CN117797072A - Use of horseradish leaf juice for inhibiting formation of glycosylated end products - Google Patents
Use of horseradish leaf juice for inhibiting formation of glycosylated end products Download PDFInfo
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- CN117797072A CN117797072A CN202410006732.5A CN202410006732A CN117797072A CN 117797072 A CN117797072 A CN 117797072A CN 202410006732 A CN202410006732 A CN 202410006732A CN 117797072 A CN117797072 A CN 117797072A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- 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
- A61P17/10—Anti-acne agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Dermatology (AREA)
- Engineering & Computer Science (AREA)
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- Pharmacology & Pharmacy (AREA)
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Abstract
The present invention provides the use of horseradish leaf juice for a composition for inhibiting the formation of a glycosylated end product.
Description
The present application is a divisional application of the invention patent application with the Chinese national application number 202010909807.2, the application date 2022, the 09 month 02 and the invention name of "the application of horseradish leaf juice for improving skin".
Technical Field
The present disclosure relates to the use of horseradish leaf juice, and more particularly to the use of horseradish leaf for preparing a composition for inhibiting the formation of a glycosylated end product.
Background
The skin is of essential importance for the protection of human individuals, which provides a first-stage protection against environmental factors such as ultraviolet radiation in sunlight, pathogens, friction and the like. The skin comprises, in order from the outside to the inside, an epidermis layer, a dermis layer mainly composed of connective tissue, and subcutaneous tissue. The dermis layer contains sufficient molecules such as collagen (collagen), elastin (elastin), and hyaluronic acid (hyaluronic acid), so that young skin has better support and elasticity.
With age or environmental factors, the skin ages and develops various damaged conditions. For example, skin may be degraded due to collagen cleavage, resulting in skin elasticity, insufficient water retention (dry skin, no luster), large pores, wrinkles, etc.; and aging such as speckle and/or yellowish brown spot due to melanin precipitation.
In addition, substances that accelerate aging, glycosylation end Products (Advanced Glycation End-Products, AGE), may be formed due to the binding of excess sugar in the blood to proteins. The accumulation of AGEs in the body also causes the skin to assume an aged, damaged condition. Especially in modern life, people often ingest excessive sugar, and factors such as lack of exercise or slow metabolism cause skin aging to be more obvious and rapid.
Disclosure of Invention
In view of the above, it is desirable to develop a composition that can slow down skin aging and damage to maintain or improve the appearance of skin.
Accordingly, it is an object of the present invention to provide the use of horseradish leaf juice for the preparation of a composition for slowing down skin aging and altering skin conditions. Horseradish (wasabia japonica), also known as wasabi, is a plant belonging to the genus behenia of the family cruciferae. Horseradish has strong pungent taste, but its pungency is different from that of capsicum, and the pungency of horseradish stimulates tongue, but the pungency of horseradish stimulates paranasal sinuses. The horseradish leaf can be eaten, and has the pungency of horseradish stem. The food processing method of the horseradish leaf comprises pickling with salt vinegar sauce overnight to obtain horseradish salad, cooking with soy sauce and boiled water to obtain dish or sushi material for eating with rice or wine, or frying with starch slurry.
Specifically, the invention aims to provide the application of horseradish leaf juice in preparing a composition for inhibiting the generation of a glycosylated end product, increasing the moisture content of skin, improving the skin firmness, reducing skin wrinkles, inhibiting the generation of skin spots, inhibiting the generation of skin melanin and inhibiting the generation of acne.
In some embodiments, the composition is a pharmaceutical composition.
In some embodiments, the composition is further prepared as a cosmetic composition, a food composition, a nutraceutical composition, or a cosmetic composition.
In some embodiments, the composition is used to prepare a composition for inhibiting the formation of skin acne.
In some embodiments, the horseradish leaf juice is obtained by extracting a horseradish leaf with an aqueous solvent.
In some embodiments, the horseradish leaf juice is used to achieve the reduction of skin aging by reducing AGE formation.
In some embodiments, the horseradish leaf juice is effective to inhibit acne formation in the skin by modulating the expression of anti-inflammatory related genes.
In some embodiments, the horseradish leaf juice is at a concentration of at least 0.5mg/mL.
In some embodiments, the horseradish leaf juice can reduce the amount of water lost through the skin, increase the moisture content of the skin, increase the elasticity of the skin, increase the firmness of the skin, lighten the spots of the ultraviolet light of the skin, improve the texture of the skin, improve the pore state of the skin, lighten the wrinkles of the skin, reduce the protoplasmic purple of the skin, or any combination of the foregoing.
As previously described, excess sugar in the blood will bind to proteins, forming AGEs. The progressive accumulation of AGE can lead to inflammatory reactions that produce free radicals that attack the skin, causing skin-related proteins to harden, fibers to break, causing the skin to lose elasticity, dull yellow, wrinkles, roughness and severe sagging, and accelerating skin aging.
Meanwhile, when grease and old waste cells on the skin accumulate, bacteria can grow, and the skin is slightly inflamed. However, over time, the hair follicle wall of the skin is damaged, causing severe inflammation, which forms acne, or what is commonly known as "acne". An important ring in inflammatory reactions is pro-inflammatory cytokines, which slow down the inflammatory reaction and achieve anti-acne effects if they slow down the secretion.
On the other hand, the ultraviolet light and the blue light also induce melanocytes in the skin to produce melanin, so that the skin produces spots and/or is darker and darker overall, and the skin is also one of common phenomena of skin aging.
The horseradish leaf juice can inhibit the formation of AGE and the pre-inflammatory cell hormone, particularly inhibit the pre-inflammatory cell hormone and inhibit the generation of melanin by regulating and controlling the expression level of IL-8 genes, so that the horseradish leaf juice can effectively delay skin aging and inhibit the formation of skin acne, and can also achieve the effects of whitening and resisting various skin aging.
The invention will now be described in more detail with reference to the drawings and specific examples, which are not intended to limit the invention thereto.
Drawings
In the drawings, "p" means that the value of p is less than 0.05, "p" means that the value of p is less than 0.01, and "p" means that the value of p is less than 0.001. The more "x" the more, the more significant the statistical difference is represented.
FIG. 1 is a graph showing comparison of results of the ratio of the amount of the glycosylated end product to the amount of the glycosylated end product in the blank control group and the experimental group.
FIG. 2 is a graph showing comparison of the results of IL-8 gene expression ratios in the blank control group and the experimental group.
FIG. 3 is a graph showing comparison of IL-8 secretion ratio results of a blank control group, an ultraviolet-B (UVB) control group and an experimental group.
FIG. 4 is a graph showing comparison of the results of the ratio of melanin relative contents in the blank control group, the control group and the experimental group.
FIG. 5 is a graph comparing the results of the relative inhibition of tyrosine in the blank control group and the experimental group.
FIG. 6 is a graph showing the comparison of the improvement ratio of skin condition (rate of percutaneous moisture loss, skin moisture retention, skin elasticity, skin firmness, spots, UV spots, skin texture, skin pores, skin wrinkles, and protoplasmic purple (acne bacillus secretion)) before and after drinking a beverage containing the horseradish leaf juice of the present invention.
Detailed Description
Some specific embodiments of the present invention are described below. The present invention may be practiced in many different forms without departing from the spirit thereof, and the scope of protection should not be limited to the conditions specifically set forth in the specification.
Statistical analysis was performed using Excel software. Data are expressed as mean.+ -. Standard Deviation (SD), and differences between groups are analyzed by student's t-test (student's s t-test).
As used herein, the values are approximations, and all experimental data are expressed in the range of plus or minus 10%, and most preferably in the range of plus or minus 5%.
The horseradish is called Wasabia japonica, also called Wasabia japonica, and belongs to the genus Behenia of the family Brassicaceae.
In some embodiments, the horseradish leaf juice can be used to slow skin aging, inhibit skin acne formation, increase skin moisture content, improve skin firmness, reduce skin wrinkles, inhibit skin wrinkle formation, inhibit skin speckle formation, inhibit skin melanin formation. Therefore, the horseradish leaf juice can be used for preparing a composition for slowing down skin aging, inhibiting skin acne formation, increasing skin moisture content, improving skin firmness, reducing skin wrinkles, inhibiting skin wrinkle generation, inhibiting skin speckle generation and inhibiting skin melanin generation.
In some embodiments, horseradish leaf juice can inhibit the formation of tyrosinase by reducing AGE formation, modulating anti-inflammatory-related gene expression, modulating IL-8 gene expression, modulating MTF gene expression.
In some embodiments, the aforementioned composition may be a pharmaceutical composition, a cosmetic composition, a food composition, a nutraceutical composition.
The pharmaceutical composition may be formulated into a dosage form suitable for enteral (enteral), parenteral (parenteral) or topical (topicaly) administration using techniques well known to those skilled in the art. For example, it may be: injectables (injection) [ e.g., sterile aqueous solutions (sterile aqueous solution) or dispersions ], sterile powders (sterile powders), external preparations (external preparation), or the like.
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more of the following: emulsifying agents (emulsifiers), suspending agents (suspending agents), disintegrating agents (decomponents), disintegrating agents (disintegrating agent), dispersing agents (binders), excipients (excipients), stabilizers (stabilizing agent), chelating agents (chelating agents), diluents (diluents), gelling agents (gelling agents), preservatives (preservatives), wetting agents (wetting agents), lubricants (lubricants), absorption retarders (absorption delaying agent), liposomes (lipomes) and the like. The choice and amount of these carriers will be readily apparent to those skilled in the art.
In some embodiments, the pharmaceutically acceptable carrier may comprise one of the following solvents: water, normal saline (PBS), phosphate buffered saline (phosphate buffered saline, PBS), aqueous alcohol-containing solutions (aqueous solution containing alcohol), and any other suitable solvent.
In some embodiments, the pharmaceutical composition may be administered by any of the parenteral routes (parenteral routes) described below: subcutaneous injection (subcutaneous injection), intraepidermal injection (intraepidermal injection), intradermal injection (intradermal injection) and intralesional injection (intralesional injection).
In some embodiments, the pharmaceutical composition may be manufactured using techniques well known to those skilled in the art as an external preparation (externalpreparation) suitable for topical application to the skin. For example, it may be any of the following, but is not limited thereto: emulsions (emulsion), gels (gels), ointments (cream), creams (stream), patches, wipes (line), powders (powder), aerosols (aerosol), sprays (spray), emulsions (condition), emulsions (serum), pastes (paste), foams (foam), drops (drop), suspensions (suspension), ointments (salve), bandages (band).
In some embodiments, the external preparation is prepared by mixing the pharmaceutical composition with a base (base) as is well known to those skilled in the art.
In some embodiments, the substrate may comprise one or more of the following additives (additives): water, alcohols, glycols, hydrocarbons such as petroleum jelly, white petrolatum]Wax (wax) [ such as paraffin wax (Paraffin) and yellow wax (yellow wax)]Preservative (preserving agents), antioxidant (antioxidants), surfactant (surfactants), absorption enhancer (absorption enhancers), stabilizer (stabilizing agents), gelling agent (gelling agents) [ such as carbopol ]974P(/>974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethyl cellulose)]Active agents (active agents), humectants (humictants), odor absorbers (odor absorbers), fragrances (fragrances), pH adjusters (pH adjusting agents), chelating agents (chelating agents), emulsifiers (emulisifers), occlusive agents (occlusive agents), softeners (emollients), thickeners (thickenes), co-solvents (solubilizing agents), permeation enhancers (penetration enhancers), anti-oxidantsIrritants (anti-irritants), colorants (color), propellants (propellants), and the like. The choice and amounts of these additives are within the skill and routine skill of those skilled in the art.
In some embodiments, the cosmetic may include an acceptable adjuvant (acceptable adjuvant) that is widely used in the art of cosmetic manufacture. For example, the acceptable adjuvant may comprise one or more of the following: solvents, gelling agents, actives, preservatives, antioxidants, sequestering agents, chelating agents, surfactants, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and the quantity of the reagents can be properly adjusted according to actual requirements.
In some embodiments, the cosmetic may be manufactured in a form suitable for skin care (skin care) or make up (make up) using techniques well known to those skilled in the art, which may be any of the following, but is not limited thereto: aqueous solutions (aqueoussolutions), aqueous-alcoholic solutions (aqueoussolutions) or oily solutions (oil solutions), emulsions in the form of oil-in-water (oil-in-oil type), water-in-oil type (water-in-oil type) or complex forms, gels, ointments, creams, masks, patches (pack), wipes, powders, aerosols, sprays, emulsions, pastes, foams, dispersions, drops, mousses (mousse), sun oils (sunscreens), lotions (water), foundations (foundation), make-up removal products (makeup removerproducts), soaps (soap) and other body cleaning products (body cleansing products), etc.
In some embodiments, the cosmetic may also be combined with one or more of the following topical agents (external use agents) of known activity: whitening agents (whitening agents) [ such as retinoic acid (tretinoin), catechin (catechin), kojic acid, arbutin and vitamin C ], moisturizers, bactericides (bactericides), ultraviolet absorbers (ultraviolet absorbers), plant extracts (plant extracts) [ such as aloe vera extracts (aloe extracts) ], skin nutrients (skin nutraceuticals), anesthetics (anesthetics), anti-acne agents (anti-acne agents), antipruritics (antipruritics), analgesics (anangetics), anti-dermatitis agents (antidermatitis agents), anti-hyperkeratosis agents (antihyperkeratolytic agents), anti-dry skin agents (anti-dry skin agents), antiperspirants (antipsoriatic agents), anti-aging agents (anti-wrinkle agents) antiwrinkle agents), anti-seborrheic agents (antiseborrheic agents), wound healing agents (wod-skin agents), corticosteroids (corticoids) (cosmetics). The choice and amount of these external agents is within the skill and routine skill of those skilled in the art.
In some embodiments, the pharmaceutical composition may be formulated as a food supplement (food additive) with any edible material for ingestion by humans and non-human animals, either by conventional methods added at the time of raw material preparation or during the manufacture of the food product.
In some embodiments, the type of food product may be, but is not limited to: beverages (beverages), fermented foods (fermented foods), baked products (bakeryproducts), health foods (health foods) and dietary supplements (dietary supplements).
In some embodiments, the "horseradish leaf juice" may be obtained by extracting the leaves of horseradish with a solvent, which may be water or an aqueous solution. In some embodiments, the extraction is performed at 70℃to 100℃for 30 minutes. In some embodiments, the "horseradish leaf juice" is obtained by extracting horseradish leaves with a suitable solvent, squeezing and removing leaf residues and finer suspended substances, and concentrating the leaf residues to a certain concentration.
In some embodiments, the "horseradish leaf juice" may also be obtained by grinding, squeezing and removing leaf residue and finer suspended matter from the horseradish leaves, and concentrating the same to a certain concentration.
The experimental procedures in the following examples were carried out at room temperature (25.+ -. 5 ℃ C.) under normal pressure (1 atm), unless otherwise specified.
EXAMPLE 1 preparation of horseradish leaf juice
The leaves of horseradish (origin: tai mountain of Taiwan and Chengdu Yi county of Chengdu) are crushed (10 speed blender of Osterizer brand) and sieved with a sieve with 10 meshes (mesh), and the oversized particles are removed to obtain horseradish leaf powder. In other embodiments, the mesh size of the screen may be 8 mesh or 12 mesh.
Then, in the heating procedure, water is used as a solvent to be mixed with horseradish leaf powder in a weight ratio of 15:1, and extraction is carried out at 85+/-5 ℃ for about 30 minutes to form a first extract containing solids.
In other embodiments, the weight ratio of solvent to horseradish leaf may be 10:1-20:1, and the soaking temperature may be 70-100deg.C for 15-45 minutes. If the solvent is too small or the soaking time is too short, the extraction efficiency will be obviously reduced; if the soaking time is too long, the effective components in the juice may be degraded.
Concentrating the first extractive solution with a concentrator (brand/model: BUCHI-Rotavapor R-100) at 60deg.C+ -5deg.C under reduced pressure until the Brix value (degreees Brix) of the solution is 8+ -0.5, and stopping concentrating to obtain herba Hordeum Ardisiae leaf juice. In other embodiments, the concentration may be performed at 45℃to 70℃under reduced pressure.
EXAMPLE 2 detection of the amount of inhibition of collagen glycosylation by horseradish leaf juice
When proteins undergo glycosylation, various glycosylation end products (Advanced glycation end products, AGEs) are produced, which are non-reducible substances and alter and affect the normal function of the proteins, thereby accelerating skin aging. Therefore, if the collagen glycosylation can be inhibited, the loss speed of the collagen can be effectively reduced, and the effect of delaying skin aging is achieved. In order to confirm whether the horseradish leaf juice has the effect of inhibiting collagen glycosylation, the collagen glycosylation amount test was performed as follows.
Preparing a medicine:
using the horseradish leaf juice obtained in the previous example 1, phosphate-Buffered Saline (PBS) at a concentration of 200mM was used, with NaH 2 PO 3 (Honeywell，#04269)、Na 2 HPO 4 (Sigma, #V 900061) with water, pH of 7.4) as solvent to obtain horseradish leaf extract solution with concentration of 0.4mg/mL。
A collagen solution having a concentration of 60mg/mL was prepared using the aforementioned phosphate buffered saline having a concentration of 200mM and collagen powder (collagen was purchased from Rousselot, model # P2000 HD). Sodium azide was added to the collagen solution so that the collagen solution contained 0.06wt% sodium azide. (sodium azide was purchased from Sigma, #S2002)
A fructose solution at a concentration of 1.5M was prepared using phosphate buffered saline at a concentration of 200mM as described above with fructose (fructose was purchased from Sigma, #F0127).
Collagen glycosylation test:
the horseradish leaf extract solution (0.2 mL) was mixed with the collagen solution (0.2 mL) and the fructose solution (0.2 mL), respectively, to prepare a sample solution.
0.2mL of deionized water was mixed with 0.2mL of the collagen solution and 0.2mL of the fructose solution to prepare a blank solution as a control group.
The fluorescence intensities (excitation wavelength 360nm, emission wavelength 460 nm) of the sample solution and the blank solution were measured using a spectrofluorimeter (manufacturer/model: bioTek FLx 800) before the collagen glycosylation reaction was performed.
The sample solution and the blank solution were allowed to react at 50 ℃ for 24 hours to allow the solution to undergo collagen glycosylation.
The fluorescence intensity (excitation wavelength 360nm, emission wavelength 460 nm) of the reacted sample solution and the blank solution were measured by a spectrofluorimeter.
The relative rate of formation of protein glycosylation end products was calculated according to the following formula:
[ (sample fluorescence intensity) After the reaction Sample fluorescence intensity Before the reaction ) Control group fluorescence intensity After the reaction Control of group fluorescence intensity Before the reaction )]×100％
The relative rates of formation of protein glycosylation end products of the samples relative to the control group are shown in figure 1. Whereas, as shown in fig. 1, horseradish leaf juice has an effect of inhibiting the production of the glycation end products of collagen. Specifically, the relative rate of protein glycosylation end products was only 41% of the control group. The horseradish leaf juice can inhibit the glycosylation reaction of the collagen, reduce the generation of glycosylation end products, further reduce the loss of the collagen in the body and achieve the effect of slowing down the skin aging.
EXAMPLE 3 cell experiment-Horseradish leaf juice Regulation of IL-8 Gene expression
Here, the medium was VIVOTM 10Serum-free hematopoietic cell medium (available from Lonza, switzerland under the number BE 02-055Q). First, human umbilical vein endothelial cells (human umbilical vein endothelial Cells, HUVEC) (obtained from the center for the preservation of biological resources, BRBC accession No. H-UV 001) were subjected to experiments on the expression level of vascular endothelial cell IL-8 gene. First at 1X 10 per well 5 Cell mass was cultured in a six-well culture dish containing 2mL of the above culture solution and cultured at 37℃for 16 hours, and HUVEC cells were then divided into the following two groups: experimental and control groups.
Experimental group: a juice-containing culture broth was prepared in a ratio of 1mg of the horseradish leaf juice prepared in the manner of example 1 (i.e., at a concentration of 1 mg/ml) per ml of culture broth, and HUVEC cells were replaced with the juice-containing culture broth for continued culture.
Control group: no treatment was performed, i.e. no additional compound was added to the culture broth containing the HUVEC cells after cultivation.
After 48 hours of culture, the cells of the experimental group and the control group after culture are respectively disrupted by cell lysates to form two groups of cell solutions.
Next, RNA from the two sets of cell solutions was collected separately using an RNA extraction reagent kit (available from Genaid corporation, taiwan, china, lotNo. FC24015-G). Next, 2000 nanograms (ng) of each extracted RNA were taken as a template byIII reverse transcriptase (available from Invitrogene, U.S. Pat. No. 18080-051) reverse transcribed with the primers shown in Table I to give the corresponding cDNA. Subsequent use of the ABI StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific)Company, usa), and KAPA SYBR FAST (purchased from Sigma company, usa, no. 38220000000), the two sets of post-reverse transcription products were subjected to quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) with the combined primers of table one, respectively, to observe the expression amounts of genes of HUVEC cells of the experimental and control groups. The apparatus for quantitative real-time reverse transcription polymerase chain reaction was set to react at 95℃for 1 second, at 60℃for 20 seconds for a total of 40 cycles, and gene quantification was performed using the 2-DeltaCt method. In this case, the mRNA expression level of each gene can be indirectly quantified by quantitative real-time reverse transcription polymerase chain reaction using cDNA, and the expression level of the protein encoded by each gene can be estimated.
List one
The relative gene expression of each gene shown in the figures described below is presented at relative magnification, where standard deviation was calculated using the STDEV formula of Excel software and analyzed in Excel software for statistically significant differences by single Student t-test (Student t-test). In the drawings, "p" means that the value of p is less than 0.05, "p" means that the value of p is less than 0.01, and "p" means that the value of p is less than 0.001. The more "x" the more, the more significant the statistical difference is represented.
As shown in FIG. 2, when the expression level of the IL-8 gene in the control group was regarded as 1 (i.e., 100%), the expression level of the IL-8 gene in the experimental group was 0.258 (i.e., 25.8%) with respect to the control group, representing that the expression level of the IL-8 gene in the experimental group was 0.258 times that in the control group.
From this, it was found that, when HUVEC cells were treated with horseradish leaf juice, the gene expression level of IL-8 gene in HUVEC cells was decreased, which means that inflammatory reaction in cells was effectively inhibited, and thus the formation of acne was effectively inhibited, and an anti-acne effect was achieved.
EXAMPLE 4 cell assay-evaluation of the efficacy of horseradish leaf juice in anti-skin inflammation
Interleukin-8 (IL-8) is known to be a cytokine that has the function of promoting inflammatory responses. When an inflammatory response occurs, the inflamed tissue releases the cytokines to attract cells of a specific function to the site of inflammation. Meanwhile, when inflammatory reaction occurs, red swelling and even pain may occur at the site. Therefore, if the secretion amount of interleukin-8 can be suppressed, the inflammatory reaction can be alleviated, and the effect of suppressing skin acne can be achieved.
Here, the change of anti-inflammatory factor in human primary skin keratinocyte HPEK-50 after treatment with horseradish leaf juice was measured by ELISA kit of human CXCL8/IL8, UV irradiation chamber and enzyme immunoassay (ELISA reader).
Materials and instruments
Cell lines: human primary skin keratinocytes (Humanprimary epidermal keratinocytes) HPEKp (CELnTEC Co., switzerland, HPEK-50).
Culture medium: serum-free keratinocyte cell culture fluid (keratinocyte-SFM) (Gibco, USA, accession # 10724-011).
Ultraviolet ray irradiation room (Vilber company, product number BLX 254)
ELISA assay kit for human CXCL8/IL 8: available from R & D systems, model D8000CD8000C, this kit contains the following reagents:
(1) Capture antibody (capture antibody)
(2) Detecting antibody (detection antibody)
(3) Streptavidin-HRP (strepitavidin-HRP)
(4) Cleaning solution (Washingbuffer): is phosphate buffered saline (PBS solution)
(5) Substrate solution (TMB)
(6) Stop solution (stop solution)
Enzyme immunoassay (ELISA reader), available from BioTek company.
Substrate solution(R&D systems)。
H2SO4, available from Sigma-Aldrich company
Cell incubator, available from Astec Inc
Oscillator (shaker) available from GenePure Inc
Horseradish leaf juice: the horseradish leaf juice used in this experiment was obtained by the preparation method described above in example 1.
Experimental procedure
The experiments were performed in three groups of 1 control group, 1 UVB irradiation group and 1 experimental group, each of which was subjected to a three-fold test:
human primary skin keratinocytes were plated at 5X 10 per well 4 In a 24-well culture dish containing 0.5ml of medium per well.
The culture dish was placed in a cell incubator and after culturing at 37℃for 24 hours, the medium was removed.
The UVB irradiation group and the experimental group were subjected to an ultraviolet irradiation chamber at an intensity of 300mJ/cm2 to induce an inflammatory reaction, and 1mg/mL horseradish leaf juice sample was simultaneously added to the cells of the experimental group, while the cells of the control group were not subjected to any treatment (neither UVB nor horseradish leaf juice was added). The above-mentioned "sample of horseradish leaf juice" was prepared by diluting the horseradish leaf juice prepared in the manner of example 1 with a medium to prepare a solution containing 1mg of horseradish leaf juice per ml.
After each group of cells was cultured at 37℃for 24 hours, 120. Mu.L of the cell culture supernatant was taken as a sample, and IL-8 in these samples was analyzed using an ELISA assay kit for human CXCL8/IL 8.
First, the capture antibody (capture antibody) was diluted with PBS, and the diluted capture antibody was applied to the bottom of a 96-well microplate in an amount of 100 μl per well, and allowed to act overnight at 4 ℃.
Next, the 96-well microdisk was washed 3 times with 200. Mu.L of wash buffer (0.05% Tween 20 (Tween 20) in PBS).
Blocking was performed with 300. Mu.L of blocking buffer (blocking buffer) (1% BSA in PBS) followed by 3 hours at 37 ℃.
Thereafter, the 96-well microdisk was washed 3 times with 200. Mu.L of wash buffer.
100. Mu.L of sample and standard (placed in dilution (0.1% BSA in PBS)) was added, followed by binding to the capture antibody for 2 hours at 37 ℃.
Next, the 96-well microdisk was washed 3 times with 200. Mu.L of wash buffer.
Then 100. Mu.L of detection antibody (detection antibody) was added, followed by detection of the capture antibody at 37℃for 2 hours.
Thereafter, the 96-well microdisk was washed 3 times with 200. Mu.L of wash buffer.
100. Mu.L of Streptavidin-HRP (strepitavidin-HRP) was added and allowed to react at room temperature for 20 minutes.
Thereafter, the 96-well microdisk was washed 3 times with 200. Mu.L of wash buffer.
Then 100. Mu.L of the substrate solution (substrate solution) (R & D systems) was added thereto, and the mixture was allowed to act at room temperature for 20 minutes.
Next, 50. Mu.L of stop solution (stop solution) was added to stop the reaction, and finally the absorbance at 450nm was measured by an enzyme immunoassay. And then performing statistical analysis by Excel software.
The statistically significant differences between the groups were determined by the Shi Tudeng t-test (Student's t-test). The results of this example are shown in FIG. 3.
FIG. 3 is a graph of data showing the efficacy of horseradish leaf juice in anti-skin inflammation according to the present invention. As can be seen from FIG. 3, the IL-8 production was significantly increased in the UVB group compared to the control group, indicating that UVB was producing an inflammatory response to human primary skin keratinocytes. In contrast, the experimental group showed a 14.3% decrease in IL-8 production compared to the UVB group. The results of this example show that the horseradish leaf juice of the present invention has the effect of anti-skin inflammation, thereby achieving the effects of inhibiting the formation of skin acne and anti-acne.
EXAMPLE 5 cell experiment-Horseradish leaf juice reduced melanogenesis
Here, the change in melanin content of melanoma cell line B16F10 after the treatment with horseradish leaf juice was measured by ELISA plate reader (enzyme-linked immunosorbent assay reader).
Materials and instruments
Cell lines: mouse melanoma cell line B16F10 (ATCC CRL-6475) was purchased from American type culture Collection (American Type Culture Collection, ATCC).
Culture medium: dulbecco's modified minimal essential medium (DMEM, available from Gibco) was added with additional ingredients to contain 1vol% antibiotic solution (Antibiotic Antimycotic Solution, available from Gibco, 15240-062), 10vol% FBS (fetal bovine Serum, available from Gibco, 10437-028).
Phosphate buffered saline (PBS solution): purchased from Gibco under product numbers 10437-028.
A1N NaOH (purchased from Sigma, product number 221465) solution was prepared in double distilled water.
ELISA reader(BioTek，FLx 800)
Horseradish leaf juice: obtained in the manner of example 1.
Experimental procedure
The experiments were divided into experimental groups and blank control groups (no horseradish leaf juice added, two groups, three replicates were performed for each group:
B16F10 cells were plated at 1.5X10 cells per well 5 In this way, the cells were inoculated into 6-well plates containing 2ml of medium per well.
The culture dish was incubated at 37℃for 24 hours in 5% CO 2.
Then, the medium per well is removed without disturbing the adherent cells.
Experimental group: after 0.0225mL of horseradish leaf juice sample was added to each well, the culture was performed at 37℃for 48 hours. Wherein, the horseradish leaf juice-like strain was prepared by diluting the horseradish leaf juice prepared in the manner of example 1 with a medium to prepare a solution containing 1mg of horseradish leaf extract per ml.
Blank control group: 2ml of medium was added to each well at 37℃and allowed to incubate at 37℃for 48 hours.
Control group: 2ml of medium was added to each well at 37℃and allowed to incubate at 37℃for 48 hours.
Then, the medium was removed and the cells were washed 2 times with PBS solution.
200 μl Trypsin (Trypsin-EDTA (10X), available from Gibco; product No. 15400-054) was added to each well and reacted for 3 minutes. After the reaction, the reaction was terminated by adding 6mL of the medium solution. The suspended cells and medium in each well were then collected into corresponding 15ml centrifugation tubes, and each tube was centrifuged at 400Xg for 5 minutes to pellet the cells.
After washing the pelleted cells twice with PBS, the cells were resuspended in 200. Mu.L of PBS.
The cell suspension was frozen with liquid nitrogen for 10 minutes and left at room temperature for about 30 minutes to completely thaw.
After complete thawing, the tube was centrifuged at 12,000g for 3 min.
After removing the supernatant and re-suspending the cell pellet with 120. Mu.L of 1N sodium hydroxide solution, the tube was dry-bathed at 60℃for 1 hour to obtain a sample to be tested.
100. Mu.L of the sample to be tested was transferred to a 96-well plate, and the optical density (OD 450) of the cell solution at 450nm was measured as the absorbance using an ELISA plate reader.
Experimental results
The relative melanin expression in the experimental, blank, and control groups was calculated according to the following formula: melanin relative expression (%) = (OD 450 value of each group/OD 450 value of blank control group) ×100%. Since the experiment was performed in triplicate, the results of the triplicate experiments were averaged and are shown in FIG. 4.
As shown in fig. 4, the results of the blank control group and the control group revealed that the expression level of melanin in the cells increased significantly after the blue light irradiation, and it was revealed that the blue light irradiation did promote the production of melanin by melanoma cells, which resulted in the generation of spots on the skin or the darkened and sunken skin as a whole. On the other hand, as shown in the results of the control group and the experimental group, when the cells were treated with the horseradish leaf juice, the expression level of melanin under blue light was lower than that of the control group not treated with the horseradish leaf juice (about 21% reduction), showing that the horseradish leaf juice was effective in reducing melanin produced by the melanocytes due to blue light.
EXAMPLE 6 cell assay-Horseradish leaf juice inhibits formation of tyrosine
Preparing a 6-hole disc, and thenInto each well 2ml of culture solution [ Dulbecco's Modified Eagle's Medium (DMEM), 1% Penicillin-streptomycin (Gibco), 10% fetal bovine serum (Gibco) containing B16F10 cells (ATCC: CRL: 6475)]With 1.5x10 per well 5 The individual cells were incubated at 37℃for 24 hours.
Thereafter, the culture medium is carefully removed without disturbing the attached cells. 2ml of fresh DMEM medium was added to the control group (three wells), and 2ml of horseradish leaf extract sample at a concentration of 1mg/ml was added to the experimental group (three other wells) and reacted for 48 hours. The "horseradish leaf juice sample" was prepared by mixing the horseradish leaf juice prepared in the manner of example 1 with the diluted horseradish leaf extract as described above to prepare a solution containing 1mg of the diluted horseradish leaf extract per ml.
Next, after the broth was removed, it was washed twice with 1 XPBS (Gibco). trypsin-EDTA was added to the cells for 3 minutes, and the suspended cells were recovered in a 15ml centrifuge tube and centrifuged at 400×g for 5 minutes to pellet the cells.
Then, the pelleted cells were washed twice with 1 XPBS, suspended in 200. Mu.l of cell lysate, mixed with shaking, and centrifuged at 12,000Xg for 20 minutes.
The supernatant was taken out into a 1.5ml centrifuge tube, and the protein concentration was measured: A. mixing Bio-rad dye with deionized water (volume ratio is 1:4), and sub-packaging 500 μl into a microcentrifuge tube; B. to the microcentrifuge tubes described above, 10, 8, 6, 4, 2, 1 and 0. Mu.l of 2mg/ml BSA were added to prepare proteins of standard concentration; C. mu.l of test sample was added for detection. 200 μl of the test sample after the above reaction was taken out and the absorbance at 595nm was measured by ELISA reader (BioTek) in a 96-well plate.
Thereafter 400. Mu.g of protein was added to each well, followed by 90. Mu.l of cell lysate, including the control group. 10ul of 10M L-Dopa was added at 37℃under dark conditions and observed every 10 minutes until the suspension became black. Then, the absorbance at 405nm was measured.
The analytical formula of the content of the tyrosinase is as follows: tyrosinase inhibition (%) = (sample absorbance/control absorbance) ×100%. The obtained values were further statistically analyzed by Student t test using microsoft EXCEL software, and the results are shown in fig. 5.
As can be seen from the results of FIG. 5, the tyrosinase activity was reduced by about 24% in the case of the horseradish leaf juice according to the example of the present invention. Therefore, the horseradish leaf juice can indeed reduce the activity of tyrosinase and further reduce the generation of melanin, so that the horseradish leaf juice can be applied to the components of the related composition for reducing skin black spots and whitening skin.
EXAMPLE 7 human body experiment-oral administration of Horseradish leaf juice
Samples were used: the beverage containing the horseradish leaf juice of the invention is 50 g/bottle (the beverage prepared by water and the horseradish leaf juice contains 3g of horseradish leaf extract in every 50g, namely, the horseradish leaf extract accounts for 6 wt%). In still other embodiments, up to 5g of horseradish leaf juice can be ingested per day. Wherein the horseradish leaf juice is prepared by the method of example 1. The Brix value (Degrees Brix) was 8.
Number of subjects: 10 subjects aged 25-40.
The experimental mode is as follows: the subjects consumed one bottle of the beverage containing the horseradish leaf juice of the present invention (each bottle contains 3g of horseradish leaf juice) daily for 56 days (i.e., 8 weeks). Before drinking (the face is clean, the day 0) and after drinking for 56 days, the numerical value of the facial skin is recorded by using corresponding instruments and measuring modes according to different detection items, and photos before and after drinking are taken. (the temperature and humidity of the test area where the subject is located are consistent when testing before and after drinking, so as to reduce the influence of external factors such as temperature and humidity on the skin).
Detecting items: skin was subjected to 1. Skin moisture loss rate, 2. Skin moisture content, 3. Skin elasticity, 4. Skin firmness, 5. Skin spots, 6. Skin uv spots, 7. Skin texture, 8. Skin pores, 9. Skin wrinkles, 10. Protoplasmic purple (acne bacillus secretion), respectively.
1. Percutaneous moisture loss rate
Skin moisture loss assay using a skin moisture loss assay available from Courage+ Khazaka electronic, germanyProbe headTM 300 (C+ K Multi Probe Adapter System, germany) was tested. The detecting probe forms a relatively stable testing environment on the surface of the skin by using a cylindrical cavity with two open ends, and the water vapor pressure gradient of different two points is measured to calculate the water content evaporated through the epidermis, so as to measure the water loss condition of the surface of the skin.
2. Moisture content of skin
Skin moisture content detection probe available from Courage+ Khazaka electronic, germanyCM825 (C+ K Multi Probe Adapter System, germany) was tested. The detection probe is based on the principle of capacitance. When the moisture content changes, the capacitance value of the skin also changes, so that the moisture content of the skin surface can be analyzed by measuring the capacitance value of the skin.
3. Skin Elasticity (Elasticity) and 4. Firmness (Firmness)
Skin elasticity test probe available from Courage+ Khazaka electronic, germanyMPA580 (C+ K Multi Probe Adapter System, germany) was tested. The testing principle is based on suction and stretching principle, negative pressure is generated on the surface of the tested skin to suck the skin into a testing probe, the depth of the skin sucked into the probe is detected through an optical testing system, and the elasticity and compactness of the skin are calculated through software analysis.
5. Skin spots
The skin images with high resolution are photographed through visible light by using a VISIA high-order digital skin detector sold by Canfield, U.S.A., and the facial skin before and after drinking are compared, and the number and area of the pigment spots are analyzed by using built-in software according to the naked eyes. The higher the measurement value, the more visible specks are indicated.
6. Skin ultraviolet ray color spot
The detection was also performed using a ViSIA high-order digital skin detector sold by Canfield, U.S., which uses UV light for facial skin imaging. Ultraviolet rays can be absorbed by melanin to improve the appearance of pigment spots, and can detect the pigment spots of the epidermis layer which are invisible to naked eyes. The higher the measured value, the more uv spots.
7. Skin texture
Skin texture was also detected using a visual high-order digital skin detector sold by Canfield, usa, which uses visible light to capture high-resolution skin images and uses built-in software to analyze roughness according to the pits and projections of the skin. The higher the measurement value, the more rough the skin.
8. Skin pore state
The skin is detected by a VISIA high-order digital skin detector sold by Canfield, U.S.A., and the facial skin before and after drinking is photographed through a high-resolution camera lens. The shadow is generated in the concave position of the pore by the irradiation of standard white light, and the pore is darker than the surrounding skin color, so that the pore can be detected, then a numerical value is obtained by software according to the analysis of the number and the area of the pore, and the higher the numerical value is, the more the number and the area of the pore are displayed.
9. Skin wrinkles
The VISIA high-order digital skin detector sold by Canfield, U.S. is used for detection, the facial skin before and after drinking is photographed through a high-resolution camera lens, and the texture position can be detected and a value can be obtained by standard white light irradiation and detection of the change of skin shadow, so that the smoothness degree of the skin can be represented.
10. Protoplasmic (acne bacillus secretion)
The acnes will secrete ultraviolet light during growth, which will produce a fluorescent response upon ultraviolet irradiation, and thus can be detected using the VISIA high-order digital skin detector sold by Canfield, usa. The instrument photographs the facial skin with UV light and detects the skin purple content. The higher the measured value, the more vigorous the acne bacillus growth, and the easier the acne.
Please refer to fig. 6. The figure shows the average percentage improvement in skin condition of subjects after drinking the beverage containing the horseradish leaf juice for 8 weeks, with the improvement ratio accounting for 100% of the total subjects. Wherein, the transdermal moisture loss rate is improved by 12.9%, the skin moisture content is increased by 12.6%, the skin elasticity is increased by 5.1%, the skin compactness is increased by 7.4%, the skin spots are improved by 14%, the skin ultraviolet spots are improved by 16%, the skin texture is improved by 20%, the skin pore state is improved by 13%, the skin wrinkles are improved by 10%, and the skin protoplasmic purple (acne bacillus secretion) is improved by 16%. Therefore, the horseradish leaf juice disclosed by the invention has the effects of spot lightening, moisturizing and improving the skin fineness, and can help to inhibit the generation of acne.
In combination with the above, the present invention not only proves that the horseradish leaf juice can inhibit the formation of the glycosylated end product, effectively delay skin aging, but also can achieve the effects of anti-inflammation and acne formation by inhibiting the expression amount of IL-8 gene and the secretion amount of IL-8. Therefore, the horseradish leaf juice of the present invention has anti-glycation and anti-acne effects. Meanwhile, the proposal also proves that the horseradish leaf juice can inhibit the tyrosine enzyme, thereby achieving the effects of whitening and inhibiting melanin formation. In the aspect of human body experiments, the proposal proves that the horseradish leaf juice can effectively improve the aging state of a plurality of skins, such as reducing the water loss through the skins, improving the moisture content of the skins, improving the elasticity and the firmness of the skins, reducing the spots of the skins, lightening the spots of the skins, improving the skin texture, improving the pore state of the skins, reducing the wrinkles of the skins and reducing the protoplasmic purple of the skins.
Of course, the present invention is capable of other various embodiments and its several details are capable of modification and variation in light of the present invention by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (12)
1. Use of horseradish leaf juice for the preparation of a composition for inhibiting the formation of a glycation end product, characterized in that said horseradish leaf juice is obtained by extraction of a horseradish leaf with an aqueous solvent.
2. The use of claim 1, wherein the inhibition of glycosylation end product production is inhibition of collagen glycosylation end product production.
3. The use of claim 2, wherein the inhibition of collagen glycosylation end product production is achieved by inhibition of collagen glycosylation reactions.
4. A use according to any one of claims 1 to 3, wherein the aqueous solvent is water.
5. The use according to any one of claims 1 to 3, characterized in that the liquid-to-solid ratio of the aqueous solvent to the horseradish leaf is between 10:1 and 20:1.
6. The use according to claim 5, wherein the liquid-to-solid ratio of the aqueous solvent to the horseradish leaf is 15:1.
7. The use according to any one of claims 1 to 3, wherein the extraction is carried out at a temperature of 70 ℃ to 100 ℃ for 15 to 40 minutes.
8. The use according to claim 7, wherein the extraction is carried out at 80-90 ℃.
9. The use according to any one of claims 1 to 3, wherein the horseradish leaf juice is formulated at a concentration of 0.4mg/mL in phosphate buffered physiological saline at a concentration of 200 mM.
10. The use according to claim 9, wherein the phosphate buffered saline comprises NaH 2 PO 3 、Na 2 HPO 4 And water, wherein the pH value of the phosphate buffer physiological saline is 7.4.
11. The use according to any one of claims 1 to 3, wherein the brix value of the horseradish leaf juice is between 7.5 and 8.5.
12. The use according to any one of claims 1 to 3, wherein the composition is a food composition, a care product composition, a health food composition, or a cosmetic composition.
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