Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof
11633443 · 2023-04-25
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K36/05
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
Y02E50/30
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
A61K31/737
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to an algal extract comprising sulphated and non-sulphated polyanionic polysaccharides, to a method for preparing an algal extract as well as to its therapeutic and prophylactic applications.
Claims
1. A method for treating anxiety, the method comprising the administration of an effective amount of an algal extract from the order of Ulvales comprising sulphated and non-sulphated polyanionic polysaccharides, the size of which is less than or equal to 50 kDa, wherein the algal extract is prepared by a process comprising the following steps: a) washing the algae and freeing the algae of sand; b) crushing the washed and freed algae to create a crushed product comprising a solid phase and a liquid phase; c) separating the solid phase of the crushed product from the liquid phase of the crushed product; d) clarifying the separated liquid phase to form a juice; e) filtering the juice with a membrane of less than 50 kDa; and f) concentrating the filtered juice; and g) drying the concentrated juice.
2. The method according to claim 1, wherein the extract is an extract of green algae of the type Ulva.
3. The method according to claim 1, wherein said polysaccharides comprise mannose and/or arabinose.
4. The method according to claim 3, wherein 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.
5. The method according to claim 4, wherein said polysaccharides comprise mannose at an amount in a range from 0.01 to 0.20% and arabinose at an amount in the range of 0.01 to 0.5%, by weight based on the weight of the total dry material of the algal extract.
6. The method according to claim 1, wherein said polysaccharides further comprise galactose, glucose, rhamnose, xylose, and glucuronic acid.
7. The method according to claim 6, wherein: the galactose is at an amount in the range of 0.05 to 0.5%; the glucose is at an amount in the range of 0.005 to 0.05%; the rhamnose is at an amount in the range of 2 to 15%; the xylose is at an amount in the range of 0.1 to 1%; and the glucuronic acid is at an amount in the range of 1 to 7%; by weight based on the weight of the total dry material of the algal extract.
8. The method according to claim 1, wherein said polysaccharides have a size of less than or equal to 15 kDa.
9. The method according to claim 1, wherein the algal extract comprises: carbon at an amount in the range of 10 to 50%; hydrogen at an amount in the range of 1 to 10%; nitrogen at an amount in the range of 1 to 5%; oxygen at an amount in the range of 20 to 50%; and sulphur at an amount in the range of 1 to 15%; in weight percentage of the total dry material of the algal extract.
10. The method according to claim 1, wherein the algal extract comprises: carbon at an amount in the range of 15 to 30%; hydrogen at an amount in the range of 3 to 6%; nitrogen at an amount in the range of 1 to 3%; oxygen at an amount in the range of 25 to 40%; and sulphur at an amount in the range of 2.5 to 10%; in weight percentage of the total dry material of the algal extract.
11. The method according to claim 1, wherein membrane is a 15 kDa membrane.
12. The method according to claim 1, wherein solid phase is separate from the liquid phase by pressing the crushed product.
13. The method according to claim 1, wherein: the algae is washed in freshwater; the algae are crushed with a refiner; the solid phase of the crushed product is separated from the liquid phase of the crushed product by pressing the crushed product with a belt press; the separated liquid phase is clarified with a clarifier with disc stacks; the membrane is a 15 kDa ceramic membrane; the filtered juice is concentrated by evaporation; and/or the concentrated juice is dried by freeze-drying or atomization.
14. The method according to claim 1, wherein the composition further comprises for at least one pharmaceutically acceptable excipient.
Description
FIGURES
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EXAMPLES
Example 1: Preparation of an Algal Extract According to the Invention
(13) One metric ton of fresh, raw, green algae of the Ulva type, is washed with fresh water and sand is removed by means of a machine for washing algae.
(14) Unless indicated otherwise, the steps of the method are carried out at room temperature.
(15) 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”). By “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.
(16) The 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.
(17) This step allows separation of the solid phase (marc) from the liquid phase (juice). The yield of obtained juice is 75%.
(18) 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”.
(19) 710 kg of a clear juice with 3.10% of dry mass (95 to 98% mass yield) and a cream (2 to 5% by mass) are thereby obtained.
(20) Subsequently, the clear juice is ultrafiltrated on a ceramic membrane (Tami Industries) of 15 kDa.
(21) 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.
(22) The filtration juice (permeate) is then dried by freeze-drying after concentration by evaporation.
(23) 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.
(24) 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%.
(25) 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.
(26) 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.
(27) 14 kg of an algal extract powder were thereby obtained.
Example 2: Determination of the Size of Sulphated and Non-Sulphated Polyanionic Polysaccharides of an Algal Extract According to the Invention by GPC (Gel Permeation Chromatography)
(28) 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.Math.10.sup.3 Da and of the 803 column: 1.7.Math.10.sup.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.
(29) The chromatogram detected by the refractometre is shown in
(30) An average size of 4.4 kDa of the polysaccharides from an algal extract according to the invention is obtained.
Example 3: Determination of the Composition of an Algal Extract According to the Invention
(31) 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.
(32) The ratio of the monosaccharides making up the polysaccharides of the algal extract according to the invention is determined according to the Kemerling method (Kemerling 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.
(33) The following reagents are used:
(34) A 3N methanol/HCl solution (Supelco);
(35) Silver carbonate;
(36) Myo-inositol;
(37) Pyridine;
(38) Sylon BFT (BSTFA+TMCS 99:1) reagent (Supelco); and
(39) Dichloromethane.
(40) The operating procedure is the following: 400 μg of the algal extract according to the invention prepared as mentioned above and 50 μg of myo-inositol are placed in a dry bath in the presence of 500 μl 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 μl of pyridine and incubated for 25 minutes at 80° C. with 80 μl of sylon (BSTFA:TMCS, 99:1, Supelco). After gentle evaporation of the excess reagents under a nitrogen jet, the trimethylsilylated methylglycosides are taken up in 500 μl 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.
(41) 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.
(42) The obtained results are shown in Table 1 below and in
(43) TABLE-US-00001 TABLE 1 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. % by weight of the % by weight of the Sample ultrafiltrate Ultrafiltration yield raw extract Ara 0.6 20.50% 0.123 Gal 1.3 20.50% 0.267 Glc 0.1 20.50% 0.021 Xyl 2.45 20.50% 0.502 Man 0.45 20.50% 0.092 Rha 39.6 20.50% 8.118 GlcA 12.9 20.50% 2.645
Example 4: Evaluation of the Effects of the Algal Extract (AE) Through a Functional Battery of Tests and of Additional Tests (Open Field, Elevated-Plus Maze, Test for Avoiding Aversive Stimulus)
(44) The effects of an algal extract according to the invention (AE) administered orally at doses of 20, 40 and 80 mg/kg for 14 days were evaluated in adult male Wistar rats.
(45) The 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.
(46) The rats used were treated according to the ethical rules dictated by ASAB and the Canadian Council for Animal Protection.
(47) Equipment and Methods
(48) Animals
(49) Twenty four (24) male Wistar Crl:WI (Han) rats (Charles River Laboratories, 69-St-Germain sur l'Arbresle, France) of 60 days of age were used. Upon receiving them, the rats were marked and distributed in groups of 2 in polycarbonate cages of the F type (48×27×20 cm, U.A.R., 91-Epinay-Sur-Orge, France). The animals were confined in an air-conditioned animal housing facility, at a temperature of 22±2° C. and with 50±20% hygrometry. The rats had standard 2016 feed (Mucedola for Harlan, Milan, Italy) and drink ad libitum and were subject to a reverse light-darkness cycle of 12 hours (light from 8 p.m. to 8 a.m.).
(50) After getting used to the laboratory conditions for one week, the rats are weighed and randomly distributed into 4 treatment groups (n=6).
(51) In order to avoid possible interferences between the various products, 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.
(52) The FOB Test
(53) The following equipment was used in this test:
(54) a transparent observation cage;
(55) a suspension device;
(56) a gripping device;
(57) an open field for locomotor and exploratory activity;
(58) an auditive stimulation apparatus emitting a standard sound stimulus;
(59) a stylus for various tactile stimulations;
(60) a digital thermometer.
(61) 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.
(62) The test included three observation phases:
(63) a direct observation phase during which the animal is not disturbed;
(64) an active observation phase during which the animal was handled;
(65) a phase dedicated to evaluating the behavioural reactions of the animals following reactivity tests.
(66) The recorded variables were the following:
(67) 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).
(68) 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.
(69) Physiological effects: salivation, lacrimation, diarrhoea, rectal temperature, cardiac-respiratory rhythm.
(70) Mortality.
(71) The following tests were conducted after two weeks of administration of the products:
(72) Elevated Plus Maze Test (LCS)
(73) 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. 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).
(74) Aversive Light Stimulus Avoidance Response (ALSAR) Test
(75) 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.
(76) 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.
(77) The 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.
(78) After 18 days of administration of the products to be tested, the rats were tested on a cognitive level for acquiring training within the scope of conditioning operating in the ALSAR model.
(79) 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.
(80) 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).
(81) Product
(82) 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.
(83) Statistical Analysis
(84) The Kruskal-Wallis test was applied, followed in the cases of heterogeneity, by comparisons by means of the Mann-Whitney U test for comparing the treated groups with the control group.
(85) 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.
(86) The statistical and graphical processing operations were carried out by means of the software package StatvieW 5 (SAS Institute, Inc., USA).
(87) Results
(88) FOB Test
(89) Comparisons of the Groups Treated with the Carrier Group in the FOBs at T0 and T14
(90) In the FOB at T0, before administering the treatment, the results of all the studied rat groups are homogeneous (Table 2, Table 3).
(91) In the FOB tests at T14 (after 14 days of administration of the treatments), the Kruskal-Wallis test showed homogeneity of the results within many variables from among the different treatment groups (Table 2).
(92) On the other hand, on certain studied variables, the AE at certain doses showed significant effects as compared with the carrier (Table 3).
(93) The main variables modified by daily administration of AE for two weeks are: The behaviour of the rats and their spontaneous locomotor activity in a housing cage: the rats treated with 3 doses are more active and more alert, The behaviour of the rats in the observation cage: there also, the rats are more active and more alert with 2 stronger doses, Interest for a shown object: at the 3 doses, AE seems to have a stimulating effect on the curiosity of the rats and their interest for novelty (increase in the exploratory activity, hedonism, antidepressant effect), The flight reactions of the rats upon approaching a finger or upon touching them are significantly reduced by AE at the 3 doses, which comes under an anti-stress/anxiolytic effect of AE, The reactions to pinching of the tail and to pinching of the paw are also significantly reduced at the 3 doses. These lowered reactions come under an anti-stress/anxiolytic effect of AE and/or an antalgic effect, The cardiac and respiratory frequencies of the rats seem to be lower than in rats treated with AE at the 3 doses, these come under an anti-stress/anxiolytic effect of AE, Agitation, nervosity and irritability of the rats were significantly lowered under administration of AE at the 2 strongest doses, coming under a calming, anti-stress and/or anti-aggressivity effect.
(94) TABLE-US-00002 TABLE 2 State of the variables before (T0) and after (T14) administration of AE for 14 days at the doses of 20, 40 and 80 mg/kg, p.o. FOB session T0 T14 Groups AE AE AE AE AE AE Items Carrier 20 40 80 Carrier 20 40 80 Displacement latency in H(ddl3) = 5.14; H(ddl3) = 4.10; the arena P = 0.16 P = 0.25 Locomotor activity H(ddl3) = 3.06; H(ddl3) = 2.43; (spaces crossed) P = 0.38 P = 0.49 Locomotor activity H(ddl3) = 4.91; H(ddl3) = 2.96; (straightening up) P = 0.18 P = 0.40 Awaken or drowsy H(ddl3) = 3.00; H(ddl3) = 0.00; condition P = 0.39 P = 1.00 Spaces crossed on an H(ddl3) = 3.00; H(ddl3) = 3.94; open branch P = 0.39 P = 0.27 Rollover reaction H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 Gripping reaction H(ddl3) = 1.11; H(ddl3) = 2.09; P = 0.77 P = 0.55 Suspension test H(ddl3) = 3.15; H(ddl3) = 1.89; P = 0.37 P = 0.59 Jump reaction to a H(ddl3) = 4.26; H(ddl3) = 2.13; sound stimulus P = 0.23 P = 0.55 Caused aggressivity H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 General muscular H(ddl3) = 3.34; H(ddl3) = 6.39; tonicity P = 0.34 P = 0.10 Limb muscular tonicity H(ddl3) = 4.60; H(ddl3) = 3.29; P = 0.20 P = 0.35 Visual placement H(ddl3) = 5.63; H(ddl3) = 1.97; reaction on the rod P = 0.13 P = 0.58 Shrieks H(ddl3) = 2.30; H(ddl3) = 4.31; P = 0.51 P = 0.23 Defecation H(ddl3) = 5.43; H(ddl3) = 3.22; P = 0.14 P = 0.36 Micturition H(ddl3) = 6.27; H(ddl3) = 2.30; P = 0.10 P = 0.51 Convulsions H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 Trembling H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 Diarrhoea H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 Exophthalmos H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Piloerection H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Salivation H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Lacrimation H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Palpebral reflex H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Strange behaviours H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Bending the trunk H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Pelvic elevation H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Elevation of the tail H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Rectal temperature H(ddl3) = 0.17; H(ddl3) = 3.67; P =0.98 P =0.30 Mortality at 24 h H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00
(95) TABLE-US-00003 TABLE 3 State of the variables before (T0) and after (T14) administration of AE for 14 days at the doses of 20, 40 and 80 mg/kg, p.o. FOB session T0 T14 Groups AE AE AE AE AE AE Items Carrier 20 40 80 Carrier 20 40 80 Behaviour of the rat in H(ddl3) = 5.15; H(ddl3) = 19.97; the housing cage P = 0.16 P = 0.0002 (D1** - D2** - D3**) Behaviour of the rat in H(ddl3) = 4.02; H(ddl3) = 7.19; the observation cage P = 0.26 P = 0.066 (D2# - D3*) Spontaneous H(ddl3) = 4.33; H(ddl3) = 15.20; locomotor activity in P = 0.23 P = 0.008 the housing cage (D1** - D2** - D3**) Interest for a shown H(ddl3) = 4.86; H(ddl3) = 6.86; object P = 0.18 P = 0.08 (curiosity) (D1* - D2# - D3*) Flight reaction upon H(ddl3) = 2.39; H(ddl3) = 6.65; approaching a finger P = 0.50 P = 0.08 (D1#- D2#- D3*) Reaction to pinching H(ddl3) = 5.50; H(ddl3) = 15.07; the tail P = 0.14 P = 0.002 (D1** - D2** - D3**) Reaction to pinching a H(ddl3) = 4.13; H(ddl3) = 9.63; hind paw P = 0.25 P = 0.02 (D1** - D2# - D3#) Flight reaction to being H(ddl3) = 2.12; H(ddl3) = 8.01; touched P = 0.55 P = 0.045 (D1* - D2# - D3#) Cardiac and H(ddl3) = 4.60; H(ddl3) = 10.78; respiratory frequency P = 0.20 P = 0.01 (D1# - D2** - D3**) Caused freezing H(ddl3) = 3.00; H(ddl3) = 6.27; P = 0.39 P = 0.09 Muscular tonicity of the H(ddl3) = 3.83; H(ddl3) = 8.52; abdomen P = 0.28 P = 0.04 (D2# - D3*) Agitation-nervosity- H(ddl3) = 1.04; H(ddl3) = 6.77; irritability P = 0.79 P = 0.08 (D2# - D3*) #P < 0.10; *P < 0.05; **P < 0.01 (Mann-Whitney Test: vs. Carrier)
Comparisons of the Different Repeated Variables Between the FOBs at T0 and T14
(96) Daily administration of AE at certain doses for 14 days seems to have an effect on certain studied main variables (Table 4): The behaviour of the rats and their spontaneous locomotor activity in the observation cage significantly increased under AE at the 3 doses (stimulating effect or anti-stress effect), The displacement latency of the control rats in the observation arena increases and their locomotor activity (crossed spaces) decreases, while in rats treated with AE, these variables remain stable between the T0 and T14 FOBs (anti-stress and antidepressant effects), The interest for a shown object: at the dose of 20 mg/kg, p.o. (per os), AE stimulates the interest of the rats for a stimulus object which is presented to them (motivation for exploring novelty, hedonism: anti-stress and antidepressant effects), The number of crossed spaces on an open branch: comparatively with the carrier, AE does not decrease the number of crossed spaces on an open branch (anti-stress effect), The flight reactions upon approaching the finger: AE at the 3 doses decreases the flight reaction upon approaching the finger of the experimenter (anti-stress effect), The reaction to pinching the tail increases in the control rat, while under 20 mg/kg of AE it decreases (anti-stress effect and/or antalgic effect), The reaction to pinching a hind paw decreases under AE, at the 3 doses (anti-stress effect and/or antalgic effect), The jump reaction to a sound stimulus significantly decreases at the AE doses of 40 and 80 mg/kg (calming and anti-stress effects), The cardiac and respiratory frequencies decrease under AE at the doses of 20 and 40 mg/kg (calming and anti-stress effects), Agitation, nervosity and irritability: at the strong dose of 80 mg/kg, AE shows a positive effect on agitation, nervosity and irritability of the rats (calming, anti-stress and/or anti-aggressivity effects).
(97) TABLE-US-00004 TABLE 4 Effects of AE on the time-dependent change of the variables between the FOBs at T0 and T14 in each treatment group Groups CARR AE/20 AE/40 AE/80 FOB session Items T0 T14 T0 T14 T0 T14 T10 T14 Behaviour of the rat N.S.
in the observation cage Spontaneous N.S.
locomotor activity in the observation cage Displacement latency
N.S. N.S. N.S. in the arena Locomotor activity
N.S. N.S. N.S. (spaces crossed) Interest for a shown N.S.
N.S. N.S. object Crossed spaces on an
N.S. N.S. N.S. open branch Flight reaction upon N.S.
approaching a finger Reaction to pinching
N.S. N.S. the tail Reaction to pinching a N.S.
hind paw Suspension test
N.S.
Jump reaction to a N.S. N.S.
sound stimulus Cardiac and N.S.
N.S. respiratory frequency Muscular tonicity of
N.S. N.S. N.S. the abdomen Agitation-nervosity- N.S. N.S. N.S.
irritability N.S.: Not significant between T0 and T14
Decreases between T0 and T14
Increases between T0 and T14 *P < 0.05; #: P < 0.10 (trend)
Elevated Plus Maze Test (LCS)
(98) In the elevated-plus maze test, after 18 days of daily treatment, AE at the doses of 20, 40 and 80 mg/kg, p.o., significantly increases the number of entries (Table 5,
(99) TABLE-US-00005 TABLE 5 Effects of AE on the number of entries into the open arms in the elevated plus maze test AE 20 AE 40 AE 80 Carrier mg/kg mg/kg mg/kg Kruskal- Products (n = 6) (n = 6) (n = 6) (n = 6) Wallis test Number of entries 1.5 4.0 5.0 4.5 H(ddl3) = into the open (1.0- (4.0- (3.0- (2.0- 6.08 branches Mean 4.0) 5.0) 5.0) 5.0) P = 0.108 (QI-QS)
(100) TABLE-US-00006 TABLE 6 Effects of AE on the time spent in the open arms in the elevated-plus maze test AE 20 AE 40 AE 80 Carrier mg/kg mg/kg mg/kg Kruskal- Products (n = 6) (n = 6) (n = 6) (n = 6) Wallis Test Time spent in the 10.0 37.5 50.5 37.0 H(ddl3) = open branches (7.0- (32.0- (38.0- (17.0- 7.76 Mean (QI-QS) 29.0) 50.0) 66.0) 45.0) P = 0.051
Aversive Light Stimulus Avoidance Response Test (ALSAR)
(101) After 18 days of daily administration of AE, the Kruskal-Wallis test showed that the number of cumulated pressing actions on both levers (active lever+inactive lever) of the rats of the different treatment groups are homogeneous at 5 minutes, 10 minutes, 15 minutes and at 20 minutes (
(102) In the test for avoiding an aversive light stimulus, at the end of the first 5 minutes of the test, after 18 days of administration of AE, the rats treated with the dose of 20 mg/kg, p.o., tend to discriminate the active lever from the inactive lever (
(103) At the end of the period of 10 minutes of the 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 (
(104) At the end of the period of 15 minutes of test, the rats treated with AE at the doses of 20 and 40 mg/kg, p.o., significantly discriminate the active lever from the inactive lever. The rats treated at the dose of 80 mg/kg, p.o., no longer discriminate both levers. The control rats still do not show any discrimination at this stage of the training test (
(105) At the end of the period of 20 minutes of test, the rats treated with AE at the doses of 20 and 40 mg/kg, p.o., still discriminate significantly the active lever from the inactive lever. The rats treated with the dose of 80 mg/kg, p.o., no longer show any discrimination and the control rats begin to exhibit a beginning of discrimination (
(106) These results show the efficiency of AE on acquiring training, and notably at the doses of 20 and 40 mg/kg, p.o.
(107) Physiological and Innocuity Data
(108) Administration of AE, regardless of the dose, did not show any influence on the weight of the rats daily treated with the doses of 20, 40 or 80 mg/kg, p.o., or on their food intake. No toxicity was detected in animals treated with AE at the doses of 20, 40 and 80 mg/kg/d, p.o., for 21 days.
(109) Conclusion
(110) The administration of 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.
Example 5: Evaluation of the Effects of the Algal Extract (AE) Through the Behavioural Despair Test
(111) The anti-depressant effects of an algal extract according to the invention (AE), administered orally as a preventive treatment with 3 doses (10, 20 and 40 mg/kg/d) were evaluated in adult male Wistar rats in the behavioural despair test (BDT).
(112) 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.
(113) The rats used were treated according to the ethical rules dictated by ASAB and the Canadian Council for Animal Protection.
(114) Equipment and Methods
(115) Animals
(116) Sixty male Wistar rats (Charles River Laboratories, France) weighing from 200-225 g were used. Upon receiving them, the rats were marked and distributed in groups of 4 in polycarbonate cages of the F type (48×27×20 cm, U.A.R., 91-Epinay-Sur-Orge, France). The animals were housed in an air-conditioned animal housing facility, at a temperature of 22±2° C. and a relative humidity of 50±20%. The rats had standard feed 2016 (Harlan, Gannat, France) and drink ad libitum. They were subject to a reverse light-darkness cycle of 12 hours.
(117) After getting used to the conditions of the laboratory for one week, the rats were weighed and randomized according to their weight in 5 treatment groups (n=12/group).
(118) Treatment Groups:
(119) Control group: treatment with spring water (Carrier),
(120) Imipramine group: treatment with Imipramine at the dose of 10 mg/kg/d (Imi),
(121) AE group 10: treatment with AE at the dose of 10 mg/kg/d (AE 10),
(122) AE group 20: treatment with AE at the dose of 20 mg/kg/d (AE 20),
(123) AE group 40: treatment with AE at the dose of 40 mg/kg/d (AE 40).
(124) In order to avoid possible interferences between the different treatments, 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.
(125) Behavioural Despair Test (BDT)
(126) 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.
(127) After 13 days of daily treatment with the products to be tested, 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.sup.th treatment.
(128) At D15, 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.
(129) 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.
(130) Product
(131) 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.
(132) 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.
(133) The animals of the Carrier group daily received spring water with an administration volume of 5 ml/kg.
(134) Statistical Analysis
(135) ANOVA in parametric mode was applied, followed, in the case of heterogeneity, by comparisons by means of the non-paired t-test for comparing the treated groups with the Carrier group and the Reference group.
(136) 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.
(137) For the food and water intakes, non-parametric mode analysis was applied: a Kruskal-Wallis test if necessary followed by a Mann Whitney U test for comparing the groups two by two.
(138) The statistical and graphic processing operations were achieved by means of the software package Statview5 (SAS Institute, Inc., USA).
(139) Results
(140) Behavioural Despair Test: Pre-Test at D14
(141) At D14, in the pre-test of the behavioural despair, ANOVA did not show any heterogeneity for the immobility times of the animals of the different treatment groups (Table 7;
(142) TABLE-US-00007 TABLE 7 Effects of AE on the immobility time(s) in the behavioural despair pre-test at D 14 (s, Average ± SME) Imipramine AE AE AE Carrier 10 mg/kg 10 mg/kg 20 mg/kg 40 mg/kg Products (n = 12) (n = 12) (n = 12) (n = 12) (n = 12) ANOVA.sub.(ddl=4) Immobility 18.7 ± 3.6 16.3 ± 4.6 12.5 ± 3.8 9.8 ± 3.2 9.3 ± 1.8 F = 1.33 duration (s) P = NS NS: non-significant.
(143) Behavioural Despair Test at D15
(144) At D15, in the behavioural despair test, ANOVA showed heterogeneity for the immobility time of the animals of the different treatment groups (Table 8).
(145) The non-paired t-test showed that the immobility times of the animals treated with Imipramine, AE 20 and AE 40 were significantly shorter than that of the animals with the Carrier (t=3.69, P=0.0013; t=2.20, P=0.039 and t=3.29, P=0.003, respectively).
(146) The non-paired t-test showed that the immobility time of the animals treated with AE 20 tended to be significantly longer than that of the animals treated with Imipramine (t=1.72, P=0.099) (Table 8;
(147) TABLE-US-00008 TABLE 8 Effects of AE on the immobility duration(s) in the behavioural despair test at D 15 (s, Average ± ESM) Imipramine AE AE AE Carrier 10 mg/kg 10 mg/kg 20 mg/kg 40 mg/kg Products (n = 12) (n = 12) (n = 12) (n = 12) (n = 12) ANOVA.sub.(ddl=4) Immobility 14.4 ± 2.7 3.8 ± 1.0 12.0 ± 5.0 7.3 ± 1.8 4.9 ± 1.0 F = 2.76 duration (s) P = 0.036 Non-paired t-test t = 3.69.sup. t = 0.42 t = 2.20 t = 3.29 (vs. Carrier) P = 0.0013 NS P = 0.039 P = 0.003 Non-paired t-test t = 1.60 t = 1.72 t = 0.75 (vs. Imipramine) P = NS.sup. T (P = 0.099) .sup. NS Non-paired t-test t = 0.98 t = 1.38 (vs. AE 10) NS NS Non-paired t-test t = 1.17 (vs. AE 20) NS NS: not significant.
Comparison of the Results of the Tests at D14 and D15
(148) The paired t-test showed that the immobility times of the rats treated with Imipramine and AE 40 significantly decrease between D14 and D15 (t=2.90, P=0.015 and t=2.42, P=0.034, respectively) (Table 9).
(149) TABLE-US-00009 TABLE 9 Comparison of the results of the tests at D 14 and D 15 (s, Average ± SME) Imipramine AE AE AE Carrier 10 mg/kg 10 mg/kg 20 mg/kg 40 mg/kg Products (n = 12) (n = 12) (n = 12) (n = 12) (n = 12) Immobility duration (s) 18.7 ± 3.6 16.3 ± 4.6 12.5 ± 3.8 9.8 ± 3.2 9.3 ± 1.8 at D 14 Immobility duration (s) 14.4 ± 2.7 3.8 ± 1.0 12.0 ± 5.0 7.3 ± 1.8 4.9 ± 1.0 at D 15 Paired t-test t= 1.41 2.90 0.11 1.10 2.42 P= NS 0.015 NS NS 0.034 NS: not significant.
Weight Evolution, Food and Water Intakes
(150) No effect of AE administered at the three doses was shown as regards the weight evolution and the food and water intakes of the animals.
(151) Behavioural Despair Test
(152) At D14: during the test for setting resignation in the animals, although the immobility duration of the animals treated with AE at the doses of 20 and 40 mg/kg is shorter than those of the animals of the other groups, no significant difference was observed between the rats treated with the Carrier, Imipramine and AE administered at the three tested doses.
(153) At D15: the anti-depressant effect of 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.
(154) Comparison of the immobility durations at D14 and D15: only the animals treated with Imipramine at the dose of 10 mg/kg and with AE at the dose of 40 mg/kg have significantly reduced their immobility duration between D14 and D15.
(155) Behaviour of the Animals and Innocuity of the Algal Extract
(156) No abnormal behaviour of the whole of the animals was observed during the experimentation for AE administered at the doses of 10, 20 and 40 mg/kg. No toxicity was detected in animals treated with AE at the doses of 10, 20 and 40 mg/kg/d for 14 days.
CONCLUSION
(157) The administration of an algal extract according to the present invention thus demonstrates an anti-depressant effect of the product.