CN113651830A - Homoharringtonine and process production method thereof - Google Patents
Homoharringtonine and process production method thereof Download PDFInfo
- Publication number
- CN113651830A CN113651830A CN202110868850.3A CN202110868850A CN113651830A CN 113651830 A CN113651830 A CN 113651830A CN 202110868850 A CN202110868850 A CN 202110868850A CN 113651830 A CN113651830 A CN 113651830A
- Authority
- CN
- China
- Prior art keywords
- homoharringtonine
- extract
- class
- eluent
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- HYFHYPWGAURHIV-UHFFFAOYSA-N homoharringtonine Natural products C1=C2CCN3CCCC43C=C(OC)C(OC(=O)C(O)(CCCC(C)(C)O)CC(=O)OC)C4C2=CC2=C1OCO2 HYFHYPWGAURHIV-UHFFFAOYSA-N 0.000 title claims abstract description 140
- HYFHYPWGAURHIV-JFIAXGOJSA-N omacetaxine mepesuccinate Chemical compound C1=C2CCN3CCC[C@]43C=C(OC)[C@@H](OC(=O)[C@@](O)(CCCC(C)(C)O)CC(=O)OC)[C@H]4C2=CC2=C1OCO2 HYFHYPWGAURHIV-JFIAXGOJSA-N 0.000 title claims abstract description 108
- 229960002230 omacetaxine mepesuccinate Drugs 0.000 title claims abstract description 108
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 56
- 238000010828 elution Methods 0.000 claims abstract description 39
- 238000001704 evaporation Methods 0.000 claims abstract description 28
- 238000003916 acid precipitation Methods 0.000 claims abstract description 24
- 239000002994 raw material Substances 0.000 claims abstract description 24
- 150000002148 esters Chemical class 0.000 claims abstract description 19
- 239000003513 alkali Substances 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 241000030995 Cephalotaxus sinensis Species 0.000 claims abstract description 15
- 230000008020 evaporation Effects 0.000 claims abstract description 12
- 238000002425 crystallisation Methods 0.000 claims abstract description 11
- 230000008025 crystallization Effects 0.000 claims abstract description 11
- 238000004440 column chromatography Methods 0.000 claims abstract description 10
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 4
- 238000003808 methanol extraction Methods 0.000 claims abstract description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 122
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 99
- 239000003480 eluent Substances 0.000 claims description 98
- 239000000284 extract Substances 0.000 claims description 90
- 239000000243 solution Substances 0.000 claims description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 46
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 44
- 239000000843 powder Substances 0.000 claims description 42
- HAVJATCHLFRDHY-UHFFFAOYSA-N Harringtonine Natural products C1=C2CCN3CCCC43C=C(OC)C(OC(=O)C(O)(CCC(C)(C)O)CC(=O)OC)C4C2=CC2=C1OCO2 HAVJATCHLFRDHY-UHFFFAOYSA-N 0.000 claims description 32
- HAVJATCHLFRDHY-JZTSUELASA-N harringtonine Chemical compound C1=C2CCN3CCC[C@]43C=C(OC)[C@@H](OC(=O)[C@](O)(CCC(C)(C)O)CC(=O)OC)[C@@H]4C2=CC2=C1OCO2 HAVJATCHLFRDHY-JZTSUELASA-N 0.000 claims description 32
- 238000003756 stirring Methods 0.000 claims description 31
- 239000012535 impurity Substances 0.000 claims description 26
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 24
- 239000002253 acid Substances 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 21
- 241000931913 Cephalotaxus fortunei Species 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 19
- 239000000463 material Substances 0.000 claims description 18
- 238000010992 reflux Methods 0.000 claims description 17
- 238000001291 vacuum drying Methods 0.000 claims description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 239000000741 silica gel Substances 0.000 claims description 16
- 229910002027 silica gel Inorganic materials 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 14
- 238000004806 packaging method and process Methods 0.000 claims description 14
- 238000005086 pumping Methods 0.000 claims description 14
- 239000013078 crystal Substances 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 239000003208 petroleum Substances 0.000 claims description 12
- 239000002585 base Substances 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 238000011049 filling Methods 0.000 claims description 10
- 238000005070 sampling Methods 0.000 claims description 10
- 238000010298 pulverizing process Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 9
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 8
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 8
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 8
- 229910052593 corundum Inorganic materials 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 8
- 238000011068 loading method Methods 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 8
- 238000004809 thin layer chromatography Methods 0.000 claims description 8
- 229910001845 yogo sapphire Inorganic materials 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000007599 discharging Methods 0.000 claims description 7
- 229910001220 stainless steel Inorganic materials 0.000 claims description 7
- 239000010935 stainless steel Substances 0.000 claims description 7
- 238000010561 standard procedure Methods 0.000 claims description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 4
- 238000010009 beating Methods 0.000 claims description 4
- 230000006837 decompression Effects 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 4
- 238000004810 partition chromatography Methods 0.000 claims description 4
- 229920006395 saturated elastomer Polymers 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 3
- 239000007779 soft material Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 abstract description 3
- 238000009826 distribution Methods 0.000 abstract 1
- 241000488899 Cephalotaxus Species 0.000 description 6
- 238000005259 measurement Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229930013930 alkaloid Natural products 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- 150000003797 alkaloid derivatives Chemical class 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 206010000871 Acute monocytic leukaemia Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 208000036566 Erythroleukaemia Diseases 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 208000035489 Monocytic Acute Leukemia Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 208000021841 acute erythroid leukemia Diseases 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000013081 microcrystal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 238000012858 packaging process Methods 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/20—Spiro-condensed systems
Abstract
The invention discloses homoharringtonine and a process production method thereof, the homoharringtonine is prepared by taking branches and leaves of cephalotaxus sinensis as preparation raw materials, the content of the homoharringtonine prepared by taking the branches and leaves of the cephalotaxus sinensis as the preparation raw materials is more than 99 percent, and the process production method comprises the following steps: the method comprises the steps of raw material pretreatment, crushing, methanol extraction, primary reduced pressure concentration, acid precipitation, extraction decoloration, ester alkali extraction, secondary reduced pressure concentration, column chromatography elution, tertiary reduced pressure concentration, distribution chromatography elution, quartic reduced pressure concentration, dissolution, evaporation to dryness, crystallization and the like.
Description
Technical Field
The invention relates to the field of medicine preparation, in particular to homoharringtonine and a process production method thereof.
Background
Homoharringtonine is an alkaloid separated from total alkaloids of cephalotaxus plants, exists in cephalotaxus plants, is a high-efficiency antitumor drug successfully developed in China, and is separated from cephalotaxus japonica and cephalotaxus fortunei for the first time by Paudler and the like in 1963; in 1969, Powell et al isolated homoharringtonine from cephalotaxus sinensis, and determined its structure, in 90 s, one of the 47 commonly used anticancer drugs was listed by Ministry of health, and loaded into 1990 edition "Chinese pharmacopoeia". The clinical application proves that the medicine has better curative effect on various acute nonlymphocytic leukemia such as acute myeloid leukemia, acute monocytic leukemia, erythroid leukemia and the like and chronic myeloid leukemia, so that the medicine has huge market development potential and broad development prospect.
The prior common preparation and production process of homoharringtonine mainly comprises the steps of preparing rhizomes, trunks and barks of harringtonine into coarse powder, adding alcohol for extraction, and carrying out column layer separation to finally obtain homoharringtonine and harringtonine, wherein the prior preparation method has the defects that: firstly, cephalotaxus plants are third-stage residual seeds, are distributed with scattered stars and stored in small quantity, have incomplete population composition, small fruiting quantity, low germination rate and very difficult updating, and are excessively harvested, so that a plurality of varieties are trapped in endangered or even extinct places, and the cephalotaxus roots and trunks are used as raw materials to prepare homoharringtonine, so that rare tree seed resources can be further greatly damaged; secondly, the yield of homoharringtonine prepared by the existing preparation method is low, and the content of homoharringtonine in the obtained product is also low, thus being not beneficial to industrial production.
Disclosure of Invention
The invention aims to: aiming at the technical problems of homoharringtonine prepared by the prior art, the invention provides homoharringtonine and a process production method thereof, the homoharringtonine uses branches and leaves of cephalotaxus sinensis as preparation raw materials, and innovation of taking the preparation raw materials solves the technical problem that rhizomes and trunks of cephalotaxus sinensis are used as raw materials to extract and prepare homoharringtonine so as to damage rare tree species resources.
The technical scheme adopted by the invention is as follows: the homoharringtonine is prepared by taking branches and leaves of cephalotaxus sinensis as preparation raw materials, and the content of the homoharringtonine prepared by taking the branches and leaves of the cephalotaxus sinensis as the preparation raw materials is more than 99%.
The homoharringtonine is prepared by the following process production method, and the specific steps are as follows:
s1: pretreatment of raw materials: processing branches and leaves of cephalotaxus sinensis according to a pretreatment SOP program, and removing impurities to obtain a clean material;
s2: crushing: crushing the cleaned material obtained in the step S1 to obtain coarse powder;
s3: methanol extraction: putting the cephalotaxus fortunei coarse powder in the step S2 into a tank from a tank top feeding port, adding excessive methanol with the concentration of more than 95% into the tank, and performing methanol heating reflux extraction on the cephalotaxus fortunei coarse powder for multiple times;
s4: primary decompression concentration: inputting the extracting solution obtained by carrying out methanol reflux extraction for multiple times in the step S3 into a tube array concentrator for concentrating to obtain an extract;
s5: acid precipitation: pouring the extract obtained by concentrating in the step S4 into an acid precipitation tank, adding 1% HCl aqueous solution for acid precipitation treatment, and collecting supernatant after the acid precipitation is finished;
s6: extraction and decoloration: placing the supernatant obtained in the step S5 in an acid precipitation tank, adding petroleum ether for extraction and decoloration, and collecting the lower-layer acid water solution;
s7: extracting ester alkali: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank for ester-base extraction, adjusting the pH value with 10% ammonia water, and extracting with chloroform;
s8: and (3) secondary reduced pressure concentration: combining the chloroform solutions obtained after the extraction in the step S7, drying the combined chloroform solutions through an anhydrous sodium sulfate drying column, inputting the dried chloroform solutions into a concentration tank, and concentrating under reduced pressure to obtain a homoharringtonine total alkali extract;
s9: and (3) column chromatography elution: and (4) performing column chromatography elution on the homoharringtonine extract obtained in the step (S8), and obtaining four types of eluents according to the classification of effective components and impurities: class I eluent, class II eluent, class III eluent and class IV eluent;
s10: and (3) carrying out vacuum concentration for three times: combining the eluents with the same classification obtained in the step S9, and respectively carrying out reduced pressure concentration to finally obtain four types of extractum: class I extract, class II extract, class III extract and class IV extract, pouring the class II extract and the class III extract into a rotary evaporator, and performing water bath vacuum concentration and evaporation to dryness to obtain class II dry extract and class III dry extract;
s11: partition chromatography elution: distributing chromatographic elution is carried out on the III-class dry paste obtained in the step S10, and the eluents obtained in different elution orders are classified and combined to respectively obtain three types of eluents: III-A eluent, III-B eluent and III-C eluent;
s12: and (3) concentrating under reduced pressure for four times: inputting the combined eluents obtained in the step S11 into a concentrator for respectively concentrating under reduced pressure to obtain corresponding III-A extract, III-B extract and III-C extract, pouring the A concentrated extract into a rotary evaporator for concentrating in a reduced pressure water bath and evaporating to dryness to obtain a fine homoharringtonine;
s13: dissolving, evaporating to dryness and crystallizing, namely adding acetone into the fine homoharringtonine product obtained in the step S12 for dissolving, concentrating and evaporating to dryness in a rotary evaporator in a reduced pressure water bath, adding ether, filtering after crystallization is complete, draining and collecting in a beaker to obtain a fine crystal product, dissolving the obtained fine crystal product with a small amount of methanol, heating in the rotary evaporator in the water bath for reduced pressure evaporation, adding ether, and obtaining the fine homoharringtonine crystal product after crystallization is completed;
s14: and (3) vacuum drying: vacuum drying the fine homoharringtonine crystal product obtained in the step S13 to obtain homoharringtonine dry powder;
s15: crushing and inner packaging: and (4) crushing the harringtonine dry powder subjected to vacuum drying in the step S14 into fine powder, and packaging according to the standard operation procedure of a packaging post in a raw material workshop, thus finishing the whole technological production process of the harringtonine.
Further, the crushed coarse powder in step S2 is 10-20 mesh, and the crushing speed is 70-100 kg/h.
Further, in the step S3, the addition amount of the methanol with a concentration of 95% or more is 3-5 times of the coarse powder of the cephalotaxus fortunei, the temperature of the methanol during heating reflux extraction is 65 ℃, the reflux time is 2 hours each time, the addition amount of the methanol with a concentration of 95% or more is 1-2 times of the coarse powder of the cephalotaxus fortunei during repeated reflux extraction, and the repeated extraction times are 2-4 times.
Further, in step S4, the vacuum degree of the reduced pressure concentration is: 0.06-0.07MPa, 40-50 deg.C, concentrating to 60 deg.C, and measuring with heat to obtain extract with relative density of 1.1.
Further, the step S5 of acid precipitation specifically includes: adding 1% HCl aqueous solution equal to the extract, stirring thoroughly for dissolving for 2 hr, adding a certain amount of water, adjusting pH to 2, standing, settling for over 24 hr, pumping out supernatant, centrifuging lower residue liquid with centrifuge at 400RPM to separate supernatant from residue, mixing supernatants, collecting in stainless steel barrel, and discarding residue.
Further, in step S6, the extracting and decoloring step includes adding one third of 60-90 ° petroleum ether into the acid precipitation tank, fully stirring and extracting for 5 minutes, standing for 0.5 hour, extracting the upper petroleum ether layer after layering, extracting for several times until the extract is almost colorless, and pumping the lower acid water solution into the extraction tank.
Further, the extracted ester base of step S7 is specifically: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank, adjusting the pH value to 5-5.5 by using 10% ammonia water, fully and uniformly stirring, adding chloroform with the same volume as the acid water solution, fully stirring and extracting for 5 minutes, standing for 0.5 hour, discharging the lower-layer chloroform layer after layering, and repeating the extraction for 8-10 times until no ester base is detected in the chloroform.
Further, in the secondary vacuum concentration step of step S8, the temperature of vacuum concentration is 55-60 ℃, the vacuum degree is 0.03MPA, and the relative density of the homoharringtonine extract obtained by vacuum concentration is 1.2.
Further, the column chromatography elution in step S9 specifically includes the following steps:
(1) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and the filter paper at the bottom of the column, and flattening;
(2) the activity is two-level 100-mesh 200-mesh Al2O3Stirring with chloroform to obtain paste, slowly pouring into column, lightly knocking the column with soft material while pouring to uniformly fill Al2O3, and circulating with chloroform for 1 hr to make Al in the column2O3Uniform and full;
(3) sample treatment: adding equal amount of chloroform into the homoharringtonine extract, stirring to dissolve completely, and stirring with equal amount of aluminum oxide;
(4) loading: adding the dissolved sample into a column to enable the surface of the sample to be smooth;
(5) and (3) elution: the chloroform in the metering cylinder was changed to chloroform: methanol 100: 0.5%, i.e. the polarity is 0.5%, then the valve is opened and added to the column for elution;
(6) controlling a valve under the column, wherein the flow rate is about 1 liter/min, collecting the product about every 5 liters as a collecting unit, sampling by using a capillary tube every 5 barrels for thin layer chromatography, detecting homoharringtonine, tracking, checking and classifying, and classifying the homoharringtonine into four classes according to effective components and impurities, wherein the four classes are respectively as follows: class I eluent, class II eluent, class III eluent and class IV eluent. The class I eluent is a precursor impurity, namely, no effective component but only impurities; the eluent of class II is crossed in front, and has both effective components and impurities; the III-class eluent is mixed ester alkali and almost all is an effective component; the IV-class eluent is post-impurity, namely, no effective component but only impurity;
(7) according to the detection result, the polarity of the eluent is changed in time, the gradient of the polarity of the eluent is three types of 0.5%, 1% and 2%, namely when the eluent is changed from type I to type II, the polarity is changed from 0.5% → 1%; when changing from class II to class III, the polarity changes from 1% → 2%.
Further, in the three reduced pressure concentration steps of step S10, the temperature of reduced pressure concentration is 55-60 ℃, the vacuum degree is 0.03MPA, the relative density of the extract is 1:1 when the reduced pressure concentration is carried out to 60 ℃ heat measurement, the temperature of water bath vacuum reduced pressure concentration and evaporation to dryness is 55-60 ℃, and the time is 0.5-1 hour.
Further, the specific step of distributing chromatography elution in step S11 is:
(1) column assembling: weighing 100-160-mesh silica gel, sufficiently and uniformly grinding the silica gel with the same amount of citric acid with pH value of 5 and disodium hydrogen phosphate buffer solution, then stirring the mixture into paste by using a proper amount of chloroform, filling the paste into a chromatographic column, and beating the column body by using a soft object to ensure that the silica gel is uniform without collusion;
(2) sample treatment: dissolving the mixed ester alkali by using chloroform with the same amount;
(3) loading: slowly adding the sample to the upper end of the column, and opening the lower valve of the column to enable the sample to be slowly adsorbed until the liquid level on the column is flush with the surface of the silica gel;
(4) adding a chloroform solution saturated by PH 5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column, eluting at the flow rate of 1 liter/min, taking 3 liters as a metering and collecting unit, sampling every 5 barrels, carrying out detection and identification on the homoharringtonine according to thin layer chromatography, classifying, combining into three parts respectively, namely III-A eluent, III-B eluent and III-C eluent, wherein the III-A eluent is homoharringtonine, the III-B eluent is a mixture of homoharringtonine and harringtonine, and the III-C eluent is harringtonine; in the above elution procedure, the first elution is of group III-A: homoharringtonine; the middle part is of the III-B type: mixtures of homoharringtonine and harringtonine; the final elution was of class III-C: harringtonine.
Further, in the step of four times of vacuum concentration in step S12, the temperature for vacuum concentration in the concentrator is 55-60 ℃, the vacuum degree is 0.03MPA, and when the extract is heated at 60 ℃, the relative density of the extract is 1.1, and the temperature for concentration in the rotary evaporator is 55-60 ℃.
Further, in the dissolving, evaporating to dryness and crystallizing steps of step S13, the temperature of concentration and evaporation to dryness in a reduced pressure water bath is 55-60 ℃ in a rotary evaporator.
Further, the vacuum drying in step S14 includes: drying in vacuum drying oven at 55-60 deg.C under 0.07MPA for 12 hr.
Further, in the pulverizing and inner packing step of step S15, the pulverized fine powder is 200 mesh.
Compared with the prior art, the invention has the beneficial effects that:
1) aiming at the technical problems existing in the prior preparation of homoharringtonine, the invention provides homoharringtonine and a process production method thereof, the homoharringtonine uses branches and leaves of cephalotaxus sinensis as preparation raw materials, and the innovation of taking the preparation raw materials solves the technical problem that the homoharringtonine is extracted and prepared by taking roots and trunks of cephalotaxus sinensis as the raw materials to damage rare tree species resources;
2) the homoharringtonine prepared by the homoharringtonine process production method provided by the invention has the content of more than 99 percent, further solves the problems of low homoharringtonine content and low yield in the existing preparation method, and is industrially produced on the premise of not influencing the normal growth of the tree species and not damaging the environment.
Drawings
FIG. 1 is a comparison graph of the IR spectroscopy test of a homoharringtonine sample prepared by the present invention and a homoharringtonine test sample;
FIG. 2 is a report of the examination of a sample of homoharringtonine prepared by the invention.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent.
Example 1
A homoharringtonine is prepared from branches and leaves of Chinese cephalotaxus, and has a content of greater than 99%.
The homoharringtonine is prepared by the following process production method, and the specific implementation steps are as follows:
s1: pretreatment of raw materials: processing branches and leaves of cephalotaxus sinensis according to a pretreatment SOP program, and removing impurities to obtain a clean material;
s2: crushing: pulverizing the purified material obtained in step S1 to obtain coarse powder, wherein the pulverized coarse powder is 10-20 meshes and the pulverizing speed is 70-100 kg/h;
s3: methanol extraction: putting the cephalotaxus fortunei coarse powder in the step S2 into a tank from a tank top feeding port, adding excessive methanol with the concentration of more than 95% into the tank, carrying out methanol heating reflux extraction on the cephalotaxus fortunei coarse powder for multiple times, wherein the adding amount of the methanol with the concentration of more than 95% is 3-5 times of that of the cephalotaxus fortunei coarse powder, the temperature of the methanol heating reflux extraction is 65 ℃, the reflux time is 2 hours each time, the adding amount of the methanol with the concentration of more than 95% is 1-2 times of that of the cephalotaxus fortunei coarse powder when the reflux extraction is repeated, and the repeated extraction times are 2-4 times;
s4: primary decompression concentration: inputting the extracting solution obtained by carrying out methanol reflux extraction for multiple times in the step S3 into a tube array concentrator for concentrating to obtain an extract, wherein the vacuum degree of the reduced pressure concentration is as follows: 0.06-0.07MPa, 40-50 deg.C, concentrating to 60 deg.C, and measuring with heat to obtain extract with relative density of 1.1.
S5: acid precipitation: pouring the extract obtained by concentrating in the step S4 into an acid precipitation tank, adding 1% HCl aqueous solution for acid precipitation treatment, and collecting supernatant after the acid precipitation is finished, wherein the steps are as follows: adding 1% HCl aqueous solution equal to the extract, stirring thoroughly for dissolving for 2 hr, adding a certain amount of water, adjusting pH to 2, standing, settling for over 24 hr, pumping out supernatant, centrifuging lower residue liquid with centrifuge at 400RPM to separate supernatant from residue, mixing supernatants, collecting in stainless steel barrel, and discarding residue;
s6: extraction and decoloration: placing the supernatant obtained in the step S5 in an acid precipitation tank, adding petroleum ether for extraction and decoloration, and collecting the lower-layer acid water solution; in step S6, the extraction and decolorization are specifically carried out by adding one third of 60-90 DEG petroleum ether into an acid precipitation tank, fully stirring and extracting for 5 minutes, standing for 0.5h, extracting the upper petroleum ether layer after layering, extracting for several times until the extract is almost colorless, and pumping the lower acid water solution into an extraction tank;
s7: extracting ester alkali: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank for ester-base extraction, adjusting the pH value with 10% ammonia water, and extracting with chloroform, wherein the ester-base extraction step specifically comprises the following steps: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank, adjusting the pH value to 5-5.5 by using 10% ammonia water, fully and uniformly stirring, adding chloroform with the same volume as the acid water solution, fully stirring and extracting for 5 minutes, standing for 0.5 hour, discharging a lower-layer chloroform layer after layering, and repeatedly extracting for 8-10 times until no ester base is detected in the chloroform;
s8: and (3) secondary reduced pressure concentration: combining the chloroform solutions obtained after the extraction in the step S7, drying the combined chloroform solutions through an anhydrous sodium sulfate drying column, inputting the dried chloroform solutions into a concentration tank, and concentrating under reduced pressure to obtain a homoharringtonine total alkali extract;
s9: and (3) column chromatography elution: and (4) performing column chromatography elution on the homoharringtonine extract obtained in the step (S8), and obtaining four types of eluents according to the classification of effective components and impurities: the method comprises the following steps of carrying out vacuum concentration on a class I eluent, a class II eluent, a class III eluent and a class IV eluent at the temperature of 55-60 ℃ and the vacuum degree of 0.03MPA to obtain a homoharringtonine extract, wherein the relative density of the homoharringtonine extract obtained by vacuum concentration is 1.2, and the column chromatography elution comprises the following specific steps:
(1) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and the filter paper at the bottom of the column, and flattening;
(2) the activity is two-level 100-mesh 200-mesh Al2O3Stirring with chloroform to obtain paste, slowly pouring into column, and gently knocking the column with soft material while pouring to obtain Al2O3Filling uniformly, and circulating up and down for 1 hour by using chloroform after filling to ensure that Al is contained2O3Uniform and full;
(3) sample treatment: adding equal amount of chloroform into the homoharringtonine extract, stirring to dissolve completely, and stirring with equal amount of aluminum oxide;
(4) loading: adding the dissolved sample into a column to enable the surface of the sample to be smooth;
(5) and (3) elution: the chloroform in the metering cylinder was changed to chloroform: methanol 100: 0.5%, i.e. the polarity is 0.5%, then the valve is opened and added to the column for elution;
(6) controlling a valve under the column, wherein the flow rate is about 1 liter/min, collecting the product about every 5 liters as a collecting unit, sampling by using a capillary tube every 5 barrels for thin layer chromatography, detecting homoharringtonine, tracking, checking and classifying, and classifying the homoharringtonine into four classes according to effective components and impurities, wherein the four classes are respectively as follows: class I eluent, class II eluent, class III eluent and class IV eluent. The class I eluent is a precursor impurity, namely, no effective component but only impurities; the eluent of class II is crossed in front, and has both effective components and impurities; the III-class eluent is mixed ester alkali and almost all is an effective component; the IV-class eluent is post-impurity, namely, no effective component but only impurity;
(7) according to the detection result, the polarity of the eluent is changed in time, the gradient of the polarity of the eluent is three types of 0.5%, 1% and 2%, namely when the eluent is changed from type I to type II, the polarity is changed from 0.5% → 1%; when changing from class II to class III, the polarity changes from 1% → 2%.
S10: and (3) carrying out vacuum concentration for three times: combining the eluents with the same classification obtained in the step S9, and respectively carrying out reduced pressure concentration to finally obtain four types of extractum: and pouring the class I extract, the class II extract, the class III extract and the class III extract into a rotary evaporator, performing water bath vacuum concentration and evaporation to dryness to obtain class II dry extract and class III dry extract, wherein the temperature of the reduced pressure concentration is 55-60 ℃, the vacuum degree is 0.03MPA, the relative density of the extracts is 1:1 when the reduced pressure concentration is performed to 60 ℃ for thermal measurement, and the temperature of the water bath vacuum concentration and evaporation to dryness is 55-60 ℃ for 0.5-1 hour.
S11: partition chromatography elution: distributing chromatographic elution is carried out on the III-class dry paste obtained in the step S10, and the eluents obtained in different elution orders are classified and combined to respectively obtain three types of eluents: the specific steps of distributing chromatographic elution for III-A eluent, III-B eluent and III-C eluent are as follows:
(1) column assembling: weighing 100-160-mesh silica gel, sufficiently and uniformly grinding the silica gel with the same amount of citric acid with pH value of 5 and disodium hydrogen phosphate buffer solution, then stirring the mixture into paste by using a proper amount of chloroform, filling the paste into a chromatographic column, and beating the column body by using a soft object to ensure that the silica gel is uniform without collusion;
(2) sample treatment: dissolving the mixed ester alkali by using chloroform with the same amount;
(3) loading: slowly adding the sample to the upper end of the column, and opening the lower valve of the column to enable the sample to be slowly adsorbed until the liquid level on the column is flush with the surface of the silica gel;
(4) adding a chloroform solution saturated by PH 5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column, eluting at the flow rate of 1 liter/min, taking 3 liters as a metering and collecting unit, sampling every 5 barrels, carrying out detection and identification on the homoharringtonine according to thin layer chromatography, classifying, combining into three parts respectively, namely III-A eluent, III-B eluent and III-C eluent, wherein the III-A eluent is homoharringtonine, the III-B eluent is a mixture of homoharringtonine and harringtonine, and the III-C eluent is harringtonine; in the above elution procedure, the first elution is of group III-A: homoharringtonine; the middle part is of the III-B type: mixtures of homoharringtonine and harringtonine; the final elution was of class III-C: harringtonine.
S12: and (3) concentrating under reduced pressure for four times: inputting the combined eluents obtained in the step S11 into a concentrator for respectively concentrating under reduced pressure to obtain corresponding III-A extract, III-B extract and III-C extract, pouring the A concentrated extract into a rotary evaporator for concentrating in a reduced pressure water bath and evaporating to obtain fine homoharringtonine, wherein the temperature for concentrating under reduced pressure in the concentrator is 55-60 ℃, the vacuum degree is 0.03MPA, and the relative density of the extract is 1.1 when the extract is heated at 60 ℃, and the temperature for concentrating in the rotary evaporator is 55-60 ℃.
S13: dissolving, evaporating to dryness and crystallizing, namely adding acetone into the fine homoharringtonine product obtained in the step S12 for dissolving, concentrating and evaporating to dryness in a rotary evaporator in a reduced pressure water bath, adding diethyl ether, filtering after crystallization is completed, draining and collecting in a beaker to obtain a fine crystal product, dissolving the obtained fine crystal product with a small amount of methanol, heating in the rotary evaporator in the water bath for reduced pressure evaporation, adding diethyl ether, after crystallization is completed, obtaining the fine homoharringtonine crystal product, and concentrating and evaporating to dryness in the rotary evaporator in the reduced pressure water bath at the temperature of 55-60 ℃.
S14: and (3) vacuum drying: vacuum drying the fine homoharringtonine crystal product obtained in the step S13 to obtain dry homoharringtonine powder, wherein the vacuum drying comprises the following specific steps: drying in vacuum drying oven at 55-60 deg.C under 0.07MPA for 12 hr.
S15: crushing and inner packaging: and (4) crushing the harringtonine dry powder subjected to vacuum drying in the step S14 into fine powder, wherein the crushed fine powder is 200 meshes, and packaging according to the standard operation procedure of a packaging post in a raw material workshop.
Example 2
In order to further verify the feasibility and the effectiveness of the technological production method of homoharringtonine provided by the invention, the specific flow is as follows, taking 808kg of homoharringtonine branches as an example for preparing and producing homoharringtonine:
1. treating 808kg branches of Gaotricuspidata according to the pretreatment SOP, and removing impurities to obtain 804kg of cleaned material;
2. putting the processed raw medicinal materials into a clean nontoxic plastic bag, sticking a material label, storing in a clean material temporary storage room, and stacking in order according to production varieties and used materials;
3. pulverizing the materials into 10-20 mesh coarse powder in a pulverizer at a pulverizing speed of 80 kg/hr to obtain 800kg coarse powder;
4. taking the coarse powder of the branches of the Gaotricuspidata fir from a temporary storage room of the clean material;
5. taking 3.2T of methanol with the concentration of more than 95 percent;
6. putting cephalotaxus fortunei coarse powder into a tank from a feeding port on the top of the tank according to 800kg, flattening, and closing the feeding port;
7. adding 4 times of methanol, and making the methanol liquid level 10cm higher than the powder;
8. opening a steam heating valve for heating, slightly boiling and refluxing the methanol in the tank, starting to count the time when the temperature of the solution in the tank reaches 65 ℃, keeping refluxing for 2 hours, and closing the heating valve;
9. opening a tank liquid discharging valve, putting the extracting solution into a temporary storage tank, and closing the liquid discharging valve after discharging;
10. adding 2 times of methanol into the extraction tank, extracting for 2 hr by the same method, and repeating the extraction for 2 times;
11. concentrating the extractive solution in a tube array concentrator under vacuum degree of 0.06-0.07MPa and temperature of 40-50 deg.C until the relative density of the extract is 1.1 (measured at 60 deg.C), and collecting the extract with an extract collecting barrel to obtain 70-75kg extract with an extract collecting rate of 2%.
The paste collection rate is the concentrated extract amount (measured at a relative density of 1.160 ℃)/the branch crude powder amount of the cephalotaxus fortunei is 100%
12. Acid precipitation:
12.1 pouring the extract 1 into an acid precipitation tank, adding 1% HCl aqueous solution with the same amount as the extract, fully stirring and dissolving for 2 hours, adding a certain amount of water, adjusting the pH value to 2, standing, and precipitating for more than 24 hours;
12.2 extracting the supernatant, centrifuging the lower residue liquid by a centrifuge at the rotation speed of 400RPM to separate the supernatant from the residue, combining the supernatants, collecting in a stainless steel barrel, and discarding the residue;
13. extraction of
13.1 decolorization
13.1.1 placing the supernatant in an acid precipitation tank, adding 60-90 deg.C petroleum ether, stirring thoroughly for 5 min, standing for 0.5h, layering, extracting the upper petroleum ether layer, and extracting for several times until the extractive solution is almost colorless. Pumping the lower layer of acid water solution into an extraction tank for extraction;
13.1.2 the combined extracts (petroleum ether layer) were passed through a 50kg anhydrous sodium sulfate drying column to absorb water, and then fed into a concentrator to be concentrated under reduced pressure to recover the reagents, which took about 0.5 hour, temperature: 55-60 ℃, vacuum degree: 0.03 MPA;
13.2 extraction of ester base: adjusting pH of the decolorized acid water solution 2 to 5-5.5 with 10% ammonia water, stirring well, adding chloroform with the same volume as the acid water solution 2, stirring well for extraction for 5 minutes, standing for 0.5 hour, discharging the lower chloroform layer after layering, and repeating the extraction for 8-10 times until no ester base is detected in the chloroform;
13.3 after the extraction, combining the chloroform solutions, drying the chloroform solutions by an anhydrous sodium sulfate drying column, inputting the dried chloroform solutions into a concentration tank for about 12 hours, and concentrating under reduced pressure until the relative density of the extract is 1.2, wherein the temperature is as follows: 55-60 ℃, vacuum degree: 0.03 MPA;
13.4, paste collection: collecting the concentrated extract in a stainless steel barrel, sampling by QA, detecting content, weighing, warehousing to obtain 6-7kg of extract 2 (homoharringtonine), and recording to obtain extraction rate of about 10%.
The extraction rate (total amount of cephalotaxus fortunei alkali)/(content of branch powder of cephalotaxus fortunei): 100%
13.5 when the pH of the acid water solution is adjusted to 9 by 10 percent ammonia water, extracting the acid water solution by using chloroform according to the same method of 4.6.2 until no alkaloid is detected;
13.6 combining the chloroform solutions, drying for 12 hours by using anhydrous NaSO4, concentrating until the relative density of the extract is 1.2 (measured at 60 ℃), the temperature is 55-60 ℃, the vacuum degree is 0.03MPa, crystallizing by using absolute ethyl alcohol, recrystallizing to obtain cephalotaxus sinensis alkali, weighing and warehousing;
14. chromatography
14.1, material preparation: taking the extract (extract 2) of the homoharringtonine from a raw material warehouse according to the production instruction, wherein the weight of the extract is about 6-7 kg;
14.2 chromatographic column installation: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and the filter paper at the bottom of the column, and flattening;
14.3 mixing 100-mesh Al2O320kg with the activity of two stages and 200 meshes with chloroform into paste, slowly pouring the paste into a column, slightly knocking the column with a soft object while pouring to ensure that the Al2O3 is uniformly filled, and circulating the filled liquid up and down for 1 hour by using chloroform to ensure that the Al2O3 is uniform and full;
14.4 sample treatment: adding equal amount of chloroform into 1kg of the homoharringtonine extract, stirring to fully dissolve, and stirring with equal amount of aluminum oxide;
14.5 loading: adding the dissolved sample into a column to enable the surface of the sample to be smooth;
14.6 elution: the chloroform in the metering cylinder was changed to chloroform: methanol 100: 0.5% (i.e. polarity of 0.5%), then open the valve, add to the column, elute;
14.7 controlling the valve under the column, the flow rate is about 1 liter/min, collecting for collecting unit about every 5 liters, sampling by capillary tube every 5 barrels to carry out thin layer chromatography, detecting homoharringtonine, tracking, checking and classifying, and classifying into four categories according to effective components and impurities, wherein the four categories are respectively: class I eluent, class II eluent, class III eluent and class IV eluent. The class I eluent is a precursor impurity, namely, no effective component but only impurities; the eluent of class II is crossed in front, and has both effective components and impurities; the III-class eluent is mixed ester alkali and almost all is an effective component; the IV-class eluent is post-impurity, namely, no effective component but only impurity;
14.8 timely replacing the polarity of the eluent according to the detection result, wherein the polarity gradient of the eluent is 0.5%, 1% and 2%, namely when the eluent is changed from class I to class II, the polarity is changed from 0.5% → 1%; when changing from class II to class III, the polarity is from 1% → 2%;
14.9 mixing the eluates of the same classification, inputting into a concentration tank, concentrating under reduced pressure until the relative density of the extract is 1:1(60 ℃ heat measurement): 55-60 ℃, vacuum degree: 0.03MPA, simultaneously recovering the reagent, discarding class I and class IV extractums, pouring concentrated extractums of class II and class III eluents into a rotary evaporator, vacuum concentrating in a water bath at 55-60 ℃ under reduced pressure until the extractums are dry, taking 0.5 hour, respectively detecting the content, weighing the attached material labels, and recording to obtain 500g of class II dry extract and 525g of class III dry extract;
the yield is (dry paste amount x content of C type)/(total alkali amount x content of high cephalotaxus fortunei) × 100%
15. Partition chromatography
15.1, material picking: taking dry paste of class III homoharringtonine from intermediate station according to production instruction.
15.2, column filling: weighing 20kg of 100-160-mesh silica gel, sufficiently and uniformly grinding the silica gel with the same amount of citric acid with pH value of 5 and disodium hydrogen phosphate buffer solution, then stirring the mixture into paste by using a proper amount of chloroform, filling the paste into a chromatographic column, and beating the column body by using a soft object to ensure that the silica gel is uniform without flow interruption;
15.3 sample treatment: dissolving the mixed ester alkali by using chloroform with the same amount;
15.4 loading: slowly adding the sample to the upper end of the column, and opening the lower valve of the column to enable the sample to be slowly adsorbed until the liquid level on the column is flush with the surface of the silica gel;
15.5 adding a chloroform solution saturated by PH 5 citric acid and disodium hydrogen phosphate buffer solution to the upper end of the column, eluting (flow rate is 1 liter/min), taking 3 liters as a metering and collecting unit, sampling every 5 barrels, carrying out high harringtonine detection and identifying according to thin layer chromatography, classifying, respectively combining into three parts, namely III-A eluent, III-B eluent and III-C eluent, wherein the III-A eluent is harringtonine, the III-B eluent is a mixture of harringtonine and harringtonine, and the III-C eluent is harringtonine; in the above elution procedure, the first elution is of group III-A: homoharringtonine; the middle part is of the III-B type: mixtures of homoharringtonine and harringtonine; the final elution was of class III-C: harringtonine;
15.6 the first to elute is homoharringtonine (class I); the middle part is a mixture of homoharringtonine and harringtonine (class II); the final elution was harringtonine (class III);
15.7 the eluates with the same classification are combined and input into a concentrator for decompression and concentration until the relative density of the extract is 1.1(60 ℃ heat measurement): 55-60 ℃, vacuum degree: 0.03MPA, and storing the II type and III type extractum in a warehouse for later use. Pouring the class I concentrated extract into a rotary evaporator, concentrating and evaporating to dryness in a reduced pressure water bath at 55-60 ℃ for about 0.5h to obtain about 180g of fine homoharringtonine 160-one, detecting, weighing, recording and sending to an intermediate station.
The yield is (refined homoharringtonine x content)/(mixed homoharringtonine of class III x content) × 100%
16. Crystallization of
16.1, material getting: picking up fine homoharringtonine from an intermediate station according to a production instruction;
16.2 crystallization: the fine homoharringtonine is dissolved in a small amount of acetone. After the dissolution is finished, concentrating and evaporating in a rotary evaporator at 55-60 ℃ in a reduced pressure water bath until the evaporation is finished, evaporating in the water bath until a small amount of acetone is generated to cause the liquid to have a wall hanging phenomenon, taking down, adding ether until micro crystal grains appear, standing overnight, and crystallizing;
16.3 after complete crystallization, filtering by using a Buchner funnel, draining and collecting in a beaker, crystallizing a fine product, dissolving by using a small amount of methanol, heating in a water bath at 55-60 ℃ in a rotary evaporator for reduced pressure evaporation after the dissolution is finished, evaporating until the methanol is a small amount, so that the solution has a wall hanging phenomenon, taking down, adding ether until crystal nuclei appear, standing overnight, and crystallizing;
16.4 repeating the operation for several times according to 4.9.3, crystallizing, recrystallizing until reaching the standard of appearance quality (i.e. white powder);
16.5 vacuum drying: placing the crystallized fine product of homoharringtonine in a stainless steel plate, placing in a vacuum drying oven, and drying at 55-60 deg.C for 12 hr. Vacuum degree of 0.07MPA to obtain homoharringtonine dry powder 70-80 g. And the QA sampling detection is carried out, and the crushing process can be carried out after the qualified product is obtained. The yield is 50-60%;
the yield is (fine homoharringtonine x content)/(dry homoharringtonine powder x content) × 100%
16.6 according to the SOP clearing of the crystallization post clearing, filling production records and clearing records;
17. pulverizing
17.1 pulverizing the harringtonine dry powder after vacuum drying into fine powder of 200 meshes in a pulverizer;
17.2, putting the crushed fine powder into a stainless steel barrel, weighing, sticking a material label, and performing an inner packaging process;
18. inner package
18.1, material getting: packaging materials from a storehouse according to a production instruction;
18.2 packaging according to the standard operation procedure of the inner packaging post of the raw material workshop, wherein the packaging specification is as follows: 50 g/bag;
18.3 the worker must sterilize the handle first, then wear the latex glove of sterilization and regard 50 g/bag as the unit, pack with the plastic film first, and then the negative pressure is pumped the surplus air, sealed;
bagging: and (3) according to the post external packing operation and the SOP operation in the raw material workshop, sleeving 1 aluminum foil bag outside each plastic bag for packaging, and sealing.
Test example 3
In the embodiment, the high harringtonine sample prepared by the process production method is compared with a high harringtonine standard test product by an infrared spectrogram, the result is shown in the attached figure 1 of the specification, and the comparison result further shows that the process preparation method of the high harringtonine provided by the invention is really feasible, and the content of the high harringtonine in the sample finally obtained by the process method for preparing the high harringtonine provided by the invention is more than 99 percent as shown in the attached figure 2 of the specification, so that the yield and the purity of the traditional process are greatly improved.
The above embodiments only express the specific embodiments of the present application, and the description thereof is specific and detailed, but it should not be understood as the limitation of the scope of the present application, and it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the technical solution idea of the present application, and these embodiments all belong to the scope of the present application.
Claims (10)
1. The homoharringtonine is characterized by being prepared by taking branches and leaves of cephalotaxus sinensis as preparation raw materials, wherein the content of the homoharringtonine prepared by taking the branches and leaves of the cephalotaxus sinensis as the preparation raw materials is more than 99%.
2. The process for producing homoharringtonine according to claim 1, wherein the process comprises the following steps: pretreating raw materials, pulverizing, extracting with methanol, concentrating under reduced pressure, precipitating with acid, decolorizing, extracting ester and alkali, concentrating under reduced pressure twice, eluting by column chromatography, concentrating under reduced pressure for three times, eluting by distributed chromatography, concentrating under reduced pressure for four times, dissolving, evaporating to dryness, and crystallizing.
3. The process for producing homoharringtonine according to claim 2, further comprising vacuum drying, pulverizing and internal packaging.
4. The process production method of homoharringtonine according to claim 2, characterized in that the process production method comprises the following steps:
s1: pretreatment of raw materials: processing branches and leaves of cephalotaxus sinensis according to a pretreatment SOP program, and removing impurities to obtain a clean material;
s2: crushing: crushing the cleaned material obtained in the step S1 to obtain coarse powder;
s3: methanol extraction: putting the cephalotaxus fortunei coarse powder in the step S2 into a tank from a tank top feeding port, adding excessive methanol with the concentration of more than 95% into the tank, and performing methanol heating reflux extraction on the cephalotaxus fortunei coarse powder for multiple times;
s4: primary decompression concentration: inputting the extracting solution obtained by carrying out methanol reflux extraction for multiple times in the step S3 into a tube array concentrator for concentrating to obtain an extract;
s5: acid precipitation: pouring the extract obtained by concentrating in the step S4 into an acid precipitation tank, adding 1% HCl aqueous solution for acid precipitation treatment, and collecting supernatant after the acid precipitation is finished;
s6: extraction and decoloration: placing the supernatant obtained in the step S5 in an acid precipitation tank, adding petroleum ether for extraction and decoloration, and collecting the lower-layer acid water solution;
s7: extracting ester alkali: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank for ester-base extraction, adjusting the pH value with 10% ammonia water, and extracting with chloroform;
s8: and (3) secondary reduced pressure concentration: combining the chloroform solutions obtained after the extraction in the step S7, drying the combined chloroform solutions through an anhydrous sodium sulfate drying column, inputting the dried chloroform solutions into a concentration tank, and concentrating under reduced pressure to obtain a homoharringtonine total alkali extract;
s9: and (3) column chromatography elution: and (4) performing column chromatography elution on the homoharringtonine extract obtained in the step (S8), and obtaining four types of eluents according to the classification of effective components and impurities: class I eluent, class II eluent, class III eluent and class IV eluent;
s10: and (3) carrying out vacuum concentration for three times: combining the eluents with the same classification obtained in the step S9, and respectively carrying out reduced pressure concentration to finally obtain four types of extractum: class I extract, class II extract, class III extract and class IV extract, pouring the class II extract and the class III extract into a rotary evaporator, and performing water bath vacuum concentration and evaporation to dryness to obtain class II dry extract and class III dry extract;
s11: partition chromatography elution: distributing chromatographic elution is carried out on the III-class dry paste obtained in the step S10, and the eluents obtained in different elution orders are classified and combined to respectively obtain three types of eluents: III-A eluent, III-B eluent and III-C eluent;
s12: and (3) concentrating under reduced pressure for four times: inputting the combined eluents obtained in the step S11 into a concentrator for respectively concentrating under reduced pressure to obtain corresponding III-A extract, III-B extract and III-C extract, pouring the A concentrated extract into a rotary evaporator for concentrating in a reduced pressure water bath and evaporating to dryness to obtain a fine homoharringtonine;
s13: dissolving, evaporating to dryness and crystallizing, namely adding acetone into the fine homoharringtonine obtained in the step S12 for dissolving, concentrating and evaporating to dryness in a rotary evaporator in a reduced pressure water bath, adding diethyl ether, filtering after complete crystallization, draining and collecting in a beaker to obtain a fine crystal product, dissolving the obtained fine crystal product with a small amount of methanol, heating in the rotary evaporator in the water bath for reduced pressure evaporation, adding diethyl ether, and obtaining the fine homoharringtonine crystal product after crystallization.
5. The process for producing homoharringtonine according to claim 4, wherein said process further comprises the steps of:
s14: and (3) vacuum drying: vacuum drying the fine homoharringtonine crystal product obtained in the step S13 to obtain homoharringtonine dry powder; s15: crushing and inner packaging: and (4) crushing the harringtonine dry powder subjected to vacuum drying in the step S14 into fine powder, and packaging according to the standard operation procedure of a packaging post in a raw material workshop, thus finishing the whole technological production process of the harringtonine.
6. The process of claim 4, wherein in step S3, the amount of methanol added is more than 95% and 3-5 times of the amount of coarse powder of Cephalotaxus fortunei, the temperature of the methanol is 65 ℃ during heating reflux extraction, the reflux time is 2h each time, the amount of methanol added is more than 95% and 1-2 times of the amount of coarse powder of Cephalotaxus fortunei during repeated reflux extraction, the number of times of repeated extraction is 2-4, and the degree of vacuum of the reduced pressure concentration in step S4 is: 0.06-0.07MPa, 40-50 deg.C, concentrating to 60 deg.C, and measuring with heat to obtain extract with relative density of 1.1.
7. The process for producing homoharringtonine according to claim 4, wherein,
the step S5 of acid precipitation specifically comprises the following steps: adding 1% HCl aqueous solution equal to the extract, fully stirring and dissolving for 2 hours, adding a certain amount of water, adjusting the pH value to 2, standing, settling for more than 24 hours, pumping out the supernatant, centrifuging the lower-layer residue liquid by a centrifugal machine at the rotation speed of 400RPM to separate the supernatant from the residue, combining the supernatants, collecting in a stainless steel barrel, discarding the residue, and in step S6, the extraction and decoloration are specifically that one third of 60-90 DEG petroleum ether is added into an acid precipitation tank, fully stirring and extracting for 5 minutes, standing for 0.5 hour, after layering, pumping out the upper-layer petroleum ether layer, and performing extraction for several times until the extract is almost colorless, and pumping the lower-layer acid water liquid into an extraction tank.
8. The process for producing homoharringtonine according to claim 4, wherein,
the extracted ester base of step S7 is specifically: pumping the lower layer of acid water solution obtained in the step S6 into an extraction tank, adjusting the pH value to 5-5.5 by using 10% ammonia water, fully and uniformly stirring, adding chloroform with the same volume as the acid water solution, fully stirring and extracting for 5 minutes, standing for 0.5 hour, discharging the lower layer of chloroform layer after layering, repeating the extraction for 8-10 times until no ester base is detected in the chloroform, wherein in the step S8 of secondary reduced pressure concentration, the temperature of reduced pressure concentration is 55-60 ℃, the vacuum degree is 0.03MPA, and the relative density of the homoharringtonine extract obtained by reduced pressure concentration is 1.2.
9. The process for producing homoharringtonine according to claim 4, wherein the step S9 comprises the following steps:
(1) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and the filter paper at the bottom of the column, and flattening;
(2) the activity is two-level 100-mesh 200-mesh Al2O3Stirring with chloroform to obtain paste, slowly pouring into column, and gently knocking the column with soft material while pouring to obtain Al2O3Filling uniformly, and circulating up and down for 1 hour by using chloroform after filling to ensure that Al is contained2O3Uniform and full;
(3) sample treatment: adding equal amount of chloroform into the homoharringtonine extract, stirring to dissolve completely, and stirring with equal amount of aluminum oxide;
(4) loading: adding the dissolved sample into a column to enable the surface of the sample to be smooth;
(5) and (3) elution: the chloroform in the metering cylinder was changed to chloroform: methanol 100: 0.5%, i.e. the polarity is 0.5%, then the valve is opened and added to the column for elution;
(6) controlling a valve under the column, controlling the flow rate to be 1 liter/min, collecting every 5 liters for a collecting unit, sampling by using a capillary tube every 5 barrels for thin layer chromatography, detecting homoharringtonine, tracking, checking and classifying, and classifying into four classes according to effective components and impurities, wherein the four classes are respectively: class I eluent, class II eluent, class III eluent and class IV eluent;
(7) according to the detection result, the polarity of the eluent is changed in time, the gradient of the polarity of the eluent is three types of 0.5%, 1% and 2%, namely when the eluent is changed from type I to type II, the polarity is changed from 0.5% → 1%; when changing from class II to class III, the polarity changes from 1% → 2%.
10. The method for producing homoharringtonine according to claim 4, wherein the step S11 of fractionating, chromatographically eluting comprises the following steps:
(1) column assembling: weighing 100-160-mesh silica gel, sufficiently and uniformly grinding the silica gel with the same amount of citric acid with pH value of 5 and disodium hydrogen phosphate buffer solution, then stirring the mixture into paste by using a proper amount of chloroform, filling the paste into a chromatographic column, and beating the column body by using a soft object to ensure that the silica gel is uniform without collusion;
(2) sample treatment: dissolving the mixed ester alkali by using chloroform with the same amount;
(3) loading: slowly adding the sample to the upper end of the column, and opening the lower valve of the column to enable the sample to be slowly adsorbed until the liquid level on the column is flush with the surface of the silica gel;
(4) adding a chloroform solution saturated by pH 5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column, eluting at the flow rate of 1 liter/min, taking 3 liters as a metering collection unit, sampling every 5 barrels, detecting and identifying the cephalotaxus fortunei by thin layer chromatography, classifying and respectively combining into three parts, namely III-A eluent, III-B eluent and III-C eluent, and firstly eluting III-A: homoharringtonine; the middle part is of the III-B type: mixtures of homoharringtonine and harringtonine; the final elution was of class III-C: harringtonine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110868850.3A CN113651830B (en) | 2021-07-29 | Homoharringtonine and process production method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110868850.3A CN113651830B (en) | 2021-07-29 | Homoharringtonine and process production method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113651830A true CN113651830A (en) | 2021-11-16 |
| CN113651830B CN113651830B (en) | 2024-05-03 |
Family
ID=
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4783454A (en) * | 1985-05-28 | 1988-11-08 | Yaguang Liu | Process for producing harringtonine and homoharringtonine |
| CA1261829A (en) * | 1986-04-30 | 1989-09-26 | Ya-Guang Liu | Process for producing harringtonine and homoharringtonine |
| US20040082565A1 (en) * | 2002-07-17 | 2004-04-29 | Chemgenex Therapeutics, Inc. | Formulations and methods of administration of cephalotaxines including homoharringtonine |
| CN102633806A (en) * | 2012-04-05 | 2012-08-15 | 贵州博丰生物科技产业开发有限公司 | Extraction and separation method of harringtonine |
| CN104402895A (en) * | 2014-12-12 | 2015-03-11 | 重庆泰濠制药有限公司 | Method for purifying homoharringtonine |
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4783454A (en) * | 1985-05-28 | 1988-11-08 | Yaguang Liu | Process for producing harringtonine and homoharringtonine |
| CA1261829A (en) * | 1986-04-30 | 1989-09-26 | Ya-Guang Liu | Process for producing harringtonine and homoharringtonine |
| US20040082565A1 (en) * | 2002-07-17 | 2004-04-29 | Chemgenex Therapeutics, Inc. | Formulations and methods of administration of cephalotaxines including homoharringtonine |
| CN102633806A (en) * | 2012-04-05 | 2012-08-15 | 贵州博丰生物科技产业开发有限公司 | Extraction and separation method of harringtonine |
| CN104402895A (en) * | 2014-12-12 | 2015-03-11 | 重庆泰濠制药有限公司 | Method for purifying homoharringtonine |
Non-Patent Citations (4)
| Title |
|---|
| 何洪源: "中国粗榧果实和枝叶中三尖杉酯碱和高三尖杉酯碱分离方法的研究", 《中草药》, vol. 32, no. 3, pages 201 - 202 * |
| 刘招祥等: "中国粗榧中三尖杉生物碱的提取与分离", 《成分化学》, pages 30 - 31 * |
| 林筱华: "抗癌物质高三尖杉酯碱的提取和研究", 龙岩师专学报, no. 03, pages 144 - 146 * |
| 邱明华等: "丽江产三尖杉的生物碱成分", 《云南植物研究》, vol. 19, no. 1, pages 97 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102319364A (en) | Manufacturing method of dendrobium candidum wallex lindll pure powder tablets | |
| CN102442916A (en) | Method for extracting natural activated products from orange drops | |
| CN106138541B (en) | Processing technology of fresh rhizoma pinelliae preparata in producing area | |
| CN108323270A (en) | The method for promoting tinosporae seed to sprout | |
| CN103540640A (en) | Preparation method of timosaponin A III | |
| CN113651830B (en) | Homoharringtonine and process production method thereof | |
| CN113651830A (en) | Homoharringtonine and process production method thereof | |
| CN1939462B (en) | Health-care Chinese medicinal preparation with anti senility function | |
| Fairbairn et al. | The alkaloids of Papaver somniferum L.—VII: Biosynthetic activity of the isolated latex | |
| CN107823235B (en) | Processing method for solid fermentation of ginseng, American ginseng and pseudo-ginseng | |
| CN101259153A (en) | Tribulus total steroid saponin and preparation thereof | |
| CN100484962C (en) | Method for preparing Codonopsis pilosula polysaccharide extractive as Codonopsis pilosula active ingredient | |
| CN102771520B (en) | Plant yield increasing agent, preparation method thereof and applications in increasing yield of vegetable plants | |
| CN111777657B (en) | Saponin compound and preparation method and application thereof | |
| CN113563356B (en) | Process preparation method of homoharringtonine | |
| CN114470101A (en) | Traditional Chinese medicine composition, preparation method thereof and oral agent containing traditional Chinese medicine composition | |
| CN101205247B (en) | Method for preparing panaxoside monomer Re,Rh1,Rh2,Rg2,Rg3 by using ginseng stem and leaves | |
| CN113563356A (en) | Process for preparing homoharringtonine | |
| CN106719899A (en) | A kind of plant flavor-absorbing disinfecting agent proportioning processes of TR 001 | |
| CN111217822A (en) | Preparation method of tetrandrine as Chinese medicinal anti-allergy agent | |
| CN103211857B (en) | Extracting, separating and purifying method of alkaloid in peanut stem leaves | |
| CN107422049B (en) | Method for extracting organic acid from asparagus rhizosphere soil | |
| CN103356796A (en) | Blood stasis removing particles and preparation method thereof | |
| CN104711312B (en) | The method that Astragaloside IV is prepared using Trichoderma viride | |
| CN102491869B (en) | Rheumlikiangense extract with liver injury protection activity and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |