WO2016037166A1 - Nouvelles compositions anti-oxydante et procédés d'administration - Google Patents
Nouvelles compositions anti-oxydante et procédés d'administration Download PDFInfo
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- WO2016037166A1 WO2016037166A1 PCT/US2015/048772 US2015048772W WO2016037166A1 WO 2016037166 A1 WO2016037166 A1 WO 2016037166A1 US 2015048772 W US2015048772 W US 2015048772W WO 2016037166 A1 WO2016037166 A1 WO 2016037166A1
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- extract
- agents
- antioxidants
- mixtures
- antioxidant
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0043—Nose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
- A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
- A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
Definitions
- the present invention relates to methods and compositions for treating respiratory symptoms caused by inhaled particulate matter.
- Air pollution is made up of a mixture of small particles in the air, and often characterized by obvious smog hanging over places where people live.
- the problem of air pollution has increased due to the rapid pace at which economies of the world are developing.
- the components that contribute to the ambient air pollution include fine particles produced during energy production (e.g., coal consumption, fuel burning), ozone (formed by chemical reactions of oxygen molecules under solar ultraviolet radiation).
- WHO World Health Organization
- the invention provides a method of treating respiratory symptoms caused by inhaled particulate matter comprising: providing an antioxidant pharmaceutical composition comprising, consisting essentially of, or consists of: a plant extract; and at least one of: vitamin A, vitamin B, vitamin C, vitamin E, minerals, ubiquinone and its derivatives, thiol- based antioxidants, saccharide-containing antioxidants, polyphenols, enzyme and small molecule antioxidants, and salts thereof; and
- the invention provides an antioxidant pharmaceutical composition
- an antioxidant pharmaceutical composition comprising, consisting essentially of, or consists of: a plant extract; and at least one of: vitamin A, vitamin B, vitamin C, vitamin E, minerals, ubiquinone and its derivatives, thiol-based antioxidants, saccharide-containing antioxidants, polyphenols, enzyme and small molecule antioxidants, and salts thereof.
- composition having a plant extract, and vitamin A, vitamin B, vitamin C, vitamin E, minerals, ubiquinone and its
- antioxidants derivatives, thiol-based antioxidants, saccharide-containing antioxidants, polyphenols, enzyme and small molecule antioxidants, mixtures thereof.
- the present invention provides methods and compositions for treating respiratory symptoms caused by inhaled particulate matter.
- the methods include the administration of the disclosed pharmaceutical compositions before, during, and after exposure to inhaled particulate matter.
- the pharmaceutical composition comprises a plant extract; and vitamin A, vitamin B, vitamin C, vitamin E, minerals, ubiquinone and its derivatives, thiol-based antioxidants, saccharide-containing antioxidants, polyphenols, plant extracts, enzyme and small molecule antioxidants, mixtures thereof, and salts thereof.
- suitable plant extracts include: acai extract, aloe vera extract, astragalus extract, auricularia auricula-judae extract, bilberry extract, bitter orange extract, black cohosh extract, bromelain extract, butterbur extract, cat's claw extract, chamomile extract, chasteberry extract, cinnamon extract, cranberry extract, dandelion extract, dendranthema morifolium extract, echinacea extract, essiac tea extract, Flor Essence tea extract, Sambucus nigra (European elder) extract, Evening Primrose extract, fenugreek extract, feverfew extract, flaxseed extract, ganoderma lucidium (reishi mushroom) extract, garlic extract, ginger extract, ginkgo biloba extract, ginseng extract, glycyrrhiza glabra (licorice) extract, goldenseal extract, grape seed extract, green tea extract, hawthorn extract, hood
- Extracts from plants may be obtained by any known means.
- thiol-based antioxidants include isothiocyanates, sulfides, glutathione and its derivatives, thioredoxin, cysteine and N-acetylcysteine, dithiol compounds, prothiol compounds, and mixtures thereof.
- dithiols include lipoic acid.
- prothiol compounds include L-2-oxothiazolidine-4-carboxylate.
- polyphenols examples include phenolic acids, ferulic acid, caffeic acid, cinnamic acid, phenylethyl ester, apigenin, pycnogenol, carotenoids, flavonoids, soy isoflavones, genistein, daidzein, equol, tretinoin, silymarin, quercetin, tannins, resveratrol, tea polyphenols, catechin, epicatechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate, and mixtures thereof.
- minerals include selenium, sodium, potassium, magnesium, calcium, zinc, copper, iron, chromium, fluorine, chlorine, iodine, phosphorous, and mixtures thereof.
- Suitable enzyme and small molecule antioxidants include superoxide dismutases, catalase, glutathione peroxidase, NADPH:quinone reductase, thioredoxin system, coenzyme Q, glutathione, bilirubin, melatonin, ferritin, L-carnitine, alpha-lipoic acid, metallothioneins, acetyl-L-carnitine, edaravone, hydroxytyrosol, tyrosol, ladostigil, mofegiline, N-acetylcysteine, N-acetylserotonin, oleocanthal, oleuropein, rasagiline, selegiline, uric acid, butylated hydroxytoluene, 2,6-di-tert-butylphenol, 1,2-diaminopropane, 2,4-dimethyl-6-tert- butylphenol, ethylenedi
- free form means the original chemical form, not the salt or any other chelating form (like dimers, or polymers).
- the pharmaceutical composition comprises an active ingredient, wherein the active ingredient consists of: a) a plant extract, and b) an antioxidant.
- the composition consists of: a) plant extract, b) an antioxidant, and/or salt thereof, and c) at least one carrier and/or excipient.
- the pharmaceutical composition consisting essentially of the active ingredients of: a) plant extract, and b) an antioxidant and/or salt thereof. That is, any other ingredients that may materially affect the basic and novel characteristics of the active ingredients of the invention are specifically excluded from the composition. Any ingredient which can potentially cause an undesirable effect/side effect, including, for example, an allergic response, may materially affect the basic and novel characteristics of the active ingredients of the invention.
- nasal decongestant and antihistamines are some examples of components which may materially affect the basic and novel characteristics of the active ingredients of the pharmaceutical compositions and may be excluded from certain embodiments of the present invention: nasal decongestant and antihistamines.
- the aforementioned ingredients may materially change the characteristics of the present pharmaceutical composition due to unwanted effects and/or potential allergic responses.
- decongestants are not desired in some embodiments of the invention.
- a decongestant is pseudoephedrine.
- unwanted potential effects of pseudoephedrine include nausea, vomiting, trouble sleeping, dizziness, headache, or nervousness may occur.
- antihistamines are not desired in some embodiments of the invention. Examples of unwanted potential effects of antihistamines include dry mouth, drowsiness, dizziness, nausea, vomiting, restlessness, moodiness, trouble urinating or not being able to urinate, blurred vision, and confusion.
- the pharmaceutical formulation does not include decongestants. In yet another embodiment, the pharmaceutical formulation does not include antihistamines.
- Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
- an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
- pharmaceutically-acceptable carriers include, but are not limited to, saline, Ringer's solution and dextrose solution.
- the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
- the following buffer pairs have been contemplated: citric acid/ potassium citrate, potassium acetate/ acetic acid, sodium hydrogen phthalate/ potassium hydroxide, potassium hydrogen phthalate / potassium dihydrogen orthophosphate, sodium hydrogen phthalate / sodium dihydrogen orthophosphate, sodium dihydrogen orthophosphate / potassium hydroxide, barbitone potassium / hydrochloric acid, tris (hydroxylmethyl) aminomethane / hydrochloric acid, potassium tetraborate/ hydrochloric acid to maintain a pH of the composition within the range of about 6.8 to 8.6.
- the preferred formulation is that of a liquid.
- excipients have been contemplated: gums, granulating agents, binders, lubricants, disintegrating agents, sweetening agents, additives, solvents, glidants, anti-adherents, anti-static agents, surfactants, viscosity enhancers, plant cellulosic material coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, antistatic agents, spheronization agents, and any combinations thereof.
- the preferred formulation is that of a dry powder or a solid aerosol. Accordingly, the following excipients have been contemplated: sugars and saccharides, preferably inhalation grade lactose, preferably alpha monohydrate lactose in the form of crystalline lactose, milled lactose or micronized lactose.
- a composition of the present invention includes aqueous nasal or oral spray composition having: 0.10-5.0% by weight/volume of an antioxidant and mixtures thereof; 0.10-5.0% by weight/volume of a plant extract and mixtures thereof; 1.0-20.0% by weight/volume of an excipient and mixtures thereof; a sufficient amount of a pharmaceutically acceptable buffer to maintain the pH of the composition within the range of about 6.8 to 8.6; and water.
- the methods of the present invention comprise the administration of a therapeutically effective amount of the composition to a human in an amount which is effective to inhibit, treat, or alleviate respiratory symptoms caused by inhaled particulate matter.
- therapeutically effective amount is defined as the amount necessary to achieve the desired outcome.
- the term “inhibit” includes “reduce and/or “prevent” and/or “shorten duration.” That is, the method of the present invention is considered to be effective if it causes one or more of: a reduction/inhibition/prevention of any symptom associated with inflammation of the respiratory tract.
- Reduction of symptoms can be assessed by comparing the magnitude and/or duration of at least one respiratory symptom in a subject at two different occasions, that is, i) when administered the pharmaceutical composition, and then the subject is exposed to inhaled particulate matter; and ii) when not administered the composition, and then the subject is exposed to inhaled particulate matter.
- the respiratory symptoms are reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.
- compositions of the present invention provide a unique and synergistic combination of effects.
- the antioxidants provide reduction/prevention of oxidative damage and the plant extracts provide immune support to the body. Accordingly, the combination provides an unexpected efficacy in the treatment of respiratory symptoms caused by inhaled particulate matter.
- PM Particulate matter
- PM refers to small solid particles and/or liquid matter existing in the air.
- PM can be sub-categorized, depending on the particle size and penetration to lung tissue, coarse particles, namely PM10 (inhalable particles less than or equal to 10 ⁇ in diameter), and fine particles, namely PM2.5 (inhalable particles less than or equal to 2.5 ⁇ in diameter), both of which are generated mostly by air pollution.
- PM2.5 comprises water-soluble inorganic ions (sulfate, nitrate, ammonium and sodium, magnesium, calcium, aluminum, etc.), organic carbon compounds and elements (iron, or lead, zinc, arsenic, cadmium, etc.).
- PM2.5 Because of the particles size of PM2.5, it can stay in air for a very long time, and increase the likelihood of respiratory symptoms of people who spend time outdoors. These symptoms may be localized to the respiratory system, including the nasal passages and the lungs, or the symptoms may be systemic.
- Symptoms localized to the respiratory system include inflammation of the lungs or nasal passages. Inflammation of the lungs includes pneumonitis, or inflammation of lung tissue, or asthma. Inflammation of the nasal passages includes sinusitis, or inflammation of nasal tissue. These symptoms may be acute or chronic.
- Lung disease includes at least for example, chronic obstructive pulmonary disease (COPD), lung cancer, and granulomatous lung diseases.
- COPD chronic obstructive pulmonary disease
- the composition is administered before the onset of symptoms.
- administration is at most about 120 minutes before exposure to inhaled particulate matter, at most about 90 minutes before exposure to inhaled particulate matter, at most about 60 minutes before exposure to inhaled particulate matter, at most about 30 minutes before exposure to inhaled particulate matter, at most about 20 minutes before exposure to inhaled particulate matter, just right before exposure to inhaled particulate matter, or simultaneously exposure to inhaled particulate matter.
- administration is at most about 120 minutes before exposure to inhaled particulate matter, at most about 90 minutes before exposure to inhaled particulate matter, at most about 60 minutes before exposure to inhaled particulate matter, at most about 30 minutes before exposure to inhaled particulate matter, at most about 20 minutes before exposure to inhaled particulate matter, just right before exposure to inhaled particulate matter, or simultaneously exposure to inhaled particulate matter.
- the composition is administered before the onset of symptoms.
- administration is at most about 120 minutes before exposure to
- composition can be administered up to about 2 hours to about 4 hours after exposure to inhaled particulate matter, but before the onset of symptoms.
- composition of the present invention can be administered by any inhalation methods known in the art.
- the pharmaceutical composition can be administered through the nose or the mouth.
- numerous specific details are set forth in order to provide a thorough understanding of the present embodiments. It will be apparent, however, to one having ordinary skill in the art that the specific detail need not be employed to practice the present
- the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof, are intended to cover a non-exclusive inclusion.
- a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.
- a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
- any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples and illustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.”
- each member may be combined with any one or more of the other members to make additional sub-groups.
- additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
- Plants (10.0 g) are added to a mixture of 500 mL of distilled water and 500 mL of methanol in a 2 L round-bottom flask and reflux for 30 minutes.
- the solution is cooled down to room temperature, filtered through a buchner funnel and the filtrate is collected, followed by passing through a 0.2 ⁇ membrane into a 2 L round-bottom flask.
- the solution is evaporated under reduced pressure on a rotary evaporator to obtain 1.0 g crude extract.
- the crude extract is dissolved in ethyl acetate (2.0 mL) and then
- Total Phenol Count (TPC) assay Folin-Ciocalteu assay is used to determine the total phenol count of the antioxidants. Folin-Ciocalteu reagent is made by dissoving 10 g sodium tungstate and 2.5 g sodium molybdate in 70 mL water, adding 5 mL 85% phosphoric acid and 10 mL concentrated hydrochloric acid, refluxing for 10 hrs, adding 15 g lithium sulfate, 5 mL water and 1 drop bromine, and refluxing for another 15 min, and cooling to room temperature and bringing to 100 mL with water, eventually forming the hexavalent phosphomolybdic-phosphotungstic acid
- O 2 scavenging activity Nitroblue tetrazolium (NBT) is used as a standard to evaluate the capacity of aliquots of antioxidants (50, 100, 200 and 300 ⁇ g) through the photochemical reduction in the riboflavin-light-NBT system. 3 mL of solution contained 50 mM phosphate buffer, 13 mM methionine, 2 ⁇ riboflavin, 100 ⁇ EDTA, 75 ⁇ NBT and 100 ⁇ ⁇ solution of various concentrations of the antioxidants. The increase in absorbance at 560 nm after 10 min illumination is monitored from a fluorescent lamp with respect to the production. H 2 O 2 scavenging activity: Antioxidants (50, 100, 200 and 300 ⁇ g) are dissolved in H 2 0
- Rats in Group 5-8 are exposed according to four different daily exposure protocols, 4 h air followed by intratracheal instillation of PM2.5 (0.2, 0.8, 3.2 mg/rat).
- Bronchoalveolar Lavage Fluids Cell Differentiation and Biochemical Analysis Twenty-four hours after the last intratracheal instillation of air or PM2.5, rats are anesthetized with chloral hydrate (30 mg/kg, i.p.). Aliquot of the recovered lavage fluid is taken and centrifuged (1500 rpm for 10 min at 4 °C) and cells are collected for cell counts by a hemocytometer and differentiation. Standard clinical laboratory method is used to determine the lactate dehydrogenase (LDH) activities in supernatants. ELISA kits are used to determine the activities of interleukin (IL)-2 and tumor necrosis factor alpha (TNF-a).
- IL interleukin
- TNF-a tumor necrosis factor alpha
- the colorimetric assay kits are used to determine the albumin and activity of alkaline phosphatase (AKP). Histopathological Examination with Hemotoxylin and Eosin (HE) Staining: The lung apex is put in a solution of 4% paraformaldehyde for at 4 °C 12 hrs. After fixation, paraffin-embedded tissues are sectioned at 5 ⁇ and are stained with HE, Cuts by ultramicrotome are mounted and stained with lead citrate, and then electron microscopy is used for observation.
- alkaline phosphatase ADP
- HE Hemotoxylin and Eosin Staining
- Buffer sodium dihydrogen orthophosphate / 0.50-1.0% potassium hydroxide
- Antioxidant vitamin E 0.15%
- Plant extract ziziphus jujuba extract 0.25%
- Buffer sodium dihydrogen orthophosphate / 0.50-1.0% potassium hydroxide
- Antioxidant Resveratrol 0.20%
- Buffer sodium dihydrogen orthophosphate / 0.50-1.0% potassium hydroxide
- Antioxidant vitamin B 20%
- Antioxidant quercetin 15%
- Plant extract tremella fuciformis extract 15%
- Plant extract 0.001-2% water soluble polyvinylpyrrolidone having an 0.05-15.00% average molecular weight of about 10,000 to
- Aromatic alcohol 0.20-5.00%
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Abstract
La présente invention concerne des compositions et des méthodes liées au traitement de symptômes respiratoires causée par des matières particulaires inhalées. Dans un mode de réalisation, le procédé comprend l'administration d'une composition comprenant un extrait de plante et un anti-oxydant par une voie d'inhalation.
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RU2717304C1 (ru) * | 2019-05-21 | 2020-03-19 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кузбасская государственная сельскохозяйственная академия" | Способ производства биологически активной добавки |
US10717825B2 (en) | 2015-07-01 | 2020-07-21 | California Instite of Technology | Cationic mucic acid polymer-based delivery system |
US11285212B2 (en) | 2013-03-01 | 2022-03-29 | California Institute Of Technology | Targeted nanoparticles |
CN115029326A (zh) * | 2022-05-27 | 2022-09-09 | 湖南福来格生物技术有限公司 | 一种酶交联聚集体及其制备方法和应用 |
US11890315B2 (en) | 2017-11-17 | 2024-02-06 | Renovion, Inc. | Stable ascorbic acid compositions and methods of using same |
US11998616B2 (en) | 2018-06-13 | 2024-06-04 | California Institute Of Technology | Nanoparticles for crossing the blood brain barrier and methods of treatment using the same |
US12097238B2 (en) | 2022-01-04 | 2024-09-24 | Renovion, Inc. | Aqueous solution comprising a glutathione salt |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US11285212B2 (en) | 2013-03-01 | 2022-03-29 | California Institute Of Technology | Targeted nanoparticles |
US10717825B2 (en) | 2015-07-01 | 2020-07-21 | California Instite of Technology | Cationic mucic acid polymer-based delivery system |
US11041050B2 (en) | 2015-07-01 | 2021-06-22 | California Institute Of Technology | Cationic mucic acid polymer-based delivery systems |
US11890315B2 (en) | 2017-11-17 | 2024-02-06 | Renovion, Inc. | Stable ascorbic acid compositions and methods of using same |
US11998616B2 (en) | 2018-06-13 | 2024-06-04 | California Institute Of Technology | Nanoparticles for crossing the blood brain barrier and methods of treatment using the same |
RU2717304C1 (ru) * | 2019-05-21 | 2020-03-19 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кузбасская государственная сельскохозяйственная академия" | Способ производства биологически активной добавки |
US12097238B2 (en) | 2022-01-04 | 2024-09-24 | Renovion, Inc. | Aqueous solution comprising a glutathione salt |
CN115029326A (zh) * | 2022-05-27 | 2022-09-09 | 湖南福来格生物技术有限公司 | 一种酶交联聚集体及其制备方法和应用 |
CN115029326B (zh) * | 2022-05-27 | 2024-01-02 | 湖南福来格生物技术有限公司 | 一种酶交联聚集体及其制备方法和应用 |
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