CN112043734A - Use of acerola cherry young fruit extract for regulating body weight, beautifying skin, resisting inflammation and resisting aging - Google Patents
Use of acerola cherry young fruit extract for regulating body weight, beautifying skin, resisting inflammation and resisting aging Download PDFInfo
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- CN112043734A CN112043734A CN201911413101.0A CN201911413101A CN112043734A CN 112043734 A CN112043734 A CN 112043734A CN 201911413101 A CN201911413101 A CN 201911413101A CN 112043734 A CN112043734 A CN 112043734A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
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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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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
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- 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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- Cosmetics (AREA)
Abstract
The invention relates to the field of plant extracts, in particular to application of an extract of acerola young fruit in weight regulation, muscle beautifying, inflammation resistance and ageing resistance. The invention discloses application of an extract of acerola young fruit to preparing a composition for regulating weight, beautifying skin, resisting inflammation and resisting aging, wherein the extract of the acerola young fruit is prepared by extracting the acerola young fruit by using water, alcohols, hydrous alcohols or a combination of the water, the alcohols and the hydrous alcohols as an extraction solvent.
Description
Technical Field
The invention relates to the field of plant extracts, in particular to application of an extract of acerola young fruit in weight regulation, muscle beautifying, inflammation resistance and ageing resistance.
Background
The skin tissue is composed of epidermis, dermis and hypodermis, wherein the dermis contains a large amount of collagen and hyaluronic acid, and is closely related to the water retention and elasticity of the skin. Human skin can have the phenomena of aging, rough skin or wrinkle generation and the like along with age, physiological factors or environmental factors, for example, the skin of normal young people has certain elasticity and tension, and when the expression muscle is relaxed, the skin can be quickly restored to eliminate the wrinkle; but in the middle age, the skin begins to age obviously, becomes thin, hard, dry and has reduced tension; dermal collagen is reduced, elastic fibers are denatured and broken, so that the tension and elasticity of the skin are reduced, and therefore, the skin cannot recover quickly after the expression muscles are relaxed, and wrinkles are formed after the skin relaxes for a long time; and with the increase of age, the skin and subcutaneous tissues are more relaxed, and in addition, the atrophy or the loss of the facial supporting tissues and the softness of muscles, the skin slides and falls under the action of gravity to form deeper wrinkles. The rough skin is a skin trouble caused by an external important factor such as dryness, ultraviolet rays, an irritant substance such as a detergent or a chemical substance, or an internal important factor such as disturbance of hormone balance, and is accompanied by phenomena such as reduction of barrier function of stratum corneum, reduction of moisture content of stratum corneum, acceleration of epidermal turnover, and roughening of cutin caused by generation of scales. Therefore, if the cells on the skin lose their elasticity and moisture-retaining function, they may cause wrinkles, dryness and loss of luster.
In another aspect. The skin is the largest barrier to protect human individuals and it has the function of combating moisture loss, pathogenic bacteria and various environmental insults. Exposure to high levels of Ultraviolet (UV), ionizing radiation, drugs or xenobiotics (xenobiotics) can cause the skin to generate Reactive Oxygen Species (ROS) and free radicals (free radials). Oxidative stress (oxidative stress) is formed when the number of reactive oxygen species and free radicals accumulated exceeds the antioxidant capacity of the cells or tissues themselves. Subsequently, the active oxygen molecules and free radicals react with intracellular compositions (including DNA, proteins, lipids, etc.), thereby causing undesirable effects on the skin.
Melanogenesis (i.e., melanin synthesis) refers to a process in which tyrosine (tyrosine) in melanocytes is converted into melanin (melanin) through catalysis of tyrosinase (tyrosine), which is a rate-limiting step of melanin production, and a series of redox reactions, after the melanocytes of skin (dermal melanophore) are induced by environmental factors such as Ultraviolet (UV) or physiological factors such as fatigue (fatigue), stress (stress), chronic inflammation (chronic inflammation), and abnormal α -melanocyte stimulating hormone (α -MSH) release in vivo. Melanin can protect the hypodermis (hypodermis) of the skin from light damage caused by ultraviolet rays (photodemage), but skin diseases (skin disorders) such as freckles (lentigines), spots (freckle), melasma (melasma), age spots (age spots), hyperpigmentation (hyperpigmentation), and the like may be caused when melanin is accumulated on the skin in a large amount or is abnormally distributed.
For skin whitening (skin whitening) and skin beautifying purposes, many melanogenesis inhibitors (melanogenesis inhibitors) have been used to lighten or remove melanin or black spots accumulated on the skin, and these melanogenesis inhibitors mostly regulate the production of melanin through the following mechanisms of action: (1) prior to melanin production: for example, inhibition of mRNA transcription (mRNA transcription) of tyrosinase (such as C2-ceramide (C2-ceramide), retinoic acid (tretinoin) and vanillic acid (vanillic acid)) and glycosylation (such as calcium D-pantethine-S-sulfonate (PasSO 3 Ca)); (2) during melanin production: for example, inhibition of tyrosinase activity (such as hydroquinone, arbutin, and kojic acid), acceleration of tyrosinase degradation (such as linoleic acid and phenylthiourea), and promotion of dopaquinone reduction (reduction) such as ascorbic acid (ascorbyl acid); and (3) after melanin production: for example, promoting melanin breakdown (such as linoleic acid), inhibiting melanosome transport (such as nicotinamide (Niacinamide) and serine protease inhibitors (serine protease inhibitors), and accelerating skin turnover (such as liquiritin (liquiritin) and glycolic acid (glycolic acid)).
Fat is an essential component in the human body, but excessive fat components cause damage to the human body. Obesity causes chronic inflammation, promotes an inflammatory response in the body, induces the transformation of adipocytes into insulin-resistant cells, and thus causes hyperglycemia, hypercholesterolemia, hypertension, and abnormal fat metabolism. Obesity is therefore a problem that is currently urgently needed to be solved.
In addition, glycosylation (glycation) is associated with skin and aging in vivo, and is also associated with diabetes which is a serious health hazard to people in China. The nature of the glycosylation reaction is known as the Maillard reaction (Maillard reaction) or carbonyl-amine browning reaction (carbonyl-amine browning), which is a non-enzymatic glycosylation reaction between the aldehyde (ketone) group of a sugar and the amine group of an amine group-containing substance (e.g., a protein, peptide, amino acid, phospholipid, nucleic acid, or derivative thereof). Chronic hyperglycemia causes glucose autooxidation and protein glycation to form highly glycosylated end products (AGEs). AGEs can cause various aging diseases such as skin wrinkles, cataract, atherosclerosis, kidney failure, etc.
When the carbohydrate for human consumption is starch, either linear or branched starches are rapidly broken down by alpha-amylase in the salivary and pancreatic glands. Amylases are lytic enzymes that hydrolyze alpha-1, 4-glucosidic bonds linked to double-stranded glucose and are classified into alpha-amylases and beta-amylases, depending on the mode of action. Alpha-amylase is an enzyme that hydrolyzes a large number of alpha-chain and alpha-chain linked polysaccharides (such as starch, glycogen) to produce glucose and maltose; alpha-amylases are found in humans and other mammals in the form of amylases (saliva, pancreas, etc.), and are also found in starch-containing seeds for storage as a food, and are secreted by many microorganisms.
Since alpha-amylase converts starch into saccharides, which in turn leads to an increase in blood glucose concentration, researchers in the field have begun to apply alpha-amylase inhibitors to the treatment of diabetes and metabolic syndrome.
Leptin is an important factor for regulating body weight, and can suppress appetite and fat hyperplasia, increase basal metabolic rate, and control body fat in a proper range. Many people with long-term obesity have the problem of insufficient secretion of lean voxels, are difficult to lose weight and are easy to regain weight.
However, the pharmaceutical, food or health product for solving the above problems (including weight control, skin care, anti-inflammation and anti-aging) is mostly made of chemical components, and is not beneficial to human health after long-term use, and the products are often expensive and not affordable for general users. In order to solve the above problems, those skilled in the art need to develop novel pharmaceuticals, food products or health products with the effects of regulating body weight, beautifying skin, anti-inflammation and anti-aging to benefit the broad population in need.
Disclosure of Invention
Accordingly, the present invention is directed to the use of an extract of acerola (Malpighia glabra) young fruit for preparing a composition for regulating body weight, beautifying skin, anti-inflammation and anti-aging, wherein the extract of acerola young fruit is prepared by extracting the acerola young fruit with water, alcohols, aqueous alcohols or a combination thereof as an extraction solvent.
In an embodiment of the present invention, the volume ratio of the acerola young fruit to the extraction solvent is between 1-5: 5 to 20.
In one embodiment of the present invention, the acerola cherry extract is present in an effective concentration of at least 0.0625 mg/mL.
In one embodiment of the present invention, the controlling of body weight comprises inhibiting alpha-amylase (alpha-amylase) activity, inhibiting lipase (lipase) activity, and increasing leptin (leptin) content.
In one embodiment of the present invention, the skin care product includes inhibiting melanogenesis in the skin, increasing collagen content in the skin, and increasing elastin content in the skin.
In one embodiment of the present invention, the skin melanin production is skin melanin production induced by blue light damage.
In one embodiment of the present invention, the anti-inflammation is inhibition of adipocyte-induced chronic inflammation.
In one embodiment of the present invention, the aging resistance is to reduce Reactive Oxygen Species (ROS) expression.
In an embodiment of the present invention, the acerola young fruit is 25 to 35 days after the acerola blooms.
In one embodiment of the present invention, the composition is a pharmaceutical, a food product or a nutraceutical.
In conclusion, the extract of the acerola young fruit of the present invention has the following effects: the effects of regulating body weight, beautifying muscles, resisting inflammation and resisting aging can be achieved by inhibiting the activity of alpha-amylase (alpha-amylase), inhibiting the activity of lipase (lipase), increasing the content of leptin (leptin), inhibiting the generation of skin melanin induced by blue light injury, increasing the content of collagen of skin, increasing the content of elastin of skin, inhibiting chronic inflammation caused by fat cells and reducing the expression of Reactive Oxygen Species (ROS). On the other hand, the acerola cherry young fruit extract can effectively prevent three highs and is suitable for developing products for beautifying muscles and managing weight.
The following examples are presented to illustrate the present invention and are not to be construed as limiting the scope of the invention, which is intended to be limited only by the appended claims.
Drawings
FIG. 1 is a data graph showing the measurement of the proanthocyanidin content of the extract of acerola cherry young fruit of the present invention;
FIG. 2 is a data graph showing the measurement of the flavone content of the extract of the acerola young fruit of the present invention;
FIG. 3 is a graph of data showing the efficacy of an extract of acerola young fruit of the present invention in inhibiting alpha-amylase activity;
FIG. 4 is a graph of data showing the efficacy of an extract of acerola young fruit of the present invention in inhibiting lipase activity;
FIG. 5 is a data plot of the efficacy of an extract of acerola young fruit of the present invention in enhancing lean voxel content;
FIG. 6 is a graph of data on the efficacy of acerola cherry young fruit extracts of the present invention in reducing AGEs-induced expression of ROS, wherein p is < 0.001;
FIG. 7 is a graph of data showing the efficacy of an extract of acerola young fruit of the present invention in inhibiting skin melanin production induced by blue light injury, wherein p is < 0.001;
fig. 8 is a data plot of the effectiveness of the acerola young fruit extract of the present invention in increasing the collagen content of the skin, wherein p represents < 0.01;
fig. 9 is a data plot of the effect of an extract of acerola young fruits of the present invention on increasing the elastin content of the skin, wherein p represents < 0.05;
fig. 10 is a graph of data on the efficacy of the acerola young fruit extract of the present invention in inhibiting adipocyte-induced chronic inflammation, wherein p is < 0.05; denotes p < 0.01.
Detailed Description
Definition of
As used herein, the numerical values are approximations and all numerical data are reported to be within the 20 percent range, preferably within the 10 percent range, and most preferably within the 5 percent range.
Statistical analysis was performed using Excel software. Data are presented as mean ± Standard Deviation (SD), and differences between these were analyzed by the stuttering t-test (student's t-test).
According to the present invention, acerola (Malpighia gladra) is a small tree of the genus Malpighiaceae (Malpighiaceae) Saxifraga (Malpighia), which is tropical in origin and edible in fruit. Also called as "Liangyejin Huwei". Acerola is native to south america, south of mexico, puerto rico, dominica, seashore, brazil, middle america, etc., but is now beginning to be grown in tropical regions of texas and asia as well. Acerola is known to contain abundant vitamin C and can be propagated by seed, cutting, etc. The growing environment favors dry sandy soil and full sunlight.
As used herein, the term "acerola cherry young fruit (Malpighia glabra fruitlet)" means a fruit 25 to 35 days after flowering of acerola cherry.
According to the present invention, the extract of the acerola young fruit may be prepared using fresh acerola young fruit, or may be prepared using acerola young fruit which has been previously subjected to a process selected from the group consisting of: drying, grinding, shredding, and combinations thereof.
As used herein, the term "anti-aging" means preventing, slowing down the aging phenomena of the human skin appearance, such as: the generation of wrinkles and loss of elasticity. The degree of evaluation to achieve this will be determined by a number of factors known to those skilled in the art, such as the general state of the consumer, age, sex, and the like.
As used herein, the terms "inhibition of melanin production" and "inhibition of melanin synthesis", "depigmentation", "lightening of melanin", "lightening of skin color", "bleaching", "whitening", "darkening", and "darkening" may be used interchangeably.
In accordance with the present invention, the drug may be manufactured using techniques well known to those skilled in the art into a dosage form suitable for parenteral (parenteral), oral (oral) or topical (topically) administration, including, but not limited to: injections (injections) [ for example, sterile aqueous solution (sterile aqueous solution) or dispersion (dispersion) ], sterile powder (sterile powder), troche (tablet), tablet (troche), buccal tablet (dosage), pill (pill), capsule (capsule), dispersible powder (dispersible powder) or granule (granule), solution, suspension (suspension), emulsion (emulsion), syrup (syrup), elixir (elixir), syrup (slurry), external preparation (external preparation), and the like.
According to the present invention, the pharmaceutical may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvents (solvent), buffers (buffer), emulsifiers (emulsifying), suspending agents (suspending agent), disintegrating agents (disintegrant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients (excipient), stabilizers (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving), wetting agents (wetting agent), lubricants (lubricating), absorption delaying agents (absorption delaying agent), liposomes (liposome) and the like. The selection and amounts of such agents are within the skill and routine skill of those skilled in the art.
According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline (normal saline), Phosphate Buffered Saline (PBS), aqueous alcohol-containing solutions (aqueous solution linking alcohol), and combinations thereof.
According to the invention, the medicament may be administered by a parenteral route (parenteral routes) selected from the group consisting of: intraperitoneal injection (intraperitoneal injection), subcutaneous injection (subcutaneous injection), intraepidermal injection (intraepithelial injection), intradermal injection (intraepithelial injection), intramuscular injection (intramucosal injection), intravenous injection (intravenous injection) and intralesional injection (intrafocal injection).
According to the present invention, pharmaceuticals 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, including, but not limited to: 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).
According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base (base) as well known to those skilled in the art.
According to the invention, the substrate may comprise one or more additives (additives) selected from the following group: water, alcohols, glycols, hydrocarbons such as petroleum jelly (jelly), white petrolatum (white) petrolatum)]Wax (wax) [ such as Paraffin and yellow wax (yellow wax)]Preservatives (preserving agents), antioxidants (antioxidants), surfactants (surfactants), absorption enhancers (absorption enhancers), stabilizers (stabilizing agents), gelling agents (gelling agents) (, ("gelling agents"), ")
974P(
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.
According to the present invention, the care product may further comprise an acceptable adjuvant (acceptable adjuvant) which is widely used in the art of care product manufacture. For example, the acceptable adjuvant may comprise one or more agents selected from the group consisting of: solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, colouring agents, thickening agents, fillers, fragrances and odour absorbers. The selection and amounts of such agents are within the skill and routine skill of those skilled in the art.
In accordance with the present invention, the cosmetic may be manufactured in a form suitable for skin care (skincare) or makeup (makeup) using techniques well known to those skilled in the art, including, but not limited to: aqueous solutions (aqueous solutions), aqueous-alcoholic solutions (aqueous-alcoholic solutions) or 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, wipes, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, mousses (mousses), sunblocks, lotions (toiletries), foundations (foundations), make-up removal products (make-up removal products), soaps (soaps) and other body cleansing products (body cleansing products), and the like.
In accordance with the present invention, the cosmetic may also be used in combination with one or more known active topical agents (external use agents) selected from the following: whitening agents (whitening agents) [ such as retinoic acid (tretinoin), catechins (catechin), kojic acid, arbutin and vitamin C ], moisturizers, anti-inflammatory agents (anti-inflammatory agents), bactericides (bacteriodes), ultraviolet absorbers (ultraviolets absorbers), plant extracts [ such as aloe vera extract (aloe vera extract) ], skin nutrients (skin nutrients), anesthetics (anesthesics), anti-acne agents (anti-acne agents), antipruritics (antipruritics), analgesics (analgesics), anti-dermatitis agents (antipermatitis agents), anti-hyperkeratotic agents (anti-hypercholesterolitic agents), anti-dry skin agents (anti-dry skin agents), anti-perspirants (anti-perspirant agents), anti-aging agents (anti-aging agents), anti-wrinkle agents (anti-rinking agents), anti-seborrheic agents (anti-anerrheic agents), wound healing agents (wound-healing agents), corticosteroids (corticosteriods), and hormones (hormones). The selection and amounts of such agents for external use are within the skill and routine skill of those skilled in the art.
According to the present invention, the food product may be used as a food additive (food additive) to be added during the preparation of the raw material or during the preparation of the food by conventional methods, and formulated with any edible material into a food product for ingestion by humans and non-human animals.
According to the present invention, the types of food products include, but are not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products), health foods (health foods) and dietary supplements (dietary supplements).
Example 1 preparation of extract of acerola (Malpighia glabra) young fruit
Firstly, cutting fruits of acerola (Malpighia glabra) (from Brazil) 25-35 days after flowering (namely acerola young fruits) into 12mm, homogenizing, and extracting the homogenized acerola young fruits by using water, alcohols, hydrous alcohols or a combination thereof as an extraction solvent, wherein the extraction temperature is 75-95 ℃, and the volume ratio of the extraction solvent to the acerola young fruits is 5-20: 1 to 5. And centrifuging the obtained product, taking the centrifuged supernatant to obtain a filtrate, and concentrating the filtrate at 50-60 ℃ under reduced pressure to obtain a concentrated product. Thereafter, the concentrated product is spray-dried to obtain an extract of the acerola young fruit of the present invention.
Example 2 measurement of proanthocyanidin and flavone contents of extract from acerola cherry young fruit
The analysis method for detecting the content of procyanidin (OPC) is mainly referred to and modified by the method described in the science of Total phenols with Phosphomopbdic-Phosphomechanical acids Reagents, V.L.Singleton, Joseph A.Rossi Am J Enol IC, January 1965,16: 144-. Taking 0.1mL of the appropriately diluted sample, adding 0.6mL of 4% vanillin methanol solution, adding 0.3mL of concentrated HCl, uniformly mixing, carrying out a light-shielding reaction at room temperature for 15 minutes, and then measuring the light absorption value at 500nm by using an ELISA Reader (ELISA Reader). The control group was replaced by an equal amount of deionized water, and 100, 200, 300, 400, 500, 600, 700 and 800 μ g/mL solutions of catechin (catechin) in methanol were prepared, and a synchronization test was performed and a standard curve was drawn as a quantitative reference. In this example, the extract of the acerola young fruit of the present invention was used as an experimental group, and the extract of the acerola mature fruit of the present invention was used as a comparative group, wherein the preparation method of the extract of the acerola mature fruit was substantially the same as that of the extract of the acerola young fruit of the present invention, except that: the mature acerola fruit is used to replace the young acerola fruit. The results of this example are shown in FIG. 1.
FIG. 1 is a data graph showing the measurement of the proanthocyanidin content of the extract of acerola young fruit of the present invention. As can be seen from fig. 1, the procyanidin content of the experimental group was significantly increased compared to the comparative group, wherein the procyanidin content of the experimental group was increased by 13.3 times compared to the comparative group. The experimental results show that the extract of acerola young fruit of the present invention releases a large amount of proanthocyanidin.
In addition, the assay for the detection of flavone content was mainly modified as described in Zhishen Jia et al, (1999), Food Chemistry,64:555-2O, after mixing well, 200. mu.L of 5% Sodium citrate (Sodium nitrate) was added, and after standing for 6 minutes, 200. mu.L of 10% Aluminum nitrate (Aluminum nitrate) was added, and then the mixture was allowed to stand for 6 minutes. Finally, 2mL of 4% Sodium hydroxide (Sodium hydroxide) and 1.4mL of H were added2And O, mixing uniformly, analyzing at 500nm, and drawing a standard curve by taking Rutin (Rutin) as a standard substance. In this example, the extract of the acerola young fruit of the present invention was used as an experimental group, and the extract of the acerola mature fruit of the present invention was used as a comparative group, wherein the preparation method of the extract of the acerola mature fruit was substantially the same as that of the extract of the acerola young fruit of the present invention, except that: the mature acerola fruit is used to replace the young acerola fruit. The results of this example are shown in FIG. 2.
FIG. 2 is a data graph showing the measurement of the flavone content of the extract of the acerola young fruit of the present invention. As can be seen from fig. 2, the flavone content in the experimental group was significantly increased compared to the comparative group, wherein the flavone content in the experimental group was increased 2.34 times compared to the comparative group. The experimental results show that the extract of the acerola young fruit of the present invention releases a large amount of flavone. Since the extract of the acerola cherry young fruit has the highest content of proanthocyanidins and flavonoids, the extract of the acerola cherry young fruit was used for the subsequent experiments.
Example 3 evaluation of the effectiveness of extracts of acerola young fruits in inhibiting the activity of alpha-amylase
The experimental method is mainly carried out by referring to the method E.Apostolidis et al, (2007), Innovative Food Science & embedding Technologies,8: 46-54. First, the following experimental materials were prepared: 0.02M sodium phosphate buffer (containing 6mM sodium chloride, pH 6.9), 2N sodium hydroxide, dinitrosalicylic acid (dinitosalicylic acid) coloring reagent (also known as terminator), 1% soluble starch (starch) and alpha-amylase (alpha-amylase).
The dinitrosalicylic acid coloring reagent is prepared in the following way: 1g of 3, 5-dinitrosalicylic acid is added to 50mL of deionized water, 30g of potassium sodium tartrate (sodium potassium tartrate) is slowly added, and 20mL of 2N sodium hydroxide is added, and the amount of deionized water is adjusted to 100 mL. The product is stored in carbon dioxide, and the storage life is limited to two weeks.
The 1% soluble starch was prepared as follows: 1g of soluble starch was dissolved in 100mL of 0.02M sodium phosphate buffer, and the solution was slowly heated to completely dissolve the starch, and then the temperature was lowered to room temperature, and the amount was determined to 100 mL. The samples were kept at room temperature for at least 4 to 5 minutes before analysis.
Alpha-amylase (5units/mL) was prepared as follows: alpha-amylase (Sigma Chemical Co., USA) is dissolved in 0.02M sodium phosphate buffer, the solid activity is 10-30 units/mg, 500KU has 50g of alpha-amylase solid per mg of 100units, 5units/mL of alpha-amylase can be prepared as 100L, and the alpha-amylase is stored in ice bath or at 4 ℃.
Next, a 0.02M sodium phosphate buffer (containing 6mM sodium chloride, pH 6.9) was used as a control group, and 2.5 wt% of acerola cherry extract was added as an experimental group, and the experimental group and the control group had a 0-minute reaction group and a 10-minute reaction group, respectively.
Then, preparing an experimental group and a control group, respectively taking 200 mu L of the experimental group and the control group, placing the two groups of the experimental group and the control group in a microcentrifuge tube, and respectively taking one tube of the two groups of the microcentrifuge tube as a 0-minute reaction group and the other tube of the microcentrifuge tube as a 10-minute reaction group; adding 200 μ L of alpha-amylase (5units/mL) dissolved in sodium phosphate buffer solution into each group, mixing well, and culturing at 25 deg.C for 10 min to allow enzyme to act; in the 0 minute reaction group, firstly adding a dinitrosalicylic acid coloring reagent (used as a terminator), and then adding 200 mu L of 1% soluble starch to avoid continuous reaction; adding 200 mu L of 1% soluble starch into a reaction group for 10 minutes, uniformly mixing, culturing at 25 ℃ for 10 minutes, adding 400 mu L of dinitrosalicylic acid coloring reagent, uniformly mixing, reacting in boiling water for 5 minutes, and cooling to room temperature. Both the 0 min and 10 min reaction groups were diluted with appropriate amounts of water to measurable ranges, for example: mu.L of reacted sample and 850. mu.L of water were measured for absorbance at 540nm with a spectrophotometric instrument, and the background was zeroed with buffer.
The calculation formula of the inhibitory ability of α -amylase activity is as follows:
percent alpha-amylase inhibition ═ 1- (a)Sample 10-ASample 0)/(AControl 10-AControl 0)]×100％
ASample 10Absorbance value of 10 min reaction set
ASample 0Absorbance value of 0 min reaction group
AControl 10Absorbance value of 10 min control reaction group
AControl 0Absorbance value of control 0 min reaction group
FIG. 3 is a graph of data showing the efficacy of an extract of acerola young fruit of the present invention in inhibiting alpha-amylase activity. As can be seen from FIG. 3, the experimental group can effectively inhibit the activity of alpha-amylase, and the inhibition percentage reaches nearly 27%. The results of this example show that the extract of acerola young fruit of the present invention can inhibit the activity of α -amylase, thereby achieving the effect of regulating body weight.
Example 4 evaluation of the effectiveness of extracts of acerola young fruits in inhibiting lipase Activity
First, buffer A was prepared, and 17mg of Structured Lipids (SLS) and 1g of Triton X-100 were quantified to 100mL of water. Next, buffer B, Tris 66mM (pH7.4), was prepared and titrated with HCl to prepare a lipase (lipase) buffer solution and a reaction terminator. Then, 1.6mM 4-Nitrophenyl laurate (4-Nitrophenyl laurate) was prepared, 51.43mg of 4-Nitrophenyl laurate was added to 100mL of buffer A, and the mixture was stirred at 65 ℃ for 15 minutes and mixed uniformly, and then cooled at room temperature until the solution became clear (storage conditions: refrigeration at 4 ℃ C., storage for 3 days; cloudy before use every other day, heating to 65 ℃ C. to make the solution clear, and then cooling for use).
Then, 150U/mL of lipase was prepared, 99.21mg of lipase was weighed and quantified with buffer B to 10mL, and the mixture was used on the day after being mixed uniformly. Then, 25. mu.L each of the blank group and 10% extract of young acerola cherry was used as a sample group, and 25. mu.L of lipase was added, followed by 50. mu.L of 1.6mM 4-nitrophenyllaurate, and after reaction at 37 ℃ for 30 minutes, 100. mu.L of buffer B was added to terminate the reaction. The absorbance was measured at 400nm and the inhibition was calculated according to the following formula (sample blank, control and blank were treated in the same manner).
FIG. 4 is a graph of data showing the efficacy of an extract of acerola young fruit of the present invention in inhibiting lipase activity. As can be seen from fig. 4, the sample group was effective in inhibiting lipase activity with a percentage of inhibition approaching 74%. The results of this example show that the acerola cherry young fruit extract of the present invention can inhibit lipase activity, thereby achieving the effect of regulating body weight.
Example 5 evaluation of the effectiveness of acerola cherry young fruit extracts in increasing lean voxel (leptin) content
First, mouse adipocytes 3T3-L1 (purchased from
CL-173TM) Cultured in preadipocyte Expansion Medium (Pre-adipocyte Expansion Medium) containing 90% Dulbecco's Modified Eagle's Medium (DMEM) (Gibco), 10% Bovine Serum (Bovine Serum, from Gibco, USA), and 1% Penicillin/streptomycin (Penicillin-streptomycin, from Gibco, USA) was added. In each of 96-well culture platesAdd 200. mu.L of medium to wells with 1X 10 of medium per well43T3-L1 cells. After incubation at 37 ℃ for 48 hours, the medium was removed. Thereafter, a Differentiation Medium (Differentiation Medium) containing 90% Dulbecco's modified Egger's Medium, 10% Fetal Bovine Serum (FBS), 1% Penicillin/streptomycin (penicilin-streptomycin), 1.0. mu.M/mL Dexamethasone (DExamethane, DEXA) (Sigma), 0.5mM/mL methyl isobutyl xanthine (IBMX) (Sigma), and 1.0. mu.g/mL Insulin (Insulin) (Sigma) was added, and fresh Differentiation Medium was replaced every 2 days. After 4 days, the differentiation medium was replaced with Adipocyte maintenance medium (Adipocyte maintenance medium) comprising 90% Dulbecco's Modified Eagle's Medium (DMEM) (Gibco), 10% Fetal Bovine Serum (FBS), 1% Penicillin/streptomycin (penicilin-streptomycin) and 1.0. mu.g/mL of insulin, and fresh Adipocyte maintenance medium was replaced every 2 days. After 7-10 days after the initiation of differentiation induction, the cells had fully differentiated. Next, the fat droplet formation was observed using a microscope.
Thereafter, 3T3-L1 cells were divided into 2 groups, including 1 experimental group and 1 control group. 0.125mg/mL of acerola young fruit extract was added to the cells of the experimental group, while the cells of the control group were not treated. After 12 days of culture (medium replacement every 48 hours), the medium was harvested and Leptin content was determined using the Mouse LEP ELISA Kit (Mouse LEP (Leptin) ELISA Kit) (Elabscience).
Fig. 5 is a data plot of the efficacy of an extract of acerola young fruit of the present invention in enhancing lean voxel content. As can be seen from FIG. 5, the leptin level in the experimental group was significantly increased (18% increase) compared to the control group. The results of this example show that the acerola cherry young fruit extract of the present invention can effectively increase the lean body mass content, thereby achieving the effect of regulating the body weight.
Example 6 evaluation of the effectiveness of acerola cherry young fruit extracts in reducing the amount of Reactive Oxygen Species (ROS) expression induced by advanced glycation end products (AGEs)
This example resists glycosylation (inhibition)Non-enzymatic browning, avoiding denaturation of functional proteins in vivo) as follows: collagen (Collagen) (60mg/mL) (containing 0.06% NaN) was prepared in 200mM phosphate buffer (pH7.4)3) And D-fructose (D-fructose) (1.5M). Thereafter, the groups were divided into 3 groups including 1 experimental group (i.e., sample), 1 AGE group, and 1 control group. The experimental and AGE groups were treated with AGEs (0.2mL collagen mixed with 0.2mL D-fructose). Then, 0.25mg/mL of the extract of the acerola young fruit was added to the experimental group, while the control group was not treated at all. Then, the reaction was carried out at 50 ℃ for 24 hours. Fluorescence detection (excitation wavelength 360nm, emission wavelength 460nm) was performed at 0.1mL each of the origin and the end of the glycosylation reaction.
Relative AGEs formation (%) was calculated as follows:
FIG. 6 is a data plot of the efficacy of the acerola young fruit extract of the present invention in reducing AGEs-induced ROS expression. As can be seen from FIG. 6, the ROS-positive cells were significantly elevated in the AGE group compared to the control group, indicating that AGEs induce cells to produce large amounts of ROS. Compared with the AGE group, the ROS positive cells in the experimental group were significantly reduced (57.1% reduction). The results of this example show that the acerola cherry young fruit extract of the present invention can effectively reduce the representation of AGEs-induced ROS, thereby achieving the anti-aging effect.
Example 7 evaluation of the effectiveness of acerola cherry young fruit extracts in inhibiting skin melanogenesis induced by blue light injury
First, mouse skin melanoma cell line B16F10 (purchased from ATCC CRL-6475) was cultured in Dulbecco's Modified Eagle's Medium (DMEM) [ supplemented with 1% penicillin/streptomycin (Gibco) and 10% FBS (Gibco)]In (1). Add 3mL of media to each well of a 6-well plate to have 1.5X 10 per well5And B16F10 cells. After incubation at 37 ℃ for 24 hours, the medium was removed.
Thereafter, B16F10 cells were divided into 4 groups, including 1 experimental group, 1 control group, 1 blue light group, and 1 comparative group. The thickness of the glass is 12J/cm2The blue light dose irradiated the cells of the blue light group, experimental group and comparative group for 1 hour. 0.0625mg/mL of acerola cherry young fruit extract was added to the cells of the experimental group, and 0.0625mg/mL of kojic acid (a melanin production inhibitor) was added to the cells of the comparative group. The cells of the control group were not treated.
After each set of cell cultures was cultured at 37 ℃ for 48 hours, the medium was removed and washed twice with 1xPBS (Gibco). Thereafter, trypsin (trypsin) was added to treat the cells for 3 minutes and the suspended cells were collected in a centrifuge tube of 15mL volume followed by spinning (spin) at 400xg/5 minutes to pellet the cells. After washing twice with 1xPBS, the cell pellet (cell pellet) was resuspended with 200. mu.L of 1 XPBS. Subsequently, the cell solution was left in liquid nitrogen for 10 minutes, and then left to stand at room temperature for 30 minutes for thawing. After thawing was complete, the rotor was spun at 12,000Xg for 30 minutes, whereupon the supernatant was removed and 120. mu.L of 1N NaOH (in ddH) was added2O). After mixing well, it was left to stand in a dry bath at 60 ℃ for 1 hour. Thereafter, a volume of 100. mu.L was taken into a 96-well plate and the absorbance (OD) of each well was read at a wavelength of 450nm with an ELISA reader450)。
The melanin content (%) was determined by measuring the absorbance (OD)450) Calculated by substituting the following equation:
melanin content (%) (OD measured for each group)450OD measured in absorbance/control group450Absorbance) × 100%
Statistically significant differences between groups were determined by the Stirling t-assay. The results of this example are shown in FIG. 7.
FIG. 7 is a graph of data showing the efficacy of an extract of acerola young fruit of the present invention in inhibiting skin melanin production induced by blue light injury. As can be seen from FIG. 7, the melanin expression was significantly increased in the blue-light group compared to the control group, indicating that the skin melanogenesis was induced by the blue-light damage. The melanin expression was significantly reduced in the experimental group compared to the blue light group and the comparative group. The results of this example show that the acerola cherry young fruit extract of the present invention can effectively inhibit the generation of melanin in the skin induced by blue light injury, thereby achieving the effect of beautifying the skin.
Example 8 evaluation of the effectiveness of extracts of acerola cherry young fruit in increasing collagen content in skin
First, human skin fibroblast (human skin fibroblast) CCD-966SK (FBS) was cultured in Minimal Essential Medium (MEM) (Gibco) supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin (penicillin)/streptomycin (streptomycin), and 1mM sodium pyruvate (sodium pyruvate)
CRL-1881) in 24-well plate, cell concentration of 500. mu.L medium 2X 104Cells/well, followed by incubation at 37 ℃ for 24 hours, were washed with PBS.
The cultured cells were then divided into 2 groups, including 1 control group and 1 experimental group. The extract of acerola young fruit obtained in example 1 was diluted with serum-free medium to a dilution having a concentration of 0.25mg/mL, and then 500. mu.L of the dilution was added to the cells of the experimental group, and 500. mu.L of serum-free medium was added to the cells of the control group. Thereafter, 1,000. mu.L of the medium was collected and transferred to a 1.5mL microcentrifuge tube, followed by addition of 200. mu.L of collagen for separation&Concentration reagent (collagen isolation)&concentration reagent) and turning the centrifuge tube 6-8 times. Subsequently, the culture was carried out overnight at 4 ℃. Thereafter, the centrifuge tube was taken out and centrifuged at 12,000rpm for 10 minutes, and then 1,000. mu.L of the supernatant was removed. Then, using SircolTMSoluble collagen assay kit (Sircol)TMThe relative percentage of Collagen in each group was analyzed by the Soluble Collagen Assay Kit (Biocolor Life Science Assays, Northern Ireland, UK). Adding 1,000. mu.L of
Stain into centrifuge tubes and gently shaken for 30 minutes followed by 12,000rpm for 10 min and the supernatant removed. Next, 750. mu.L of an acid-salt washing reagent (acid-washing reagent) was added, and centrifuged at 12,000rpm for 10 minutes. Thereafter, the supernatant was removed and the tube was inverted, and the liquid in the tube was removed with a cotton swab. Then, 250. mu.L of an alkaline reagent was added thereto and sufficiently mixed with shaking, and then the absorbance at a wavelength of 555nm was measured by a spectroscopic signal sensor, thereby calculating the collagen secretion rate, wherein the collagen secretion rate of the control group was used as a 100% reference. The results of this example are shown in FIG. 8.
Fig. 8 is a data graph showing the effect of the extract of acerola young fruit of the present invention on increasing the collagen content in the skin. As can be seen from FIG. 8, the relative percentage of collagen measured in the experimental group was significantly increased compared to the control group. The results of this example show that the extract of acerola young fruit of the present invention can increase the collagen content in the skin, thereby achieving the effect of beautifying the skin.
Example 9 evaluation of the effectiveness of extracts of acerola cherry young fruit in elevating the level of elastin (elastin) in the skin
The experiment is referred to FastinTMElastin assay kit (Fastin)TMElastin Assay kit). First, human dermal fibroblasts CCD-966SK are cultured in Minimal Essential Medium (MEM) (Gibco) supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin and 1mM sodium pyruvate (Gibco) ((II))
CRL-1881) in 6-well plate, cell concentration of 1X 10 in 2mL of medium5Cells/well, followed by incubation at 37 ℃ for 24 hours.
The cultured cells were then divided into 2 groups, including 1 control group and 1 experimental group. The extract of acerola young fruit obtained in example 1 was diluted with a medium to a dilution having a concentration of 0.25mg/mL, and then 2mL of the dilution was added to the cells of the experimental group, and 2mL of the medium was added to the cells of the control group. After culturing each group of cells for 48 hours, the medium was removed and washed once with 1X PBS. Next, each group of cells was treated with trypsin (trypsin) for 3 minutes, and then the trypsin activity was terminated with the medium and transferred to a test tube of 1.5mL volume. Thereafter, cells were harvested by trypsinization and transferred to a 1.5mL volume microcentrifuge tube, followed by centrifugation at 300Xg for 5 minutes. Next, the cell pellet was retained in about 300. mu.L of PBS (Gibco), and then 100. mu.L of 1.0M oxalic acid (oxalic acid) was added to 300. mu.L of the cell suspension and allowed to act in a heat meter at 100 ℃ for 1 hour. Thereafter, an equal volume of Elastin precipitation Reagent (Elastin Precipitating Reagent) was added to each tube, the tubes were capped and vortexed briefly, and allowed to sit for 15 minutes to complete the precipitation. Next, centrifuge at 10,000xg for 10 minutes, then drain the liquid contents of the tube, by gently patting the inverted tube onto a paper towel to remove most of the fluid remaining in the tube.
Next, 1mL of dye reagent was added, the contents were mixed by inverting the tube, and then the tube was placed on a mechanical shaker for 90 minutes. After that, the unbound dye in the tube is drained. Elastin-dye complexes can be observed as reddish brown deposits in the bottom and inner lower wall of the tube. Next, 250 μ L of dye dissociation reagent was added to each tube, and the tube was capped to release the dye into solution by vortex mixer to ensure that all bound dye had entered the solution. Thereafter, the contents of each tube were transferred to a 96-well plate, and the plate was placed in an ELISA reader to measure absorbance at 513 nm. The results of this example are shown in FIG. 9.
Fig. 9 is a data graph showing the effect of the extract of acerola young fruit of the present invention on increasing the content of elastin in the skin. As can be seen from FIG. 9, the elastin content measured in the experimental group is significantly increased compared to the control group. The results of this example show that the acerola cherry young fruit extract of the present invention can increase the elastin content in the skin, thereby achieving the effect of beautifying the skin.
Example 10 evaluation of the effectiveness of acerola cherry young fruit extract in inhibiting adipocyte-induced chronic inflammation
Collecting differentiated fat fineCell OP9(
CRL-2749TM) The medium (conditioned medium), which induces adipocyte inflammation and secretion of inflammatory factors, is ready for use. In 96 hole plate each hole 1X 104RAW264.7(
TIB-71TM) After culturing in an incubator at 37 ℃ for 24 hours, the original medium was aspirated, replaced with adipocyte-conditioned medium, added to a 96-well plate at 200. mu.L each, and the sample to be tested was treated at the same time.
OP9 cells were then divided into 3 groups, including 1 experimental group, 1 pathological control group, and 1 blank control group. The cells of the experimental and pathological control groups were treated with OP-9 conditioned medium to induce an inflammatory response. Next, 0.25mg/mL of the extract of acerola young fruit was added to the cells of the experimental group, and the cells of the blank group were added with DMEM (Gibco), 1% penicillin/streptomycin (Gibco), and 4mM L-glutamic acid amide (L-glutamine) (Gibco). After the reaction was returned to the 37 ℃ incubator for 18 hours, 150. mu.L of the solution was removed from each well and transferred to a new 96-well plate, and 130. mu.L of ddH was added to each well2O, then 10. mu.L each of the agent A and the agent B in the Gilisha Reagent Kit (Griess Reagent Kit) (Invitrogen) was added to each well, and the mixture was reacted at room temperature for 30 minutes, and finally the absorbance at 548nm was measured using a total spectrum optical analyzer (BioTek).
Fig. 10 is a data graph showing the efficacy of the acerola young fruit extract of the present invention in inhibiting chronic inflammation caused by adipocytes. As can be seen from FIG. 10, the inflammation (NO content) of the pathological control group was significantly increased compared to that of the blank control group, indicating that the adipocytes induce inflammatory response. Compared with the pathological control group, the degree of inflammation (NO content) of the experimental group is significantly reduced (by 8.5%). The results of this example show that the acerola cherry young fruit extract of the present invention can effectively inhibit chronic inflammation caused by adipocytes, thereby achieving anti-inflammatory effects.
In summary, the acerola cherry young fruit extract of the present invention can achieve the effects of regulating body weight, beautifying skin, anti-inflammation and anti-aging by inhibiting α -amylase activity, inhibiting lipase activity, increasing leptin content, inhibiting skin melanin generation induced by blue light injury, increasing collagen content in skin, increasing elastin content in skin, inhibiting chronic inflammation caused by adipocytes, and reducing ROS expression level. On the other hand, the acerola cherry young fruit extract can effectively prevent three highs and is suitable for developing products for beautifying muscles and managing weight.
The foregoing is by way of example only, and not limiting. It is intended that all equivalent modifications or variations without departing from the spirit and scope of the present invention shall be included in the appended claims.
Claims (10)
1. Use of an extract of acerola young fruit for preparing a composition for regulating body weight, beautifying skin, anti-inflammation and anti-aging, wherein the extract of acerola young fruit is prepared by extracting the acerola young fruit with water, alcohols, aqueous alcohols or a combination thereof as an extraction solvent.
2. The use according to claim 1, wherein the volume ratio of the acerola young fruit to the extraction solvent is 1-5: 5 to 20.
3. The use according to claim 1, wherein the effective concentration of the extract of acerola young fruit is at least 0.0625 mg/mL.
4. The use of claim 1, wherein said modulating body weight comprises inhibiting alpha-amylase activity, inhibiting lipase activity, and increasing leptin levels.
5. The use according to claim 1, wherein said skin-beautifying includes inhibiting melanogenesis of the skin, increasing collagen content of the skin and increasing elastin content of the skin.
6. Use according to claim 5, wherein the skin melanogenesis is skin melanogenesis induced by blue light damage.
7. The use according to claim 1, wherein the anti-inflammation is inhibition of adipocyte-induced chronic inflammation.
8. Use according to claim 1, characterized in that the anti-ageing is a reduction of the active oxygen molecule expression.
9. The use according to claim 1, wherein the acerola young fruit is 25 to 35 days after the acerola blooms.
10. The use according to claim 1, wherein the composition is a pharmaceutical, a food product or a nutraceutical.
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| Publication number | Publication date |
|---|---|
| TWI734332B (en) | 2021-07-21 |
| TW202042830A (en) | 2020-12-01 |
| CN114886952A (en) | 2022-08-12 |
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