KR101453448B1 - Composition for preventing or treating depression comprising fucoidan - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating depression comprising fucoidan as an active ingredient, and a health functional food for preventing or ameliorating depression. The fucoidan of the present invention is contained as an active ingredient in a pharmaceutical composition or a health functional food and can be used for the development of a therapeutic agent or an improvement agent for depression.

Description

[0001] The present invention relates to a composition for preventing or treating depression including fucoidan,

The present invention relates to a composition for preventing or treating depression including fucoidan, and more particularly, to a pharmaceutical composition for preventing or treating depression comprising fucoidan as an active ingredient, and a health functional food for preventing or improving depression.

Depression is a global problem in humans due to a relatively high lifetime prevalence and significant associated disability, often associated with sleep deprivation, low self-esteem, feelings of guilt, and a tendency to become obsessed with suicidal thoughts. Thus, the disease is considered a complex disorder, and its underlying mechanism of action remains unclear. In addition, depression is a disease that is close to us as it is called a cold of the mind, but depression causes difficulties in everyday life, which is a socially and economically disastrous disease. In addition, 60-70% of suicides, one of the causes of sudden death, are caused by depression, leading to death.

Chronic exposure to stressful life events is an important risk factor for the maintenance and development of many psychological or impotent conditions in humans, such as major depression, and has long lasting deleterious effects on brain function. The most important physiological, neuro-endocrinological and behavioral abnormalities in this disease are hyperactivation of the hypothalamic-pituitary-adrenal axis (HPA). The activation response to acute stress leads to systemic alterations that enhance the native function of the organism in order to maintain homeostasis and minimize the effects of the threat. Excessive stress, however, damages the ability to control physiology and behavioral responses to brain function and subsequent stressors, resulting in abnormal physiological changes. Several animal studies with chronic stress have shown that morphological changes in the hypothalamus, hippocampus, amygdala, changes in various neurotransmitters, BDNF in the hippocampus and serum, brain-derived neurotrophic induced hypothalamic-pituitary-adrenal axis (HPA), including changes in expression levels, weight loss, and behavioral changes.

Previous studies have reported repeated depressive stress related to depression. An animal model with symptoms such as depression in response to repeated restraint stress stimuli is useful in determining candidate antidepressant efficacy in drug screening. Thus, it has been used as a preclinical test to confirm its efficacy in the development of various antidepressants.

The antidepressants currently under development include monoamine oxidase inhibitors (MAOs), selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors Have been developed and used clinically. In addition, representative examples of commonly used antidepressants include benzodiazepine. Diazepam, oxazepam, lorazepam, alprazolam, helazepam, clonazepam, and the like. However, most antidepressants are not effective against very diverse and complex depressive symptoms, most of which lead to serious side effects. Therefore, based on a new understanding of the pathogenesis of depression, a new therapeutic strategy using natural products or medicinal plants is urgently needed.

Under these circumstances, the present inventors have made intensive efforts to find a cure for depression, and as a result, confirmed that depression symptoms are reduced when fucoidan is administered to an animal model that induces depression, thereby completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating depression comprising fucoidan as an active ingredient.

It is another object of the present invention to provide a health functional food for preventing or ameliorating depression comprising fucoidan as an active ingredient.

In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating depression comprising fucoidan as an active ingredient.

The term "fucoidan (FCN)" used in the present invention is a sulfated polysaccharide having a sticky, viscous structure, and is a component contained in brown algae such as common seaweed and sea tangle, and has an average molecular weight of 20 KDa. Fucose) is a substance in which basic sugar and sulfate group are combined. The fucoidans specifically include Sugar Kelp ( Laminaria) saccharina), L. Dizzy Tata (L. digitata), Pacific Sargassum (Fucus distichus , and Cladosiphon okamuranus .

Such fucoidan has been reported to have a variety of physiological and biological activities such as antioxidants, anticoagulants, anticancer agents, antibiotics, anti-inflammatory activity, and potential value proposals for medical use. In particular, anti-inflammatory and antioxidant activities have attracted much attention in recent years. However, it has not yet been reported whether fucoidan can improve depression. In the present invention, it has been found that fucoidan is effective for preventing, treating or improving depression.

Specifically, in one embodiment of the present invention, a depressive animal model having depressive symptoms was prepared by applying restraint stress to rats for 2 weeks. Using this, a weight change measurement, a serum corticosterone (CORT) measurement, a forced swimming load Experiments such as behavioral tests, immunoreactivity testing of adrenocorticotropic hormone releasing factor (CRF) and tyrosine hydroxylase (TH), and measurement of BDNF mRNA expression in the hippocampus were used to identify various factors associated with depression Respectively.

As a result, it was confirmed that the group administered with fucoidan alleviated the symptoms deteriorating in the group of depressed animal models caused by restraint stress throughout all the examples. Specifically, the administration of fucoidan alleviated weight loss due to depression, inhibited the increase of serum CORT content, and the forced swimming load test showed that the immobility time increased by depression and the decreased climbing behavior time increased Respectively. In addition, the expression of adrenocorticotropic hormone releasing factor (CRF) and tyrosine hydroxylase (TH), which is increased by depression, is restored and decreased, and the hippocampal BDNF mRNA is also decreased by depression, but increased in the fucoidan administration group .

These results show that fucoidan has an antidepressant effect on depression induced by restraint stress.

As a positive control, fluoxetine (FLX), a serotonin reuptake inhibitor, has been used as a positive control, and the result of the STR + FCN50 group administered with 50 mg / kg of fucoidan is STR + FLX The inventors of the present invention found that the fucoidan of the present invention has an effect that is not inferior to the conventional antidepressants at all.

The term "depression" as used in the present invention refers to a disease that causes various cognitive and psychosomatic symptoms with deterioration in desensitization and depression as main symptoms, resulting in deterioration of daily function. However, It is a disease caused by various biochemical, genetic and environmental factors.

Antidepressants manufactured by conventional chemical methods have serious side effects compared to the effects thereof. Therefore, the present invention utilizes fucoidan obtained from natural raw materials to confirm the antidepressant effect. The fucoidan of the present invention has an antidepressant effect, that is, prevention, improvement, or therapeutic effect of depression.

In the present invention, "prevention" means any action that inhibits or delays depression by the administration of the pharmaceutical composition according to the present invention, and "treatment" means that the administration of the pharmaceutical composition causes suspicion of depression, Means any act that improves or benefits.

The pharmaceutical composition comprising the fucoidan of the present invention as an active ingredient may further comprise a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like can be used for oral administration. In case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used.

Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other solutions, suspensions, tablets, herbal medicines, capsules, sustained-release preparations, and the like. Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In addition, the composition of the present invention can be administered in various ways such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, transdermal, and the dose varies depending on the age, sex, And can be readily determined by one skilled in the art. Preferably, the composition of the present invention may be administered orally or parenterally. Also, since the dosage of the composition can be determined by those skilled in the art depending on the kind of the active ingredient, along with various related factors such as route of administration, degree of disease, sex, body weight and age, Is not limited.

The dosage of the pharmaceutical composition of the present invention is not particularly limited and may be varied depending on the body's absorption, body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, . In general, the daily dosage may preferably be from 10 to 100 mg / kg, more preferably from 20 to 70 mg / kg, and even more preferably from 20 to 50 mg / kg, based on the amount of fucoidan, kg. < / RTI > The pharmaceutical composition of the present invention is prepared in consideration of an effective dose range, and the unit dosage formulations thus formulated are used according to the judgment of the expert who monitors or observes the administration of the drug as required, and a specialized dosage regimen Or several times at predetermined time intervals. In one embodiment of the present invention, the experimental groups administered with fucoidan at 10, 20 and 50 mg / kg, respectively, were used, and it was confirmed that the effect was enhanced in a dose-dependent manner and that the 50 mg / kg administration group showed the best effect Respectively.

In another aspect, the present invention provides a health functional food for preventing or improving depression comprising fucoidan as an active ingredient. The above-mentioned fucoidan is as described above and may be added to health functional foods for the purpose of preventing or improving depression.

When the composition of the present invention is used as a health functional food additive, the composition may be added as it is or may be used together with other health functional foods or health functional food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use. Generally, the composition of the present invention may be added in an amount of preferably not more than 15 parts by weight, more preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term intake intended for health control and hygiene, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount in the above range.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the composition can be added include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen and other noodles, gums, ice cream, Alcoholic beverages, and vitamin complexes, and may include all the health functional foods in the conventional sense, and foods used as feeds for animals.

In addition, when the health functional food composition of the present invention is used in the form of a drink, it may contain various sweetening agents, flavoring agents, or natural carbohydrates as additional components such as ordinary beverages. The natural carbohydrates may be polysaccharides such as disaccharides such as monosaccharides such as glucose and fructose, maltose, sucrose, dextrin, cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be about 0.01 to 0.04 g, more preferably 0.02 to 0.03 g per 100 ml of the composition of the present invention. The sweeteners may be natural sweeteners such as tau martin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

The fucoidan of the present invention is contained as an active ingredient in a pharmaceutical composition or a health functional food and can be used for the development of a therapeutic agent or an improvement agent for depression.

Figures 1 and 2 show the experimental schedule for observing depressive behavior due to restraint stress. FIG. 1 shows the weight change measurement and CORT measurement schedule, and FIG. 2 shows the forced swimming load test, immunohistochemistry and RT-PCR schedule.
FIG. 3 shows changes in body weight for each group during two weeks of restraint stress.
FIG. 4 shows the result of measuring the corticosterone (CORT) content of serum for each group after restraint stress.
FIG. 5 shows measurement results of the floating time (A), the climbing action time (B), and the swimming time (C) for each group in the forced swimming load test.
FIG. 6 shows the results of immunohistochemistry of CRF expression in the hypothalamus (PVN) of each group after confinement stress. A: SAL group, B: STR group, C: STR + FCN50 group, D: STR + FLX group.
FIG. 7 shows the results of immunohistochemistry (FIG. 7) showing TH expression in the basal lateral nucleus (BLA, FIG. 7E to H) . A, E: SAL group, B, F: STR group, C, G: STR + FCN50 group, D, H: STR + FLX group.
FIG. 8 shows the results of measurement of BDNF mRNA expression in the hippocampus of each group after confinement stress.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  1: Material preparation

The positive control group fluoxetine (FLX) and the experimental group fucoidan (FCN) were purchased from Sigma Chemical Co. (St. Louis, Mo., USA).

A male adult SD (Sprague-Dawley) rat of 260-280 g was used as an experimental animal. Mature SD rats (260-280 g) were housed in a temperature-controlled room (22 ± 2 ° C) for at least one week before the experiment, allowed free access to food and water, and maintained a 12 h day / night cycle.

Example  2: Experimental group

2-1. Corticosterone ( corticosterone , CORT ) Measure

Experimental animal rats were subjected to restraint stress for 2 weeks. Experimental animal rats were randomly divided into 6 groups of 7 rats per each group as shown in Table 1 below. Among them, in order to measure the concentration of corticosterone (CORT), only 4 rats per each group were tested for blood of rats.

The positive control group, fluoxetine (FLX) and the experimental group, fucoidan (FCN), were dissolved in 0.9% physiological saline solution and treated by intraperitoneal injection 30 minutes before the daily stress for 2 weeks. All drugs were prepared fresh immediately before each experiment. As shown in Figure 1, the restraint stress progression was performed once daily for two hours between 10 am and 12 am for two weeks in a rodent immobilization bag.

Specifically, the rats were placed in a conical, transparent plastic tube (20 x 7 cm) with one 3-mm hole and one open and the other for respiration, giving restraint stress. The experimental animal has enough air in the tube, but is floating. Parameters such as weight change (measured from the beginning of restraint stress) and serum CORT content (measured after repeated restraint stress) were measured to monitor the effect of stress. All rat groups except SAL group (negative control, unstressed group) applied the same confinement stress.

2-2. Behavioral and neurobiological assessment

For the treatment of depression in experimental animal models with repeated depression-induced depression-like symptoms, efficacy was assessed using behavioral and neurobiological methodologies by administering fucoidan (FCN). The rats were randomly divided into six groups of 7 rats per each group as described in Table 1 above. Exercise function was assessed after the last 24 h of the stress protocol. The schedule of confinement stress and behavioral experiment is as shown in Fig.

Example  3: Measurement of weight change

To determine if repeated confinement stress induced weight loss, weight changes in rats were measured daily for 2 weeks of confinement stress.

Rats exposed to repeated confinement stress began to lose weight on the first day of imprisonment, and this initial weight loss lasted for that period without recovery, and even deteriorated. As shown in FIG. 3, the STR group receiving restraint stress showed a progressive weight loss over 2 weeks when compared to the no-stressed negative control (SAL group). On the other hand, the rat group receiving fucoidan (FCN) during this period showed that weight loss due to restraint stress was inhibited, and in particular, the rat group administered 50 mg / kg fucoidan showed almost no difference compared to the STR group And showed no change in body weight.

These results indicate that weight loss effects are caused by restraint stress, but when fucoidan is administered, these weight loss effects are lost and normal weight changes are recovered.

Example  4: Corticosterone  Measure

When repeated stress is applied to the rat, the serum CORT content, which is gradually decreased due to the adrenal habituation, is greatly increased by acute confinement stress. Serum CORT content was measured in each group after exposure to repeated confinement stress for 2 weeks.

The experimental animals were sacrificed by decaptitation after stress induction, and blood samples were collected for the measurement of serum corticosterone (CORT) content. To this end, the anesthetized rats rapidly cut their necks and blood was drawn rapidly through the abdominal aorta. Blood was centrifuged at 4000g for 10 minutes and the serum thus obtained was stored at -20 ° C until use. CORT concentrations were measured by competitive enzyme-linked immunoassay (ELISA) using a rabbit polyclonal CORT antibody (OCTEIA Corticosterone kit; Alpco Diagnostics Co., Windham, NH, USA) ). A sample (or a standard) and a conjugate were added to each well, and the plate was allowed to stand at room temperature for 1 hour without blocking. The wells were then washed several times with buffer and developed with appropriate color and analyzed for optical density (OD) at 450 nm using an enzyme immunoassay reader (MutiRead 400, Authos Co., Vienna, Austria) Respectively.

As a result, the serum CORT concentration in the rat group subjected to repeated confinement stress was significantly increased to 462.45% (P <0.05) when compared to the no-stressed negative control (SAL group). On the other hand, when fucoidan (FCN) was administered daily, it was confirmed that the increase in the serum CORT induced by repeated confinement stress was somewhat inhibited (FIG. 4).

Example  5: Forced swimming load test Forced swimming test )

FST (forced swimming test), a representative behavioral test for depression, has been commonly used to assess potential antidepressant activity in rodent models. Depressed rats are forced to submerged in water for extended periods of time to exhibit immersive behavior. At this time, administration of the antidepressant reduces immobility behavior by increasing escape responses such as climbing and swimming.

To test whether depression was induced by the repeated confinement stress and whether fucoidan could reduce such depressive symptoms, forced swimming load experiments were performed using the experimental groups.

A transparent Plexiglas cylinder (20 cm diameter x 50 cm high) was filled to 25 cm with water to a depth of 30 cm. At this depth, the rat does not touch the bottom of the cylinder with its tail or hind legs. On the 15th day, all groups of rats were placed in a water-filled cylinder and trained for 15 minutes. On day 16, the experimental animals were forced swimming for 5 minutes and monitored for escape behavior (climbing and swimming). In addition, the dwell time was recorded for a 5 minute test time. Climbing was defined as the movement of the forefoot in the upward direction along the edge of the swim chamber, and swimming was considered to be an overall motion including traversing the quadrant of the swim chamber. Floating behavior was calculated by the length of time that the experimenter did not exhibit escape behavior (total test time minus the time spent climbing and swiming in total test time; total test time - time spent in climbing and swimming activities). The behavior of the experimental animals was continuously recorded over the entire test time using an overhead video camera. After the test, the rats were taken out of the tank, dried with a towel, and then returned to us for the first time. The water in the swimming tank was exchanged between the rats.

As a result, rats subjected to repeated restraint stress for 2 weeks showed significant depressive symptoms when compared to negative control (SAL) administered with physiological saline solution, immobility time increased during forced swimming load test, Showed a decreasing characteristic. On the other hand, in the group administered with fucoidan, it was confirmed that depression symptoms were relieved in a concentration-dependent manner, that is, the immobility time was decreased and the climbing action time was increased. Especially, in STR + FCN50 group, FLX group (positive control group).

Specifically, as shown in Fig. 5A (floating time), during the forced swimming load test, rats in the STR group had more immobilization time than the negative control SAL group, and this effect was confirmed to be recovered in the fucoidan administration group , Indicating that depression symptoms are reduced by fucoidan administration.

As shown in Fig. 5B (climbing behavior time), the STR group rats were reduced in the climbing behavior, which is a typical escape response behavior for depression evaluation, as compared with the SAL group, which is a negative control group, , Indicating that depressed mood symptoms are reduced by fucoidan administration.

On the other hand, as shown in C (swimming time) in FIG. 5, there was no significant difference in swimming responses such as swimming among all the groups.

Example  6: Adrenocortical stimulating hormone Release factor  ( 코르 티 - releasing factor , CRF ) And tyrosine hydroxylase ( tyrosine hydroxylase , TH ) Immunohistochemistry ( immunohistochemistry , IHC ) Examination of tissue

Following this behavioral experiment, the corticotrophin-releasing factor (CRF) and tyrosine hydroxylase (TH) immunoreactivity associated with depression and neurotransmission were identified. The CRF is known to be involved in hyperactivation of hypothalamic pituitary contraction (HPA) and depression-like behavior, and TH is also known as an enzyme involved in psychopathological conditions such as depression.

For immunohistochemical staining, three rats in each group were anesthetized with sodium pentobarbital (80 mg / kg, ip), perfused with 0.9% physiological saline solution along the ascending aorta , Followed by perfusion with 300 mL of formalin (4% paraformaldehyde, 4% paraformaldehyde) diluted to 4% in 0.1 M phosphate-buffered saline (PBS). The brains of rats were randomly excised, fixed overnight and cryopreserved in 20% sucrose solution in 0.1 M phosphate buffered saline (PBS) at 4 ° C. Using a cryostat (cryostat: Leica CM1850; Leica Microsystems Ltd., Nussloch, Germany), the hypothalamus, the locus coeruleus (LC), the basolateral nucleus of the amygdala , And BLA) crossed each other. To confirm CRF and TH expression, the sections were immunostained using the avidin-biotin-peroxidase complex (ABC). The sections were incubated with goat anti-CRF antibody (1: 500 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and sheep anti-TH antibody (1: 2000 dilution; Chemicon International Inc., Temecula, CA, USA) was diluted with PBST (PBS plus 0.3% Triton X-100) and left at 4 ° C for 72 hours. Secondary antibodies were obtained from Vector Laboratories Co. (Burlingame, CA, USA) and diluted 1: 200 in PBST containing 2% serum. To visualize the immunoreactivity, the sections were incubated with ABC reagent (Vectastain Elite ABC kit; Vector) for 90 minutes and incubated with 3,3'-diaminobenzidine (DAB; Sigma-Aldrich Chemical Co., St , And 0.01% hydrogen peroxide (H ₂ O ₂ ) for 1 min. Finally, tissues were rinsed with PBS, followed by brief washing with water and then individually placed on slides. Images were captured using the AxioVision 3.0 imaging system (Carl Zeiss Inc., Oberkochen, Germany) and processed using Adobe Photoshop (Adobe Systems Inc., San Jose CA, USA). The sections were observed at 200x magnification, and the number of CRF- and TH-labeled cells was calculated from the basolateral nucleus (BLA) of the hypothalamus, cerebellum (LC) and amygdala by an experimenter unknown to the experimental group. Three specimens were randomly assigned to one of three groups (100 x 100 ㎛ ² ) distributed in the hypothalamus, locus coeruleus (LC) and basolateral nucleus of the amygdala (BLA) The number of immunopositive cells was confirmed. Immunopoietic cells were defined as having a higher staining intensity than background values, and distinct brown points representing CRF- and TH-immunopositive cells were observed in the hypothalamus, LC (LC) and basolateral nucleus (BLA) of the amygdala. The brightness and contrast differences of the original images were not adjusted to exclude the possibility of subjective selection of immunopositive cells.

Corticotropin releasing factor (CRF) immunoreactivity was analyzed in cell bodies of hypothalamic regions including paraventricular nucleus (PVN) as shown in FIG. 6. As a result, the number of CRF immunopositive fibers in the thread brains (PVN) in the rat brain of the STR group was increased by 151.03%. Specifically, the number of CRF immunopositive neurons was analyzed and it was confirmed that CRF expression was significantly increased in the rats of the STR group (FIG. 6B) as compared with the SAL group (A of FIG. 6) + FCN50 group (C in Fig. 6) is significantly reduced compared with the STR group (B in Fig. 6). This indicates that CRF-immunoreactivity is increased by repeated restraint stress and is significantly restored by the administration of fucoidan (FCN). The number of CRF-immunoreactive neurons in the STR + FCN50 group (FIG. 6C) was similar to the STR + FLX group (FIG. 6D).

In addition, the immunoreactivity of tyrosine hydroxylase (TH) was analyzed in adrenergic regions, including brain (LC) and basolateral nucleus (BLA) of amygdala, as shown in FIG. The number of immunostimulatory fibers of tyrosine hydroxylase (TH) in the cerebral cortex (LC) and the basal lateral nucleus (BLA) of the STR group increased to 139.80% and 147.74%, respectively. Specifically, analysis of the number of TH immunocompetent neurons showed that the expression of TH in the rats of the STR group (B and F in Fig. 7) was significantly increased as compared with the SAL group (A and E in Fig. 7) , And it was confirmed that STR + FCN50 group (C and G in FIG. 7) was significantly reduced compared with the STR group (B and F in FIG. 7). This indicates that an increased number of TH-immunopositive neurons in rats subjected to repeated confinement stresses are significantly restored by the administration of fucoidan (FCN). In addition, the number of TH-immunopositive neurons in the STR + FCN50 group (C and G in Figure 7) was similar to the STR + FLX group (D and H in Figure 7).

Example  7: Seahorse BDNF  ( Brain derived  However, brain - derived  노로 트rophic factor ) mRNA  Expression evaluation

MRNA expression of BDNF as a protein marker associated with depression was assessed. BDNF is a protein produced in the brain that acts on both neurons in the central nervous system and peripheral nervous system and is known to be closely related to depression.

Expression levels of BDNF (brain-derived neurotrophic factor) mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR). The rat brain hippocampus was isolated from 4 rats per group. After monocotylosis, the brain was rapidly removed and stored at -80 ° C until use. Total RNA (total RNA) was extracted from brain tissue using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) according to the supplier's instructions. Complementary DNA was first synthesized from total RNA using reverse transcriptase (Takara Co., Shiga, Japan) and PCR was performed using a PTC-100 programmable thermal controller (MJ Research Inc., Watertown, MA, USA). The conditions of operation are as follows: 30 cycles of denaturation at 95 ° C for 30 sec, annealing at 58 ° C for 30 sec, extension at 72 ° C for 30 sec for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) second; 27 cycles for BDNF, denaturation at 95 ° C for 30 seconds, annealing at 57 ° C for 30 seconds, extension at 72 ° C for 30 seconds. All primers were amplified using primer design software provided by the published mRNA sequence and the Whitehead Institute for Biomedical Research (Cambridge, Mass., USA; http://www.genome.wi.mit.edu) Primer 3 was used. ATC CCA TCA CCA TCT TCC AG-3 '(SEQ ID NO: 1) and (reverse) 5'-CCT GCT (SEQ ID NO: 1) in the case of GAPDH (406 bp) TCA CCA CCT TCT TG-3 '(SEQ ID NO: 2); For BDNF (153 bp), 5'-CAG GGG CAT AGA CAA AAG-3 'and (reverse) 5'-CTT CCC CTT TTA ATG GTC-3' (SEQ ID NO: 4). The PCR product was isolated from 1.2% agarose gel and stained with ethidium bromide (EtBr). The density of each band was quantified using an image-analyzing system (i-Max ^™ , CoreBio System Co., Seoul, Korea). The degree of expression was compared by calculating the concentration of each target band, such as BDNF, relative to GAPDH.

As shown in FIG. 8, the expression of BDNF mRNA was significantly reduced in the rat hippocampus of the STR group as compared with that of the SAL group. However, the reduced amount of BDNF mRNA expression in the STR group was effectively restored in the STR + FCN50 group. The recovered expression level was almost similar to that of the normal rats of the SAL group, and was almost equal to that of the positive control group STR + FLX.

When these various results are combined, it can be seen that fucoidan has the effect of relieving depression.

Example  8: Statistical processing

All measurements were performed by independent researchers who were unaware of the experimental conditions, and the results in the figures were expressed as mean ± standard error (SE). Analysis of variance (ANOVA) and Tukey's post-hoc test were performed using SPSS (Version 13.0; SPSS Inc., Chicago, IL, USA) to compare the significance of each result. Statistical significance was set at p <0.05.

Claims (4)

A pharmaceutical composition for preventing or treating depression, which comprises fucoidan and is administered at 20 to 50 mg of fucoidan / kg body weight.
The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier.
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