DESCRIPTION

The present invention relates to an extraction process of active ingredients from the alga Ascophyllum nodosum for obtaining a stable aqueous extract of Ascophyllum nodosum

Ascophyllum alga-based preparations (Ascophyllum nodosum, Laminaria digitata) are commonly used as adjuvant products in diets due to their high content of alginate fibres and sulfurated polysaccharides (fucoidans). Ascophyllum also contains a high concentration of vitamins, in particular vitamin C, beta-carotene, Bl, B2, B3, B6, B12, E, H, K, polyphenols antioxidants and a high concentration of minerals and iodine. There are numerous methods for the treatment and extraction of active ingredients from the alga Ascophyllum nodosum for uses intended for the human diet. For example, patent application US2010/0056473 Al describes a method for the extraction of fucoidans starting from Ascophyllum nodosum whereas patent application US 2008/0280994 Al provides extracts of Ascophyllum nodosum which have significant pharmacological characteristics. Each of these extraction methods favours conditions suitable for the concentration of at least one of the active ingredients contained in the alga. For example, the method described in patent US2010/0056473 Al enables a fraction enriched in fucoidans to be obtained, whereas the method described in patent US 2008/0280994 Al favours the obtaining of a fraction enriched in anti-oxidising polyphenols. In other methods, on the other hand, the aim is to obtain solid extracts containing all or almost all the active ingredients of the alga. The extraction techniques of active ingredients from Acophyllum nodosum currently in use are neither efficient nor cheap for the preparation of stable liquid extracts that can be used both as food additives and as additives for the textile industry. In fact, preparations enriched in fucoidans, for example obtained through the method described in patent US2010/0056473 Al have an excellent solubility and stability profile in aqueous solutions but are obtained starting from chromatographic separations, with an absolutely unsustainable production cost for use as a liquid preparation for uses other than medical application. The preparations enriched in polyphenols, as described in patent US 2008/0280994 Al, are obtained by extraction with organic solvents and are therefore virtually free from fucoidans as well as all the other water soluble components of Ascophyllum (including water soluble vitamins). The solid extracts contain all the active ingredients but also a high concentration of specific proteins of the plant, a component with potential allergenic effects. Furthermore, the alginate rich component of the solid extracts determines the formation of gelatinous deposits during the resuspension in aqueous solutions making the liquid preparation unusable.

Patent CN 102 525 838 A describes a skin care formulation containing complex polysaccharides and a preparation method of such formulation. The product obtained with the method described in CN 102 525 838 A contains complex polysaccharides such as polysaccharides of Polygonatum odoratum and polysaccharides of oats, but also contains fats and oils that make such product not suitable for dietary use. The method for the preparation of the formulation further provides a centrifugation system. Such centrifugation systems do not enable the proteins contained in the algae to be removed. As is known, proteins are one of the main sources of allergies and inflammatory reactions, also in the dietary field, and therefore the products obtained through centrifugation systems are not very useful in the dietary field. Furthermore, centrifugation systems are extremely expensive and complex and require high energy consumption for their operation.

The object of patent CN 102 525 838 A is to provide a formulation for cosmetic use with antioxidant activity to slow down skin ageing and overcome free radicals and reduce the production of lipid peroxides.

The technical task of the present invention is to obviate the above-described technical drawbacks of the prior art, in particular by forming a new extraction process of active ingredients from Ascophyllum nodosum which enables stable liquid formulations to be obtained suitable both for dietary use and as preparations for the treatment of fabrics with bioactive properties.

Within the scope of this technical task, an object of the invention is to provide a stable product that can be used as an additive in the food or textile industry.

Another object of the invention is to provide an extraction method for the preparation of stable liquid products that does not provide the use of organic solvents or chemical additives.

A further object of the invention is to provide a method for the extraction of active ingredients from Ascophyllum nodosum which is cheap, offers high energy savings and can be easily applied to the industrial scale. A further object of the invention is to provide a method for the extraction of active ingredients from Ascophyllum nodosum which allows a hypoallergenic product to be obtained and having an immunological and anti-inflammatory effect.

Finally the object of the invention is to provide a method for the extraction of active ingredients from Ascophyllum nodosum that allows a protein-free product to be obtained that can be used in the food and textile industry.

The technical task, as well as these and other objects, according to the present invention, are attained by an extraction process of active ingredients from the alga Ascophyllum nodosum characterised in that it comprises in succession the steps of:

- obtaining a homogeneous product through the micronisation of a quantity of fresh or dried Ascophyllum nodosum alga

- detaching the active ingredients from the fibres by treating said homogeneous product in an ultrasonic bath at a controlled pH at a value of less than 6 for the microbiological stabilisation of the said homogeneous product

- eliminating the fibres through a first product filtration process

- removing the proteins from the product for its physical stabilisation

- eliminating odorous and/or oily substances through a second product filtration process

- sterilising the product in order to obtain a stable aqueous extract through a third filtration process

Preferably during the micronisation step the dried alga is rehydrated in a minimum volume of water. In particular the pH is corrected through the addition of citric acid, preferably in a pH interval comprised between 4.5 and 5.5

Preferably the first filtration process is performed on a refining filter press.

Preferably the second filtration process is performed on active carbon.

Preferably the third filtration process is performed on a sterile filter.

In particular the treatment in the ultrasonic bath is performed for 24 hours at 25 °C. Preferably the step of removing the proteins is performed by means of the combined effect of ethanol and bentonite.

In particular, the step of removing the proteins is performed through the addition to the product of ethanol up to a maximum of 20% in volume and through the subsequent incubation of the product with bentonite at a temperature comprised between 2 °C and 25 °C.

Preferably the step of removing the proteins is performed through dialysis against water at a temperature comprised between 2 °C and 25 °C in constant agitation for 24 hours.

In particular, after the sterilisation step, a preservation step is provided of the stable aqueous extract in sealed containers without the addition of preservatives or stabilisers.

The invention further discloses a stable aqueous extract of Ascophyllum nodosum obtained through the extraction process described above.

Preferably the stable aqueous extract may comprise ethanol up to a maximum of 20% in volume, and sulfurated polysaccharides with high molecular weight.

Preferably the stable aqueous extract is free from ethanol and comprises sulfurated polysaccharides with a low molecular weight.

The invention finally discloses the use of the stable aqueous extract as an industrial additive for the impregnation of fabrics or as a food additive.

Further characteristics and advantages of the invention will more fully emerge from the description of a preferred but not exclusive embodiment of the extraction process according to the invention, illustrated by way of non-limiting example in the accompanying figures of the drawings, in which:

figure 1 shows a block diagram of the process in compliance with a first embodiment of the invention;

figure 2 shows a block diagram of the process in compliance with a second embodiment of the invention;

figure 3 shows the data obtained with the experiment performed on cultures of peripheral blood mononuclear cells of coeliac patients;

figure 4 shows the analysis of the expression of the CD45 cluster of differentiation surface markers for all the lymphocytes. The characterisation of the lymphocytes is performed through flow cytometry;

figure 5 shows the loading of the samples into the wells of the plate for performing the anti-inflammatory tests with extracts of Ascophyllum nodosum;

figure 6 shows the tryptophan and neopterin calibrations carried out for performing the anti-inflammatory tests;

figures 7 and 8 show the effects of the inhibition of the degradation of tryptophan and the inhibition of the production of neopterin obtained with the simple dilution of the liquid extract of Ascophyllum nodosum. The term "removing" used in the present invention has the same meaning of eliminating.

The extraction process can be defined as a cold aqueous extraction, potentially containing up to 20% in volume of ethanol, at a controlled pH in a range comprised between 4.5-5-5, followed by a protein removal step and finally a sterile filtration on oenological press filters.

According to a first embodiment of the invention, the process provides the use of raw materials such as alga of the Ascophyllum nodosum genus, fresh or dried and chopped, ethanol, citric acid, bentonite for oenological use, active carbon and carbon and diatom filters for oenological use.

The process is performed with tools such as a high speed cutting mill (final mesh < 100 micron), ultrasonic bath and press filter.

The process according to the first embodiment of the invention provides the micronisation of the alga.

In the event that the alga is previously dried, the micronisation is performed in a minimum volume of distilled water so as to allow the rehydration of the alga itself (5 1 water per kg of dry alga) until the complete homogeneity of the product. The homogenate thus obtained is directly transferred into the ultrasonic bath for 24 hours at 25 °C controlling the pH (4.5 - 5.5) and potentially correcting with citric acid or directly adding 0.1 % of citric acid. After the ultrasonic treatment, ethanol (0 to 20 %) is added to the homogenate, filtered on a "refining" filter (00 filters) and incubated at a temperature comprised between 2 and 25 °C, preferably cold (2- 8 °C), with bentonite (lg bentonite per 1 of homogenate) previously suspended in a minimum volume of water. The suspension is left to settle for 15 days. If necessary, the possibility to decant or refilter the homogenate on refining filters is provided, then passing everything through an active carbon cartridge which contributes to eliminating odorous or oily substances. Finally, the homogenate is filtered on a sterile press (0.4 micron), or, if still cloudy, on a 2-8 micron filter before being passed onto a sterile filter. The product thus obtained is kept at room temperature (2-25 °C), preferably cold (2-8 °C), in sealed polypropylene containers without adding preservatives or stabilisers.

In a second embodiment of the invention, the process provides the use of raw materials such as alga of the Ascophyllum nodosum genus, fresh or dried and chopped, citric acid, dialysis tubes, carbon and diatom filters for oenological use. The process is performed with tools such as a high speed cutting mill (final mesh < 100 micron), ultrasonic bath and press filter.

In the second embodiment the process provides the micronisation of the alga. In the event that the alga is previously dried, the micronisation is performed in a minimum volume of distilled water so as to allow the rehydration of the alga itself (5 1 water per kg of dry alga) until the complete homogeneity of the product. The homogenate thus obtained is directly transferred into the ultrasonic bath for 24 hours at 25 °C in the presence of citric acid as a pH corrector. After the ultrasonic treatment, the homogenate is filtered on a "refining" press filter (00 filters) and transferred into dialysis tubes. The dialysis tubes are placed in a water bath (3-4 volumes of water per volume of homogenate) at a temperature comprised between 2-25 °C, preferably cold (2-8 °C) in constant agitation for 24 hours. In this case, the dialyzate is recovered while the contents of the dialysis tubes are eliminated (fibres, proteins and other non-diffusible elements) or reused for different purposes (i.e. fertilizer). The dialyzate is passed through an active carbon cartridge which contributes to eliminating odorous and oily substances. Finally, the filtration is performed on the sterile press filter (0.4 micron). The product thus obtained is kept at room temperature (2-25 °C), preferably cold (2-8 °C), in sealed polypropylene containers without adding preservatives or stabilisers.

The correction of the pH is an extremely important step in the extraction process of active ingredients from the alga. In fact, the pH correction allows the microbiological stabilisation of the product to be obtained since the maintenance of the pH in a range comprised between 4.5 and 5.5 allows the growth of any microorganisms that should not be present to be eliminated.

The correction of the pH is performed with citric acid, or alternatively can also be performed through acetic acid.

The step of removing the proteins, on the other hand, allows a physical stabilisation of the product to be obtained.

In fact, the elimination of the proteins reduces the risk of the formation of precipitates from the liquid phase.

The treatment in the ultrasonic bath allows the active ingredients adhered to the fibres of the micronised alga to be detached so as to effectively collect the active ingredients and eliminate the fibres through the filtration step on a refining press filter.

Table 1, shown below, highlights the results obtained in the process experiment according to the first and second embodiment of the invention with different solvent compositions. The following were analysed: the protein content with the Bradford (Sigma Aldrich) method and the sulfurated polysaccharide content with IR spectroscopy as well as the colour (polyphenols and pigments) via UV-Vis spectroscopy. The best conditions (polysaccharide/highest protein ratio) are those that have led to the definition of the process according to the present invention. The process according to the first embodiment of the invention, in the presence of ethanol at 15-20%, generates a more concentrated extract in which 70 mg of protein are present per litre of extract (approximately half those present in wine). The process according to the second embodiment of the invention, on the other hand, generates a more diluted extract, in which, however, there are virtually no proteins.

The extraction process of active ingredients from Ascophyllum nodosum according to the present invention guarantees the preparation of stable liquid formulations, suitable both for dietary use in the form of a drink and/or additive for carbohydrates (bread and pasta) or, as preparations for the treatment of fabrics with bioactive properties. In fact, the procedures used provide cold treatments in the absence of organic solvents or chemical additives of any kind, with the exception of citrate and ethanol, which are however "food grade" products. In this way, the extraction of all the water soluble vitamins, mineral salts, iodine, polyphenols, soluble terpenoid fraction and a significant fraction of polysaccharides with low molecular weight, is guaranteed whereas no fibres, alginates, lipids and particularly proteins are extracted. The entire process is low cost, such that it can be performed with the typical equipment for oenology and therefore easy to be applied to an industrial scale.

The product obtained from the procedure according to the present invention can be defined as a concentrated aqueous extract to be added as a nutritional complement to drinks and/or preparations based on carbohydrates or as a preparation to be impregnated or sprayed onto fabrics in order to give them bioactive properties. The optimal dilution for the final use must consider the ethanol content. In fact, the extract containing 20% of ethanol is more concentrated in active ingredients and must be diluted approximately 20 times in the final product. The extract free from ethanol may, on the other hand, be diluted 1 :2 up to 1 :5. The process according to the first preferred embodiment leads to the production of a product containing sulfurated polysaccharides with high molecular weight, which is more suitable for impregnating fabrics while the procedure according to the second preferred embodiment of the invention guarantees a product free from proteins but which contains only sulfurated polysaccharides with low molecular weight, which are less suitable for impregnating fabrics but have a more favourable profile for use as food additives.

The experiments performed to demonstrate how removing the proteins through the method described by the present invention allows a product to be obtained with an anti-inflammatory and immunological effect to be used in the dietary and fabric field, are shown below.

EXAMPLE 1

Immunological and anti-inflammatory effect of the extract of Alga Ascophyllum nodosum on cultures of peripheral blood mononuclear cells of coeliac patients.

Coeliac disease is one of the most common systemic autoimmune disorders both in Europe and the United States and manifests itself in genetically predisposed individuals following the ingestion of foods containing gluten, a lipoprotein substance that originates from two types of proteins (gliadin and glutenin) present in most cereals.

Abundant experimental evidence suggests that the coeliac disease is caused by an innate response and, subsequently, T cell mediated directed against some gliadin peptides at the level of the duodenal mucosa until the latter is damaged (atrophy of the intestinal villi, hyperplasia of the crypts of Lieberkuhn and/or local intraepithelial lymphocytosis). Experiment

Patients.

The coeliac patients used in the trial were made aware of the aims and methods of the trial protocol in order to obtain informed written consent. The patients enrolled in the trial respected the following criteria:

- gluten free diet (GFD) for the last 6 to 12 months, first negativisation of the serum anti-endomysial (EMA) and anti-tTG antibodies, age over 18.

- diagnosis made in accordance with the guidelines recently established by the "European Society for Pediatric Gastroenterology, Hepatology, and Nutrition" (ESPGHAN).

- absence of other autoimmunity, inflammatory and tumorous diseases. Methods.

The peripheral blood mononuclear cells (hereinafter PBMC) of the patients were isolated from whole blood samples, of each patient, by means of a standard cell extraction method performed with the aid of Lympholyte®-H.

The PBMCs obtained from each patient ( ~ 4x10 ⁶ cells per patient) were cultivated for 48 hours, at 37 °C and in the presence of an atmosphere comprising 95% air and 5% C0 ₂ with respect for the following trial conditions:

- Only growth medium (internal negative control)

- Growth medium + peptic-tryptic digest (PT) of a-gliadin (internal positive control)

- Growth medium + peptic-tryptic digest (PT) of a-gliadin + alga extract. Preliminary results

The data shown in Figure 3 suggests an effect of the Ascophyllum nodosum extract in PBMC cultures of coeliac patients which can be translated into an imbalance T. The increase in lymphocytes T and the decrease in lymphocytes B, both induced by the alga, indicate a shift of the specific immunity of the coeliac disease towards the cell-mediated response to the damage of the humoral response. The humoral response is responsible for the production of specific antibodies and therefore for the onset of coeliac disease. Therefore the inhibition of such humoral response induced by the Ascophyllum nodosum extract has a beneficial effect on the course of the disease.

Furthermore, the data shown in Figure 4 show that the expression analysis of the surface markers for regulatory T lymphocytes (CD3+, CD4+, CD25 ^bright , CD127 ^Iow , highlighted a slight decrease in such cells after culture with Ascophyllum nodosum extract

EXAMPLE 2

ANTI-INFLAMMATORY TESTS ON ASCOPHYLLUM NODOSUM EXTRACTS

The test is based on the dosing of the degradation of tryptophan coupled to the production assay of neopterin as indicators of the inflammatory process in progress (see attached publication and references cited). In short, a solution of polymorphonucleated leukocytes (PBMC) is subjected to pro-inflammatory stimulation with phytohemagglutinin so as to stimulate the degradation of tryptophan and the production of neopterin. After 48 hours incubation at 37 °C the two analytes are dosed with an ELISA test with the appropriate negative controls. The inhibition of the consumption of tryptophan and production of neopterin indicates an anti-inflammatory action.

The anti-inflammatory activity of the extracts of the alga Ascophyllum nodosum was highlighted in vitro on leukocytes using the inhibition effect of the degradation of tryptophan and the inhibition of the production of neopterin in the presence of phytohemagglutinin.

The ELISA test was performed on the following samples:

- LI: LOT No: xx/12 ASCO M100. Water/alcohol extract 85/15 V/V.

Sterile filtrate. Stored between 2-8 °C. Lot 1 refers to a sample of dried algae treated with the method of the present invention.

- L2: Lot 1 diluted 1 :2 (formulated with glycerol on a fabric impregnated at high concentration)

- L3: Lot 1 diluted 1 :4 (formulated with glycerol on a fabric impregnated at medium concentration)

- L4: Lot 1 diluted 1 :8 (formulated with glycerol on a fabric impregnated at low concentration)

- SI, S2 and S3: fresh human serum (three samples of human serum obtained from three different subjects).

The fabrics used in the anti-inflammatory test were cotton (e.g. cotton gauze) as an example of possible products with medical purposes and non-woven fabric as an example of products for disposable fabric purposes (e.g. nappies and sanitary towels). The fabric used in the test was left to soak for 4 hours with the alga extract obtained through the process according to the present invention and then dried in the air.

The ELISA tests performed on said samples were:

- Nl - neopterin ELISA test RE59321 (BioSupplyUK) Neopterin Elisa Kit, lot 073-12. Immunoenzymatic assay for the diagnostic quantitative determination in vitro of neopterin in human serum, plasma and urine.

- Wl- Kit ELISA tryptophan K7730 (BiosupplyUK) Tryptophan ELISA Kit, lot 085-12

- FT- phytohemagglutinin L 1668 (Sigma).

The ELISA tests are standard tests and were performed as shown in the manufacturer's instructions.

The diagram drawn up for the positioning of the samples in the plate was in the form of a scattered matrix with equally spaced controls so as to minimise any position effects in the reading of the plate (Figure 5).

The isolated leukocytes from the serum were present in the quantity of lxl0 ⁶ /ml in all the wells. In particular SI in columns 1-4, S2 in columns 5-8 and S3 in columns 9-12.

The controls used are: addition of buffer (Bu), addition of phytohemagglutinin (FT) or addition of salicylate (Sal). The controls were also performed in combination (FT+Sal+Bu etc.).

The readings were performed on a plate reader with UV-Vis Biorad absorption both for the tryptophan degradation plate (320 nm) and for the neopterin production one (405 nra).

In a separate ELISA plate portion, the tryptophan and neopterin calibrations were performed as reported in Figure 6. Below are the recordings of the data obtained (absorbance values) in the different wells of the ELISA tests indicated above.

Results

For reading the results shown in the graphs of Figures 7, 8 and in Table 6 it is necessary to consider that with absorbance values close to 2 there is no production of neopterin (or degradation of tryptophan) which therefore highlights the absence of inflammation. With absorbance values close to 0, on the other hand, the production of neopterin is maximum, which means high inflammation. It can therefore be noted how the inhibition effect of the degradation of tryptophan and the inhibition of the production of neopterin is very clear after the addition of the liquid extract of Ascophyllum nodosum up to dilutions of 1 :8. The effect is very clear also in the solutions incubated with fabric impregnated with increasing doses of alga extract (Table 6).

Table 6

The table below shows the effects of inhibition of the degradation of tryptophan and inhibition of the production obtained on samples of impregnated fabrics

The experiments show how the extract of Ascophyllum Nodosum alga obtained with the method that is the subject matter of the present invention can be applied, with numerous advantages for the human organism, in various market segments. In fact, thanks to the fact that the extract is almost colourless, inodorous and flavourless, it can be used in the textile industry (e.g. on yarns), in the curative- medical industry (e.g. on disposal non-woven fabric products such as nappies and sanitary towels) and in the dietary industry (e.g. in the production of baked products such as bread, biscuits and pasta).

The process as conceived herein is susceptible to many modifications and variations, all falling within the scope of the invented concept; furthermore, all the details are replaceable by technically equivalent elements.

The materials used, as well as the dimensions, may in practice be of any type according to requirements and the state of the art.