Quassin compounds extracted from euphorbia pulex and their application in the preparation of drugs against neurodegenerative diseases

Abstract

The present invention provides quassin compounds extracted from safflower and their application in the preparation of anti-neurodegenerative disease drugs, belonging to the field of pharmaceutical technology. The present invention separates and identifies 7 new quassin compounds from safflower. In vitro neuroprotective activity experiments show that compounds 1, 2, 4 and 5 have significant protective activity against 6-OHDA-induced PC12 cells; in vivo neuroprotective activity experiments show that compounds 2 and 4 can increase PINK1 B9 The present invention shows that compounds 2 and 4 simultaneously exhibit significant in vivo and in vitro neuron protection activities and can be further developed for the treatment of neurodegenerative diseases such as Parkinson's disease.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to quassin compounds extracted from euphorbia cerasifera and application thereof in preparing medicines for resisting neurodegenerative diseases. Background Art

[0002] Parkinson's disease (PD), also known as Parkinson's paralysis, is the second largest neurodegenerative disease after Alzheimer's disease. The main pathological characteristics of PD are the loss of dopaminergic neurons in the substantia nigra compacta of the midbrain, the aggregation of α-synuclein in neurons to form Lewy bodies, and the lack of dopamine in the striatum; its clinical symptoms are divided into motor symptoms (bradykinesia, resting tremor and posture and gait disorders, etc.) and non-motor symptoms (loss of smell, constipation, cognitive dysfunction and sleep disorders, etc.). Methods for treating Parkinson's disease include drug therapy, surgical treatment, stem cell transplantation and gene therapy, among which drug therapy is the mainstream treatment method but can only treat symptoms, cannot delay or prevent disease tension, and long-term use will produce serious side effects. Therefore, there is an urgent need to develop new and more effective drugs targeting new targets.

[0003] PD is divided into two categories: sporadic and familial, of which the former accounts for 90% of all patients. Its pathogenic mechanism has not yet been elucidated, and it may be the result of the combined effects of abnormal cell-autonomous regulation and cell-non-autonomous regulation. Six genes have been found to be associated with autosomal dominant or recessive forms of PD, namely SNCA, PARKIN, PINK1 / PARK6, PARK8 / LRRK2, DJ-1 and UCH-L1. Among them, PINK1 gene mutation is an important cause of autosomal recessive early-onset PD, which exists in both sporadic and familial PD patients. The PINK1 protein it encodes is a mitochondrial-targeted serine / threonine kinase that plays an important role in mitochondrial autophagy. It can continuously detect the state of mitochondria, identify damaged mitochondria, and eliminate them through mitochondrial autophagy to maintain mitochondrial homeostasis. When the PINK gene is mutated or its function is impaired, it will lead to mitochondrial dysfunction, reduced function, structural damage and abnormal accumulation, affecting neuronal function, thereby causing PD and other neurodegenerative diseases. Therefore, PINK1 is not only an important protein that regulates mitochondrial autophagy, but also a potential target for the development of anti-PD drugs.

[0004] Harrisonia perforata (Bl.) Merr. is a plant of the genus Harrisonia in the family Simulaceae. There are four species of this genus in the world, distributed in tropical Africa, tropical Asia and Oceania. Only one species is produced in my country, mainly distributed in Fujian, Guangdong and Hainan; it is commonly found in low-altitude shrubs and sparse forests. So far, there has been no research report on the anti-PD activity of Harrisonia perforata. Summary of the invention

[0005] In view of this, the present invention aims to provide quassin compounds extracted from euphorbia cerevisiae and their use in the preparation of drugs against neurodegenerative diseases. The quassin compounds extracted from euphorbia cerevisiae provided by the present invention have good in vivo and in vitro neuroprotective activity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a quassin compound extracted from oxalis fruit, which has a structure shown in any one of compounds 1 to 7:

[0008] Compound 1; in compound 1, R1=OMe, R2=OAc;

[0009] Compound 2; in compound 2, R1 = OMe, R2 = OH;

[0010] Compound 3; in compound 3, R1=OH, R2=H;

[0011] Compound 4; in compound 4, R=OH;

[0012] Compound 5;

[0013] Compound 6;

[0014] Compound 7.

[0015] The present invention provides a method for extracting quassin compounds extracted from the above-mentioned euphorbia pulex, comprising the following steps:

[0016] (1) using methanol to reflux extract the ground taro branches to obtain a methanol extract;

[0017] (2) concentrating the methanol extract under reduced pressure to obtain a crude extract;

[0018] (3) dispersing the crude extract with water, performing a first extraction on the obtained dispersion using petroleum ether to obtain a petroleum ether extract; and performing a second extraction on the petroleum ether extract using ethyl acetate to obtain an ethyl acetate extract;

[0019] (4) subjecting the ethyl acetate extract to small pore resin chromatography and performing a first gradient elution, wherein the procedure of the first gradient elution comprises: sequentially eluting with a 30% by volume methanol aqueous solution, a 50% by volume methanol aqueous solution, a 70% by volume methanol aqueous solution, a 90% by volume methanol aqueous solution and anhydrous methanol to obtain five eluents, which are named Fr1, Fr2, Fr3, Fr4 and Fr5 respectively;

[0020] (5) subjecting the Fr3 to silica gel column chromatography and performing a second gradient elution, wherein the procedure of the second gradient elution comprises: sequentially using a petroleum ether-ethyl acetate solution with a volume ratio of 10:1, a petroleum ether-ethyl acetate solution with a volume ratio of 5:1, a petroleum ether-ethyl acetate solution with a volume ratio of 3:1, a petroleum ether-ethyl acetate solution with a volume ratio of 2:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:2, a petroleum ether-ethyl acetate solution with a volume ratio of 1:4, pure ethyl acetate and methanol for elution to obtain nine fractions, which are named Fr3-1, Fr3-2, Fr3-3, Fr3-4, Fr3-5, Fr3-6, Fr3-7, Fr3-8 and Fr3-9 in sequence;

[0021] (6) performing a first chromatography separation on the Fr3-3 using Sephadex LH-20 methanol gel column chromatography to obtain a first chromatographic solution; performing semi-preparative high performance liquid chromatography separation on the first chromatographic solution, and performing a third gradient elution to obtain compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6;

[0022] The eluent of the first chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1;

[0023] The procedure of the third gradient elution includes: eluting with 30% by volume acetonitrile aqueous solution, 35% by volume acetonitrile aqueous solution, 40% by volume acetonitrile aqueous solution, 45% by volume acetonitrile aqueous solution, 50% by volume acetonitrile aqueous solution, and 55% by volume acetonitrile aqueous solution in sequence;

[0024] (7) performing a second chromatography separation on Fr3-2 using a cross-linked dextran LH-20 gel column to obtain a second chromatographic solution; performing high performance liquid chromatography separation on the second chromatographic solution, and performing a fourth elution to obtain compound 7;

[0025] The eluent for the second chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1;

[0026] The eluent for the fourth elution is an acetonitrile aqueous solution with a volume fraction of 54%;

[0027] There is no time sequence restriction between steps (6) and (7).

[0028] Preferably, in step (4), the conditions for small-pore resin chromatography separation include:

[0029] Small pore resin column size: 120mm*1000mm;

[0030] Eluent flow rate: 25 mL / min;

[0031] Elution flow rate: 10 L per gradient.

[0032] Preferably, in step (6), the conditions for the first chromatographic separation include:

[0033] Gel column size: 20mm*200mm;

[0034] The flow rate of the eluent was 0.3 mL / min;

[0035] The amount of eluent used is 1L;

[0036] The conditions for the semi-preparative HPLC separation include:

[0037] Chromatographic column: Waters XSelect CSH C-18 column, specification: 5 μm, 10×250 mm;

[0038] The mobile phase comprises: mobile phase A and mobile phase B; the mobile phase A is pure water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 70-45%; the volume fraction of the mobile phase B is 30-55%; each elution is 10 minutes, and then 5% of the mobile phase B is added; elution is performed for another 10 minutes, and then 5% of the mobile phase B is added, and so on, until the mobile phase B is increased to 55% and eluted for 10 minutes;

[0039] The flow rate of the mobile phase was 3.0 mL min -1 ;

[0040] The column temperature was 25°C;

[0041] The injection volume was 30 μL each time.

[0042] Preferably, in step (7), the conditions for the second chromatographic separation include:

[0043] Gel column size: 20mm*200mm;

[0044] The flow rate of the eluent was 0.3 mL / min;

[0045] The amount of eluent used is 1L;

[0046] The conditions for the HPLC separation include:

[0047] Chromatographic column: Waters XSelect CSH C-18 column, specification: 5 μm, 10×250 mm;

[0048] The mobile phase includes: mobile phase A and mobile phase B; the mobile phase A is pure water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 54%; the volume fraction of the mobile phase B is 46%;

[0049] The flow rate of the mobile phase was 3.0 mL min -1 ;

[0050] The column temperature was 25°C;

[0051] The injection volume was 30 μL each time.

[0052] The invention provides application of quassin compounds extracted from mulberry and euphorbia pulex fruit in preparing drugs for resisting neurodegenerative diseases.

[0053] Preferably, the anti-neurodegenerative disease drug is an anti-Parkinson's disease drug.

[0054] The present invention provides an anti-Parkinson's disease pharmaceutical composition, comprising an active ingredient and a pharmaceutically acceptable carrier and / or excipient;

[0055] The active ingredients include the quassin compounds extracted from the above-mentioned cow tendon fruit.

[0056] Preferably, the mass content of the active ingredient in the anti-Parkinson's disease pharmaceutical composition is 0.1-99%.

[0057] Preferably, the dosage form of the anti-Parkinson's disease pharmaceutical composition is one or more of a liquid preparation, a solid preparation, a spray and an aerosol.

[0058] The present invention provides quassin compounds extracted from the fruit of the Chinese yam, which have the structure shown in any one of compounds 1 to 7. The present invention separates and identifies 7 new quassin compounds from the fruit of the Chinese yam, and in vitro experiments show that they all have certain protective activity against 6-OHDA-induced PC12 cells, among which compounds 1, 2, 4 and 5 have significant activity; in vivo experiments show that compounds 2 and 4 can increase the expression of PINK1 B9 The present invention shows that compounds 2 and 4 exhibit significant in vivo and in vitro neuroprotective activities and can be further developed for the treatment of neurodegenerative diseases such as Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1is the protective effect of the compound on 6-OHDA-induced PC12 cells;

[0060] Figure 2 Compounds for PINK1 B9 Protection effect on fruit flies. DETAILED DESCRIPTION

[0061] The present invention provides a quassin compound extracted from oxalis fruit, which has a structure shown in any one of compounds 1 to 7:

[0062] Compound 1; in compound 1, R1=OMe, R2=OAc; Compound 2; in compound 2, R1 = OMe, R2 = OH; Compound 3; in compound 3, R1=OH, R2=H;

[0063] Compound 4; in compound 4, R=OH;

[0064] Compound 5;

[0065] Compound 6;

[0066] Compound 7.

[0067] The present invention provides a method for extracting quassin compounds extracted from the above-mentioned euphorbia pulex, comprising the following steps:

[0068] (1) using methanol to reflux extract the ground taro branches to obtain a methanol extract;

[0069] (2) concentrating the methanol extract under reduced pressure to obtain a crude extract;

[0070] (3) dispersing the crude extract with water, performing a first extraction on the obtained dispersion using petroleum ether to obtain a petroleum ether extract; and performing a second extraction on the petroleum ether extract using ethyl acetate to obtain an ethyl acetate extract;

[0071] (4) subjecting the ethyl acetate extract to small pore resin chromatography and performing a first gradient elution, wherein the procedure of the first gradient elution comprises: sequentially eluting with a 30% by volume methanol aqueous solution, a 50% by volume methanol aqueous solution, a 70% by volume methanol aqueous solution, a 90% by volume methanol aqueous solution and anhydrous methanol to obtain five eluents, which are named Fr1, Fr2, Fr3, Fr4 and Fr5 respectively;

[0072] (5) subjecting the Fr3 to silica gel column chromatography and performing a second gradient elution, wherein the procedure of the second gradient elution comprises: sequentially using a petroleum ether-ethyl acetate solution with a volume ratio of 10:1, a petroleum ether-ethyl acetate solution with a volume ratio of 5:1, a petroleum ether-ethyl acetate solution with a volume ratio of 3:1, a petroleum ether-ethyl acetate solution with a volume ratio of 2:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:2, a petroleum ether-ethyl acetate solution with a volume ratio of 1:4, pure ethyl acetate and methanol for elution to obtain nine fractions, which are named Fr3-1, Fr3-2, Fr3-3, Fr3-4, Fr3-5, Fr3-6, Fr3-7, Fr3-8 and Fr3-9 in sequence;

[0073] (6) performing a first chromatography separation on Fr3-3 using Sephadex LH-20 methanol gel column chromatography to obtain a first chromatographic solution; performing semi-preparative high performance liquid chromatography separation on the first chromatographic solution, and performing a third gradient elution to obtain compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6;

[0074] The eluent of the first chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1;

[0075] The procedure of the third gradient elution includes: eluting with 30% by volume acetonitrile aqueous solution, 35% by volume acetonitrile aqueous solution, 40% by volume acetonitrile aqueous solution, 45% by volume acetonitrile aqueous solution, 50% by volume acetonitrile aqueous solution, and 55% by volume acetonitrile aqueous solution in sequence;

[0076] (7) performing a second chromatography separation on Fr3-2 using a cross-linked dextran LH-20 gel column to obtain a second chromatographic solution; performing high performance liquid chromatography separation on the second chromatographic solution, and performing a fourth elution to obtain compound 7;

[0077] The eluent for the second chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1;

[0078] The eluent for the fourth elution is an acetonitrile aqueous solution with a volume fraction of 54%;

[0079] There is no time sequence restriction between steps (6) and (7).

[0080] The present invention uses methanol to reflux extract the ground material of the schizonepeta truncatum branches to obtain a methanol extract. In the present invention, the particle size of the ground material of the schizonepeta truncatum is preferably 80 to 100 meshes. In the present invention, the concentration of the methanol is preferably 100%. In the present invention, the temperature of the reflux extraction is preferably the reflux temperature of methanol.

[0081] In the present invention, the number of reflux extraction is preferably three times, and the methanol extracts are combined after three refluxes. In the present invention, the time of a single reflux extraction is preferably 2.8 to 3.2 hours, more preferably 3 hours.

[0082] After obtaining the methanol extract, the present invention performs reduced pressure concentration on the methanol extract to obtain a crude extract. In the present invention, the pressure of the reduced pressure concentration is preferably 55 to 65 bar, more preferably 60 bar; the time is preferably 9.5 to 10.5 hours, more preferably 10 hours. The present invention recovers the methanol solvent through the reduced pressure concentration to obtain an extract, i.e., a crude extract. In the present invention, based on 1 kg of Beauveria oleifera branches, the mass of the extract is preferably 180 to 220 g, more preferably 200 g.

[0083] After obtaining the crude extract, the present invention disperses the crude extract with water, performs a first extraction on the obtained dispersion using petroleum ether to obtain a petroleum ether extract; and performs a second extraction on the petroleum ether extract using ethyl acetate to obtain an ethyl acetate extract. In the present invention, the mass ratio of the crude extract to water is preferably 1:8 to 1:12, more preferably 1:10. In the present invention, the volume ratio of the petroleum ether to the dispersion is preferably 1:0.8 to 1:1.2, more preferably 1:1; the role of the petroleum ether extraction is to remove low-grade grease.

[0084] In the present invention, the volume ratio of the ethyl acetate to the petroleum ether extract is preferably 1:0.8-1.2, more preferably 1:1, and the ethyl acetate extraction serves to enrich the quassin-containing fraction.

[0085] After obtaining the ethyl acetate extract, the present invention performs small-pore resin chromatography on the ethyl acetate extract and performs a first gradient elution. The procedure of the first gradient elution includes: sequentially using a methanol aqueous solution with a volume fraction of 30%, a methanol aqueous solution with a volume fraction of 50%, a methanol aqueous solution with a volume fraction of 70%, a methanol aqueous solution with a volume fraction of 90% and anhydrous methanol for elution, and collecting 5 parts of eluent, which are named Fr1, Fr2, Fr3, Fr4 and Fr5 in sequence. In the present invention, the small-pore resin is preferably an MCI small-pore resin. In the present invention, the mass ratio of the ethyl acetate extract to the volume of the small-pore resin is preferably 1:10. In the present invention, the volume of the eluent used in each gradient in the first gradient elution is preferably 10L, and the flow rate of the eluent is preferably 25mL / min.

[0086] After obtaining the Fr3, the present invention performs silica gel column chromatography separation on the Fr3 and performs a second gradient elution, wherein the second gradient elution procedure includes: sequentially using a petroleum ether-ethyl acetate solution with a volume ratio of 10:1, a petroleum ether-ethyl acetate solution with a volume ratio of 5:1, a petroleum ether-ethyl acetate solution with a volume ratio of 3:1, a petroleum ether-ethyl acetate solution with a volume ratio of 2:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:2, a petroleum ether-ethyl acetate solution with a volume ratio of 1:4, pure ethyl acetate and methanol for elution to obtain nine fractions, which are named Fr3-1, Fr3-2, Fr3-3, Fr3-4, Fr3-5, Fr3-6, Fr3-7, Fr3-8 and Fr3-9 in sequence. In the present invention, the conditions for the silica gel column chromatography separation preferably include:

[0087] Silica gel column size: 120mm*800mm;

[0088] Eluent flow rate: 50 mL / min;

[0089] Elution flow rate: 30 L per gradient.

[0090] In the present invention, the mass ratio of Fr3 to silica gel in the silica gel column is preferably 30:1.

[0091] In the present invention, the volume of the eluent used in each gradient in the second gradient elution is preferably 30 L, and the flow rate of the eluent is preferably 50 mL / min.

[0092] The present invention uses Sephadex LH-20 methanol gel column chromatography to perform a first chromatographic separation on Fr3-3 to obtain a first chromatographic liquid; the first chromatographic liquid is repeatedly subjected to semi-preparative high performance liquid chromatography separation, and a third gradient elution is performed to obtain compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6. In the present invention, the eluent of the first chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1; in the present invention, the conditions of the first chromatographic separation include:

[0093] Gel column size: 20mm*200mm;

[0094] The flow rate of the eluent was 0.3 mL / min;

[0095] The eluent was a 1:1 dichloromethane-methanol solution;

[0096] The amount of eluent used is 1L;

[0097] In the present invention, the third gradient elution procedure includes: the third gradient elution procedure includes: sequentially using 30% by volume acetonitrile aqueous solution, 35% by volume acetonitrile aqueous solution, 40% by volume acetonitrile aqueous solution, 45% by volume acetonitrile aqueous solution, 50% by volume acetonitrile aqueous solution, and 55% by volume acetonitrile aqueous solution for elution. In the present invention, the conditions for semi-preparative high performance liquid chromatography separation include:

[0098] Chromatographic column: Waters XSelect CSH C-18 column (5 μm, 10 × 250 mm);

[0099] The mobile phase includes: mobile phase A and mobile phase B; the mobile phase A is pure water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 70-45%; the volume fraction of the mobile phase B is 30-55%; each elution is 10 minutes, and then 5% of the mobile phase B is added; elution is performed for another 10 minutes, and then 5% of the mobile phase B is added, and so on, until the mobile phase B is increased to 55% and eluted for 10 minutes.

[0100] The flow rate of the mobile phase was 3.0 mL min -1 ;

[0101] The column temperature was 25°C;

[0102] The injection volume was 30 μL each time.

[0103] The present invention uses a cross-linked dextran LH-20 gel column to perform a second chromatographic separation on Fr3-2 to obtain a second chromatographic solution; the second chromatographic solution is subjected to high performance liquid chromatography separation, and a fourth elution is performed to obtain compound 7. In the present invention, the eluent of the second chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1; the conditions of the second chromatographic separation include:

[0104] Gel column size: 20mm*200mm;

[0105] The flow rate of the eluent was 0.3 mL / min;

[0106] The eluent was a 1:1 dichloromethane-methanol solution;

[0107] The amount of eluent used is 1L;

[0108] In the present invention, the eluent of the fourth elution is an acetonitrile aqueous solution with a volume fraction of 54%. The conditions for the high performance liquid chromatography separation include:

[0109] Chromatographic column: Waters XSelect CSH C-18 column (5 μm, 10 × 250 mm);

[0110] The mobile phase includes: mobile phase A and mobile phase B; the mobile phase A is pure water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 54%; and the volume fraction of the mobile phase B is 46%.

[0111] The flow rate of the mobile phase was 3.0 mL min -1 ;

[0112] The column temperature was 25°C;

[0113] The injection volume was 30 μL each time.

[0114] The present invention provides the quassin compounds extracted from the above-mentioned oxalis fruit or the use of the quassin compounds extracted from the above-mentioned oxalis fruit in the preparation of drugs for resisting neurodegenerative diseases.

[0115] In the present invention, the anti-neurodegenerative disease drug is preferably an anti-Parkinson's disease drug.

[0116] The present invention provides an anti-Parkinson's disease pharmaceutical composition, comprising an active ingredient and a pharmaceutically acceptable carrier and / or excipient. In the present invention, the active ingredient comprises the quassin compounds extracted from the above-mentioned oxalis fruit or the quassin compounds extracted from the oxalis fruit obtained by the above-mentioned extraction method. In the present invention, the mass content of the active ingredient in the anti-Parkinson's disease pharmaceutical composition is preferably 0.1 to 99%, more preferably 0.5 to 90%. The present invention has no special requirements for the specific types of the pharmaceutically acceptable carrier and / or excipient, and can use pharmaceutically acceptable, non-toxic and inert pharmaceutical carriers and / or excipients well known to those skilled in the art.

[0117] In the present invention, the dosage form of the anti-Parkinson's disease pharmaceutical composition is preferably one or more of a liquid preparation, a solid preparation, a spray and an aerosol. In the present invention, the liquid preparation is preferably one of an injection, a suspension, an emulsion, a solution and a syrup, and the solid preparation is preferably one of a tablet, a capsule, a granule and an granule.

[0118] In the present invention, the administration method of the anti-Parkinson's disease pharmaceutical composition is preferably one or more of injection, oral administration, sublingual administration and mucosal dialysis, and the injection is preferably one or more of intravenous injection, intravenous drip, intramuscular injection and subcutaneous injection.

[0119] The quassin compounds extracted from the cassia twig fruit provided by the present invention and their application in the preparation of anti-neurodegenerative disease drugs are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0120] Example 1

[0121] The plant sample of the twig ... The Fr3 (45.7 g) portion was chromatographed on a silica gel column with a size of 120 mm*800 mm. It was eluted with petroleum ether-ethyl acetate (10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:4), pure ethyl acetate and methanol. The eluent flow rate was 50 mL / min and the eluent flow rate was 30 L for each gradient. Nine fractions (Fr3-1 to Fr3-9) were obtained. Fr3-3 was further separated by Sephadex LH-20 methanol gel column chromatography (dichloromethane-methanol volume ratio of 1:1) and semi-preparative HPLC acetonitrile-water (30%-55%) to obtain compounds 1 (1.1 mg), 2 (0.9 mg), 3 (6 mg), 4 (72 mg), 5 (1.2 mg) and 6 (11.7 mg), wherein the size of the Sephadex LH-20 methanol gel column was 20 mm*200 mm, the flow rate of the eluent was 0.3 mL / min, and the amount of the eluent was 1 L; the semi-preparative HPLC column was Waters XSelect CSH C-18 column (5 μm, 10×250 mm), and the flow rate of the mobile phase was 3.0 mL·min -1 , column temperature was 25°C, and injection volume was 30 μL each time. Fr3.2 used cross-linked dextran LH-20 (dichloromethane-methanol volume ratio of 1:1) and HPLC acetonitrile-water (54%) to obtain 7 (5.9 mg), wherein the cross-linked dextran LH-20 gel column size was 20 mm*200 mm, the flow rate of the eluent was 0.3 mL / min, and the amount of eluent was 1 L; the HPLC column was Waters XSelect CSH C-18 column (5 μm, 10×250 mm), and the flow rate of the mobile phase was 3.0 mL·min -1 ; Column temperature was 25°C; injection volume was 30 μL each time.

[0122] The structures and characterization data of isolated compounds 1 to 7 are as follows:

[0123]

[0124] Compound 1: white amorphous powder; [α] 23 D -64.8 (c 0.08, MeOH); IR (KBr) ν max 3435, 2959, 2927, 2856, 1731, 1675, 1637, 1657, 1462, 1442, 1383, 1275, 1231, 1167, 1124, and 1038 cm -1 ; ESI-MS(+) m / z 418 [M+Na] + ; HR-ESI-MS(+) m / z 441.1879 [M+Na] + (calcd for C 23 H 30 O7Na, 441.1884). 1 1H NMR (CDCl3, 800 MHz) δ H : 4.60 (1H, dd, J = 12.3 Hz, 7.0 Hz, H-2), 1.34 (1H, dd, J = 12.3 Hz, 12.3 Hz, H-3α), 2.35 (1H, m, H-3β), 2.08 (1H, m, H-4), 1.50 (1H, m, H-5), 2.04 (1H, m, H-6α), 1.82 (1H, m, H-6β), 4.32 (1H, t, H-7), 3.30 (1H, s, H-9), 5.83 (1H, d, J = 1.20, H-12), 2.40 (1H, dd, J = 12.5, 6.6, H-14), 2.61 (1H, dd, J = 18.7 Hz, 12.5 Hz, H-15α), 2.95 (1H, d, J = 18.7 Hz, 6.6 Hz, H-15β), 0.96 (3H, d, J = 6.6 Hz, H-18), 4.74 (1H, d, J = 13.0 Hz, H-19a), 4.46 (1H, d, J = 13.0 Hz, H-19b), 2.12 (3H, s, 19-OAc(Me)), 1.25 (3H, s, H-20), 1.94 (3H, d, J = 1.2 Hz, H-21), 3.46 (3H, s, O-CH3); 13C NMR (CDCl3, 800MHz) δ: 207.2 (C-1), 81.9 (C-2), 44.4 (C-3), 29.2 (C-4), 47.4 (C-5) ,27.3(C-6),82.1(C-7),37.2(C-8),47.1(C-9),52.6(C-10),194.1(C-11),126.6( C-12),156.8(C-13),48.0(C-14),31.2(C-15),168.7(C-16),18.9(C-18),62.3(C -19),170.5(19-OAc),21.4(19-OAc(Me)),23.0(C-20),22.5(C-21),58.1(O-CH3).

[0125] Compound 2: white amorphous powder; [α] 23 D -46.2(c 0.06,MeOH); IR(KBr)νmax 3439,2963,2927,2873,2856,1723,1671,1636,1440,1383,1275,1251,1229,1059,992,978,890,and811cm-1; ESI-MS(+)m / z 347[M+H] + ;HR-ESI-MS(+)m / z 347.1840[M+H] + (calcd for C 20 H 27 O5,347.1853). 1H NMR (CDCl3, 600MHz) δ: 4.80-4.91 (1H, m, H-2), 1.14 (1H, d, J = 12.1Hz H-3α),2.04(1H,m,H-3β),1.85(1H,m,H-4),1.79-1.92(1H,m,H-5),2.48(1H,ddd,J=12.4Hz,6.8Hz,4.0Hz,H -6α),2.36(1H,dd,J=12.4Hz,6.8Hz,H-6β),4.31(1H,t,J=2.7Hz,H-7),3.20(1H,s,H-9),5.85(1H,d,J=1.20 ,H-12),2.02(1H,dd,J=6.7,12.4,H-14),2.60(1H,dd,J=18.7Hz,12.4Hz,H-15α),2.96(1H,dd,J=18.7Hz,6. 7Hz,H-15β),0.93(3H,d,J=6.5Hz,H-18),1.44(2H,s,H-19),1.19(3H,s,H-20),1.96(3H,d,J=1.0Hz,H-21); 13 C NMR (CDCl3, 600MHz) δ: 213.5 (C-1), 69.9 (C-2), 46.5 (C-3), 28.4 (C-4), 47.5 (C-5), 26.4 (C-6), 82.3 (C-7), 37.2 (C-8), 51.0 (C-9), 47.7 (C- 10),194.9(C-11),126.6(C-12),157.2(C-13),47.8(C-14),31.2(C-1 5),169.0(C-16),18.4(C-18),15.2(C-19),23.1(C-20),22.4(C-21).

[0126] Compound 3: white amorphous powder; [α] 23 D -93.2(c 0.05,MeOH); IR(KBr)νmax 3424,2961,2924,2853,1725,1661,1638,1469,1440,1383,1315,1256,1226,1110,1037,985,970,864,and 810cm-1; ESI-MS(+)m / z 399[M+Na] + ;HR-ESI-MS(+)m / z 399.1767[M+Na] + (calcd for C 21 H 28O6Na, 399.1778). 1 1H NMR (CDCl3, 800 MHz) δ: 5.17 (1H, dd, J = 12.7 Hz, 1.7 Hz, H-2), 1.31 (1H, d, J = 12.7 Hz H-3α), 2.33 (1H, ddd, J = 12.7 Hz, 6.6 Hz, 4.1 Hz, H-3β), 2.17 (1H, m, H-4), 1.38 (1H, m, H-5), 2.17 (1H, m, H-6α), 1.58 (1H, m, H-6β), 4.58 (1H, dd, J = 12.9 Hz, 6.6 Hz, H-7), 3.40 (1H, s, H-9), 5.94 (1H, d, J = 1.20, H-12), 2.43 (1H, dd, J = 12.7, 6.5, H-14), 2.43 (1H, dd, J = 18.6 Hz, 12.7 Hz, H-15α), 2.95 (1H, dd, J = 18.6 Hz, 6.5 Hz, H-15β), 0.99 (3H, d, J = 6.4 Hz, H-18), 4.12 (1H, d, J = 12.6 Hz, H-19a), 4.06 (1H, d, J = 12.6 Hz, H-19b), 2.01 (1H, s, 19-OH), 1.23 (3H, s, H-20), 2.00 (3H, d, J = 1.0 Hz, H-21), 3.43 (3H, s, O-CH3); 13C NMR (CDCl3, 800 MHz) δ: 208.8 (C-1), 82.1 (C-2), 43.3 (C-3), 29.4 (C-4), 46.1 (C-5), 27.0 (C-6), 82.2 (C-7), 37.2 (C-8), 48.4 (C-9), 51.1 (C-10), 200.2 (C-11), 126.7 (C-12), 158.3 (C-13), 48.5 (C-14), 30.7 (C-15), 168.4 (C-16), 18.9 (C-18), 60.8 (C-19), 23.8 (C-20), 22.4 (C-21), 58.0 (O-CH3).

[0127] Compound 4: White amorphous powder; [α] 23 D +88.5 (c 0.05, MeOH); IR (KBr) νmax 3428, 2960, 2925, 2876, 2852, 1732, 1670, 1664, 1638, 1461, 1440, 1383, 1250, 1114, 1080, 1040, 939, 868, and 860 cm -1 ; ESI-MS(+) m / z 375 [M + H]+ HR-ESI-MS(+)m / z 375.1794[M+H] + (calcdfor C 21 H 27 O6,375.1802). 1 H NMR(CDCl3,500MHz)δ:4.60(1H,dd,J12.7Hz,7.0Hz,H-3),4.31(1H,dd,J6). 12.7Hz,3.5Hz,1.4Hz,H-4),1.81(1H,ddd,J4.5Hz,9.4Hz,2.1Hz,H-5),2.0 9(1H,dt,J14.5Hz,3.5Hz,H-6α),1.58(1H,dd,J14.5Hz,2.1Hz,H-6β),4. 06(1H,t,J11.8Hz,H-7),3.24(1H,s,H-9),5.39(1H,d,J2.7,H-12),2.41( 1H,dd,J12.3,6.8,H-14),2.62(1H,dd,J18.6Hz,12.3Hz,H-15α),2.96(1). H,dd,J18.6Hz,6.8Hz,H-15β),1.16(3H,d,J6.8Hz,H-18),5.07(1H,dd,J) 11.9Hz,2.4Hz,H-19a),3.93(1H,dd,JS11.9Hz,2.4Hz,H-19b),1.67(1H,s, 19-OH), 1.21(3H,s,H-20), 1.98(3H,d,J1.0Hz,H-21),3.62(3H,s,O-CH3) 13 C NMR(CDCl3,500MHz)δ:199.5(C-1), 157.1(C-2), 150.5(C-3), 41.2(C-4), 38.0(C-5), 26.8(C-6), 60.9(C-7), 30.8(C-8), 32.4(C-9), 47.8(C-10),1 95.5(C-11), 115.9(C-12), 126.9(C-13), 47.4(C-14), 52.4(C-15), (C-16), 20.4(C-18), 81.8(C-19), 23.0(C-20), 22.2(C-21), 55.5(O-CH3).

[0128] Picture 5: Environmental Protection [α] 23 D-44.6(c 0.08,MeOH):IR(KBr)νmax 3435,2964,2926,2853,1743,1671,1647,1441,1384,1301,1279,1263,1230,1196,1114,1072,1053,952,and 860cm-1ESI-MS(+)m / z 379[M+H] + HR-ESI-MS(+)m / z 379.1511[M+Na] + (calcd.for C). 21 H 24 O5Na,379.1516). 1 H NMR(CD3OD,800MHz)δ:6.15(1H,s,H-3),3.24(1H,dd,J15.5Hz,6.7Hz,H-6α),2.58(1H,d,J15.5 Hz,H-6β), 4.46(1H,t,J2.8Hz,H-7),3.35(1H,s,H-9),5.82(1H,s,H-12),2.97(1H,dd,J16.9Hz, 2.0Hz,H-14),2.70(1H,dd,J16.9Hz,2.0Hz,H-15a),3.00(1H,dd,J16.9Hz,2.0Hz,H-15b),1.24 (3H,s,H-18),1.92(3H,s,H-19),1.64(3H,s,H-20),1.96(3H,d,J1.7,H-21),3.74(3H,s,O-CH3) 13 C NMR(CD3OD,800MHz)δ: 202.1(C-1), 158.3(C-2), 133.7(C-3), 150.7(C-4), 117.3(C-5), 33.5(C-6), 82.5(C-7), 28.3(C-8), 39.5(C-9), 49.3(C-10), 198.8(C-11), 125.1(C-12), 127.2(C-13), 49.4(C-14), 28.2(C-15), (C-16), 55.8(C-18), 18.3(C-19), 24.7(C-20), 21.4(C-21), 62.3(O-CH3).

[0129] Picture 6: Environmental Protection [α] 23 D+171.0(c 0.09,MeOH); IR(KBr)νmax 3434,3096,2934,2853,1733,1699,1678,1638,1622,1599,1439,1383,1247,1234,1218,1171,1044,977,930,and 892cm-1; ESI-MS(+)m / z 357[M+Na] + ;HR-ESI-MS(+)m / z 357.1704[M+H] + (calcd for C 21 H 25 O5,357.1697). 1 H NMR (CDCl3, 500MHz) δ: 6.07 (1H, s, H-3), 2.11 (1H, d, J = 2.8 Hz, H-5), 2.85 (1H, m, H-6α), 2.09 (1H, t, J = 2.8, H- 6β),4.38(1H,t,J=2.8Hz,H-7),3.04(1H,s,H-9),5.84(1H,S,H-12),2.37(1H,dd,J=12.0Hz,7.2Hz,H-14),2 .641(1H,dd,J=18.8Hz,12.0Hz,H-15a),2.97(1H,dd,J=18.8Hz,7.2Hz,H-15b),5.18(1H,d,J=1.7Hz,H-18a) ,4.95(1H,d,J=1.7HZ,H-18b),1.18(3H,s,H-19),1.38(3H,s,H-20),1.93(3H,s,H-21),3.36(3H,s,O-CH3); 13 CNMR (CDCl3, 500MHz) δ: 196.9 (C-1), 155.6 (C-2), 114.2 (C-3), 149.3 (C-4) ,50.9(C-5),40.6(C-6),82.0(C-7),37.6(C-8),45.2(C-9),46.3(C-10),1 95.0(C-11),127.0(C-12),141.5(C-13),47.3(C-14),31.1(C-15),169.0( C-16),113.2(C-18),13.6(C-19),25.6(C-20),22.3(C-21),55.5(O-CH3).

[0130] Compound 7: white amorphous powder; [α] 23 D-9.2(c 0.07,MeOH); IR(KBr)νmax 3411,2974,2918,2850,1754,1743,1684,1635,1576,1446,1415,1436,1326,1202,1046,and 986cm-1; ESI-MS(+)m / z 353[M+Na] + ;HR-ESI-MS(+)m / z353.1365[M+Na] + (calcd for C 19 H 22 O5Na,353.1359). 1 H NMR (CDCl3, 500MHz) δ: 5.88 (1H, dd, J = 3.0Hz, 1.5Hz, H-3), 4.68 (1H, s, H-5), 2.39 (1H, d, J = 15. 6Hz,H-6α),2.15(1H,dd,J=15.6Hz,5.0Hz,H-6β),4.62(1H,d,J=5.0Hz,H-7),2.91(1H,s,H-9), 5.77(1H,d,J=1.3Hz,H-12),2.42-2.49(1H,m,H-14),2.65(1H,dd,J=18.4Hz,7.9Hz,H-15a),2 .97(1H,dd,J=18.4Hz,7.9Hz,H-15b),2.19(3H,s,H-18),1.31(3H,s,H-20),1.94(3H,s,H-21); 13 C NMR(CDCl3,500MHz)δ:194.9(C-2),120.5(C-3),166.5(C-4),91.3(C-5),44.3(C-6),86.9(C-7),22.1(C-8),52.1(C-9),44.8(C-10), 172.0(C-11),127.7(C-12),157.0(C-13),45.1(C-14),30.1(C-15),168.4(C-16),44.5(C-18),20.0(C-19),16.5(C-20),21.2(C-21).

[0131] In vitro neuroprotective activity of the compound of Test Example 1

[0132] PC12 cells were cultured at 1×10 4 / well was inoculated in a 96-well cell culture plate, 100 μL of DMEM complete culture medium was added to each well, and after culturing in a cell culture incubator for 24 hours, the compound was prepared to a dosage concentration of 20 μg / mL (containing 0.2% DMSO). The experiment set up a control group (control, positive control rasagiline), a model group (6-OHDA) and a schizophyllum compound group (6-OHDA+compound). The control group first added 100 μL of complete culture medium (containing 0.2% DMSO), incubated for 2 hours, and then incubated for 24 hours without 6-OHDA treatment; the model group first added 100 μL of complete culture medium (containing 0.2% DMSO), incubated for 2 hours, and then added 1 μL of 6-OHDA (300 μM) to treat the cells for 24 hours; the schizophyllum compound group first added 100 μL of 20 μg / mL compound solution, incubated for 2 hours, and then added 1 μL of 6-OHDA (300 μM) to treat the cells for 24 hours.

[0133] CCK-8 cell viability test:

[0134] After the culture in the cell culture incubator was completed, 10 μL of CCK8 solution was added to each well and incubated in the incubator for 1 h. The absorbance value at 450 nm was measured with an enzyme-labeled instrument to calculate the cell survival rate.

[0135] The protective effects of the compounds on 6-OHDA-induced PC12 cells are as follows Figure 1 shown. Figure 1 The concentration of the compound in A was 20 μg / mL, and the concentrations of the compound in B and C were 25, 50, and 150 μM.

[0136] The results showed that compounds 1, 2, 4 and 5 exhibited certain neuroprotective activity, among which compounds 1 and 5 had the most significant activity, with cell survival rates of 86.31 and 85.61%, respectively, which were comparable to the activity of the positive control rasagiline (89.03%). Figure 1 B in the figure shows that at different concentrations (25, 50 and 150 μM), compounds 1, 2, 4 and 5 have no cytotoxicity to PC12 cells. C shows that compound 2 (50 μM) has the highest neuroprotective activity (92.84%), even higher than the positive control rasagiline.

[0137] In vivo neuroprotective activity of the compounds of Test Example 2

[0138] (1) Cultivation of Drosophila

[0139] W1118(WT) and PINK1 B9[w(*)Pink1(B9)] flies were cultured in a fruit fly incubator (60% relative humidity, 25°C, and a 12-h light / dark cycle). The formula of the conventional culture medium for fruit flies is as follows: 90 g yeast, 240 g corn flour, 32 g agar, 189 g glucose, 98 g sucrose, and 25 mL preservative (10% methyl paraben), which were evenly mixed in 3.5 L distilled water and then boiled. The experimental medium was prepared by adding 100 μM of the compounds (1, 2, 4, and 5). By collecting WT and PINK1 B9 Male fruit flies were subjected to the following experiment. The experimental fruit flies were divided into three groups: ① WT fruit flies fed with conventional feed; ② PINK1 fruit flies fed with conventional culture medium B9 Drosophila; ③Experimental culture medium feeding PINK1 B9 Fruit fly.

[0140] (2) Fruit fly crawling experiment

[0141] Each group consisted of 30 fruit flies, and the fruit flies' crawling ability was tested using a fruit fly infrared behavior recorder. The fruit flies were first anesthetized with CO2, transferred to a glass tube (15×1.5 cm), and adapted to room temperature for 30 minutes. The glass tube was gently tapped to place the fruit flies at the bottom, and placed in a behavior recorder. The fruit flies' crawling ability was recorded every 5 minutes, and the record was made at the same time every day for at least 4 hours.

[0142] (3) Determination of ATP content in Drosophila

[0143] Each group consisted of 5 fruit flies. The fruit flies were first anesthetized with CO2, and the thorax of the fruit flies was removed under a stereomicroscope, homogenized in 50 μL pre-cooled PBS buffer solution, and then centrifuged at 4°C for 15 min (12000 rpm). Finally, the ATP content in the supernatant was detected using an ELISA kit (Beyotime, Jiangsu, China).

[0144] (4) Determination of dopamine content in Drosophila

[0145] Each group consisted of 30 fruit flies, which were first anesthetized with CO2, collected into centrifuge tubes, and then frozen in liquid nitrogen. The heads of the fruit flies were collected through a filter after rapid tapping, homogenized in precooled 50 μL of citric acid acetate buffer (50 mM, pH = 6.5), and then centrifuged at 4 ° C for 15 min (12000 rpm), and the supernatant was collected. Finally, the dopamine content in the samples was detected using an ELISA kit (MEIMian, Jiangsu, China).

[0146] Compounds against PINK1 B9 The protective effect of fruit flies is as follows Figure 2 As shown, Figure 2In the figure, A is the crawling experiment test result, B is the ATP content measurement result, and C is the dopamine content measurement result.

[0147] The experimental results show that: ① As shown in A, compared with wild-type fruit flies, PINK1 B9 The crawling ability of Drosophila was significantly reduced (18.46%), but after treatment with compounds 2 and 4 (100 μM), PINK1 B9 The crawling ability of fruit flies increased to 40.00 and 47.33% on the 6th day; ② As shown in B, PINK1 B9 The ATP content of Drosophila was 42.41%, but after treatment with compounds 2 and 4 (100 μM), PINK1 B9 The ATP content of Drosophila increased to 66.27 and 67.70%. ③ As shown in C, PINK1 B9 The dopamine content of Drosophila was 54.78%. However, after 18 days of treatment with compounds 2 and 4 (100 μM), PINK1 B9 The flies' dopamine levels increased to 71.99 and 89.45 percent, respectively.

[0148] Experimental results and conclusions:

[0149] The present invention relates to the separation and identification of 7 new quassin compounds from the fruit of the Chinese yam. The in vitro neuroprotective activity experiment shows that compounds 1, 2, 4 and 5 have significant protective activity against 6-OHDA-induced PC12 cells; the in vivo neuroprotective activity experiment shows that compounds 2 and 4 can increase the expression of PINK1 B9 The crawling ability of fruit flies was improved, and the content of ATP and dopamine was increased. This study showed that compounds 2 and 4 exhibited significant neuroprotective activity in vitro and in vivo, and can be further developed for the treatment of diseases such as Parkinson's disease.

[0150] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The quassin compounds extracted from the fruit of the Chinese yam have the structure shown in any one of compounds 1, 2, 4, and 5: In compound 1, R1=OMe, R2=OAc; In compound 2, R1=OMe, R2=OH; In compound 4, R = OH; 2. The method for extracting quassin compounds from the schizonepeta tenaca according to claim 1, comprising the following steps: (1) using methanol to reflux extract the ground taro branches to obtain a methanol extract; (2) concentrating the methanol extract under reduced pressure to obtain a crude extract; (3) dispersing the crude extract with water, and performing a first extraction on the obtained dispersion using petroleum ether to obtain a petroleum ether extract; Performing a second extraction on the petroleum ether extract using ethyl acetate to obtain an ethyl acetate extract; (4) subjecting the ethyl acetate extract to small pore resin chromatography and performing a first gradient elution, wherein the procedure of the first gradient elution comprises: sequentially eluting with a 30% by volume methanol aqueous solution, a 50% by volume methanol aqueous solution, a 70% by volume methanol aqueous solution, a 90% by volume methanol aqueous solution and anhydrous methanol to obtain five eluents, which are named Fr1, Fr2, Fr3, Fr4 and Fr5 respectively; (5) subjecting the Fr3 to silica gel column chromatography and performing a second gradient elution, wherein the procedure of the second gradient elution comprises: sequentially using a petroleum ether-ethyl acetate solution with a volume ratio of 10:1, a petroleum ether-ethyl acetate solution with a volume ratio of 5:1, a petroleum ether-ethyl acetate solution with a volume ratio of 3:1, a petroleum ether-ethyl acetate solution with a volume ratio of 2:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:1, a petroleum ether-ethyl acetate solution with a volume ratio of 1:2, a petroleum ether-ethyl acetate solution with a volume ratio of 1:4, pure ethyl acetate and methanol for elution to obtain nine fractions, which are named Fr3-1, Fr3-2, Fr3-3, Fr3-4, Fr3-5, Fr3-6, Fr3-7, Fr3-8 and Fr3-9 in sequence; (6) performing a first chromatography separation on the Fr3-3 using Sephadex LH-20 methanol gel column chromatography to obtain a first chromatographic solution; performing semi-preparative high performance liquid chromatography separation on the first chromatographic solution, and performing a third gradient elution to obtain compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6; In compound 1, R1=OMe, R2=OAc; In compound 2, R1=OMe, R2=OH; In compound 3, R1=OH, R2=H; In compound 4, R = OH; The eluent of the first chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1; The procedure of the third gradient elution includes: eluting with 30% by volume acetonitrile aqueous solution, 35% by volume acetonitrile aqueous solution, 40% by volume acetonitrile aqueous solution, 45% by volume acetonitrile aqueous solution, 50% by volume acetonitrile aqueous solution, and 55% by volume acetonitrile aqueous solution in sequence; (7) performing a second chromatography separation on Fr3-2 using a cross-linked dextran LH-20 gel column to obtain a second chromatographic solution; performing high performance liquid chromatography separation on the second chromatographic solution, and performing a fourth elution to obtain compound 7; The eluent for the second chromatographic separation is a dichloromethane-methanol solution with a volume ratio of 1:1; The eluent for the fourth elution is an acetonitrile aqueous solution with a volume fraction of 54%; There is no time sequence restriction between steps (6) and (7).

3. The extraction method according to claim 2, characterized in that In the step (4), the conditions for small-pore resin chromatography separation include: Small pore resin column size: 120mm*1000mm; Eluent flow rate: 25 mL / min; Elution flow rate: 10 L per gradient.

4. The extraction method according to claim 2, characterized in that In the step (6), the conditions for the first chromatographic separation include: Gel column size: 20mm*200mm; The flow rate of the eluent was 0.3 mL / min; The amount of eluent used is: 1L; The conditions for the semi-preparative HPLC separation include: Chromatographic column: Waters XSelect CSH C-18 column, specification: 5 μm, 10×250 mm; The mobile phase comprises: mobile phase A and mobile phase B; the mobile phase A is pure water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 70-45%; the volume fraction of the mobile phase B is 30-55%; each elution is 10 minutes, and then 5% of the mobile phase B is added; elution is performed for another 10 minutes, and then 5% of the mobile phase B is added, and so on, until the mobile phase B is increased to 55% and eluted for 10 minutes; The flow rate of the mobile phase was 3.0 mL min -1 ; The column temperature was 25°C; The injection volume was 30 μL each time.

5. The extraction method according to claim 2, characterized in that In the step (7), the conditions for the second chromatographic separation include: Gel column size: 20mm*200mm; The flow rate of the eluent was 0.3 mL / min; The amount of eluent used is: 1L; The conditions for the HPLC separation include: Chromatographic column: Waters XSelect CSH C-18 column, specification: 5 μm, 10×250 mm; The mobile phase includes: mobile phase A and mobile phase B; the mobile phase A is pure water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 54%; the volume fraction of the mobile phase B is 46%; The flow rate of the mobile phase was 3.0 mL min -1 ; The column temperature was 25°C; The injection volume was 30 μL each time.

6. Use of the quassin compounds extracted from the kiwifruit as claimed in claim 1 or the quassin compounds extracted from the kiwifruit obtained by the extraction method as claimed in any one of claims 2 to 5 in the preparation of drugs against neurodegenerative diseases.

7. The use according to claim 6, characterized in that: The anti-neurodegenerative disease drug is an anti-Parkinson's disease drug.

8. An anti-Parkinson's disease pharmaceutical composition, comprising the quassin compounds extracted from the kiwifruit of claim 1 or the quassin compounds extracted from the kiwifruit of claim 2 obtained by the extraction method of any one of claims 2 to 5, and also comprising a pharmaceutically acceptable carrier and / or excipient.

9. The anti-Parkinson's disease pharmaceutical composition according to claim 8, characterized in that: The dosage form of the anti-Parkinson's disease pharmaceutical composition is one or more of a liquid preparation, a solid preparation, a spray and an aerosol.

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