CN114028316A - Bletilla striata dedifferentiated cell extract, application thereof and cosmetic containing same - Google Patents
Bletilla striata dedifferentiated cell extract, application thereof and cosmetic containing same Download PDFInfo
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- CN114028316A CN114028316A CN202110352693.0A CN202110352693A CN114028316A CN 114028316 A CN114028316 A CN 114028316A CN 202110352693 A CN202110352693 A CN 202110352693A CN 114028316 A CN114028316 A CN 114028316A
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- cell extract
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- culture
- dedifferentiated cell
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- 235000001705 insufficient nutrition Nutrition 0.000 description 1
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- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
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- 229910052603 melanterite Inorganic materials 0.000 description 1
- RCYSGAYIEFAJTG-UHFFFAOYSA-N methyl acetate;propanoic acid Chemical compound CCC(O)=O.COC(C)=O RCYSGAYIEFAJTG-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
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- 239000000419 plant extract Substances 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- RUOJZAUFBMNUDX-UHFFFAOYSA-N propylene carbonate Chemical compound CC1COC(=O)O1 RUOJZAUFBMNUDX-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 230000008786 sensory perception of smell Effects 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- SFZCNBIFKDRMGX-UHFFFAOYSA-N sulfur hexafluoride Chemical compound FS(F)(F)(F)(F)F SFZCNBIFKDRMGX-UHFFFAOYSA-N 0.000 description 1
- 229960000909 sulfur hexafluoride Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- JOHWNGGYGAVMGU-UHFFFAOYSA-N trifluorochlorine Chemical compound FCl(F)F JOHWNGGYGAVMGU-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Images
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- 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
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- 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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
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- 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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
Abstract
The invention provides a bletilla striata dedifferentiated cell extract, application thereof and cosmetics containing the bletilla striata dedifferentiated cell extract. Specifically, the invention provides a supercritical carbon dioxide extract of bletilla striata dedifferentiated cells, which has excellent triple beauty functions of whitening, antioxidation and anti-wrinkle and has the advantage of low skin irritation. The invention also provides the application of the supercritical carbon dioxide extract and cosmetics containing the supercritical carbon dioxide extract so as to meet all skin beautifying requirements of consumers all at once.
Description
Technical Field
The invention relates to a beauty maintaining component, and application and a product thereof, in particular to a beauty maintaining component containing plant extracts.
Background
With the improvement of science and technology and the improvement of living standard, the interaction between people is frequent, and in the process of social activities, people pay more and more attention to the appearance of the people and pursue a younger and healthy appearance, thereby bringing the activation of the skin care market.
The beauty products on the market are full of precious minerals, however, most products with stronger functionality are more irritating to the skin of the human body, for example: although the fruit acid or aldehyde A has excellent anti-wrinkle effect, the fruit acid or aldehyde A has burning sensation when being used, and is easy to cause skin red swelling, dryness and desquamation; although the benzenediol has excellent whitening effect and can be used for lightening black spots, the benzenediol has the side effects of irritation and photosensitivity; and levo-vitamin C has excellent antioxidant function, but belongs to an acidic substance, and the irritation to skin is still to be improved. Therefore, active ingredients with low skin irritation have yet to be developed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a plant dedifferentiated cell extract, which is obtained by supercritical extraction; wherein the plant is a plant of the family Orchidaceae (Orchidaceae) and genus Bletilla (Bletilla), and the solvent for supercritical extraction is carbon dioxide.
The plant dedifferentiated cell extract has the triple beautifying functions of whitening, antioxidation and anti-wrinkle, and has the advantage of low skin irritation.
The dedifferentiated cells of the invention comprise plant cells which have not been differentiated or retrodifferentiated (differentiation).
Preferably, the dedifferentiated cells are obtained from any one or a combination of seeds (Seed), Embryoid bodies (embryo), Somatic embryos (solar embryos), protospheres (Protocorm), protospheroid-like bodies (PLBs), calli (calli).
Preferably, the seed comprises any one of Embryo (Embryo), Embryo (Germ) or a combination thereof.
According to the present invention, the cosmetic efficacy of the above plant dedifferentiated cell extract is superior to that of the differentiated cell extract, and is milder without skin or eye irritation.
In one embodiment, the dedifferentiated cells are obtained from a tissue culture obtained by induction culturing of seeds.
The culture medium and method for induction culture described above are well known in the art and can be adjusted according to the requirements, for example: MS Medium (Murashige & Skoog Medium) or B5 Medium (B5 Medium) can be used.
Preferably, the number of days of induction culture is from 40 days to 70 days, for example: 40 days, 50 days, 60 days or 70 days.
Preferably, the pH value of the induction medium used for the induction culture is 5.7 to 5.9; more preferably, the pH is 5.8.
In one embodiment, the induction medium comprises sucrose and coconut water. Preferably, the coconut water is present in an amount of 10 to 15 volume percent based on the total volume of the induction medium, for example: 10 volume percent, 11 volume percent, 12 volume percent, 13 volume percent, 14 volume percent, or 15 volume percent; and the sucrose content is from 20g/L to 30g/L, for example: 20g/L, 22g/L, 24g/L, 26g/L, 28g/L or 30 g/L.
Preferably, the induction medium is further added selected from the group consisting of: 6-Benzylaminopurine (6-benzamidopurine, 6-BA), 2,4-dichlorophenoxyacetic acid (2, 4-dichlorphenoxyacetic acid, 2,4-D) and naphthylacetic acid (NAA); more preferably, the content of 6-BA, 2,4-D or NAA is 0.1mg/mL to 5mg/mL based on the total volume of the induction medium, for example: 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, or 5 mg/mL.
Preferably, the tissue culture is a somatic embryo, a protosphere, a spheroid or a callus as described above.
The culture medium and the culture method for inducing and culturing the seeds to generate somatic embryos, protospheroids, protospheroid-like bodies or callus tissues can be adjusted according to requirements. Since the induction of seed to generate dedifferentiated cells belongs to the mature technology and has operation stability, the invention is adopted, but not limited thereto.
Preferably, the dedifferentiated cells are obtained by further subjecting the tissue culture to proliferation culture.
The culture medium and the propagation culture method of the propagation culture can be adjusted according to requirements.
In one embodiment, the culture medium for the proliferation culture is the same as the induction culture medium.
Preferably, the number of days of the propagation culture is 30 to 45 days, for example: 30 days, 35 days, 40 days or 45 days. The proliferation culture days are determined according to the growth condition of the dedifferentiated cells, and harvest is carried out when the dedifferentiated cells grow full or reach the required amount; in one embodiment, the enrichment culture in suspension in liquid medium is typically harvested after about 30 days; in another embodiment, the proliferation culture is performed in a solid medium and typically harvest occurs after about 45 days.
Preferably, the proliferation culture is a culture in a dark environment.
Preferably, the proliferation culture is further inoculated with subculture to fresh proliferation medium every 15 to 40 days, for example: 15 days, 20 days, 25 days, 30 days, 35 days or 40 days. Before the dedifferentiated cells grow full or reach the required amount, if the dedifferentiated cells have insufficient nutrition, the dedifferentiated cells can be inoculated to a fresh proliferation culture medium, and if the amount of the cells is enough, the cells can be subcultured or harvested at any time. In one embodiment, the multiplication culture in suspension in liquid medium is generally harvested or subcultured about 15 days later; in another embodiment, the proliferation culture in solid medium is generally harvested or subcultured about 35 days later.
The above-mentioned supercritical extraction is a conventional technique, and can be used to extract targets with different characteristics by adjusting parameters. Experiments prove that the extract obtained by the invention has unpredictable effects: whitening, oxidation resistance, wrinkle resistance and low irritation, and the extraction conditions are as follows.
Preferably, the pressure of the supercritical extraction is 2000psi to 7000psi, for example: 2000psi, 2500psi, 3000psi, 3500psi, 4000psi, 4500psi, 5000psi, 5500psi, 6000psi, 6500psi, or 7000 psi. More preferably, the pressure of the supercritical extraction is 4000psi to 5000 psi.
Preferably, the temperature of the supercritical extraction is from 40 ℃ to 60 ℃, for example: 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃.
Preferably, the time of the supercritical extraction is 5 hours to 10 hours, such as: 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours.
Preferably, the flow rate of the solvent used for supercritical extraction is 4L/min to 6L/min, for example: 4L/min, 4.2L/min, 4.4L/min, 4.6L/min, 4.8L/min, 5L/min, 5.2L/min, 5.4L/min, 5.6L/min, 5.8L/min, or 6L/min.
In one embodiment, the supercritical extraction further uses an entrainer; preferably, the entrainer comprises any one or combination of methanol, ethanol, propanol, hexanol, propylene glycol, ethylene glycol, n-butanol, acetone, butanone, formic acid, acetic acid, ethyl acetate, cyclohexane, n-hexane, methyl chloride, dioxane, dimethyl sulfoxide, dimethylacetamide, ethylene glycol monomethyl ether, acetonitrile, sulfur hexafluoride, chlorine trifluoride, sulfur dioxide, carbon disulfide, tetrahydrofuran, propylene carbonate, chloroform and petroleum ether; more preferably, the entrainer is ethanol.
In one embodiment, the plant is Bletilla striata (Bletilla formosana (Hayata) Schltr.) in Taiwan.
According to the present invention, the effective concentration of the plant dedifferentiated cell extract is 0.005 wt% or more, preferably, the effective concentration is 0.005 wt% to 30 wt%, for example: 0.005 weight percent, 0.008 weight percent, 0.01 weight percent, 0.03 weight percent, 0.05 weight percent, 0.08 weight percent, 0.1 weight percent, 0.2 weight percent, 0.3 weight percent, 0.4 weight percent, 0.5 weight percent, 0.6 weight percent, 0.7 weight percent, 0.8 weight percent, 0.9 weight percent, 1 weight percent, 1.5 weight percent, 2 weight percent, 2.5 weight percent, 3 weight percent, 3.5 weight percent, 4 weight percent, 4.5 weight percent, 5 weight percent, 10 weight percent, 15 weight percent, 20 weight percent, 25 weight percent, or 30 weight percent. More preferably, the effective concentration is an effective concentration for human use.
According to a specific embodiment of the present invention, the zebrafish embryo treated with 10mM kojic acid (about 1421 μ g/mL) has a melanin production inhibition rate of about 55%, and the bletilla striata dedifferentiated cell extract of the present invention treated with 100 μ g/mL has a melanin production inhibition rate of about 66%, i.e., the bletilla striata dedifferentiated cell extract of the present invention has a better melanin inhibition effect at a concentration of 1/14 of kojic acid. "inhibition of production of コウジ acid at メラニンと and various pigmentation disorders に, する are responsible for the effect of treating the effect (inhibition of melanin production and treatment of various pigmentation by kojic acid)" published in 1994, which discloses that 1% kojic acid can significantly inhibit melanin pigmentation in male and female subjects. It is known that the effective concentration of the plant dedifferentiated cell extract of the present invention is 1/14%, i.e., 0.07 wt%, it can exhibit whitening effect superior to kojic acid, and the effective concentration for human use can be 0.005 wt% to 30 wt%.
The invention also provides application of the plant dedifferentiated cell extract in whitening, antioxidation or anti-wrinkle.
Preferably, the whitening comprises: inhibiting or preventing melanin production, or degrading melanin.
Preferably, the antioxidant comprises: scavenging free radicals, reducing or slowing oxidative stress, or increasing gene expression of catalase or superoxide dismutase.
Preferably, the anti-wrinkle comprises: increasing the expression level of an elastin-producing gene, or inhibiting or reducing the production of matrix metal chelating protease; more preferably, the matrix metal chelating protease is matrix metal chelating protease-1 (MMP-1).
Preferably, the above plant dedifferentiated cell extract has low skin irritation; more preferably, the plant dedifferentiated cell extract has low eye irritation.
The invention further provides a cosmetic comprising the plant dedifferentiated cell extract, wherein the formulation of the cosmetic comprises a solution, a suspension, a spray, a liquid lotion, a mousse, a essence, a gel, an emulsion, a microemulsion, a cream, an ointment, a stick, a soap block, a powder or a patch.
In one embodiment, the cosmetic product may be a facial cleanser, makeup remover, soap, bath milk, hair wash, lotion, cream, pre-makeup milk, sun block, makeup base, foundation, pack, patch, hand cream, body lotion, or mask.
The "cosmetic" according to the regulation of item 1 of item 3 of the cosmetic hygiene and safety management method, item 1, modified by 2018, 05, 02, of taiwan, means "a preparation applied to the external part of the human body, teeth or oral mucosa for moisturizing the skin, stimulating the sense of smell, improving body odor, modifying the appearance or cleansing the body. However, the term "to be used with drugs" is not limited to "in accordance with other regulations". Therefore, the cosmetic of the present invention includes only the cosmetic specified in item 1, item 3 of the sanitary and safety management of cosmetics, but does not include the cosmetic identified as a drug by other statutes.
The invention also provides the application of the cosmetic, which is used for whitening, resisting oxidation or resisting wrinkles, wherein the cosmetic comprises the plant dedifferentiated cell extract; preferably, the whitening comprises: inhibiting or preventing melanin production, or degrading melanin; the antioxidant comprises: scavenging free radicals, reducing or slowing oxidative stress, or increasing gene expression of catalase or superoxide dismutase; or the anti-wrinkle comprises: increasing the expression level of an elastin-producing gene, or inhibiting or reducing the production of matrix metal chelating protease.
In conclusion, the plant dedifferentiated cell extract has the advantages of whitening, oxidation resistance, wrinkle resistance and low skin irritation, can avoid the uncomfortable body feeling and side effect of skin of a consumer, meets multiple beauty and maintenance requirements of the consumer, and reduces the complicated process that a single product only has a single effect and needs to adopt a plurality of maintenance products with different beauty functions. In other words, the invention has three advantages of effectiveness, efficiency and safety, can satisfy all the skin beauty requirements of consumers all at once in all directions, and has great market competitiveness.
Drawings
FIG. 1 is a high performance liquid chromatography analysis of different extracts.
Detailed Description
Several modes of operation are provided below in order to illustrate embodiments of the present invention; those skilled in the art can readily appreciate from the disclosure of the present invention that the advantages and features of the present invention may be realized and attained by various modifications and changes without departing from the spirit and scope of the invention.
Preparation example 1: callus of bletilla striata seeds
The experiment adopts seeds of bletilla striata of Taiwan to carry out induction culture so as to obtain callus. Firstly, cleaning shells of capsules and fruit pods which develop for 2.5 months after pollination by using a cleaning agent, disinfecting the shells by using 75% alcohol for 1 minute, disinfecting the shells by using 1% to 3% sodium hypochlorite solution for 30 minutes, cleaning the shells for 3 times by using sterile water in a sterile operation table, taking out seeds, and culturing the seeds for 50 to 70 days by using an induction culture medium with the pH value of 5.8 to obtain callus; wherein the components of the induction medium are shown in Table 1; wherein 6-BA, 2,4-D and NAA are added to the induction medium alternatively or alternatively, the addition amount of each is 0.1 to 5mg/L, and the obtained callus tissues are mixed for standby.
Table 1: induction medium
| Kind of ingredient | Content (mg/L) |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| KH2PO4 | 170 |
| ZnSO4·7H2O | 8.6 |
| MnSO4·H2O | 16.9 |
| CuSO4·5H2O | 0.025 |
| KI | 0.083 |
| CoCl2·6H2O | 0.025 |
| H3BO3 | 62 |
| Na2MoO4·2H2O | 0.25 |
| Na2EDTA | 37.3 |
| FeSO4·7H2O | 27.8 |
| Pyridoxine hydrochloride (vitamin B6) | 0.5 |
| Thiamine hydrochloride (vitamin B1) | 0.400 |
| Nicotinic acid (vitamin B3) | 0.500 |
| Glycine | 1.000 |
| Inositol | 100 |
| Agar | 7000 |
| Coconut water | 100 to 150mL/L |
| Sucrose | 20000 |
| 6-BA/2,4-D/NAA | 0.1 to 10 |
Preparation example 2: bletilla striata dedifferentiated cells
Inoculating the callus of preparation example 1 to a fresh solid proliferation medium, and performing proliferation culture in a dark environment at room temperature of 25 ℃ to obtain a tissue culture containing bletilla striata dedifferentiated cells; wherein, in the process of proliferation culture, a part of tissue culture is taken for subculture on day 35, and the rest tissue culture is continuously subjected to proliferation culture or is harvested simultaneously. The proliferation medium is the same as the induction medium described above.
Preparation example 3: rhizoma bletillae extract
Example 1: supercritical carbon dioxide extracts of dedifferentiated cells
Freeze-drying and grinding a tissue culture which is cultured for 35 to 45 days and contains the bletilla striata dedifferentiation cells of preparation example 2 to obtain cell freeze-dried powder, extracting for 5 to 10 hours by using supercritical carbon dioxide fluid as a solvent under the pressure of 4000 to 5000psi, the temperature of 40 to 60 ℃ and the flow rate of the carbon dioxide fluid of 4 to 6L/min to obtain pasty supercritical carbon dioxide fluid extract, namely the supercritical carbon dioxide extract of the bletilla striata dedifferentiation cells.
Comparative example 1: ethanol extracts of dedifferentiated cells
The tissue culture including bletilla striata dedifferentiated cells, which was cultured for 35 to 45 days and included in preparation example 2, was freeze-dried and then pulverized to obtain cell lyophilized powder. Mixing the freeze-dried powder and a 50% ethanol water solution according to the weight ratio of 1: 50, oscillating for 1 hour by ultrasonic wave, filtering the obtained extract, concentrating under reduced pressure, and freeze-drying to obtain the ethanol extract of the bletilla striata dedifferentiation cells.
Comparative example 2: ethanol extract of medicinal materials
And grinding the dried rhizoma bletillae roots to obtain the rhizoma bletillae powder. Mixing rhizoma bletilla powder and 50% ethanol water solution according to the weight ratio of 1: mixing the raw materials according to the weight-volume ratio of 50, oscillating for 1 hour by ultrasonic waves, filtering the obtained extract, concentrating under reduced pressure, and freeze-drying to obtain the ethanol extract of the medicinal materials.
Analytical example 1: high performance liquid chromatography analysis
The high performance liquid chromatography used in the experiment is Waters 600; the chromatography column is C-18, inert sil 5ODS, 4.6 x 250 mm; the wavelength is UV 200nm to 400 nm; the flow rate is 1.0 mL/min; the liquid A of the mobile phase is H2O (0.1% phosphoric acid), the liquid B is acetonitrile, the gradient elution mode is 0-15 minutes (95-85: 5-15), 15-50 minutes (85-40: 15-60), 50-55 minutes (40-95: 60-5), 55-60 minutes (95: 5), the sample injection amount is as follows: 20 mu L of the solution; the column temperature was room temperature.
10 mg of each of example 1, comparative example 1 and comparative example 2 was dissolved in 1mL of a solvent, wherein the solvent of example 1 was ethanol and the solvents of comparative example 1 and comparative example 2 were water, to obtain a sample stock solution, and the sample stock solution was dissolved by ultrasonic oscillation and filtered, and then analyzed, and the results are shown in FIG. 1; in fig. 1, the components of example 1, comparative example 1 and comparative example 2 are different, and from the peaks falling at about 40 to 45 minutes and 55 minutes in example 1, it is understood that example 1 contains many components having small polarity.
Analysis example 2: analysis of Total Polyphenol content
10 mg of each of example 1, comparative example 1 and comparative example 2 was dissolved in 1mL of a solvent, wherein the solvent of example 1 was ethanol and the solvents of comparative example 1 and comparative example 2 were water, to obtain a sample stock solution. Diluting 100 μ L of the sample stock solution with solvent 10 times, adding 2mL of water and 0.25mL of 1N Folin-Ciocalteu reagent, mixing well, standing for 5 min, adding 0.25mL of 20% Na2CO3The solution is mixed evenly and reacted for 2 hours, then
3 multifunctional micro-disk spectrometer, measuring the absorbance at 750nm, using Gallic acid (Gallic acid) as the reference standard, and Gallic acid equivalent (Gallic acid equivalent) mg contained in each group per gram as the total polyphenol content, the results are shown in table 2.
Table 2: total polyphenol content of each group
| Group of | Total polyphenol content (mg gallic acid eq./g) |
| Example 1 | 23.74±0.75 |
| Comparative example 1 | 25.07±0.97 |
| Comparative example 2 | 26.10±0.62 |
As can be seen from table 2, the total polyphenol contents of example 1, comparative example 1 and comparative example 2 are similar; of these, the total polyphenol content measured in example 1 was the lowest.
Test example 1: cell experiments for whitening efficacy
Cytotoxicity assay of mouse melanocytes
Mouse melanocytes B16-F10 cultured in DMEM medium (
CRL-6475TM) Planting into 96-well culture plate with density of 5 × 10 per well3The cells were cultured for 24 hours and attached to a culture dish. Each control group was supplemented with solvent in DMEM medium, where example 1 used DMSO as solvent and the control group of example 1 added the same amount of DMSO as the solvent in example 1 as control-0.5% DMSO based on medium; comparative examples 1 and 2, in which water was used as a solvent and the control group of the comparative example was also added with the same amount of water as the amount of the solvent in each of comparative examples 1 and 2 as a control-0.25% water based on the culture medium, each of experimental groups was added with different concentrations of example 1, comparative example 1 and comparative example 2, after 48 hours of culture 500. mu.g/mL of MTT solution was added, and after 2 hours of culture at 37 ℃, the MTT solution was removed, 200. mu.L of DMSO was added to dissolve crystals, and the absorbance at 570nm was measured, each group was duplicated, and the relative cell survival rate of the experimental group was calculated based on the survival rate of the control group, i.e., the survival rate was 100%, with the results shown in Table 3.
Table 3: relative cell viability results for experimental groups
From the results shown in Table 3, the survival rate of melanocytes of mice in each group was similar and was over 90%.
(II) test for inhibition of melanogenesis by mouse melanocyte
Inoculating the mouse melanocyte into 6-well culture plate with density of 3 × 10 per well4After culturing the cells for 24 hours to attach them to the culture dish, the fresh DMEM medium was replaced, and 100nM of alpha-Melanocyte stimulating hormone (alpha-MSH) was added to each of the groups except the control group. Each negative control group was further added with a solvent, wherein DMSO was used as a solvent in example 1, and the negative control group in example 1 was added with the same amount of DMSO as the solvent in example 1 as a control — 0.4% DMSO based on the culture medium; comparative examples 1 and 2 used water as a solvent, and the negative control group of the comparative example also added water in the same amount as the solvent in each of comparative examples 1 and 2 as a control, 0.2% water based on the culture medium, and each of the experimental groups added example 1, comparative example 1, and comparative example 2 in different concentrations. Arbutin (Arbutin) was added to the positive control group. Culturing each group for 48 hours, washing with PBS once, adding trypsin to make the mouse melanocyte shed, collecting the mouse melanocyte, centrifuging and removing supernatant, adding 1M NaOH and 10% DMSO, heating in a 80 ℃ dry bath for 2 hours, measuring the light absorption value of 400nm, repeating each group two times, and calculating the relative melanin content of the other groups by taking the melanin content of the respective negative control group as a reference, namely the melanin content is 100%, wherein the results are shown in Table 4; wherein, the comparison objects of the T assay are respective negative control groups.
Table 4: relative melanin content results for each group
As can be seen from the results shown in Table 4, the melanin content was reduced to 49.4% in example 1 at a concentration of 200. mu.g/mL, and to 74.0% in example 1 at a concentration of 50. mu.g/mL, indicating that the melanin production inhibition rate of example 1 was about 25% to 50%; in contrast, the melanin production inhibition rates of comparative examples 1 and 2 were only about 10% to 20%. Therefore, the efficacy of example 1 in inhibiting melanin production is significantly better than that of comparative examples 1 and 2.
Second, the efficacy of example 1 in inhibiting melanogenesis was not due to cytotoxicity, due to similar survival rates of melanocytes in the groups of mice. Finally, based on the similarity of the total phenol content in example 1, comparative example 1 and comparative example 2, the better melanogenesis inhibitory effect of example 1 is also independent of the total phenol content.
Test example 2: zebra fish experiment for whitening efficacy
Stimulating the wild zebra fish to lay eggs, collecting the eggs laid within one hour, and screening embryos which accord with the fertilization development state by a microscope 9 hours after fertilization. Each group had 12 fish embryos, the control group was E3 broth (i.e., zebrafish embryo broth), the positive control group was supplemented with Kojic acid (Kojic acid), and each experimental group was supplemented with example 1, comparative example 1, and comparative example 2; wherein, because the zebra fish embryo melanin can be generated 24 hours after fertilization, the treatment is completed before the melanin generation, and the melanin formation condition of the fish body after hatching is observed 72 hours after fertilization: first, the fish body was anesthetized and placed on a plane, and after photographing in a top view, the melanin plaques were analyzed by using software (ImageJ), and the relative melanin contents of the remaining groups were calculated based on the melanin plaque intensity of the control group, i.e., the melanin content was 100%, with the results shown in table 5.
Table 5: relative melanin content results for each group
| Group of | Concentration of | Average (%) | Inhibition rate |
| Positive control group | 10mM | 48.6％±9.3 | 51.40％ |
| Example 1 | 100μg/mL | 33.40％±6.2 | 66.60％ |
| Comparative example 1 | 100μg/mL | 65.95％±9.3 | 34.05％ |
| Comparative example 2 | 100μg/mL | 68.9％±5.9 | 31.10％ |
As can be seen from Table 5, the inhibition rate of example 1 to the generation of melanin in zebrafish reaches 66.60% at a concentration of 100. mu.g/mL, which is better than 34.05% of comparative example 1 at the same concentration and 31.10% of comparative example 2 at the same concentration. In addition, 100 u g/mL example 1 concentration is converted into 0.1g/L, inhibition of 66.60%, 10mM positive control group concentration is converted into 1.42g/L, inhibition of 51.40%; it is understood that example 1 can achieve a melanin production-inhibiting effect superior to that of the commercially available kojic acid, as well as a significantly lower concentration.
Test example 3: DPPH test for Oxidation resistance
In a 96-well culture plate, 50 μ L of solvent was added to a blank of each experimental group (examples and comparative examples), wherein example 1 uses DMSO as the solvent; comparative examples 1 and 2 with waterAs a solvent. The experimental group was added 50. mu.L of the sequence dilution solutions of example 1, comparative example 1 and comparative example 2, each group was repeated at least three times, and the absorbance at 515nm was measured as a background value before adding 150. mu.L of the PPH solution. After adding DPPH and mixing uniformly, after reacting at room temperature for 20 minutes, the absorbance at 515nm was measured, and the concentration (IC) at which DPPH clearance reaches 50% was calculated50) I.e., the half clearance concentration of DPPH; wherein DPPH clearance rate is [ (A-A0) - (B-B0)/(A-A0)]X is 100%; a is the light absorption value obtained by the blank group; b is the light absorption value obtained by the experimental group; a0 is background absorbance value of blank group; b0 is the background absorbance of the experimental or control group, and the results are shown in table 6.
Table 6: half clearance concentration of DPPH
| Group of | IC50(μg/mL) |
| Example 1 | 1079.6±85.7 |
| Comparative example 1 | 1376.4±44.2 |
| Comparative example 2 | 1223.0±149.4 |
As can be seen from Table 6, the half-scavenging concentrations of DPPH were similar in the three groups, and therefore the antioxidant capacity of the three groups was similar.
Test example 4: antioxidant ABTS free radical scavenging experiments
In a 96-well culture plate, 100. mu.L of solvent was added to a blank of each experimental group (examples and comparative examples), in which example 1 was DMSOAs a solvent; comparative examples 1 and 2 used water as a solvent. Experimental group 100. mu.L of the dilution series solutions of example 1, comparative example 1 and comparative example 2 were added, each group was repeated at least three times, and before adding 100. mu.L of 280. mu.M ABTS (2,2' -azino-bis (3-ethylbenzoazoline-6-sulfonic acid) solution, the absorbance at 734nm was measured as a background value, after adding ABTS solution and mixing uniformly, after reacting at 25 ℃ for 15 minutes, the absorbance at 734nm was measured, and the concentration at which the ABTS radical scavenging rate reached 50% (IC) was calculated (IC)50) The calculation formula is as in test example 3, and the results are shown in Table 7.
Table 7: half scavenging concentration of ABTS free radical
| Group of | IC50(μg/mL) |
| Example 1 | 60.69±0.50 |
| Comparative example 1 | 39.01±0.62 |
| Comparative example 2 | 55.81±1.80 |
As can be seen from table 7, the ABTS radical scavenging efficacy of example 1 was not superior to that of comparative examples 1 and 2.
Test example 5: human fibroblast metabolic activity oxide assay
(ii) cytotoxicity assay of human fibroblasts
Human fibroblast CCD-966SK cultured in MEM medium (
CRL-1881TM) Planting into 96-well culture plate with density of 5 × 10 per well3The cells were cultured for 24 hours and attached to a culture dish.
Solvent was added to MEM medium for each control group, with DMSO as solvent for example 1, and the control group of example 1 added the same amount of DMSO as the solvent for example 1 as a control — 1% DMSO based on medium, with different concentrations of example 1 added for each experimental group. After 24 hours of culture, 500. mu.g/mL of MTT solution was added, and after 2 hours of culture at 37 ℃, the MTT solution was removed, 200. mu.L of DMSO was added to dissolve crystals, absorbance at 570nm was measured, each group was triplicated, and the relative cell viability of the experimental group was calculated based on the control cell viability, i.e., the viability was 100%, and the results are shown in Table 8.
Table 8: relative cell viability results for experimental groups
As is clear from Table 8, the cell viability reached 90% or more at the concentration of 250. mu.g/mL or less in example 1.
(II) metabolic activity oxide assay of human fibroblasts
The human fibroblasts were seeded into 96-well culture plates at a density of 5X 10 cells per well3The cells were cultured for 24 hours and attached to a culture dish, and then 500. mu.M H was added2O2After waiting for 24 hours, the MEM medium was replaced with fresh one. Control group in MEM medium added solvent, wherein, example 1 in DMSO as solvent, and example 1 control group added as example 1 solvent the same amount of DMSO as control-based on the culture medium 0.5% DMSO, each experimental group added different concentrations of example 1. After each group was cultured for 24 hours,the cells were divided into two portions, and after one portion was washed twice with PBS, a solution containing 10. mu.M DCFH-DA fluorescent dye was added and reacted at 37 ℃ for 30 minutes, and then DCF fluorescence intensity was measured to represent the content of cellular active oxides. Repeating the two groups, and calculating the relative cellular active oxide content of the experimental group by taking the fluorescence intensity of the control group as a reference, namely the cellular active oxide content is 100%; the other part was subjected to cytotoxicity test of human fibroblasts and reacted with 500. mu.g/mL MTT solution at 37 ℃ for 3 hours, absorbance at 570nm was measured to represent cell activity, and the results were repeated for two groups as shown in Table 9; wherein, the comparison object of the T-test is a control group.
Table 9: relative cell viability and relative DCF fluorescence intensity results for the experimental groups
As can be seen from Table 9, the cell viability was similar at each concentration, and the DCF fluorescence intensity was significantly lower than that of the control group at a concentration of 250. mu.g/mL, which suppressed the oxidative stress by almost 35%, indicating that example 1 indeed reduced the active oxide and reduced or slowed the oxidative stress.
Test example 6: catalase and superoxide dismutase gene expression experiment of human fibroblast
The human fibroblasts were seeded in 6-well plates at a density of 1.5X 10/well5After culturing the cells for 24 hours to attach the cells to the culture dish, the MEM medium was replaced with fresh one. Control group in MEM medium was added solvent, where example 1 was DMSO as solvent and control group of example 1 was added the same amount of DMSO as the solvent in example 1 as control-0.4% DMSO based on medium. The experimental group was treated with 200. mu.g/mL of example 1 for 24 hours. After washing with PBS once, collecting cells and purifying RNA, after reverse transcription of cDNA, real-time quantitative polymerase chain reaction (real-time PCR) is carried out, and catalase and superoxide dismutase gene expression in the human fibroblast cells treated in example 1 are converted by using catalase and superoxide dismutase gene expression in the control group as referenceRelative gene expression level of the disproportionation enzyme; wherein, the experiments are respectively by Quick-RNATMThe Miniprep Kit purified RNA, reverse transcribed cDNA using the Magic RT cDNA synthesis Kit, and performed PCR using KAPA SYBR Fast qPCR Master Mix, the results of which are shown in Table 10.
Table 10: catalase and superoxide dismutase relative gene expression fold after treatment in example 1
| Group of | Concentration of | Catalase (Bei) | Superoxide dismutase (Bei) |
| Example 1 | 200μg/mL | 2.3±0.22 | 2.0±0.26 |
As can be seen from Table 10, the relative gene expression levels of catalase and superoxide dismutase in example 1 are 2 times that in the control group, which indicates that example 1 can indeed increase the expression levels of catalase and superoxide dismutase genes, and that example 1 can increase the antioxidant capacity of human fibroblast cells, resist environmental pathogenic factors, and further increase the skin defense.
Test example 7: anti-wrinkle elastin-producing gene (ELN2) expression experiment
The human fibroblasts were seeded in 6-well plates at a density of 1.5X 10/well5Culturing the cells for 24 hours to attach the cells to a culture dishAfter that, the MEM medium was replaced with fresh one. Control group in MEM medium was added solvent, where example 1 was DMSO as solvent and control group of example 1 was added the same amount of DMSO as the solvent in example 1 as control-0.4% DMSO based on medium. The experimental group was treated with 200. mu.g/mL of example 1 for 24 hours. Washing with PBS once, collecting cells, purifying RNA, performing reverse transcription to obtain cDNA, performing real-time quantitative polymerase chain reaction, and converting relative gene expression level of ELN2 in human fibroblast cells treated in example 1 with reference to elastin protogenic gene (ELN2) expression level of control group; wherein, the experiments are respectively by Quick-RNATMThe Miniprep Kit purified RNA, reverse transcribed cDNA using the Magic RT cDNA synthesis Kit, and performed PCR using KAPA SYBR Fast qPCR Master Mix, the results of which are shown in Table 11.
Table 11: example 1 fold relative Gene expression amount of elastin-producing Gene
| Group of | Concentration of | Elastin producing gene (Bei) |
| Example 1 | 200μg/mL | 1.8±0.26 |
As is clear from Table 11, the expression level of the elastin growth gene in example 1 is 1.8 times that of the control group, which indicates that example 1 can increase skin structural protein, and thus can improve anti-wrinkle and skin defense.
Test example 8: anti-wrinkle matrix metalloproteinase-1 (MMP-1) protein experiment
(ii) cytotoxicity assay of human fibroblasts
Human fibroblast Hs68 (cultured in DMEM medium)
CRL-1635TM) Planting into 96-well culture plate with density of 1 × 10 per well4The cells were cultured for 24 hours and attached to a culture dish. Control group was supplemented with solvent in DMEM medium, where example 1 uses DMSO as solvent and the control group of example 1 was supplemented with the same amount of DMSO as the solvent in example 1 as control-0.2% DMSO based on medium; comparative examples 1 and 2 use water as a solvent, and the control group of the comparative examples also adds the same amount of water as the solvent of each group of comparative examples 1 and 2 as a control-10% water based on the culture medium; the experimental groups were supplemented with 20. mu.g/mL of example 1, 1000. mu.g/mL of comparative example 1, and 1000. mu.g/mL of comparative example 2. After 24 hours of incubation, 500. mu.g/mL of MTT solution was added, and after 2 hours of incubation at 37 ℃, the MTT solution was removed, 200. mu.L of DMSO was added to dissolve the crystals, the absorbance at 570nm was measured, and the relative cell viability of the experimental groups was calculated based on the control survival rate, i.e., the survival rate was 100%, and the results are shown in Table 12.
As can be seen from Table 12, example 1 showed high survival at a concentration of 20. mu.g/mL, and comparative examples 1 and 2 showed high survival at a concentration of 1000. mu.g/mL.
(II) MMP-1 assay for human fibroblasts
The human fibroblasts were seeded into 96-well culture plates at a density of 1X 10 cells per well4After 24 hours of culture, the cells were attached to the culture dish, and then 10ng/mL TNF-. alpha.was added after replacing fresh DMEM medium. Control group was supplemented with solvent in DMEM medium, where example 1 uses DMSO as solvent and the control group of example 1 was supplemented with the same amount of DMSO as the solvent in example 1 as control-0.2% DMSO based on medium; comparative examples 1 and 2 used water as solvent, and the control group of the comparative examples also added the same amount of water as the solvent in each group of comparative examples 1 and 2 as control-medium as culture medium10% water on a baseline; the experimental group was added with 20. mu.g/mL of example 1, 1000. mu.g/mL of comparative example 1 and 1000. mu.g/mL of comparative example 2, and after waiting for 18 hours, the cell culture fluid was collected and analyzed for MMP-1 content by immunoenzyme assay (ELISA). The relative MMP-1 content of the experimental group was calculated based on the MMP-1 content of the control group, i.e., 100% MMP-1 content, and the results are shown in Table 12.
Table 12: results of relative cell viability and relative MMP-1 content in experimental groups
As is clear from Table 12, in example 1, the MMP-1 production inhibitory effect was observed at a concentration of 20. mu.g/mL; in contrast to comparative examples 1 and 2, the inhibitor was not effective even at 50-fold concentration, and the MMP-1 production was increased. It is known that example 1 can inhibit the degradation of skin structural proteins, and thus achieve the effects of anti-wrinkle and improving skin defense, and the inhibitory effect is not due to cytotoxicity.
Test example 9: security analysis
(first) evaluation experiment of eye irritation
This experiment was tested by reconstructing a model of human corneal cells (RhCE), following OECD TG492 specifications, and the kit used was EpiOcular certified by ECVAM (the European Reference Laboratory for alternative to animal testing)TMEye Irritation Test (OCL-200-EIT) set.
Water was added to the negative control group and methyl acetate (methyl acetate) was added to the positive control group in this experiment, and 50mg of each of example 1 and comparative example 1 was added to each of the experiment groups, and after 6 hours of incubation, the culture solution was replaced with fresh one. After further incubation for 18 hours, 1mg/mL MTT solution was added to react for 3 hours, and crystals were dissolved with isopropanol (isopropanol) to measure the absorbance at 570 nm. In this experiment, the light absorption value of the negative control group was used as a reference, that is, the light absorption value of the negative control group was 100%, and if the light absorption value of the remaining groups was less than 60%, it was judged to have eye irritation, and the results are shown in table 13.
Table 13: results of determination of relative cell viability and ocular irritation in each group
| Group of | Relative cell survival (%) | Eye irritation |
| Positive control group | 47.6％±4.17 | O |
| Example 1 | 73.0％±1.20 | X |
| Comparative example 1 | 5.7％±0.33 | O |
As is clear from table 13, in the case of example 1 and comparative example 1, the eye irritation was not observed in example 1 when the same amount of addition was used, but the eye irritation was observed in comparative example 1, so that the eye irritation was low in example 1, and the use was safe for human body.
(II) evaluation test of skin irritation
The experiment was performed by reconstituting a human keratinocyte (RhE) skin model, following the OECD TG439 specification, and the set used was In Vitro EpiDerm certified by ECVAMTMSkin Iritation Test (EPI-200-SIT) set.
The DPBS solution was added to the negative control group, the 5% SDS aqueous solution was added to the positive control group, and 25mg of each of example 1 and comparative example 1 was added to the test group, and after 1 hour of incubation, the culture medium was replaced with fresh one. After further incubation for 42 hours, 1mg/mL MTT solution was added to react for 3 hours, and crystals were dissolved with isopropanol to measure the absorbance at 570 nm. In this experiment, the light absorption value of the negative control group was used as a reference, that is, the light absorption value of the negative control group was 100%, and if the light absorption value of the remaining group was less than 50%, the skin irritation was judged to have been observed, and the results are shown in table 14.
Table 14: relative survival rate of each group of cells and skin irritation determination results
| Group of | Relative cell survival (%) | Skin irritation |
| Positive control group | 1.6±0.14 | O |
| Example 1 | 98.0±0.30 | X |
| Comparative example 1 | 98.4±0.59 | X |
As is clear from table 14, in example 1, the cell viability was similar to that of comparative example 1 at the same addition amount, and was similar to 100% of the negative control group, so that example 1 had no skin irritation and was suitable for external use.
In conclusion, the plant dedifferentiated cell extract has the triple beauty functions of whitening, oxidation resistance and wrinkle resistance, and has the advantages of low skin and eye irritation.
Claims (12)
1. A plant dedifferentiated cell extract is obtained by supercritical extraction; wherein the plant is a plant of the family Orchidaceae (Orchidaceae) and genus Bletilla (Bletilla), and the solvent for supercritical extraction is carbon dioxide.
2. The plant dedifferentiated cell extract according to claim 1, wherein the dedifferentiated cells are taken from any one of seeds (Seed), Embryoid bodies (embryo), Somatic embryos (solar embryos), protospheres (Protocorm), protospheroid-like bodies (PLBs), Callus (calli) or combinations thereof.
3. The plant dedifferentiated cell extract according to claim 1, wherein the dedifferentiated cells are obtained from a tissue culture obtained by induction culture of seeds.
4. The plant dedifferentiated cell extract according to claim 1, wherein the dedifferentiated cells are obtained by proliferation culture: the number of days of the proliferation culture is 30-45 days; or the pH value of the multiplication medium used for the multiplication culture is 5.7 to 5.9.
5. The plant dedifferentiated cell extract according to claim 1, wherein the pressure of the supercritical extraction is 2000psi to 7000psi, the temperature is 40 ℃ to 60 ℃, and the time is 5 hours to 10 hours; and the flow rate of the solvent is 4L/min to 6L/min.
6. The plant dedifferentiated cell extract according to claim 1, wherein the supercritical extraction additionally uses an entrainer.
7. The plant dedifferentiated cell extract according to claim 1, wherein the plant is Bletilla striata (Bletilla formosana (Hayata) Schltr.) in taiwan.
8. The plant dedifferentiated cell extract according to claim 1, in an effective concentration of 0.005 to 30% by weight.
9. Use of the plant dedifferentiated cell extract according to any one of claims 1 to 8 for whitening, antioxidation or anti-wrinkle.
10. The use of claim 9, wherein the whitening comprises: inhibiting or preventing melanin production, or degrading melanin; the antioxidant comprises: scavenging free radicals, reducing or slowing oxidative stress, or increasing gene expression of catalase or superoxide dismutase; or the anti-wrinkle comprises: increasing the expression level of an elastin-producing gene, or inhibiting or reducing the production of matrix metal chelating protease.
11. A cosmetic comprising the plant dedifferentiated cell extract according to any one of claims 1 to 8, wherein the formulation of the cosmetic comprises a solution, a suspension, a spray, a liquid lotion, a mousse, a serum, a gel, an emulsion, a microemulsion, a cream, an ointment, a stick, a soap bar, a powder or a patch.
12. The cosmetic according to claim 11, which is a facial cleanser, makeup removing cream, soap, bath milk, hair wash, lotion, cream, pre-makeup milk, sun block, makeup base, foundation, patch, body lotion, or pack.
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