CN103087070B - The preparation of high purity nitidine chloride and quality controlling means thereof - Google Patents
The preparation of high purity nitidine chloride and quality controlling means thereof Download PDFInfo
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Abstract
A kind of preparation of high purity nitidine chloride and quality controlling means thereof, it is characterized in that: first the two sides crown is pulverized, use acidic ethanol heating and refluxing extraction, ethanol extraction grinds repeatedly through chloroform, gained precipitation dissolve with methanol, hydrochloric acid adjusts pH, recrystallization, obtain nitidine chloride coarse crystallization, use recrystallizing methanol again, further raising purity, be separated by reverse phase silica gel C-18 Column preparation high performance liquid chromatography, analysis mode high performance liquid chromatograph is utilized to detect to collected every a elutriant, merge the identical and purity of retention time between 90% ~ 98% and the nitidine chloride component of more than 98%, concentrating under reduced pressure, the nitidine chloride component that the nitidine chloride of purity between 90% ~ 98% and purity are greater than 98% can be obtained respectively, and adopt thin-layer chromatographic analysis method and liquid phase chromatography analytical method to carry out quality control.The method can not only improve nitidine chloride purity, and can reduce production cost.
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 nitidine chloride.
Background technology
Shinyleaf Pricklyash Root (Zanthoxylum nitidum (Roxb.) DC.), belong to the dry root of 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, for one of Guangxi large main product advantage medicinal material, there is 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, be the first-selected conventional medicine of medical family, be widely used in tcm prescription, Chinese patent medicine and fine chemical product.Annual requirement increases day by day in recent years, and the product using Shinyleaf Pricklyash Root medicinal material to make raw material has multiple kind 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 Guangxi special medicinal material, its nature and flavor are pungent, bitter, tepor, mild toxicity, there is the effects such as dispelling wind and removing obstruction in the collateral, removing dampness to relieve pain, subduing swelling and detoxicating, modern study proves that Shinyleaf Pricklyash Root has swelling and pain relieving, antibacterial isoreactivity, has significant pharmacological action for cardiovascular systems, neural system and unstriated muscle etc.; There is Development volue at anticancer aspect again simultaneously.Guangxi Shinyleaf Pricklyash Root industry annual sales revenue once reached more than 10 hundred million yuan, and Shinyleaf Pricklyash Root product has become one of specialty industries 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 Shinyleaf Pricklyash Root medicinal material is very low, 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 nitidine chloride chemical reference substance, is apparent, has great practical significance and learning value to the modern effect of Chinese medicine.
Open source literature reports the method that many nitidine chlorides extract, as: 1.[inscribes one's name] 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 time pharmaceutical college of Guangxi Medical University [periodical name], 2010,21(11): 2779-2781.[digest] optimum resin of object screening enriching and purifying nitidine chloride and optimised process.Method is by the method for Static Adsorption-desorb, and with the adsorption rate of nitidine chloride and total alkaloids and desorption efficiency for index, Comprehensive Evaluation determines best purifying process.The separating effect of result Ls006 resin to nitidine chloride is best.After 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 given prominence to.2.[inscribes one's name] to wear intelligence in extraction process [author] the thunder roc Liu Shao Li Xin of the preferred Shinyleaf Pricklyash Root of orthogonal experiment bright for brave yellow dawn, Xiang Ya institute of Central South University [periodical name] Chinese materia medica Leader, 2005,11(5): 71-74.[digest] adopt orthogonal experiment method, with the nitidine chloride rate of transform for evaluation index, the extraction process of Shinyleaf Pricklyash Root is carried out preferably.Find use water extraction Shinyleaf Pricklyash Root, the rate of transform of nitidine chloride is quite low, and less than 30%, and with extraction using alcohol, the rate of transform of nitidine chloride can reach 70%, therefore determines extraction solvent ethanol.Select alcohol concn, solvent consumption, extraction time 3 factors, each factor establishes 3 levels.Orthogonal experiment results shows, optimal extract process is Shinyleaf Pricklyash Root medicinal material 60% extraction using alcohol twice, for the first time 9 times amount, extracts 2h, and second time 7 times amount, extract 1.5h.3.[inscribes one's name] 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): 133-136.[digest] optimum extraction process of the preferred Shinyleaf Pricklyash Root medicinal material of object.Method adopts orthogonal test, using volume fraction of ethanol, ethanol consumption, extraction time and extraction time as preferred factor, with paste-forming rate and the nitidine chloride rate of transform for overall target, determines rational ethanol-extracted technique.Result optimal extract process is: volume fraction of ethanol is 70%, and ethanol consumption is 8 times, extracts 4 times, each 1.5h.The extraction process that conclusion optimization obtains is stablized feasible, provides experimental basis for producing.4[inscribes one's name] research [author] Huang of the antitumor effective constituent of Shinyleaf Pricklyash Root control merit Li Zhi and, Guangxi institute of Pharmaceutical Research [periodical name] chemical journal, 1980,38(6): 535-542.[digest] crown 95% ethanol in two sides extracts four times 60 DEG C of temperature, each 4h, filter, merging filtrate, decompression recycling ethanol obtains medicinal extract.This cream 5% acetic acid is pinched molten repeatedly, leaves standstill filtrate, separates out precipitation, and decompression elimination precipitation, precipitation uses 95% washing with alcohol, obtains blue or green yellow mercury oxide.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 backflow, filtrate is placed in 60 DEG C of vacuum flask crystallizations, separates out yellow needles.Leaching crystallization, through recrystallizing methanol, obtains nitidine chloride, and yield is 0.149%.5[inscribes one's name] 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): 342.[digest] use methyl alcohol circumfluence distillation, reclaim under reduced pressure methyl alcohol obtains medicinal extract.Repeatedly grind medicinal extract with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH is 2, and separate out light green needle crystal after placing, be nitidine chloride, yield is 0.2%-0.3%.
Although it is more that current people study the method extracting nitidine chloride, various method is different, but a lot of method extracting nitidine chloride also 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, namely purity is greater than 98%, and there is no nitidine chloride purity and quality controlling means, the needs of high purity nitidine chloride chemical reference substance cannot be met.
Summary of the invention
The preparation method of a kind of high purity nitidine chloride that object of the present invention is developed specially for deficiency existing in above-mentioned prior art just and quality controlling means, this preparation method can not only improve nitidine chloride purity, and can production cost be reduced, the nitidine chloride purity obtained is greater than 98%, quality is good, can as chemical reference substance or standard substance, control of ensuring the quality of products.
The present invention is that its chemical name, molecular formula, structural formula are as follows through extracting and developing, refining, purifying and obtained nitidine chloride from the dry root of Rutaceae xanthoxylum Shinyleaf Pricklyash Root (Zanthoxylum nitidum (Roxb.) DC.):
Chinese name: nitidine chloride
Chemical name: 2,3-dimethoxy-12-methyl-(1,3)-benzo two dislikes luxuriant also (5,6-C) 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 object of the invention is to be realized by following scheme:
The preparation method of high purity nitidine chloride of the present invention, this preparation method comprises following technological process: with the two sides crown after pulverizing for raw material, add water-alcohol solution refluxing extraction three times, first time Plus acidic ethanol 10-15 times amount, reflux 2-5 hour, second time and for the third time Plus acidic ethanol 5-8 times amount, respectively reflux 2-5 hour, merge acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into paste, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH regulator is 2-3, pistac needle crystal is separated out after placing, by recrystallizing methanol, nitidine chloride coarse crystallization can be obtained, use dissolve with methanol again, ultrasonic 30-60 minute, be heated to 80-100 DEG C, filtered while hot, filtrate lets cool, leave standstill 24 hours recrystallizations, separate out the faint yellow fine needle crystal of purity 90-95%, again coarse crystallization is used preparative high performance liquid chromatography separation and purification, chromatographic column is C-18 post, utilize acetonitrile-0.1% trifluoroacetic acid for moving phase wash-out, flow velocity is 5 ~ 10 mL/min, determined wavelength is 271nm, column temperature is 25-35 DEG C, collect 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 the purity nitidine chloride that is greater than more than 98%, concentrating under reduced pressure, obtains between purity 90%-98% and is greater than the nitidine chloride of more than 98%.
The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and 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 is detected as: chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid; Determined wavelength: 271nm; Flow velocity: 0.6 mL/min ~ 1.5mL/min.
The quality controlling means of high purity nitidine chloride of the present invention is: (1) thin-layer chromatographic analysis method; (2) HPLC analytical procedure.
1, described employing thin-layer chromatographic analysis method carries out 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-ethanol ratio (10:1-5:1); System (3) ethyl acetate-ethanol ratio (15:1-8:1).Point sample: on the same plate, by the concentration gradient point sample that 20mg, 40mg, 60mg, 80mg, 100mg are different.Put the saturated 15min of expansion cylinder, launch respectively, exhibition distance: 15cm.Location: spray with 10% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting UV-light (365nm).Result is in thin-layer chromatography, and visible lurid single fluorescence spot, 3 kinds of developping agent systems, the gradient point sample of 5 different concns, is single spot, has no impurity spot, meets 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; Area normalization standard measure.System (1) moving phase: acetonitrile: 0.1% trifluoroacetic acid solution ratio (33:67-20:80); Determined wavelength: 271nm; System (2) moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio (33:67-20:80); Determined wavelength: 254nm; System condition (3) moving phase: methyl alcohol: 0.1% trifluoroacetic acid ratio (50:50-30:70); Determined wavelength: 271nm.
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 and make the solution of every 1ml containing 1mg, under condition determination, sample introduction 20ml(is about equivalent to 20mg), injection liquid chromatography, record color atlas respectively to more than 2.5 times that go out peak retention time of principal constituent by two mobile phase solvent systems, calculate content by area normalization method, result systems measurement reference substance content is all more than 98%.Determination of foreign matter, desolventizes outside 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, Peak homogeneity is carried out 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 completely, are indicated as single pure substance peak.
Result: the nitidine chloride chemical reference substance of separation of the present invention, purifying, detects through infrared spectra, UV spectrum, nucleus magnetic resonance, mass spectrum and physico-chemical property and confirms chemical structure.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.
Compared with prior art, the substantive distinguishing features that the present invention gives prominence to significant progress is:
1, the present invention prepares the supply problem that purity meets the nitidine chloride chemical reference substance of chemical reference substance requirement (content more than 98%) first from Shinyleaf Pricklyash Root, and the quality control for Shinyleaf Pricklyash Root medicinal material provides provides scientific basic and guarantee.
2, the present invention is reasonable in design, technique is simple, alcohol water solvent is utilized to extract, after chloroform process, recrystallization can obtain the higher nitidine chloride of purity, avoids conventional column chromatography method, and organic solvent consumption and energy consumption reduce all greatly, decrease environmental pollution simultaneously, very be applicable to suitability for industrialized production, prepare finally by preparative high performance liquid chromatography the nitidine chloride chemical reference substance that purity reaches more than 98%, method is simple.
3, velocity of separation of the present invention is fast, with short production cycle, has good application prospect.
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 studying nitidine chloride chemical reference substance, set up the analysis determining method of the batch extracting technique of nitidine chloride chemical reference substance, purity and content and determination of foreign matter, thus 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 guarantee.Result of study can be the Essential Chemistry foundation that nitidine chloride 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 nitidine chloride, set up normalized analysis test method, formulate Testing index and the analytical procedure of the control nitidine chloride 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.
Accompanying drawing explanation
Fig. 1 is preparation technology's schema of high purity nitidine chloride;
Fig. 2 is nitidine chloride chromatographic peak uv absorption spectra;
Fig. 3 is nitidine chloride chromatographic peak purity test 5 spectrograms;
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:
With two sides crown root for raw material merges acid alcohol extracting solution through pulverizing--Plus acidic extraction using alcohol--, reclaim ethanol,--repeatedly grinding with chloroform--gained precipitation dissolve with methanol that is concentrated into paste, adding hydrochloric acid to pH regulator is separate out pistac needle crystal after 2-3--places--by recrystallizing methanol,----------filtrate lets cool, and------again by the separation and purification of coarse crystallization preparative high performance liquid chromatography,--chromatographic column is C-18 post--to be utilized acetonitrile-0.1% trifluoroacetic acid for moving phase wash-out and--collects the nitidine chloride component concentrating under reduced pressure that--utilizes HPLC to detect to collected every a elutriant--filtered while hot ultrasonic 30-60 minute to separate out purity 90-95% faint yellow fine needle crystal to leave standstill 24 hours recrystallizations to be heated to 80-100 DEG C to use dissolve with methanol again can to obtain nitidine chloride coarse crystallization, obtain between purity 90%-98% and be greater than the nitidine chloride of 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, containing unsaturated conjugated system in description architecture, UV spectrum conforms to 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 completely, 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 pure substance peak.
As can be seen from Figure 5, retention time about 9 minutes is principal constituent nitidine chloride, and principal constituent content is greater than 98.0%, and each impurity peak area summation is no more than 2.0%, and the HPLC detection color atlas peak result data information of its quality control is as follows:
| 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, first time Plus acidic ethanol 10 times amount, reflux 2 hours; Second time and for the third time Plus acidic ethanol 5 times amount, respectively reflux 2 hours; Merge acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into paste, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH is 2, and separating out pistac needle crystal after placing, by recrystallizing methanol, is nitidine chloride coarse crystallization.The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and ethanol weight content is 40-80%, pH=3-4.
(2) nitidine chloride crude product uses dissolve with methanol again, and ultrasonic 30 minutes, be heated to 90 DEG C, filtered while hot, filtrate let cool, and leave standstill 24 hours recrystallizations, separate out faint yellow fine needle crystal, purity reaches 90-95%.
(3) coarse crystallization is used preparative high performance liquid chromatography separation and purification, chromatographic column is C-18 post, and utilize acetonitrile-0.1% trifluoroacetic acid (30:70) for moving phase wash-out, flow velocity is 5 mL/min, and determined wavelength is 271nm, and column temperature is 25 DEG C; Collect 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); Determined wavelength: 271nm; Flow velocity: 1.0 mL/min, merge the identical and purity of retention time between 90%-98% and be greater than more than 98% nitidine chloride, concentrating under reduced pressure, obtain purity between 90%-98% and be greater than more than 98% nitidine chloride.
Embodiment two: the two sides crown is ground into meal by (1), adds 70% alcohol reflux three times of pH=3, first time Plus acidic ethanol 12 times amount, reflux 3 hours; Second time and for the third time Plus acidic ethanol 6 times amount, respectively reflux 3 hours; Merge acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into paste, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH is 2, and separating out pistac needle crystal after placing, by recrystallizing methanol, is nitidine chloride coarse crystallization.The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and ethanol weight content is 40-80%, pH=3-4.
(2) nitidine chloride crude product uses dissolve with methanol again, and ultrasonic 40 minutes, be heated to 95 DEG C, filtered while hot, filtrate let cool, and leave standstill 24 hours recrystallizations, separate out faint yellow fine needle crystal, purity reaches 90-95%.
(3) coarse crystallization is used preparative high performance liquid chromatography separation and purification, chromatographic column is C-18 post, and utilize acetonitrile-0.1% trifluoroacetic acid (25:75) for moving phase wash-out, flow velocity is 7 mL/min, and determined wavelength is 271nm, and column temperature is 25 DEG C; Collect 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); Determined wavelength: 271nm; Flow velocity: 1.0 mL/min) merge the identical and purity of retention time between 90%-98% and be greater than more than 98% nitidine chloride, concentrating under reduced pressure, obtain purity between 90%-98% and be greater than more than 98% nitidine chloride.
Embodiment three: the two sides crown is ground into meal by (1), adds 50% alcohol reflux three times of pH=4, first time Plus acidic ethanol 15 times amount, reflux 2 hours; Second time and for the third time Plus acidic ethanol 5 times amount, respectively reflux 5 hours; Merge acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into paste, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH is 3, and separating out pistac needle crystal after placing, by recrystallizing methanol, is nitidine chloride coarse crystallization.The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and ethanol weight content is 40-80%, pH=3-4.
(2) nitidine chloride crude product uses dissolve with methanol again, and ultrasonic 60 minutes, be heated to 80 DEG C, filtered while hot, filtrate let cool, and leave standstill 24 hours recrystallizations, separate out faint yellow fine needle crystal, purity reaches 90-95%.
(3) coarse crystallization is used preparative high performance liquid chromatography separation and purification, chromatographic column is C-18 post, and utilize acetonitrile-0.1% trifluoroacetic acid (35:65) for moving phase wash-out, flow velocity is 6 mL/min, and determined wavelength is 271nm, and column temperature is 25 DEG C; Collect 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); Determined wavelength: 271nm; Flow velocity: 6.0 mL/min; Merge the identical and purity of retention time between 90%-98% and be greater than more than 98% nitidine chloride, concentrating under reduced pressure, obtain purity between 90%-98% and be greater than more than 98% nitidine chloride.
The quality controlling means of high purity nitidine chloride:
[TLC distinguish] is got this product and is added methyl alcohol and make every 1ml containing the solution of 1mg, on same silica gel g thin-layer plate, press the concentration gradient point sample that 20mg, 40mg, 60mg, 80mg, 100mg are different respectively, with chloroform-methanol (12:1), chloroform-ethanol (10:1), ethyl acetate-ethanol (15:1) three kinds of systems for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects 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.
[assay] measures according to high performance liquid chromatography (Chinese Pharmacopoeia version annex 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; Determined wavelength is 271nm.Number of theoretical plate calculates should be not less than 3000 by nitidine chloride peak.
Assay method
it is appropriate that precision takes the reference substance being dried to constant weight in 105 DEG C, add methyl alcohol and make the solution of every 1ml containing 1mg, shake up, precision measures 20ml injection liquid chromatography, record color atlas is to more than 2.5 times that go out peak retention time of principal constituent, content is calculated, principal constituent (nitidine chloride C by area normalization method
21h
18nO
4cl) peak must not be less than 98.0%, if any impurity peaks, desolventizes outside peak, and each impurity peak area summation must not more than 2.0%.
Claims (2)
1. a nitidine chloride preparation method, is characterized in that, processing step is as follows: with the two sides crown after pulverizing for raw material, Plus acidic ethanolic soln refluxing extraction three times, first time Plus acidic ethanol 10-15 times amount, reflux 2-5 hour, second time and for the third time Plus acidic ethanol 5-8 times amount, respectively reflux 2-5 hour, merge acid alcohol extracting solution, reclaim ethanol, continue to be concentrated into paste, repeatedly grind with chloroform, gained precipitation dissolve with methanol, adding hydrochloric acid to pH regulator is 2-3, pistac needle crystal is separated out after placing, by recrystallizing methanol, nitidine chloride coarse crystallization can be obtained, use dissolve with methanol again, ultrasonic 30-60 minute, be heated to 80-100 DEG C, filtered while hot, filtrate lets cool, leave standstill 24 hours recrystallizations, separate out the faint yellow fine needle crystal of purity 90-95%, again coarse crystallization is used preparative high performance liquid chromatography separation and purification, chromatographic column is C-18 post, utilize acetonitrile-0.1% trifluoroacetic acid for moving phase wash-out, flow velocity is 5-10mL/min, determined wavelength is 271nm, column temperature is 25-35 DEG C, collect 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 the purity nitidine chloride component that is greater than more than 98%, concentrating under reduced pressure, obtains between purity 90%-98% and is greater than the nitidine chloride of more than 98%,
The hydrochloric acid weight content of described acidic ethanol is 10-30% hydrochloric acid, and ethanol weight content is 40-80%, pH=3-4;
Described acetonitrile-0.1% trifluoroacetic acid moving phase wash-out weight proportion is 20:80 ~ 40:60;
Described HPLC is detected as: chromatographic column: C-18 post; Moving phase: acetonitrile: 0.1% trifluoroacetic acid; Determined wavelength: 271nm; Flow velocity: 0.6mL/min ~ 1.5mL/min.
2. a detection method for nitidine chloride, is characterized in that: adopt thin-layer chromatographic analysis method or HPLC analytical procedure to detect;
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 is 12:1-6:1; System (2) chloroform-ethanol ratio is 10:1-5:1; System (3) ethyl acetate-ethanol ratio is 15:1-8:1, point sample: on the same plate, by 20 μ g, 40 μ g, 60 μ g, 80 μ g, concentration gradient point sample that 100 μ g are different, put the saturated 15min of expansion cylinder, launch respectively, exhibition distance: 15cm, location: spray with 10% 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;
Described HPLC analytical procedure: chromatographic condition: C-18 post, 4.6 × 25cm; Flow velocity: 0.6-1.5ml/min; Sample size: 10-20 μ l; Area normalization standard measure; System (1) moving phase: acetonitrile: 0.1% trifluoroacetic acid solution ratio is 33:67-20:80; Determined wavelength: 271nm; System (2) moving phase: acetonitrile: 0.1% trifluoroacetic acid ratio is 33:67-20:80; Determined wavelength: 254nm; System condition (3) moving phase: methyl alcohol: 0.1% trifluoroacetic acid ratio is 50:50-30:70; Determined wavelength: 271nm;
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 and make the solution of every 1ml containing 1mg, under condition determination, injection liquid chromatography, record color atlas respectively to more than 2.5 times that go out peak retention time of principal constituent by two mobile phase solvent systems, calculate content by area normalization method, result measures reference substance content all more than 98%. determination of foreign matter, desolventize outside 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, Peak homogeneity is carried out 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 completely, are indicated as single pure substance peak.
Priority Applications (1)
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| CN106970167A (en) * | 2017-04-28 | 2017-07-21 | 广西壮族自治区梧州食品药品检验所 | A kind of method of Nitidine Chloride content in measure shiny pricklyash toothpaste |
| CN107417692B (en) * | 2017-07-21 | 2019-03-12 | 广东罗浮山国药股份有限公司 | A kind of method of purification of chlorinated nitidine |
| CN108467399A (en) * | 2018-03-16 | 2018-08-31 | 广西壮族自治区中医药研究院 | A method of preparing Nitidine Chloride from total alkali extract of Radix zanthoxyli stem branch |
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| CN101361850A (en) * | 2008-09-26 | 2009-02-11 | 广西医科大学 | Method for extracting and separating total alkaloids in prickly ash |
| CN101768163A (en) * | 2010-01-26 | 2010-07-07 | 广西医科大学 | Method for separating purified nitidine chloride by using cation exchange resin |
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| CN101361850A (en) * | 2008-09-26 | 2009-02-11 | 广西医科大学 | Method for extracting and separating total alkaloids in prickly ash |
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