CN112386627A - Application of rosemary callus juice and culture medium for inducing rosemary callus - Google Patents
Application of rosemary callus juice and culture medium for inducing rosemary callus Download PDFInfo
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- CN112386627A CN112386627A CN202010823733.0A CN202010823733A CN112386627A CN 112386627 A CN112386627 A CN 112386627A CN 202010823733 A CN202010823733 A CN 202010823733A CN 112386627 A CN112386627 A CN 112386627A
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention provides an application of rosemary callus juice, wherein the composition is used for inhibiting the generation of Reactive Oxygen Species (ROS), improving the activity of mitochondria and/or improving the expression amount of moisturizing genes. The invention also provides a culture medium for inducing the rosemary callus.
Description
Technical Field
The present invention relates to the application of rosemary callus juice, and is especially the use of rosemary callus juice in preparing skin aging inhibiting composition and the culture medium for inducing rosemary callus.
Background
The skin provides a first stage of protection for human subjects against environmental factors such as Ultraviolet (UV) radiation, pathogens, friction in the sun. The skin comprises, in order from the outside to the inside, an epidermal layer, a dermal layer mainly composed of connective tissue, and a subcutaneous tissue. Among them, the dermis contains enough molecules such as collagen (collagen), elastin (elastin), and hyaluronic acid (hyaluronic acid), so that the young skin has better support and elasticity.
The skin will age normally with age, resulting in various damaged skin conditions. For example, the skin has aging symptoms such as decreased skin elasticity, insufficient water retention (dry and dull skin), large pores, wrinkles, blotches and/or dullness on the whole, and yellow wax.
However, under certain environmental factors, the process of skin aging is accelerated. For example, natural factors such as ultraviolet rays in sunlight are generally common. When exposed to ultraviolet light, the ultraviolet light causes the aforementioned skin aging conditions to accelerate.
Disclosure of Invention
In view of the above, the present invention provides an application of rosemary callus juice, which is used for preparing a composition for resisting skin aging.
In some embodiments, the use of rosemary callus juice for the preparation of a composition for inhibiting the production of reactive oxygen species.
In some embodiments, the composition is used to increase skin moisture, improve skin redness, improve skin texture, reduce skin blemishes, or a combination thereof.
In some embodiments, use of rosemary callus juice in the preparation of a composition for increasing mitochondrial activity.
In some embodiments, the use of rosemary callus juice in the preparation of a composition for increasing the expression of moisturizing genes, wherein the moisturizing genes are at least one of: KRT10 gene, KRT14 gene, KRT1 gene, AQP3 gene, FLG gene, SMPD1 gene and GBA gene.
In some embodiments, the aforementioned composition is a pharmaceutical composition.
In some embodiments, the aforementioned composition is a food composition or a cosmetic composition.
In some embodiments, the aforementioned composition is an internal composition or an external composition.
In some embodiments, the rosemary callus fluid is obtained from rosemary callus induced by a culture medium comprising potassium nitrate (KNO3), calcium chloride dihydrate (CaCl)2.2H2O), magnesium sulfate (MgSO)4.7H2O), potassium dihydrogen phosphate (KH)2PO4) 2, 4-dichlorophenoxyacetic acid, and 6-benzylaminopurine.
The rosemary callus juice can effectively prevent skin aging and maintain or improve skin appearance because the rosemary callus juice can inhibit the generation of free radicals and/or inhibit the generation of melanin.
In addition, the scheme also proves that the rosemary callus juice can improve the activity of mitochondria and the expression of moisturizing genes. Therefore, the rosemary callus juice can passively reduce the loss of skin fibroblasts influenced by free radicals, and can also actively improve the activity of mitochondria and the expression of moisturizing genes, so that the skin is healthier.
Drawings
FIG. 1 is a graph comparing the results of total flavonoids in control group, control group and experimental group.
FIG. 2 is a graph showing comparison of results of expression amounts of moisturizing genes in control group, control group and experimental group.
FIG. 3 is a graph showing the comparison of the results of the relative amounts of Reactive Oxygen Species (ROS) produced in the control group, the control group and the experimental group.
Figure 4 is a graph comparing the results of relative JC-1 aggregation for control versus experimental groups.
FIG. 5 is a graph comparing the change of redness of each group before and after applying a placebo and a mask containing essence of rosemary callus juice to the facial skin of a subject. P value <0.01, compared to baseline.
FIG. 6 is a graph comparing the change in redness of each group of subjects before applying placebo and the serum containing rosemary callus sap to the facial skin and after 4 weeks of continuous use.
Fig. 7 is a graph comparing changes in skin moisture content of each group before and after applying a placebo and a mask containing essence of rosemary callus sap to the facial skin of a subject.
Fig. 8 is a graph comparing the change in skin moisture content of each group of subjects before (week 0) and after 4 weeks (week 4) of continuous use of placebo and the serum containing rosemary callus sap.
Fig. 9 is a graph comparing the change in skin texture of each group of subjects before (week 0) and after 4 weeks (week 4) of continuous use of placebo and the essence containing rosemary callus sap.
Fig. 10 is a graph comparing the change of skin spots in each group before (week 0) and after 4 weeks (week 4) of continuous use of the placebo and the essence containing rosemary callus sap on the face skin of the subjects. P value <0.05, compared to baseline. # p value <0.05, compared to placebo.
Detailed Description
Some embodiments of the present disclosure will be described below. The present disclosure may be embodied in many different forms without departing from the spirit thereof, and the scope of protection should not be limited to the details set forth in the specification.
Statistical analysis was performed using Excel software. Data are expressed as mean ± Standard Deviation (SD) and differences between groups were analyzed by student's t-test (student's t-test). In the drawings, the term "indicates a p value of less than 0.05, the term" indicates a p value of less than 0.01, and the term "indicates a p value of less than 0.001. As more "x", the more significant the statistical difference.
As used herein, the numerical values are approximate and all experimental data are shown to be within a range of plus or minus 10%, and more preferably within a range of plus or minus 5%.
Rosemary (Rosmarinus officinalis) is Labiatae (Labiatae), is a perennial evergreen shrub of Rosmarinus, is native to Europe and the coast of the Mediterranean sea, is cultivated in France, Spain, Italian, Nanslav, Tunisia, Morocco and other countries, and is distributed in the United states, China and other regions.
In some embodiments, rosemary callus juice obtained from rosemary callus may have at least one of the following capabilities: inhibiting free radical generation, inhibiting melanin generation, increasing mitochondrial activity, and increasing moisturizing gene expression level. Thus, rosemary callus juice may be used to prepare a composition having at least one of the following capabilities: inhibiting free radical generation, inhibiting melanin generation, increasing mitochondrial activity, and increasing moisturizing gene expression level.
Wherein, the moisturizing gene can be at least one of the following: KRT10(keratin 10) gene, KRT14(keratin 14) gene, KRT1(keratin 1) gene, AQP3(Aquaporin-3) gene, FLG (Flagellar basal-body rod protein) gene, SMPD1(Sphingomyelin phospholipid 1) gene and GBA (beta-Glucocerebrosidase) gene.
In some embodiments, rosemary callus juice is effective in preventing skin aging phenomena, thereby maintaining or improving skin appearance.
In some embodiments, the rosemary callus juice is effective in preventing skin aging by at least one of the following actions: inhibiting melanin generation, inhibiting active oxygen substance generation, improving skin cell antioxidant ability, and improving mitochondrial activity.
Since the active oxygen species are highly active, they react with collagen molecules in the skin, thereby cleaving collagen. Since collagen, such as "scaffold" in the skin, is used to provide support and elasticity to the skin, skin aging is accelerated in the absence of collagen, and skin aging phenomena such as decreased elasticity, insufficient water retention (dry and dull skin), enlarged pores, and wrinkles occur. On the other hand, the melanocytes in the skin produce melanin, which also causes skin spots and/or aging phenomena such as overall darkness or yellow wax.
In other words, the rosemary callus juice is effective in preventing at least one of the following skin aging phenomena: inhibiting decrease of skin elasticity, preventing insufficient water retention (dry and lusterless skin), inhibiting pore enlargement, inhibiting wrinkle generation, inhibiting skin speckle, and preventing skin darkness or yellow wax.
In some embodiments, the composition can be a pharmaceutical composition, a cosmetic composition, or a food composition.
Wherein the medicinal composition comprises rosemary callus juice with effective content.
In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) that is widely used in pharmaceutical manufacturing technology. For example, a pharmaceutically acceptable carrier can comprise one or more of the following carriers: emulsifying agents (emulsifying agent), suspending agents (suspending agent), disintegrating agents (disintegrant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients (excipient), stabilizing agents (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving), wetting agents (wetting agent), lubricants (lubricating agent), absorption delaying agents (absorbing delaying agent), liposomes (liposome), and the like. The choice and amount of such carriers can be readily selected by one of ordinary skill in the art.
In some embodiments, the pharmaceutically acceptable carrier may further comprise a solvent selected from the group consisting of: water, normal saline (normal saline), Phosphate Buffered Saline (PBS), aqueous alcohol-containing solution (aqueous solution stabilizing alcohol), and any other suitable solvent.
In some embodiments, the pharmaceutical composition may be formulated into a dosage form suitable for enteral (enterrally), parenteral (parentrally), oral, or topical (topically) administration using techniques well known to those skilled in the art. The dosage form of the pharmaceutical composition may be, for example: injections (injection) [ e.g., sterile aqueous solution (sterile aqueous solution) or dispersion (dispersion) ], sterile powders (sterile powder), external preparations (external preparation), or the like.
In some embodiments, the pharmaceutical composition may be administered by any of the parenteral routes (parenteral routes) described below: subcutaneous injection (subecanal injection), intradermal injection (intraepithelial injection), and intralesional injection (intralesion).
In some embodiments, the pharmaceutical composition can be manufactured into an external preparation (external preparation) suitable for topical application to the skin using techniques well known to those skilled in the art. For example, the external agent may be any of the following, but is not limited thereto: creams (lotions), liniments (liniments), powders (powders), aerosols (aerogels), sprays (sprays), emulsions (positions), serums (serums), pastes (pastes), foams (foams), drops (drops), suspensions (suspensions), ointments (salves), and bandages (bandages).
In some embodiments, the external preparation is prepared by mixing the effective amount of rosemary callus juice with a base (base) as known to those skilled in the art.
In some embodiments, the substrate may comprise additives (additives) of one or more of the following: water, alcohols, glycols, hydrocarbons such as petroleum jelly and white petrolatum]Wax (wax) [ such as Paraffin and yellow wax (yellow wax)]Preserving agents (preserving agents), antioxidants (antioxidants), surfactants (surf)acts), absorption enhancers (absorption enhancers), stabilisers (stabilizing agents), gelling agents (gelling agents) [ such as carbopol ]
(
974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethyl cellulose)]Active agents (actives), moisturizers (humectants), odor absorbers (odor absorbers), perfumes (fragrans), pH adjusting agents (pH adjusting agents), chelating agents (chelating agents), emulsifiers (emulsifiers), occlusive agents (occlusive agents), softeners (emulsifiers), thickeners (thickeners), solubilizing agents (solubilizing agents), penetration enhancers (penetration enhancers), anti-irritants (anti-irritants), colorants (colorants), and propellants (propellants). The selection and amounts of such additives are within the skill and routine skill of those skilled in the art.
Wherein the cosmetic composition comprises rosemary callus juice in an effective amount.
In some embodiments, the care composition may further comprise an acceptable adjuvant (acceptable adjuvant) that is widely used in care manufacturing technology. For example, an acceptable adjuvant may comprise one or more of the following adjuvants: solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, colouring agents, thickening agents, fillers, fragrances and odour absorbers. The selection and the amount of the reagents can be properly adjusted according to actual requirements.
In some embodiments, the cosmetic composition may be manufactured in a form suitable for skin care (skincare) or make-up (makeup) using techniques well known to those skilled in the art. Among them, the form suitable for skin care or make-up may be any one of the following, but is not limited thereto: aqueous solutions (aqueous solutions), aqueous-alcoholic solutions (aqueous-alcoholic solutions), oily solutions (oil solutions), emulsions in the form of oil-in-water type, water-in-oil type or compound type, gels, ointments, creams, masks (masks), patches (packs), liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, mousses (mousses), sunblocks, lotions (toiletries), foundations (foundations), make-up removal products (makeup remover products), soaps (soaps), and other body cleansing products (body cleansing products).
In some embodiments, the cosmetic composition may further comprise one or more of the following known active topical agents (external use agents): whitening agents (whitening agents) [ such as vitamin a acid (tretinoin), catechin (catechin), kojic acid, arbutin and vitamin C ], moisturizing agents, bactericides (bacteriodes), ultraviolet absorbers (ultravirosomes), plant juices (plant extracts) [ such as aloe vera juice (aloe extract) ], skin nutrients (skin nutrients), anesthetics (anesthesics), antiacne agents (anti-acne agents), antipruritics (antipruritics), analgesics (analgesis), anti-dermatitis agents (anti-dermatitis agents), anti-hypercholesterolitics (anti-xerosis agents), anti-dry skin agents (anti-dry skin agents), antiperspirants (antipsoriatics), anti-aging agents (anti-aging agents), anti-aging agents (anti-inflammation agents), anti-wrinkle agents (anti-inflammation agents), anti-aging agents (anti-wrinkle agents), anti-aging agents (anti-seborrheic agents), anti-aging agents (anti-corticosteroid), and anti-corticosteroid (anti-aging agents). The selection and amounts of such agents for external use are within the skill and routine skill of those skilled in the art.
Wherein the food composition comprises rosemary callus juice in an effective amount.
In some embodiments, the food composition may be administered to the subject orally. Wherein the food composition is in the form of powder, granule, solution, colloid, or paste. The subject may be a human.
In some embodiments, the food composition may be a food product or food additive.
In some embodiments, the dietary supplement may be formulated with any edible material into a food product for ingestion by humans and non-human animals by conventional methods either during raw material preparation or during food preparation.
In some embodiments, the type of food product may be, but is not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products), health foods (health foods) or dietary supplements (dietary supplements).
In other words, the composition may be an internal composition or an external composition depending on the use mode.
In some embodiments, the rosemary callus growth may be induced by an induction medium comprising potassium nitrate (KNO)3) Calcium chloride dihydrate (CaCl)2.2H2O), magnesium sulfate (MgSO)4.7H2O), potassium dihydrogen phosphate (KH)2PO4) 2, 4-dichlorophenoxyacetic acid, and 6-benzylaminopurine.
In some embodiments, rosemary callus is obtained as follows: firstly, cultivating rosemary seeds into rosemary plants, then sterilizing the rosemary plants to remove microorganisms on the surfaces of the plants, and then washing the rosemary plants with sterilized water. Then, the surface of the leaves of the rosemary plant is cut, torn or sheared to generate a wound. Thereafter, the leaf with wound was cultured in the induction medium until callus was generated. Finally, the rosemary callus was stored at-20 ℃ for subsequent use.
In some embodiments, the sterilization treatment can be washing the rosemary plant with sterile water, such as 2 vol% sodium hypochlorite solution, and the aforementioned step of washing the rosemary plant with sterile water can be repeated as necessary.
In some embodiments, the culturing of rosemary callus takes about 1 to 3 months, as the case may be.
In some embodiments, the "rosemary callus juice" may be an extract obtained by extracting rosemary callus with a suitable extraction solvent at a specific extraction temperature for a suitable extraction time. For example, rosemary callus may be soaked in water (i.e., extraction solvent) at a specific extraction temperature for a suitable soaking time (i.e., extraction time), and cooled to room temperature (i.e., 25 ℃ -30 ℃) after soaking to obtain rosemary callus juice.
In some embodiments, the callus of rosemary may be intact callus, or may be separated into fragments, granules or powder by physical pretreatment. Wherein the physical pretreatment may include at least one of: cutting, shearing, mashing and grinding.
The experimental procedures in the following examples were carried out at room temperature (25 ℃ C. -30 ℃ C.) and normal pressure (1atm), unless otherwise specified.
[ example 1] preparation of Induction Medium
In this example, induction media were formulated according to tables 1A to 1C below:
TABLE 1A (Induction Medium composition)
The induction medium was prepared to 4000mL according to the above ingredients so that it contained 2,4D at a concentration of 0.5mg/L, 0.1mg/L BA, 30g/L sugar, and 6.5g/L agar. Wherein 2,4-D is 2, 4-dichlorophenoxyacetic acid, BA is 6-benzylaminopurine, and agar is agar.
The formulations of 2,4-D and BA are shown in Table 1B, SOL1, SOL2, SOL3 and SOL4 in Table 1C.
TABLE 1B (2,4-D with BA composition)
TABLE 1C (SOL1, SOL2, SOL3 and SOL4 components)
[ example 2] preparation of Rosemary callus juice
Materials and instruments
1. Rosemary seeds (from vegetable suppliers, trade code: M07791579).
2. Induction medium (prepared in example 1).
Experimental procedure
In this example, rosemary callus juice was prepared as follows:
1. cultivating the rosemary seeds into rosemary plants.
2. The rosemary plants were subjected to a sterilization treatment to remove microorganisms on the surface of the plants, after which the rosemary plants were rinsed with sterilized water.
3. The surface of the leaves of the rosemary plant was wounded.
4. Then, the callus obtained in step 3 was cultured in the induction medium prepared in example 1at 18 to 25 ℃ and 45 to 75% humidity.
5. Thereafter, water was mixed with the rosemary callus of step 4 at a ratio of 10: 1 (water: callus) in a weight ratio.
6. The mixture obtained in step 5 is subjected to microwave extraction (for example, heating to 75 ℃ at a strength of 1500Hz, then maintaining the temperature at 75 ℃ at 300Hz, setting the stirring speed of the juicer at 5, for 20 minutes, to form a first extract containing solids) at 75 ℃.
7. The mixture obtained in step 8 (i.e., the first extract) was filtered through a 400-mesh filter to provide a filtrate.
8. The filtrate obtained from step 7 was heated to 95 ℃ and maintained for 30 minutes for sterilization.
9. And (4) cooling the filtrate obtained in the step (8) to obtain rosemary callus juice, and refrigerating and storing the rosemary callus juice for subsequent experiments.
[ example 3] callus Effect quantitative test-measurement of flavonoid Compound content
Purpose of experiment
In order to verify the effectiveness of rosemary callus, the concentrations of total flavonoids (flavonoids) in the common rosemary leaf juice and rosemary callus juice were compared to establish the effectiveness of rosemary callus.
Materials and instruments
1. Sodium nitrate (purchased from Sigma, product No. 31443).
2. Aluminum nitrate (available from Alfa Aesar, product No. 12360).
3. Sodium hydroxide (available from Macron, product number 7708-10).
4. Rutin (available from Chroma Dex, product number ASB-00018440).
5. Spectrophotometer (product model: Jasco V-730).
Experimental procedure
In this example, rosemary leaf juice was prepared as follows:
1. rosemary seeds are cultivated into rosemary plants, and rosemary leaves (hereinafter referred to as rosemary leaves) are obtained from the rosemary plants by cutting, tearing or shearing.
2. Mixing the rosemary leaves with rosemary leaves: water 1.5: 10 by weight ratio, homogenizing with a juicer, heating to 75 deg.C, and extracting at the same temperature for 20 min to obtain a first extractive solution containing solids.
3. Cooling the first extract to room temperature.
4. Filtering the first extractive solution with 400 mesh sieve to obtain rosemary leaf juice.
In this example, a test sample of the efficacy of rosemary leaf juice (hereinafter referred to as a first efficacy test sample) was prepared as follows:
1. after diluting rosemary leaf juice with water by 10 times according to the volume, 200. mu.L of rosemary leaf juice is put into a test tube.
2. 200. mu.L of a 5 wt% aqueous solution of sodium nitrate was added to the test tube, and the mixture was mixed well and allowed to stand for reaction for 6 minutes.
3. Then, 200. mu.L of a 10 wt% aqueous aluminum nitrate solution was added, and after uniformly mixing, the mixture was allowed to stand for reaction for 6 minutes, and then 2mL of a 4 wt% aqueous sodium hydroxide solution was added to uniformly mix the mixture.
4. Finally, 1.4mL of water is added into the test tube and mixed uniformly to obtain a first efficacy test sample.
5. 200 μ L of the first validation test sample in the test tube was placed in a 96-well reaction plate and absorbance was detected with a spectrophotometer at a wavelength of 500 nm.
In this example, a test sample of effectiveness of rosemary callus sap (hereinafter referred to as the second test sample) was prepared as follows:
1. the rosemary callus obtained in example 2 was diluted 10 times by volume with water, and 200. mu.L of the solution was taken out and placed in a test tube.
2. 200. mu.L of a 5 wt% aqueous solution of sodium nitrate was added to the test tube, and the mixture was mixed well and allowed to stand for reaction for 6 minutes.
3. Then, 200. mu.L of a 10 wt% aqueous aluminum nitrate solution was added, and after uniformly mixing, the mixture was allowed to stand for reaction for 6 minutes, and then 2mL of a 4 wt% aqueous sodium hydroxide solution was added to uniformly mix the mixture.
4. Finally, 1.4mL of water is added into the test tube and mixed uniformly to obtain a second efficacy test sample.
5. 200 μ L of the second validation sample in the test tube was placed in a 96-well reaction plate and absorbance was detected with a spectrophotometer at a wavelength of 500 nm.
In this example, the preparation steps of the standard of the efficacy test are as follows (in this case, rutin equivalent is expressed as the relative content of total flavonoids):
1. firstly, 200 mu g/mL of rutin methanol solution is prepared to be used as the rutin standard solution.
2. Adding 0 μ L, 200 μ L, 400 μ L, 600 μ L, 800 μ L and 1000 μ L of the standard liquid into different test tubes, and adding water into the test tubes to make the total volume of the test tubes be 1200 μ L.
3. Then, 200. mu.L of the diluted rutin standard solution of step 2 was taken out from each tube and put into another new tube, and 200. mu.L of 5 wt% sodium nitrate aqueous solution was added into the tube, mixed well and allowed to stand for reaction for 6 minutes.
4. Then, 200. mu.L of a 10 wt% aqueous aluminum nitrate solution was added, and after uniformly mixing, the mixture was allowed to stand for reaction for 6 minutes, and then 2mL of a 4 wt% aqueous sodium hydroxide solution was added to uniformly mix the mixture.
5. Finally, 1.4mL of water is added into the test tube and mixed evenly to obtain a standard substance, 200 mu L of the standard substance in the test tube is put into a 96-hole reaction plate, and the light absorption value is detected by a spectrophotometer under the wavelength of 500 nm. And drawing the absorbance values of the six rutin solutions with different concentrations according to the same method to obtain a detection line.
6. And then, converting the light absorption values of the first and second effectivity test samples into the total flavone content by using a calibration curve. In this way, the total flavone content of the rosemary callus juice before dilution (i.e., the rosemary callus juice obtained in example 2) was estimated to be 3000 ppm.
Results of the experiment
As shown in FIG. 1, the concentration of total flavonoids in rosemary callus sap was about 2.5 times that in common rosemary leaf sap. It is obvious that the effective components of the juice extracted from the rosemary callus are higher than those extracted from common rosemary.
It should be noted that while flavone is not known to be an active ingredient for the applications described herein, it is known that flavone may have other applications (e.g., prevention of cardiovascular diseases, etc.), and previous experiments have shown that rosemary callus juice according to the present disclosure may have a higher total flavone content than rosemary leaf extract, and thus may have various composite applications. In addition, in some embodiments, the total flavone content can also be used as a criterion for determining the concentration endpoint.
[ example 4] Gene test
Materials and instruments
1. Serum-free medium specific for keratinocytes (Keratinocyte-SFM; purchased from Thermo, product No. 17005042).
2. Human epidermal keratinocytes (hereinafter referred to as HPEK-50 cells; available from CELLnTEC).
RNA extraction reagent kit (from Geneaid corporation, product No. FC 24015-G).
4.Super
III reverse transcriptase (from Invitrogene, product No. 18080-.
ABI StepOnePlusTM Real-Time PCR system (available from Thermo Fisher Scientific Co.).
KAPA SYBR FAST (available from Sigma, accession number 38220000000).
Experimental procedure
The experimental procedure for cell culture was as follows:
in this case, a serum-free medium dedicated to keratinocytes was used as the medium.
First, the HPEK-50 cells were subjected to experiments of expression levels of KRT10 gene, KRT14 gene, KRT1 gene, AQP3 gene, FLG gene, SMPD1 gene and GBA gene in keratinocytes. First, 1X 105 cells per well were cultured in a six-well plate containing 2mL of the above-mentioned culture solution and cultured at 37 ℃ for 16 hours, and then the HPEK-50 cells were divided into the following three groups: experimental, control and control groups.
Experimental groups: a sap-containing culture solution was prepared in a proportion of 0.25mg/mL of rosemary callus sap prepared in the manner of example 2 (i.e., concentration of 0.25mg/mL), and culturing was continued by replacing HPEK-50 cells with the sap-containing culture solution.
Control group: a sap-containing culture solution was prepared in a proportion of 0.25mg per mL of rosemary leaf sap prepared in the manner of example 2 (i.e., concentration of 0.25mg/mL), and culturing was continued by replacing HPEK-50 cells with the sap-containing culture solution.
Control group: no treatment was performed, i.e.no additional compounds were added to the culture broth containing the cultured HPEK-50 cells.
After culturing the experimental group, the control group and the control group for 48 hours, the cells of the experimental group, the control group and the control group after culturing are respectively broken the cell membranes by the cell lysate of the RNA extraction reagent kit to form two groups of cell solutions.
The experimental procedure for the polymerase chain reaction is as follows:
1. RNA in the two sets of cell solutions was collected separately using RNA extraction reagent kits.
2. Then, 2000 nanograms (ng) of the extracted RNA were used as templates in each group and were processed by Super
III reverse transcriptase was performed by reverse transcription with primer (primer) cohesively in Table 2 to generate the corresponding cDNA.
3. Then, two sets of reverse transcription products were subjected to quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) using ABI StepOnePlusTM Real-Time PCR system and KAPA SYBR FAST, respectively, with the combined primers shown in Table 2, to observe the expression amounts of the genes of the HPEK-50 cells of the experimental group and the control group. The apparatus for quantitative real-time reverse transcription polymerase chain reaction was set to react at 95 ℃ for 1 second and 60 ℃ for 20 seconds for a total of 40 loops, and gene quantification was performed using the 2- Δ Ct method. In this case, the quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression level of each gene, and the expression level of the protein encoded by each gene can be estimated.
TABLE 2
Results of the experiment
As shown in fig. 2, compared with the control group and the control group (rosemary leaf juice), the gene expression levels of KRT10 gene, KRT14 gene, KRT1 gene, AQP3 gene, FLG gene, SMPD1 gene and GBA gene in the experimental group (rosemary callus juice) were significantly increased. Wherein, KRT10 gene, KRT14 gene and KRT1 gene are cutin-synthesizing genes to prevent excessive loss of skin moisture; AQP3 is a cell membrane aquaporin (aquaporin) gene; FLG is a gene related to formation of Natural Moisturizing Factors (NMF); SMPD1 is an enzyme gene that metabolizes lipid molecules (sphingomyelins) to ceramide; GBA is enzyme for synthesizing ceramide, and can fill lipid in intercellular space of skin stratum corneum, enhance moisture retention, and prevent water loss.
[ example 5] cell experiment-Rosemary callus juice inhibits ROS production (Hydrogen peroxide treatment)
In this case, the change of the content of active oxygen substances in Human skin fibroblasts (Human skin fibroblasts) CCD-966sk cells after rosemary callus sap treatment and hydrogen peroxide reaction was measured by using a fluorescent probe DCFH-DA in combination with a flow cytometer.
Materials and instruments
1. Cell lines: human dermal fibroblast cells CCD-966sk (purchased from Taiwan center for biological resources preservation and research (BCRC), No.60153), hereinafter referred to as CCD-966sk cells.
2. Cell culture medium: basal medium containing 10 vol% FBS (total bone serum, from Gibco). Wherein the basal medium is prepared from Eagle's minimal essential medium (MEM, available from Gibco under product number 15188-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco.
DCFH-DA solution: dichlorodihydrofluorescein diacetate (2, 7-dichoro-dihydro-fluoroescein diacetate, DCFH-DA; purchased from Sigma) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 5mg/mL solution of DCFH-DA.
5. Hydrogen peroxide (H2O 2): purchased from Sigma-Aldrich, product number 95299-1L.
6. Trypsin (Trypsin-EDTA): 10 XTrypsin-EDTA (from Gibco) was diluted 10-fold with 1 XPBS solution.
7. Flow cytometry: available from Beckman, product model 660519.
Experimental procedure
The experiment will be divided into three groups of an experimental group, a control group (the group without adding rosemary callus juice and without being treated by hydrogen peroxide) and a control group (the group without adding rosemary callus juice and being treated by hydrogen peroxide) and each group is respectively subjected to two repeated experiments:
1. CCD-966sk cells were seeded at 1X 105 cells per well in 6-well plates containing 2mL of cell culture medium per well.
2. The culture plate was incubated at 37 ℃ with 5% CO2 for 24 hours.
3. The cell culture medium was removed.
4. 2mL of the experimental medium was added to each well of the plate and incubated at 37 ℃ for 1 hour.
The experimental medium in the experimental group was 2mL of cell culture medium supplemented with 5. mu.L of the rosemary callus juice obtained in example 2 (i.e., the rosemary callus juice was 0.25% by volume of the cell culture medium).
The experimental medium for the control group was 2mL of cell culture medium alone (i.e. without rosemary callus juice).
The experimental medium for the control group was 2mL of cell culture medium alone (i.e. without rosemary callus juice).
5. Add 5mg/mL DCFH-DA solution 2 u L each hole in the cell culture medium, DCFH-DA treatment of cells for 15 minutes.
6. After DCFH-DA treatment, H2O2 was added to the experimental medium of the experimental group and the experimental medium of the control group, respectively, and reacted at 37 ℃ for 1 hour. Specifically, 35% wt of hydrogen peroxide was diluted to 100mM (10. mu.L of hydrogen peroxide was added to 990. mu.L of redistilled water), and then 20. mu.L of 100mM hydrogen peroxide was added to 2mL of cell culture plates.
7. After reaction, each well was rinsed 2 times with 1mL of 1 XPBS solution.
8. 200 μ L of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, 6mL of cell culture medium was added to terminate the reaction.
9. The cells and cell culture medium in each well were collected into individual corresponding 15mL centrifuge tubes, and the centrifuge tubes containing the cells and medium were centrifuged at 400xg for 10 minutes.
10. After centrifugation, the supernatant was removed and the cell pellet was back-lysed with 1X PBS solution.
11. Centrifuge at 400Xg for 10 min.
12. After centrifugation, the supernatant was removed and the cells were resuspended in 1mL of 1 XPBS solution in the dark to obtain the test cell fluid.
13. Detecting DCFH-DA fluorescence signals in the test cell fluid of each well by using flow cytometry. The excitation wavelength for fluorescence detection is 450-490nm, and the emission wavelength is 510-550 nm. Since DCFH-DA is hydrolyzed into DCFH (dichlorodihydrofluorescein) and then oxidized into DCF (dichlorofluorescein) capable of emitting green fluorescence by the active oxygen substance after entering the cells, the fluorescence intensity of the DCFH-DA treated cells can reflect the content of the active oxygen substance in the cells, and thus the ratio of the number of the cells highly expressed by the active oxygen substance in the cells to the original number of the cells can be known. Since the experiment was conducted in duplicate, the measurement results of duplicate experiments of each group were averaged to obtain an average value, and then the average values of the control group and the experimental group were converted into relative ROS production amounts by taking the average value of the control group as 100%, as shown in fig. 3.
Results of the experiment
As shown in fig. 3, the results of comparison between the control group and the control group revealed that the amount of ROS produced (high fluorescence) was significantly increased (about 5-fold) after the hydrogen peroxide treatment; it shows that hydrogen peroxide treatment can indeed lead to the production of reactive oxygen species in the cells, which in turn can cause subsequent damage to skin fibroblasts. On the other hand, according to the results of comparing the control group with the experimental group, it can be known that the relative ROS production is reduced by about 84% after the cells are treated by rosemary callus juice, even lower than that of the control group; it was shown that rosemary callus sap was effective in reducing the production or accumulation of reactive oxygen species in cells. In other words, rosemary callus juice can act as an active oxygen scavenger. That is, the rosemary callus juice can reduce the content of active oxygen substances in cells, and reduce the oxidative damage of the cells caused by the active oxygen substances and the like.
[ example 6] cell assay-assay for the mitochondrial Activity of skin cells
To investigate the effect of rosemary callus juice on the mitochondrial function of skin cells, this example uses a flow cytometer to evaluate the change of mitochondrial activity of human skin fibroblasts CCD-966sk after being treated with rosemary callus juice.
Materials and instruments
1. Cell lines: human dermal fibroblasts CCD-966sk (BCRC No. 60153).
2. Cell culture medium: basal medium containing 10 vol% FBS (from Gibco). . Wherein the basal medium is prepared from Eagle's minimal essential medium (MEM, available from Gibco under product number 15188-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco.
4. Mitochondrial membrane potential detection kit (BDTMMitoScreen (JC-1) kit, model 551302). Wherein the mitochondrial membrane potential detection kit comprises JC-1 dye (lyophilized) and 10 Xanalysis buffer. Prior to use, 10X assay buffer was diluted 10-fold with 1X PBS to form 1X assay buffer. Add 130. mu.L DMSO to JC-1 stain (lyophilized) to form a JC-1 stock solution. The JC-1 stock solution was then diluted with 1 Xassay buffer to form a JC-1working solution (JC-1working solution; i.e., JC-1 mitochondrial specific stain). Dilution ratio JC-1 stock solution to 1 Xassay buffer was 1: 100.
5. Trypsin: 10 XTrypsin-EDTA (from Gibco) was diluted 10-fold with 1 XPBS solution.
6. Flow cytometry: purchased from BD Pharmingen, model BDTM Accuri C6 Plus.
Experimental procedure
The experiment will be carried out in two groups, an experimental group and a control group (group without rosemary callus juice), each group being subjected to three replicates:
1. CCD-966sk cells were seeded at 1X 105 cells per well in 6-well plates containing 2mL of cell culture medium per well.
2. The medium in each well of the plate was changed to 2mL of experimental medium. Among them, the experimental medium of the experimental group was a cell culture medium containing 5. mu.L of the rosemary callus juice obtained in example 2. The experimental medium for the control group was a simple cell culture medium (i.e., without rosemary callus juice).
3. The culture plate was incubated at 37 ℃ with 5% CO2 for 24 hours.
4. The experimental medium in the culture dish was removed and rinsed 2 times with 1mL of 1X PBS solution.
5. 200 μ L of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, the reaction was terminated by adding a cell culture medium. The cells and cell culture medium in each well were collected into individual corresponding 15mL centrifuge tubes, and the centrifuge tubes containing the cells and cell culture medium were centrifuged at 400xg for 10 minutes.
6. After centrifugation, the supernatant was removed and the cell pellet was either re-lysed with 1mL of 1 XPBS solution or transferred to a 1.5mL centrifuge tube to form a cell suspension.
7. Centrifuge tubes containing the cell suspension at 400Xg for 5 minutes.
8. After centrifugation, the supernatant was removed from each tube and 100. mu.L of JC-1working reagent was added to each tube.
9. The cell pellet in each centrifuge tube was vortexed uniformly with JC-1working reagent and incubated for 15 minutes in the dark.
After 10.15 minutes, each tube was centrifuged at 400Xg for 5 minutes.
11. After centrifugation, the supernatant in each centrifuge tube was removed, the cell pellet in each tube was redissolved in 1mL of 1X PBS solution and centrifuged at 400xg for 5 minutes.
12. After centrifugation, the supernatant in each centrifuge tube was removed, the cell pellet in each centrifuge tube was redissolved in 1mL of 1X PBS solution and centrifuged at 400xg for 5 minutes.
13. After centrifugation, the supernatant was removed from each tube and the cells were resuspended in 500. mu.L of 1 XPBS to obtain the test cell fluid.
14. And measuring the membrane potential of the cell mitochondria in the fine liquid to be detected of each hole by using a flow cytometer so as to analyze the activity of the mitochondria. Since the experiment was conducted in duplicate, the results of duplicate experiments for each group were averaged to obtain an average value, and then the average value of the experimental group was converted to a relative JC-1 aggregation amount by taking the average value of the control group as a relative JC-1 aggregation amount of 100%, as shown in FIG. 6.
Results of the experiment
As can be seen from the results in FIG. 4, the amount of accumulation of the callus juice of rosemary in the experimental group was about 119.02% relative to JC-1. In other words, the mitochondrial activity of human dermal fibroblasts in the experimental group was increased (about 1.19-fold) compared to the control group. The rosemary callus juice can improve the activity of the mitochondria of the skin cells, thereby achieving the effect of improving the activity of the skin cells.
Example 7 human experiment-use of Rosemary callus juice in an external mode
Using samples
1. Essence liquid: the liquid mixture contains rosemary callus juice (hereinafter referred to as juice essence) and placebo (liquid mixture without rosemary callus juice).
Juice essence components: 0.2 wt% of xanthan gum, 0.6 wt% of jasminorange ketone, and a thickening agent U21 (type number)
Ultrez 21polymer)0.1 wt%, 1, 3-butanediol 5 wt%, triethanolamine 0.1 wt%, hexanediol 0.6 wt%, rosemary callus extract 1 wt%, and water in balance.
Placebo ingredients: 0.2 wt% of xanthan gum, 0.6 wt% of jasminorange ketone, and a thickening agent U21 (type number)
Ultrez 21polymer)0.1 wt%, 1, 3-butanediol 5 wt%, triethanolamine 0.1 wt%, hexanediol 0.6 wt%, and the balance water.
2. Face pack: a facial mask containing a juice essence (obtained by soaking a facial mask cloth in the juice essence to be fully absorbed, hereinafter referred to as essence facial mask) and a placebo facial mask (obtained by soaking a facial mask cloth of the same type in the placebo to be fully absorbed, hereinafter referred to as placebo facial mask) and then fully absorbing the facial masks. Here, each mask contained 20mL of the essence or placebo described in point 1.
The number of subjects: 8 subjects between 30-55 years of age.
Experimental mode
Instantaneous effect experiment: after the whole face of 8 subjects was cleaned, the measurement values of specific skin test items (i.e., skin red pigment and skin moisture content) of the left and right half faces before the mask was applied were recorded or photographs before the application were taken using corresponding instruments and measurement methods. Then, the juice mask and the placebo mask were applied to the right half face and the left half face of the subject, respectively, and after 15 minutes, the subject was removed, and then the left half face and the right half face were slightly rubbed with the fingers and the abdomen. After the facial mask is removed for about 5 minutes, measuring specific skin detection items or taking photos by using corresponding instruments and measuring modes, and comparing the photos with the numerical values of the original left half face or the original right half face.
And (3) long-acting experiment: after 8 subjects cleansed their faces in the morning and evening, the placebo and sap solutions were applied to the left and right half faces, respectively, and slightly rubbed on the abdomen to promote absorption, and the test was performed before use (i.e., before first application of the placebo and sap solutions, considered as week 0) and after continued use for 4 weeks (week 4).
Before and after use, when the skin temperature and humidity of a test area where a subject is located are required to be consistent, so that the influence of factors such as external temperature and humidity on the skin is reduced.
Detecting items
1. Skin red pigment detection
The skin red pigment was detected using a multifunctional skin tester (C + K Multi Probe Adapter System) purchased from Courage + Khazaka electronic, Germany, and equipped with
MX18 skin red pigment index detection probe. In the test process, a probe connected to a skin tester is contacted with a skin area to be tested of a subject, and the content of hemoglobin in the skin is determined by measuring the reflection amount of light with a specific wavelength irradiated on the skin of the human body. Higher measured values indicate higher levels of red pigment in the skin. And analyzing the test result by a software attached to the skin tester to obtain the skin red pigment content of the area to be tested.
2. Skin moisture content detection
The moisture content of the skin was measured using a multifunctional skin tester, available from Courage + Khazaka electronic, Germany, in combination with a moisture meter
CM 825 probe. During the test, the probe of the measuring instrument is contacted with the skin area to be measured of the subject, and the moisture content of the epidermal layer of the area to be measured is measured. The method adopts a capacitance method to test the moisture content of human skin, the principle is that the difference of dielectric constants of water and other substances is obvious, the capacitance value of the skin is different according to the difference of the moisture content of the skin, and the observation value can represent the moisture value of the skin.
3. Skin texture state detection
The detection of the texture state of the skin is performed by using a VISIA high-order digital skin quality detector (VISIA complex Analysis System) purchased from Canfieldscientific, USA, the facial skin before and after the essence is used is photographed by a high-resolution camera lens, and the texture position can be detected by standard white light irradiation and the change of the skin shadow can be detected, so that a value can be obtained, which represents the smoothness of the skin.
4. Skin speckle state detection
The detection of skin speckle state is carried out by taking pictures of facial skin before and after the essence is used with high-resolution camera lens, irradiating with standard white light, detecting macroscopic pigment speckles on skin, and analyzing the quantity and area with software.
Results of the experiment
As can be seen from fig. 5, in the transient test, the redness of the skin was improved immediately after the serum mask was used compared to the group using the placebo mask (the redness of the skin was reduced by about 6.4% compared to the group using the serum mask).
As can be seen from fig. 6, in the long-term experiment, the redness of the skin was improved compared to the group using the placebo after the juice essence was used (the redness of the skin was reduced by about 24.8% compared to the group using the placebo).
Therefore, the rosemary callus juice applied to the skin in an external mode can improve the condition of the skin redness.
As can be seen from fig. 7, in the transient test, the moisture content of the skin was increased immediately after the serum mask was used compared to the group using the placebo mask (the moisture content of the skin was increased by about 6.5% compared to the group using the serum mask). As can be seen from fig. 8, in the long-term experiment, the moisture content of the skin was increased after the juice essence was used compared to the group using the placebo (the moisture content of the skin was increased by about 8.8% compared to the group using the placebo). Therefore, the rosemary callus juice applied to the skin in an external mode can improve the moisture retention of the skin.
As can be seen from fig. 9, the skin texture was significantly improved by about 8.9% after the continuous use of the sap essence for 4 weeks, compared to before the use of the sap essence. Based on this, it was found that when the rosemary callus sap was applied to the skin in an external application, the skin texture was reduced and the smoothness of the skin was improved.
As can be seen from fig. 10, the spot condition of the skin was improved by about 5.7% after the continuous use of the sap essence for 4 weeks, compared to before the use of the sap essence. Based on this, it was found that when the rosemary callus sap was applied to the skin in an external form, skin spots could be surely reduced.
The experimental result shows that the rosemary callus juice can improve the skin condition so as to achieve the effect of effectively preventing skin aging.
In summary, the present application proves that the rosemary callus juice can inhibit the generation of free radicals and/or inhibit the generation of melanin, so that the rosemary callus juice can effectively prevent the skin aging phenomenon and maintain or improve the skin appearance. In addition, the present case also proves that the rosemary callus juice can improve the activity of mitochondria and the expression of moisturizing genes. Therefore, the rosemary callus juice can passively reduce the loss of skin fibroblasts influenced by free radicals, and can also actively improve the activity of mitochondria and the expression of moisturizing genes, so that the skin is healthier.
The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Dajiang biomedical corporation Ltd
<120> use of rosemary callus juice and culture medium for inducing rosemary callus
<130> NA
<150> US 62/886,404
<151> 2019-08-14
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
Claims (9)
1. Use of rosemary callus juice for the preparation of a composition for inhibiting the production of Reactive Oxygen Species (ROS).
2. The use of claim 1, wherein the composition is used to increase skin moisture, improve skin redness, improve skin texture, reduce skin blotchiness, or a combination thereof.
3. Use of Rosmarinus officinalis callus juice for preparing composition for promoting activity of mitochondria is provided.
4. Use of rosemary callus juice for preparing a composition for increasing expression of moisturizing genes, wherein the moisturizing genes are at least one of the following: KRT10(keratin 10) gene, KRT14(keratin 14) gene, KRT1(keratin 1) gene, AQP3(Aquaporin-3) gene, FLG (Flagellar basal-body rod protein) gene, SMPD1(Sphingomyelin phospholipid 1) gene and GBA (beta-Glucocerebrosidase) gene.
5. The use of any one of claims 1 to 4, wherein the composition is a pharmaceutical composition, a cosmetic composition or a food composition.
6. The use of any one of claims 1 to 4, wherein the composition is an oral composition.
7. The use as claimed in any one of claims 1 to 4, wherein the composition is a topical composition.
8. The use according to any one of claims 1-4, wherein the rosemary callus juice is obtained from a rosemary callus induced by a culture medium comprising potassium nitrate (KNO)3) Calcium chloride dihydrate (CaCl)2.2H2O), magnesium sulfate (MgSO)4.7H2O), potassium dihydrogen phosphate (KH)2PO4) 2, 4-dichlorophenoxyacetic acid, and 6-benzylaminopurine.
9. A culture medium for inducing herba Rosmarini officinalis callus comprises potassium nitrate (KNO)3) Calcium chloride dihydrate (CaCl)2.2H2O), magnesium sulfate (MgSO)4.7H2O), potassium dihydrogen phosphate (KH)2PO4) 2, 4-dichlorophenoxyacetic acid, and 6-benzylaminopurine.
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| US201962886404P | 2019-08-14 | 2019-08-14 | |
| US62/886,404 | 2019-08-14 |
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| CN201911264809.4A Pending CN112386655A (en) | 2019-08-14 | 2019-12-11 | Use of phalaenopsis amabilis extract for preparing composition for resisting saccharification and improving skin appearance |
| CN201911307950.8A Pending CN112386643A (en) | 2019-08-14 | 2019-12-18 | Application of Chinese violet extract in preparation of anti-aging composition |
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| CN113398186B (en) * | 2021-08-20 | 2021-11-02 | 山东中医药大学附属医院 | Viola yedoensis makino extract and preparation method and application thereof |
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| TWI772723B (en) | 2022-08-01 |
| CN112386534A (en) | 2021-02-23 |
| TW202106323A (en) | 2021-02-16 |
| TWI729618B (en) | 2021-06-01 |
| TW202106330A (en) | 2021-02-16 |
| CN112386643A (en) | 2021-02-23 |
| TWI819235B (en) | 2023-10-21 |
| CN112386665B (en) | 2022-05-06 |
| TW202106324A (en) | 2021-02-16 |
| CN112386655A (en) | 2021-02-23 |
| CN112386665A (en) | 2021-02-23 |
| TW202106329A (en) | 2021-02-16 |
| TW202106328A (en) | 2021-02-16 |
| TWI809299B (en) | 2023-07-21 |
| CN115227774A (en) | 2022-10-25 |
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