CN112807736A - Ultrasonic-assisted enzyme extraction technology for fresh extraction of plants - Google Patents
Ultrasonic-assisted enzyme extraction technology for fresh extraction of plants Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
- B01D11/0265—Applying ultrasound
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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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
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Abstract
The invention discloses an ultrasonic assisted enzyme extraction technology for fresh extraction of plants. The method mainly comprises the following steps: (1) crushing: cleaning fresh plants to be extracted, and removing mildewed, damaged and scab parts; processing in a beater to obtain a mixture of juice and pulp; (2) ultrasonic enzymolysis: adding water, cellulase and pectinase, starting an ultrasonic generator, and carrying out ultrasonic treatment under stirring; (3) squeezing: putting the materials into a squeezer and squeezing to obtain residues; (4) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with a solvent; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation. Due to the obvious synergistic effect and permeability, the yield of the total flavone is higher than that of the conventional process.
Description
Technical Field
The invention relates to the field of cosmetics, in particular to an Ultrasonic assisted Enzyme Extraction technology (Ultrasonic-assisted Enzyme Extraction technology) for fresh Extraction of plants.
Background
The history of using natural plants to make up and beautify is very long in China, and people make the red and blue flower and leaf juice and coagulated fat growing in the swallow field into the rouge beautifying and making up in the period of invar; the Huangdi's Canon and Huatuo Shen Yi Mi Chuan of the three kingdoms of Qin and Han both recorded many medicines with skin caring effect; in Tang dynasty, the variety of cosmetics became abundant day by day, Sun Simiao says "cream hand ointment, Jiaxiang Guo, Shirengui Sheng" in Qianjin wing prescription. "
With the gradual rise of the society's tendency to return to nature', people are increasingly demanding on active ingredients or active parts contained in biological organisms such as animals, plants, microorganisms, etc., which have a positive effect on human health care.
Biological organisms are composed of complex chemical components, but contain few effective active ingredients. If the glycyrrhiza glabra root contains the effective component with whitening and anti-inflammatory effects, the glabridin content is only about 0.1-0.5%.
Usually, the extraction of the active ingredient is carried out by the solvent method. When the solvent is added into the extract, the solvent gradually enters into the cell through the cell wall or cell membrane due to diffusion and osmosis to dissolve soluble substances, so that the concentration difference between the inside and the outside of the cell is caused, the solution inside and the outside of the cell is continuously exchanged repeatedly until the concentration of the solution inside and the outside of the cell reaches dynamic balance, the saturated solution is separated, and a new solvent is continuously added, so that most of the required components can be dissolved.
In the extraction process, the selection of the solvent and the extraction method are key. The traditional extraction method mainly comprises a decoction method, an immersion method and a percolation method. But has the disadvantages of unstable technological process and product heat, long production period and large solvent loss.
The total flavone is generally biological total flavone, which refers to a flavonoid compound, is a large class of natural products, and is a main component in plants, especially in many Chinese herbal medicines. Its efficacy and action mainly depend on its composition, generally its ingredients include flavone and flavonol, also have flavanone, isoflavone, biflavone, etc., look at its efficacy is to protect hematopoietic system, and have protective action to immune system, can also scavenge free radical, resist oxidation, protect DNA, etc..
The present inventors have made extensive studies in the aforementioned field of plant active extract and, as a result, have found that the yield of total flavonoids is higher than that of the conventional process due to its remarkable synergy and permeability properties by combining several specific processes known per se in the art within a well-defined parameter range, thereby completing the present invention.
Disclosure of Invention
The invention aims to provide a method for extracting total flavonoids from natural plants.
The invention is implemented by the following steps:
an ultrasonic-assisted enzyme extraction method for fresh plant extraction comprises the following steps:
(1) crushing: cleaning fresh plants to be extracted, and removing mildewed, damaged and scab parts; processing in a beater to obtain a mixture of juice and pulp;
(2) ultrasonic enzymolysis: adding water, cellulase and pectinase, starting an ultrasonic generator, and carrying out ultrasonic treatment under stirring;
(3) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(4) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with a solvent; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
The cellulase can be a commercial product, and the enzyme activity of the cellulase can be 1 ten thousand to 10 ten thousand U/g and 10 ten thousand to 180 ten thousand U/g. The amount of cellulase is suitably adjusted according to the cellulose content of the extracted plant, and is generally added in an amount of 0.1 to 5 wt%.
The pectinase can be a commercially available product, and the enzyme activity of the pectinase can be 1 ten thousand to 10 ten thousand U/g and 10 ten thousand to 30 ten thousand U/g. The amount of pectinase is adjusted according to the pectin content of the extracted plant, and is generally 0.001-0.1 wt%.
The working frequency of the ultrasonic generator can be 20-40 KHZ.
The solvent can be ethanol, propylene glycol, methanol, butanediol, and their aqueous solution.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention adopts fresh plant raw materials, and the operation is carried out at low temperature, so that the damage to active matters is reduced to the minimum;
(2) the method adopts cellulase and pectinase in combination with ultrasonic treatment of plant raw materials, is favorable for forming a loose structure and is easy for subsequent effective substances to seep out;
(3) flexibility: different solvents and process parameters can be selected according to the difference of chemical components in the extracted matter for selective extraction.
(4) The yield is as follows: experiments prove that compared with the conventional process, the method provided by the invention has high flavone yield.
Detailed Description
The embodiments in the description are only for illustrating the present invention and do not limit the scope of the present invention. The scope of the present invention is defined only by the appended claims, and any omissions, substitutions, or modifications made based on the embodiments disclosed herein will fall within the scope of the present invention.
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.
For the purpose of enhancing understanding of the present invention, the present invention will be further described in detail with reference to the following examples, which are provided for illustration only and are not to be construed as limiting the scope of the present invention.
Example 1
A method for extracting bamboo leaf flavone comprises the following steps:
(1) crushing: cleaning fresh bamboo leaves, and removing mildew, worm damage and scab parts; adding appropriate amount of water, and processing in pulping machine to obtain mixture of juice and meat;
(2) ultrasonic enzymolysis: adding water, 10 ten thousand U/g of cellulase and 10 ten thousand U/g of pectinase, starting a 30KHZ ultrasonic generator, and carrying out ultrasonic treatment under stirring; wherein the total weight of the water is 10 times of the bamboo leaves; the adding weight of the cellulase is 1 percent of that of water; the added weight of the pectinase is 0.01 percent of that of water;
(3) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(4) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with 95% (V/V) ethanol water solution; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
Example 2
A method for preparing ginkgetin comprises the following steps:
(1) crushing: cleaning fresh folium Ginkgo, and removing mildew, worm damage and scar; adding appropriate amount of water, and processing in pulping machine to obtain mixture of juice and meat;
(2) ultrasonic enzymolysis: adding water, 10 ten thousand U/g of cellulase and 10 ten thousand U/g of pectinase, starting a 30KHZ ultrasonic generator, and carrying out ultrasonic treatment under stirring; wherein the total weight of water is 8 times of that of folium Ginkgo; the adding weight of the cellulase is 1 percent of that of water; the added weight of the pectinase is 0.01 percent of that of water;
(3) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(4) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with 85% (V/V) methanol water solution; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
Example 3
A method for preparing blueberry flavone comprises the following steps:
(1) crushing: cleaning fresh blueberry leaves, and removing mildew, worm-eaten and scab parts; processing in a beater to obtain a mixture of juice and pulp;
(2) ultrasonic enzymolysis: adding water, 10 ten thousand U/g of cellulase and 10 ten thousand U/g of pectinase, starting a 30KHZ ultrasonic generator, and carrying out ultrasonic treatment under stirring; wherein the total weight of the water is 1 time of that of the blueberry leaves; the adding weight of the cellulase is 1 percent of that of water; the added weight of the pectinase is 0.01 percent of that of water;
(3) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(4) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with 85% (V/V) ethanol water solution; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
Comparative example 1
A method for extracting bamboo leaf flavone comprises the following steps:
(1) crushing: cleaning fresh bamboo leaves, and removing mildew, worm damage and scab parts; adding appropriate amount of water, and processing in pulping machine to obtain mixture of juice and meat;
(2) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(3) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with 95% (V/V) ethanol water solution; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
Comparative example 2
A method for preparing ginkgetin comprises the following steps:
(1) crushing: cleaning fresh folium Ginkgo, and removing mildew, worm damage and scar; adding appropriate amount of water, and processing in pulping machine to obtain mixture of juice and meat;
(2) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(3) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with 85% (V/V) methanol water solution; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
Comparative example 3
A method for preparing blueberry flavone comprises the following steps:
(1) crushing: cleaning fresh blueberry leaves, and removing mildew, worm-eaten and scab parts; processing in a beater to obtain a mixture of juice and pulp;
(2) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(3) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with 85% (V/V) ethanol water solution; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
Example 4
The samples prepared in examples 1-3 and comparative examples 1-3 were tested for total flavonoid content according to the method described in section 1.2.4 of the reference (advanced dawn, Marvinl, Shihuili, Guomeng, Poplar-Behnken model for optimizing the extraction process and antioxidant activity analysis [ J/OL ] for general flavone of kenaf and food industry science: 1-10[2020-12-20].
The specific test method comprises the following steps: (1) weighing rutin which is dried in vacuum to constant weight and is compared with 0.0210g, placing the rutin in a 100mL volumetric flask, adding 3mL absolute ethyl alcohol for dissolving, adding distilled water to scale, and shaking up to obtain a rutin control solution of 0.21 mg/mL. (2) 0.3mL5% sodium nitrite solution is respectively added into 0.0, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 and 7.0 mL 0.21mg/mL rutin contrast solution in a 25mL colorimetric tube with a plug, the mixture is shaken up and kept stand for 6min, then 0.3mL5% aluminum nitrate solution is added, the mixture is shaken up and kept stand for 6min, 4.0mL 4% sodium hydroxide solution is added, 30% ethanol solution is used for constant volume to 25mL, the mixture is shaken up and kept stand, and then the absorbance A is measured at 500 nm. (3) And drawing a rutin standard curve regression equation of A =12.7671C-0.0421 and R2=0.9992 by taking the concentration (mg/mL) of rutin as an abscissa and the absorbance as an ordinate. (4) The samples prepared in examples 1 to 3 and comparative examples 1 to 3 were dissolved in ethanol at a feed-to-liquid ratio of 1:100, respectively, and then 1.0mL of the sample was accurately transferred to a 25mL colorimetric tube with a stopper, and the absorbance thereof was measured according to the test method for preparing a standard curve. The total flavone extraction rate in the sample was calculated according to the following formula: the total flavone yield (%) = (c × M × 10-3)/M × 100. Wherein C (mg/g): calculating the mass concentration of the flavone in the test sample according to the absorbance and the dilution times; m (g): the weight of the sample extract; m (g): weighing the weight of the extract to be extracted. The test results are shown in Table 1.
TABLE 1 flavone yield for each test group
As can be seen from Table 1, the extracts obtained in examples 1 to 3 of the present invention had significantly higher flavone contents than those obtained by the conventional methods.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. An ultrasonic-assisted enzyme extraction method for fresh plant extraction is characterized by comprising the following steps:
(1) crushing: cleaning fresh plants to be extracted, and removing mildewed, damaged and scab parts; processing in a beater to obtain a mixture of juice and pulp;
(2) ultrasonic enzymolysis: adding water, cellulase and pectinase, starting an ultrasonic generator, and carrying out ultrasonic treatment under stirring;
(3) squeezing: putting the materials into a squeezer and squeezing to obtain residues;
(4) column chromatography: transferring the residue into a chromatographic column, filling the column and compacting; then eluting with a solvent; collecting eluate, centrifuging to obtain supernatant, and removing solvent by rotary evaporation.
2. The method for ultrasonic-assisted enzymatic extraction of fresh plant extracts according to claim 1, wherein the enzymatic activity of the cellulase is 1 ten thousand to 10 ten thousand U/g, 10 ten thousand to 180 ten thousand U/g.
3. The ultrasonic-assisted enzymatic extraction method for fresh-extraction of plants according to claim 2, wherein the cellulase is used in an amount of 0.1 to 5 wt%.
4. The method for ultrasonic-assisted enzyme extraction of fresh-extract plants according to claim 1, wherein the enzyme activity of the pectinase is 1 ten thousand to 10 ten thousand U/g, 10 ten thousand to 30 ten thousand U/g.
5. The method for ultrasonic-assisted enzyme extraction of fresh plant extracts according to claim 4, wherein the amount of pectinase is 0.001-0.1 wt%.
6. The ultrasonic assisted enzymatic extraction method for fresh-extraction of plants according to claim 1, wherein the working frequency of the ultrasonic generator is 20-40 KHZ.
7. The ultrasonic assisted enzyme extraction method for fresh-extract plants of claim 1, wherein the solvent is ethanol, propylene glycol, methanol, butylene glycol, or their aqueous solution.
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Citations (6)
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| JP2006008645A (en) * | 2004-06-28 | 2006-01-12 | Wasoshin Kk | Method for producing extract of inonotus obliquus |
| CN102206550A (en) * | 2011-05-05 | 2011-10-05 | 武汉工业学院 | Method for preparing osmanthus concrete and osmanthus total flavonoid through enzyme hydrolysis technology |
| CN102948758A (en) * | 2012-11-26 | 2013-03-06 | 陕西科技大学 | Method for extracting buckwheat flavone from buckwheat bran |
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| CN109966211A (en) * | 2019-05-05 | 2019-07-05 | 上海悦目化妆品有限公司 | A kind of fresh extraction technology of plant and its application in cosmetic mask |
-
2020
- 2020-12-28 CN CN202011574411.3A patent/CN112807736A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006008645A (en) * | 2004-06-28 | 2006-01-12 | Wasoshin Kk | Method for producing extract of inonotus obliquus |
| CN102206550A (en) * | 2011-05-05 | 2011-10-05 | 武汉工业学院 | Method for preparing osmanthus concrete and osmanthus total flavonoid through enzyme hydrolysis technology |
| CN102948758A (en) * | 2012-11-26 | 2013-03-06 | 陕西科技大学 | Method for extracting buckwheat flavone from buckwheat bran |
| CN105146520A (en) * | 2015-09-25 | 2015-12-16 | 西藏藏真堂藏药产业有限公司 | Fructus phyllanti enzyme and preparing method thereof |
| CN108210555A (en) * | 2018-01-25 | 2018-06-29 | 安徽诚亚生物科技有限公司 | A kind of homogenate extraction method of glycyrrhiza total flavonoid |
| CN109966211A (en) * | 2019-05-05 | 2019-07-05 | 上海悦目化妆品有限公司 | A kind of fresh extraction technology of plant and its application in cosmetic mask |
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