FR2900343A1 - PROTEASES INHIBITING COMPOSITION, IN PARTICULAR PAPAINEE, CATHEPSIN K AND CATHEPSIN B, AND PROCESS FOR PURIFYING PROTEASE INHIBITING MOLECULES - Google Patents
PROTEASES INHIBITING COMPOSITION, IN PARTICULAR PAPAINEE, CATHEPSIN K AND CATHEPSIN B, AND PROCESS FOR PURIFYING PROTEASE INHIBITING MOLECULES Download PDFInfo
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- 239000000203 mixture Substances 0.000 title claims abstract description 25
- 239000004365 Protease Substances 0.000 title claims abstract description 23
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 20
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 15
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract 7
- 102000035195 Peptidases Human genes 0.000 title description 13
- 108010084457 Cathepsins Proteins 0.000 title description 3
- 102000005600 Cathepsins Human genes 0.000 title description 3
- 239000000284 extract Substances 0.000 claims abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- 239000003480 eluent Substances 0.000 claims description 22
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 10
- 239000003960 organic solvent Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 241000237852 Mollusca Species 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims 2
- 239000004480 active ingredient Substances 0.000 claims 1
- 235000019419 proteases Nutrition 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000712 Cathepsin B Proteins 0.000 description 3
- 102000004225 Cathepsin B Human genes 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 241000237502 Ostreidae Species 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 235000020636 oyster Nutrition 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 101000898454 Bos taurus Cathepsin B Proteins 0.000 description 1
- 229910021532 Calcite Inorganic materials 0.000 description 1
- 108090000625 Cathepsin K Proteins 0.000 description 1
- 102000004171 Cathepsin K Human genes 0.000 description 1
- 208000001490 Dengue Diseases 0.000 description 1
- 206010012310 Dengue fever Diseases 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 241000490567 Pinctada Species 0.000 description 1
- 241001464019 Pinctada margaritifera Species 0.000 description 1
- 206010036030 Polyarthritis Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 208000025729 dengue disease Diseases 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 208000030428 polyarticular arthritis Diseases 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
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- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Marine Sciences & Fisheries (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
L'invention concerne une composition inhibitrice de protéases comportant des molécules extraites de nacre ayant une masse moléculaire comprise entre 100 Da et 400 Da.L'invention concerne également un procédé d'extraction de molécules inhibitrices de protéases comportant les étapes d'extraire des molécules à partir de poudre de nacre au moyen d'une solution aqueuse, de purifier l'extrait obtenu selon des fractions de différents poids moléculaires, et de sélectionner des fractions ayant une activité inhibitrice de protéases.The invention relates to a protease-inhibiting composition comprising molecules extracted from mother-of-pearl having a molecular mass of between 100 Da and 400 Da. The invention also relates to a process for extracting protease-inhibiting molecules comprising the steps of extracting molecules. from mother-of-pearl powder with an aqueous solution, to purify the extract obtained in fractions of different molecular weights, and to select fractions having a protease inhibitory activity.
Description
La présente invention concerne une composition inhibitrice de protéases etThe present invention relates to a protease inhibiting composition and
un procédé d'extraction de molécules inhibitrices de protéases. ARRIERE PLAN DE L'INVENTION On. sait que les protéases peuvent avoir un effet négatif sur la santé des humains ou des animaux en provo-quant soit un développement anormal soit une dégradation des cellules humaines ou animales, notamment dans certaines pathologies telles que l'ostéoporose, l'arthrose, la polyarthrite rhumatoïde en dégradant la matrice osseuse et/ou cartilagineuse, ou encore dans le développement de maladies virales telle que la dengue ou parasitaires tel-les que le paludisme. Il existe donc un besoin constant de trouver de nouvelles molécules inhibitrices de protéases plus particulièrement adaptées à certaines protéases et pouvant être utilisées soit directement telles qu'obtenues par la mise en ouvre d'un procédé d'extraction soit synthétisées. Pour qu'une synthèse soit plus accessible, il est en outre préférable de rechercher des molécules de faible poids moléculaire. OBJET DE L'INVENTION Un but de l'invention est de proposer une composition inhibitrice de protéases, notamment des protéases à csytéine, telles que la papaïne, la cathépsine K ou la cathépsine B, sans que cette liste soit exhaustive. RESUME DE L'INVENTION A cet effet, on propose selon l'invention une composition inhibitrice de protéases comportant le prin- cipe actif comprenant des molécules extraites de nacre ou de coquille de mollusque et ayant une masse moléculaire comprise entre 100 Da et 400 Da. Il a été en effet constaté qu'une composition de ce type permet d'obtenir une inhibition pouvant aller jusqu'à 90% sur la papaïne et la cathépsine B. a method for extracting protease inhibitory molecules. BACKGROUND OF THE INVENTION knows that proteases can have a negative effect on the health of humans or animals by causing abnormal development or degradation of human or animal cells, especially in certain pathologies such as osteoporosis, osteoarthritis, polyarthritis rheumatoid by degrading the bone matrix and / or cartilage, or in the development of viral diseases such as dengue or parasitic such as malaria. There is therefore a constant need to find new protease inhibiting molecules more particularly adapted to certain proteases and which can be used either directly as obtained by the implementation of an extraction process or synthesized. For a synthesis to be more accessible, it is furthermore preferable to search for molecules of low molecular weight. OBJECT OF THE INVENTION An object of the invention is to provide an inhibitory protease composition, especially csyteine proteases, such as papain, cathesins K or cathepsin B, without this list being exhaustive. SUMMARY OF THE INVENTION To this end, a protease inhibiting composition comprising the active principle comprising molecules extracted from mother-of-pearl or mollusk shell and having a molecular mass of between 100 Da and 400 Da is proposed according to the invention. It has indeed been found that a composition of this type makes it possible to obtain an inhibition of up to 90% on papain and cathepsin B.
Selon un autre aspect de l'invention, celle-ci concerne un procédé d'extraction de molécules inhibitrices de protéases comportant les étapes d'extraire les molécules à partir de poudre de nacre ou de coquille de mollusque au moyen d'une solution aqueuse, de purifier l'extrait obtenu selon des fractions de différents poids moléculaires et de sélectionner des fractions ayant une activité inhibitrice de protéases. De préférence, la solution aqueuse initiale corn- prend 40 à 60 % de solvant organique, en particulier un solvant organique choisi dans le groupe comprenant l'éthanol, l'isopropanol, l'acétone et le n-butanol. DESCRIPTION DETAILLEE DE L'INVENTION D'autres caractéristiques et avantages de l'in- vention apparaîtront à la lecture de la description qui suit d'un mode de mise en oeuvre particulier non limitatif du procédé d'extraction selon l'invention et de la composition obtenue. A titre d'exemple, le procédé selon l'invention a été mis en oeuvre de la façon suivante . des coquilles d'huîtres Pinctada margaritifera débarrassées de leur calcite ont été réduites en poudre selon une granulométrie comprise entre 1 et 200 m. 500 grammes de cette poudre ont. été mélangés avec un litre de solution compor- tant 50 % d'eau et 50 % d'isopropanol. Le mélange obtenu a été agité pendant trente minutes à température ambiante puis la suspension a été centrifugée et le filtrat a été évaporé sous vide à 40 . Les inhibiteurs de protéases présents dans l'ex- trait sec ainsi obtenu ont été purifiés selon trois étapes de purification : Etape 1 L'extrait sec obtenu a été mélangé avec une solution comprenant un volume d'isopropanol pour neuf volumes d'eau, à raison de 10 mg d'extrait sec pour 1 ml de solu- tion. Un volume de ce mélange a été introduit dans une colonne chromatographique en phase inversée de type C8 qui a par ailleurs été alimentée avec un éluant A comprenant un mélange à 0,1% d'acide trifluoroacétique dans de l'eau pure, et un éluant B comprenant un mélange à 0,1 % d'acide trifluoroacétique dans de l'acétonitrile. L'élution a été commencée avec un solvant contenant 5 % d'éluant B à un débit de 2 ml/mn et un gradient d'élution amenant le pourcentage d'éluant B à 40 % au bout de 60 mn. Des fractions ont été prélevées toutes les minutes. Les fractions obtenues ont été lyophilisées. Les résidus sont dissous dans l'eau puis un échantillon de chaque fraction est soumis au test d'inhibition de la papaïne, de la cathépsine B et de la cathépsine K. Les meilleurs résultats d'inhibition ont été obtenus avec les fractions correspondant à une élution comprise entre 5 % et 6 %. Ces fractions ont été lyophilisées. La. colonne chromatographique a ensuite été lavée de façon connue en soi en portant l'élution à 80 % d'éluant B puis en réduisant sa proportion à 5 % pour préparer la colonne à la deuxième étape. Etape 2 L'extrait sélectionné dans l'étape 1 a été redilué à raison de 20 mg/ml dans l'éluant A et un volume de ce mélange a été introduit dans la colonne chromatographique avec un éluant A comprenant une solution à 0,1 % d'acide trifluoroacétique dans de l'eau pure et un éluant B comprenant un mélange à 0,1 % d'acide trifluoroacétique dans de l'acétonitrile. L'élution a été commencée à un taux de 5 % d'éluant B avec un débit de 0,7 ml/mn, c'est-à-dire environ un quart du débit de l'étape 1. L'élution a été maintenue à un taux de 5 % d'éluant B pendant 15 mn puis progressivement augmentée jusqu'à 20 % au bout de 45 mn. Des fractions ont été prélevées toutes les 30 se- condes, soit à une fréquence double de l'étape 1. Les 4 fractions obtenues ont été lyophilisées puis des échantillons ont été redilués et soumis au test d'inhibition des protéases. L'inhibition la plus forte a été obtenue avec la fraction correspondant à une élution comprise en- tre 5 % et 6 %. Les fractions retenues ont été lyophilisées. La colonne chromatographique a été lavée comme précédemment puis ramenée à un taux d'élution de 5 % et un débit de 0,7 ml/mn. According to another aspect of the invention, this relates to a process for extracting protease inhibiting molecules comprising the steps of extracting the molecules from mother-of-pearl powder or mollusk shell using an aqueous solution, to purify the obtained extract in fractions of different molecular weights and to select fractions having a protease inhibitory activity. Preferably, the initial aqueous solution comprises 40 to 60% of organic solvent, in particular an organic solvent selected from the group consisting of ethanol, isopropanol, acetone and n-butanol. DETAILED DESCRIPTION OF THE INVENTION Other features and advantages of the invention will become apparent on reading the following description of a particular, non-limiting embodiment of the extraction process according to the invention and of the composition obtained. By way of example, the process according to the invention has been implemented as follows. Pinctada margaritifera oyster shells cleared of their calcite have been reduced to powder with a grain size between 1 and 200 m. 500 grams of this powder have. mixed with one liter of solution containing 50% water and 50% isopropanol. The resulting mixture was stirred for thirty minutes at room temperature and then the suspension was centrifuged and the filtrate was evaporated in vacuo at 40 ° C. The protease inhibitors present in the dry extract thus obtained were purified according to three purification steps: Step 1 The dry extract obtained was mixed with a solution comprising one volume of isopropanol for nine volumes of water, 10 mg of solids per 1 ml of solution. A volume of this mixture was introduced into a C8 reversed phase chromatographic column which was further fed with an eluent A comprising a mixture of 0.1% trifluoroacetic acid in pure water and an eluent. B comprising a mixture of 0.1% trifluoroacetic acid in acetonitrile. Elution was started with a solvent containing 5% eluent B at a flow rate of 2 ml / min and an elution gradient bringing the percentage of eluent B to 40% after 60 min. Fractions were taken every minute. The fractions obtained were lyophilized. The residues are dissolved in water and then a sample of each fraction is subjected to the inhibition test of papain, cathepsin B and cathepsin K. The best results of inhibition were obtained with the fractions corresponding to one elution between 5% and 6%. These fractions were lyophilized. The chromatographic column was then washed in a manner known per se by eluting at 80% eluent B and then reducing its proportion to 5% to prepare the column at the second step. Step 2 The extract selected in step 1 was rediluted at 20 mg / ml in eluent A and a volume of this mixture was introduced into the chromatographic column with an eluent A comprising a 0.1 solution. % trifluoroacetic acid in pure water and eluent B comprising a mixture of 0.1% trifluoroacetic acid in acetonitrile. Elution was started at a rate of 5% eluent B with a flow rate of 0.7 ml / min, ie about a quarter of the flow of step 1. The elution was maintained at a rate of 5% eluent B for 15 minutes and then gradually increased to 20% after 45 minutes. Fractions were taken every 30 seconds, twice the frequency of step 1. The 4 fractions obtained were freeze-dried and then samples were rediluted and tested for protease inhibition. The strongest inhibition was obtained with the fraction corresponding to an elution of between 5% and 6%. The retained fractions were lyophilized. The chromatographic column was washed as before and then brought to an elution rate of 5% and a flow rate of 0.7 ml / min.
Etape 3 La dernière étape de purification est réalisée à un pH de 7 sur une colonne chromatographique en phase in-versée de type Ça qui est alimentée avec un éluant A comprenant un mélange de 95 % d'acétate de triéthylamine 100 mM ajusté à un pH 7 par de l'acide acétique, et de 5 % d'acétonitrile, et un éluant B comprenant un mélange de 5 % d'acétate de triéthylamine 100 mM, pH 7 et de 95 % d'acétonitrile. Les fractions contenant les inhibiteurs de pro- téases recueillis à l'issue de l'étape 2 sont introduites dans la colonne chromatographique. L''éluti.on a été commencée à un taux de 5 % d'éluant B et maintenue pendant 15 mn à un débit de 0,7 ml/mn, le taux d'élution a ensuite été augmenté réguliè-rement pour atteindre 12 % d'éluant B au bout de 30 mn. Une fraction a été prélevée toutes les 30 secondes puis les tests d'inhibition des protéases ont été effectués de la même façon que précédemment avec les différentes fractions prélevées. Les tests les plus favora- bles ont été obtenus avec les fractions 44 et 45 correspondant à une élution comprise entre 7 % et 9 %. Les fractions finalement sélectionnées se sont révélées efficaces lors de tests d'inhibition effectués sur la cathépsine B bovine et la cathépsine K humaine re- combinante. Step 3 The last purification step is carried out at a pH of 7 on a Ca-type in-phase chromatographic column which is fed with an eluent A comprising a mixture of 95% 100 mM triethylamine acetate adjusted to a pH 7 with acetic acid, and 5% acetonitrile, and eluent B comprising a mixture of 5% 100 mM triethylamine acetate, pH 7 and 95% acetonitrile. The fractions containing the protease inhibitors collected at the end of step 2 are introduced into the chromatographic column. The eluate was started at a level of 5% eluent B and held for 15 minutes at a flow rate of 0.7 ml / min, the elution rate was then increased steadily to 12 % eluent B after 30 minutes. A fraction was taken every 30 seconds and the protease inhibition tests were carried out in the same way as previously with the various fractions removed. The most favorable tests were obtained with fractions 44 and 45 corresponding to an elution of between 7% and 9%. The finally selected fractions were found to be effective in inhibition tests on bovine cathepsin B and recombinant human K cathepsin.
Des mesures effectuées au spectromètre de masse ont révélé que ces fractions correspondaient à des molécules ayant un poids moléculaire compris entre 100 Da et 400 Da. Measurements made with the mass spectrometer revealed that these fractions corresponded to molecules having a molecular weight between 100 Da and 400 Da.
Bien entendu, l'invention n'est pas limitée au mode de mise en œuvre du procédé décrit et est susceptible de variantes de réalisation qui apparaîtront à l'homme de métier sans sortir du cadre de l'invention tel que défini par les revendications. Of course, the invention is not limited to the mode of implementation of the method described and is capable of variants that will occur to those skilled in the art without departing from the scope of the invention as defined by the claims.
En particulier, bien que l'invention ait été décrite en relation avec un extrait de nacre d'huître Pinctada marga:ritifera, on peut également utiliser de la poudre de coquille entière de mollusques nacriers ou non nacriers tels que des gastéropodes. In particular, although the invention has been described in connection with an oyster mother-of-pearl extract Pinctada marga: ritifera, it is also possible to use whole shell powder of nacreous or non-nacreous molluscs such as gastropods.
Le procédé selon l'invention peut également être mis en oeuvre en utilisant un solvant organique compris dans le groupe comprenant l'éthanol, l'acétone ou ne nbutanol. Bien que des meilleurs résultats d'inhibition aient été obtenus avec une solution aqueuse d'extraction comprenant 40 % à 60 % de solvant organique dans l'eau, une activité inhibitrice a également été révélée après extraction avec de l'eau pure ou une solution comportant un mélange d'eau et de solvant organique selon un taux compris entre 0 et 80 %. Au-delà de 80 %, le rendement d'extraction devient très faible voire nul. Bien que l'invention ait été décrite en relation avec des étapes de purification réalisées immédiatement après l'extraction, on peut prévoir une étape de dialyse entre l'extraction et la purification afin de sélection- ner une gamme de poids moléculaire déterminée préalable- ment à la purification. Une étape de purification sur une résine échangeuse d'ions (anions ou cations) peut être introduite dans le procédé entre l'étape d'extraction et les étapes de purification ou entre deux étapes de purification afin d'éliminer des contaminants et enrichir les extraits en inhibiteurs. Bien que le procédé selon l'invention ait été décrit selon une mise en oeuvre permettant d'isoler des mo- lécules actives ayant un poids moléculaire compris entre 100 Da et 400 Da, le même procédé peut être mis en oeuvre selon des conditions adaptées pour isoler des molécules actives de poids moléculaire plus élevé pouvant aller jusqu'à 100.000 Da voire même 200.000 Da. The process according to the invention can also be carried out using an organic solvent included in the group comprising ethanol, acetone or n-butanol. Although better inhibition results were obtained with an aqueous extraction solution comprising 40% to 60% of organic solvent in water, inhibitory activity was also revealed after extraction with pure water or a solution. comprising a mixture of water and organic solvent at a level between 0 and 80%. Above 80%, the extraction yield becomes very low or even zero. Although the invention has been described in connection with purification steps performed immediately after extraction, a dialysis step between extraction and purification may be provided to select a previously determined molecular weight range. to purification. A purification step on an ion exchange resin (anions or cations) can be introduced in the process between the extraction step and the purification steps or between two purification steps in order to remove contaminants and enrich the extracts. inhibitors. Although the process according to the invention has been described according to one embodiment making it possible to isolate active molecules having a molecular weight between 100 Da and 400 Da, the same process can be carried out under conditions adapted to isolate active molecules of higher molecular weight up to 100,000 Da or even 200,000 Da.
Claims (6)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0603712A FR2900343B1 (en) | 2006-04-26 | 2006-04-26 | PROTEASES INHIBITING COMPOSITION, IN PARTICULAR PAPAINEE, CATHEPSIN K AND CATHEPSIN B, AND PROCESS FOR PURIFYING PROTEASE INHIBITING MOLECULES |
| PCT/FR2007/000667 WO2007128896A2 (en) | 2006-04-26 | 2007-04-20 | Protease inhibitor compound, notably papain, cathepsin k and cathepsin b, and a procedure for the purification of protease inhibitor molecules |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0603712A FR2900343B1 (en) | 2006-04-26 | 2006-04-26 | PROTEASES INHIBITING COMPOSITION, IN PARTICULAR PAPAINEE, CATHEPSIN K AND CATHEPSIN B, AND PROCESS FOR PURIFYING PROTEASE INHIBITING MOLECULES |
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| Publication Number | Publication Date |
|---|---|
| FR2900343A1 true FR2900343A1 (en) | 2007-11-02 |
| FR2900343B1 FR2900343B1 (en) | 2008-07-04 |
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| FR0603712A Expired - Fee Related FR2900343B1 (en) | 2006-04-26 | 2006-04-26 | PROTEASES INHIBITING COMPOSITION, IN PARTICULAR PAPAINEE, CATHEPSIN K AND CATHEPSIN B, AND PROCESS FOR PURIFYING PROTEASE INHIBITING MOLECULES |
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| FR (1) | FR2900343B1 (en) |
| WO (1) | WO2007128896A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010116060A1 (en) * | 2009-04-10 | 2010-10-14 | Centre National De La Recherche Scientifique -Cnrs- | Method for producing and using active principles of limestone |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2743075A1 (en) * | 1995-12-28 | 1997-07-04 | Centre Nat Rech Scient | PROCESS FOR THE PREPARATION OF ACTIVE SUBSTANCES FROM NACRE, PRODUCTS OBTAINED USEFUL IN PARTICULAR AS MEDICAMENTS |
| FR2777190A1 (en) * | 1998-04-14 | 1999-10-15 | Georges Camprasse | EXTRACTION PROCESS, IDENTIFICATION OF THE ACTIVE INGREDIENTS CONTAINED IN THE INTERNAL AND EXTERNAL SHELLS OF SHELLFISH, THEIR USE IN PEOPLE-BASED THERA, DIAGNOSIS AND COSMETIC PREPARATIONS |
| FR2785503A1 (en) * | 1998-11-06 | 2000-05-12 | Pharma Futura | Aragonite orthorhombic calcium carbonate from mother of pearl is used to make foods for the prevention and treatment of osteoporosis |
| FR2856303A1 (en) * | 2003-06-20 | 2004-12-24 | Innovation Et De Rech Applique | THERAPEUTIC USE OF LIPIDS EXTRACTED FROM NACRE |
-
2006
- 2006-04-26 FR FR0603712A patent/FR2900343B1/en not_active Expired - Fee Related
-
2007
- 2007-04-20 WO PCT/FR2007/000667 patent/WO2007128896A2/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2743075A1 (en) * | 1995-12-28 | 1997-07-04 | Centre Nat Rech Scient | PROCESS FOR THE PREPARATION OF ACTIVE SUBSTANCES FROM NACRE, PRODUCTS OBTAINED USEFUL IN PARTICULAR AS MEDICAMENTS |
| FR2777190A1 (en) * | 1998-04-14 | 1999-10-15 | Georges Camprasse | EXTRACTION PROCESS, IDENTIFICATION OF THE ACTIVE INGREDIENTS CONTAINED IN THE INTERNAL AND EXTERNAL SHELLS OF SHELLFISH, THEIR USE IN PEOPLE-BASED THERA, DIAGNOSIS AND COSMETIC PREPARATIONS |
| FR2785503A1 (en) * | 1998-11-06 | 2000-05-12 | Pharma Futura | Aragonite orthorhombic calcium carbonate from mother of pearl is used to make foods for the prevention and treatment of osteoporosis |
| FR2856303A1 (en) * | 2003-06-20 | 2004-12-24 | Innovation Et De Rech Applique | THERAPEUTIC USE OF LIPIDS EXTRACTED FROM NACRE |
Non-Patent Citations (1)
| Title |
|---|
| XUE ET AL: "A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus", COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. PART B, BIOCHEMISTRY AND MOLECULAR BIOLOGY, ELSEVIER,OXFORD, GB, vol. 145, no. 1, September 2006 (2006-09-01), pages 16 - 26, XP005618757, ISSN: 1096-4959 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010116060A1 (en) * | 2009-04-10 | 2010-10-14 | Centre National De La Recherche Scientifique -Cnrs- | Method for producing and using active principles of limestone |
| FR2944214A1 (en) * | 2009-04-10 | 2010-10-15 | Centre Nat Rech Scient | OBTAINING AND USE OF ACTIVE PRINCIPLES OF LIMESTONE |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2007128896A3 (en) | 2008-03-13 |
| WO2007128896A2 (en) | 2007-11-15 |
| FR2900343B1 (en) | 2008-07-04 |
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