CN113651830B - Homoharringtonine and process production method thereof - Google Patents
Homoharringtonine and process production method thereof Download PDFInfo
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- 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 94
- 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 87
- 229960002230 omacetaxine mepesuccinate Drugs 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 31
- 230000008569 process Effects 0.000 title claims abstract description 27
- YMNCVRSYJBNGLD-KURKYZTESA-N cephalotaxine Chemical compound C([C@@]12C=C([C@H]([C@H]2C2=C3)O)OC)CCN1CCC2=CC1=C3OCO1 YMNCVRSYJBNGLD-KURKYZTESA-N 0.000 claims abstract description 62
- DSRNKUZOWRFQFO-UHFFFAOYSA-N cephalotaxine Natural products COC1=CC23CCCN2CCc4cc5OCOc5cc4C3=C1O DSRNKUZOWRFQFO-UHFFFAOYSA-N 0.000 claims abstract description 61
- 239000002994 raw material Substances 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 23
- 241000030995 Cephalotaxus sinensis Species 0.000 claims abstract description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 120
- 239000003480 eluent Substances 0.000 claims description 104
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 96
- 239000000284 extract Substances 0.000 claims description 71
- 238000000605 extraction Methods 0.000 claims description 52
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 44
- 239000000843 powder Substances 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 42
- 238000003756 stirring Methods 0.000 claims description 31
- 239000012535 impurity Substances 0.000 claims description 28
- 238000010828 elution Methods 0.000 claims description 27
- 238000003916 acid precipitation Methods 0.000 claims description 26
- 239000003513 alkali Substances 0.000 claims description 25
- 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
- 150000002148 esters Chemical class 0.000 claims description 24
- 238000001704 evaporation Methods 0.000 claims description 24
- 239000000463 material Substances 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 20
- 239000002253 acid Substances 0.000 claims description 19
- 238000011068 loading method Methods 0.000 claims description 16
- 238000010992 reflux Methods 0.000 claims description 16
- 238000001291 vacuum drying Methods 0.000 claims description 15
- 230000006837 decompression Effects 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 238000004806 packaging method and process Methods 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- 239000003208 petroleum Substances 0.000 claims description 12
- 239000000741 silica gel Substances 0.000 claims description 12
- 229910002027 silica gel Inorganic materials 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 239000013078 crystal Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 241000931913 Cephalotaxus fortunei Species 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 9
- 238000004810 partition chromatography Methods 0.000 claims description 9
- 238000005086 pumping 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
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 8
- 238000002425 crystallisation Methods 0.000 claims description 8
- 230000008025 crystallization Effects 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
- 239000008055 phosphate buffer solution Substances 0.000 claims description 8
- 239000007779 soft material Substances 0.000 claims description 8
- 238000004809 thin layer chromatography Methods 0.000 claims description 8
- 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 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000010009 beating Methods 0.000 claims description 7
- 230000001276 controlling effect Effects 0.000 claims description 7
- 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 7
- 238000005070 sampling 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
- 239000004480 active ingredient Substances 0.000 claims description 6
- 230000008020 evaporation Effects 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- 239000002585 base Substances 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 4
- 238000007599 discharging Methods 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
- 229920006395 saturated elastomer Polymers 0.000 claims description 4
- 238000003808 methanol extraction Methods 0.000 claims description 3
- 238000004042 decolorization Methods 0.000 claims description 2
- 241000894007 species Species 0.000 abstract description 6
- 241000488899 Cephalotaxus Species 0.000 description 10
- 238000002156 mixing Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 229930013930 alkaloid Natural products 0.000 description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 229910052593 corundum Inorganic materials 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 229910001845 yogo sapphire Inorganic materials 0.000 description 3
- 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
- BYFGZMCJNACEKR-UHFFFAOYSA-N aluminium(i) oxide Chemical compound [Al]O[Al] BYFGZMCJNACEKR-UHFFFAOYSA-N 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
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 206010000871 Acute monocytic leukaemia Diseases 0.000 description 1
- 208000031261 Acute myeloid 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
- 208000031637 Erythroblastic Acute Leukemia Diseases 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
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 235000012054 meals Nutrition 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
- 238000012946 outsourcing Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention discloses a homoharringtonine and a process production method thereof, wherein the homoharringtonine is prepared by taking branches and leaves of cephalotaxus sinensis as a preparation raw material, the content of the homoharringtonine prepared by taking the branches and leaves of cephalotaxus sinensis as the preparation raw material is more than 99 percent, and the process production method comprises the following steps: the preparation method adopts the technical scheme that the preparation raw materials are innovated, so that the technical problem that the existing technology for extracting and preparing the cephalotaxine by using the rhizome and trunk of cephalotaxine as raw materials to destroy the rare tree species resources is solved, the content and yield of the cephalotaxine are further improved by innovating the technological production method, and the problem that the content and yield of the cephalotaxine are lower by using the rhizome and trunk of cephalotaxine as raw materials is solved.
Description
Technical Field
The invention relates to the field of medicine preparation, in particular to homoharringtonine and a process production method thereof.
Background
The cephalotaxine is an alkaloid separated from the total alkaloids of cephalotaxus plants, exists in cephalotaxus plants, is a high-efficiency antitumor drug developed successfully in China, and is separated from cephalotaxus japonica and cephalotaxus fortunei for the first time in 1963 by Paudler and the like; in 1969, powell et al first separated homoharringtonine from Cephalotaxus sinensis, determined its structure, and listed in one of 47 anticancer drugs commonly used by Ministry of health in the 90 s, and loaded into the 1990 edition of Chinese pharmacopoeia. Clinical application shows that the composition has good curative effect on acute myelogenous leukemia, acute monocytic leukemia, erythroblastic leukemia and other acute nonlymphoblastic leukemia and chronic myelogenous leukemia, and thus, the composition has huge market development potential of homoharringtonine and broad development prospect.
The existing common preparation and production process of homoharringtonine mainly comprises the steps of preparing rhizome, trunk and bark of cephalotaxus fortunei into coarse powder, adding alcohol for extraction, and separating column layers to finally obtain homoharringtonine and cephalotaxine, wherein the existing preparation method has the following defects: the cephalotaxine is a third-period residual seed, the distribution star powder and the preservation quantity are small, the population composition is incomplete, the setting quantity is small, the germination rate is low, the updating is very difficult, in addition, excessive harvest is carried out, so that a plurality of species are involved in endangered or even extinct places, the cephalotaxine is prepared by using the cephalotaxine rhizome and trunk as raw materials, and the rare tree seed resources are further greatly destroyed; secondly, the yield of the homoharringtonine prepared by the existing preparation method is low, and the content of the homoharringtonine in the obtained product is low, so that the method is not beneficial to industrial production.
Disclosure of Invention
The invention aims at: aiming at the technical problems of the preparation of the homoharringtonine in the prior art, the invention provides the homoharringtonine and the process production method thereof, the homoharringtonine uses branches and leaves of cephalotaxus sinensis as the preparation raw material, the innovation of the material taking of the preparation raw material solves the technical problems of destroying rare tree species resources by extracting and preparing the homoharringtonine by using rhizome and trunk of cephalotaxus sinensis as the raw material, the process production method of the homoharringtonine provided by the invention has higher content of the produced homoharringtonine and further solves the problems of lower content and lower yield of the homoharringtonine prepared by the existing preparation method, and the process production method of the homoharringtonine provided by the invention carries out industrial production on the premise of not influencing normal growth of tree species and not damaging the environment.
The technical scheme adopted by the invention is as follows: a homoharringtonine is prepared from branches and leaves of cephalotaxus sinensis as a preparation raw material, and the content of the homoharringtonine prepared from the branches and leaves of cephalotaxus sinensis as the preparation raw material is more than 99%.
The homoharringtonine is prepared by the following process production method, and comprises the following specific steps:
S1: pretreatment of raw materials: the branches and leaves of cephalotaxus sinensis are treated according to a pretreatment SOP program, and impurities are removed to obtain a clean material;
s2: crushing: crushing the net 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 of more than 95% into the tank, and carrying out methanol heating reflux extraction on the cephalotaxus fortunei coarse powder, and repeating for a plurality of times;
S4: concentrating under reduced pressure: the extracting solution obtained by the methanol reflux extraction in the step S3 is input into a tube array concentrator for concentration to obtain extractum;
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 completed;
S6: extracting and decoloring: placing the acid precipitation supernatant obtained in the step S5 into an acid precipitation tank, adding petroleum ether for extraction and decoloration, and collecting a lower acid water solution;
s7: extraction of ester base: pumping the lower acid water solution obtained in the step S6 into an extraction tank for ester alkali extraction, adjusting the pH with 10% ammonia water, and extracting with chloroform;
s8: secondary decompression 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, and concentrating the dried chloroform solutions under reduced pressure in a concentrating tank to obtain cephalotaxine total alkali extract;
S9: column chromatography elution: performing column chromatography elution on the cephalotaxine extractum obtained in the step S8, and obtaining four types of eluents according to classification of active ingredients and impurities: class I eluent, class II eluent, class III eluent, class IV eluent;
S10: three times of decompression concentration: and (3) merging the eluents with the same classification obtained in the step (S9), and respectively concentrating under reduced pressure to finally obtain four types of extractum: pouring the class II extract and the class III extract into a rotary evaporator, and carrying out water bath vacuum reduced pressure concentration and evaporation to obtain class II dry extract and class III dry extract;
s11: partition chromatography elution: and (3) performing partition chromatography elution on the class III dry paste obtained in the step (S10), classifying and combining eluents obtained in different elution sequences to respectively obtain three types of eluents: III-A eluent, III-B eluent and III-C eluent;
S12: four times of decompression concentration: inputting the combined eluents of each category 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-category concentrated extract into a rotary evaporator for concentrating and evaporating to dryness in reduced pressure water bath to obtain a refined homoharringtonine product;
S13: dissolving, evaporating and crystallizing, namely adding acetone into the refined 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 crystallization is completed, extracting and collecting the refined homoharringtonine in a beaker to obtain a crystallized refined product, dissolving the obtained crystallized refined product with a small amount of methanol, heating in a water bath in the rotary evaporator for evaporating under reduced pressure, adding diethyl ether, and obtaining the refined homoharringtonine crystallized product after crystallization is completed;
S14: vacuum drying: vacuum drying the refined homoharringtonine crystal product obtained in the step S13 to obtain dry homoharringtonine powder;
s15: crushing and inner packaging: and (3) crushing the dried homoharringtonine powder subjected to vacuum drying in the step (S14) into fine powder, and packaging according to a standard operation procedure of a packaging post in a raw material workshop, thereby completing the whole process production process of the homoharringtonine.
Further, the crushed coarse powder in the step S2 is 10-20 meshes, and the crushing speed is 70-100 kg/hour.
Further, in the step S3, the amount of methanol added is 3-5 times of the cephalotaxus fortunei coarse powder, the temperature during heating reflux extraction of methanol is 65 ℃, the reflux time is 2 hours each time, and the amount of methanol added is 1-2 times of the cephalotaxus fortunei coarse powder 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, controlling the temperature at 40-50 ℃, concentrating to 60 ℃ for thermal measurement, wherein the relative density of the extract is 1.1.
Further, the step S5 of acid precipitation is specifically 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 more than 24 hr, extracting supernatant, centrifuging the lower residue with a centrifuge at 400RPM to separate supernatant from residue, mixing the supernatant with the residue, collecting in stainless steel barrel, and discarding the residue.
In step S6, adding one third of 60-90 degree 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 in the step S7 specifically includes: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank, regulating the pH value to be 5-5.5 by using 10% ammonia water, fully stirring uniformly, 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 repeating the extraction for 8-10 times until no ester alkali is detected in the chloroform.
Further, in the secondary reduced pressure concentration step of the step S8, the reduced pressure concentration temperature is 55-60 ℃, the vacuum degree is 0.03MPA, and the relative density of the cephalotaxine extractum obtained by the reduced pressure concentration is 1.2.
Further, the specific column chromatography elution step in the step S9 is as follows:
(1) And (3) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and filter paper on the bottom of the column, and flattening;
(2) Stirring 100-200 mesh Al 2O3 with chloroform to paste, slowly pouring into column, pouring while lightly beating the column with soft material to make Al2O3 filling uniform, and circulating with chloroform for 1 hr to make Al 2O3 uniform and filled;
(3) Sample treatment: adding equal amount of chloroform into cephalotaxine extract, stirring to dissolve thoroughly, and stirring with equal amount of aluminum oxide;
(4) Loading: adding the dissolved sample into a column to level the surface of the mixed sample;
(5) Eluting: chloroform in the metering cylinder was changed to chloroform: methanol=100: 0.5, namely the polarity is 0.5%, then opening a valve, adding the valve into a column, and eluting;
(6) Controlling the valve under the column, collecting about every 5L as a collecting unit, sampling every 5 barrels by using a capillary tube to perform thin layer chromatography, detecting cephalotaxine, tracking, checking and classifying, and dividing the effective components and impurities into four types: class I eluent, class II eluent, class III eluent, class IV eluent. The I-type eluent is a precursor impurity, namely no effective component and only impurities are contained; the class II eluent is crossed before, and has active ingredients and impurities; the III-class eluent is mixed ester alkali, and almost all the III-class eluent is an effective component; the IV-class eluent is a post impurity, namely, no effective component and only impurities are contained;
(7) According to the detection result, the polarity of the eluent is changed in time, the polarity gradient of the eluent is three of 0.5%, 1% and 2%, namely, when the eluent is changed from class I to class II, the polarity is from 0.5% to 1%; when the class II is changed into the class III, the polarity is from 1% -2%.
Further, in the three reduced pressure concentration steps of the step S10, the reduced pressure concentration temperature 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 ℃, the water bath vacuum reduced pressure concentration and evaporation temperature is 55-60 ℃, and the time is 0.5-1 hour.
Further, the specific steps of the partition chromatography elution in the step S11 are as follows:
(1) And (3) column loading: weighing 100-160 mesh silica gel, grinding and homogenizing with equal amount of citric acid and disodium hydrogen phosphate buffer solution with pH=5, stirring with chloroform to paste, loading into chromatographic column, beating with soft material to make silica gel uniform and free of hook flow;
(2) Sample treatment: dissolving the mixed ester alkali with equal amount of chloroform;
(3) Loading: slowly adding the sample at the upper end of the column, opening a valve below the column to enable the sample to be slowly adsorbed until the liquid level on the column is level with the silica gel surface;
(4) Adding chloroform solution saturated by PH=5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column for eluting, wherein the flow rate is 1 liter/min, 3 liters is taken as a metering and collecting unit, high cephalotaxine detection is carried out every 5 barrels for sampling, identification is carried out according to thin layer chromatography, classification is respectively combined into three parts, namely III-A eluent, III-B eluent and III-C eluent, wherein the III-A eluent is high cephalotaxine, the III-B eluent is a mixture of high cephalotaxine and cephalotaxine, and the III-C eluent is cephalotaxine; summarizing the elution procedure described above, the first elution is of class III-A: homoharringtonine; the middle part is III-B: a mixture of homoharringtonine and harringtonine; finally, III-C: cephalotaxine.
Further, in the four-time reduced pressure concentration step of the step S12, the reduced pressure concentration is performed in a concentrator at a temperature of 55-60 ℃, the vacuum degree is 0.03mpa, the relative density of the extract is 1.1 when the temperature is 60 ℃, and the concentration temperature is 55-60 ℃ in a rotary evaporator.
Further, in the dissolving, evaporating and crystallizing steps of the step S13, the evaporating and evaporating temperature is 55-60 ℃ in a rotary evaporator by concentrating in a reduced pressure water bath.
Further, the vacuum drying in step S14 specifically includes the steps of: in a vacuum drying oven, the vacuum degree is 0.07MPA, and the drying is carried out for 12 hours at 55-60 ℃.
Further, in the pulverizing and inner packaging step of step S15, the pulverized fine powder was 200 mesh.
Compared with the prior art, the invention has the beneficial effects that:
1) Aiming at the technical problems existing in the existing preparation of homoharringtonine, the invention provides the homoharringtonine and the process production method thereof, wherein the homoharringtonine uses branches and leaves of cephalotaxus sinensis as the preparation raw material, and the innovation of the raw material preparation material solves the technical problems that the existing preparation method using rhizome and trunk of the homoharringtonine as the raw material extracts the homoharringtonine to destroy the rare tree species resource;
2) The content of the homoharringtonine prepared by the process production method of the homoharringtonine provided by the invention is more than 99%, the problems of low content and low yield of the homoharringtonine prepared by the existing preparation method are further solved, and the process production method of the homoharringtonine provided by the invention is used for industrial production on the premise of not influencing the normal growth of tree species and not damaging the environment.
Drawings
FIG. 1 is a graph of infrared spectrum test comparison of a homoharringtonine sample and a homoharringtonine test sample prepared by the invention;
FIG. 2 is a test report of homoharringtonine samples prepared by the present 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 cephalotaxus sinensis as raw materials, and the content of homoharringtonine is 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: the branches and leaves of cephalotaxus sinensis are treated according to a pretreatment SOP program, and impurities are removed to obtain a clean material;
S2: crushing: crushing the net material obtained in the step S1 to obtain coarse powder, wherein the crushed coarse powder is 10-20 meshes, and the crushing speed is 70-100 kg/hour;
S3: methanol extraction: putting the cephalotaxus coarse powder in the step S2 into a tank from a tank top feeding port, adding excessive methanol of more than 95% into the tank, carrying out methanol heating reflux extraction on the cephalotaxus coarse powder, repeating for a plurality of times, wherein the adding amount of the methanol of more than 95% is 3-5 times of that of the cephalotaxus coarse powder, the temperature of the methanol during heating reflux extraction is 65 ℃, the reflux time is 2 hours each time, and the adding amount of the methanol of more than 95% is 1-2 times of that of the cephalotaxus coarse powder and the repeated extraction times are 2-4 times;
S4: concentrating under reduced pressure: and (3) inputting the extracting solution obtained by the repeated methanol reflux extraction in the step (S3) into a tube array concentrator for concentration to obtain an extract, wherein the vacuum degree of reduced pressure concentration is as follows: 0.06-0.07MPa, controlling the temperature at 40-50 ℃, concentrating to 60 ℃ for thermal measurement, wherein the relative density of the extract is 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 completed, wherein the steps specifically comprise: 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 more than 24 hr, extracting supernatant, centrifuging the lower residue with a centrifuge at 400RPM to separate supernatant from residue, mixing the supernatant with the residue, collecting in a stainless steel barrel, and discarding the residue;
S6: extracting and decoloring: placing the acid precipitation supernatant obtained in the step S5 into an acid precipitation tank, adding petroleum ether for extraction and decoloration, and collecting a lower acid water solution; in the step S6, the specific steps of extraction and decolorization are that one third of petroleum ether with 60-90 degrees is added into an acid precipitation tank, the mixture is fully stirred and extracted for 5 minutes, the mixture is stood for 0.5 hour, after layering, an upper petroleum ether layer is extracted, extraction is carried out for several times until an extract liquid is almost colorless, and a lower acid water solution is pumped into the extraction tank;
S7: extraction of ester base: pumping the lower acid water solution obtained in the step S6 into an extraction tank for ester alkali extraction, adjusting the pH with 10% ammonia water, and extracting with chloroform, wherein the step of extracting ester alkali comprises the following steps: pumping the lower-layer acid water solution obtained in the step S6 into an extraction tank, regulating the pH value to be 5-5.5 by using 10% ammonia water, fully stirring uniformly, 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 alkali is detected in the chloroform;
s8: secondary decompression 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, and concentrating the dried chloroform solutions under reduced pressure in a concentrating tank to obtain cephalotaxine total alkali extract;
s9: column chromatography elution: performing column chromatography elution on the cephalotaxine extractum obtained in the step S8, and obtaining four types of eluents according to classification of active ingredients and impurities: the specific steps of column chromatography elution are as follows:
(1) And (3) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and filter paper on the bottom of the column, and flattening;
(2) Stirring 100-200 mesh Al 2O3 with chloroform to paste, slowly pouring into column, pouring while lightly beating the column with soft material to make Al 2O3 filled uniformly, and circulating with chloroform for 1 hr to make Al 2O3 uniform and filled;
(3) Sample treatment: adding equal amount of chloroform into cephalotaxine extract, stirring to dissolve thoroughly, and stirring with equal amount of aluminum oxide;
(4) Loading: adding the dissolved sample into a column to level the surface of the mixed sample;
(5) Eluting: chloroform in the metering cylinder was changed to chloroform: methanol=100: 0.5, namely the polarity is 0.5%, then opening a valve, adding the valve into a column, and eluting;
(6) Controlling the valve under the column, collecting about every 5L as a collecting unit, sampling every 5 barrels by using a capillary tube to perform thin layer chromatography, detecting cephalotaxine, tracking, checking and classifying, and dividing the effective components and impurities into four types: class I eluent, class II eluent, class III eluent, class IV eluent. The I-type eluent is a precursor impurity, namely no effective component and only impurities are contained; the class II eluent is crossed before, and has active ingredients and impurities; the III-class eluent is mixed ester alkali, and almost all the III-class eluent is an effective component; the IV-class eluent is a post impurity, namely, no effective component and only impurities are contained;
(7) According to the detection result, the polarity of the eluent is changed in time, the polarity gradient of the eluent is three of 0.5%, 1% and 2%, namely, when the eluent is changed from class I to class II, the polarity is from 0.5% to 1%; when the class II is changed into the class III, the polarity is from 1% -2%.
S10: three times of decompression concentration: and (3) merging the eluents with the same classification obtained in the step (S9), and respectively concentrating under reduced pressure to finally obtain four types of extractum: pouring the class I extract, the class II extract, the class III extract and the class IV extract into a rotary evaporator, carrying out water bath vacuum reduced pressure concentration and evaporation to dryness to obtain class II dry extract and class III dry extract, wherein the reduced pressure concentration temperature 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 ℃, and the water bath vacuum reduced pressure concentration and evaporation to dryness temperature is 55-60 ℃ for 0.5-1 hour.
S11: partition chromatography elution: and (3) performing partition chromatography elution on the class III dry paste obtained in the step (S10), classifying and combining eluents obtained in different elution sequences to respectively obtain three types of eluents: the specific steps of the III-A eluent, the III-B eluent and the III-C eluent are as follows:
(1) And (3) column loading: weighing 100-160 mesh silica gel, grinding and homogenizing with equal amount of citric acid and disodium hydrogen phosphate buffer solution with pH=5, stirring with chloroform to paste, loading into chromatographic column, beating with soft material to make silica gel uniform and free of hook flow;
(2) Sample treatment: dissolving the mixed ester alkali with equal amount of chloroform;
(3) Loading: slowly adding the sample at the upper end of the column, opening a valve below the column to enable the sample to be slowly adsorbed until the liquid level on the column is level with the silica gel surface;
(4) Adding chloroform solution saturated by PH=5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column for eluting, wherein the flow rate is 1 liter/min, 3 liters is taken as a metering and collecting unit, high cephalotaxine detection is carried out every 5 barrels for sampling, identification is carried out according to thin layer chromatography, classification is respectively combined into three parts, namely III-A eluent, III-B eluent and III-C eluent, wherein the III-A eluent is high cephalotaxine, the III-B eluent is a mixture of high cephalotaxine and cephalotaxine, and the III-C eluent is cephalotaxine; summarizing the elution procedure described above, the first elution is of class III-A: homoharringtonine; the middle part is III-B: a mixture of homoharringtonine and harringtonine; finally, III-C: cephalotaxine.
S12: four times of decompression concentration: concentrating the combined eluents obtained in the step S11 in a concentrator under reduced pressure to obtain corresponding III-A extract, III-B extract and III-C extract respectively, pouring the concentrated extract A into a rotary evaporator for concentrating and evaporating to dryness in a reduced pressure water bath to obtain refined homoharringtonine, concentrating under reduced pressure in the concentrator at 55-60 ℃ at a vacuum degree of 0.03MPA and a temperature of 55-60 ℃ when heated at 60 ℃, wherein the relative density of the extract is 1.1, and concentrating in the rotary evaporator at a temperature of 55-60 ℃.
S13: dissolving, evaporating and crystallizing, namely adding acetone into the high cephalotaxine essence 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, extracting and collecting the crystals in a beaker to obtain a crystal essence, dissolving the obtained crystal essence with a small amount of methanol, heating in a water bath in the rotary evaporator for evaporating under reduced pressure, adding diethyl ether, crystallizing to obtain the high cephalotaxine essence, and concentrating and evaporating to dryness in a reduced pressure water bath in the rotary evaporator at 55-60 ℃.
S14: vacuum drying: vacuum drying the refined homoharringtonine crystal product obtained in the step S13 to obtain dry homoharringtonine powder, wherein the vacuum drying comprises the following specific steps of: in a vacuum drying oven, the vacuum degree is 0.07MPA, and the drying is carried out for 12 hours at 55-60 ℃.
S15: crushing and inner packaging: and (3) crushing the homoharringtonine 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 effectiveness of the process production method of the homoharringtonine provided by the invention, taking 808kg of homoharringtonine branches as an example, the specific flow is as follows:
1. 808kg of cephalotaxus fortunei branches are treated according to the pretreatment SOP, and the impurities are removed to obtain 804kg of clean materials;
2. placing the processed raw materials into a clean nontoxic plastic bag, attaching a material label, storing in a clean material temporary storage room, and stacking orderly according to the production variety and the used materials;
3. Pulverizing the materials into coarse powder of 10-20 meshes in a pulverizer at a pulverizing speed of 80 kg/hr to obtain coarse powder of 800kg;
4. Picking up cephalotaxus fortunei branch coarse powder from a clean material temporary storage room;
5. getting more than 95% of methanol 3.2T;
6. putting the cephalotaxus meal into a tank from a tank top feeding port according to 800kg, flattening, and closing the feeding port;
7. Adding 4 times of methanol, and making the methanol liquid level higher than the powder surface by 10cm;
8. opening a steam heating valve for heating, enabling methanol in the tank to slightly boil and reflux, starting counting when the temperature of the solution in the tank reaches 65 ℃, keeping reflux for 2 hours, and closing the heating valve;
9. Opening a tank drain valve, putting the extracting solution into a temporary storage tank, and closing the drain valve after the extracting solution is put;
10. adding 2 times of methanol into the extraction tank, extracting for 2 hours by the same method, and repeating the extraction for 2 times;
11. concentrating the extractive solution in tube concentrator under vacuum of 0.06-0.07MPa at 40-50deg.C until the relative density of extract is 1.1 (60deg.C), collecting extract with an extract collecting barrel to obtain extract 70-75kg with an extract yield of about 2%.
Yield = concentrated extract amount (relative density 1.1 ℃ measured at 60 ℃)/cephalotaxus branch coarse powder amount 100%
12. Acid precipitation:
12.1 Pouring the extract 1 into an acid precipitation tank, adding 1% HCl aqueous solution equivalent to the extract, fully stirring and dissolving for 2 hours, adding a certain amount of water, adjusting the pH value to 2, standing, and settling for more than 24 hours;
12.2 Extracting supernatant, centrifuging the lower residue liquid with a centrifuge at 400RPM to separate supernatant from residue, mixing the supernatants, collecting in a stainless steel barrel, and discarding residue;
13. Extraction
13.1 Decoloring (decoloring)
13.1.1 Placing the above supernatant in an acid precipitation tank, adding one third of 60-90 deg. petroleum ether, stirring and extracting for 5 min, standing for 0.5 hr, layering, extracting upper petroleum ether layer, and extracting several times until the extract is almost colorless. Pumping the lower acid water solution into an extraction tank for extraction;
13.1.2 the extracts (petroleum ether layer) were combined, passed through a 50kg dry column of anhydrous sodium sulfate to absorb water, and then fed into a concentrator to concentrate under reduced pressure to recover the reagent for about 0.5 hour at a temperature of: 55-60 ℃, vacuum degree: 0.03MPA;
13.2 Extraction of ester base: regulating pH of the decolorized acid water solution 2 to be 5-5.5 with 10% ammonia water, stirring thoroughly, adding chloroform of the same volume as the acid water solution 2, stirring thoroughly, extracting for 5min, standing for 0.5 hr, layering, discharging lower chloroform layer, repeating extraction for 8-10 times until no ester alkali is detected in chloroform;
13.3 After the extraction is completed, the chloroform solutions are combined and dried by an anhydrous sodium sulfate drying column, the dried chloroform solution is input into a concentration tank for reduced pressure concentration until the relative density of the extract is 1.2 for about 12 hours, and the temperature is as follows: 55-60 ℃, vacuum degree: 0.03MPA;
13.4 And (3) collecting paste: collecting the concentrated extract in a stainless steel barrel, sampling by QA, detecting content, weighing, and warehousing to obtain about 6-7kg of extract 2 (cephalotaxine) with an extraction rate of about 10%.
Extraction ratio = (content of total cephalotaxine)/(content of cephalotaxus branch coarse powder) 100%
13.5 Adjusting the pH of the acid water solution to be 9 with 10% ammonia water, and extracting with chloroform according to the same method of 4.6.2 until no alkaloid is detected;
13.6 Mixing chloroform solutions, drying with anhydrous NaSO4 for 12 hr, concentrating to obtain extract with relative density of 1.2 (60 deg.C), temperature of 55-60 deg.C, vacuum degree of 0.03MPa, crystallizing with anhydrous ethanol, recrystallizing to obtain cephalotaxine, weighing, and warehousing;
14. Chromatography
14.1 And (3) material receiving: taking cephalotaxine total alkali extract (extract 2) from the raw materials warehouse according to production instruction, about 6-7kg;
14.2 And (3) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and filter paper on the bottom of the column, and flattening;
14.3 Stirring 320kg of 100-200 mesh Al2O with activity of two stages with chloroform to form paste, slowly pouring into a column, lightly knocking the column with soft material while pouring to make Al2O3 filling uniform, and circulating with chloroform for 1 hr to make Al2O3 uniform and full;
14.4 Sample treatment: adding equal amount of chloroform into 1kg of cephalotaxine total alkali extractum, stirring to dissolve thoroughly, and stirring with equal amount of aluminum oxide;
14.5 Loading: adding the dissolved sample into a column to level the surface of the mixed sample;
14.6 Eluting: chloroform in the metering cylinder was changed to chloroform: methanol=100: 0.5 (i.e., 0.5% polarity), then opening the valve, adding into the column, and eluting;
14.7 controlling the valve under the column, the flow rate is about 1 liter/min, about every 5 liters is collected as a collecting unit, every 5 barrels is sampled by a capillary tube to carry out thin layer chromatography, the cephalotaxine is detected, and the valve is tracked, checked and classified, and can be divided into four types according to the effective components and impurities, namely: class I eluent, class II eluent, class III eluent, class IV eluent. The I-type eluent is a precursor impurity, namely no effective component and only impurities are contained; the class II eluent is crossed before, and has active ingredients and impurities; the III-class eluent is mixed ester alkali, and almost all the III-class eluent is an effective component; the IV-class eluent is a post impurity, namely, no effective component and only impurities are contained;
14.8 According to the detection result, the polarity of the eluent is changed in time, and the polarity gradient of the eluent is three of 0.5%, 1% and 2%, namely, the polarity is from 0.5% to 1% when the eluent is changed from class I to class II, and the polarity is from 1% to 2% when the eluent is changed from class II to class III;
14.9 Mixing eluents with the same classification, inputting into a concentration tank, and concentrating under reduced pressure until the relative density of the extract is 1:1 (60 ℃ for thermal measurement): 55-60 ℃, vacuum degree: 0.03MPA, meanwhile, carrying out reagent recovery, discarding class I and class IV extractum, pouring class II and class III eluent concentrated extractum into a rotary evaporator, concentrating in a water bath at 55-60 ℃ under vacuum and decompressing to dryness, respectively detecting the content about 0.5 hour, weighing and pasting material labels, and recording to obtain class II dry extractum 500g and class III dry extractum 525g;
yield = (class C dry extract amount x content)/(homoharringtonine total amount x content) ×100%
15. Partition chromatography
15.1 And (3) material receiving: and (3) taking the III-class cephalotaxine mixed ester alkali dry paste from the intermediate station according to the production instruction.
15.2 And (3) column loading: weighing 20kg of 100-160 mesh silica gel, fully and uniformly grinding with equal amount of citric acid with PH=5 and disodium hydrogen phosphate buffer solution, stirring with chloroform to form paste, loading into chromatographic column, and beating the column with soft material to make the silica gel uniform without flow hooking;
15.3 Sample treatment: dissolving the mixed ester alkali with equal amount of chloroform;
15.4 Loading: slowly adding the sample at the upper end of the column, opening a valve below the column to enable the sample to be slowly adsorbed until the liquid level on the column is level with the silica gel surface;
15.5 Adding chloroform solution saturated by PH=5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column, eluting (the flow rate is 1 liter/min), taking 3 liters as a metering and collecting unit, sampling every 5 barrels, carrying out high cephalotaxine detection, identifying according to thin layer chromatography, classifying and combining into three parts respectively, namely III-A eluent, III-B eluent and III-C eluent, wherein the III-A eluent is high cephalotaxine, the III-B eluent is a mixture of high cephalotaxine and cephalotaxine, and the III-C eluent is cephalotaxine; summarizing the elution procedure described above, the first elution is of class III-A: homoharringtonine; the middle part is III-B: a mixture of homoharringtonine and harringtonine; finally, III-C: cephalotaxine;
15.6 The first to elute is homoharringtonine (class I); the middle part is a mixture of homoharringtonine and harringtonine (II type); finally, cephalotaxine (class III) is eluted;
15.7 The eluents with the same classification are combined and input into a concentrator for decompression concentration until the relative density of the extract is 1.1 (60 ℃ for heat measurement) temperature: 55-60 ℃, vacuum degree: 0.03MPA, and storing the class II and class III extractum in a warehouse for standby. Pouring the I-type 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 160-180g of refined homoharringtonine, detecting, weighing, recording and sending to an intermediate station.
Yield = (high cephalotaxine essence x content)/(high cephalotaxine class III mixed ester alkali amount x content) ×100%
16. Crystallization
16.1 And (3) material receiving: picking up the refined homoharringtonine from the intermediate station according to the production instruction;
16.2 And (3) crystallization: the refined homoharringtonine is dissolved with a small amount of acetone. Concentrating in a reduced pressure water bath at 55-60deg.C in a rotary evaporator, evaporating to dryness until acetone is small and wall hanging phenomenon of liquid exists, taking off, adding diethyl ether until trace crystal grains appear, standing overnight, and crystallizing;
16.3 Filtering with Buchner funnel after crystallization is completed, draining, collecting in beaker, dissolving with small amount of methanol, heating in 55-60deg.C water bath in rotary evaporator for evaporation under reduced pressure until methanol is small, removing, adding diethyl ether until crystal nucleus appears, standing overnight, and crystallizing;
16.4 Repeating the operation for several times according to 4.9.3 method, crystallizing, recrystallizing until reaching the appearance quality standard (namely white powder);
16.5 Vacuum drying: placing the crystallized refined homoharringtonine crystal in a stainless steel plate, placing in a vacuum drying oven, and drying at 55-60deg.C for 12 hr. Vacuum degree is 0.07MPA, and about 70-80g of cephalotaxine dry powder is obtained. And the sample is detected by QA, and the crushing process can be carried out after the sample is qualified. The yield is 50-60%;
Yield = (high cephalotaxine essence x content)/(high cephalotaxine dry powder amount x content) ×100%
16.6 According to the crystal post, clearing SOP, filling in production records and clearing records;
17. crushing
17.1, Drying the dried homoharringtonine powder in vacuum, and crushing the homoharringtonine powder into 200 meshes of fine powder in a crusher;
17.2 Placing the crushed fine powder into a stainless steel barrel for weighing, attaching a material label, and carrying out a wrapping process;
18. inner package
18.1 And (3) material receiving: packing materials from the warehouse collar according to production instructions;
18.2 Packaging according to standard operation procedures of packaging posts in a raw material workshop, wherein the packaging specification is as follows: 50 g/bag;
18.3 The staff must sterilize the handle, wear the sterilized latex glove with 50 g/bag as unit, pack with plastic film first, then suck the superfluous air under negative pressure, seal;
Bagging: and (3) performing post operation according to the outsourcing of a raw material workshop, performing SOP operation, sleeving 1 aluminum foil bag package on each plastic bag, and sealing.
Test example 3
In the embodiment, the infrared spectrogram of the homoharringtonine sample and the homoharringtonine standard test product prepared by the process production method is compared, the result is shown in the attached figure 1 of the specification, the comparison result further shows that the process preparation method of the homoharringtonine provided by the invention is truly feasible, and the content of the homoharringtonine in the sample finally obtained by the process preparation method of the homoharringtonine provided by the invention is more than 99 percent, so that the yield and purity of the traditional process are greatly improved.
The above examples merely illustrate specific embodiments of the present application, which are described in detail and are not to be construed as limiting the scope of the application, it should be noted that modifications and improvements can be made by those skilled in the art without departing from the technical scope of the application.
Claims (5)
1. A process for preparing homoharringtonine is characterized in that,
The cephalotaxine is prepared by taking branches and leaves of cephalotaxine as a preparation raw material, and the content of the cephalotaxine prepared by taking branches and leaves of cephalotaxine as the preparation raw material is more than 99%;
the process production method specifically comprises the following steps:
S1: pretreatment of raw materials: the branches and leaves of cephalotaxus sinensis are treated according to a pretreatment SOP program, and impurities are removed to obtain a clean material;
s2: crushing: crushing the net material obtained in the step S1 to obtain coarse powder;
S3: methanol extraction: putting the 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 carrying out methanol heating reflux extraction on the coarse powder, wherein the repeated extraction times are 2-4 times;
S4: concentrating under reduced pressure: the extracting solution obtained by the methanol reflux extraction in the step S3 is input into a tube array concentrator for concentration to obtain extractum;
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 completed;
S6: extracting and decoloring: placing the acid precipitation supernatant obtained in the step S5 into an acid precipitation tank, adding petroleum ether for extraction and decoloration, and collecting a lower acid water solution;
S7: extraction of ester base: pumping the lower acid water solution obtained in the step S6 into an extraction tank for ester alkali extraction, adjusting the pH to 5-5.5 by using 10% ammonia water, and extracting by using chloroform;
s8: secondary decompression 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, and concentrating the dried chloroform solutions under reduced pressure in a concentrating tank to obtain cephalotaxine total alkali extract;
S9: column chromatography elution: performing column chromatography elution on the cephalotaxine extractum obtained in the step S8, and obtaining four types of eluents according to classification of active ingredients and impurities: class I eluent, class II eluent, class III eluent, class IV eluent; wherein the eluent of class I is front impurity, the eluent of class II is front cross, the eluent of class III is mixed ester alkali, and the eluent of class IV is rear impurity;
S10: three times of decompression concentration: and (3) merging the eluents with the same classification obtained in the step (S9), and respectively concentrating under reduced pressure to finally obtain four types of extractum: pouring the class II extract and the class III extract into a rotary evaporator, and carrying out water bath vacuum reduced pressure concentration and evaporation to obtain class II dry extract and class III dry extract;
s11: partition chromatography elution: and (3) performing partition chromatography elution on the class III dry paste obtained in the step (S10), classifying and combining eluents obtained in different elution sequences to respectively obtain three types of eluents: III-A eluent, III-B eluent and III-C eluent;
S12: four times of decompression concentration: inputting the combined eluents of each category 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-category concentrated extract into a rotary evaporator for concentrating and evaporating to dryness in reduced pressure water bath to obtain a refined homoharringtonine product;
S13: dissolving, evaporating and crystallizing, namely adding acetone into the refined 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 crystallization is completed, extracting and collecting the refined homoharringtonine in a beaker to obtain a crystallized refined product, dissolving the obtained crystallized refined product with a small amount of methanol, heating in a water bath in the rotary evaporator for evaporating under reduced pressure, adding diethyl ether, and obtaining the refined homoharringtonine crystallized product after crystallization is completed;
the specific column chromatography elution step in the step S9 comprises the following steps:
(1) And (3) installing a chromatographic column: fixing the column on a frame, keeping the column vertical in the horizontal direction, putting the cut absorbent cotton and filter paper on the bottom of the column, and flattening;
(2) Stirring 100-200 mesh Al 2O3 with chloroform to paste, slowly pouring into column, pouring while lightly beating the column with soft material to make Al 2O3 filled uniformly, and circulating with chloroform for 1 hr to make Al 2O3 uniform and filled;
(3) Sample treatment: adding equal amount of chloroform into cephalotaxine extract, stirring to dissolve thoroughly, and stirring with equal amount of aluminum oxide;
(4) Loading: adding the dissolved sample into a column to level the surface of the mixed sample;
(5) Eluting: chloroform in the metering cylinder was changed to chloroform: methanol=100: 0.5, namely the polarity is 0.5%, then opening a valve, adding the valve into a column, and eluting;
(6) Controlling the valve under the column, wherein the flow rate is 1 liter/min, collecting every 5 liters as a collecting unit, sampling every 5 barrels by using a capillary tube to perform thin layer chromatography, detecting cephalotaxine, tracking, checking and classifying, and dividing the sample into four types according to effective components and impurities: class I eluent, class II eluent, class III eluent, class IV eluent;
(7) According to the detection result, the polarity of the eluent is changed in time, the polarity gradient of the eluent is three of 0.5%, 1% and 2%, namely, when the eluent is changed from class I to class II, the polarity is from 0.5% to 1%; when the class II is changed into the class III, the polarity is from 1 percent to 2 percent;
the specific steps of the partition chromatography elution in the step S11 are as follows:
(1) And (3) column loading: weighing 100-160 mesh silica gel, grinding and homogenizing with equal amount of citric acid and disodium hydrogen phosphate buffer solution with pH=5, stirring with chloroform to paste, loading into chromatographic column, beating with soft material to make silica gel uniform and free of hook flow;
(2) Sample treatment: dissolving the mixed ester alkali with equal amount of chloroform;
(3) Loading: slowly adding the sample at the upper end of the column, opening a valve below the column to enable the sample to be slowly adsorbed until the liquid level on the column is level with the silica gel surface;
(4) Adding chloroform solution saturated by PH=5 citric acid and disodium hydrogen phosphate buffer solution into the upper end of the column, eluting at a flow rate of 1 liter/min, taking 3 liters as a metering and collecting unit, carrying out cephalotaxine detection every 5 barrels of samples, identifying according to thin layer chromatography, classifying and combining into three parts respectively, namely III-A eluent, III-B eluent and III-C eluent, wherein the first elution is III-A: homoharringtonine; the middle part is III-B: a mixture of homoharringtonine and harringtonine; finally, III-C: cephalotaxine.
2. The process for the production of homoharringtonine of claim 1, further comprising the steps of:
S14: vacuum drying: vacuum drying the refined homoharringtonine crystal product obtained in the step S13 to obtain dry homoharringtonine powder; s15: crushing and inner packaging: and (3) crushing the dried homoharringtonine powder subjected to vacuum drying in the step (S14) into fine powder, and packaging according to a standard operation procedure of a packaging post in a raw material workshop, thereby completing the whole process production process of the homoharringtonine.
3. The process production method of homoharringtonine according to claim 1, wherein in the step S3, the addition amount of methanol is 3-5 times of the harringtonine coarse powder, the temperature during heating reflux extraction of methanol is 65 ℃, the reflux time is 2 hours each time, and the addition amount of methanol is 1-2 times of the harringtonine coarse powder during repeated reflux extraction, and in the step S4, the vacuum degree of reduced pressure concentration is: 0.06-0.07MPa, controlling the temperature at 40-50 ℃, concentrating to 60 ℃ for thermal measurement, wherein the relative density of the extract is 1.1.
4. The process for the production of homoharringtonine according to claim 1, wherein,
The step S5 of acid precipitation 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, regulating the pH value to 2, standing, settling for more than 24 hours, extracting supernatant, centrifuging lower-layer residue liquid by using a centrifugal machine at the rotating speed of 400RPM, separating the supernatant from the residue, merging the supernatant and collecting the supernatant in a stainless steel barrel, discarding the residue, and in the step S6, the extraction and decolorization are specifically that 60-90 DEG petroleum ether is added into an acid precipitation tank, fully stirring and extracting is carried out for 5 minutes, standing for 0.5 hour, after layering, extracting an upper-layer petroleum ether layer for several times, pumping lower-layer acid water liquid into an extraction tank until the extract is almost colorless.
5. The process for the production of homoharringtonine according to claim 1, wherein,
The extraction ester alkali in the step S7 specifically comprises the following steps: pumping the lower acid water solution obtained in the step S6 into an extraction tank, regulating the pH value to be 5-5.5 by using 10% ammonia water, fully stirring uniformly, 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 chloroform layer after layering, repeating the extraction for 8-10 times until no ester alkali is detected in the chloroform, wherein in the step S8 of the secondary decompression concentration, the decompression concentration temperature is 55-60 ℃, the vacuum degree is 0.03MPa, and the relative density of the cephalotaxus fortunei total alkali extract obtained by the decompression concentration is 1.2.
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| WO2004009092A1 (en) * | 2002-07-17 | 2004-01-29 | Chemgenex Pharmaceuticals Limited | Formulations and methods of administration of cephalotaxines, including homoharringtonine |
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