US20180344627A1 - Skin care applications of extracellular metabolites from bacillus coagulans - Google Patents

Skin care applications of extracellular metabolites from bacillus coagulans Download PDF

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US20180344627A1
US20180344627A1 US15/996,725 US201815996725A US2018344627A1 US 20180344627 A1 US20180344627 A1 US 20180344627A1 US 201815996725 A US201815996725 A US 201815996725A US 2018344627 A1 US2018344627 A1 US 2018344627A1
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bacillus coagulans
skin
composition
extracellular metabolite
partially purified
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Muhammed Majeed
Kalyanam Nagabhushanam
Sivakumar Arumugam
Furqan Ali
Shaheen Majeed
Lakshmi Mundkur
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Sami Chemicals and Extracts Ltd
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Assigned to SAMI LABS LIMITED reassignment SAMI LABS LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MAJEED, SHAHEEN, MR
Assigned to SAMI LABS LIMITED reassignment SAMI LABS LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: Majeed, Shaheen, ALI, FURQAN, ARUMUGAM, SIVAKUMAR, MAJEED, MUHAMMED, MUNDKUR, Lakshmi, NAGABHUSHANAM, KALYANAM
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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
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers

Definitions

  • the invention in general relates to probiotics. More specifically, the present invention discloses the skin care applications of extracellular metabolite isolated from Bacillus coagulans MTCC 5856.
  • ROS reactive oxygen species
  • inflammatory markers increasing the oxidative stress and inflammation in the skin cells thereby damaging the cellular biomolecules (lipids, sugars, proteins, and polynucleotides). Protection against these agents will eventually improve the skin texture and lead to graceful aging.
  • the skin cells inherently possess cellular defense systems against ROS and inflammatory agents, which include nonenzymatic molecules (glutathione, vitamins A, C, and E, and several antioxidants present in foods) and enzymatic scavengers of ROS, with superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX).
  • nonenzymatic molecules glutathione, vitamins A, C, and E, and several antioxidants present in foods
  • SOD superoxide dismutase
  • CAT catalase
  • GPX glutathione peroxidase
  • Probiotics are now garnering much attention in the skin care arena.
  • Probiotic of the genera Lactobacillus and Bifidobacteria are reported for treatment of acne, rosacea, protection against aging, photo damage and UV:
  • Probiotics strains alone or in combination with food have shown antioxidant activity and reduce damages caused by oxidation ((Yang Wang, Yapping Wu, Yuanyuan Wang, Han Xu, Xiaogiang Mei, Dongyou Yu, Yibing Wang and Weifen Li, Antioxidant Properties of Probiotic Bacteria, Nutrients. 2017 May; 9(5): 521).
  • the extracelluar metabolities of probiotics are also reported to confer skin protection against microbial infections (U.S. Pat. No. 7,544,363B2). These products are now increasing being used in skin care applications ( Bacillus Ferment, Ganeden Inc, https://cosmetics.specialchem.com/inci/bacillus-ferment, accessed 28 May 2018).
  • the present invention fulfills the above mentioned objectives and provides further related advantages.
  • the present invention discloses the skin care applications of a composition containing, the partially purified extracellular metabolite isolated from Bacillus coagulans MTCC 5856. More specifically, the invention discloses the ability of the extracellular metabolite isolated from Bacillus coagulans MTCC 5856 to confer protection to the skin fibroblasts against UV induced cell damage and apoptosis, oxidative stress and inflammation.
  • FIG. 1 a shows fibroblast cells stained with crystal violet incubated with different concentration of the partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856, conferring UV protection.
  • FIG. 1 b is the graphical representation showing the % protection against UV A exposure by the partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856
  • FIG. 1 c is the graphical representation showing the % protection against UV B exposure by the partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856
  • FIG. 2 a is the graphical representation showing the % reduction in apoptotic cells by the partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856
  • FIG. 2 b shows dot plot of UV-B exposed cells treated with different concentration of partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856 (A) and its histogram (B)
  • FIG. 3 is the graphical representation showing the % ROS scavenging by the partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856
  • FIG. 4 is the graphical representation showing the % DPPH scavenging by the partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.
  • the present invention discloses a method of protecting skin fibroblasts from UV induced damage, said method comprising steps of brining into contact skin fibroblast cells with effective concentration of a composition containing partially purified extracellular metabolite preparation from Bacillus coagulans , to bring about the effect of protection against UV-A and UV-B induced cell damage and apoptosis.
  • the strain of Bacillus coagulans is Bacillus coagulans MTCC 5856.
  • effective concentration of the partially purified extracellular metabolite preparation is 0.01% v/v to 2.0% v/v of the total composition.
  • the invention discloses a composition containing partially purified extracellular metabolite preparation from Bacillus coagulans for use as a skin care agent, wherein the composition confers skin protection by a) protecting the skin against UV-A and UV-B induced cell damage and apoptosis, b) reducing oxidative stress in the skin by scavenging ROS and c) reducing inflammation by decreasing the levels inflammatory markers in the skin.
  • the strain of Bacillus coagulans is Bacillus coagulans MTCC 5856.
  • effective concentration of the partially purified extracellular metabolite preparation is 0.01% v/v to 2.0% v/v of the total composition.
  • composition is formulated with pharmaceutically/cosmeceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and/or incorporated into formulations containing skin care ingredients and administered topically in the form of creams, gels, lotions, powder, serum, oil, suspensions, ointments, soaps, scrubs, emulsions, and compacts.
  • the invention discloses a composition containing partially purified extracellular metabolite isolated from Bacillus coagulans for use as an anti-oxidant and anti-inflammatory agent.
  • the strain of Bacillus coagulans is Bacillus coagulans MTCC 5856.
  • the composition is formulated with pharmaceutically/cosmeceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, and preservatives and/or incorporated into formulations containing skin care ingredients and administered topically in the form of creams, gels, lotions, powder, serum, oil, suspensions, ointments, soaps, scrubs, emulsions, and compacts.
  • the extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861.
  • the product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.
  • UVA radiation The amount of UVA radiation on earth's surface is approximately 20-fold higher than that of UVB. It is responsible for the tanning effects of human skin and had been considered mostly harmless for many years.
  • UV radiation has broad spectrum, ranging from 40 to 400 nm (30-3 eV), which is divided into Vacuum UV (40-190 nm), Far UV (190-220 nm), UVC (220-290 nm), UVB (290-320), and UVA (320-400 nm), of which the latter two are medically important.
  • Vacuum UV 40-190 nm
  • UVC 220-290 nm
  • UVB 290-320
  • UVA 320-400 nm
  • UVA The amount of exposure to UVA usually remains constant, whereas UVB exposure occurs more in the summer UV radiations are responsible for high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma.
  • UVB irradiation has been demonstrated to produce ROS in the cells and skin, which induces the synthesis of matrix metalloproteinases (MMPs), causing photoaging effects in skin.
  • MMPs matrix metalloproteinases
  • UVA up regulates the formation of matrix metalloproteinase (MMPs), enzymes that degrade the matrix protein's elastin and collagen, which, if not prevented, can result in marked reduction in skin elasticity and increased wrinkling UVA can penetrate deeper into the skin in comparison to UVB and contributes to photoaging, photocarcinogenesis and photodermatosis and increase in reactive oxygen species (ROS) in fibroblasts and cells which are deeper inside the skin.
  • MMPs matrix metalloproteinase
  • ROS reactive oxygen species
  • HDFa Human dermal fibroblasts
  • Balb/C 3T3 Human dermal fibroblasts
  • DMEM Dulbeccos modified minimal essential medium
  • FBS heat-inactivated foetal bovine serum
  • penicillin 100 ⁇ g/mL streptomycin (Life technologies) at 37° C. in a humidified 5% CO 2 incubator.
  • HDFa cells were maintained in Fibroblast media from Thermo Fisher.
  • Dulbeccos modified minimal essential medium DMEM
  • Fetal bovine serum FBS
  • Crystal violet Neutral red dye
  • Neutral Red is a weak cationic dye that readily penetrates cell membranes by non-diffusion, accumulating intracellularly in lysosomes. Alterations of the cell surface of the sensitive lysosomal membrane brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged or dead cell. Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the vital dye Neutral Red when measured 24 hours after treatment with the test chemical and irradiation. Exposure to UVA or UVB causes cell death which can also be visualized by imaging under high resolution phase contrast microscope. The viable cells are then stained using crystal violet which stains the nuclei. The cells are imaged in a bright field microscope to visualize the live cells.
  • the extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861.
  • the product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.
  • UV light can accelerate premature ageing and DNA damage.
  • UVB-induced apoptosis is the programmed cell death of cells that become damaged by ultraviolet rays. Apoptosis is a physiological process that promotes cell death which can be termed as active suicide of cells. Failure of the body to remove DNA damaged cells increases the risk of skin cancer. Reduction in apoptosis and cell death is hallmark of anti ageing molecule.
  • Skin cells Skin cells (dermal fibroblasts, keratinocytes or melanocytes) purchased from American Type Culture Collection (ATCC, Manassas, Va.) and maintained as a monolayer culture in Dulbeccos modified minimal essential medium (DMEM) Life technologies, CA, USA) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS; GIBCO/Invitrogen, Carlsbad, Calif.), 100 units/mL penicillin and 100 ⁇ g/mL streptomycin (Life technologies) at 37° C. in a humidified 5% CO 2 incubator.
  • DMEM Dulbeccos modified minimal essential medium
  • Annexin-FITC Propidium Iodide—Apoptosis detection kit, Biovision, USA DMEM, FBS phosphate buffer (pH7.4)
  • apoptosis Exposure of skin cells to UVB light (290-320 nm) triggers cell death and fragmentation of DNA (apoptosis).
  • the cells which are undergoing apoptotic death can be specifically stained using an antibody to a protein called Annexin.
  • Annexin phosphatidylserines (PS, membrane phospholipids) are held on the inner layer of the cell membrane, so Annexin V does not attach to the cells.
  • PS phosphatidylserines
  • the PS are exposed on the outer layer, where they attach to the FITC-labelled Annexin V and stain the cell surface green.
  • PI propidium iodide
  • Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells. The cells were analysed by flow cytometry to determine the cell death and the apoptotic cells
  • the extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861.
  • the product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.
  • UV radiation is the most important environmental factor in the development of skin cancer and skin aging.
  • Oxidative stress is developed in skin when the UV-induced generation of ROS exceeds the ability of endogenous defence mechanism.
  • the reduction of oxidative stress can be achieved either by lowering exposure to UVR and/or by increasing levels of antioxidant defence mechanism.
  • Melanin present in the skin and the anti oxidant enzymatic reactions protect our skin from oxidative stress.
  • Many studies have shown treatment with antioxidants prior to UV exposure can prevent oxidative damage to cellular biomolecules.
  • HDFa Human dermal fibroblasts
  • Balb/C 3T3 ThermoFisher Scientific/Mouse fibroblast cell line (Balb/C 3T3) purchased from National centre of cell collection (NCCS, Pune, India). Cells were maintained in fibroblast media (Gibco) at 37° C. in a humidified 5% CO 2 incubator.
  • Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen atom/molecule and include H 2 O 2 (hydrogen peroxide), NO (nitric oxide), O 2 ⁇ (oxide anion), peroxynitrite (ONOO ⁇ ), hydrochlorous acid (HOCl), and hydroxyl radical (OH ⁇ ).
  • CM-H2DCFDA 5-(and 6)-chloromethyl-20,70-dichlorohydrofluorescein diacetate (CM-H2DCFDA) freely permeates the plasma membrane and is hydrolyzed in the cytosol to form the DCFH carboxylate anion. Oxidation results in the formation of fluorescent DCF, which is maximally excited at 495 nm and emits at 520 nm.
  • Fibroblast cells were seeded at 50,000 cells per well in 96 well black plates and allowed to grow as a monolayer for 24 hours. Cells were pretreated with different concentration of extracellular metabolite for one hour. The plate was exposed to UVA radiation for 1 hour. Freshly prepared DCFH-DA reagent was added to all the wells (2 ug/well). The plate was incubated at 37° C. for 30 mins. Fluorescence was recorded at 485:520, Ex:Em wavelength in FluoStar Optima microplate reader
  • the extracellular metabolite preparation from Bacillus coagulans MTCC 5856 showed dose dependent reduction in ROS induced by UV exposure in fibroblasts. ( FIG. 3 ).
  • the extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861.
  • the product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.
  • ROS Reactive oxygen species
  • DPPH ⁇ , ⁇ -diphenyl- ⁇ -picrylhydrazyl
  • Reagents 0.1 mM of DPPH in ethanol, 0.1M phosphate buffered saline (pH7.4)
  • Microtitre plates 96 well microtitre plates (Corning, USA)
  • DPPH is a stable free radical in a methanolic solution with an absorbance at 520 nm. If the free radicals are scavenged by an anti oxidant molecule, the resulting solution appears yellow. The hydrogen atoms or electrons donation ability of the extracellular metabolite was measured by the bleaching of purple coloured DPPH methanol solution.
  • the extracellular metabolite preparation was diluted in PBS.
  • 20 ⁇ L of extracellular metabolite preparation was mixed with 180 ⁇ L of DPPH in methanol in a 96 well plate following the method as described earlier (Clarke et al., 2013). The plate was kept in the dark for 15 min, after which the absorbance of the solution was measured at 540 nm using a microplate reader (TECAN Ltd, Gurnnedorf, Switzerland). Blanks (DMSO, methanol) and standard (Trolox solution in DMSO) were recorded simultaneously. The extracts were screened with variable concentrations to establish the inhibition concentration (IC 50 , the concentration reducing DPPH absorbance by 50%).
  • the free radical scavenging activity was calculated as follows,
  • the extracellular metabolite preparation showed dose dependent anti oxidant activity comparable to Trolox®.
  • the concentration at which 50% of enzyme activity in inhibited (IC 50 ) was found to be 0.43% for the extracellular metabolite preparation. ( FIG. 4 )
  • Example 5 Anti Inflammatory Activity of Extracellular Metabolite Preparation from Bacillus coagulans MTCC 5856
  • the extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861.
  • the product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.
  • Skin inflammation can result from exposure to UV or ionizing radiation, allergens, or by contact with chemical irritants or allergens.
  • an inflammatory agent When the skin is exposed to an inflammatory agent, the cells in the skin produce an inflammatory messenger known as cytokine.
  • cytokine a inflammatory messenger
  • cytokines trigger an inflammatory cascade which produce other cytokines activate the blood cells, produce free radicals which damage the skin.
  • THP1 human monocytes purchased from American Type Culture Collection (ATCC, Manassas, Va.) and maintained as a monolayer culture in Rosewell park memorial institute Medium (RPMI Life technologies, CA, USA) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS; GIBCO/Invitrogen, Carlsbad, Calif.), 100 units/mL penicillin and 100 ⁇ g/mL streptomycin (Life technologies) at 37° C. in a humidified 5% CO 2 incubator.
  • FBS foetal bovine serum
  • FBS heat-inactivated foetal bovine serum
  • penicillin 100 ⁇ g/mL streptomycin
  • Reagents and buffers Lipopolysaccharide (LPS, Sigma chemicals, USA), Phosphate buffered saline, RPMI, FBS
  • TNF- ⁇ Tumour necrosis factor
  • ELISA Enzyme linked Immunosorbent assay
  • THP-1 cells were stimulated with 100 ng with lipopolysacharide (LPS, 0.1 ⁇ g/mL) to induce TNF- ⁇ secretion.
  • LPS lipopolysacharide
  • Cells were pre treated with different concentrations extracellular metabolite preparation before LPS treatment.
  • the cell supernatants were collected 24 hour after treatment and secreted TNF- ⁇ as estimated by cytokine ELISA as described by the manufacturer. Unstimulated cells were used as negative control. The limit of detection was ⁇ 1 pg/mL.
  • Example 6 Formulations Containing Extracellular Metabolite Preparation from Bacillus coagulans for Skin Care
  • composition containing the extracellular metabolite from Bacillus coagulans MTCC 5856 may be formulated with pharmaceutically/cosmeceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and/or incorporated into formulations containing anti-aging ingredients and administered topically in the form of creams, gels, lotions, powder, serum, oil, suspensions, ointments, soaps, scrubs, emulsions, and compacts.
  • one or more skin care ingredients are selected from the group consisting of, but not limited to, Alpha Lipoic Acid, oxyresveratrol, Beet root extract, Boswellia serrata Extract, ⁇ boswellic acids, Boswellia serrata oil, Centella asiatica Extract, triterpenes, Garcinia indica extract, anthocyanins, Cocos nucifera extract and juice, Coleus forskohlii Extract, forskolin, Coleus forskohlii Oil, Tetrahydropiperine, Ellagic Acid, Gallnut Extract, polyphenols, Galanga Extract, Glycyrrhizinic Acid, Green Tea Extract, Epigallocatechin Gallate, Licorice extract, MonoAmmonium Glycyrrhizinate, Limonoids, Oleanolic Acid, Cosmetic peptides (Oleanolic acid linked to Lys-Thr-Thr-Lys-Ser, Oleanolic acid linked to Lys-Val-Lys), Ol
  • one or more anti-oxidants and anti-inflammatory agents are selected from the group consisting of, but not limited to, vitamin A, D, E, K, C, B complex, rosmarinic acid, Alpha Lipoic Acid, oxyresveratrol, Ellagic Acid, Glycyrrhizinic Acid, Epigallocatechin Gallate, plant polyphenols, Glabridin, moringa oil, oleanolic acid, Oleuropein, Carnosic acid, urocanic acid, phytoene, lipoid acid, lipoamide, ferritin, desferal, billirubin, billiverdin, melanins, ubiquinone, ubiquinol, ascorbyl palmitate, Mg ascorbyl phosphate, ascorbyl acetate, tocopherols and derivatives such as vitamin E acetate, uric acid, ⁇ -glucosylrutin, calalase and the superoxide dismutase, glutathione, selenium compounds,
  • one or more bioavailability enhancers are selected from the group, but not limited to, piperine, tetrahydropiperine, quercetin, Garlic extract, ginger extract, and naringin.
  • Tables 2-6 provide illustrative examples of skin care formulations containing partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856 ( Bacillus ferment filtrate extract)

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CN112545963A (zh) * 2020-12-30 2021-03-26 上海澄穆化妆品有限公司 一种具有美白效果的组合物、美白复合制剂及美白复合制剂的制备方法
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