TW201440765A - Composition for reducing D-galactose induced cranial neuron damage - Google Patents

Abstract

The present invention provides a composition for reducing D-galactose induced cranial neuron damage, which at least contains ergothioneine (EGT). The applied ratio is that the amount of EGT applied for each kg of individual is less than 2 mg. Preferably, the composition and the melatonin may have the synergetic effect, and the applied ratio is that the amount of melatonin applied for each kg of individual is less than 10 mg. Preferably, the applied ratio of EGT in the composition is that the amount of EGT applied for each kg of individual is greater than 0.5 mg. In summary, the present invention utilizes EGT to reduce D-galactose induced cranial neuron damage and achieves the purpose of cranial neuron protection.

Description

A composition for reducing damage of brain nerve cells induced by D-galactose

The present invention provides an evaluation of ergothioneine (EGT) to reduce the ability of DG to induce damage to brain nerve cells, and in particular to a composition which can improve the damage caused by DG-induced brain nerve cells such as Alzheimer's disease.

The brain is a nervous system control center composed of numerous nerve cells, which controls the normal operation of internal organs and organs, the response to pain, touch, temperature, pressure, etc., the coordination of external behaviors, and such as cognition and affection. Mental activities such as memory and learning. However, various parts of the body are distributed with various nervous systems. The nervous systems are composed of many nerve cells and are directly or indirectly connected to the brain. The brain message or the message is sent to the brain, so that the body can complete the behaviors and actions required for daily life. The normal operation of the brain depends on the communication between the nerve cells to achieve the purpose of completing mental activities, such as thinking and memory. Learning, normal nerve cells connect with synapses and transmit messages.

However, with aging or oxide accumulation, the nerve cells in the brain gradually lose function or even die, causing memory and other brain functions to decline, causing behavioral and mental problems such as anxiety, agitation, insomnia and hallucinations. Because the brain's nerve cells are responsible for thinking, remembering, and acting, as time goes on, aging and oxidation are intensifying, and even the most basic daily life, such as brushing, dressing, and bathing, may be lost.

The pathological hallmark of Alzheimer's disease is the neurofibrillary tangle (NFT), the senile plaques and the severe loss of neuronal cells in the cerebral cortex, which is a kind of neurodegenerative diseases; One of the main reasons why nerve cells can not function properly or lose their function is the occurrence of senile plaques. The plaques are composed of β-amvloid peptide (Aβ), which accumulate outside the nerve cells and increase with age. , the content of senile plaques will also increase, and the amyloid deposition will also As a result, amyloid protein can be used as an indicator of aging, and the proportion of Aβ-42/Aβ-40 in the brain of Alzheimer's patients will increase, so Aβ-42 excess has been considered to cause Alzheimer's disease. One of the reasons is also considered to be a more toxic Aβ form, which is cytotoxic in neural cell culture and its cytotoxicity depends on its length and degree of polymerization. In recent years, many scientific studies have clearly pointed out that the main cause of memory loss in Alzheimer's patients is the neurotransmitter of hippocampal neurons in the brain--the brain message substance called acetylcholine. A large number of studies have shown that Aβ antagonists increase the concentration of acetylcholine in hippocampal neurons of 12-month-old mice, so it is known that the formation of Aβ should be the main cause of the decrease in acetylcholine concentration in the brain. Studies have found that acetylcholinesterase is also involved in the partial decomposition of acetylcholine. Therefore, the main therapeutic agent for Alzheimer's disease is to use an inhibitor of acetylcholinesterase to increase the concentration of acetylcholine in the brain. And function to achieve improved performance.

In the existing mechanism, there is no specific biochemical indicator for the diagnosis of Alzheimer's disease. It is still necessary to rely on brain dissection or section to observe whether there are a large number of depositions of amyloid plaques and neurofibrillary tangles, and there is no specific drug. Radical. The current drugs to improve patient intelligence are mainly acetylcholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor inhibitors (to prevent the stimulating toxicity of glutamate). Currently, the US FDA is legally used to treat Alzheimer's disease. There are four kinds of drugs: Tacrine, Donepezil, Rivastigmine and Memantine. The first three are acetylcholinesterase inhibitors, and the last one is non-competitive NMDA receptors. , will cause some side effects. These drugs do not completely cure Alzheimer's disease, and not every patient has an effect. In clinical trials, only about one-third to one-half of the patients have an effect, and most of the effects are stable or slow the progression of the disease. Not to return the patient to a normal condition. Although the drug for the treatment of Alzheimer's disease has made great progress, it is still in symptomatic treatment with limited effect.

Oxidative stress can lead to the decomposition of antioxidants such as glutathione (GSH) and vitamin E in the body. Studies have shown that plasma antioxidants in patients with Alzheimer's disease are significantly reduced, so oxidative stress is also considered to cause Alzheimer's disease. An important reason for the formation. In addition, Aβ also causes neuronal apoptosis and synapse reduction, and its pathological mechanism of inducing neuronal apoptosis to induce Alzheimer's disease is thought to be related to its increased oxidative stress, ie, promoting free radicals (eg: O2 -), formation of reactive oxygen species (eg H2O2) and lipid peroxides (eg 4-hydroxynonenal (HNE), etc. It is known that reactive oxygen species (nitrogen) and its products reactive with proteins and lipids (such as Protein carbonyl) induce neuronal apoptosis by activating protein kinase. The mechanism of Aβ-induced neuronal apoptosis is: Aβ induces H2O2 and HNE production, which activates SAPKS (stress-activated protein kinases), and then SAPKS reactivates p53 (an apoptotic transcription factor), and finally triggers related pro-apoptotic factors. (Promotes apoptotic factors), the granulosa gland releases cyochrome c (cytochrome c), promotes caspase-3 activation (serine degrading enzyme) and decomposes poly ADP-ribose polymerase (PARP) to promote apoptosis.

In recent years, therapeutic drugs for Alzheimer's disease have begun to focus on the development of antioxidant drugs, because many literatures indicate that neurodegeneration and cognitive impairment in Alzheimer's disease are related to oxidative stress. There are two ways to avoid the oxidative stress caused by ROS in brain tissue and cells. One is to induce the elevation of antioxidant enzymes in brain tissue and cells, such as glutathione peroxidase (GPx), hemeoxygenase (HO-1) and Mn-. Superoxidase (Mn-SOD), etc., to reduce intracellular ROS production to reduce cytotoxicity; the other is to regulate apoptosis pathways such as inhibition of mitogen-activated protein kinase (MAPKs) to reduce apoptosis, important MAPK signaling cascades Including cellular regulated kinase (ERK), c-jun amino-terminal kinase (JNK) and p38 MAPK.

There are four conditions for the treatment of Alzheimer's disease. One must be able to be absorbed and utilized in the living body, the other must pass the brain-blood barrier (BBB), and the third must not have strong side effects. Finally, it must be synergistic with other drugs or ingredients to reduce side effects. In the existing research, it is known that ergot sulfur EGT has the ability to freely pass through the BBB, enter the central nervous system, and has EGT transporter-organic cation transporter-1 (OCTN1) on the cell membrane, so it can freely enter and exit cells, and if the cell lacks OCTN1, It is impossible to accumulate and absorb EGT in the body (Gründemann, 2012). Furthermore, there is currently a literature on the side effects of EGT, so EGT has considerable potential to be a health product for preventing Alzheimer's disease.

Melatonin is an indolamine compound that is an endogenous antioxidant with good antioxidant capacity. There are many advantages to using melatonin. First, it is soluble in both polar and non-polar systems, so it is easy to enter cells. Secondly, melatonin can pass through the blood-brain barrier and enter the central nervous system. Studies have shown that the content of Melatonin in the cerebrospinal fluid of Alzheimer's patients is significantly reduced; Melatonin has inhibition. Aβ induces apoptosis of neuronal cells such as PC12 and C6. The results of the experiment showed that 10 mg/kg of Melatonin and four-month-old transgenic mice, TBARS content and pro-apoptotic proteins such as Bax and caspase-3 were continuously administered. The performance of proteins such as Par-4 was significantly reduced, and the content of Glutathione (GSH) was significantly increased.

D-galactose (DG) is a physiologically available nutrient, but when excessive DG is ingested, it causes abnormal metabolism of non-enzymatic glycation and produces a large amount of active oxygen, which destroys the body's resistance. The equilibrium state of oxidation/oxidation causes peroxidation damage to cells. Injecting DG into rodents for 6-10 weeks will cause the animal to progressively reduce learning ability and memory, and produce higher amounts of free radicals in the brain tissue, leading to oxide accumulation, and then induce Aβ formation, resulting in oxidative stress. In addition, it inhibits and blocks the function of communication and message transmission between nerve cells, resulting in functional impairment of the brain, deteriorating learning and memory, and serious or even loss of life. Therefore, this study used DG subcutaneous injection (s.c) to induce mouse aging patterns to induce aging in mice.

The present invention is a composition for reducing D-galactose-induced brain nerve cell damage, the composition comprising at least ergothione, administered in a ratio of less than 2 mg per gram of ergothione. Preferably, wherein the composition is synergistic with melatonin in a ratio of less than 10 mg of melatonin administered per kg of individual. Preferably, the ergot sulfur of the composition is applied in a ratio of greater than 0.5 mg per gram of individual ergothione.

As described above, ergothione is utilized to reduce brain damage caused by D-galactose and to protect nerve cells.

Figure 1 is a graph showing the body weight trend of mice in the present invention after injection of DG.

Fig. 2 is a trend diagram and path diagram of the avoidance reaction time of the mouse of the present invention in the active avoidance test.

Figure 3 is a graph showing the number and area of A[beta] plaque formation induced by DG in mice of the present invention.

Figure 4 is a bar graph of the results of mice infected with DG caused by brain nerve injury.

Based on the study of ergothione, the present invention is to further investigate whether the administration of ergothione and melatonin can improve the learning and memory ability of aging mice induced by DG. The present invention is to further study the effective dose and synergistic ratio of ergothione and melatonin to improve the learning and memory ability of aged mice.

The present invention provides a composition for reducing damage of brain nerve cells induced by D-galactose, the composition comprising at least ergothione or melatonin, wherein ergot sulfur can be absorbed and utilized in living organisms, and passes through blood. The characteristics of the brain barrier and no side effects, so the use of ergot sulfur in combination with other antioxidants (such as melatonin) can help improve brain damage caused by DG in mice. Ergosine has a proprietary transporter (OCTN1), so it can enter the blood-brain barrier in the brain, promote the increase of antioxidant content in the brain, reduce the plaques induced by DG, and reduce the damage of DG on mouse nerve cells. Ergostin has a fairly good effect on preventing or improving the learning and memory damage caused by aging of DG-induced mice.

In the present invention, 0.5 mg of ergothione is administered per kg of the individual, and the administration method is mainly by oral administration, and the ergothione extracted from the mushroom waste can be administered to the individual in any manner. And applied to the corresponding amount of ergothione according to the weight of the individual, such as: the extract is directly supplied to the individual for consumption, or processed and then applied to the individual, and the processing method can be: A tablet is prepared by using an appropriate amount of ergothione for individual use or addition to other health foods, or synergistically with other drugs or ingredients. In the examples, the ergothionin and melatonin are mainly used in synergy with each other, and the above individuals may be humans that are healthy or have been induced by DG (saccharification) to cause nerve cell damage; the following examples are based on mice. The subject, more specifically, a mouse which was induced by DG to cause nerve injury was used as an experimental object to verify the effect of the composition of the present invention.

The following is an example to illustrate the efficacy of ergothione in a mouse model of brain injury induced by DG in mice, and it has been experimentally confirmed that ergothione can improve DG on mouse brain. The damage caused by nerve cells and the ergot sulfur and melatonin can be used synergistically and multiply.

In the present invention, male 16-week-old mice are used as experimental animals, and the mice are grouped in two experiments. The number of mice in each group is 10, and the food and water of the mice are self-study. From feeding, the mice will be fed standard feed (Purina chow) during the test, the temperature of the animal room is controlled at 22±2 ° C, the illumination time is controlled by an automatic timer, and the cycle is 12 hours, the relative humidity is 50-70%, in the present invention. The mouse strains used in each group except the blank group may be mice that have been injured by Aβ-type amyloid-induced nerve injury, or the following surgery is performed on healthy mice for use in the experimental group. .

Implementation design:

Male 16-week-old C57BL/6 strain mice (National Animal Center) were used as experimental animals (10 mice per group). The food and water of the mice were free to eat. During the test, the mice were fed standard feed (Purina chow). # 5001). Further determine the dose of ergot sulfur, the test environment for the animal room air conditioning temperature is controlled at 22 ± 2 ° C, the light time is controlled by an automatic timer, 12 hours cycle, relative humidity 50 ~ 70%. The group design is shown in Table 1.1, and the experimental design is shown in Table 1.2.

86 re-cognitive test. : Active learning trials; long-term memory is performed every seven days (64th, 71st, and 78th days), and short-term memory is 64 to 66 for three consecutive days, once a day.

Protective effect of EGT on learning and memory impairment induced by DG in mice:

Referring to Figure 1, after the second week of injection of DG, the activity of the mice was significantly lower than that of the blank group, and the body gradually showed a downward trend in weight, but the body weight of the mice fed the melatonin group and the EGT group was the weight of the control group. There was no significant difference compared to the comparison.

Please refer to Figure 2, the active avoidance test is conducted for three consecutive days after Melatonin and EGT administration (two weeks after DG injection). The higher the number of successful avoidance reactions and the shorter the active avoidance time, the more learning and memory ability of mice. good. On the first day, there was no difference in the time of each group's avoidance (about 8.5-9.5 seconds), and the time of active avoidance began to decrease significantly on the second day. Among them, C57BL/6 mice fed the low-dose EGT group and the melatonin group. The most significant decrease (about 7 seconds) was not significantly different from the blank control group (p>0.05), but the active avoidance time of the EGT high dose group was higher than that of the positive control group (Aβ induction group) (about 8.7 seconds). However, on the third day, the EGT high-dose group began to gradually reduce the time of active avoidance (about 8.2 seconds), while the active avoidance time of the DG-induced group did not show a downward trend, indicating that mice fed ergot sulfur had more The ability to learn and remember, so in the active avoidance learning test and Mohs water maze test, it was found that whether feeding ergot sulfur or melatonin did improve the effect of mice injured by DG induced nerve injury and can improve Learning and memory of aging mice.

Referring to Figure 3, it was found in immunohistochemical staining analysis that mice fed ergothione significantly reduced the number and area of Aβ plaque formation induced by DG. Figure 4 also shows the ergot sulfur and ergot fed. Sulfur and melatonin do improve the effects of brain-induced nerve injury in mice induced by DG. It can also be found that ergot and melatonin can be used synergistically without mutual impairment; Figure 4 shows The ergot thiosis content and ectopic sulphur receptor (OCTN1) in ergothiosis mice in brain tissue were significantly higher than those in the control group (P<0.05), indicating that ergot sulfur can enter the brain. Inside BBB.

In addition, referring to Table 2, feeding ergot sulfur and melatonin on the regulation of acetylcholine activity in the brain of aging mice and oxidative stress (eg, reducing lipid peroxide, increasing SOD enzyme activity and GSH/GSSG ratio) The reduction also has a significant improvement effect, thus protecting the mouse brain Nerve cells should have considerable effects.

Claims (3)

A composition for reducing D-galactose-induced damage to brain nerve cells, the composition comprising at least ergothione, administered in a ratio of less than 2 mg per gram of ergothione. A composition for reducing D-galactose-induced brain nerve cell injury according to the first aspect of the patent application, wherein the composition has synergistic effect with melatonin, and the ratio of administration is less than 10 mg per meginal administration of melatonin. . The composition for reducing the D-galactose-induced brain nerve cell injury according to the first aspect of the patent application, wherein the composition of the ergot sulfur is applied in a ratio of more than 0.5 mg per gram of individual ergothione.