CN113367242A - Method for extracting alkaloid from nettle and application of alkaloid as growth promoting additive - Google Patents
Method for extracting alkaloid from nettle and application of alkaloid as growth promoting additive Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G5/00—Alkaloids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention provides a method for extracting alkaloid from nettle, which has the advantages of simple process, high efficiency, rapidness, safety, environmental protection, high yield and purity, finds that the alkaloid has the effect of promoting the growth of animals and can be used as a growth promoting additive to be applied to the growth and reproduction of the animals, and the method for extracting the alkaloid from the nettle comprises the pretreatment of the nettle and the supercritical N2O extraction, coarse extraction and fine purification, and the extraction is performedN of (A)2The O as the fluid can be recycled, has no solvent residue, can provide an inert environment, effectively avoids product oxidation, does not denature the effective components of the alkaloid, and effectively ensures the quality and activity of the extracted alkaloid.
Description
Technical Field
The invention belongs to the technical field of extraction of active ingredients of traditional Chinese medicines, and particularly relates to a method for extracting alkaloid from nettle and application of the alkaloid as a growth promoting additive.
Background
Alkaloid (alkaloid) is a natural organic compound containing nitrogen heterocycle with alkalinity and obvious physiological activity, widely exists in various plants in nature, and is an effective component of a plurality of Chinese herbal medicines and medicinal plants. So far, various alkaloids are separated from plants for clinical use, such as vinblastine, camptothecin, leonurine, colchicine, ephedrine and the like, and researches on extraction, separation and purification methods and processes of the alkaloids have become the focus of attention on the basis that the alkaloids have the characteristics of tumor resistance, low toxicity, cancer resistance and low cost.
The nettle family plant is a traditional wild plant which can be used as both medicine and food, has long medicinal history, and has multiple efficacies of dispelling wind and eliminating dampness, calming liver and arresting convulsion, detoxifying and the like. At present, proper solvents such as acid water, acid alcohol, alkali alcohol and the like are adopted, and the alkaloid components are extracted by using the technologies such as soaking, percolation, decoction, reflux, ultrasound and the like, and then are further subjected to coarse extraction and purification refining for extraction. Such as large amount of starch, gum, pectin, mucilage, pigment, etc., which is not easy to be processed by the subsequent steps of coarse extraction, purification and refining.
Patent CN102327303A discloses a method for efficiently separating picrasma quassioides alkaloid, which adopts supercritical extraction technology combined with supercritical fluid reverse extraction crystallization technology to extract and purify picrasma quassioides alkaloid, and has the advantages of short separation and purification time, high separation efficiency and simple process. Compared with complex, energy-consuming and time-consuming processes such as thermal reflux extraction, organic solvent extraction, chromatographic column purification and the like, the method has industrial significance.
Zhang Haiyue et al (research on adsorption and separation of alkaloids in Urtica angustifolia by macroporous resin, Zhang Haiyue et al, China brewing, vol. 31, No. 1 of 2012) extracted alkaloids in the whole herb of Urtica angustifolia by alcohol extraction, extracted with petroleum ether after reflux extraction with 85% ethanol, and then primarily separated and purified by using weak-polar AB-8 macroporous adsorption resin. Research results show that the AB-8 macroporous adsorption resin has better adsorption capacity on alkaloid; the desorption effect is best when 40% vol ethanol is eluted, and the sample loading amount and the eluent dosage are 1: the best effect is obtained when the dosage is 2.
Therefore, in order to overcome the problems of the extraction method and the separation and purification method for extracting the alkaloid from the nettle, the technical personnel in the field need to solve the problems of developing a simple and rapid extraction and purification method which can reduce the energy consumption and the production cost and improving the purity of the alkaloid in the nettle.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for extracting alkaloid from nettle, which has the advantages of simple process, high efficiency, rapidness, safety, environmental protection, high yield and purity, discovers that the alkaloid has the effect of promoting the growth of animals and can be used as a growth promoting additive to be applied to the growth and reproduction of the animals, and the method for extracting the alkaloid from the nettle comprises the pretreatment of the nettle and the supercritical N2And O, extracting, removing coarse substances and fine substances, and purifying and refining.
In order to achieve the purpose, the invention adopts the following technical scheme that the method for extracting the alkaloid from the nettle comprises the following steps:
step 1, cleaning, airing and crushing whole nettle, sieving the whole nettle with a 100-mesh 200-mesh standard sieve to obtain nettle powder, adding an alkaline pretreatment solution which is 1-2 times of the weight of the nettle powder, carrying out ultrasonic treatment for 10-30min, and filtering and airing the mixture to obtain pretreated nettle powder;
step 2, mixing the pretreated nettle powder obtained in the step 1 with an alkaline substanceAccording to the weight ratio of 100: 3-5, adding entrainer 10-15% of the nettle powder, and performing supercritical N2O extraction, wherein the extraction process comprises the following steps: pressure of 25-35Mpa, temperature of 40-60 deg.C, N2O flow is 2-5L/h, extraction time is 60-80min, and concentrated solution is obtained after the extract is concentrated;
step 3, dissolving the concentrated solution obtained in the step 2 with hydrochloric acid, filtering, adjusting the pH value of the filtrate to 9-10 with ammonia water, extracting with chloroform for 3-4 times, combining the extract solutions, and performing rotary evaporation to obtain a crude extract;
and 4, dispersing the crude extract obtained in the step 3 by using water to obtain a dispersion liquid with the concentration of 0.1-0.2g/mL, adjusting the pH value to 5-6, selecting macroporous adsorption resin for upper column separation, wherein the flow rate of the upper column is 3-7BV/h, then eluting by using water for 1-3BV to remove impurities, eluting by using 30-50 wt% of ethanol for 4-10BV, wherein the elution flow rate is 2-5BV/h, collecting eluent, concentrating and drying to obtain the nettle alkaloid.
Further, the nettle is one of Urtica dioica, Urtica angustifolia, Urtica canescens, Urtica fissa, Urtica dioica and Urtica macrorrhiza.
The alkaline pretreatment solution is prepared from 20-30 wt% of ammonia water, methanol and water according to the weight ratio of 30-60: 10-20: 40-50 of the above-mentioned materials.
The alkaline substance is selected from one or more of calcium hydroxide, sodium carbonate and potassium carbonate. The entrainer is selected from one of 95 wt% ethanol or 95 wt% methanol.
The hydrochloric acid concentration in the step 3 is 0.05-0.2mol/L, and the nonpolar macroporous adsorption resin is selected from one of D-101, X-5, HPD-100, D3520 and AB-8.
In the step 4, 30-50 wt% of ethanol and 20-30 wt% of ammonia water are selected according to the weight ratio of 100: 5-10 as eluent.
The invention also aims to provide application of the nettle alkaloid, which is prepared by the method, is added with conventional auxiliary materials to prepare any pharmaceutically acceptable conventional dosage form, including capsules, tablets, pills, granules and sustained release agents, is used as a growth promoting additive and is applied to animal feed, such as: pigs, chickens, sheep, fish, etc.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for extracting alkaloid from nettle, which has the advantages of simple operation, high yield and N for extraction2The O as the fluid can be recycled, has no solvent residue, can provide an inert environment, effectively avoids product oxidation, does not denature the effective components of the alkaloid, and effectively ensures the quality and activity of the extracted alkaloid. The prepared alkaloid can be used as growth promoting additive to be added into animal feed to promote animal growth.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow. All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
Example 1
A method for extracting alkaloid from herba Urticae Cannabinae comprises the following steps:
step 1, cleaning, airing and crushing whole plant of the nettle of different strains, sieving the whole plant of the nettle with a standard sieve of 200 meshes to obtain nettle powder, adding an alkaline pretreatment solution which is 2 times of the weight of the nettle powder, carrying out ultrasonic treatment for 1min, and then filtering and airing the mixture to obtain pretreated nettle powder; the alkaline pretreatment solution is prepared from 30 wt% of ammonia water, methanol and water according to a weight ratio of 60: 10: 50, mixing the components;
step 2, mixing the pretreated nettle powder obtained in the step 1 with an alkaline substance calcium hydroxide according to a weight ratio of 100: 3 mixing, adding 95 wt% ethanol 15 wt% of herba Urticae Cannabinae powder as entrainer, and performing supercritical N2O extraction, wherein the extraction process comprises the following steps: pressure 35Mpa, temperature 40 deg.C, N2O flow of 4L/h, extraction time of 80min, concentrating the extract to obtain concentrated solution;
step 3, dissolving the concentrated solution obtained in the step 2 with 0.1mol/L hydrochloric acid, filtering, adjusting the pH value of the filtrate to 10 with ammonia water, extracting with chloroform for 4 times, combining the extract liquor, and performing rotary evaporation to obtain a crude extract;
and 4, dispersing the crude extract obtained in the step 3 by using water to obtain a dispersion liquid with the concentration of 0.2g/mL, adjusting the pH value to 6, selecting macroporous adsorption resin D-101 for upper column separation, wherein the flow rate of the upper column is 7BV/h, eluting by using water for 3BV to remove impurities, eluting by using 50 wt% of ethanol for 8BV, and the elution flow rate is 2BV/h, collecting eluent, concentrating and drying to obtain the nettle alkaloid.
Example 2
A method for extracting alkaloid from herba Urticae Cannabinae comprises the following steps:
step 1, cleaning, airing and crushing whole plant of the urtica angustifolia, sieving the cleaned and crushed whole plant of the urtica angustifolia by a 150-mesh standard sieve to obtain urtica powder, adding an alkaline pretreatment solution which is 1.5 times of the weight of the urtica powder, carrying out ultrasonic treatment for 20min, and then filtering and airing the mixture to obtain pretreated urtica powder; the alkaline pretreatment solution is prepared from 25 wt% of ammonia water, methanol and water according to a weight ratio of 50: 15: 50, mixing the components;
step 2, mixing the pretreated nettle powder obtained in the step 1 with an alkaline substance sodium carbonate according to a weight ratio of 100: 4 mixing, adding methanol 95 wt% of herba Urticae Cannabinae powder 12 wt% as entrainer, and performing supercritical N2O extraction, wherein the extraction process comprises the following steps: pressure of 30MPa, temperature of 50 deg.C, N2O flow rate of 3.5L/h, extraction time of 70min, concentrating the extract to obtain concentrated solution;
step 3, dissolving the concentrated solution obtained in the step 2 by using 0.2mol/L hydrochloric acid, filtering, adjusting the pH value of the filtrate to 9 by using ammonia water, extracting for 3 times by using chloroform, combining the extract liquor, and carrying out rotary evaporation to obtain a crude extract;
and 4, dispersing the crude extract obtained in the step 3 by using water to obtain a dispersion liquid with the concentration of 0.15g/mL, adjusting the pH value to 5.5, selecting macroporous adsorption resin X-5 for upper column separation at the flow rate of 4BV/h, eluting by using water for 2BV to remove impurities, eluting by using 40 wt% of ethanol for 10BV at the flow rate of 2.5BV/h, collecting eluent, concentrating and drying to obtain the nettle alkaloid.
Example 3
A method for extracting alkaloid from herba Urticae Cannabinae comprises the following steps:
step 1, cleaning, airing and crushing whole plant of the nettle schizophyllum, sieving the whole plant of the nettle schizophyllum with a standard sieve of 100 meshes to obtain nettle powder, adding an alkaline pretreatment solution which is 2 times of the weight of the nettle powder, carrying out ultrasonic treatment for 25min, and then filtering and airing the mixture to obtain pretreated nettle powder; the alkaline pretreatment solution is prepared from 20 wt% of ammonia water, methanol and water according to a weight ratio of 60: 20: 40 are mixed to form
Step 2, mixing the pretreated nettle powder obtained in the step 1 with an alkaline substance potassium carbonate according to a weight ratio of 100: 5 mixing, adding 95 wt% ethanol 11 wt% of herba Urticae Cannabinae powder as entrainer, and performing supercritical N2O extraction, wherein the extraction process comprises the following steps: pressure 25Mpa, temperature 60 ℃, N2O flow rate of 5L/h, extraction time of 80min, concentrating the extract to obtain concentrated solution;
step 3, dissolving the concentrated solution obtained in the step 2 by using 0.1mol/L hydrochloric acid, filtering, adjusting the pH value of the filtrate to 10 by using ammonia water, extracting for 4 times by using chloroform, combining the extract liquor, and carrying out rotary evaporation to obtain a crude extract;
and 4, dispersing the crude extract obtained in the step 3 by using water to obtain a dispersion liquid with the concentration of 0.1g/mL, adjusting the pH value to 6, selecting macroporous adsorption resin AB-8 for upper column separation, wherein the flow rate of the upper column is 5BV/h, eluting by using water for 3BV to remove impurities, eluting by using 35 wt% of ethanol for 8BV, and the elution flow rate is 4BV/h, collecting eluent, concentrating and drying to obtain the nettle alkaloid.
Example 4
A method for extracting alkaloid from herba Urticae Cannabinae comprises the following steps:
step 1, cleaning, airing and crushing whole rough-root nettle, sieving by a 200-mesh standard sieve to obtain nettle powder, adding an alkaline pretreatment solution 2 times of the weight of the nettle powder, carrying out ultrasonic treatment for 15min, and filtering and airing to obtain pretreated nettle powder; the alkaline pretreatment solution is prepared from 30 wt% of ammonia water, methanol and water according to a weight ratio of 30: 10: 50 are mixed to form
Step 2, mixing the pretreated nettle powder obtained in the step 1 with an alkaline substance calcium hydroxide according to a weight ratio of 100: 3.5 mixingMixing, adding methanol 95 wt% of herba Urticae Cannabinae powder 12 wt% as entrainer, and performing supercritical N2O extraction, wherein the extraction process comprises the following steps: pressure 28Mpa, temperature 55 deg.C, N2O flow rate of 4L/h, extraction time of 70min, concentrating the extract to obtain concentrated solution;
step 3, dissolving the concentrated solution obtained in the step 2 with 0.05mol/L hydrochloric acid, filtering, adjusting the pH value of the filtrate to 10 with ammonia water, extracting with chloroform for 4 times, combining the extract liquor, and performing rotary evaporation to obtain a crude extract;
and 4, dispersing the crude extract obtained in the step 3 by using water to obtain a dispersion liquid with the concentration of 0.15g/mL, adjusting the pH value to 6, selecting macroporous adsorption resin D3520 for upper column separation, wherein the flow rate of the upper column is 6BV/h, eluting 2BV by using water to remove impurities, and then using 40 wt% of ethanol and 20 wt% of ammonia water to prepare a dispersion liquid according to the weight ratio of 100: 10 as eluent to elute 7BV, eluting at the flow rate of 3BV/h, collecting the eluent, concentrating and drying to obtain the nettle alkaloid.
Comparative example 1
Selective use of supercritical CO2Fluid extraction replacement of supercritical N2Fluid O extraction, the remaining steps and process were exactly the same as in example 3.
Comparative example 2
No alkaline substance potassium carbonate was added in step 2, and the remaining steps and processes were completely the same as in example 3.
Comparative example 3
The crude extract obtained in step 3 was dispersed with water to give a dispersion of 0.1g/mL, and the pH was adjusted to 7, the remaining steps and process being identical to those of example 3.
After testing, the yield and purity of the nettle alkaloid obtained in examples 1-4 and comparative examples 1-3 are shown in Table 1.
TABLE 1
| Numbering | Example 1 | Example 2 | Example 3 | Example 4 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Yield/% | 1.35％ | 1.46％ | 1.42％ | 1.39％ | 1.03％ | 1.25％ | 1.32％ |
| Purity/%) | 79.3％ | 80.2％ | 82.4％ | 81.2％ | 81.5％ | 80.4％ | 78.3％ |
From the experimental data in table 1, it can be found that the method for extracting alkaloid from nettle provided by the present invention can obtain purity and yieldHigh yield of alkaloid and supercritical N2Fluid O is supercritical CO2The fluid is more suitable for extracting alkaloid from nettle, and the yield of alkaloid can be improved by applying alkaline substance during extraction process.
Testing the effect of the nettle alkaloid as the growth promoting additive, selecting 300 healthy pigs, randomly dividing the pigs into 3 groups (A, B, C three groups, each group is 100), feeding the group A with common feed, and adding the nettle alkaloid prepared in the example 1 and the nettle alkaloid prepared in the example 3 into the common feed in the groups B and C respectively, wherein the weight ratio of the nettle alkaloid to the corncob meal is 1: 10 to prepare granules in an amount of 20 kg/ton (feed) and the results are shown in table 2 after feeding for 30 days.
TABLE 2
| Test items | Meat ratio of materials | Daily gain/g | Area per cm of eye volume2 | Intramuscular fat/% | Incidence rate/%) |
| Group A | 3.2 | 815 | 39.2 | 1.8 | 13 |
| Group B | 2.89 | 908 | 48.3 | 2.3 | 2 |
| Group C | 2.93 | 913 | 48.6 | 2.2 | 3 |
From the test data in table 2, it can be found that the nettle alkaloid can promote the growth of pigs, reduce the incidence rate of pigs, and can be used as a growth promotion additive.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for extracting alkaloid from nettle is characterized by comprising the following steps: the method comprises the following steps:
step 1, cleaning, airing and crushing whole nettle, sieving the whole nettle with a 100-mesh 200-mesh standard sieve to obtain nettle powder, adding an alkaline pretreatment solution which is 1-2 times of the weight of the nettle powder, carrying out ultrasonic treatment for 10-30min, and filtering and airing the mixture to obtain pretreated nettle powder;
step 2, mixing the pretreated nettle powder obtained in the step 1 with an alkaline substance according to the weight ratio of 100: 3-5, adding entrainer 10-15% of the nettle powder, and performing supercritical N2O extraction, wherein the extraction process comprises the following steps: pressure of 25-35Mpa, temperature of 40-60 deg.C, N2O flow rate of 2-5L/h, extraction time of 60-80min, and concentrating the extract to obtain concentrated extractLiquid condensation;
step 3, dissolving the concentrated solution obtained in the step 2 with hydrochloric acid, filtering, adjusting the pH value of the filtrate to 9-10 with ammonia water, extracting with chloroform for 3-4 times, combining the extract solutions, and performing rotary evaporation to obtain a crude extract;
and 4, dispersing the crude extract obtained in the step 3 by using water to obtain a dispersion liquid with the concentration of 0.1-0.2g/mL, adjusting the pH value to 5-6, selecting macroporous adsorption resin for upper column separation, wherein the flow rate of the upper column is 3-7BV/h, then eluting by using water for 1-3BV to remove impurities, eluting by using 30-50 wt% of ethanol for 4-10BV, wherein the elution flow rate is 2-5BV/h, collecting eluent, concentrating and drying to obtain the nettle alkaloid.
2. The method of claim 1, wherein the alkaloid is extracted from nettle by: the nettle is one of Urtica dioica, Urtica angustifolia, Urtica canescens, Urtica fissa, Urtica dioica and Urtica macrorrhiza.
3. The method of claim 1, wherein the alkaloid is extracted from nettle by: in the step 1, the alkaline pretreatment solution is prepared from 20-30 wt% of ammonia water, methanol and water according to a weight ratio of 30-60: 10-20: 40-50 of the above-mentioned materials.
4. The method of claim 1, wherein the alkaloid is extracted from nettle by: the alkaline substance is selected from one or more of calcium hydroxide, sodium carbonate and potassium carbonate.
5. The method of claim 1, wherein the alkaloid is extracted from nettle by: the entrainer is selected from one of 95 wt% ethanol or 95 wt% methanol.
6. The method of claim 1, wherein the alkaloid is extracted from nettle by: the concentration of hydrochloric acid in the step 3 is 0.05-0.2 mol/L.
7. The method of claim 1, wherein the alkaloid is extracted from nettle by: the macroporous adsorption resin is selected from one of D-101, X-5, HPD-100, D3520 and AB-8.
8. The method of claim 1, wherein the alkaloid is extracted from nettle by: in the step 4, 30-50 wt% of ethanol and 20-30 wt% of ammonia water are selected according to the weight ratio of 100: 5-10 as eluent.
9. The alkaloid of nettle according to claim 1, wherein the alkaloid is obtained from nettle by a process.
10. The use of a nettle alkaloid according to claim 9 wherein: adding the nettle alkaloid into conventional auxiliary materials to prepare any pharmaceutically acceptable conventional dosage form, and applying the dosage form to animal feed as a growth promoting additive; the preparation comprises capsules, tablets, pills, granules and sustained release agents.
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