WO2014107106A1 - Use of the molecules of a novel endophytic filamentous bacterium as antioxidant and anticancer product - Google Patents

Use of the molecules of a novel endophytic filamentous bacterium as antioxidant and anticancer product Download PDF

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WO2014107106A1
WO2014107106A1 PCT/MA2013/000051 MA2013000051W WO2014107106A1 WO 2014107106 A1 WO2014107106 A1 WO 2014107106A1 MA 2013000051 W MA2013000051 W MA 2013000051W WO 2014107106 A1 WO2014107106 A1 WO 2014107106A1
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amycolatopsis
antioxidant
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inhibiting
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Rafik Errakhi
Ahmed Lebrihi
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Universite Moulay Ismail
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • Actinomycetes are gram-positive filamentous bacteria, present in various ecological environments and particularly in soils. These microorganisms play an important role in improving the quality of agricultural soil, in particular by contributing to the processes of recycling and biodegradation of organic matter and mineral elements (El-tarabily et al, 2008). Actinomycetes have a great power of transformation of complex organic substances difficult or non-degradable by other microorganisms such as complex polymers, polysaccharides, lignocellulose, chitin (Goodfellow and Williams, 1983, Zaitlin and Watson, 2006). In agronomy, these bacteria play an important role in improving growth and protecting plant crops.
  • the RC21 strain was obtained according to a selection process comprising the following steps:
  • strain RC21 Isolation of strain RC21 and / or a mutant strain, depending on its ability to invade the plant tissue and live indoors without causing disease.
  • the isolation of the strain according to the invention can be carried out from samples of an endemic plant in Morocco, which grows only atlas medium.
  • Samples of the root of the endemic medium-atlas plant are obtained sterile after removing 15 cm from the root of the soil.
  • the samples taken are placed in sterile closed polyethylene bags and stored at 4 ° C.
  • the actinomycete strains are subsequently isolated on SEA Soil Extract Agar medium according to four types of successive treatments: treatment by grinding, heat, agitation and centrifugation.
  • actinomycete strains are isolated by microscopic recognition on the basis of their morphological characteristics. Actinomycetes are transferred to ISP2 medium. The pure isolines are kept at 4 ° C for 2 months. Alternatively, the cultures are resuspended and stored in glycerol at -20 ° C.
  • the strain RC21 according to the invention is selected for its ability to inhibit gram-positive and gram-negative bacteria. It is recognizable by these phenotypic, physiological and molecular characteristics described below.
  • the bacterium forms a broadly branched substrate mycelium on the culture media tested (ISP2, ISP3, and Bennet).
  • the aerial mycelium is fragmented in the form of a stem, which is specific to strains of the genus Amycolatopsis.
  • the aerial mycelium is white on ISP2 and Bennett agar media, but is brown to gray-brown on other media.
  • the substrate mycelium varies from yellow brown to gray brown depending on the medium. Spore chains is long with irregular arrows a lot of spores (10-50). Spores are smooth and not mobile.
  • Strain RC21 does not produce meloid pigment.
  • Growth temperatures are between 12 and 37 ° C (optimal is between 28 and 32 ° C). Good growth occurs at pH 5-9. NaCl is not tolerated up to a concentration of 5% (w / v).
  • Strain RC21 uses for its growth, D-glucose, mannitol, lactose, sucrose, maltose and dextrain.
  • glactose, xylose, inositol, sorbose, fructose, arabinose, raffinose, rhamnose and cellulose are not used.
  • Starch is not hydrolysed. Nitrates are reduced to nitrite, gelatin is not liquefied, it also degrades adenine, Tween 20, sodium acetate.
  • L alanine, L - proline, phenylanine, L-theronine and D-novalin are used as the sole source of carbon.
  • Amplification of the 16s rDNA portion was performed using two universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5'-GGTTACCTTGTTACGACTT-3').
  • sequence 16s rDNA is the following: SEQ No. 1
  • the phylogenetic analysis of the sequence 16s rDNA shows that the said endophytic bacterium RC21 belongs to the genus Amycolatopsis. This analysis also showed that it is close to 99% of the following strains Amycolatopsis rifamycinica DSM 46095 and Amycolatopsis pretoriensis NRRL B-24133.
  • the present invention also relates to the mutant strains of strain RC21.
  • mutant strain is meant any RC21 strain obtainable by selective mutation from the strain RC21.
  • the mutation techniques consist in putting the RC21 strain in the presence of a physical mutagenic agent, such as radiation, or a physical mutagenic agent, such as radiation, or a chemical mutagenic agent, and then to select in a suitable medium the mutants of interest remaining using their antibiotic spectrum.
  • the strain RC21 according to the invention or its mutant strains show important biological activities. These are varied and multiple. It involves the production of antifungal, antibacterial, anticancer and antioxidant molecules.
  • the strain RC21 according to the invention or its mutant strains has shown an activity against Mycobacterium tubercolosis and Mycobacterium phlei this shows that the RC21 strain can be effective against the disease of tuberculosis. It also showed very positive antibacterial activities against Echerichia coli, Pseudomonas aerogenosa, Staphylococcus aureus, as well as antifungal activities against Mucor ramaniamus and Candidat albicans ( Figure 1 and 2).
  • the strain RC21 according to the invention or its mutant strains has shown an activity against breast cancer cells (L1) and kidney cells (L2).
  • Active compounds that may have been produced from strain RC21
  • the strain RC21 according to the invention is capable of producing active compounds.
  • the present invention provides a process for extracting and purifying these active compounds derived from strain RC21.
  • the extraction and purification process according to the invention comprises the following steps:
  • the invention relates to the active compounds or antibiotics obtained by this process.

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Abstract

This invention relates to the use of the Amycolatopsis sp. RC21 strain, of a mutant strain or of an active compound for inhibiting the growth of the causal agent of tuberculosis, and/or inhibiting the proliferation of cancer cells and/or for an antioxidant effect. Likewise, the subject matter of the present invention is a novel actinomycete strain of endophytic nature, Amycolatopsis sp. RC21, characterized by a 16S rDNA sequence SEQ No. 1 deposited in the Pubmed/NCBI nucleotide database, or a mutant strain thereof, and also a method for selecting this strain. The invention is also directed towards a method for extracting and purifying active compounds from the Amycolatopsis sp. RC21 strain or from a mutant strain, and also the active compounds obtained.

Description

Utilisation des molécules d'une nouvelle bactérie endophyte filamenteuse comme produit antioxydant et anticancéreuse  Use of the molecules of a new filamentous endophytic bacteria as an antioxidant and anti-cancer product
Les actinomycètes sont des bactéries filamenteuses Gram-positives, présentes dans divers milieux écologiques et en particulier dans les sols. Ces microorganismes jouent un rôle important dans l'amélioration de la qualité du sol agricole, notamment en contribuant aux processus de recyclage et de biodégradation de la matière organique et des éléments minéraux (El-tarabily et al, 2008). Les actinomycètes ont un grand pouvoir de transformation des substances organiques complexes difficilement ou non dégradables par les autres microorganismes comme les polymères complexes, les polysaccharides, les lignocelluloses, la chitine (Goodfellow et Williams, 1983; Zaitlin et Watson, 2006). En agronomie, ces bactéries jouent un rôle important dans l'amélioration de la croissance et la protection des cultures de plantes. Actinomycetes are gram-positive filamentous bacteria, present in various ecological environments and particularly in soils. These microorganisms play an important role in improving the quality of agricultural soil, in particular by contributing to the processes of recycling and biodegradation of organic matter and mineral elements (El-tarabily et al, 2008). Actinomycetes have a great power of transformation of complex organic substances difficult or non-degradable by other microorganisms such as complex polymers, polysaccharides, lignocellulose, chitin (Goodfellow and Williams, 1983, Zaitlin and Watson, 2006). In agronomy, these bacteria play an important role in improving growth and protecting plant crops.
La où elles sont très connus, c'est dans le domaine pharmaceutique et de cosmétique. Tel que 60% des antibiotiques naturels dans le monde sont produites par les actinomycètes. Par exemple ; deux nouvelles substances Thiazostatins A et B ayant un effet anti-oxydant puissant ont été isolés d'une souche d'actinomycète Streptomyces tolurosus 1368-MT1 (Shido et al 1989). Un autre exemple de trois isoflavonoïdes antioxydantes 4',7,8-Trihydroxyisoflavone, 3',4'.,7-Trihydroxyisoflavone et 8-chloro-3',4',5,7-tetrahydroxyisoflavone (3) ont été isolés à partir de Streptomyces sp. OH- 1049.  Where they are well known is in the pharmaceutical and cosmetic field. As 60% of the world's natural antibiotics are produced by actinomycetes. For example ; two new substances Thiazostatin A and B with a strong antioxidant effect were isolated from an actinomycete strain Streptomyces tolurosus 1368-MT1 (Shido et al 1989). Another example of three antioxidant isoflavonoids 4 ', 7,8-Trihydroxyisoflavone, 3', 4 ', 7-Trihydroxyisoflavone and 8-chloro-3', 4 ', 5,7-tetrahydroxyisoflavone (3) were isolated from of Streptomyces sp. OH-1049.
Caractérisation de la souche selon l'invention Characterization of the strain according to the invention
Procédé d'isolement de la souche endophyte RC21  Process for isolating RC21 endophytic strain
La souche RC21a été obtenue selon un procédé de sélection comprenant les étapes suivantes :  The RC21 strain was obtained according to a selection process comprising the following steps:
Mise en présence d'un échantillon biologique susceptible de contenir la souche RC21 et/ou une souche mutante, avec un milieu approprié pour la sélection de souches d' actinomycètes,  Bringing together a biological sample likely to contain the strain RC21 and / or a mutant strain, with a medium suitable for the selection of actinomycete strains,
- Traitement dudit échantillon pour isoler les souches actinomycètes présents, et  Treatment of said sample to isolate the actinomycete strains present, and
Isolement de souche RC21 et/ou d'une souche mutante, en fonction de sa capacité à envahir le tissu végétale et de vivre à l'intérieur sans causer de maladie.  Isolation of strain RC21 and / or a mutant strain, depending on its ability to invade the plant tissue and live indoors without causing disease.
L'isolement de la souche selon l'invention peut être effectué à partir d'échantillons d'une plante endémique au Maroc, qui ne pousse qu'au moyen atlas.  The isolation of the strain according to the invention can be carried out from samples of an endemic plant in Morocco, which grows only atlas medium.
Le protocole décrit en suivant.  The protocol described next.
Des échantillons de la racine de la plante endémique de moyen atlas sont obtenus de façon stérile après avoir retirer 15 cm de la racine du sol.  Samples of the root of the endemic medium-atlas plant are obtained sterile after removing 15 cm from the root of the soil.
LES échantillons prélevés sont placés dans des sachets stériles en polyéthylènes fermés et stockés à 4°C.  The samples taken are placed in sterile closed polyethylene bags and stored at 4 ° C.
Les souches actinomycètes sont en suite isolées sur milieu SEA Soil Extract Agar selon quatre types de traitements successifs : traitement par le broyage, la chaleur, agitation et centrifugation.  The actinomycete strains are subsequently isolated on SEA Soil Extract Agar medium according to four types of successive treatments: treatment by grinding, heat, agitation and centrifugation.
Après traitements des échantillons, on isole les souches d' actinomycètes par reconnaissance au microscope sur la base de leurs caractéristiques morphologiques. Les actinomycètes sont transférés sur le milieu ISP2. Les isolais purs sont maintenus à 4°C pendant 2 mois. Alternativement, les cultures sont resuspendues et conservées dans le glycérol à -20°C La souche RC21 selon l'invention est sélectionnée pour sa capacité de d'inhibition des bactéries à gram positif et à gram négatif. Elle est reconnaissable par ces caractéristiques phénotypiques, physiologiques et moléculaire décrites ci-après. After treatment of the samples, the actinomycete strains are isolated by microscopic recognition on the basis of their morphological characteristics. Actinomycetes are transferred to ISP2 medium. The pure isolines are kept at 4 ° C for 2 months. Alternatively, the cultures are resuspended and stored in glycerol at -20 ° C. The strain RC21 according to the invention is selected for its ability to inhibit gram-positive and gram-negative bacteria. It is recognizable by these phenotypic, physiological and molecular characteristics described below.
Etude morphologique Morphological study
La bactérie forme un mycélium de substrat largement ramifié sur les milieux de culture testés (ISP2, ISP3, et Bennet). Le mycélium aérien est fragmenté en forme de tige, caractère spécifique aux souches du genre Amycolatopsis.  The bacterium forms a broadly branched substrate mycelium on the culture media tested (ISP2, ISP3, and Bennet). The aerial mycelium is fragmented in the form of a stem, which is specific to strains of the genus Amycolatopsis.
Le mycélium aérien est blanc sur les milieux gélosés ISP2 et Bennett, mais il est brun à gris brun sur d'autres milieux. Le mycélium de substrat varie de jaune brun à gris brun selon le milieu. Spore chaînes est longue avec irrégulière flèches beaucoup de spores (10-50). Les spores sont lisses et non mobiles.  The aerial mycelium is white on ISP2 and Bennett agar media, but is brown to gray-brown on other media. The substrate mycelium varies from yellow brown to gray brown depending on the medium. Spore chains is long with irregular arrows a lot of spores (10-50). Spores are smooth and not mobile.
Etude physiologique  Physiological study
La souche RC21 ne produit pas de pigment méloïdés.  Strain RC21 does not produce meloid pigment.
Les températures de croissance sont entre 12 et 37°C (optimale est comprise entre 28 et 32°C). Bonne croissance se produit à un pH 5-9. NaCl n'est pas toléré jusqu'à une concentration de 5% (p/v).  Growth temperatures are between 12 and 37 ° C (optimal is between 28 and 32 ° C). Good growth occurs at pH 5-9. NaCl is not tolerated up to a concentration of 5% (w / v).
La souche RC21 utilise pour sa croissance, D-le glucose, le mannitol, le lactose, le saccharose, le maltose et le dextrain. Par contre le glactose, le xylose, l'inositol, le sorbose, le fructose, l'arabinose, le raffinose, le rhamnose et la cellulose, ne sont pas utilisés. L'Amidon n'est pas hydrolysé. Les nitrates sont réduits en nitrites, la gélatine n'est pas liquéfiée, elle dégrade également l'adénine, la Tween 20, de l'acétate de sodium. Aussi, l'alanine L, L - la proline, La phenylanine, L-theronine et D-novaline sont utilisés comme unique source de carbone.  Strain RC21 uses for its growth, D-glucose, mannitol, lactose, sucrose, maltose and dextrain. On the other hand, glactose, xylose, inositol, sorbose, fructose, arabinose, raffinose, rhamnose and cellulose are not used. Starch is not hydrolysed. Nitrates are reduced to nitrite, gelatin is not liquefied, it also degrades adenine, Tween 20, sodium acetate. Also, L alanine, L - proline, phenylanine, L-theronine and D-novalin are used as the sole source of carbon.
Etude moléculaire  Molecular study
L'amplification de la partie ADNr 16s a été effectué en utilisant deux amorces universels 27f (5'-AGAGTTTGATCCTGGCTCAG-3') et 1492r (5'-GGTTACCTTGTTACGACTT-3'). Amplification of the 16s rDNA portion was performed using two universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5'-GGTTACCTTGTTACGACTT-3').
La séquence 16s ADNr est la suivante : SEQ n°l The sequence 16s rDNA is the following: SEQ No. 1
GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCT GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCT
GCGATTACTAGCGACTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCGCGATTACTAGCGACTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATC
CGAACTGAGACCGGCTTTAAGGGATTCGCTCCACCTCGCGGTATCGCAGCCGAACTGAGACCGGCTTTAAGGGATTCGCTCCACCTCGCGGTATCGCAGC
CCTCTGTACCAGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGGGCACCTCTGTACCAGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGGGCA
TGATGACTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTTGATGACTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCT
CCCACGAGTCCCCGCCATAACGCGCTGGCAACGTAGGATAAGGGTTGCGCCCCACGAGTCCCCGCCATAACGCGCTGGCAACGTAGGATAAGGGTTGCGC
TCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCA
TGCACCACCTGTACACCAACCACAAGGGAAGCCCCATCTCTGGGGATGTCTGCACCACCTGTACACCAACCACAAGGGAAGCCCCATCTCTGGGGATGTC
TGGCGCATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCTGGCGCATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATC
CACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAG
CCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCA
CGGACAACGTGGATGTCGCCCACACCTAGCGCCCAACGTTTACAGCGTGGCGGACAACGTGGATGTCGCCCACACCTAGCGCCCAACGTTTACAGCGTGG
ACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGC
GTCAGTATCGGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATA TCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTACCGAACT GTCAGTATCGGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATA TCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTACCGAACT
CAAGTCTGCCCGTATCGACCGCACGCTCCACGTTAAGCGTGGAGATTTCA CAAGTCTGCCCGTATCGACCGCACGCTCCACGTTAAGCGTGGAGATTTCA
CGGCCGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAATAAATCCGGCGGCCGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAATAAATCCGG
ACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCC
GGTGCTTCTTATCCAGGTACCGTCACTTGCGCTTCGTCCCTGGCGAAAGAGGTGCTTCTTATCCAGGTACCGTCACTTGCGCTTCGTCCCTGGCGAAAGA
GGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGG
CTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTATGNGTATGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTATGNGTATG
GGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGCCGGCTACCGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGCCGGCTACC
CGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCCGCGCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCCGCG
GGTTCATCCTGCACCGCCGGAACTTTCAACAACCCCCCATGCGAGAGATTGGTTCATCCTGCACCGCCGGAACTTTCAACAACCCCCCATGCGAGAGATT
GTGATATCCGGTATTAGACCTCGTTTCCAAGGCTTATCCCAGAGTGCAGGGTGATATCCGGTATTAGACCTCGTTTCCAAGGCTTATCCCAGAGTGCAGG
GCAGATTACCCACGTGTTACTCACCCGTTCGCCACTCATCCCCACCCGA GCAGATTACCCACGTGTTACTCACCCGTTCGCCACTCATCCCCACCCGA
Cette séquence est déposée dans la banque de donnée de nucléotides Pubmed/NCBI. This sequence is deposited in the Pubmed / NCBI nucleotide database.
L'analyse phylogénétique de la séquence 16s rADN montre que la dite bactérie endophyte RC21 appartient au genre de Amycolatopsis. Cette analyse a montré aussi qu'elle est proche 99% des souches suivantes Amycolatopsis rifamycinica DSM 46095 et Amycolatopsis pretoriensis NRRL B-24133.  The phylogenetic analysis of the sequence 16s rDNA shows that the said endophytic bacterium RC21 belongs to the genus Amycolatopsis. This analysis also showed that it is close to 99% of the following strains Amycolatopsis rifamycinica DSM 46095 and Amycolatopsis pretoriensis NRRL B-24133.
Des tests physiologiques et l'Hybridation ADN- ADN ont montré qu'elle correspond à une nouvelle espèce dans le groupe des Actinomycètes.  Physiological tests and DNA-DNA hybridization showed that it corresponds to a new species in the Actinomycetes group.
La présente invention vise également les souches mutantes de la souche RC21. Par « souche mutante », on entend désigner toute souche RC21 pouvant être obtenue par mutation sélective à partir de la souche RC21. Les techniques de mutation consistent à mettre la souche RC21 en présence d'un agent mutagène physique, tel qu'un rayonnement, ou d'un agent mutagène physique, tel qu'un rayonnement, ou d'un agent mutagène chimique, puis à sélectionner dans un milieu approprié les mutants d'intérêt restant en utilisant leur spectre antibiotique.  The present invention also relates to the mutant strains of strain RC21. By "mutant strain" is meant any RC21 strain obtainable by selective mutation from the strain RC21. The mutation techniques consist in putting the RC21 strain in the presence of a physical mutagenic agent, such as radiation, or a physical mutagenic agent, such as radiation, or a chemical mutagenic agent, and then to select in a suitable medium the mutants of interest remaining using their antibiotic spectrum.
La souche RC21 selon l'invention ou ses souches mutantes, montrent des activités biologiques importantes. Ces dernières sont variés et multiples. Il s'agit de la production des molécules antifongiques, antibactériennes, anticancéreuses et antioxydants.  The strain RC21 according to the invention or its mutant strains, show important biological activities. These are varied and multiple. It involves the production of antifungal, antibacterial, anticancer and antioxidant molecules.
La souche RC21 selon l'invention ou ses souches mutantes, a montré une activité contre Mycobacterium tubercolosis et Mycobacterium phlei ceci montre que la souche RC21 peut être efficace contre la maladie de la tuberculeuse. Elle a montré également des activités antibactériennes très positives contre Echerichia coli, Pseudomonas aerogenosa, staphylococcus aureus, de même des activités antifongiques contre Mucor ramaniamus et Candidat albicans (figure 1 et 2).  The strain RC21 according to the invention or its mutant strains, has shown an activity against Mycobacterium tubercolosis and Mycobacterium phlei this shows that the RC21 strain can be effective against the disease of tuberculosis. It also showed very positive antibacterial activities against Echerichia coli, Pseudomonas aerogenosa, Staphylococcus aureus, as well as antifungal activities against Mucor ramaniamus and Candidat albicans (Figure 1 and 2).
La souche RC21 selon l'invention ou ses souches mutantes, a montré une activité contre les cellules cancéreuses de sein (Ll) et cellules du rein (L2).  The strain RC21 according to the invention or its mutant strains, has shown an activity against breast cancer cells (L1) and kidney cells (L2).
Des tests de l'effet anti-oxydatives ont été effectués également. Ils ont montré que la souche RC21 selon l'invention présente un effet anti-oxydatives.  Antioxidative effect tests were also performed. They showed that the strain RC21 according to the invention has an anti-oxidative effect.
Le spectre d'activité de la souche RC21 selon l'invention est résumé dans le tableau ci- dessous.  The spectrum of activity of the strain RC21 according to the invention is summarized in the table below.
+ ^ Activité faible Activité moyenne + ^ Low activity Average activity
+++ Activité élevée  +++ High activity
Figure imgf000005_0001
Figure imgf000005_0001
Composes actifs susceptibles s'être produits à partir de la souche RC21 Active compounds that may have been produced from strain RC21
La souche RC21 selon l'invention est capable de produire des composés actifs. La présente invention propose un procédé d'extraction et de purification de ces composées actifs issus de la souche RC21. The strain RC21 according to the invention is capable of producing active compounds. The present invention provides a process for extracting and purifying these active compounds derived from strain RC21.
Le procédé d'extraction et de purification selon l'invention comprend les étapes suivantes : The extraction and purification process according to the invention comprises the following steps:
- Fermentation de la souche RC21  - Fermentation of strain RC21
Sonication de la culture produite de la souche RC21  Sonication of the culture produced of strain RC21
- Centrifugation du filtrat  - Centrifugation of the filtrate
Récupération du surnagent  Recovery of the supernatant
- Extraction en utilisant des solvants organiques hydrophiles et hydrophobes  - Extraction using hydrophilic and hydrophobic organic solvents
Evaporation des solvants - Purification des composés actifs Evaporation of solvents - Purification of active compounds
L'invention vise les composés actifs ou antibiotiques obtenus par ce procédé.  The invention relates to the active compounds or antibiotics obtained by this process.
L'activité des composés actifs de la souche RC21 selon les solvants d'extraction est représentée dans le tableau ci-dessous.  The activity of the active compounds of strain RC21 according to the extraction solvents is shown in the table below.
Activité Activité Activité anticancéreuse antimicrobienne antioxydant  Activity Activity Anticancer Anti-microbial Activity Antioxidant
Souche  Strain
Acétate Acétate Acétate  Acetate Acetate Acetate
Hexane Hexane Hexane d'éthyle d'éthyle d'éthyle  Hexane Hexane Ethyl ethyl ethyl hexane
RC21 ++++ ++ + + +++ ++  RC21 ++++ ++++ +++ ++

Claims

Les revendications The revendications
1. Souche Amycolatopsis sp. RC21, caractérisée par une séquence de l'ADNr 16S SEQ n°l . 1. Amycolatopsis strain sp. RC21, characterized by a sequence of 16S SEQ ID No. 1 rDNA.
2. Procédé de séléction d'une souche selon la revendication 1, caractérisé en ce qu'il comprend les étapes suivantes  2. A method of selecting a strain according to claim 1, characterized in that it comprises the following steps
Mise en présence d'un échantillon végétale d'une plante endémique de moyen atlas susceptible de cointenir la souche Amycolatopsis sp. RC21, avec un milieu approprié pour la sélection de souches actinomycètes,  Placing a plant sample of a plant endemic medium atlas likely to coinain strain Amycolatopsis sp. RC21, with a medium suitable for the selection of actinomycete strains,
Traitement dudit échantillon pour isoler les souchtes d' actinomycètes présentes, et Isolement de la souche Amycolatopsis sp. RC21, en fonction de sa capacité à produire des molécules anticancéreuses, antrioxydantes et antituberculeuses.  Treatment of said sample to isolate actinomycetes strains present, and Isolation of Amycolatopsis sp. RC21, depending on its ability to produce anticancer, anti-oxidant and antituberculous molecules.
3. Utilisation d'une souche selon la revendication 1, pour inhiber la croissance de l'agent causal de la tuberculeuse  3. Use of a strain according to claim 1 for inhibiting the growth of the causative agent of tuberculosis
4. Utilisation d'une souche selon la revendication 1 , pour inhiber la prolifération des cellules cancéreuses de sein (Ll) et des cellules cancéreuses de rein (Ll).  4. Use of a strain according to claim 1 for inhibiting the proliferation of breast cancer cells (L1) and kidney cancer cells (L1).
5. Utilisation d'une souche selon la revendication 1, pour avoir un effet antioxydant.  5. Use of a strain according to claim 1 for having an antioxidant effect.
PCT/MA2013/000051 2013-01-04 2013-12-09 Use of the molecules of a novel endophytic filamentous bacterium as antioxidant and anticancer product WO2014107106A1 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
G. Y. A. TAN: "Amycolatopsis australiensis sp. nov., an actinomycete isolated from arid soils", INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 56, no. 10, 1 October 2006 (2006-10-01), pages 2297 - 2301, XP055118931, ISSN: 1466-5026, DOI: 10.1099/ijs.0.64260-0 *
SAINTPIERRE-BONACCIO DANIELLE ET AL: "Amycolatopsis plumensis sp nov., a novel bioactive actinomycete isolated from a New-Caledonian brown hypermagnesian ultramafic soil", INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 55, no. Part 5, September 2005 (2005-09-01), pages 2057 - 2061, XP002724691, ISSN: 1466-5026 *

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