TWI276439B - Application of sesamin and sesamolin on stroke prevention and protection from nervous degeneration - Google Patents

Abstract

This invention relates to provide pharmaceutical application of sesamin and sesamolin on stroke prevention and protection from nervous degeneration and medical composition containing sesamin and/or sesamolin.

Description

—1276439 · V. INSTRUCTIONS (1) I, [Background of the Invention] 'Cerebrovascular disease currently accounts for the second highest mortality rate in the Bay, even if the stroke is mild, it causes the patient and family to be extremely negatively economically and mentally. The main cause of stroke is the brain tissue ischemia caused by embolization, which causes local neurons to be hypoxic and die due to ischemia. At present, we have learned that the pathological mechanism of partial central nervous injury death is cerebral ischemia/hypoxia, the energy supply system of neurons is disturbed, and a large amount of neurotransmitter (glutamate) is released, and the device is activated by activating NMDA. Affects the calcium ion constant system, producing free radicals and nitric oxide, causing secondary damage to the cells. In the last few years, it has been found that free radicals produced by oxidation are also important causes of cell death, and many antioxidant genes and reaction mechanisms have also been discovered. It is a nerve energy, and the injury is currently called sesame sterol, sex, and the national nutrient fruit is shown in the research system that prevents us from its constituents. The neuron in the brain lesions is used for neuroimmunology. The Journal of Liver Protection for Immune Protection 1 8 Whether it is sesame stroke and nerves on sesame seeds has been well understood with little non-time release and its axis response, nourishing with chemicals, etc. ( :1-11 for proteins of neurocytosis, but For a variety of cell processes in neurons, the help is to help a variety of therapeutic effects and alcohol. See (Ϊ 993)) Glue cell-changing and degenerative genes have their anti-oxidative astrocyte cells and microglia cells, which are involved in brain metabolism. There are a variety of kinesins (white matter) responsible for repairing the tissue, which is the basis for the recovery of stroke patients. Including, for example, reducing gallbladder detoxification, anti-oxidant activity, for example, camp and Aki, medium, but no studies have been protected, or available, and no studies have yet been conducted. By a series of research results, the protective cells have various effects on the cells.

-1276439 V. INSTRUCTIONS (2) ' • r·.丨' , still unclear, so we will discuss the protective effects of various antioxidants, including sesamin and sesamin, in Chiba, confirming that it is indeed It has the effect of preventing pre-stroke and protecting nerves. SUMMARY OF THE INVENTION One aspect of the present invention relates to the use of sesamin and sesamol in for the prevention of secondary medicine and the protection of neurodegenerative medicines. 0 Another aspect of the present invention relates to sesamin Use with sesameol for the manufacture of pharmaceuticals for the prevention of stroke and protection of neurodegeneration.

A further aspect of the invention relates to a pharmaceutical composition for preventing and/or treating a stroke comprising an effective amount (ingredient) of sesamin and/or linolenic acid. Another aspect of the present invention relates to a pharmaceutical composition for preventing neuropathy and degeneration, which comprises an effective amount of sesamin and/or sesame alcohol, and a prime of 0 [schematic description of the illustration] i 7 : f τ antioxidant, (chicken phenol, 7 fertility •, sesamin and zhi 2 it: stimulated BV-2 cell strain production - inhibition of nitrogen oxide

;^理仃 represents the untreated group·, the second line represents the 1 microgram LPS M: search f table LPS + sesame (respectively 20, 5 〇 and 1 〇〇 2) $ rational:: 仃 represents LPS + tocopherol ( 20, 50, and 100, respectively, the treatment group, the 9th-U line represents the Lps + sesamin 100 μΜ treatment group; the 12th 14th 疋 “5 (] and 2〇, 5〇 read the state represents the LPS+ hemp Alcohol (respectively

Page 6 1276439 V. INSTRUCTIONS (3) Figure 2 shows the activity of each antioxidant in different concentrations (20, 50 and 100 #Μ) on the sputum 202-stimulated BV-2 cell line in two replicate experiments. Inhibition of oxygen (ROS) - Figure 3 shows the inhibitory effects of various antioxidants (sesame phenol, plant-tocopherol, sesamin and cisplatin) on the production of nitric oxide by LPS-stimulated f-class glial cells. Figure 4 shows a photograph of serial sections of the brain of control gerbils and sesamin-treated squirrels after TTC staining. [Description of the Invention] In addition to the mainly contained ~6 fatty acids, sesame oil also contains some lignans which are beneficial to the health of the body, including sesamin (sesara^n), sesamolin, sesame phenol. (sesamin〇l), episepithelin glial cells and micro-gel

Se=mo 1 in has a protective nerve ^ microgel cell V1 as a chemical substance s = amiη and hemp and sesame alcohol sin and the ancient box κ Ms. Gate, according to this, it is inferred that the aim of the prevention of stroke and protection The sesamin and the sesame of the present invention have the function of chemical conversion. The scope of music theory. Their role can be achieved by = non-predictable, free radicals released after use, and - nitrogen oxides, = = glial cell injury, gamma 1 : This can be used in the treatment of heart A Dark eight, ritual ~ (five) For example, such as for high blood pressure, phlegm treatment, "blood disability, heart palpitations, and unsteady angina, peripheral, / 0 treatment and palpitations are not _ door and cardiovascular disorders, heart rhythm

Cardiovascular tissue is subjected to secondary arsenic via cells and brain or ''έ

Incomplete, for the treatment of thromboembolism, shock, transient ischemic attacks, and so on. - obstruction and peripheral ischemia, such as myocardial infarction, prevention of restenosis, low cytoplasmic cytoplasmic - 6 and tumor damage as a mid-term repair may be suitable for lowering sleep disorders for treating Alzheimer's disorders, such as coke and/or With stimulants and

The sesamin and sesamin of the present invention can also be interspersed with various intercellular substances released by brain lesions: : scorpion? Liver 1), etc.) reduces inflammation and promotes repair of t-neurosystemic disorders. In particular, it eliminates cognitive deficiencies and improves learning ’ ‘, ^ ^, Xing 5 recall, and Helm's disease. They are also suitable for sputum and mouth _ treatment of central nervous system "m force and inhibition t, middle (four) meridian _ cause L function of this obstacle 'and for the adjustment of pathologically eating disorders related to the use of music. Also suitable for prevention and brain cerebral ischemia, cerebral ischemia, and cranial brains can be appropriate for those skilled in the art, such as Rev. in Remington Medical Sciences, Ginaro, and Mike Publishing Remington: The Science of Medicine and Conventional compositions may be presented in conventional forms, such as liquid, suspension or topical coatings: Obstruction (brain stroke), such as control of sequelae of traumatic trauma, and according to accepted conventional techniques, 1 985 or Ray Minton Medical Sciences Division, Easton, PA, 1 990, or surgery, 19th edition (1995). These groups of capsules, tablets, sprays, pharmaceutical carriers or diluents used for dissolution may be conventional solids. Or liquid carrier. Solid, examples of carriers are lactose, gypsum powder, Yan sugar, cyclodextrin, talc, gelatin, agar, money, arab #, stearic acid, stearic acid or fiber, low carbon number of element Topographical puzzle. Liquid carrier Examples are syrup, peanuts

1276439

Disc grease, fat · give five, the date of the month (5) acid, fatty acid amide, polyoxyethylene oil, olive oil or water. Similarly, the carrier or diluent ^^ _ continuous release material, such as single hard: =: already; vinegar used in this technique, used alone with me. Glycan 4, or bisglyceryl distearate The compositions of the invention are typically used for oral administration. For oral administration, sesamin and/or zhi's are administered orally, for example, in the form of tablets or capsules, and are mixed with a carrier and molded into tablets. Suitable carriers for use include starch, sugars, dicalcium phosphate, and the like. Such compositions may include 1 Ge 2, iron stearate emulsifier: emulsifier: preservative-stabilizer 1 color additive I, for example, pharmaceutical composition is administered daily or t per week for a pharmaceutical composition The effective amount is the one that can provide clinically significant efficacy. , the type will be (-partial) depending on the particular condition to be treated, the age of the patient, etc.: and the general health status, and other habits of the two factors. Preferably, the unit dosage form is in solid form ("' in liquid form (a solution, suspension, lotion, etc. capsule), or in the form of a sterile injectable solution. The conventional method of the ancient medicine school is equipped with fr. The conventional excipients are suitable for parenteral or oral administration. The compound is pharmaceutically acceptable for the production of harmful reactions. "Mechanical:: and physical substances. Machine or inorganic carrier

_1276439 V. INSTRUCTIONS (6) Examples of such carriers are water, salt solutions, alcohols, glycols, polyhydroxyethoxylated castor oil, #浆, peanut oil, olive oil, gelatin, 'lactose , gypsum powder, sucrose, agar, fruit acid, gum arabic, starch sugar, magnesium stearate, talc, tannic acid, stearic acid, fatty acid monoglycerides, season

Pentaerythritol fatty acid vinegar, hydroxymethine cellulose and polyethylene? Pyrrolidone and calcium phosphate. :, U Pharmaceutical preparations may be sterilized and, if desired, adjuvants which do not adversely react with the active compound, such as adhesives, lubricants, preservatives, disintegrating agents, stabilizers, wetting agents, emulsifiers, It is used to mix salts, buffers and/or coloring substances that affect osmotic pressure. For parenteral administration, it is especially suitable as an injectable solution or suspension, preferably an aqueous solution having the active compound dissolved in the polymethylated castor oil. For oral administration, tablets, troches, or capsules having talc and/or a carbohydrate carrier or an adhesive are preferred, and the carrier is preferably lactose or calcium phosphate and/or corn starch and/or horse. Potato starch. A syrup, elixir or the like can be used when a sweetener excipient can be used.

[Examples] The present invention is further illustrated by the following examples, which are intended to be illustrative only, and are not intended to limit the scope of the invention. Any person skilled in the art can make any modifications and changes without departing from the spirit and scope of the present invention, and should be included in the scope of the present invention. Example 1 · Ex vivo experiment

1276439 V. INSTRUCTIONS. (7) (1) BV-2 cell line The mouse BV~2 cell line was first prepared by Dr. B〇cchni, which is a standard mouse microcapsule V-raf/v-rayc carcinogenic soil due to the retrovirus (J 2 ) immortalized cell line (b 1 as i et al '). The cells were cultured to fullness prior to the experiment and then treated with trypsin for τ and fruit, which was then washed twice with warm DMEM and then treated in serum-free medium. Chiba kitchen antioxidants (sesame, sputum - fertility, sesamin, and so on, i, LPS-stimulated ^-2 cell line produces nitric oxide ... έ to '; in this experiment, cells First treatment with 1 0 0 ng/mL LPS, mouth /, 2 and ^ not - the same concentration (2 〇, 50 and 1 〇 0, in Figure 1 is the number (^ieSS reverse; ft antioxidant Incubate for 24 hours, then use... P to take 100 μl of culture supernatant and isotonic reagent (1 part 0·1% Ν-η兹盆w ^ , /, 寻合积格ness H3P04 ) ^ , (Neptidyl) Ethylamine, 1 part of 1% sulfonamide, dissolved in 5%, trough tissue culture plate in the sunny place for anti-caries, measuring at 540 nm, 叨本府死,~, 疋4丁久 should ι 〇 / Knife clock, Ί 妹 罢 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ = strain, can indeed inhibit or reduce its production - oxidizing gas (= Γ 2 ; ; (2. In the two repetitions, each antioxidant in the experiment with different concentrations of living oxygen (R0S) inhibition,, field strain Inducing birth for 24 hours, with 1 H2(), , Tian cell in the 96 ~ trough flat plate H202 order processing for 60 minutes, then add 1 〇 "

Page 11 1276439 V. Description of the invention (8) H2DCF-dA and various antioxidants. After reacting at 37 ° C for 60 minutes, the amount of fluorescence of the cells was detected at an excitation wavelength of 485 - ί : ηιη and a wavelength of 538 nm emission. From the results of Figure 2, each of the antioxidants can reduce the induced reactive oxygen species, even up to the active oxygen value measured in the BV-2 control group (line 1) without the Η202-treatment. Degree (lines 4, 7, and 10). Tables 1 and 2 show the anti-inflammatory effects of sesamin and/or sesame. As described above, BV-2 cells were cultured in a 96-well plate, and the number of cells reached 2 X 105 cells/well' induced the production of inflammatory factors such as wIL-6 and TNF_^ by LPS (1 μg). The effects of sesamin and sesamin on the reduction of 116 and TNF-^ production were observed. The IL-6 and TNF-α levels were tested according to the standard ELISA method using anti-T!^ or anti-TN^-α antibodies, and based on the obtained 〇D45〇: the amount of IL 6 and TNFi present was calculated. . The results are shown on the table and in Table 2, the results of 0 LPS Ϊ 1匕 table show that 'sesamin and sesamin can reduce the inflammatory factors induced by LPS (such as the reduction effect of ^6 nalexin, Go to the mountain + ; ^ 80 value, and the TNF-α-based dans-treated group measured by the LPS treatment group compared with the LPS control group, IL-6 and inflammatory factors damage ^ j is not in the protection of nerve cells from protection: force, and sesamin on the nerve cells under hypoxia... cell strain culture = = = in the oxygen test, the system will be under hypoxic conditions (谇In the treatment medium of sodium |S sugar, then placed at 85%), at: c〇wm 〇<, to simulate the cells in hypoxia

Page 12

I —1276439 V. Description of invention (9). After the completion of the cultivation, each treatment group is subjected to MTT bifurcation containing thiazolidine blue, a dye which can be crystallized by the granular material and wind in the living cells, and the crystalline substance can be dissolved in the presence of Cold agent one Aya? (DMS0), and can be counted by the color absorbance value of 1 in the experiment I's the treatment group cells in the treated culture supernatant, MTi solution (0.3 mg / ml) was added to the culture two: After incubation for 1 hour at 37 C, the hydrazine solution was aspirated, DMSO was added, shaken for 10 minutes, and the absorbance (OD540) was measured at a wavelength of 54 Torr (10). When the cell dies, it releases a stable enzyme called lactate dehydration (LDH), which can be converted into a red product by LDH by adding the added tetrazolium salt (INT). The value was determined by the value (OD490). The survival rate and cytotoxicity of the BV-2 cell lines of each treatment group are shown in Table 3, without the control group of the hypoxic-treated control cell line (which is regarded as the normal cell without hypoxia treatment). And 4 in. The semi- and fruit-harvest columns The alcoholic cells of Tables 3 and 4 have higher survival values for the nerve cells but not for the separatase cells. Both sucrose and sesame can be treated in a low group, especially when the value is not protected with anoxicin and sesame.

The results showed that under the condition of hypoxia, Sesamin and Shiba had the effect of protecting the cells, and under the condition of anoxic conditions, the cells treated with Sesamin, the treated group (Table 3), indicated that the cells were damaged. In terms of cytotoxicity measured by LDH, C. sinensis protects BV-2 cells from being treated with sesamin (50 / zM), and the LDII assay is almost the same (Table 4). The temple protects the nerve cells from damage caused by hypoxic conditions.

1276439 V. Inventions (ίο) have significant effects. (2) PC12 cell strain The present invention also uses a PC12 cell strain (which is a neuron-like cell strain) as an in vitro test material to study the dehydrogenated plum of lactate induced by sesame and sesamin on the sputum and the sputum 202- The impact of ldji) release. As described above for the treatment of BV-2 cells, LDH was induced by PC12 cells with Ατρ (5 〇〇 幻 or (10) (10 00 VM), and the culture supernatant was taken 1 〇 0 micron.

Add the same amount of LDH solution, and then measure the absorbance ((^ value) of the sample at wavelengths of 49 () rim and 630 nm, and calculate the cell-mediated cytotoxicity percentage (%). Shown in Tables 5 and 6. The results of Tables 5 and 6 show that sesamin and sesamin significantly reduce the release of LM induced by ATP and H202, and reduce cell-mediated cytotoxicity. Sesamin and Sesamin have the effect of protecting nerve cells. Example 2 · "Primary cuiture" Analytical consumption: Energy: In vitro experiments further simulate the preservation of primary cultures closer to living cells, analysis. Rat primary The primary glial culture is as in the prior art (Wang et al., 2001). The *q will be prepared as a 妯妳腴 & 6 & I gel cells are separated from the neuron tissue knife. The cells are cultured in supplements with 1% fetal bovine serum, υ/πιί

Page 14

And in DMEM medium of 100 μg/mL streptomycin for 10-14 days. The flask was shaken at 240 rpm and shaken at 37 °C to separate the glial cells. Incubate for 20 minutes at 37 t, remove non-adherent cells and add "fresh medium. Check the purity of the glial cell culture with 0X42 antibody. The cells were cultured for 4 days before the experiment. According to the above Example 1, test Each of the oxidants (sesame phenol, r-sodium phenol, sesamin, and sesame ketone) inhibited the production of nitric oxide by LPS-stimulated primary glial cells. The results are not shown in Figure 3, although the oxidants are Compared with the Lps-induced group, a lower amount of nitric oxide was produced, and the effects of sesamin and sesamin were more apparent (10th to 14th lines). This result also supports sesamin and sesame alcohol. The cytoprotective effect of the cells. Example 3. Living experiment This example uses the gerbil as the recording mode, and the effect of the sedative on the repair of brain-deficient blood damage 1 feeds a microliter containing 3.5 mg of sesamin and / Or the composition of sesamin: Jade: oil, the control animals only fed 20 〇 microliters of Wang, 士, + 8:3. -9: 〇., for three days; = two: food time For the early morning ligation for 2 hours to simulate the occurrence of adiabatic ischemia: when the sputum, the right cerebral artery fork The gerbils were removed from the gerbils. The brains were sectioned with TTC (gasification two smiles A ..., Manjin light knife v recorded l 1 匕 one base tetrazolium nitrogen) and the total infarct was calculated according to the OPTIMA 6.2 system.达# I _ 薇μ, from + _ & ^ product. Figure 4 is the control group of gerbil and zebra treatment: " mouse brain serial section staining picture shows the necrotic fine F Λ that can not be stained by TTC / Θ Θ 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨

$15页1276439 V. Description of invention (12) The total infarct size accounts for 6.86%, which is 61% lower than that of the control group. The combination of anesthetic and sesamin has a total infarct size of f - 0. It has also been confirmed that the damage caused by sesamin and sesame alcohol ^^ monthly blood infarction has preventive and therapeutic effects. Therefore, the present invention utilizes the high degree of natural law creation, which can be extremely and ambiguously intended. The present invention is an unprecedented design and has the effect of being used. Therefore, the above 13⁄4 manufacture has met the invention patent. Highly creative. , 提起 filed an invention of the Shen Shi according to law, and asked for an early patent, to the sense

1276439 Brief description of the diagram u Figure 1 shows the inhibition and effect of various antioxidants (sesame, 7 - fertility, sesamin and cedar, linolein) on the production of nitric oxide by LPS-stimulated p_2 cells. The first row represents the untreated group; the second row represents the 1 μg lps treatment group; the 3-5th row represents the LPS + sesame phenol (2 〇, 50 and 1 〇〇 / / M, respectively) treatment group; Lines -8 represent LPS+tocopherol (2〇, 5〇 and 1〇〇//M, respectively) treatment groups; Lines 9-11 represent jLPS+sesin (2〇, 5〇 and 100#M, respectively) treatment groups Lines 12-14 represent the LPS + Sesameol 20, 50 and 100 //M) treatment groups. v v is J疋 Figure 2 shows the antioxidants in two repeated experiments.

(20, 50, and 100 pairs of H202-stimulated BV~21⁄2

Inhibition of living oxygen (ROS) | ' Cell-induced S Figure 3 shows the inhibitory effects of various antioxidants (sesame phenol, γ-tocopherol, sphingosine) on LPS-stimulated primary glial cells. Nitric Oxide Figure 4 shows a continuous slice of the brain of the control gerbil and the squirrel of the control group of TTC itch itch. < 厥素处理沙

Page 17 1276439 Table 1, BV-2 cell line LPS-induced TNF-α test results LPS-induced TNF-α (pg) in the experimental group 603 LPS (1 μ§) 874 LPS + sesamin, without LPS treatment 10 μΜ 595 LPS + Sesamin, 50 μΜ 813 Table 2. IL-6-induced TNF-α test results in BV-2 cell line LPS-induced IL-6 (pg) in the experimental group 2467 LPS in the LPS-free group (1 pg) 7884 LPS+ Sesamin, 100 μΜ 3837 LPS + Sesamin, 50 μΜ 7579 1276439 Table 3. MTT test results of BV-2 cell strain under hypoxia. Experimental group MTT 〇D survival rate % without hypoxia Treatment group 1.40 100% Anoxic condition 0.84 60% Hypoxia + Sesamin, 20 μΜ 1.02 7.3% Hypoxia + Sesamin, 50 μΜ 1.23 88% Hypoxia + Sesamin, 20 μΜ 1.11 79% Hypoxia + Sesame Alcohol, 50 μΜ 1.06 76% Table 4. Test results of LDH induced by BV-2 cell strain under hypoxia. Experimental group LDH OD Cytotoxicity % No treatment by hypoxia 1.81 100% Hypoxia treatment 3.10 171% Hypoxia + Sesamin, 20 μΜ 2.56 141% Hypoxia + Sesamin, 50 μΜ 1.87 103% Hypoxia + Sesamin, 20 μΜ 2.28 126% Hypoxia + Sesameol素素, 50 μΜ 2.73 150% 1276439 Table 5. ATP-induced LDH test results of PC12 cell line LDH OD cytotoxicity of experimental group % ATP-treated group 0.429 100% ATP (500 μΜ) 1.463 341% ΑΤΡ + Sesamin, 20 μΜ 0.664 155% ΑΤΡ + Sesamin, 50 μΜ 0.962 224% ΑΤΡ + Sesamin, 20 μΜ 0,487 114% ΑΤΡ + Sesamin, 50 μΜ 0.437 102% Table 6. PC12 cell line H2〇2-induced LDH test results Group LDH OD Cytotoxicity % Not treated with H2O2 0.425 H2〇2 (1000 μΜ) 1.936 100% Η2〇2+ Sesamin, 20 μΜ 1.287 66% Η2〇2+ Sesamin, 50 μΜ 1.221 63% Η2〇2+ Sesame, 20 μΜ 1.055 54% Η2〇2+ Sesame, 50 μΜ 0.840 43%

Claims (1)

1276439 6. Patent application scope C|Lp#^ 1 · A use of sesamin and sesameol for the manufacture of a medicament for prevention and/or: wind 1, wherein the manufacture comprises sesamin and sesamin with a pharmaceutical An acceptable carrier, excipient or diluent is blended to form a pharmaceutical product. 2. The use of sesamin and sesamin for the manufacture of a medicament for the protection of neurodegenerative, wherein the kit comprises: sesamin and sesamin with a pharmaceutically acceptable carrier, excipient or ~# The release agents are blended to form a pharmaceutical product. Page 18 1276439 The present invention relates to the use of sesamin and sesam〇Hn for the manufacture of a medicament for preventing stroke and protecting nerve degeneration, and a pharmaceutical composition comprising anesthetic and/or sesamin. . (1) The representative figure of the case is ····· (2), the representative symbol of the representative figure of the case is a simple description: the first line represents the untreated group; the second line represents the 1 microgram Lps processing group; -5 lines represent LPS + sesame phenol (2 〇, 5 〇 and 1 〇〇 #m respectively), group; lines 6-8 represent LPS + tocopherol (2 〇, 5 〇 and 1 〇 () vM respectively) 'Lines 9-11 represent Lps + Sesamin (2 〇, 5 〇 and 1 〇〇 = 分别, respectively, 丄 -12-12 lines represent LPS + Sesamin (20, 50 and 100 / M, respectively) .