• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present disclosure provides use of rutin as a GABAA receptor inhibitor, and belongs to the field of GABAA receptor inhibitors. The rutin can bind to a GABAA receptor. With a binding rate of 100% with the GABAA receptor, rutin achieves 5 a comparable effect to a positive control drug of diazepam. Through the cell patch clamp experiment, it is found that rutin can inhibit the chloride ion current of GABAA channels. Based on the above experiments, it is believed that rutin can be used as a GABAA receptor inhibitor for use in the treatment 10 of neurological disorders.
    公开号:NL2027853A
    申请号:NL2027853
    申请日:2021-03-29
    公开日:2021-06-07
    发明作者:Song Chunhong；Qiao Mingqi；Xia Xiaowen；Wang Jieqiong；Xue Ling；Wang Haijun；Wang Xiangjun；Ma Mingyu
    申请人:Univ Shandong Traditional Chinese Medicine；
    IPC主号:
    专利说明:

    USE OF RUTIN IN PREPARATION OF GABA, RECEPTOR INHIBITOR
    TECHNICAL FIELD The present disclosure belongs to the field of GABA, receptor inhibitors, and particularly relates to use of rutin in the preparation of a GABA, receptor inhibitor.
    BACKGROUND Rutin is also known as rutoside, vitamin P, quercetin rutinoside, violaquercitrin, birutan, sophorin, rutin trihydrate, or eldrin. Rutin is present in many plants, such as Ruta graveolens leaves, Zizyphus jujuba, Prunus armeniaca, orange peel, Solanum lycopersicum, and Fagopyrum esculentum flowers. With a wide range of sources, rutin can be extracted from plants or can be synthesized artificially. Rutin can be used as an edible antioxidant, a nutritional enhancer, and the like. Rutin has the vitamin P-like, anti- inflammatory, hypolipidemic, and antiviral effects.
    The GABA, receptor, also known as y-aminobutyric acid type A receptor, is an ionotropic receptor and a ligand- gated ion channel. GABA is widely distributed in the brain of mammals and regulates various functions of the brain via a protein complex called GABA, receptor. The GABA, receptor causes changes in chloride conductivity and membrane polarization. Many types of GABAa receptor ligands are disclosed at present, including zolpidem and zaleplon among non-benzodiazepines; zopiclone among pyrrolones; and so on.
    There is no report that rutin can be used as a GABA, receptor inhibitor.
    SUMMARY In view of this, the present disclosure is intended to provide use of rutin in the preparation of a GABA, receptor inhibitor. Rutin can bind to a GABA, receptor, with a binding rate of 100%.
    In order to realize the objective of the present disclosure, the present disclosure provides the following technical solutions.
    The present disclosure provides use of rutin in the preparation of a GABA, receptor inhibitor. Preferably, the GABA, receptor inhibitor may be a liquid preparation. Preferably, the rutin may have a concentration S 10% mol/L in the GABA, receptor inhibitor. Preferably, the rutin may have a concentration of 10% mol/L to 10% mol/L in the GABA, receptor inhibitor. Preferably, a solvent for the GABA, receptor inhibitor may be dimethyl sulfoxide (DMSO). Preferably, the rutin may have a purity of 2 99%. The present disclosure provides rutin for use in the treatment of neurological disorders. Preferably, the neurological disorders may include anxiety, insomnia, premenstrual dysphoric disorder (PMDD), and depression. Beneficial effects of the present disclosure: The present disclosure provides use of rutin in the preparation of a GABA, receptor inhibitor, and the rutin can bind to a GABA, receptor. With a binding rate of 100% with the GABA, receptor, rutin achieves a comparable effect to a positive ligand of diazepam. Therefore, rutin can be used as a GABA, receptor inhibitor for use in the treatment of neurological disorders.
    BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows an agonistic effect of the endogenous ligand GABA on the GABA receptor current and a recovery time curve thereof, where, A shows the effect of the perfusion of GABA at different concentrations on the GABA, receptor current; B shows a fitting curve; C shows the influence of different washing times on the current; and D is a broken line graph illustrating current changes at different washing times. FIG. 2 shows the gradient inhibition of the positive compound (bicuculline) on the GABAA receptor currents, where, A shows the effect of bicuculline at different concentrations on the GABA-induced GABA. receptor current;
    and B shows a bicuculline concentration-current fitting curve. FIG. 3 shows the inhibitory effect of rutin on the GABA, receptor current, where, A shows the effect of rutin at different concentrations on the GABA-induced GABA. receptor current; and B is a statistical chart illustrating the inhibitory effect of rutin at different concentrations on the GABA-induced GABA, receptor current.
    DETAILED DESCRIPTION The present disclosure provides use of rutin in the preparation of a GABA, receptor inhibitor. In the present disclosure, the GABA, receptor inhibitor may preferably be a liquid preparation in use. The present disclosure has no specific limitations on a solvent for the GABA, receptor inhibitor, and in a specific implementation of the present disclosure, a solvent for the GABA, receptor inhibitor may preferably be DMSO. The DMSO may preferably be purchased from Solarbio, with Item No. D8371. In the present disclosure, the rutin may have a purity 2 99%. In the present disclosure, the rutin may preferably be purchased from Shanghai Yuanye Biological Co., Ltd., with batch No. Y16M9S61523. In the present disclosure, the rutin may be dissolved in DMSO to obtain the GABA, receptor inhibitor. In the present disclosure, in the GABA, receptor inhibitor, the rutin may have a concentration preferably S 107% mol/L and more preferably of 10% mol/L to 10% mol/L; and specifically may have a concentration of 107% mol/L, 10% mol/L, 106 mol/L, 107 mol/L, or 107% mol/L. The present disclosure also provides rutin for use in the treatment of neurclogical disorders. In the present disclosure, the rutin GABA; receptor inhibitor can treat neurological disorders, and the neurological disorders may preferably include anxiety and insomnia. The technical sclutions provided by the present disclosure will be described in detail below with reference to examples, but the examples should not be construed as limiting the claimed scope of the present disclosure.
    Example 1 Single rutin component: purity: 99%, purchased from Shanghai Yuanye Biological Co., Ltd., batch No. Y16M2561523; which was used for a ligand-receptor binding experiment. The rutin component was dissolved in DMSO (Solarbio, D8371) to obtain a solution with a concentration of 2 x 107% mol/L to 2 x 107% mol/L for the experiment.
    Male or female rats at 180 g to 220 g: purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license No.: SCXK (Jing) 2012-0001.
    The rats were sacrificed by cervical dislocation, and the whole brain was collected, frozen in liquid nitrogen, and then placed in a 0.32 mol/L sucrose solution. The sucrose solution and the whole brain tissue have a volume-mass ratio of 20 mL : 1 g. Then the tissue was homogenized for 10 s with a tissue homogenizer (Optima, L100xp) and then placed on ice for 30 s, and the operation was repeated 3 to 4 times. A resulting homogenate was centrifuged for 10 min at 4°C and 1,000 g, and a resulting precipitate was discarded. A resulting supernatant was centrifuged for 20 min at 4°C and 20,000 g, a resulting supernatant was discarded, and a precipitate was washed once with water. A resulting solution was centrifuged once again for 20 min at 20,000 g, a resulting supernatant was discarded, and a precipitate was dissolved in 50 mmol/L Tris-HCl (pH 7.4) to achieve a protein concentration of 0.8 mg/mL to 1.6 mg/mL, so that a rat cortical neuronal membrane was obtained, which was immediately cryopreserved at -80°C. The protein concentration was determined by a protein concentration determination kit (BCA kit).
    1. The prepared rat cortical neuronal membrane was taken and added, as a membrane receptor, to various reaction solutions according to the following order and proportions: a: total binding tube: 48 uL of membrane receptor + 1 uL of DMSO + 1 uL of 3H-flunitrazepam (PerkinElmer, USA, 2461442), concentration: 2.64 x 107% mol/L;
    b: non-specific tube: 48 uL of membrane receptor + 1 uL of diazepam (1076 mol/L) + 1 uL of 3H-flunitrazepan, concentration: 2.64 x 10% mol/L; c: single traditional Chinese medicine (TCM) component 5 tube: 48 ul of membrane receptor + 1 uL of single TCM component (drug concentration: 108 mol/L to 107% mol/L) + 1 uL of 3H-flunitrazepam (PerkinElmer, USA, 2461442), concentration: 2.64 x 107% mol/L.
    2. After the radicligand of 3H-flunitrazepam was added to each of the above tubes, the tubes were immediately incubated for 2 h in a shaker at 37°C.
    3. The solution in the tube was subjected to suction filtration to obtain a filter cake on glass fiber filter paper {Shanghai Xingya Purification Material Factory, Q/IEFJ01-1997), and the filter cake was dried for 0.5 h and added with a scintillation solution (preparation: 2-phenyl- 5-(4-biphenyl)-1, 3, 4-oxadiazole (PBD) was dissolved in xylene to obtain a solution with a concentration of 8 g/L). Then counting was conducted with an XH-6925 liquid scintillation counter (Xi'an Nuclear Instrument Factory, No. 022).
    4. An inhibition rate of each compound against the isotopic ligand by binding was calculated according to the following formula: inhibition rate = (total binding tube (cpm) - TCM component tube (cpm))}/{total binding tube (cpm) - non- specific binding tube (cpm) ) Results were shown in Table 1. During a concentration range of 10% mol/L to 10% mol/L, rutin achieved an inhibition rate close to 1, exhibiting a comparable effect to the positive control drug of diazepam.
    Table 1 Inhibition rates of rutin during a concentration range of 107% mol/L to 107% mol/L
    Molar Rutin Rutin Rutin Rutin Rutin Diazepam concentration/mol/L 5 9 Blank (2 x 107%) | (2 x 1075) | (2 x 1076) | (2 x 1077) | (2 =x 107%) (107%) 2490 1 1854 + 2202 = 2322 1 1832 + 7971 + {1873.5 14 Radiocactivity/CPM
    545.89 38.18 670.33 441.23 21.21 1076.22 204.35 ene | |e EEN
    : It can be known from the above example that the rutin of the present disclosure can bind to a GABA, receptor, and with a binding rate of 100% with the GABA, receptor, the rutin achieves a comparable effect to the positive drug of diazepam. Therefore, the rutin can be used as a GABA, receptor inhibitor for use in the treatment of neurological disorders.
    Cell patch clamp experiment data Experimental method Cultivation of primary neuronal cells 1) Glass slides were treated with 0.1% gelatin on a clean bench, which were cleaned 3 times with a serum-free medium before use and then placed in a 3.5 cm petri dish for later use.
    2) The head skin of newborn rats at an age of 1 d to 3 d was sterilized with 75% ethanol, and then the skin, muscle, and subcutaneous fascia were cut open successively to take out the brain under aseptic conditions, which was placed into a pre-cooled balanced salt solution (BSS) of artificial cerebrospinal fluid (ACSF).
    3) The meninges were removed under a dissecting microscope, and the embryonic cerebral cortex (3 to 4 pairs of cerebral cortex) was isolated and taken out.
    4) The cortex was transferred to another small clean petri dish with 1 ml of ACSF, and the tissue was cut to a size of about 1 mm.
    5) 2 ml of a 0.253 trypsin-EDTA digestion solution was added, and a resulting solution was subjected to digestion for 5 min in a 37°C water bath.
    6) The digestion solution was taken out, an inoculation medium was added to stop the digestion, and a resulting mixture was placed in a 37°C incubator.
    7) The steps 4) and 5) were repeated 3 to 4 times for incompletely-digested parts.
    8) Mixture solutions of tissue blocks and cells obtained from the digestions each were filtered through a
    70 um filter mesh, and a resulting filtrate was collected in a new petri dish.
    9) Cell suspensions obtained from the filtration were combined and transferred to a 15 ml centrifuge tube, and then centrifuged at 800 rpm for 5 min. A resulting supernatant was discarded, then 5 ml to 10 ml of fresh medium was added to the tube, and a resulting mixture was gently pipetted up and down to obtain a single cell suspension.
    10) Cells were counted, and according to a cell density adjusted to 1 x 10%ml, the cells were inoculated to the petri dish with a glass slide and then cultivated in an incubator at 37°C and 5% CO». 24 h later, the medium was replaced with a maintenance medium.
    11} 7 d after the cultivation, the patch clamp experiment could be conducted.
    Patch clamp experiment 1) A rutin solution was serially diluted with extracellular fluid (ECF) to 100 uM, 300 uM, and 1,000 uM, and a positive control drug (bicuculline) solution was diluted with ECF to 30 uM, with an inhibition rate of 50.74 t 2.63% (n = 26).
    2) At room temperature (25°C), a glass slide was taken and cleaned twice with ECF, and then placed on an object stage of an inverted microscope.
    3) The whole-cell suction-breaking patch clamp technology was adopted, a resistance at the tip of a borosilicate glass microelectrode was set to 2 MQ to 6 MQ, and in the Gap-free mode, a membrane potential was clamped at -60 mV. After the clamp was completed, recording was stably conducted for 1 min in the ECF. When a GABA, current amplitude decreases and desensitization begins after administration by paracellular perfusion, it is considered that a drug action reaches a steady state. During the operation, it should be ensured that, in the experiment, a membrane resistance is greater than 1,000 MQ, and a leakage current is less than 10% of an ion channel current.
    Data Analysis Raw data were recorded using Clampex 10.7 and stored as *abf files in a computer network system of the Scope Research Institute.
    Data were acquired and analyzed with pCLAMP 10.7 software.
    Peak current values before the compound was added were used to calculate an average peak value, which was adopted as a control current amplitude.
    Peak current values after the compound was added were used to calculate an average peak value, which was adopted as a current amplitude after inhibition.
    An inhibition rate of a test compound against the GABA receptor current was calculated according to the following equation: inhibition rate (3) = {1- (residual current amplitude) / {control current amplitude)} * 100. The above calculation method was used to obtain an inhibition rate of a test compound against the GABA, receptor current (mean + standard error).
    Experimental results Table 2 Compound information Name of test Weight Molecular | Solvent | Concentration Max imum Inhibition rate compound (mg) weight of stock detection at maximum (g/mol) solution (mM) concentration detection concentration (%)
    610.52 | DMSO 1000 u 34.01 # 3.24 y—aminobutyric | 20 103.1 H:© 30 acid
    The perfusion of GABA could induce a significant inward current. The successive perfusion of 1 uM, 3 uM, 10 uM, 30 uM, and 100 uM GABA solutions could activate GABA, receptor currents in a gradient. According to a fitting curve, the EC: was 3.95 + 10.63 uM, and in this experiment, the current induced at 30 uM GABA was always adopted as a control. The GABA, receptor was a ligand-gated ion channel. In order to determine a recovery time after desensitization, 30 pM GABA was administered, then washing was conducted for 1 min, 2 min, 3 min, 4 min, and 5 min, and 30 pM GABA was administered once again. Currents after the second administration were compared with that after the first administration in size, and it can be seen that the GABA was completely washed away after 5 min of washing, as shown in FIG. 1.
    The perfusion of GABA could induce a significant inward current. The successive perfusion of 1 uM, 3 uM, 10 uM, 30 uM, and 100 uM bicuculline could inhibit GABA, receptor currents in a gradient. According to a fitting curve, the IC was 38.20 + 37.60 uM, as shown in FIG. 2.
    The perfusion of 30 uM GABA could induce a significant inward current. During the simultaneous perfusion of 30 uM GABA and rutin {100/300/1,000 uM), an induced current was significantly reduced, as shown in FIG. 3.
    In summary, it can be seen from the whole-cell patch clamp detection that rutin exhibits an inhibitory effect on the GABA, receptor current to some degree; and the positive control compound (30 pM bicuculline) exhibits an inhibition rate of 50.74 + 2.63% (n = 26) on the GABA, receptor channel current.
    The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.
    权利要求:
    Claims (8)
    [1]
    Use of rutin in the preparation of a GABAa receptor inhibitor.
    [2]
    Use according to claim 1, wherein the GABA receptor inhibitor is a liquid preparation.
    [3]
    Use according to claim 2, wherein the rutin has a concentration of £107% mol/L in the GABAx receptor inhibitor.
    [4]
    Use according to claim 3, wherein the rutin has a concentration of 107% mol/L to 10% mol/L in the GABA4 receptor inhibitor.
    [5]
    Use according to claim 2, wherein a solvent for the GABA 1 receptor inhibitor is dimethyl sulfoxide (DMSO).
    [6]
    Use according to claim 1, wherein the rutin has a purity of 2 99%.
    [7]
    7. Rutin for use in treating neurological disorders.
    [8]
    The rutin for use according to claim 7, wherein the neurological disorders include anxiety, insomnia, premenstrual dysphoric disorder (PMDD), and depression.
    -0-0-0-0-
    类似技术:
    公开号 | 公开日 | 专利标题
    Lord et al.1974|Inhibitors of haemopoietic cell proliferation?: Specificity of action within the haemopoietic system
    CN106344595A|2017-01-25|Application of sodium alginate oligose and derivative to treatment of pain
    NL2027853A|2021-06-07|Use of rutin in preparation of gabaa receptor inhibitor
    CN105418789A|2016-03-23|Method for preparing low-molecular oyster polysaccharide with immune adjustment and anti-tumor activity
    TW201416084A|2014-05-01|Immunemodulating agent
    Rabot et al.2007|Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue
    Richardson et al.1959|Action of polylysine on some ascites tumors in mice.
    JP2003504410A|2003-02-04|Algal proteoglycan extract and method of using same
    KR101097231B1|2011-12-21|A composition for proliferation and differenciation of neural stem cells or neural precursor cells
    CN104587274B|2018-09-14|A kind of leaf of bamboo homo-orientin extract
    CN111228255A|2020-06-05|Application of quercetin in preparation of GABAA receptor inhibitor
    CN110772481A|2020-02-11|Application of hydrogen sulfide modified mesenchymal stem cell outer vesicle in hypoxic ischemic brain injury
    Zhang et al.2020|Transplantation of microencapsulated neural stem cells inhibits neuropathic pain mediated by P2X7 receptor overexpression
    RU2414914C1|2011-03-27|Agent for supporting therapy and method for making agent of frozen-dried urchin hardroe
    US20180318291A1|2018-11-08|Use of aminothiazole myd88 specific inhibitor tjm2010-5
    JP6969041B2|2021-11-24|Vascular Endothelial Nitric Oxide Synthetic Enzyme Production Promoter and Oral Composition
    CN107375479A|2017-11-24|Dachengqi decoction is preparing the application in improving the medicine for senile dementia as caused by maincenter inflammation
    JP2002537267A|2002-11-05|High molecular weight extract of CONVOLVULUSARVENSIS
    CN111991412B|2021-11-05|Medicine for treating Parkinson's disease
    JP6868854B2|2021-05-12|Method for manufacturing anti-obesity agent and food and drink composition
    CN109701024B|2020-12-11|New application of BK channel opener
    CN107151695B|2021-11-09|PiRNA combination for detecting acute myocardial ischemia diseases and detection method and application thereof
    CN113304088A|2021-08-27|Preparation method of bletilla striata extract with repairing and whitening effects and bletilla striata extract
    CN112843237A|2021-05-28|Novel leukocyte extract mixed factor and preparation method thereof
    CN112315947A|2021-02-05|Application of exosome loaded with curcumin in preparation of skin care product or skin injury repairing medicine
    同族专利:
    公开号 | 公开日
    CN111297881A|2020-06-19|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN202010243129.0A|CN111297881A|2020-03-31|2020-03-31|Application of rutin in preparation of GABAA receptor inhibitor|
    [返回顶部]