CN104892620B - A kind of preparation method of high-purity karanjin - Google Patents
A kind of preparation method of high-purity karanjin Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of high-purity karanjin, its method is：The dry root of Fordia cauliflora is crushed, with ethanol heating and refluxing extraction, merges extract solution, concentration, suspension petroleum ether extraction, stands, merge petroleum ether layer, be concentrated to give petroleum ether extract.Petroleum ether extract uses petroleum ether through silica gel column chromatography：Ethyl acetate：Acetone system gradient elution, is detected with thin-layer chromatography, collects the eluent containing karanjin, is merged, is concentrated under reduced pressure, and is recrystallized, is obtained karanjin coarse crystallization.Isolated and purified again with preparing high-efficient liquid phase technique, analytic type high performance liquid chromatography detection is utilized to collected every a eluent, respectively obtain the karanjin component that karanjin and purity of the purity between 90%~98% are more than 98%.This is reasonable in design, and technique simple separation speed is fast, with short production cycle, is adapted to industrialized production.
Description
Technical field
The present invention relates to field of compound preparation, and in particular to a kind of preparation method of high-purity karanjin.
Background technology
Chemical reference substance is also known as standard items, is the control in kind of quality standards in Chinese drugs research, quality testing and quality control,
The research of traditional Chinese chemical contrast, is a very important part of Chinese medicine Standardization Research, and the quality evaluation to product is special
It is not that traditional Chinese chemical contrast plays extremely important effect in the quality control of pharmaceutical production, is that traditional Chinese medicine quality controls
Basis and core.
Karanjin is a kind of furoflavone chemical composition, is one of active components of plants, and many plants and medicine
The index composition of product standard quality control.Current not yet corresponding national drug standards material, both at home and abroad in karanjin
The system research of medicine chemical reference substance has no report, with reference to traditional Chinese chemical contrast（For assay）Technical requirements, it is right
Karanjin chemical reference substance is studied, establish batch extracting technique, purity and the content of karanjin chemical reference substance with
And the analysis determining method of determination of foreign matter, so as to establish the technical standard of karanjin chemical reference substance, be its as Chinese medicine
Learn reference substance and the quality standard research of medicinal material and preparation provides scientific basic and guarantee.
Karanjin is from legume Fordia caulifloraFordia caulifloraHems1Dry Tofu pudding leaf plant separates
The active material arrived, from known to open source literature, the extraction separation process of document report karanjin and content assaying method, such as：
1.【Autograph】The research of Indian beech chemical composition：The dry kg of Indian beech leaf coarse powder 8, extracted 3 times with 75 ethanol of 10 times of amounts,
Total kg of medicinal extract 2.1 is obtained after extract solution concentration.Medicinal extract is suspended in water, it is substantially colorless to extract with petroleum ether extraction, merge
And concentrated extract, obtain oil ether moiety (124 g).The g of petroleum ether extract 75 is separated through silica gel column chromatography repeatedly, uses stone
Oily ether, the ethyl acetate gradient elution of petroleum ether one, obtain karanjin monomer.2.【Autograph】The chemical composition analysis of beautiful youth's umbrella：Take
Beautiful youth's umbrella coarse powder, with 95% ethanol (suitable 10 times of amounts of medicinal material) diacolation, collect percolate and be concentrated to give yellow mercury oxide (yield
0.27%), flowed back to obtain chloroform leachable (yield 0.16%) through chloroform.Above-mentioned chloroform leachable is taken, with 100~200 mesh silica gel
Low temperature drying after uniform mixing, upper silicagel column (wet method dress post), with chloroform, the methanol elution gradient of chloroform one, is collected into after finely ground
73 components, TLC inspections are known, and merge spot same composition, are prepared through column chromatography repeatedly and thin layer separation, with acetone repeated recrystallize
Purifying, isolated karanjin monomer.3.【Autograph】Strong medicine Fordia cauliflora branches and leaves chemical constitution study：Fordia cauliflora branches and leaves 15Kg,
Dry in the shade, crush, extracted 3 times with 95% ethanol cold soaking, merge extract solution and be concentrated under reduced pressure, obtain the g of ethanol extract about 500.Gained medicinal extract
Water is added to post-process to obtain the g of oil ethereal extract 30 with the L of petroleum ether 2 extractions successively after being suspended, the L of ethyl acetate 2 extractions post-process to obtain vinegar
The g of acetoacetic ester medicinal extract 50, the L of n-butanol 2 extraction post-process to obtain the g of n-butanol medicinal extract 45, and extraction rear solution is concentrated to give water section
200g.The g of ethyl acetate medicinal extract 45 is taken through silica gel column chromatography, with the methanol (100 of chloroform one：0 one 0：100) gradient elution, every part
200 mL, through the close position of TLC thin layer combining data detection compositions, Fr.III is through silica gel column chromatography, the ethyl acetate (20 of petroleum ether one：
1) elute, collect flow point, pillar layer separation obtains karanjin monomer repeatedly by C18, SephadexLH-20 etc..4.【Topic
Name】Indian beech chemical composition and antioxidation activity research：To Indian beech leaf coarse powder using 75% ethanol extract, then by petroleum ether,
The opposed polarity solvent extraction such as chloroform, ethyl acetate and n-butanol, it is divided into four opposed polarity positions.Then silica gel column layer is used
Analysis, polyamide column chromatography, macroporous absorbent resin and sephadex（Sephadex LH-20）Obtained etc. a variety of chromatography means
To karanjin.5.【Autograph】The content of karanjin in RP-HPLC methods measure Indian beech medicinal material：Establish RP-HPLC measure water
The method of Indian beech cellulose content in Calusena lansium medicinal material.6.【Autograph】HPLC methods determine the content of karanjin in beautiful youth's umbrella：Establish high
The method that effect liquid phase chromatogram method determines Indian beech cellulose content in beautiful youth's umbrella.7.【Autograph】Indian beech in the peaceful tincture of HPLC methods measure swelling and pain
The content of element：Establish the content of karanjin in the peaceful tincture of HPLC methods measure swelling and pain.8.【Autograph】The XRF of thin-layer chromatography one
Determine the research of Indian beech cellulose content in water sieve umbrella medicinal material：Establish the fluorescence spectrophotometry water sieve umbrella reclaimed water of thin-layer chromatography one
The method of Calusena lansium cellulose content.9.【Autograph】The discriminating of water sieve umbrella medicinal material and the assay of karanjin：Using character, it is micro-,
The methods such as TLC differentiate water sieve umbrella medicinal material true and false；The content of karanjin in water sieve umbrella medicinal material, medicinal material inspection are determined using HPLC methods
Deng item with reference to Chinese Pharmacopoeia method measure, the quality standard for strengthening medicinal material water sieve umbrella is worked out.10.【Title】A kind of high content Indian beech
The method of purification of element, the application for a patent for invention of application number 201010263044.5 disclose a kind of purification of high content karanjin
Method, specific method include:Dried flower beanstalk branch is crushed, extracted with finite concentration ethanol, concentration, petroleum ether extraction twice, extracts
Macroreticular resin in extraction raffinate, upper polyamide column isolates and purifies again for eluent concentration, and 95% ethyl alcohol recrystallization produces.
The above method discusses the extraction separation of karanjin from different perspectives, and separation purity is higher, but most of purity
The not up to requirement of traditional Chinese chemical contrast, i.e. purity are more than 98%, can not meet the need of high-purity karanjin chemical reference substance
Will.
By studying karanjin chemical reference substance, the batch extracting work of karanjin chemical reference substance is established
The analysis determining method of skill, purity and content and determination of foreign matter, so as to establish the technical standard of karanjin chemical reference substance,
Scientific basic and guarantee are provided for its quality standard research as traditional Chinese chemical contrast and medicinal material and preparation.Result of study
More complete Essential Chemistry foundation can be provided for karanjin chemical reference substance, grasp its chemical information and analysis and testing technology,
Be advantageous to the further utilization of Related product, and for developing the peculiar product in China, exploitation adds with high-tech, height
The product of value, improve the market competitiveness, it will produce potential and immeasurable social benefit and economic benefit.Produce from now on
Various products, either at home or into international market, be required for leaning on high-caliber quality standard and high-caliber point
Measuring technology is analysed to be developed and improved, otherwise will lose market, the quality standard and detection method of product become more and more
Important, the medication standard of " safely, effectively with quality controllable " has been become a consensus of the international community, and pharmaceutical production should be around this center exhibition
Open, its core is quality standard controlled level, and chemical reference substance then plays a key role.But current most of Chinese medicinal material and
Its preparation, because chemical composition is not clear or without chemical reference substance, can not illustrate the chemical substance basis of its effect, can not also carry out matter
Amount control, and can not be received by modern civilization society, also it is difficult to enter international drug market as Chinese herbal medicine and natural drug
Restriction it is crucial, technology barriers bring difficulty to development Chinese Medicine Industry, therefore, carry out research and the matter of Chemical Constituents of Chinese Traditional And Folk Medicine
Amount standardization research is the only way which must be passed of modernization of Chinese medicine development, material base and medium-height grass to illustrating Chinese herbal medicine effect
The formulation of the production and processing technology of medicine preparation, the discriminating of low-quality goods etc. have great importance.Certainly, to Indian beech
Element carries out quality standard research, establishes the analysis test method of standardization, formulates the horizontal control karanjin quality of high-tech
Testing index and analysis method, be allowed to it is scientific, standardization, enhance our international competitiveness, for Chinese medicine enter international market creation
Condition, there is great practical significance and learning value.
Chemical reference substance of the karanjin as plant, medicinal material and products thereof, is the key problem in technology of quality control, Zhong Duoqi
Industry, scientific research and testing department are required for the karanjin reference substance of high-purity, and its market demand is very big, because karanjin is in medicine
Content in material is low, and extraction and separation technology requires very high, difficulty is very big.The present invention carries out karanjin Chemistry for Chinese Traditional Medicine standard items system
The research of standby and its Quality Control Technology, solves the problems, such as high-purity karanjin chemical reference substance, the effect to the modernization of Chinese medicine
It is it will be apparent that with great practical significance and learning value.
The content of the invention
It is an object of the invention to provide a kind of preparation method of high-purity karanjin.
The present invention is from the dry Tofu pudding of legumeFordia caulifloraHems1Extracted in root, separation, essence
Prepared by making, purifying karanjin, its chemical name, molecular formula, structural formula are as follows：
Chinese name：Karanjin
Chemical name：3- methoxyl groups -2- phenyl -4H- furans [2,3-H] -1- benzofuran -4- ketone（3-methoxy-2-
phenyl-4H-furo[2,3-H]-1-benzopyran-4-one）
English name：Karanjin
Molecular formula：C18 H12 O4
Structural formula is as follows：
The purpose of the present invention is realized by following technical scheme：
The preparation method of high-purity karanjin of the present invention, specifically comprises the following steps：
1) dry root of dry Tofu pudding is extracted with alcohol reflux, extract solution filtration, merging filtrate, is reclaimed ethanol, is obtained medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0~A minutes, elution system
For petroleum ether；B~C minutes, elution system are petroleum ether-ethyl acetate-acetone, ratio 10:1:2~6:4:1；The A is
10~25, the B are 11~26, and the C is 50~70；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, obtain between purity 90%-98% and more than 98% with
On karanjin.
The step 1）The addition of middle ethanol is 5-10 times, concentration of alcohol 50-90% of medicinal material weight, refluxing extraction
Number is 2-8 times.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, and methanol-water elutes for mobile phase, and flow velocity is
5~10 mL/min, Detection wavelength 304nm, column temperature are 25-35 DEG C.
The step 4）TLC detection methods are：Lamellae：Silica G；3 kinds of solvent systems：System（1）Petroleum ether-acetone
Part by weight 7:3, system（2）Dichloromethane-acetone part by weight 9.8:0.2, system（3）Petroleum ether-ethyl acetate-acetone weight
Amount ratio 8:1:1；Point sample：With methanol 1mg/mL solution, on same silica G plate, by different point sample amount gradient point samples,
Point sample amount is respectively 20 ug, 40 ug, 60 ug, 80 ug, 100 ug；Expansion cylinder is put respectively to deploy, open up away from：15cm；Positioning：Spray
With 5% ethanol solution of sulfuric acid, dry, it is clear to be heated to spot development at 105 DEG C, puts uviol lamp（365nm）Under inspect；As a result exist
In thin-layer chromatography, it is seen that the single fluorescence spot of yellow green, 3 kinds of solvent systems, the gradient point sample of 5 various concentrations,
For single spot, impurity spot is had no.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
0.6-1.5ml/min；Sample size：10~20ul；Area normalization standard measure；System condition for following three conditions wherein it
One：
Condition（1）Mobile phase：The phosphoric acid solution of methanol -0.2% part by weight 30:70-90:10, Detection wavelength：260nm；
Condition（2）Mobile phase：The phosphoric acid solution of acetonitrile -0.2% part by weight 30:70-80:20, Detection wavelength：260nm；
Condition（3）Mobile phase：The phosphoric acid solution of acetonitrile -0.2% part by weight 20:80-80:20, Detection wavelength：304nm.
Preferably, the step 6）System condition（3）Mobile phase：The phosphoric acid solution of acetonitrile -0.2% part by weight 30:70-
80:20；Detection wavelength：304nm.
Content and purity testing：Precision weigh in 105 DEG C dry to constant weight reference substances it is appropriate, add mobile phase solution to be made
Per solution of the 1ml containing 1mg, under condition determination, sample introduction 20ul（It is approximately equivalent to 20ug）, liquid chromatograph is injected, with 3 flowings
Phase solvent system records chromatogram to more than 2.5 times of appearance retention time of principal component respectively, is calculated with area normalization method
Content, as a result system measurement reference substance content is equal more than 98%.Determination of foreign matter, respectively in the chromatogram of different system record,
In addition to solvent peak, impurity peak area summation result is respectively less than 2.0%.
Peak purity detects：Take reference substance appropriate, by flow phase system, on high performance liquid chromatograph, use diode array
DAD detectors carry out Peak homogeneity, the HPLC chromatogram peak of karanjin>98%, its chromatographic peak uv absorption spectra is three-dimensional
Collection of illustrative plates and 5 spectrograms are completely superposed, and are shown to be single pure material peak.
As a result：Present invention separation, the karanjin chemical reference substance of purifying, it is common through infrared spectrum, ultraviolet spectra, nuclear-magnetism
Shake, mass spectrum and Physico-chemical tests confirm chemical constitution.TLC detections through 3 development systems, 5 various concentrations, 2 mobile phase systems
The HPLC detections of system and 2 different wave lengths, while purity test is done with DAD to chromatographic peak, show to meet Chinese medicine assay use
The requirement of chemical reference substance, content are more than 98%.
A kind of high-purity karanjin, above-mentioned preparation method are prepared, and its purity is more than 98%.
A kind of preparation method of high-purity karanjin provided by the invention has advantages below：
1st, the present invention is reasonable in design, and technique is simple, is extracted using alcohol aqueous solvent, is recrystallized after a silica gel column chromatography
It can obtain the higher karanjin of purity, most prepare the water that purity reaches more than 98% through preparative high performance liquid chromatography afterwards
Calusena lansium element chemical reference substance, method is simple.
2nd, separating rate of the present invention is fast, with short production cycle, is adapted to industrialized production, there is good application prospect.
3rd, the present invention carries out purity test, assay and quality control using thin-layer chromatography and high performance liquid chromatography, really
Protect the quality of product.
4th, the present invention prepares purity from dry Tofu pudding and meets chemical reference substance requirement, Indian beech of the content more than 98%
Element, solve the supply problem of karanjin chemical reference substance, offer science base is provided for the quality control of dry Tofu pudding medicinal material
Plinth and guarantee.
Brief description of the drawings
Fig. 1 is the preparation technology flow chart of high-purity karanjin；
Fig. 2 is karanjin chromatographic peak uv absorption spectra；
Fig. 3 is karanjin DAD Peak homogeneity HPLC chromatograms；
Fig. 4 is the HPLC detection chromatograms of karanjin quality control.
Embodiment
The present invention is further illustrated below by embodiment.It should be understood that embodiments of the invention are to be used to illustrate
The present invention rather than limitation of the present invention.The present invention is belonged to according to the simple modifications that the essence of the present invention is carried out to the present invention
Claimed scope.Unless otherwise indicated, the percentage of the amount of alcohol in the present invention is percentage by volume, and v/v represents solution
Volume ratio.
Comparative example
The dry root 5kg of dry Tofu pudding, 6 times 85% of addition ethanol heating and refluxing extraction 4 times after crushing, 2 hours every time, merge
Extract solution；Extract solution is concentrated into the 1/4 of original volume, with the petroleum ether ultrasonic extraction 2 times of equal volume amounts, discards extract, will
AB-8 large pore resin absorption columns on raffinate, first it is eluted with water to colourless, is then eluted with 6 times of methanol of column volume 80%, is collected,
Reagent is recovered under reduced pressure, concentrates, gained medicinal extract by polyamide column chromatography, successively with ethyl acetate, ethyl acetate-methanol (9:
1), acetate-methanol (9: 4) gradient elution, eluent is collected, reclaim reagent, coarse-grain is concentrated to dryness to obtain, with 95% ethanol
Recrystallize 5 times repeatedly, obtain the karanjin product of purity 98.8%.
Embodiment 1：
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 3 times with 90% alcohol reflux of 8 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-20min, elution system
For petroleum ether；21-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 8：2：1；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, and obtains the karanjin that purity is 92% and 98.6%.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：65:35 be that mobile phase elutes,
Flow velocity is 5mL/min, and Detection wavelength 304nm, column temperature is 25 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
0.6ml/min；Sample size：10ul；System condition：The phosphoric acid 68 of mobile phase acetonitrile -0.2%：32, Detection wavelength 304nm；Area is returned
One, which changes method, calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, except solvent peak
Outside, each impurity peak area summation must not exceed 2.0%.
Embodiment 2：
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 4 times with 80% alcohol reflux of 9 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-25min, elution system
For petroleum ether；26-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 7：3：1；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, and obtains the karanjin that purity is 95% and 98.8%.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：70:30 be that mobile phase elutes,
Flow velocity is 6mL/min, and Detection wavelength 304nm, column temperature is 30 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
1.2ml/min；Sample size：10ul；System condition：The phosphoric acid 72 of mobile phase acetonitrile -0.2%：28, Detection wavelength 304nm；Area is returned
One, which changes method, calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, except solvent peak
Outside, each impurity peak area summation must not exceed 2.0%.
Embodiment 3：
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 5 times with 70% alcohol reflux of 10 times of weight after the dry root 6kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-15min, elution system
For petroleum ether；16-60min, elution system are petroleum ether-ethyl acetate-acetone, ratio 9：1：1；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, and obtains the Indian beech that purity is 96.6% and 99.3%
Element.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：75:25 be that mobile phase elutes,
Flow velocity is 5mL/min, and Detection wavelength 304nm, column temperature is 35 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
1ml/min；Sample size：10ul；System condition：The phosphoric acid 80 of mobile phase acetonitrile -0.2%：20, Detection wavelength 304nm；Area normalization
Change method calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, in addition to solvent peak,
Each impurity peak area summation must not exceed 2.0%.
Embodiment 4
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 2 times with 60% alcohol reflux of 7 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-10min, elution system
For petroleum ether；11-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 6：4：1；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, and obtains the Indian beech that purity is 96.5% and 99.2%
Element.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：15:85 be that mobile phase elutes,
Flow velocity is 10mL/min, and Detection wavelength 304nm, column temperature is 35 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
0.8ml/min；Sample size：10ul；System condition：The phosphoric acid solution of methanol -0.2% 30:70, Detection wavelength 260nm；Area normalization
Change method calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, in addition to solvent peak,
Each impurity peak area summation must not exceed 2.0%.
Embodiment 5
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 8 times with 50% alcohol reflux of 6 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-20min, elution system
For petroleum ether；21-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 8：1：2；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, and obtains the Indian beech that purity is 95.2% and 99.0%
Element.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：30:70 be that mobile phase elutes,
Flow velocity is 8mL/min, and Detection wavelength 304nm, column temperature is 30 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
1ml/min；Sample size：20ul；System condition：The phosphoric acid solution of methanol -0.2% 90:10, Detection wavelength 260nm；Area normalization
Method calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, in addition to solvent peak, respectively
Impurity peak area summation must not exceed 2.0%.
Embodiment 6
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 7 times with 60% alcohol reflux of 5 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：Condition of gradient elution is：0-
15min, elution system are petroleum ether；16-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 9：3：1；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, and obtains the Indian beech that purity is 94.3% and 99.1%
Element.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：40:60 be that mobile phase elutes,
Flow velocity is 6mL/min, and Detection wavelength 304nm, column temperature is 30 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
1ml/min；Sample size：20ul；System condition：The phosphoric acid solution of acetonitrile -0.2% 30:70, Detection wavelength 260nm；Area normalization
Method calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, in addition to solvent peak, respectively
Impurity peak area summation must not exceed 2.0%.
Embodiment 7
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 4 times with 75% alcohol reflux of 8 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-20min, elution system
For petroleum ether；21-60min, elution system are petroleum ether-ethyl acetate-acetone, ratio 10：1：2；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, obtains the karanjin of purity 93.8% and 98.5%.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：60:40 be that mobile phase elutes,
Flow velocity is 7mL/min, and Detection wavelength 304nm, column temperature is 30 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
1ml/min；Sample size：10ul；System condition：The phosphoric acid solution of acetonitrile -0.2% 80:20, Detection wavelength 260nm；Area normalization
Method calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, in addition to solvent peak, respectively
Impurity peak area summation must not exceed 2.0%.
Embodiment 8
A kind of preparation method of high-purity karanjin, is concretely comprised the following steps：
1) extracted 3 times with 65% alcohol reflux of 10 times of weight after the dry root 5kg of dry Tofu pudding is crushed, extract solution filtration,
Merging filtrate, ethanol is reclaimed, obtains medicinal extract；
2）Medicinal extract through petroleum ether extraction, is stood again, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3）Petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collection contains
The flow point of karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-25min, elution system
For petroleum ether；26-70min, elution system are petroleum ether-ethyl acetate-acetone, ratio 7：2：1；
4）Flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90%
Coarse crystallization；
5）Karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6）Collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-
Between 98% and purity is more than more than 98% karanjin, is concentrated under reduced pressure, obtains the karanjin of purity 92.3% and 99.7%.
The step 5）The chromatographic column of preparative high-performance liquid chromatographic is C-18 posts, methanol-water：75:25 be that mobile phase elutes,
Flow velocity is 7mL/min, and Detection wavelength 304nm, column temperature is 30 DEG C.
The step 4）TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, same
On one silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, 100 ug different concentration gradient point samples, with oil
Ether-acetone（7：3）, dichloromethane-acetone（9.8:0.2）, petroleum ether-ethyl acetate-acetone（8:1:1）Three kinds of systems are expansion
Agent, deploy, take out, dry, spray with 5% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts ultraviolet light
（365nm）Under inspect.Three kinds of solvent systems, the gradient point sample of five various concentrations, it is single spot in thin-layer chromatography
Point, have no impurity spot.
The step 6）HPLC detection methods be：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
1ml/min；Sample size：10ul；System condition：The phosphoric acid solution of acetonitrile -0.2% 30:70, Detection wavelength 304nm；Area normalization
Method calculates content, principal component（Karanjin C18 H12 O4）Peak must not be less than 98.0%, if any impurity peaks, in addition to solvent peak, respectively
Impurity peak area summation must not exceed 2.0%.
Although above the present invention is made to retouch in detail with general explanation, embodiment and experiment
State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed
Scope.
Claims (1)
1. a kind of preparation method of high-purity karanjin, it is characterised in that methods described specifically comprises the following steps：
1) extracted 5 times, extract solution filtration, closed with 70% alcohol reflux of 10 times of weight after the dry root 6kg of dry Tofu pudding is crushed
And filtrate, ethanol is reclaimed, obtains medicinal extract；
2) medicinal extract is stood again through petroleum ether extraction, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3) petroleum ether extract, with petroleum ether-ethyl acetate-acetone system gradient elution, collects aqueous Huang through silica gel column chromatography
Pi Su flow point, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-15min, elution system are stone
Oily ether；16-60min, elution system are petroleum ether-ethyl acetate-acetone, ratio 9：1：1；
4) flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90% and slightly tie
It is brilliant；
5) karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6) collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-98%
Between and purity be more than more than 98% karanjin, be concentrated under reduced pressure, obtain purity be 96.6% and 99.3% Indian beech
Element；
The chromatographic column of the step 5) preparative high-performance liquid chromatographic is C-18 posts, methanol-water：75:25 be that mobile phase elutes, flow velocity
For 5mL/min, Detection wavelength 304nm, column temperature is 35 DEG C；
Step 4) the TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, in same silicon
On glue G lamellaes, respectively by 20ug, 40ug, 60ug, 80ug, 100ug different concentration gradient point samples, using part by weight as 7：3
Petroleum ether-acetone, part by weight 9.8:0.2 dichloromethane-acetone, part by weight 8:1:1 petroleum ether-acetic acid second
Ester-acetone, three kinds of systems are solvent, are deployed, and take out, dry, spray with 5% ethanol solution of sulfuric acid, spot is heated at 105 DEG C
Colour developing is clear, puts and is inspected under 365nm ultraviolet lights；In thin-layer chromatography, three kinds of solvent systems, the gradient point of five various concentrations
Sample, it is single spot, has no impurity spot；
The HPLC detection methods of the step 6) are：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：1ml/
min；Sample size：10ul；System condition：The phosphoric acid 80 of mobile phase acetonitrile -0.2%：20, Detection wavelength 304nm；Area normalization method
Content is calculated, karanjin peak must not be less than 98.0%；
Or methods described specifically comprises the following steps：
1) extracted 2 times, extract solution filtration, merged with 60% alcohol reflux of 7 times of weight after the dry root 5kg of dry Tofu pudding is crushed
Filtrate, ethanol is reclaimed, obtains medicinal extract；
2) medicinal extract is stood again through petroleum ether extraction, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3) petroleum ether extract, with petroleum ether-ethyl acetate-acetone system gradient elution, collects aqueous Huang through silica gel column chromatography
Pi Su flow point, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-10min, elution system are stone
Oily ether；11-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 6：4：1；
4) flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90% and slightly tie
It is brilliant；
5) karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6) collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-98%
Between and purity be more than more than 98% karanjin, be concentrated under reduced pressure, obtain purity be 96.5% and 99.2% Indian beech
Element；
The chromatographic column of the step 5) preparative high-performance liquid chromatographic is C-18 posts, methanol-water：15:85 be that mobile phase elutes, flow velocity
For 10mL/min, Detection wavelength 304nm, column temperature is 35 DEG C；
Step 4) the TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, in same silicon
On glue G lamellaes, respectively by 20ug, 40ug, 60ug, 80ug, 100ug different concentration gradient point samples, using part by weight as 7：3
Petroleum ether-acetone, part by weight 9.8:0.2 dichloromethane-acetone, part by weight 8:1:1 petroleum ether-acetic acid second
Three kinds of systems of ester-acetone are solvent, are deployed, and take out, dry, spray with 5% ethanol solution of sulfuric acid, spot is heated at 105 DEG C
Colour developing is clear, puts and is inspected under 365nm ultraviolet lights；In thin-layer chromatography, three kinds of solvent systems, the gradient of five various concentrations
Point sample, it is single spot, has no impurity spot；
The HPLC detection methods of the step 6) are：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：
0.8ml/min；Sample size：10ul；System condition：The phosphoric acid solution of methanol -0.2% 30:70, Detection wavelength 260nm；Area normalization
Change method calculates content, and karanjin peak must not be less than 98.0%；
Or methods described specifically comprises the following steps：
1) extracted 8 times, extract solution filtration, merged with 50% alcohol reflux of 6 times of weight after the dry root 5kg of dry Tofu pudding is crushed
Filtrate, ethanol is reclaimed, obtains medicinal extract；
2) medicinal extract is stood again through petroleum ether extraction, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3) petroleum ether extract, with petroleum ether-ethyl acetate-acetone system gradient elution, collects aqueous Huang through silica gel column chromatography
Pi Su flow point, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-20min, elution system are stone
Oily ether；21-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 8：1：2；
4) flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90% and slightly tie
It is brilliant；
5) karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6) collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-98%
Between and purity be more than more than 98% karanjin, be concentrated under reduced pressure, obtain purity be 95.2% and 99.0% Indian beech
Element；
The chromatographic column of the step 5) preparative high-performance liquid chromatographic is C-18 posts, methanol-water：30:70 be that mobile phase elutes, flow velocity
For 8mL/min, Detection wavelength 304nm, column temperature is 30 DEG C；
Step 4) the TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, in same silicon
On glue G lamellaes, respectively by 20ug, 40ug, 60ug, 80ug, 100ug different concentration gradient point samples, using part by weight as 7：3
Petroleum ether-acetone, part by weight 9.8:0.2 dichloromethane-acetone, part by weight 8:1:1 petroleum ether-acetic acid second
Ester-acetone, three kinds of systems are solvent, are deployed, and take out, dry, spray with 5% ethanol solution of sulfuric acid, spot is heated at 105 DEG C
Colour developing is clear, puts and is inspected under 365nm ultraviolet lights.In thin-layer chromatography, three kinds of solvent systems, the gradient point of five various concentrations
Sample, it is single spot, has no impurity spot；
The HPLC detection methods of the step 6) are：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：1ml/
min；Sample size：20ul；System condition：The phosphoric acid solution of methanol -0.2% 90:10, Detection wavelength 260nm；Area normalization method meter
Content is calculated, karanjin peak must not be less than 98.0%；
Or methods described specifically comprises the following steps：
1) extracted 7 times, extract solution filtration, merged with 60% alcohol reflux of 5 times of weight after the dry root 5kg of dry Tofu pudding is crushed
Filtrate, ethanol is reclaimed, obtains medicinal extract；
2) medicinal extract is stood again through petroleum ether extraction, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3) petroleum ether extract, with petroleum ether-ethyl acetate-acetone system gradient elution, collects aqueous Huang through silica gel column chromatography
Pi Su flow point, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：Condition of gradient elution is：0-
15min, elution system are petroleum ether；16-50min, elution system are petroleum ether-ethyl acetate-acetone, ratio 9：3：1；
4) flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90% and slightly tie
It is brilliant；
5) karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6) collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-98%
Between and purity be more than more than 98% karanjin, be concentrated under reduced pressure, obtain purity be 94.3% and 99.1% Indian beech
Element；
The chromatographic column of the step 5) preparative high-performance liquid chromatographic is C-18 posts, methanol-water：40:60 be that mobile phase elutes, flow velocity
For 6mL/min, Detection wavelength 304nm, column temperature is 30 DEG C；
Step 4) the TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, in same silicon
On glue G lamellaes, respectively by 20ug, 40ug, 60ug, 80ug, 100ug different concentration gradient point samples, using part by weight as 7：3
Petroleum ether-acetone, part by weight 9.8:0.2 dichloromethane-acetone, part by weight 8:1:1 petroleum ether-acetic acid second
Ester-acetone, three kinds of systems are solvent, are deployed, and take out, dry, spray with 5% ethanol solution of sulfuric acid, spot is heated at 105 DEG C
Colour developing is clear, puts and is inspected under 365nm ultraviolet lights；In thin-layer chromatography, three kinds of solvent systems, the gradient of five various concentrations
Point sample, it is single spot, has no impurity spot；
The HPLC detection methods of the step 6) are：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：1ml/
min；Sample size：20ul；System condition：The phosphoric acid solution of acetonitrile -0.2% 30:70, Detection wavelength 260nm；Area normalization method meter
Content is calculated, karanjin peak must not be less than 98.0%；
Or methods described specifically comprises the following steps：
1) extracted 3 times, extract solution filtration, closed with 65% alcohol reflux of 10 times of weight after the dry root 5kg of dry Tofu pudding is crushed
And filtrate, ethanol is reclaimed, obtains medicinal extract；
2) medicinal extract is stood again through petroleum ether extraction, is merged petroleum ether layer, is concentrated to give petroleum ether extract；
3) petroleum ether extract, with petroleum ether-ethyl acetate-acetone system gradient elution, collects aqueous Huang through silica gel column chromatography
Pi Su flow point, described petroleum ether-ethyl acetate-acetone system condition of gradient elution are：0-25min, elution system are stone
Oily ether；26-70min, elution system are petroleum ether-ethyl acetate-acetone, ratio 7：2：1；
4) flow point TLC combining data detections, concentrate, recrystallization, produce the karanjin that purity is weight content 80~90% and slightly tie
It is brilliant；
5) karanjin coarse crystallization is isolated and purified with preparative high performance liquid chromatography, collects karanjin component；
6) collected every a eluent is detected using HPLC, merging retention time is identical and purity is in 90%-98%
Between and purity be more than more than 98% karanjin, be concentrated under reduced pressure, obtain the karanjin of purity 92.3% and 99.7%；
The chromatographic column of the step 5) preparative high-performance liquid chromatographic is C-18 posts, methanol-water：75:25 be that mobile phase elutes, flow velocity
For 7mL/min, Detection wavelength 304nm, column temperature is 30 DEG C；
Step 4) the TLC detection methods are：Karanjin crude product is taken to add methanol that solution of every 1ml containing 1mg is made, in same silicon
On glue G lamellaes, respectively by 20ug, 40ug, 60ug, 80ug, 100ug different concentration gradient point samples, using part by weight as 7：3
Petroleum ether-acetone, part by weight 9.8:0.2 dichloromethane-acetone, part by weight 8:1:1 petroleum ether-acetic acid second
Ester-acetone, three kinds of systems are solvent, are deployed, and take out, dry, spray with 5% ethanol solution of sulfuric acid, spot is heated at 105 DEG C
Colour developing is clear, puts and is inspected under 365nm ultraviolet lights；In thin-layer chromatography, three kinds of solvent systems, the gradient of five various concentrations
Point sample, it is single spot, has no impurity spot；
The HPLC detection methods of the step 6) are：Chromatographic condition：Chromatographic column C-18,4.6 × 250mm, 10um；Flow velocity：1ml/
min；Sample size：10ul；System condition：The phosphoric acid solution of acetonitrile -0.2% 30:70, Detection wavelength 304nm；Area normalization method meter
Content is calculated, karanjin peak must not be less than 98.0%.
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| CN102241686A (en) * | 2011-05-06 | 2011-11-16 | 南京泽朗农业发展有限公司 | Preparation method of karanjin |
| CN102372720A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Method for purifying high-content karanjin |
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| CN102372720A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Method for purifying high-content karanjin |
| CN102241686A (en) * | 2011-05-06 | 2011-11-16 | 南京泽朗农业发展有限公司 | Preparation method of karanjin |
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| 刘金磊等.壮药干花豆枝叶化学成分研究.《中草药》.2012,第43卷(第6期),1071-1074. * |
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