Use of menthol in the preparation of a topical composition for ameliorating neurodegenerative diseases and stroke

Abstract

The present invention provides a use of menthol or a menthol isomer for preparing an external composition for ameliorating neurodegenerative diseases or strokes caused by damage and degeneration of brain neurons or a lack of dopamine in the brain. The external composition is a product made of a patch, liquid, cream, oil, powder, gel, spray, or other combination to be applied to the skin, or a product for covering the limbs. The product for covering the limbs is a glove, foot cover, sock, or an extended portion or layer of clothing that ensures continuous contact of the menthol with the skin. The present invention also provides a use of menthol or a menthol isomer for preparing an external composition for ameliorating diseases or symptoms caused by damage and degeneration of brain neurons, a lack of dopamine in the brain, or a stroke.

Description

[Technical field]

[0001] The present invention belongs to the field of improving neurodegenerative diseases and stroke, and provides a use of a menthol composition for external application and clarifies the scope of its application. [Background technology]

[0002] Neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease are global problems in which neurons in the brain and spinal cord continuously degenerate or die over time, causing ataxia and mental dysfunction (dementia), and currently there is no cure or treatment to prevent neurodegenerative diseases. Parkinson's disease is a representative disease also known as paralysis agitans, and clinical tests show that the dopamine content in the brain is generally low, and there is a phenomenon in which two pigmented nuclei in the brain stem (substantia nigra and locus ceruleus) deteriorate. In addition, since the part of the substantia nigra that degenerates corresponds to a decrease in the dopamine content in the striatum, Parkinson's disease is thought to be caused by the deterioration of the dopamine pathway in which the substantia nigra compacta is projected to the striatum, and symptoms include 1) involuntary fibrillation at rest, 2) muscle rigidity, 3) difficulty initiating movement, and 4) behavioral retardation. Parkinson's disease cannot currently be cured, and symptoms are mainly alleviated by taking medication. However, medications often have side effects such as dyskinesia, and as the disease worsens, the amount of medication required must be increased, increasing the risk of developing psychiatric disorders.

[0003] Stroke is caused by the interruption or reduction of blood flow supply to the brain, and clinical stroke is divided into three main types: ischemic stroke, hemorrhagic stroke, and transient ischemic attack. Ischemic stroke accounts for more than 85% of all stroke incidence. When a stroke occurs, brain tissue does not receive enough oxygen and nutrients, which leads to further brain cell death, and local or global brain dysfunction progresses clinically rapidly, and if it persists for more than 24 hours, it may even lead to death. After the occurrence of a stroke, neuroinflammation of brain tissue is induced, causing further brain damage. Clinical therapeutic drugs that delay neuroinflammation are often used to prevent or alleviate post-stroke complications. Currently, the standard treatment for stroke is tissue-type plasminogen activator (tPA), but the system for evaluating whether it can be administered intravenously to patients is strict, and there is a high risk of causing intracranial hemorrhage.

[0004] Current patents relating to the field of the present invention are: Taiwan Patent Publication No. TW200848063 discloses a method and composition for treating neurodegenerative disorders, which uses an oral inhibitor to block the flow of ions through ion channels to treat neurodegenerative disorders. However, although the patent confirmed its efficacy through cell line ion channel experiments, it did not evaluate the behavioral improvement of each indication, and the clinical efficacy is unclear. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above, an object of the present invention is to provide an efficient, simple and side-effect-free method for ameliorating neurodegenerative diseases and stroke. [Means for solving the problem]

[0006] The present invention provides a use of menthol or a menthol isomer for the preparation of an external composition for ameliorating neurodegenerative diseases or stroke. The menthol isomers described herein are structural isomers or stereoisomers, and the weight percentage of the menthol or the menthol isomers is between 1% and 20%.

[0007] The topical compositions described herein are prepared as patches, liquids, creams, oils, powders, gels, sprays or other combinations that are applied to the skin or as extremity coverings, such as gloves, foot covers, socks, garment extensions or layers that ensure continuous contact of the menthol with the skin.

[0008] The neurodegenerative disease described herein is caused by damage degeneration of brain neurons or lack of dopamine in the brain, such as Parkinson's disease and Alzheimer's disease. The stroke is ischemic stroke. The external composition can be used to improve symptoms caused by damage degeneration of brain neurons, lack of dopamine or stroke, including but not limited to cognitive dysfunction, motor dysfunction, depression, limb weakness, hemiplegia, headache, dizziness, aphasia or slurred speech, blurred or impaired vision, palpitations, convulsions, nausea and vomiting, lethargy, salivation, difficulty swallowing, facial nerve disorder, and fecal incontinence.

[0009] The present invention also provides menthol or a menthol isomer for use in preparing an external composition for ameliorating diseases or symptoms caused by damage and degeneration of brain neurons, a lack of dopamine in the brain, or a stroke.

[0010] According to an embodiment of the present invention, in a mouse model of Parkinson's disease, it has been demonstrated that external application of menthol improves motor coordination in Parkinson's disease, enhances the content of dopamine in the brain, prevents damage to dopamine neurons in the substantia nigra, and thus has neuroprotective effects of enhancing the content of TH.In addition, according to another embodiment, it has been demonstrated that external application of menthol can reduce cerebral infarction volume and improve sensory-motor nerve damage in a mouse model of ischemic stroke for 7 days. Effect of the Invention

[0011] The present invention breaks through the barriers of conventional intractable neurodegenerative diseases and strokes and the severe side effects of drugs, and achieves the following beneficial effects: (1) It is an external preparation, so it is easy to use The present invention breaks through the conventional drug treatment forms for neurodegenerative diseases and strokes, eliminating the discomfort of injection or oral administration and the inconvenience of the need for regular administration or condition evaluation, and is the first external treatment form that can achieve its effect by applying, spraying, soaking or covering the skin, which is very convenient to use and not limited by location. (2) No side effects Current drugs for treating neurodegenerative diseases and strokes often have side effects, and require careful consideration of dosage and the use of other drugs to suppress side effects. However, menthol is safe to use and has no side effects, making it very flexible when combined with other drugs and preparations. (3) Simultaneously improves both neurodegeneration and stroke The new use of menthol provided by the present invention has been proven through rigorous and thorough animal testing to have neuroprotective effects, intracerebral dopamine enhancing effects, cerebral infarction improving effects, etc., that is, it can be applied externally to neurodegeneration and cerebral stroke, which are the causes of the above-mentioned diseases, and there is no discomfort or side effects caused by having to take various medicines for these diseases at once, so that the quality of life of patients can be improved. (4) It can be widely applied to improve illnesses. Menthol has been proven to have neuroprotective effects, increase dopamine in the brain, and improve cerebral infarction. Therefore, in addition to neurodegeneration and stroke, it is expected to have an improving effect on other diseases caused by the above-mentioned causes. It can be applied to many diseases and symptoms. Combined with the above-mentioned low side effects and advantages for external use, it has great potential for market development and high industrial application value. [Brief description of the drawings]

[0012] [Figure 1A] FIG. 1 is a diagram showing the experimental design of embodiment 1. [Figure 1B]FIG. 1 shows the time spent on the rotarod by each group of Parkinson's disease mice. [Figure 1C] FIG. 1 shows the striatal dopamine content in each group of Parkinson's disease mice. [Figure 1D] FIG. 1 shows the expression state of tyrosine hydroxylase (TH) in the substantia nigra of Parkinson's disease mice of each group. [Figure 2A] FIG. 1 shows the experimental design of embodiment 2. [Figure 2B-1] FIG. 1 shows the residence time on the rotarod of each group of ischemic stroke mice. [Figure 2B-2] FIG. 1 shows the residence time on the rotarod of each group of ischemic stroke mice. [Figure 2C-1] FIG. 13 shows the time it took for each group of ischemic stroke mice to find the tape. [Figure 2C-2] FIG. 13 shows the time when each group of ischemic stroke mice removed the tape. [Figure 2D-1] FIG. 1 shows the quantified cerebral infarction volume in each group of ischemic stroke mice. [Figure 2D-2] FIG. 1 shows samples of ischemic stroke mice from each group stained with brain TTC (2,3,5-triphenyltetrazolium chloride). [Figure 3A] FIG. 1 shows a glove, one of the products for which the menthol topical composition can be used. [Figure 3B] FIG. 1 shows a second product that can be made using the menthol topical composition, a foot cover. [Figure 4A] This is a diagram showing an experimental design in which mice with ischemic stroke were administered menthol orally or immersed in the limbs or backs. [Figure 4B-1] FIG. 1 shows the residence time on the rotarod of each group of ischemic stroke mice. [Figure 4B-2] FIG. 1 shows the residence time on the rotarod of each group of ischemic stroke mice. [Figure 4C-1]FIG. 13 shows the time it took for each group of ischemic stroke mice to find the tape. [Figure 4C-2] FIG. 13 shows the time it took for each group of ischemic stroke mice to find the tape. [Figure 4D-1] FIG. 1 shows the quantified cerebral infarction volume in each group of ischemic stroke mice. [Figure 4D-2] FIG. 1 shows the quantified cerebral infarction volume in each group of ischemic stroke mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] All technical and scientific terms used herein have the meanings shared by experts in the field unless otherwise defined. The term "MPTP" used herein means 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is a Parkinson's neurotoxin that destroys dopamine-producing neurons in the substantia nigra (SN) in the brain. Those skilled in the art will create an animal model of Parkinson's disease mice by injecting MPTP into the mouse body. The full name of the term "LC-MS / MS" is liquid chromatography-tandem mass spectrometry. The full name of the term "TH" is tyrosine hydroxylase, which is an enzyme required to produce dopamine in cells. The term "substantia nigra" (SN) is a group of nerve nuclei in the midbrain, which has cells containing TH, which produce dopamine, and whose apoptosis leads to Parkinson's disease. The term "MCAO" is Middle Cerebral Artery Occlusion, a technique used to build an animal model of ischemic stroke. The full name of the term "TTC" is 2,3,5-triphenyltetrazolium chloride, which is an oxidation-reduction indicator that is reduced to the red compound TPF (1,3,5-triphenylformazan) by dehydrogenases in viable cells (especially succinate dehydrogenase in mitochondria). After tissue necrosis, the dehydrogenase activity is lost and TTC cannot be reduced, so the necrotic tissue appears pale white and the normal tissue appears deep red. TTC can be used as a stain to detect ischemic infarcted tissue. The following is a description of specific embodiments to further illustrate the technical features and effects of the present invention.

[0014] Embodiment 1 Improvement of Parkinson's disease with external menthol

[0015] 1. Experimental design and construction of a mouse model of Parkinson's disease

[0016] The experimental mice were divided into three groups: a control group (normal mice), an MPTP group (a disease model of Parkinson's mice constructed by MPTP), and an MPTP+8%ME group (a disease model of Parkinson's mice constructed by MPTP + immersion of the front paws in 8% menthol). The experimental schedule is shown in Figure 1A. For administration of menthol, the two front paws of the MPTP+8%ME group mice were immersed in a menthol solution with a weight percentage concentration of 8% for 10 seconds every day on days 1 to 5 and days 8 to 10. For construction of the disease model of Parkinson's disease mice, MPTP was administered daily at 10 mg / kg (day) to the MPTP group and MPTP+8%ME mice by intraperitoneal injection (intraperitoneal, ip) on days 3 to 5, and the animal model of Parkinson's disease mice was completed. Finally, on the 10th day, an exercise endurance test, dopamine content measurement, and immunohistochemical staining were performed to observe the symptom-improving effect of menthol on Parkinson's disease.

[0017] 2. Exercise endurance test - Rotarod test

[0018] The mice in each group were measured on the first and tenth days by the rotarod test. On the automatic accelerating rotarod, the rotarod speed was started at 4 rpm (revolutions per minute), the rotarod continued to accelerate, and reached a maximum rotarod speed of 40 rpm for about 5 minutes, after which the test was continued at the maximum rotarod speed of 40 rpm, and the mouse's dwell time on the rotarod was calculated (Figure 1B). The ΔLatency to fall shown on the vertical axis in the figure indicates the subtraction of the dwell time on the first day from the dwell time on the 10th day. As a result, the dwell time of the MPTP group was significantly shorter in the test on the 10th day compared to the first day, and the mice showed symptoms of decreased motor coordination in Parkinson's disease. Similar to Parkinson's disease, in the MPTP+8%ME group soaked in menthol, the number of seconds the mice stayed in the dwell time was longer than on the first day, indicating that dwelling in menthol can recover from the decrease in motor coordination caused by Parkinson's disease, and even recover to the same level as normal mice (control group).

[0019] 3. Measurement of Dopamine Content

[0020] To measure the dopamine content in the brain, the mice were sacrificed after the completion of the rotarod test, their brains were removed, and the dopamine content in the striatum of the mice was measured by LC-MS / MS (Figure 1C). As a result, the dopamine content in the brain of the MPTP group was significantly decreased on the 10th day, and typical symptoms of Parkinson's disease were observed. In the MPTP+8%ME group, the dopamine content was equivalent to that of the control group, indicating that menthol can restore the symptoms of decreased dopamine content in the brain of Parkinson's disease mice.

[0021] 4. Immunohistochemical (IHC) staining

[0022] Subsequently, IHC was used to detect the TH content in the substantia nigra region of the mouse brain (Figure 1D). The results showed that compared with the typical decline in TH content of Parkinson's disease in the MPTP group, the TH content of the MPTP + 8% ME group was restored to the same level as the control group. Mice with Parkinson's disease contain TH, and dopamine-producing nerve cells are destroyed. The results of this part indicate that menthol has a neuroprotective effect and avoids the decline of dopamine.

[0023] The first part of the Parkinson's disease trial showed that topical menthol improved motor disability caused by Parkinson's disease, restored brain dopamine content, and had neuroprotective effects, and could therefore be used as a pathway to improve neurodegenerative diseases.

[0024] Embodiment 2 Improvement of stroke with external menthol

[0025] 1. Experimental Design and Construction of the Ischemic Stroke Mouse Disease Model

[0026] Mice were divided into four groups, namely, control group (mice with ischemic stroke constructed by MCAO treatment), water group (mice with ischemic stroke constructed by MCAO treatment + immersion of limbs in water), MCAO + 8% ME group (mice with ischemic stroke constructed by MCAO treatment + immersion of limbs in 8% menthol), and MCAO + 16% ME group (mice with ischemic stroke constructed by MCAO treatment + immersion of limbs in 16% menthol). All menthol was prepared with 95% ethanol. The experimental schedule is shown in Figure 2A, and mice were sacrificed for follow-up analysis on the 7th day. Mice underwent MCAO surgery on day 0 and developed ischemic stroke. The first dose of menthol was administered once a day after MCAO until day 6. On the day of MCAO treatment, mice were immersed in menthol for 1 minute 20 minutes after MCAO surgery. From the second to sixth day, the mice were immersed in menthol for only 15 seconds. The water group served as a control and was immersed in water for the same period. Sensory-motor nerve function tests were performed one day before MCAO treatment (indicated as "pre-MCAO") and on the first, third, and sixth days after MCAO treatment, and cerebral infarction volume was measured on the seventh day after MCAO treatment to observe the ameliorative effect of menthol on stroke.

[0027] 2. Sensorimotor Nerve Function Testing

[0028] 2.1 Rotarod test

[0029] The rotarod test was performed on the 3rd and 6th days of each group on an automatic accelerating rotarod (4-40 rpm) to measure the mouse residence time (Figure 2B-1, Figure 2B-2). The results of the day were taken as the standard for pre-MCAO, and each group was tested three times. One-way ANOVA was used for multiple comparison analysis (Dunnett's test), and the statistical differences between groups were (*p<0.05). Asterisks * indicate significant differences between the control group, water group, and MCAO+8%ME group (*p<0.05; **p<0.01). As a result, the residence time of the MCAO group was significantly shortened on the 3rd and 6th days, confirming the symptoms of sensorimotor impairment due to ischemic stroke. Meanwhile, the residence time of the mice treated with menthol was prolonged compared to the MCAO group and the water group, indicating that menthol improves the function of sensorimotor neurons in stroke mice.

[0030] 2.2 Adhesive removal test

[0031] The mice were subjected to the tape removal test on the first, third, and sixth days, and the results on the day of pre-MCAO were used as the standard. The experimental procedure was as follows: first, before placing the mice in the test cage, a 4 (mm) x 4 (mm) tape was attached to the front paws of the mice, and the time it took the mice to find and remove the tape was measured (Figure 2C-1, Figure 2C-2). Statistical analysis was also performed using the method described in 2.1 (*p<0.05; **p<0.01; ***p<0.001). The statistical analysis between groups showed that the mice administered menthol had a significantly shorter tape removal time than the control group and water group, and on the sixth day, the time they found the tape was significantly shorter than the water group, and their reaction speed was significantly better, indicating that administration of menthol can improve sensorimotor dysfunction caused by stroke.

[0032] 3. Measurement of cerebral infarct volume

[0033] One week after MCAO, the mice in each group were sacrificed by perfusion, and brain specimens were presented for vital TTC staining (Figure 2D-2) to evaluate the cerebral infarct volume (mm 3) was measured (Figure 2D-1). In addition, the measured volumes were subjected to one-way ANOVA with multiple comparison analysis (Tukey's test), and the statistical differences between groups were (*p<0.05). The results showed that the use of menthol significantly reduced cerebral infarction volume compared to the control group and water group.

[0034] The present invention shows, in a Parkinson's disease mouse model, that external application of menthol improves the motor coordination ability of Parkinson's disease, increases the content of dopamine in the brain, and has a neuroprotective effect of preventing the damage of dopamine neurons in the substantia nigra, thereby increasing the content of TH.The present invention shows, in a Parkinson's disease mouse model, that external application of menthol can reduce the cerebral infarction volume within 7 days and improve the sensory-motor nerve deficit.

[0035] The present invention has revealed that Parkinson's disease and stroke can be improved by administering menthol as an external preparation.In addition, in this method, menthol has the effects of increasing intracerebral dopamine, protecting neurons, and reducing cerebral infarct volume, and therefore has a neuroprotective effect, and can be used to improve other neurodegenerative diseases such as Alzheimer's.Menthol isomers and compounds similar thereto are sufficient for those skilled in the art to assume that they have similar effectiveness, and are included in the scope of the claims of the present invention.

[0036] As is evident from the embodiments of the present invention, menthol is prepared as an external composition that can be used to improve neurodegenerative diseases or stroke, and can be used to improve symptoms caused by brain neuron damage degeneration, dopamine deficiency or stroke. The symptoms include, but are not limited to, cognitive dysfunction, motor dysfunction, depression, limb weakness, hemiplegia, headache, dizziness, aphasia or slurred speech, blurred or impaired vision, palpitations, convulsions, nausea and vomiting, lethargy, salivation, difficulty swallowing, facial nerve disorder, and fecal incontinence. According to the embodiments of the present invention, the effective range of the percentage of menthol is 1% to 20% when the external composition is administered to the human body by converting it into different animal models.

[0037] The topical composition may contain a pharma- ceutically acceptable adjuvant, diluent, or carrier. Due to the cooling effect of menthol itself, after adding an active ingredient for cosmetics or skin care, it can be made into a topical composition that has uses such as disease improvement, cooling sensation, and skin care and cosmetics. The topical composition may be a patch, liquid, cream, oil, powder, gel, spray, or other combination product applied to the skin, or a product for covering the extremities. The product for covering the extremities may be a glove (FIG. 3A), a foot cover (FIG. 3B), a sock, an extension design part of clothing, or a layered product, which can ensure continuous contact between the skin and menthol. The examples of FIG. 3A and FIG. 3B are one of the possibilities for implementing the present invention, and any implementation, conversion, change of the covering material, or modification of the appearance that does not depart from the above concept is included in the scope of the claims of the present invention.

[0038] 4. Efficacy differences between topical and oral forms

[0039] In this experiment, mice were divided into five groups: water group (ischemic stroke mice constructed by MCAO treatment + limb immersion treatment with water), 8% ME group (ischemic stroke mice constructed by MCAO treatment + limb immersion treatment with 8% menthol), 16% ME group (ischemic stroke mice constructed by MCAO treatment + limb immersion treatment with 16% menthol), back ME group (ischemic stroke mice constructed by MCAO treatment + back immersion treatment with Mintol), and oral ME group (ischemic stroke mice constructed by MCAO treatment + oral administration of menthol). All menthol was treated with 95% ethanol. For the experimental design and data analysis method, please refer to the present specification, and for the experimental schedule, please refer to Figure 4A. Mice were sacrificed for follow-up analysis on the 7th day. As shown in Figure 4B-1 and Figure 4B-2, the measurement results were subjected to one-way ANOVA with multiple comparison analysis (Duncan's method), and the statistical differences between groups were (p<0.05, *: compared with water group, †: compared with 16% ME group, △: compared with 8% ME group). In the rotarod test, the groups such as external application to the back or oral administration had no effect on improving the residence time, and the groups of mice whose limbs were immersed in menthol had a longer residence time than the groups such as back immersion, water immersion, or oral administration, indicating that external menthol applied to the limbs can improve the function of sensory motor neurons in stroke mice. In the tape removal test, the test results showed that the limbs were immersed in menthol, and ... The group of mice immersed in menthol had significantly less time to find the tape than the other groups, and their reaction speed was significantly better, indicating that menthol in the form of topical application can improve the sensory-motor dysfunction caused by stroke. In comparison, the method of topical application to the back or oral administration did not have the above effect. In the test to measure the cerebral infarction volume, the test results shown in Figures 4D-1 and 4D-2 showed that the group of mice whose limbs were immersed in menthol could significantly reduce the cerebral infarction volume. Meanwhile, the groups immersed in back, water, or oral administration did not have the effect of reducing the cerebral infarction volume.Therefore, by applying the menthol of the present invention to the limbs in an external form, it is possible to improve the sensory motor neuron function and motor dysfunction of stroke mice and reduce the cerebral infarction volume. However, when applied externally to the back or orally, it does not have a significant effect of improving ischemic stroke symptoms, so it is shown that application by a special route, that is, an external form, is necessary to achieve the effects of the present invention.

[0040] The present invention further provides an external composition for improving the diseases or symptoms caused by brain neuron damage degeneration, brain dopamine deficiency, or stroke.The concept of applying menthol or menthol isomers in an external manner to improve the diseases and symptoms described in the present invention are all considered as equivalent implementations and are included in the scope of the claims declared by the present invention.

[0041] The above embodiments are intended to explain the technical ideas and features of the present invention, and the purpose is to enable those skilled in the art to understand and implement the contents of the present invention, and are not intended to limit the scope of the claims of the present invention. In other words, any equivalent changes or modifications made according to the spirit disclosed by the present invention should be included in the scope of the claims of the present invention.

Claims

1. 1. Use of menthol in the preparation of an external composition for improving Parkinson's disease or stroke, comprising: The topical composition is applied to an extremity, The weight percentage of the menthol in the topical composition is 1% to 20%.

2. The use according to claim 1 ,

2. The frequency of administration of the topical composition is 1 to 2 times a day.

2. The use according to claim 1 .

3. The use according to claim 1, characterized in that the topical composition is a patch, a liquid, a cream, an oil, a powder, a gel, a spray or a product for coating the limb, which is applied to the skin.

4. The limb covering product is a glove, a foot cover, a sock, an extension or layer of clothing.

4. The use according to claim 3.

5. The extremity coating product ensures continuous contact of menthol with the skin.

4. The use according to claim 3.

6. The stroke is an ischemic stroke.

6. Use according to claim 1 or 5.

7. The topical composition is used to improve symptoms caused by damage and degeneration of brain neurons, dopamine deficiency, or stroke, including cognitive dysfunction, motor dysfunction, depression, limb weakness, hemiplegia, headache, dizziness, aphasia or slurred speech, blurred or impaired vision, palpitations, convulsions, nausea and vomiting, lethargy, salivation, difficulty swallowing, facial nerve disorder, and fecal incontinence.

2. The use according to claim 1 .

8. 1. Use of menthol in the preparation of an external composition to be applied to the extremities to ameliorate disease or symptoms caused by Parkinson's disease.

9. 1. Use of menthol in the preparation of an external composition to be applied to a limb to ameliorate disease or symptoms resulting from stroke.

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