CN102627641A - Method for extracting multiple alkaloids from rauvolfia vomitoria - Google Patents
Method for extracting multiple alkaloids from rauvolfia vomitoria Download PDFInfo
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Abstract
The invention discloses a method for extracting multiple alkaloids from rauvolfia vomitoria. The method comprises the following steps of: adding acid-containing ethanol aqueous solution into root powder of the rauvolfia vomitoria, stirring and filtering to obtain leach liquor; concentrating the leach liquor, adsorbing with non-polar macroporous adsorption resin, washing and eluting with 40 percent ethanol and 80 percent ethanol; collecting eluate respectively, concentrating and then adsorbing with weakly acidic cation exchange resin, washing and eluting with 50 percent ethanol; collecting eluate respectively, adjusting pH to be 3 by using concentrated acid, concentrating and then adsorbing with a C18 reversed phase chromatography medium and eluting with the ethanol aqueous solution with gradually increasing concentration; and collecting the eluate in stages respectively, combining the parts with higher single alkaloid purity in the eluate respectively, concentrating to obtain solid, adding an organic solvent to crystallize to obtain crystals of yohimbine, ajmaline, reserpoid, reserpine and rescinnamine. According to the method, five alkaloids can be extracted from a plant at most at one time; and the method has the advantages of few steps, less dosage of the used organic solvent, low pollution and simple required equipment.
Description
Technical field
What the present invention relates to is a kind of medicine separation method of medical technical field, specifically is from plant Rauvolfia vomitoria Afzel. (Rauwolfia vomitoria Afz.), to extract Yohimbine (yohimbine), rauwolfine (ajmaline), Serpophite (serpentine), serpentine (reserpine), sharp new amine 5 kinds of alkaloidal methods such as (rescinnamine).
Background technology
Radix Rauvolfiae is an Apocynaceae Rauwolfia plant, contains multiple effective component.Rauvolfia vomitoria Afzel. is a kind of of Radix Rauvolfiae, introduces from Africa, wherein contains multiple vegeto-alkalis such as serpentine, rauwolfine, Yohimbine, Serpophite, and these vegeto-alkalis can be used as medicine, have clinically multiple treatment of diseases effect.
Through the prior art literature search is found, from Radix Rauvolfiae (comprising Rauvolfia vomitoria Afzel.), extract effective component, report more is the extraction serpentine, adopts benzene to extract in early days, uses macroporous resin extraction later on.From Rauvolfia vomitoria Afzel., extract the report of several kinds of components simultaneously, the research of extracting serpentine and Yohimbine is arranged, but the Yohimbine purity that extracts lower (Liu Yu, Feng Lei, Zhao Fengsheng, medicine biotechnology, 2008,15 (5): 393-397).From Rauvolfia vomitoria Afzel., extract the report of serpentine, Reserpilline, the new alkali of Radix Rauvolfiae in addition simultaneously; But extraction step is more, and spended time is long, and (Zhou Xueqing, Feng Yuhong, Zhang Chong, Chen Sili, woods are strong, University Of Hainan's journal natural science edition; 2008,26 (2): 145-148).
Summary of the invention
The present invention is directed to the above-mentioned deficiency that prior art exists; A kind of 5 kinds of alkaloidal methods of from Rauvolfia vomitoria Afzel., once can extracting at most are provided; Combine with technology such as acidic ethanol lixiviate, macroporous resin adsorption, weakly acidic cation-exchange resin extraction, reversed phase chromatography separation, the vegeto-alkali that extracts all has higher purity.
The present invention realizes through following technical scheme:
The present invention provides a kind of multiple alkaloidal method of from Rauvolfia vomitoria Afzel., extracting, and promptly extracts Yohimbine, rauwolfine, Serpophite, serpentine, sharp new amine simultaneously; Specifically comprise the steps:
(1) get the Rauvolfia vomitoria Afzel. root and pulverize the powder that forms, add acidiferous aqueous ethanolic solution, stirring, lixiviate, filtration obtain vat liquor, and program repeats 2-3 time like this;
(2) vat liquor with gained merges, and the decompression heating concentrates, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin; Use water washing resin then, use 40% ethanol elution subsequently, collect elutriant (being designated as A1); Use 80% ethanol elution again, collect elutriant (being designated as B1);
(3) two portions elutriant A1 of above-mentioned collection and the B1 heating of reducing pressure is respectively concentrated, the elutriant A1 after concentrating adsorbs with weakly acidic cation-exchange resin, uses water washing resin then, uses 50% ethanol elution subsequently, collects elutriant (being designated as A2); Elutriant B1 after concentrating adsorbs with weakly acidic cation-exchange resin, uses water washing resin then, uses 50% ethanol elution subsequently, collects elutriant (being designated as B2);
(4) two portions elutriant A2 and the B2 with above-mentioned collection is adjusted to pH about 3 with concentrated acid respectively, and the decompression heating concentrates, and the elutriant A2 after concentrating uses C
18The absorption of reversed phase chromatography medium is used the aqueous ethanolic solution wash-out, Fractional Collections elutriant (being designated as A3) then; Elutriant B2 after concentrating uses C
18The absorption of reversed phase chromatography medium is used the aqueous ethanolic solution wash-out, Fractional Collections elutriant (being designated as B3) then;
(5) elutriant A3 and the B3 with above-mentioned Fractional Collections measures wherein various alkaloids content with high performance liquid chromatograph, merges respectively containing the higher part of Yohimbine or rauwolfine and purity among the elutriant A3; Merge respectively containing Serpophite or serpentine or sharp new amine and the higher part of purity among the elutriant B3; The decompression heating concentrates respectively, to the liquid evaporate to dryness, adds the organic solvent crystallization, obtains the crystal of Yohimbine, rauwolfine, Serpophite, serpentine, sharp new amine.
Acidiferous aqueous ethanolic solution described in the step (1) contains the water (volume percent) of 10%-60%, contains acid simultaneously, and the concentration of acid is 0.02-0.2mol/L, and acid can be sulfuric acid, hydrochloric acid or acetate;
Described lixiviate, the volume mL of each acidiferous aqueous ethanolic solution that adds is 3-10 a times of Rauvolfia vomitoria Afzel. root powder dry weight g.
In the step (2), with the vat liquor merging of gained, the decompression heating concentrates; With nonpolar macroporous adsorption resin absorption, the volume mL of nonpolar macroporous adsorption resin is the 40%-50% of Rauvolfia vomitoria Afzel. root powder dry weight g to vat liquor after concentrating, uses water washing resin then at normal temperatures; The volume of water is 1-5 a times of nonpolar macroporous adsorption resin volume, uses 40% ethanol elution subsequently, and 40% alcoholic acid volume is 2-5 a times of nonpolar macroporous adsorption resin volume; Collect elutriant A1; Use 80% ethanol elution again, 80% alcoholic acid volume is 2-5 a times of nonpolar macroporous adsorption resin volume, collects elutriant B1;
The described water washing resin of using is to feed in the post with the import from post of deionized water or pure water, flows out from the outlet of post again, and washing time is 0.5-3h;
Described 40% ethanol wherein contains volume percent and is 59% water and volume percent and be 1% hydrochloric acid;
Described 80% ethanol wherein contains volume percent and is 19% water and volume percent and be 1% hydrochloric acid.
In the step (3); Two portions elutriant A1 that collects and the B1 heating of reducing pressure respectively is concentrated; At normal temperatures with weakly acidic cation-exchange resin absorption, the volume mL that adsorbs the used weakly acidic cation-exchange resin of each part elutriant is the 20%-30% of Rauvolfia vomitoria Afzel. root powder dry weight g, uses water washing resin then respectively for elutriant A1 after concentrating and B1; The volume of water is 1-5 a times of weakly acidic cation-exchange resin volume; Use 50% ethanol elution subsequently, 50% alcoholic acid volume is 3-5 a times of weakly acidic cation-exchange resin volume, collects elutriant A2 and B2;
The described water washing resin of using is to feed in the post with the import from post of deionized water or pure water, flows out from the outlet of post again, and washing time is 0.5-2h;
Described 50% ethanol wherein contains volume percent and is 45% water and volume percent and be 5% ammoniacal liquor.
In the step (4), the elutriant A2 and the B2 that collect are adjusted to pH about 3 with concentrated acid respectively, the decompression heating concentrates; Elutriant A2 after concentrating uses C at normal temperatures
18The absorption of reversed phase chromatography medium, C
18The volume mL of reversed phase chromatography medium is the 10%-20% of Rauvolfia vomitoria Afzel. root powder dry weight g, uses 5%, 10%, 15%, 20%, 95% aqueous ethanolic solution wash-out then successively, and the volume summation of aqueous ethanolic solution is C
18The 10-30 of reversed phase chromatography medium volume times, Fractional Collections 10-20 part elutriant A3; Elutriant B2 after concentrating uses C at normal temperatures
18The absorption of reversed phase chromatography medium, C
18The volume mL of reversed phase chromatography medium is the 10%-20% of Rauvolfia vomitoria Afzel. root powder dry weight g, uses 15%, 20%, 25%, 30%, 35%, 40%, 55%, 95% aqueous ethanolic solution wash-out then successively, and the volume summation of aqueous ethanolic solution is C
18The 10-30 of reversed phase chromatography medium volume times, Fractional Collections 10-20 part elutriant B3;
Described concentrated acid is the vitriol oil or concentrated hydrochloric acid;
Described Fractional Collections elutriant is that elution time or elution volume are divided into plurality of sections, and the elutriant in each section elution time or the elution volume is collected as 1 part.
Adding organic solvent crystallization described in the step (5) for the Serpophite among the Yohimbine among the elutriant A3, rauwolfine or the elutriant B3, adds methyl alcohol, stirs to make the solid dissolving; The methyl alcohol add-on can make solid all dissolve, but unsuitable excessive, adds ether again; The adding volume is 2-5 a times of methyl alcohol volume, stirs 1-3h, places 10-20h in 0-5 ℃; Separate out crystallization, filter or centrifugal removal mother liquor, obtain crystallization; For serpentine among the elutriant B3 or sharp new amine, add ETHYLE ACETATE, stir and make the solid dissolving; The ETHYLE ACETATE add-on can make solid all dissolve, but unsuitable excessive, adds methyl alcohol again; The adding volume is 2-5 a times of ETHYLE ACETATE volume, stirs 1-3h, places 10-20h in 0-5 ℃; Separate out crystallization, filter or centrifugal removal mother liquor, obtain crystallization; Put into 40-50 ℃ of baking oven by above step gained crystal and dry by the fire 5-10h, till constant weight;
In the step (5); The elutriant A3 of Fractional Collections and B3 measure wherein various alkaloids content with high performance liquid chromatograph; If certain alkaloidal purity is not high enough, need to purify once more, can be with containing the alkaloidal elutriant merging that desire is purified once more among elutriant A3 or the B3; As A2 or B2, operate once again according to step (4).
It all is under temperature 45-65 ℃, the condition of vacuum tightness 0.08-0.10M Pa, to carry out that above-described decompression heating concentrates, remove specify, generally with about half of liquid concentration to volume originally.
In the aforesaid method of the present invention, can only get 40% ethanol eluate A1 and carry out respective operations in said step (3), (4), (5), obtain A2, A3 successively, and final vegeto-alkali Yohimbine, rauwolfine; Also can only get 80% ethanol eluate B1 and carry out respective operations in the said step (3), (4), (5), obtain B2, B3 successively, and final vegeto-alkali Serpophite, serpentine, sharp new amine.
Compared with prior art; The present invention combines technology such as acidic ethanol lixiviate, macroporous resin adsorption, weakly acidic cation-exchange resin extraction, reversed phase chromatography separation; Extract Yohimbine, rauwolfine, Serpophite, serpentine, sharp new 5 kinds of vegeto-alkalis of amine simultaneously, the vegeto-alkali that extracts all has higher purity.
Embodiment
Elaborate in the face of embodiments of the invention down: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.Do not have the operation of detailed description in following examples, can adopt routine operation of the prior art.
Embodiment 1
Get exsiccant Rauvolfia vomitoria Afzel. root powder 2500g, add and contain the 50% ethanolic soln lixiviate of 0.025mol/L vitriolic, lixiviate is 2 times altogether; The liquid volume that twice lixiviate adds is respectively 25000mL, 12500mL; Each extraction time is 2h, extraction time have a rest stirrings, lixiviate after-filtration at every turn.
2 portions of vat liquors are merged, and volume is 31000mL, and decompression heating simmer down to 16000mL filters.The 1200mL HZ-818 nonpolar macroporous adsorption resin (East China University of Science China shake Science and Technology Ltd. system) that filtrating feeding is contained in the post adsorbs co-adsorption 20h.Feed in the resin column with the 1500mL deionized water then and wash 1.5h.With carrying out wash-out, elution time 4h in 2400mL 40% ethanolic soln (containing 1% hydrochloric acid) the feeding resin column.
Collect elutriant, the decompression heating is concentrated into 840mL.Liquid concentrator feeds the 700mL D152 weakly acidic cation-exchange resin (Chemical Plant of Nankai Univ.'s system) that is contained in the post and adsorbs co-adsorption 4h.Feed in the resin column with the 800mL deionized water then and wash 1.5h.With carrying out wash-out, elution time 4h in 2100mL 50% ethanolic soln (containing 5% ammoniacal liquor) the feeding resin column.
Collect elutriant, using 10mol/L sulfuric acid to regulate pH is 3, and the decompression heating is concentrated into 1100mL.Liquid concentrator feeds the 500mL C that is contained in the post
18Reversed phase chromatography medium (Japanese YMC corporate system) adsorbs co-adsorption 2h.Use 2000mL 5% ethanolic soln, 4000mL 10% ethanolic soln, 1000mL 15% ethanolic soln, 1000mL 20% ethanolic soln, 500mL 95% ethanolic soln (all containing 0.01% trifluoroacetic acid) to feed in the post then successively and carry out wash-out, elution time 15h.Elutriant is divided into 13 parts of collections.
The elutriant of Fractional Collections is measured through high performance liquid chromatograph, and wherein Yohimbine content is higher in 2 of 5% ethanolic soln wash-out parts of elutriants, merges volume 1000mL.The decompression heating is concentrated into dried, adds 10mL dissolve with methanol solid, adds the 40mL ether again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the Yohimbine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get Yohimbine 14mg, purity 52%.
Rauwolfine content is higher in 3 parts of elutriants of 10% ethanolic soln wash-out, merges volume 2700mL.The decompression heating is concentrated into dried, adds 15mL dissolve with methanol solid, adds the 50mL ether again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the rauwolfine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get rauwolfine 406mg, purity 63%.
Embodiment 2
Rauvolfia vomitoria Afzel. lixiviate, non-polar macroporous resin absorption are identical with embodiment 1 with wash-out, weakly acidic cation-exchange resin absorption and wash-out, crystallization and exsiccant implementation step, and difference is:
C
18After the higher part of Yohimbine content merges in the elutriant that reversed phase chromatography is collected, volume 1000mL, the decompression heating is concentrated into 700mL, feeds the 500mL C that is contained in the post again
18The reversed phase chromatography medium adsorbs, co-adsorption 2h.Use 2000mL 5% ethanolic soln, 2000mL 10% ethanolic soln, 500mL 15% ethanolic soln, 500mL 20% ethanolic soln, 500mL 95% ethanolic soln (all containing 0.01% trifluoroacetic acid) to feed in the post then successively and carry out wash-out, elution time 10h.Elutriant is divided into 10 parts of collections.
Measure through high performance liquid chromatograph, wherein Yohimbine content is higher in 2 of 5% ethanolic soln wash-out parts of elutriants, merges volume 800mL.Crystallization gets Yohimbine 6mg, purity 87%.
C
18After the higher part of rauwolfine content merges in the elutriant that reversed phase chromatography is collected, volume 2700mL, the decompression heating is concentrated into 1500mL, feeds the 500mL C that is contained in the post again
18The reversed phase chromatography medium adsorbs, co-adsorption 2.5h.Use 2000mL 5% ethanolic soln, 2000mL 10% ethanolic soln, 500mL 15% ethanolic soln, 500mL 20% ethanolic soln, 500mL 95% ethanolic soln (all containing 0.01% trifluoroacetic acid) to feed in the post then successively and carry out wash-out, elution time 10h.Elutriant is divided into 10 parts of collections.
Measure through high performance liquid chromatograph, wherein rauwolfine content is higher in 2 of 10% ethanolic soln wash-out parts of elutriants, merges volume 700mL.Crystallization gets rauwolfine 230mg, purity 90%.
Embodiment 3
Get exsiccant Rauvolfia vomitoria Afzel. root powder 2500g, add and contain the 50% ethanolic soln lixiviate of 0.025mol/L vitriolic, lixiviate is 2 times altogether; The liquid volume that twice lixiviate adds is respectively 25000mL, 12500mL; Each extraction time is 2h, extraction time have a rest stirrings, lixiviate after-filtration at every turn.
2 portions of vat liquors are merged, and volume is 30000mL, and decompression heating simmer down to 15000mL filters.The 1200mL HZ-818 nonpolar macroporous adsorption resin that filtrating feeding is contained in the post adsorbs co-adsorption 18h.Feed in the resin column with the 1500mL deionized water then and wash 1.5h.With carrying out wash-out, elution time 4h in 2500mL 40% ethanolic soln (containing 1% hydrochloric acid) the feeding resin column.Use 3000mL 80% ethanolic soln (containing 1% hydrochloric acid) to feed in the resin column again and carry out wash-out, elution time 6h.
Collect 80% ethanolic soln elutriant, the decompression heating is concentrated into 680mL.Liquid concentrator feeds the 600mLD152 weakly acidic cation-exchange resin that is contained in the post and adsorbs co-adsorption 4h.Feed in the resin column with the 800mL deionized water then and wash 1.5h.With carrying out wash-out, elution time 6h in 3000mL 50% ethanolic soln (containing 5% ammoniacal liquor) the feeding resin column.
Collect elutriant, using 10mol/L sulfuric acid to regulate pH is 3, and the decompression heating is concentrated into 1200mL.Liquid concentrator feeds the 500mL C that is contained in the post
18The reversed phase chromatography medium adsorbs, co-adsorption 2h.Use 800mL 15% ethanolic soln, 800mL 20% ethanolic soln, 600mL 25% ethanolic soln, 600mL 30% ethanolic soln, 600mL35% ethanolic soln, 600mL 40% ethanolic soln, 600mL 55% ethanolic soln, 500mL 95% ethanolic soln (all containing 0.01% trifluoroacetic acid) to feed in the post then successively and carry out wash-out, elution time 10h.Elutriant is divided into 10 parts of collections.
The elutriant of Fractional Collections is measured through high performance liquid chromatograph, and wherein Serpophite content is higher in 3 of 20%, 25%, 30% ethanolic soln wash-out parts of elutriants, merges volume 1600mL.The decompression heating is concentrated into dried, adds 15mL dissolve with methanol solid, adds the 50mL ether again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the Serpophite deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get Serpophite 780mg, purity 95%.
Reserpine content is higher in 1 part of elutriant of 35% ethanolic soln wash-out, volume 600mL.The decompression heating is concentrated into dried, adds 10mL acetic acid ethyl dissolution solid, adds 45mL methyl alcohol again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the serpentine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get serpentine 107mg, purity 98%.
Sharp new amine content is higher in 1 part of elutriant of 40% ethanolic soln wash-out, volume 300mL.The decompression heating is concentrated into dried, adds 10mL acetic acid ethyl dissolution solid, adds 45mL methyl alcohol again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain sharp new amine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, the new amine 8mg that gets profit, purity 98%.
Embodiment 4
Get exsiccant Rauvolfia vomitoria Afzel. root powder 5000g, add and contain the 50% ethanolic soln lixiviate of 0.05mol/L vitriolic, lixiviate is 2 times altogether; The liquid volume that twice lixiviate adds is respectively 50000mL, 25000mL; Each extraction time is 2h, extraction time have a rest stirrings, lixiviate after-filtration at every turn.
2 portions of vat liquors are merged, and volume is 60000mL, and decompression heating simmer down to 30000mL filters.The 2300mL HZ-818 nonpolar macroporous adsorption resin that filtrating feeding is contained in the post adsorbs co-adsorption 20h.Feed in the resin column with the 3000mL deionized water then and wash 2h.With carrying out wash-out in 5000mL 40% ethanolic soln (containing 1% hydrochloric acid) the feeding resin column, elution time 5h collects elutriant.Use 6000mL 80% ethanolic soln (containing 1% hydrochloric acid) to feed in the resin column again and carry out wash-out, elution time 6h collects elutriant.
The elutriant decompression heating of 40% ethanolic soln is concentrated into 2400mL.Liquid concentrator feeds the 1200mLD152 weakly acidic cation-exchange resin that is contained in the post and adsorbs co-adsorption 4h.Feed in the resin column with the 1400mL deionized water then and wash 1.5h.With carrying out wash-out, elution time 6h in 4500mL 50% ethanolic soln (containing 5% ammoniacal liquor) the feeding resin column.
Collect elutriant, using 10mol/L sulfuric acid to regulate pH is 3, and the decompression heating is concentrated into 2000mL.Liquid concentrator feeds the 500mL C that is contained in the post
18The reversed phase chromatography medium adsorbs, co-adsorption 3.5h.Use 6000mL 5% ethanolic soln, 3500mL 10% ethanolic soln, 500mL 15% ethanolic soln, 1000mL 20% ethanolic soln, 1000mL 95% ethanolic soln (all containing 0.01% trifluoroacetic acid) to feed in the post then successively and carry out wash-out, elution time 20h.Elutriant is divided into 15 parts of collections.
The elutriant of Fractional Collections is measured through high performance liquid chromatograph, and wherein Yohimbine content is higher in 3 of 5% ethanolic soln wash-out parts of elutriants, merges volume 3300mL.The decompression heating is concentrated into dried, adds 20mL dissolve with methanol solid, adds the 80mL ether again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the Yohimbine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get Yohimbine 26mg, purity 55%.
Rauwolfine content is higher in 3 parts of elutriants of 10% ethanolic soln wash-out, merges volume 3500mL.The decompression heating is concentrated into dried, adds 30mL dissolve with methanol solid, adds the 100mL ether again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the rauwolfine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get rauwolfine 1027mg, purity 71%.
The HZ-818 nonpolar macroporous adsorption resin is concentrated into 1600mL with the elutriant decompression heating that 80% ethanolic soln wash-out obtains.Liquid concentrator feeds the 1200mL D152 weakly acidic cation-exchange resin that is contained in the post and adsorbs co-adsorption 4h.Feed in the resin column with the 1500mL deionized water then and wash 1.5h.With carrying out wash-out, elution time 6h in 6000mL 50% ethanolic soln (containing 5% ammoniacal liquor) the feeding resin column.
Collect elutriant, using 10mol/L sulfuric acid to regulate pH is 3, and the decompression heating is concentrated into 3000mL.Liquid concentrator feeds the 500mL C that is contained in the post
18The reversed phase chromatography medium adsorbs, co-adsorption 5h.Use 1500mL 15% ethanolic soln, 1500mL 20% ethanolic soln, 3500mL 25% ethanolic soln, 1500mL 30% ethanolic soln, 1200mL 35% ethanolic soln, 600mL 40% ethanolic soln, 600mL 55% ethanolic soln, 700mL 95% ethanolic soln (all containing 0.01% trifluoroacetic acid) to feed in the post then successively and carry out wash-out, elution time 21h.Elutriant is divided into 20 parts of collections.
The elutriant of Fractional Collections is measured through high performance liquid chromatograph, and wherein Serpophite content is higher in 3 of 20%, 25% ethanolic soln wash-out parts of elutriants, merges volume 1800mL.The decompression heating is concentrated into dried, adds 30mL dissolve with methanol solid, adds the 100mL ether again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the Serpophite deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get Serpophite 1564mg, purity 97%.
Reserpine content is higher in 2 parts of elutriants of 35% ethanolic soln wash-out, merges volume 1200mL.The decompression heating is concentrated into dried, adds 20mL acetic acid ethyl dissolution solid, adds 80mL methyl alcohol again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain the serpentine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, get serpentine 309mg, purity 99%.
Sharp new amine content is higher in 1 part of elutriant of 40% ethanolic soln wash-out, volume 600mL.The decompression heating is concentrated into dried, adds 10mL acetic acid ethyl dissolution solid, adds 45mL methyl alcohol again, stirs 1h, places 12h in 4 ℃.At the centrifugal 10min of 4000r/min, obtain sharp new amine deposition, put into 40 ℃ of baking ovens and dry by the fire 10h, the new amine 21mg that gets profit, purity 98%.
More than be the preferred embodiments of the present invention, the present invention also has other attainable modes.Although content of the present invention has been done detailed introduction through above-mentioned preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art has read foregoing, for multiple modification of the present invention with to substitute all will be conspicuous.Therefore, protection scope of the present invention should be limited appended claim.
Claims (17)
1. one kind is extracted multiple alkaloidal method from Rauvolfia vomitoria Afzel., it is characterized in that, specifically comprises the steps:
(1) get the Rauvolfia vomitoria Afzel. root and pulverize the powder that forms, add acidiferous aqueous ethanolic solution, stirring, lixiviate, filtration obtain vat liquor, and program repeats 2-3 time like this;
(2) vat liquor with gained merges, and the decompression heating concentrates, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin, uses water washing resin then, uses 40% ethanol elution subsequently, collects elutriant A1, uses 80% ethanol elution again, collects elutriant B1;
(3) two portions elutriant A1 of above-mentioned collection and the B1 heating of reducing pressure is respectively concentrated, the elutriant A1 after concentrating and B1 use water washing resin then respectively with weakly acidic cation-exchange resin absorption, use 50% ethanol elution subsequently, collection elutriant A2, B2;
(4) two portions elutriant A2 and the B2 with above-mentioned collection is adjusted to pH about 3 with concentrated acid respectively, and the decompression heating concentrates, and elutriant A2 and B2 after concentrating use C respectively
18The absorption of reversed phase chromatography medium is used the aqueous ethanolic solution wash-out, Fractional Collections elutriant A3, B3 then;
(5) elutriant A3 and the B3 with above-mentioned Fractional Collections measures wherein various alkaloids content with high performance liquid chromatograph, merges respectively containing the higher part of Yohimbine or rauwolfine and purity among the elutriant A3; Merge respectively containing Serpophite or serpentine or sharp new amine and the higher part of purity among the elutriant B3; The decompression heating concentrates respectively, to the liquid evaporate to dryness, adds the organic solvent crystallization, obtains the crystal of Yohimbine, rauwolfine, Serpophite, serpentine, sharp new amine.
2. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, the acidiferous aqueous ethanolic solution described in the step (1), containing volume percent is the acid of water and the 0.02-0.2mol/L of 10%-60%.
3. according to claim 2ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that described acid is sulfuric acid, hydrochloric acid or acetate.
4. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, in the step (1), during each lixiviate used acidiferous aqueous ethanolic solution volume mL be Rauvolfia vomitoria Afzel. root powder dry weight g 3-10 doubly.
5. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, in the step (2); The vat liquor of gained is merged, and the decompression heating concentrates, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin at normal temperatures; The volume mL of nonpolar macroporous adsorption resin is the 40%-50% of Rauvolfia vomitoria Afzel. root powder dry weight g, uses water washing resin then, and the volume of water is 1-5 a times of nonpolar macroporous adsorption resin volume; Use 40% ethanol elution subsequently; 40% alcoholic acid volume is 2-5 a times of nonpolar macroporous adsorption resin volume, collects elutriant A1, uses 80% ethanol elution again; 80% alcoholic acid volume is 2-5 a times of nonpolar macroporous adsorption resin volume, collects elutriant B1.
6. describedly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method according to claim 1 or 5, it is characterized in that, 40% ethanol described in the step (2) wherein contains volume percent and is 59% water and volume percent and be 1% hydrochloric acid; Described 80% ethanol wherein contains volume percent and is 19% water and volume percent and be 1% hydrochloric acid.
7. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method; It is characterized in that; In the step (3), two portions elutriant A1 that collects and the B1 heating of reducing pressure is respectively concentrated, elutriant A1 and B1 after concentrated adsorb with weakly acidic cation-exchange resin respectively at normal temperatures; The volume mL that adsorbs the used weakly acidic cation-exchange resin of each part elutriant is the 20%-30% of Rauvolfia vomitoria Afzel. root powder dry weight g; Use water washing resin then, the volume of water is 1-5 a times of weakly acidic cation-exchange resin volume, uses 50% ethanol elution subsequently; 50% alcoholic acid volume is 3-5 a times of weakly acidic cation-exchange resin volume, collects elutriant A2 and B2.
8. describedly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method according to claim 1 or 7, it is characterized in that, 50% ethanol described in the step (3) wherein contains volume percent and is 45% water and volume percent and be 5% ammoniacal liquor.
9. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that in the step (4), the elutriant A2 and the B2 that collect are adjusted to pH 3 with concentrated acid respectively, the decompression heating is concentrated; Elutriant A2 after concentrating uses C at normal temperatures
18The absorption of reversed phase chromatography medium, C
18The volume mL of reversed phase chromatography medium is the 10%-20% of Rauvolfia vomitoria Afzel. root powder dry weight g, uses 5%, 10%, 15%, 20%, 95% aqueous ethanolic solution wash-out then successively, and the volume summation of aqueous ethanolic solution is C
18The 10-30 of reversed phase chromatography medium volume times, Fractional Collections 10-20 part elutriant A3; Elutriant B2 after concentrating uses C at normal temperatures
18The absorption of reversed phase chromatography medium, C
18The volume mL of reversed phase chromatography medium is the 10%-20% of Rauvolfia vomitoria Afzel. root powder dry weight g, uses 15%, 20%, 25%, 30%, 35%, 40%, 55%, 95% aqueous ethanolic solution wash-out then successively, and the volume summation of aqueous ethanolic solution is C
18The 10-30 of reversed phase chromatography medium volume times, Fractional Collections 10-20 part elutriant B3.
10. according to claim 9ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method; It is characterized in that; 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 95% aqueous ethanolic solution described in the step (4); Wherein contain volume percent respectively and be 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 45%, 5% water, and to contain volume percent be 0.01% trifluoroacetic acid.
11. describedly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method according to claim 1 or 9, it is characterized in that the concentrated acid described in the step (4) is the vitriol oil or concentrated hydrochloric acid.
12. describedly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method according to claim 1 or 9; It is characterized in that; Fractional Collections elutriant described in the step (4) is that elution time or elution volume are divided into plurality of sections, and the elutriant in each section elution time or the elution volume is collected as 1 part.
13. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, the adding organic solvent crystallization described in the step (5), concrete operations are: for Yohimbine, rauwolfine or Serpophite; Add methyl alcohol, stir and make the solid dissolving, the methyl alcohol add-on can make solid all dissolve, and adds ether again; The adding volume is 2-5 a times of methyl alcohol volume, stirs, in 0-5 ℃ of placement; Separate out crystallization, remove mother liquor, obtain crystallization; For serpentine or sharp new amine, add ETHYLE ACETATE, stir and make the solid dissolving, the ETHYLE ACETATE add-on can make solid all dissolve; Add methyl alcohol again, the adding volume is 2-5 a times of ETHYLE ACETATE volume, stirs, in 0-5 ℃ of placement; Separate out crystallization, remove mother liquor, obtain crystallization; Till the gained crystal is put into 40-50 ℃ of baking oven and is dried to constant weight.
14. describedly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method according to claim 1 or 13, it is characterized in that, in the step (5); The elutriant A3 of Fractional Collections and B3 measure wherein various alkaloids content with high performance liquid chromatograph; If certain alkaloidal purity is not high enough, need to purify once more, can be with containing the alkaloidal elutriant merging that desire is purified once more among elutriant A3 or the B3; As A2 or B2, operate once again according to step (4).
15. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that it is under temperature 45-65 ℃, the condition of vacuum tightness 0.08-0.10M Pa, to carry out that said decompression heating concentrates.
16. each describedly extracts multiple alkaloidal method from Rauvolfia vomitoria Afzel. according to claim 1-15; It is characterized in that; Only get 40% ethanol eluate A1 and carry out respective operations in said step (3), (4), (5), obtain A2, A3 successively, and final vegeto-alkali Yohimbine, rauwolfine.
17. each describedly extracts multiple alkaloidal method from Rauvolfia vomitoria Afzel. according to claim 1-15; It is characterized in that; Only get 80% ethanol eluate B1 and carry out respective operations in said step (3), (4), (5); Obtain B2, B3 successively, and final vegeto-alkali Serpophite, serpentine, sharp new amine.
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