CN112390847A - Method for extracting toosendanin from toosendan fruit - Google Patents
Method for extracting toosendanin from toosendan fruit Download PDFInfo
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- CN112390847A CN112390847A CN202011249785.8A CN202011249785A CN112390847A CN 112390847 A CN112390847 A CN 112390847A CN 202011249785 A CN202011249785 A CN 202011249785A CN 112390847 A CN112390847 A CN 112390847A
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- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
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Abstract
The invention discloses a method for extracting toosendanin from toosendan fruit, which comprises the following steps: crushing the toosendan fruit, immersing the toosendan fruit in water, adding compound enzyme for enzymolysis, extracting the enzymolysis liquid by adopting a high-voltage pulse electric field, adding a clarifying agent into the obtained extracting solution for removing impurities, decoloring by using active carbon, concentrating to obtain an extract, applying macroporous adsorption resin to the extract, concentrating the obtained eluent under reduced pressure to obtain a crude extract, dissolving the crude extract, adding petroleum ether for precipitation, washing and drying the precipitate, dissolving and drying again to obtain the toosendanin. The method extracts the toosendanin through the steps of pretreatment of complex enzyme, extraction of high-voltage pulse electric field, clarification of clarifying agent, decoloration of active carbon, adsorption of macroporous adsorption resin, precipitation of petroleum ether and the like, can effectively improve the purity and extraction rate of the toosendanin, and has the advantages of high yield, less impurities and high purity of the extracted toosendanin.
Description
Technical Field
The invention relates to the technical field of extraction of active ingredients of medicaments, in particular to a method for extracting toosendanin from toosendan fruit.
Background
Melia toosendan (Melia tosendan Sieb. et Zucc.) is a subspecies of Melia toosendan, deciduous tree, as high as 10 m. Toosendanin is a tetracyclic triterpene compound extracted from root bark, branch bark and fruit of Melia plant. Toosendanin has effects of expelling parasites, killing parasite, etc., and has good anti-sarcoma effect, and can induce cell differentiation and apoptosis and inhibit proliferation of various tumor cells of human and animal. At present, the extraction method of toosendanin mainly comprises an extraction method, a Soxhlet extraction method, an ultrasonic extraction method, a microwave-assisted extraction method, a supercritical carbon dioxide extraction method and the like.
The existing method for extracting toosendanin from toosendan fruit trees adopts extraction and recrystallization methods, which are simple, but the extraction time is long, the efficiency is low, and the method can not meet the requirement of industrial production. Or cleaning dried mature fructus Toosendan, pulverizing, grinding into powder, oven drying, and adding 60% ethanol solvent; adjusting the pH value of the solution to 4-6, and adding cellulase for enzymolysis; placing the extracting solution in a double-frequency ultrasonic extractor to obtain an enzymolysis solution; inactivating the enzymolysis liquid at high temperature to obtain an extracting solution, centrifuging, and taking supernatant; extracting with chloroform, drying with anhydrous sodium sulfate, vacuum filtering, concentrating, adding ethanol, heating to dissolve, dripping petroleum ether until the mixture becomes turbid, standing, and separating out light yellow solid, i.e. toosendanin. The method can improve the content of toosendanin by enzymolysis and double-frequency ultrasonic extraction, but has insufficient separation and purification effects, and the obtained toosendanin has low purity.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for extracting toosendanin from toosendan fruit. The method extracts the toosendanin through the steps of pretreatment of complex enzyme, extraction of high-voltage pulse electric field, clarification of clarifying agent, decoloration of active carbon, adsorption of macroporous adsorption resin, precipitation of petroleum ether and the like, can effectively improve the purity and extraction rate of the toosendanin, and has the advantages of high yield, less impurities and high purity of the extracted toosendanin.
The invention is realized by the following technical scheme:
a method for extracting toosendanin from fructus Toosendan comprises the following steps:
(1): cleaning fructus Toosendan, air drying, and pulverizing;
(2): immersing the crushed material in the step (1) with water, adding complex enzyme accounting for 0.01-0.05% of the weight of the material, and performing enzymolysis for 1-3 hours to obtain an enzymolysis liquid;
(3): mixing the enzymolysis liquid in the step (2) with absolute ethyl alcohol according to a material-liquid ratio of 1:15-20, and putting the mixture into a high-voltage pulse electric field for extraction for 6-12min to obtain a crude extract;
(4): adding natural Chinese medicine clarifying agent KBT-ZTC into the crude extract in step (3), filtering when flocculate is not increased, adding 0.1-0.3g/L active carbon into the filtrate for decolorizing for 10-20min, centrifuging, and concentrating supernatant by rotary evaporation to obtain extract;
(5): dissolving the extract in the step (4) with ethanol, adsorbing the solution by macroporous adsorption resin, removing impurities by using distilled water during desorption, eluting by using methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a crude extract;
(6): dissolving the crude extract obtained in the step (5) by using methanol, adding petroleum ether until the solution becomes turbid, standing overnight, washing the separated precipitate by using distilled water, and drying to obtain a crude product of toosendanin;
(7): and (4) dissolving the crude toosendanin product obtained in the step (6) by using 3-6 times of anhydrous ethanol, performing rotary evaporation, and recovering ethanol to obtain a pure toosendanin product.
As a further description of the present invention, the Toosendan fruit of step (1) is pulverized to 60-100 mesh.
As a further illustration of the invention, the complex enzyme in the step (2) is prepared by mixing cellulase, hemicellulase and pectinase in any proportion.
As a further explanation of the invention, the electric field intensity of the high-voltage pulse electric field in the step (3) is 25-40kV/cm, and the number of pulses is 6-10.
As a further illustration of the invention, the natural Chinese medicine clarifying agent KBT-ZTC produced in the step (4) is a clarifying agent produced by Kelier Biotechnology Co., Ltd, and the addition amount is 2-5 g/L.
As a further illustration of the present invention, the formaldehyde in step (5) is 60-100% formaldehyde.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the invention treats the crushed material of the melia toosendan fruits with complex enzyme to destroy the cell walls of the material, and then extracts the material by combining a high-voltage pulse electric field, and the magnetic field generated by the pulse electric field and the pulse electric field alternately act to increase the permeability of the cell membranes of the material, intensify the oscillation and finally make the substances in the cell membranes flow out. The invention can maximize the leaching rate of the toosendanin by an enzymolysis technology and a high-voltage pulse electric field technology, effectively improve the extraction rate of the toosendanin, and greatly shorten the extraction time compared with the conventional extraction time.
2. The natural traditional Chinese medicine clarifying agent KBT-ZTC is introduced, so that a good clarifying effect is achieved on macromolecular impurities such as protein, grease, starch and the like in the ethanol extraction process; the extract is decolorized by active carbon, so that the pigment is removed, and a good basis is provided for subsequent separation and purification.
3. The invention separates and purifies toosendanin by using macroporous resin, separates toosendanin from the crude extract, and reduces the content of impurities in toosendanin.
4. The method extracts the toosendanin through the steps of pretreatment of complex enzyme, extraction of high-voltage pulse electric field, clarification of clarifying agent, decoloration of active carbon, adsorption of macroporous adsorption resin, precipitation of petroleum ether and the like, can effectively improve the purity and extraction rate of the toosendanin, and has the advantages of high yield, less impurities and high purity of the extracted toosendanin.
5. The extraction rate of the toosendanin is 1.3-1.6mg/g, and the purity is more than 96%.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the scope of the present invention.
Example 1
A method for extracting toosendanin from fructus Toosendan comprises the following steps:
(1): cleaning fructus Toosendan, air drying, and pulverizing to 60 mesh;
(2): immersing the crushed material in the step (1) with water, adding complex enzyme accounting for 0.01% of the weight of the material, and performing enzymolysis for 3 hours to obtain an enzymolysis liquid; the complex enzyme is prepared by mixing cellulase, hemicellulase and pectinase in any proportion;
(3): mixing the enzymolysis liquid in the step (2) with absolute ethyl alcohol according to a material-liquid ratio of 1:15, and putting the mixture into a high-voltage pulse electric field for extraction for 12min to obtain a crude extract; the electric field intensity of the high-voltage pulse electric field is 25kV/cm, and the number of pulses is 6;
(4): adding a natural traditional Chinese medicine clarifying agent KBT-ZTC into the crude extract obtained in the step (3), wherein the natural traditional Chinese medicine clarifying agent KBT-ZTC is a clarifying agent produced by Kelail biotechnology limited and the addition amount is 2 g/L; when the flocculate is not increased, filtering, adding 0.3g/L active carbon into the filtrate for decolorizing for 10min, centrifuging, and concentrating the supernatant by rotary evaporation to obtain extract;
(5): dissolving the extract in the step (4) with ethanol, adsorbing the solution by macroporous adsorption resin, removing impurities by using distilled water during desorption, eluting by using 60% methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a crude extract;
(6): dissolving the crude extract obtained in the step (5) by using methanol, adding petroleum ether until the solution becomes turbid, standing overnight, washing the separated precipitate by using distilled water, and drying to obtain a crude product of toosendanin;
(7): and (4) dissolving the crude toosendanin product obtained in the step (6) by using 3 times of anhydrous ethanol, performing rotary evaporation, and recovering ethanol to obtain a pure toosendanin product.
According to the detection of high performance liquid chromatography, the extraction rate of the toosendanin in the embodiment is 1.31%, and the purity is 96.4%.
Example 2
A method for extracting toosendanin from fructus Toosendan comprises the following steps:
(1): cleaning fructus Toosendan, air drying, and pulverizing to 100 mesh;
(2): immersing the crushed material in the step (1) with water, adding complex enzyme accounting for 0.01-0.05% of the weight of the material, and performing enzymolysis for 1 hour to obtain an enzymolysis liquid; the complex enzyme is prepared by mixing cellulase, hemicellulase and pectinase in any proportion;
(3): mixing the enzymolysis liquid in the step (2) with absolute ethyl alcohol according to a material-liquid ratio of 1:20, and putting the mixture into a high-voltage pulse electric field for extraction for 6min to obtain a crude extract; the electric field intensity of the high-voltage pulse electric field is 40kV/cm, and the number of pulses is 10;
(4): adding a natural traditional Chinese medicine clarifying agent KBT-ZTC into the crude extract obtained in the step (3), wherein the natural traditional Chinese medicine clarifying agent KBT-ZTC is a clarifying agent produced by Kelail biotechnology limited, and the addition amount is 5 g/L; when the flocculate is not increased, filtering, adding 0.1g/L active carbon into the filtrate for decolorizing for 20min, centrifuging, and concentrating the supernatant by rotary evaporation to obtain extract;
(5): dissolving the extract in the step (4) with ethanol, adsorbing the solution by macroporous adsorption resin, removing impurities by using distilled water during desorption, eluting by using 100% methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a crude extract;
(6): dissolving the crude extract obtained in the step (5) by using methanol, adding petroleum ether until the solution becomes turbid, standing overnight, washing the separated precipitate by using distilled water, and drying to obtain a crude product of toosendanin;
(7): and (4) dissolving the crude toosendanin product obtained in the step (6) by using absolute ethyl alcohol with the weight 6 times that of the crude toosendanin product, performing rotary evaporation, and recovering the ethyl alcohol to obtain a pure toosendanin product.
According to the detection of high performance liquid chromatography, the extraction rate of the toosendanin in the embodiment is 1.40%, and the purity is 96.5%.
Example 3
A method for extracting toosendanin from fructus Toosendan comprises the following steps:
(1): cleaning fructus Toosendan, air drying, and pulverizing to 80 mesh;
(2): immersing the crushed material in the step (1) with water, adding complex enzyme accounting for 0.02% of the weight of the material, and performing enzymolysis for 2 hours to obtain an enzymolysis liquid; the complex enzyme is prepared by mixing cellulase, hemicellulase and pectinase in any proportion;
(3): mixing the enzymolysis liquid in the step (2) with absolute ethyl alcohol according to a material-liquid ratio of 1:18, and putting the mixture into a high-voltage pulse electric field for extraction for 10min to obtain a crude extract; the electric field intensity of the high-voltage pulse electric field is 30kV/cm, and the number of pulses is 8;
(4): adding a natural traditional Chinese medicine clarifying agent KBT-ZTC into the crude extract obtained in the step (3), wherein the natural traditional Chinese medicine clarifying agent KBT-ZTC is a clarifying agent produced by Kelail biotechnology limited, and the addition amount is 3 g/L; when the flocculate is not increased, filtering, adding 0.2g/L active carbon into the filtrate for decolorizing for 15min, centrifuging, and concentrating the supernatant by rotary evaporation to obtain extract;
(5): dissolving the extract in the step (4) with ethanol, adsorbing the solution by macroporous adsorption resin, removing impurities by using distilled water during desorption, eluting by using 75% methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a crude extract;
(6): dissolving the crude extract obtained in the step (5) by using methanol, adding petroleum ether until the solution becomes turbid, standing overnight, washing the separated precipitate by using distilled water, and drying to obtain a crude product of toosendanin;
(7): and (4) dissolving the crude toosendanin product obtained in the step (6) by using 5 times of anhydrous ethanol, performing rotary evaporation, and recovering ethanol to obtain a pure toosendanin product.
According to the detection of high performance liquid chromatography, the extraction rate of the toosendanin in the embodiment is 1.56%, and the purity is 97.1%.
Example 4
A method for extracting toosendanin from fructus Toosendan comprises the following steps:
(1): cleaning fructus Toosendan, air drying, and pulverizing to 80 mesh;
(2): immersing the crushed material in the step (1) with water, adding complex enzyme accounting for 0.04% of the weight of the material, and performing enzymolysis for 2 hours to obtain an enzymolysis liquid; the complex enzyme is prepared by mixing cellulase, hemicellulase and pectinase in any proportion;
(3): mixing the enzymolysis liquid in the step (2) with absolute ethyl alcohol according to a material-liquid ratio of 1:17, and putting the mixture into a high-voltage pulse electric field for extraction for 8min to obtain a crude extract; the electric field intensity of the high-voltage pulse electric field is 35kV/cm, and the number of pulses is 9;
(4): adding a natural traditional Chinese medicine clarifying agent KBT-ZTC into the crude extract obtained in the step (3), wherein the natural traditional Chinese medicine clarifying agent KBT-ZTC is a clarifying agent produced by Kelail biotechnology limited, and the addition amount is 4 g/L; when the flocculate is not increased, filtering, adding 0.3g/L active carbon into the filtrate for decolorizing for 15min, centrifuging, and concentrating the supernatant by rotary evaporation to obtain extract;
(5): dissolving the extract in the step (4) with ethanol, adsorbing the solution by macroporous adsorption resin, removing impurities by using distilled water during desorption, eluting by using 80% methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a crude extract;
(6): dissolving the crude extract obtained in the step (5) by using methanol, adding petroleum ether until the solution becomes turbid, standing overnight, washing the separated precipitate by using distilled water, and drying to obtain a crude product of toosendanin;
(7): and (4) dissolving the crude toosendanin product obtained in the step (6) by using absolute ethyl alcohol with the weight 4 times that of the crude toosendanin product, performing rotary evaporation, and recovering the ethyl alcohol to obtain a pure toosendanin product.
The extraction rate of toosendanin in this example was 1.53% and the purity was 97.0% as determined by high performance liquid chromatography.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method for extracting toosendanin from toosendan fruit is characterized by comprising the following steps:
(1): cleaning fructus Toosendan, air drying, and pulverizing;
(2): immersing the crushed material in the step (1) with water, adding complex enzyme accounting for 0.01-0.05% of the weight of the material, and performing enzymolysis for 1-3 hours to obtain an enzymolysis liquid;
(3): mixing the enzymolysis liquid in the step (2) with absolute ethyl alcohol according to a material-liquid ratio of 1:15-20, and putting the mixture into a high-voltage pulse electric field for extraction for 6-12min to obtain a crude extract;
(4): adding natural Chinese medicine clarifying agent KBT-ZTC into the crude extract in step (3), filtering when flocculate is not increased, adding 0.1-0.3g/L active carbon into the filtrate for decolorizing for 10-20min, centrifuging, and concentrating supernatant by rotary evaporation to obtain extract;
(5): dissolving the extract in the step (4) with ethanol, adsorbing the solution by macroporous adsorption resin, removing impurities by using distilled water during desorption, eluting by using methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a crude extract;
(6): dissolving the crude extract obtained in the step (5) by using methanol, adding petroleum ether until the solution becomes turbid, standing overnight, washing the separated precipitate by using distilled water, and drying to obtain a crude product of toosendanin;
(7): and (4) dissolving the crude toosendanin product obtained in the step (6) by using 3-6 times of anhydrous ethanol, performing rotary evaporation, and recovering ethanol to obtain a pure toosendanin product.
2. The method of claim 1, wherein the fruits of Melia toosendan are pulverized to 60-100 mesh in step (1).
3. The method for extracting toosendanin from toosendan fruit as claimed in claim 1, wherein the complex enzyme in step (2) is a mixture of cellulase, hemicellulase and pectinase at any ratio.
4. The method according to claim 1, wherein the high voltage pulse electric field in step (3) has an electric field strength of 25-40kV/cm and a number of pulses of 6-10.
5. The method as claimed in claim 1, wherein the natural Chinese medicinal clarifying agent KBT-ZTC produced by Kelail Biotechnology limited in step (4) is 2-5 g/L.
6. The method of claim 1, wherein the formaldehyde in step (5) is 60-100% formaldehyde.
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| CN202011249785.8A CN112390847A (en) | 2020-11-10 | 2020-11-10 | Method for extracting toosendanin from toosendan fruit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057826A (en) * | 2021-12-22 | 2022-02-18 | 上海诗丹德标准技术服务有限公司 | Preparation method of toosendanin reference substance |
| CN114716502A (en) * | 2022-04-15 | 2022-07-08 | 菏泽学院 | Method for extracting phellinus linteus triterpenoid |
-
2020
- 2020-11-10 CN CN202011249785.8A patent/CN112390847A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057826A (en) * | 2021-12-22 | 2022-02-18 | 上海诗丹德标准技术服务有限公司 | Preparation method of toosendanin reference substance |
| CN114057826B (en) * | 2021-12-22 | 2023-10-17 | 上海诗丹德标准技术服务有限公司 | Preparation method of reference substance of toosendanin |
| CN114716502A (en) * | 2022-04-15 | 2022-07-08 | 菏泽学院 | Method for extracting phellinus linteus triterpenoid |
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