KR101676828B1 - A composition comprising an extract of Hippophae rhamnoides for treating or preventing a physiological stress, depression or dementia - Google Patents

Abstract

The present invention relates to a composition containing a vitamin A tree extract, wherein the level of dopamine, noradrenaline, and corticosterone in the brain and blood of a mouse in an animal experiment using a mouse causing mental stress level and lowering the level of serotonin, thereby confirming that the compound is useful for inhibiting, preventing and treating mental stress related diseases.

Description

TECHNICAL FIELD The present invention relates to a composition for treating and preventing mental stress, depression or dementia, which contains vitamin A extract as an active ingredient,

The composition containing the vitamin-tree extract of the present invention as an active ingredient can be used as an active ingredient by reducing the levels of brain and blood dopamine, noradrenaline, serotonin (5-HT), and corticosterone in mice in an animal experiment using a mouse that induced mental stress , And is effective in suppressing, preventing and improving stress-related diseases or fatigue because it is excellent in resistance to mental stress.

[1] Charney, D. S.: Neuroanatomical circuits moculating fear and anxiety behaviors. Acta. Psychiatr. Scand. Suppl. 417, 38 (2003);

[2] Millan, M. J.: The neurobiology and control of anxious states. Prog. Neurobiol. 70, 83 (2003)

[3] Sugimoto, Y, et al.,: Effects of extracts and nephrine from the embryo of Nelumbo nucifera seeds on the central nervous system. Phytomedicin. 15,1117 (2008);

[4] Arago, G. F., et al.,: A possible mechanism for anxiolytic and antidepressant effects of alpha and beta-amyrin from Protium heptaphyllum (Aubl.) March. Pharmacol. Biochem. Behav. 85, 827 (2006).

[Literature 5] Kalivas, P. W. and Duffy, P.: Selective activation of dopamine transmission in the nucleus accumbens by stress. Brain Res. 675, 325 (1995);

[6] Tidey, J. W. and Miczek, K. A. Social defeat stress selectively alters mesocorticolimbic dopamine release: an in vivo microdialysis study. Brain Res. 721,140 (1996).

[7] Gesto, M., Soengas, JL and Miguez, JM: Acute and prolonged stress responses of brain monoaminergic activity and plasma cortisol levels in rainbow trout modified by PAHs (naphthalene, beta-naphthoflavone and benzo (a) pyrene) treatment. Aquat. Toxicol. 86, 341 (2008).

[8] Buffalari, D. M. and Grace, A. A. Chronic cold stress increases excitatory effects of norepinephrine on spontaneous and evoked activity of basolateral amygdala neurons. Int. J. Neuropsychopharmacol. 12, 95 (2009). [9] Saito, T., et al.,: Effect of chronic cold exposure on noradrenergic modulation in the preoptic area of thermoregulation in freely moving rats. Life Sci. 83, 79 (2008).

[Literature 10] Lapiz-bluhm, M. D., et al.,: Chronic intermittent cold stress and serotonin depletion induce deficits of reversal learning in an attentional set-shifting test in rats. Psychopharmacology (Berl) 202,329 (2009).

Parker, C. C. et al.,: Restraint stress and exogenous corticosterone differentially altered sensitivity to the sedative-hypnotic effects of ethanol in inbred long-sleep and inbred short-sleep mice. Antistress effect through neurotransmitter regulation of green tea extract and theanine complex 249 Vol. 53, No. 5, 2009 Alcohol. 42, 477 (2008)

[12] Swiergiel, A. H., et al.,: Effects of chlordiazepoxide on footshock- and corticotropin-releasing factor-induced increases in cortical and hypothalamic norepinephrine secretion in rats. Neurochem. Int. 52, 1220 (2008)

[Literature 13] Komiya, M., Takeuchi, T. and Harada, E.: Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and activities in mice. Behav. Brain Res. 17, 172, 240 (2006).

[14] Seo, J. et al.,: Anxiolytic -like effects of obovatol isolated from Magnolia obovata: Involvement of GABA / benzodiazepine receptors complex. Progress in Neuro-Psychopharmacology & Biological Psychiatry 31, 1363 (2007)

[Document 15] Korean Patent Registration No. 10-0008014

[Document 16] Korean Patent Registration No. 10-0012695

[Document 17] Korean Patent Registration No. 10-0008675

[Literature 18] Journal of antimicrobial chemotherapy, L. C. Chiang, W. Chiang, M. C. Liu and C. C. Lin, Vol. 52, pp 194-198, 2003

[Literature 19] J. Agric. Food Chem., Vol. 47, pp 3480-3488

[Literature 20] J. of Chromatography A, Chu Chen, 1154, pp. 250 to 259

The present invention relates to a composition for the treatment and prevention of mental stress-related diseases containing vitamin-tree extract as an active ingredient.

Stress has been referred to as the source of all diseases since ancient times. Especially in modern society, social factors such as academic work, work, marriage, child care, environmental factors such as weather and traffic, And it is recognized as a very important social problem.

As our society rapidly develops and diversifies, the number of people who complain of generalized anxiety disorder and mental illness due to various stresses are increasing as the roles required for modern people are increased. According to the 2006 Mental Disease Epidemiology Survey released by the Ministry of Health and Welfare, the prevalence rate of mental illness in the past year was 17.1%. This is one in six adults between the ages of 18 and 64, and in 2006, the lifetime prevalence of mental illness, which is the proportion of people with at least one mental illness during the lifetime, was 30%, or one in three adults. Considering the recent trend of excessive mental illness due to excessive school-age or various stresses, the prevalence rate of the whole population is higher.

Anxiety and stress are closely related to in vivo neurotransmitters and hormones. In response to external stimuli and stress, the body controls the secretion of neurotransmitters and hormones in the hypothalamus. The signal from the hypothalamus to the central nervous system causes dopamine and noradrenaline, serotonin serotonin, and the like, thereby regulating physiological activities such as emotional state, heart rate, blood pressure, and blood flow increase of skeletal muscle. The hormone secretion from the hypothalamus to the pituitary gland and the adrenal glands is a circulation system through the hypothalamic-pituitary-adrenal (HPA) axis system. When any external stimulus or stress is applied, Secretion of corticotropin-releasing factor (CRF). When the hormone binds to a receptor that specifically binds to the CRF of the pituitary gland, it secretes adrenocorticotropic hormone (ACTH). ACTH arrives at the adrenal gland through the blood and lymph nodes, releasing corticosterone-like steroid hormones and catecholamine-like neurotransmitters from the cortex to the blood, thereby releasing heart rhythm, blood pressure and energy metabolism And regulate the secretion of these factors through negative feedback regulation (1,2 Kharney, DS: Neuroanatomical circuits: moculating fear and anxiety behaviors. Acta. Psychiatr. Scand. 417, 38 (2003), 2) Millan, MJ: The neurobiology and control of anxious states. Prog. Neurobiol. 70, 83 (2003).)

In clinical practice, medication is combined with long-term psychotherapy to treat anxiety. In the case of medication, diazepam, lorazepam, clonazepam, alprazolam, Benzodiazepine-based anti-anxiety drugs are mainly used, and azapirone-based buspirone selectively acts on serotonin receptors to selectively relieve post-anxiety symptoms. . Recently, studies on stress-regulating substances derived from natural materials that can complement the side effects of these drugs have been actively carried out. 3,4 Sugimoto, Y, et al.: Effects of extracts and nephrine from the embryo of Nelumbo nucifera seeds on the central nervous system. Phytomedicin. 15,1117 (2008); 4) Arago, G. F., et al.,: A possible mechanism for anxiolytic and antidepressant effects of alpha and beta-amyrin from Protium heptaphyllum (Aubl.) March. Pharmacol. Biochem. Behav. 85, 827 (2006).) Dopamine and serotonin have been studied as hormones as the main regulators.

The relationship between stress-related neurotransmitter changes and anxiety / stress is as follows: First, dopamine is a type of catecholamine-type neurotransmitter that acts as an excitatory transfer of neuronal cells, , it is known to regulate the motivation state. Excessive secretion leads to diseases such as schizophrenia. If it is insufficient, it is known to cause movement disorders such as Parkinson's disease. The dopamine system is sensitive to stress and various acute stresses have been reported to increase dopamine release, 7, Kalivas, PW and Duffy, P.: Selective activation of dopamine transmission in the shell of the nucleus accumbens by stress. Brain Res. 675, 325 (1995); 8) Tidey, J. W. and Miczek, K. A. Social defeat stress selectively alters mesocorticolimbic dopamine release: an in vivo microdialysis study. Brain Res. 721, 140 (1996).) Even when exposed to chronic and endotoxin-induced stresses such as naphthalene, betanaphthoflavone and benzopyrene, dopamine and dopamine DOPAC (3,4-dihydroxyphenylacetic acid), which is an amine metabolite of dopamine, has been reported to increase in the brain. [9] Gesto, M., Soengas, JL and Miguez, JM: Acute and prolonged stress responses of brain monoaminergic activity and plasma cortisol levels in rainbow trout are modified by PAHs (naphthalene, beta-naphthoflavone and benzo (a) pyrene) treatment. Aquat. Toxicol. 86, 341 (2008).)

Noradrenaline is also a neurotransmitter secreted by the adrenal cortex to adapt to stress by the HPA axis system. It activates the sympathetic nervous system and causes physiological symptoms when stress or anxiety such as blood pressure, heart rate, . It has been reported that inducing stress in experimental animals through cold stress increases norepinephrine release and activates neuronal activity of basolateral amygdala. 10Buffalari, DM and Grace, AA: Chronic cold stress increases excitatory effects of norepinephrine on spontaneous and evoked activity of basolateral amygdala neurons. Int. J. Neuropsychopharmacol. In a study by Saito et al. (2004), it was reported that noradrenalin secretion increased in acute cold stress and maintained the body temperature by controlling the medial preoptic area.1111 Saito, T., et al., Effects of chronic cold exposure on noradrenergic modulation in the preoptic area of thermoregulation in freely moving rats. Life Sci. 83, 79 (2008).

Serotonin has been reported to decrease serotonin levels when intermittent cold stress is induced, and serotonin deficiency may decrease learning and attention. 12Lapiz-bluhm, MD, et al. ,: Chronic intermittent cold stress and serotonin depletion induce deficits of reversible learning in an attentional set-shifting test in rats. Psychopharmacology (Berl) 202, 329 (2009).) The effects of corticosterone levels and sedative effects of ethanol on restraint stress induced by long-sleeping mice and short mice The results of the present study showed that corticosterone levels were elevated in acute and repeated constipation experiments. 13 Parker, CC et al.,: Restraint stress and exogenous corticosterone differentially altered sensitivity to the sedative-hypnotic effects of ethanol in inbred long-sleep and inbred short-sleep mice. Antistress effect through neurotransmitter regulation of green tea extract and theanine complex 249 Vol. 53, No. 5, 2009 Alcohol. 42, 477 (2008)).

In the present study, we measured the levels of neurotransmitters measured using the benzodiazepine drugs, which are commonly used antibiotics, clinically. In this study, we used electric foot shock and microdialysis, , Norepinephrine was increased by more than 300% compared with the control group, and it was found that treatment with 5 mg / kg of chlordiazepoxide, a representative benzodiazepine-based drug, significantly decreased elevated norepinephrine levels .14 Swiergiel, AH, et al.,: Effects of chlordiazepoxide on footshock- and corticotropin-releasing factor-induced increases in cortical and hypothalamic norepinephrine secretion in rats. Neurochem. Int. 52, 1220 (2008).) However, these drugs cause excessive sedative effects, muscle relaxation, insomnia, drug dependence, and so on. Komiya et al. Measured anti-stress efficacy using lemon oil flavor by measuring serotonin and dopamine activity in mice. As a result, lemon oil inhibited the activity of dopamine, increased serotonergic neuron activity, and showed antistress efficacy. 15) Oh16Komiya, M., Takeuchi, T. and Harada, E.: Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and activities in mice. Behav. Brain Res. 17, 172, 240 (2006) reported an anxiolytic efficacy associated with the GABA / benzodiazepine receptor in Magnolia obovata extract. (16) Seo, J. et al., Anxiolytic -like effects of obovatol isolated from Magnolia obovata: Involvement of GABA / benzodiazepine receptors complex. Progress in Neuro-Psychopharmacology & Biological Psychiatry 31, 1363 (2007). Comparing the results of these studies suggests that levels of dopamine, noradrenaline and corticosterone levels increase and levels of serotonin decrease when stress / anxiety is induced .

Korean Patent Registration No. 10-0008014 discloses a method for decreasing the basal activity of the hypothalamus-pituitary-adrenal axis and influencing sleep- and sleep-related behaviors with conventional techniques, Relationship. Korean Patent Registration No. 10-0012695 discloses a food composition for improving stress relief, fatigue recovery, or exercise performance, which comprises a caspase extract. Korean Patent Registration No. 10-0008675 discloses a composition containing a caspase, Fatigue recovery, or stress. However, techniques and researches related to sleep deprivation and stress using natural plant resources are insignificant.

Hippophae rhamnoides, a plant of the Elaeagnaceae family, is found on the sand dunes of the eastern and southeastern coasts of Britain. It is a mountainous region in Europe and Asia, the Inner Mongolia region of China , And Siberia (Russia), and the Canadian Outback. The pharmacological activities of plant-derived flavonoids and glycosylated flavonoids are well known (Journal of antimicrobial chemotherapy, LC Chiang, W. Chiang, MC Liu and CC Lin, Vol. 52, pp 194-198, 2003). Many researchers have been conducting researches on active ingredients of lumber trees. Especially, the contents of various natural vitamins (A, B, C, E, P and K) were analyzed by quantitative and qualitative analysis and the contents of various amino acids and contents of various metals were analyzed. In addition, studies on the content of various nutrients and the study of fruit-related properties have been carried out as basic data for using the fruit of Sanjay as food (J. Agric. Food Chem., Vol. 47, pp 3480-3488). And mainly in China, Sanjay tree Hippophae rhamnoides ssp. Sinensis 15 species, Hippophae rhamnoides ssp. Seven species of yunnanensis, Hippophae rhamnoides ssp. 5 species of wolongensis, Hippophae rhamnoides ssp. Four species of stellatopilosa, Hippophae rhamnoides ssp. Various flavonoids derived from three species of tibetana were classified into 12 kinds of flavonoids by HPLC standardization (J. of Chromatography A, Chu Chen, 1154, pp 250 to 259). In addition, the content of 12 flavonoids isolated from various wild plants grown in various regions of China was studied.

None of the above documents, however, discloses or teaches the inhibitory activity against mental stress-related diseases containing vitamin-A extract as an active ingredient.

Accordingly, the present inventors have found that natural extracts which are excellent for suppressing stress caused by stress can be used as an antioxidant. The inventors of the present invention have found that vitamin A extract is effective in suppressing the stress caused by mental stress in animal experiments using mouse brain and blood dopamine, it has been confirmed that it is useful for inhibiting, preventing and treating mental stress related diseases by confirming that the levels of noradrenaline and corticosterone are lowered and the level of serotonin is increased Thus completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for treating and preventing mental stress-related diseases containing vitamin-tree extract as an active ingredient.

The present invention also provides a health functional food for improving and preventing mental stress-related diseases containing vitamin-tree extract as an active ingredient.

The vitamin tree as defined herein includes fruit, stem, leaf, flower, root, outpost, preferably stem or leaf part, more preferably leaf part.

The extract as defined herein is a polar solvent selected from water, alcohol, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water and a mixed solvent, more preferably water or 20 to 90% water and alcohol Includes extracts soluble in mixed solvents.

The stress-related disease as defined herein refers to a disease caused by mental stress, and specifically includes depression, anxiety, insomnia, hypersomnia, narcolepsy, difficulty sleeping in sleep, circadian rhythm sleep disorder, nightmares, Sleep disorders such as depression, more specifically depression, anxiety or insomnia.

Hereinafter, the present invention will be described in detail.

A volume of about 1 to 100 times, preferably about 2 to 20 times the weight of the sample after freeze-drying the vitamins, stems, flowers, roots and outposts of the present invention, preferably stem or leaf material, Preferably a water and alcohol mixed solvent, more preferably water or a mixture of water and alcohol mixed in a ratio of 1: 1 to 10, selected from the group consisting of water, alcohol, lower alcohol having 1 to 4 carbon atoms, Preferably at a temperature of 20 to 120 캜, preferably 30 to 80 캜 for about 1 to 72 hours, preferably for 2 to 12 hours, using an extraction method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction Can be extracted with hot water, extracted, filtered under reduced pressure and concentrated to obtain the vitamin tree extract of the present invention.

The present invention also provides a pharmaceutical composition and a health functional food for the treatment and prevention of mental stress related diseases containing the vitamin tree extract prepared by the above production method and the above production method as an effective ingredient.

The vitamin tree extracts prepared above reduce levels of dopamine, noradrenaline, and corticosterone in the brain and blood of mice in an animal experiment using a mouse that induced mental stress, It was confirmed that the level of serotonin was increased and thus it was found to be useful for inhibiting, preventing and treating mental stress related diseases.

The composition of the present invention contains the herbal extract in an amount of 0.01 to 99% by weight based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal or intravenous administration.

The present invention also provides a health functional food for improving and preventing mental stress-related diseases containing vitamin-tree extract as an active ingredient.

The health functional food containing the extract of the present invention can be used variously for medicines, foods and beverages for the improvement and prevention of stress related diseases. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complex, leach tea, health supplement foods and the like, and they are in the form of powder, granule, tablet, capsule or beverage Can be used.

Accordingly, the present invention also provides a health supplement for improving and preventing mental stress-related diseases containing vitamin-tree extract as an active ingredient.

Accordingly, the present invention also provides a food additive for improving and preventing mental stress-related diseases containing vitamin-tree extract as an active ingredient.

The food forms to which the extract of the present invention can be added include various foods of candy, beverages, gums, tea, a vitamin complex, or foods that are health supplement foods.

The extract of the present invention can be added to food or beverage for the purpose of suppressing stress and preventing it. At this time, the amount of the extract in the food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above extract as an essential ingredient in the indicated ratios, have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The vitamin-tree extract of the present invention lowers levels of dopamine, noradrenaline and corticosterone in the brain and blood of mice in an animal experiment using a mouse causing mental stress, the present invention provides a pharmaceutical composition or health functional food useful for inhibiting, preventing and treating mental stress related diseases by confirming that the level of serotonin is increased.

1 is a view showing a drug treatment process;
FIG. 2 is a graph showing the effect of vitamin-tree extract on brain and blood dopamine levels in stress-induced mice induced by electrical shock (data are expressed as means (mean latencies) ± SD (n = 10) * P <0.001, ** P <0.01, * P <0.05 vs control);
FIG. 3 is a graph showing the effect of vitamin-tree extract on brain and blood noradrenaline levels in stress-induced mice induced by electrical shock; data are expressed as mean latencies ± SD (n = 10) ** P <0.001, ** P <0.01, * P <0.05 vs control);
FIG. 4 is a graph showing the effect of vitamin-tree extracts on brain and blood 5-HT levels in stress-induced mice induced by electrical shock; data are expressed as mean latencies ± SD (n = 10). *** P <0.001, ** P <0.01, * P <0.05 vs control);
FIG. 5 is a graph showing the effect of vitamin-tree extract on brain and blood corticosterone levels in stress-induced mice induced by electrical shock; data are expressed as mean latencies ± SD (n = 10). *** P <0.001, ** P <0.01, * P <0.05 vs control);

Hereinafter, the present invention will be described in detail with reference to the following Reference Examples and Experimental Examples.

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Example  One. Vitamin tree  Manufacture of stem and leaf extracts

1-1. Preparation of water extract

30 kg of Vitamin tree leaf (Jeongnam Jangheung Co., Ltd.) was lyophilized for 48 hours, dried at low temperature, and powdered after finishing the selection process, water of 20 times the weight of the vitamin tree leaf powder was added, (COSMOS 660, Kyungseo Machinery Industry), filtered through a filter equipped with a 100-mesh filter, and the supernatant was concentrated under reduced pressure. The extract was lyophilized to give a hot-water extract of vitamin A (hereinafter referred to as HPW) Was used as a sample for the following experiment.

1-2. Manufacture of alcohol extracts

30 kg of Vitamin tree leaf (Jeongnam Jangheung Co., Ltd.) was lyophilized for 48 hours and dried at low temperature. After the selection process, it was powdered, and 40% of the weight of vitamin tree leaf powder was added to 40% (COSMOS660, Kyungshee Machinery Industry) for 5 hours. The filtrate was filtered through a filter equipped with a 100-mesh filter, and the supernatant was reduced in pressure and concentrated. The extract was freeze-dried to obtain Vitamin C truncate extract ) Was obtained and used as a sample in the following experiment.

Experimental Example  One. Vitamin tree  Identification of stress relieving effect of extract

Measurement of the content of stress-related neurotransmitters and hormones (dopamine, noradrenaline, serotonin, corticosterone) in the serum and brain main tissues after inducing stress by electric foot shock of the vitamin extracts obtained in the above Examples (Sang-Ki Park et al., Combination of Green Tea Extract and L-Theanine Alleviates Electric Foot Shock Induced Stress by Modulating) was applied in the following manner by applying the method described in the literature Neurotransmitters in Mice Yakhak Hoeji Vol. 53, No. 52, pp41 ~ 249 (2009))

1-1. Laboratory animals and samples

Six - week - old male ICR mice (20-25 g) were purchased from the central laboratory animal, and general symptoms were observed for 1 week. The body weight was measured and only healthy ones were selected. Experimental animals were housed, managed, administered, and tested according to GLP level management standards at the Laboratory Animal Research Support Center of Chungbuk National University. The experimental animals were housed three times per cage for isolation. The environmental conditions of the cage were 23 ± 3 ℃, 55 ± 10%, 12 hours (7:00 am to 7:00 pm), 150 To 200 lux. The water and animal feeds were freely available and the average daily water intake was measured to dissolve the sample in water.

1-2. Test substance dosing and dosing (see Table 1 and Figure 1)

The animals were divided into two groups: diazepam (Diazepam, Cat # D0899, Sigma, St. Louis, USA) at a dose of 2 mg / kg. b.w. Dose was intraperitoneally administered once 30 minutes before the induction of stress. In the case of teanin and vitamin A leaf hot water extract, it was orally administered once a day for a total of 31 days.

treatment schedule Group 전기 충격 concentration Normal - 비성 처리 Control + 비성 처리 Diazepam + 2 mg / kg, b.w. (I.P.) * Teanin + 4 mg / kg, b.w low + 50 mg / kg, b.w middle + 150 mg / kg, b.w high + 300 mg / kg, b.w * bw: body weight
** IP: intraperitoneally

1-3. Electrical Stimulation for Stress Induction

As a method of inducing stress in an experimental animal, a method of electric foot shock described in the literature was used. (Sang-Ki Park et al., Combination of Green Tea Extract and L-Theanine Alleviates Electric Foot Shock Induced Stress by Modulating Neurotransmitters in Mice Yakhak Hoeji Vol. 53, No. 52, pp41 ~

After the end of the test substance administration, stress was applied to the foot by electric shock of 0.6 mA for 1 second at intervals of 5 seconds for 30 minutes, and then mice were autopsied, blood and brain were excised.

Samples (theanin and vitamin A hydrothermal extracts) were orally administered for 31 days on the day before the experiment, and diazepam was administered intraperitoneally only on the 31st day of electric shock.

1-4 Collection and storage of blood and brain

After the induction of stress, the animals were anesthetized with ether and the abdomen was incised. The blood was collected from the abdominal artery to the immediate post-cardiac arrest with a 1-ml syringe, and the heparin-coated blood collection vacutainer (BD, And the mixture was allowed to stand at room temperature for 2 hours. The supernatant was centrifuged at 15,000 rpm for 15 minutes at 4 ° C, and the supernatant was separated and frozen (-20 ° C).

The brain was extracted from the blood-removed mouse and 200-500 μl of extraction buffer (0.3 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 10 mM HEPES (pH 7.9), 1.5 mM MgCl 2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 0.1% protease inhibitor, 0.1% phosphatase inhibitor, 0.5% NP 40) and homogenized. The resulting mixture was thoroughly mixed with extraction buffer to a final volume of 1 ml and lysed at 4 ° C for 2 hours. After the reaction was completed, the sample was placed in a 1.5 ml tube, vortexed for 5 minutes at 3 ° C, centrifuged at 15,000 rpm for 15 minutes at 4 ° C, and the supernatant was taken.

1-5. Biochemical analysis

The contents of dopamine (DA), noradrenaline (NE), serotonin (5-HT) and corticosterone (CS) in the blood and brain tissues were measured by ELISA. Dopamine (Dopamine ELISA Kit, Cat # KA1887, Abnova Corp , Serotonin (Serotonin ELISA Kit, Cat # KA1894, Abnova Corp, Taipei City, Taiwan), Corticosterone (Corticosterone ELISA Kit, Taipei City, Taiwan), Norepinephrine (Norepinephrine ELISA Kit, Cat # KA1891, Abnova Corp, Ki, Cat # KA0468, Abnova Corp, Taipei City, Taiwan) were assayed according to the manufacturer's method using each kit.

1-6. Statistical analysis

Statistical significance was assessed using Student's t-test for differences between groups, p values <0.05 (* p <0.05), <0.01 (** p < 0.01) and less than 0.001 (*** p <0.001), and statistical significance was determined.

1-7. Experiment result

As a result, the dopamine levels in the blood were not significantly different before and after the stress stimulation, and the positive control group, DZP theanin, did not show any significant change. There was no significant change in the vitamin - tree leaf hydrothermal extract group. In the control group (86.72 ± 5.21 ng / mg protein), the dopamine levels in the brain tissue were significantly higher than those in the control group (51.32 ± 3.15 ng / mg protein ) Compared with the control group. In addition, when treated with the extract of vitamin wood hydrothermal extract, the concentration of dopamine decreased by 80.41 ± 3.42, 77.43 ± 3.94 and 71.42 ± 3.41 ng / mg protein in the Low, Middle and High groups, respectively . Significant reductions in dopamine levels were also observed in the positive control DZP (63.52 ± 4.72 ng / mg protein) and theanine (73.31 ± 5.15 ng / mg protein). The levels of dopamine increased by stress were significantly decreased by vitamin A hydrothermal extract. (See Fig. 2)

After applying mental stress to Electric Foot Shock, the effect of vitamin A hydrothermal extract on serum noradrenalin levels was measured. In the control group (71.52 ± 7.32 ng / mg protein), which had only electrical stimulation for 30 minutes, the level of noradrenalin in the blood was significantly higher than that of the untreated group (32.51 ± 4.74 ng / mg protein) Respectively. The concentration of noradrenalin was decreased by 56.41 ± 3.94 and 54.17 ± 4.12 ng / mg protein in the middle and high groups, respectively. Increased noradrenalin levels were also significantly reduced in the positive control DZP (45.73 ± 4.32 ng / mg protein) and theanine (53.35 ± 3.24 ng / mg protein). The level of noradrenalin in the brain tissue was significantly increased in the control group (105.11 ± 7.52 ng / mg protein) compared to the no-treatment group (31.47 ± 3.15 ng / mg protein) Respectively. When the extracts of Vitamin A hydrothermal extract were treated at different concentrations, 87.43 ± 5.15 88.13 ± 3.94 and 78.32 ± 4.51 ng / mg protein in the Low, Middle and High groups showed a decrease in noradrenaline levels in a concentration-dependent manner. Significant reductions in noradrenalin levels were also observed in the positive controls, DZP (62.72 ± 6.14 ng / mg protein) and theanine (81.12 ± 5.15 ng / mg protein). The level of noradrenalin increased by stress was significantly decreased by vitamin hydrothermal extract. (See Fig. 3)

When 5-HT levels in serum were measured, the 5-HT levels in the brain in the control group (10.33 ± 2.46 ng / mg protein), which only received 30 minutes of electrical stimulation, Compared with the control group (21.31 ± 2.11 ng / mg protein). When 5-HT levels of 17.12 ± 3.14 ng / mg protein were significantly increased in the high-group treated with the vitamin-tree hydrothermal extract, The decrease in 5-HT levels was also significantly increased in the positive control DZP (28.43 ± 3.31 ng / mg protein) and theanine (16.64 ± 2.14 ng / mg protein). The levels of 5-HT in brain tissues were significantly higher in the control group (14.43 ± 5.21 ng / mg protein) than in the control group (34.42 ± 3.15 ng / mg) in the brain without stress stimulation protein) significantly decreased compared to the control group. The 5-HT levels were significantly increased by 23.35 ± 3.94 and 32.35 ± 3.41 ng / mg protein in the middle and high groups, respectively. The decrease in 5-HT levels was also significantly increased in the positive control DZP (42.36 ± 4.72 ng / mg protein) and theanine (28.42 ± 3.36 ng / mg protein). In particular, in the case of DZP, the 5-HT content was significantly increased at the level of the untreated group, and the 5-HT level decreased by the stress was significantly increased again by the vitamin hydrothermal extract. (See Fig. 4)

After applying mental stress with Electric Foot Shock, the effect of vitamin A hydrothermal extract on serum corticosterone levels was measured. In the control group (32.25 ± 2.89 ng / mg protein), which had only 30 minutes of electrical stimulation, corticosterone levels in the brain were significantly higher than those in the untreated group (9.51 ± 3.15 ng / mg protein) Respectively. When corticosterone extracts were treated at the concentrations of 24, 233 ± 3.94 and 24.43 ± 3.41 ng / mg protein in the middle and high groups, the corticosterone levels were significantly decreased. The increase in corticosterone levels was also significantly reduced in the positive control group, DZP (13.36 ± 2.35 ng / mg protein) and theanine (21.14 ± 3.24 ng / mg protein). The level of corticosterone in brain tissue was significantly higher in the control group (1562 ± 140.32 ng / mg protein) than in the electrical stimulation group for 30 minutes (154.35 ± 35.36 ng / mg protein) protein significantly increased compared to the control group. When the extracts of Vitamin A hydrothermal extract were treated at different concentrations, the levels of corticosterone were significantly decreased in the Low, Middle and High groups by 1251.36 ± 84.42, 1152.36 ± 84.35, and 1095.45 ± 79.64 ng / mg protein, respectively . The increase in corticosterone levels was also significantly reduced in the positive control group, DZP (625.32 ± 105.35 ng / mg protein) and theanine (984.43 ± 94.35 ng / mg protein). The corticosterone levels decreased by stress were significantly decreased by vitamin A hydrothermal extract. (See Fig. 5)

In conclusion, vitamin A extracts lowered levels of mouse brain and blood dopamine, noradrenaline and corticosterone levels in mice in mental stress-induced animals, and serotonin serotonin levels in the brain, and thus it is considered to be a functional material effective for suppressing mental stress. The present invention can be used as a functional material by performing additional metabolic mechanism studies.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but will be specifically described.

Formulation example  One. Sanje  Produce

HPW ------------------------------------------------- 200 mg

Lactose ------------------------------------------------ 100 mg

Talc ------------------------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

HPL ------------------------------------------------- 200 mg

Corn starch ------------------------------------------ 100 mg

Lactose ------------------------------------------------ 100 mg

Magnesium stearate ----------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

HPW ------------------------------------------------- 200 mg

Crystalline cellulose - 3 mg

Lactose ------------------------------------------- 14.8 mg

Magnesium Stearate ------------------------------- 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

HPL ------------------------------------------------- 200 mg

Mannitol ---------------------------------------------- 180 mg

Sterile sterile distilled water for injection --------------------------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O ------------------------------------------ 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

HPW ------------------------------------------------- 200 mg

Isolation Party ---------------------------------------------- 10 g

Mannitol ------------------------------------------------- 5 g

Purified water ------------------------------------------------

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

HPL ------------------------------------------------ 1000 mg

Vitamin mixture -----------------------------------------

Vitamin A Acetate ---------------------------------- 70 g

Vitamin E -------------------------------------------- 1.0 mg

Vitamin B1 ------------------------------------------ 0.13 mg

Vitamin B2 ------------------------------------------ 0.15 mg

Vitamin B6 ------------------------------------------- 0.5 mg

Vitamin B12 ------------------------------------------ 0.2 g

Vitamin C --------------------------------------------- 10 mg

Biotin ----------------------------------------------- 10 μg

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------------- 50 [mu] g

Calcium pantothenate --------------------------------------- 0.5 mg

Inorganic mixture -----------------------------------------

Ferrous sulfate ------------------------------------------ 1.75 mg

Zinc oxide - 0.82 mg

Magnesium carbonate --------------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Calcium Phosphate - 55 mg

Potassium citrate ------------------------------------------- 90 mg

Calcium carbonate -------------------------------------------- 100 mg

Magnesium Chloride --------------------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

HPW ----------------------------------------------- 1000 mg

Citric acid -------------------------------------------- 1000 mg

Oligosaccharides -------------------------------------------- 100 g

Vitamin tree concentrate -------------------------------------- 2 g

Taurine ------------------------------------------------ 1 g

Add purified water - 900 ml total

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (8)

Vitamin tree leaf material (true vitamins, Jangheung) was lyophilized and ground, extracted with hot water at 20 to 120 ° C for 1 to 72 hours in a volume of 2 to 20 times the weight of the sample, filtered and concentrated under reduced pressure A pharmaceutical composition for treating and preventing a mental stress related disease selected from anxiety or insomnia containing the obtained vitamin tree leaf water extract as an active ingredient. delete delete delete Vitamin tree leaf material (true vitamins, Jangheung) was lyophilized and ground, extracted with hot water at 20 to 120 ° C for 1 to 72 hours in a volume of 2 to 20 times the weight of the sample, filtered and concentrated under reduced pressure A health functional food for improving and preventing mental stress related diseases selected from anxiety or insomnia containing the obtained vitamin tree leaf water extract as an active ingredient. delete Vitamin tree leaf material (true vitamins, Jangheung) was lyophilized and ground, extracted with hot water at 20 to 120 ° C for 1 to 72 hours in a volume of 2 to 20 times the weight of the sample, filtered and concentrated under reduced pressure A health supplement for improving and preventing mental stress related diseases selected from anxiety or insomnia containing the obtained vitamin tree leaf water extract as an active ingredient. delete

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