CN101974005B - Preparation method of high-content sesquiterpenoids tripterygium alkaloid - Google Patents
Preparation method of high-content sesquiterpenoids tripterygium alkaloid Download PDFInfo
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- CN101974005B CN101974005B CN2010102694201A CN201010269420A CN101974005B CN 101974005 B CN101974005 B CN 101974005B CN 2010102694201 A CN2010102694201 A CN 2010102694201A CN 201010269420 A CN201010269420 A CN 201010269420A CN 101974005 B CN101974005 B CN 101974005B
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Abstract
The invention discloses a method of separately preparing high-content sesquiterpenoids tripterygium alkaloid monomers by using semi-preparation chromatography, comprising the following steps of: with tripterygium ethyl acetate extract waste liquid subjected to the removal of triptolide and tripdiolide as a raw material, leaching by chloralkane; recrystallizing by using lower alcohol as a solvent; next, preparing the high-content sesquiterpenoids tripterygium alkaloid monomers by adopting inverse high-efficiency liquid phase semi-preparation chromatography; adopting on-line ultraviolet detection; collecting objectives; and obtaining four sesquiterpenoids tripterygium alkaloid monomers (wilfortrine, wilfordine, tripterygiumgyrine and wilforine) with content which is greater than 99.0 percent after freezing and drying. Due to the adoption of the disclosed preparation method, the separated product has the advantages of high purity, short process, low cost and easy industrialization.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of preparation method of high-load sesquiterpenoids trypterygine alkaloid monomer.
Background technology
Trypterygine (Tripterygium wilfordii Hook.f.) is the Celastraceae tripterygium plant; Be used as medicine with root, leaf, flower and fruit;,, significant curative effect is all arranged like rheumatoid arthritis, chronic nephritis, lupus erythematosus, contact dermatitis etc. to the treatment autoimmune disorder.According to " middle traditional Chinese medicines are planted will " record; Wilforine, wilfordine and diterpenoid-lactone, galactitol all have tangible anti-tumor activity; Mouse leukemia and human nasopharyngeal carcinoma all there are restraining effect, can make the blood flow increasing of arteriole diastole on every side, lower the resistance of peripheral blood flow; The obstruction of microcirculation improvement, the effect of invigorating blood circulation.Multiple abnormalism reaction there is the hormonelike anti-inflammatory action with non-bacteria inflammation.
The trypterygine complex chemical composition, pharmacological action is extensive, and important clinical application value is arranged.But, limited the popularization of clinical application because the trypterygine toxic side effect is big.Should further strengthen active ingredients of thunder god vine is efficiently extracted, is purified into to trypterygine pharmaceutical chemistry, pharmacological action and Study of Clinical Application, for the further development and use research of the quality monitoring of the trypterygine prepared slices of Chinese crude drugs and trypterygine Chinese medicinal materials lays the foundation.
The trypterygine vegeto-alkali is one of main active ingredient of trypterygine; It is the main object that people study always; Wherein the higher relatively trypterygine vegeto-alkali of content particularly, as: wilfortrine (wilfortrine), wilfordine (wilfordine), Wilforgine 20 (wilforgine) and Wilforine (wilforine) etc.The preparation of trypterygine alkaloid monomer at present mainly adopt liquid/liquid extraction method column chromatography chromatography (Lin Sui, Li Yuanchao, Sakurai letter, Guo Shunmin, Deng Fuxiao. the research of trypterygine sesquiterpene alkaloids, Botany Gazette, 2001,43 (6): 647-649; Chen Junyuan, Xia Zhilin, Deng Fuxiao. the improvement of trypterygine alkaloid extracting technique; Chinese Journal of Pharmaceuticals; 1989,20 (11): 484-485) and high-speed countercurrent chromatography (High-speed countercurrent chromatography, HSCCC) (CN100422188C) etc.; Main employing trypterygine rhizome is a raw material; The trypterygine alkaloid monomer content that obtains generally hangs down about 95% or more, and its purity is difficult to satisfy the requirement of the pharmaceutical reference substance of trypterygine alkaloid monomer, also is unfavorable for the further research of trypterygine sesquiterpenoids vegeto-alkali pharmacology.
Summary of the invention
The objective of the invention is to deficiency, a kind of half preparative hplc separation purification method of high-load trypterygine alkaloid monomer is provided to existing trypterygine alkaloid monomer technology of preparing.
The preparing method's of high-content sesquiterpenoids trypterygine alkaloid monomer concrete steps are following:
1) will remove trypterygine vinyl acetic monomer medicinal extract waste liquid behind triptolide and the Triptodiolide; Easy volatile organic acid with 5～15g/L; Or the easy volatile alkali regulator solution pH value of 5～20g/L carries out recrystallization to 6-10.5, and the solvent of recrystallization is a lower alcohol; The temperature of recrystallization is 0～40 ℃, obtains the thick total alkaloids of trypterygine;
2) with the thick total alkaloids of trypterygine; Using volume ratio is after 55: 45～99: 1 acetonitrile dissolves with water mixed solvent; Using volume ratio is that 55: 45～99: 1 acetonitrile and water mixed solvent is moving phase; With the flow velocity of 0.2～2.0mL/min, in 0～40 ℃ of TR, on C18 anti-phase half preparative hplc post, realize separating of wilfortrine, wilfordine, Wilforgine 20 and Wilforine; Detect through on-line ultraviolet, obtain wilfortrine, wilfordine, Wilforgine 20 and Wilforine.
Described wilfortrine, wilfordine, Wilforgine 20 and Wilforine are the sesquiterpenoids vegeto-alkali, the alkaloidal molecular structural formula of sesquiterpenoids:
Ac＝acetate，Bz＝benzoyl，Fur＝furanoyl。
Described C18 chromatographic column is common C18 post.Described C18 chromatographic column is preferably Hypersil C18 chromatographic column.
Described lower alcohol is methyl alcohol, ethanol or Virahol.Described lower alcohol is preferably methyl alcohol.The ratio of described acetonitrile and water is 75: 25, and described flow rate of mobile phase is 1.0mL/min, and described column temperature is 30 ℃.Described easy volatile organic acid is acetic acid, hydrochloric acid or formic acid.Described easy volatile organic acid is preferably acetic acid, and acetate concentration is 10g/L.Described easy volatile alkali is ammoniacal liquor, and the concentration of ammoniacal liquor is 10g/L.
The used raw material of the present invention is the trypterygine vinyl acetic monomer medicinal extract waste liquid that removes triptolide and Triptodiolide, and the cost of material cost is low, belongs to utilization of waste material.Used various chemical reagent is general, be prone to purchase.Chromatographic separation and collect and can realize robotization, easy and simple to handle, the wilfortrine of acquisition, wilfordine, Wilforgine 20 and Wilforine product content high (＞99.0%) are applicable to half preparative chromatograph of various models.
Below in conjunction with embodiment and accompanying drawing the present invention is done further explanation, but be not limited to the scope of present embodiment.
Description of drawings
Fig. 1 is that trypterygine vegeto-alkali half preparative hplc of embodiment 1 flows out the ultraviolet color atlas; Moving phase be acetonitrile/water=(60: 40, v/v) HPLC figure; Among the figure, peak 1,2,3,4 is respectively wilfortrine, wilfordine, Wilforgine 20 and Wilforine;
Fig. 2 is that trypterygine vegeto-alkali half preparative hplc of embodiment 3 flows out the ultraviolet color atlas; Moving phase be acetonitrile/water=(75: 25, v/v) HPLC figure; Among the figure, peak 1,2,3,4 is respectively wilfortrine, wilfordine, Wilforgine 20 and Wilforine;
Fig. 3 (a) is the one-level mass spectrum of the trypterygine vegeto-alkali wilfortrine under the positive ion mode of APCI atmospheric pressure chemical ionization source (APCI) that obtains with the direct injection mode that obtained by embodiment 3;
Fig. 3 (b) is the one-level mass spectrum of the trypterygine vegeto-alkali wilfordine under the positive ion mode of APCI atmospheric pressure chemical ionization source (APCI) that obtains with the direct injection mode that obtained by embodiment 3;
Fig. 3 (c) is the one-level mass spectrum of the trypterygine vegeto-alkali Wilforgine 20 under the positive ion mode of APCI atmospheric pressure chemical ionization source (APCI) that obtains with the direct injection mode that obtained by embodiment 3;
Fig. 3 (d) is the one-level mass spectrum of the trypterygine vegeto-alkali Wilforine under the positive ion mode of APCI atmospheric pressure chemical ionization source (APCI) that obtains with the direct injection mode that obtained by embodiment 3.
Embodiment
Embodiment 1:
Get 5.0g oneself remove the trypterygine vinyl acetic monomer medicinal extract waste liquid of triptolide and Triptodiolide, volatilize solvent after, take by weighing 2.0g in beaker, use the 10mL chloroform extraction at every turn, repeat 3 times, the combined chloroform layer obtains chloroform medicinal extract after volatilizing; Chloroform medicinal extract merges acetic acid extraction liquid, again with 10g/L ammoniacal liquor adjusting pH to 10 with the acetic acid extraction of 10g/L 3 times then; Cross the filter cake thing with deionized water wash 5 times; Oven dry obtains thick total alkaloids, adds the dissolve with methanol of 30.0mL then; At room temperature carry out recrystallization, the raw material that obtains with recrystallization carries out chromatographic separation.
The thick total alkaloids of trypterygine that recrystallization is obtained; Using volume ratio is after 60: 40 acetonitrile dissolves with water mixed solvent, and using volume ratio is that 60: 40 acetonitrile and water mixture is moving phase, with the flow velocity of 1.0mL/min; When 30 ℃ of temperature; On Hypersil C18 anti-phase semipreparative column, separate, adopt online ultraviolet detection, the detection wavelength is 230nm; Collect the corresponding compound of each chromatographic peak respectively, get wilfortrine, wilfordine, Wilforgine 20 and 4 kinds of sesquiterpenoids trypterygines of Wilforine vegeto-alkali after the vacuum-drying.
Embodiment 2:
Get 5.0g oneself remove the trypterygine vinyl acetic monomer medicinal extract waste liquid of triptolide and Triptodiolide, volatilize solvent after, take by weighing 2.0g in beaker, use the 10mL chloroform extraction at every turn, repeat 3 times, the combined chloroform layer obtains chloroform medicinal extract after volatilizing; Chloroform medicinal extract merges formic acid extraction liquid, again with 15g/L formic acid ammoniacal liquor adjusting pH to 6 with the formic acid extraction of 10g/L 3 times then; Cross the filter cake thing with deionized water wash 5 times; Oven dry obtains thick total alkaloids, adds the Virahol dissolving of 20.0mL then; At room temperature carry out recrystallization, the raw material that obtains with recrystallization carries out chromatographic separation.
The thick total alkaloids of trypterygine that recrystallization is obtained; Using volume ratio is after 99: 1 acetonitrile dissolves with water mixed solvent, and using volume ratio is that 99: 1 acetonitrile and water mixture is moving phase, with the flow velocity of 0.2mL/min; When 40 ℃ of temperature; On Hypersil C18 anti-phase semipreparative column, separate, adopt online ultraviolet detection, the detection wavelength is 230nm; Collect the corresponding compound of each chromatographic peak respectively, get wilfortrine, wilfordine, Wilforgine 20 and 4 kinds of sesquiterpenoids trypterygines of Wilforine vegeto-alkali after the vacuum-drying.
Embodiment 3:
Get 10.0g oneself remove the trypterygine vinyl acetic monomer medicinal extract waste liquid of triptolide and Triptodiolide, volatilize solvent after, take by weighing 2.0g in beaker, use the 10mL chloroform extraction at every turn, repeat 3 times, the combined chloroform layer obtains chloroform medicinal extract after volatilizing; Chloroform medicinal extract merges acetic acid extraction liquid, again with 8g/L ammoniacal liquor adjusting pH to 10 with the acetic acid extraction of 10g/L 3 times then; Cross the filter cake thing with deionized water wash 5 times; Oven dry obtains thick total alkaloids, adds the dissolve with methanol of 10.0～50.0mL then; At room temperature carry out recrystallization, the raw material that obtains with recrystallization carries out chromatographic separation.
The thick total alkaloids of trypterygine that recrystallization is obtained; Using volume ratio is after 75: 25 acetonitrile dissolves with water mixed solvent, and using volume ratio is that 75: 25 acetonitrile and water mixture is moving phase, with the flow velocity of 1.0mL/min; When 25 ℃ of temperature; On Hypersil C18 anti-phase semipreparative column, separate, adopt online ultraviolet detection, the detection wavelength is 230nm; Collect the corresponding compound of each chromatographic peak respectively, get wilfortrine, wilfordine, Wilforgine 20 and 4 kinds of sesquiterpenoids trypterygines of Wilforine vegeto-alkali after the vacuum-drying.
Embodiment 4:
Get 5.0g oneself remove the trypterygine vinyl acetic monomer medicinal extract waste liquid of triptolide and Triptodiolide, volatilize solvent after, take by weighing 2.0g in beaker, use the 10mL chloroform extraction at every turn, repeat 3 times, the combined chloroform layer obtains chloroform medicinal extract after volatilizing; Chloroform medicinal extract merges hydrochloric acid extraction liquid with the hydrochloric acid extraction of 10g/L 3 times then, again with 5g/L hydrochloric acid adjusting pH to 6; Cross the filter cake thing with deionized water wash 5 times; Oven dry obtains thick total alkaloids, adds the dissolve with ethanol of 20.0mL then; At room temperature carry out recrystallization, the raw material that obtains with recrystallization carries out chromatographic separation.
The thick total alkaloids of trypterygine that recrystallization is obtained; Using volume ratio is after 55: 45 acetonitrile dissolves with water mixed solvent, and using volume ratio is that 55: 45 acetonitrile and water mixture is moving phase, with the flow velocity of 2.0mL/min; When 0 ℃ of temperature; On Hypersil C18 anti-phase semipreparative column, separate, adopt online ultraviolet detection, the detection wavelength is 230nm; Collect the corresponding compound of each chromatographic peak respectively, get wilfortrine, wilfordine, Wilforgine 20 and 4 kinds of sesquiterpenoids trypterygines of Wilforine vegeto-alkali after the vacuum-drying.
Embodiment 5:
Get 10.0g oneself remove the trypterygine vinyl acetic monomer medicinal extract waste liquid of triptolide and Triptodiolide, volatilize solvent after, take by weighing 2.0g in beaker, use the 10mL chloroform extraction at every turn, repeat 3 times, the combined chloroform layer obtains chloroform medicinal extract after volatilizing; Chloroform medicinal extract merges acetic acid extraction liquid, again with 12g/L ammoniacal liquor adjusting pH to 10.5 with the acetic acid extraction of 15g/L 3 times then; Cross the filter cake thing with deionized water wash 5 times; Oven dry obtains thick total alkaloids, adds a certain amount of dissolve with methanol then; At room temperature carry out recrystallization, the raw material that obtains with recrystallization carries out chromatographic separation.
The thick total alkaloids of trypterygine that recrystallization is obtained; Using volume ratio is after 75: 25 acetonitrile dissolves with water mixed solvent, and using volume ratio is that 75: 25 acetonitrile and water mixture is moving phase, with the flow velocity of 1.0mL/min; When 30 ℃ of temperature; On Hypersil C18 anti-phase semipreparative column, separate, adopt online ultraviolet detection, the detection wavelength is 230nm; Collect the corresponding compound of each chromatographic peak respectively, get wilfortrine, wilfordine, Wilforgine 20 and 4 kinds of sesquiterpenoids trypterygines of Wilforine vegeto-alkali after the vacuum-drying.
The foregoing description is used for the present invention that explains, rather than limits the invention, and in the protection domain of spirit of the present invention and claim, to any modification and the change that the present invention makes, all is included into protection scope of the present invention.
Embodiment 6:
Get 5.0g oneself remove the trypterygine vinyl acetic monomer medicinal extract waste liquid of triptolide and Triptodiolide, volatilize solvent after, take by weighing 2.0g in beaker, use the 10mL chloroform extraction at every turn, repeat 3 times, the combined chloroform layer obtains chloroform medicinal extract after volatilizing; Chloroform medicinal extract merges formic acid extraction liquid, again with 20g/L ammoniacal liquor adjusting pH to 10 with the formic acid extraction of 10g/L 3 times then; Cross the filter cake thing with deionized water wash 5 times; Oven dry obtains thick total alkaloids, adds the dissolve with ethanol of 20.0mL then; At room temperature carry out recrystallization, the raw material that obtains with recrystallization carries out chromatographic separation.
The thick total alkaloids of trypterygine that recrystallization is obtained; Using volume ratio is after 55: 45 acetonitrile dissolves with water mixed solvent, and using volume ratio is that 55: 45 acetonitrile and water mixture is moving phase, with the flow velocity of 0.2mL/min; When 30 ℃ of temperature; On Hypersil C18 anti-phase semipreparative column, separate, adopt online ultraviolet detection, the detection wavelength is 230nm; Collect the corresponding compound of each chromatographic peak respectively, get wilfortrine, wilfordine, Wilforgine 20 and 4 kinds of sesquiterpenoids trypterygines of Wilforine vegeto-alkali after the vacuum-drying.
Claims (3)
1. alkaloidal preparation method of high-content sesquiterpenoids trypterygine is characterized in that concrete steps are following:
1) will remove trypterygine vinyl acetic monomer medicinal extract waste liquid behind triptolide and the Triptodiolide; Easy volatile organic acid with 5～15g/L; Or the easy volatile alkali regulator solution pH value of 5～20g/L carries out recrystallization to 6-10.5, and the solvent of recrystallization is a lower alcohol; The temperature of recrystallization is 0～40 ℃, obtains the thick total alkaloids of trypterygine;
2) with the thick total alkaloids of trypterygine; Using volume ratio is after 55: 45～99: 1 acetonitrile dissolves with water mixed solvent; Using volume ratio is that 55: 45～99: 1 acetonitrile and water mixed solvent is moving phase; With the flow velocity of 0.2～2.0mL/min, in 0～40 ℃ of TR, on C18 anti-phase half preparative hplc post, realize separating of wilfortrine, wilfordine, Wilforgine 20 and Wilforine; Detect through on-line ultraviolet, obtain wilfortrine, wilfordine, Wilforgine 20 and Wilforine;
Described lower alcohol is methyl alcohol, ethanol or Virahol, and the easy volatile organic acid is acetic acid, hydrochloric acid or formic acid, and easy volatile alkali is ammoniacal liquor, and the concentration of ammoniacal liquor is 10g/L; The molecular structural formula of described wilfortrine, wilfordine, Wilforgine 20 and Wilforine is:
The wilfortrine Wilforine
The wilfordine Wilforgine 20
Wherein: Ac representative
is an ethanoyl.
2. the alkaloidal preparation method of a kind of high-content sesquiterpenoids trypterygine according to claim 1 is characterized in that described C18 chromatographic column is a Hypersil C18 chromatographic column.
3. the alkaloidal preparation method of a kind of high-content sesquiterpenoids trypterygine according to claim 1, the ratio that it is characterized in that described acetonitrile and water is 75: 25, and described flow rate of mobile phase is 1.0mL/min, and described column temperature is 30 ℃.
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| CN104817565B (en) * | 2015-03-17 | 2017-04-26 | 宁波市疾病预防控制中心 | Method for efficient extraction of tripterygium wilfordii alkaloids by ionic liquid |
| CN112250624A (en) * | 2020-11-06 | 2021-01-22 | 重庆市中药研究院 | Nicotinic acid derivative VII with anti-inflammatory, anti-platelet aggregation and immunosuppressive activities and application thereof |
| CN112225694A (en) * | 2020-11-06 | 2021-01-15 | 重庆市中药研究院 | Nicotinic acid derivative I with anti-inflammatory, immunosuppression and platelet aggregation resistance and application thereof |
| CN112225693A (en) * | 2020-11-06 | 2021-01-15 | 重庆市中药研究院 | Nicotinic acid derivative IV with immunosuppression, platelet aggregation inhibition and blood glucose reduction activities and application thereof |
| CN112194619A (en) * | 2020-11-09 | 2021-01-08 | 重庆市中药研究院 | Isonicotinic acid derivative VIII with platelet aggregation resisting, anti-tumor and immunosuppressive activities and application thereof |
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| CN1911932A (en) * | 2006-08-25 | 2007-02-14 | 浙江大学 | Method for separating preparing tripterygium wilfordii monomer from tripterygium wilfordii by countercurrent flow chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1911932A (en) * | 2006-08-25 | 2007-02-14 | 浙江大学 | Method for separating preparing tripterygium wilfordii monomer from tripterygium wilfordii by countercurrent flow chromatography |
Non-Patent Citations (2)
| Title |
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| 黄文华 等.雷公藤药材中 个生物碱成分的含量测定及其质量评价.《第五届全国雷公藤学术会议论文汇编》.2008,133-138. |
| 黄文华 等.雷公藤药材中 个生物碱成分的含量测定及其质量评价.《第五届全国雷公藤学术会议论文汇编》.2008,133-138. * |
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