CN101775419A - Method for extracting suquassine from Lespedeza kunmingensis of Leguminosae - Google Patents
Method for extracting suquassine from Lespedeza kunmingensis of Leguminosae Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the field of natural organic chemistry, and relates to a method for enriching and purifying sukushinamine in lespedeza bicolor by utilizing a staged extraction method. The method is characterized in that: the invention adopts the sectional extraction technology, which is different from the single product of the prior art, and the extract is divided into 3 sections according to the difference of PH to obtain 3 main products, so that lespedeza bicolor is comprehensively utilized, and the value of economic crop lespedeza bicolor is greatly improved. The product purity is high: the purity of the Nigeriline reaches 99.5 percent, the purity of the laspezil reaches more than 99 percent, the purity of the Suqiansihizi alkali reaches 99 percent, and the Nigeriline reaches the requirement of medicine, or the derivative of the Suqiansihizi alkali can be prepared. The extraction rate is high, and the production steps are simple: the plant cell wall part is decomposed by adopting a biological enzymolysis technology and an ultrasonic technology, so that the filtering speed and the purification effect in the production process are improved, and the subsequent separation and purification steps are simplified. Compared with the traditional extraction method, the method has the advantages that the amount of acid, alkali and organic solvent is greatly reduced, so that the method has no pollution to the environment and low cost.
Description
Technical field
The invention belongs to the natural organic chemistry field, relate to a kind of method of from leguminous plants Xingan Stem or leaf of Shrub Lespedeza, extracting the bitter western alkali of Soviet Union.
Background technology
Stem or leaf of Shrub Lespedeza (Lespdeza bicolor Turcz) has another name called apricot bar, three look Stem or leaf of Shrub Lespedeza, tea with an army etc., belongs to kind surplus the pulse family lespedeza Lespdeza Michx plant about 60, is distributed in South East Asia to Australian northeast not as good as the North America.China produces 26 kinds, except that Xinjiang, extensively in each provinces and regions, the whole nation.For the pharmaceutical use of lespedeza plant, people early have understanding, become to grade at people's terpene, organic acid and sterols.Stem or leaf of Shrub Lespedeza total flavones part is to reducing residual nitrogen content in the blood, and daring to delete renal function has sure curative effect, can be used for treating symptoms such as chronic glomerulus, uremia, pyelonephritis, renal tuberculosis and renal atrophy.The chemical ingredients of isolation identification comprises flavones, alkaloid, triterpene, organic acid and sterols etc. from this platymiscium.Chemical ingredients, over-ground part separate obtain orientin (orientin), isorientin, isorientin-2 "-xyloside, isovitexin (isovitex-in) isovitexin-2 "-xyloside Isorhamnetol-3-O-rutinoside, isoshaftoside diosmetin-7-o xyloside compositions such as (diosrmetin-7-o-xyloside)
Ni Gelin alkali, Lars skin alkali and the bitter western alkali alkaloid of reviving are active components that is separated to the earliest from the lespedeza plant, but up to the present from this platymiscium isolation identification 7 alkaloid compounds.Nineteen sixty-eight, people such as Japanese scholar GotOr separate from spend more Stem or leaf of Shrub Lespedeza L.florbundaBunge and obtain Ni Gelin (Nb Nb-dimethyltryptamine), and identifies it by chemical process.Nineteen sixty-five, people such as Morimoto get a new alkaloids from this plant, utilize what chemical process of UV.IR, NMR to identify its structure, the bitter western alkali of called after Lars skin alkaloid lespedamine and Soviet Union. modern pharmacological research and clinical showing, the Stem or leaf of Shrub Lespedeza alkaloid has multiple biological activity and curative effect, Ni Gelin alkali is the specifics of treatment cardiovascular and cerebrovascular and senile dementia, and the dysmnesia that dementia patients and brain organic pathology are caused also have the improvement effect; The bitter western alkali of reviving also has the anticancer analgesic effect; Its derivative alkaloid Lars skin alkaloid ammonium salt is used for the treatment of digestive tract cancer, liver cancer, ovarian cancer, lung cancer, incidence cancer, malignant lymphoma etc., and is wherein better to cancer of the stomach, ovarian cancer curative effect.
At present, be raw material with the Stem or leaf of Shrub Lespedeza in the prior art, through loaded down with trivial details extraction separation purification step, only produce a kind of Stem or leaf of Shrub Lespedeza Ni Gelin alkali product, and the alkaloid that is rich in Soviet Union bitter western alkali and Lars skin basic active composition is fully not sharp, and production cycle length, productive rate yield height, product far also do not reach the high purity requirement of pharmaceutical grade.In a word, existing is that the alkaloidal method of raw material production has the following disadvantages with the Stem or leaf of Shrub Lespedeza: product is single, step is cumbersome, the cycle is long, the product recovery rate is not high, safety coefficient is low, cost is high, the Stem or leaf of Shrub Lespedeza resource utilization is not high and environmental pollution is serious, and is difficult for industrialization.
Summary of the invention
The object of the present invention is to provide a kind of alkaloidal method of from pulse family lespedeza plant, comprehensively extracting, this method product recovery rate height, cost is low, is convenient to suitability for industrialized production, can obtain 3 kinds of products simultaneously, and environmental pollution is little.
Provided by the inventionly extract alkaloidal method from pulse family lespedeza plant, its step comprises:
(1) adding volume in the Xingan Stem or leaf of Shrub Lespedeza after chopping is the process water of 6 times of amounts, add complex cellulase again, the mass percent of complex cellulase and leguminous plants Xingan Stem or leaf of Shrub Lespedeza is 0.1 ~ 0.5%, regulate pH value to 5 ~ 8, at 20 ~ 60 ℃ of following enzymolysis 12-36 of temperature hours, with lespedeza vegetable cell fragmentation behind the enzymolysis, filter again, obtain extracting solution;
(2) concentrate said extracted liquid, obtain extract concentrated solution, striking point is between 1.05～1.10 for the proportion of this concentrated solution when measuring for 20 ℃;
(3) by pH value substep said extracted thing concentrated solution is carried out ultrasonic extraction;
(4) will extract each product of obtaining of step is that 1: 70～1: 20 amount is carried out column chromatography respectively with thick product than filler, again with carrying out gradient elution;
(5) same composition that column chromatography for separation obtained merges, recrystallization repeatedly, Ni Gelin alkali, Lars skin alkali and the bitter western alkali high-purity monomer of reviving.
The inventive method has been carried out following optimization to technique scheme,
The component and the mass ratio of complex cellulase are in the step (1): cellulase 40-50%, polygalacturonase 30-40%, hemicellulase 20-30%.
Step (2) extracts by following process:
The first step extraction: the pH value of extract concentrated solution behind the enzymolysis is transferred to 3-5, adds 10～15 times of volume organic solvents,, extract 1～3 time with frequency 40KHz supersound extraction 50-65min.Extract 3 extraction liquids and merge organic solvent phase, the reclaim under reduced pressure organic solvent gets paste, and proportion is the first step extraction product between 1.05-1.1.
Second step extraction: the first step extraction product is added the pH value be transferred to 6.5-7.2, add 10～15 times of volume organic solvents, with frequency 40KHz supersound extraction 50-55min, extract 1-3 time, merge organic solvent phase, the reclaim under reduced pressure organic solvent gets paste, proportion is the second step extraction product between 1.05-1.1.
The 3rd step extraction: the pH value of the second step extraction product extract concentrated solution is transferred to 10-12.5, add 10～15 times of volume organic solvents, with frequency 40KHz supersound extraction 45-55min, extract 1-3 time, merge organic solvent phase, the reclaim under reduced pressure organic solvent gets paste, proportion is the 3rd step extraction product between 1.05-1.1.
The organic solvent of above-mentioned extraction usefulness is a kind of in methyl alcohol, ethanol, propyl carbinol, primary isoamyl alcohol, chloroform, the ethyl acetate.
The inventive method has following technique effect:
(1) extraction yield height, production stage is simple: adopt biological enzymolysis technology and ultrasonic technology that the vegetable cell wall part is decomposed, utilize again and adjust mechanical shear stress and whipping force, destroy plant cell tissue rapidly, the chemical ingredients of histocyte inside is fully contacted with solvent, make solution transfer effectively very much fast, reach inside and outside dissolution equilibrium in short period of time at the utmost point, both increased substantially alkaloidal stripping in the plant, impurity stripping when avoiding high temperature extraction again, improve filtyration velocity and purification effect in the production process, simplify the later separation purification step.The present invention and traditional extraction process relatively use the amount of soda acid and organic solvent to significantly reduce, thereby environmentally safe, cost are low.
(2) the stage extraction technology that adopts of the present invention with handicraft product was single different in the past, is divided into 3 sections according to the difference of PH with extract, obtains 3 kinds of main productss, and the comprehensive utilization Stem or leaf of Shrub Lespedeza has been improved the value of cash crop Stem or leaf of Shrub Lespedeza greatly.
(3) product purity height of the present invention: Ni Gelin alkali purity reaches 99.5%, Lars skin alkali purity reaches more than 99%, and the bitter western alkali alkali purity of reviving can reach 99%, reaches medicinal requirements, maybe can prepare medicinal its derivative.
Embodiment
Be illustrated for example below, these embodiment are just to the describing in further detail of the inventive method, and do not mean that any limitation of the invention.
Embodiment 1
(1) enzymolysis
Get the 10kg Stem or leaf of Shrub Lespedeza, pulverize the 10-20 order, add the 20kg aqueous solution, in multi-function extractor, transfer PH6.8, temperature is 40 ℃-45 ℃ of room temperatures, adds cellulase 6.25g, polygalacturonase 4.8g, hemicellulase 3.5g, enzymolysis 72 hours, after enzymolysis finishes, adopt the Mechanical Crushing technology that plant tissue is smashed to pieces, filter, filtrate is that 0.08-0.09MPa, 60 ℃ concentrate in vacuum tightness, obtain 1.2L proportion and the be 1.08 crude extract concentrated solution of (20 ℃ time).
The Mechanical Crushing technology can be that conventional organization is smashed homogenate technology and solid sample crushing technology to pieces.
(2) ultrasonic extraction
The first step extraction: transfer the PH to 6.8 of enzymolysis crude extract concentrated solution, add ether, consumption is respectively 1.8L, 1.2L, 0.9L, each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 3 times merges ether phase reclaim under reduced pressure ether, the step extraction product of winning;
The extraction of second step: the first step extraction product pH value to 9-10, is added ethyl acetate, and each consumption is 0.8L, each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 2 times, combined ethyl acetate phase reclaim under reduced pressure ethyl acetate gets the second step extraction product;
The extraction of the 3rd step: the second step extraction product pH value adds chloroform to 10.5-11.5, and consumption is respectively 1L, 0.8L, and each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 2 times, combined chloroform phase reclaim under reduced pressure chloroform get the 3rd step extraction product;
(3) separation and purification
The first step extraction product adds small amount of methanol dissolving, with product than 1: 40 upper prop of silica gel, sherwood oil: ethyl acetate (10: 1-1: 10) gradient elution, thin layer are followed the tracks of and are detected, and merge same composition, methyl alcohol-chloroform recrystallization, dry 1.8g Ni Gelin alkali.
The second step extraction product adds small amount of methanol dissolving, is 1: 10 upper prop with product than silica gel, and respectively with water, 40% methyl alcohol, 65% methyl alcohol, pure methyl alcohol, ethanol elution, thin layer are followed the tracks of and detected, and merge same composition, the n-propyl alcohol recrystallization, dry 1.7g Lars skin alkali.
The 3rd step extraction product adds small amount of methanol solution, is 1: 60 upper prop with product than diatomite, and with sherwood oil, ethyl acetate, acetone, methyl alcohol wash-out respectively, thin layer is followed the tracks of to detect and merged same composition, the n-propyl alcohol recrystallization, dry the 6.8g bitter western alkali of reviving.
Embodiment 2
(1) enzymolysis
Get the dried Stem or leaf of Shrub Lespedeza of 10kg, the 30kg aqueous solution, in multi-function extractor, transfer PH between 5-6, temperature is 40 ℃, adds cellulase 4.2g, polygalacturonase 3.3g, hemicellulase 2.5g, enzymolysis after 24 hours using-system smash the broken sample of homogenate to pieces, filter, filtrate is that 0.08-0.09MPa, 65 ℃ concentrate in vacuum tightness, obtain 1.26L proportion and be 1.05 extract concentrated solution of (20 ℃ time).
(2) ultrasonic extraction
The first step extraction: transfer the PH to 5.5 of extract concentrated solution, add propyl carbinol, consumption is respectively 0.5L, 0.4L, 0.3L, each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 3 times merges the propyl carbinol phase, the reclaim under reduced pressure propyl carbinol, the step extraction product of winning;
The extraction of second step: the first step extract accent pH value to 8-10, is added diethyl ether, and consumption is respectively 0.6L, 0.4L, 0.4L, and each assisting with frequency 40KHz supersound extraction 50-55min merges ether phase reclaim under reduced pressure ether, gets the second step extraction product;
The extraction of the 3rd step: the second step extraction product pH value adds chloroform to 11.5-12.5, and consumption is respectively 1L, 0.8L, and each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 2 times, combined chloroform phase reclaim under reduced pressure chloroform get the 3rd step extraction product;
(3) separation and purification
The first step extraction product adds the small amount of methanol dissolving, ammoniacal liquor alkalizes to PH8.5, ethyl acetate extraction 3 times, and it all is 0.6L that each consumption divides, combined ethyl acetate phase reclaim under reduced pressure, with product than 1: 60 upper prop of macroporous resin, water, 30%, 50%, 70% ethanol elution, thin layer is followed the tracks of and is detected, merge same composition, the ether recrystallization, dry 1.4g Ni Gelin alkali.
The second step extraction product adds small amount of methanol dissolving, is 1: 10 upper prop with product than silica gel, and respectively with sherwood oil, chloroform, dehydrated alcohol wash-out, thin layer are followed the tracks of and detected, and merge same composition, ethyl alcohol recrystallization, dry 7.8g Lars skin alkali.
The 3rd step extraction product adds small amount of methanol dissolving, is 1: 50 upper prop with the product ratio aluminum oxide, and with sherwood oil, acetone, methyl alcohol wash-out respectively, thin layer is followed the tracks of and detected, and merges same composition, the n-propyl alcohol recrystallization, dry the 15.6g bitter western alkali of reviving.
Embodiment 3
(1) enzymolysis
Get the 10kg Stem or leaf of Shrub Lespedeza, the 40kg aqueous solution, transfer PH between 7-8, temperature is 60 degree, adds cellulase 11 g, polygalacturonase 7g, hemicellulase 5g, enzymolysis 12 hours, used the broken sample of high-shear flash extracter 30 minutes, and filtered, filtrate is at vacuum tightness power 0.08-0.09MPa, 70 ℃ concentrate, obtain 1.3L proportion and be 1.01 extract concentrated solution of (20 ℃ time).
(2) ultrasonic extraction
The first step extraction: transfer the PH to 7.0 of enzymatic hydrolyzed extract concentrated solution, add chloroform, each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 3 times, each chloroform consumption is 0.4L, combined chloroform phase, reclaim under reduced pressure chloroform, the step extraction product of winning;
The extraction of second step: the first step extract is transferred PH to 10.5-10.8, and ethyl acetate extraction 3 times, consumption are 0.6L, 0.5L and 0.4L, each auxiliary with frequency 40KHz supersound extraction 50-55min, number of times 3 times, combined ethyl acetate phase, the reclaim under reduced pressure ethyl acetate obtains the second step extract;
The extraction of the 3rd step: the second step extract is transferred PH to 10.8-11.2, extracted with diethyl ether, and consumption is 0.4L, and it is auxiliary at every turn that number of times 3 times merges the ether phase with frequency 40KHz supersound extraction 50-55min, and the reclaim under reduced pressure ether obtains the 3rd step extract;
(3) separation and purification
The first step extraction product adds the small amount of methanol dissolving, ammoniacal liquor alkalizes to PH10,3 extractions of ethyl acetate, combined ethyl acetate phase reclaim under reduced pressure ethyl acetate, with 1: 45 upper prop of product ratio aluminum oxide, sherwood oil, ethyl acetate, methyl alcohol is wash-out respectively, thin layer is followed the tracks of and is detected, merge same composition, the alcohol-water recrystallization is dried to such an extent that 2.1g Ni Gelin subtracts alkali.
The second step extraction product adds small amount of methanol dissolving, is 1: 40 upper prop with product than silica gel, and respectively with sherwood oil, chloroform, dehydrated alcohol wash-out, thin layer are followed the tracks of and detected, and merge same composition, and ethyl alcohol recrystallization is dried to such an extent that the 7.3g bitter west of reviving subtracts.
The 3rd step extraction product adds less water dissolving, is 1: 30 upper prop with the product ratio aluminum oxide, and with sherwood oil, acetone, methyl alcohol wash-out respectively, thin layer is followed the tracks of and detected, and merges same composition, the n-propyl alcohol recrystallization, dry the 11.6g lycoramine.
Claims (8)
1. one kind is utilized the alkaloidal method of extraction in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that its step is as follows:
(1) adding volume in the Xingan Stem or leaf of Shrub Lespedeza after chopping is the process water of 6 times of amounts, add complex cellulase again, the mass percent of complex cellulase and leguminous plants Xingan Stem or leaf of Shrub Lespedeza is 0.1 ~ 0.5%, regulate pH value to 5 ~ 8, at 20 ~ 60 ℃ of following enzymolysis 12-36 of temperature hours, with lespedeza vegetable cell fragmentation behind the enzymolysis, filter again, obtain extracting solution;
(2) concentrate said extracted liquid, obtain extract concentrated solution, striking point is between 1.05～1.10 for the proportion of this concentrated solution when measuring for 20 ℃;
(3) by pH value substep said extracted thing concentrated solution is carried out ultrasonic extraction;
(4) will extract each product of obtaining of step is that 1: 70～1: 20 amount is carried out column chromatography respectively with thick product than filler, again with carrying out gradient elution;
(5) same composition that column chromatography for separation obtained merges, recrystallization repeatedly, Ni Gelin alkali, Lars skin alkali and the bitter western alkali high-purity monomer of reviving.
2. according to claim 1 and 2 utilize in the stage extraction enriching and purifying lespedeza plant extracted its speciality of alkaloidal method and is: in the described step (1), cross 10～80 order teachers behind the Stem or leaf of Shrub Lespedeza powder and carry out enzymolysis again.
3. according to claim 1 and 2 the utilization extracted alkaloidal method in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that: in affiliated step (1), the used enzyme of enzymolysis is a complex cellulase, and enzymolysis time is 12～36 hours.
4. according to claim 3 the utilization extracted alkaloidal method in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that: in the described step (1), enzymolysis process is pickling process, reflux extraction, one or more in percolation, enzymolysis process, the continuous extraction.
5. according to claim 3 the utilization extracted alkaloidal method in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that; In the affiliated step (1), the mass percent of complex cellulase and leguminous plants Xingan Stem or leaf of Shrub Lespedeza is 0.1 ~ 0.5%.
6. according to claim 5 the utilization extracted alkaloidal method in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that: in described step (3), regulating the used alkali of pH value is sodium hydroxide and ammoniacal liquor.
7. according to claim 6 the utilization extracted alkaloidal method in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that: will extract each product of obtaining of step in the step (4) is that 1: 70～1: 20 amount is carried out column chromatography respectively with thick product than filler, again with carrying out gradient elution;
8. according to claim 7 the utilization extracted alkaloidal method in the stage extraction method enriching and purifying lespedeza plant, it is characterized in that: in step (5), the same composition that column chromatography for separation is obtained merges, recrystallization repeatedly, Ni Gelin alkali, Lars skin alkali and the bitter western alkali high-purity monomer of reviving.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102550544A (en) * | 2011-11-30 | 2012-07-11 | 新疆汇通旱地龙腐植酸有限责任公司 | Dry alhagi sparsifolia alkaline essential oil and extraction and application thereof |
| CN103202884A (en) * | 2013-04-03 | 2013-07-17 | 西安交通大学 | Method for extracting hovenaia dulcis total alkaloid by semi-bionic-enzymic method |
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| CN102550544A (en) * | 2011-11-30 | 2012-07-11 | 新疆汇通旱地龙腐植酸有限责任公司 | Dry alhagi sparsifolia alkaline essential oil and extraction and application thereof |
| CN103202884A (en) * | 2013-04-03 | 2013-07-17 | 西安交通大学 | Method for extracting hovenaia dulcis total alkaloid by semi-bionic-enzymic method |
| CN103202884B (en) * | 2013-04-03 | 2014-09-03 | 西安交通大学 | Method for extracting hovenaia dulcis total alkaloid by semi-bionic-enzymic method |
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