CN104892620A - Preparation method for high purity Karanjin - Google Patents
Preparation method for high purity Karanjin Download PDFInfo
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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Abstract
The invention relates to a preparation method for high purity Karanjin. The method includes: crushing the dried root of Fordia cauliflora Hemsl, using ethanol to conduct heating reflux extraction, merging the extracted liquid, performing concentration, extracting the suspension with petroleum ether, conducting standing, merging the petroleum ether layer, and performing concentration to obtain a petroleum ether extract, subjecting the petroleum ether extract to silica gel column chromatography, carrying out gradient elution with a petroleum ether-ethyl acetate-acetone system, conducting detection by thin layer chromatography (TLC), collecting eluent containing Karanjin, carrying out merging, vacuum concentration and recrystallization to obtain Karanjin coarse crystals, then conducting separation and purification by high performance liquid chromatography, and detecting each of the collected eluent by analytical type high performance liquid chromatogram, thus obtaining Karanjin with purity of 90%-98% and Karanjin component with purity of over 98% respectively. The method provided by the invention has the characteristics of reasonable design, simple process and fast separation speed, and short production cycle, thus being suitable for industrial production.
Description
Technical field
The present invention relates to field of compound preparation, be specifically related to a kind of preparation method of high purity karanjin.
Background technology
Chemical reference substance is also known as standard substance, it is the material object contrast of quality standards in Chinese drugs research, quality examination and quality control, the research of traditional Chinese chemical contrast, it is a very important part of Chinese medicine Standardization Research, to the quality evalution of product, particularly in the quality control of pharmaceutical production, traditional Chinese chemical contrast plays earth shaking effect, is basis and the core of traditional Chinese medicine quality control.
Karanjin is a kind of furoflavone chemical composition, is one of active components of plants, is also the index composition of many plants and drug standard quality control.Not yet corresponding national drug standards material at present, report is had no to the systematic study of karanjin traditional Chinese chemical contrast both at home and abroad, with reference to the technical requirements of traditional Chinese chemical contrast (for assay), karanjin chemical reference substance is studied, set up the analysis determining method of the batch extracting technique of karanjin chemical reference substance, purity and content and determination of foreign matter, thus set up the technological standard of karanjin chemical reference substance, for its quality standard research as traditional Chinese chemical contrast and medicinal material and preparation provides scientific basic and guarantee.
Karanjin is from leguminous plants F. cauliflora Hemsl.
fordia caulifloraHems1. dry Tofu pudding leaf plant is separated the active substance obtained, known to open source literature, the extraction and isolation process of bibliographical information karanjin and content assaying method, as: 1.[inscribes one's name] research of Pongamia glabra chemical composition: dry Pongamia glabra leaf meal 8 kg, with 75 extraction using alcohol 3 times of 10 times amount, after extracting solution is concentrated total medicinal extract 2.1 kg.Be suspended in by medicinal extract in water, substantially colourless to extraction liquid with petroleum ether extraction, merge and concentrated extract, (124 g) to obtain sherwood oil part.By petroleum ether extract 75 g through silica gel column chromatography separation repeatedly, with sherwood oil, sherwood oil one vinyl acetic monomer gradient elution, obtain karanjin monomer.2.[inscribes one's name] chemical composition analysis of YULANGSAN: get YULANGSAN meal, with 95% ethanol (suitable medicinal material 10 times amount) diacolation, collect that percolate is concentrated to obtain yellow mercury oxide (yield 0.27%), to reflux to obtain chloroform leachable (yield 0.16%) through chloroform.Get above-mentioned chloroform leachable, with cryodrying after 100 ~ 200 order silica gel uniform mixings, upper silicagel column (wet method dress post) after porphyrize, with chloroform, chloroform one methanol elution gradient, collect 73 components, TLC examines knowledge, merge spot same composition, through column chromatography and Thin-layer separation preparation repeatedly, with acetone repeatedly recrystallization purifying, be separated and obtain karanjin monomer.3.[inscribes one's name] strong medicine F. cauliflora Hemsl. branches and leaves chemical constitution study: F. cauliflora Hemsl. branches and leaves 15Kg, dries in the shade, pulverizes, extract 3 times, united extraction liquid concentrating under reduced pressure, obtain ethanol extract about 500 g with 95% ethanol cold soaking.Gained medicinal extract adds water and extracts aftertreatment with sherwood oil 2 L successively after suspendible and obtain sherwood oil medicinal extract 30 g, vinyl acetic monomer 2 L extracts aftertreatment and obtains vinyl acetic monomer medicinal extract 50 g, propyl carbinol 2 L extracts aftertreatment and obtains propyl carbinol medicinal extract 45 g, and extraction rear solution concentrates to obtain water section 200g.Get vinyl acetic monomer medicinal extract 45 g through silica gel column chromatography, with chloroform one methyl alcohol (100:0 mono-0:100) gradient elution, every part of 200 mL, through the close position of TLC thin layer combining data detection composition, Fr.III is through silica gel column chromatography, sherwood oil one vinyl acetic monomer (20:1) wash-out, collect flow point, by C18, SephadexLH-20 etc. repeatedly pillar layer separation obtain karanjin monomer.4.[inscribe one's name] Pongamia glabra chemical composition and anti-oxidant activity research: 75% extraction using alcohol is adopted to Pongamia glabra leaf meal, then through opposed polarity solvent extractions such as sherwood oil, chloroform, ethyl acetate and propyl carbinols, is divided into into four opposed polarity positions.Then the multiple chromatographic separation means such as silica gel column chromatography, polyamide column chromatography, macroporous adsorbent resin and dextrane gel (Sephadex LH-20) are used to obtain karanjin.5.[inscribes one's name] RP-HPLC method measures the content of karanjin in Pongamia glabra medicinal material: set up the method that RP-HPLC measures karanjin content in Pongamia glabra medicinal material.6.[inscribes one's name] HPLC method measures the content of karanjin in YULANGSAN: the method setting up karanjin content in high effective liquid chromatography for measuring YULANGSAN.7.[inscribes one's name] HPLC method measures the content of the karanjin in peaceful tincture of swelling and ache: set up the content that HPLC method measures karanjin in the peaceful tincture that swells and ache.8.[inscribes one's name] thin-layer chromatography one fluorometry to measure in water sieve umbrella medicinal material karanjin containing quantifier elimination: the method setting up karanjin content in thin-layer chromatography one fluorescence spectrophotometry water sieve umbrella.9.[inscribes one's name] discriminating of water sieve umbrella medicinal material and the assay of karanjin: adopt the methods such as proterties, micro-, TLC to differentiate water sieve umbrella medicinal material true and false; Adopt HPLC method to measure the content of karanjin in water sieve umbrella medicinal material, the items such as medicinal material inspection measure with reference to Chinese Pharmacopoeia method, work out the quality standard of strong medicinal material water sieve umbrella.10.[title] a kind of method of purification of high-content karanjin, the application for a patent for invention of application number 201010263044.5 discloses a kind of method of purification of high-content karanjin, concrete grammar comprises: pulverized by dried flower beanstalk branch, with finite concentration extraction using alcohol, concentrate, petroleum ether extraction twice, macroporous resin on raffinate, elutriant is concentrated goes up polyamide column separation and purification again, 95% ethyl alcohol recrystallization and get final product.
Aforesaid method discusses the extraction and isolation of karanjin from different perspectives, and separation purity is higher, but purity great majority do not reach the requirement of traditional Chinese chemical contrast, and namely purity is greater than 98%, cannot meet the needs of high purity karanjin chemical reference substance.
By studying karanjin chemical reference substance, set up the analysis determining method of the batch extracting technique of karanjin chemical reference substance, purity and content and determination of foreign matter, thus set up the technological standard of karanjin chemical reference substance, for its quality standard research as traditional Chinese chemical contrast and medicinal material and preparation provides scientific basic and guarantee.Result of study can be the Essential Chemistry foundation that karanjin chemical reference substance provides more complete, grasp its chemical information and analysis and testing technology, be conducive to the further exploitation of related products, and for the peculiar product of exploitation China, exploitation has hi-tech, high value-added product, improve the market competitiveness, potential and immeasurable social benefit and economic benefit will be produced.The various products produced from now on, no matter be at home or enter world market, all need obtain development by high-caliber quality standard and high-caliber analysis and testing technology and improve, otherwise will market be lost, the quality standard of product and detection method become more and more important, and the medication standard of " safe, effective and quality controllable " is become a consensus of the international community, and pharmaceutical production should round this center deployment, its core is quality standard level of control, and chemical reference substance then plays a key role.But current most of Chinese medicinal material and preparation thereof, because chemical composition is not clear or without chemical reference substance, the chemical substance basis of its effect cannot be illustrated, also quality control cannot be carried out, and can not by modern civilization society accept, also becoming herbal medicine and natural drug, to be difficult to enter the restriction of international drug market crucial, technology barriers bring difficulty to development Chinese Medicine Industry, therefore, research and the quality standard standardized study of carrying out Chemical Constituents of Chinese Traditional And Folk Medicine are the only way which must be passed that the modernization of Chinese medicine develops, to the formulation of the production and processing technology of the basic substance and Chinese herbal and crude drugs preparations of illustrating herbal medicine effect, discriminating of low-quality goods etc. has great importance.Certainly, quality standard research is carried out to karanjin, set up normalized analysis test method, formulate Testing index and the analytical procedure of the control karanjin quality of hi-tech level, make it scientific, standardize, enhance our international competitiveness, create conditions for Chinese medicine enters world market, there is great practical significance and learning value.
Karanjin is as the chemical reference substance of plant, medicinal material and products thereof, it is the key problem in technology of quality control, numerous enterprises, scientific research and inspection department all need highly purified karanjin reference substance, its market requirement is very large, because the content of karanjin in medicinal material is low, extraction and separation technology requirement is very high, difficulty is very large.The present invention carries out the preparation of karanjin Chemistry for Chinese Traditional Medicine standard substance and Quality Control Technology research thereof, solves the problem of high purity karanjin chemical reference substance, is apparent, has great practical significance and learning value to the modern effect of Chinese medicine.
Summary of the invention
The object of this invention is to provide a kind of preparation method of high purity karanjin.
The present invention is from the dry Tofu pudding of leguminous plants
fordia caulifloraHems1. root in through extracting and developing, refining, purifying and obtained karanjin, its chemical name, molecular formula, structural formula are as follows:
Chinese name: karanjin
Chemical name: 3-methoxyl group-2-phenyl-4H-furans [2,3-H]-1-cumarone-4-ketone (3-methoxy-2-phenyl-4H-furo [2,3-H]-1-benzopyran-4-one)
English name: Karanjin
Molecular formula: C
18h
12o
4
Structural formula is as follows:
The object of the invention is by following technical scheme realize:
The preparation method of high purity karanjin of the present invention, specifically comprises the steps:
1) the dry root alcohol reflux of dry Tofu pudding, extracting solution filters, merging filtrate, reclaims ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0 ~ A minute, and elution system is sherwood oil; B ~ C minute, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 10:1:2 ~ 6:4:1; Described A is 10 ~ 25, and described B is 11 ~ 26, and described C is 50 ~ 70;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains between purity 90%-98% and is greater than the karanjin of more than 98%.
In described step 1), the add-on of ethanol is 5-10 times of medicinal material weight, and alcohol concn is 50-90%, and refluxing extraction number of times is 2-8 time.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water is moving phase wash-out, and flow velocity is 5 ~ 10 mL/min, and determined wavelength is 304nm, and column temperature is 25-35 DEG C.
Described step 4) TLC detection method is: thin layer plate: silica gel G; 3 kinds of developping agent systems: system (1) sherwood oil-acetone part by weight 7:3, system (2) dichloromethane-acetone part by weight 9.8:0.2, system (3) petroleum ether-ethyl acetate-acetone part by weight 8:1:1; Point sample: with the solution of Methanol 1mg/mL, on same silica gel 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; Put expansion cylinder to launch respectively, exhibition distance: 15cm; Location: spray with 5% ethanol solution of sulfuric acid, dry, be heated to spot development at 105 DEG C clear, inspect under putting ultraviolet lamp (365nm); Result is in thin-layer chromatography, and visible yellowish green single fluorescence spot, 3 kinds of developping agent systems, the gradient point sample of 5 different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: 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 is one of them of following three conditions:
Condition (1) moving phase: methyl alcohol-0.2% phosphoric acid solution part by weight 30:70-90:10, determined wavelength: 260nm;
Condition (2) moving phase: acetonitrile-0.2% phosphoric acid solution part by weight 30:70-80:20, determined wavelength: 260nm;
Condition (3) moving phase: acetonitrile-0.2% phosphoric acid solution part by weight 20:80-80:20, determined wavelength: 304nm.
Preferably, system condition (3) moving phase of described step 6): acetonitrile-0.2% phosphoric acid solution part by weight 30:70-80:20; Determined wavelength: 304nm.
Content and purity testing: it is appropriate that precision takes the reference substance being dried to constant weight in 105 DEG C, add moving phase solution and make the solution of every 1ml containing 1mg, under condition determination, sample introduction 20ul(is about equivalent to 20ug), injection liquid chromatography, record color atlas respectively to more than 2.5 times that go out peak retention time of principal constituent by 3 mobile phase solvent systems, calculate content by area normalization method, result systems measurement reference substance content is equal more than 98%.Determination of foreign matter, respectively in the color atlas of different system record, desolventizes outside peak, and impurity peak area summation result is all less than 2.0%.
Peak purity detects: get reference substance appropriate, by flow phase system, on high performance liquid chromatograph, Peak homogeneity is carried out with diode array DAD detector, the HPLC chromatographic peak >98% of karanjin, its chromatographic peak uv absorption spectra, three-dimensional collection of illustrative plates and 5 spectrograms overlap completely, are indicated as single pure substance peak.
Result: the karanjin chemical reference substance of separation of the present invention, purifying, confirms chemical structure through infrared spectra, UV spectrum, nucleus magnetic resonance, mass spectrum and Physico-chemical tests.Detect through the TLC of 3 development systems, 5 different concns, the HPLC of 2 flow phase system and 2 different wave lengths detects, and do purity test to chromatographic peak DAD, show the requirement meeting Chinese medicine assay chemical reference substance, content is greater than 98% simultaneously.
A kind of high purity karanjin, above-mentioned preparation method is prepared from, and its purity is greater than 98%.
The preparation method of a kind of high purity karanjin provided by the invention has the following advantages:
1, the present invention is reasonable in design, technique is simple, and utilize alcohol water solvent to extract, after a silica gel column chromatography, recrystallization can obtain the higher karanjin of purity, prepare finally by preparative high performance liquid chromatography the karanjin chemical reference substance that purity reaches more than 98%, method is simple.
2, velocity of separation of the present invention is fast, with short production cycle, is applicable to suitability for industrialized production, has good application prospect.
3, the present invention adopts thin-layer chromatography and high performance liquid chromatography to carry out purity test, assay and quality control, guarantees the quality of product.
4, the present invention prepares purity and meets chemical reference substance requirement from dry Tofu pudding, and the karanjin of content more than 98%, solves the supply problem of karanjin chemical reference substance, and the quality control for dry Tofu pudding medicinal material provides and provides scientific basic and guarantee.
Accompanying drawing explanation
Fig. 1 is preparation technology's schema of high purity karanjin;
Fig. 2 is karanjin chromatographic peak uv absorption spectra;
Fig. 3 is karanjin DAD Peak homogeneity HPLC color atlas;
Fig. 4 is that the HPLC of karanjin quality control detects color atlas.
Embodiment
The present invention is further illustrated below by embodiment.It should be understood that embodiments of the invention are for illustration of the present invention instead of limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.Except as otherwise noted, the percentage ratio of the amount of alcohol in the present invention is percent by volume, and v/v represents the volume ratio of solution.
Comparative example
The dry root 5kg of dry Tofu pudding, adds 6 times of 85% alcohol heating reflux and extracts 4 times, each 2 hours, united extraction liquid after pulverizing, extracting solution is concentrated into 1/4 of original volume, with the sherwood oil ultrasonic extraction 2 times of equal volume amounts, discard extraction liquid, by AB-8 macroporous adsorptive resins on raffinate, first wash with water to colourless, then 6 times of column volume 80% methanol-eluted fractions are used, collect, reclaim under reduced pressure reagent, concentrated, gained medicinal extract passes through polyamide column chromatography, use ethyl acetate successively, ethyl acetate-methyl alcohol (9: 1), acetate-methanol (9: 4) gradient elution, collect elutriant, reclaim reagent, be concentrated into dry coarse-grain, with 95% ethanol recrystallization 5 times repeatedly, obtain purity 98.8% karanjin product.
Embodiment 1:
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 90% of 8 times of weight alcohol reflux 3 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-20min, and elution system is sherwood oil; 21-50min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 8:2:1;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin that purity is 92% and 98.6%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 65:35 is moving phase wash-out, and flow velocity is 5mL/min, and determined wavelength is 304nm, and column temperature is 25 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 0.6ml/min; Sample size: 10ul; System condition: moving phase acetonitrile-0.2% phosphoric acid 68:32, determined wavelength 304nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 2:
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 80% of 9 times of weight alcohol reflux 4 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-25min, and elution system is sherwood oil; 26-50min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 7:3:1;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin that purity is 95% and 98.8%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 70:30 is moving phase wash-out, and flow velocity is 6mL/min, and determined wavelength is 304nm, and column temperature is 30 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 1.2ml/min; Sample size: 10ul; System condition: moving phase acetonitrile-0.2% phosphoric acid 72:28, determined wavelength 304nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 3:
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 6kg of dry Tofu pudding pulverize after with 70% of 10 times of weight alcohol reflux 5 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-15min, and elution system is sherwood oil; 16-60min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 9:1:1;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin that purity is 96.6% and 99.3%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 75:25 is moving phase wash-out, and flow velocity is 5mL/min, and determined wavelength is 304nm, and column temperature is 35 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 1ml/min; Sample size: 10ul; System condition: moving phase acetonitrile-0.2% phosphoric acid 80:20, determined wavelength 304nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 4
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 60% of 7 times of weight alcohol reflux 2 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-10min, and elution system is sherwood oil; 11-50min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 6:4:1;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin that purity is 96.5% and 99.2%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 15:85 is moving phase wash-out, and flow velocity is 10mL/min, and determined wavelength is 304nm, and column temperature is 35 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 0.8ml/min; Sample size: 10ul; System condition: methyl alcohol-0.2% phosphoric acid solution 30:70, determined wavelength 260nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 5
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 50% of 6 times of weight alcohol reflux 8 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-20min, and elution system is sherwood oil; 21-50min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 8:1:2;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin that purity is 95.2% and 99.0%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 30:70 is moving phase wash-out, and flow velocity is 8mL/min, and determined wavelength is 304nm, and column temperature is 30 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 1ml/min; Sample size: 20ul; System condition: methyl alcohol-0.2% phosphoric acid solution 90:10, determined wavelength 260nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 6
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 60% of 5 times of weight alcohol reflux 7 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: condition of gradient elution is: 0-15min, and elution system is sherwood oil; 16-50min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 9:3:1;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin that purity is 94.3% and 99.1%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 40:60 is moving phase wash-out, and flow velocity is 6mL/min, and determined wavelength is 304nm, and column temperature is 30 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 1ml/min; Sample size: 20ul; System condition: acetonitrile-0.2% phosphoric acid solution 30:70, determined wavelength 260nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 7
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 75% of 8 times of weight alcohol reflux 4 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-20min, and elution system is sherwood oil; 21-60min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 10:1:2;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin of purity 93.8% and 98.5%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 60:40 is moving phase wash-out, and flow velocity is 7mL/min, and determined wavelength is 304nm, and column temperature is 30 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 1ml/min; Sample size: 10ul; System condition: acetonitrile-0.2% phosphoric acid solution 80:20, determined wavelength 260nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Embodiment 8
A preparation method for high purity karanjin, concrete steps are:
1) the dry root 5kg of dry Tofu pudding pulverize after with 65% of 10 times of weight alcohol reflux 3 times, extracting solution filters, merging filtrate, and recovery ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0-25min, and elution system is sherwood oil; 26-70min, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 7:2:1;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains the karanjin of purity 92.3% and 99.7%.
The chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, and methanol-water: 75:25 is moving phase wash-out, and flow velocity is 7mL/min, and determined wavelength is 304nm, and column temperature is 30 DEG C.
Described step 4) TLC detection method is: get karanjin crude product and add methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, respectively by 20 ug, 40 ug, 60 ug, 80 ug, concentration gradient point sample that 100 ug are different, with sherwood oil-acetone (7:3), dichloromethane-acetone (9.8:0.2), petroleum ether-ethyl acetate-acetone (8:1:1) three kinds of systems for developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns, is single spot, has no impurity spot.
The HPLC detection method of described step 6) is: chromatographic condition: chromatographic column C-18,4.6 × 250mm, 10um; Flow velocity: 1ml/min; Sample size: 10ul; System condition: acetonitrile-0.2% phosphoric acid solution 30:70, determined wavelength 304nm; Area normalization method calculates content, principal constituent (karanjin C
18h
12o
4) peak must not be less than 98.0%, if any impurity peaks, desolventize outside peak, each impurity peak area summation must not more than 2.0%.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. a preparation method for high purity karanjin, is characterized in that, described method specifically comprises the steps:
1) the dry root alcohol reflux of dry Tofu pudding, extracting solution filters, merging filtrate, reclaims ethanol, obtains medicinal extract;
2) medicinal extract is again through petroleum ether extraction, leaves standstill, and merges petroleum ether layer, concentrates to obtain petroleum ether extract;
3) petroleum ether extract is through silica gel column chromatography, and with petroleum ether-ethyl acetate-acetone system gradient elution, collect the flow point containing karanjin, described petroleum ether-ethyl acetate-acetone system condition of gradient elution is: 0 ~ A minute, and elution system is sherwood oil; B ~ C minute, elution system is petroleum ether-ethyl acetate-acetone, and ratio is 10:1:2 ~ 6:4:1; Described A is 10 ~ 25, and described B is 11 ~ 26, and described C is 50 ~ 70;
4) flow point TLC combining data detection, concentrated, recrystallization, obtains the karanjin coarse crystallization that purity is weight content 80 ~ 90%;
5) by the preparative high performance liquid chromatography separation and purification of karanjin coarse crystallization, karanjin component is collected;
6) utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and the purity karanjin that is greater than more than 98%, concentrating under reduced pressure, obtains between purity 90%-98% and is greater than the karanjin of more than 98%.
2. the preparation method of high purity karanjin as claimed in claim 1, is characterized in that, in described step 1), the add-on of ethanol is 5-10 times of medicinal material weight, and alcohol concn is 50-90%, and refluxing extraction number of times is 2-8 time.
3. the preparation method of high purity karanjin as claimed in claim 1, it is characterized in that, the chromatographic column of described step 5) preparative high-performance liquid chromatographic is C-18 post, methanol-water is moving phase wash-out, flow velocity is 5 ~ 10 mL/min, and determined wavelength is 304nm, and column temperature is 25-35 DEG C.
4. the preparation method of high purity karanjin as claimed in claim 3, it is characterized in that, described methanol-water mobile phase methanol-water proportioning is: 15:85 ~ 80:20.
5. the preparation method of high purity karanjin as claimed in claim 1, it is characterized in that, described step 4) TLC detection method is: thin layer plate: silica gel G; 3 kinds of developping agent systems: system (1) sherwood oil-acetone part by weight 7:3, system (2) dichloromethane-acetone part by weight 9.8:0.2, system (3) petroleum ether-ethyl acetate-acetone part by weight 8:1:1; Point sample: with the solution of Methanol 1mg/mL, on same silica gel 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; Put expansion cylinder to launch respectively, exhibition distance: 15cm; Location: spray with 5% ethanol solution of sulfuric acid, dry, be heated to spot development at 105 DEG C clear, inspect under putting ultraviolet lamp (365nm); Result is in thin-layer chromatography, and visible yellowish green single fluorescence spot, 3 kinds of developping agent systems, the gradient point sample of 5 different concns, is single spot, has no impurity spot.
6. the preparation method of high purity karanjin as claimed in claim 1, it is characterized in that, the HPLC detection method of described step 6) is: 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 is one of them of following three conditions:
Condition (1) moving phase: methyl alcohol-0.2% phosphoric acid solution part by weight 30:70-90:10, determined wavelength: 260nm;
Condition (2) moving phase: acetonitrile-0.2% phosphoric acid solution part by weight 30:70-80:20, determined wavelength: 260nm;
Condition (3) moving phase: acetonitrile-0.2% phosphoric acid solution part by weight 20:80-80:20, determined wavelength: 304nm.
7. the preparation method of high purity karanjin as claimed in claim 6, is characterized in that, described system condition (3) moving phase: acetonitrile-0.2% phosphoric acid solution part by weight 30:70-80:20; Determined wavelength: 304nm.
8. a high purity karanjin, is prepared from by preparation method according to claim 1, and its purity is greater than 98%.
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| CN115915934A (en) * | 2020-04-03 | 2023-04-04 | 特尔维瓦股份有限公司 | Water Chinese wampee oil composition, preparation method and application thereof |
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Application publication date: 20150909 Assignee: Guangxi Houde Pharmaceutical Co.,Ltd. Assignor: GUANGXI INSTITUTE OF CHINESE MEDICINE & PHARMACEUTICAL SCIENCE Contract record no.: X2022450000405 Denomination of invention: A Preparation Method of High Purity Aquaxanthin Granted publication date: 20180410 License type: Common License Record date: 20221227 |
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