CN103083361B - Fairy shrimp egg extract, preparation method and uses thereof - Google Patents
Fairy shrimp egg extract, preparation method and uses thereof Download PDFInfo
- Publication number
- CN103083361B CN103083361B CN201110332791.4A CN201110332791A CN103083361B CN 103083361 B CN103083361 B CN 103083361B CN 201110332791 A CN201110332791 A CN 201110332791A CN 103083361 B CN103083361 B CN 103083361B
- Authority
- CN
- China
- Prior art keywords
- brine shrimp
- shrimp ovum
- extract
- composition
- ovum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a fairy shrimp egg extract, a preparation method and uses thereof. The present invention further relates to uses of the fairy shrimp egg extract in treatment and/or prevention of inflammation related skin abnormality and excessive nitric oxide production related diseases.
Description
Technical field
The present invention relates to brine shrimp ovum extract and preparation method thereof.The invention still further relates to this extract in the purposes treating and/or preventing in the skin abnormality relevant to inflammation and the disease relevant with the excessive generation of nitric oxide.
Background technology
Nitric oxide (NO) is unsettled free radical in organism, has become now one of mankind's physiology and pathology important indicator.
Nitric oxide is formed by the nitrogen-atoms of the end guanidine radicals of L-spermine acid by nitric oxide synthetase.Under physiological situation, the NO of low amount continues to show to such an extent that the special-shaped state of Ca-dependent NOS (cNOS) is produced by being present in various kinds of cell (comprising endothelial cell and neuron).The nitric oxide of described low amount is relevant to multiple physiology adjusting, the adjusting of the control of for example blood pressure, the transmission of nerve signal and function of immune system; On the other hand, under pathological condition, the NO of a large amount is produced by the special-shaped state of Ca-dependent NOS (iNOS) that can bring out performance in various kinds of cell kenel (comprising endothelial cell, smooth muscle cell and macrophage).
The stimulating factor that can bring out the generation of nitric oxide a large amount is quite various, transcribing of the nitrous oxide synthetase gene that the excessive manufacture (as TNF (TNF), il-1 (IL-1), interferon) of for example struvite cytokine can be brought out, and then generation a large amount nitric oxide, and then bring out the excessive generation of inflammatory cell factor or other inflammation GAP-associated protein GAP (for example, TNF α, IL-6, IL-1, IL-8, ICAM-1, VCAM-1 and VEGF).
Now, the whole world is because of climatic variation or various pollutant, and such as global warming, greenhouse effects, ozone layer broken hole, ultraviolet ray are excessive, motor vehicle emission pernicious gas, indoor and outdoor waste gas and second-hand-cigarette etc., and serious threat is to the mankind's health.Especially pass by over 100 years, the temperature in Taiwan 1.3 degree that risen, are 2 times of global mean value, estimate by very long hot again, to make the interaction aggravation of climatic factor and pollutant, the change of these external environments following summer, to produce harm to human body, especially with the skin of defence forefront for.
When skin metabolism slows down, sunlight, ultraviolet ray, pollution sources, the even invasion and attack of life style and pressure, cause it to lose gradually due reparation reaction and poised state, and now microgroove, spot, the phenomenon such as lax, coarse, sagging are appeared in one's mind one by one.Causing one of reason of these aging phenomena is that skin is accepted many interior external stimulus, can produce in inside inflammatory reaction.According to characteristic with start send out a reason can be divided into chronic inflammatory reaction and nerve inflammatory reaction.Skin connects kind accepts the impacts of two kinds of inflammatory reactions, can be subject to aging reaction aggravation, wrinkle and do sth. in advance appearance and intensification, skin and lose the puzzlement of the problems such as the degree of packing.Scytitis reaction has become newly aobvious learning of anti-ageing trend, in addition, the melanic generation of skin is a very complicated physiological reaction, the inflammation of skin, oxidation reaction, also can cause melanin to generate, when occurring inflammation in cell, meeting secretory cell hormone, stimulates " tyrosinase " to impel melanin to form.
Moreover, (particularly iNOS) is relevant with following disease for known nitric oxide synthetase: platelet aggregation, homeostasis process, tissue damage, inflammation situation, shock state, immune disorders, gastrointestinal motility illness and central nervous system disease, diabetic neuropathy (Epstein, The New England Journal of Medicine, 1993,329 (27): 2002-2011; The people such as Hobbs, Annu.Rev.Pharmacol.Toxicol, 1999,39:191-220; The people such as Pfeilschfter, Cell Biology International, 1996,20 (1): 51-58).Prior art is also pointed out neurodegeneration and closely related (the N Engl J Med of diabetic ulcer, Volume 343 (11), September 14,2000, pp.787-793 and J.Fam.Pract, Volume 49 (11) Supplement.November 2000S40-S48).Nitric oxide synthetase is also relevant with the situation being caused by nitric oxide, such as tissue damage, brain ischemic, epilepsy, immune response and inflammatory reaction (people such as Mulligan, Proc.Natl.Acad.Sci.USA, 1991,88:6338-6342).
Many serious and the human diseases of refractory is relevant with the dysregulation of inflammatory process, this class disease comprises the disease that is not considered as traditionally struvite symptom, such as cancer, atherosclerotic and diabetes.Similarly, such as the autoimmune disease of rheumatoid arthritis, lupus, psoriasis and multiple sclerosis relate to illing tissue in the activation of improper waiting a moment property of inflammatory process, it is caused the dysfunction of non-self identification and reaction mechanism by self identification in immune system and reaction mechanism.In the neurodegenerative disorders such as Alzheimer's disease (Alzheimer ' s disease) and Parkinson's disease (Parkinson ' s disease), neurotrosis for example, raises relevant with the activation of Microglial cell and the content of proinflammatory albumen (inducible nitric oxide synthase (iNOS)).
Came from before 400,000,000 years halomereid genus artemia (formal name used at school: Artemia) the most original on the earth, be commonly called as shrimps (Artemia Salina), halogen worm, extra large monkey, boiled shrimps with shell in salt water, salt insect, water horse red horse etc., without unique genus under Notostraca Lu Chong section, can treat as raising pets, also can be used as fish meal, is one of Major Foods of soft coral.And in history is recorded and is carried, when European enters in North American continent at first, some Indian aboriginals that just once found northeast U.S. just with the brine shrimp ovum that dries as complementary food.
Shrimps through very long biological evolution, has been found its extremely resistance to various rugged environments, and the salt content that shrimps can also cannot be survived fish exceedes in the waters of six times, seawater survives.Brine shrimp ovum is called as durable ovum, can resist for a long time the injury of unfavorable factor in external environment, protects the embryo of wherein dormancy, when by the time environment is applicable to shrimps breeding existence, then broken wall and go out procreation race.Find at present, brine shrimp ovum under long-term high-intensity ultraviolet ray, irradiation with radiation, high temperature and drought, and in toxic solvent, all show mirable vitality and energy.Brine shrimp ovum is served outer space by France in 1978 and Russian scientists, reaches 96 hours and accept outer space utmost point rugged environment, for example extremely temperature, in a large number cosmic radiation irradiations etc.When these ovum are brought back after the earth, in ensuing ten or twenty year, do a large amount of research, comprise the chemical composition change of incubation rate and ovum etc., result shows that the brine shrimp ovum of taking back has the incubation rate up to 96%.
According to the history of life of shrimps and the process from adult to larva thereof; there is the molecule of the extraneous adverse environment of various antagonism; tide over extreme hostile environment with protection brine shrimp ovum; for example p26, Artesunate (Artemin), two guanosine tetraphosphates (diguanosine tetraphosphate, GP4G) and trehalose (Trehalose).Wherein two guanosine tetraphosphates have confirmed to have multiple curative effect, and for example European patent discloses it No. 1049455 and can strengthen skin immune system, chafe cytothesis, and supply ultraviolet protection by the assisted Extraction that improves cutin interlayer simultaneously; TaiWan, China patent discloses and discloses it for No. 200640475 and can stimulate corium or the large molecule synthesis of epidermis and/or prevent its degraded; Japanese Patent Publication discloses two guanosine tetraphosphates for No. 204238297 and has the effect of protection skin antagonism external pressure, and can promote the generation of heat shock protein; No. 204238297th, Japanese Patent Publication, United States Patent (USP) disclose No. 2009148544, United States Patent (USP) the 7th, 615,546 and 7,544, No. 375 and French Patent (FRP) disclose two guanosine tetraphosphates for No. 2834887 and have activating and regenerating cell function, improve Skin Cell state, prevent pachylosis can smoothing wrinkle and anti-aging; TaiWan, China patent discloses its adjustable intercellular contact for No. I281413; European patent discloses for No. 1336402 and can promote natural on-off cycles of hair growth and/or reduce hair loss.
Because the composition of brine shrimp ovum is abundant, have treatment potentiality, therefore the present invention pays close attention to the novel brine shrimp ovum extract of exploitation, its manufacture method and uses thereof.
Summary of the invention
The invention provides a kind of method of preparing brine shrimp ovum extract, it comprises the following step:
A) provide brine shrimp ovum;
B) adverse circumstance is processed described brine shrimp ovum; With
C) homogenization step brine shrimp ovum b).
The present invention also provides a kind of brine shrimp ovum extract.
The present invention provides again a kind of composition that comprises brine shrimp ovum extract of the present invention.
The present invention also provides a kind of brine shrimp ovum extract according to the present invention in the purposes preventing and/or treating in the skin abnormality relevant to inflammation.
The present invention also provides a kind of brine shrimp ovum extract according to the present invention in the purposes preventing and/or treating in the disease relevant to the excessive generation of nitric oxide.
The present invention describes in detail in following part.Other features, objects and advantages of the present invention can be found apparently in the specific embodiment of the present invention and claim.
Unless otherwise defined herein, otherwise the scientific and technical terminology using in conjunction with the present invention should have the implication that those skilled in the art conventionally understand.The implication of term and category should be clear and definite; But if there is any potential ambiquity that obtains, definition provided in this article has precedence over any dictionary or external definition.
Unless the context requires otherwise, otherwise singular references should comprise plural number, and plural term also should comprise odd number.
Unless otherwise instructed, otherwise be interpreted as thering is following implication according to the application's following term used:
Term used herein " prevention " represents the generation of the initial or reduction illness of the symptom that delays easy diseased individuals.
Term used herein " treatment " represents to reduce and/or improve the symptom of diseased individuals.
Term used herein " individuality " represents animal, particularly mammal.According to a preferred embodiment of the invention, " individuality " refers to the mankind.
Consumption when term used herein " treatment effective dose " represents active component separately or merges use with other treatment/medicine, this consumption is enough to show therapeutic efficiency.
Term used herein " carrier " or " pharmaceutically acceptable carrier " refer to diluent, excipient, acceptor or analog, and it is known by the those skilled in the art in pharmaceutical composition preparing technical field.
Preparation method
According to the present invention, the method for preparing brine shrimp ovum extract can comprise the following step:
A) provide brine shrimp ovum;
B) adverse circumstance is processed described brine shrimp ovum; With
C) homogenization step brine shrimp ovum b).
Term used herein " brine shrimp ovum " refers to the ovum of Artemia Salina or Brine Shrimp, and shrimps also claims halogen worm, extra large monkey, boiled shrimps with shell in salt water, salt insect, water horse red horse, belongs to without Notostraca Lu Chong section genus artemia.
According to the collection method of brine shrimp ovum of the present invention, obtain during and the condition that obtains be well known to those skilled in the art, there is no particular restriction.
According to the present invention, by collection and brine shrimp ovum stand adverse circumstance, in a concrete preferred embodiment of the present invention, described adverse circumstance is salt adverse circumstance, UV adverse circumstance, hot adverse circumstance and sour adverse circumstance.
In a concrete preferred embodiment of the present invention, described salt adverse circumstance is by approximately 2% to approximately 5% saline treatment approximately 6 to approximately 12 hours; Preferably use approximately 3% saline treatment approximately 12 hours.Described salt is sodium chloride, magnesium chloride or potassium chloride preferably.
In a concrete preferred embodiment of the present invention, described UV adverse circumstance is to irradiate approximately 12 to approximately 24 hours with UV, and exposure intensity is approximately 6000 to about 10000mW/cm
2; Preferably irradiate approximately 18 hours.
In a concrete preferred embodiment of the present invention, described hot adverse circumstance is to process approximately 3 to approximately 7 minutes in approximately 95 DEG C to approximately 105 DEG C; Being preferable over approximately 100 DEG C boils approximately 5 minutes.
In a concrete preferred embodiment of the present invention, described sour adverse circumstance is to process approximately 20 to approximately 40 minutes to the hydrogen ion concentration of about 4N with about 1N; Preferably, described sour adverse circumstance is to process approximately 30 minutes by the hydrogen ion concentration of about 2N.Described hydrogen ion is preferably provided by sulfuric acid or hydrochloric acid.
The method according to this invention, step c) homogenization step brine shrimp ovum b) is the treatment step for obtaining extract.Preferably, through step b) adverse circumstance brine shrimp ovum after treatment first after filtration impurity and clean after, then homogenize.The means of described impurity screening and cleaning are that those skilled in the art can complete.Can be general method of cell disruption according to the method for homogeneous brine shrimp ovum of the present invention, for example, use cell crushing instrument or ultrasonic vibrating instrument to carry out.Can conveniently carry out for making to homogenize, can first will be added to the water through the brine shrimp ovum of adverse circumstance processing, preferably, be with the water blend step of approximately 3 to approximately 10 times brine shrimp ovum b), to obtain brine shrimp ovum aqueous mixtures; More preferably, be with the water blend step of approximately 6 times brine shrimp ovum b).
In a concrete preferred embodiment of the present invention, the method according to this invention also comprises removes the c) cell fragment of the brine shrimp ovum that homogenizes of gained of step, and obtains clarified solution.The means of described removal cell fragment are that those skilled in the art can complete, for example, use centrifugal or filter type removal.
In a specific embodiment of the present invention, step c) comprises:
C1) with the brine shrimp ovum of the water blend step (b) of approximately 3 to approximately 10 times, to obtain brine shrimp ovum aqueous mixtures;
C2) homogenization step c1) brine shrimp ovum aqueous mixtures; With
C3) remove step c2) cell fragment homogenizing in brine shrimp ovum aqueous mixtures of gained, and obtain clarified solution; Preferably, step c3) by centrifugal removal step c2) cell fragment homogenizing in brine shrimp ovum aqueous mixtures of gained.
The method according to this invention, preferably also separately comprise steps d), to concentrate described brine shrimp ovum extract, its method that can anyly know concentrates brine shrimp ovum extract, for example freeze drying or evaporation (J.Pharmacol.Sci., 99:294-300,2005 and J.Chromatography, 932:91-95,2001) to obtain through concentrated brine shrimp ovum extract, and selectively add excipient, for example, with the common freeze drying of methylcellulose.
In a concrete preferred embodiment of the present invention, described treated brine shrimp ovum is through filtering the solvent being added with Transformatin process, and the method for described filtration is that those skilled in the art can implement.For example use general Filter paper filtering, after filtration, water cleans, then smashes cell after mixing with the water containing protease inhibitors of approximately 3 to approximately 10 times of amounts; Preferably smash cell with after the mixing containing the water of protease inhibitors of approximately 6 times of amounts, then within centrifugal 20 minutes, get its supernatant in 4 DEG C of 4500rpm, after filtration, concentrated, freeze drying, be stored in-20 DEG C of refrigerator-freezers for subsequent use.
Extract
The invention provides a kind of brine shrimp ovum extract, it is according to above-mentioned that method makes.
In specific embodiments of the invention, extract is carried out to high pressure lipuid chromatography (HPLC), the chromatographic column of use is
250-4HPLC-Cartridge NH
2, 5 μ m, column oven temperature is 25 DEG C, and sampling volume is 10 μ L, and flow velocity is 1.0mL/min, and be 30min running time, mobile phase is CH
3cN: H
2o=4: 1 (volume ratio), extract according to the present invention is to locate to have absworption peak in 2.43 to 2.45,3.12 to 3.15,4.59 to 4.61,5.05 and 5.57 to 5.63 minutes in retention time.
In a concrete preferred embodiment of the present invention, by salt adverse circumstance processing for brine shrimp ovum, and make extract according to above-mentioned preparation method, described extract obtains high pressure liquid chromatography figure as shown in Figure 1 through above-mentioned condition analysis, and it is within 1.92,2.45,3.12,4.59,5.05 and 5.57 minutes, to locate to have absworption peak in retention time.
In another concrete preferred embodiment of the present invention, by UV adverse circumstance processing for brine shrimp ovum, and make extract according to above-mentioned preparation method, through the high pressure liquid chromatography figure of above-mentioned condition analysis as shown in Figure 2, it is within 1.92,2.45,3.12,4.59,5.05 and 5.57 minutes, to locate to have absworption peak in retention time to described extract.
In another concrete preferred embodiment of the present invention, by hot adverse circumstance processing for brine shrimp ovum, and make extract according to above-mentioned preparation method, through the high pressure liquid chromatography figure of above-mentioned condition analysis as shown in Figure 3, it is within 1.92,2.43,3.15,4.61,5.05 and 5.60 minutes, to locate to have absworption peak in retention time to described extract.
In another concrete preferred embodiment of the present invention, by sour adverse circumstance processing for brine shrimp ovum, and make extract according to above-mentioned preparation method, through the high pressure liquid chromatography figure of above-mentioned condition analysis as shown in Figure 4, it is within 2.45,3.12,3.63,4.61,5.05 and 5.63 minutes, to locate to have absworption peak in retention time to described extract.
Composition
The invention provides a kind of composition that comprises the brine shrimp ovum extract of the present invention for the treatment of effective dose.The present composition can be used as food compositions, medical composition or cosmetic composition.
The present composition can be applied to individuality by any suitable approach, for example Orally administered.The suitable formulation of the present composition includes, but are not limited to lozenge, lozenge, rigid or soft capsule, water-based or oily suspensions, emulsion, dispersibles powder or particle, syrup or elixir.As needs, can by described composition sterilizing or with any pharmaceutically acceptable carrier, the such as mixing such as stabilizing agent, wetting agent.
The present composition can be by any method of knowing and the pharmaceutically acceptable mixed with excipients of knowing.Therefore, can comprise for the oral present composition, for example one or more colouring agent, sweetener, flavoring agent and/or anticorrisive agent.
The drug combination that the present composition can use at present with other one or more, to treat the disease relevant to the excessive generation of nitric oxide.
Preferably be stored in water-based liquid medium according to cosmetic composition of the present invention.
In essence, water can comprise water; Its also can comprise water and with the mixture of water mutual solvents (at 25 DEG C for being greater than the water of the 50 % by weight power of dissolving each other), for example contain the low-carbon (LC) monohydric alcohol of 1 to 5 carbon atom, as ethanol or isopropyl alcohol, containing the dihydroxylic alcohols of 2 to 8 carbon atoms, as propane diols, ethylene glycol, 1,3-butanediol or DPG, C
3-C
4ketone and C
2-C
4aldehyde, and glycerine.
Described waterborne compositions can be used as skin skin toning agent, wetting agent or NMF, with to being exposed in environmental factor or pathologic condition or impaired skin performance promotes skin shielding to repair and reply the function of normal skin shielding.
Selectively, cosmetic composition of the present invention can also be the form of emulsion or newborn cream composition.It can comprise emulsifying surfactant, can be selected from anion and non-ionic surface active agent.For character and the function (emulsification) of surfactant, can reference paper " Encyclopedia of Chemical Technology, Kirk-Othmer ", the 22nd volume, 333-432 page, the 3rd edition, 1979, Wiley; For anion and non-ionic surface active agent, especially, 347-377 page that can reference data.
The surfactant that is preferred for the present composition is selected from: non-ionic surfactant: aliphatic acid, fatty alcohol, poly-ethoxyquin or Pegylation fatty alcohol, as poly-ethoxyquin stearyl alcohol or cetyl stearyl alcohol, the fatty acid ester of sucrose, alkyl glucose ester, particularly C
1-C
6the polyoxyethylene fatty acid ester of alkyl glucose, and composition thereof; Teepol: through the C of amine, ammoniacal liquor or alkali salt neutralization
16-C
30aliphatic acid, and composition thereof.Preferably use can obtain the surfactant of oil-in-water or water bag wax emulsion.
Selectively, composition of the present invention is the form of aqueous gel or hydrogel composition.Hydrogel composition comprises thickener so that liquid solution thickening.The example of thickener includes but not limited to that carbon compound, cellulose are main material, glue, seaweeds, guar gum, pectin, carragenines, gelatin, mineral or mineral thickener, polyethylene glycol and many alcohol, polyacrylamide and other polymeric viscosifier through sex change.Preferably use composition is invested to stability and the thickener of suitable flow behavior.
Composition of the present invention can further comprise the physiologically acceptable antioxidant of effective dose, natural or sex change amino acid, natural or sex change sterol compounds, natural or sex change glue protein, silk-fibroin or soybean protein.
Composition of the present invention is preferably prepared into and is applied to part, as the keratin material of skin, hair, eyelashes or nail.It can be the form that is just being usually used in any form of expression, particularly water-based of this type of application or oily solution, oil-in-water or water-in-oil emulsion, poly-silica emulsion, microemulsion or nanoemulsions, water-based or oil-base gel or liquid, pulpous state or solid-state hydrated product.
The present invention can be fluid conventionally and can have white or the outward appearance of coloured breast frost, ointment, milk, lotion, slurries, slurry, mousse or gel.It is selectively applied on skin partly with the form of aerosol, paster or powder.It can be also solid-state form, for example bar-shaped form.It can be used as skin maintenance product and/or cosmetic product uses.Or it can be prepared into shampoo or hair conditioner.
In known way, composition of the present invention also can comprise cosmetics and commonly use to obtain additive and adjuvant, as hydrophily or lipophilicity gelling agent, anticorrisive agent, antioxidant, solvent, spices, filler, pigment, odor absorbent and dyestuff.
Cosmetic composition of the present invention can comprise fatty material, for example oil, and particularly mineral oil (petroleum liquid glue), plant origin oil (Avocado Oil, evening primrose oil, safflower oil, soybean oil, wheat-germ oil, almond kernel oil), animal origin oil (lanolin), artificial oil (perhydro MF59), silicone (ring first silicone grease) and fluorocarbon oil (perfluor polyether).Fatty alcohol (as cetanol), aliphatic acid, wax and glue, particularly poly-silicone jelly, is also representational fatty material.
Cosmetic composition of the present invention can comprise emulsifying agent and co-emulsifier, and it comprises the fatty acid ester of polyethylene glycol, as PEG-100 stearate, PEG-50 stearate and PEG-40 stearate; The fatty acid ester of polyalcohol, for example, as glycol stearate, glucitol tristearate and oxygen ethylidene glucitol stearate, commercially available trade (brand) name Tween
tM20 or Tween
tM60; And composition thereof.
Cosmetic composition of the present invention can comprise hydrophily gelling agent, especially, it comprises carboxyl vinyl polymer (carbon compound), acrylic copolymer (as acrylate/alkyl acrylate copolymer), polyacrylamide, polysaccharide, natural gum and clay.Especially, the example of lipophilicity gelling agent comprises slaine and the hydrophobic silicone of sex change clay (for example bentonite), aliphatic acid.
Cosmetic composition according to the present invention is particularly suitable for being applied topically to keratan matrix/material, as skin, keratan fiber (hair and eyelashes) and nail.
Purposes
The invention provides a kind of brine shrimp ovum extract according to the present invention in the purposes preventing and/or treating in the skin abnormality relevant to inflammation.
In the preferred specific embodiment of the present invention, the skin abnormality relevant to inflammation comprises: as aging, pachylosis, skin or trichochromes precipitation disease, dry skin, high seborrhagia, high seborrhagia related defects, sensitive, dandruff, nature hair loss, bald head, abnormal acne, blackhead or polymorphic, old acne, daylight and medicine or professional acne, rose spot, the psoriasis of extensive and/or severe form, ichthyosis and class ichthyosis state, Darier's disease, palm keratosis, albinism and albinoidism state, lichen planus, all optimum or pernicious skin hyperplasias, extensive or serious rheumatoid psoriasis, itch, erythema, atopic dermatitis, contact dermatitis, contact eczema, lichen planus, pruigo, nettle rash, pruigo contact dermatitis and hyperkeratinization.
Term used herein " aging " refers to wrinkle and microgroove, lose the consolidation of skin and/or elasticity, atrophoderma, existence expand pore more irregular skin grain, lose skin brilliance and/or pigementation vestige.
The symptom of term used herein " sensitive " generally includes the pain of skin area experience, as sting pain, shouting pain, itch or tinea is itched, Burning Pain, rubescent, heat bitterly, uncomfortable, tight etc.
The invention provides a kind of brine shrimp ovum extract in the purposes preventing and/or treating in the disease relevant to the excessive generation of nitric oxide.
In the preferred specific embodiment of the present invention, the disease relevant to the excessive generation of nitric oxide comprises: as inflammation, septic shock, hemorrhagic shock, traumatic shock, anaphylactic shock, toxicogenic shock syndrome, hypovolemic shock, ischemic, cerebral ischemia, cytokine administration, cytokine excessively shows, ulcer, inflammatory bowel disease (as ulcerative colitis or Crohn disease (Crohn ' s disease)), diabetes, metabolic syndrome, nephrosis, diabetic neuropathy and diabetic ulcer, arthritis, rheumatoid arthritis, asthma, Alzheimer's disease, parkinsons disease, multiple sclerosis, cirrhosis, heteroplastic transplantation repulsive interaction, encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis, lymph corpuscle Choriomeningitis, glomerule nephritis, uveitis, ileitis, inflammation is (as liver inflammation, kidney inflammation etc.), burn, infect and (comprise bacterium, virus, fungi and parasitiation), influenza, the respiratory inflammation that the infection of the upper respiratory tract is relevant, haemodialysis, chronic fatigue syndrome, apoplexy, cancer is (as breast cancer, melanocytoma, solid tumor, leukaemia, myeloma, lymthoma etc.), cardio pulmonary bypass, ischemic damage and reperfusion damage, thrombotic or hemorrhagic stroke, subarachnoid hemorrhage, cerebral angiospasm, miocardial infarction, shock, gastritis, ulcerative colitis, adult breathes painful syndrome, cachexia, myocarditis, Autoimmune Disorders, eczema, psoriasis, heart failure, heart disease, artery sclerosis, miocardial infarction, acute coronary syndrome, ISR after vascular surgery, hypertension and vasculitis, dermatitis, atopic dermatitis, acne, nettle rash, systemic lupus erythematosus, AIDS, AIDS dementia, chronic neurodegenerative disease, chronic ache, priapism, tumour fibre modification, amyotrophic lateral sclerosis, ALS and muscular dystrophy, myodystony, myasthenia gravis (M.G.), schizophrenia, depressed, syndrome before passing through, anxiety, habituation is (as medicine, alcohol and nicotine dependence), the tolerance that morphine causes and withrawal symptom, bipolarity illness, stress disorders after wound, anorexia nervosa, ADHD and general self-closing disease obstacle, antimigraine, inflammatory pain and neuralgia, Huntington's disease, epilepsy, nerve degeneration imbalance, gastrointestinal motility imbalance, obesity, hyperingestion, solid tumour (as neuroblastoma), malaria, leukemia, spinal cord fibre modification, injury of lungs, transplant host disease, head damage, CNS wound, hepatitis, kidney failure, pancreatitis, liver disease (as chronic hepatitis C), drug-induced injury of lungs (as Paraquat), macular degeneration, diabetic retinopathy, glaucoma, the retinitis, hearing disability, tinnitus, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, osteoporosis, osteoarthritis, other degenerative symptom of os osseum and cartilage, acute or chronic graft failure or rejection and transplanting versus-host disease, the mucositis relevant with cancer therapy (comprising radiotherapy or chemotherapy), severe burn, platelet aggregation deficiency, the imbalance of homeostasis process, tissue damage etc.
In the preferred specific embodiment of the present invention, with brine shrimp ovum extract-treated macrophage, can obviously reduce and produce and be dose-dependent effect because of the nitric oxide of induced by LPS.
Preferably, the disease relevant to the excessive generation of nitric oxide according to the present invention is inflammation; More preferably, it is chafing.
Inflammation is relevant to many cytohormones, wherein interleukin-6 (Interleukin-6, IL-6) mainly secreted by macrophage and T cell, can stimulate the hyperplasia differentiation of T cell and B cell and the increase of acute protein matter, the important cells hormone of Earlier period of inflammation reaction (acute phase reaction) during for infection.
In the preferred specific embodiment of the present invention, with brine shrimp ovum extract-treated macrophage, the generation of IL-6 is also suppressed by brine shrimp ovum extract, and the generation that particularly can suppress IL-6 reaches 38%.And described brine shrimp ovum extract is not by due to cytotoxicity on the impact of proinflammatory disease cytohormone.Therefore can effectively increase phagocytic activity according to brine shrimp ovum extract of the present invention, and reduce disengaging of inflammation hormone, can strengthen immunity and can suppress inflammation.
Brine shrimp ovum extract according to the present invention is feasible healing potion; it does not cause toxicity to normal cell; in the preferred specific embodiment of the present invention; with brine shrimp ovum extract with do not add extract group and process normal skin fibroblast; brine shrimp ovum extract-treated groups has higher cell to attach and survival ratio compared to not adding extract group, and known brine shrimp ovum extract has the effect of protection normal cell growth.
Those skilled in the art can select suitable route of administration and dosage to treat without difficulty.According to the present invention, preferred approach is Orally administered.Dosage is determined according to the essence of disease and situation thereof, the individual situation such as age and health status, route of administration and pre-treatment.Those skilled in the art should understand dosage and can change along with individual age, body weight, health status and other correlative factor.
Following non-limiting example contributes to those skilled in the art to implement the present invention.Described embodiment should not be considered as exceedingly limiting the present invention.Those skilled in the art can modify and change embodiment discussed in this article in the situation that not deviating from spirit of the present invention or category, and still belong to scope of the present invention.
Brief description of the drawings
Fig. 1 has shown the 2nd group of room temperature high pressure liquid chromatography figure that extract carries out in embodiment 1.
Fig. 2 has shown the 3rd group of room temperature high pressure liquid chromatography figure that extract carries out in embodiment 1.
Fig. 3 has shown the 4th group of room temperature high pressure liquid chromatography figure that extract carries out in embodiment 1.
Fig. 4 has shown the 5th group of room temperature high pressure liquid chromatography figure that extract carries out in embodiment 1.
Fig. 5 a has shown the result figure examining under a microscope with the normal skin fibroblast that does not add extract-treated.
Fig. 5 b has shown the result figure examining under a microscope with the normal skin fibroblast that the 2nd group of extract (1mg/mL) in embodiment 1 processed.
Fig. 5 c has shown the result figure examining under a microscope with the normal skin fibroblast that does not add extract-treated.
Fig. 5 d has shown the result figure examining under a microscope with the normal skin fibroblast that the 2nd group of extract (2mg/mL) in embodiment 1 processed.
Fig. 6 has shown with each group extract, trehalose in embodiment 1 or has not added after extract-treated macrophage, through the nitric oxide growing amount analysis result figure of induced by LPS.
Fig. 7 has shown with the 2nd group of extract in embodiment 1 or has not added after extract-treated macrophage, through the IL-6 of induced by LPS growing amount analysis result figure.
Fig. 8 has shown with the 2nd group of extract in embodiment 1 or has not added after extract-treated macrophage, through the MTT of induced by LPS analysis of cell proliferation result figure.
Detailed description of the invention
The preparation of embodiment 1, brine shrimp ovum extract
The present embodiment provides respectively five groups of extracts:
The 1st group: untreated brine shrimp ovum
The 2nd group: brine shrimp ovum is placed in to 3% salt solution 6 hours
The 3rd group: brine shrimp ovum is irradiated 18 hours under UV
The 4th group: brine shrimp ovum is boiled 10 minutes in 100 DEG C
The 5th group: by brine shrimp ovum at 2N H
2sO
4in leave standstill 30 minutes
Every group weighs 5g, after processing respectively, filter and clean with pure water, mix with the water containing 1: 1000 protease inhibitors of 6 times of amounts respectively, and with cell crushing instrument (Planetary micro mill machine) in 500rpm process 5 minutes, smash, and in centrifugal 20 minutes of 4 DEG C of 4500rpm, extract after filtration, concentrated, freeze drying, be stored in-20 DEG C of refrigerator-freezers for subsequent use.
Embodiment 2, the survival rate analysis of brine shrimp ovum extract to normal skin fiber mother cell
The mouse macrophage strain RAW264.7 that the present embodiment uses and NIH 3T3 normal skin fibroblast be purchased from Hsinchu Foodstuff Industrial and Development Inst., and Growth of Cells is including in the cell culture fluid of the DMEM of 10% hyclone and 1mM Sodium Pyruvate (Dulbecco ' s modified Eagles medium).Above-mentioned cell is placed in 37 DEG C of incubators and is cultivated.
By 6 × 10
4nIH 3T3 normal skin fibroblast be inoculated in six porose discs, and overnight incubation, then use 1 to 2mg/ml as in embodiment 1 the 2nd group, or do not add extract-treated 5 days, under microscope, take a picture (10X), its result is as shown in Fig. 5 a to Fig. 5 d.
Lacking CO
2state under, brine shrimp ovum extract-treated groups cell kenel and do not add extract group and have variation slightly, the cell of the dendron shape of the 2nd group forms less, and it is in the majority to present the cell of round shape; But with having after brine shrimp ovum extract-treated, higher cell attaches, the ratio of tiling, and has more tree-shaped cynapse to produce.Fibroblast to adhere to stretching be necessary for hyperplasia, the fibroblast president under stable state goes out that filopodia, cytoplasm are netted, the kenel of tiling and the number of growth increases significantly.Therefore add the cell of brine shrimp ovum extract to continue and stably growth, under harsh environment, add brine shrimp ovum extract and there is the Normocellular effect of protection.
By 6 × 10
4nIH 3T3 normal skin fibroblast be inoculated in six porose discs, and overnight incubation, then use 1 to 2mg/ml as in embodiment 1 the 2nd group, or do not add extract-treated 5 days.In the culture plate of removal clarified solution, every hole adds 2% crystal violet of 300 μ L, and in room temperature dyeing 15 minutes, then cleans to remove dead cell with mobile running water, and dry 3 hours.Use again 30% acetate dissolution of 1mL, and read with ELISA instrument under OD 580nm, and calculate cell survival rate.Its result is as shown in table 1.
Table 1:
Lacking CO
2state under, after the brine shrimp ovum extract-treated NIH3T3 normal skin fibroblast of variable concentrations 5 days, the standard of comparison of extract group as cell survival rate will do not added, can find out from crystal violet method test result, brine shrimp ovum extract-treated groups has higher cell to attach and survival ratio compared to not adding extract group.Described result is presented at and lacks CO
2state under, brine shrimp ovum extract have protection normal cell growth effect.
Embodiment 3, brine shrimp ovum extract are to suppressing nitric oxide production generation analysis
Suppress one of main method that is generated as assessment anti-inflammatory activity of nitric oxide free radical (NO), its principle is for utilizing lipopolysaccharides (lipopolysaccharide, LPS) stimulate mouse macrophage RAW264.7, simulation when inflammatory reaction by inducible nitric oxide synthase (inducible nitric oxide synthase, iNOS) produce a large amount of free radicals, and the ability of utilizing extract to remove NO free radical is assessed composition and whether is had anti-inflammatory activity.
The analysis determining method of nitrite concentration is with Griess reagent [containing 0.1%N-(1-naphthyl) ethylenediamine (N-(1-naphthyl) ethylenediamine) and 1% sulfanilamide (SN) (Sulfanilamide) in 5% phosphoric acid] and nitrite reaction, the change color generating, under 548nm, measure trap, after contrast trap-concentration standard curve, can obtain NO
2content in tissue culture medium.Because the half-life of NO is very short, can be oxidized to rapidly nitrite, more further be oxidized to nitrate, therefore can use at short notice Griess reactant to measure the amount of nitrite, come burst size (people such as Paul, 1994, the Anal Biochem.1982Oct of secondary indication NO; 126 (1): 131-8).
By 6 × 10
4rAW264.7 macrophage be inoculated in six porose discs, and overnight incubation, again with 0.25 to 1mg/ml as the 2nd group to the 4th group, trehalose in embodiment 1 or do not add extract-treated 1 hour, add the lipopolysaccharides of 100ng/mL to process 2 days, and gather in the crops clarified solution sample.
First mix N-(1-naphthyl) ethylenediamine of 5 μ L and the sulfanilamide (SN) of 5 μ L, add again sample or the standard items (nitrite of 0 to 25 μ M) of 75 μ L, and using ultra-pure water as blank group, add again after the ultra-pure water of 65 μ L, cultivate described mixture 30 minutes in room temperature lucifuge, then read the light absorption value of OD 548nm with ELISA instrument.
Its result as shown in Figure 6.
In macrophage by the 4th group of extract-treated, NO produces obviously and reduces; The brine shrimp ovum extract relatively adding with variable concentrations (0.5mg/ml and 0.25mg/mL), 0.5mg/ml has the ability that suppresses better NO generation, this has confirmed to obtain according to the present invention extract and has had the effect that suppresses NO generation, and is dose-dependent effect.
Using iNOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) as positive controls, 10 μ M L-NAME have the activity that suppresses RAW264.7 macrophage generation NO as a result simultaneously.
Trehalose is one of composition having in shrimps the effect of protection resting egg, but trehalose does not have the activity that significantly suppresses RAW264.7 macrophage generation NO in described embodiment.
Embodiment 4, the impact of brine shrimp ovum extract on proinflammatory hormone IL-6
Mouse macrophage strain RAW264.7 is implanted in 24 porocyte culture plates, in 37 DEG C of incubators, cultivate after 3 hours, add as in embodiment 1 the 2nd group, or do not add the cell culture fluid of extract and lipopolysaccharides (100ng/mL), cultivate respectively after 18 hours, collecting cell culture supernatant, and store the analysis in order to follow-up cytohormone in-80 DEG C of refrigerators.
It is to adopt mouse IL-6ELISA kit (R & D system, USA) that cytohormone analysis is measured.By ELISA instrument detection OD 450nm trap, in cell culture supernatant, IL-6 concentration is tried to achieve with interpolation calibration curve.
The result of IL-6 as shown in Figure 7.Result shows, along with the increase of brine shrimp ovum extract doses, can obviously reduce the nitric oxide generation because of induced by LPS.And the generation of IL-6 is also suppressed by brine shrimp ovum extract.PRELIMINARY RESULTS shows the brine shrimp ovum extract that adds variable concentrations (2mg/ml, 1mg/ml and 0.5mg/mL), and 2mg/ml can suppress the generation of IL-6 better.The generation that brine shrimp ovum extract (the 2nd group) suppresses IL-6 in the time that concentration is 2mg/mL reaches 32.
For confirming that extract is not by due to cytotoxicity, therefore carry out Cytotoxic test with MTT analytic approach on the impact of proinflammatory cytohormone.3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl tetrazole bromine salt (MTT, [3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide]) analytic approach is mainly tetrazolium nitrogen salt (tetrazolium salt) can be reduced into formazan (formazan) according to the succinate dehydrogenase (succinate dehydrogenase) in grain line body in living cells, the growing amount of the formazan of blue brown is directly proportional to living cells number, can utilize enzyme linked immunosorbent assay (ELISA) instrument (ELISAreader) to read trap in 570nm wavelength, the trap of each groove represents cell survival rate with the ratio of reference cell trap.
By 6 × 10
4rAW264.7 macrophage be inoculated in six porose discs, and overnight incubation, use again 0.25 to 1mg/ml as in embodiment 1 the 2nd group, or do not add extract-treated 1 hour, add the lipopolysaccharides of 100ng/mL to process 2 days, to remove after every hole PBS cleaning of culture plate of clarified solution, add the MTT solution (0.5mg/ml PBS) of 500 μ L, and in 37 DEG C of dyeing 30 minutes, remove after supernatant, add the DMSO of 500 μ L to process 5 minutes, and read with ELISA instrument under OD 570nm, and calculate cell survival rate.Its result as shown in Figure 8.
After processing RAW264.7 macrophage 2 days with the brine shrimp ovum extract of variable concentrations (1,0.5 and 0.25mg/mL), not add the standard of comparison of extract group as cell survival rate, with MTT analysis of cell proliferation method test result.Brine shrimp ovum extract-treated groups can't affect the survival ratio of RAW264.7 cell.It is shown as extract is not by due to cytotoxicity on the impact of proinflammatory cytohormone.
Statistical analysis
Adopt Statistical Analysis System (SAS) software to carry out statistical analysis, with variance analysis (Analysis of variance, ANOVA) carry out statistical disposition, and with significant difference degree (p < 0.05) between the more each factor of Duncan ' s test, the data obtained represents with mean value ± standard deviation (Mean ± SD).
Claims (11)
1. a purposes for brine shrimp ovum extract, it is for the preparation of the medicine that prevents and/or treats the disease relevant to the excessive generation of nitric oxide.
2. purposes according to claim 1, the wherein said disease relevant to the excessive generation of nitric oxide is inflammation.
3. purposes according to claim 1 and 2, the method for wherein preparing described brine shrimp ovum extract comprises the following step:
A) provide brine shrimp ovum;
B) adverse circumstance is processed described brine shrimp ovum; With
C) homogenization step brine shrimp ovum b).
4. purposes according to claim 3, wherein step adverse circumstance b) is selected the group of free salt adverse circumstance, UV adverse circumstance, hot adverse circumstance and sour adverse circumstance composition.
5. purposes according to claim 3, it also comprises removes the c) cell fragment of the brine shrimp ovum that homogenizes of gained of step, and obtains clarified solution.
6. purposes according to claim 3, wherein step c) comprises:
C1) with the water blend step of approximately 3 to approximately 10 times brine shrimp ovum b), to obtain brine shrimp ovum aqueous mixtures;
C2) homogenization step c1) brine shrimp ovum aqueous mixtures; With
C3) remove step c2) cell fragment homogenizing in brine shrimp ovum aqueous mixtures of gained, and obtain clarified solution.
7. purposes according to claim 6, wherein step c3) by centrifugal removal step c2) cell fragment homogenizing in brine shrimp ovum aqueous mixtures of gained.
8. purposes according to claim 1 and 2, wherein said brine shrimp ovum extract carries out high pressure lipuid chromatography (HPLC), and the chromatographic column of use is
250-4HPLC-Cartridge NH
2, 5 μ m, column oven temperature is 25 DEG C, and sampling volume is 10 μ L, and flow velocity is 1.0mL/min, and be 30min running time, mobile phase is CH
3cN:H
2o=4:1 volume ratio, retention time is to locate to have absworption peak in 2.43 to 2.45,3.12 to 3.15,4.59 to 4.61,5.05 and 5.57 to 5.63 minutes.
9. the purposes of a composition that contains brine shrimp ovum extract, it is for the preparation of the medicine that prevents and/or treats the disease relevant to the excessive generation of nitric oxide, and described composition comprises brine shrimp ovum extract and selectable pharmaceutically acceptable carrier, diluent or the excipient for the treatment of effective dose.
10. purposes according to claim 9, the wherein said disease relevant to the excessive generation of nitric oxide is inflammation.
11. according to the purposes described in claim 9 or 10, and wherein said composition is food compositions, medical composition or cosmetic composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110332791.4A CN103083361B (en) | 2011-10-28 | 2011-10-28 | Fairy shrimp egg extract, preparation method and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110332791.4A CN103083361B (en) | 2011-10-28 | 2011-10-28 | Fairy shrimp egg extract, preparation method and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103083361A CN103083361A (en) | 2013-05-08 |
| CN103083361B true CN103083361B (en) | 2014-12-03 |
Family
ID=48196841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110332791.4A Active CN103083361B (en) | 2011-10-28 | 2011-10-28 | Fairy shrimp egg extract, preparation method and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103083361B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104974207B (en) * | 2014-04-03 | 2017-07-28 | 珀莱雅化妆品股份有限公司 | A kind of method that GP4G is extracted in the artemia cysts from salt water |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5104668A (en) * | 1991-01-24 | 1992-04-14 | Salt Lake Brine Shrimp, Inc. | Process for treating unhatchable Artemia brine shrimp cysts |
| CN1122458C (en) * | 1998-10-30 | 2003-10-01 | 芦政运 | Edible salted shrimp protein powder and its processing method |
| CN1137631C (en) * | 2000-10-16 | 2004-02-11 | 中国科学院动物研究所 | Fengnian sweet potato chips with shrimp and their production process |
| RU2346697C9 (en) * | 2006-07-03 | 2010-10-27 | Общество с ограниченной ответственностью "Геонек-Сиб" | Biologically active supplement based on cysts of maxillopod artemia salina |
-
2011
- 2011-10-28 CN CN201110332791.4A patent/CN103083361B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103083361A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100336521C (en) | Composition comprising melissa leaf extract for anti-angiogenic and matrix metalloproteinase inhibitory activity | |
| JP2003171310A (en) | Skin barrier function-reinforcing agent | |
| TW200423961A (en) | Composition for promoting production of type I collagen and/or elastin | |
| JP4931356B2 (en) | Glutamate decarboxylase activator | |
| AU2021236518B2 (en) | A highly concentrated seawater mineral extract and uses thereof | |
| CN105476950B (en) | Moisturizing foam mildy wash and preparation method thereof | |
| CN109517748A (en) | Black food small-molecular peptides are repairing the medical application in hepatic injury | |
| KR20210021376A (en) | Method for obtaining aqueous extract of Dunaliella salina and cosmetic use thereof | |
| CN103083361B (en) | Fairy shrimp egg extract, preparation method and uses thereof | |
| WO2012117170A2 (en) | Extract of winter branches of hippophae and use thereof in a cosmetic composition | |
| CN1607956A (en) | Topical cosmetic composition having a natural plant active ingredient and method of using same | |
| CN103251652B (en) | Adversity brine shrimp egg extract, preparation method and application thereof | |
| JP4109099B2 (en) | Screening method for active ingredient of hair restorer and its use | |
| CN105943430A (en) | Cosmetic composition having function of removing acne | |
| KR100475196B1 (en) | The health-supporting food being effective the hairloss preventation or hair-growth and its preparation method | |
| CN105581965B (en) | Conditioning-type women private portion conditioning liquid is sterilized in nutrition | |
| CN107712880A (en) | A kind of peony pollen nutrient tablet and preparation method thereof | |
| JPH10120558A (en) | Skin preparation for improving sputum for external use | |
| TWI504398B (en) | Extracts of artemia cysts, preparation method thereof and use of the same | |
| JP2007119433A (en) | Useful material such as cosmetic or health food obtained for formulating sake lees extract with rhodiola rosea extract and the like | |
| Pannu et al. | The green blood” wheatgrass juice, a health tonic having antibacterial potential | |
| JP2010024190A (en) | Skin barrier function ameliorator | |
| JP2011057596A (en) | Tryptase activity inhibitor and use thereof | |
| KR102448962B1 (en) | Manufacturing method of hypermineral ionized water, hypermineral ionized water manufactured by the same and cosmetic composition for skin keratin comprising the same | |
| JPS5995210A (en) | Cosmetic |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |