WO2023236453A1 - Method for preparing albiflorin-6'-o-benzenesulfonate - Google Patents
Method for preparing albiflorin-6'-o-benzenesulfonate Download PDFInfo
- Publication number
- WO2023236453A1 WO2023236453A1 PCT/CN2022/133498 CN2022133498W WO2023236453A1 WO 2023236453 A1 WO2023236453 A1 WO 2023236453A1 CN 2022133498 W CN2022133498 W CN 2022133498W WO 2023236453 A1 WO2023236453 A1 WO 2023236453A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- paeoniflorin
- benzenesulfonate
- glycoside
- solvent
- mixed solvent
- Prior art date
Links
- 229940077388 benzenesulfonate Drugs 0.000 title claims abstract description 166
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000001035 drying Methods 0.000 claims abstract description 76
- 239000012046 mixed solvent Substances 0.000 claims abstract description 69
- 238000006243 chemical reaction Methods 0.000 claims abstract description 48
- 238000000926 separation method Methods 0.000 claims abstract description 41
- 239000000463 material Substances 0.000 claims abstract description 21
- CSKNSYBAZOQPLR-UHFFFAOYSA-N benzenesulfonyl chloride Chemical compound ClS(=O)(=O)C1=CC=CC=C1 CSKNSYBAZOQPLR-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000004440 column chromatography Methods 0.000 claims abstract description 19
- 239000002253 acid Substances 0.000 claims abstract description 18
- 239000011230 binding agent Substances 0.000 claims abstract description 17
- 239000003054 catalyst Substances 0.000 claims abstract description 17
- 238000000605 extraction Methods 0.000 claims abstract description 16
- 238000001179 sorption measurement Methods 0.000 claims abstract description 13
- 229910052751 metal Inorganic materials 0.000 claims abstract description 6
- 239000002184 metal Substances 0.000 claims abstract description 6
- YKRGDOXKVOZESV-UHFFFAOYSA-N paeoniflorin Natural products O1C(C)(C2(CC34)OC5C(C(O)C(O)C(CO)O5)O)CC3(O)OC1C24COC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-UHFFFAOYSA-N 0.000 claims description 131
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 128
- -1 paeoniflorin glycosides Chemical class 0.000 claims description 108
- 229930182470 glycoside Natural products 0.000 claims description 99
- 239000002904 solvent Substances 0.000 claims description 90
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 69
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 60
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 54
- 239000000047 product Substances 0.000 claims description 49
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 44
- 238000003756 stirring Methods 0.000 claims description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 238000010898 silica gel chromatography Methods 0.000 claims description 37
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 claims description 36
- 239000003480 eluent Substances 0.000 claims description 33
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 32
- IEJIGPNLZYLLBP-UHFFFAOYSA-N dimethyl carbonate Chemical compound COC(=O)OC IEJIGPNLZYLLBP-UHFFFAOYSA-N 0.000 claims description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 28
- 239000000741 silica gel Substances 0.000 claims description 28
- 229910002027 silica gel Inorganic materials 0.000 claims description 28
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 21
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 20
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 claims description 17
- 244000236658 Paeonia lactiflora Species 0.000 claims description 17
- 235000008598 Paeonia lactiflora Nutrition 0.000 claims description 17
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 15
- 238000002425 crystallisation Methods 0.000 claims description 14
- 230000008025 crystallization Effects 0.000 claims description 14
- JMMWKPVZQRWMSS-UHFFFAOYSA-N isopropanol acetate Natural products CC(C)OC(C)=O JMMWKPVZQRWMSS-UHFFFAOYSA-N 0.000 claims description 13
- 229940011051 isopropyl acetate Drugs 0.000 claims description 13
- GWYFCOCPABKNJV-UHFFFAOYSA-N isovaleric acid Chemical compound CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims description 13
- 239000007810 chemical reaction solvent Substances 0.000 claims description 11
- 239000012043 crude product Substances 0.000 claims description 11
- 150000002148 esters Chemical class 0.000 claims description 11
- 238000007710 freezing Methods 0.000 claims description 11
- 230000008014 freezing Effects 0.000 claims description 11
- 238000000859 sublimation Methods 0.000 claims description 11
- 230000008022 sublimation Effects 0.000 claims description 11
- 241000736199 Paeonia Species 0.000 claims description 10
- 235000006484 Paeonia officinalis Nutrition 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 238000010992 reflux Methods 0.000 claims description 10
- 150000002576 ketones Chemical class 0.000 claims description 9
- 239000003463 adsorbent Substances 0.000 claims description 8
- 125000002524 organometallic group Chemical group 0.000 claims description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 8
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical group CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 229940092714 benzenesulfonic acid Drugs 0.000 claims description 6
- 238000010981 drying operation Methods 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 4
- GJRQTCIYDGXPES-UHFFFAOYSA-N iso-butyl acetate Natural products CC(C)COC(C)=O GJRQTCIYDGXPES-UHFFFAOYSA-N 0.000 claims description 4
- FGKJLKRYENPLQH-UHFFFAOYSA-M isocaproate Chemical compound CC(C)CCC([O-])=O FGKJLKRYENPLQH-UHFFFAOYSA-M 0.000 claims description 4
- OQAGVSWESNCJJT-UHFFFAOYSA-N isovaleric acid methyl ester Natural products COC(=O)CC(C)C OQAGVSWESNCJJT-UHFFFAOYSA-N 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 4
- 235000011181 potassium carbonates Nutrition 0.000 claims description 4
- 238000005377 adsorption chromatography Methods 0.000 claims description 3
- LJQKCYFTNDAAPC-UHFFFAOYSA-N ethanol;ethyl acetate Chemical compound CCO.CCOC(C)=O LJQKCYFTNDAAPC-UHFFFAOYSA-N 0.000 claims description 3
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical group OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 150000002338 glycosides Chemical class 0.000 claims description 3
- 150000008282 halocarbons Chemical class 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 claims description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 claims description 2
- 238000003808 methanol extraction Methods 0.000 claims description 2
- 238000000874 microwave-assisted extraction Methods 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
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- 238000002791 soaking Methods 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 229910001887 tin oxide Inorganic materials 0.000 claims description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims 2
- 150000001298 alcohols Chemical class 0.000 claims 1
- 125000000217 alkyl group Chemical group 0.000 claims 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims 1
- 235000017557 sodium bicarbonate Nutrition 0.000 claims 1
- AFNRRBXCCXDRPS-UHFFFAOYSA-N tin(ii) sulfide Chemical class [Sn]=S AFNRRBXCCXDRPS-UHFFFAOYSA-N 0.000 claims 1
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- 238000009776 industrial production Methods 0.000 abstract description 6
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- QQUHMASGPODSIW-UHFFFAOYSA-N Albiflorin Natural products C=1C=CC=CC=1C(=O)OCC12C(=O)OC3(C)CC(O)C1CC32OC1OC(CO)C(O)C(O)C1O QQUHMASGPODSIW-UHFFFAOYSA-N 0.000 abstract description 4
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the field of compounds, and specifically relates to a preparation method of paeoniflorin-6'-O-benzenesulfonate.
- White peony root is the root of Paeonia lactiflora, which has antipyretic and analgesic effects. Modern pharmacology shows that white peony extract also has various pharmacological activities such as anti-inflammatory and immunomodulatory, detoxifying and liver-protecting, anti-mutagenic and anti-tumor. Total glycosides of white peony are also the first to be used in the clinical treatment of rheumatoid joints. It is an anti-inflammatory immunomodulatory drug for inflammation, and has the characteristics of good curative effect, few adverse reactions, and good tolerance.
- Albiflorin also has a variety of pharmacological effects, such as anti-epileptic, analgesic, detoxification, anti-vertigo, etc. It can also be used to treat rheumatoid arthritis and bacterial infections. Dysentery, enteritis, viral hepatitis, senile diseases, etc.
- Paeoniflorin is the second most abundant active ingredient in the traditional Chinese medicine white peony root [1].
- paeoniflorin glycosides have unique pharmacodynamic characteristics that are different from paeoniflorin. It has better pharmacological effects than paeoniflorin in terms of blood enrichment, antidepressant, and potential treatment of diabetes. characteristics, and has anti-convulsant, anti-inflammatory and liver-protecting effects [2].
- Patent CN102603827 studies the preparation method of the similar compound paeoniflorin-6'-O-benzenesulfonate, using 98% paeoniflorin as the raw material, using pyridine as the acid compress and reaction solvent, DMAP as the catalyst, and slowly adding peony root.
- the molar ratio of glycosides to benzene sulfonyl chloride dissolved in methylene chloride is 20 hours, after post-treatment, the yield is about 40%.
- the compound paeoniflorin-6'-O-benzenesulfonate described in this patent can also be synthesized by this method, but there are the following shortcomings: 1.
- the purity of paeoniflorin required for the reaction raw materials is not less than 98%.
- Sources are limited, and there are currently no kilogram-level raw materials with a purity of more than 98% on the market; 2.
- the reaction yield is low, the catalyst, reaction solvent and subsequent purification solvent required for the reaction are large, and the reaction process is not economical and environmentally friendly; 3.
- the reaction raw materials need to be added step by step, and the raw materials need to be added slowly during the reaction process.
- the reaction time for large-scale preparation is long and the process is complicated, which is not conducive to industrial production.
- paeoniflorin-6'-O-benzenesulfonate has the characteristics of strong lipophilicity, difficulty in crystallization, strong hygroscopicity, and poor thermal stability, it significantly increases the difficulty of the drying process. After adopting the process of directly drying the precipitates, the solvent encapsulation phenomenon exists in the finished product, causing the solvent residue to seriously exceed the standard. If the sample is dissolved and dried directly under vacuum, paeoniflorin-6'-O-benzenesulfonate is prone to alcohol degradation, resulting in a significant increase in related substances, which undoubtedly brings challenges to the research on drying processes.
- the material is heated and dried under low temperature and vacuum conditions, which is suitable for the preparation of heat-sensitive materials.
- Materials that are soluble and stable in aqueous solution can use water as the sole solvent for this drying procedure, but paeoniflorin-6'-O-benzenesulfonate is not suitable because it is slightly soluble in water.
- Tert-butanol which has high melting point, high vapor pressure, non-toxicity and is miscible with water, is an excellent choice as a drying solvent for drugs that are difficult to dissolve in water and are heat-labile.
- Tert-butanol-water co-solvent has been used in a variety of injectable drugs.
- the production process can improve drying efficiency and shorten drying time, but paeoniflorin-6'-O-benzenesulfonate is unstable in alcohol-containing solvents and prone to degradation, resulting in a greatly reduced quality of the finished product that does not meet the requirements.
- the present invention provides for the first time a preparation route for paeoniflorin-6'-O-benzenesulfonate.
- medicinal materials rich in paeoniflorin such as white peony root as raw materials, 80% is obtained through extraction, adsorption and silica gel column chromatography.
- % or more of paeoniflorin glycosides, through esterification reaction obtain the crude paeoniflorin glycoside-6'-O-benzenesulfonate, and then through chromatographic column separation, purification and drying, finally obtain the medicinal paeoniflorin glycosides -6'-O-benzenesulfonate.
- the present invention adopts the following technical solutions:
- a preparation method of paeoniflorin-6’-O-benzenesulfonate comprising the following steps:
- an organic metal catalyst, an acid binding agent and a chemically active benzenesulfonyl chloride are used to react with paeoniflorin glycoside in S2, so that the reaction can produce paeoniflorin glycoside-6'-O-benzenesulfonic acid.
- the ester reaches more than 80%, and then is separated and purified by column chromatography and then refined to obtain more than 98% of paeoniflorin-6'-O-benzenesulfonate ester.
- step S4 the purity of the obtained paeoniflorin-6'-O-benzenesulfonate fine product B has reached more than 98% after testing.
- the liquid phase solvent does not produce peaks or is subtracted from the background, premium product B actually contains more solvents used in the refining process.
- the residues of various solvents used in the present invention in the finished drug should basically be controlled below 0.5%, so further drying of S5 is required.
- step S5 the present invention proposes a ternary solvent system of dimethyl carbonate, C3-4 ketones, and water with a proportion within a certain range.
- the freezing point is -10-0°C, which meets the solvent requirements of the programmed drying process and is expected to Unexpectedly, while successfully solving the drying problem of fat-soluble paeoniflorin-6'-O-benzenesulfonate, the related substances in the finished product no longer increase compared with before drying, and the solvent residues (including: the good quality used for refining
- the solvent isopropyl acetate or isobutyl acetate, the drying solvent dimethyl carbonate, acetone can all be lower than the specified value.
- the mixed solvent consists of dimethyl carbonate, acetone and water, or consists of dimethyl carbonate, butanone and water. Under this combination, the mixed solvent has better solubility for paeoniflorin-6'-O-benzenesulfonate, which can better reduce solvent residues and reduce the increase in related substance content after the drying process.
- the total solvent residue of paeoniflorin-6'-O-benzenesulfonate is no more than 0.6%.
- the carbonate residue of paeoniflorin-6'-O-benzenesulfonate is not higher than 0.5%, and the residue of C3 ⁇ 4 ketones is not higher than 0.2%.
- the content of related substances in paeoniflorin-6'-O-benzenesulfonate increases by no more than 0.2%.
- steps S1-2 and S3 those skilled in the art know that when column chromatography is used for separation, the starting point for collecting the eluent is when the main component appears, and the end point is when impurities appear or the spots of the main component become smaller.
- the medicinal materials include one or more of white peony root, red peony root, paeoniae bark, dog head, peony root, and purple peony root, using 80% to 100% ethanol or 80% to 100% ethanol.
- the extraction method includes one of cold soaking, thermal reflux or microwave-assisted extraction; in step S1-2, the column chromatography separation is silica gel column chromatography separation, and the eluent is an alkane derivative-lower alcohol mixed solvent ;
- the organometallic catalyst includes one or more of C1 to C4 alkyl tin halides, aryl tin halides, C1 to C4 alkyl tin oxides, and C1 to C4 alkyl tin sulfides
- the acid binding agent includes one or more of triethylamine, N, N'-diisopropylethylamine, pyridine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate
- the reaction solvent includes One or more of acetonitrile, tetrahydrofuran, acetone, pyridine, methyl ethyl ketone, N,N'-dimethylformamide, 1,4-di
- step S1-2 compared with the haloalkane-alcohol system, when the alkane derivative-alcohol system is used in the present invention to separate the crude paeoniflorin glycosides by column chromatography, its elution capacity is stronger, and the separation effect is achieved at the same time , the amount of eluent is relatively small, and it is less toxic than chloroform, making the production safer and more environmentally friendly.
- step S2 chemically active benzenesulfonyl chloride is used as the substrate.
- Pyridine can be used as both a reaction solvent for S2 and an acid-binding agent.
- the reaction solvent can be organic solvents such as acetone and acetonitrile
- the acid binding agent can be organic acid binding agents such as triethylamine, or inorganic acid binding agents such as potassium carbonate.
- S2 also includes extracting the product with ethyl acetate and water after the reaction is terminated, and then concentrating to obtain the crude paeoniflorin-6'-O-benzenesulfonate ester.
- the reaction temperature was room temperature, and the reaction time was 2 to 5 hours. It is best to use artificial control to terminate the reaction process. If the reaction is not terminated and the reaction time is too long, more impurities will be produced by side reactions. Add water and ethyl acetate and stir to terminate the reaction and perform extraction at the same time. After stirring, let it stand and separate the layers to take the organic layer.
- the present invention creatively uses the method of dissolving paeoniflorin-6'-O-benzenesulfonate fine product A in a good solvent and then adding a poor solvent for crystallization in step S4, which can improve the purity of the product and reduce the Impurities.
- the medicinal material includes one or more of white peony root and red peony root, and is extracted using 95% to 100% ethanol or 95% to 100% methanol.
- the extraction method includes thermal reflux;
- Step S1 -2 the mass ratio of the silica gel used for the silica gel column chromatography separation to the crude paeoniflorin glycoside is 10 to 20:1, and the mass ratio of the alkane derivative-lower alcohol mixed solvent to the crude paeoniflorin glycoside
- the volume to mass ratio is 40 to 65 ml: 1 g; in step S2, the dosage of the benzenesulfonyl chloride is 20% to 60% of the mass of the paeoniflorin glycoside, and the dosage of the organic metal catalyst is the paeoniflorin glycoside.
- the mass ratio of the silica gel used for the silica gel column chromatography separation to the crude paeoniflorin-6'-O-benzenesulfonate is 6 to 12:1, and the halogenated hydrocarbon-lower alcohol mixed solvent and the crude paeoniflorin-6'-O-benzenesulfonate are
- the volume to mass ratio of the crude paeoniflorin-6'-O-benzenesulfonate is 55 to 80 ml: 1 g
- the mass ratio of ethanol or methanol to the medicinal material is 5 to 20:1; in step S1-2, the mass of the silica gel used for silica gel column chromatography separation and the crude paeoniflorin glycosides The ratio is 10-15:1, and the volume-to-mass ratio of the alkane derivative-lower alcohol mixed solvent and the crude paeoniflorin glycoside is 50-60ml:1g; in step S2, the amount of benzene sulfonyl chloride is 25% to 30% of the mass of the paeoniflorin glycoside, the dosage of the organometallic catalyst is 0.5% to 5.0% of the mass of the paeoniflorin glycoside, and the molar ratio of the acid binding agent to the paeoniflorin glycoside It is 1.2 ⁇ 1.6:1; in step S5, the volume mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1 ⁇ 15mL:1
- step S1-2 when the amount of filler is larger, the separation effect is better.
- the amount is 10 to 15 times that of the crude paeoniflorin glycoside, the amount of eluent is relatively small while ensuring a better separation effect. .
- the mass ratio of ethanol or methanol to the medicinal material is 5 to 10:1; in step S1-2, the mesh number of the silica gel used for silica gel column chromatography separation is 60 to 400 mesh, and the pore size is 60 to 400 mesh.
- the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 1 to 10:1; in step S2, the amount of the organometallic catalyst is 0.6% to 0.6% of the mass of the paeoniflorin glycosides.
- step S3 the mesh number of the silica gel used for the silica gel column chromatography separation is 60 to 400 mesh, and the pore diameter is The volume ratio of the halogenated hydrocarbon to the lower alcohol in the halogenated hydrocarbon-lower alcohol mixed solvent is 6 to 12:1; in step S4, the paeoniflorin-6'-O-benzenesulfonate fine product A After dissolving with the good solvent at 50-60°C, the temperature is lowered to 10-20°C and the poor solvent is added for crystallization; in step S5, the programmed drying includes pre-freezing, sublimation drying and analytical drying steps. The temperatures of pre-freezing, the sublimation drying and the analytical drying are increased in sequence.
- the adsorption includes one or more of stirring adsorption and column chromatography adsorption
- the adsorbent includes one or more of silica gel and alumina, and is eluted with pure ethanol.
- the crude paeoniflorin glycoside is obtained; in step S1-2, the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 3 to 7:1; in step S2, the reaction is carried out at room temperature , the reaction time is 2 to 5h; in step S4, after crystallization, it also includes the process of filtration, rinsing, and re-filtration to obtain the paeoniflorin glycoside-6'-O-benzenesulfonate fine product B; in step S5, The temperature of the pre-freezing is -60 ⁇ -10°C, the temperature of the sublimation drying is -10 ⁇ 0°C, and the temperature of the analytical drying is 5 ⁇ 65°C.
- step S1-1 generally speaking, in addition to more than 20% of paeoniflorin glycosides, the crude paeoniflorin glycosides also contain a large amount of tannins, sugar gums and other impurities. If column chromatography is used directly for separation, it will not only affect The purity of paeoniflorin glycosides also has a great impact on the separation efficiency. Therefore, using adsorbents to remove impurities such as tannins and sugar gums in paeoniflorin glycosides can further improve the purity and separation efficiency of the resulting paeoniflorin glycosides.
- the adsorbents used in the present invention are low in price and have good separation effects, and can reduce the cost of industrial production of paeoniflorin-6'-O-benzenesulfonate.
- the crude paeoniflorin glycosides can be dissolved in a solvent consistent with the eluent used in column chromatography before column chromatography.
- the mass ratio of the adsorbent to the medicinal material extract is 1-4:1, and the volume-mass ratio of pure ethanol to the adsorbent is 2-6ml:1g; step S1-
- the crude paeoniflorin derivative is dissolved in the alkane derivative-lower alcohol mixed solvent, and the alkane derivative in the alkane derivative-lower alcohol mixed solvent includes ethyl acetate.
- the lower alcohol in the alkane derivative-lower alcohol mixed solvent includes one or more of methanol, ethanol, and isopropyl alcohol.
- step S3 before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin-6'-O-benzenesulfonate crude product; in step S4, use the Rinse with poor solvent; in step S5, the pre-freezing operation is to cool down to -60 ⁇ -10°C and keep it warm for 30 ⁇ 300min, and the sublimation drying operation is to raise the temperature to -10 ⁇ 0°C within 30 ⁇ 180min and keep it warm for 30 ⁇ 180min. The temperature is maintained for 180 to 720 minutes.
- the analytical drying operation is to raise the temperature to 5 to 20°C within 30 to 240 minutes and keep the temperature for 60 to 600 minutes, and then raise the temperature to 50 to 65°C within 30 to 240 minutes and keep the temperature for 300 to 1440 minutes.
- the alkane derivative-lower alcohol mixed solvent is used to dissolve the crude paeoniflorin glycoside in a volume mass ratio of 1 to 3 ml: 1 g, and the alkane derivative is
- the substance-lower alcohol mixed solvent is an ethyl acetate-methanol mixed solvent or an ethyl acetate-ethanol mixed solvent, and the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 4 to 6:1;
- step S3 before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the crude paeoniflorin-6'-O-benzenesulfonate ester in a volume mass ratio of 1 to 3 ml: 1 g.
- step S5 the pre-freezing operation is to lower the temperature to -40 ⁇ -10°C and keep it warm for 90 ⁇ 300min, and the sublimation drying operation is to raise the temperature to -10 ⁇ 0°C within 60 ⁇ 180min and keep it warm for 240 ⁇ 600min.
- the operation of analytical drying is to raise the temperature to 5-15°C within 60-180 min and keep it warm for 90-540 min, and then raise the temperature to 50-60°C within 60-180 min and keep it warm for 480-1440 min.
- a kind of paeoniflorin-6’-O-benzenesulfonate obtained by the preparation method of paeoniflorin-6’-O-benzenesulfonate has the following structure:
- the obtained paeoniflorin-6'-O-benzenesulfonate has good blood-enriching effect.
- the present invention proposes for the first time a process route for preparing paeoniflorin-6'-O-benzenesulfonate.
- the route is simple, the conditions are mild, and it is suitable for industrial production.
- the synthesis method in the present invention has the advantages of high yield, small amount of catalyst and organic solvent, short reaction time, and no need to drop raw materials.
- the elution system of ethyl acetate-methanol or ethyl acetate-ethanol is preferably used.
- the dosage of remover can also improve the safety of people and industrial production.
- the drying process of the present invention can ensure that the solvent residue of the finished product of paeoniflorin-6'-O-benzenesulfonate is qualified without increasing the content of related substances and ensure that the paeoniflorin-6'-O-benzenesulfonate product is qualified. High quality of finished acid ester products.
- Figure 1 is a hydrogen spectrum of the product of Example 1 of the present invention.
- Figure 2 is a carbon spectrum of the product of Example 1 of the present invention.
- a paeoniflorin-6'-O-benzenesulfonate is prepared.
- the specific process is:
- A Take 500g of white peony slices, add 4L of 95% ethanol for hot reflux extraction, filter and concentrate to obtain 31.9g of medicinal extract, then add 63.8g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 100 mL ethanol to elute, collect the eluate, and concentrate to obtain 15.4 g of crude paeoniflorin glycoside, with a purity of 25.3%.
- Example 1 the hydrogen spectrum of the product is shown in Figure 1 and the carbon spectrum is shown in Figure 2. After identification, it was determined that the product had the following structure:
- a paeoniflorin-6'-O-benzenesulfonate is prepared.
- the specific process is:
- A Take 500g of white peony slices, add 600ml of 80% ethanol for thermal reflux extraction, filter and concentrate to obtain 37.6g of medicinal extract, then add 112.8g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 150 mL ethanol to elute, collect the eluate, and concentrate to obtain 17.95g of crude paeoniflorin glycoside, with a purity of 22.6%.
- a paeoniflorin-6'-O-benzenesulfonate is prepared.
- the specific process is:
- A Take 1kg of white peony slices, add 6L of absolute ethanol for thermal reflux extraction, filter and concentrate to obtain 31g of medicinal extract, then add 183g of neutral alumina for stirring and adsorption, add the adsorbed neutral alumina to the chromatography column, and use Elute with 180 mL of ethanol, collect the eluate, and concentrate to obtain 36.4 g of crude paeoniflorin glycoside, with a purity of 27.3%.
- a paeoniflorin-6'-O-benzenesulfonate is prepared.
- the specific process is:
- A Take 5kg of white peony slices, add 50L of 95% ethanol for thermal reflux extraction, filter and concentrate to obtain 401.4g of medicinal extract, then add 600g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column. Use 1.2L ethanol to elute, collect the eluate, and concentrate to obtain 208.5g of crude paeoniflorin glycoside, with a purity of 28.4%.
- the eluate was collected and concentrated under reduced pressure to obtain 46.25g of paeoniflorin glycoside with a purity of 93.6%.
- a paeoniflorin-6'-O-benzenesulfonate is prepared.
- the specific process is:
- A Take 10kg of white peony slices, add 70L of 90% ethanol for thermal reflux extraction, filter and concentrate to obtain 903g of medicinal extract, then add 1.8kg of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column. Use 3.6 L of ethanol to elute, collect the eluate, and concentrate to obtain 505 g of crude paeoniflorin glycoside, with a purity of 23.6%.
- a paeoniflorin-6'-O-benzenesulfonate is prepared.
- the specific process is:
- A Take 50kg of white peony slices, add 500L of 95% ethanol for thermal reflux extraction, filter and concentrate to obtain 4.0kg of medicinal extract, then add 8.0kg of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 16.0L ethanol to elute, collect the eluate, and concentrate to obtain 1.8 kg of crude paeoniflorin glycoside, with a purity of 20.3%.
- This comparative example uses paeoniflorin glycoside (purity 98%) instead of peony extract as the starting material to prepare paeoniflorin glycoside-6'-O-benzenesulfonate, which undergoes the following processes:
- step D is omitted.
- step D is omitted.
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- Steps A to C are the same as in Embodiment 4, except that step D is as follows:
- the residues of various solvents used in the present invention in the finished drug should basically be controlled below 0.5%. From the comparison between Example 4 and Comparative Examples 2 to 7, it can be found that using dimethyl carbonate, acetone and water, or a combination of dimethyl carbonate, methyl ethyl ketone and water as the solvent, the effect of paeoniflorin-6-O' - The benzenesulfonate ester is dissolved and then dried in a programmed manner. The solvent residue can meet the standards and there is no significant increase in related substances. The quality of the dried product is guaranteed.
- Comparative Example 6 a mixed solvent of dimethyl carbonate and acetone was used to perform programmed drying of paeoniflorin-6-O'-benzenesulfonate, and the solvent residue could not be reduced below the standard.
- Comparative Example 7 used a mixed solvent of tert-butyl alcohol and water to perform programmed drying of paeoniflorin-6-O'-benzenesulfonate. Although the solvent residue could meet the standards, related substances increased significantly. It can be seen that only by using the specific mixed solvent of the present invention can the requirements that the dissolved residues meet the standards and there is no increase in related substances can be met.
- Comparative Example 8 when a mixed solvent composed of dimethyl carbonate, acetone and water is used as the solvent for programmed drying, not all proportions can obtain qualified finished products.
- the results of solvent residues and related substances in the finished product are investigated.
- the acetone:water ratio in the mixed solvent is less than 1:4, as shown in groups 1, 2, and 8 in the table, the mixed system will appear stratified, and the dimethyl carbonate in the finished product will exceed the limit, affecting the drying effect; and when the mixture When the acetone content in the system exceeds 10%, as shown in Group 4, the residual acetone in the finished product exceeds the limit.
- the mixed solvent system does not stratify, the solvent residues of dimethyl carbonate and acetone meet the specified requirements, and there is no obvious relevant substance after drying Increase. It can be seen that only by using a mixed solvent of dimethyl carbonate, C3 ⁇ 4 ketones and water in a specific ratio can the dissolved residues meet the standards and there is no increase in related substances.
- mice were randomly divided into 9 groups according to body weight, half male and half female, namely control group, model group, positive drug group, paeoniflorin glycoside, and paeoniflorin glycoside-6'-O-
- control group model group
- positive drug group paeoniflorin glycoside
- 0.5 mL of blood was bled from the fundus venous plexus and administered intragastrically 24 hours later.
- the positive drug group was intragastrically administered compound donkey-hide gelatin slurry at 20 ml/kg
- the control and model groups were intragastrically administered distilled water at 120 mg/kg.
- 30 ⁇ L of blood was collected from the orbit to detect the number of red blood cells (see Table 14).
- paeoniflorin-6'-O-benzenesulfonate and paeoniflorin can effectively increase the number of red blood cells in mice, and the effect of paeonilide-6'-O-benzenesulfonate is greater. It is better than paeoniflorin glycoside, and the effect of high dose of paeoniflorin glycoside-6'-O-benzenesulfonate is close to that of compound donkey-hide gelatin. Compared with paeoniflorin, the reason why paeonilide-6'-O-benzenesulfonate is more effective may be due to increased lipophilicity and improved in vivo bioavailability.
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Abstract
The present invention belongs to the field of compounds, and discloses a method for preparing albiflorin-6'-O-benzenesulfonate. The method comprises the following steps: taking a medicinal material that is rich in albiflorin as a starting material, subjecting same to extraction, adsorption, separation and purification to obtain albiflorin with a relatively high purity; and reacting an organic metal catalyst, an acid-binding agent and chemically active benzenesulfonyl chloride with albiflorin, carrying out column chromatography separation and refining on the product to obtain more than 98% of albiflorin-6'-O-benzenesulfonate, dissolving same in a mixed solvent, and carrying out programmed drying to finally obtain a finished product of albiflorin-6'-O-benzenesulfonate. The method has a high reaction yield, a simple operation, and mild conditions, and is very suitable for industrial production of albiflorin-6'-O-benzenesulfonate.
Description
本发明属于化合物领域,具体涉及一种芍药内酯苷-6’-O-苯磺酸酯的制备方法。The invention belongs to the field of compounds, and specifically relates to a preparation method of paeoniflorin-6'-O-benzenesulfonate.
白芍为毛崀科芍药的根,具有解热镇痛等功效。具现代药理学表明,白芍提取物还具有抗炎免疫调、解毒保肝、抗诱变和抗肿瘤节等多种药理活性,白芍总苷也是第一个用于临床治疗类风湿性关节炎的抗炎免疫调节药,且具有疗效好,不良反应少,耐受性好等特点。芍药内酯苷(Albiflorin)作为白芍的主要活性成分之一,同样具有多种药理功效,例如:抗癫痫、镇痛、戒毒、止眩晕等,也可用以治疗类风湿性关节炎、细菌性痢疾、肠炎、病毒性肝炎、老年性疾病等。White peony root is the root of Paeonia lactiflora, which has antipyretic and analgesic effects. Modern pharmacology shows that white peony extract also has various pharmacological activities such as anti-inflammatory and immunomodulatory, detoxifying and liver-protecting, anti-mutagenic and anti-tumor. Total glycosides of white peony are also the first to be used in the clinical treatment of rheumatoid joints. It is an anti-inflammatory immunomodulatory drug for inflammation, and has the characteristics of good curative effect, few adverse reactions, and good tolerance. As one of the main active ingredients of white peony root, Albiflorin also has a variety of pharmacological effects, such as anti-epileptic, analgesic, detoxification, anti-vertigo, etc. It can also be used to treat rheumatoid arthritis and bacterial infections. Dysentery, enteritis, viral hepatitis, senile diseases, etc.
芍药内酯苷是中药白芍中含量位居第二的活性成分[1]。近年来,随着对其药效学研究的深入,发现芍药内酯苷具有区别于芍药苷的独特的药效特点,在补血、抗抑郁、糖尿病潜在治疗方面,具有优于芍药苷的药效特点,并具有抗惊厥、抗炎护肝的作用[2]。进一步地,在研究芍药内酯苷药代动力学中发现,ICR小鼠口服芍药内酯苷后,除胃、肠、肾外,其他组织含量均较低,且清除速率快[3],这可能与芍药内酯苷的脂溶性差有关。因此,需要对芍药内酯苷进行衍生化改造,提高芍药内酯苷的脂溶性,从而提高芍药内酯苷的吸收和生物利用率,更好的发挥其药效作用。Paeoniflorin is the second most abundant active ingredient in the traditional Chinese medicine white peony root [1]. In recent years, with in-depth research on its pharmacodynamics, it has been found that paeoniflorin glycosides have unique pharmacodynamic characteristics that are different from paeoniflorin. It has better pharmacological effects than paeoniflorin in terms of blood enrichment, antidepressant, and potential treatment of diabetes. characteristics, and has anti-convulsant, anti-inflammatory and liver-protecting effects [2]. Furthermore, in the study of the pharmacokinetics of paeoniflorin glycosides, it was found that after oral administration of paeoniflorin glycosides to ICR mice, except for the stomach, intestine, and kidney, the contents of other tissues were lower and the clearance rate was rapid [3]. This is It may be related to the poor fat solubility of paeoniflorin glycosides. Therefore, it is necessary to carry out derivatization of paeoniflorin glycosides to improve the fat solubility of paeoniflorin glycosides, thereby improving the absorption and bioavailability of paeoniflorin glycosides and better exerting its medicinal effects.
专利CN102603827对相似化合物芍药苷-6'-O-苯磺酸酯的制备方法进行了研究,以98%芍药苷为原料,采用吡啶作为敷酸剂和反应溶剂,DMAP作为催化剂,缓慢滴加芍药苷2倍摩尔比的溶解于二氯甲烷的苯磺酰氯,反应20小时情况下,经后处理,产率在40%左右。本专利所述化合物芍药内酯苷-6'-O-苯磺酸酯也可用此方法合成,但是存在以下缺陷:1.反应原料所需的芍药内酯苷纯度要求不低于98%,原料来源受限,市场暂无纯度在98%以上的公斤级别原料销 售;2.反应产率偏低,反应所需的催化剂、反应溶剂以及后续纯化溶剂用量大,反应工艺不经济环保等;3.反应原料需要分步骤加入,且反应过程需缓慢滴加原料,大量制备反应时间长,工艺复杂,不利于工业化生产。Patent CN102603827 studies the preparation method of the similar compound paeoniflorin-6'-O-benzenesulfonate, using 98% paeoniflorin as the raw material, using pyridine as the acid compress and reaction solvent, DMAP as the catalyst, and slowly adding peony root. When the molar ratio of glycosides to benzene sulfonyl chloride dissolved in methylene chloride is 20 hours, after post-treatment, the yield is about 40%. The compound paeoniflorin-6'-O-benzenesulfonate described in this patent can also be synthesized by this method, but there are the following shortcomings: 1. The purity of paeoniflorin required for the reaction raw materials is not less than 98%. Sources are limited, and there are currently no kilogram-level raw materials with a purity of more than 98% on the market; 2. The reaction yield is low, the catalyst, reaction solvent and subsequent purification solvent required for the reaction are large, and the reaction process is not economical and environmentally friendly; 3. The reaction raw materials need to be added step by step, and the raw materials need to be added slowly during the reaction process. The reaction time for large-scale preparation is long and the process is complicated, which is not conducive to industrial production.
另一方面,由于芍药内酯苷-6'-O-苯磺酸酯具有亲脂性强、难以结晶、吸湿性强、热稳定性差等特点,显著增加了干燥工艺的难度。采用将析出物直接干燥的工艺后,成品存在溶剂包裹现象致使溶剂残余严重超标。若将样品溶解后直接真空干燥,芍药内酯苷-6'-O-苯磺酸酯则易发生醇降解,导致有关物质显著增加,这些都无疑给干燥工艺的研究带来了挑战。利用升华的原理,在低温和真空条件下对物料进行程序式加热干燥,适用于热敏性物料的制备。在水溶液中可溶且稳定的物料可采用水作为唯一溶剂进行这一干燥程序,但芍药内酯苷-6'-O-苯磺酸酯由于微溶于水而不适用。具有高熔点、高蒸汽压,无毒且与水互溶特点的叔丁醇是难溶于水且不耐热药物干燥溶剂的优良选择,叔丁醇-水共溶剂已被用于多种注射药物的生产过程,可提高干燥效率,缩短干燥时间,但芍药内酯苷-6'-O-苯磺酸酯在含醇溶剂中不稳定,容易发生降解,致使成品质量大大降低而不符合要求。On the other hand, because paeoniflorin-6'-O-benzenesulfonate has the characteristics of strong lipophilicity, difficulty in crystallization, strong hygroscopicity, and poor thermal stability, it significantly increases the difficulty of the drying process. After adopting the process of directly drying the precipitates, the solvent encapsulation phenomenon exists in the finished product, causing the solvent residue to seriously exceed the standard. If the sample is dissolved and dried directly under vacuum, paeoniflorin-6'-O-benzenesulfonate is prone to alcohol degradation, resulting in a significant increase in related substances, which undoubtedly brings challenges to the research on drying processes. Using the principle of sublimation, the material is heated and dried under low temperature and vacuum conditions, which is suitable for the preparation of heat-sensitive materials. Materials that are soluble and stable in aqueous solution can use water as the sole solvent for this drying procedure, but paeoniflorin-6'-O-benzenesulfonate is not suitable because it is slightly soluble in water. Tert-butanol, which has high melting point, high vapor pressure, non-toxicity and is miscible with water, is an excellent choice as a drying solvent for drugs that are difficult to dissolve in water and are heat-labile. Tert-butanol-water co-solvent has been used in a variety of injectable drugs. The production process can improve drying efficiency and shorten drying time, but paeoniflorin-6'-O-benzenesulfonate is unstable in alcohol-containing solvents and prone to degradation, resulting in a greatly reduced quality of the finished product that does not meet the requirements.
因此为解决上述问题,开发一条适合产业化制备芍药内酯苷-6'-O-苯磺酸酯的工艺路线迫在眉睫。Therefore, in order to solve the above problems, it is urgent to develop a process route suitable for the industrial preparation of paeoniflorin-6'-O-benzenesulfonate.
发明内容Contents of the invention
本发明首次提供了一条芍药内酯苷-6'-O-苯磺酸酯制备路线,以白芍等富含芍药内酯苷的药材为原料,通过提取、吸附和硅胶柱层析,得到80%以上的芍药内酯苷,经过酯化反应,得到芍药内酯苷-6'-O-苯磺酸酯粗品,再经过色谱柱分离、精制及干燥,最终得到可药用的芍药内酯苷-6'-O-苯磺酸酯。The present invention provides for the first time a preparation route for paeoniflorin-6'-O-benzenesulfonate. Using medicinal materials rich in paeoniflorin such as white peony root as raw materials, 80% is obtained through extraction, adsorption and silica gel column chromatography. % or more of paeoniflorin glycosides, through esterification reaction, obtain the crude paeoniflorin glycoside-6'-O-benzenesulfonate, and then through chromatographic column separation, purification and drying, finally obtain the medicinal paeoniflorin glycosides -6'-O-benzenesulfonate.
为了解决上述技术问题,本发明采用如下技术方案:In order to solve the above technical problems, the present invention adopts the following technical solutions:
一种芍药内酯苷-6’-O-苯磺酸酯的制备方法,包括如下步骤:A preparation method of paeoniflorin-6’-O-benzenesulfonate, comprising the following steps:
S1:从药材中提取芍药内酯苷;S1: Extract paeoniflorin glycosides from medicinal materials;
S2:按比例向所述芍药内酯苷中加入有机金属催化剂、缚酸剂、苯磺酰氯进行反应,反应终止后浓缩反应产物得到芍药内酯苷-6'-O-苯磺酸酯粗品;S2: Add an organic metal catalyst, an acid binding agent, and benzenesulfonyl chloride to the paeoniflorin glycoside in proportion to carry out the reaction. After the reaction is terminated, the reaction product is concentrated to obtain a crude paeoniflorin glycoside-6'-O-benzenesulfonate ester;
S3:将所述芍药内酯苷-6'-O-苯磺酸酯粗品分离后得到芍药内酯苷-6'-O-苯磺酸酯精品A;S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate product to obtain paeonilide-6'-O-benzenesulfonate fine product A;
S4:将所述芍药内酯苷-6'-O-苯磺酸酯精品A用良溶剂溶解后,加入不良溶剂析晶,析晶后得到芍药内酯苷-6'-O-苯磺酸酯精品B;S4: After dissolving the paeoniflorin-6'-O-benzenesulfonic acid ester boutique A in a good solvent, add a poor solvent for crystallization, and obtain paeoniflorin-6'-O-benzenesulfonic acid after crystallization. Ester Boutique B;
S5:将所述芍药内酯苷-6'-O-苯磺酸酯精品B溶解于混合溶剂中,所述混合溶剂包括碳酸二甲酯、C3~4酮类和水,进行程序式干燥,最终得到芍药内酯苷-6'-O-苯磺酸酯成品。S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in a mixed solvent, the mixed solvent includes dimethyl carbonate, C3~4 ketones and water, and perform programmed drying. Finally, the finished product of paeoniflorin-6'-O-benzenesulfonate is obtained.
根据本发明的方法,S2中采用有机金属催化剂、缚酸剂和化学性质活泼的苯磺酰氯与芍药内酯苷进行反应,能使得反应生成的芍药内酯苷-6'-O-苯磺酸酯达到80%以上,再经柱层析分离纯化后精制即可得到98%以上的芍药内酯苷-6'-O-苯磺酸酯。According to the method of the present invention, an organic metal catalyst, an acid binding agent and a chemically active benzenesulfonyl chloride are used to react with paeoniflorin glycoside in S2, so that the reaction can produce paeoniflorin glycoside-6'-O-benzenesulfonic acid. The ester reaches more than 80%, and then is separated and purified by column chromatography and then refined to obtain more than 98% of paeoniflorin-6'-O-benzenesulfonate ester.
在步骤S4精制结束后,获得的芍药内酯苷-6'-O-苯磺酸酯精品B经检测纯度已经达到了98%以上。但由于纯度检测中,液相溶剂是不出峰或者当背景扣除的,精品B实际还裹挟着较多精制过程中所用的溶剂。而按药典中的规定,本发明所用的各种溶剂在成品药中的残留基本都应该控制在0.5%以下,因此需要进行S5的进一步干燥处理。After the refining in step S4, the purity of the obtained paeoniflorin-6'-O-benzenesulfonate fine product B has reached more than 98% after testing. However, since in the purity test, the liquid phase solvent does not produce peaks or is subtracted from the background, premium product B actually contains more solvents used in the refining process. According to the provisions of the Pharmacopoeia, the residues of various solvents used in the present invention in the finished drug should basically be controlled below 0.5%, so further drying of S5 is required.
在步骤S5中,本发明提出的比例在一定范围内的碳酸二甲酯、C3~4酮类、水三元溶剂体系,凝固点为-10~0℃,符合程序式干燥工艺溶剂要求,并预料不到地在成功地解决了脂溶性芍药苷-6'-O-苯磺酸酯干燥难题的同时,使成品的有关物质相较干燥前不再增加,并且溶剂残留(包括:精制所用的良溶剂乙酸异丙酯或乙酸异丁酯、干燥所用溶剂碳酸二甲酯、丙酮)均能低于规定值。In step S5, the present invention proposes a ternary solvent system of dimethyl carbonate, C3-4 ketones, and water with a proportion within a certain range. The freezing point is -10-0°C, which meets the solvent requirements of the programmed drying process and is expected to Unexpectedly, while successfully solving the drying problem of fat-soluble paeoniflorin-6'-O-benzenesulfonate, the related substances in the finished product no longer increase compared with before drying, and the solvent residues (including: the good quality used for refining The solvent isopropyl acetate or isobutyl acetate, the drying solvent dimethyl carbonate, acetone) can all be lower than the specified value.
在本发明的一些实施方式中,所述混合溶剂由碳酸二甲酯、丙酮和水组成,或由碳酸二甲酯、丁酮和水组成。在该组合下,混合溶剂对芍药苷-6'-O-苯磺酸酯具有更好的溶解性能,达到更好的降低溶剂残留、减少干燥工艺后有关物质含量的增加。In some embodiments of the present invention, the mixed solvent consists of dimethyl carbonate, acetone and water, or consists of dimethyl carbonate, butanone and water. Under this combination, the mixed solvent has better solubility for paeoniflorin-6'-O-benzenesulfonate, which can better reduce solvent residues and reduce the increase in related substance content after the drying process.
在本发明的一些实施方式中,程序式干燥后,芍药苷-6'-O-苯磺酸酯的总溶剂残留不高于0.6%。优选地,程序式干燥后,芍药苷-6'-O-苯磺酸酯的碳酸酯残留不高于0.5%,C3~4酮类残留不高于0.2%。In some embodiments of the present invention, after programmed drying, the total solvent residue of paeoniflorin-6'-O-benzenesulfonate is no more than 0.6%. Preferably, after programmed drying, the carbonate residue of paeoniflorin-6'-O-benzenesulfonate is not higher than 0.5%, and the residue of C3~4 ketones is not higher than 0.2%.
在本发明的一些实施方式中,程序式干燥后,芍药苷-6'-O-苯磺酸酯中有关物质的含量增长不高于0.2%。In some embodiments of the present invention, after programmed drying, the content of related substances in paeoniflorin-6'-O-benzenesulfonate increases by no more than 0.2%.
优选的,包括如下步骤:Preferably, it includes the following steps:
S1:包括如下步骤:S1: Includes the following steps:
S1-1:药材用醇提取,提取液过滤浓缩后得到药材提取物,经过吸附洗脱得到芍药内酯苷粗品;S1-1: The medicinal materials are extracted with alcohol, the extract is filtered and concentrated to obtain the medicinal material extract, and the crude paeoniflorin glycoside is obtained through adsorption and elution;
S1-2:将所述芍药内酯苷粗品进行柱层析分离,从而得到所述芍药内酯苷,所述芍药内酯苷的纯度在80%以上;S1-2: The crude paeoniflorin glycoside is separated by column chromatography to obtain the paeoniflorin glycoside, and the purity of the paeoniflorin glycoside is above 80%;
S2:按比例向所述芍药内酯苷中加入所述有机金属催化剂、所述缚酸剂、所述苯磺酰氯,在反应溶剂中进行反应,之后加入有机相和水相终止反应并进行萃取,浓缩后得到所述芍药内酯苷-6'-O-苯磺酸酯粗品;S2: Add the organometallic catalyst, the acid binding agent, and the benzenesulfonyl chloride to the paeoniflorin glycoside in proportion, carry out the reaction in the reaction solvent, and then add the organic phase and the aqueous phase to terminate the reaction and perform extraction. , after concentration, the crude paeoniflorin-6'-O-benzenesulfonate ester is obtained;
S3:将所述芍药内酯苷-6'-O-苯磺酸酯粗品经柱层析分离,收集洗脱液,得到所述芍药内酯苷-6'-O-苯磺酸酯精品A,所述芍药内酯苷-6'-O-苯磺酸酯精品A的纯度在95%以上;S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate ester through column chromatography, collect the eluent, and obtain the paeonilide-6'-O-benzenesulfonate fine product A , the purity of the paeoniflorin-6'-O-benzenesulfonate fine product A is above 95%;
S4:将所述芍药内酯苷-6'-O-苯磺酸酯精品A用所述良溶剂溶解后,加入所述不良溶剂析晶,析晶后得到芍药内酯苷-6'-O-苯磺酸酯精品B,所述芍药内酯苷-6'-O-苯磺酸酯精品B的纯度在98%以上;S4: After dissolving the paeoniflorin-6'-O-benzenesulfonate fine product A with the good solvent, adding the poor solvent for crystallization, and obtaining paeonilide-6'-O after crystallization. -Exquisite benzenesulfonate B, the purity of the paeoniflorin-6'-O-benzenesulfonate elite B is above 98%;
S5:将所述芍药内酯苷-6'-O-苯磺酸酯精品B溶解于所述混合溶剂中,所述混合溶剂包括碳酸二甲酯、C3~4酮类和水体按积比4~11:0.2~0.6:1混合而成,进行程序式干燥,最终得到芍药内酯苷-6'-O-苯磺酸酯成品。S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in the mixed solvent, which includes dimethyl carbonate, C3~4 ketones and water in a volume ratio of 4 ~11:0.2~0.6:1 is mixed, and programmed drying is performed to finally obtain the finished product of paeoniflorin-6'-O-benzenesulfonate.
在步骤S1-2和步骤S3中,本领域技术人员知晓,当采用柱层析分离时,收集洗脱液的起点为主要成分出现时,终点为出现杂质或主要成分斑点变小时。In steps S1-2 and S3, those skilled in the art know that when column chromatography is used for separation, the starting point for collecting the eluent is when the main component appears, and the end point is when impurities appear or the spots of the main component become smaller.
优选的,步骤S1-1中,所述药材包括白芍、赤芍、丹皮、狗头、牡丹根、紫牡丹中的一种或多种,使用80%~100%乙醇或80%~100%甲醇提取,提取方式包括冷浸、热回流或微波辅助提取中的一种;步骤S1-2中,所述柱层析分离为硅胶柱色谱分离,洗脱剂为烷烃衍生物-低级醇混合溶剂;步骤S2中,所述有机金属催化剂包括C1~C4的烷基卤化锡、芳基卤化锡、C1~C4的烷基氧化锡、C1~C4的烷基硫化锡中的一种或多种,所述缚酸剂包括三乙胺、N,N'-二异丙基乙胺、吡啶、碳酸钾、碳酸氢钾、碳酸钠、碳酸氢钠中的一种或多种,所述反应溶剂包括乙腈、四氢呋喃、丙酮、吡啶、甲基乙基酮、N,N’-二甲基甲酰胺、1,4-二氧六环、二甲亚砜中的一种或多种,所述有机相为乙酸乙酯;步骤S3中,所述柱层析分离为硅胶柱色谱分离,洗脱剂为卤代烃-低级醇混合溶剂;步骤S4 中,所述良溶剂包括乙酸异丙酯、乙酸异丁酯中的一种或多种,所述不良溶剂包括正己烷、石油醚、异丙醚、异辛烷中的一种或多种;步骤S5中,述混合溶剂包括碳酸二甲酯、C3~4酮类和水体按积比5~8:0.3~0.5:1混合而成。Preferably, in step S1-1, the medicinal materials include one or more of white peony root, red peony root, paeoniae bark, dog head, peony root, and purple peony root, using 80% to 100% ethanol or 80% to 100% ethanol. Methanol extraction, the extraction method includes one of cold soaking, thermal reflux or microwave-assisted extraction; in step S1-2, the column chromatography separation is silica gel column chromatography separation, and the eluent is an alkane derivative-lower alcohol mixed solvent ; In step S2, the organometallic catalyst includes one or more of C1 to C4 alkyl tin halides, aryl tin halides, C1 to C4 alkyl tin oxides, and C1 to C4 alkyl tin sulfides, The acid binding agent includes one or more of triethylamine, N, N'-diisopropylethylamine, pyridine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate, and the reaction solvent includes One or more of acetonitrile, tetrahydrofuran, acetone, pyridine, methyl ethyl ketone, N,N'-dimethylformamide, 1,4-dioxane, and dimethyl sulfoxide, the organic phase is ethyl acetate; in step S3, the column chromatography separation is silica gel column chromatography separation, and the eluent is a halogenated hydrocarbon-lower alcohol mixed solvent; in step S4, the good solvent includes isopropyl acetate, isopropyl acetate One or more of butyl ester, the poor solvent includes one or more of n-hexane, petroleum ether, isopropyl ether, isooctane; in step S5, the mixed solvent includes dimethyl carbonate, C3 ~4 Ketones and water are mixed in a volume ratio of 5~8:0.3~0.5:1.
在步骤S1-2中,相对于卤代烷烃-醇体系,本发明中采用烷烃衍生物-醇体系对芍药内酯苷粗品进行柱层析分离时,其洗脱能力更强,在达到分离效果同时,洗脱剂用量相对较少,且比三氯甲烷毒性小,生产更安全环保。In step S1-2, compared with the haloalkane-alcohol system, when the alkane derivative-alcohol system is used in the present invention to separate the crude paeoniflorin glycosides by column chromatography, its elution capacity is stronger, and the separation effect is achieved at the same time , the amount of eluent is relatively small, and it is less toxic than chloroform, making the production safer and more environmentally friendly.
在步骤S2中,采用化学性质活泼的苯磺酰氯作为底物,为提高反应安全性,需要再无水条件下使用。而吡啶既可作为S2的反应溶剂,又可作为缚酸剂,但是吡啶的具有生殖毒性,不适合工业化大量使用。从低毒低成本的角度出发,工业化生产中反应溶剂可以选择丙酮、乙腈等有机溶剂,缚酸剂可以选择有机缚酸剂如三乙胺,或无机缚酸剂如碳酸钾等。In step S2, chemically active benzenesulfonyl chloride is used as the substrate. In order to improve the safety of the reaction, it needs to be used under anhydrous conditions. Pyridine can be used as both a reaction solvent for S2 and an acid-binding agent. However, pyridine is reproductively toxic and is not suitable for large-scale industrial use. From the perspective of low toxicity and low cost, in industrial production, the reaction solvent can be organic solvents such as acetone and acetonitrile, and the acid binding agent can be organic acid binding agents such as triethylamine, or inorganic acid binding agents such as potassium carbonate.
在本发明的一些实施方式中,S2还包括在所述反应终止后,采用乙酸乙酯和水对产物进行萃取,再浓缩得到所述芍药内酯苷-6'-O-苯磺酸酯粗品。反应温度室温,反应时间2~5h。反应的进程是最好采用人为控制终止反应的方式。如果不终止反应,反应时间过长的话副反应产生的杂质就会变多。加入水和乙酸乙酯搅拌就是在终止反应,同时进行萃取,搅拌之后静置分层取有机层。S2中反应结束后,一般会有副产物、有机盐、缚酸剂以及未完全反应的底物存在,使用乙酸乙酯和水进行萃取获得萃取液,能有效除去部分副产物和其他杂质,从而得到纯度更高的芍药内酯苷-6'-O-苯磺酸酯粗品。In some embodiments of the present invention, S2 also includes extracting the product with ethyl acetate and water after the reaction is terminated, and then concentrating to obtain the crude paeoniflorin-6'-O-benzenesulfonate ester. . The reaction temperature was room temperature, and the reaction time was 2 to 5 hours. It is best to use artificial control to terminate the reaction process. If the reaction is not terminated and the reaction time is too long, more impurities will be produced by side reactions. Add water and ethyl acetate and stir to terminate the reaction and perform extraction at the same time. After stirring, let it stand and separate the layers to take the organic layer. After the reaction in S2, there will generally be by-products, organic salts, acid binding agents and incompletely reacted substrates. Using ethyl acetate and water to extract the extract can effectively remove some of the by-products and other impurities, thereby A crude paeoniflorin-6'-O-benzenesulfonate product with higher purity is obtained.
实验发现,如果不加入不良溶剂,直接在加入良溶剂溶解芍药内酯苷-6'-O-苯磺酸酯精品A后降温析晶,发现无法获得固态晶体,只能获得一种黏糖浆状物质,无法进行过滤将固液分离,这可能与芍药内酯苷-6'-O-苯磺酸酯的特殊性质有关。于是本发明创造性地在步骤S4中,利用芍药内酯苷-6'-O-苯磺酸酯精品A在良溶剂中溶解后,再加入不良溶剂析晶的方式,能提高产物的纯度,减少杂质。Experiments have found that if you do not add a poor solvent, directly add a good solvent to dissolve Paeoniflorin-6'-O-benzenesulfonate Premium A and then cool it down to crystallize, you will find that you cannot obtain solid crystals and can only obtain a viscous syrupy state. Substance, solid-liquid separation cannot be separated by filtration, which may be related to the special properties of paeoniflorin-6'-O-benzenesulfonate. Therefore, the present invention creatively uses the method of dissolving paeoniflorin-6'-O-benzenesulfonate fine product A in a good solvent and then adding a poor solvent for crystallization in step S4, which can improve the purity of the product and reduce the Impurities.
优选的,步骤S1-1中,所述药材包括白芍、赤芍中的一种或多种,使用95%~100%乙醇或95%~100%甲醇提取,提取方式包括热回流;步骤S1-2中,所述硅胶柱色谱分离所用的硅胶与所述芍药内酯苷粗品的质量比为10~20:1,所述烷烃衍生物-低级醇混合溶剂与所述芍药内酯苷粗品的体积质量比为40~65ml:1g;步骤S2中,所述苯磺酰氯的用量为所述芍药内酯苷质量的 20%~60%,所述有机金属催化剂的用量为所述芍药内酯苷质量的0.05%~20%,所述缚酸剂与所述芍药内酯苷的摩尔比为1~2:1,所述反应溶剂与所述芍药内酯苷的体积质量比为2~10ml:1g,所述有机相与所述芍药内酯苷的体积质量比为2.5~7.5ml:1g,所述水相与所述芍药内酯苷的体积质量比为5~15ml:1g;步骤S3中,所述硅胶柱色谱分离所用的硅胶与所述芍药内酯苷-6'-O-苯磺酸酯粗品的质量比为6~12:1,所述卤代烃-低级醇混合溶剂与所述芍药内酯苷-6'-O-苯磺酸酯粗品的体积质量比为55~80ml:1g;步骤S4中,所述良溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品A的体积质量比为10~20mL:1g,所述不良溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品A的体积质量比为8~12mL:1g;步骤S5中,所述混合溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品B的体积质量比为1~20mL:1g。Preferably, in step S1-1, the medicinal material includes one or more of white peony root and red peony root, and is extracted using 95% to 100% ethanol or 95% to 100% methanol. The extraction method includes thermal reflux; Step S1 -2, the mass ratio of the silica gel used for the silica gel column chromatography separation to the crude paeoniflorin glycoside is 10 to 20:1, and the mass ratio of the alkane derivative-lower alcohol mixed solvent to the crude paeoniflorin glycoside The volume to mass ratio is 40 to 65 ml: 1 g; in step S2, the dosage of the benzenesulfonyl chloride is 20% to 60% of the mass of the paeoniflorin glycoside, and the dosage of the organic metal catalyst is the paeoniflorin glycoside. 0.05% to 20% of the mass, the molar ratio of the acid binding agent to the paeoniflorin glycoside is 1 to 2:1, and the volume to mass ratio of the reaction solvent to the paeoniflorin glycoside is 2 to 10 ml: 1g, the volume and mass ratio of the organic phase and the paeoniflorin glycoside is 2.5-7.5ml: 1g, the volume and mass ratio of the aqueous phase and the paeoniflorin glycoside is 5-15ml: 1g; in step S3 , the mass ratio of the silica gel used for the silica gel column chromatography separation to the crude paeoniflorin-6'-O-benzenesulfonate is 6 to 12:1, and the halogenated hydrocarbon-lower alcohol mixed solvent and the crude paeoniflorin-6'-O-benzenesulfonate are The volume to mass ratio of the crude paeoniflorin-6'-O-benzenesulfonate is 55 to 80 ml: 1 g; in step S4, the good solvent and the paeonilide-6'-O-benzenesulfonate are The volume-to-mass ratio of the fine acid ester A is 10 to 20 mL: 1 g, and the volume-to-mass ratio of the poor solvent to the fine paeoniflorin-6'-O-benzenesulfonate ester A is 8 to 12 mL: 1g; steps In S5, the volume to mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1 to 20 mL: 1 g.
优选的,步骤S1-1中,乙醇或甲醇与所述药材质量比为5~20:1;步骤S1-2中,所述硅胶柱色谱分离所用的硅胶与所述芍药内酯苷粗品的质量比为10~15:1,所述烷烃衍生物-低级醇混合溶剂与所述芍药内酯苷粗品的体积质量比为50~60ml:1g;步骤S2中,所述苯磺酰氯的用量为所述芍药内酯苷质量的25%~30%,所述有机金属催化剂的用量为所述芍药内酯苷质量的0.5%~5.0%,所述缚酸剂与所述芍药内酯苷的摩尔比为1.2~1.6:1;步骤S5中,所述混合溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品B的体积质量比为1~15mL:1g。Preferably, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 20:1; in step S1-2, the mass of the silica gel used for silica gel column chromatography separation and the crude paeoniflorin glycosides The ratio is 10-15:1, and the volume-to-mass ratio of the alkane derivative-lower alcohol mixed solvent and the crude paeoniflorin glycoside is 50-60ml:1g; in step S2, the amount of benzene sulfonyl chloride is 25% to 30% of the mass of the paeoniflorin glycoside, the dosage of the organometallic catalyst is 0.5% to 5.0% of the mass of the paeoniflorin glycoside, and the molar ratio of the acid binding agent to the paeoniflorin glycoside It is 1.2~1.6:1; in step S5, the volume mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1~15mL:1g.
步骤S1-2中,当填料的用量越大,分离效果越好,当用量为芍药内酯苷粗品的10~15倍时,在保证分离效果较佳的前提下,洗脱剂用量相对较少。In step S1-2, when the amount of filler is larger, the separation effect is better. When the amount is 10 to 15 times that of the crude paeoniflorin glycoside, the amount of eluent is relatively small while ensuring a better separation effect. .
优选的,步骤S1-1中,乙醇或甲醇与所述药材质量比为5~10:1;步骤S1-2中,所述硅胶柱色谱分离所用的硅胶的目数为60~400目,孔径为
所述烷烃衍生物-低级醇混合溶剂中烷烃衍生物与低级醇的体积比为1~10:1;步骤S2中,所述有机金属催化剂的用量为所述芍药内酯苷质量的0.6%~3.0%;步骤S3中,所述硅胶柱色谱分离所用的硅胶的目数为60~400目,孔径为
所述卤代烃-低级醇混合溶剂中卤代烃与低级醇的体积比为6~12:1;步骤S4中,将所述芍药内酯苷-6'-O-苯磺酸酯精品A在50~60℃中用所述良溶剂溶解后,降温至10~20℃加入所述不良溶剂析晶;步骤S5中,所述程序式干燥包括预冻、升华干燥和解析干燥步骤,所述预冻、所述升华干燥和所述解析干燥的温度依次增高。
Preferably, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 10:1; in step S1-2, the mesh number of the silica gel used for silica gel column chromatography separation is 60 to 400 mesh, and the pore size is 60 to 400 mesh. for The volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 1 to 10:1; in step S2, the amount of the organometallic catalyst is 0.6% to 0.6% of the mass of the paeoniflorin glycosides. 3.0%; in step S3, the mesh number of the silica gel used for the silica gel column chromatography separation is 60 to 400 mesh, and the pore diameter is The volume ratio of the halogenated hydrocarbon to the lower alcohol in the halogenated hydrocarbon-lower alcohol mixed solvent is 6 to 12:1; in step S4, the paeoniflorin-6'-O-benzenesulfonate fine product A After dissolving with the good solvent at 50-60°C, the temperature is lowered to 10-20°C and the poor solvent is added for crystallization; in step S5, the programmed drying includes pre-freezing, sublimation drying and analytical drying steps. The temperatures of pre-freezing, the sublimation drying and the analytical drying are increased in sequence.
优选的,步骤S1-1中,所述吸附包括搅拌吸附、柱层析吸附中的一种或多种,吸附剂包括硅胶、氧化铝中的一种或多种,用纯乙醇进行洗脱,得到所述芍药内酯苷粗品;步骤S1-2中,所述烷烃衍生物-低级醇混合溶剂中烷烃衍生物与低级醇的体积比为3~7:1;步骤S2中,在室温进行反应,反应时间为2~5h;步骤S4中,析晶后,还包括过滤、漂洗、再过滤过程,得到所述芍药内酯苷-6'-O-苯磺酸酯精品B;步骤S5中,所述预冻的温度为-60~-10℃,所述升华干燥的温度为-10~0℃,所述解析干燥的的温度为5~65℃。Preferably, in step S1-1, the adsorption includes one or more of stirring adsorption and column chromatography adsorption, the adsorbent includes one or more of silica gel and alumina, and is eluted with pure ethanol. The crude paeoniflorin glycoside is obtained; in step S1-2, the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 3 to 7:1; in step S2, the reaction is carried out at room temperature , the reaction time is 2 to 5h; in step S4, after crystallization, it also includes the process of filtration, rinsing, and re-filtration to obtain the paeoniflorin glycoside-6'-O-benzenesulfonate fine product B; in step S5, The temperature of the pre-freezing is -60~-10°C, the temperature of the sublimation drying is -10~0°C, and the temperature of the analytical drying is 5~65°C.
在步骤S1-1中,一般来说,芍药内酯苷粗品中除了含有超过20%的芍药内酯苷外,还含有大量鞣质、糖胶等杂质,如直接采用柱层析进行分离不仅影响芍药内酯苷的纯度,还对分离效率有较大的影响。因此采用吸附剂除去芍药内酯苷中鞣质、糖胶等杂质,能进一步提高所得芍药内酯苷的纯度和分离效率。In step S1-1, generally speaking, in addition to more than 20% of paeoniflorin glycosides, the crude paeoniflorin glycosides also contain a large amount of tannins, sugar gums and other impurities. If column chromatography is used directly for separation, it will not only affect The purity of paeoniflorin glycosides also has a great impact on the separation efficiency. Therefore, using adsorbents to remove impurities such as tannins and sugar gums in paeoniflorin glycosides can further improve the purity and separation efficiency of the resulting paeoniflorin glycosides.
而本发明中所用吸附剂如硅胶、氧化铝等,价格低廉且具有较好的分离效果,能降低芍药内酯苷-6'-O-苯磺酸酯产业化生产的成本。为进一步提高芍药内酯苷的分离效率,在柱层析前可采用与柱层析所用洗脱剂一致的溶剂对芍药内酯苷粗品进行溶解。The adsorbents used in the present invention, such as silica gel, alumina, etc., are low in price and have good separation effects, and can reduce the cost of industrial production of paeoniflorin-6'-O-benzenesulfonate. In order to further improve the separation efficiency of paeoniflorin glycosides, the crude paeoniflorin glycosides can be dissolved in a solvent consistent with the eluent used in column chromatography before column chromatography.
优选的,步骤S1-1中,所述吸附剂与所述药材提取物的质量比为1~4:1,纯乙醇与所述吸附剂的体积质量比为2~6ml:1g;步骤S1-2中,所述硅胶柱色谱分离前,用所述烷烃衍生物-低级醇混合溶剂溶解所述芍药内酯苷粗品,所述烷烃衍生物-低级醇混合溶剂中的烷烃衍生物包括乙酸乙酯、乙酸异丙酯、三氯甲烷、二氯甲烷中的一种或多种,所述烷烃衍生物-低级醇混合溶剂中的低级醇包括甲醇、乙醇、异丙醇中的一种或多种;步骤S3中,所述硅胶柱色谱分离前,用所述卤代烃-低级醇混合溶剂溶解所述芍药内酯苷-6'-O-苯磺酸酯粗品;步骤S4中,用所述不良溶剂进行漂洗;步骤S5中,所述预冻的操作为降温至-60~-10℃并保温30~300min,所述升华干燥的操作为在30~180min内升温至-10~0℃并保温180~720min,所述解析干燥的操作为在30~240min内升温至5~20℃并保温60~600min,接着在30~240min内升温至50~65℃并保温300~1440min。Preferably, in step S1-1, the mass ratio of the adsorbent to the medicinal material extract is 1-4:1, and the volume-mass ratio of pure ethanol to the adsorbent is 2-6ml:1g; step S1- In 2, before the silica gel column chromatography separation, the crude paeoniflorin derivative is dissolved in the alkane derivative-lower alcohol mixed solvent, and the alkane derivative in the alkane derivative-lower alcohol mixed solvent includes ethyl acetate. , one or more of isopropyl acetate, chloroform, and dichloromethane, and the lower alcohol in the alkane derivative-lower alcohol mixed solvent includes one or more of methanol, ethanol, and isopropyl alcohol. ; In step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin-6'-O-benzenesulfonate crude product; in step S4, use the Rinse with poor solvent; in step S5, the pre-freezing operation is to cool down to -60~-10°C and keep it warm for 30~300min, and the sublimation drying operation is to raise the temperature to -10~0°C within 30~180min and keep it warm for 30~180min. The temperature is maintained for 180 to 720 minutes. The analytical drying operation is to raise the temperature to 5 to 20°C within 30 to 240 minutes and keep the temperature for 60 to 600 minutes, and then raise the temperature to 50 to 65°C within 30 to 240 minutes and keep the temperature for 300 to 1440 minutes.
优选的,步骤S1-2中,所述硅胶柱色谱分离前,用所述烷烃衍生物-低级醇混合溶剂按体积质量比1~3ml:1g溶解所述芍药内酯苷粗品,所述烷烃衍生物-低级醇混合溶剂为乙酸乙酯-甲醇混合溶剂或乙酸乙酯-乙醇混合溶剂,所述烷烃衍生物-低级醇混合溶剂中烷烃衍生物与低级醇的体积比为4~6:1;步骤S3中, 所述硅胶柱色谱分离前,用所述卤代烃-低级醇混合溶剂按体积质量比1~3ml:1g溶解所述芍药内酯苷-6'-O-苯磺酸酯粗品;步骤S5中,所述预冻的操作为降温至-40~-10℃并保温90~300min,所述升华干燥的操作为在60~180min内升温至-10~0℃并保温240~600min,所述解析干燥的操作为在60~180min内升温至5~15℃并保温90~540min,接着在60~180min内升温至50~60℃并保温480~1440min。Preferably, in step S1-2, before the silica gel column chromatography separation, the alkane derivative-lower alcohol mixed solvent is used to dissolve the crude paeoniflorin glycoside in a volume mass ratio of 1 to 3 ml: 1 g, and the alkane derivative is The substance-lower alcohol mixed solvent is an ethyl acetate-methanol mixed solvent or an ethyl acetate-ethanol mixed solvent, and the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 4 to 6:1; In step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the crude paeoniflorin-6'-O-benzenesulfonate ester in a volume mass ratio of 1 to 3 ml: 1 g. ; In step S5, the pre-freezing operation is to lower the temperature to -40~-10°C and keep it warm for 90~300min, and the sublimation drying operation is to raise the temperature to -10~0°C within 60~180min and keep it warm for 240~600min. , the operation of analytical drying is to raise the temperature to 5-15°C within 60-180 min and keep it warm for 90-540 min, and then raise the temperature to 50-60°C within 60-180 min and keep it warm for 480-1440 min.
一种如上所述芍药内酯苷-6’-O-苯磺酸酯的制备方法获得的芍药内酯苷-6’-O-苯磺酸酯,具有如下结构:A kind of paeoniflorin-6’-O-benzenesulfonate obtained by the preparation method of paeoniflorin-6’-O-benzenesulfonate has the following structure:
获得的芍药内酯苷-6'-O-苯磺酸酯具有较好的补血作用。The obtained paeoniflorin-6'-O-benzenesulfonate has good blood-enriching effect.
与现有技术相比较,实施本发明,具有如下有益效果:Compared with the prior art, the implementation of the present invention has the following beneficial effects:
本发明中首次提出芍药内酯苷-6'-O-苯磺酸酯制备的工艺路线,路线简单、条件温和,适合于工业化生产。本发明中的合成方法具有产率高、催化剂及有机溶剂用量少、反应时间短、无需滴加原料等优点。本发明中的芍药内酯苷硅胶柱层析纯化步骤中优选采用的是乙酸乙酯-甲醇或乙酸乙酯-乙醇的洗脱体系,相对于含有卤代烃的洗脱体系,不仅能减少洗脱剂的用量,还能提高人身及工业生产的安全性。本发明的干燥工艺,能够在保证芍药内酯苷-6'-O-苯磺酸酯成品的溶剂残余合格的同时,不增加有关物质含量,确保芍药内酯苷-6'-O-苯磺酸酯成品的高质量。The present invention proposes for the first time a process route for preparing paeoniflorin-6'-O-benzenesulfonate. The route is simple, the conditions are mild, and it is suitable for industrial production. The synthesis method in the present invention has the advantages of high yield, small amount of catalyst and organic solvent, short reaction time, and no need to drop raw materials. In the silica gel column chromatography purification step of paeoniflorin glycosides in the present invention, the elution system of ethyl acetate-methanol or ethyl acetate-ethanol is preferably used. Compared with the elution system containing halogenated hydrocarbons, it can not only reduce the elution The dosage of remover can also improve the safety of people and industrial production. The drying process of the present invention can ensure that the solvent residue of the finished product of paeoniflorin-6'-O-benzenesulfonate is qualified without increasing the content of related substances and ensure that the paeoniflorin-6'-O-benzenesulfonate product is qualified. High quality of finished acid ester products.
图1为本发明实施例1产物的氢谱图。Figure 1 is a hydrogen spectrum of the product of Example 1 of the present invention.
图2为本发明实施例1产物的碳谱图。Figure 2 is a carbon spectrum of the product of Example 1 of the present invention.
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the present invention clearer, the concept and technical effects of the present invention will be clearly and completely described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without exerting creative efforts are all protection scope of the present invention.
实施例1Example 1
本实施例制备了一种芍药内酯苷-6'-O-苯磺酸酯,具体过程为:In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:
A:取白芍饮片500g,加入4L 95%乙醇热回流提取,过滤浓缩,得到药材提取物31.9g,再加入63.8g中性氧化铝搅拌吸附,将吸附后的中性氧化铝加入层析柱,使用100mL乙醇洗脱,收集洗脱液,浓缩,得到15.4g芍药内酯苷粗品,芍药内酯苷纯度为25.3%。将所得芍药内酯苷粗品使用30mL溶剂(三氯甲烷:异丙醇=3:1)溶解,填装200g硅胶,洗脱剂为三氯甲烷:异丙醇=5:1,经硅胶柱层析洗脱,共洗脱1L,收集洗脱液,减压浓缩,得到3.1g芍药内酯苷,纯度为91.2%。A: Take 500g of white peony slices, add 4L of 95% ethanol for hot reflux extraction, filter and concentrate to obtain 31.9g of medicinal extract, then add 63.8g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 100 mL ethanol to elute, collect the eluate, and concentrate to obtain 15.4 g of crude paeoniflorin glycoside, with a purity of 25.3%. The obtained crude paeoniflorin glycoside was dissolved in 30 mL of solvent (chloroform: isopropanol = 3:1), filled with 200g of silica gel, and the eluent was chloroform: isopropanol = 5:1, and passed through a silica gel column layer The elution was analyzed, and a total of 1 L was eluted. The eluate was collected and concentrated under reduced pressure to obtain 3.1 g of paeoniflorin glycoside with a purity of 91.2%.
B:使用12.2mL丙酮将所得芍药内酯苷充分溶解,加入0.061g二甲基二氯化锡和0.75mL三乙胺,再加入0.775g的苯磺酰氯,搅拌反应。待反应2h后,加入25mL乙酸乙酯和18.5mL纯水,搅拌5min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品3.75g,纯度为84.7%。使用10mL溶剂(三氯甲烷:甲醇=10:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装40g硅胶,洗脱剂为三氯甲烷:甲醇=9:1,经硅胶柱层析洗脱,TLC监控,共洗脱225ml洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩得到2.65g的芍药内酯苷-6'-O-苯磺酸酯,纯度为96.3%。B: Use 12.2 mL acetone to fully dissolve the obtained paeoniflorin glycoside, add 0.061 g dimethyltin dichloride and 0.75 mL triethylamine, then add 0.775 g benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 25 mL of ethyl acetate and 18.5 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 3.75 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 84.7%. Use 10 mL of solvent (chloroform: methanol = 10:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate and fill it with 40 g of silica gel. The eluent is chloroform: methanol = 9:1. , eluted by silica gel column chromatography and monitored by TLC, a total of 225ml of eluent was eluted, and the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate was collected and concentrated under reduced pressure to obtain 2.65g of paeoniflorin. Glycoside-6'-O-benzenesulfonate, purity 96.3%.
C:使用47.7mL乙酸异丙酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入26.5mL正己烷搅拌析晶,过滤,用正己烷漂洗,再过滤,得到2.35g纯度为98.4%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 47.7 mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 26.5 mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 2.35 g Paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4%.
D:将上一步得到的2.35g纯度为98.4%的芍药内酯苷-6'-O-苯磺酸酯,加入混合溶剂(9.4mL碳酸二甲酯、0.59mL丙酮和1.76mL水),搅拌使完全溶解,置于盘内,按照表1参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Add 2.35g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4% obtained in the previous step to the mixed solvent (9.4mL dimethyl carbonate, 0.59mL acetone and 1.76mL water), and stir Dissolve it completely, place it in a plate, and perform the drying procedure according to the parameters in Table 1. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表1 实施例1干燥参数及样品质量检验表Table 1 Example 1 drying parameters and sample quality inspection table
以实施例1为例,产物的氢谱图如图1所示,碳谱图如图2所示。经鉴定,确定产物具有如下结构:Taking Example 1 as an example, the hydrogen spectrum of the product is shown in Figure 1 and the carbon spectrum is shown in Figure 2. After identification, it was determined that the product had the following structure:
实施例2Example 2
本实施例制备了一种芍药内酯苷-6'-O-苯磺酸酯,具体过程为:In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:
A:取白芍饮片500g,加入600ml 80%乙醇热回流提取,过滤浓缩,得到药材提取物37.6g,再加入112.8g中性氧化铝搅拌吸附,将吸附后的中性氧化铝加入层析柱,使用150mL乙醇洗脱,收集洗脱液,浓缩,得到17.95g芍药内酯苷粗品,芍药内酯苷纯度为22.6%。将所得芍药内酯苷粗品使用36mL溶剂(乙酸 乙酯:甲醇=4:1)溶解,填装215.5g硅胶,洗脱剂为乙酸乙酯:甲醇=4:1,经硅胶柱层析洗脱,共洗脱750ml,收集洗脱液,减压浓缩,得到3.9g芍药内酯苷,纯度为85.4%。A: Take 500g of white peony slices, add 600ml of 80% ethanol for thermal reflux extraction, filter and concentrate to obtain 37.6g of medicinal extract, then add 112.8g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 150 mL ethanol to elute, collect the eluate, and concentrate to obtain 17.95g of crude paeoniflorin glycoside, with a purity of 22.6%. The obtained crude paeoniflorin glycoside was dissolved in 36 mL of solvent (ethyl acetate: methanol = 4: 1), filled with 215.5 g of silica gel, and the eluent was ethyl acetate: methanol = 4: 1, and was eluted by silica gel column chromatography. , a total of 750 ml was eluted, the eluate was collected, and concentrated under reduced pressure to obtain 3.9 g of paeoniflorin glycoside with a purity of 85.4%.
B:使用8mL乙腈将所得芍药内酯苷充分溶解,加入0.078g二叔丁基二氯化锡和1.315g碳酸氢钠,再加入1.17g的苯磺酰氯,搅拌反应。待反应2h后,加入39mL乙酸乙酯和19.5mL纯水,搅拌5min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品4.15g,纯度为80.6%。使用8mL溶剂(三氯甲烷:乙醇=8:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装37.5g硅胶,洗脱剂为三氯甲烷:乙醇=8:1,经硅胶柱层析洗脱,TLC监控,共洗脱290ml洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩得到2.85g的芍药内酯苷-6'-O-苯磺酸酯,纯度为95.2%。B: Use 8 mL of acetonitrile to fully dissolve the obtained paeoniflorin glycoside, add 0.078g di-tert-butyltin dichloride and 1.315g sodium bicarbonate, then add 1.17g benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 39 mL of ethyl acetate and 19.5 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 4.15 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 80.6%. Use 8 mL of solvent (chloroform: ethanol = 8: 1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 37.5 g of silica gel. The eluent is chloroform: ethanol = 8: 1. Elute through silica gel column chromatography and monitor by TLC. Elute a total of 290ml of eluent. Collect the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate and concentrate under reduced pressure to obtain 2.85g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 95.2%.
C:使用45mL乙酸异丁酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入28.5mL正己烷搅拌析晶,过滤,正己烷漂洗,再过滤,得到2.5g纯度为98.1%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 45 mL of isobutyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 28.5 mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 2.5 g of purity It is 98.1% paeoniflorin-6'-O-benzenesulfonate.
D:将上一步得到的2.5g纯度为98.1%的芍药内酯苷-6'-O-苯磺酸酯,加入混合溶剂(20mL碳酸二甲酯、1.26mL丁酮和3.76mL水),搅拌使完全溶解,置于盘内,按照表2参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Add 2.5g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.1% obtained in the previous step to the mixed solvent (20mL dimethyl carbonate, 1.26mL methyl ethyl ketone and 3.76mL water), and stir Dissolve it completely, place it in a plate, and perform the drying procedure according to the parameters in Table 2. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表2 实施例2干燥参数及样品质量检验表Table 2 Example 2 drying parameters and sample quality inspection table
经鉴定,确定产物具有如下结构:After identification, it was determined that the product had the following structure:
实施例3Example 3
本实施例制备了一种芍药内酯苷-6'-O-苯磺酸酯,具体过程为:In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:
A:取白芍饮片1kg,加入6L无水乙醇热回流提取,过滤浓缩,得到药材提取物31g,再加入183g中性氧化铝搅拌吸附,将吸附后的中性氧化铝加入层析柱,使用180mL乙醇洗脱,收集洗脱液,浓缩,得到36.4g芍药内酯苷粗品,芍药内酯苷纯度为27.3%。将所得芍药内酯苷粗品使用72.8mL溶剂(乙酸异丙酯:甲醇=6:1)溶解,填装400g硅胶,洗脱剂为乙酸异丙酯:甲醇=6:1,经硅胶柱层析洗脱,共洗脱1.82L,收集洗脱液,减压浓缩,得到8.86g芍药内酯苷,纯度为87.3%。A: Take 1kg of white peony slices, add 6L of absolute ethanol for thermal reflux extraction, filter and concentrate to obtain 31g of medicinal extract, then add 183g of neutral alumina for stirring and adsorption, add the adsorbed neutral alumina to the chromatography column, and use Elute with 180 mL of ethanol, collect the eluate, and concentrate to obtain 36.4 g of crude paeoniflorin glycoside, with a purity of 27.3%. The obtained crude paeoniflorin glycoside was dissolved in 72.8 mL of solvent (isopropyl acetate: methanol = 6:1), filled with 400 g of silica gel, and the eluent was isopropyl acetate: methanol = 6:1, and subjected to silica gel column chromatography. After elution, a total of 1.82L was eluted. The eluate was collected and concentrated under reduced pressure to obtain 8.86g of paeoniflorin glycoside with a purity of 87.3%.
B:使用18mL二甲亚砜将所得芍药内酯苷充分溶解,加入0.0266g二甲基硫化锡和1.96g碳酸钾,再加入2.4g的苯磺酰氯,搅拌反应。待反应2h后,加入100mL乙酸乙酯和60mL纯水,搅拌5min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品7.2g,纯度为88.2%。使用18mL溶剂(二氯甲烷:乙醇=10:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装57.6g硅胶,洗脱剂为二氯甲烷:乙醇=10:1,经硅胶柱层析洗脱,TLC监控,共洗脱920ml洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩得到5.8g的芍药内酯苷-6'-O-苯磺酸酯,纯度为96.7%。B: Use 18 mL of dimethyl sulfoxide to fully dissolve the obtained paeoniflorin glycoside, add 0.0266 g of dimethyl tin sulfide and 1.96 g of potassium carbonate, then add 2.4 g of benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 100 mL of ethyl acetate and 60 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 7.2 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 88.2%. Use 18 mL of solvent (dichloromethane: ethanol = 10: 1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate and fill it with 57.6 g of silica gel. The eluent is dichloromethane: ethanol = 10: 1. Elute through silica gel column chromatography and monitor by TLC. Elute a total of 920ml of eluent. Collect the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate and concentrate under reduced pressure to obtain 5.8g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 96.7%.
C:使用69mL乙酸异丙酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入46mL异丙醚搅拌析晶,过滤,异丙醚漂洗,再过滤,得到5.2g纯度为98.2%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 69 mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 46 mL of isopropyl ether to stir and crystallize, filter, rinse with isopropyl ether, and filter again to obtain 5.2 g. Paeoniflorin-6'-O-benzenesulfonate with a purity of 98.2%.
D:将上一步得到的5.2g纯度为98.9%的芍药内酯苷-6'-O-苯磺酸酯,加入混合溶剂(20.8mL碳酸二甲酯、2.6mL丙酮和2.6mL水),搅拌使完全溶解, 置于盘内,按照表3参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Add 5.2g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.9% obtained in the previous step to the mixed solvent (20.8mL dimethyl carbonate, 2.6mL acetone and 2.6mL water), and stir Dissolve it completely, place it in a plate, and proceed with the drying procedure according to the parameters in Table 3. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表3 实施例3干燥参数及样品质量检验表Table 3 Example 3 drying parameters and sample quality inspection table
经鉴定,确定产物具有如下结构:After identification, it was determined that the product had the following structure:
实施例4Example 4
本实施例制备了一种芍药内酯苷-6'-O-苯磺酸酯,具体过程为:In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:
A:取白芍饮片5kg,加入50L 95%乙醇热回流提取,过滤浓缩,得到药材提取物401.4g,再加入600g中性氧化铝搅拌吸附,将吸附后的中性氧化铝加入层析柱,使用1.2L乙醇洗脱,收集洗脱液,浓缩,得到208.5g芍药内酯苷粗品,芍药内酯苷纯度为28.4%。将所得芍药内酯苷粗品使用417mL溶剂(二氯甲烷:乙醇=6:1)溶解,填装2.5kg硅胶,洗脱剂为二氯甲烷:乙醇=6:1,经硅胶柱层析洗脱,共洗脱11.5L,收集洗脱液,减压浓缩,得到46.25g芍药内酯苷,纯度为93.6%。A: Take 5kg of white peony slices, add 50L of 95% ethanol for thermal reflux extraction, filter and concentrate to obtain 401.4g of medicinal extract, then add 600g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column. Use 1.2L ethanol to elute, collect the eluate, and concentrate to obtain 208.5g of crude paeoniflorin glycoside, with a purity of 28.4%. The obtained crude paeoniflorin glycoside was dissolved in 417 mL of solvent (methylene chloride: ethanol = 6:1), filled with 2.5 kg of silica gel, and the eluent was methylene chloride: ethanol = 6:1, and was eluted by silica gel column chromatography. , a total of 11.5L was eluted. The eluate was collected and concentrated under reduced pressure to obtain 46.25g of paeoniflorin glycoside with a purity of 93.6%.
B:使用92.5mL乙腈将所得芍药内酯苷充分溶解,加入0.46g二甲基基二 氯化锡和11.2ml三乙胺,再加入11.5g的苯磺酰氯,搅拌反应。待反应2h后,加入465mL乙酸乙酯和230mL纯水,搅拌5min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品39g,纯度为92.5%。使用80mL溶剂(三氯甲烷:乙醇=8:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装234g硅胶,洗脱剂为三氯甲烷:乙醇=9:1,经硅胶柱层析洗脱,TLC监控,共洗脱2.73L洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩得到27.5g的芍药内酯苷-6'-O-苯磺酸酯,纯度为96.9%。B: Use 92.5 mL of acetonitrile to fully dissolve the obtained paeoniflorin, add 0.46 g of dimethyltin dichloride and 11.2 ml of triethylamine, then add 11.5 g of benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 465 mL of ethyl acetate and 230 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 39 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 92.5%. Use 80mL of solvent (chloroform: ethanol = 8:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 234g of silica gel. The eluent is chloroform: ethanol = 9:1. , eluted by silica gel column chromatography and monitored by TLC, a total of 2.73L of eluent was eluted, and the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate was collected and concentrated under reduced pressure to obtain 27.5g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 96.9%.
C:使用550mL乙酸异丙酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入330mL石油醚搅拌析晶,过滤,石油醚漂洗,再过滤,得到24g纯度为99.1%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 550mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 330mL of petroleum ether to stir and crystallize, filter, rinse with petroleum ether, and filter again to obtain 24g with a purity of 99.1 % of paeoniflorin-6'-O-benzenesulfonate.
D:将上一步得到的24g纯度为99.1%的芍药内酯苷-6'-O-苯磺酸酯,加入混合溶剂(57.6mL碳酸二甲酯、3.6mL丙酮和10.8mL水),搅拌使完全溶解,置于盘内,按照表4参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Add 24g of paeoniflorin-6'-O-benzenesulfonate with a purity of 99.1% obtained in the previous step to the mixed solvent (57.6mL dimethyl carbonate, 3.6mL acetone and 10.8mL water), and stir until Completely dissolve, place in a plate, and perform drying procedures according to the parameters in Table 4. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表4 实施例4干燥参数及样品质量检验表Table 4 Example 4 drying parameters and sample quality inspection table
经鉴定,确定产物具有如下结构:After identification, it was determined that the product had the following structure:
实施例5Example 5
本实施例制备了一种芍药内酯苷-6'-O-苯磺酸酯,具体过程为:In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:
A:取白芍饮片10kg,加入70L 90%乙醇热回流提取,过滤浓缩,得到药材提取物903g,再加入1.8kg中性氧化铝搅拌吸附,将吸附后的中性氧化铝加入层析柱,使用3.6L乙醇洗脱,收集洗脱液,浓缩,得到505g芍药内酯苷粗品,芍药内酯苷纯度为23.6%。将所得芍药内酯苷粗品使用1000mL溶剂(乙酸乙酯:甲醇=6:1)溶解,填装6.0kg硅胶,洗脱剂为乙酸乙酯:甲醇=6:1,经硅胶柱层析洗脱,共洗脱29L,收集洗脱液,减压浓缩,得到153g芍药内酯苷,纯度为87.5%。A: Take 10kg of white peony slices, add 70L of 90% ethanol for thermal reflux extraction, filter and concentrate to obtain 903g of medicinal extract, then add 1.8kg of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column. Use 3.6 L of ethanol to elute, collect the eluate, and concentrate to obtain 505 g of crude paeoniflorin glycoside, with a purity of 23.6%. The obtained crude paeoniflorin glycoside was dissolved in 1000 mL of solvent (ethyl acetate: methanol = 6: 1), filled with 6.0 kg of silica gel, and the eluent was ethyl acetate: methanol = 6: 1, and was eluted by silica gel column chromatography. , a total of 29L was eluted, the eluate was collected, and concentrated under reduced pressure to obtain 153g of paeoniflorin glycoside with a purity of 87.5%.
B:使用380mL丙酮将所得芍药内酯苷充分溶解,加入4.6g二甲基二氯化锡和15ml三乙胺,再加入42.8g的苯磺酰氯,搅拌反应。待反应2h后,加入1.5L乙酸乙酯和750mL纯水,搅拌5min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品162g,纯度为85.2%。使用400mL溶剂(三氯甲烷:甲醇=10:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装1.8kg硅胶,洗脱剂为三氯甲烷:甲醇=10:1,经硅胶柱层析洗脱,TLC监控,共洗脱11.2L洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩得到123g的芍药内酯苷-6'-O-苯磺酸酯,纯度为95.1%。B: Use 380 mL of acetone to fully dissolve the obtained paeoniflorin glycoside, add 4.6 g of dimethyltin dichloride and 15 ml of triethylamine, then add 42.8 g of benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 1.5L ethyl acetate and 750mL pure water, stir for 5 minutes, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 162 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 85.2%. Use 400 mL of solvent (chloroform: methanol = 10: 1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 1.8 kg of silica gel. The eluent is chloroform: methanol = 10: 1. Elute through silica gel column chromatography and monitor by TLC. A total of 11.2L of eluent is eluted. The fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate is collected and concentrated under reduced pressure to obtain 123g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 95.1%.
C:使用1845mL乙酸异丁酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入1353mL正己烷搅拌析晶,过滤,正己烷漂洗,再过滤,得到106g纯度为98.5%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 1845mL of isobutyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 1353mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 106g with a purity of 98.5 % of paeoniflorin-6'-O-benzenesulfonate.
D:将上一步得到的106g纯度为98.5%的芍药内酯苷-6'-O-苯磺酸酯,加入混合溶剂(1.272L碳酸二甲酯、79.5mL丁酮和238.5mL水),搅拌使完全溶解, 置于盘内,按照表5参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Add 106g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.5% obtained in the previous step to the mixed solvent (1.272L dimethyl carbonate, 79.5mL methyl ethyl ketone and 238.5mL water), and stir Dissolve it completely, place it in a plate, and perform the drying procedure according to the parameters in Table 5. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表5 实施例5干燥参数及样品质量检验表Table 5 Example 5 drying parameters and sample quality inspection table
经鉴定,确定产物具有如下结构:After identification, it was determined that the product had the following structure:
实施例6Example 6
本实施例制备了一种芍药内酯苷-6'-O-苯磺酸酯,具体过程为:In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:
A:取白芍饮片50kg,加入500L 95%乙醇热回流提取,过滤浓缩,得到药材提取物4.0kg,再加入8.0kg中性氧化铝搅拌吸附,将吸附后的中性氧化铝加入层析柱,使用16.0L乙醇洗脱,收集洗脱液,浓缩,得到1.8kg芍药内酯苷粗品,芍药内酯苷纯度为20.3%。将所得芍药内酯苷粗品使用3.6L溶剂(乙酸乙酯:乙醇=6:1)溶解,填装20kg硅胶,洗脱剂为乙酸乙酯:乙醇=6:1,经硅胶柱层析洗脱,共洗脱90L,收集洗脱液,减压浓缩,得到382g芍药内酯苷,纯度为85.2%。A: Take 50kg of white peony slices, add 500L of 95% ethanol for thermal reflux extraction, filter and concentrate to obtain 4.0kg of medicinal extract, then add 8.0kg of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 16.0L ethanol to elute, collect the eluate, and concentrate to obtain 1.8 kg of crude paeoniflorin glycoside, with a purity of 20.3%. The obtained crude paeoniflorin glycoside was dissolved in 3.6L solvent (ethyl acetate: ethanol = 6:1), filled with 20kg silica gel, and the eluent was ethyl acetate: ethanol = 6:1, and was eluted by silica gel column chromatography. , a total of 90L was eluted, the eluate was collected, and concentrated under reduced pressure to obtain 382g of paeoniflorin glycoside with a purity of 85.2%.
B:使用900mL丙酮将所得芍药内酯苷充分溶解,加入10.6g二乙基二氯化 锡和60ml三乙胺,再加入103g的苯磺酰氯,搅拌反应。待反应2h后,加入3.8L乙酸乙酯和1.9L纯水,搅拌5min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品402g,纯度为82.8%。使用800mL溶剂(三氯甲烷:乙醇=8:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装4.0kg硅胶,洗脱剂为三氯甲烷:乙醇=8:1,经硅胶柱层析洗脱,TLC监控,共洗脱30L洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩得到307g的芍药内酯苷-6'-O-苯磺酸酯,纯度为95.6%。B: Use 900 mL acetone to fully dissolve the resulting paeoniflorin glycoside, add 10.6 g diethyl tin dichloride and 60 ml triethylamine, then add 103 g benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 3.8L ethyl acetate and 1.9L pure water, stir for 5 minutes, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 402 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 82.8%. Use 800mL of solvent (chloroform: ethanol = 8:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 4.0kg of silica gel. The eluent is chloroform: ethanol = 8: 1. Elute through silica gel column chromatography and monitor by TLC. Elute a total of 30L of eluent. Collect the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate and concentrate under reduced pressure to obtain 307g of paeoniflorin. Glycoside-6'-O-benzenesulfonate, purity 95.6%.
C:使用4.0L乙酸异丙酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入2.8L异丙醚搅拌析晶,过滤,异丙醚漂洗,再过滤,得到286g纯度为98.4%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 4.0L isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 2.8L isopropyl ether to stir and crystallize, filter, rinse with isopropyl ether, and filter again to obtain 286g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4%.
D:将上一步得到的286g纯度为98.4%的芍药内酯苷-6'-O-苯磺酸酯,加入混合溶剂(1.144L碳酸二甲酯、71.5mL丙酮和214.5mL水),搅拌使完全溶解,置于盘内,按照表6参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Add 286g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4% obtained in the previous step to the mixed solvent (1.144L dimethyl carbonate, 71.5mL acetone and 214.5mL water), and stir until Completely dissolve, place in a plate, and perform drying procedures according to the parameters in Table 6. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表6 实施例6干燥参数及样品质量检验表Table 6 Example 6 drying parameters and sample quality inspection table
经鉴定,确定产物具有如下结构:After identification, it was determined that the product had the following structure:
对比例1Comparative example 1
本对比例采用芍药内酯苷(纯度98%)而非芍药提取物作为起始原料来制备芍药内酯苷-6'-O-苯磺酸酯,分别经过如下工艺过程:This comparative example uses paeoniflorin glycoside (purity 98%) instead of peony extract as the starting material to prepare paeoniflorin glycoside-6'-O-benzenesulfonate, which undergoes the following processes:
现有工艺:Existing technology:
B:取芍药内酯苷6g(纯度98%)加入反应瓶中,加入240mL(吡啶:三氯甲烷=1:1)混合溶液溶解,再加入228mg的4-二甲氨基吡啶活化30min。在3h内,将3ml苯磺酰氯溶于240mL三氯甲烷后滴于反应液进行反应,反应温度为25℃,滴加完毕后继续反应12h,反应结束后,用水洗涤三次,每次80ml,保留有机层用无水硫酸钠干燥、过滤后浓缩得4.9g,纯度50.6%,产率为32.6%。B: Add 6g of paeoniflorin glycoside (purity 98%) into the reaction bottle, add 240 mL (pyridine: chloroform = 1:1) mixed solution to dissolve, and then add 228 mg of 4-dimethylaminopyridine for activation for 30 minutes. Within 3 hours, dissolve 3 ml of benzene sulfonyl chloride in 240 mL of chloroform and drop it into the reaction solution for reaction. The reaction temperature is 25°C. After the dropwise addition, continue the reaction for 12 hours. After the reaction is completed, wash with water three times, 80 ml each time, and keep The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain 4.9 g, with a purity of 50.6% and a yield of 32.6%.
C:使用12mL溶剂(三氯甲烷:无水乙醇=3:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,使用36g硅胶进行柱层析分离(洗脱剂为三氯甲烷:甲醇=25:1),共洗脱1L,得1.68g的芍药内酯苷-6'-O-苯磺酸酯,纯度为97.3%。C: Use 12 mL of solvent (chloroform: absolute ethanol = 3:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and use 36 g of silica gel for column chromatography separation (eluent is tris Methyl chloride: methanol = 25:1), a total of 1 L was eluted, and 1.68 g of paeoniflorin-6'-O-benzenesulfonate was obtained, with a purity of 97.3%.
为更清晰地看出现有制备工艺与本发明工艺的区别,省略步骤D。In order to more clearly see the difference between the existing preparation process and the process of the present invention, step D is omitted.
本发明工艺:The process of the present invention:
B:取芍药内酯苷6g(纯度98%),使用25mL丙酮充分溶解,带溶解完全后,加入0.04g二甲基二氯化锡10min,再依次加入2.5mL三乙胺,再加入2g苯磺酰氯,搅拌反应2h,TLC检测。反应完全后,加入60mL乙酸乙酯和30mL纯水,搅拌15min,终止反应。静置0.5h,取有机层,减压浓缩,得到芍药内酯苷-6'-O-苯磺酸酯粗品6.82g,纯度为92.6%,产率为83.1%。使用13.64mL溶剂(三氯甲烷:无水乙醇=3:1)将芍药内酯苷-6'-O-苯磺酸酯粗品溶解,填装54.6g硅胶,洗脱剂为三氯甲烷:乙醇=12:1,经硅胶柱层析洗脱,TLC监控,共洗 脱410mL洗脱剂,收集只含芍药内酯苷-6'-O-苯磺酸酯的馏分,减压浓缩,得到5.18g的芍药内酯苷-6'-O-苯磺酸酯,纯度为97.7%。B: Take 6g of paeoniflorin glycoside (purity 98%), use 25mL of acetone to fully dissolve it, add 0.04g of dimethyltin dichloride for 10 minutes, then add 2.5mL of triethylamine, and then add 2g of benzene Sulfonyl chloride, stir the reaction for 2 hours, and detect by TLC. After the reaction is complete, add 60 mL of ethyl acetate and 30 mL of pure water, stir for 15 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 6.82 g of paeoniflorin-6'-O-benzenesulfonate crude product, with a purity of 92.6% and a yield of 83.1%. Use 13.64mL of solvent (chloroform: absolute ethanol = 3:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 54.6g of silica gel. The eluent is chloroform: ethanol. =12:1, eluted by silica gel column chromatography and monitored by TLC, a total of 410mL of eluent was eluted, and the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate was collected and concentrated under reduced pressure to obtain 5.18 g of paeoniflorin-6'-O-benzenesulfonate, with a purity of 97.7%.
C:使用72.5mL乙酸异丙酯将上述芍药内酯苷-6'-O-苯磺酸酯溶解完全,再加入51.8mL正己烷搅拌析晶,过滤,正己烷漂洗,再过滤,得到4.93g纯度为99.2%的芍药内酯苷-6'-O-苯磺酸酯。C: Use 72.5 mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 51.8 mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 4.93 g. Paeoniflorin-6'-O-benzenesulfonate with a purity of 99.2%.
为更清晰地看出现有制备工艺与本发明工艺的区别,省略步骤D。In order to more clearly see the difference between the existing preparation process and the process of the present invention, step D is omitted.
对比例2Comparative example 2
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,置于真空干燥箱中,按表7参数进行真空干燥。干燥结束后,收集样品,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, place it in a vacuum drying oven, and perform vacuum drying according to the parameters in Table 7. After drying, samples are collected, sampled and tested for solvent residues and related substances.
表7 对比例2干燥参数及样品质量检验表Table 7 Comparative Example 2 drying parameters and sample quality inspection table
对比例3Comparative example 3
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,加入20mL甲醇搅拌使完全溶解,置于真空干燥箱中,按表7参数进行真空干燥。干燥结束后,收集样品,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add 20 mL of methanol and stir to dissolve completely, place it in a vacuum drying oven, and perform vacuum drying according to the parameters in Table 7. After drying, samples are collected, sampled and tested for solvent residues and related substances.
表8 对比例3干燥参数及样品质量检验表Table 8 Comparative Example 3 drying parameters and sample quality inspection table
对比例4Comparative example 4
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,加入20mL丙酮搅拌使完全溶解,置于真空干燥箱中,按表8参数进行真空干燥。干燥结束后,收集样品,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add 20 mL of acetone and stir to dissolve completely, place it in a vacuum drying box, and perform vacuum drying according to the parameters in Table 8. After drying, samples are collected, sampled and tested for solvent residues and related substances.
表9 对比例4干燥参数及样品质量检验表Table 9 Comparative Example 4 drying parameters and sample quality inspection table
对比例5Comparative example 5
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,加入溶剂(100mL碳酸二甲酯),搅拌使完全溶解,置于盘内,按照表9参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add solvent (100 mL of dimethyl carbonate), stir to completely dissolve, place in a plate, and perform drying procedures according to the parameters in Table 9. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表10 对比例5干燥参数及样品质量检验表Table 10 Comparative Example 5 drying parameters and sample quality inspection table
对比例6Comparative example 6
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,加入混合溶剂(85mL碳酸二甲酯和15mL丙酮),搅拌使完全溶解,置于盘内,按照表10参数进行干燥程序。干燥结束后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add mixed solvent (85mL dimethyl carbonate and 15mL acetone), stir to completely dissolve, place in a plate, and dry according to the parameters in Table 10 program. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表11 对比例6干燥参数及样品质量检验表Table 11 Comparative Example 6 drying parameters and sample quality inspection table
对比例7Comparative example 7
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,加入混合溶剂(80mL叔丁醇+20mL水)搅拌使完全溶解,置于盘内,按照表11参数进行干燥程序。干燥结束 后,收集样品粉末,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add mixed solvent (80mL tert-butyl alcohol + 20mL water) and stir until completely dissolved, place it in a plate, and perform the drying procedure according to the parameters in Table 11. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.
表12 对比例7干燥参数及样品质量检验表Table 12 Comparative Example 7 drying parameters and sample quality inspection table
对比例8Comparative example 8
步骤A~C同实施例4,区别在于,步骤D如下:Steps A to C are the same as in Embodiment 4, except that step D is as follows:
D:称取芍药内酯苷-6'-O-苯磺酸酯20g,按照表12加入不同比例的由碳酸二甲酯、丙酮和水组成的混合溶剂100mL,搅拌使完全溶解,进行程序式干燥,收集样品粉末,取样并检验溶剂残余和有关物质。D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add 100 mL of a mixed solvent composed of dimethyl carbonate, acetone and water in different proportions according to Table 12, stir to completely dissolve, and perform the program formula Dry, collect sample powder, take samples and test for solvent residues and related substances.
表13 不同比例混合溶剂状态及样品质量检验表Table 13 Mixed solvent status and sample quality inspection table with different proportions
效果例1Effect example 1
从实施例1~实施例2可以看出在芍药内酯苷提纯步骤中,氯仿-醇类体系分离效果较好,但使用的溶剂倍量高,选用乙酸乙酯-醇类体系更适合大量制备。It can be seen from Examples 1 to 2 that in the purification step of paeoniflorin glycosides, the chloroform-alcohol system has a better separation effect, but the solvent used is high. The ethyl acetate-alcohol system is more suitable for large-scale preparation. .
从实施例1~实施例6可看出,逐步放大反应,所需要的反应溶剂、洗脱剂用量均在权力要求范围内,且具有良好的稳定性。It can be seen from Examples 1 to 6 that by gradually amplifying the reaction, the required dosages of reaction solvents and eluents are within the scope of the claims and have good stability.
从对比例1中可发现,若本工艺直接采用纯度高于98%的芍药内酯苷为原料,产率可达80%以上,远高于现有的工艺,且反应的羟基选择性较好,产物纯度更高,杂质比现有工艺更少,更易通过硅胶柱层析制备得到高纯度的芍药内酯苷-6'-O-苯磺酸酯。所述柱层析将洗脱剂为三氯甲烷:甲醇=6:1~12:1比现有工艺洗脱剂(三氯甲烷:甲醇=20:1~45:1)具有更大的极性,洗脱能力更强,在保证得到高纯度芍药内酯苷-6-O'-苯磺酸酯的同时,更能节省溶剂用量以及加快分离效率。It can be found from Comparative Example 1 that if this process directly uses paeoniflorin glycoside with a purity higher than 98% as raw material, the yield can reach more than 80%, which is much higher than the existing process, and the hydroxyl selectivity of the reaction is better. , the product has higher purity and fewer impurities than the existing process, and it is easier to prepare high-purity paeoniflorin-6'-O-benzenesulfonate through silica gel column chromatography. The column chromatography uses the eluent of chloroform: methanol = 6:1 to 12:1 to have a greater polarity than the existing process eluent (chloroform: methanol = 20:1 to 45:1). properties and stronger elution capacity. While ensuring high purity of paeoniflorin-6-O'-benzenesulfonate, it can save solvent consumption and speed up separation efficiency.
按药典中的规定,本发明所用的各种溶剂在成品药中的残留基本都应该控制在0.5%以下。从实施例4与对比例2~7相比,可以发现使用碳酸二甲酯、丙酮和水,或碳酸二甲酯、丁酮和水的组合作为溶剂,对芍药内酯苷-6-O'-苯磺酸酯进行溶解后进行程序式干燥,其溶剂残留能够符合标准,且有关物质无明显上升,干燥后的成品质量有保障。而使用甲醇为溶剂,对芍药内酯苷-6-O'-苯磺酸酯进行真空干燥,溶剂残留虽然能够符合标准,但有关物质明显增加。对比例4中使用丙酮为溶剂,对芍药内酯苷-6-O'-苯磺酸酯进行真空干燥,虽然有关物质无明显增加,但丙酮的溶剂残留无法降至标准之下。对比例5中只使用碳酸二甲酯为溶剂,对芍药内酯苷-6-O'-苯磺酸酯进行程序式干燥,碳酸二甲酯溶剂残留无法降至标准以下。同样,对比例6用碳酸二甲酯和丙酮混合溶剂,对芍药内酯苷-6-O'-苯磺酸酯进行程序式干燥,溶剂残留都无法降至标准以下。对比例7使用叔丁醇-水混合溶剂对对芍药内酯苷-6-O'-苯磺酸酯进行程序式干燥,溶剂残留虽然能够符合标准,但有关物质明显增加。可见,只有使用本发明特定的混合溶剂,才能满足溶残符合标准且有关物质无增加的要求。According to the provisions of the Pharmacopoeia, the residues of various solvents used in the present invention in the finished drug should basically be controlled below 0.5%. From the comparison between Example 4 and Comparative Examples 2 to 7, it can be found that using dimethyl carbonate, acetone and water, or a combination of dimethyl carbonate, methyl ethyl ketone and water as the solvent, the effect of paeoniflorin-6-O' - The benzenesulfonate ester is dissolved and then dried in a programmed manner. The solvent residue can meet the standards and there is no significant increase in related substances. The quality of the dried product is guaranteed. When methanol is used as the solvent to vacuum-dry paeoniflorin-6-O'-benzenesulfonate, although the solvent residue can meet the standards, the related substances increase significantly. In Comparative Example 4, acetone was used as the solvent to vacuum-dry paeoniflorin-6-O'-benzenesulfonate. Although there was no significant increase in related substances, the solvent residue in acetone could not be reduced below the standard. In Comparative Example 5, only dimethyl carbonate was used as the solvent, and the paeoniflorin-6-O'-benzenesulfonate was dried in a programmed manner. The dimethyl carbonate solvent residue could not be reduced below the standard. Similarly, in Comparative Example 6, a mixed solvent of dimethyl carbonate and acetone was used to perform programmed drying of paeoniflorin-6-O'-benzenesulfonate, and the solvent residue could not be reduced below the standard. Comparative Example 7 used a mixed solvent of tert-butyl alcohol and water to perform programmed drying of paeoniflorin-6-O'-benzenesulfonate. Although the solvent residue could meet the standards, related substances increased significantly. It can be seen that only by using the specific mixed solvent of the present invention can the requirements that the dissolved residues meet the standards and there is no increase in related substances can be met.
从对比例8可以看出,采用由碳酸二甲酯、丙酮和水组成的混合溶剂作为程序式干燥的溶剂,并非任意比例均能获得质量合格的成品。通过设置不同比例下的混合溶剂,进行成品溶剂残余和有关物质的结果考察。混合溶剂中丙酮:水比例小于1:4时,如表中组别1、2、8所示,混合体系出现分层现象,成品中碳酸二甲酯超过限度,影响干燥效果;而当混合体系统中丙酮含量超过10%时,如组别4所示,则成品中丙酮残余超过限度。当碳酸二甲酯、丙酮和水的体积比为4~11:0.2~0.6:1时,混合溶剂体系不分层,碳酸二甲酯和丙酮的溶剂残余符合规定要求,干燥后有关物质无明显增加。可见,只有采用特定比例下的碳酸二甲酯、C3~4酮类和水混合溶剂才能达到溶残符合标准且有关物质无增加的目的。It can be seen from Comparative Example 8 that when a mixed solvent composed of dimethyl carbonate, acetone and water is used as the solvent for programmed drying, not all proportions can obtain qualified finished products. By setting mixed solvents at different proportions, the results of solvent residues and related substances in the finished product are investigated. When the acetone:water ratio in the mixed solvent is less than 1:4, as shown in groups 1, 2, and 8 in the table, the mixed system will appear stratified, and the dimethyl carbonate in the finished product will exceed the limit, affecting the drying effect; and when the mixture When the acetone content in the system exceeds 10%, as shown in Group 4, the residual acetone in the finished product exceeds the limit. When the volume ratio of dimethyl carbonate, acetone and water is 4~11:0.2~0.6:1, the mixed solvent system does not stratify, the solvent residues of dimethyl carbonate and acetone meet the specified requirements, and there is no obvious relevant substance after drying Increase. It can be seen that only by using a mixed solvent of dimethyl carbonate, C3~4 ketones and water in a specific ratio can the dissolved residues meet the standards and there is no increase in related substances.
效果例2Effect example 2
芍药内酯苷-6'-O-苯磺酸酯补血实验Blood enrichment experiment of paeoniflorin-6'-O-benzenesulfonate
1.适应性喂养后,将90只小鼠按体重随机分成9个组,雌雄各半,即对照组、模型组、阳性药组、芍药内酯苷、芍药内酯苷-6'-O-苯磺酸酯分别设置高中低3个剂量组(120,60,30mg/kg),每组10只。对除对照外的其他七组由眼底静脉丛放血0.5mL,24h后灌胃给药,阳性药组按20ml/kg灌胃复方阿胶浆,对照组与模型组按120mg/kg灌胃蒸馏水。于给药第0,5,10天眼眶采血30μL,检测其红细胞数量(见表14)。1. After adaptive feeding, 90 mice were randomly divided into 9 groups according to body weight, half male and half female, namely control group, model group, positive drug group, paeoniflorin glycoside, and paeoniflorin glycoside-6'-O- There are three dose groups (120, 60, 30mg/kg) of benzenesulfonate, high, medium and low respectively, with 10 animals in each group. For the other seven groups except the control group, 0.5 mL of blood was bled from the fundus venous plexus and administered intragastrically 24 hours later. The positive drug group was intragastrically administered compound donkey-hide gelatin slurry at 20 ml/kg, and the control and model groups were intragastrically administered distilled water at 120 mg/kg. On days 0, 5, and 10 after administration, 30 μL of blood was collected from the orbit to detect the number of red blood cells (see Table 14).
2.统计学方法:实验数据采用SPSS软件进行One-way ANOVA统计学分析,结果用x±s表示。2. Statistical methods: The experimental data were analyzed statistically by One-way ANOVA using SPSS software, and the results were expressed as x±s.
表14 芍药内酯苷-6'-O-苯磺酸酯及芍药内酯苷对血虚小鼠红细胞数的影响(x±s,n=8)Table 14 Effects of paeoniflorin-6'-O-benzenesulfonate and paeoniflorin on the number of red blood cells in anemia mice (x±s, n=8)
注:与0天比较,*P<0.05,**P<0.01。Note: Compared with day 0, *P<0.05, **P<0.01.
从实验结果可知,芍药内酯苷-6'-O-苯磺酸酯和芍药内酯苷都能有效提高小鼠红细胞数量,芍药内酯苷-6'-O-苯磺酸酯的效果要更好于芍药内酯苷,并且高剂量的芍药内酯苷-6'-O-苯磺酸酯效果接近于复方阿胶浆。对比于芍药内酯苷,芍药内酯苷-6'-O-苯磺酸酯效果更好的原因可能是增加了亲脂性,提高了体内生物利用度。It can be seen from the experimental results that both paeoniflorin-6'-O-benzenesulfonate and paeoniflorin can effectively increase the number of red blood cells in mice, and the effect of paeonilide-6'-O-benzenesulfonate is greater. It is better than paeoniflorin glycoside, and the effect of high dose of paeoniflorin glycoside-6'-O-benzenesulfonate is close to that of compound donkey-hide gelatin. Compared with paeoniflorin, the reason why paeonilide-6'-O-benzenesulfonate is more effective may be due to increased lipophilicity and improved in vivo bioavailability.
以上所揭露的仅为本发明的较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。What is disclosed above is only the preferred embodiment of the present invention. Of course, it cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope of the present invention.
Claims (10)
- 一种芍药内酯苷-6’-O-苯磺酸酯的制备方法,其特征在于,包括如下步骤:A preparation method of paeoniflorin-6’-O-benzenesulfonate, which is characterized in that it includes the following steps:S1:从药材中提取芍药内酯苷;S1: Extract paeoniflorin glycosides from medicinal materials;S2:按比例向所述芍药内酯苷中加入有机金属催化剂、缚酸剂、苯磺酰氯进行反应,反应终止后浓缩反应产物得到芍药内酯苷-6'-O-苯磺酸酯粗品;S2: Add an organic metal catalyst, an acid binding agent, and benzenesulfonyl chloride to the paeoniflorin glycoside in proportion to carry out the reaction. After the reaction is terminated, the reaction product is concentrated to obtain a crude paeoniflorin glycoside-6'-O-benzenesulfonate ester;S3:将所述芍药内酯苷-6'-O-苯磺酸酯粗品分离后得到芍药内酯苷-6'-O-苯磺酸酯精品A;S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate product to obtain paeonilide-6'-O-benzenesulfonate fine product A;S4:将所述芍药内酯苷-6'-O-苯磺酸酯精品A用良溶剂溶解后,加入不良溶剂析晶,析晶后得到芍药内酯苷-6'-O-苯磺酸酯精品B;S4: After dissolving the paeoniflorin-6'-O-benzenesulfonic acid ester boutique A in a good solvent, add a poor solvent for crystallization, and obtain paeoniflorin-6'-O-benzenesulfonic acid after crystallization. Ester Boutique B;S5:将所述芍药内酯苷-6'-O-苯磺酸酯精品B溶解于混合溶剂中,所述混合溶剂包括碳酸二甲酯、C3~4酮类和水,进行程序式干燥,最终得到芍药内酯苷-6'-O-苯磺酸酯成品。S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in a mixed solvent, the mixed solvent includes dimethyl carbonate, C3~4 ketones and water, and perform programmed drying. Finally, the finished product of paeoniflorin-6'-O-benzenesulfonate is obtained.
- 如权利要求1所述制备方法,其特征在于,包括如下步骤:The preparation method according to claim 1, characterized in that it includes the following steps:S1:包括如下步骤:S1: Includes the following steps:S1-1:药材用醇提取,提取液过滤浓缩后得到药材提取物,经过吸附洗脱得到芍药内酯苷粗品;S1-1: The medicinal materials are extracted with alcohol, the extract is filtered and concentrated to obtain the medicinal material extract, and the crude paeoniflorin glycoside is obtained through adsorption and elution;S1-2:将所述芍药内酯苷粗品进行柱层析分离,从而得到所述芍药内酯苷,所述芍药内酯苷的纯度在80%以上;S1-2: The crude paeoniflorin glycoside is separated by column chromatography to obtain the paeoniflorin glycoside, and the purity of the paeoniflorin glycoside is above 80%;S2:按比例向所述芍药内酯苷中加入所述有机金属催化剂、所述缚酸剂、所述苯磺酰氯,在反应溶剂中进行反应,之后加入有机相和水相终止反应并进行萃取,浓缩后得到所述芍药内酯苷-6'-O-苯磺酸酯粗品;S2: Add the organometallic catalyst, the acid binding agent, and the benzenesulfonyl chloride to the paeoniflorin glycoside in proportion, carry out the reaction in the reaction solvent, and then add the organic phase and the aqueous phase to terminate the reaction and perform extraction. , after concentration, the crude paeoniflorin-6'-O-benzenesulfonate ester is obtained;S3:将所述芍药内酯苷-6'-O-苯磺酸酯粗品经柱层析分离,收集洗脱液,得到所述芍药内酯苷-6'-O-苯磺酸酯精品A,所述芍药内酯苷-6'-O-苯磺酸酯精品A的纯度在95%以上;S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate ester through column chromatography, collect the eluent, and obtain the paeonilide-6'-O-benzenesulfonate fine product A , the purity of the paeoniflorin-6'-O-benzenesulfonate fine product A is above 95%;S4:将所述芍药内酯苷-6'-O-苯磺酸酯精品A用所述良溶剂溶解后,加入所述不良溶剂析晶,析晶后得到芍药内酯苷-6'-O-苯磺酸酯精品B,所述芍药内酯苷-6'-O-苯磺酸酯精品B的纯度在98%以上的;S4: After dissolving the paeoniflorin-6'-O-benzenesulfonate fine product A with the good solvent, adding the poor solvent for crystallization, and obtaining paeonilide-6'-O after crystallization. - Fine benzenesulfonate ester B, the purity of the paeoniflorin-6'-O-benzenesulfonate fine ester B is above 98%;S5:将所述芍药内酯苷-6'-O-苯磺酸酯精品B溶解于所述混合溶剂中,所述混合溶剂包括碳酸二甲酯、C3~4酮类和水体按积比4~11:0.2~0.6:1混合而成,进行程序式干燥,最终得到芍药内酯苷-6'-O-苯磺酸酯成品。S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in the mixed solvent, which includes dimethyl carbonate, C3~4 ketones and water in a volume ratio of 4 ~11:0.2~0.6:1 is mixed, and programmed drying is performed to finally obtain the finished product of paeoniflorin-6'-O-benzenesulfonate.
- 如权利要求2所述制备方法,其特征在于,步骤S1-1中,所述药材包括白芍、赤芍、丹皮、狗头、牡丹根、紫牡丹中的一种或多种,使用80%~100%乙醇或80%~100%甲醇提取,提取方式包括冷浸、热回流或微波辅助提取中的一种;步骤S1-2中,所述柱层析分离为硅胶柱色谱分离,洗脱剂为烷烃衍生物-低级醇混合溶剂;步骤S2中,所述有机金属催化剂包括C1~C4的烷基卤化锡、芳基卤化锡、C1~C4的烷基氧化锡、C1~C4的烷基硫化锡中的一种或多种,所述缚酸剂包括三乙胺、N,N'-二异丙基乙胺、吡啶、碳酸钾、碳酸氢钾、碳酸钠、碳酸氢钠中的一种或多种,所述反应溶剂包括乙腈、四氢呋喃、丙酮、吡啶、甲基乙基酮、N,N’-二甲基甲酰胺、1,4-二氧六环、二甲亚砜中的一种或多种,所述有机相为乙酸乙酯;步骤S3中,所述柱层析分离为硅胶柱色谱分离,洗脱剂为卤代烃-低级醇混合溶剂;步骤S4中,所述良溶剂包括乙酸异丙酯、乙酸异丁酯中的一种或多种,所述不良溶剂包括正己烷、石油醚、异丙醚、异辛烷中的一种或多种;步骤S5中,述混合溶剂包括碳酸二甲酯、C3~4酮类和水体按积比5~8:0.3~0.5:1混合而成。The preparation method according to claim 2, characterized in that in step S1-1, the medicinal materials include one or more of white peony root, red peony root, paeoniae root, dog head, peony root, and purple peony root, and 80% is used. ~100% ethanol or 80% ~ 100% methanol extraction, the extraction method includes one of cold soaking, hot reflux or microwave-assisted extraction; in step S1-2, the column chromatography separation is silica gel column chromatography separation, elution The agent is an alkane derivative-lower alcohol mixed solvent; in step S2, the organometallic catalyst includes C1 to C4 alkyl tin halide, aryl tin halide, C1 to C4 alkyl tin oxide, C1 to C4 alkyl One or more tin sulfides, the acid binding agent includes one of triethylamine, N, N'-diisopropylethylamine, pyridine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate. One or more, the reaction solvent includes acetonitrile, tetrahydrofuran, acetone, pyridine, methyl ethyl ketone, N,N'-dimethylformamide, 1,4-dioxane, and dimethyl sulfoxide. One or more, the organic phase is ethyl acetate; in step S3, the column chromatography separation is silica gel column chromatography, and the eluent is a halogenated hydrocarbon-lower alcohol mixed solvent; in step S4, the The good solvent includes one or more of isopropyl acetate and isobutyl acetate, and the poor solvent includes one or more of n-hexane, petroleum ether, isopropyl ether, and isooctane; in step S5, The mixed solvent includes dimethyl carbonate, C3-4 ketones and water mixed in a volume ratio of 5-8:0.3-0.5:1.
- 如权利要求3所述制备方法,其特征在于,步骤S1-1中,所述药材包括白芍、赤芍中的一种或多种,使用95%~100%乙醇或95%~100%甲醇提取,提取方式包括热回流;步骤S1-2中,所述硅胶柱色谱分离所用的硅胶与所述芍药内酯苷粗品的质量比为10~20:1,所述烷烃衍生物-低级醇混合溶剂与所述芍药内酯苷粗品的体积质量比为40~65ml:1g;步骤S2中,所述苯磺酰氯的用量为所述芍药内酯苷质量的20%~60%,所述有机金属催化剂的用量为所述芍药内酯苷质量的0.05%~20%,所述缚酸剂与所述芍药内酯苷的摩尔比为1~2:1,所述反应溶剂与所述芍药内酯苷的体积质量比为2~10ml:1g,所述有机相与所述芍药内酯苷的体积质量比为2.5~7.5ml:1g,所述水相与所述芍药内酯苷的体积质量比为5~15ml:1g;步骤S3中,所述硅胶柱色谱分离所用的硅胶与所述芍药内酯苷-6'-O-苯磺酸酯粗品的质量比为6~12:1,所述卤代烃-低级醇混合溶剂与 所述芍药内酯苷-6'-O-苯磺酸酯粗品的体积质量比为55~80ml:1g;步骤S4中,所述良溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品A的体积质量比为10~20mL:1g,所述不良溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品A的体积质量比为8~12mL:1g;步骤S5中,所述混合溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品B的体积质量比为1~20mL:1g。The preparation method according to claim 3, characterized in that in step S1-1, the medicinal materials include one or more of white peony root and red peony root, using 95% to 100% ethanol or 95% to 100% methanol. Extraction, the extraction method includes thermal reflux; in step S1-2, the mass ratio of the silica gel used for the silica gel column chromatography separation and the crude paeoniflorin glycoside is 10 to 20:1, and the alkane derivative-lower alcohol is mixed The volume to mass ratio of the solvent to the crude paeoniflorin glycoside is 40-65ml:1g; in step S2, the dosage of benzenesulfonyl chloride is 20%-60% of the mass of the paeoniflorin glycoside, and the organic metal The amount of catalyst used is 0.05% to 20% of the mass of the paeoniflorin glycoside, the molar ratio of the acid binding agent to the paeoniflorin glycoside is 1 to 2:1, and the reaction solvent and the paeoniflorin glycoside are The volume to mass ratio of the glycoside is 2 to 10 ml: 1 g, the volume to mass ratio of the organic phase to the paeoniflorin glycoside is 2.5 to 7.5 ml: 1 g, and the volume to mass ratio of the aqueous phase to the paeoniflorin glycoside is is 5~15ml:1g; in step S3, the mass ratio of the silica gel used for the silica gel column chromatography separation and the crude paeoniflorin-6'-O-benzenesulfonate ester is 6~12:1, the The volume to mass ratio of the halogenated hydrocarbon-lower alcohol mixed solvent and the crude paeoniflorin glycoside-6'-O-benzenesulfonate is 55-80ml:1g; in step S4, the good solvent and the paeoniflorin glycoside-6'-O-benzenesulfonate crude product are The volume-to-mass ratio of the paeoniflorin-6'-O-benzenesulfonate boutique A is 10 to 20 mL: 1 g, and the volume of the poor solvent and the paeoniflorin-6'-O-benzenesulfonate boutique A The mass ratio is 8 to 12 mL: 1 g; in step S5, the volume to mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1 to 20 mL: 1 g.
- 如权利要求3所述制备方法,其特征在于,步骤S1-1中,乙醇或甲醇与所述药材质量比为5~20:1;步骤S1-2中,所述硅胶柱色谱分离所用的硅胶与所述芍药内酯苷粗品的质量比为10~15:1,所述烷烃衍生物-低级醇混合溶剂与所述芍药内酯苷粗品的体积质量比为50~60ml:1g;步骤S2中,所述苯磺酰氯的用量为所述芍药内酯苷质量的25%~30%,所述有机金属催化剂的用量为所述芍药内酯苷质量的0.5%~5.0%,所述缚酸剂与所述芍药内酯苷的摩尔比为1.2~1.6:1;步骤S5中,所述混合溶剂与所述芍药内酯苷-6'-O-苯磺酸酯精品B的体积质量比为1~15mL:1g。The preparation method according to claim 3, characterized in that, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 20:1; in step S1-2, the silica gel used for silica gel column chromatography separation The mass ratio to the crude paeoniflorin glycoside is 10 to 15:1, and the volume to mass ratio of the alkane derivative-lower alcohol mixed solvent to the crude paeoniflorin glycoside is 50 to 60 ml: 1g; in step S2 , the dosage of benzenesulfonyl chloride is 25% to 30% of the mass of the paeoniflorin glycoside, the dosage of the organometallic catalyst is 0.5% to 5.0% of the quality of the paeoniflorin glycoside, and the acid binding agent The molar ratio to the paeoniflorin glycoside is 1.2-1.6:1; in step S5, the volume to mass ratio of the mixed solvent to the paeoniflorin glycoside-6'-O-benzenesulfonate fine product B is 1 ~15mL: 1g.
- 如权利要求3所述制备方法,其特征在于,步骤S1-1中,乙醇或甲醇与所述药材质量比为5~10:1;步骤S1-2中,所述硅胶柱色谱分离所用的硅胶的目数为60~400目,孔径为 所述烷烃衍生物-低级醇混合溶剂中烷烃衍生物与低级醇的体积比为1~10:1;步骤S2中,所述有机金属催化剂的用量为所述芍药内酯苷质量的0.6%~3.0%;步骤S3中,所述硅胶柱色谱分离所用的硅胶的目数为60~400目,所述卤代烃-低级醇混合溶剂中卤代烃与低级醇的体积比为6~12:1;步骤S4中,将所述芍药内酯苷-6'-O-苯磺酸酯精品A在50~60℃中用所述良溶剂溶解后,降温至10~20℃加入所述不良溶剂析晶;步骤S5中,所述程序式干燥包括预冻、升华干燥和解析干燥步骤,所述预冻、所述升华干燥和所述解析干燥的温度依次增高。 The preparation method according to claim 3, characterized in that, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 10:1; in step S1-2, the silica gel used for silica gel column chromatography separation The mesh number is 60~400 mesh, and the aperture is The volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 1 to 10:1; in step S2, the amount of the organometallic catalyst is 0.6% to 0.6% of the mass of the paeoniflorin glycosides. 3.0%; in step S3, the mesh number of the silica gel used for the silica gel column chromatography separation is 60 to 400 mesh, and the volume ratio of the halogenated hydrocarbon to the lower alcohol in the halogenated hydrocarbon-lower alcohol mixed solvent is 6 to 12: 1; In step S4, after dissolving the paeoniflorin-6'-O-benzenesulfonate fine product A with the good solvent at 50 to 60°C, the temperature is lowered to 10 to 20°C and the poor solvent is added. Crystallization; in step S5, the programmed drying includes the steps of pre-freezing, sublimation drying and analytical drying, and the temperatures of the pre-freezing, sublimation drying and analytical drying increase in sequence.
- 如权利要求6所述制备方法,其特征在于,步骤S1-1中,所述吸附包括搅拌吸附、柱层析吸附中的一种或多种,吸附剂包括硅胶、氧化铝中的一种或多种,用纯乙醇进行洗脱,得到所述芍药内酯苷粗品;步骤S1-2中,所述烷烃衍生物-低级醇混合溶剂中烷烃衍生物与低级醇的体积比为3~7:1;步骤S2中, 在室温进行反应,反应时间为2~5h;步骤S4中,析晶后,还包括过滤、漂洗、再过滤过程,得到所述芍药内酯苷-6'-O-苯磺酸酯精品B;步骤S5中,所述预冻的温度为-60~-10℃,所述升华干燥的温度为-10~0℃,所述解析干燥的的温度为5~65℃。The preparation method according to claim 6, characterized in that in step S1-1, the adsorption includes one or more of stirring adsorption and column chromatography adsorption, and the adsorbent includes one or more of silica gel and alumina. A variety of methods are used to elute with pure ethanol to obtain the crude paeoniflorin glycoside; in step S1-2, the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 3 to 7: 1; In step S2, the reaction is carried out at room temperature, and the reaction time is 2 to 5 hours; in step S4, after crystallization, it also includes the process of filtration, rinsing, and re-filtration to obtain the paeoniflorin-6'-O-benzene. Sulfonate ester boutique B; in step S5, the pre-freezing temperature is -60~-10°C, the sublimation drying temperature is -10~0°C, and the analytical drying temperature is 5~65°C.
- 如权利要求6所述制备方法,其特征在于,步骤S1-1中,所述吸附剂与所述药材提取物的质量比为1~4:1,纯乙醇与所述吸附剂的体积质量比为2~6ml:1g;步骤S1-2中,所述硅胶柱色谱分离前,用所述烷烃衍生物-低级醇混合溶剂溶解所述芍药内酯苷粗品,所述烷烃衍生物-低级醇混合溶剂中的烷烃衍生物包括乙酸乙酯、乙酸异丙酯、三氯甲烷、二氯甲烷中的一种或多种,所述烷烃衍生物-低级醇混合溶剂中的低级醇包括甲醇、乙醇、异丙醇中的一种或多种;步骤S3中,所述硅胶柱色谱分离前,用所述卤代烃-低级醇混合溶剂溶解所述芍药内酯苷-6'-O-苯磺酸酯粗品;步骤S4中,用所述不良溶剂进行漂洗;步骤S5中,所述预冻的操作为降温至-60~-10℃并保温30~300min,所述升华干燥的操作为在30~180min内升温至-10~0℃并保温180~720min,所述解析干燥的操作为在30~240min内升温至5~20℃并保温60~600min,接着在30~240min内升温至50~65℃并保温300~1440min。The preparation method according to claim 6, wherein in step S1-1, the mass ratio of the adsorbent to the medicinal material extract is 1 to 4:1, and the volume to mass ratio of pure ethanol to the adsorbent is 2 to 6 ml: 1 g; in step S1-2, before the silica gel column chromatography separation, use the alkane derivative-lower alcohol mixed solvent to dissolve the crude paeoniflorin glycoside, and the alkane derivative-lower alcohol mixed solvent Alkane derivatives in the solvent include one or more of ethyl acetate, isopropyl acetate, chloroform, and methylene chloride, and lower alcohols in the alkane derivative-lower alcohol mixed solvent include methanol, ethanol, One or more isopropyl alcohols; in step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin-6'-O-benzenesulfonic acid The crude ester product; in step S4, rinse with the poor solvent; in step S5, the pre-freezing operation is to cool down to -60~-10°C and keep it warm for 30~300min, and the sublimation drying operation is to Raise the temperature to -10~0°C within 180min and keep it warm for 180~720min. The operation of analytical drying is to raise the temperature to 5~20°C within 30~240min and keep it warm for 60~600min, and then raise the temperature to 50~65°C within 30~240min. ℃ and keep warm for 300~1440min.
- 如权利要求6所述制备方法,其特征在于,步骤S1-2中,所述硅胶柱色谱分离前,用所述烷烃衍生物-低级醇混合溶剂按体积质量比1~3ml:1g溶解所述芍药内酯苷粗品,所述烷烃衍生物-低级醇混合溶剂为乙酸乙酯-甲醇混合溶剂或乙酸乙酯-乙醇混合溶剂,所述烷烃衍生物-低级醇混合溶剂中烷烃衍生物与低级醇的体积比为4~6:1;步骤S3中,所述硅胶柱色谱分离前,用所述卤代烃-低级醇混合溶剂按体积质量比1~3ml:1g溶解所述芍药内酯苷-6'-O-苯磺酸酯粗品;步骤S5中,所述预冻的操作为降温至-40~-10℃并保温90~300min,所述升华干燥的操作为在60~180min内升温至-10~0℃并保温240~600min,所述解析干燥的操作为在60~180min内升温至5~15℃并保温90~540min,接着在60~180min内升温至50~60℃并保温480~1440min。The preparation method according to claim 6, characterized in that, in step S1-2, before the silica gel column chromatography separation, the alkane derivative-lower alcohol mixed solvent is used to dissolve the alkane derivative-lower alcohol mixed solvent at a volume mass ratio of 1 to 3 ml: 1 g. Paeoniflorin crude product, the alkane derivative-lower alcohol mixed solvent is an ethyl acetate-methanol mixed solvent or an ethyl acetate-ethanol mixed solvent, and the alkane derivative and lower alcohol in the alkane derivative-lower alcohol mixed solvent are The volume ratio is 4 to 6:1; in step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin- 6'-O-benzenesulfonate crude product; in step S5, the pre-freezing operation is to cool down to -40~-10°C and keep the temperature for 90~300min, and the sublimation drying operation is to heat up to -10~0°C and kept at 240~600min. The analytical drying operation is to raise the temperature to 5~15°C within 60~180min and keep it at 90~540min, and then raise the temperature to 50~60°C within 60~180min and keep it at 480°C. ~1440min.
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CN103044503A (en) * | 2012-12-24 | 2013-04-17 | 浙江工业大学 | Method for rapidly and efficiently extracting paeoniflorin and albiflorin |
CN103951717A (en) * | 2014-04-11 | 2014-07-30 | 浙江工业大学 | Method for extracting and preparing benzoylpaeoniflorin and benzoyl albiflorin |
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CN102603827A (en) * | 2012-02-10 | 2012-07-25 | 魏伟 | Paeoniflorin aromatic ester derivative, preparation method and applications thereof |
CN103044503A (en) * | 2012-12-24 | 2013-04-17 | 浙江工业大学 | Method for rapidly and efficiently extracting paeoniflorin and albiflorin |
CN103951717A (en) * | 2014-04-11 | 2014-07-30 | 浙江工业大学 | Method for extracting and preparing benzoylpaeoniflorin and benzoyl albiflorin |
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