WO2001073112A1 - Procede d'identification des composes filtres anti-uv - Google Patents

Procede d'identification des composes filtres anti-uv Download PDF

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Publication number
WO2001073112A1
WO2001073112A1 PCT/IN2000/000038 IN0000038W WO0173112A1 WO 2001073112 A1 WO2001073112 A1 WO 2001073112A1 IN 0000038 W IN0000038 W IN 0000038W WO 0173112 A1 WO0173112 A1 WO 0173112A1
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WO
WIPO (PCT)
Prior art keywords
protection
compounds
bactena
benzoate
radiation
Prior art date
Application number
PCT/IN2000/000038
Other languages
English (en)
Inventor
Suman Preet Singh Khanuja
Krishna Kumar Agarwal
Tiruppadiripuliyur Ranganathan Santha Kumar
Atique Ahmad
Ajit Kumar Shasany
Mahendra Pandurang Darokar
Sushil Kumar
Original Assignee
Council Of Scientific And Industrial Research
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Council Of Scientific And Industrial Research filed Critical Council Of Scientific And Industrial Research
Priority to AU2000255630A priority Critical patent/AU2000255630A1/en
Priority to PCT/IN2000/000038 priority patent/WO2001073112A1/fr
Publication of WO2001073112A1 publication Critical patent/WO2001073112A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)

Definitions

  • the present invention relates to the development and use of a novel bacterial system based protocol to screen the efficacy of the chemical compounds for Ultraviolet radiation protection.
  • the invention further is related to the new use of a compound menthyl benzoate suitable for providing protection against the harmful effects of ultraviolet radiation.
  • the extent of protection by menthyl benzoate is much higher than the known UV protectants like dibenzoyl methane and benzyl benzoate.
  • the exposure to sunlight can pose a variety of damages to the skin and the damaging effects may result in hazardous effects like sunburn, which primarily result from exposure to UVB radiation within the sunlight spectrum having a wavelength of about 290 to 320 nm. But continuous exposure over the long run may also lead to malignant cancerous cells on the skin surface. Studies have demonstrated a strong relationship between sunlight exposure and human skin cancer. Other hazards of ultra violet radiation exposure include premature aging of the skin, which is primarily caused by the UVA radiation having a wavelength of from about 320 nm to about 400 nm. This condition is characterized by wrinkling and pigments changes of the skin, along with other physical changes such as cracking, telangiectasis, solar dermatoses, ecchymoses, and loss of elasticity etc.
  • UVB domain UVB domain
  • UVA domain UVA domain
  • UVA absorbing sunscreen actives or UV sieve compounds is conspicuous among the products, which are both commercially available and also approved for global use
  • One class of the available sunscreen actives includes dibenzoylmethane compounds which provide broad-spectrum UV protection such as 4-tert-butyl-4'- methoxydibenzoylmethane
  • these sunscreens tend to photodegrade upon exposure to UV radiation thereby reducing their UVA efficacy Consequently, sunscreen products, which include these compounds, are typically difficult to formulate due to the inherent lack of photostabihty of dibenzoylmethane compounds
  • modification of these chemicals may be hazardous to the skin or making them environmentally unsafe
  • the compounds known to be safe for human use can be screened isolated and modified for use as sunscreen Sunlight is required for health, and a sunscreen composition absorbing 100% of the sunlight is not desirable
  • the desirable feature is that the damaging wavelength range of sunlight spectrum should be reduced to the safe threshold level which is skin and tissue safe by using the compounds which are harmless to the biological system
  • the invention provides a method for screening compounds for effective protection against the harmful ultraviolet radiation, thereby providing UV screen to prevent the harmful affect of the radiation on the biological system which comprises, a Inoculating a single colony is inoculated in 5 ml Luria broth and incubated at 37°C temperature for at least 16 hour in a water bath shaker and is growing in presence of test compounds with plain LB as growth medium, b harvesting the bacterial culture is harvested by centrifugation and pellet is suspended in equal amount of sterile distilled water, c transferring atleast 0 3 ml of the broth culture of the bacteria is transferred to pet ⁇ plates forming a thin layer at the bottom (in replicates), d optionally covering the plates are covered with UV transparent film, which is coated with different concentrations of the test compounds, e placing a UV source is placed at least 10 cm above the plates (which may vary depending upon the experiment), f switching on the light source is switched on for different time intervals for the predetermined le
  • the invention provide a novel use of a semi-synthetic compound menthyl benzoate which can be used as an UV protectant filter providing better protection, by screening the ultra violet rays, thereby providing protection to the biological systems including human skin
  • the invention provides a formulation comprising menthyl benzoate ranging between 0 002-0 02% (w/w) in glycerin, cold cream, skin cream, antibiotic ointments, sunscreen lotions and any other body care compositions used for external UV protection applications
  • Menthyl Benzoate A solution of l-menthol(l gram) in benzoyl chloride (1 ml) was stirred at room temperature for 72 hours This solution yielded menthyl benzoate of 89 5% purity Further purification of this product through column chromatography resulted to increase in purity level to at least 98 8% This fraction yielded colourless crystals and confirmed for purity by 'HNMR, 13 CNTvIR, IR and Mass spectra
  • Benzyl Benzoate A mixture of 1 ml benzyl alcohol and 1 ml benzyl chloride was stirred at room temperature for 72 hours This solution yielded benzyl benzoate of 87 0% purity Further purification of this product by column chromatography gave at least 99% benzyl benzoate
  • Dibenzoyl methane This commercial chemical was obtained from M S Lancaster Synthesis Ltd, Eastgate, White Lund, Morecambe, Lancashire LA3 3DY, UK (Catalogue no2104) chemicals with 98% purity level UV protection assays UV light is not only harmful to humans but also to all other biological systems including microorganisms Majority of the effect studies in microbes for its modes of action have shown the formation of thymidine dimers, which cause mutations and excessive exposure thus leads to death of the bacteria As bacteria are devoid of cellulose or protein wall and are single celled, they are much more sensitive to the UV light and hence efficient bio- mdicators of UV damage Therefore, the assays involving the screening of potential UV protectant compounds against bacterial system exposure in presence and absence of test compound is easy, less time consuming and environmentally safe We carried out simple experiments involving the bacterium Escherichia coll (Wild type) (Kumar, S 1976 Journal of Bacteriology, 125 545-555) as the test system Escherichia col is a
  • Example 1 Exposure of the bacteria to UV light through a UV transparent film coated with the compound Step A Growing bacterial cultures The wild type strain, CA 8000 of Escherichia coli was used in the assays A single colony was inoculated in 5 ml Luria broth and incubated at 37°C temperature for 16 hour in a water bath shaker Bacteria were grown in presence of test compounds with plain LB as growth medium
  • Step B The bacterial culture was harvested by centrifugation and pellet was suspended in equal amount of sterile distilled water The bacterial pellet was harvested by centrifuging at 5000 RPM for 10 minute and suspended in 25 ml sterile distilled water
  • Step C About 0 3 ml of the broth culture of the bacteria was transferred to petriplates forming a thin layer at the bottom (in replicates) At least 5 replicates per treatment were used along with control plates containing bacteria Step D The plates were covered with UV transparent film, which was coated with different concentrations of the test compounds In this invention, we employed market available food wraps (Cling Films) which were found to be 100% transparent
  • Step E In the dark room a UV source was placed at least 10 cm above the plates (which may vary depending upon the experiment) In this invention, the UV torch emitting two- fixed wavelength (312 nm and 365 nm) was used
  • Step F Light source was switched-on for different time intervals for the predetermined lethality levels based on the killing curve of control bacteria determined by UV exposure through uncoated film
  • Step G The viable cfu (colony forming unit) count was determined by serially diluting the exposed and control bacteria and then plating on the Luria agar plates followed by counting the viable units of the bacteria forming colonies (Table 1)
  • Step A Growing bacterial cultures
  • the wild type strain CA 8000 of Escherichia coli was in the assays
  • a single colony was inoculated in 5 ml Luria broth and incubated at 37°C temperature for 16 hour in a water bath shaker
  • Bacteria were grown in presence of test compounds with plain LB as growth medium.
  • Step B Determining killing percentage of bacteria in relation to UV exposure time. This was done in the following steps
  • the broth culture was diluted to 10 times in sterile water b About 0 3 ml of the diluted bacterial suspension was transferred to the petriplates forming a thin layer at the bottom (in replicates) c In the dark room a UV source was placed at least 10 cm above the plates (This distance may vary depending upon the need of the experiment) d The cfu (colony forming unit) count was determined by serially diluting the exposed and control bacteria and plating on the Luria agar plate followed by counting the viable units of the bacteria forming colonies upon incubation at 37°C overnight
  • the bacterial cultures were grown in presence of different concentrations of the test compounds, exposed in the same way as in step B and viable cfu count was determined In these assays it was found that 50 ⁇ g/ml concentration of the compound menthyl benzoate provided a significant protection in comparison to other known UV protactants (Table 2)
  • Table 2 Comparative UV protection activity of benzoate compounds against Escherichia coh as assayed by the protocol described above
  • the compound of invention was found to be safe to the bacterial system as found out in our invention which as such does not kill the bacterial cell and the cfu remain same for treated and untreated cultures and no decrease of titre was observed by growing the bacteria in presence of the compound
  • a bacterial model system could be constructed with the simple method to employ in rapid large scale screening of compounds which can protect the cells against UV damage (312 nm to 365nm) either in form of a sieve or in vicinity of target cells
  • UV damage 312 nm to 365nm
  • a variety of compounds including several monoterpenes and their derivatives were screened using Benzyl benzoate and Dibenzoyal methane as control UV screen molecules
  • the procedure is rapid, novel, most convenient and environmentally compatible not requiring human or animal subject and/or complexities of cell culture in skin testing Results in this procedure, can be obtained overnight and are 100%) interpretable as evident from the control compounds and additionally provide quantifiable data on extent of protection
  • menthyl benzoate was found to provide significant protection with excellent efficiency for both UV radiation (UVA and UVB) and hence making its use as UV protectant evident ADVANTAGES

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

La présente invention concerne un protocole très rapide de recherche systématique à base d'un système bactérien innovant pour détecter les molécules protectrices anti-UV et pour déterminer leur efficacité par rapport à leur pouvoir de protection contre les rayonnements ultraviolets dans les systèmes biologiques. L'invention concerne également la découverte d'une nouvelle utilisation du menthyl-benzoate en composé comme protection anti-UV efficace capable d'assurer une protection contre les effets délétères du rayonnement ultraviolet. Le pouvoir protecteur du menthyl-benzoate est bien supérieur à ceux des protections anti-UV connues à base de dibenzoyl-méthane et de benzyl-benzoate.
PCT/IN2000/000038 2000-03-29 2000-03-29 Procede d'identification des composes filtres anti-uv WO2001073112A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2000255630A AU2000255630A1 (en) 2000-03-29 2000-03-29 Method for identifying uv-filter compounds
PCT/IN2000/000038 WO2001073112A1 (fr) 2000-03-29 2000-03-29 Procede d'identification des composes filtres anti-uv

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IN2000/000038 WO2001073112A1 (fr) 2000-03-29 2000-03-29 Procede d'identification des composes filtres anti-uv

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107300597A (zh) * 2017-08-08 2017-10-27 山东西格玛检测技术服务有限公司 一种土壤中2‑苯基苯并咪唑‑5‑磺酸的检测方法
CN110157772A (zh) * 2019-05-20 2019-08-23 西南医科大学 一种抗紫外产品的紫外防护性能的微生物评价方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691158A (en) * 1993-10-15 1997-11-25 Mary Kay Cosmetics, Inc. System and method for determining efficacy of sunscreen formulations
EP0848944A2 (fr) * 1996-11-21 1998-06-24 The C.P. Hall Company Compositions antisolaires comprenant des benzoates ou hydroxybenzoates d'alkyle à chaine longue et ramifiée
WO1998058255A1 (fr) * 1997-06-17 1998-12-23 Universite Laval Evaluation des lesions cutanees provoquees par l'exposition a des agents nocifs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691158A (en) * 1993-10-15 1997-11-25 Mary Kay Cosmetics, Inc. System and method for determining efficacy of sunscreen formulations
EP0848944A2 (fr) * 1996-11-21 1998-06-24 The C.P. Hall Company Compositions antisolaires comprenant des benzoates ou hydroxybenzoates d'alkyle à chaine longue et ramifiée
WO1998058255A1 (fr) * 1997-06-17 1998-12-23 Universite Laval Evaluation des lesions cutanees provoquees par l'exposition a des agents nocifs

Non-Patent Citations (7)

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Title
CHETELAT A ET AL: "Photomutagenesis test development: I. 8-Methoxypsoralen, chlorpromazine and sunscreen compounds in bacterial and yeast assays.", MUTATION RESEARCH, vol. 292, no. 3, 1993, pages 241 - 250, XP000974951, ISSN: 0027-5107 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1988, SHIMOI K ET AL: "EFFECT OF UV ABSORBERS ON UV-INDUCED MUTAGENESIS IN ESCHERICHIA-COLI B-R", XP002156094, Database accession no. PREV198885121720 *
DEAN S W ET AL: "DEVELOPMENT OF ASSAYS FOR THE DETECTION OF PHOTOMUTAGENICITY OF CHEMICALS DURING EXPOSURE TO UV LIGHT I. ASSAY DEVELOPMENT", MUTAGENESIS, vol. 6, no. 5, 1991, pages 335 - 342, XP000974946, ISSN: 0267-8357 *
DEAN S W ET AL: "DEVELOPMENT OF ASSAYS FOR THE DETECTION OF PHOTOMUTAGENICITY OF CHEMICALS DURING EXPOSURE TO UV LIGHT II. RESULTS OF TESTING THREE SUNSCREEN INGREDIENTS", MUTAGENESIS, vol. 7, no. 3, 1992, pages 179 - 182, XP000974546, ISSN: 0267-8357 *
EISEI KAGAKU, vol. 34, no. 1, 1988, pages 21 - 24, ISSN: 0013-273X *
JONES ROBERT W ET AL: "A microbiological assay for the sun protection factor of sunscreen products.", JOURNAL OF PHARMACY AND PHARMACOLOGY, vol. 50, no. SUPPL., 1998, 135th Meeting of the British Pharmaceutical Conference;Eastbourne, England, UK; September 8-11, 1998, pages 138, XP000974958, ISSN: 0022-3573 *
MARROT L: "In vitro evaluation of solar UV genotoxicity: Photoprotection by the sunscreen MEXORYL SX.", MUTATION RESEARCH, vol. 379, no. 1 SUPPL. 1, 1997, 7th International Conference on Environmental Mutagens;Toulouse, France; September 7-12, 1997, pages S188, XP000974963, ISSN: 0027-5107 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107300597A (zh) * 2017-08-08 2017-10-27 山东西格玛检测技术服务有限公司 一种土壤中2‑苯基苯并咪唑‑5‑磺酸的检测方法
CN107300597B (zh) * 2017-08-08 2019-08-13 山东西格玛检测技术服务有限公司 一种土壤中2-苯基苯并咪唑-5-磺酸的检测方法
CN110157772A (zh) * 2019-05-20 2019-08-23 西南医科大学 一种抗紫外产品的紫外防护性能的微生物评价方法

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