CN103087070A - Preparation method for high-purity nitidine chloride as well as quality control method of high-purity nitidine chloride - Google Patents
Preparation method for high-purity nitidine chloride as well as quality control method of high-purity nitidine chloride Download PDFInfo
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Abstract
The invention discloses a preparation method for high-purity nitidine chloride as well as a quality control method of high-purity nitidine chloride. The preparation method is characterized by comprising the following steps of: crushing radix zanthoxyli roots, heating up, refluxing and extracting by using acid alcohol; repeatedly grinding alcohol extract by chloroform; dissolving the obtained precipitates by using methanol, adjusting pH of the solution by using hydrochloric acid, and obtaining nitidine chloride coarse crystals by recrystallization; carrying out recrystallization by using methanol for further improving the purity; preparing by using reverse phase silica gel C-18 and separating by using high-performance liquid chromatography; detecting every part of collected eluant by utilizing an analysis type high performance liquid chromatograph; combining nitidine chloride components with the same retention time, and purity of 90%-98% and more than 98%; concentrating under reduced pressure to obtain nitidine chloride components with purity of 90%-98% and nitidine chloride components with purity bigger than 98%; and carrying out quality control by adopting a thin-layer chromatographic analysis method and a liquid-phase chromatographic analysis method. According to the preparation method disclosed by the invention, not only can the purity of the nitidine chloride be improved, but also the production cost can be reduced.
Description
Technical field
The present invention relates to a kind of natural medicine field, be specifically related to a kind of preparation and quality controlling means thereof of high purity chlorination nitidine.
Background technology
Shinyleaf Pricklyash Root (Zanthoxylum nitidum (Roxb.) DC.), the dry root that belongs to Rutaceae Shinyleaf Pricklyash Root plant, it is the endemic plant of south China, it is the conventional Chinese medicine that the Pharmacopoeia of the People's Republic of China records, be one of Guangxi large main product advantage medicinal material, having good promoting the circulation of QI to relieve pain, promoting blood circulation and removing blood stasis, dispelling wind and removing obstruction in the collateral effect, is medicine man medicine first-selected commonly used, is widely used in tcm prescription, Chinese patent medicine and fine chemical product.Annual requirement increases day by day in recent years, and the product that uses the Shinyleaf Pricklyash Root medicinal material to make raw material has a plurality of kinds such as national reputable brand " Shinyleaf Pricklyash Root Chinese medicinal toothpaste ", " SANJIU WEITAI ", " JINJI JIAONANG ", " compound Radix zanthoxyli lozenge ", " Shinyleaf Pricklyash Root analgesia sheet ", " bone-setting liquor ", " ZHONGHUA DIEDA WAN ", " Huoluozhitong pills ".Shinyleaf Pricklyash Root is the Guangxi special medicinal material, its nature and flavor are hot, bitter, tepor, mild toxicity, have the effects such as dispelling wind and removing obstruction in the collateral, removing dampness to relieve pain, subduing swelling and detoxicating, modern study proof Shinyleaf Pricklyash Root has swelling and pain relieving, antibiotic isoreactivity, for cardiovascular systems, neural system and unstriated muscle etc., significant pharmacological action is arranged; There is exploitation to be worth at anticancer aspect again simultaneously.Guangxi Shinyleaf Pricklyash Root industry annual sales revenue once reached more than 10 hundred million yuan, and the Shinyleaf Pricklyash Root product has become one of specialty industries of revitalizing local economy.Nitidine chloride is one of Shinyleaf Pricklyash Root activeconstituents, it is Pharmacopoeia of the People's Republic of China set quota composition, nitidine chloride is as the chemical reference substance of Shinyleaf Pricklyash Root and products thereof, it is the key problem in technology of quality control, numerous enterprises, scientific research and inspection department all need highly purified nitidine chloride reference substance, its market requirement is very large, and because the content of nitidine chloride in the Shinyleaf Pricklyash Root medicinal material is very low, the extraction and separation technology requirement is very high, difficulty is very large.The present invention carries out the preparation of nitidine chloride Chemistry for Chinese Traditional Medicine standard substance and Quality Control Technology thereof, solves the problem of high purity chlorination nitidine chemical reference substance, and modern effect is apparent to Chinese medicine, has great practical significance and learning value.
Open source literature has been reported the method that many nitidine chlorides extract, as: 1.[autograph] nitidine chloride [author] Lu Ling Chun Fanglinqiaolong Shengjing city in resin cation (R.C.) purifying Shinyleaf Pricklyash Root, precious traditional Chinese medical science traditional Chinese medicines during pharmaceutical college of Guangxi Medical University [periodical name], 2010,21(11): the 2779-2781.[digest] optimum resin and the optimised process of purpose screening enriching and purifying nitidine chloride.The method of method by Static Adsorption-desorb, take the adsorption rate of nitidine chloride and total alkaloids and desorption efficiency as index, the comprehensive judge determined best purifying process.The Ls006 resin is best to the separating effect of nitidine chloride as a result.After the Ls006 resin separation purification, in finished product, the purity of nitidine chloride is greater than 90%, and retention rate reaches 56.84%.Conclusion adopts nitidine chloride in Ls006 resin cation (R.C.) separation and purification Shinyleaf Pricklyash Root, and simple to operate, purification effect is outstanding.2.[autograph] to wear intelligence in extraction process [author] the thunder roc Liu Shao Li Xin of the preferred Shinyleaf Pricklyash Root of orthogonal experiment bright brave yellow dawn, the refined institute in Central South University Hunan [periodical name] Chinese materia medica Leader, 2005,11(5): the 71-74.[digest] adopt orthogonal experiment method, take the nitidine chloride rate of transform as evaluation index, the extraction process of Shinyleaf Pricklyash Root is carried out preferably.Find to use the water extraction Shinyleaf Pricklyash Root, the rate of transform of nitidine chloride is quite low, less than 30%, and uses extraction using alcohol, and the rate of transform of nitidine chloride can reach 70%, therefore determine to extract solvent ethanol.Select alcohol concn, solvent consumption, extraction time 3 factors, each factor is established 3 levels.Orthogonal experiment results shows, optimal extract process is the Shinyleaf Pricklyash Root medicinal material with 60% extraction using alcohol twice, and 9 times of amounts, extract 2h for the first time, and 7 times of amounts, extract 1.5h for the second time.3.[autograph] the U.S. suitable Zhou Yisheng of the right beam of the intelligent yellow rhythm of Shinyleaf Pricklyash Root Study on extraction [author] Lv Jie Lu Xiao, Guangdong Pharmaceutical University's [periodical name] ACAD J GCP, 2011,27(2): the 133-136.[digest] optimum extraction process of the preferred Shinyleaf Pricklyash Root medicinal material of purpose.Method adopts orthogonal test, as preferred factor, take paste-forming rate and the nitidine chloride rate of transform as overall target, determines rational ethanol-extracted technique with volume fraction of ethanol, ethanol consumption, extraction time and extraction time.Optimal extract process is as a result: volume fraction of ethanol is 70%, and the ethanol consumption is 8 times, extracts 4 times, each 1.5h.The extraction process that conclusion optimization obtains is stablized feasible, for production provides experimental basis.4[autograph] research [author] Huang of the antitumor effective constituent of Shinyleaf Pricklyash Root control merit Li Zhi and, the chemical journal of Guangxi institute of Pharmaceutical Research [periodical name], 1980,38(6): the 535-542.[digest] the two sides crown extracts four times 60 ℃ of temperature with 95% ethanol, each 4h, filter, merging filtrate, decompression recycling ethanol gets medicinal extract.This cream is pinched molten repeatedly with 5% acetic acid, standing filtrate is separated out precipitation, decompression elimination precipitation, and precipitation is used 95% washing with alcohol, gets bluish yellow look precipitation.This precipitation is dissolved in warm water ammoniacal liquor ammonification, chloroform extraction.Reclaim chloroform, residue is dissolved in hydrochloric acid, places for some time and separates out coarse crystallization.Coarse crystallization is dissolved in hot methanol, decolorizing with activated carbon, filtered while hot after water-bath refluxes, filtrate is placed in 60 ℃ of vacuum flask crystallizations, separates out yellow needle crystal.The leaching crystallization through recrystallizing methanol, gets nitidine chloride, and yield is 0.149%.5[autograph] the research I of Shinyleaf Pricklyash Root chemical composition: structural research [author] Wang Xinmei [periodical name] the Zhongshan Medical College journal with the alkaloidal separation of antitumour activity and alkaloid third, 1980,1(4): the 342.[digest] extract with the methyl alcohol thermal backflow, reclaim under reduced pressure methyl alcohol gets medicinal extract.Repeatedly grind medicinal extract with chloroform, gained precipitation is used dissolve with methanol, add hydrochloric acid to pH be 2, separate out light green needle crystal after placement, be nitidine chloride, yield is 0.2%-0.3%.
Although it is more that people study the method for extracting nitidine chloride at present, the whole bag of tricks is different, but the method for much extracting nitidine chloride exists the weak point that purity is low, yield is low or production cost is high, purity does not all reach the requirement of traditional Chinese chemical contrast, be that purity is greater than 98%, and there is no nitidine chloride purity and quality controlling means, can't satisfy the needs of high purity chlorination nitidine chemical reference substance.
Summary of the invention
Purpose of the present invention is just for existing deficiency in above-mentioned prior art and preparation method and the quality controlling means of a kind of high purity chlorination nitidine of special development, this preparation method can not only improve nitidine chloride purity, and can reduce production costs, the nitidine chloride purity that obtains greater than 98%, quality is good, can be used as chemical reference substance or standard substance, the control of ensuring the quality of products.
The present invention be from the dry root of Rutaceae xanthoxylum Shinyleaf Pricklyash Root (Zanthoxylum nitidum (Roxb.) DC.) through extract, separate, refining, purifying and the nitidine chloride that makes, its chemical name, molecular formula, structural formula are as follows:
Chinese name: nitidine chloride
Chemical name: 2,3-dimethoxy-12-methyl-(1,3)-benzo two dislikes luxuriant also (5,6-C) the phenanthridines muriate (2,3-dimethoxy-12-methyl-[1,3] benzodioxolo[5,6-c] phenanthridin-12-ium chloride)
English name: Nitidine chloride
Molecular formula: C
21H
18NO
4Cl
Structural formula:
The objective of the invention is to realize by following scheme:
the preparation method of high purity chlorination nitidine of the present invention, this preparation method comprises following technological process: the two sides crown after to pulverize adds the water-alcohol solution refluxing extraction three times as raw material, adds for the first time acid ethanol 10-15 and doubly measures, reflux 2-5 hour, add for the second time and for the third time acid ethanol 5-8 and doubly measure, reflux is 2-5 hour respectively, merge the acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into the thick paste shape, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH regulator is 2-3, separate out pistac needle crystal after placement, use recrystallizing methanol, can obtain the nitidine chloride coarse crystallization, use again dissolve with methanol, ultrasonic 30-60 minute, be heated to 80-100 ℃, filtered while hot, filtrate lets cool, standing 24 hours recrystallizations, separate out the faint yellow fine needle crystal of purity 90-95%, again coarse crystallization is used the preparative high performance liquid chromatography separation and purification, chromatographic column is the C-18 post, utilize acetonitrile-0.1% trifluoroacetic acid to be the moving phase wash-out, flow velocity is 5～10 mL/min, the detection wavelength is 271nm, column temperature is 25-35 ℃, collect the nitidine chloride component, and utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and purity greater than the nitidine chloride more than 98%, concentrating under reduced pressure obtains between purity 90%-98% and greater than the nitidine chloride more than 98%.
The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and the ethanol weight content is 40-80%, pH=3-4.
Described acetonitrile-0.1% trifluoroacetic acid moving phase wash-out proportioning is: 20:80～40:60.
Described HPLC detects: chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid; Detect wavelength: 271nm; Flow velocity: 0.6 mL/min～1.5mL/min.
The quality controlling means of high purity chlorination nitidine of the present invention is: (1) thin-layer chromatographic analysis method; (2) HPLC analytical procedure.
1, described employing thin-layer chromatographic analysis method is carried out the nitidine chloride analytical procedure: thin layer plate: silica gel G-0.8% Xylo-Mucine.3 kinds of developping agent systems: system (1) chloroform-methanol ratio (12:1-6:1); System (2) chloroform-proportion of ethanol (10:1-5:1); System (3) ethyl acetate-ethanol ratio (15:1-8:1).Point sample: on same plate, press 20mg, 40mg, 60mg, 80mg, concentration gradient point sample that 100mg is different.Put the saturated 15min of expansion cylinder, launch respectively, exhibition distance: 15cm.The location: spray is heated to the spot colour developing at 105 ℃ clear with 10% ethanol solution of sulfuric acid, puts under UV-light (365nm) and inspects.Result in thin-layer chromatography, visible lurid single fluorescence spot, 3 kinds of developping agent systems, the gradient point sample of 5 different concns is single spot, has no the impurity spot, meets the chemical reference substance requirement.
2, described HPLC analytical procedure: chromatographic condition: C-18 post 4.6 * 25cm; Flow velocity: 1ml/min; Sample size: 10-20ml; The area normalization standard measure.System (1) moving phase: acetonitrile: 0.1% trifluoroacetic acid solution ratio (33:67-20:80); Detect wavelength: 271nm; System (2) moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio (33:67-20:80); Detect wavelength: 254nm; System condition (3) moving phase: methyl alcohol: 0.1% trifluoroacetic acid ratio (50:50-30:70); Detect wavelength: 271nm.
Content and purity testing: precision takes in 105 ℃ of reference substances that are dried to constant weight appropriate, add moving phase and make the solution that every 1ml contains 1mg, under condition determination, sample introduction 20ml(approximately is equivalent to 20mg), the injection liquid chromatography, with two mobile phase solvent systems record respectively color atlas to principal constituent go out more than 2.5 times of peak retention time, calculate content with area normalization method, systems measurement reference substance content is all more than 98% as a result.Determination of foreign matter desolventizes outside the peak, and impurity peak area summation result is all less than 2.0%.
Peak purity detects: get reference substance appropriate, by system (1), on high performance liquid chromatograph, carry out Peak homogeneity with diode array DAD detector, HPLC color atlas (〉 98%), the uv absorption spectra of its chromatographic peak, three-dimensional collection of illustrative plates and 5 spectrograms overlap fully, are indicated as single pure substance peak.
Result: the nitidine chloride chemical reference substance of separation of the present invention, purifying, detect through infrared spectra, UV spectrum, nucleus magnetic resonance, mass spectrum and physico-chemical property and confirm chemical structure.TLC through 5 different concns of 3 development systems detects, and the HPLC of 2 flow phase system and 2 different wave lengths detects, and simultaneously chromatographic peak is done purity test with DAD, shows to meet the requirement that the Chinese medicine assay is used chemical reference substance, and content is greater than 98%.
Compared with prior art, the substantive distinguishing features given prominence to of the present invention and significant progress are:
1, the present invention prepares the supply problem that purity meets the nitidine chloride chemical reference substance of chemical reference substance requirement (content is more than 98%) first from Shinyleaf Pricklyash Root, for the quality control of Shinyleaf Pricklyash Root medicinal material provides scientific basic and assurance.
2, the present invention is reasonable in design, technique is simple, utilize pure water solvent to extract, after processing through chloroform, recrystallization can obtain the higher nitidine chloride of purity, has avoided column chromatography method commonly used, and organic solvent consumption and energy consumption all reduce greatly, reduced simultaneously environmental pollution, be fit to very much suitability for industrialized production, prepare purity finally by preparative high performance liquid chromatography and reach nitidine chloride chemical reference substance more than 98%, method is simple.
3, velocity of separation of the present invention is fast, and is with short production cycle, and good application prospect is arranged.
4, 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.
By the nitidine chloride chemical reference substance is studied, set up batch extracting technique, purity and the content of nitidine chloride chemical reference substance and the analysis determining method of determination of foreign matter, thereby set up the technological standard of nitidine chloride chemical reference substance, for its quality standard research as traditional Chinese chemical contrast and medicinal material and preparation provides scientific basic and assurance.Result of study can be the nitidine chloride chemical reference substance more complete Essential Chemistry foundation is provided, 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, will produce potential and immeasurable social benefit and economic benefit.The various products of producing from now on, no matter be at home or enter the world market, all need obtain development and improve by high-caliber quality standard and high-caliber analysis and testing technology, otherwise will lose market, it is more and more important that the quality standard of product and detection method become, and the medication standard of " safe, effective and quality controllable " is become a consensus of the international community, and pharmaceutical production should be round this center deployment, its core is quality standard control level, and chemical reference substance plays a key role.but present most of Chinese medicinal material and preparation thereof, not clear or without chemical reference substance because of chemical composition, can't illustrate the chemical substance basis of its effect, also can't carry out quality control, and can not be accepted by modern civilization society, also become the restriction key that herbal medicine and natural drug are difficult to enter international drug market, technology barriers have brought difficulty for the 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 of modernization of Chinese medicine development, formulation to the production and processing technology of the basic substance of illustrating the herbal medicine effect and Chinese herbal and crude drugs preparations, discriminating of low-quality goods etc. has great importance.Undoubtedly, nitidine chloride is carried out quality standard research, set up normalized analysis test method, formulate detection index and the analytical procedure of the control nitidine chloride quality of hi-tech level, make it scientific, standardization, enhance our international competitiveness, create conditions for Chinese medicine enters the world market, have great practical significance and learning value.
Description of drawings
Fig. 1 is preparation technology's schema of high purity chlorination nitidine;
Fig. 2 is nitidine chloride chromatographic peak uv absorption spectra;
Fig. 3 is 5 spectrograms of nitidine chloride chromatographic peak purity test;
Fig. 4 is nitidine chloride DAD Peak homogeneity HPLC color atlas;
Fig. 5 is that the HPLC of nitidine chloride quality control detects color atlas.
Recognize from Fig. 1, concrete technology is:
take two sides crown root as raw material, through pulverizing--adding acid alcohol extract--, merge the acid alcohol extract, reclaim ethanol,--repeatedly grinding with chloroform--gained precipitation is dissolved with methyl alcohol to be concentrated into the thick paste shape, adding hydrochloric acid is adjusted to after 2-3--places and separates out pistac acicular crystal to pH--use recrystallizing methanol, can obtain the Nitidine Chloride coarse crystallization and--dissolve with methyl alcohol again that--ultrasonic 30-60 minute--is heated to 80-100 ℃ and--filters while hot--filtrate lets cool--standing 24 hours recrystallizations------chromatographic column is the C-18 post------collected every a eluent is utilized to the HPLC detection--reduced pressure concentration of collecting the Nitidine Chloride component that utilizes acetonitrile-0.1% trifluoroacetic acid to be the mobile phase wash-out of again coarse crystallization use to the preparative high performance liquid chromatography separation and purification of separating out the faint yellow fine needle crystal of purity 90-95%, obtain between purity 90%-98% and greater than the Nitidine Chloride more than 98%.
As can be seen from Figure 2, the UV spectrum of nitidine chloride 271, there is maximum absorption band at 329 ± 1nm place, contains unsaturated conjugated system in description architecture, UV spectrum conforms to the nitidine chloride chemical structure.
As can be seen from Figure 3, the spectral purity inspection of 5 chromatographic peaks of nitidine chloride, 5 spectrum overlap fully, are indicated as single pure substance peak.
As can be seen from Figure 4, the diode array DAD Peak homogeneity HPLC color atlas of nitidine chloride is single peak, is indicated as the pure substance peak.
As can be seen from Figure 5, retention time is the principal constituent nitidine chloride about 9 minutes, and principal constituent content is greater than 98.0%, and each impurity peak area summation is no more than 2.0%, and it is as follows that the HPLC of its quality control detects color atlas peak result data information:
| Sequence number | Retention time (minute) | Peak area | Result (%) | Theoretical plate number |
| 1 | 4.85 | 34658 | 0.10 | 14419 |
| 2 | 6.44 | 2863 | 0.01 | 9973 |
| 3 | 7.09 | 36346 | 0.11 | 6315 |
| 4 | 7.86 | 64554 | 0.19 | 17039 |
| 5 | 8.43 | 7141 | 0.02 | 19163 |
| 6 | 9.68 | 33760526 | 99.21 | 3299 |
| 7 | 12.77 | 2588 | 0.01 | 17078 |
| 8 | 14.00 | 89879 | 0.26 | 18343 |
| 9 | 19.51 | 32021 | 0.09 | 18605 |
Embodiment
Embodiment one:
(1) the two sides crown is ground into meal, adds 80% alcohol reflux three times of pH=3, add for the first time 10 times of amounts of acid ethanol, reflux 2 hours; Add for the second time and for the third time 5 times of amounts of acid ethanol, reflux is 2 hours respectively; Merge the acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into the thick paste shape, repeatedly grind with chloroform, the gained precipitation is used dissolve with methanol, add hydrochloric acid to pH be 2, separate out pistac needle crystal after placement, use recrystallizing methanol, be the nitidine chloride coarse crystallization.The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and the ethanol weight content is 40-80%, pH=3-4.
(2) the nitidine chloride crude product is used dissolve with methanol again, and ultrasonic 30 minutes, be heated to 90 ℃, filtered while hot, filtrate lets cool, and standing 24 hours recrystallizations are separated out faint yellow fine needle crystal, and purity reaches 90-95%.
(3) coarse crystallization is used the preparative high performance liquid chromatography separation and purification, chromatographic column is the C-18 post, and utilizing acetonitrile-0.1% trifluoroacetic acid (30:70) is the moving phase wash-out, and flow velocity is 5 mL/min, and the detection wavelength is 271nm, and column temperature is 25 ℃; Collect the nitidine chloride component, and utilize HPLC to detect to collected every a elutriant, chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio (33:67); Detect wavelength: 271nm; Flow velocity: 1.0 mL/min, merge the identical and purity of retention time between 90%-98% and greater than the nitidine chloride more than 98%, concentrating under reduced pressure obtains purity between 90%-98% and greater than the nitidine chloride more than 98%.
Embodiment two: (1) is ground into meal with the two sides crown, adds 70% alcohol reflux three times of pH=3, adds for the first time 12 times of amounts of acid ethanol, reflux 3 hours; Add for the second time and for the third time 6 times of amounts of acid ethanol, reflux is 3 hours respectively; Merge the acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into the thick paste shape, repeatedly grind with chloroform, the gained precipitation is used dissolve with methanol, add hydrochloric acid to pH be 2, separate out pistac needle crystal after placement, use recrystallizing methanol, be the nitidine chloride coarse crystallization.The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and the ethanol weight content is 40-80%, pH=3-4.
(2) the nitidine chloride crude product is used dissolve with methanol again, and ultrasonic 40 minutes, be heated to 95 ℃, filtered while hot, filtrate lets cool, and standing 24 hours recrystallizations are separated out faint yellow fine needle crystal, and purity reaches 90-95%.
(3) coarse crystallization is used the preparative high performance liquid chromatography separation and purification, chromatographic column is the C-18 post, and utilizing acetonitrile-0.1% trifluoroacetic acid (25:75) is the moving phase wash-out, and flow velocity is 7 mL/min, and the detection wavelength is 271nm, and column temperature is 25 ℃; Collect the nitidine chloride component, and utilize HPLC to detect to collected every a elutriant, (chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio (30:70); Detect wavelength: 271nm; Flow velocity: 1.0 mL/min) merge the identical and purity of retention time between 90%-98% and greater than the nitidine chloride more than 98%, concentrating under reduced pressure obtains purity between 90%-98% and greater than the nitidine chloride more than 98%.
Embodiment three: (1) is ground into meal with the two sides crown, adds 50% alcohol reflux three times of pH=4, adds for the first time 15 times of amounts of acid ethanol, reflux 2 hours; Add for the second time and for the third time 5 times of amounts of acid ethanol, reflux is 5 hours respectively; Merge the acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into the thick paste shape, repeatedly grind with chloroform, the gained precipitation is used dissolve with methanol, add hydrochloric acid to pH be 3, separate out pistac needle crystal after placement, use recrystallizing methanol, be the nitidine chloride coarse crystallization.The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and the ethanol weight content is 40-80%, pH=3-4.
(2) the nitidine chloride crude product is used dissolve with methanol again, and ultrasonic 60 minutes, be heated to 80 ℃, filtered while hot, filtrate lets cool, and standing 24 hours recrystallizations are separated out faint yellow fine needle crystal, and purity reaches 90-95%.
(3) coarse crystallization is used the preparative high performance liquid chromatography separation and purification, chromatographic column is the C-18 post, and utilizing acetonitrile-0.1% trifluoroacetic acid (35:65) is the moving phase wash-out, and flow velocity is 6 mL/min, and the detection wavelength is 271nm, and column temperature is 25 ℃; Collect the nitidine chloride component, and utilize HPLC to detect to collected every a elutriant, chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio (35:75); Detect wavelength: 271nm; Flow velocity: 6.0 mL/min; Merge the identical and purity of retention time between 90%-98% and greater than the nitidine chloride more than 98%, concentrating under reduced pressure obtains purity between 90%-98% and greater than the nitidine chloride more than 98%.
The quality controlling means of high purity chlorination nitidine:
[thin layer discriminating] got this product and added methyl alcohol and make the solution that every 1ml contains 1mg, on same silica gel g thin-layer plate, press respectively 20mg, 40mg, 60mg, 80mg, concentration gradient point sample that 100mg is different, take chloroform-methanol (12:1), chloroform-ethanol (10:1), ethyl acetate-ethanol (15:1) three kinds of systems as developping agent, launch, take out, dry, spray is heated to the spot colour developing at 105 ℃ clear with 10% ethanol solution of sulfuric acid, puts under UV-light (365nm) and inspects.In thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns is single spot, has no the impurity spot.
[assay] measured according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix VI D in 2010).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are weighting agent, acetonitrile: 0.1% trifluoroacetic acid solution (33:67) is moving phase; The detection wavelength is 271nm.Number of theoretical plate should be not less than 3000 by nitidine chloride peak calculating.
Assay method
Precision takes in 105 ℃ of reference substances that are dried to constant weight appropriate, add methyl alcohol and make the solution that every 1ml contains 1mg, shake up, precision measures 20ml injection liquid chromatography, record color atlas and go out more than 2.5 times of peak retention time to principal constituent, calculate content with area normalization method, principal constituent (nitidine chloride C
21H
18NO
4Cl) peak must not be less than 98.0%, if any impurity peaks, desolventizes outside the peak, and each impurity peak area summation must not surpass 2.0%.
Claims (6)
1. high purity chlorination nitidine preparation method, it is characterized in that: comprise following technological process: the two sides crown after to pulverize is as raw material, add acid ethanolic soln refluxing extraction three times, add for the first time acid ethanol 10-15 and doubly measure, reflux 2-5 hour, add for the second time and for the third time acid ethanol 5-8 and doubly measure, reflux is 2-5 hour respectively, merge the acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into the thick paste shape, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH regulator is 2-3, separate out pistac needle crystal after placement, use recrystallizing methanol, can obtain the nitidine chloride coarse crystallization, use again dissolve with methanol, ultrasonic 30-60 minute, be heated to 80-100 ℃, filtered while hot, filtrate lets cool, standing 24 hours recrystallizations, separate out the faint yellow fine needle crystal of purity 90-95%, again coarse crystallization is used the preparative high performance liquid chromatography separation and purification, chromatographic column is the C-18 post, utilize acetonitrile-0.1% trifluoroacetic acid to be the moving phase wash-out, flow velocity is 5-10 mL/min, the detection wavelength is 271nm, column temperature is 25-35 ℃, collect the nitidine chloride component, and utilize HPLC to detect to collected every a elutriant, merge the identical and purity of retention time between 90%-98% and purity greater than the nitidine chloride component more than 98%, concentrating under reduced pressure obtains between purity 90%-98% and greater than the nitidine chloride more than 98%,
The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and the ethanol weight content is 40-80%, pH=3-4.
2. high purity chlorination nitidine preparation method according to claim 1, it is characterized in that: described acetonitrile-0.1% trifluoroacetic acid moving phase wash-out weight proportion is 20:80～40:60.
3. high purity chlorination nitidine preparation method according to claim 1 is characterized in that: described HPLC detects and is: chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid; Detect wavelength: 271nm; Flow velocity: 0.6 mL/min～1.5mL/min.
4. the quality controlling means of a high purity chlorination nitidine, is characterized in that: adopt thin-layer chromatographic analysis method and HPLC analytical procedure.
5. the quality controlling means of high purity chlorination nitidine according to claim 4, it is characterized in that: described thin-layer chromatographic analysis method is: thin layer plate: silica gel G-0.8% Xylo-Mucine, three kinds of developping agent systems: system (1) chloroform-methanol ratio (12:1-6:1); System (2) chloroform-proportion of ethanol (10:1-5:1); System (3) ethyl acetate-ethanol ratio (15:1-8:1), point sample: on same plate, press 20mg, 40mg, 60mg, 80mg, concentration gradient point sample that 100mg is different, put the saturated 15min of expansion cylinder, launch respectively, exhibition distance: 15cm, the location: spray is with 10% ethanol solution of sulfuric acid, be heated to the spot colour developing at 105 ℃ clear, put under UV-light (365nm) and inspect, in thin-layer chromatography, three kinds of developping agent systems, the gradient point sample of five different concns is single spot, has no the impurity spot.
6. the quality controlling means of according to claim 4 with 5 described high purity chlorination nitidines, is characterized in that: described HPLC analytical procedure: chromatographic condition: C-18 post, 4.6 * 25cm; Flow velocity: 0.6-1.5ml/min; Sample size: 10-20ml; The area normalization standard measure; System (1) moving phase: acetonitrile: 0.1% trifluoroacetic acid solution ratio (33:67-20:80); Detect wavelength: 271nm; System (2) moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio (33:67-20:80); Detect wavelength: 254nm; System condition (3) moving phase: methyl alcohol: 0.1% trifluoroacetic acid ratio (50:50-30:70); Detect wavelength: 271nm;
Content and purity testing: precision takes in 105 ℃ of reference substances that are dried to constant weight appropriate, add moving phase and make the solution that every 1ml contains 1mg, under condition determination, the injection liquid chromatography, with two mobile phase solvent systems record respectively color atlas to principal constituent go out more than 2.5 times of peak retention time, calculate content with area normalization method, result is measured reference substance content all more than 98%. determination of foreign matter, desolventize outside the peak, impurity peak area summation result is all less than 2.0%;
Described peak purity detects: get reference substance appropriate, by system (1), on high performance liquid chromatograph, carry out Peak homogeneity with diode array DAD detector, HPLC color atlas (〉 98%), the uv absorption spectra of its chromatographic peak, three-dimensional collection of illustrative plates and 5 spectrograms overlap fully, are indicated as single pure substance peak.
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