CN115212245A - Composition and capsule and tablet containing the same - Google Patents

Abstract

The present invention provides a composition, and a capsule and a tablet containing the composition, which can greatly enhance the efficacy of each substance in addition to the synergistic effect of the effects of each substance constituting the composition. The composition contains extracts of centella asiatica, emblica officinalis, salacia reticulata and Hericium erinaceum, and diosgenin and divalent ferric salt as effective components. By including a divalent ferric salt, the efficacy of each of the materials that make up the composition itself is greatly enhanced beyond expectations.

Description

Composition and capsule and tablet containing the same

Technical Field

The present invention relates to compositions having multiple functions. More particularly, the present invention relates to a composition having, for example, a cognitive dysfunction prevention function, a health promotion function, a nutrition support function, a beauty function, and other various functions and effects.

Background

Conventionally, as a supplement (beauty supplement) having a beauty function, for example, a supplement (food composition) containing collagen, centella asiatica, and deep ocean water has been proposed (for example, see patent document 1).

According to the food composition (beauty tonic) of patent document 1, it is considered that the combination of centella, deep seawater and collagen exerts a beauty effect of improving the metabolism of the skin and preventing the skin aging by utilizing the action of each component.

In addition, as a supplement (health food) having a health promoting function, for example, a health supplement (health food) prepared by adding centella asiatica (centella asiatica) to emblica and mixing the mixture has been proposed (for example, see patent document 2). According to the tonic of patent document 2, it is considered that the effect of the active ingredient of emblic leafflower fruit is improved by the synergistic action of emblic leafflower fruit and centella asiatica (centella asiatica), and the health promoting effect is thereby improved.

On the other hand, the present inventors have proposed that, as a novel active water having pharmacological/physiological effects, active water obtained by adding a divalent trivalent iron salt and an organic germanium compound to water is invented (patent document 3), and as a novel bioremediation agent having further improved pharmacological/physiological effects as compared with the active water, a bioremediation agent containing a divalent trivalent iron salt and an organic germanium compound in water in combination with royal jelly or/and propolis is invented (patent document 4).

The former "active water" has been evaluated for its effectiveness in the united states and patented (patent document 5).

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 3495712

Patent document 2: japanese patent laid-open No. 2007-135493

Patent document 3: japanese laid-open patent publication No. 10-66982

Patent document 4: japanese laid-open patent publication No. 10-114666

Patent document 5: U.S. Pat. No. 6203821

Disclosure of Invention

Problems to be solved by the invention

The supplements described in patent documents 1 and 2 can provide supplements having various properties (document 1 is a food composition, and document 2 is a health food).

Among them, patent document 1 utilizes a synergistic effect (effect) of the original effects of each component (substance) of centella asiatica (centella asiatica), deep seawater, and collagen. Patent document 2 also utilizes a synergistic effect of the effects inherent in each component (substance) of emblic leafflower fruit and centella asiatica (centella asiatica). Therefore, the effect of improving each component itself cannot be expected.

In view of the above circumstances, the present invention has been made to provide a composition having a plurality of functions which have not been achieved so far, taking advantage of the results of the research and development carried out by the present inventors so far, in order to greatly improve the functions (effects) of each substance itself beyond expectations, in addition to the synergistic effects (effects) of the effects of each substance (component) constituting the composition.

Means for solving the problems

The present inventors have continued research and experiments to solve the above problems and as a result, have reached their object, and have provided their inventions.

That is, one invention (first invention) of the present invention is a composition characterized by containing extracts of centella asiatica, phyllanthus emblica, salacia reticula, and hericium erinaceum, as active ingredients, diosgenin, and a divalent ferric salt.

Another invention (second invention) of the present invention is characterized in that the composition of the first invention is used for the prevention or/and treatment of cognitive dysfunction.

Still another invention (third invention) of the present invention is characterized in that the composition of the first or second invention further contains zinc as an active ingredient.

In the present invention, the composition can be contained in the form of a capsule as a content.

In the present invention, the centella asiatica and other substances contained in the composition may be mixed in an oily liquid mixture and contained as contents in a capsule.

In the present invention, a tablet can be constituted with the composition via a binder.

Here, each composition will be described in detail.

The divalent ferric salt is an active material developed in recent years, and the active material (divalent ferric salt) is considered to be a single compound showing an intermediate property between divalent iron and trivalent iron or a single compound in which divalent iron and trivalent iron coexist. The divalent or trivalent iron salt can be industrially produced (see, for example, japanese patent publication No. Hei 3-63593 and Japanese patent publication No. Hei 4-27171).

Examples of the divalent or trivalent iron salt include compounds represented by the following formula:

Fe ⁺² _m Fe ⁺³ _n Cl _2m+3n (in the formula, m and n represent positive integers).

The divalent or trivalent iron salt is obtained, for example, as a transport system when ferrous chloride is put into an aqueous solution of a strong base such as sodium hydroxide, calcium hydroxide, potassium hydroxide, or lithium hydroxide to cause valence conversion (first method). A specific production method by the first method is, for example, obtained by the following steps. Specifically, the method comprises a step of dissolving ferrous chloride in an aqueous solution of a strong alkali, a step of neutralizing the solution, and a step of concentrating the neutralized solution.

The divalent or trivalent iron salt may be obtained by mixing a trivalent iron salt with a divalent metal salt to obtain a solution. Specifically, for example, the method can be performed by adding a ferrous salt to a dilute aqueous solution of a predetermined concentration containing a ferric salt and a divalent metal salt at a predetermined ratio, dissolving the ferrous salt, and concentrating the resulting solution (second method). As the ferrous salt, for example, ferrous chloride, ferrous sulfate, and ferrous nitrate can be used. Examples of the divalent metal salt include calcium chloride, magnesium chloride, zinc chloride, magnesium sulfate, calcium nitrate, magnesium nitrate, and zinc nitrate.

M in the formula of the divalent trivalent iron salt: the ratio of n is a specific value depending on the kind of the substance used for producing the compound.

The divalent ferric salt greatly enhances the action (potency) possessed by the metrorrhagia (snowmobile) bowls and other individual substances constituting the composition. Therefore, for example, the function (action) of each of the substances is enhanced by mixing a large bowl and each of the other substances into an oily mixed solution and adding and mixing the divalent or trivalent iron salt thereto. The above-mentioned effects are exerted by adding the divalent trivalent iron salt in a very small amount. This result is confirmed by experiments performed by the present inventors. The composition of the present invention can be used, for example, as an internal preparation.

Centella asiatica (gotukola), also known as "big bowl", is a herb well known in japan as centella asiatica (centella asiatica).

Centella asiatica (centella asiatica) is used as a remedy for skin erosion and ulcer caused by infection such as leprosy in india, which is a traditional medical ayurvedic bark. In addition, it is also used for promoting blood circulation, purifying blood, enhancing immunity, improving intelligence, and improving memory. As the components, triterpenes and saponins such as madecassoside, asiaticoside, asiatic acid and madecassic acid have been confirmed. In bangladesh, thailand, srilanka, centella asiatica (centella asiatica) is eaten as a leafy vegetable, and no problem is recognized in terms of safety.

Emblic leafflower (known by the scientific name pbyllanthus embroca) is a deciduous small tree of the phyllanthaceae family, also known as "currant of indian origin". In Sanskrit, the expression "Amalaki" is used according to the meaning of "nursing women" and in English, the expression "Indian goose weberry" is used.

Phyllanthus emblica has effects of preventing diabetes and hypertension, lowering cholesterol value, beautifying muscle, improving constipation, preventing anemia, improving stress resistance, and preventing canities and alopecia.

Salacia reticulata (scientific name) is a lianoid plant of Salacia of Celastraceae of Celastrales, has a diabetes preventive effect, and is used as a therapeutic agent for diabetes in ayurvedic rodgersia indica of India, and the effect thereof was recognized by WHO in 2002.

Diosgenin is a natural component contained in sweet potatoes, has been used since ancient times as a traditional crude drug beneficial to invigoration and strengthening, and is known to have various effects such as an anticancer effect, a food allergy improvement effect, an anti-aging effect, a diet effect and the like.

Hericium erinaceum (a scientific name: hericium erinaceus) is one of edible mushrooms of the genus Hericium of the family Hericium erinaceum, and attracts attention in the treatment of cognitive disorders. "Hericione (Hericione)" which is a component contained in Hericium erinaceum has recently attracted attention as a specific drug for cognitive disorders. In addition, it is expected to exert various effects on infectious diseases, circulatory system, digestive system, endocrine, and the like.

Zinc is 1 of 16 essential minerals in human body, and keeps normal taste. Activating and resisting oxidation. Improving immunity. Support development and growth. In addition, it is expected to have an effect of maintaining beauty of skin and hair and an effect of alleviating depression.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, it is possible to provide a composition which can greatly enhance the function (efficacy) of each substance itself beyond expectations, in addition to the synergistic effect (effect) of the effects possessed by centella asiatica and other substances (components) constituting the composition.

Drawings

Fig. 1 is an explanatory view illustrating a test method of an embodiment of the present invention.

Fig. 2A to 2C show the test results (1).

Fig. 3A and 3B show the test result (2).

Fig. 4 shows the test results (3).

Fig. 5 shows the test result (4).

Fig. 6A to 6D show the test results (5).

Detailed Description

An example of an embodiment of the composition of the present invention will be described below.

The composition of the present embodiment employs centella asiatica, emblica officinalis, salacia reticulata, diosgenin (sweet potato extract), various extracts of hericium erinaceum, and divalent ferric salt (MRN-100) as constituent substances (components).

The composition (antisa) can be produced by, for example, forming centella asiatica, emblic leafflower fruit, and other individual substances (components) into a powder-like form (powder-like to granular form) and preparing them by a desired method.

The mixing ratio of the above-mentioned constituent components of the composition is not particularly limited, and examples thereof include divalent trivalent iron salt (MRN-100): about 1.5 to 4.0, centella asiatica: about 20.0 to 28.0, emblic leafflower fruit: about 13.0 to 18.0, salacia reticulata: 20.0-26.0, diosgenin: about 20.0 to 26.0, hericium erinaceus: about 10.0 to 15.0 (unit weight%).

In addition, zinc may be added to and mixed with the constituent materials (components) of the composition in a very small amount as required. The blending ratio of zinc is not particularly limited, and may be, for example, about 0.5 to 1.5% by weight based on the total amount of the composition.

(examples)

Next, an embodiment of the present invention will be explained. The following examples are disclosed as examples thereof, but it is needless to say that the present invention is not limited to these examples.

In the following examples, a divalent ferric salt (MRN-100) was used as prepared below.

1.0mg of ferrous chloride was added to 100ml of a 0.5N aqueous solution of caustic soda, dissolved with stirring and left to stand for 24 hours. Insoluble materials formed in the solution were removed, the solution was neutralized with hydrochloric acid, concentrated under reduced pressure and crystallized by drying in a dryer. The obtained crystals were redissolved by adding 50ml of an 80 wt% aqueous solution of 2-propanol, concentrated under reduced pressure to remove the solvent, and dried, and the redissolution, concentration, and drying were repeated several times to obtain 0.25mg of crystals. The crystals are dissolved in water (distilled water or pure water) and diluted to about 100 times to about 10000 times to obtain an aqueous solution of about 0.01% to about 1% of the divalent ferric salt (a stock solution of the divalent ferric salt). Then, the stock solution is powdered to produce a divalent trivalent iron salt, and the powdered divalent trivalent iron salt is used.

In this example, as the composition, a composition (Antia) prepared by blending the substances (powder particles) of Nos. 1 to 6 in the following tables in respective blending amounts was used as a sample.

[ Table 1]

The composition produced in the above manner can be provided in the form of a capsule or tablet containing the composition as a content.

In the oily liquid mixture used in the form of capsules, safflower seed oil, an emulsifier, beeswax, and the like can be used. The blending ratio of the safflower oil, the emulsifier and the beeswax is not particularly limited, and for example, the emulsifier may be used in a range of about 7w% to about 8w% and the beeswax may be used in a range of about 7w% to about 8w% based on the weight of the safflower oil.

Here, an outline of the present example using the above-described sample will be described.

(background)

Numerous neurodegenerative diseases such as alzheimer's disease are associated with oxidative stress. Therefore, antioxidant therapy is proposed for the prevention and treatment of neurodegenerative diseases.

(purpose)

The ability of antioxidant Antiia (referred to as this sample. The same below) exerting a protective effect on Sporadic Alzheimer's Disease (SAD) induced in mice was verified. Antiia is an antioxidant substance obtained by adding MRN-100 (divalent ferric salt) to extracts extracted from edible Hericium erinaceus, centella asiatica and Salacia reticulata, diosgenin (extract from tuber of Dioscorea opposita) and Phyllanthus emblica (Indian gooseberry).

(method)

In inducing SAD in mice, a single Intracerebroventricular (ICV) injection of Streptozotocin (STZ) (3 mg/kg) was used. Antiia was injected Intraperitoneally (IP) in three doses (25, 50, 100 mg/kg/day) for 21 days. Neuro-behavioral examinations were performed within 24 hours from the final day of injection. Thereafter, the mice were sacrificed by cervical dislocation and decapitation. For biochemical marker determination, hippocampus was rapidly excised, weighed and homogenized.

(results)

Treatment with Antiia significantly improved the performance of mice in the Mosley (Morris) water maze. Furthermore, biochemical analysis shows that Antia exerts protective effects on a plurality of compounds such as GSH, MDA, NF-kappa B, IL-6, TNF-alpha, amylin-beta and the like. Further studies using western blotting showed the protective effect of Antia on the JAK2/STAT3 pathway.

(conclusion)

Antiia exerts a meaningful protection against cognitive dysfunction induced by ICV-STZ injection. This effect is achieved by starch peptide (Amyloid) formation, inflammation and the targeting of oxidative stress pathways. The JAK2/STAT3 pathway plays a role in protecting neuroinflammatory diseases such as SAD and neurodegenerative diseases.

Next, the present embodiment will be described in detail.

(preface)

Age-related neurological disorders such as Alzheimer's Disease (AD) are increasing. AD is a neurodegenerative disorder characterized by progressive decline in memory and cognition, and is the most common cause of cognitive disorders, accounting for 60 to 80% of all cases. Sporadic Alzheimer's Disease (SAD), the most prevalent form of AD in the elderly, is associated with progressive neurodegeneration in the central nervous system. Several pathways including oxidative stress, starch peptide formation, inflammation, and autophagy pathways were explored as possible targets for SAD.

The appearance of markers of oxidative stress is one of the earliest changes in AD brain, occurring before macroscopic starch peptide precipitation and accumulation of neurofibrillary changes. Oxidative stress is associated with numerous disorders such as chronic inflammation, AD, parkinson's disease, and the like. Neurons of the brain show high oxygen consumption and high energy production, and therefore the risk of Reactive Oxygen Species (ROS) and overproduction of oxidative damage becomes very high.

In AD brain, usually solid amylin- β (Α β) and Tau proteins are aggregated in amylin-like filaments called spots (plaques) and tangles (kinks). Now, although the pathway for a β to accumulate in the central nervous system to initiate cell disease is not yet solved, a β has the possibility of damaging neurons, leading to neuronal death, and in the proposed mechanism, ROS generation involving self-concentration of a β is involved. When this occurs in vitro (in vitro), at the membranes of neurons, associated with depolarization of the final synaptic membrane, excessive calcium influx, and mitochondrial dysfunction.

Neurodegenerative diseases such as AD are also diseases accompanied by neuroinflammation. The transcription factor NF-. Kappa.B is known to play an important role in the inflammatory response of neurons. Under normal physiological conditions, NF-. Kappa.B forms a cytoplasmic complex with its inhibitor IkB. Alpha., and when it is stimulated, NF-. Kappa.B induces the transcription of inflammatory target genes such as cyclooxygenase-2 (COX-2), interleukin-1. Beta., (IL-1. Beta.), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-. Alpha.). Further, neuroinflammation is associated with autophagy in neurodegenerative diseases. Pathological disruption of autophagy may lead to the initiation or worsening of neuroinflammation, which in turn may lead to autophagic disorders that worsen neurodegeneration. Autophagy is known to decrease and contribute to the pathological accumulation of Tau aggregates in human AD as well as in mouse models of AD. Autophagy is known to be regulated by mammalian targeting of rapamycin to mTOR, and inhibition of mTOR has been shown to prevent neuroinflammation in a mouse model of cerebral anesthesia. Furthermore, GSK-3 β has been shown to inhibit cortical neuroinflammation in rats subjected to ischemic brain injury by activating autophagy.

Up to now, the pharmacological management of AD has been limited. It is believed that long term use of non-steroidal anti-inflammatory drugs (NSAIDs) was thought to be associated with a reduction in the likelihood of AD onset in 2007. Evidence also suggests that NSAIDs can reduce inflammation associated with starch peptide plaques, but trials have been discontinued due to the frequent occurrence of adverse events. There are no drugs, supplements that show a reduced risk of AD and, unfortunately, current FDA-approved AD treatments provide only symptomatic relief and do not delay or cure the disease.

Recently, antioxidant substances have been attracting attention in preventing the onset of AD by reducing oxidative stress damage. Furthermore, it has been found that phytochemicals whose use in conventional medicine has been confirmed are beneficial to health, and the use and search of plant-derived drugs and nutritional supplements have been accelerated in recent years. The components of the traditional Chinese medicinal mushroom called hericium erinaceus promote the synthesis of nerve growth factors of cultured astrocytes and improve mild cognitive impairment of human. Centella asiatica has been traditionally used for cognitive disorders and memory improvement, and extracts thereof improve memory retention in rodents, showing that oxidative stress that alters and regulates the pathology of amyloid peptide β in hippocampus of mouse model of AD is associated with neurodegeneration resulting from AD. Diosgenin, which is a plant-derived steroid sapogenin, is shown to exert an anticancer effect, improve cognitive impairment accompanying aging, and alleviate diabetic neuropathy. Recently, conditions have been demonstrated in which diosgenin improves memory function, reducing axonal degeneration in AD mouse models. Emblic leafflower fruit, currant of india, was shown to have a potent free radical scavenging effect, exhibiting a high degree of neuroprotective capacity in panels of bioassays targeted by oxidative stress, carbonyl stress, protein glycation, Α β fibrosis, acetylcholinesterase inhibition, neuroinflammation. In addition, cognitive function, brain antioxidant enzyme and acetylcholinesterase activity in rat models of AD were improved. Finally, salacia reticulata (Salacia reticulata) shows detrimental cognitive changes in streptozotocin-induced young diabetic rats and protection of mercury toxicity in mouse hippocampus.

In this example, the cognitive protective effect of an antioxidant product called Antia comprising Hericium erinaceum, centella asiatica, diosgenin, phyllanthus emblica, salacia reticulata, etc. was verified. These components are treated with the hydrogen ferrite solution MRN-100 in order to produce Antia. In previous studies of MRN-100, protection against oxidative stress associated with aging and oxidative damage of endothelial cells, mouse and human leukemia cells was shown. In recent studies related to Antia, the ability to repair oxidative stress-induced mitochondrial dysfunction in human peripheral blood lymphocytes has been shown. In view of the above neuroprotective effects of plant components of Antia, it is hypothesized that Antia has beneficial effects on pathways associated with AD, namely oxidative stress, amylopeptization, inflammation and autophagy pathways. The effect of antisa on SAD-induced mice via Intracerebroventricular (ICV) injection of Streptozotocin (STZ) was studied. This is a complete animal model of SAD based on brain resistance to insulin, and is a pathological model that mimics memory impairment, oxidative stress, neuroinflammation, neurodegeneration, etc., and aging associated with human SAD. Here, behavioral, biochemical and western blot experiments supporting the hypothesis are presented.

(method)

< animals >

Male albino mice weighing 25-30 g were provided from animal facilities of Kairou university, egypt, and acclimatized 1 week before the study. Animals were kept under controlled environmental conditions of a certain temperature (25 + -2 deg.C), a relative humidity of 60 + -10%, and a light-dark cycle (12/12 hours). Standard meals and water can be ingested freely. To minimize the pain experienced by the animals, great effort is expended to reduce the number of animals used. The study was approved by the animal experimental ethics committee (the medical office of the university of korea), following the recommendations of the national institutes of health for experimental animal care and use-related guidelines (2011).

< chemicals >

STZ was purchased from Sigma-Aldrich co.

STZ was dissolved in physiological saline (0.9% NaCl), and ICV was injected into 10. Mu.L by the manual method. The dissolution of Antia in physiological saline was carried out in three amounts: corresponding to 25mg/kg, 50mg/kg and 100mg/kg of the adult dose (4 tablets on 1 day). Next, intraperitoneal (i.p.) administration was performed at a volume of 0.1ml/20g mouse. Experiments fresh drug solutions were prepared daily. The control group received saline injections in the same volume of the same route of administration. The other chemicals were the highest analytical grade.

< sample (Antia) >)

Antiia comprises edible Hericium erinaceus, centella asiatica, and Salacia reticulata, diosgenin (an extract from tubers of sweet potatoes), and Phyllanthus emblica (Indian gooseberry), which are natural compounds derived from a wide variety of mushrooms and plants. The material was subjected to a liquid treatment of iron base called MRN-100. MRN-100 is made from phytosin and is an iron-based compound derived from ferrous and trivalent ferrite (ferrate fluid). The correct chemical composition of Antia is now under active investigation. Antiia is supplied by ACM Co., ltd, japan. The concentration of Antiia from MRN-100 is about 2X 10 ^-12 And preparing mol/L of Distilled Water (DW).

< Induction of SAD >

SAD was induced by injecting STZ (3 mg/kg) into the lateral ventricles of mice with ICV in free-hand order and with renewal by warneck et al. This is to avoid the possibility of cerebral vein crossing. After anesthetizing the mice with thiopental (5 mg/kg, ip), the head was fixed with pressure from above the ear downward, and the right triangle between the ventricles was visualized directly through the skin and the skull, whereby the position of bregma was designated at the center of the eye and skull of the targeted lateral ventricle insertion needle, and the needle was inserted approximately 1mm from this point on the outer side. Mice acted normally after 1 minute of injection.

< Experimental plan >)

The experimental plan is shown in figure 1. Mice were arbitrarily divided into 5 groups of 12 animals each. Group I (control): mice were injected with ICV once and intraperitoneally (i.p.) with normal saline continuously for 21 days, and used as a normal control group. Group II (STZ): STZ (3 mg/kg, ICV) was administered once to mice and used as a model of SAD. Group III (STZ + antisa 1): mice were given Antia (25 mg/kg, i.p.) after STZ (3 mg/kg, ICV) daily for 21 consecutive days after 5 hours. Group IV (STZ + antisia 2): mice were dosed 5 hours later with STZ (3 mg/kg, ICV), followed by Antia (50 mg/kg, i.p), followed by 21 consecutive days of daily dosing. Group V (STZ + antisia 3): mice were given Antia (100 mg/kg, i.p.) after 5 hours, 21 consecutive days of daily STZ (3 mg/kg, ICV). 24 hours after the end of the treatment, a neuro-behavioral test including an object recognition test and a Moseri Water Maze (MWM) test was performed, and the test was performed in order from a low stress test to a high stress test. To limit the possibility of circadian variation to a minimum, all tests were carried out in the light cycle of the animals under overhead lighting.

< action evaluation >

And (4) performing object identification testing. The object recognition test is used to evaluate long-term memory, and to infer cognition. In this study, the tests performed were performed continuously over 3 days. On day 1 (acclimation period), each mouse was adjusted to the surrounding environment at a size of 30X 30cm ³ Was placed in the wooden box for 30 minutes. Day 2 two wooden cubes of the same shape, color and size were placed in the corners of opposite sides of a box with a wall thickness of 2cm for acquaintance or training. Each mouse was placed in the center of each box and placed for 10 minutes to explore the two objects. The test was performed on day 3. One of the two identical cubes is replaced with a new object of different shape, size, color. Both objects were revealed again for 5 minutes for each mouse. The additional objects were washed with 70% ethanol between experiments with animals in order to prevent their movements from being induced by odor cues. All objects and locations are location specific or adjusted in a manner that reduces potential bias due to object tilt. The mouse cannot move the object and the subject is placed in a box always facing the same wall. The animal's actions were videotaped, and the following indices were calculated:

1) Identification index: the result varies between +1 and-1 by dividing the difference between the search times of the new and familiar objects by the value of the total time spent in the search of the two objects. A positive score indicates the time spent by a new object, a negative score indicates the time spent by a familiar object, and a score of 0 indicates a preference difference of 0.

2) Identification indexes are as follows: the time it takes for the animal to explore a new object. Ratio of total exploration time for two objects.

Moseri water maze test. The MWM test was used to investigate spatial learning and memory in experimental mice. The maze consists of a circular groove of stainless steel (diameter 210cm, height 51 cm) divided into four quadrants, up to 35cm in depth and filled with water (25. + -. 2 ℃). A blackened submersible platform (10 cm wide, 28cm high) is set within a target quadrant of 2cm below the water surface. The platform remains in a consistent position between training and testing. In order to make the platform invisible, a non-toxic dye for making water opaque purple is added. The memory acquisition attempts (120 seconds/attempt) are performed for 4 consecutive days, with at least a 15 minute interval between attempts, 1 day. At each attempt, the animal was free to find the platform hidden in the target quadrant. If the mouse finds a plateau, it is allowed to rest for an additional 20 seconds, and if within 120 seconds the animal does not yet reach the plateau it is gently induced to the plateau and maintained for 20 seconds. The average evasion latency is calculated as the time required for each mouse to find the hidden platform and used as an index for acquisition or learning. On day 5, mice were subjected to an exploratory experimental course. Thus, the platform was removed from the pool, allowing each mouse to explore the pool within 60 seconds. The time spent by each mouse in the subject quadrant with the hidden platform pre-placed is recorded as an index for retrieval or memory.

< treatment of brain >

After the behavioral test, mice were euthanized by cervical dislocation, brains were dissected rapidly, and washed with ice-cold physiological saline. Hippocampus was excised from each brain on ice-cold glass plates (n = 6). Hippocampus is homogenized in ice-cold physiological saline to prepare a 10% homogeneous solution, which is divided into several aliquots and stored at-80 deg.C. The other hippocampus was stored at-80 ℃ for western blot analysis.

< Biochemical assay >

Determination of biomarkers of oxidative stress and inflammation. Lipid peroxidation of hippocampus was postulated by measuring the level of Malondialdehyde (MDA). MDA was measured by measuring thiobarbituric acid-reactive substances according to the method described by neishan and sanyuan. Furthermore, the brain Glutathione (GSH) content was determined spectrophotometrically using Ellman's reagent according to the method described by Beutler et al. The results are expressed as Mmol/mg protein.

Enzyme immunoassay. Levels of TNF- α and IL-6 in the hippocampus were estimated using mouse ELISA kits purchased from RayBiotech inc (norcrots, georgia, usa) and R & D Systems inc (minneapolis, usa), respectively. The sequence is carried out as instructed by the manufacturer. The results are presented as pg/mg protein for both TNF- α and IL-6.

Western blot analysis. After extracting a protein solution from brain tissue, an equivalent amount of protein (20 to 30. Mu.g of total protein) was separated by SDS-PAGE (10% acrylamide gel), and a Bio-Rad Trans-Blot system transcribed to a polyvinylidene fluoride membrane (Pierce, rockford, IL, USA) was used. The Western blotting was performed by incubating the membrane for 1 hour at room temperature with a blocking solution containing 20mM Tris-Cl, pH 7.5, 150mM NaCl, 0.1 % Tween 20, and 3% bovine serum albumin. Membranes were incubated with one of the primary antibodies P-JAK2 (Tyr 1007/1008), P-STAT3 (Tyr 705), I κ B- α, GSK-3 β, mTOR, COX-2, β for one night at 4 ℃ with actin purchased from Thermo Fisher Scientific Inc. (Rockford, ill., U.S.A.). After washing, a peroxidase-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 hour. The band intensities were analyzed using a ChemiDoc imaging system equipped with Image LabTM Software Version 5.1 (Bio-Rad Laboratories, inc. Hercules, calif., USA). Results are expressed in arbitrary units after normalization to levels of β -actin.

And (4) determining the protein content. The protein content was determined according to Bradford (Bradford) method. All results are expressed as tissue concentration in mg per protein.

< statistical analysis >

Data are presented as mean ± standard error. Data were analyzed after one-way analysis of variance (ANOVA) using Tukey-Kramer multiple comparison test. Statistical analysis was performed using GraphPad Prism software (Version 6, graphPad software, inc. San diego, california, usa) and presented as a chart. The level of significance was set at p <0.05 in all statistical tests.

(results)

The effect of Antiia on the locomotion and biochemical functions of ICV-STZ treated mice was determined by neurolocomotion testing and biochemical analysis of hippocampal content. The effects of STZ and antis (25, 50 and 100 mg/kg) on neurobehavioral testing were performed within 24 hours from the final day of antis injection. The potential for protective effects of anti-treatment on ICV-STZ-injected mice was investigated using the Mosley water maze. As shown in fig. 2A of Mean Escape Latency (MEL), different groups of mice required different times for escape on day 2. Alzheimer's disease mice required 1.63 times the time to wean from day 2. On the other hand, alzheimer's disease mice for Antia only required 1.08 times the time at day 2 of shedding from control mice at day 2. These results were further confirmed on day 3 and day 4. In the quadrant targeted by the moslem's water maze, the study of the effect of anti on the time spent by mice (fig. 2B) revealed that the mice with alzheimer's disease consumed only 25.4% of the time in the quadrant compared to the control mice, while the mice with alzheimer's disease administered with 25, 50 and 100mg/kg of anti consumed 72.5%, 75.8% and 85.4% of the time in the quadrant compared to the control mice, respectively.

The effects of STZ and anti were further investigated by selection of new and different object identification tests and preference indicators. STZ induced a decrease in the recognition index of SAD mice when compared to the control group, and dose dependence appeared after anti administration (25, 50 and 100 mg/kg), with a significant increase compared to the STZ group. Furthermore, the time taken to search for new objects was reduced by 63% compared to the control group when injected into ICV-STZ mice. Normalization of the preference index for anti-dosing (25, 50 and 100 mg/kg) showed that anti-dosed mice were dose dependent and preferred for new subjects compared to familiar subjects (figure 2C).

To investigate the ability of anti to attenuate amylin formation, inflammation, autophagy, and oxidative stress pathways, several biochemical analyses of the content of the hippocampus of ICV-STZ-treated mice were performed. A study of the protective effect of anti-therapy on the hippocampal content of Glutathione (GSH) and Malondialdehyde (MDA) was conducted. The results in fig. 3A show that GSH levels in alzheimer's disease mice are 15.5% of the GSH levels in control mice. On the other hand, the Alzheimer's disease mice of Antia showed a dose-dependent increase in GSH content, with 78.7% of the control GSH level being maximal for 100mg/kg of Antia treatment. The results of the level of hippocampal content of MDA showed that mice injected with ICV-STZ had significantly higher MDA levels by 4.3-fold compared to control mice. On the other hand, in the case of the Alzheimer's disease mice with Antia, MDA content of the mice administered with Antia in amounts of 25, 50 and 100mg/kg was increased by 3.5 times, 2.5 times and 1.8 times, respectively (FIG. 3B).

The effect of ICV-STZ injection on the level of anti-inflammatory cytokines in the hippocampus was also investigated by the presence or absence of anti-treatment. Two cytokines, TNF-. Alpha.and IL-6 were investigated. The results in FIG. 4 show that STZ model mice showed significant increases in TNF- α and IL-6 cytokine expression compared to control mice, but that therapeutic dose-dependent inhibition of Antia achieved 100mg/kg of control levels. The same trend also occurs in the hippocampal content of NF-. Kappa.Bp 65. The results in FIG. 4 show that the level of NF- κ Bp65 in Alzheimer's mice increased and that the level of NF- κ Bp65 gradually decreased in Alzheimer's mice with Antia.

Since amyloid peptide β is usually a solid protein assembled into amyloid peptide-like filaments constituting plaques in alzheimer's disease, the effect of anti on the contents of the hippocampus of amyloid peptide β 1-42 in mice injected with ICV-STZ was examined. The results depicted in fig. 5 show that the expression of amylin β was increased about 4-fold in STZ model mice compared to control mice. Interestingly, the level of amylin β was significantly reduced in alzheimer's mice with anti. The effect is dose dependent, with 100mg/kg reaching a minimum level.

Further, protein expression was investigated. Phosphorylation levels of STAT and JAK protein expression are established methods used in the study of alzheimer's disease. Whether treatment with Antia inhibited phosphorylation of STAT expression in STZ mice was investigated. As expected, the phosphorylation levels of STAT protein expression were significantly reduced compared to control mice. However, the phosphorylation level of STAT3 was significantly inhibited after STZ mice were treated with anti (fig. 6A). The same results were also observed for the expression of JAK2 protein. By treatment with antisa, the phosphorylation level of JAK2 was greatly inhibited by STZ injection (fig. 6A). These results show the protective effect of Antia on the JAK2/STAT3 pathway.

In previous studies it has been shown that glycogen synthase kinase-3 (GSK-3) phosphorylates Tau proteins, the major component of neurofibrillary tangles. Inhibition of GSK-3a provides a new pathway to reduce the formation of both of the pathological features of alzheimer's disease, namely, starch peptide plaques and neurofibrillary tangles. The results in FIG. 6B show that the levels of GSK-3 β in Alzheimer's disease mice are expressed 7-fold higher than the levels of GSK-3 β in control mice. On the other hand, after the treatment of Antia, the expression of GSK-3 beta is severely inhibited, which is about 3 times that of the control. The results in FIG. 6B show that the IKB-alpha expression in Alzheimer's mice is about 6.5 times greater than the IKB-alpha level in control mice. On the other hand, after the treatment with Antia, the expression of IKB-alpha was severely inhibited, about 2.8 times that of the control.

Several studies have shown that mammalian rapamycin targeting (mTOR) has the potential to be associated with amyloid peptide beta as well as Tau-induced neurodegeneration. In previous studies, the level of mTOR phosphorylated by Ser2481 in medial lateral cephalic cortex was high in AD cases compared to control cases. The results in figure 6C show significant levels of increase in mTOR and p-AKT protein expression, 5-fold and 6-fold greater, respectively, in STZ-injected mice compared to the levels in control mice, but treatment with anti reversed the increase, approaching each other.

Finally, COX-2 is an important enzyme in the inflammatory process. The results in FIG. 6D show that Alzheimer's mice show significant induction of COX-2 expression. This was 600% of the COX-2 levels in control mice. However, treatment with Antia reduced COX-2 expression by a large 150% to 300%.

(discussing)

The results of this study show the ability of the antioxidant Antiia to exert a protective effect on SAD induced in mice. For a long time, the components of Antia have been shown to have a variety of neuroregenerative and protective properties. Hericium erinaceus shows synthesis of nerve growth factor. The centella asiatica extract reduces the levels of amyloid peptide beta in the brain of Alzheimer's disease in experimental animals. Diosgenin enhances cognitive ability in mice. Emblic leafflower fruit functions as a powerful antioxidant having a powerful neuroprotective effect and cognitive enhancing properties. In addition, salacia reticula protected the young diabetic rats from harmful cognitive changes and mercury toxicity in the mouse hippocampus. It is shown herein that Antia reduces cognitive dysfunction in a mouse model by targeting several linked pathways including amylin formation, inflammation, autophagy, and oxidative stress pathways.

In this study, induction of SAD by STZ mice induced a significant cognitive function reduction in the moseri water maze and the new foreign object recognition test. ICV injection of STZ is an experimental model that mimics the progressive pathology of SAD similar to the human brain. From the inability of mice to recognize familiar and new objects and to recognize xenobiotic recognition tasks in the moss water maze, STZ-treated mice showed significant learning and memory impairment. This is consistent with previous studies. However, by taking a large rise in escape latency in the trial, time spent in the target quadrant in the exploration trial of the moss water maze test, and an increase in the difference and preference index in the new foreign object recognition test, the antisa prevents spatial memory and short-term memory impairment caused by STZ. The improvement in memory deficit for object recognition may be due to previously proven effects of several components of Antia. For example, diosgenin has an anti-amyloidogenic effect, and spirulina shows a strong neuroprotective effect on neuronal loss and cognition in AD. Further, it was confirmed that orally administered powdered dried hericium erinaceus is effective in improving mild cognitive impairment in humans.

STZ administration showed a significant increase in expression of NF- κ B and the hippocampal content of the anti-inflammatory cytokines, i.e., TNF- α and IL-6. NF-kB plays an important role in the neuron inflammatory reaction induced by the transcription of inflammatory target genes such as COX-2, IL-1 beta, IL-6, TNF-alpha and the like. TNF- α is associated with systemic inflammation, in particular, not only with amyloid peptide formation regulated via β -secretase, but also with AD-associated brain neuritis. Further, severe neuropathological changes of parkinson's disease/alzheimer's disease and the like are associated with an increase in IL-6 expression in the brain. NF-. Kappa.B has also been shown to modulate the expression level of the rate-limiting enzyme associated with the production of amyloid peptide beta, namely BACE-1. The Janus Kinase/Signal Transducers and Activators and Transcription (JAK/STAT) signaling pathway was introduced in the 1980 s as a mediating pathway for interferon signaling. Neuroinflammation is associated with diseases, and activation of the JAK2/STAT3 pathway causes pathogenic inflammation. Therefore, targeting of the JAK2/STAT3 pathway can be used as a protective therapy for neuroinflammatory diseases such as AD and neurodegenerative diseases.

In this study, as shown, the levels of all inflammatory cytokines measured were reduced, the expression of phosphorylated STAT3 and JAK2 was severely inhibited, and administration of anti showed a significant anti-inflammatory effect. The STAT3/JAK2 pathway is linked to the production of TNF-. Alpha.. The apparent inhibition of TNF- α as well as NF-kB may be due to the action of Hericium erinaceus, which is known as Hericium erinaceus. This is the adhesion molecule as well as IL-6/TNF-. Alpha..

Neuroinflammation is associated with a deficiency in autophagy, which has the potential to contribute to neurodegeneration. Mammalian target of rapamycin (mTOR) is known to regulate autophagy in conjunction with protein kinase B (Akt). Several studies have highlighted the close association between mTOR signaling and the presence of amyloid peptide β plaques and cognitive impairment of AD. Furthermore, it is known that autophagy activation is linked to GSK-3 β inhibitors in studies in humans as well as rats in AD, which indeed contribute to the pathological accumulation of Tau aggregates.

Treatment with Antia reversed the expression of elevated levels of mTOR, akt, IKB- α, and GSK-3 β following STZ injection, approaching that of the control. It has been shown in recent reports that a significant improvement in cognitive function is caused by an increase in axonal density of neurons of diosgenin. This can be achieved by modulation of the PI3K-Akt pathway, which is known to regulate local protein translation via the mTOR pathway, playing an important role in axonal regeneration.

The results of this study showed that Antia increased GZ and decreased lipid peroxidation in STZ-treated mice. Previous studies have shown that the generation of ROS via self-aggregation of amyloid peptide β damages neurons, with the potential to cause neuronal death. Lipid peroxidation has been reported to be one of the main effects of free radical mediated damage that directly damages membranes, and increased lipid peroxidation in the brain of AD patients. Treatment of mice treated with STZ by Antia improved the oxidative stress index. This may be due to the known ability to reverse oxidative stress-induced mitochondrial dysfunction and apoptosis. Centella asiatica, which is widely known as centella asiatica, exhibits remarkable radical elimination properties, reduction of lipid peroxidation, and protection of DNA fragmentation caused by oxidative stress, and has been found to provide a plurality of mechanisms for changing the brain pathology of alzheimer's disease. In previous studies, treatment of the component of Antia increased brain levels of GSH, superoxide dismutase, catalase and glutathione peroxidase, suppressed nitrogen at the level of oxidative stress, and ferrate solution MRN-100 used for the inhibition of biomarkers of oxidative stress such as total free radicals showed beneficial antioxidant properties. GSH has the ability to protect against damage caused by ROS, and is an antioxidant with the potential to protect the oxidation of the oligomeric amylopeptin β, as well as the neurotoxic degeneration.

(conclusion)

From this study it can be concluded that Antia exerts a significant protective effect on sporadic AD induced by ICV injection of STZ. This effect is achieved by targeting amylin formation, inflammation and oxidative stress pathways. The JAK2/STAT3 pathway is regulated and induced by the regulation of Akt/mTOR/GSK-3 beta pathway to play a role in protecting neuroinflammation. This is the first example, as far as the applicant knows, for the purpose of pursuing the protective effect of anti on neurodegenerative diseases such as SAD.

Claims (6)

1. A composition comprises extracts of centella asiatica, phyllanthus emblica, salacia reticulata, and Hericium erinaceum, and diosgenin and a divalent ferric salt as effective ingredients.

2. The composition according to claim 1, for the prevention or treatment of cognitive dysfunction.

3. The composition according to claim 1 or 2, further comprising zinc as an active ingredient.

4. A capsule characterized by containing the composition according to any one of claims 1 to 3 as an content.

5. A capsule characterized by containing centella asiatica and other substances contained in the composition according to any one of claims 1 to 3 as a content by mixing the components in an oily mixture.

6. A tablet comprising the composition of any one of claims 1 to 3.

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