CN102600247A - Corydalis saxicola bunting alkaloid extract and preparation method thereof, as well as extraction method of dehydrocavidine - Google Patents
Corydalis saxicola bunting alkaloid extract and preparation method thereof, as well as extraction method of dehydrocavidine Download PDFInfo
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- CN102600247A CN102600247A CN2012101042074A CN201210104207A CN102600247A CN 102600247 A CN102600247 A CN 102600247A CN 2012101042074 A CN2012101042074 A CN 2012101042074A CN 201210104207 A CN201210104207 A CN 201210104207A CN 102600247 A CN102600247 A CN 102600247A
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Abstract
The invention discloses a corydalis saxicola bunting alkaloid extract and a preparation method thereof, as well as an extraction method of dehydrocavidine on the basis the alkaloid extract. According to the method, in the raw material pretreatment, pectinase and cellulose perform enzyme hydrolysis to the medicament, so that the cellulose component and pectin component in plant tissues are destroyed, the solubility of effective components is improved, and the content of total alkaloids in the extract is greatly increased. The content of the total alkaloids extracted by using a twice chromatography method is over 95 percent, and only ethanol is used as a solvent; and the dehydrocavidine monomer is extracted from the total alkaloids by adopting a high-speed counter-current chromatography technology, and the purity is over 99 percent.
Description
Technical field
The present invention relates to biological technical field, especially, relate to the method for distilling of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract and method for preparing, deydrokaividing.
Background technology
Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) (Corydalis saxicola Bunting), Papaveraceae Corydalis plant, perennial herb.Have that significant analgesia is stable, antibiotic, antiinflammatory, antitumor, antiviral, function of gallbladder promoting, immunoregulation effect, can be used for treatment of diseases such as acute icterohepatitis, viral hepatitis, chronic hepatitis, hyperbilirubinemia, liver cirrhosis, hepatocarcinoma clinically.Its effective ingredient is that (Corydalis Saxicolae dehydrocavidine) is main multiple alkaloid, and the alkaloid height directly influences the quality and the curative effect of medical material with deydrokaividing.
The Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) plant distributes and is confined to Limestone Mountain Areas, belongs to the tor endemic species.Now resource is few, only is grown in county such as Fengshan and the Guizhou of northwest, osmanthus, the dark and damp grotto mouth in limestone area of south of Yunnan under the wild state.Because habitat conditions is abominable, its natural propagation rate is very low, population development difficulty, and the resource reserves are very limited.In recent years,, start with from the grotto artificial culture through scientific and technical personnel's research, the characteristic unique according to Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae), the natural imitation environment carries out artificial culture, has obtained success, makes the Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) production of medicine that stable raw material sources arranged.Process at present the injection and the tablet of total alkaloids of meadowrueleaf corydalis root, be used for clinically, become the current particularly special effect medicine of hepatitis B, liver cirrhosis, hepatocarcinoma etc. of hepatitis that cures mainly.
Traditional Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extracting technique is: Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) is cleaned dried, be ground into coarse powder, with 5 times of amount 90% ethanol; Flooded reflux, extract, 4 times, each 1 hour 24 hours; Merge extractive liquid,, decompression recycling ethanol also is concentrated to the clear paste that relative density is 0.92～1.02 (60 ℃), puts cold; Filter, filtrating is concentrated into the thick paste that relative density is 1.16～1.20 (60 ℃).In thick paste, add an amount of distilled water, regulate pH value 2～3, stir fully, filter, wash 4～5 times with 10% hydrochloric acid solution with 10% hydrochloric acid solution; Merge acid liquid, regulate pH value 9～10 with sodium carbonate, separate out brown precipitation, filter, filtering residue is with 5 times of chloroform extraction 5～6 times; Combined chloroform liquid adds anhydrous sodium sulfate dehydration, filters, and reclaims chloroform and does near; Add a small amount of ethanol again, decompression and solvent recovery, drying promptly gets.This technology exists that yield is low, labor intensity is big, be prone to cause shortcoming such as environmental pollution, and has used chloroform in the technology, and the serious harm producers' is healthy.
Summary of the invention
Technical problem to be solved by this invention provides the method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract, and this method for preparing has improved the dissolution rate of effective ingredient, thereby has improved total alkaloid contents in the extract greatly.
Technical problem to be solved by this invention also is the Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract that provides method for preparing to obtain, and the total alkaloid content in this Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract is more than 95%, and the solvent of use has only ethanol.
Technical problem to be solved by this invention also is to provide the method for distilling of deydrokaividing, the deydrokaividing monomer that this method for distilling obtains, and purity is greater than 99%.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: the method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract comprises:
Step 1) is ground into coarse powder with Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae), drops in the extraction container; Heat up after the buffer of 20 times of weight of adding, add compound enzyme and obtain the pretreatment mixture, the pH of said buffer is 2.5～6.0; The consumption of said compound enzyme is 2～5%; Make said pretreatment mixture in 35～65 ℃ of temperature, insulation 1～16 hour, with dilute hydrochloric acid said pretreatment mixture pH value is adjusted to 2～3 then, in 35～65 ℃ of temperature, insulation 1～10 hour; Then 4000r/min is centrifugal, collects supernatant as extracting solution; Said compound enzyme is the compositions that comprises cellulase and pectase;
Step 2), make extracting solution that said step 1) obtains through macroporous adsorbent resin, using concentration is that 20%～80% alcoholic solution is resolved as eluent, collects the solution after the desorbing, obtains thick total alkaloids after the drying.
In the technique scheme, said extraction container can slective extraction jar or other suitable containers that extracts.The consumption 2～5% of said compound enzyme be meant said compound enzyme weight with respect to the weight of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) and buffer and ratio, i.e. the weight of compound enzyme/(weight of the weight+buffer of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae))=2～5%.Said compound enzyme is the compositions that comprises cellulase and pectase, the ratio preferred cellulose enzyme of wherein said cellulase and pectase: pectase=1: 0.5～4.5.In this proportion, the dissolution rate of effective ingredient significantly improves, thereby has improved total alkaloid contents in the extract greatly.
Said buffer is selected from a kind of or its combination in sodium phosphate buffer, kaliumphosphate buffer, glycine-hydrochloride buffer, acetic acid-sodium acetate buffer, citric acid-sodium hydroxide-hydrochloride buffer, the citric acid-sodium citrate buffer.
The model of said macroporous adsorbent resin is selected from any among HZ-806, AB-8, DA-201, HPD-400, NKA-9, S-8, the HPD-500.
The method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract of the present invention also comprises step 3), with said step 2) the thick total alkaloids that obtains dissolves with buffer, on Sephadex LH-20 detached dowel, makes with extra care, and obtains the total alkaloids after refining; Said buffer is selected from a kind of or its combination in sodium phosphate buffer, kaliumphosphate buffer, glycine-hydrochloride buffer, acetic acid-sodium acetate buffer, citric acid-sodium hydroxide-hydrochloride buffer, the citric acid-sodium citrate buffer.Said purified concrete steps are: thick total alkaloids is used the eluent eluting, the eluting peak of collection of biological alkali under the monitoring of detector then with buffer dissolving back upper prop.
The present invention solves the problems of the technologies described above the technical scheme that is adopted and also is, the Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract adopts method for preparing to obtain.
Alkaloid in the said Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract is more than 95%.
The present invention solves the problems of the technologies described above the technical scheme that is adopted and also is; The method for distilling of deydrokaividing; Total alkaloids behind prepared obtain refining of said method is separated through high speed adverse current chromatogram, obtain the deydrokaividing monomer, purity reaches more than 99%; The solvent system of said high speed adverse current chromatogram is selected from any in normal hexane-methanol-water, normal hexane-ethyl acetate-methanol-water, normal hexane-acetonitrile-water, n-butyl alcohol-methanol-water, n-butyl alcohol-acetic acid-water, ethyl acetate-methanol-water, the ethyl acetate-n-butanol-water.
In sum, the present invention compared with prior art has following advantage:
(1), in raw-material pretreatment; Cooperate pectase that medical material is carried out enzyme hydrolysis with cellulase; Destroy cellulose components and the pectin composition in the plant tissue, improved the dissolution rate of effective ingredient, thereby improved total alkaloid contents in the extract greatly.
(2), the total alkaloids that obtains behind the refining purification, content reaches more than 95%, the solvent of use has only ethanol.
(3), adopt high-speed countercurrent chromatography from total alkaloids, to extract the deydrokaividing monomer, purity is greater than 99%.
Description of drawings
Fig. 1 is a process chart of the present invention.
The specific embodiment
Following embodiment can make those skilled in the art more fully understand the present invention, but does not limit the present invention in any way.
Embodiment
The preparation of the thick total alkaloids of embodiment 1 Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae)
After cleaning, exsiccant Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) pulverized with pulverizer is preliminary, drop into extractor, add 20 times of weight sodium phosphate buffer (pH4.0,0.2mol/L), temperature is risen to 45 ℃; (cellulase: pectase=1: 1), consumption is 2%, slowly stirs to add compound enzyme; After 45 ℃ of temperature kept 8 hours, stopped reaction was adjusted to 2.0 with dilute hydrochloric acid with pH value; Be incubated 8 hours, 4000r/min is centrifugal, collects extracting solution.Extracting solution is crossed AB-8 macroporous adsorptive resins absorption total alkaloids, and the desorbing eluent is 55% ethanol water.Solution after the desorbing obtains thick total alkaloids after reclaiming ethanol, drying.Total alkaloid content in this thick total alkaloids is 65.8%.Wherein, the thick total alkaloids quality * 100% of total alkaloid contents=total alkaloids amount/elute from resin
Wherein, said AB-8 macroporous adsorptive resins can use any model among HZ-806, DA-201, HPD-400, NKA-9, S-8, the HPD-500 to substitute.Said sodium phosphate buffer also can be substituted by a kind of or its combination in kaliumphosphate buffer, glycine-hydrochloride buffer, acetic acid-sodium acetate buffer, citric acid-sodium hydroxide-hydrochloride buffer, the citric acid-sodium citrate buffer.
The preparation of the thick total alkaloids of embodiment 2 Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae)s
After cleaning, exsiccant Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) pulverized with pulverizer is preliminary, drop into extractor, add 25 times of weight sodium acetate-acetate buffer (pH4.5,0.2mol/L), temperature is risen to 45 ℃; (cellulase: pectase=1: 1), consumption is 3%, slowly stirs to add compound enzyme; Kept 8 hours for 45 ℃ in temperature, stopped reaction is adjusted to 2.0 with dilute hydrochloric acid with pH value; Be incubated 8 hours, 4000r/min is centrifugal, collects extracting solution.Extracting solution is crossed DA-201 macroporous adsorptive resins absorption total alkaloids, and the desorbing eluent is 50% ethanol water.Solution after the desorbing obtains thick total alkaloids after reclaiming ethanol, drying.Total alkaloid content in this thick total alkaloids is 66.2%.
The embodiment 3 refining total alkaloidss that obtain
The thick total alkaloids that embodiment 1 or embodiment 2 obtain is with the glycine-hydrochloride buffer dissolving (pH3.0 of 10 times of amounts; 0.1mol/L); On Sephadex LH-20 detached dowel, make with extra care; The eluting peak of collection of biological alkali under the monitoring of detector, the alkaloid after obtaining refining purification after desalination, the drying, content is 96.8%.
The monomeric method for distilling of embodiment 4 deydrokaividings
With embodiment 3 obtain refining after total alkaloids be sample, further separating dehydrogenated Ka Weiding on high-speed counter-current chromatograph, the solvent system of employing is normal hexane-methanol-water; Rotating speed 800r/min; Flow velocity 2.0mL/min, 35 ℃ of temperature, separating medium obtains the deydrokaividing monomer after reclaiming solvent, drying; Detect through HPLC, monomer purity is 99.3%.
The monomeric method for distilling of embodiment 5 deydrokaividings
With the total alkaloids is sample, further separating dehydrogenated Ka Weiding on high-speed counter-current chromatograph, and the solvent system of employing is normal hexane-acetonitrile-water; Rotating speed 850r/min; Flow velocity 2.0mL/min, 35 ℃ of temperature, separating medium obtains the deydrokaividing monomer after reclaiming solvent, drying; Detect through HPLC, monomer purity is 99.6%.
The ratio of cellulase and pectase is to the influence of total alkaloid content in embodiment 6 compound enzymes
After cleaning, exsiccant Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) pulverized with pulverizer is preliminary, drop into extractor, add 20 times of weight sodium phosphate buffer (pH4.0,0.2mol/L), temperature is risen to 45 ℃; Add compound enzyme (ratio of cellulase and pectase is seen table 1), consumption is 4%, slowly stirs; After 55 ℃ of temperature kept 10 hours, stopped reaction was adjusted to 2.0 with dilute hydrochloric acid with pH value; Be incubated 10 hours, 4000r/min is centrifugal, collects extracting solution.Extracting solution is crossed AB-8 macroporous adsorptive resins absorption total alkaloids, and the desorbing eluent is 55% ethanol water.Solution after the desorbing obtains thick total alkaloids after reclaiming ethanol, drying.
| Cellulase: pectase | Total alkaloid contents | |
| Experiment 1 | 1∶0.5 | 61.2％ |
| Experiment 2 | 1∶2 | 65.3％ |
| Experiment 3 | 1∶3 | 66.4％ |
| Contrast 1 | 1∶0.3 | 52.1％ |
| Contrast 2 | 1∶5 | 55.3％ |
| Contrast 3 | 1∶5.5 | 53.5％ |
In the last table, the ratio of said cellulase and pectase is its mass ratio.
The thick total alkaloids quality * 100% of total alkaloid contents=total alkaloids amount/elute from resin
Said total alkaloid contents is meant the total alkaloid contents in the resultant thick total alkaloids, and it has reacted the dissolution rate of effective ingredient in the preparation process.
So in the compound enzyme according to the invention, the ratio preferred cellulose enzyme of cellulase and pectase: pectase=1: 0.5～4.5.In this proportion, the dissolution rate of effective ingredient significantly improves, thereby has improved total alkaloid contents greatly.
As stated, realize the present invention preferably.
Claims (7)
1. the method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract is characterized in that, comprising:
Step 1) is ground into coarse powder with Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae), drops in the extraction container; Heat up after the buffer of 20 times of weight of adding, add compound enzyme and obtain the pretreatment mixture, the pH of said buffer is 2.5～6.0; The consumption of said compound enzyme is 2～5%; Make said pretreatment mixture in 35～65 ℃ of temperature, insulation 1～16 hour, with dilute hydrochloric acid said pretreatment mixture pH value is adjusted to 2～3 then, in 35～65 ℃ of temperature, insulation 1～10 hour; Then 4000r/min is centrifugal, collects supernatant as extracting solution; Said compound enzyme is the compositions that comprises cellulase and pectase;
Step 2), make extracting solution that said step 1) obtains through macroporous adsorbent resin, using concentration is that 20%～80% alcoholic solution is resolved as eluent, collects the solution after the desorbing, obtains thick total alkaloids after the drying.
2. the method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract according to claim 1; It is characterized in that said buffer is selected from a kind of or its combination in sodium phosphate buffer, kaliumphosphate buffer, glycine-hydrochloride buffer, acetic acid-sodium acetate buffer, citric acid-sodium hydroxide-hydrochloride buffer, the citric acid-sodium citrate buffer.
3. the method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract according to claim 2 is characterized in that, the model of said macroporous adsorbent resin is selected from any among HZ-806, AB-8, DA-201, HPD-400, NKA-9, S-8, the HPD-500.
4. the method for preparing of Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract according to claim 3; It is characterized in that, also comprise step 3), with said step 2) the thick total alkaloids that obtains dissolves with buffer; On Sephadex LH-20 detached dowel, make with extra care the total alkaloids after obtaining making with extra care; Said buffer is selected from a kind of or its combination in sodium phosphate buffer, kaliumphosphate buffer, glycine-hydrochloride buffer, acetic acid-sodium acetate buffer, citric acid-sodium hydroxide-hydrochloride buffer, the citric acid-sodium citrate buffer.
5. the Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract is characterized in that, adopts aforesaid right to require each method of 1-4 to prepare.
6. Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract according to claim 5 is characterized in that, the total alkaloid content in the said Corydalis thalictrifolia Franch. (Radix Corydalis Thalictrifoliae) alkaloid extract is more than 95%.
7. the method for distilling of deydrokaividing is characterized in that, requires the 4 prepared total alkaloidss that obtain to separate through high speed adverse current chromatogram aforesaid right, obtains the deydrokaividing monomer, and purity reaches more than 99%; The solvent system of said high speed adverse current chromatogram is selected from any in normal hexane-methanol-water, normal hexane-ethyl acetate-methanol-water, normal hexane-acetonitrile-water, n-butyl alcohol-methanol-water, n-butyl alcohol-acetic acid-water, ethyl acetate-methanol-water, the ethyl acetate-n-butanol-water.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105055534A (en) * | 2015-08-07 | 2015-11-18 | 赵明东 | Production method of high purity Corydalis saxicola Bunting alkaloid |
| CN108014517A (en) * | 2017-12-20 | 2018-05-11 | 泰州医药城国科化物生物医药科技有限公司 | A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying |
| CN109125258A (en) * | 2018-10-25 | 2019-01-04 | 华润三九（南昌）药业有限公司 | A kind of preparation method of intensified loquet distillate |
| CN114191467A (en) * | 2021-12-06 | 2022-03-18 | 广西师范大学 | Method for extracting high-purity alkaloid from corydalis saxicola bunting |
| CN114402978A (en) * | 2021-12-07 | 2022-04-29 | 广西壮族自治区药用植物园 | Method for improving dehydrocavidine content in corydalis saxicola bunting |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105055534A (en) * | 2015-08-07 | 2015-11-18 | 赵明东 | Production method of high purity Corydalis saxicola Bunting alkaloid |
| CN105055534B (en) * | 2015-08-07 | 2018-08-10 | 赵明东 | The production method of high-purity meadowrueleaf corydalis root alkaloid |
| CN108014517A (en) * | 2017-12-20 | 2018-05-11 | 泰州医药城国科化物生物医药科技有限公司 | A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying |
| CN109125258A (en) * | 2018-10-25 | 2019-01-04 | 华润三九（南昌）药业有限公司 | A kind of preparation method of intensified loquet distillate |
| CN109125258B (en) * | 2018-10-25 | 2020-09-18 | 华润三九（南昌）药业有限公司 | Preparation method of strong loquat syrup |
| CN114191467A (en) * | 2021-12-06 | 2022-03-18 | 广西师范大学 | Method for extracting high-purity alkaloid from corydalis saxicola bunting |
| CN114191467B (en) * | 2021-12-06 | 2023-06-06 | 广西师范大学 | Method for extracting high-purity alkaloid from corydalis saxicola bunting |
| CN114402978A (en) * | 2021-12-07 | 2022-04-29 | 广西壮族自治区药用植物园 | Method for improving dehydrocavidine content in corydalis saxicola bunting |
| CN114402978B (en) * | 2021-12-07 | 2023-02-28 | 广西壮族自治区药用植物园 | Method for improving dehydrocavidine content in corydalis saxicola bunting |
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