FR2705685A1 - Microalgal biomass, its extracts, SOD, compositions containing them and process involving these products - Google Patents

Abstract

The invention relates to an essentially pure microalgal biomass, characterised in that it consists of cyanobacteria of the species Mastigocladus laminosus. It also relates to a microalgae extract and a thermostable and/or thermotolerant SOD obtained by extraction. The invention also relates to dermocosmetic or pharmaceutical compositions, to matured muds or food additives containing microalgae or their extracts. It finally relates to processes for improving the condition of healthy skin or of a healthy subject and his locomotive or general comfort.

Description

i The present invention relates to a phytoplankton, present in particular

  in thermal waters, and which has a set of characteristics making it particularly suitable for

  uses in the therapeutic and cosmetological fields.

  Thermomineral waters are by definition natural mineral waters of interest in which at least two factors contribute to pharmacological activities. On the one hand, mineralization is currently the best defined domain and, on the other hand, thermal plankton which has so far been little studied from the point of view of its

  biological composition and its active compounds.

  In all thermomineral waters one meets plankton in more or less quantity. It is therefore a living environment. We know that in a living environment (freshwater, seawater, terrestrial environment) multiple interactions (competition, self or heteroantagonism) define the balance of the environment. The thermal environment as a living environment is no exception to the rule of competition between species. We also know that the interactions defined above are carried out using chemical mediators (terpenes, peptides, alkaloids, etc.). These molecules act on the biochemical systems of neighboring species, either by stimulating their growth (stimulin), or more often by inhibiting them (antimitotics) or eliminating them (toxins). In the latter case, these molecules are defense substances. Such chemical mediators affect more advanced organisms like humans and can

  lead to therapeutic activities.

  It has been shown that the skin of mammals (including humans) is permeable not only to mineral substances but also to certain organic substances. In this case certain molecules contained in thermal water and excreted by plankton are likely to be absorbed by the skin. This has already been shown by Professor Lefèvre from 1964 to 1975 when he notes that a planktonic species collected at the stations of Dax and Barèges (Phormidium uncinatum) and cultivated in his Laboratory had bactericidal and healing properties. Such a result was obtained both from plankton itself and from culture juice, which demonstrated that this species excreted chemicals in the medium capable of diffusing through the human body. Professor Lefévre gave the name cyanostimulins to substances excreted by Phormidium uncinatum because they allowed wounds to be cleaned and tissues to regenerate. But

  the latter could not isolate the molecule or molecules responsible for this activity.

  No researcher has yet been able to isolate molecules with biological activity

in warm waters.

  It would therefore be desirable to be able to have a thermophilic phytoplankton, the properties of which would be maintained under the conditions of use of hydrotherapy, that is to say at higher temperatures.

  or equal to 370 C, for a sufficiently long time.

  This is why the present invention relates to an essentially pure microalgal biomass, characterized in that it consists

  of cyanobacteria of the Mastigocladus laminosus species.

  Mastigocladus laminosus is a cyanobacterium whose spongy thallus reaches a maximum of development between 30 and 60 C. It is

  present in the surface thermal waters of Amélie-les-Bains (France).

  The algae generally used in cosmetology are obtained by marine aquatic harvesting or by culture in a lagoon system. They are therefore always ill-defined products because practically

  impossible to purify and not thermophilic.

  On the contrary, the microalgal biomass according to the invention, which can be obtained, for example, by culture in a tubular photoreactor closed at

  continuous flow, consists of a single species, thermophilic.

  Mastigocladus laminosus synthesizes thermostable molecules, with a high yield, which also allows their extraction on an industrial scale. It is a filamentous cyanobacterium which can withstand, on the one hand, the constraints of use such as body heat without partially degrading and losing its effectiveness; this allows it to be used either pure in local skin application, or in balneotherapy or to enrich mud. On the other hand, it can withstand the various manipulations necessary for the preparation and / or preservation of

  raw products or extracts without denaturing the molecules of interest.

  Among the components of interest in the biomass of Mastigocladus laminosus, mention should be made of pigments: phycoerythrocyanin and

  phycocyanin, which are phycobiliproteins, as well as carotenoids.

  Furthermore, the microalgal biomass according to the invention exhibits a thermostable and / or thermotolerant superoxide dismutase activity. By

  thermostable and / or thermotolerant, we mean a superoxide activity

  dismutase which persists at least partially after treatment at a temperature above 60 C for approximately 10 min. Superoxide dismutase (SOD) activity exerts on ions

  superoxides which it induces dismutation and confers anti-

  radicals to products. It has been shown for example in FR 2 287 899 that this SOD activity is sought in cosmetics, and in particular for

  its anti-aging or UV protection properties.

  The anti-radical activity of SOD makes it particularly

  suitable in two major types of applications, namely as anti

  inflammatory and to fight against aging.

  However, it is generally a heat-inactivated enzyme. One of the advantages of the SOD activity manifested by the biomass of Mastigocladus laminosus, is its thermotolerance, the activity being maintained at around 60% after treatment at a temperature of 64 C. It therefore exhibits optimal activity at temperatures d use of about 40 C, either during local applications, on human skin, pure or in mixture with thermal mud, or for the seeding of

peloid to mature.

  The biomass can then be treated with a view to its conservation, by sterilization and / or lyophilization. In addition, the extraction and / or separation techniques of the active fractions using heating can

be used without damage.

  Finally, another advantage of the biomass according to the invention is its high SOD content, which is of the order of 3 to 6 NBT units / mg of material.

  dry, measured according to the technique of Winterbourn et al (J. Lab. Clim. med.

  (2) 337-341, 1975). The young biomass of M. laminosus is richer than

  biomass in stationary growth phase.

  The present invention therefore also relates to an extract of microalgae characterized in that it is derived from cyanobacteria of the species Mastigocladus laminosus. More particularly, it relates to an extract which exhibits superoxide dismutase activity resistant to a temperature of approximately 64 C.

  The invention includes aesthetic, anti

  aging, anti-inflammatory and improvement of general well-being

  of Mastigocladus laminosus biomass or its extracts.

  The pure biomass of Mastigocladus laminosus or extracts,

  can be used to prepare dermo-cosmetic compositions.

  By dermo-cosmetic composition is meant preparations intended to be applied to the skin of the face or of the body. They can be carried out extemporaneously and consist essentially of the biomass of Mastigocladus laminosus, which is kept in contact with the skin at a temperature of the order of 35 to 45 C. At this temperature, the properties of thermoplankton do not are not altered. Its action on the skin is exerted in particular in the case of adipose overload (which it contributes to reduce while improving the unsightly appearance in orange peel ") and / or aging, and / or locomotor comfort or

of well-being.

  The invention also includes thermal mud enriched with Mastigocladus laminosus biomass or its extracts having SOD activity, and / or matured after sowing with said

  biomass. The indications remain the same.

  Finally, it includes compositions in the form of lotions, creams, packaged ointments and containing Mastigocladus laminosus biomass or extracts having thermotolerant and / or thermostable SOD activity, optionally in combination with other

  active ingredients and / or cosmetologically acceptable excipients.

  Such compositions can in particular be used to delay or prevent aging of the skin, or as sunscreen to help combat damage caused to the skin by UV radiation and / or with the aim of promoting locomotor comfort and

well be general.

  These compositions can advantageously be sterilized by heat to improve their preservation while maintaining the properties and

especially SOD activity.

  The microalgal biomass of Mastigoclodus laminosus or its extracts exhibiting thermotolerant SOD activity are also useful as medicaments. This is why the present invention also relates to a pharmaceutical composition containing such a biomass or

its extracts.

  It will be particularly useful for the treatment of dermatological conditions and / or as an adjunct treatment in rheumatology (anti-inflammatory action), in particular in a subject suffering from degenerative arthropathy, with a view to reducing or controlling the pain

  secondary and lesion evolution.

  The invention also relates to a food additive characterized in that it contains essentially pure microalgal biomass, consisting of cyanobacteria of the species Mastigocladus laminosus preferably having a thermostable and / or thermotolerant SOD activity, an extract derived from cyanobacteria of l Mastigocladus laminosus and having a superoxide dismutase activity resistant to a temperature of approximately 64 C, or a purified SOD prepared

from these products.

  It also relates to a process for improving the condition of healthy skin, in particular aging skin, characterized in that a composition is applied locally containing at least microalgae of the species Mastigocladus laminosus, or an extract of said microalgae having superoxide activity -dismutase, at a temperature of about 35 to 45 C. It relates to a process for improving the performance of a healthy subject characterized in that one administers by local and / or oral route

  a microalgal biomass, an extract, or a SOD as defined above

  above, especially in the form of a dermo-cosmetic, pharmaceutical or locally matured mud composition; food additives obtained from Mastigocladus laminosus promote the physical or intellectual resistance of healthy subjects exposed to conditions

of aggression.

  Finally, it relates to a process for enriching waters intended for balneotherapy, characterized in that an essentially pure microalgal biomass, consisting of cyanobacteria of the species Mastigocladus laminosus, is introduced into the water or the fluid in which the subject will be immersed. preferably having a thermostable and / or thermotolerant SOD activity, an extract from cyanobacteria of

  the species Mastigocladus laminosus and having a superoxide activity-

  dismutase resistant to a temperature of around 64 C, or a purified SOD

  prepared from these products.

  Other characteristics and advantages of the present invention will appear on reading the example which follows. In this example, reference will be made to the appended figure which represents the assay spectrum by chromatography of the biomass of Mastigocladus laminosus Example: Obtaining and characterization of a biomass of Mastigocladus laminosus The strain of Mastigocladus laminosus from the collection of the Institut Pasteur (reference 7521) has been cultured in reactors

aerated two liters.

  An axenic culture is obtained. This filamentous Cynaobacterium occurs in liquid medium in the form of star aggregates of 5 to 15mm in diameter. These in turn can join together giving birth to large clusters of several centimeters. At the beginning, this microalga tends to stick to the wall of the reactor by forming veils which can detach with bubbling and participate in the formation of clusters, in particular on the surface of the culture; after acclimatization, it is more

  homogeneous, which allows its culture in continuous mode.

  The optimal culture conditions are as follows: nutritive medium BGII Composition of the BGII medium NaNO3 1.5 g / 1 MgSO4, 7H20 0.075 g / 1 CaCl2, 2H20 0.036 g / 1 5. K2IPO4, 3H20 0.04 g / 1 citric acid 0.006 g / 1 Na2CO3 0.02 g / 1 Fe-EDTA 0.02 g / 1 Microelements i ml / 1 Solution of microelements H3BO3 2.860 g MnCI2, 4H20 1.810 g ZnSO4, 7H20 0, 220 g CuS04, 5H20 0.080 g MoO3 85 % 0, 036 g CoSO4, 7H20 0.090 g * Distilled water qs 1000 ml temperature = 45 C, - light = 5000 lux without photoperiod - C02 = 1% mixed in air, - aeration = 10 air volume / culture volume / hour (VVH) - pH8 Under these conditions, the multiplying factor after 9 days of culture is 17.5. The concentration obtained is then 0.378 g of material

dryer / liter of culture.

  The expected productivity in tubular photoreactors is therefore 10 g / m2 / day. The biomass obtained with this culture was used to measure the

SOD and phycobiliproteins.

  The attached figure shows the spectrum obtained by chromatographic analysis. It shows the existence of 3 peaks and 2 shoulders. Peaks 1 and 5 respectively at wavelengths 438 and 678 nm (nanometers)

  indicate the presence of Chlorophyll a.

  The No. 2 shoulder at 494 nm corresponds to the carotenoids. These,

  although present, are in the minority.

  Finally, the shoulder n 3 and the peak n 4, at the respective wavelengths 582 and 618 nm, correspond to the absorbance of the phycobiliproteins.

  Peak 4 is that of phycocyanin, a blue pigment.

  Shoulder No. 3, according to the literature (KINGSLEY S. ROWAN, Photosynthetic pigments of algae, Cambridge University Press, Cambridge), seems to resemble the absorbance of phycoerythrocyanine. This molecule, from the phycobiliprotein family, is a red pigment found in Cyanobacteria. The pigments were extracted by grinding the cells with a French press (2 passes) and centrifugation (10 min at 5000 revolutions per minute). The dotted curve is the spectrum of the pellet obtained after centrifugation. It contains cellular debris and shows that all of the pigments have been extracted except for a residue of chlorophyll (small peak at 438 and 680 nm). The quantitative determination of phycocyanin and phycoerythrocyanin necessitates the search for specific extinction coefficients in the literature or the total purification of these pigments by chromatography methods. Indeed, these coefficients are specific to

  each species and even vary according to the extraction protocol.

  The SOD assay was carried out according to the NBT method. Under the culture conditions described above, SOD is present in an amount of 4 units per mg of dry matter. By way of comparison, Porphyridium cruentum, one of the microalgaes richest in SOD, in

contains 5.

  It is important to note that the excellent behavior of Mastigocladus laminosus, observed in a ventilated reactor of two liters (conditions which are as close as possible to the tubular reactors) makes it possible to envisage favorably the culture in photoreactor. Indeed, the size of the aggregates, much smaller than the diameter of the tubing,

is not an obstacle.

  The type of material currently used on photoreceptors does not allow a culture temperature greater than 40-45. This should not be incompatible with the culture of thermophilic Cyanobacteria, hence the advantage of determining the minimum temperature for which

  observes optimal growth of the strain.

Claims (13)

  1. Essentially pure microalgal biomass, characterized in that it consists of cyanobacteria of the species Mastigocladus laminosus. 2. Microalgal biomass according to claim 1, characterized in that it has a thermotolerant superoxide dismutase activity and / or thermostable.   3. Microalgae extract characterized in that it comes from cyanobacteria of the species Mastigocladus laminosus and has a   superoxide dismutase activity resistant to a temperature of approximately 64 C.   4. Superoxide dismutase (SOD) obtained by extraction from a   biomass according to one of claims 1 or 2.   5. Dermo-cosmetic composition characterized in that it contains   a microalgal biomass according to one of claims 1 or 2, or a   extract according to claim 3 or an SOD according to claim 4.   6. Dermo-cosmetic composition according to claim 5, characterized in that it consists essentially of microalgae from the species Mastigocladus laminosus.   7. dermo-cosmetic composition according to claim 5, characterized in that it also contains excipients cosmetologically acceptable.   8. Matured mud, characterized in that it is seeded with a   microalgal biomass according to one of claims 1 and 2.   9. Pharmaceutical composition characterized in that it contains   a microalgal biomass according to one of claims 1 or 2, an extract   according to claim 3 or a SOD according to claim 4.   10. Food additive characterized in that it contains   microalgal biomass according to one of claims 1 or 2, an extract   according to claim 3 or a SOD according to claim 4.   11. A method for improving the condition of healthy skin, characterized in that a composition is applied locally containing at least microalgae of the species Mastigocladus laminosus, or an extract of said microalgae having superoxide dismutase activity, at a temperature about 35 to 45 C. 12. Method for improving the performance of a healthy subject characterized in that it is administered locally at a temperature of 35 to     C and / or oral a product according to one of claims i to 4, or a   composition according to one of claims 5 to 8.   13. Process for enriching water intended for balneotherapy, characterized in that a biomass is introduced according to one of the   claims i and 2, an extract according to claim 3 or a superoxide- dismutase according to claim 4.