CN113842407A - Extraction method of sweet osmanthus extract and product thereof - Google Patents
Extraction method of sweet osmanthus extract and product thereof Download PDFInfo
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- CN113842407A CN113842407A CN202111369859.6A CN202111369859A CN113842407A CN 113842407 A CN113842407 A CN 113842407A CN 202111369859 A CN202111369859 A CN 202111369859A CN 113842407 A CN113842407 A CN 113842407A
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- osmanthus
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- osmanthus fragrans
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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Abstract
The invention discloses an extraction method of an osmanthus fragrans extract and a product thereof, and relates to the technical field of plant component extraction. The extraction steps are as follows: (1) ultrasonically extracting sweet osmanthus with ethanol, and freeze-drying to obtain a sweet osmanthus crude extract; (2) leaching the crude extract of the osmanthus fragrans in glycerol aqueous solution, centrifuging and filtering after leaching is finished, and concentrating to obtain concentrated solution; (3) extracting the concentrated solution by tert-butanol, then carrying out ethyl acetate extraction on the extracted water phase, and concentrating the ethyl acetate phase to remove the solvent to obtain the osmanthus fragrans extract. The invention overcomes the problem that the existing extraction method is difficult to extract and obtain the osmanthus fragrans extract with high content of total phenols and total ketones, and the obtained extract has strong oxidation resistance and broad-spectrum antibacterial property, and meanwhile, the extraction method is simple and has great popularization and application prospects.
Description
Technical Field
The invention relates to the technical field of plant component extraction, in particular to an extraction method of an osmanthus fragrans extract and a product thereof.
Background
Osmanthus fragrans (Thunb.) Lour) belongs to the genus Osmanthus (Oleaceae), also known as laurel and rhinoceros, commonly called as Osmanthus fragrans, is one of ten traditional flowers in China and has a thousand-year cultivation history. The osmanthus fragrans is evergreen shrub or small arbor, temperate tree species and leaf pair, and is mostly oval or oblong; the flowers grow in clusters, and the corolla splits into basic milk, which is milky white, yellow, orange red and the like.
Osmanthus fragrans has long been an excellent landscaping tree species, can be used as a landscape plant, has high edible and medicinal values, and is widely applied to foods, cosmetics and medicines. In the past, researches on chemical components in the osmanthus fragrans have mostly focused on analysis of essential oil aroma components, and in recent years, with intensive researches on the osmanthus fragrans, the extraction of total phenols, total ketones and essential oil substances in the osmanthus fragrans has attracted more and more attention, and in addition, some reports on nitrite removing effect of osmanthus fragrans pigment and bacterial inhibition of pericarp pigment thereof exist.
In the current stage, when the osmanthus fragrans is extracted, the traditional extraction, crushing extraction and the like are usually adopted, the content of total phenols and total ketones in the extracted osmanthus fragrans extract is low, the antioxidant and bacteriostatic effects of the extract are not ideal, the extract only has an inhibiting effect on specific strains (mainly bacillus and aspergillus niger), and the broad-spectrum antibacterial property is poor.
In view of the above current situation, it is an urgent technical problem to provide a new method for extracting osmanthus fragrans, so that the extracted osmanthus fragrans extract has high total phenol and ketone contents, strong oxidation resistance and broad-spectrum antibacterial property.
Disclosure of Invention
The invention aims to provide an extraction method of an osmanthus extract and a product thereof, which are used for solving the problems in the prior art, remarkably improving the contents of total phenols and total ketones of the osmanthus extract and enabling the osmanthus extract to have stronger oxidation resistance and broad-spectrum antibacterial property.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides an extraction method of an osmanthus extract, which comprises the following steps:
(1) ultrasonically extracting sweet osmanthus with ethanol, and freeze-drying to obtain a sweet osmanthus crude extract;
(2) leaching the osmanthus crude extract in a glycerol aqueous solution, centrifuging and filtering after leaching is finished, and concentrating to obtain a concentrated solution;
(3) and extracting the concentrated solution by tert-butyl alcohol, then carrying out ethyl acetate extraction on the extracted water phase, and concentrating the ethyl acetate phase to remove the solvent to obtain the osmanthus fragrans extract.
Further, the ultrasonic extraction time in the step (1) is 10-30 min.
Further, the adding amount of the ethanol is 5-30 times of the mass of the osmanthus fragrans.
Further, the mass concentration of the glycerol aqueous solution is 20-60%.
Further, the feed-liquid ratio of the osmanthus crude extract to the glycerol aqueous solution is 1g:10-50 g.
Further, the leaching time in the step (2) is 6-8 h.
The invention also provides the sweet osmanthus extract extracted by the extraction method.
The invention discloses the following technical effects:
firstly, the osmanthus fragrans is subjected to crude extraction by using an ethanol solution, then the crude extract is leached by using a glycerol aqueous solution with a specific concentration, the leaching liquor is sequentially subjected to two-step extraction by using tert-butyl alcohol and ethyl acetate, and then the osmanthus fragrans extract can be obtained in an ethyl acetate phase, and the osmanthus fragrans extract has high total phenol content and total ketone content, and has strong oxidation resistance and broad-spectrum antibacterial property.
The invention overcomes the problem that the existing extraction method is difficult to extract and obtain the osmanthus fragrans extract with high content of total phenols and total ketones, and the obtained extract has strong oxidation resistance and broad-spectrum antibacterial property, and meanwhile, the extraction method is simple and has great popularization and application prospects.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a gallic acid standard curve;
FIG. 2 is a rutin standard curve;
FIG. 3 is an ascorbic acid standard curve.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The osmanthus fragrans used in the embodiment of the invention is osmanthus fragrans, and is purchased in a large pharmacy of holiday Jierkang.
Example 1
An extraction method of sweet osmanthus extract comprises the following steps:
(1) grinding sweet osmanthus into powder, weighing 10g of the powder, adding the powder into a reaction bottle, adding 95% ethanol with the mass being 5 times of that of the powder, carrying out ultrasonic extraction for 10min, carrying out reduced pressure concentration on the obtained extracting solution, removing the solvent, and carrying out freeze drying to obtain a crude extract of the sweet osmanthus;
(2) placing the crude extract of the osmanthus fragrans into a glycerol aqueous solution with the mass concentration of 20% according to the material-liquid ratio of 10g to 100g, and leaching at normal temperature for 6 hours; after leaching, transferring the leaching liquor into a centrifuge cup, centrifuging for 5min at 4000r/min, collecting upper-layer liquid, transferring the upper-layer liquid into a vacuum suction filter for suction filtration, collecting filtrate, placing the filtrate in a pear-shaped bottle, concentrating under reduced pressure, and removing a solvent to obtain a concentrated solution;
(3) dissolving the obtained concentrated solution with 2 times of water by mass, extracting with 5 times of tert-butanol, extracting the water phase with 3 times of ethyl acetate, concentrating the ethyl acetate phase under reduced pressure, and removing solvent to obtain 14mL of flos Osmanthi Fragrantis extract.
Example 2
The extraction method of the osmanthus fragrans extract is the same as that in example 1, except that 23mL of the osmanthus fragrans extract is obtained when the mass concentration of the glycerol aqueous solution is 40%.
Example 3
The extraction method of the osmanthus fragrans extract is the same as that in example 1, except that the glycerol aqueous solution has a mass concentration of 60% to obtain 24mL of the osmanthus fragrans extract.
Example 4
An extraction method of sweet osmanthus extract comprises the following steps:
(1) grinding sweet osmanthus into powder, weighing 10g of the powder, adding the powder into a reaction bottle, adding 30 times of 95% ethanol by mass, carrying out ultrasonic extraction for 30min, carrying out reduced pressure concentration on the obtained extracting solution, removing the solvent, and carrying out freeze drying to obtain a crude extract of the sweet osmanthus;
(2) placing the crude extract of the osmanthus fragrans into a glycerol aqueous solution with the mass concentration of 20% according to the material-liquid ratio of 10g to 200g, and leaching for 7 hours at normal temperature; after leaching, transferring the leaching liquor into a centrifuge cup, centrifuging for 5min at 4000r/min, collecting upper-layer liquid, transferring the upper-layer liquid into a vacuum suction filter for suction filtration, collecting filtrate, placing the filtrate in a pear-shaped bottle, concentrating under reduced pressure, and removing a solvent to obtain a concentrated solution;
(3) dissolving the obtained concentrated solution with 8 times of water by mass, extracting with tert-butyl alcohol of the same volume as the water solution for 3 times, extracting the water phase with ethyl acetate of 5 times of the water solution for 3 times, concentrating the ethyl acetate phase under reduced pressure, and removing the solvent to obtain 38mL of sweet osmanthus flower extract.
Example 5
The extraction method of the osmanthus fragrans extract is the same as that in example 4, except that 59mL of the osmanthus fragrans extract is obtained when the mass concentration of the glycerol aqueous solution is 40%.
Example 6
The extraction method of the osmanthus fragrans extract is the same as that in example 4, except that the mass concentration of the glycerol aqueous solution is 60%, and 74mL of the osmanthus fragrans extract is obtained.
Example 7
An extraction method of sweet osmanthus extract comprises the following steps:
(1) grinding sweet osmanthus into powder, weighing 10g of the powder, adding the powder into a reaction bottle, adding 95% ethanol with the mass being 20 times of that of the powder, carrying out ultrasonic extraction for 20min, carrying out reduced pressure concentration on the obtained extracting solution, removing the solvent, and carrying out freeze drying to obtain a crude extract of the sweet osmanthus;
(2) placing the crude extract of the osmanthus fragrans into glycerol aqueous solution with the mass concentration of 20% according to the material-liquid ratio of 10g to 500g, and leaching at normal temperature for 8 hours; after leaching, transferring the leaching liquor into a centrifuge cup, centrifuging for 5min at 4000r/min, collecting upper-layer liquid, transferring the upper-layer liquid into a vacuum suction filter for suction filtration, collecting filtrate, placing the filtrate in a pear-shaped bottle, concentrating under reduced pressure, and removing a solvent to obtain a concentrated solution;
(3) dissolving the obtained concentrated solution with 6 times of water by mass, extracting with 3 times of tertiary butanol with volume being 3 times of that of the water solution, extracting the water phase with 3 times of ethyl acetate with volume being equal to that of the water solution, carrying out decompression concentration on the ethyl acetate phase, and removing the solvent to obtain 81mL of the sweet osmanthus flower extract.
Example 8
The extraction method of the osmanthus fragrans extract is the same as that in example 7, except that the mass concentration of the glycerol aqueous solution is 40%, and 155mL of the osmanthus fragrans extract is obtained.
Example 9
The extraction method of the osmanthus fragrans extract is the same as that in example 7, except that the glycerin aqueous solution with the mass concentration of 60% is used for obtaining 227mL of the osmanthus fragrans extract.
Comparative example 1
The difference from example 1 is that the concentration of glycerin was adjusted to 80%.
Comparative example 2
The difference from example 1 is that the glycerol concentration was adjusted to 15%.
Comparative example 3
The difference from example 1 is that the treatment process of step (3) is not performed.
Comparative example 4
The difference from the example 1 is that the treatment process of the step (1) is not performed, and the glycerin aqueous solution leaching is directly performed by using the crushed sweet osmanthus as a raw material.
Comparative example 5
The difference from the embodiment 1 is that the osmanthus fragrans is replaced by the osmanthus fragrans.
Effect verification example 1 Total phenol content of extract
1. Standard curve for preparing gallic acid
Preparing a reference gallic acid into a 0.1mg/mL standard solution, accurately measuring the standard solution into a stoppered test tube with the volume of 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL and 1.20-10.0 mL, adding 1.00mL of forskolin phenol color developing agent, uniformly mixing, adding 5.0mL of 1mol/L sodium carbonate solution, fixing the volume to a scale by using distilled water, uniformly mixing, standing for 1h at room temperature and in a dark place, measuring the absorbance value at 760nm, and drawing a standard curve by taking the concentration of gallic acid as an abscissa and the absorbance value as an ordinate. The standard curve of gallic acid is shown in figure 1.
2. Determining the total phenol content of the extract
Accurately transferring 1.00mL of glycerol extract to be tested into a 10.0mL test tube, measuring the absorbance value according to the method, obtaining a corresponding value in a gallic acid standard curve according to the absorbance value, wherein the total phenol content of the sample is expressed by the relative amount of gallic acid (mg GAE/100g), and the measurement result is shown in Table 1.
TABLE 1
| Total phenol content (mg GAE/100g) | |
| Example 1 | 4.603 |
| Example 2 | 4.3275 |
| Example 3 | 4.3385 |
| Example 4 | 4.349 |
| Example 5 | 4.2745 |
| Example 6 | 4.349 |
| Example 7 | 4.0155 |
| Example 8 | 3.777 |
| Example 9 | 3.5495 |
| Comparative example 1 | 2.878 |
| Comparative example 2 | 2.727 |
| Comparative example 3 | 3.6005 |
| Comparative example 4 | 3.1015 |
| Comparative example 5 | 3.627 |
Effect verification example 2 Total Ketone content of extract
1. Preparation of rutin Standard Curve
Preparing a reference substance rutin into a standard solution of 0.1mg/mL by using methanol, accurately sucking 0.0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL and 1.2mL of the standard solution, placing the standard solution in a 10mL graduated test tube, adding 0.3mL of 5% sodium nitrite, shaking uniformly, standing for 6min, adding 0.3mL of 10% aluminum nitrate, shaking uniformly, standing for 6min, adding 4mL of 4% sodium hydroxide solution, diluting with distilled water to a constant volume, shaking uniformly, standing for 15min, respectively measuring the light absorption value at 510nm, and drawing a standard curve by using the concentration of rutin as a horizontal coordinate and the light absorption value as a vertical coordinate. The standard rutin curve is shown in FIG. 2.
2. Determining the total flavone content of the extract
1.0mL of the extract solution was placed in a 10mL graduated tube and the absorbance value was measured as described above. Obtaining corresponding value in rutin standard curve according to absorbance value, wherein the total flavone content of the sample is expressed by rutin relative content (mg CAE/100g), and the measuring result is shown in Table 2.
TABLE 2
| Total Flavonoids content (mg CAE/100g) | |
| Example 1 | 36.525 |
| Example 2 | 20.6 |
| Example 3 | 17.15 |
| Example 4 | 21.025 |
| Example 5 | 11.325 |
| Example 6 | 10.575 |
| Example 7 | 8.35 |
| Example 8 | 4.825 |
| Example 9 | 3.725 |
| Comparative example 1 | 3.3425 |
| Comparative example 2 | 3.2412 |
| Comparative example 3 | 3.1890 |
| Comparative example 4 | 2.7214 |
| Comparative example 5 | 2.6365 |
Effect verification example 3 Total antioxidant Properties of extract
1. Preparation of a Standard Curve
0.1mL of ascorbic acid standard solution with different concentrations, 0%, 1%, 2%, 3%, 4% and 5%, is put into a test tube with a plug, 5mL of mixed solution (containing 0.6mol/L sulfuric acid, 28mmol/L sodium phosphate and 4mmol/L ammonium molybdate) is respectively added, the plug is added, the test tube is heated in a water bath at 95 ℃ for 90min, flowing water is taken out and cooled to room temperature, the absorbance value at 695nm wavelength is measured, and a standard curve is drawn. The ascorbic acid standard curve is shown in figure 3.
2. Determination of total antioxidant Properties of extract
Taking 0.1mL of extract sample solution in a 10mL test tube, measuring the absorbance value according to the method, and checking the quantity of 1mg of sample corresponding to Vc on an ascorbic acid standard curve, namely: XmgVc/mg sample. The results are shown in Table 3.
TABLE 3
Effect verification example 4 DPPH radical scavenging
0.1mL of the extract solution was added to 3.9mL of 1X 10-4A solution of DPPH in mol/L. Starting from the time when the DPPH solution is added, placing the mixture at room temperature in the dark for 90min, measuring the absorbance at the wavelength of 517nm, and recording the absorbance as A1. The same procedure was followed except that 0.1mL of distilled water was used instead of the extract solution, and the absorbance was recorded as A0. The clearance was calculated as follows:
the results are shown in Table 4:
TABLE 4
Effect verification example 5 bacteriostatic effect of extract
The experimental strains are shown in table 5:
TABLE 5
| Bacterial species name | Strain numbering |
| Escherichia coli | BNCC185254 |
| Staphylococcus aureus | BNCC186335 |
| Bacillus cereus | BNCC103930 |
| Bacillus subtilis | BNCC109047 |
| Yeast | BNCC157508 |
| Aspergillus niger | BNCC186380 |
1. Activation of the Strain
Respectively using inoculating loops to pick out Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, yeast and Aspergillus niger from a strain plate in a superclean bench, marking on the solid culture medium plate, and inversely culturing in a biochemical incubator at 37 ℃ for 14h (culturing for 3 days by Aspergillus niger).
2. Seed liquid preparation
And (3) selecting two rings of bacterial colonies from the activated escherichia coli, staphylococcus aureus, bacillus cereus, bacillus subtilis and yeast flat plate, inoculating the two rings of bacterial colonies into 50mL of liquid culture medium, marking, placing in a 37 ℃ constant temperature incubator, carrying out shake culture for 14h-16h at the rotating speed of 180r/min until the absorbance of the two rings of bacterial colonies reaches 0.8 at the wavelength of 620nm of a spectrophotometer, and stopping culture.
And (3) cleaning the activated aspergillus niger flat plate with sterile water, collecting bacterial liquid, sucking the bacterial liquid by using an injector, filtering the bacterial liquid by using a needle filter, dropping a proper amount of filter liquid on a blood counting chamber, and placing the filter liquid under an optical microscope to count the spore number of the bacterial liquid.
Number of cells per ml-average number of cells per cell × 4 × 106X dilution factor
The number of spores is 105-107The bacterial liquid of (2) is used as seed liquid.
3. Detection of sample bacteriostatic effect
The filter paper was punched into a 0.6cm diameter disc with a punch, and the disc was placed in a petri dish and sterilized by sealing. Under aseptic conditions, the filter paper disc was immersed in the sample solution for 30 minutes with aseptic forceps for use.
Under aseptic operation, 1mL of seed bacterium liquid is added into every 300mL of sterilized culture medium which is cooled to 55 ℃, the seed bacterium liquid is fully shaken up and poured into a sterile culture dish, after the culture medium is cooled and solidified, a filter paper sheet for soaking samples is placed in a solidified test bacterium flat plate, and two samples are made for each sample in parallel. Standing for 20 minutes, placing the mixture in a constant-temperature incubator at 37 ℃ for culturing for 16 hours, and measuring the size of the inhibition zone. The bacteriostatic effect of the aspergillus niger is treated in the same way. The results are shown in Table 6 (unit: cm).
TABLE 6
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (7)
1. The extraction method of the sweet osmanthus extract is characterized by comprising the following steps of:
(1) ultrasonically extracting sweet osmanthus with ethanol, and freeze-drying to obtain a sweet osmanthus crude extract;
(2) leaching the osmanthus crude extract in a glycerol aqueous solution, centrifuging and filtering after leaching is finished, and concentrating to obtain a concentrated solution;
(3) and extracting the concentrated solution by tert-butyl alcohol, then carrying out ethyl acetate extraction on the extracted water phase, and concentrating the ethyl acetate phase to remove the solvent to obtain the osmanthus fragrans extract.
2. The extraction method according to claim 1, wherein the ultrasonic extraction time in step (1) is 10-30 min.
3. The extraction method according to claim 1, wherein the amount of the ethanol added is 5-30 times of the mass of the osmanthus fragrans.
4. The extraction method according to claim 1, wherein the concentration by mass of the glycerin aqueous solution is 20 to 60%.
5. The extraction method of claim 1, wherein the feed-to-liquid ratio of the crude extract of osmanthus fragrans to the aqueous glycerol solution is 1g:10-50 g.
6. The extraction process according to claim 1, wherein the leaching time in step (2) is 6-8 h.
7. The sweet osmanthus extract extracted by the extraction method of any one of claims 1 to 6.
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| CN101664423A (en) * | 2008-09-04 | 2010-03-10 | 河南大学 | Hypoglycemic active part of osmanthus fragrans, preparation method and application thereof |
| CN110787218A (en) * | 2019-11-14 | 2020-02-14 | 科丽思化妆品（上海）有限公司 | Extraction process of sweet osmanthus extract |
| WO2021208947A1 (en) * | 2020-04-14 | 2021-10-21 | 云南英格生物技术有限公司 | Coffee silverskin extract, preparation method therefor and use thereof |
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| CN101664423A (en) * | 2008-09-04 | 2010-03-10 | 河南大学 | Hypoglycemic active part of osmanthus fragrans, preparation method and application thereof |
| CN110787218A (en) * | 2019-11-14 | 2020-02-14 | 科丽思化妆品（上海）有限公司 | Extraction process of sweet osmanthus extract |
| WO2021208947A1 (en) * | 2020-04-14 | 2021-10-21 | 云南英格生物技术有限公司 | Coffee silverskin extract, preparation method therefor and use thereof |
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