CN104147089A - Opopanax extract, preparation method and application thereof - Google Patents
Opopanax extract, preparation method and application thereof Download PDFInfo
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- CN104147089A CN104147089A CN201310179899.3A CN201310179899A CN104147089A CN 104147089 A CN104147089 A CN 104147089A CN 201310179899 A CN201310179899 A CN 201310179899A CN 104147089 A CN104147089 A CN 104147089A
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Abstract
The invention provides an opopanax extract. The extract is prepared by extracting opopanax by a polar solvent, adsorbing the crude extract by virtue of adsorption resin, and eluting by eluent composed of water and ethanol. The opopanax extract is prepared by using a single raw material opopanax, is simple and effective, is capable of inhibiting the activity of tyrosinase, can be applied to cosmetics, and can be used for producing foods, animal feeds, medicines, quasi-drugs and the like.
Description
Technical field
The present invention relates to a Plant Extracts, the extract, preparation method that relates in particular to a kind of Acacia farnesiana Willd. plant with and at cosmetics, or the application in food, animal feed, medicine and quasi drug etc.
Background technology
The colour of skin is that people examine one of standard of beauty.For Asia women, the pale colour of skin is a kind of especially generally to be pursued.For this reason, various products arise at the historic moment, as: whitening products, anti-ultraviolet product and film product etc.
After deliberation, skin color contained various pigments in Skin Cell comprehensively determine.In numerous pigments, in melanocyte, contained melanin amount is topmost determiner.Further studies confirm that, tryrosinase (EC1.14.18.1) is the synthetic key enzyme of melanin.Thus, active by restraint of tyrosinase, to reduce melanin, generate, thereby the skin effect that reaches whitening becomes one of the important evidence of research and development skin-protection product (Chinese journals of practical medicine [J], 2005,21,1364-1366; Xiamen University's journal (natural science edition) [J], 2007,46,274-282).In these products conventional whitening agent as: hydroquinone, kojic acid, arbutin and Vc derivant etc. are several, these materials or less stable and be difficult to realize the effect of removing active oxygen, or have suitable cytotoxicity.Therefore, the safety of tyrosinase inhibitor is the factor that needs in whitening and skin-protecting R & D of complex to lay equal stress on and consider.
Japanese patent laid-open 10-25238 discloses a kind of skin preparations for extenal use, should for preparation of external preparation for skin comprise by by use the solvents such as the bark of the black chaste tree of sallee (Acacia mearnsii De Wild.) and the heartwood of larch-tree as: the means such as methanol, acetone, ethanol or the extraction of water equal solvent are separated with post obtain extract.Wherein, black chaste tree extract has restraint of tyrosinase activity.
Chinese invention patent ZL200680055582.7 discloses a kind of antioxidant composition containing from the material of Acacia bark, with water, alcohol or alcoholic solution, extracts the bark of sallee and obtains.Said composition can be applicable to food, animal feed, medicine, quasi drug and cosmetics etc.But also unexposed said composition has tyrosinase inhibitory action, and also unexposed sallee has whitening function.
The material with tyrosinase inhibitor effect or the compositions that derive from natural plants have the advantages such as safe, with low cost, just day by day obtain the attention of skin protection industry.
Summary of the invention
One object of the present invention is to provide a kind of preparation method of Acacia farnesiana Willd. extract.
Another object of the present invention is to provide a kind of Acacia farnesiana Willd. extract, has the effect of restraint of tyrosinase activity.
Another object of the present invention is to provide the extract of a kind of Acacia farnesiana Willd., with this nursing skin, has whitening effect, uses safety, without any side effects to human body skin.
A further object of the present invention is to provide the extract of a kind of Acacia farnesiana Willd., is applied to the products such as food, animal feed, medicine, quasi drug and cosmetics as active component.
Alleged " Acacia farnesiana Willd. " of the present invention refers to plant Cortex Acacia decurrens (Acacia dealbata link.), especially its alabastrum, bark and leaves.
As conventionally to understand in technical staff, improve the dissolution rate that surface area is conducive to material, thereby improve the extraction effect of active component.Therefore, processing method, as: be cut into piece, sheet and bar, especially make the Acacia farnesiana Willd. that the modes such as powder (as: mean diameter is less than 2.5mm) make and also should be included in the present invention.
Alleged " extract " of the present invention refers to one or more compositions with restraint of tyrosinase active substance or the mixture that contain that extract from Acacia farnesiana Willd..Each active substance simply mixes in proportion, or forms another kind of form with other material, as: but be not limited only to micelle, liposome and suspension etc.
" active substance " that the present invention is alleged or " active component " refer to by vitro enzyme suppress to experimental results show that can restraint of tyrosinase activity compound, polymer, nucleic acid, polypeptide, protein and separately with complex and the conjugate of the formation of chemical bonds such as ionic bond, covalent bond, coordinate bond or hydrophobic interaction.
" processing " that the present invention is alleged or " preprocessing " are interpreted as decomposition or the fractionation raw material carried out with physics mode, (as: cut, saw, press, grind, stir or pulverizing etc.) is divided into the larger material of volume the material of small volume in every way, as: section shape, bulk, lamellar, strip, graininess, powdery or powder.
Alleged " polar solvent " of the present invention refers to the organic solvent that contains hydroxyl or carbonyl isopolarity group, as: but be not limited only to water, Methanamide, dimethyl formamide, hexamethyl phosphoramide, tetramethylethylenediamine, triethylamine, tri-n-butylamine, trioctylamine, acetic acid, trifluoroacetic acid, acetonitrile, methyl formate, ethyl acetate, DMC dimethyl carbonate, dimethyl sulfoxine, acetone, butanone, dioxane, pyridine, oxolane, methanol, ethanol, isopropyl alcohol, n-butyl alcohol and ether etc. one or more, preferentially select ethanol water, concentration is 30v/v%-90v/v%, or is 50v/v%-90v/v%, or is 30v/v%-39v/v%, 40v/v%-49v/v%, one or more of 50v/v%-70v/v% and 70v/v%-90v/v%, or be 50v/v%, 51v/v%, 52v/v%, 53v/v%, 54v/v%, 55v/v%, 60v/v%, 65v/v%, 66v/v%, 67v/v%, 68v/v%, 69v/v%, 70v/v%, 71v/v%, 72v/v%, 73v/v%, 74v/v%, 75v/v%, 76v/v%, 77v/v%, 78v/v%, 79v/v%, 80v/v%, 81v/v%, 82v/v%, 83v/v%, 84v/v%, 85v/v%, 86v/v%, 87v/v%, 88v/v%, one or more of 89v/v% and 90v/v%.
Alleged " adsorbent resin " of the present invention is the macroporous adsorbent resin that persons skilled in the art are understood, preferential selection adsorbent resin low pole or middle polarity, as: but be not limited only to X-5, AB-8, D101, XDA-6, LX-21, NK-9, D3520 and WLD etc., especially AB-8 resin.
In the present invention, Acacia farnesiana Willd. extract polar solvent amount used is not particularly limited, and should make Acacia farnesiana Willd. fully contact with polar solvent.Be applicable to polar solvent consumption of the present invention, as: but be not limited only to operating weight 5-60 doubly, or 10-20 is doubly, or 10-50, or 20-50 doubly can obtain good extraction effect to Acacia farnesiana Willd. raw material to the polar solvent of Acacia farnesiana Willd..Weight multiple as: but be not limited only to 10,15,20,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 and 60, can be applicable to preferably the present invention and obtain extraction effect.
Polar solvent can infiltrate Acacia farnesiana Willd., but the leaching rate of active substance or dissolution rate are still lower, be necessary to adopt further method as: but be not limited only to decoct, boil, reflux, stirring, homogenate and the extracting mode such as ultrasonic, to improve working performance.These methods can be used alone or in combination, also can be repeatedly.Said extracted mode through the appropriate time (being no more than 2 hours) as: but be not limited only to 0.5-1.0 hour, 0.5-2.0 hour and 1.0-2.0 hour etc. can increase leaching rate or the dissolution rate of active substance.
Eluent eluting is that the present invention obtains one of committed step of active substance, contribute to the raising of extract to tryrosinase inhibition, but the concrete mode of its eluting, as: linear gradient elution, isocratic elution and staged gradient elution etc. must not limit the present invention.In order to adapt to the needs of production scale and separating medium (as: adsorbent resin), those skilled in the art can select and be suitable for any one or more type of elution, and collect corresponding eluent.Type of elution provided by the invention, as: with water and ethanol, form eluent, with water, 20v/v% ± 5v/v% ethanol, 40v/v% ± 5v/v% ethanol, 60v/v% ± 5v/v% ethanol and 80v/v% ± 5v/v% ethanol, carry out eluting successively, forming total gradient is 20v/v%-80v/v% ethanol elution mode, and collects respectively eluent; And for example: with water and ethanol, form eluent, with water, 40v/v% ± 5v/v% ethanol and 80v/v% ± 5v/v% ethanol, carry out eluting successively, and collect respectively eluent; For another example: with water and ethanol, form eluent, carry out 20v/v%-80v/v% ethanol, 20v/v%-60v/v% ethanol or 30v/v%-50v/v% ethanol linear gradient elution, and collect eluent.
The preparation method of a kind of Acacia farnesiana Willd. extract provided by the invention, use polar solvent to extract Acacia farnesiana Willd., as: but be not limited only to impose decoct, boil, reflux, stirring, homogenate and one or more ultrasonic extracting modes process and Acacia farnesiana Willd. crude extract, again after adsorbent resin absorption, the eluent eluting forming with water and ethanol, and collect eluent and obtain.
After measured, make the extract of various Acacia farnesiana Willd. with method provided by the invention, have the effect of restraint of tyrosinase activity, significantly check melanin generates.Further experiment checking, has whitening effect with this nursing skin, can be applicable to the products such as food, animal feed, medicine, quasi drug and cosmetics.
The beneficial effect that technical solution of the present invention realizes:
It is raw material that single Acacia farnesiana Willd. plant is take in the present invention, and especially the alabastrum of Acacia farnesiana Willd. plant, bark and leaves, make product quality controlled, and effectiveness is stable.
The present invention first uses polar solvent to extract, and gained crude extract makes Acacia farnesiana Willd. extract after adsorbent resin absorption eluting.This technical scheme is simply efficient, is suitable for industrialized mode, the active substance in Acacia farnesiana Willd. plant being extracted, being utilized and develops.
It is reliable that the present invention produces the method safety of extract, is convenient to the application in the products such as food, animal feed, medicine, quasi drug and cosmetics.
Accompanying drawing explanation
Fig. 1 is the inhibitory action that Acacia farnesiana Willd. extract of the present invention generates melanin; Wherein, A represents the inhibition situation that this hurdle generates melanin for the extract that adopts embodiment 2 and make; B represents that this hurdle is the inhibition situation that the extract that makes of embodiment 3 generates melanin; C represents the inhibition situation that extract that embodiment 4 makes generates melanin.
The specific embodiment
Below in conjunction with accompanying drawing, describe technical scheme of the present invention in detail.The embodiment of the present invention is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.
Embodiment 1 Acacia farnesiana Willd. extraction conditions preferably
Fresh Acacia farnesiana Willd. 10g, adopts L
9(3
4) orthogonal test (drug research common mathematical method and computer program [P], Guizhou science and technology publishing house, 1997) carries out the optimization of extraction process, take solid-liquid ratio (A), concentration of alcohol (B), extraction time (C) is experimental factor, and blank column (D).The extraction ratio of take carries out positive quadraturing design test, Optimized Extraction Process condition as index.Factor level design is in Table 1, and wherein, from production cost consideration, extraction time is generally no more than 2 hours, the results are shown in Table 2.
Table 1 orthogonal test factor level
Table 2L
9(3
4) orthogonal experiment plan takes into account result table
Table 3 orthogonal test intuitive analysis table
Table 4 orthogonal test analysis of variance table
F
0.05(2,2)＝19.0
Orthogonal test variance analysis shows: confidence level is 0.95 o'clock, and concentration of alcohol and extraction time are significant factors, and solid-liquid ratio is not remarkable factor in selected three levels.Intuitive analysis can obtain, and A3B1C3 i.e. 50 times of solid-liquid ratios, 50% concentration of alcohol and 2 hours extraction times, are the optimum extraction conditions of Acacia farnesiana Willd. extract.Because solid-liquid ratio is not remarkable to extraction ratio impact effect, the consideration based on solvent cost, can also adopt 20 times of solid-liquid ratios in actual production.
Producing of embodiment 2 Acacia farnesiana Willd. alabastrum extracts
After 100g Acacia farnesiana Willd. alabastrum is cleaned, mix with 2L70v/v% alcoholic solution, at room temperature, supersound extraction 1 hour, isolates crude extract, through decompression, steams after alcoholic solution, obtains Acacia farnesiana Willd. alabastrum crude extract 6g.
Depletion Herba Albiziae alabastrum crude extract 1g, with after deionized water dissolving, be loaded to AB-8 resin filling post, use successively deionized water, 20v/v% ethanol, 40v/v% ethanol, 60v/v% ethanol and 80v/v% ethanol gradient elution, collect respectively eluent, decompression steams second alcohol and water, obtains respectively washing the Acacia farnesiana Willd. alabastrum extract at position, 20v/v% ethanol position, 40v/v% ethanol position, 60v/v% ethanol position and 80v/v% ethanol position, freezing preservation at-20 ℃.
Producing of embodiment 3 wattle peel extract
After 50g Acacia farnesiana Willd. bark is cleaned, mix with 1L70v/v% alcoholic solution, by circulation countercurrent extraction method, extract 2 hours, isolate crude extract, through decompression, steam after deionized water, obtain Acacia farnesiana Willd. bark crude extract 5g.
Depletion Herba Albiziae bark crude extract 5g, with after deionized water dissolving, be loaded to X-5 resin filling post, use successively deionized water, 20v/v% ethanol, 40v/v% ethanol, 60v/v% ethanol and 80v/v% ethanol gradient elution, collect respectively eluent, decompression steams second alcohol and water, obtains respectively washing the wattle peel extract at position, 20v/v% ethanol position, 40v/v% ethanol position, 60v/v% ethanol position and 80v/v% ethanol position, freezing preservation at-20 ℃.
Producing of embodiment 4 wattle leaf extracts
After 200g Acacia farnesiana Willd. leaves is cleaned, mix with 4L50v/v% alcoholic solution, at room temperature, stir and extract 1.5 hours, isolate crude extract, through decompression, steam after alcoholic solution, obtain wattle Leave extract 8g.
Depletion silk tree Leave extract 1g, with after deionized water dissolving, be loaded to AB-8 resin filling post, use successively deionized water, 20v/v% ethanol, 40v/v% ethanol, 60v/v% ethanol and 80v/v% ethanol gradient elution, collect respectively eluent, decompression steams second alcohol and water, obtains respectively washing the wattle leaf extract at position, 20v/v% ethanol position, 40v/v% ethanol position, 60v/v% ethanol position and 80v/v% ethanol position, freezing preservation at-20 ℃.
The detection of embodiment 5 tyrosinase inhibitory activities
According to the sample of embodiment 2, embodiment 3 and embodiment 4 preparations, be dissolved in deionized water respectively, be mixed with aqueous solution, for measuring tyrosinase inhibitory activity, take arbutin as object of reference.Determination step is: soda acid buffer solution (pH6.8,30mM) 70 μ L, 1.66mM tyrosine solution 80 μ L, sample solution 80 μ L mix homogeneously, in 37 ℃ of thermostatic water bath, incubation is more than 5 minutes, add tryrosinase solution (1000U/ml) 10 μ L, after 37 ℃ of constant temperature 10min, by microplate reader, measure the absorbance A of 475nm wavelength
475.With deionized water, replace sample aqueous solution, as reference solution, measure equally absorbance, the results are shown in Table 5.
Tyrosinase activity suppression ratio computing formula is: suppression ratio (%)=(A
0-(A
475-B))/A
0* 100%, A wherein
0the absorbance of reference solution, A
475be the absorbance of sample solution, B is the absorbance after sample aqueous solution dilutes 3 times.
The measurement result of table 5 tyrosinase inhibitory activity
From table 5, the extract obtaining from the alabastrum of Acacia farnesiana Willd. plant, bark and leaves respectively, after adsorbent resin separation, gained 20v/v% ethanol, 40v/v% ethanol, 60v/v% ethanol and 80v/v% alcohol elution all can be realized the inhibition to tyrosinase activity, and its 40v/v% alcohol elution is the strongest to the inhibitory action of tyrosinase activity.
The detection that embodiment 6 removes free radical activity
The Acacia farnesiana Willd. extract preparing by embodiment 2, example 3 and example 4 methods respectively, measures it by DPPH method and removes free radical activity, with V
cfor object of reference.Determination step is: accurately pipette extracting solution 2mL in 10mL tool plug test tube, add people 2mL DPPH alcoholic solution (2 * 10
-4mol/L), fully mix, room temperature is standing, uses the absorbance A of spectrophotometric determination 517nm wavelength after 30min
517(take ethanol as reference); Measure 2mL DPPH solution and the mixed absorbance A of 2mL ethanol simultaneously
c, and 2mL extracting solution and the mixed absorbance A of 2mL alcoholic solution
0.Parallel assay three times, averages, and according to following formula, calculates free radical scavenging activity.
Free radical scavenging activity=(1-(A
517-A
0)/A
c) * 100%.
Table 6DPPH method is removed the testing result of free radical activity
From table 6, the extract obtaining from the alabastrum of Acacia farnesiana Willd. plant, bark and leaves respectively, after adsorbent resin separation, gained 20v/v% ethanol, 40v/v% ethanol, 60v/v% ethanol and 80v/v% alcohol elution all can be realized the removing to DPPH free radical, and its 40v/v% alcohol elution is the strongest to the scavenging action of free radical.
The inhibitory action that embodiment 7 Acacia farnesiana Willd. extracts generate melanin
According to the 40v/v% alcohol elution sample of embodiment 2, embodiment 3 and embodiment 4 preparations, be dissolved in deionized water respectively, be mixed with 1mg/mL aqueous solution, for measuring the inhibitory action that melanin is generated, the arbutin solution of same concentrations of take is object of reference.Step is: soda acid buffer solution (pH6.8,30mM) 70 μ L, 1.66mM tyrosine solution 80 μ L, sample solution 80 μ L mix homogeneously, in 37 ℃ of thermostatic water bath, incubation more than 5 minutes, adds tryrosinase solution (1000U/ml) 10 μ L, 37 ℃ of constant temperature 4 hours, the results are shown in Figure 1.
As seen from Figure 1, with negative blank with add the positive control of arbutin to compare, add the sample color of embodiment 2, embodiment 3 and embodiment 4 prepared extracts to be brown, show the significantly generation of check melanin.The inhibitory action that the extract of Acacia farnesiana Willd. plant alabastrum (being embodiment 2) generates melanin is (it is light brown that sample is) the most by force.
Claims (11)
1. produce a method for Acacia farnesiana Willd. extract, it is characterized in that using polar solvent to carry out extraction process to Acacia farnesiana Willd. and Acacia farnesiana Willd. crude extract, then after adsorbent resin absorption, the eluent eluting forming with water and ethanol, and collect eluent.
2. the method for producing Acacia farnesiana Willd. extract according to claim 1, is characterized in that described polar solvent is ethanol water.
3. the method for producing Acacia farnesiana Willd. extract according to claim 2, the concentration that it is characterized in that described ethanol water is 50v/v%-90v/v%.
4. the method for producing Acacia farnesiana Willd. extract according to claim 1, the consumption that it is characterized in that described polar solvent be Acacia farnesiana Willd. weight 5-60 doubly.
5. the method for producing Acacia farnesiana Willd. extract according to claim 1, is characterized in that described eluent is 20v/v%-80v/v% ethanol.
6. the method for producing Acacia farnesiana Willd. extract according to claim 1, is characterized in that described adsorbent resin is AB-8.
7. the Acacia farnesiana Willd. extract obtaining according to the method for producing Acacia farnesiana Willd. extract one of claim 1-6 Suo Shu.
8. one kind contains Acacia farnesiana Willd. extract claimed in claim 7 in the application in whitening field.
9. one kind contains Acacia farnesiana Willd. extract claimed in claim 7 for removing free radical.
10. one kind contains the application of Acacia farnesiana Willd. extract claimed in claim 7 in restraint of tyrosinase activity.
The application of one of 11. according to Claim 8-10 described Acacia farnesiana Willd. extracts, is characterized in that for food prepared therefrom, animal feed, medicine, quasi drug or cosmetics.
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| CN105342916A (en) * | 2015-11-19 | 2016-02-24 | 伽蓝（集团）股份有限公司 | Plant extract product composition, and preparation method and application thereof |
| CN112544815A (en) * | 2020-11-27 | 2021-03-26 | 广西壮族自治区畜牧研究所 | Feed for enhancing liver function of chicken and application thereof |
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| CN105342916A (en) * | 2015-11-19 | 2016-02-24 | 伽蓝（集团）股份有限公司 | Plant extract product composition, and preparation method and application thereof |
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| CN112544815A (en) * | 2020-11-27 | 2021-03-26 | 广西壮族自治区畜牧研究所 | Feed for enhancing liver function of chicken and application thereof |
| CN112544815B (en) * | 2020-11-27 | 2023-10-20 | 广西壮族自治区畜牧研究所 | Feed for enhancing liver function of chickens and application of feed |
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