material Herba Ixeritis Sonchifoliae itself to carry out compound has no report at home and abroad.
Background technology
Herba Ixeritis Sonchifoliae, is commonly called as Herba Ixeritis Sonchifoliae, magnitude all over the sky, is 2 years raw dry aerial parts of composite family gutweed Lepidium Herba Ixeritis Sonchifoliae [Ixerissonchifolia (Bge) Hance], and main product is in China northeast, North China and other places." the Sanitation Ministry medicine standard Inner Mongol fascicle " recorded, and Herba Ixeritis Sonchifoliae bitter, pungent is sharp, cool, tool appetizing, and removing toxic substances, synthetism, goes ruffian effect, for anorexia, removing toxic substances, fracture, toothache.Among the peoplely be mainly used in treating the diseases such as ecphyaditis, tonsillitis, nameless gall.
Research to Ixeris sonchifolia Hance and preparation had a great development in recent years, and Herba Ixeritis Sonchifoliae contains the chemical compositions such as flavones, adenosine, lactone according to the literature.Pharmacological research shows that flavones ingredient is one of main active ingredient classification wherein, and chemical research shows that Lxeris sonchifolia (bunge) hance flavone constituents is higher with luteolin-7-O-β-D-Glucose aldehydic acid glycosides, luteolin 7-O-β-D glucoside, apigenin 7-O-β-D glucuronide, apigenin 7-O-β-D glucoside and other several flavones ingredient content.
The method of research Herba Ixeritis Sonchifoliae effective constituent can be summarized as following several at present:
1, first water or ethanol-extracted, is suspended in the extract reclaiming after solvent in water, uses respectively chloroform, vinyl acetic monomer, n-butanol extraction, is divided into several Extraction parts.Recycle silicon glue post or high performance liquid phase semipreparative column chromatography, be separated into single component material.Aforesaid method is the common analysis of the single flavone component of laboratory study, is not suitable for medicine industryization and produces Herba Ixeritis Sonchifoliae extractive of general flavone.
2, the patent of invention of application number 200510082836.1 " luteolin-7-O-β-D phranoglucuronide and extracting method and purposes ", disclose and from Herba Ixeritis Sonchifoliae medicinal material, extracted a kind of method that separates luteolin-7-O-β-D phranoglucuronide, successively adopt calcium-sulphur method to obtain crude flavonoid powder extract, separate preparation with HPLC through silica gel column chromatography again, obtain yellow unformed powder.This preparation method is only applicable to lab analysis type technique, and the product amount of acquisition is nanogram(ng) level, and complicated operation, and product yield is low, and cost is large, is not suitable for suitability for industrialized production.
3, the application for a patent for invention of application number 200610072744.X " Herba Ixeritis Sonchifoliae total flavones and preparation method thereof and freeze-dried powder and quality controlling means containing it ".The preparation method of Herba Ixeritis Sonchifoliae extract is disclosed, adopt alcohol liquid acid adjustment repeatedly to adjust alkaline purification method, because flavonoid compound is unstable in basic solution, easily there is oxidation and ring-opening reaction, and a large amount of weak acid yellow ketone compounds in Herba Ixeritis Sonchifoliae are difficult for separating out in acidic solution, cause yield low, a large amount of activeconstituentss run off.Meanwhile, although this patent provides extract luteolin 7-O-β-D-Glucose aldehydic acid glycosides content is only 8% left and right.
4, the patent of invention " a kind of preparation that contains Herba Ixeritis Sonchifoliae extract and preparation method thereof " of application number 200410078168.0, disclose a kind of total flavones composition that contains and be no less than 80, wherein luteolin 7-O-glucuronic acid is no less than 24, adenosine is no less than 0.18 Herba Ixeritis Sonchifoliae extract and preparation, what adopt is that extracting solution adds flocculation agent or directly carries out column chromatography, flocculation process complicated operation on the one hand, product yield is low, and easily bring non-medication impurity into, on the other hand, the extracting solution of simple water or alcohol processing carries out column chromatography, foreign matter content is many, affect separating effect, cause and produce load greatly, and finished product content is low.
5, Herba Ixeritis Sonchifoliae tradition usage and theory all think that Herba Ixeritis Sonchifoliae flavonoid glycoside is its main effective constituent, and working method focuses mostly in the research of flavonoid glycoside.But along with the checking of clinical application and herbal pharmacology model, the pharmacological action of sowthistle-leaf ixeris seedling flavonoid aglycone class material more and more comes into one's own, there is antisepsis and anti-inflammation as Herba Ixeritis Sonchifoliae Main Flavonoids aglycon apigenin and luteolin are proved to be, antiviral, anti-infective, anticoagulation, removing free radical and the multiple pharmacological effect such as anti-oxidant, antitumor, evidence luteolin and apigenin have the effect that suppresses transplanted tumor in nude mice, kinds of tumor cells growth in vitro.In Herba Ixeritis Sonchifoliae crude drug, contain a large amount of flavonoid glycoside compounds, and aglycon class material natural content is only less than 0.5%, by the technique of Herba Ixeritis Sonchifoliae flavonoid glycoside purification employing acid hydrolysis production sowthistle-leaf ixeris seedling flavonoid aglycone, not only productive rate is very low, and there is inevitable environmental issue, therefore find a kind of efficient, environmental protection, sowthistle-leaf ixeris seedling flavonoid aglycone production technique is just of great practical significance and using value easily.The orthogonal test of system has been done in this research to the spontaneous enzymatic hydrolysis flavonoid glycoside of the very high Herba Ixeritis Sonchifoliae of hydrolysis efficiency, result shows, within the scope of test investigation, taking medicinal material as hydrolysis substrate, by spontaneous enzymic hydrolysis effect, can make Herba Ixeritis Sonchifoliae flavonoid glycoside percent hydrolysis reach more than 90%, and the phenomenon that medicinal material does not go mouldy in hydrolytic process, prove compared with acid hydrolysis, producing sowthistle-leaf ixeris seedling flavonoid aglycone class material by spontaneous enzymatic hydrolysis Herba Ixeritis Sonchifoliae flavonoid glycoside, is a kind of environmental protection, economy, feasible method.
Summary of the invention
The object of this invention is to provide a kind of preparation method of Herba Ixeritis Sonchifoliae total flavones aglycon.
Another object of the present invention is to provide the Chinese medicine preparation that contains Herba Ixeritis Sonchifoliae total flavones aglycon, is used for the treatment of the purposes of coronary heart diseases and angina pectoris, tumour and cerebral infarction.
Although it is general flavone glycoside compounds that many bibliographical information Herba Ixeritis Sonchifoliaes are used for the treatment of the main chemical compositions of cardiovascular and cerebrovascular diseases, but contriver is through experiment showed, that to pharmacokinetics in Herba Ixeritis Sonchifoliae total flavones glycosides body the effective constituent that Herba Ixeritis Sonchifoliae is mainly used in treating cardiovascular and cerebrovascular diseases is Flavone aglycone compounds in Herba Ixeritis Sonchifoliae.Pharmacological testing and clinical observation result of study show, the KUDIEZI ZHUSHEYE (being called for short Herba Ixeritis Sonchifoliae B) that KUDIEZI ZHUSHEYE (being called for short Herba Ixeritis Sonchifoliae A) and the Herba Ixeritis Sonchifoliae total flavones glycosides made from Herba Ixeritis Sonchifoliae total flavones aglycon extract made relatively, difference highly significant, P < 0.001.
After the invention reside in the new enzymolysis process of employing, obtain high purity Herba Ixeritis Sonchifoliae total flavones aglycon extract, belong to effective part group new in Herba Ixeritis Sonchifoliae, mainly taking luteolin and apigenin as main, both total contents exceed the more than 50% of total solids, steady quality, determined curative effect, safety is controlled, meets the requirement of preparing traditional Chinese medicine.Pharmacodynamics and clinical test results show, the KUDIEZI ZHUSHEYE that Herba Ixeritis Sonchifoliae total flavones aglycon constituents is made has the pharmacological action of obvious treatment coronary heart diseases and angina pectoris and cerebral infarction, for further improving the quality product of KUDIEZI ZHUSHEYE, better quality is provided, active stronger, new basic substance and production process route that security is higher.
The object of the present invention is achieved like this
A kind of preparation method of sowthistle-leaf ixeris seedling flavonoid aglycone is ground into Herba Ixeritis Sonchifoliae after 5-20 order, enzymolysis.Extract to such an extent that sowthistle-leaf ixeris seedling flavonoid aglycone weight percentage is more than 20% crude extract medicinal extract to the medicinal material after enzymolysis, then through macroporous resin column chromatography and solvent recrystallization method, sowthistle-leaf ixeris seedling flavonoid aglycone crude extract is carried out purifying and obtains sowthistle-leaf ixeris seedling flavonoid aglycone sterling.Sowthistle-leaf ixeris seedling flavonoid aglycone can be used as in the medicament that bulk drug is applied to various relative diseases.
Described enzymolysis is according to self lytic enzyme enzymolysis in Herba Ixeritis Sonchifoliae, and does not need to add in addition lytic enzyme, and the condition of enzymolysis is: the consumption of (1) water: 3 one 15 times of Ixeris sonchifolia Hances; (2) temperature of enzymolysis: 35-50 DEG C; (3) enzymolysis time is 3.0-18 hour.
Described sowthistle-leaf ixeris seedling flavonoid aglycone content is the preparation method of 20% above crude extract medicinal extract, and it is characterized in that has two kinds to the extracting method of medicinal material after enzymolysis: (1) alcohol extracting method: enzymolysis solution is concentrated, and adding ethanol, to be made into determining alcohol be 60-80% solution; Refluxing extraction 2 hours, filters, then uses 60-80% alcohol water extraction 2-3 time, and united extraction liquid, obtains sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract through concentrating under reduced pressure; (2) permeating extraction: enzymolysis solution filters, ethyl acetate extraction, obtain sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract (A), medicinal material after enzymolysis, adopt 60-80% alcohol water diacolation, decompression recycling ethanol, obtains sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract (B), medicinal extract (A) and medicinal extract (B) merge to obtain total sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract, and the condition of percolation is: total liquid measure: the 3-6 of dry medicinal material weight doubly; Temperature: 25-35 DEG C; Flow: 2-5L/10min.
Described silica gel column chromatography refers to one or many column chromatography, and packing material is macroporous resin AB-8, and the 5-10 that consumption is sample doubly measures, and eluent is 60-95% ethanolic soln.
Described recrystallization solvent used is a kind of or its mixture in ethanol, acetone and water.
Described sowthistle-leaf ixeris seedling flavonoid aglycone can be used as in the medicament that bulk drug is applied to various relative diseases, comprises the oral and various formulations of injection, is particularly useful for the preparation of novel KUDIEZI ZHUSHEYE.
The purity of described sowthistle-leaf ixeris seedling flavonoid aglycone is more than or equal to 50% (weight ratio), more than yield is calculated as 2% (weight ratio) by medicinal material.
Because sowthistle-leaf ixeris seedling flavonoid aglycone has many-sided pharmacological action, the sowthistle-leaf ixeris seedling flavonoid aglycone of this patent gained can be applied in the medicament of various relative diseases as bulk drug separately or with other medicines combination.
First the invention discloses a kind of method of utilizing the lytic enzyme hydrolysis Herba Ixeritis Sonchifoliae glycosides itself existing in Herba Ixeritis Sonchifoliae to make it to be converted into its Flavone aglycone.Detailed process comprises pulverizing and two unit operations of enzymolysis of medicinal material, and method is simple to operate, and cost is low, is easy to suitability for industrialized production and uses.Due to enzymatic hydrolysis condition gentleness, the lactonic ring of sowthistle-leaf ixeris seedling flavonoid aglycone can open loop not produce the product that optically-active changes.
Fig. 1 is GPLC figure before the hydrolysis of reference substance luteolin.
Fig. 2 is sample HPLC figure before Herba Ixeritis Sonchifoliae enzymolysis.
Fig. 3 is sample HPLC figure after Herba Ixeritis Sonchifoliae enzymolysis.
Fig. 4 is luteolin canonical plotting.
Next the invention discloses the extracting method of sowthistle-leaf ixeris seedling flavonoid aglycone.Can adopt the method with hydrous alcohol extraction or carbon dioxide upercritical fluid extraction to the medicinal material after enzymolysis.
The screening of enzymatic hydrolysis condition
Test design, according to trial test result, is chosen the water yield, three factors of temperature and time, respectively gets three levels, selects L9 (3) orthogonal table to test.
Table 1 is level of factor table
Process of the test and evaluation of result are got 9 parts of Herba Ixeritis Sonchifoliae meal equivalent, by table 2 test design successively enzymolysis, oven dry, measure the content of the luteolin of each sample with luteolin liquid phase method.
Result shows, taking total-flavonoid aglycone content as index, the factor that affects Herba Ixeritis Sonchifoliae self enzymatic hydrolysis effect is followed successively by C > A > B, factor c, factor A have significant impact (P < 0.05) to Herba Ixeritis Sonchifoliae self enzymolysis Herba Ixeritis Sonchifoliae glycosides, and factor B is secondary cause.Consider the practical situation of enzymatic hydrolysis cost according to test-results, determine that optimum process condition is A
2b
2c
3, the material of getting it filled, adds 5 times of water gagings, in 35 DEG C, and enzymatic hydrolysis 12h, for subsequent use after drying.
Proof test
Due to the definite optimum process condition of orthogonal test, A
2b
2c
3therefore, need to carry out proof test.Take
Appropriate 3 parts of Ixeris sonchifolia Hance powder, carries out parallel test by optimum process respectively, and with the total content of the luteolin apigenin in HPLC method working sample, after the enzymatic hydrolysis of optimised process, medicinal material all aglucone content is 84.3%, 81.2% and 88.0%.
Enzymatic hydrolysis efficiency test
Get medicinal material before and after enzymolysis, measure respectively luteolin and Quantitative Determination of Apigenin after the flavonoid glycoside compound content of luteolin and apigenin and hydrolysis, result Herba Ixeritis Sonchifoliae is by after spontaneous enzymatic hydrolysis, has 95.6% Herba Ixeritis Sonchifoliae flavonoid glycoside compound to be hydrolyzed.Before and after hydrolysis, medicinal material HPLC figure is relatively shown in Fig. 1, Fig. 2 and Fig. 3.
Pharmacological evaluation checking
The test of [experimental example 1] dog myocardial ischemia
Experiment purpose:
By Pharmacodynamic test of active extract, the pharmacological action of the KUDIEZI ZHUSHEYE that more total phenolic acid method and stone-sulphur method are produced, KUDIEZI ZHUSHEYE technical study provides experimental basis.
On the impact of myocardial infarction due to anesthetized dog coronary ligation
Get 20 of dogs, ♀ ♂ dual-purpose, body weight 7.5-10kg for test.Be divided at random four groups.(1) pseudo-operation group: physiological saline 1.0ml/kg; (2) model group: physiological saline 1.0ml/kg; (3) total flavones group: 1.0mg/kg; (4) general flavone glycoside tuple: 1.0mg/kg., be fixed on operating table with after 3% vetanarcol 1ml/kg anesthesia by the hidden Venule of forelimb.In left side, fourth, fifth intercostal is opened chest, exposes heart, and meets breathing apparatus.Separate right common femoral artery, femoral vein, femoral arteriography is measured arterial pressure (BP) and heart rate (HR), and femoral venous catheter is in order to intravenous drip medicine. and open pericardium, be sewn in the wall of the chest, make pocket cradle.With No. 0 upper 1/3 place of silk thread ligation ramus descendens anterior arteriae coronariae sinistrae, the not ligation of threading of pseudo-operation group.Near selecting infarct, 10 of positions mapping point seam is put epicardial electrogram electrode, and is selecting a control point away from infarct, Yong Ba road physiograph chest leads mapping epicardial electrogram (EECG).Administration after ligation 15min.Record before ischemic, after ischemic 15min and administration 15,30,60,120, the EECG of 180min, blood pressure (SAP, DAP, MAP), heart rate (HR).Add up ST field offset ∑-ST of each mapping point EECG, and raise >=more than 2mv N-ST of ST section.After each group administration 180min, femoral vein is got blood, measures serum CPK, LDH, MDA, SOD value.After getting blood, take out immediately heart, wash away after blood with physiological saline, take ventricular weight, and ventricle crosscut is become to 5, be placed in 37 DEG C of 0.25%NBT solution 15min that dyes, cut off the non-infarct that each myocardium sheet is colored, undyed infarct cardiac muscle is weighed, obtain heavily respectively infarction size divided by heavy or ventricle whole-heartedly and account for heavy or account for the %. that ventricle is heavy whole-heartedly
The results are shown in Table 2 one tables 3.
Table 2 is impact (x ± s) of myocardial infarction CPK, LDH due to anesthetized dog coronary ligation, MDA, SOD
Note: n=5, Student-t examines face; Each group compared with model group: * p < 0.05, * * p < 0.01;
Each group compared with puppet operation group: #p < 0.05, ##p < 0.01.
The impact (X ± S) that table 3 is the total phenolic acid of Herba Ixeritis Sonchifoliae on myocardial infarct size due to anesthetized dog coronary ligation
Note: n=5, Student-t inspection, each group compared with model group group: * p < 0.05, * * p < 0.01; Each group compared with puppet operation group:
#p < 0.05,
##p < 0.01.
3, experimental result shows: Herba Ixeritis Sonchifoliae total flavones and Flavone aglycone intravenously administrable all obviously alleviate myocardial ischemia degree; Can reduce CPK in serum, LDH, MDA release, enhance SOD activity, with significantly (P < 0.05,0.01) of model group comparing difference; And can dwindle myocardial infarct size (P < 0.05,0.01), the heart rate quickening (P < 0.05,0.01) of slowing down and causing because of ischemic.Prompting sowthistle-leaf ixeris seedling flavonoid aglycone improves significantly to dog myocardial ischemia due to coronary ligation, and effect is better than Herba Ixeritis Sonchifoliae total flavones glycosides.
[test example 2] impact on anesthetized dog cerebral blood flow (CBF)
Test method: get 18 of hybrid dogs, body weight 7-11kg, male and female half and half, are divided into 3 groups at random, 6 every group.Be grouped into: (1) solvent control group (1.0ml/kg); (2) Herba Ixeritis Sonchifoliae flavonoid glycoside group (1.0mg/kg); (3) sowthistle-leaf ixeris seedling flavonoid aglycone group (1.0mg/kg).When experiment with 3% vetanarcol 30mg/kg, the outer saphena injecting anesthetic dog of back leg, separate left common carotid artery, isolate left vertebral artery to chest lock mastoid process direction again along arteria carotis communis, isolate separately left side internal carotid artery, the cassette that puts appropriate diameter at internal carotid artery and vertebral artery is respectively popped one's head in and is connected in MFV mono-1200 type electromagnetic flowmeter survey internal carotid arterys and vertebral artery amount.The equal synchronous recording of each index is in the intelligent eight road physiographs of SLY-808 and be input to the processing of PowerLab8Sp data Collection & Processing System.The posterior vein injecting heparin 10mg/kg that performed the operation makes animal whole body heparinization.The about 30min of balance, after observed indices is stable, each treated animal intravenous drip administration (40/min), capacity 5.4ml/kg.Record respectively before administration and administration after 15,30,6o, 90,120 and the each desired value of 180min.
After experiment finishes, taking out dog brain weighs, taking ICAF amount and vertebral artery amount sum again divided by 1/2 brain recast as cerebral blood flow (CBF), calculate cerebral blood flow (CBF) and cerebral vascular resistance (mean arterial blood pressure/cerebral blood flow (CBF)) that every 100g brain is heavy, and be worth the front value of an administration after obtaining each index change percentage in arid Δ %[(administration) the front value of/administration] × 100%.
The all data of statistical procedures represent with mean ± standard deviation (X ± S), adopt t inspection processing data.
Table 4 is impact (X ± S n=6) unit: the ml (100gmin) on anesthetized dog cerebral blood flow (CBF)
-1
Note: with the comparison of solvent control group, *: P < 0.05, * *: P < 0.01, * * *: P < 0.001; Compare #p < 0.05, ##p < 0.01, ###p < 0.001. with KUDIEZI ZHUSHEYE B group
Result shows, after administration, when 30-180min, Herba Ixeritis Sonchifoliae flavonoid glycoside (1.0mg/kg) compared with solvent control group, has significant difference with sowthistle-leaf ixeris seedling flavonoid aglycone group dog cerebral blood flow (CBF); Cerebral blood flow (CBF) 30-180min after administration has significance different compared with solvent control group, P < 0.01 or P < 0.001.Herba Ixeritis Sonchifoliae total flavones glycosides group and the comparison of sowthistle-leaf ixeris seedling flavonoid aglycone group, difference highly significant, P < 0.001, sowthistle-leaf ixeris seedling flavonoid aglycone group can obviously increase anesthetized dog cerebral blood flow (CBF), and is obviously better than Herba Ixeritis Sonchifoliae flavonoid glycoside.
Embodiment
Embodiment mono-
A kind of preparation method of sowthistle-leaf ixeris seedling flavonoid aglycone comprises following processing step:
1, Ixeris sonchifolia Hance is ground into 10-20 order, then enzymolysis, described enzymolysis is according to self lytic enzyme enzymolysis in Herba Ixeritis Sonchifoliae, and do not need to add in addition lytic enzyme, the condition of enzymolysis is: add the doubly water of (weight ratio) of Ixeris sonchifolia Hance 3-15, enzymolysis 1.5-12 hour at 25-50 DEG C of temperature;
2, the enzymolysis solution of step 1 gained is concentrated, add ethanol to be made into the solution that alcoholic degree is 50-95% (volume ratio), refluxing extraction 1-3 hour, filter, the extraction using alcohol that is 50-95% by volume ratio again 2-3 time extracts 1-2 hour at every turn, united extraction liquid, obtain sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract through concentrating under reduced pressure, this medicinal extract is more than 30% containing sowthistle-leaf ixeris seedling flavonoid aglycone by weight percentage;
3, sowthistle-leaf ixeris seedling flavonoid aglycone medicinal extract obtained above, again through the method for macroporous resin column chromatography and recrystallization, carries out purifying and obtains sowthistle-leaf ixeris seedling flavonoid aglycone sterling it; Macroporous resin column chromatography described in the purification process of above-mentioned sowthistle-leaf ixeris seedling flavonoid aglycone crude extract refers to one or many column chromatography, and packing material is macroporous resin AB-8, and doubly, eluent is aqueous ethanolic solution to the 10-20 that consumption is sample size; Above-mentioned recrystallization solvent used is methyl alcohol, ethanol, acetone, chloroform, the one in water or mixture.
Embodiment bis-
A preparation method for sowthistle-leaf ixeris seedling flavonoid aglycone, is to get Ixeris sonchifolia Hance 100kg, is ground into meal, adds water 500 liters, and under 35 DEG C of conditions, enzymolysis 6 hours, static, filters.Filtrate decompression is concentrated into 1/5th left and right, adds the aqueous solution that ethanol is joined 60% ethanol, adds in medicinal material residue, and refluxing extraction 1 hour, filters.Residue continues with 60% extraction using alcohol twice, each one hour.United extraction liquid, concentrating under reduced pressure.Then carry out macroporous resin column separation.Merge the flow point that contains sowthistle-leaf ixeris seedling flavonoid aglycone, evaporate to dryness.With methyl alcohol or ethanol (95%, analytical pure) recrystallization repeatedly, obtaining purity is the sowthistle-leaf ixeris seedling flavonoid aglycone 2.1kg of 86.5% content.
Embodiment tri-
A preparation method for sowthistle-leaf ixeris seedling flavonoid aglycone, is to get Ixeris sonchifolia Hance 100kg, is ground into meal, adds water 500 liters, and under 35 DEG C of conditions, enzymolysis 12 hours, static, filters.Filtrate decompression is concentrated into 1/5th left and right, adds ethanol and be made into the aqueous solution of 70% ethanol, adds in medicinal material residue, and refluxing extraction 3 hours, filters.Residue continues with 60% extraction using alcohol twice, each one hour.United extraction liquid, concentrating under reduced pressure.Then carry out macroporous resin column separation, merge the flow point that contains sowthistle-leaf ixeris seedling flavonoid aglycone, evaporate to dryness.With methyl alcohol or ethanol (95%, analytical pure) recrystallization repeatedly, obtaining purity is the sowthistle-leaf ixeris seedling flavonoid aglycone 2.4kg of 89.2% content.
Embodiment tetra-
A preparation method for sowthistle-leaf ixeris seedling flavonoid aglycone, is to get Ixeris sonchifolia Hance double centner, pulverizes, and crosses 10 mesh sieves, adds water 600 liters, and under 40 DEG C of conditions, enzymolysis 8 hours, static, filters.Filtrate decompression is concentrated near dry, adds 95% aqueous ethanolic solution, adds in medicinal material residue, and refluxing extraction 3 hours, filters.Residue continues with 95% extraction using alcohol twice, each one hour.United extraction liquid, concentrating under reduced pressure.Then carry out column chromatography, merge the flow point that contains sowthistle-leaf ixeris seedling flavonoid aglycone, evaporate to dryness.With methyl alcohol or ethanol (95%, analytical pure) recrystallization repeatedly, obtain purity and be 2.8 kilograms of the sowthistle-leaf ixeris seedling flavonoid aglycones of 90.3% content.
Embodiment five
The assay of sowthistle-leaf ixeris seedling flavonoid aglycone
1. instrument and reagent
Shimadzu LC-10A liquid chromatograph, electronic analytical balance (1/100000), autoclave sterilization pot, luteolin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 11520-200504), KUDIEZI ZHUSHEYE (Shuangding Pharmaceutical Co., Ltd., Shenyang), acetonitrile, methyl alcohol (chromatographic grade), water (water for injection).
2. method and result
1. method system suitability is investigated
Determine that through repetition test chromatographic condition is: octadecylsilane chemically bonded silica, moving phase: acetonitrile: 0.4% phosphoric acid water (10: 90, v/v), flow velocity: 1.0ml/min, column temperature: 30 DEG C, wavelength: 350nm, sample size: 10 μ l.Theoretical plate number is calculated and is not less than 5000 by luteolin, and the resolution of itself and adjacent chromatographic peak is greater than 1.5, and tailing factor is 0.95～1.3.Under above-mentioned chromatographic condition, Fig. 1 is shown in by the collection of illustrative plates of reference substance and sample.
2. linearity range is investigated
Precision takes luteolin reference substance 10.4mg, put in 10ml volumetric flask, add dissolve with methanol and be diluted to scale, shake up, the accurate 0.5ml that draws puts in 25ml measuring bottle, add methyl alcohol and be diluted to scale, shake up, make the reference substance solution of every 1ml containing 0.0208mg, accurate 2.0,4.0,6.0,8.0,10.0, the 12.0 μ l injection high performance liquid chromatographs of drawing, measure chromatographic peak area, measurement result is in table 5.
Table 5 linearity range is investigated
Taking sample size as X-coordinate, chromatographic peak area is ordinate zou, drawing standard curve, and through linear regression, regression equation is: Y=3894460.85X-11779.67, r=0.9999.
Good in 0.0416 μ g-0.2496 μ g scope internal linear relation by measurement result prompting luteolin sample size, see Fig. 4.
Samples contg determination data
Through adopting preceding method, measure the content of luteolin in 20 batches of sowthistle-leaf ixeris seedling flavonoid aglycones, measurement result is in table 6.
Table 6 different batches Herba Ixeritis Sonchifoliae total flavones aglycon assay result (n=2)
Embodiment six
The preparation method of novel KUDIEZI ZHUSHEYE: get Herba Ixeritis Sonchifoliae total flavones aglycon 2.0kg, inject water 20kg, add 20% sodium hydroxide solution and regulate pH value to 5.5～6.5, fully be stirred to dissolve, filter, filtrate injecting is diluted with water to every 1ml and is equivalent to 1.0mg sowthistle-leaf ixeris seedling flavonoid aglycone, filters, and filtrate adds 0.1～0.2% medical active carbon powder, boil 20 minutes, put-5 DEG C of following placements and reach 10 hours, filter, regulate pH value to 7.0～8.0, with millipore filtration, (aperture 0.22 μ m) filters, embedding, sterilizing, to obtain final product.
Claims (1)
1. the preparation method of a sowthistle-leaf ixeris seedling flavonoid aglycone, after it is characterized in that Herba Ixeritis Sonchifoliae is pulverized, according to self intrinsic lytic enzyme enzymolysis, extract to such an extent that sowthistle-leaf ixeris seedling flavonoid aglycone weight percentage is more than 20% crude extract medicinal extract to the medicinal material after enzymolysis, through macroporous resin column chromatography and solvent recrystallization method, sowthistle-leaf ixeris seedling flavonoid aglycone crude extract is carried out purifying and obtains sowthistle-leaf ixeris seedling flavonoid aglycone extract again, Flavone aglycone content is greater than 80%, and sowthistle-leaf ixeris seedling flavonoid aglycone is applied to as bulk drug in the medicament of various relative diseases;
Described enzymolysis is according to self lytic enzyme enzymolysis in Herba Ixeritis Sonchifoliae, and does not need to add in addition lytic enzyme, and the condition of enzymolysis is: the consumption of (1) water: 3 one 15 times of Ixeris sonchifolia Hances; (2) temperature of enzymolysis: 35-50 DEG C; (3) enzymolysis time is 3.0 1 18 hours;
Described sowthistle-leaf ixeris seedling flavonoid aglycone content is that the preparation process of 20% above crude extract medicinal extract has two kinds: (1) alcohol extracting method: enzymolysis solution is concentrated, and adding ethanol, to be made into determining alcohol be 60-80% solution; Refluxing extraction 2 hours, filters, then uses 60-80% alcohol water extraction 2-3 times, and united extraction liquid, obtains sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract through concentrating under reduced pressure; (2) permeating extraction: enzymolysis solution filters, ethyl acetate extraction, obtain sowthistle-leaf ixeris seedling flavonoid aglycone crude extract extractum A, medicinal material after enzymolysis, adopt 60-80% alcohol water diacolation, decompression recycling ethanol, obtains sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract B, extractum A and medicinal extract B merge to obtain total sowthistle-leaf ixeris seedling flavonoid aglycone crude extract medicinal extract, and the condition of percolation is: total liquid measure: the 3-6 of dry medicinal material weight doubly; Temperature: 25-35 DEG C; Flow: 2-5L/10min;
Described macroporous resin column chromatography refers to one or many column chromatography, and packing material is macroporous resin AB-8,5-10 times of amounts that consumption is sample, and eluent is 60-95% ethanolic soln;
Described recrystallization solvent used is at least one in ethanol, acetone and water.
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