US20150320811A1 - Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof - Google Patents

Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof Download PDF

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US20150320811A1
US20150320811A1 US14/651,345 US201314651345A US2015320811A1 US 20150320811 A1 US20150320811 A1 US 20150320811A1 US 201314651345 A US201314651345 A US 201314651345A US 2015320811 A1 US2015320811 A1 US 2015320811A1
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ulva
algal extract
amount
extract
range
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Hervé Demais
Isabelle Le Cheviller
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Amadeite SAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • the present invention relates to an algal extract comprising sulphated and non-sulphated polyanionic polysaccharides, and to a method for preparing an algal extract, as well as to its therapeutic and prophylactic applications.
  • Ulva species are abundant algae found in the intertidal or foreshore area. They colonize hard substrates, anchored by an attachment disc, but certain species may also give rise to free drifting living forms. Ulva algae are fast growing algae and opportunistic as to space and absorption of nutrients. Their growth in the water column is particularly observed in eutrophised coastal waters and in lagoons where Ulva sp. proliferates in the form of green tides (Fletcher, 1996). Often mass production and mass strandings, producing noxious gases when they accumulate, result from this (Morand and Briand, 1996).
  • the use of green algae is essentially based on uses giving the possibility of opening new routes for removing these algae considered as undesirable.
  • the contemplated application routes concern the production of biogas (Briand & Morand 1997, Morand & Briand 1999, Morand et al., 2006), biomaterials (bioplastics) (Chiellini et al., 2008), biodegradable cups (Sassi et al., 2008), papers and cardboards for packaging (Nicolucci & Monegata et al., 1993), charge nano-composites (Demais et al., 2006a, 2006b), animal nutrition: detoxifying feed for animals (Demais et al., 2006), proteins and appetence factors for fish farming (DeBose et al., 2008, Kut Guroy et al., 2007) and cosmetics (surfactants) (Ranson et al., 2008).
  • the present invention relates to an extract from algae of the order of the Ulvales, in particular an extract from green algae of the Ulva type, comprising sulphated and non-sulphated polyanionic polysaccharides, the size of which is less than or equal to 50 kDa.
  • said polysaccharides of the algal extract according to present invention comprise mannose and/or arabinose. More particularly, said polysaccharides comprise at least 0.005% of mannose and/or at least 0.005% of arabinose, by weight based on the weight of the total dry material of the algal extract, notably at least 0.01% of mannose and/or at least 0.01% of arabinose. Still more particularly, said polysaccharides comprise mannose in an amount ranging from 0.01 to 0.20% and arabinose in an amount ranging from 0.01 to 0.5%, by weight based on the weight of the total dry material of the algal extract, notably mannose in an amount ranging from 0.03 to 0.15% and arabinose in an amount ranging from 0.01 to 0.2%.
  • said polysaccharides further comprise:
  • said polysaccharides comprise:
  • algal extract according to the invention comprising:
  • the algal extract according to the present invention comprises sulphated and non-sulphated polyanionic polysaccharides for which the size is less than 40, 30, 20 or 15 kDa. Still more particularly, the sulphated and non-sulphated polyanionic polysaccharides of the algal extract according to the invention has a size of less than or equal to 15 kDa.
  • a dalton (Da) is a mass unit defined as being equal to one-twelfth of the mass of a carbon 12 atom, a mass which will subsequently be estimated from a mixture of several isotopes (mainly carbon 12 and carbon 13, respectively having 6 and 7 neutrons in addition to the 6 protons like any carbon atom).
  • One dalton is, with quite good accuracy, the mass of a hydrogen atom, the exact value being 1.00794 amu (atomic mass unit).
  • the kilodalton (kDa) is equal to 1,000 Da.
  • the masses mentioned in kDa are determined by any method usually used by one skilled in the art, in particular the masses of the sulphated and non-sulphated polyanionic polysaccharides of the algal extracts according to the present invention may be discriminated by ultrafiltration on membranes only letting through molecules of predetermined sizes.
  • green algae of the Ulva type are meant the green algae grouped in the genus Ulva from the family of Ulvaceae, of the order of the ulvales. Mention may notably be made of the following species and sub-species: Ulva acanthophora, Ulva anandii, Ulva angusta, Ulva arasakii, Ulva armoricana, Ulva atroviridis, Ulva attenuata, Ulva beytensis, Ulva bifrons, Ulva brevistipitata, Ulva bulbosa, Ulva burmanica, Ulva byssoides, Ulva californica, Ulva chaetomorphoides, Ulva clathrata, Ulva coccinea, Ulva compressa, Ulva conglobata, Ulva cornucopiae, Ulva cornuta, Ulva covelongensis, Ulva crassa, Ulva crassimembrana, Ulva curvata, Ulva dact
  • the algal extract according to the invention is an extract from algae of the Ulva armoricana species.
  • the algal extract comprises:
  • the algal extract comprises:
  • the other chemical elements present in the dry material of the extract are notably represented by the minerals (Ca, K, Na, Mg, Al, Cl, I, P, Fe, etc).
  • the algal extract according to the invention is characterized by the 1 H NMR spectrum shown in FIG. 1 .
  • This 1 H NMR spectrum was recorded at 298 K on a Bruker Avance 500 spectrometer equipped with an inverse TCI cryogenic probe 5 mm 1 H/ 13 C/ 15 N. Before the analysis, the samples were dissolved in 99.97% of D 2 O atoms. The chemical shifts are expressed in ppm relatively to an external standard (trimethylsilylpropionic acid). No suppression of the HOD signal was achieved.
  • the present invention relates to a method for producing an extract from algae of the order of ulvales, in particular an extract from green algae of the Ulva type, wherein:
  • the algae are washed with fresh water.
  • Sand may be removed from them by any means made available to one skilled in the art.
  • Said algae are then milled, notably by means of a milling machine, such as for example a refiner or a cutter.
  • the solid phase of the milled product, the marc is separated from its liquid phase, the juice, by pressing the milled product, for example by means of a belt or plate press or by centrifugation.
  • juice is meant the cytoplasm juice which includes the wall structure of the double structure of the cells of the algae.
  • the obtained liquid phase is then clarified, for example with a clarifier with disk stacks, or by centrifugation, decantation or filtration (for example with a pocket or a plate).
  • the obtained juice is then ultrafiltrated.
  • ultrafiltration is carried out on a membrane of less than 50 kDa, notably on a 40, 30, 20 or 15 kDa membrane. More particularly, the membrane will be a 15 kDa membrane.
  • This membrane may for example be a ceramic membrane or an organic membrane. More particularly, the membrane will be a ceramic membrane.
  • the obtained filtration juice may then be concentrated, for example by reverse osmosis, evaporation or precipitation, and then for example dried by freeze-drying or atomization.
  • the obtained extract may then again be crushed, in order to obtain a homogeneous powder in terms of grain size.
  • the method according to the invention partly takes place at room temperature.
  • room temperature is meant a temperature comprised between 5 and 25° C.
  • the method according to the invention partly takes place at a temperature comprised between 4 and 10° C., this in order to avoid microbial growth.
  • This method differs from most methods described in the prior art because of the absence of a step involving precipitation of the algal extract. It is also distinguished from the previous extraction methods by the absence of any use of solvents, in particular organic solvents, which represents a major advantage from an ecological point of view.
  • the present invention relates to an algal extract which may be obtained by the method as described earlier.
  • the present invention relates to an algal extract as defined above which may be obtained by the method as described earlier.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an algal extract as described earlier as well as to a pharmaceutically acceptable excipient.
  • the present invention also relates to an algal extract as described earlier for its use as a drug.
  • the present invention also relates to an algal extract as described earlier for its use in preventing and/or treating neurological disorders, depression, stress, anxiety, as an agent allowing improvement of memory notably in preventing and/or treating ageing or Alzheimer's disease, in preventing and/or treating neuropathic and inflammatory chronic pain, skin pain (of the skin, of subcutaneous tissues and associated organs) or body pain.
  • the present invention also relates to the use of an algal extract as described earlier as a drug.
  • the present invention also relates to the use of an algal extract as described earlier in preventing and/or treating neurological disorders, depression, stress, anxiety, as an agent allowing improvement of memory notably in preventing and/or treating ageing or Alzheimer's disease, in preventing and/or treating neuropathic and inflammatory chronic pain, cutaneous pain (of the skin, subcutaneous tissues and associated organs) or body pain.
  • neurological disorders are meant diseases of the nervous system, in particular of the brain.
  • diseases of the nervous system in particular of the brain.
  • diseases mention may notably be made of brain pathologies such as Parkinson's disease, multiple sclerosis, Alzheimer's disease, dementia, migraines, headaches.
  • depression By depression is meant a mood disorder essentially characterized by a condition with loss of motivation or vital momentum in an individual, either associated or not with different symptoms. The most characteristic symptoms are a loss of hope, of desire, of self-esteem. Other signs may occur, such as fatigue, sadness, negative thoughts, dark thoughts, suicidal intentions, anxiety or fear or in certain extremely rare cases, hallucinations.
  • depression This term of depression is again found under the names of recurrent depressive disorder , nervous breakdown , clinical depression , unipolar depression , major depressive and characterized episode or further depressive syndrome .
  • the term of depressions designates the whole of the types of depression.
  • the terms of depressivity or depressive feeling are also used. Common language also mentions feeling low , which has similar symptoms, but more attenuated.
  • stress is meant the whole of the responses of an organism subject to pressures or constraints from its environment. These responses always depend on the perception which the individual has of the pressures which he/she feels. This is a complex sequence of events causing physiological and psychosomatic responses.
  • anxiety is meant a psychological and physiological condition characterized by somatic, emotional, cognitive and behavioural components. In the absence or in the presence of psychological stress, anxiety may generate feelings of fear, restlessness, difficulty and worries. Anxiety is considered as a normal reaction in a stressing situation. When anxiety becomes excessive, it may be classified under the name of anxiety disorder .
  • an algal extract according to the present invention may be used both in veterinary applications, such as for example for preventing and/or treating stress in livestock and pets or as an anti-depressant agent for livestock or pets, and in applications intended for humans, for example via food supplements with a health purpose without any secondary effects or a drug, in order for example to prevent and/or treat disorders related to stress, to anxiety, to depression, as well as an agent allowing improvement in memory, notably in students, for combating ageing or further within the scope of treating Alzheimer's disease.
  • the pharmaceutically acceptable excipients are selected according to the pharmaceutical form and to the desired administration route, from among usual excipients which are known to one skilled in the art.
  • the algal extract as defined above may be administered as unit doses, mixed with conventional pharmaceutical excipients to animals and to human beings, for preventing or treating the aforementioned disorders.
  • the suitable administration forms comprise oral forms such as tablets, soft or hard gelatin capsules, powders, granules or oral solutions or suspensions, sublingual, buccal, intratracheal, intraocular, intranasal administration forms, by inhalation, topical, parenteral such as transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms and implants.
  • oral forms such as tablets, soft or hard gelatin capsules, powders, granules or oral solutions or suspensions, sublingual, buccal, intratracheal, intraocular, intranasal administration forms, by inhalation, topical, parenteral such as transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms and implants.
  • the compounds according to the invention may be used in creams, gels, ointments or lotions.
  • the main active ingredient may be mixed with a pharmaceutical carrier such as gelatin, starch, lactose, magnesium stearate, talcum, gum arabic or the like.
  • the tablets may also be coated with saccharose, a cellulose derivative, or other suitable materials or further they may be treated so that they have a prolonged or delayed activity and continuously release a predetermined amount of active ingredient.
  • a preparation as gelatin capsules may for example be obtained by mixing the active ingredients with a diluent and by pouring the obtained mixture into soft or hard gelatin capsules.
  • compositions containing an algal extract according to the invention may also appear in liquid from, for example, as solutions, emulsions, suspensions or syrups, and notably in a suitable form for oral or intranasal administration for example.
  • suitable liquid supports may for example be water, organic solvents such as glycerol or glycols, as well as their mixtures, in varied proportions, in water.
  • a preparation as a syrup or elixir or for administration in drop form may also contain the active ingredient together with an acaloric sweetener for example, methylparabene or propylparabene as an antiseptic, as well as an agent providing taste and a suitable colouring agent.
  • an acaloric sweetener for example, methylparabene or propylparabene as an antiseptic, as well as an agent providing taste and a suitable colouring agent.
  • the powders or the granules dispersible in water may for example contain the active ingredient mixed with dispersion agents or wetting agents, or suspension agents, such as polyvinylpyrrolidone, also with sweeteners or flavour correctors.
  • the dose administered daily in humans may attain 0.5 to 25 mg/kg, in one or several takings, in particular from 1.5 to 18 mg/kg, and still more particularly from 3 to 15 mg/kg.
  • the daily administered dose in animals may attain 5 to 150 mg/kg, in one or several takings, in particular from 10 to 100 mg/kg and even more particularly from 20 to 80 mg/kg.
  • the daily dose of the algal extract according to the invention will be the smallest effective dose of the algal extract capable of producing a therapeutic effect.
  • effective dose is meant any amount of a composition which improves one or several of the characteristic parameters of the disease or of the disorder to be treated.
  • the suitable dosage for each patient, human or animal is determined by the physician or the veterinary surgeon according to the administration route, the weight and the response of said patient.
  • an algal extract according to the present invention may be used for preventing and/or treating the aforementioned pathologies in a food composition.
  • food composition is for example meant any type of nutraceutical, of food products as a yogurt or a drink, notably a dairy drink, any type of raw material, technological additive or auxiliary, in the form of pre-mixes, either medicinal or not, intended to be incorporated into foodstuffs, any type of full or supplement foodstuffs, intended for humans or animals.
  • the present invention also relates to a method for treating the pathologies indicated above which comprises administration to a patient of an effective dose of an algal extract according to the invention.
  • FIG. 1 1 H NMR spectrum of an algal extract (AE) according to the present invention.
  • FIG. 2 Chromatogram obtained with the algal extract according to the invention separated on two Shodex 802 and 803 columns.
  • FIG. 3 Chromatogram obtained after analysis of trimethylsilylated derivatives of the sample of the algal extract according to the invention by gas chromatography.
  • Ara Arabinose
  • Gal Galactose
  • Glc Glucose
  • Xyl Xylose
  • Man Mannose
  • Rha Rhamnose
  • GlcA Glucuronic acid.
  • FIG. 4 Effects of AE on the number of entries into the open arms in the elevated plus-maze test.
  • FIG. 5 Effects of AE on the time spent in the open arms in the elevated plus-maze test.
  • FIG. 6 Effects of AE on the total number of cumulated pressing actions on both levers in the learning test for avoiding an aversive light stimulus (ALSAR for Aversive Light Stimulus Avoidance Response).
  • ALSAR Aversive Light Stimulus Avoidance Response
  • FIG. 7 Effects of AE on the total number of pressing actions on the active levers (LA) and inactive levers (LI) at 5 minutes in the ALSAR test.
  • FIG. 8 Effects of AE on the total number of pressing actions on the active levers (LA) and inactive levers (LI) at 10 minutes in the ALSAR test.
  • FIG. 9 Effects of AE on the total number of pressing actions on the active levers (LA) and inactive levers (LI) at 15 minutes in the ALSAR test.
  • FIG. 10 Effects of AE on the total number of pressing actions on the active levers (LA) and inactive levers (LI) at 20 minutes in the ALSAR test.
  • FIG. 11 Effects of AE on the duration of immobility(ies) in the pre-test of behavioural despair at D14 (s, Average ⁇ SME).
  • FIG. 12 Effects of AE on the duration of immobility(ies) in the behavioural despair test at D15 (s, Average ⁇ SME).
  • the steps of the method are carried out at room temperature.
  • the algae (1 metric ton of drained algae with 8% dry material) are then crushed into fine particles by means of an industrial refiner (Inotec brand type “I175CDI-75D”).
  • fine particles are meant particles for which the size is comprised between 50 and 1,000 nm, with two populations, the first for which the sizes are comprised between 50 and 200 nm, the second for which the sizes are comprised between 600 and 1,000 nm.
  • crushed material was then pressed by means of an industrial belt press of the brand Flottweg type “B FRU 800 HK”, at a throughput of about 1 metric ton/hour.
  • This step allows separation of the solid phase (marc) from the liquid phase (juice).
  • the yield of obtained juice is 75%.
  • the 750 kg of raw juice obtained are then clarified by means of a clarifier with disc stacks of the brand Flottweg type “AC 2000”.
  • the clear juice is ultrafiltrated on a ceramic membrane (Tami Industries) of 15 kDa.
  • a permeate and a retentate are thereby obtained.
  • the permeate is kept until 640 kg of filtration juice (91% of volume yield) with 2.2% of dry material are obtained.
  • the filtration juice (permeate) is then dried by freeze-drying after concentration by evaporation.
  • the concentration is achieved on a simple effect evaporator (EVA 1000, Pignat) with the following parameters: forced reflow, supply flow rate 10 L/h, vapour pressure of 1 bar, vacuum pressure of 0.3 bars and evaporation temperature of 90° C.
  • EVA 1000, Pignat simple effect evaporator
  • a first concentration is achieved with an evaporated water flow of 8 L/h and the Brix degree rises from 5.5 (equal to a dry material concentration of 4.5%) to 14.7.
  • This solution is then concentrated a second time with an evaporated water flow of 5-6 L/h and the Brix degree rises up to 34.
  • the dry material concentration of the solution is determined at 38.4%.
  • Freeze-drying is then carried out by means of a Bioblock scientific apparatus (version CHRIST alpha 1-4 LSC) at a freezing temperature of ⁇ 80° C. which is also the minimum temperature during this step.
  • a Bioblock scientific apparatus version CHRIST alpha 1-4 LSC
  • the obtained powder is then crushed with a planetary milling machine MiniMill of the Philips brand.
  • the product was introduced into milling bowls (10 g of product in each milling bowl with 4 zirconia balls). The whole was set into rotation for 15 minutes at speed 10.
  • the algal extract according to the invention and prepared according to Example 1 is ultrafiltrated on a 1,000 Da membrane and is dissolved at a concentration of 0.5 g/L in water. It is then injected on two Shodex 802 and 803 columns placed in series (fractionation domain of the 802 column: 4.10 3 Da and of the 803 column: 1.7.10 5 Da).
  • the eluent used is 0.1 M sodium nitrate with 0.2% sodium azide at a flow rate of 0.5 ml/min. Detection is achieved via a Wyatt refractometre and an 18-angle light scattering detector Wyatt. The do/dc are assumed to be equal to 0.150 ml/g.
  • the chromatogram detected by the refractometre is shown in FIG. 2 .
  • the algal extract according to the invention and prepared according to Example 1 is purified by front ultrafiltration in amicon cells operating with stirring. A regenerated cellulose membrane with a cut-off threshold of 1,000 Da is used. 572.1 mg of a sample of algal extract according to the invention are dissolved in 150 ml of ultrapure milli-Q water. Five litres of water are used for removing all the molecules with a mass of less than 1,000 Da. The retentate is freeze-dried. 117 mg of sample are weighed. The ultrafiltration yield is therefore 20.5% (w/w). The following analyses were carried out on ultrafiltrated samples.
  • the ratio of the monosaccharides making up the polysaccharides of the algal extract according to the invention is determined according to the Kamerling method (Kamerling et al., 1975) modified by Montreuil (Montreuil et al., 1986).
  • the identification and dosage of the monosaccharides requires hydrolysis by methanolysis of the polymer, in order to only obtain monomers.
  • the glycoside residues are then trimethylsilylated in order to make them volatile. They are thus identified and assayed by gas chromatography in the form of o-trimethylsilylated methylglycosides.
  • the operating procedure is the following: 400 ⁇ g of the algal extract according to the invention prepared as mentioned above and 50 ⁇ g of myo-inositol are placed in a dry bath in the presence of 500 ⁇ l of a 3 N methanol/hydrochloric acid mixture (Supelco) for 4 hours at 100° C. After cooling down to room temperature, the methanolysate is neutralized with silver carbonate. The samples are centrifuged for 15 minutes at 3,000 rpm and the supernatant is evaporated under a nitrogen jet. The compounds are then dissolved in 80 ⁇ l of pyridine and incubated for 25 minutes at 80° C. with 80 ⁇ l of sylon (BSTFA: TMCS, 99:1, Supelco).
  • BSTFA TMCS, 99:1, Supelco
  • the trimethylsilylated methylglycosides are taken up in 500 ⁇ l of dichloromethane and then injected into gas chromatography (injection into a column, FID detector: flame ionization).
  • the carrier gas is nitrogen.
  • the column, of the HP-5MS type (30 m, inner diameter 0.25 mm), is apolar.
  • the programme for the rise in temperature is the following: 120° C. maintained for 1 minute, and then a gradient of 1.5° C./min up to 180° C., followed by a gradient of 2° C./min up to 200° C.
  • Each monosaccharide is identified by comparing its relative retention times relatively to the internal standard, with those of pure monosaccharides treated under the same conditions.
  • a response coefficient is calculated for each monosaccharide relatively to the internal standard in order to define the proportion of each monosaccharide within the polysaccharides of the algal extract according to the invention.
  • composition of the algal extract according to the invention obtained after analysis of trimethylsilylated derivatives by gas chromatography, expressed by weight based on the total weight of the algal extract; with Ara: Arabinose; Gal: Galactose; Glc: Glucose; Xyl: Xylose; Man: Mannose; Rha: Rhamnose, GlcA: Glucuronic Acid.
  • AE effects of AE were evaluated on groups of 6 rats as compared with a control batch treated with the carrier (spring water) through a functional battery of tests (FOB). Additional tests, open field, elevated-plus maze and the test for avoiding an aversive light stimulus, were conducted for completing the evaluation of the effects of AE.
  • the rats used were treated according to the ethical rules dictated by ASAB and the Canadian Council for Animal Protection.
  • the rats of a same cage all received the same treatment.
  • the rats of the different groups were all handled in the same way and under the same conditions.
  • thermometer a digital thermometer
  • the FOB test was conducted as a blind test before administering the product (T0) and after a period of 14 days of treatment (T14) via an oral route at doses of 20, 40 and 80 mg/kg.
  • the test included three observation phases:
  • Behavioural effects spontaneous locomotor activity, locomotor behaviour disorders, flight reaction to being touched, irritability, caused aggressive and freezing behaviours, drowsiness, micturition and defecation, sensorimotor responses (visual placement, pinching the paw, pinching the tail and reaction to a sound stimulus).
  • Neurological effects pupil response, palpebral reflex, pelvic elevation, position of the tail, muscular tonicity of the limbs and of the abdomen, rollover test, gripping test, trembling and piloerection.
  • Physiological effects salivation, lacrimation, diarrhoea, rectal temperature, cardiac-respiratory rhythm.
  • Elevated plus maze test (LCS)
  • the experimental device with the shape of a cross, is elevated to 80 cm above the ground. It comprises four arms with a length of 50 cm and a width of 15 cm. Two opposite arms are closed by vertical walls with a height of 40 cm, the two other arms being open to empty space, and, consequently represent anxiogenic locations.
  • the animal On day 18, one hour after administration of the treatment, the animal was placed at the centre of the cross of the device and was able to freely access the whole of the four arms. The behaviour of the animal was recorded by means of the ANYMAZE system for 5 minutes.
  • the variables studied in this test were the number of entries into the open arms, as well as the time spent in these open arms. A small number of entries and a short time spent in the open arms are considered as indicating anxiety (Lister, 1987).
  • ALSAR Aversive Light Stimulus Avoidance Response
  • This model uses aversion of the rat for a strongly illuminated environment.
  • the rat learns to master its aversive light environment within the scope of conditioning which operates by pressing on an active lever in order to obtain darkness periods of 30 seconds as a positive reinforcement.
  • the experimental device consists in a strongly illuminated isolated cage (50 ⁇ 40 ⁇ 37 cm), and including two levers: one active one, allowing when it is actuated, 30 seconds of darkness to be obtained followed by return of the light, while the other lever is inactive (does not cause any positive reinforcement).
  • the pressing actions on the active lever, during the darkness period do not provide additional darkness periods.
  • the rat is placed in the cage for 20 minutes and the number of pressing actions on each lever is counted during the experimentation.
  • test battery consisting of 4 conditioning devices is entirely automated and controlled via a computer. Thus, no experimenter is present in the room during the test.
  • the rats were tested on a cognitive level for acquiring training within the scope of conditioning operating in the ALSAR model.
  • the recorded variables were the total number of pressing actions on the active (LA) and inactive (LI) levers and the numbers of pressing actions on each of both levers during the light phase.
  • the numbers of active and inactive pressing actions allowed evaluation of the level of manipulatory activity during the test session. Acquiring training (discrimination between both levers) was evaluated by comparing the number of pressure actions on each of the two levers during the light phase (LA vs. LI).
  • the product is an AE (algal extract) as a powder representing a freeze-dried and crushed concentrated fraction of a water-soluble algal extract prepared according to Example no. 1.
  • the AE is dissolved in spring water and administered orally 60 minutes before the FOB tests and the additional behavioural tests by means of an intragastric feeding probe.
  • Wilcoxon's test was used for two-by-two comparisons of the repeated FOB measurements, between T0 and T14 in each of the groups and for discriminating between both ALSAR levers in an illuminated environment.
  • the main variables modified by daily administration of AE for two weeks are:
  • ALSAR Aversive Light Stimulus Avoidance Response Test
  • the rats treated with AE at the dose of 40 mg/kg, p.o. significantly discriminate the active lever from the inactive lever and those treated with doses of 20 and 80 mg/kg, p.o., tend to operate a discrimination between both levers. This is not the case of the control rats which do not show any trend towards discrimination ( FIG. 8 ).
  • an algal extract according to the present invention thus shows the neurological effects of the product, notably the anti-stress/anxiolytic, anti-depressant, antalgic activities as well as an early acquisition activity of training and of facilitating memorization.
  • AE algal extract according to the invention
  • 3 doses (10, 20 and 40 mg/kg/d) were evaluated in adult male Wistar rats in the behavioural despair test (BDT).
  • the extract was daily administered as a semi-chronic treatment for 14 days.
  • the effects of the extract were evaluated in the BDT on groups of 12 rats as compared with a control batch treated with the carrier (spring water) and a reference batch treated with Imipramine at the dose of 10 mg/kg/d.
  • the rats used were treated according to the ethical rules dictated by ASAB and the Canadian Council for Animal Protection.
  • the rats of a same cage all receive the same dose of AE.
  • the rats of the different groups are all handled in the same way and under the same conditions.
  • the experimental devices consisted in Plexiglas cylinders with a height of 50 cm and a diameter of 20 cm filled with water at 25° C. up to a height of 30 cm.
  • the rats were placed at D14 in the devices for a period of 15 minutes in order to evaluate the immobility time of each animal. Immediately after this first test, the rats received their 14 th treatment.
  • the rats received two treatments: the first, 5 hours before the test and the second 1 hour before the test. This procedure is usually used in the literature for testing the efficiency of a product against depression. The test was conducted under the same conditions as the day before for a period of 5 minutes.
  • the recorded immobility duration during the first 5 minutes of both tests (at D14 and D15) reflects the capacity of the animal of resigning itself when it is confronted with a situation from which it cannot escape. This resignation behaviour is assimilated with depression.
  • the product is an AE (algal extract) as a powder representing a freeze-dried and crushed concentrated fraction of a water-soluble algal extract prepared according to Example no. 1.
  • the extract to be tested was dissolved in spring water and was administered to the rats orally at one of the 3 tested doses for 15 successive days with an administration volume of 5 ml/kg.
  • the animals were weighed every two or three days in order to adapt their specific treatments to their weight. Their food and water intakes were noted every two or three days during the whole duration of the study.
  • the animals of the Reference group receive daily 10 mg/kg/d of Imipramine hydrochloride (Sigma Aldrich) dissolved in spring water with an administration volume of 5 ml/kg.
  • Imipramine hydrochloride Sigma Aldrich
  • the animals of the Carrier group daily received spring water with an administration volume of 5 ml/kg.
  • Paired ANOVA was used, followed, in the case of heterogeneity, by comparisons with the paired t-test for two by two comparisons of repeated measurements of BDT in each of the groups.
  • AE administered at the doses of 20 and 40 mg/kg was ascertained since the immobility durations of the animals treated at these doses were significantly shorter than those of the animals treated with the Carrier.
  • AE administered at the dose of 40 mg/kg gave the possibility of obtaining an immobility duration similar to that of the animals treated with Imipramine at 10 mg/kg.
  • a dose-dependent effect was observed in animals treated with AE at the doses of 10, 20 and 40 mg/kg.
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KR101833667B1 (ko) * 2017-02-14 2018-03-12 주식회사씨에스텍 청각 추출물을 유효성분으로 포함하는 관절연골 보호 또는 치료용 약학적 조성물
FR3087338B1 (fr) * 2018-10-22 2020-10-09 Amadeite Extrait d'algues pour son utilisation pour le traitement ou la prevention de l'immunosuppression post-traumatique
CN110420216A (zh) * 2019-08-09 2019-11-08 天津科技大学 石莼多糖在抑制β-淀粉样蛋白聚集中的用途
CN114796261A (zh) * 2022-03-04 2022-07-29 天津科技大学 石莼多糖在制备抗帕金森症药物、保健品或食品中的应用

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HUE045232T2 (hu) 2019-12-30
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US11633443B2 (en) 2023-04-25
PT2931295T (pt) 2019-10-02
US20180303887A1 (en) 2018-10-25
US20210038662A1 (en) 2021-02-11
ES2746106T3 (es) 2020-03-04
BR112015013567A8 (pt) 2019-10-15
FR2999084B1 (fr) 2015-03-13

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