CN105726410A - Dianella ensifolia active extract as well as preparation method and application thereof - Google Patents
Dianella ensifolia active extract as well as preparation method and application thereof Download PDFInfo
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- CN105726410A CN105726410A CN201610187409.8A CN201610187409A CN105726410A CN 105726410 A CN105726410 A CN 105726410A CN 201610187409 A CN201610187409 A CN 201610187409A CN 105726410 A CN105726410 A CN 105726410A
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- villous themeda
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- 239000000284 extract Substances 0.000 title claims abstract description 103
- 238000000605 extraction Methods 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 244000058946 Dianella ensifolia Species 0.000 title abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 77
- 239000003480 eluent Substances 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000000287 crude extract Substances 0.000 claims abstract description 18
- 239000000843 powder Substances 0.000 claims abstract description 14
- 239000002537 cosmetic Substances 0.000 claims abstract description 13
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 99
- 241000931197 Themeda Species 0.000 claims description 90
- 241000233855 Orchidaceae Species 0.000 claims description 56
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000006166 lysate Substances 0.000 claims description 12
- 239000003208 petroleum Substances 0.000 claims description 10
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- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 206010040829 Skin discolouration Diseases 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 4
- NIQQIJXGUZVEBB-UHFFFAOYSA-N methanol;propan-2-one Chemical compound OC.CC(C)=O NIQQIJXGUZVEBB-UHFFFAOYSA-N 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- NBEMQPLNBYYUAZ-UHFFFAOYSA-N ethyl acetate;propan-2-one Chemical compound CC(C)=O.CCOC(C)=O NBEMQPLNBYYUAZ-UHFFFAOYSA-N 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 18
- 210000002752 melanocyte Anatomy 0.000 abstract description 12
- 230000002087 whitening effect Effects 0.000 abstract description 11
- 241000196324 Embryophyta Species 0.000 abstract description 8
- 239000003795 chemical substances by application Substances 0.000 abstract description 5
- 238000002386 leaching Methods 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 238000005377 adsorption chromatography Methods 0.000 abstract description 2
- 239000013049 sediment Substances 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 28
- 239000000523 sample Substances 0.000 description 18
- 230000001629 suppression Effects 0.000 description 18
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 238000013461 design Methods 0.000 description 11
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 8
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- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 6
- NCCWCZLEACWJIN-UHFFFAOYSA-N orsellinic acid methyl ester Chemical compound COC(=O)C1=C(C)C=C(O)C=C1O NCCWCZLEACWJIN-UHFFFAOYSA-N 0.000 description 6
- 210000003491 skin Anatomy 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 5
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
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- 238000004809 thin layer chromatography Methods 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- RFKMWWMZUHXFBA-UHFFFAOYSA-N 1-(2,4-dihydroxy-6-methoxy-3-methylphenyl)ethanone Chemical compound COC1=CC(O)=C(C)C(O)=C1C(C)=O RFKMWWMZUHXFBA-UHFFFAOYSA-N 0.000 description 2
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 2
- -1 Resorcin compound Chemical class 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
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- 238000010992 reflux Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- ZTVIKZXZYLEVOL-DGKWVBSXSA-N 2-hydroxy-2-phenylacetic acid [(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] ester Chemical group C([C@H]1CC[C@@H](C2)N1C)C2OC(=O)C(O)C1=CC=CC=C1 ZTVIKZXZYLEVOL-DGKWVBSXSA-N 0.000 description 1
- URHMWJDVTSXLST-UHFFFAOYSA-N CC=1C(=C(C(=C(C(=O)O)C1C)O)C)O.COC(C1=C(C(=C(C=C1C)O)C)O)=O Chemical compound CC=1C(=C(C(=C(C(=O)O)C1C)O)C)O.COC(C1=C(C(=C(C=C1C)O)C)O)=O URHMWJDVTSXLST-UHFFFAOYSA-N 0.000 description 1
- ODINCKMPIJJUCX-UHFFFAOYSA-N Calcium oxide Chemical compound [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000035126 Facies Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 241000130764 Tinea Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 208000010247 contact dermatitis Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
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- 201000003265 lymphadenitis Diseases 0.000 description 1
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- 239000012567 medical material Substances 0.000 description 1
- 150000002730 mercury Chemical class 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
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- 229960005322 streptomycin Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- 239000000052 vinegar Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- 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
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a dianella ensifolia active extract as well as a preparation method and application thereof and belongs to the field of plant separation and extraction. The preparation method comprises the following steps: leaching and extracting, namely soaking dianella ensifolia powder into alkalescent water with the pH value of 8-10, so as to obtain leaching liquor, then regulating pH value of the leaching liquor to be 2-6, then standing to obtain sediments, and drying, so that dianella ensifolia crude extract is obtained; carrying out extraction, namely dissolving the dianella ensifolia crude extract with water to obtain dianella ensifolia dissolved solution, and then adding an extracting agent into the dianella ensifolia dissolved solution for extracting, so that extracts are obtained; and carrying out separation, namely separating the extracts by adopting adsorption chromatography, and carrying out gradient elution by taking mixed liquor of organic solvents in different ratios as an eluent, so that the dianella ensifolia active extract is obtained. The obtained dianella ensifolia active extract has antityrosinase activity and inhibitory effect on B16 melanocytes and can be applied to whitening cosmetics.
Description
Technical field
The present invention relates to plant separation and Extraction field, in particular to the work that a kind of mountain villous themeda is blue
Property extract and its preparation method and application.
Background technology
In recent years, along with the cosmetics of the most various chemosynthesis classes, poisoning thing occurs time and again
Part, the plant that people increasingly advocate some pure naturals selecting cosmetics when is other
Cosmetics.Drug effect is gentle, ill effect is little and obtains the wide of consumer because it has for Chinese herbal medicine
General approval.But, the Chinese herbal medicine of China is of a great variety, and the property of medicine is changeable, and in every taste medical material
All containing multiple chemical composition, this is just to finding the sky that exploitation is a kind of efficiently, side effect is little
So Chinese herbal medicine skin-lightening cosmetic causes great difficulty.
Mountain villous themeda orchid is that Liliaceae mountain villous themeda belongs to the dry of herbaceous plant mountain villous themeda (Dianella ensifolia)
Dry herb, is to be distributed widely in Chinese yunnan, Sichuan, Dong people, Guangxi, Guangdong
The common herbs in south, South Jiangxi, Coastal Area in Zhejiang Province, Fujian and Taiwan.Medicine
On being worth, after the root stock mill dry powder that mountain villous themeda is blue, adjust vinegar external application, can control ulcerative carbuncle abscess, tinea,
Lymphadenitis etc..
Resorcin compound to the blue chemical constitution study isolated of mountain villous themeda in recent years
Have: 2,4-dihydroxy-3,6-dimethylbenzoate methyl ester (methyl 2,4-dihydroxy-3,
6-dimethylbenzoate), 2, the 4-dihydroxy i.e. methyl orsellinate of-6-methyl toluate
(methyl 2,4-dihydroxy-6-methylbenzoate, methylorsellinate) 2 Hes
4-dihydroxy-6-methoxyl group-3-methyl acetophenone (2,
4-dihydroxy-6-methoxy-3-methylacetophenone)。
Comprehensive existing documents and materials, do not find that mountain villous themeda is blue and ingredient has whitening function
Relevant report.
Summary of the invention
The first object of the present invention is the activity extract providing a kind of mountain villous themeda blue.This mountain
The blue activity extract of villous themeda has antityrosinase activity have B16 melanocyte to be pressed down
Make use, and it is little to the side effect of skin, it is possible to as natural plant whitening formulation application
In various skin-lightening cosmetics.
The second object of the present invention is the preparation side of the activity extract providing a kind of mountain villous themeda blue
Method.Adopt the purity of effective ingredient in the activity extract that the mountain villous themeda prepared in this way is blue high,
And it is big to the inhibitory action of tryrosinase and B16 melanocyte.
The third object of the present invention is the application of the activity extract providing a kind of mountain villous themeda blue,
Will the blue activity extract of mountain villous themeda as the active component of skin-lightening cosmetic.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
The preparation method of the activity extract that a kind of mountain villous themeda is blue, comprises the following steps:
Leach step: mountain villous themeda orchid powder is soaked in the week-base water that pH value is 8-10 and carries out
Extraction, then removes mountain villous themeda orchid powder and obtains lixiviating solution, then the pH value regulating lixiviating solution is
2-6, then standing processes, then the supernatant removing lixiviating solution is precipitated thing, by precipitate
Obtain mountain villous themeda orchid crude extract after drying；
Extraction step: mountain villous themeda orchid crude extract water dissolution is obtained mountain villous themeda orchid lysate, then
In the villous themeda orchid lysate of mountain, add extractant extract, be extracted thing, wherein extractant
Volume be 0.5-2 times of volume of mountain villous themeda orchid lysate；
Separating step: separate extract by adsorption charomatography, with having of different ratio
Machine solvent mixed liquor carries out gradient elution as eluant, obtains eluent, removes
Eluant in eluent, obtains the activity extract that mountain villous themeda is blue.Whitening agent can suppress cheese
Propylhomoserin enzymatic activity or blocking-up tyrosine generate melanic oxidative pathway, thus reduce black
Element generates the effect reaching skin whitening.It is raw that skin-whitening agents acts on dermal melanin exactly
In one-tenth, metabolic process, suppression melanin generates and the material of compliant.But it is traditional
Skin-whitening agents, often uses chemical substance, as having the mercury salt of whitening effect and having
The hydroquinone of effect of dispelling spots.Although these compounds have the effect being rapidly achieved whitening, but to skin
Skin has bigger toxic and side effects, the especially damage to sensitive skin even more serious, makes for a long time
With causing contact dermatitis or cause the ill effects such as permanent decolouring, many countries by
Disabling.The skin-whitening agents currently required that should comply with two standards: one is to tryrosinase
Activity has higher suppression ratio, can significantly reduce melanic generation；Two have higher
Safety, nontoxic non-stimulated to human body skin.
In the present invention, using mountain villous themeda this traditional medicinal plants blue is object of research, obtains
A kind of mountain villous themeda that there is antityrosinase activity and B16 melanocyte is had inhibitory action
Blue activity extract.Mountain villous themeda orchid is natural plants, and whitening effect is good, and the property of medicine is gentle, to skin
The zest of skin is little, and side effect is little.Therefore, this mountain villous themeda orchid extract can be as U.S.
The active component of albefaction cosmetic, is applied in skin-lightening cosmetic.
When preparing the activity extract of this mountain villous themeda orchid, carry out initially with alkali extraction and acid precipitation
Thick extraction.This extracting method is advantageous in that the week-base water that pH is 8-10 can increase mountain
Acid ingredient and the dissolution rate of alkalescence composition in villous themeda orchid, thus improve extraction efficiency.Subsequently
The purpose that pH value is 2-6 of regulation gained lixiviating solution is, makes the acid ingredient in solution
Reduce with the dissolubility of alkalescence composition, separate out from lixiviating solution, static after be precipitated,
Thus remove the partial impurities in aqueous extract, further up to isolated and purified effect.
Subsequently, extract with extractant, according to similar compatibility principle, mountain villous themeda orchid is dissolved
Chemical composition in liquid carries out segmentation according to polarity size, and can remove mountain villous themeda orchid lysate
In the biggest or the least chemical composition of some polarity.The extract of gained is used absorption
Chromatography is carried out the most after purification, in the activity extract that gained mountain villous themeda is blue, effectively
The content of active substance increases, and purity increases, its antityrosinase activity and to B16 black
The inhibitory action of element cell is obviously improved.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but this
Skilled person is it will be appreciated that the following example is merely to illustrate the present invention, and should not regard
For limiting the scope of the present invention.Unreceipted actual conditions person in embodiment, according to normal condition
Or the condition of manufacturer's suggestion is carried out.Agents useful for same or instrument unreceipted production firm person, all
For the conventional products that can be obtained by commercially available purchase.
The activity extract that a kind of mountain villous themeda is blue, is prepared by the method comprised the steps:
Leach step: mountain villous themeda orchid powder is soaked in the week-base water that pH value is 8-10 and carries out
Extraction, then removes mountain villous themeda orchid powder and obtains lixiviating solution, then the pH value regulating lixiviating solution is
2-6, then standing processes, then the supernatant removing lixiviating solution is precipitated thing, by precipitate
Obtain mountain villous themeda orchid crude extract after drying.
In some preferred embodiments of the present invention, mountain villous themeda orchid is powder, added during extraction
The volume ratio of week-base water and mountain villous themeda orchid powder be 1:6-1:10.Further it is preferably,
Use supercritical ultrasonics technology to extract, extract 1-3 time, every time with 20MHz-50MHZFrequency
The ultrasonic 30min-50min of rate, merging filtrate after filtration, obtain lixiviating solution.This extracting method
The dissolution rate of active substance in the villous themeda orchid of mountain can be increased, be further able to improve mountain villous themeda orchid and slightly carry
Thing is to tryrosinase and the inhibitory activity of B16 cell.
Extraction step: mountain villous themeda orchid crude extract water dissolution is obtained mountain villous themeda orchid lysate, then
In the villous themeda orchid lysate of mountain, add extractant extract, be extracted thing, wherein extractant
Volume be 0.5-2 times of volume of mountain villous themeda orchid lysate.
In some preferred embodiments of the present invention, extractant is ethyl acetate, obtains second
Acetoacetic ester extract.Owing to ethyl acetate belongs to the organic solvent of middle polarity, according to similar
Compatibility principle, it is possible to remove the chemical composition that some polarity in Aqueous extracts are the biggest or the least,
Reservation can be dissolved in the chemical composition in ethyl acetate.Further, the present invention's
In some preferred embodiments, extractant includes petroleum ether, chloroform, ethyl acetate, extraction
Step for mountain villous themeda orchid lysate is extracted by petroleum ether, chloroform, ethyl acetate successively,
Obtain petroleum ether extract, chloroform extract, acetic acid ethyl ester extract.This extracting process
Impurity contained in the acetic acid ethyl ester extract obtained is less, thus reduces impurity to tyrosine
The impact of the inhibitory activity of enzyme and B16 cell.Separating step: by adsorption charomatography to extraction
Thing separates, and carries out gradient with the organic solvent mixed liquor of different ratio as eluant and washes
De-, obtain eluent, remove the eluant in eluent the most again, obtain the work that mountain villous themeda is blue
Property extract.In some preferred embodiments of the present invention, carry out point by adsorption charomatography
From extract be acetic acid ethyl ester extract, according to extract to tryrosinase and B16 cell
Inhibitory activity follow the tracks of find, tryrosinase and B16 cell are pressed down by acetic acid ethyl ester extract
System activity is big, therefore, directly acetic acid ethyl ester extract is carried out adsorption chromatography separation, favorably
In reducing operation, reducing time and Financial cost, the most i.e. can obtain tyrosine
The activity extract that the inhibitory activity of enzyme and B16 cell is best.
In some preferred embodiments of the present invention, this adsorption charomatography is silica gel column chromatography
Method.The good separating effect of silica gel column chromatography, it is possible to the chemical constituent in the villous themeda orchid of mountain is carried out more
The most isolated and purified, good to the inhibitory activity of tryrosinase and B16 cell to obtain
Active site.It is further preferred that in chromatographic column equipped with silica gel and acetic acid ethyl ester extract
Dry weight between weight ratio be 40～50:1, in this proportion, it is possible to substantially carry
The post effect of high silicagel column, improves while shortening disengaging time, saving eluting agent and divides
From effect.
Further, in preferred embodiment of the present invention, eluant is: chloroform-acetone mixes
Close liquid, ethyl acetate-acetone mixed liquor, acetone-methanol mixed liquor and methanol-water mixture,
Wherein in chloroform-acetone mixture, the volume ratio of chloroform and acetone is 10～30:1, acetic acid second
In ester-acetone mixture, the volume ratio of ethyl acetate and acetone is 20～40:1, acetone-methanol
In mixed liquor, the volume ratio of acetone and methanol is 2～10:1, in methanol-water mixture methanol and
The volume ratio of water is 10～20:1.
Further preferred, the gradient elution program of column chromatography is as shown in table 1, obtains 32
Bottle eluent, every part of 250mL.Use thin layer chromatography analysis, merge contained compound component
Close eluent, i.e. merges 10-15 bottle eluent and recycling design obtains the first activity and carries
Take thing, merge 16-27 bottle eluent and recycling design obtains the second activity extract, merge
28-32 bottle eluent recycling design obtain the 3rd activity extract.
Elution program in table 1 silica gel column chromatography
The first activity extract of this gradient elution program gained, the second activity is used to extract
In thing and the 3rd activity extract, the purity of active substance is higher, thin to tryrosinase and B16
The inhibitory activity of born of the same parents is more preferable, and wherein the second activity extract is to tryrosinase and B16 cell
Inhibitory activity is optimal.
The mountain villous themeda orchid obtained by said method extracts activity extract, has antityrosinase
Activity also has inhibitory action to B16 melanocyte.It is that a kind of natural plant whitening carries
Take thing, human body is had no side effect, have application potential in skin-lightening cosmetic field.This mountain villous themeda
Blue active site can be used for preparing the cosmetics with whitening function: as astringent, cosmetic water,
Emulsion, cream, cleansing cream etc..
Embodiment 1
1. extraction step:
The mountain villous themeda orchid dried powder taking 250g is put in vessel, adds 1L pure water, uses Calx
After the PH of breast regulation solution is 8, vessel are put into ultrasonic 30min in Ultrasound Instrument, ultrasonic
The frequency of ripple is 20MHz.After being then passed through filter, obtain lixiviating solution, with HCl regulation extraction
The PH of liquid to 6, after standing overnight, removes supernatant, is precipitated, vacuum drying,
To mountain villous themeda orchid crude extract.Take 1mg mountain villous themeda orchid crude extract and be configured to 0.5mg/mL solution, put
Save backup in 4 DEG C.
2. extraction step:
Remaining mountain villous themeda orchid crude extract 100mL pure water is dissolved, is subsequently adding 50mL's
Ethyl acetate extracts, and extracts three times, the ethyl acetate every time extracting gained is merged mutually,
Concentrating under reduced pressure reclaims ethyl acetate solvent, obtains acetic acid ethyl ester extract, about 200mg.Take
The acetic acid ethyl ester extract of 1mg is configured to the solution of 0.5mg/mL, is placed in 4 DEG C and saves backup.
3. separating step:
Silica gel column chromatography is used to separate above-mentioned acetic acid ethyl ester extract.First 8g silica gel is weighed
(specification is 100-200 mesh) carries out filling post, and the column volume of chromatographic column is 1L；Then by institute
The acetic acid ethyl ester extract obtained dissolves, and enters with 200mg silica gel (specification is 200-300 mesh)
Row mixes sample；Use a dry method on a sample subsequently and acetic acid ethyl ester extract is placed in the superiors in silicagel column
Silica Surface, use organic solvent to carry out gradient elution, eluting order is as shown in table 1,
Obtain 32 bottles of eluents, every part of 250mL.Use thin layer chromatography analysis, merge containedization
The eluent that polymer component is close, i.e. merges 6-12 bottle eluent and recycling design obtains first
Activity extract, merges 13-21 bottle eluent and recycling design obtains the second activity extract,
Merge 22-28 bottle eluent and recycling design obtains the 3rd activity extract.
Take first activity extract of 1mg, the second activity extract and the 3rd activity respectively to carry
Take thing and be configured to the solution of 0.5mg/mL, be placed in 4 DEG C and save backup.
Embodiment 2
1. extraction step:
The mountain villous themeda orchid dried powder taking 250g is put in vessel, adds 1.5L pure water, uses stone
After the PH of ash breast regulation solution is 10, vessel are put into ultrasonic 30min in Ultrasound Instrument, super
The frequency of sound wave is 50MHz.After being then passed through filter, obtain lixiviating solution, with HCl regulation leaching
The PH of extract to 2, after standing overnight, removes supernatant, is precipitated, vacuum drying,
Obtain mountain villous themeda orchid crude extract.Mountain villous themeda orchid crude extract is configured to 0.5mg/mL solution, is placed in
4 DEG C save backup.
2. extraction step:
Remaining mountain villous themeda orchid crude extract 100mL pure water is dissolved, is subsequently adding 200mL
Ethyl acetate extract, extract three times, the ethyl acetate every time extracting gained be harmonious
And, concentrating under reduced pressure reclaims ethyl acetate solvent, obtains acetic acid ethyl ester extract, about 220mg.
The acetic acid ethyl ester extract taking 1mg is configured to the solution of 0.5mg/mL, is placed in 4 DEG C of preservations
Standby.
3. separating step:
Silica gel column chromatography is used to separate above-mentioned acetic acid ethyl ester extract.First 11g silicon is weighed
Glue (specification is 100-200 mesh) carries out filling post, and the column volume of chromatographic column is 1L；Then will
The acetic acid ethyl ester extract of gained dissolves, with 220mg silica gel (specification is 200-300 mesh)
Carry out mixing sample；Use a dry method on a sample subsequently to be placed in silicagel column acetic acid ethyl ester extract and go up most
The Silica Surface of layer, uses organic solvent to carry out gradient elution, and eluting order is as shown in table 1,
Obtain 32 bottles of eluents, every part of 250mL.Use thin layer chromatography analysis, merge containedization
The eluent that polymer component is close, i.e. merges 10-15 bottle eluent and recycling design obtains
One activity extract, merges 16-27 bottle eluent and recycling design obtains the second activity and extracts
Thing, merges 28-32 bottle eluent and recycling design obtains the 3rd activity extract.
Take first activity extract of 1mg, the second activity extract and the 3rd activity respectively to carry
Take thing and be configured to the solution of 0.5mg/mL, be placed in 4 DEG C and save backup.
Embodiment 3
The method of the activity extract of the preparation mountain villous themeda orchid that the present embodiment is provided is with embodiment 2
Unanimously, difference is:
In extraction step, remaining mountain villous themeda orchid crude extract 100mL pure water is dissolved, so
After extract with petroleum ether, chloroform, ethyl acetate and n-butyl alcohol successively.These four extracts
Agent all extracts 3 times, each 200mL.Respectively the organic facies of 3 extraction gained is merged,
Concentrating under reduced pressure recycling design, respectively obtains petroleum ether extract (about 12mg), chloroform extraction
Thing (about 23mg), acetic acid ethyl ester extract (about 80mg) and n-butyl alcohol extract is (about
29mg).Take the petroleum ether extract of 1mg, chloroform extract, ethyl acetate extraction respectively
Thing and n-butyl alcohol extract are configured to 0.5mg/mL solution, are placed in 4 DEG C and save backup.
Embodiment 4
The method of the activity extract of the preparation mountain villous themeda orchid that the present embodiment is provided is with embodiment 2
Unanimously, difference is, in separating step, uses silica gel column chromatography separating acetic acid
The gradient elution program used during ethyl ester extract is as shown in table 2:
Elution program in table 2 silica gel column chromatography
Embodiment 5
Take 250g medicinal powder, add the methanol solution 1.5L of 70%, heating and refluxing extraction 2
Secondary, the time of each solvent boiling is 2h, merging filtrate, after recycling design, and vacuum drying,
Obtain mountain villous themeda orchid crude extract.Take 1mg mountain villous themeda orchid crude extract and be configured to 0.5mg/mL solution,
It is placed in 4 DEG C to save backup.
Experimental example 1
The sample of gained in embodiment 1-5 is done tyrosinase inhibitory activity aptitude tests:
Experimental technique:
1) with vitamin C as positive control, blank group, sample sets, the positive are set up respectively
Matched group and negative control group.
2) in the test tube of sample sets and negative control group, it is separately added into the variable concentrations of 1mL
Sample solution, the test tube of blank group and positive group adds the phosphate buffer of 1mL respectively
(pH=6.8).
3) in the test tube of sample sets and positive controls, 0.5mL tryrosinase it is separately added into
Solution (enzyme position 125u/mL alive), 0.5mL phosphorus in the test tube of blank group and negative control group
Acid buffer (pH=6.8), shake all test tubes, makes sample and tryrosinase fully mix,
10 minutes are hatched at 37 DEG C.
4) respectively in blank group, sample sets, positive controls and all examinations of negative control group
Pipe adds 0.03wt% tyrosine solution, reacts 10min after mix homogeneously, be i.e. engraved in
Absorbance (T) is measured at 475nm.
Suppression ratio I (%)=[1-(T-T0)/(C-C0)] × 100%
Wherein, T is the absorbance of sample sets；T0Absorbance for feminine gender group；C is positive
The absorbance of group；C0Absorbance for blank group.
Calculate the suppression ratio of each sample by above-mentioned formula, suppression ratio is the biggest, antityrosinase
Activity is the strongest, and whitening effect is the best.The results are shown in Table 3
Experimental example 2
The sample of gained in embodiment 1-5 is done B16 black cell inhibitory action determination experiment,
Vitamin C with concentration as 0.5mg/mL is as positive control.
Will with the DMEM cell culture fluid containing 10% hyclone, penicillin and streptomycin
B16 melanocyte density is adjusted to 1 × 105Individual/mL, is inoculated in 6 holes with every hole 3mL thin
In born of the same parents' culture plate, at 37 DEG C, the CO of 5%2Under the conditions of cultivate after 24h at each Kong Zhongtian
Add testing sample and matched group.After cultivating 3 days, abandon supernatant and wash, the most often with PBS
Hole adds 0.5mL trypsin digestion cell reaction 3min, adds 2mL cell culture fluid and terminates
Digestion reaction, counting often organizes cell.After reject cell suspension, add 1mo1 L-1NaOH
Solution 0.5mL, centrifugal 5min after vibrations 1h, rotating speed time centrifugal is 1500r min-1, point
Do not take 200 μ l and move on to 96 porocyte culture plates, with solution in each hole of microplate reader detection at ripple
Absorbance at a length of 450nm.Calculating B16 cell suppression ratio:
Suppression ratio=[1-(A/B)/(C/D)] × 100%
Wherein, A is sample well absorbance, and B is sample well cell number, and C is control wells
Absorbance, D is control wells cell number.The results are shown in Table 3.
In table 3 embodiment 1-5, gained sample is to tryrosinase and the suppression ratio of B16 melanocyte
It is appreciated that from table 3:
1., when the extract that extraction mountain villous themeda orchid is thick, compared to heating reflux method, use ultrasonic
Extraction method can improve obtained mountain villous themeda orchid crude extract to the suppression ratio of tryrosinase (from
18% brings up to 27%-28%), and to the suppression ratio of B16 melanocyte (from 12%
Bring up to 21%-22%), therefore use the extracting method in the present invention, it is possible to make mountain villous themeda blue
In effective active matter dissolution rate increase.
2., by compared with positive controls, the acetic acid ethyl ester extract of mountain villous themeda orchid is black to B16
Chromatophorous suppression ratio (suppression ratio is 88%) is better than positive control medicine vitamin C
B16 melanocyte is had suppression ratio (suppression ratio is 84%), and the acetic acid second that mountain villous themeda is blue
Ester extract to the inhibitory action of tryrosinase with positive control medicine vitamin C to tyrosine
The suppression ratio effect of enzyme is close.
3. the activity of each sample in comparison embodiment 3, it is found that acetic acid second after extraction
Ester extract is to the suppression ratio (bringing up to 53% from 18%) of tryrosinase and to B16
The suppression ratio (bringing up to 45% from 21%) of melanocyte is all significantly increased.Additionally,
Petroleum ether extract, chloroform extract and n-butyl alcohol extract are to tryrosinase and B16 black
The suppression ratio of element cell is respectively less than 30%, illustrates in the villous themeda orchid of mountain tryrosinase and B16 black
Element cell has the composition of inhibitory action to be soluble in ethyl acetate, after ethyl acetate extracts, and institute
The active substance content of the acetic acid ethyl ester extract obtained increases.
4. being found by embodiment 2 and embodiment 3 contrast, contrast finds, in extraction step,
After using petroleum ether, chloroform, ethyl acetate and n-butanol extraction successively, the ethyl acetate of gained
The extract inhibitory activity (respectively 53% and 45%) to tryrosinase and B16 melanocyte
For higher than the acetic acid ethyl ester extract being directly extracted with ethyl acetate rear gained in embodiment 2
Activity (respectively 61% and 52%).
5. being found by embodiment 2 and embodiment 4 contrast, in embodiment 4, the first activity carries
Take thing, the second activity extract and the 3rd activity extract to tryrosinase and B16 melanin
The inhibitory activity of cell is significantly lower than the corresponding site in embodiment 2, and this illustrates table of the present invention
Gradient elution program in 1 is to antityrosinase and the work of B16 melanocyte in the villous themeda orchid of mountain
Property material separating effect is more preferable.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that
May be made that without departing from the spirit and scope of the present invention many other change and
Amendment.It is, therefore, intended that include in the following claims belonging in the scope of the invention
All such changes and modifications.
Claims (10)
1. the preparation method of the activity extract that a mountain villous themeda is blue, it is characterised in that include with
Lower step:
Leach step: mountain villous themeda orchid powder is soaked in the week-base water that pH value is 8-10 and carries out
Extraction, then removes described mountain villous themeda orchid powder and obtains lixiviating solution, then regulate described lixiviating solution
PH value is 2-6, and then standing processes, then the supernatant removing described lixiviating solution is precipitated
Thing, will obtain mountain villous themeda orchid crude extract after described drying precipitate；
Extraction step: described mountain villous themeda orchid crude extract water dissolution is obtained mountain villous themeda orchid lysate,
Then in the villous themeda orchid lysate of described mountain, add extractant extract, be extracted thing, its
Described in the volume of extractant be 0.5-2 times of volume of described mountain villous themeda orchid lysate；
Separating step: by adsorption charomatography, described extract is separated, use different ratio
Organic solvent mixed liquor carry out gradient elution as eluant, obtain eluent, the most again
Remove the described eluant in described eluent, obtain the activity extract that described mountain villous themeda is blue.
The preparation method of the activity extract that mountain the most according to claim 1 villous themeda is blue, its
Being characterised by, described extractant is ethyl acetate.
The preparation method of the activity extract that mountain the most according to claim 1 villous themeda is blue, its
Being characterised by, described extractant includes petroleum ether, chloroform and ethyl acetate, in described extraction
Step carries out extraction with petroleum ether and chloroform the most successively remove in the villous themeda orchid lysate of described mountain
Impurity, then extract by ethyl acetate, obtain acetic acid ethyl ester extract.
The preparation method of the activity extract that mountain the most according to claim 1 villous themeda is blue, its
Being characterised by, described adsorption charomatography is silica gel column chromatography.
The preparation method of the activity extract that mountain the most according to claim 4 villous themeda is blue, its
Be characterised by, in described silica gel column chromatography, use a dry method on a sample, in chromatographic column equipped with
Silica gel and the dry weight of described extract between weight ratio be 40～50:1.
The preparation method of the activity extract that mountain the most according to claim 4 villous themeda is blue, its
Being characterised by, in described silica gel column chromatography, described eluant includes: chloroform-acetone mixes
Close liquid, ethyl acetate-acetone mixed liquor, acetone-methanol mixed liquor and methanol-water mixture,
In wherein said chloroform-acetone mixture, the volume ratio of chloroform and acetone is 10～30:1, institute
Stating the volume ratio of ethyl acetate and acetone in ethyl acetate-acetone mixed liquor is 20～40:1,
In described acetone-methanol mixed liquor, the volume ratio of acetone and methanol is 2～10:1, described methanol
In-water mixed liquid, the volume ratio of first alcohol and water is 10～20:1.
The preparation method of the activity extract that mountain the most according to claim 1 villous themeda is blue, its
It is characterised by, in described leach step, while extraction, described week-base water is carried out Under Ultrasonic Vibration
Swinging, the frequency of described sonic oscillation is 20MHz-50MHZ, the time of described sonic oscillation
For 30min-50min.
The preparation method of the activity extract that mountain the most according to claim 1 villous themeda is blue, its
It is characterised by, in described leach step, described week-base water and the volume of described mountain villous themeda orchid powder
Ratio is 6～10:1.
9. the mountain villous themeda orchid prepared according to the preparation method described in any one of claim 1-8
Activity extract.
10. an application for the activity extract that mountain according to claim 9 villous themeda is blue,
It is characterized in that, activity extract blue for described mountain villous themeda is become as the activity of skin-lightening cosmetic
Point.
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| CN113831352A (en) * | 2020-12-16 | 2021-12-24 | 顺德职业技术学院 | Novel flavane compound of dianella root and preparation method and application thereof |
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| CN1993114A (en) * | 2004-05-28 | 2007-07-04 | 尤尼根制药公司 | Diarylalkanes as potent inhibitors of binuclear enzymes |
| KR20110002209A (en) * | 2009-07-01 | 2011-01-07 | 학교법인 동의학원 | Extraction method of asiasari radix containing whitening active components |
| CN105267580A (en) * | 2015-11-27 | 2016-01-27 | 广西中医药大学 | Application of dianella ensifolia or extracts thereof |
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| CN113831352A (en) * | 2020-12-16 | 2021-12-24 | 顺德职业技术学院 | Novel flavane compound of dianella root and preparation method and application thereof |
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