WO2001000789A2

WO2001000789A2 - Cyanobacterial strain of the genus nostoc and bioactive molecules obtained therefrom

Info

Publication number: WO2001000789A2
Application number: PCT/EP2000/006066
Authority: WO
Inventors: Raffaella Piccardi; Andrea Frosini; Alba Ena; Maria Cristina Margheri; Alessandro Degli Innocenti; Luca Calamai; Mario Roberto Tredici
Original assignee: Universita' Degli Studi Di Firenze; Cnr Consiglio Nazionale Delle Ricerche; Sogesca S.R.L.
Priority date: 1999-06-29
Filing date: 2000-06-29
Publication date: 2001-01-04
Also published as: IT1307320B1; AU6430200A; ITPI990038A1; WO2001000789A3

Abstract

A new cyanobacterial strain of the genus Nostoc, suitable for exploitation in agriculture, pharmaceutics and cosmetics. A molecule having molecular weight of 356 present in cultures, biomasses and extracts of Nostoc KONA97, has shown marked antifungal and antitumor properties. Particularly, lipophilic extracts of KONA97 completely inhibit the growth of Colletotrichum trifolii, Penicillium expansum and Rhizoctonia solani, while lower levels of inhibition have been found against Candida albicans, Phoma sp. and Fusarium roseum. The lipophilic extracts have shown marked toxicity against Artemia salina and Brachionus sp., also inhibiting the growth of HCT8 tumour cell lines.

Description

TITLE

CYANOBACTERIAL STRAIN OF THE GENUS NOSTOC, CULTURES,

BIOMASSES, EXTRACTS, BIOACTIVE MOLECULES OBTAINED

THEREFROM AND USE THEREOF DESCRIPTION

Field of the invention

The present invention relates to a new cyanobacterial strain of the genus Nostoc .

This invention further relates to cultures, biomasses, extracts and bioactive molecules derived from this strain and their use in agriculture, pharmaceutics and cosmetics.

Particularly, but not exclusively, this invention is related to antifungal and antitumor uses of these cultures, biomasses, extracts and bioactive molecules.

Background of the invention

The present invention is the result of studies carried out in the field of antifungal, antitumor and antibiotic substances from microorganisms. In particular in agricultural application, new antifungal substances are needed to inhibit the growth of pathogenic fungi .

Besides, in the pharmaceutical field, new agents are necessary to treat neoplastic diseases. Moreover, the antimicrobial properties of these cultures, biomasses, extracts and bioactive molecules, should be useful in cosmetic formulations as preservatives .

Summary of the invention The present invention provides a new cyanobacterial strain of the genus Nostoc, suitable for exploitation in agriculture, pharmaceutics and cosmetics.

A sample of the culture of the microorganism according to the invention, called KONA97, has been deposited under the Budapest Treaty in the American Type Culture Collection (ATCC) , 10801 University Boulevard - Manassas, VA 20110 - 2209 USA. The sample has been assigned the accession number ATCC PTA 251. According to this invention, cultures, biomasses and extracts obtained from the above mentioned KONA97, and bioactive molecules present in these cultures, biomasses and extracts, are provided.

In particular, a molecule having molecular weight of 356 present in cultures, biomasses and extracts of Nostoc KONA97, has shown marked antifungal and antitumor properties .

Another aspect of the invention, is the agricultural, pharmaceutical and cosmetic use of cultures, biomasses, extracts and bioactive molecules from KONA97.

Particularly, lipophilic extracts of KONA97 completely inhibit the growth of Colletotrichum trifolii ,

Penicillium expansum and Rhizoctonia solani , while lower levels of inhibition have been found against Candida albicans , Phoma sp . and Fusarium roseum .

The lipophilic extracts have shown marked toxicity against Artemia salina and Brachionus sp . , also inhibiting the growth of HCT8 tumour cell lines.

Obviously, this invention includes cyanobacterial strains obtained from mutations, variations, recombination of Nostoc KONA97, as well as bioactive molecules obtained from new micro-organisms or via synthetic procedures, having the same characteristics of those derived from KONA97. Brief description of the drawings and tables

Further aspects of the cyanobacterium will be made clearer by the following description of its isolation, its characteristics and properties, the production methodology thereof and subsequent extraction techniques of its active substances, with reference to the attached drawings, wherein:

- Table 1 shows the composition of the mineral medium used for the cultivation of KONA97; - Table 2 summarizes the elution method to obtain the first active fraction during the process of chemical characterization of the active molecules in the extracts;

- Table 3 summarizes the elution method carried out for the analytical separation of impure fractions from previous semi-preparative analyses;

- Figure 1 is a graphic representation of the elution method of tab.3, and shows in ordinates the percentage of acetonitrile in acid water, and in abscissas the time, wherein the gradient in sigmoidal curve is obtained joining two quadratics.

- Figure 2 shows the ^XH NMR spectrum of the isolated active molecule.

- Figure 3 shows the ¹³C NMR spectrum of the isolated active molecule. Detailed description of the invention a) isolation of the microorganism

Nostoc strain KONA97 has been isolated from a fragment of volcanic rock taken from Kilauea volcano (Big Island, Hawaii) , applying the following method. Small rock fragments were placed in an Erlemeyer flask containing 100ml of BG-11₀ medium, enriched with NaHC0₃ (see tab.l) and added with cycloheximide (200 mg L^"1) , to reduce chlorophyceae and fungi growth.

The enriched culture has been incubated at 25 °C under continuous light (20 μmol photon m^"2 s^"1) . After about 40 days, little green colonies have developed. These colonies were constituted of cyanobacterial filaments whose morphological characteristics were to be related to Nostoc genus. Subsequently, the isolation was carried out spreading dilutions of the enriched culture on BG-11₀ agarized. Then, the Petri dishes were incubated, under the above described conditions, until cyanobacterial colonies appeared. The colonies were constituted by a unialgal Nostoc (Nostoc sp. K0NA97) .

The purification of the culture from bacteria was obtained using the ability of the organism to produce akinetes (thermo-resistant cells) during the stationary phase of growth.

A culture of Nostoc sp . KONA97 with akinetes was treated for 10 minutes at 55 °C, and then diluted and spread on agarized medium. Through microscopical observation (120x) , pure cyanobacterial colonies were detected and isolated.

Microbiological purity was checked by growing Nostoc sp. KONA97 on agarized BG-11₀, added with glucose (5 g L^"1) and casaminic acids (2 g L^"1) . The cultures have been incubated at 30 °C for 15 days, in darkness. The absence of bacteria has been evaluated both macroscopically, checking the clearness of the suspension, and by phase- contrast microscopy. b) Description of Nostoc strain KONA97

Morphological characteristics Under microscopic examination the organism shows unbranched, flexuous and twisted filaments, surrounded by a hyaline capsule. The mature filaments are composed by chains of cells (5.2-6.5 μm in diameter) with heterocysts

(5.6-6.8 μm in diameter) in intercalary, or rarely terminal position. Akinetes are round, 8-10.2 μm in diameter, with granular content, and form in distal position with respect to the heterocyst . Hormogonia are mobile, formed by cylindrical cells, 3.5 μm in diameter.

Reproduction occurs by hormogonia in addition to random trichome breakage, especially near the akinetes.

Culture characteristics

Colonies on agar appear raised, with broken or fimbriate margins, slightly ondulating and bright surface. Under microscopic examination, colonies appear constituted of long heterocystous filaments, embedded in abundant mucilage. The color is bright green. c) Biomass production

The culture of Nostoc PTA 251 has been carried out in batch, in an orbital incubator at 30 °C. The cultures were illuminated with 80 μmol photon m^"2 s^"1 and maintained in a air:C0₂ (95:5; v:v) atmosphere. All culture phases were carried out axenically.

The biomass has been harvested at a cell concentration of about 1.5 g L^"1 by filtration on a 12 μm pores size nylon cloth and frozen at -20 °C. d) Extraction process

The extraction was carried out on the lyophilised and powdered biomass. Two different procedures were used: the first, to evaluate the range of bioactivity of the cyanobacterial extracts during the screening phase, and the second for the chemical characterisation of the active molecules . e) Screening phase In the screening phase, each gram of powdered biomass was sequentially extracted with a mixture ethanol :water (3:7, v:v) to obtain a hydrophilic extract and then with 40ml dichloromethane : isopropanol (1:1, v:v) to obtain a lipophilic extract. Both extracts were dried in vacuum and resuspended in a small amount of water (crude hydrophilic extract) or of ethanol (crude lipophilic extract) . f) Characterization phase

A second method of extraction was set up for the chemical characterization of the active lipophilic extract obtained as described above. For each gram of Nostoc KONA97 powdered biomass, 100ml of methanol were added, and stirred overnight. The suspension was then filtered and the methanol extract was added with 50ml of water. This solution was then extracted by partitioning twice with 50ml of dichloromethane . The organic phase was dried in Rotavapor ( T < 35 °C) to achieve dark green solid. Suspending this residue in ethanol, an extract was yielded, which was stored at -20 °C and used for the following purification procedures, g) Bioactivity tests

Both the hydrophilic and lipophilic extracts were tested for their antifungal (against Aspergillus flavus , Candida albicans, Colletotrichum trifolii , Fusarium roseum, Phoma sp . , Penicillium expansum, Rhizoctonia solani ) , antibacterial (against Escherichia coli , Pseudomonas aeruginosa, Staphylococcus aureus) and cytotoxic activity (against Artemia salina, Brachionus sp . and the tumor cell line HCT8) .

The hydrophilic extract didn't inhibit any of the tested organisms .

The lipophilic extract completely inhibited Colletotrichum trifolii , Penicillium expansum and Rhizoctonia solani , whereas lower levels of inhibition were observed for Candida albicans , Phoma sp . and Fusarium roseum . This extract was found to be lethal for both Artemia salina and Brachionus sp . , and also inhibited HCT8 growth in vitro. No activity was detected against bacteria. h) Isolation and purification Semi-preparative HPLC/DAD analysis

A semi -preparative column was used to achieve a good purification of the active substances, and to obtain adequate amounts for further analysis.

HPLC analysis were made by a liquid chromatograph (Beckman Programmable Solvent Module 126) , with photo- diode detector (Beckman Diode Array Detector Module 168) , managed by the apposite System Gold software (Beckman) . The column was a Kromasil 5C18 (Technicol) , 8 mm x 25 cm, with a pre-column ODS 5 μm (Beckman), 4.6 mm x 4.5 cm, of the same stationary phase and a 100 μL loop for the injection. The elution was made using H₂0 and methanol, while the rinsing was made on methanol 100%. The elution method was a one-step linear gradient, with constant flow at 1ml min^"1. The detection was ensured by a DAD set with two reading channels at 210±4 and 250±4 nm. This allowed to isolate and accumulate an active fraction, with retention time 19-20 minutes, containing the active molecule .

Analytical HPLC/DAD analysis

This analysis has been done to obtain a better resolution of the active substance's peak, so as to be able to analyse it in MS/MS.

A Macherey-Nagel column , CC 250/4 Nucleosil 100-5 C18 AB, 4 x 250 mm, was used, with a pre-column of the same stationary phase. For the injection a 20 μL loop was used. The eluting mixture was H₂0/H⁺ (pH=3 with HCOOH 1.5b) and acetonitrile. The elution method was a single step sigmoidal gradient, with constant flow at 1ml min^"1 (tab. 3, fig. 1)

The DAD detector (diode array detector) has been set for two reading channels at 210±4 and 250±4 nm. The second value was chosen to follow the active substance, having a peak apex at about 254 nm in methanol. Spectra in the range 190-600 nm on peak apex were accumulated during the whole chromatographic run. Applying this method, it was possible to determine an active peak at 19.7 min.

Using the DAD it was possible to obtain the UV-vis. spectrum (190-600 nm) of the active substance, which showed a maximum of absorbance at 212 nm, and a higher second at 254 nm. These data have been confirmed by the analysis made on the pure substance.

HPLC/MS/MS analysis

To define the molecular mass of the active molecule, two kind of MS analysis were carried out. First the active fraction was analysed directly in MS, then an HPLC/MS/MS was made on the active fraction obtained by semi- preparative HPLC.

The molecular weight of the active substance was 356 m/z, being the molecular ion present at 357 m/z. This molecule was characterised by the ¹H and ¹³C NMR spectra reported in figures 2 and 3, respectively.

The foregoing description will so fully reveal the invention according to the conceptual point of view, so that others, by applying current knowledge, will be able to modify and/or adapt for the various examples without further research and without parting from the invention, and it is therefore to be understood that such adaptations and modifications will have to be considered as equivalent to the specific examples. The means to realise the different functions described herein could have a different nature without, for this reason, departing from the field of the invention. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. 10801 University Blvd ^• Manassas, VA 20110-2209 • Telephone: 703-365-2700 • FAX: 703-

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7.3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10.2

To: (Name and Address of Depositor or Attorney)

University of Florence Attn: Mario Tredici DISTAM - P.le delle Cascine, 27 50144 Firenze, Italy

Deposited on Behalf of: University of Florence - National Research Council of Italy - Sogesca S.r.l.

Identification Reference by Depositor: Patent Deposit Designation

Cyanobacterial strain of the genus NOSTOC, KONA97 PTA-251

The deposit was accompanied by: a scientific description X a proposed taxonomic description indicated above.

The deposit was received June 18. 1999 by this International Depository Authority and has been accepted.

AT YOUR REQUEST: X_ We will inform you of requests for the strain for 30 years.

The strain will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive, or if a U.S. Patent is issued citing the strain, and ATCC is instructed by the United States Patent & Trademark Office or the depositor to release said strain.

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same.

The strain will be maintained for a period of at least 30 years from date of deposit, or five years after the most recent request for a sample, whichever is longer. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested July 23. 1999. On that date, the culture was viable.

International Depository Authority: American Type Culture Collection, Manassas, VA 201 10-2209 USA.

S siiggnnaattuurree ooft ppeerrssoonn h haavviinngg aauutthhoorriittyy, t too rreepprreesent ATCC:

Date: July 23, 1999

Barbara M. Hailey, Administrator, Patent Dep/rsϊtory cc: Dott. Ing. Marco Celestino

Claims

1. A new cyanobacterial strain of the genus Nostoc, called Nostoc sp. KONA97, and deposited at ATCC as PTA 251.

2. Any cyanobacterial strain obtained by mutation, variation, recombination of the strain of claim 1.

3. Cultures, biomasses and related extracts, obtained from strains of claims 1 or 2.

4. Bioactive molecules present in the cultures or biomasses of Nostoc sp . KONA97 or related extracts of claims 3.

5. Bioactive molecules of claim 4, with molecular weight 356.

6. Bioactive molecules of claims 4 or 5 obtained from other organisms or by chemical synthesis .

7. The use in agriculture of cultures, biomasses, extracts, bioactive molecules from Nostoc sp . KONA97 of claims from 1 to 6.

8. The use of cultures, biomasses, extracts, bioactive molecules from Nostoc sp . K0NA97 of claim 7, as antifungal, antibacterial, insecticidal , nemathocidal and antimicrobical agents.

9. The use of lipophilic extracts of Nostoc sp . KONA97 of claim 7 against Aspergillus flavus, Candida albicans, Colletotrichum trifolii , Fusarium roseum, Phoma sp . , Penicillium expansum, Rhizoctonia solani , Artemia salina, Brachionus sp .

10. The use of cultures, biomasses, extracts, bioactive molecules from Nostoc sp. KONA97 of claims from 1 to 6, in the pharmaceutical field.

11. The use of cultures, biomasses, extracts, bioactive molecules from Nostoc sp . KONA97 of claim 10, as antineoplastic agents.

12. The use of lipophilic extracts of Nostoc sp . KONA97 of claim 7, to inhibit the growth of the tumor cell line HCT8

13. A method for producing biomass of Nostoc sp . KONA97 of claims 1 or 2 , comprising the following phases:

- inoculation of the culture with the cyanobacterium; - incubation in an orbital incubator, at 30 °C, under continuous illumination and in air/C02 atmosphere;

- mass culture in closed photobioreactors and/or open systems .

14. The use in the cosmetic field of cultures, biomasses, extracts, bioactive molecules from Nostoc sp . K0NA97 of claims from 1 to 6 , as preservatives.