TWI466667B - Use of sesamin derivates in protection from nerve injury - Google Patents

Description

Use of sesamin derivatives for anti-nerve damage

The invention claims the invention patent application No. 101120989 as the priority basis of the case.

The present invention relates to the use of a sesamin derivative for anti-nerve damage, and more particularly, the present invention relates to a sesamin derivative for protecting against epilepsy or by abnormal accumulation of a starch-like protein (Aβ). The use of nerve cell damage.

Sesamin has been confirmed by medical research to protect the liver, resist inflammation, resist hypertension, lower cholesterol, prevent aging, and inhibit cancer. In many research reports at home and abroad, it is also pointed out that sesamin is an antioxidant, has the ability to scavenge free radical ROS, and can effectively inhibit the nitric oxide inflammatory reaction of microglia cells after being stimulated by endotoxin. To achieve the role of protecting the brain. Recent studies have found that sesamin has protective neuronal effects on PC12 cells and BV-2 microglia cells; and sesame meal fermentation can improve the learning and memory ability of SAMP8 mice, enhance the antioxidant defense system in vivo, and reduce lipids and proteins. Peroxidation and total deposition of brain amyloid (Aβ peptide).

The sesamin derivative of the present invention is described in U.S. Patent No. 4,343,796, in which SD-1 (2,3-bis(3-methoxybenzyl)butane-1,4-diol) belongs to an enterolactone. The derivative can be synthesized synthetically (Kise, Naoki et al., J. Org. Chem. 2000 , 65(2), 464-468). It is a compound that can pass the blood-brain barrier and has anti-oxidation, anti-inflammatory and anti-aging effects.

Epilepsy, commonly known as sheep halo and sheep epilepsy, is a chronic brain disease caused by congenital or acquired factors, characterized by abnormal discharge of brain cells causing repeated attacks. Most scholars define epilepticus as a seizure of at least 30 minutes. Causes of status epilepticus include acute nerves, acute systemic diseases, or interruption of anti-epileptic drugs. Status epilepticus can cause brain damage or even death of the patient. Repeated episodes of epilepsy can also cause brain tissue damage and affect intelligence, often causing learning disabilities in children. In animal experiments, electrical stimulation and chemical-induced status epilepticus can cause brain neuronal death (Hsieh PF, 1999, Neurol Res 21 : 399-403; Gupta YK et al., 2002, Pharmacol Biochem Behav 71 (1- 2): 245-49). Status epilepticus can also cause medial temporal lobe sclerosis and spontaneous recurrent episodes (epilepsy) (Wasterlain CG et al., 1996, Epilepsy Res 26 : 255-65).

In the past two decades, many new anti-epileptic drugs have been invented, making 80% of patients unable to suffer from seizures. The main purpose of drug treatment is to control the onset of the drug so that it does not happen at all, or to retreat to make the number and severity of the attack significantly lower. Regular use can inhibit seizures, allowing patients to lead a normal life. Different types of epilepsy require different anti-epileptic drugs. Doctors will take medicine according to the patient's condition. Some patients may need to take multiple drugs.

Commonly used anti-epileptic drugs include dilantin, tegretol, valproic acid, mysoline, phenobabital, clobazam (Frisium), and Clonazepam (Rivotril). Etc. In addition, there is a Valium anal suppository, which is quite useful for children to have an anal attack. After taking the anti-epileptic drugs, the blood is absorbed into the brain, so that the area causing abnormal discharge is stable and the epilepsy is controlled. To control epilepsy well, the concentration of the drug in the blood can be maintained stable day and night. Taking medicine drops blood levels, often causing multiple episodes; if you take too many drugs, it is surprising that it may also cause seizures. Antiepileptic drugs are not addictive, but sometimes have side effects. Side effects include lethargy, weight gain, temporary hair loss, skin rash, gum swelling, imbalance, and gastrointestinal discomfort.

One of the main pathological features of Alzheimer's disease is a class derived from amyloid precursor protein (APP). The senile plaque formed by the abnormal accumulation of amyloid β peptide (Aβ). In addition to Aβ, the main components of senile plaques contain degraded nerve cells, activated astrocyte cells and small glial cells. However, whether activation of astrocyte cells will aggravate or protect the death of nerve cells remains to be elucidated.

At present, there are two main types of drugs for the treatment of Alzheimer's disease on the market: one is cholinesterase inhibitors; the other is neuroprotective agents. The choline esterase inhibitor is a key enzyme in the decomposition of acetylcholine, which inhibits acetylcholine by blocking the action of cholinesterase. Decompose to increase the content of acetylcholine in the brain. Another type of neuroprotective agent, such as Memantine (Namenda), blocks glutamate damage to brain cells, thereby slowing down the loss of life skills, and is currently the only drug for the treatment of moderate to severe dementia. Both drugs delay the loss of memory and help the patient perform the actions required for daily living. It is very important that these drugs do not cure Alzheimer's disease, they can only alleviate the symptoms of Alzheimer's disease.

The invention first proves that the sesamin derivative SD-1 has the effect of protecting the nerve cells from excessive excitability by the in vitro nerve cell protection experiment and the animal experiment, and the neuron cell protection effect is better than that of the sesamin. It is especially useful for protecting against excessive excitation of nerve cells caused by epilepsy. The invention combines high-purity sesamin sesamin and sesamin derivative SD-1 into a series of experiments such as blood-brain barrier of animals and pharmacokinetics in animals to design and develop new treatments or A small molecule drug that alleviates excessive cell damage or Alzheimer's disease.

In one aspect, the invention relates to a neuroprotective composition comprising a sesamin derivative. The sesamin derivative has the following chemical structural formula:

Wherein R ¹ and R ^{2 are} each independently selected from the group consisting of hydroxy, (C _1-6 )alkyl, (C _1-6 )alkoxy, O-(C _1-6 )alkyl, and COR ^c ; and R ³ and R ^{4 is} each independently selected from the group consisting of a hydroxyl group, a (C _1-6 )alkyl group, and a (C _1-6 ) alkoxy group.

In one embodiment of the present invention, the sesamin derivative is 2,3-bis(3-methoxybenzyl)butane-1,4-diol having a molecular formula of C ₂₀ H ₂₆ O ₄ , The molecular weight is 330.42.

In one embodiment of the invention, the composition is for protecting nerve cells from excessive excitation damage. In another embodiment of the invention, the composition is for protecting nerve cell damage caused by epilepsy.

In another embodiment of the invention, the composition is for use in protecting against neuronal damage caused by the amylin-like protein A[beta]. In some embodiments of the invention, the composition is used to combat the lethal toxicity of the amyloid-like protein A[beta] to nerve cells.

In yet another embodiment of the invention, the composition is for use in the treatment or prevention of Alzheimer's disease, particularly for the treatment or prevention of Alzheimer's disease.

Figure 1 shows the results of the PC12 cell damage (Cytotoxicity, %) test. The degree of PC12 cell damage (Cytotoxicity, %) was 100% in the experimental group with 150 μM of kainic acid. CK: control group, no added any agent; KA: experimental group plus carbamic acid 150 μM; SA: Kaying Acid 150 μM plus sesamin 10 μM; SD1: kainic acid 150 μM plus sesamin derivative SD-1, 10 μM.

Figure 2 shows the results of Cell Viability (%) test under cadmium acid 150 μM stress treatment. The PC12 cell survival rate (Cell Viability, %) was 100% in the control group. CK: control group, no added drug; KA: experimental group plus carbamic acid 150 μM; SA: kainic acid 150 μM plus sesamin 10 μM; SD1: kainic acid 150 μM plus sesamin derivative SD-1, 10 μM.

Figure 3 shows the release of calcium ions from the sesamin derivative SD-1 for the protection of neuronal hyperexcitability by kainic acid. CK: control group, no added drug; KA: experimental group plus carbamic acid 150 μM; SA: kainic acid 150 μM plus sesamin 10 μM; SD1: kainic acid 150 μM plus sesamin derivative SD-1, 10 μM.

Figure 4 shows that the sesamin derivative SD-1 has protection against PC12 cells by the amyloid beta peptide (A-β _1-42 5 μM) compared to the untreated control. Group number 1: control group, no added starch-like peptide and any drug; 2: starch-like peptide β (A-β _1-42 5 μM); 3: starch-like peptide β + sesamin 10 μM; 4: Amyloid beta peptide + sesamin 50 μM; 5: amyloid beta peptide + Ebixa 10 μM; 6: amyloid beta peptide + Ebixa 50 uM; 7: starch-like beta peptide + Exelon 10 uM; 8: starch-like beta peptide + Exelon 50 μM; 9: starch-like peptide β + sesamin derivative SD-1 10 μM; and 10: starch-like peptide β + sesamin derivative SD-1 50 μM.

Figure 5 shows that the sesamin derivative SD-1 has a reduced release of nitric oxide from one of the LPS-treated BV-2 microgel cells compared to the untreated control group. Group number 1: control group, no LPS and any drug added; 2: LPS (1 ng); 3: LPS + sesamin 10 μM; 4: LPS + sesamin 50 μM; 5: LPS + Ebixa 10 μM; 6: LPS + Ebixa 50 uM; 7: LPS + Exelon 10 uM; 8: LPS + Exelon 50 μM; 9: LPS + sesamin derivative SD-1 10 μM; and 10: LPS + sesamin derivative SD-1 50 μM .

Figure 6 shows that the sesamin derivative SD-1 has an excellent protective effect on the degree of lipid peroxidation caused by the neuropeptide PC12 due to the amyloid beta peptide compared to the untreated control group. Group number 1: control group, no added starch-like peptide and any drug; 2: starch-like peptide β (A-β _1-42 5 μM); 3: starch-like peptide β + sesamin 10 μM; 4: Amyloid beta peptide + sesamin 50 μM; 5: amyloid beta peptide + Ebixa 10 μM; 6: amyloid beta peptide + Ebixa 50 uM; 7: starch-like beta peptide + Exelon 10 uM; 8: starch-like beta peptide + Exelon 50 μM; 9: starch-like peptide β + sesamin derivative SD-1 10 μM; and 10: starch-like peptide β + sesamin derivative SD-1 50 μM.

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Implementation example

Kainate (KA) is a similar substance of glutamate, which has strong neuromuscular excitability and is often used to induce marginal lobe epilepsy in animals including status epilepticus. Injection of kainic acid into the peritoneal cavity or brain of rats can cause damage to neurons in the hippocampus and amygdala, and develop into seizures, which induce tonic-clonic seizures and marginal motor signs. These include wet dog shakes (wet dog shakes), facial myoclonia and paw tremor.

In the experiments of the following examples, the neural cell strain PC12 used was purchased from the Bioresource Collection and Research Center (BCRC, Hsin-Chu, Taiwan), number BCRC 60048. The BV-2 cell line was provided by Dr. Zheng Qiqing from Taichung Rong.

Synthesis of Sesamin Derivative SD-1

3-Methoxy-benzaldehyde 1 (0.1 mol), malonic acid (0.4 mol) and hexahydropyridine (1.0 g / 15 ml) were dissolved in pyridine, and then the solution was heated to reflux under nitrogen for 18 hr. The reaction mixture was then cooled to 0 ° C and the pH was adjusted to 1.0 by the addition of aqueous 1.0 N hydrochloric acid. The solution was filtered, and the obtained white solid was washed with water and dried under vacuum to give 3-(3-methoxy-phenyl)-acrylic acid ( 2 ) in a yield of 91.5%.

3-(3-Methoxy-phenyl)-acrylic acid ( 2 , 0.1 mol) was stirred with 10% Pd/C in methanol at 30 psi overnight. The solution was washed twice with brine and concentrated under reduced pressure. The residue was purified via a vermiculite gel column to give 3-(3-methoxy-phenyl)-propionic acid ( 3 ).

THF was added to a 500 ml two-necked flask and allowed to stand at 0 ° C for 10 minutes under nitrogen. Then diisopropylamine (160 mmol) was added, then 2.5 M 16.0 ml n- BuLi (170 mmol) was added to the solution, and the reaction mixture was stirred at 0 ° C for 30 min. The solution was cooled to -78 ° C and stirred for a further 30 minutes. At -78 ℃, dissolved in THF of 3- (3-methoxy - phenyl) - propionic acid (3, 80 mmol) Jia Ruda 1.0 hours. The reaction mixture was warmed to 0 ° C for 3.0 h and then warmed to room temperature for 2.0 h. The reaction mixture was then cooled again to -78 ° C, and iodine (80 mmol) dissolved in THF was slowly added to the solution. The reaction mixture was warmed to room temperature and stirred for 18 h. The solution was then quenched with water and extracted twice with EA. The organic layer was extracted NaHSO ₃ solution and brine, dried by MgSO _4, concentrated and placed under reduced pressure. The residue was purified via a vermiculite gel column to give 2,3-bis(3-methoxy-benzyl)succinic acid ( 4 ), yield 37%.

The 2,3-bis (3-methoxy-benzyl) - acid (4, 40 mmol) and sulfuric acid As methanol and heated to reflux for 18 hours under nitrogen. The reaction mixture was then quenched with water and extracted with EA. The organic layer was extracted NaCO ₃ solution and brine, dried by MgSO _4, concentrated and placed under reduced pressure. The residue was purified via a vermiculite gel column to give dimethyl 2,3-bis(3-methoxy-benzyl)succinate ( 5 ), yield 78%.

First LAH (90 mmol) in a round bottom flask on a vacuum pump for 2 hours, the THF was dissolved 2,3-bis (3-methoxy-benzyl) - dimethyl succinate (5, 30 mmol), Slowly drip into a round bottom bottle under an ice bath at 0 °C. The reaction mixture was then heated to reflux for 18 hours. The solution was then quenched with water and extracted with EA, dried over MgSO _4, and concentrated under reduced pressure placed. The residue was purified via a silica gel chromatography column to give the product 2,3-bis(3-methoxybenzyl)butyl-1,4-diol in a yield of 78%.

Protective effect of sesamin derivative SD-1 on neuronal damage induced by kainic acid

Carbonic acid causes nerve cell swelling, organelle disruption or even death, as well as glial cell reactions, such as the activation and proliferation of astrocytes and microglia, and the response of neuroglial cells. It appears earlier than the stellate cells. Taniwaki et al. also found that activated neuroglial cells were observed in the brain of rats with kainic acid, and the phenomenon of activation may be due to nerve damage or excessive nerve excitation. When kainic acid was directly injected into the hippocampus or amygdala, CA3 (3 parts of the golden calf, Cornu Ammonis 3, the dentate gyrus of the hippocampus of the cerebral cortex) showed a greater degree of neuronal damage than CA1 (golden calf 1) , Cornu Ammonis 1, dentate gyrus of the hippocampus of the cerebral cortex) or CA4 (Golden calf 4, Cornu Ammonis 4, hippocampal formation of the cerebral cortex) The dentate gyrus CA4 is more severe. Thus, this example utilizes the addition of kainic acid to the neuronal cell line PC12 to elicit an excitotoxicity test to confirm that the sesamin derivative SD-1 has the effect of reducing the damage of the nerve cell strain PC12.

Sesamin derivative SD-1 tested by Pampa-BBB

For drugs that treat or slow down brain cell damage, being able to penetrate the blood-brain barrier should be a must. According to the PAMPA-BBB method of LI et al., 4 μL of PBL solution (20 mg/mL PBL dissolved in dodecane) was taken up, coated on a filter, and allowed to stand for 5 minutes to dry. A 75 μL sample of sesamin derivative compound (2 mg/mL) was taken and 1425 μL of universal buffer was added to prepare a working solution at a concentration of 100 μg/mL. Pipette 300 μL of the prepared working solution into step 2 of the PAMPA-BBB experimental device. The PBL coated filter disc is mounted above the chassis. Pipette 200 μL of universal buffer and add it to the PAMPA-BBB membrane well. Finally, cover a layer of plastic cover to avoid evaporation of water and let it stand at room temperature for 18 hours. After the reaction time is over, the samples in the chassis and the filter disk well are respectively taken up, and the sample concentration is quantified by a micro disk reader.

Calculate the effective permeability coefficient (Pe) by the formula:

Where C _A (t) = sample concentration in the pores of the filter disk at the end of the experiment

C _D (0), C _D (t) = sample concentration in the chassis slot at the beginning and end of the experiment

V _A = filter disc hole volume = 0.2 cm ³

V _D = chassis hole volume = 0.3 cm ³

A = filter surface area = 0.24 cm ²

t = reaction time (unit: second)

t _lag = filter steady state time (average 1200 seconds).

R = loss factor (proportion of sample adsorption by filter)

Sesamin is not stable in the test buffer, so the effective penetration coefficient cannot be measured. The two commercially available Alzheimer's drugs, Exelon and Ebixa, have undoubtedly have blood-brain barrier function. It is obvious that for drugs that treat or slow Alzheimer's disease, the ability to penetrate the blood-brain barrier should be The necessary conditions. The sesamin derivative SD-1 of the present invention also exhibits a function of blood-brain barrier penetration, and is considered to be a potential drug candidate.

Cell culture and cell viability analysis were determined by the sesamin derivative SD-1 as shown in Table 1 above, which can pass the blood-brain barrier. Therefore, whether it is possible to pass the blood-brain barrier by comparing the sesamin derivative SD-1 with sesamin It can better protect the nerves through the blood-brain barrier than sesamin. The mouse nerve cell strain PC12 was cultured in a DMEM medium (10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin) in a 37 ° C incubator containing 5% CO ₂ . 24 hours before the kainic acid treatment, the cells were seeded in a 24-well plate, and 5 × 10 ⁵ cells were cultured in 1 ml of the culture solution in each well. The cells were treated with kainic acid (150 μM), sesamin (10 μM) or sesamin derivative SD-1 (10 μM) in an aqueous solution, and after 24 hours of incubation, stained with Trypan blue dye. Viable cell counting was performed under a microscope. The cells in which no agent was added were used as a control group.

As for the sesamin derivative SD-1, in order to protect the nerve cells from the damage caused by the carbaryl acid, the supernatant can be taken after the cell experimental treatment to test the release of lactate dehydrogenase (LDH) to test the nerve cell receptor. The extent of damage; while the cells were tested for succinic acid in the mitochondria of living cells by 4-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Dehydroquinone converts the tetrazolium of MTT to the blue product MTT formazan to test neuronal survival.

As shown in Fig. 1 and Fig. 2, the sesamin derivative SD-1 can reduce the damage rate of the neuronal cell line PC12, and even the sesamin derivative SD-1 can cause nerve cells to be hardly damaged by kainic acid. When the sesamin derivative SD-1 was compared with sesamin, the neuroprotective effect of the sesamin derivative SD-1 was significantly better than that of sesamin.

Determination of calcium ion concentration in cytoplasm The apoptotic effect of nerve cells can be triggered by the release of a large amount of calcium ions, which activates calcium and magnesium ion-dependent endonucleases due to a large increase in intracellular calcium ions, resulting in deoxyribonucleic acid. Neuronal apoptosis of the fragment. The kainic acid initiates depolarization of the membrane of the nerve cell by activating the channel of the kainic acid acceptor (the glutamic acid acceptor subtype), releasing calcium ions from the channel regulated by the calcium potential. Kanada et al. used Ginkgo biloba extract to reduce the release of calcium ions from neutrophils induced by kainic acid to protect cerebellar nerve cells in rats ( Biol Pharm Bull. 28 : 934-6, 2005). Red lotus extract can also reduce the release of calcium ions from nerve cells induced by kainic acid to protect the primary cells of rat cerebral cortex.

It is known that the sesamin derivative SD-1 has antioxidant activity. Therefore, this experiment tests the release of calcium ions induced by the addition of kainic acid to the nerve cell strain PC12 to test the inhibitory nerve of the sesamin derivative SD-1. The degree of release of cellular calcium ions was further tested for the degree of protection against apoptosis by neuronal and neuroglial cells induced by kainic acid.

PC12 cells were inoculated on slides before the test, and cultured in a constant temperature incubator at 37 ° C for 24 hours. The cells were incubated with 2 μM fura-2 acetoxymethyl ester (fura-2 AM) in a 37 ° C incubator for 30 minutes in the incubator to allow fura-2 AM to enter the cytoplasm. Observe under UV illumination and record the color change of fura-2 per second in the cells. 30 seconds after the start of recording of the data, 150 μM of kainic acid (as a comparison group) and an aqueous solution of sesamin (10 μM) or sesamin derivative SD-1 (10 μM) were added and recording was continued for 10 minutes. The scanner is used to detect excitation wavelengths of 340 nm and 380 nm alternating once per second with an absorption wavelength of 510 nm. After the PC12 cells were treated with kainic acid or sesamin or sesamin derivative SD-1 aqueous solution, the supernatant was tested for calcium ion concentration test.

From the results shown in Fig. 3, the sesamin derivative SD-1 has a protective effect against kainic acid causing excessive excitation damage of nerve cells. When the sesamin derivative SD-1 was compared with sesamin, the neuroprotective effect of the sesamin derivative SD-1 was significantly better than that of sesamin.

It was confirmed by the above-mentioned lactate dehydrogenase (LDH), tetramethylthiazole blue (MTT) and the cytoplasmic calcium ion concentration test that the degree of neuronal damage was confirmed, and the sesamin derivative SD-1 had a neuroprotective effect. Moreover, the neuroprotective effect of the sesamin derivative SD-1 is more remarkable than that of sesamin.

Sesamin derivative SD-1 reduces the degree of epilepsy in injured animals. It is known that kainic acid causes persistent seizures and irreversible damage to nerve cells. Carnitine can also cause swelling of nerve cells, broken organelles or even death, as well as neuroglial cell reactions, and activation may be due to nerve damage or excessive nerve excitation.

This experiment was observed in animal model, and the sesamin derivative SD-1 was effective in reducing persistent seizures caused by kainic acid and preventing irreversible nerve cell damage caused by epilepsy. Adult male FVB/NJNarl mice (age 8-9 weeks, body weight 30-35 grams) via forced mouth The gastric tube was infused with sesamin derivative SD-1 (dose 10 mg/kg, n=20 per group) for 3 days, and the fourth day was kainic acid (red alanine, kainic acid, 35 mg). /kg) Subcutaneous injection induces status epilepticus. The first control group: normal mice plus carbaryl (35 mg / kg) subcutaneous injection. The second experimental group: SD-1 was administered, and gacaic acid (35 mg/kg) was injected subcutaneously. According to Racine, the behavior of status epilepticus can be divided into: stage 0, normal activity; stage 1, rest; stage 2, posture stiffness; stage 3, nod; stage 4, cramp + fall; stage 5 , continuous cramps + fall; and the sixth stage, the whole body is strong and cramps. The animals were allowed to recover naturally after observing the behavioral severity of the status epilepticus for 5 hours.

The results are shown in Table 2 below. Among them, the severity level is represented by I (initial, minor, severe series: 1-2), M (middle, severe series: 3), and C (critical, severe series: 4-6), I<M< C; severity series: 1 < 2 < 3 < 4 < 5 < 6.

It was confirmed by the above animal experiments that the sesamin derivative SD-1 significantly reduced the degree of epilepsy; feeding SD-1 (10 mg/kg) reduced the number of 17 FVB mice with the highest severity of epilepsy to only 3 only.

Lactic dehydrogenase activity and MTT analysis One of the main pathological features of Alzheimer's disease is the abnormal starch-like protein (Aβ) derived from amyloid precursor protein (APP). Accumulated into a senile plaque. Its main components are amyloid-like proteins and degenerating nerve cells that activate astrocyte cells and small glial cells. Therefore, activated astrocyte cells should play a role in Aβ-induced toxicity. PC12 is known to be an internationally recognized cell line that can represent nerve cells. Therefore, this experiment is to determine the effect of amyloid-like protein on the cellular damage of nerve cell PC12, and add sesamin and sesamin derivatives to explore the neuroprotective function of sesamin and sesamin derivatives on nerve cells in 24 hours of stress. Variety.

Cell viability was determined by reduction of MTT [tetramethylthiazole blue]. In living cells, yellow MTT is metabolized into blue formazan via the dehydrogenase of the mitochondria, and the amount of viable cells can be reflected by measuring the absorbance after the reaction. PC12 cells 5 x 105/ml were pre-incubated in 24-well plates (24-well) for 12 hours and then rinsed with phosphate buffered saline (1 x PBS). The medium was treated with different concentrations of Sesamin, Ebixa, Exelon and Sesamin derivative SD1 (10, 50 μM) in a concentration of 5 μM Aβ1-42 peptide and the control medium for 24 hours, and grown at 0.5 °C at 37 °C. In ml MTT, after 60 minutes, 200 ml of DMSO (Dimethyl sulfoxide) dissolution solution was added to each well and the absorbance was read on an automated SpectraMax 340 (Sunnyvale, CA, USA) microtiter plate reader. Results The data shown is the average percentage of viable cells relative to control cells.

According to the results of Fig. 4, the toxicity of the sesamin derivative SD-1 to PC12 cells is comparable to that of the currently marketed drugs Ebixa and Exelon, indicating that the sesamin derivative SD-1 is safe and non-toxic to nerve cells. Moreover, compared with the untreated control group, it has the effect of protecting nerve cells from damage caused by amyloid.

Effect of Sesamin and Sesamin Derivatives on the Release of Nitric Oxide For the central nervous system, nitric oxide is one of the cell signaling factors, and oxygen and nitric oxide radicals can cause ester peroxidation and nerve cells. The casualties, therefore, we can detect the release of nitric oxide from one of the BV-2 microgel cells as a means of determining the cellular or cytopathic response of the glial cells. Method for the determination of nitric oxide: The determination of Nitrite is a quantitative analysis using a Griess reaction in a color reaction, in which two different solutions (1% sulfanilamide and 0.1% naphthylenediamine dissolved in 5% phosphoric acid) are respectively included: The ratio of 1 is mixed, and then mixed with the cell culture solution at the same volume ratio. After the mixing reaction, the OD micrograph reader is used to measure the absorbance OD (540) and the relative concentration at a wavelength of 540 nm. Conversion. And using NaNO ₂ as a standard, the concentration standard calibration line was used in the concentration range of 0-100 μM to determine the amount of nitrogen oxide released from the glial cells.

From the results shown in Fig. 5, the sesamin derivative SD1 is equivalent to the known antioxidants sesamin, Ebixa and Exelon in suppressing the release of one of the nitrogen oxides caused by LPS, and shows that the sesamin derivative SD1 protects nerve cells from injury. In terms of it, it does have significant effects.

The degree of lipid peroxidation of sesamin and sesamin derivatives is due to the large amount of unsaturated fatty acids in the nerve cell membrane, which are easily denatured by free radical attack; the oxidation of lipids is a chain reaction initiated by free radicals, and the lipid oxidation is cleavage. The oxidation products (especially malondialdehyde, MDA molecules) are highly active in nature and are highly reactive with biomolecules. Moreover, it is known that the starch-like protein β (Aβ protein) causes an increase in the concentration of lipid peroxide in the brain. Therefore, in this experiment, we tested the lipid oxide malondialdehyde MDA in the PC12 by the treatment group and the control group. The difference in content indicates the extent and efficacy of sesamin in inhibiting the formation of peroxides in brain neurons.

The results are shown in Figure 6. The results of this experiment confirmed that the concentration of malondialdehyde (MDA) in the neuronal cell PC12 treated with the sesamin derivative SD1 was higher than that of the untreated and sesamin (10 μM) or Exelon (10, 50 μM). The treated nerve cell PC12 was significantly reduced, confirming that the sesamin derivative SD1 has an excellent anti-lipid peroxidation function.

Based on the above results, the sesamin derivative SD1 has excellent protective effects on nerve cells, and is particularly useful for protecting nerve cell damage caused by epilepsy and for protecting abnormal accumulation of starch-like protein (Aβ) in nerve cells. Caused by nerve damage.

Claims (7)

A use of a sesamin derivative for the preparation of a neuroprotective drug, wherein the sesamin derivative is 2,3-bis(3-methoxybenzyl)butan-1,4-diol. The use according to the first aspect of the patent application, wherein the neuroprotective system is selected from the group consisting of protecting against excessive excitation damage of nerve cells, increasing nerve cell survival rate, inhibiting calcium ion release from nerve cells, inhibiting nerve cell apoptosis, and inhibiting glial cells. Nitric oxide release and inhibition of neuronal lipid peroxidation. According to the use of the first aspect of the patent application, it is used for the preparation of a medicament for protecting nerve cell damage caused by epilepsy. According to the use of the first aspect of the patent application, it is used for the preparation of a medicament for treating epilepsy. According to the use of the first aspect of the patent application, wherein the neuroprotective system is selected from the group consisting of protecting nerve cells from abnormal damage caused by abnormal accumulation of starch β (Aβ) protein and increasing abnormal accumulation of starch-like protein. A group of nerve cell viability. According to the use of the first aspect of the patent application, it is for the preparation of a medicament for alleviating the toxicity of Aβ protein to nerve cells. According to the use of the first aspect of the patent application, it is for the preparation of a medicament for preventing and/or treating Alzheimer's disease.