CN111202777B - A Rosa tenuifolia callus cell extract and its application in preparing skin external preparation - Google Patents
A Rosa tenuifolia callus cell extract and its application in preparing skin external preparation Download PDFInfo
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- CN111202777B CN111202777B CN202010150699.5A CN202010150699A CN111202777B CN 111202777 B CN111202777 B CN 111202777B CN 202010150699 A CN202010150699 A CN 202010150699A CN 111202777 B CN111202777 B CN 111202777B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/738—Rosa (rose)
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Botany (AREA)
- Birds (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Alternative & Traditional Medicine (AREA)
- Medical Informatics (AREA)
- Mycology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to an application of a rosa minutissima callus cell extract in preparing a skin external preparation, which is characterized in that the rosa minutissima callus cell extract is used as an anti-aging active ingredient and a whitening active ingredient in the skin external preparation; the invention has the advantages that experiments show that the rosa tenuifolia callus cell extract has the effects of eliminating skin free radicals, increasing skin cell proliferation, protecting skin cells from oxidative damage, protecting skin cells from damage caused by UVA, and inhibiting the tyrosinase activity of human epidermal melanocyte B16 and the generation amount of melanin; the callus cells of the rosa tenuifolia are used as raw materials, so that the rosa tenuifolia has the advantages of unlimited proliferation, stable quality of substances in each batch, short culture period, environmental friendliness and easiness in batch culture regardless of seasons, can be produced and manufactured without damaging plant bodies, and has great value and significance for sustainable development and utilization of plant resources.
Description
The invention relates to the field of plant extract application, in particular to a rosa multiflora callus cell extract and application thereof in preparing a skin external preparation.
Background
Rosa plants are various in species, and not only can be used as ornamental plants to be introduced into gardens, but also can be used as medicinal plants, and roots, stems, leaves and flowers of the Rosa plants can be used as medicines. However, because the seeds of roses have deep dormancy characteristics, the roses are difficult to germinate in the same year, and the garden introduction is difficult. Therefore, by utilizing the plant tissue cell culture technology, a large amount of plant cells or callus can be cultured under the manually controllable and repeated conditions, related raw materials such as enzyme preparations, medical products, additives and the like can be provided for human beings, land and resources are not occupied, and the defects that plant resources cannot be reasonably and fully used and medicinal material quality is different due to different growth conditions such as plant germination or geographical environment, climate, soil and the like are overcome. Rosa tenuifolia (Rosa graciliflora Rehd. Et Wils), also called chestnut, mostly grows in hillside with elevation of 3300-4500 m, under spruce forest or forest side bush, and is distributed only in parts of Yunnan, sichuan, tibet and Shaanxi. The rose flower is gorgeous in color and pleasant in fragrance, is an excellent landscaping material, and roots and fruits of the rose flower can be used as medicines, so that the rose flower not only can improve the immunity, but also has an indispensable effect on certain diseases, and is a rose flower which is worthy of attention and deep development.
The study of secondary metabolites in plant tissues (including organs and cells) has been carried out for over 30 years. The plant cell culture can produce high-value secondary metabolites and has wide application prospect in food, medicines and cosmetics. Research reports that some plant cell extracts (such as grapes, swiss apples and the like) are applied to cosmetics and have the effects of resisting oxidation, protecting ultraviolet injury, resisting inflammation, resisting aging and the like. However, no report is made on the application of the callus cells of rosa capillata in cosmetics.
CN106456526B relates to a cosmetic composition for skin whitening or skin wrinkle improvement, and it is mentioned that 3,5-di-O-methyl-gamboge is extracted from white rose leaves and is used as an active ingredient for whitening/anti-wrinkle. The rosa plants have 2 subgenus and more than 200 subgenus all over the world, and the components of the rosa plants are greatly different from each other. The patent adopts white rose, which belongs to the cultivated variety.
The tyrosinase inhibitory activity mentioned in this patent is ethanol, butanol and ethyl acetate chromatography and the compound 3,5-di-O-methyl-gamboge, and there is no data on the inhibitory activity in the aqueous layer, and the applicant speculates from the expert knowledge that this compound is poorly soluble in water.
Disclosure of Invention
The applicant has previously conducted research on the culture stage of rosa minutissima, such as 2019100603660, a tissue culture method of rosa minutissima. Subsequent research and detection show that the rosa multiflora callus cell extract has the effects of resisting aging and whitening skin. In particular, anti-aging active ingredients which scavenge free radicals from the skin, increase the proliferation of skin cells, protect skin cells from oxidative damage, and protect skin cells from damage caused by UVA; and a whitening active ingredient which inhibits the tyrosinase activity and the melanin production of the human epidermal melanocyte B16.
In the skin external preparation, the content of the rosa minutissima callus cell extract is 0.001% -1%, preferably 0.1% -1%, and the percentage refers to the percentage of the mass of the rosa minutissima callus cell extract in the total mass of the skin external preparation.
The culture method of the rosa minutissima callus cells is any one of solid culture and liquid culture.
Wherein the solid culture comprises the following steps:
A. taking a reagent MS culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), sucrose, agar and water;
B. preparing a culture medium by using water as a solvent according to MS, 2,4-D3.0mg/L, KT0.5mg/L, sucrose 30g/L and agar 6 g/L;
C. adjusting the pH value of the solid culture medium to 5.7-5.8, and sterilizing in a sterilization pot at 121 ℃ for 20min to obtain the solid culture medium;
D. adding a solid culture medium with the volume of 10-20% of the bottle volume into a culture container, inoculating callus cells with the volume of 1-2% of the culture medium after the solid culture medium is solidified, wherein the culture conditions are that the temperature is 19-21 ℃, the humidity is 60-75%, the illumination intensity is 1500Lx-2000Lx, and the illumination is 12 hours every day;
E. the callus cells of the rosa minutissima are harvested after 20 days of culture.
Wherein the liquid culture comprises the following steps:
A. taking a reagent MS culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), sucrose and water;
B. preparing a culture medium by using water as a solvent according to MS, 2,4-D3.0mg/L, KT0.5mg/L and 30g/L of cane sugar;
C. adjusting the pH value of the culture medium to 5.7-5.8, and sterilizing in a sterilizer at 121 deg.C for 20min to obtain liquid culture medium;
D. adding a liquid culture medium with a bottle volume of 20-30% into a culture container, inoculating callus cells with a volume of 1-2% of the liquid culture medium, and placing the cells into a shaking table for culture at a temperature of 24-26 ℃ and a rotating speed of 110r/min;
E. the callus cells of the rosa minutissima are harvested after 20 days of culture.
The water is deionized water or glacier water, and the glacier water is prepared from multi-Ji Qu Dengni Ma on the north slope of Himalayas, the altitude is 5128 m:
the extraction of the callus cells of the rosa tenuifolia obtained by the solid culture or the liquid culture comprises the following steps:
A. weighing callus cells of rosa tenuifolia, grinding and crushing;
B. adding deionized water with the volume 5 times that of the callus cells of the rosa tenuifolia, and immersing and stirring;
C. ultrasonic extracting at 500W for 2 times, each for 30min, centrifuging at 5000rmp for 10min, collecting supernatant, filtering, and mixing filtrates;
D. freeze drying to obtain the callus cell extract of rosa tenuifolia.
The invention has the advantages that the experiments show that the rosa tenuifolia callus cell extract has the effects of eliminating skin free radicals, increasing skin cell proliferation, protecting skin cells from oxidative damage, protecting skin cells from damage caused by UVA, and inhibiting the tyrosinase activity and the melanin generation amount of human epidermal melanocyte B16. Therefore, the rosa tenuifolia callus cell extract is applied to the skin external preparation as an anti-aging and whitening active ingredient. In addition, the callus cells of the rosa tenuifolia are used as raw materials, so that the rosa tenuifolia has the advantages of unlimited proliferation, stable quality of substances in each batch, short culture period, environmental friendliness and easiness in batch culture regardless of seasons, can be produced and manufactured without damaging plant bodies, and has great value and significance for sustainable development and utilization of plant resources.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
EXAMPLE I culture of callus cells of Rosa capillipes
1. Solid culture
Taking a reagent MS culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), sucrose, agar and glacier water, preparing the culture medium according to a formula of MS +2,4-D3.0 mg/L + KT0.5mg/L + sucrose 30g/L + agar 6g/L, adjusting the pH value to 5.7-5.8, and sterilizing for 20min at 121 ℃ in a sterilizing pot for later use. An appropriate amount of callus cells were inoculated in the culture flask, and in this example, 0.3 to 0.5g of rosa multiflora callus cells were selectively inoculated according to the capacity of 270ML of the PC culture flask. The culture conditions are that the temperature is 20 +/-1 ℃, the humidity is 60% -75%, the illumination intensity is 1500Lx-2000Lx, the illumination is 12 hours every day, and the cells are harvested after 20 days of culture.
2. Liquid culture
Taking a reagent MS culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), cane sugar and glacier water, preparing the culture medium according to a formula of MS +2,4-D3.0 mg/L + KT0.5mg/L + cane sugar 30g/L, adjusting the pH to about 5.8, and sterilizing in a sterilizer at 121 ℃ for 20min for later use. In this example, according to the capacity of 100ml of triangular flask, 0.3g-0.5g of callus cells are inoculated, then 30ml of liquid culture medium is added, and the mixture is placed into a shaking table for culturing at 25 +/-1 ℃ and 110r/min, and the cells are harvested after 20 days of culturing.
Wherein, the glacier water is used as a solvent for preparing the culture medium and can be replaced by deionized water.
Example II extraction of callus cells of Rosa minutissima
1. Extraction of callus cells of rosa minutissima
The callus cells of the rosa tenuifolia obtained by cultivation in the first example are weighed respectively, 10g of the callus cells are weighed respectively, ground and crushed, 5 times of deionized water is added, immersed and stirred, 500W ultrasonic extraction is carried out for 2 times, 30min is carried out each time, 50010 rmp centrifugation is carried out for 10min, supernatant is taken and filtered, filtrate is combined, freeze drying is carried out, and after the extract is obtained, weighing is carried out and yield is calculated (see table 1).
The following batches refer to samples taken at different times under the same experimental conditions.
TABLE 1 extraction yield of Rosa tenuifolia callus cells
2. Determination of polyphenol content in rosa tenuifolia callus cell extract
The total polyphenol content is detected by Folin-Ciocalteu method and gallic acid as reference substance. Taking 0.1mL gallic acid standard solution and sample solution to be tested, paralleling for 3 times, adding 0.5 mL 10% Folin-Ciocalteu reagent, mixing, standing at room temperature for 5min, adding 0.4 mL 7.5% NaCO3 solution, mixing, standing at room temperature for 60 min, and determining the OD value at 765 nm. A standard curve was prepared from OD520 of each tube of the standard solution for the content of nutgall, and the polyphenol content in the sample was calculated from the standard curve (see Table 2).
TABLE 2 Polyphenol content of Rosa tenuifolia callus cells
| Sample (I) | Polyphenol content (%) |
| Solid culture-batch 1 | 1.55 |
| Solid culture-batch 2 | 1.19 |
| Solid culture-batch 3 | 1.35 |
| Solid culture-batch 4 | 1.59 |
| Liquid culture-batch 1 | 1.48 |
| Liquid culture-batch 2 | 1.41 |
EXAMPLE III Rosa stringbush callus cell extract assay
1. Effect of callus cell extract of Rosa capillipes on radical scavenging rate
1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable nitrogen-centered organic radical. The DPPH method was proposed in 1958 and widely used to quantitatively determine the anti-aging ability of biological samples, classified substances and foods. The method is based on the characteristic that DPPH free radicals have single electron, have strong absorption at 517nm, and alcoholic solution is purple. When the free radical scavenger exists, the absorption of the free radical scavenger disappears gradually due to the pairing with a single electron of the free radical scavenger, and the fading degree of the free radical scavenger and the number of the electrons received by the free radical scavenger form a quantitative relation, so that a spectrophotometer can be used for carrying out rapid quantitative analysis to detect the free radical scavenging condition, thereby evaluating the anti-aging capability of the sample.
Adding 0.lmL of the extract sample solution into a test tube, adding 0.1ml of DPPH ethanol solution, mixing, reacting in dark at room temperature for 30min in a dark place, measuring the OD value at 525nm, and calculating the clearance rate according to the following formula: clearance I (%) = [1- (T-T0)/(C-C0) ] × 100%, wherein: t0: absorbance of 0.1ml sample solution added with 0.1ml 95% ethanol; t: absorbance of 0.1ml sample plus 0.1ml DPPH solution; c0: absorbance of 0.1ml water plus 0.1ml 95% ethanol; c: absorbance of 0.1ml water plus 0.1ml DPPH solution.
As can be seen from the results in Table 3, the callus extract of Rosa strigosa has a certain effect of scavenging free radicals.
TABLE 3 DPPH clearance of Rosa tenuifolia callus cells
| Sample (I) | Test concentration (mg/mL) | DPPH clearance (%) |
| Solid culture-batch 1 | 0.5 | 56 |
| Solid culture-batch 2 | 0.5 | 45 |
| Solid culture-batch 3 | 0.5 | 49 |
| Solid culture-batch 4 | 0.5 | 60 |
| Liquid culture-batch 1 | 0.5 | 50 |
| Liquid culture-batch 2 | 0.5 | 48 |
2. Effect of callus cell extract of Rosa capillipes on skin cell proliferation
Keratinocytes are the most important cells in the epidermis, and are involved in forming the physical barrier of the skin, preventing the invasion of undesirable factors such as physical, chemical and microbial factors, and protecting the skin. One of the main characteristics of skin aging is thinning of the epidermis and a slow rate of wound healing, which is mainly caused by a reduction in the regenerative capacity of keratinocytes in the epidermis and a reduction in the proliferative capacity of the keratinocyte system. Human skin fibroblasts are the most important cells in the dermal reticulum of the skin and are one of the major repair cells after skin aging and cell damage. It can promote the migration, proliferation and differentiation of epidermal cells, secrete great amount of collagen, elastic fiber protein and cell repairing factors, and has powerful self-renewing capacity to repair aged skin.
The prepared sample is prepared into a solution by deionized water or DMSO, and added into a culture solution of human immortalized epidermal Hacat cells or human primary Fibroblast (FB), and a solvent without the sample is used as a blank control. After 48 hours of culture, after staining the cells by MTT method, absorbance at 550nm was measured by microplate reader, the proliferation rate of blank control was 100%, and the effect on Hacat or FB cell proliferation was evaluated with reference to blank control.
Wherein, the human immortalized epidermis HaCaT is an immortalized human keratinocyte cell line, and is widely applied to scientific research because the cell shape and the function of the immortalized human keratinocyte line are similar to those of normal human keratinocyte cells and can be transmitted for at least 140 generations; compared with cell lines, human primary fibroblasts can simulate the functions of human skin better. As can be seen from the results in Table 4, the callus cell extract of Rosa tenuifolia has certain proliferation promoting effect on Hacat cells and fibroblasts in the epidermis of skin cells.
TABLE 4 Effect of Rosa centifolia callus cells on skin cell proliferation
| Sample(s) | Test concentration (mg/mL) | Hacat cell proliferation Rate (%) | FB cell proliferation Rate (%) |
| Positive control (calf serum) | 50 | 120 | 140 |
| Solid culture-batch 1 | 0.1 | 103 | 113 |
| Solid culture-batch 2 | 0.1 | 109 | 115 |
| Solid culture-batch 3 | 0.1 | 108 | 118 |
| Solid culture-batch 4 | 0.1 | 110 | 122 |
| Liquid culture-batch 1 | 0.1 | 105 | 110 |
| Liquid culture-batch 2 | 0.1 | 106 | 112 |
| Solid culture-batch 1 | 0.5 | 109 | 139 |
| Solid culture-batch 2 | 0.5 | 115 | 145 |
| Solid culture-batch 3 | 0.5 | 118 | 132 |
| Solid culture-batch 4 | 0.5 | 120 | 148 |
| Liquid culture-batch 1 | 0.5 | 110 | 135 |
| Liquid culture-batch 2 | 0.5 | 113 | 137 |
3. Protection effect of rosa tenuifolia callus cell extract on oxidative damage of skin cells
Free radicals can cause irreversible oxidative damage to organism at cellular level, molecular level or tissue and organ level, accelerate aging process of organism cells or whole organism, and induce various diseases related to aging. Hydrogen peroxide (H) 2 0 2 ) Is an important oxygen free radical in cells, and can cause direct oxidative stress reaction and oxidative damage caused by oxidative stress on skin cells of a human body. In vitro utilization of H 2 0 2 Can induce and accelerate the aging process of cells caused by oxidative stress, and simulate the pathological process of oxidative damage in vivo.
Human immortalized epidermal Hacat cells were seeded at 5000 cells/well in 96-well plates and cultured for 72 hours at 37 ℃ in a 5% CO2 cell incubator. Cells were cultured for 6 hours in serum-free starvation medium before sample treatment, after which the cells were cultured for 24 hours in starvation medium containing the sample. Then, the cells were treated with a starvation medium containing 500uM hydrogen peroxide (H2O 2) for 1 hour, and then with a medium containing the sample to be tested, and the cells were cultured for 24 hours and then assayed for viability by the MTT method. As a control (-), one treated without H2O2 was used. VE was used as a positive control.
As can be seen from the results in Table 5, the callus cell extract of Rosa strigosa has an obvious protective effect on oxidative damage of cells caused by hydrogen peroxide.
TABLE 5 protective action of callus cells of Rosa tenuifolia on oxidative damage of skin
| Sample (I) | Test concentration (mg/mL) | Cell viability (%) |
| Blank control (No H2O2 treatment) | - | 100 |
| Blank control (H2O 2 treatment) | - | 65 |
| Positive control VE | 1 | 74 |
| Solid culture-batch 1 | 0.1 | 73 |
| Solid culture-batch 2 | 0.1 | 83 |
| Solid culture-batch 3 | 0.1 | 72 |
| Solid culture-batch 4 | 0.1 | 77 |
| Liquid culture-batch 1 | 0.1 | 75 |
| Liquid culture-batch 2 | 0.1 | 76 |
| Solid culture-batch 1 | 0.5 | 80 |
| Solid culture-batch 2 | 0.5 | 84 |
| Solid culture-batch 3 | 0.5 | 92 |
| Solid culture-batch 4 | 0.5 | 90 |
| Liquid culture-batch 1 | 0.5 | 83 |
| Liquid culture-batch 1 | 0.5 | 85 |
4. Ultraviolet injury protection effect of rosa minutissima callus cell extract
Aging of the skin is classified into intrinsic aging and extrinsic aging, and sunlight, especially Ultraviolet (UV) irradiation, is a major factor in the formation of extrinsic aging, so extrinsic aging is also called photoaging. The major contributors to skin photoaging are UVA (320 nm-400 nm) and a small amount of UVB (280-320 nm). Many data indicate that 95% of the uv light that strikes the skin is absorbed by keratinocytes, so we have established a model of photodamage to the skin by UVA irradiation of human immortalized epidermal HaCaT cells, and use this model to evaluate the protective effect of actives on uv damage to the skin.
Human immortalized epidermal HaCaT cells were seeded at 5000 cells/well in 96-well plates and cultured for 72 hours at 37 ℃ in a 5% CO2 cell incubator. Cells were cultured for 6 hours in starvation medium without serum before sample treatment, after which the cells were cultured in medium containing the sample for 24 hours. After changing the medium to PBS, UVA (365 nm) was irradiated at a dose of 10J/cm2. And adding a culture medium containing a sample to be detected, continuously culturing for 24 hours, and detecting the cell viability by using an MTT method. The group without UVA irradiation served as a control group (UVA-), and the group with UVA irradiation served as a control group (UVA +). Vc was used as a positive control.
As can be seen from the results in Table 6, the callus cell extract of Rosa strigosa has an obvious protective effect on cell damage caused by UVA.
TABLE 6 protective Effect of Rosa tenuifolia callus cells on UVA damage of skin
| Sample (I) | Test concentration (mg/mL) | Cell viability (%) |
| Blank control (No UVA treatment) | - | 100 |
| Blank control (UVA treatment) | - | 87 |
| Positive control Vc | 0.1 | 111 |
| Solid culture-batch 1 | 0.1 | 91 |
| Solid culture-batch 2 | 0.1 | 100 |
| Solid culture-batch 3 | 0.1 | 94 |
| Solid culture-batch 4 | 0.1 | 99 |
| Liquid culture-batch 1 | 0.1 | 90 |
| Liquid culture-batch 2 | 0.1 | 89 |
| Solid culture-batch 1 | 0.5 | 95 |
| Solid culture-batch 2 | 0.5 | 97 |
| Solid culture-batch 3 | 0.5 | 99 |
| Solid culture-batch 4 | 0.5 | 101 |
| Liquid culture-batch 1 | 0.5 | 96 |
| Liquid culture-batch 2 | 0.5 | 97 |
5. Whitening effect of rosa tenuifolia callus cell extract
Recent studies have demonstrated that pigmentation is caused by ultraviolet activation of melanin-producing enzymes in melanocytes in the epidermis to produce pigments. Melanocytes located in the basal layer of the human epidermis contact and associate with surrounding keratinocytes to form an "epidermal melanin unit", in which melanin is a nitrogen-containing complex that is synthesized in the melanosome. Generally, melanin is an important factor in determining skin color in terms of quantity and quality of melanin. During melanin production, tyrosinase converts tyrosine into dopa and dopaquinone, which are then polymerized by non-enzymatic oxidation to produce macromolecular melanin. Therefore, the melanin generation can be limited and the skin pigmentation can be improved by inhibiting the activity of tyrosinase.
The color of the skin comes from melanin stored within keratinocytes. Generally, people who store melanin-rich skin tone is darker and more protected from solar radiation. Melanin is an important factor in determining skin color in terms of quantity and quality of melanin. Tyrosinase is a copper-containing oxidoreductase with a complex structure, and is widely present in microorganisms, animals, plants, and human bodies. Tyrosinase is a key enzyme in the synthesis of melanin by the skin in humans.
The inhibition of the sample on tyrosinase was determined by L-Dopa oxidation. Mouse melanoma B16 cells were cultured in a 96-well plate at a density of 1X105, after 24 hours, a sample powder of the extract was dissolved in PBS and sterilized by filtration, and then added to the cells to culture for 48 hours, the culture solution was removed, 100. Mu.L of PBS buffer containing 1% TritonX-100 was added to each well, 50. Mu.L of 0.2 mg/mL L-DOPA was then added thereto, and after 3 hours of treatment at 37 ℃, the absorbance of 490 nm was measured. The enzyme activity was calculated as follows: tyrosinase inhibition = [1- (experimental OD value/control OD value ] × 100% ] while the MTT method was used to determine the effect of the sample on B16 cell viability.
And (3) measuring the content of melanin in the cells by adopting a NaOH cracking method. Mouse melanoma B16 cells were cultured in 12-well plates at a density of 1X105, after 24 hours, a sample powder of the extract was dissolved in PBS, filtered and sterilized, added to the cells, cultured for 48 hours, the supernatant was discarded, trypsinized, centrifuged to collect the cells in a centrifuge tube, 150. Mu.L of a 1mol/L NaOH solution (containing 10% DMSO) was added thereto, the cells were lysed sufficiently at 80 ℃ for 1h, and the absorbance of 405 nm was measured. The protein concentration of B16 cells was determined by BCA method for correction of absorbance values. Data statistics are expressed as a percentage of the control group.
As is clear from the results in Table 7, the callus extract of Rosa strigosa has no effect on the viability of B16 cells, but inhibits the tyrosinase activity and melanogenesis of B16 cells.
TABLE 7 Effect of Rosa centifolia callus cells on tyrosinase activity and melanogenesis in mouse melanoma B16 cells
Example four skin preparation for external use containing extract of callus cells of Rosa capillata Thunb
The extract of the callus cells of rosa minutissima prepared in example two was used for the preparation of an external preparation for skin. The skin external preparation is preferably a cosmetic composition such as a lotion, essence, cream, etc. The weight percentage of the rosa multiflora callus cell extract in the skin external preparation is 0.001% -1%, preferably 0.1% -1%. The following are examples of specific applications of the callus cell extract of rosa capillaris in skin external preparations, and "-" in the following tables indicates no additive.
1. Toning lotion containing rosa tenuifolia callus cell extract
Taking the rosa minutissima callus cell extract prepared in the second example to prepare the toning lotion with anti-aging and whitening effects, wherein the toning lotion comprises the following components in percentage by weight as shown in table 8:
TABLE 8
2. Essence containing callus cell extract of Rosa capillipes
Taking the rosa minutissima callus cell extract prepared in the second embodiment to prepare essence with anti-aging and whitening effects, wherein the essence comprises the following components as shown in table 9:
TABLE 9
3. Emulsion/cream containing callus cell extract of rosa capillata
Taking the rosa minutissima callus cell extract prepared in the second example to prepare emulsion/cream with anti-aging and whitening effects, wherein the emulsion/cream comprises the following components as shown in table 10:
watch 10
In the above experiment, the callus cells of Rosa tenuifolia were induced from Rosa tenuifolia in Yunnan Shangrila in the laboratory, glacial water was collected from Himalaya Tibet Qu Dengni Ma, local multi-Ji Qu Dengni Maquan was located in Himalaya mountain north slope, elevation 5128 m, spring mouth temperature was kept at 1.5-2 ℃ throughout the year, MS medium and auxin were purchased from Phyto Tech company, sucrose and agar were purchased from Chinese medicinal company. Wherein wild Rosa tenuifolia fruits were collected from Shangrira Yunnanensis by professor Long Chunlin, central university, and the species was identified as Rosa tenulifolia (Rosa graciliflora); glacier water was collected from the polygiy Qu Dengni ma in the north slope of himalayas, where the elevation was 5128 meters and the water source temperature was kept at 1.5-2 ℃ throughout the year.
The technical solution of the present invention has been explained by focusing on the above embodiments. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or measurement to the teachings of the invention without departing from the essential scope thereof. Accordingly, the disclosed embodiments are not to be considered in a limiting sense, but rather are intended to be illustrative. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (6)
1. The application of the rosa minutissima callus cell extract in preparing the skin external preparation is characterized in that the rosa minutissima callus cell extract is used as an anti-aging active ingredient and a whitening active ingredient in the skin external preparation, and the culture method of the rosa minutissima callus cells is any one of solid culture and liquid culture; wherein the solid culture comprises the following steps:
A. taking a reagent MS culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), sucrose, agar and glacier water;
B. preparing a culture medium by taking glacier water as a solvent according to MS, 2,4-D3.0mg/L, KT0.5mg/L, sucrose 30g/L and agar 6 g/L;
C. adjusting the pH value of the solid culture medium to 5.7-5.8, and sterilizing in a sterilizer at 121 deg.C for 20min to obtain solid culture medium;
D. adding a solid culture medium with a bottle volume of 10-20% into a culture container, inoculating callus cells with a culture medium volume of 1-2% after the solid culture medium is solidified, wherein the culture conditions are that the temperature is 19-21 ℃, the humidity is 60-75%, the illumination intensity is 1500Lx-2000Lx, and the illumination is 12 hours per day;
E. harvesting callus cells of the rosa minutissima after culturing for 20 days;
the liquid culture comprises the following steps:
A. taking a reagent MS culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), sucrose and glacier water; B. preparing a culture medium by using glacier water as a solvent according to MS, 2,4-D3.0mg/L, KT0.5mg/L and 30g/L of cane sugar;
C. adjusting the pH value of the culture medium to 5.7-5.8, and sterilizing in a sterilizer at 121 deg.C for 20min to obtain liquid culture medium;
D. adding a liquid culture medium with a bottle volume of 20-30% into a culture container, inoculating callus cells with a volume of 1-2% of the liquid culture medium, and placing the cells into a shaking table for culture at a temperature of 24-26 ℃ and a rotating speed of 110r/min;
E. harvesting callus cells of the rosa minutissima after culturing for 20 days;
the method for extracting the rosa minutissima callus cells comprises the following steps:
A. weighing the callus cells of the rosa tenuifolia, grinding and crushing;
B. adding deionized water with the volume 5 times of the volume of the callus cells of the rosa multiflora, and immersing and stirring;
C. ultrasonic extracting at 500W for 2 times, each for 30min, centrifuging at 5000rmp for 10min, collecting supernatant, filtering, and mixing filtrates;
D. freeze drying to obtain the callus cell extract of rosa tenuifolia.
2. The use of an extract from callus cells of rosa multiflora thunb for the preparation of an external preparation for skin according to claim 1, wherein the anti-aging ingredient is an anti-aging active ingredient for scavenging free radicals of the skin, increasing the proliferation of skin cells, protecting skin cells from oxidative damage, and protecting skin cells from damage caused by UVA.
3. The use of the extract from callus cells of rosa multiflora according to claim 1, wherein the whitening active ingredient is a whitening active ingredient that inhibits the tyrosinase activity and the melanin production of human epidermal melanocyte B16.
4. Use of the callus cell extract of rosa capillaris according to claims 2 to 3 for preparing a skin preparation for external use, wherein the content of the callus cell extract of rosa capillaris is 0.001% to 1% by mass of the callus cell extract of rosa capillaris in the skin preparation for external use.
5. Use of the callus of rosa capillaris as claimed in claim 4 for preparing an external preparation for skin, wherein the content of the callus of rosa capillaris is 0.1% to 1%.
6. The use of a callus cell extract of rosa capillata according to claim 1, wherein the glacier water is selected from the group consisting of polyghatti Qu Dengni ma, north slope of himalayas, elevation 5128 m.
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