CN108392540B - Method for extracting total alkaloids of datura flower - Google Patents
Method for extracting total alkaloids of datura flower Download PDFInfo
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- CN108392540B CN108392540B CN201810336919.6A CN201810336919A CN108392540B CN 108392540 B CN108392540 B CN 108392540B CN 201810336919 A CN201810336919 A CN 201810336919A CN 108392540 B CN108392540 B CN 108392540B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
Abstract
The invention relates to a method for extracting total alkaloids from datura flower, which comprises the following steps: pulverizing 100g flos Daturae Metelis, and sieving; mixing 1-butyl-3-methylimidazole lactate 3-8 g, PEG 300-1000 2-6 g and deionized water 200g, and adjusting the pH value to 3-5 by using lactic acid to obtain an acid leaching solution; adding the acid leaching solution into flos daturae, adding 0.8-1.5 g of acid cellulase and 0.5-0.8 g of acid pectinase, uniformly mixing, keeping the temperature at 35-55 ℃, stirring for 1-4 h, filtering, washing filter residues with hydrochloric acid aqueous solution with the pH of 2-3, and combining filtrates; adding 1% ammonium sulfate and 2% NaCl into the filtrate, adding ethyl acetate, extracting for 3 times, removing an ethyl acetate phase, adjusting the pH of an alkaloid-enriched phase to 9-11 with ammonia water, extracting for 3 times with chloroform, and recovering the chloroform under reduced pressure to obtain total alkaloids.
Description
Technical Field
The invention relates to a method for extracting total alkaloids of datura flower.
Background
Flos Daturae Metelis is dried flower of Datura stramonium L of Solanaceae, has effects of relieving asthma and cough, relieving pain, and relieving spasm, contains tropane alkaloid as main ingredient, has strong physiological activity, and also contains withanolide steroid and flavonoid as main ingredient, and is difficult to separate in extraction. How to fully extract the effective components of the alkaloid and improve the utilization rate is the key for improving the quality of the finished product. The common researches on extracting the datura flower alkaloid mainly focus on immersion extraction, reflux extraction and microwave extraction (Chinese patent medicine, 34 th volume of 5 months, 2012, 5 th period), wherein the immersion extraction is based on that free alkaloid can be dissolved in alcohol, chloroform, ether and other organic solvents, and alkaloid salts, particularly inorganic acid salts, are easily soluble in water and insoluble or difficultly soluble in common organic solvents; by utilizing the properties of alkaloid, dissolving the alcohol extract with acid water, removing insoluble impurities, alkalifying with alkali liquor to convert alkaloid salt into free alkali, extracting with organic solvent, mixing organic solvent extractive solutions, and concentrating to obtain total alkaloid. The research shows that the extraction rate is not ideal, and in recent years, the research on how to improve the extraction rate of the alkaloid in the datura flower is not many.
CN201610422547.X discloses a method for extracting and purifying scopolamine from flos Daturae Metelis, which comprises obtaining leaching solution of flos Daturae Metelis; filtering the leaching solution by a ceramic membrane, concentrating by a nanofiltration membrane, and recovering ethanol to obtain a mixture; dissolving the mixture with dilute hydrochloric acid, and filtering to obtain filtrate; extracting the filtrate and keeping the aqueous solution; after the pH value of the water phase is adjusted, extracting for four times to obtain a chloroform solution; drying the chloroform solution, and recovering chloroform to obtain total alkaloids; separating the total alkaloids by basic alumina column chromatography, and recovering chloroform to obtain scopolamine.
CN201610756028.7 discloses an optimal microwave extraction method of flos Daturae Metelis effective components, which adopts parameters such as microwave power, microwave radiation time, solvent dosage, soaking time, extraction temperature, etc. to extract dry extract, total alkaloids and scopolamine of flos Daturae Metelis; the contents of total alkaloids and scopolamine are determined by acid dye colorimetry and high performance liquid chromatography, and the yield of dry extract is determined by mass method. The optimal process condition for microwave extraction of flos Daturae Metelis is to soak 10 times of medicinal materials in 75% ethanol for 2.0h, and continuously radiate for 25min under the microwave condition of 730W power and 65 deg.C temperature.
In view of the above, it is necessary to invent a method for extracting total alkaloids from datura flower with high purity.
Disclosure of Invention
The invention aims to provide a method for extracting total alkaloids in datura flower, which is simple to operate and high in extraction rate.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) pulverizing 100g flos Daturae Metelis, and sieving;
(2) mixing 1-butyl-3-methylimidazole lactate 3-8 g, PEG 300-1000 2-6 g and deionized water 200g, and adjusting the pH value to 3-5 by using lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos daturae treated in the step (1), adding 0.8-1.5 g of acid cellulase and 0.5-0.8 g of acid pectinase, uniformly mixing, keeping the temperature at 35-55 ℃, stirring for 1-4 h, filtering, washing filter residues with hydrochloric acid aqueous solution with the pH of 2-3, and combining filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, adding ethyl acetate, extracting for 3 times, removing an ethyl acetate phase, adjusting the pH of an alkaloid-enriched phase to 9-11 with ammonia water, extracting for 3 times with chloroform, and recovering chloroform under reduced pressure to obtain the total alkaloids.
As a further improvement of the invention, the pH value of the step (2) is 3.5-4.5.
As a further improvement of the invention, the mass ratio of the 1-butyl-3-methylimidazole lactate to the PEG to the deionized water in the step (2) is 5.5:4: 200.
As a further improvement of the invention, the adding amount of the acid cellulase in the step (3) is 1.2g, and the adding amount of the acid pectinase is 0.7 g.
As a further improvement of the invention, the pH in the step (4) is 10.
As a further improvement of the invention, the volume ratio of the ethyl acetate used for 3 times to the filtrate is respectively as follows: 2-3: 2-2.5: 1.5-2.5.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:
the scheme provided by the invention improves the extraction rate of the total alkaloids in the datura flower by utilizing the principle of acid dissolution and alkali precipitation. Through carrying out wall breaking on the datura flower under the acidic condition, and matching with the 1-butyl-3-methylimidazole lactate and PEG, the activity of enzyme is ensured, meanwhile, the full dissolution of active ingredients can be effectively ensured, the wall breaking and the extraction are carried out simultaneously, the dissolubility of alkaloid is ensured in the suitable acidic environment, and the extraction separation is more facilitated.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail and fully with reference to the following embodiments.
Example 1
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, respectively extracting with 2 times volume amount of ethyl acetate of the filtrate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume amount of chloroform for 3 times, recovering chloroform under reduced pressure, and drying to obtain total alkaloid dry extract 16.2g with total alkaloid extraction rate of 0.447%.
Example 2
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 3g, PEG800 2g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, respectively extracting with 2 times volume amount of ethyl acetate of the filtrate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume amount of chloroform for 3 times, recovering chloroform under reduced pressure, and drying to obtain total alkaloid dry extract 15.6g with total alkaloid extraction rate of 0.428%.
Example 3
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 8g, PEG1000 6g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 40 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with chloroform for 3 times, recovering chloroform under reduced pressure, and drying to obtain total alkaloid dry extract 16.4g with total alkaloid extraction rate of 0.449%.
Example 4
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG300 4g and deionized water 200g, and adjusting pH to 4.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 0.8g of acid cellulase and 0.5g of acid pectinase, mixing uniformly, keeping the temperature at 55 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, respectively extracting with 2 times volume of ethyl acetate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 11 with ammonia water, extracting with chloroform for 3 times, recovering chloroform under reduced pressure, and drying to obtain total alkaloid dry extract 16.0g with total alkaloid extraction rate of 0.439%.
Example 5
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG600 4g and deionized water 200g, and adjusting pH to 5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.5g of acid cellulase and 0.8g of acid pectinase, mixing uniformly, keeping the temperature at 35 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, respectively extracting with 2 times volume of ethyl acetate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 9 with ammonia water, extracting with chloroform for 3 times, recovering chloroform under reduced pressure, and drying to obtain total alkaloid dry extract 15.4g with total alkaloid extraction rate of 0.421%.
Example 6
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 8g, PEG400 2g and deionized water 200g, and adjusting pH to 4 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 35 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH3, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, respectively extracting with 2 times volume amount of ethyl acetate of the filtrate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume amount of chloroform for 3 times, recovering chloroform under reduced pressure, and drying to obtain total alkaloid dry extract 15.7g with total alkaloid extraction rate of 0.433%.
Comparative example 1
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) taking 200g of deionized water, and adjusting the pH value to 3.5 by using lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 2
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 3
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 4g of PEG400 and 200g of deionized water, and adjusting the pH to 3.5 by using lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 4
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with hydrochloric acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 5
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG1500 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 6
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 10g, PEG300 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 7
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG300 8g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 8
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 9
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) adding flos Daturae Metelis into 200g deionized water, adding 1.2g cellulase and 0.7g pectinase, stirring for 2.5 hr, adjusting pH to 3.5 with hydrochloric acid, adding 5.5g 1-butyl-3-methylimidazole lactate and 4g PEG400, stirring, filtering, washing the residue with hydrochloric acid aqueous solution of pH2.5, and mixing filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 10
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with water solution, and combining the filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, respectively, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 11
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) extracting with 2 times volume of ethyl acetate of the filtrate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 12
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 3% ammonium sulfate into the filtrate, extracting with 2 times volume of ethyl acetate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
Comparative example 13
A method for extracting total alkaloids from flos Daturae Metelis comprises the following steps:
(1) crushing 100g of hindu datura flower, and sieving the crushed hindu datura flower by a sieve of 80 meshes for later use;
(2) mixing 1-butyl-3-methylimidazole lactate 5.5g, PEG400 4g and deionized water 200g, and adjusting pH to 3.5 with lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos Daturae Metelis treated in the step (1), adding 1.2g of acid cellulase and 0.7g of acid pectinase, mixing uniformly, keeping the temperature at 50 ℃, stirring for 2.5h, filtering, washing the filter residue with hydrochloric acid aqueous solution with pH of 2.5, and combining the filtrates;
(4) adding 3% NaCl into the filtrate, extracting with 2 times volume of ethyl acetate for 3 times, removing ethyl acetate phase, adjusting pH of alkaloid-enriched phase to 10 with ammonia water, extracting with 1.5 times volume of chloroform for 3 times, and recovering chloroform under reduced pressure to obtain total alkaloids.
The acidic cellulose used in the examples and comparative examples of the present invention was a conventional commercially available product, and the acidic pectinase was obtained from Shandong Jinning Baiyi company.
Effect example 1
The method for measuring the content of the total alkaloids comprises the following steps:
1. preparing an atropine sulfate reference solution:
taking 10 mg of atropine sulfate dried at 102 ℃ to constant mass, precisely weighing, placing in a 100mL measuring flask, adding water to dissolve to constant volume to scale, and shaking up to obtain 0.1 g/L atropine sulfate reference solution.
2. 0.5g·mL-1Preparing a bromocresol green solution of an acid dye:
precisely weighing 50mg of bromocresol green and 1.021g of potassium hydrogen phthalate, and adding 0.2 mol.L-1Dissolving in 6.0 mL sodium hydroxide solution, diluting with water to 100mL, shaking, and filtering if necessary to obtain 0.5 g/mL-1Bromocresol green solution.
3. Preparing an atropine sulfate acid dye spectrophotometry standard curve:
precisely absorbing 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of atropine sulfate reference solution, respectively placing in a separating funnel, sequentially adding 3.0 mL and 10.0 mL of chloroform of 0.500 g/L acid dye bromocresol green solution, supplementing water to equal volume, shaking for extraction for 2min, standing for 5min, separating chloroform layer solution, adding 0.5g anhydrous sodium sulfate, and standing for 20 min. Another separating funnel which is added with 10.0 mL of trichloromethane precisely is taken, 1.0 mL of water is added precisely, a blank control solution is prepared by the same method, the absorbance is measured at the wavelength of 420 nm, and the regression equation Y =0.0562X +0.0067 and r = 0.9999 is obtained by taking the absorbance as the ordinate, which shows that atropine sulfate has good linear relation in the range of 20-100 mu g.
4. Method for measuring total alkaloid content in extract
Taking 0.2 g of total alkaloid dry paste, precisely weighing, adding 10mL of water for dissolving, adjusting the pH to 2-3, filtering, adjusting the pH of filtrate to 9-10 by using ammonia test solution, extracting for 4 times by using trichloromethane, taking 10mL of trichloromethane each time, combining the trichloromethane, fixing the volume to 100mL, and shaking up to obtain a test solution; precisely sucking 1.0 mL of the test solution, measuring absorption value according to 3 items, and calculating total alkaloid mass concentration and total alkaloid extraction rate.
Total alkaloid extraction rate (%) = [ (total alkaloid mass concentration x 100 x dry paste amount)/dry paste sample amount in test sample ] × 100/medicinal material amount
As shown in the table, the extraction effect is not considerable without adding 1-butyl-3-methylimidazole lactate and PEG300, and the extraction rate of total alkaloids is improved without adding 1-butyl-3-methylimidazole lactate or PEG300, but is still not ideal. The 1-butyl-3-methylimidazolium lactate and the PEG300 are added before wall breaking, so that the extraction rate of the total alkaloids can be effectively promoted in the wall breaking process. The more and better the 1-butyl-3-methylimidazole lactate and PEG300 were added, the significantly lower the extraction rate as can be seen from comparative examples 6 and 7. The molecular weight of PEG, also has an effect on the extraction yield, and the results were disappointing when PEG1500 was added. Lactic acid is more effective at adjusting pH than hydrochloric acid, presumably because hydrochloric acid affects enzyme activity. The extraction rate is promoted by using ammonium sulfate and NaCl together. The method provided by the invention obviously improves the extraction rate of the total alkaloids.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. The method for extracting the total alkaloids from the flos chrysanthemi, which is characterized by comprising the following steps of:
(1) pulverizing 100g flos Daturae Metelis, and sieving;
(2) mixing 1-butyl-3-methylimidazole lactate 3-8 g, PEG 300-1000 2-6 g and deionized water 200g, and adjusting the pH value to 3-5 by using lactic acid to obtain an acid leaching solution;
(3) adding the acid leaching solution obtained in the step (2) into the flos daturae treated in the step (1), adding 0.8-1.5 g of acid cellulase and 0.5-0.8 g of acid pectinase, uniformly mixing, keeping the temperature at 35-55 ℃, stirring for 1-4 h, filtering, washing filter residues with hydrochloric acid aqueous solution with the pH of 2-3, and combining filtrates;
(4) adding 1% ammonium sulfate and 2% NaCl into the filtrate, adding ethyl acetate, extracting for 3 times, removing an ethyl acetate phase, adjusting the pH of an alkaloid-enriched phase to 9-11 with ammonia water, extracting for 3 times with chloroform, and recovering chloroform under reduced pressure to obtain the total alkaloids.
2. The extraction method of total alkaloids from flos Daturae Metelis according to claim 1, wherein the pH value in step (2) is 3.5-4.5.
3. The method for extracting total alkaloids from flos Daturae Metelis as claimed in claim 1, wherein the mass ratio of 1-butyl-3-methylimidazole lactate, PEG and deionized water in step (2) is 5.5:4: 200.
4. The method for extracting total alkaloids from flos Daturae Metelis as claimed in claim 1, wherein the amount of acidic cellulase added in step (3) is 1.2g, and the amount of acidic pectinase added is 0.7 g.
5. The method for extracting total alkaloids from flos Daturae Metelis as claimed in claim 1, wherein the pH in step (4) is 10.
6. The method for extracting the total alkaloids from the flos chrysanthemi, as claimed in claim 1, wherein the volume ratio of the ethyl acetate to the filtrate for 3 times is respectively: 2-3: 2-2.5: 1.5-2.5.
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