CN102627641B - 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 from plant Rauvolfia vomitoria Afzel. (Rauwolfia vomitoria Afz.), extracts 5 kinds of alkaloidal methods such as Yohimbine (yohimbine), rauwolfine (ajmaline), Serpophite (serpentine), serpentine (reserpine), rescinnamine (rescinnamine).
Background technology
Radix Rauvolfiae is Apocynaceae Rauwolfia plant, contains multiple effective component.Rauvolfia vomitoria Afzel. is a kind of of Radix Rauvolfiae, from Africa, introduces, and wherein contains the multiple alkaloids such as serpentine, rauwolfine, Yohimbine, Serpophite, and these alkaloids can be used as medicine, have clinically the therapeutic action to various diseases.
Through prior art literature search is found, from Radix Rauvolfiae (comprising Rauvolfia vomitoria Afzel.), extract effective component, reporting more is to extract serpentine, adopts in early days benzene to extract, and uses macroporous resin extraction later.From Rauvolfia vomitoria Afzel., extract the report of several components simultaneously, have the research of extracting serpentine and Yohimbine, but the Yohimbine purity extracting lower (Liu Yu, Feng Lei, Zhao Fengsheng, Pharmaceutical Biotechnology, 2008,15 (5): 393-397).From Rauvolfia vomitoria Afzel., extract in addition the report of serpentine, Reserpilline, the new alkali of Radix Rauvolfiae simultaneously, but extraction step is more, spended time is long, and (Zhou Xueqing, Feng Yuhong, Zhang Chong, Chen Sili, woods are strong, the Journal of Hainan University's natural science edition, 2008,26 (2): 145-148).
Summary of the invention
The present invention is directed to prior art above shortcomings, a kind of 5 kinds of alkaloidal methods of once can extracting at most from Rauvolfia vomitoria Afzel. are provided, by technology such as acidic ethanol lixiviate, macroporous resin adsorption, weakly acidic cation-exchange resin extraction, reversed phase chromatography separation, combine, the alkaloid extracting all has higher purity.
The present invention is achieved through the following technical solutions:
The invention provides a kind of multiple alkaloidal method of extracting from Rauvolfia vomitoria Afzel., extract Yohimbine, rauwolfine, Serpophite, serpentine, rescinnamine simultaneously; Specifically comprise the steps:
(1) get Rauvolfia vomitoria Afzel. root and pulverize the powder forming, add containing sour aqueous ethanolic solution, stirring, lixiviate, filtration, obtain vat liquor, and program repeats 2-3 time like this;
(2) vat liquor of gained is merged, decompression heating is concentrated, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin, then wash resin with water, use subsequently 40% ethanol elution, collect elutriant (being designated as A1), use again 80% ethanol elution, collect elutriant (being designated as B1);
(3) two portions elutriant A1 of above-mentioned collection and B1 are reduced pressure respectively heating is concentrated, and the elutriant A1 after concentrated adsorbs with weakly acidic cation-exchange resin, then washes resin with water, uses subsequently 50% ethanol elution, collects elutriant (being designated as A2); Elutriant B1 after concentrated adsorbs with weakly acidic cation-exchange resin, then washes resin with water, uses subsequently 50% ethanol elution, collects elutriant (being designated as B2);
(4) two portions elutriant A2 of above-mentioned collection and B2 are adjusted to pH 3 left and right with concentrated acid respectively, decompression heating is concentrated, the elutriant A2 C after concentrating
18the absorption of reversed phase chromatography medium, then uses aqueous ethanolic solution wash-out, Fractional Collections elutriant (being designated as A3); Elutriant B2 C after concentrated
18the absorption of reversed phase chromatography medium, then uses aqueous ethanolic solution wash-out, Fractional Collections elutriant (being designated as B3);
(5) the elutriant A3 of above-mentioned Fractional Collections and B3 are measured to wherein various alkaloidal content with high performance liquid chromatograph, by containing Yohimbine or rauwolfine and the higher part of purity in elutriant A3, merge respectively; By containing Serpophite or serpentine or rescinnamine and the higher part of purity in elutriant B3, merge respectively; Decompression heating is concentrated respectively, to liquid evaporate to dryness, adds organic solvent crystallization, obtains the crystal of Yohimbine, rauwolfine, Serpophite, serpentine, rescinnamine.
Described in step (1) containing sour aqueous ethanolic solution, the water that contains 10%-60% (volume percent) contains acid simultaneously, the concentration of acid is 0.02-0.2mol/L, acid can be sulfuric acid, hydrochloric acid or acetic acid;
Described lixiviate, the volume mL containing sour aqueous ethanolic solution at every turn adding is 3-10 times of Rauvolfia vomitoria Afzel. root powder dry weight g.
In step (2), the vat liquor of gained is merged, decompression heating is concentrated, vat liquor after concentrated 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, then wash resin with water, the volume of water is 1-5 times of nonpolar macroporous adsorption resin volume, use subsequently 40% ethanol elution, the volume of 40% ethanol is 2-5 times of nonpolar macroporous adsorption resin volume, collect elutriant A1, use again 80% ethanol elution, the volume of 80% ethanol is 2-5 times of nonpolar macroporous adsorption resin volume, collect elutriant B1,
The described resin that washes with water, is to pass in post from the import of post with deionized water or pure water, then flows out from the outlet of post, and washing time is 0.5-3h;
40% described ethanol, wherein contains volume percent and is the hydrochloric acid that 59% water and volume percent are 1%;
80% described ethanol, wherein contains volume percent and is the hydrochloric acid that 19% water and volume percent are 1%.
In step (3), two portions elutriant A1 of collection and the B1 heating of reducing pressure respectively is concentrated, elutriant A1 and B1 after concentrated adsorb with weakly acidic cation-exchange resin respectively at normal temperatures, the volume mL that adsorbs every a part of elutriant weakly acidic cation-exchange resin used is the 20%-30% of Rauvolfia vomitoria Afzel. root powder dry weight g, then wash resin with water, the volume of water is 1-5 times of weakly acidic cation-exchange resin volume, use subsequently 50% ethanol elution, the volume of 50% ethanol is 3-5 times of weakly acidic cation-exchange resin volume, collect elutriant A2 and B2,
The described resin that washes with water, is to pass in post from the import of post with deionized water or pure water, then flows out from the outlet of post, and washing time is 0.5-2h;
50% described ethanol, wherein contains volume percent and is the ammoniacal liquor that 45% water and volume percent are 5%.
In step (4), the elutriant A2 of collection and B2 are adjusted to pH 3 left and right with concentrated acid respectively, decompression heating is concentrated; Elutriant A2 after concentrated 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, then uses successively 5%, 10%, 15%, 20%, 95% aqueous ethanolic solution wash-out, 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 concentrated 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, then uses successively 15%, 20%, 25%, 30%, 35%, 40%, 55%, 95% aqueous ethanolic solution wash-out, 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 some sections, and the elutriant in each section of elution time or elution volume is collected as 1 part.
Described in step (5), add organic solvent crystallization, the Serpophite in the Yohimbine in elutriant A3, rauwolfine or elutriant B3, adds methyl alcohol, stirring makes dissolution of solid, methyl alcohol add-on can make solid all dissolve, but unsuitable excessive, then adds ether, add volume be methyl alcohol volume 2-5 doubly, stir 1-3h, in 0-5 ℃, place 10-20h, crystallization, filter or centrifugal removal mother liquor, obtain crystallization; For the serpentine in elutriant B3 or rescinnamine, add ethyl acetate, stir and make dissolution of solid, ethyl acetate add-on can make solid all dissolve, but unsuitable excessive, add again methyl alcohol, add volume be ethyl acetate volume 2-5 doubly, stir 1-3h, in 0-5 ℃, place 10-20h, crystallization, filters or centrifugal removal mother liquor, obtains crystallization; By above step gained crystal, put into 40-50 ℃ of baking oven and dry 5-10h, till constant weight;
In step (5), the elutriant A3 of Fractional Collections and B3 measure wherein various alkaloidal content with high performance liquid chromatograph, if certain alkaloidal purity is not high enough, need to again purify, can merge containing the alkaloidal elutriant that wish purifies again in elutriant A3 or B3, as A2 or B2, according to step (4), operate once again.
Above-described decompression heating is concentrated is all to carry out under temperature 45-65 ℃, the condition of vacuum tightness 0.08-0.10M Pa, except special instruction, generally by liquid concentration to about half of original volume.
In aforesaid method of the present invention, can only get 40% ethanol eluate A1 and carry out respective operations in described step (3), (4), (5), obtain successively A2, A3, and final alkaloid Yohimbine, rauwolfine; Also can only get 80% ethanol eluate B1 and carry out respective operations in described step (3), (4), (5), obtain successively B2, B3, and final alkaloid Serpophite, serpentine, rescinnamine.
Compared with prior art, the present invention combines the technology such as acidic ethanol lixiviate, macroporous resin adsorption, weakly acidic cation-exchange resin extraction, reversed phase chromatography separation, extract Yohimbine, rauwolfine, Serpophite, serpentine, 5 kinds of alkaloids of rescinnamine, the alkaloid extracting all has higher purity simultaneously.
Embodiment
Below embodiments of the invention are elaborated: the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.In following examples, there is no the operation describing in detail, can adopt routine operation of the prior art.
Embodiment 1
Get dry Rauvolfia vomitoria Afzel. root powder 2500g, add the 50% ethanolic soln lixiviate containing 0.025mol/L sulfuric acid, total immersion is carried 2 times, the liquid volume that twice lixiviate adds is respectively 25000mL, 12500mL, each extraction time is 2h, and extraction time stirrings of having a rest filtered after lixiviate 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 (Hua Zhen Science and Technology Ltd. of East China University of Science system) that filtrate passing into is contained in post adsorbs co-adsorption 20h.Then with 1500mL deionized water, pass into and in resin column, wash 1.5h.With 2400mL 40% ethanolic soln (containing 1% hydrochloric acid), pass into and in resin column, carry out wash-out, elution time 4h.
Collect elutriant, decompression heating is concentrated into 840mL.Concentrated solution passes into the 700mL D152 weakly acidic cation-exchange resin (Chemical Plant of Nankai Univ.'s system) being contained in post and adsorbs, co-adsorption 4h.Then with 800mL deionized water, pass into and in resin column, wash 1.5h.With 2100mL 50% ethanolic soln (containing 5% ammoniacal liquor), pass into and in resin column, carry out wash-out, elution time 4h.
Collecting elutriant, is 3 with 10mol/L sulphur acid for adjusting pH, and decompression heating is concentrated into 1100mL.Concentrated solution passes into the 500mL C being contained in post
18reversed phase chromatography medium (Japanese YMC company system) adsorbs co-adsorption 2h.Then use successively 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 pass into and in post, 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 in 2 of 5% ethanolic soln wash-out parts of elutriants, Yohimbine content is higher, merges volume 1000mL.Decompression heating is concentrated into dry, adds 10mL dissolve with methanol solid, then adds 40mL ether, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain Yohimbine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain Yohimbine 14mg, purity 52%.
In 3 parts of elutriants of 10% ethanolic soln wash-out, rauwolfine content is higher, merges volume 2700mL.Decompression heating is concentrated into dry, adds 15mL dissolve with methanol solid, then adds 50mL ether, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain rauwolfine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain rauwolfine 406mg, purity 63%.
Embodiment 2
Rauvolfia vomitoria Afzel. lixiviate, non-polar macroporous resin absorption and wash-out, weakly acidic cation-exchange resin absorption and wash-out, crystallization and dry implementation step are identical with embodiment 1, and difference is:
C
18after the part that in the elutriant that reversed phase chromatography is collected, Yohimbine content is higher merges, volume 1000mL, decompression heating is concentrated into 700mL, then passes into the 500mL C being contained in post
18reversed phase chromatography medium adsorbs, co-adsorption 2h.Then use successively 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 pass into and in post, carry out wash-out, elution time 10h.Elutriant is divided into 10 parts of collections.
Through high performance liquid chromatograph, measure, wherein in 2 of 5% ethanolic soln wash-out parts of elutriants, Yohimbine content is higher, merges volume 800mL.Crystallization, obtains Yohimbine 6mg, purity 87%.
C
18after the part that in the elutriant that reversed phase chromatography is collected, rauwolfine content is higher merges, volume 2700mL, decompression heating is concentrated into 1500mL, then passes into the 500mL C being contained in post
18reversed phase chromatography medium adsorbs, co-adsorption 2.5h.Then use successively 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 pass into and in post, carry out wash-out, elution time 10h.Elutriant is divided into 10 parts of collections.
Through high performance liquid chromatograph, measure, wherein in 2 of 10% ethanolic soln wash-out parts of elutriants, rauwolfine content is higher, merges volume 700mL.Crystallization, obtains rauwolfine 230mg, purity 90%.
Embodiment 3
Get dry Rauvolfia vomitoria Afzel. root powder 2500g, add the 50% ethanolic soln lixiviate containing 0.025mol/L sulfuric acid, total immersion is carried 2 times, the liquid volume that twice lixiviate adds is respectively 25000mL, 12500mL, each extraction time is 2h, and extraction time stirrings of having a rest filtered after lixiviate 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 filtrate passing into is contained in post adsorbs, co-adsorption 18h.Then with 1500mL deionized water, pass into and in resin column, wash 1.5h.With 2500mL 40% ethanolic soln (containing 1% hydrochloric acid), pass into and in resin column, carry out wash-out, elution time 4h.Use again 3000mL 80% ethanolic soln (containing 1% hydrochloric acid) to pass into and in resin column, carry out wash-out, elution time 6h.
Collect 80% ethanolic soln elutriant, decompression heating is concentrated into 680mL.Concentrated solution passes into the 600mLD152 weakly acidic cation-exchange resin being contained in post and adsorbs, co-adsorption 4h.Then with 800mL deionized water, pass into and in resin column, wash 1.5h.With 3000mL 50% ethanolic soln (containing 5% ammoniacal liquor), pass into and in resin column, carry out wash-out, elution time 6h.
Collecting elutriant, is 3 with 10mol/L sulphur acid for adjusting pH, and decompression heating is concentrated into 1200mL.Concentrated solution passes into the 500mL C being contained in post
18reversed phase chromatography medium adsorbs, co-adsorption 2h.Then use successively 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 pass into and in post, 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 in 3 of 20%, 25%, 30% ethanolic soln wash-out parts of elutriants, Serpophite content is higher, merges volume 1600mL.Decompression heating is concentrated into dry, adds 15mL dissolve with methanol solid, then adds 50mL ether, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain Serpophite precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain Serpophite 780mg, purity 95%.
In 1 part of elutriant of 35% ethanolic soln wash-out, reserpine content is higher, volume 600mL.Decompression heating is concentrated into dry, adds 10mL acetic acid ethyl dissolution solid, then adds 45mL methyl alcohol, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain serpentine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain serpentine 107mg, purity 98%.
In 1 part of elutriant of 40% ethanolic soln wash-out, rescinnamine content is higher, volume 300mL.Decompression heating is concentrated into dry, adds 10mL acetic acid ethyl dissolution solid, then adds 45mL methyl alcohol, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain rescinnamine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain rescinnamine 8mg, purity 98%.
Embodiment 4
Get dry Rauvolfia vomitoria Afzel. root powder 5000g, add the 50% ethanolic soln lixiviate containing 0.05mol/L sulfuric acid, total immersion is carried 2 times, the liquid volume that twice lixiviate adds is respectively 50000mL, 25000mL, each extraction time is 2h, and extraction time stirrings of having a rest filtered after lixiviate 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 filtrate passing into is contained in post adsorbs, co-adsorption 20h.Then with 3000mL deionized water, pass into and in resin column, wash 2h.With 5000mL 40% ethanolic soln (containing 1% hydrochloric acid), pass in resin column and carry out wash-out, elution time 5h, collects elutriant.Use 6000mL 80% ethanolic soln (containing 1% hydrochloric acid) to pass in resin column and carry out wash-out, elution time 6h, collects elutriant again.
The elutriant decompression heating of 40% ethanolic soln is concentrated into 2400mL.Concentrated solution passes into the 1200mLD152 weakly acidic cation-exchange resin being contained in post and adsorbs, co-adsorption 4h.Then with 1400mL deionized water, pass into and in resin column, wash 1.5h.With 4500mL 50% ethanolic soln (containing 5% ammoniacal liquor), pass into and in resin column, carry out wash-out, elution time 6h.
Collecting elutriant, is 3 with 10mol/L sulphur acid for adjusting pH, and decompression heating is concentrated into 2000mL.Concentrated solution passes into the 500mL C being contained in post
18reversed phase chromatography medium adsorbs, co-adsorption 3.5h.Then use successively 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 pass into and in post, 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 in 3 of 5% ethanolic soln wash-out parts of elutriants, Yohimbine content is higher, merges volume 3300mL.Decompression heating is concentrated into dry, adds 20mL dissolve with methanol solid, then adds 80mL ether, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain Yohimbine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain Yohimbine 26mg, purity 55%.
In 3 parts of elutriants of 10% ethanolic soln wash-out, rauwolfine content is higher, merges volume 3500mL.Decompression heating is concentrated into dry, adds 30mL dissolve with methanol solid, then adds 100mL ether, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain rauwolfine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain rauwolfine 1027mg, purity 71%.
The elutriant decompression heating that HZ-818 nonpolar macroporous adsorption resin obtains with 80% ethanolic soln wash-out is concentrated into 1600mL.Concentrated solution passes into the 1200mL D152 weakly acidic cation-exchange resin being contained in post and adsorbs, co-adsorption 4h.Then with 1500mL deionized water, pass into and in resin column, wash 1.5h.With 6000mL 50% ethanolic soln (containing 5% ammoniacal liquor), pass into and in resin column, carry out wash-out, elution time 6h.
Collecting elutriant, is 3 with 10mol/L sulphur acid for adjusting pH, and decompression heating is concentrated into 3000mL.Concentrated solution passes into the 500mL C being contained in post
18reversed phase chromatography medium adsorbs, co-adsorption 5h.Then use successively 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 pass into and in post, 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 in 3 of 20%, 25% ethanolic soln wash-out parts of elutriants, Serpophite content is higher, merges volume 1800mL.Decompression heating is concentrated into dry, adds 30mL dissolve with methanol solid, then adds 100mL ether, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain Serpophite precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain Serpophite 1564mg, purity 97%.
In 2 parts of elutriants of 35% ethanolic soln wash-out, reserpine content is higher, merges volume 1200mL.Decompression heating is concentrated into dry, adds 20mL acetic acid ethyl dissolution solid, then adds 80mL methyl alcohol, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain serpentine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain serpentine 309mg, purity 99%.
In 1 part of elutriant of 40% ethanolic soln wash-out, rescinnamine content is higher, volume 600mL.Decompression heating is concentrated into dry, adds 10mL acetic acid ethyl dissolution solid, then adds 45mL methyl alcohol, stirs 1h, in 4 ℃, places 12h.At the centrifugal 10min of 4000r/min, obtain rescinnamine precipitation, put into 40 ℃ of baking ovens and dry 10h, obtain rescinnamine 21mg, purity 98%.
Be more than 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 by above preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.Those skilled in the art has read after foregoing, for multiple modification of the present invention with to substitute will be all apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (15)
1. from Rauvolfia vomitoria Afzel., extract a multiple alkaloidal method, it is characterized in that, specifically comprise the steps:
(1) get Rauvolfia vomitoria Afzel. root and pulverize the powder forming, add containing sour aqueous ethanolic solution, stirring, lixiviate, filtration, obtain vat liquor, and program repeats 2-3 time like this;
(2) vat liquor of gained is merged, decompression heating is concentrated, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin, then washes resin with water, uses subsequently 40% ethanol elution, collects elutriant A1, then uses 80% ethanol elution, collects elutriant B1; 40% described ethanol, wherein contains volume percent and is the hydrochloric acid that 59% water and volume percent are 1%; 80% described ethanol, wherein contains volume percent and is the hydrochloric acid that 19% water and volume percent are 1%;
(3) two portions elutriant A1 of above-mentioned collection and B1 are reduced pressure respectively heating is concentrated, and the elutriant A1 after concentrated and B1, respectively with weakly acidic cation-exchange resin absorption, then wash resin with water, use subsequently 50% ethanol elution, collect elutriant A2, B2; 50% described ethanol, wherein contains volume percent and is the ammoniacal liquor that 45% water and volume percent are 5%;
(4) two portions elutriant A2 of above-mentioned collection and B2 are adjusted to pH3 left and right with concentrated acid respectively, decompression heating is concentrated, and elutriant A2 and B2 after concentrating use respectively C
18reversed phase chromatography medium absorption, then with containing the aqueous ethanolic solution wash-out that volume percent is 0.01% trifluoroacetic acid, Fractional Collections elutriant A3, B3;
(5) the elutriant A3 of above-mentioned Fractional Collections and B3 are measured to wherein various alkaloidal content with high performance liquid chromatograph, by containing Yohimbine or rauwolfine and the higher part of purity in elutriant A3, merge respectively; By containing Serpophite or serpentine or rescinnamine and the higher part of purity in elutriant B3, merge respectively; Decompression heating is concentrated respectively, to liquid evaporate to dryness, adds organic solvent crystallization, obtains the crystal of Yohimbine, rauwolfine, Serpophite, serpentine, rescinnamine.
2. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, described in step (1) containing sour aqueous ethanolic solution, 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 acetic acid.
4. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, in step (1), during each lixiviate used containing sour 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 step (2), the vat liquor of gained is merged, decompression heating is concentrated, vat liquor after concentrated 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, then wash resin with water, the volume of water is 1-5 times of nonpolar macroporous adsorption resin volume, use subsequently 40% ethanol elution, the volume of 40% ethanol is 2-5 times of nonpolar macroporous adsorption resin volume, collect elutriant A1, use again 80% ethanol elution, the volume of 80% ethanol is 2-5 times of nonpolar macroporous adsorption resin volume, collect elutriant B1.
6. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, in step (3), two portions elutriant A1 of collection and the B1 heating of reducing pressure respectively is concentrated, elutriant A1 and B1 after concentrated adsorb with weakly acidic cation-exchange resin respectively at normal temperatures, the volume mL that adsorbs every a part of elutriant weakly acidic cation-exchange resin used is the 20%-30% of Rauvolfia vomitoria Afzel. root powder dry weight g, then wash resin with water, the volume of water is 1-5 times of weakly acidic cation-exchange resin volume, use subsequently 50% ethanol elution, the volume of 50% ethanol is 3-5 times of weakly acidic cation-exchange resin volume, collect elutriant A2 and B2.
7. according to claim 1ly from Rauvolfia vomitoria Afzel., extract multiple alkaloidal method, it is characterized in that, in step (4), elutriant A2 and the B2 of collection are adjusted to pH3 with concentrated acid respectively, decompression heating is concentrated; Elutriant A2 after concentrated 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, then uses successively 5%, 10%, 15%, 20%, 95% aqueous ethanolic solution wash-out, 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 concentrated 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, then uses successively 15%, 20%, 25%, 30%, 35%, 40%, 55%, 95% aqueous ethanolic solution wash-out, 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.
8. according to claim 7ly 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 step (4), wherein contain respectively volume percent and be 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 45%, 5% water, and to contain volume percent be 0.01% trifluoroacetic acid.
9. according to extracting multiple alkaloidal method described in claim 1 or 7 from Rauvolfia vomitoria Afzel., it is characterized in that, the concentrated acid described in step (4) is the vitriol oil or concentrated hydrochloric acid.
10. according to extracting multiple alkaloidal method described in claim 1 or 7 from Rauvolfia vomitoria Afzel., it is characterized in that, Fractional Collections elutriant described in step (4), is that elution time or elution volume are divided into some sections, and the elutriant in each section of elution time or elution volume is collected as 1 part.
11. according to claim 1ly extract multiple alkaloidal method from Rauvolfia vomitoria Afzel., it is characterized in that, described in step (5), add organic solvent crystallization, concrete operations are: for Yohimbine, rauwolfine or Serpophite, add methyl alcohol, stir and make dissolution of solid, methyl alcohol add-on can make solid all dissolve, add again ether, add volume be methyl alcohol volume 2-5 doubly, stir, in 0-5 ℃ of placement, crystallization, removes mother liquor, obtains crystallization; For serpentine or rescinnamine, add ethyl acetate, stir and make dissolution of solid, ethyl acetate add-on can make solid all dissolve, add again methyl alcohol, add volume be ethyl acetate volume 2-5 doubly, stir, in 0-5 ℃ of placement, crystallization, removes mother liquor, obtains crystallization; Till gained crystal is put into 40-50 ℃ of baking oven and is dried to constant weight.
12. according to extracting multiple alkaloidal method described in claim 1 or 11 from Rauvolfia vomitoria Afzel., it is characterized in that, in step (5), the elutriant A3 of Fractional Collections and B3 measure wherein various alkaloidal content with high performance liquid chromatograph, if certain alkaloidal purity is not high enough, need to again purify, can merge containing the alkaloidal elutriant that wish purifies again in elutriant A3 or B3, as A2 or B2, according to step (4), operate once again.
13. according to claim 1ly extract multiple alkaloidal method from Rauvolfia vomitoria Afzel., it is characterized in that, described decompression heating is concentrated is to carry out under temperature 45-65 ℃, the condition of vacuum tightness 0.08-0.10M Pa.
14. 1 kinds are extracted multiple alkaloidal method from Rauvolfia vomitoria Afzel., its feature in, specifically comprise the steps:
(1) get Rauvolfia vomitoria Afzel. root and pulverize the powder forming, add containing sour aqueous ethanolic solution, stirring, lixiviate, filtration, obtain vat liquor, and program repeats 2-3 time like this;
(2) vat liquor of gained is merged, decompression heating is concentrated, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin, then washes resin with water, uses subsequently 40% ethanol elution, collects elutriant A1; 40% described ethanol, wherein contains volume percent and is the hydrochloric acid that 59% water and volume percent are 1%;
(3) the elutriant A1 decompression heating of above-mentioned collection is concentrated, the elutriant A1 after concentrating adsorbs with weakly acidic cation-exchange resin, then washes resin with water, uses subsequently 50% ethanol elution, collects elutriant A2; 50% described ethanol, wherein contains volume percent and is the ammoniacal liquor that 45% water and volume percent are 5%;
(4) the elutriant A2 of above-mentioned collection is adjusted to pH3 left and right with concentrated acid, decompression heating is concentrated, the elutriant A2 C after concentrating
18reversed phase chromatography medium absorption, then with containing the aqueous ethanolic solution wash-out that volume percent is 0.01% trifluoroacetic acid, Fractional Collections elutriant A3;
(5) the elutriant A3 of above-mentioned Fractional Collections is measured to wherein various alkaloidal content with high performance liquid chromatograph, by containing Yohimbine or rauwolfine and the higher part of purity in elutriant A3, merge respectively; Decompression heating is concentrated, to liquid evaporate to dryness, adds organic solvent crystallization, obtains the crystal of Yohimbine, rauwolfine.
15. 1 kinds are extracted multiple alkaloidal method from Rauvolfia vomitoria Afzel., its feature in, specifically comprise the steps:
(1) get Rauvolfia vomitoria Afzel. root and pulverize the powder forming, add containing sour aqueous ethanolic solution, stirring, lixiviate, filtration, obtain vat liquor, and program repeats 2-3 time like this;
(2) vat liquor of gained is merged, decompression heating is concentrated, and the vat liquor after concentrating adsorbs with nonpolar macroporous adsorption resin, then washes resin with water, uses subsequently 40% ethanol elution, collects elutriant A1, then uses 80% ethanol elution, collects elutriant B1; 40% described ethanol, wherein contains volume percent and is the hydrochloric acid that 59% water and volume percent are 1%; 80% described ethanol, wherein contains volume percent and is the hydrochloric acid that 19% water and volume percent are 1%;
(3) the elutriant B1 decompression heating of above-mentioned collection is concentrated, the elutriant B1 after concentrating adsorbs with weakly acidic cation-exchange resin, then washes resin with water, uses subsequently 50% ethanol elution, collects elutriant B2; 50% described ethanol, wherein contains volume percent and is the ammoniacal liquor that 45% water and volume percent are 5%;
(4) the elutriant B2 of above-mentioned collection is adjusted to pH3 left and right with concentrated acid, decompression heating is concentrated, the elutriant B2 C after concentrating
18reversed phase chromatography medium absorption, then with containing the aqueous ethanolic solution wash-out that volume percent is 0.01% trifluoroacetic acid, Fractional Collections elutriant B3;
(5) the elutriant B3 of above-mentioned Fractional Collections is measured to wherein various alkaloidal content with high performance liquid chromatograph, by containing Serpophite or serpentine or rescinnamine and the higher part of purity in elutriant B3, merge respectively; Decompression heating is concentrated, to liquid evaporate to dryness, adds organic solvent crystallization, obtains the crystal of Serpophite, serpentine, rescinnamine.
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| CN104513236A (en) * | 2014-12-30 | 2015-04-15 | 广州白云山汉方现代药业有限公司 | Method of utilizing supercritical CO2 extraction and macroporous resin adsorption separation to extract reserpine from rauvolfia verticillate |
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