Bisindole alkaloid of aralia as well as preparation method and application of bisindole alkaloid

Abstract

The invention discloses aralia bisindole alkaloid as well as a preparation method and application of the aralia bisindole alkaloid. The prepared aralia plant extract contains at least one of bisindole alkaloids Z-1-Z-21, monomer extraction and cell and animal experiment verification show that the bisindole alkaloid compound can protect nerve cell injury caused by glutamic acid, can improve the epilepsy state and prolong the epilepsy incubation period in an animal model, and can be used for preparing a medicine for treating the epilepsy. The cell arrangement in the brain sea horse area of the epilepsy mouse is improved; the neuron deformation is reduced. In addition, the series of bisindole alkaloid compounds have chemical structure types different from those of existing epilepsy treatment targeted drugs, and have wide application prospects in preparation of novel epilepsy treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of natural compounds, in particular to a Hunteria bisindole alkaloid, a preparation method and application thereof. BACKGROUND

[0002] Neurodegenerative diseases are diseases caused by the progressive degeneration of neurons in structure or function, which can lead to progressive loss of brain function and overlapping clinical syndromes. According to the severity of the disease, it can be divided into acute neurodegenerative diseases and chronic neurodegenerative diseases. The former includes cerebral ischemia (CI), brain injury, epilepsy (EP); the latter includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and different degrees of spinocerebellar ataxia (SCA) and the like. Modern medicine believes that the pathogenesis of neurodegenerative diseases is related to oxidative stress, mitochondrial dysfunction, excitatory toxin, immune inflammation and the like. Due to the complexity of pathogenic factors and the irreversibility of pathological changes, so far no effective cure has been proposed for neurodegenerative diseases, and the drugs used in clinical practice can only improve symptoms and cannot delay disease progression and fundamentally reverse the progressive neurodegeneration. Therefore, the development of drugs for preventing and treating neurodegenerative diseases has become an important research content and a major problem to be solved.

[0003] Epilepsy is an acute neurodegenerative disease, which is a sudden, repeated and transient central nervous system dysfunction caused by abnormal discharge of brain neurons. Repeated seizures or long-term seizures can cause neuronal damage and cell death, thereby causing damage to the patient's motor, sensory, consciousness and mental functions, and seriously affecting the patient's quality of life. Although there are many drugs and treatment options in clinical practice, one-third of the drugs will develop drug resistance after long-term use, and most of the drugs have serious side effects. Therefore, there is an urgent need to develop new low-toxicity and high-efficiency antiepileptic drugs. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a Hunteria bisindole alkaloid.

[0005] Another purpose of the present application is to provide the application of the Hunteria bisindole alkaloid described above. The purpose of the present application is achieved by the following technical solutions:

[0006] A case of olive tree bisindole alkaloids, the structural formula is as follows at least one:

[0007] The said case of olive tree bisindole alkaloids, at least one of the following compounds:

[0008] Pleiomutinine (Z-1), 19'-epi-pleiomutinine (Z-2), 16-epi-pleiomutinine (Z-3), huncaniterine A (Z-4), 19'-epi-huncaniterine A (Z-5), huncaniterine B (Z-6), 19'-epi-huncaniterine B (Z-7), N4-chloromethyl-pleiomutinine (Z-8), pycnanthine (Z-9), 19'-epi-pleiomutinine N-oxide (Z-10), huntezeyline A (Z-11), 19'-epi-huntezeyline A (Z-12), contortarine A (Z-13), contortarine A N 4' -oxide (Z-14), contortarine A N4-oxide (Z-15), contortarine A N-oxide (Z-16), hunterlanine U (Z-17), hunterlanine V (Z-18), hunterizeyline B (Z-19), hunterzeynine A (Z-20), hunterzeynine B (Z-21).

[0009] A case of olive tree alkaloid composition, containing at least one of the above-mentioned case of olive tree alkaloids.

[0010] A preparation method of a case of olive tree alkaloid composition, comprising the following steps:

[0011] The dry case of olive tree branches and leaves are ground to obtain medicinal powder, and then extracted by percolation. The pH value of the extract is adjusted to be acidic. After extraction with an organic solvent, the organic layer is concentrated under reduced pressure to obtain an extract. The extract is dissolved in a mixed solvent and then extracted with pure water. The water layer is concentrated appropriately, and finally chromatographed with a chromatography column. The first eluted part is collected as a case of olive tree alkaloid composition containing bisindole alkaloids.

[0012] The extraction solvent is at least one of water, acidic water, absolute ethanol, or an ethanol aqueous solution with any concentration.

[0013] The solution used in the percolation extraction is a 1-2% hydrochloric acid aqueous solution.

[0014] The pH is adjusted to 1-2.

[0015] The organic solvent is chloroform or dichloromethane.

[0016] The mixed solvent is a solvent obtained by mixing CH3OH and CH2Cl2 at a volume ratio of 1:2-5.

[0017] The chromatography column is a Sephadex LH-20 chromatography column.

[0018] The Hunteria plant of the present application refers to a Hunteria plant of the Apocynaceae family.

[0019] The Hunteria alkaloid or the Hunteria alkaloid composition described above is used for preparing a medicine for protecting nerve cells.

[0020] The Hunteria alkaloid or the Hunteria alkaloid composition described above is used for preparing a medicine for treating neurodegenerative diseases.

[0021] The Hunteria alkaloid or the Hunteria alkaloid composition described above is used for preparing an anti-epilepsy medicine.

[0022] The medicine comprises the bisindole alkaloid compound and derivatives thereof, and / or pharmaceutically acceptable salts thereof, and / or stereoisomers thereof, and / or prodrug molecules thereof of the present application.

[0023] The "prodrug" refers to a prodrug that is converted in vivo into the structure of the compound and the pharmaceutically acceptable salt thereof involved in the present application.

[0024] The medicine comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0025] Preferably, the excipients include any one or a combination of at least two of carriers, wetting agents, disintegrating agents, emulsifying agents, co-solvents, solubilizing agents, osmotic pressure adjusting agents, surfactants, coating materials, coloring agents, pH adjusting agents, antioxidants, bacteriostatic agents, or buffers.

[0026] Preferably, the dosage form of the medicine includes any one of tablets, capsules, dripping pills, granules, injections, powder injections, or aerosols.

[0027] The present application has the following advantages and effects relative to the prior art:

[0028] 1、The extract of the plant of the genus Hunteria obtained by extraction and separation contains bisindole alkaloids, and further separation and purification obtains active Hunteria alkaloids Z-1 to Z-21, and the chemical structures of the Hunteria alkaloids Z-1 to Z-21 are determined through experiments. Cell and animal models prove that the series of compounds can protect nerve cells from glutamic acid-induced damage, improve the status of epilepsy, prolong the latency of epilepsy, improve the cell arrangement in the hippocampus of the brain of the epilepsy mouse and reduce the deformation of neurons, and play a role in treating epilepsy through multiple mechanisms. In addition, the series of bisindole alkaloid compounds have a different chemical structure type from existing epilepsy treatment target drugs, and have a wide application prospect in the preparation of new epilepsy treatment drugs.

[0029] 2、The extract of the plant of the genus Hunteria obtained by extraction and separation contains bisindole alkaloids, and the extraction and purification method provided by the present application has few steps and low economic cost. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a chemical structural formula of compounds Z1 to Z10.

[0031] Figure 2 is a chemical structural formula of compounds Z11 to Z21.

[0032] Figure 3 Water maze quadrant division diagram.

[0033] Figure 4 is RT-PCR detection of the expression of some genes in the epilepsy model mouse treated with compound Z-1; wherein, A is detection of the expression of GAD65 gene; B is detection of the expression of GAD67 gene; C is detection of the expression of GAT-1 gene; D is detection of the expression of SOD gene; E is detection of the expression of GABA gene; and F is detection of the expression of BDNF gene.

[0034] Figure 5 is a hippocampus neuron pathological section of the epilepsy model mouse, wherein, A is a negative control, B is a PTZ model group, and C is a compound Z-1 intervention group. DETAILED DESCRIPTION

[0035] The present application will be further described in detail below in combination with examples and drawings, but the embodiments of the present application are not limited thereto.

[0036] In the following embodiments, if specific experimental conditions are not specified, the general experimental conditions or the experimental conditions recommended by the reagent company are usually used. If not specified, the materials, reagents and the like used are reagents and materials obtained from commercial channels.

[0037] Example 1 Extraction and separation method of the genus Olea europaea extract

[0038] Dried branches and leaves of the genus Eleutherodactyla are pulverized to obtain a total of 10 kg of medicinal powder. The extract is then extracted by percolation with 500 liters of a 2% aqueous hydrochloric acid solution, and the pH of the extract is adjusted to 1-2. After extraction with dichloromethane, a dichloromethane layer and an acidic aqueous layer are obtained. The obtained dichloromethane layer is concentrated under reduced pressure to obtain an extract, which is collectively referred to as total alkaloids. The extract is dissolved in 1.5 liters of a mixed solvent of CH3OH-CH2Cl2 (1:3), and pure water is added in a 1:1 volume ratio for three-phase extraction. The obtained aqueous layer is appropriately concentrated and finally chromatographed on a Sephadex LH-20 column using CH3OH-H2O (4:1) as the eluent. The first eluting fractions are collected, analyzed by TLC, and combined to obtain Eleutherodactyla extract 1.

[0039] Example 2 Isolation of Bisindole Alkaloids from the Extract of Euphorbia cerevisiae

[0040] The extract 1 of the genus Euphorbia obtained in Example 1 was subjected to column chromatography on 200-300 mesh silica gel using chloroform-methanol as the mobile phase for gradient elution to obtain a total of five fractions, AE. After TLC and HPLC analysis, the following compounds and their retention times on analytical HPLC were obtained by repeated ODS, Sephadex LH-20 column chromatography, and high-performance liquid chromatography: Compound Z-1 (methanol:water:diethylamine, 75:25:0.2%) was isolated from fraction C. R =28.3min), Z-2 (methanol: water: diethylamine, 78:22:0.2%, t R =20.5min), Z-3 (methanol: water: diethylamine, 75:25:0.2%, t R =26.0min), Z-4 (methanol: water: diethylamine, 68:32:0.2%, t R =16.7min), Z-5 (methanol:water:diethylamine, 70:30:0.2%, t R =15.8min), Z-10 (methanol: water: diethylamine, 65:35:0.2%, t R =29.2min), Z-11 (methanol: water: diethylamine, 85:15:0.2%, t R =17.2min), Z-12 (methanol: water: diethylamine, 83:17:0.2%, t R =20.7min), Z-13 (methanol: water: diethylamine, 85:15:0.2%, t R =16.4min). Compound Z-6 (acetonitrile:water:diethylamine, 55:45:0.2%, t R= 26.4 min), Z-7 (methanol:water:diethylamine, 70:30:0.2%, t R = 22.3 min), Z-8 (methanol:water:diethylamine, 73:27:0.2%, t R = 17.8 min), Z-9 (methanol:water:diethylamine, 70:30:0.2%, t R = 20.5 min), Z-14 (methanol:water:diethylamine, 70:30:0.2%, t R = 24.6 min), Z-15 (methanol:water:diethylamine, 70:30:0.2%, t R = 19.5 min), Z-16 (methanol:water:diethylamine, 60:40:0.2%, t R = 21.9 min), Z-17 (methanol:water:diethylamine, 78:22:0.2%, t R = 20.6 min), Z-18 (methanol:water:diethylamine, 75:25:0.2%, t R = 19.7 min), Z-19 (methanol:water:diethylamine, 78:22:0.2%, t R = 20.8 min), Z-20 (methanol:water:diethylamine, 80:20:0.2%, t R = 17.6 min), Z-21 (methanol:water:diethylamine, 80:20:0.2%, t R = 15.9 min).

[0041] Structure identification of bisindole alkaloids in example 3

[0042] The compound isolated in Example 2 was identified as 16 bisindole alkaloids by physicochemical data and spectral methods, which are pleiomutinine (Z-1), 19'-epi-pleiomutinine (Z-2), 16-epi-pleiomutinine (Z-3), huncaniterine A (Z-4), 19'-epi-huncaniterine A (Z-5), huncaniterine B (Z-6), 19'-epi-huncaniterine B (Z-7), N4-chloromethyl-pleiomutinine (Z-8), pycnanthine (Z-9), 19'-epi-pleiomutinine N-oxide (Z-10), huntezeyline A (Z-11), 19'-epi-huntezeyline A (Z-12), contortarine A (Z-13), contortarine A N-oxide (Z-14), contortarine A N4-oxide (Z-15) and contortarine A N-oxide (Z-16), hunterlanine U (Z-17), hunterlanine V (Z-18), hunterizeyline B (Z-19), hunterzeynine A (Z-20), hunterzeynine B (Z-21), the structural formula of which is shown in 4' Figures 1-2 The characterization data are as follows:

[0043] Compound Z-1

[0044] Red-brown powder; [a]25D+243.7 (c 0.4, CH3OH); modified BiCl3reaction was orange-red spot; UV (CH3OH) λ max (log e) 209 (3.74), 248 (3.37), 300 (3.01) nm; IR (KBr) v max 2923, 2854, 1748, 1652, 1608, 1471, 1375, 1244, 1103, 1029, 753 cm -1 ; HR-ESI-MS: m / z: 615.3675 [M+H] + (calcd for C 40 H 47 N4O2, 615.3694); 1 H NMR (500 MHz, CD3OD) and​13 C NMR (125 MHz, CD3OD) see Table 1.

[0045] Compound Z-2

[0046] Yellow powder; [a]25D+213.13 (c 1.07, CH3OH); modified BiI3 reaction as orange-red spots; UV (CH3OH) λ max (log e) 208 (3.67), 253 (2.26) 307 (2.83); IR (KBr) v max 3404, 2936, 1745, 1605, 1451, 1350, 1207, 747 cm -1 ; HR-ESI-MS: m / z: 615.3675 [M+H] + (calcd for C 40 H 47 N4O2, 615.3694). 1 H NMR (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) see Table 2.

[0047] Compound Z-5

[0048] Brown powder; [a]25D+75.27 (c 1.00, CH3OH); modified BiI3 reaction as orange-red spots; UV (CH3OH) λ max (log e) 208 (3.67), 253 (2.26) 307 (2.83); IR (KBr) v max 3413, 2946, 1725, 1644, 1466, 1382, 1206, 754 cm -1 ; HR-ESI-MS: m / z: 631.4018 [M+H] + (calcd for C 40 H 47 N4O3, 631.4010). 1 H NMR (500 MHz, CD3OD) and 13 C NMR (125 MHz, CD3OD) see Table 3.

[0049] Compound Z-8

[0050] Yellow gum; (c 1.2, CH3OH); modified BiI3 reaction as orange-red spots; UV (CH3OH) λ max(log ε) 202 (4.89), 220 (4.38), 257 (4.02), 288 (3.66) nm; IR (KBr) v max 3413, 2946, 1725, 1644, 1466, 1382, 1206, 754 cm -1 ; HR-ESI-MS m / z: 663.3458 [M] + , calcd for C 41 H 48 N4O2Cl, 663.3460; ECD (CH3OH) λ max (Δε) 217 (-24.27), 231 (-2.57), 240 (-5.25), 260 (13.60), 279 (1.64), 298 (3.90), 311 (1.75), 327 (2.42) nm; 1 H NMR (600 MHz, CDC13) and 13 CNMR (150 MHz, CDC13) data are listed in Table 4.

[0051] Compound Z-10

[0052] Light brown powder; (c 1.01, CH3OH); modified BiI3 / KI reaction as orange-red spot; UV (CH3OH) λ max (log ε) 206 (3.35), 251 (2.87); IR (KBr) v max 3392, 2955, 1745, 1482, 1453, 1355, 1210, 752 cm -1 ; HR-ESI-MS: m / z: 647.3590 [M + H] + (calcd for C 40 H 47 N4O4, 647.3592); ECD (CH3CN) λ max (Δε) 334 (+1.4), 305 (+4.5), 264 (+12.1), 243 (-4.4), 220 (-18.9), 206 (+19.5); 1 H NMR (500 MHz, CD3OD) and 13 C NMR (125 MHz, CD3OD) are listed in Table 5.

[0053] Compound Z-13

[0054] Brown powder; (c 1.1, CH3OH); modified potassium bismuth iodide reaction produces orange-red spots; UV(CH3OH)λ max (logε)203(4.94),253(4.46),303(4.29),328(4.10)nm; IR(KBr)v max 3278,2930,2869,1715,1661,1611,1455,1336,1231,1142,1012cm -1 ; HR-ESI-MS m / z: 615.3695[M+H] + ,calcd for C 40 H 47 N4O2,615.3694;ECD(CH3OH)λ max (Δε)221(26.21),234(8.20),266(27.74),284(4.59),301(10.26),320(1.90)nm; 1 H NMR (600 MHz, CDCl3) and 13 C NMR (150 MHz, CDCl3) data are shown in Table 6.

[0055] Compound Z-17

[0056] Light brown powder; (c 1.14, CH3OH); modified potassium bismuth iodide reaction produces orange-red spots; UV(CH3OH)λ max (logε)206(3.81),225(3.76),283(3.24)nm; IR(KBr)ν max 3508,3399,2950,1723,1629,1573,1462,1378,1347,1282,1155,1058,740cm -1 ; HR-ESI-MS m / z 765.3846[M+H] + (calcd for C 44 H 53 N4O8,765.3858);ECD(CH3OH)λ max (Δε)223(-22.10),235(15.50)nm; 1 HNMR (600 MHz, CDCl3) and 13 C NMR (150 MHz, CDCl3) data are shown in Table 7.

[0057] Compound Z-18

[0058] Yellow-brown powder; (c 0.94, CH3OH); UV (CH3OH) λ max (log ε) 206 (3.54), 228 (3.36), 291 (2.94), 326 (2.86) nm; IR (KBr) v max 3366, 2943, 1714, 1626, 1608, 1458, 1385, 1252, 1057, 746 cm -1 HR-ESI-MS m / z 765.3859 [M+H] + (calcd for C 44 H 53 N4O8, 765.3858); ECD (CH3OH) λ max (Δε) 203 (+12.80), 225 (-10.20), 248 (+2.50), 311 (-3.10) nm; 1 H NMR (500 MHz, CD3OD) and 13 C NMR (125 MHz, CD3OD) data are listed in Table 8.

[0059] Compound Z-20

[0060] Yellow powder; (c 1.57, CH3OH); UV (CH3OH) λ max (log ε) 207 (4.60), 228 (4.49), 255 (4.04), 287 (3.97) nm; IR (KBr) v max 3364, 2932, 1608, 1454, 1365, 1326, 1186, 743 cm -1 HR-ESI-MS m / z 559.3794 [M+H] + (calcd for C 38 H 47 N4, 559.3795); ECD (CH3OH) λ max (Δε) 202 (+32.10), 220 (-9.00), 238 (-17.50) nm; 1 H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) data are listed in Table 9.

[0061] Table 1 1 H and 13 C NMR data of Z-1 (δ in ppm, J in Hz) a

[0062]

[0063] a Overlapped

[0064] Table 2 1 H and 13 C NMR data of Z-2 (δ in ppm, J in Hz) a

[0065]

[0066]

[0067] a Overlapped

[0068] Table 3 1 H and 13 C NMR data of Z-5 (δ in ppm, J in Hz) a

[0069]

[0070]

[0071] a Overlapped

[0072] Table 4 1 H and 13 C NMR data of Z-8 (δ in ppm, J in Hz) a

[0073]

[0074] a Overlapped

[0075] Table 5 NMR data of Z-10 (CD3OD, δ in ppm, J in Hz)

[0076]

[0077]

[0078] a Overlapped

[0079] Table 6 1 H and 13C NMR data of Z-13(δin ppm,J in Hz) a

[0080]

[0081] a Overlapped

[0082] Table 7 1 H and 13 C NMR data of Z-17(δin ppm,J in Hz) a

[0083]

[0084]

[0085] a Overlapped

[0086] Table 8 1 H and 13 C NMR data of Z-18(δin ppm,J in Hz) a

[0087]

[0088] a Overlapped

[0089] Table 9 1 H and 13 C NMR data of Z-20(δin ppm,J in Hz) a

[0090]

[0091] a Overlapped

[0092] Example 4 Protective Effects of Bisindole Alkaloids on Glutamate-Induced HT-22 Neuronal Cell Damage

[0093] 4.1 Experimental methods

[0094] HT22 cells in the logarithmic growth phase were selected and digested with trypsin to prepare a cell suspension. The concentration of the cell suspension was adjusted to 10 5 / ml, 100 μL per well was inoculated in 96-well cell culture plates, 6 replicates were set for each treatment, and the HT22 cells were divided into control group, Glu (glutamic acid) treatment group, Glu + drug treatment group. After 24 h of culture, the cells entered the logarithmic growth phase, the culture solution was removed, and the cells were washed twice with PBS buffer. The drug treatment group was added with different concentrations of the tested drug and positive drug, and the culture was continued for 1 h. Then, 5 mM / L Glu (glutamic acid) was added to each well of the treatment group, and the culture was continued for 24 h before observing the cell morphology.

[0095] After the cells were treated for the treatment time, 20 μL of MTT solution with a concentration of 5 mg / ml was added to each well, and the cells were cultured in the cell culture box for 4 h. After 4 h, the culture was terminated, the culture solution in the wells was carefully removed, 150 μL of DMSO was added to each well, and the microplate shaker was placed on low speed for 10 min to fully dissolve the crystals. The absorbance (OD) of each well was measured at 570 nm wavelength on an enzyme-linked immunoassay detector.

[0096] The cell survival rate was calculated according to the following formula: cell survival rate (%) = (OD value of drug treatment group - OD value of control group) / (OD value of Glu treatment group - OD value of control group) x 100%.

[0097] 4.2 Experimental results

[0098] The experimental results are shown in Table 10. Compared with the Glu-induced damage group, compounds Z-1 to Z-21 and total alkaloids can improve the survival rate of Glu-induced HT22 cells in a concentration-dependent manner, but have no obvious inhibitory effect on the survival rate of normal HT22 cells, indicating that the compounds have a protective effect on Glu-induced HT22 cell damage. The half inhibitory concentration IC 50 of the representative compound Z-1 is 1.60 ± 0.41 μM, which is better than the neuroprotective activity of the positive drug quercetin.

[0099] Table 10 Protective effect of compounds Z-1 to Z-21 on glutamate-induced HT22 cell damage (n = 5-6)

[0100]

[0101] Example 5 Effect of compound Z-1 on the epilepsy grade score and epilepsy latency of mice in an epilepsy model

[0102] 5.1 Experimental method

[0103] 30 mice of 20-25 g were randomly divided into negative control group, pentylenetetrazol (PTZ) group and Z-1 group according to body weight, 10 mice per group. The mice in each group were given corresponding doses of test samples by intraperitoneal injection at 10 mL / kg of body weight, and the negative control group was given the same amount of normal saline by intraperitoneal injection, 1 time / day. 30 min after administration, the mice in each group were given PTZ solution by intraperitoneal injection at 10 mL / kg of body weight, and the negative control group was given the same amount of normal saline by intraperitoneal injection, 2 times / day. PTZ was injected for a total of 15 times, and the operation was completed at 8:30-10:00 every day. The mice were closely observed for behavioral changes within 30-60 min after administration.

[0104] Epilepsy grade scoring and recording: After each PTZ intraperitoneal injection, the mice were placed in empty cages and observed and recorded for 1 hour of behavior, and the epilepsy grade and epilepsy latency were recorded according to the Racine grading standard:

[0105] 0: no behavioral changes, normal behavior;

[0106] 1: facial muscle spasm, including frequent and rhythmic whisker shaking and chewing;

[0107] 2: nodding-like neck muscle clonus;

[0108] 3: longitudinal twitching along the body or unilateral forelimb clonus;

[0109] 4: bilateral forelimb clonus;

[0110] 5: generalized tonic clonic grand mal seizures, or death.

[0111] The mouse latency time recording and seizure grade standard are as follows: the mouse behavior was observed for 30 min after PTZ injection, and the seizure latency was the time from injection to grade 2 seizure. The seizure grade was recorded as grade 5 found within 60 min. The mouse was judged to be successfully modeled if it showed grade 2 or higher for 5 consecutive days during modeling.

[0112] 5.2 Experimental results

[0113] As shown in Tables 11-12, the epilepsy grade score: compared with the negative control group, the epilepsy grade score of the PTZ model group mice increased on D1, D5, D10, D15, D20, D25 and D30, and the difference was statistically significant (P<0.05 or P<0.01); compared with the PTZ model group, the epilepsy grade score of the Z-1 group mice at each detection time point had no statistical significance (P>0.05).

[0114] Latency of epilepsy: Compared with the negative control group, the latency of epilepsy of the PTZ model group was shortened on D5, D10, D15, D20, D25 and D30, all with statistical differences (P<0.05 or P<0.01). Compared with the PTZ model group, the latency of epilepsy of the Z-1 group was prolonged on D10, D20 and D30, all with statistical significance (P<0.05).

[0115] Table 11 epilepsy grade score of mice in each group

[0116]

[0117] Note: T test was used for analysis; compared with the negative control group, "▲" P<0.05 and "▲▲" P<0.01, compared with the PTZ group, "*" P<0.05 and "**" P<0.01.

[0118] Table 12 latency of epilepsy of mice in each group seconds

[0119]

[0120] Note: T test was used for analysis; compared with the negative control group, "▲" P<0.05 and "▲▲" P<0.01, compared with the PTZ group, "*" P<0.05 and "**" P<0.01.

[0121] Example 6 Effect of compound Z-1 on the Morris water maze positioning navigation test of epilepsy model mice

[0122] 6.1 Experimental setting of Morris water maze method

[0123] The Morris water maze method was divided into 1, 2, 3, 4 quadrants, and the quadrant opposite to the target platform was quadrant 1, and the quadrant where the target platform was located was quadrant 3. The mice were tested in a 1.2-meter-diameter circular tank, and the platform (14-centimeter-diameter) was submerged 2 centimeters below the water surface.

[0124] 6.2 Positioning navigation ability experiment

[0125] The experimental animals were treated according to the method of 5.1 in Example 5. On days 1-5, the platform was fixed in quadrant 3, and the entry point was placed in quadrants 1, 2, 3 and 4 in order, and the entry point of the mice was in quadrant 1. The mice were placed in the pool slowly with their backs to the platform, and the time was counted for 120 seconds. If the mouse failed to find the target platform within the specified 120 seconds, it was manually guided to find the target platform, and after it was controlled to stay on the platform for 30 seconds, it was taken out of the pool. The time required for each experimental mouse to find the target platform in the pool was recorded, and each experimental mouse was tested once a day.

[0126] 6.3 Experimental results

[0127] The experimental results are shown in Tables 13-17. Compared with the negative control group, the escape latency of the PTZ model group mice in the D2 third quadrant was shortened, the escape latency in the D3 second quadrant was prolonged, the escape latency in the D4 first quadrant and fourth quadrant was shortened, all with statistical differences (P<0.05). Compared with the PTZ model group, the escape latency of the compound Z-1 group mice in the D2 first quadrant and third quadrant was prolonged and the escape latency in the D3 second quadrant was shortened, all with statistical differences (P<0.05).

[0128] Table 13 Escape latency of mice in each group in D1 four quadrants seconds

[0129]

[0130] Note: T test was used for analysis; compared with the negative control group, “▲” P<0.05 and “▲▲” P<0.01, compared with the PTZ group, “*” P<0.05 and “**” P<0.01.

[0131] Table 14 Escape latency of mice in each group in D2 four quadrants seconds

[0132]

[0133] Note: T test was used for analysis; compared with the negative control group, “▲” P<0.05 and “▲▲” P<0.01, compared with the PTZ group, “*” P<0.05 and “**” P<0.01.

[0134] Table 15 Escape latency of mice in each group in D3 four quadrants seconds

[0135]

[0136] Note: T test was used for analysis; compared with the negative control group, “▲” P<0.05 and “▲▲” P<0.01, compared with the PTZ group, “*” P<0.05 and “**” P<0.01.

[0137] Table 16 Escape latency of mice in each group in D4 four quadrants seconds

[0138]

[0139] Note: T test was used for analysis; compared with the negative control group, “▲” P<0.05 and “▲▲” P<0.01, compared with the PTZ group, “*” P<0.05 and “**” P<0.01.

[0140] Table 17 D5 escape latency of each group of mice in four quadrants seconds

[0141]

[0142] Note: T-test was used for analysis; "▲" P<0.05 and "▲▲" P<0.01 compared with the negative control group; "*" P<0.05 and "**" P<0.01 compared with the PTZ group.

[0143] Example 7 RT-PCR detection results of compound Z-1 on mice with epilepsy model

[0144] 7.1 Experimental method

[0145] After the experiment in Example 5 ended on D30, 4 mice were randomly selected from each group, decapitated, and brain tissue stripping was performed on an ice surface in a glass container. The hippocampal tissue of the mice was stripped and collected, and the surface of the tissue was washed with pre-cooled PBS to remove possible red blood cells. The tissue was quickly frozen in liquid nitrogen for 3-5 s, placed on a tin foil paper to avoid light, and stored. The tin foil paper was then transferred to a 1.5 mL centrifuge tube and numbered, and the centrifuge tube was quickly transferred to a -80°C refrigerator for storage. The tissue homogenate was used for RT-PCR detection, GAPDH was used as an internal standard gene, and the expression levels of GAD65, GAD67, GAT-1, SOD, GABA, and BDNF were detected.

[0146] 7.2 Experimental results

[0147] The experimental results are shown in Table 19 and Figure 4 Compared with the negative control group, the expression of GAD65, GAD67, GAT-1, SOD, GABA, and BDNF in the PTZ group of mice was significantly decreased (P<0.01). Compared with the PTZ group, the expression of GAD65, GAD67, GAT-1, SOD, GABA, and BDNF in the Z-1 group of mice was significantly increased (P<0.05).

[0148] Table 18 Primer sequences

[0149]

[0150] Table 19 RT-PCR detection results

[0151]

[0152] Note: T-test was used for analysis; compared with the negative control group, "▲" P<0.05 and "▲▲" P<0.01, compared with the PTZ group, "*" P<0.05 and "**" P<0.01.

[0153] Results of pathological detection of the compound Z-1 in Example 8 on the epilepsy model mice

[0154] 8.1 Experimental method

[0155] After the experiment D30 in Example 5 was completed, 3 mice were randomly taken from each group, and the whole brain of the mice was taken out by decapitation and skull dissection, and then was fixed in a neutral formaldehyde solution at room temperature for 24 hours, and the volume ratio of the fixing solution to the brain tissue was 1:10. The hippocampus part was taken, and Nissl staining was used to observe the neuron damage in the hippocampus region.

[0156] 8.2 Experimental results

[0157] Some neuron pathological sections are shown in Figure 5 , and the specific results are as follows:

[0158] The negative control group: the cells in the whole hippocampus region were closely arranged, and the morphology was regular, and no abnormal condition was found.

[0159] The PTZ model group: the number of cells in the whole hippocampus region was reduced, and the distance was increased, and a large number of neurons were deformed, and the cell nuclei were pyknosis and the staining was deepened.

[0160] The Z-1 group: the cells in the whole hippocampus region were closely arranged, and the morphology was relatively regular, and only a small amount of neurons were deformed, and the cell nuclei were pyknosis and the staining was deepened.

[0161] The pathological results show that the drug Z-1 can improve the cell arrangement in the hippocampus region of the epilepsy mice and reduce the neuron deformation to a certain extent.

[0162] The above experimental results reveal that the extract of the Olea genus plant has the neuroprotective activity, and can be applied to the treatment of epilepsy and other neurodegenerative diseases.

[0163] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application should be equivalent replacement modes, and all are included in the protection scope of the present application.

Claims

1. A bisindole alkaloid of Elaeagnus truncatula, characterized in that at least one of the following compounds:

2. The vilmorinan bisindole alkaloid of claim 1, wherein at least one of the following compounds: Pleiomutinine (Z-1), 19'-epi-pleiomutinine (Z-2), 16-epi-pleiomutinine (Z-3), huncaniterine A (Z-4), 19'-epi-huncaniterine A (Z-5), huncaniterine B (Z-6), 19'-epi-huncaniterine B (Z-7), N4-chloromethyl-pleiomutinine (Z-8), pycnanthine (Z-9), 19'-epi-pleiomutinine N-oxide (Z-10), huntezeyline A (Z-11), 19'-epi-huntezeyline A (Z-12), contortarine A (Z-13), contortarine AN 4' oxide (Z-14), contortarine AN4-oxide (Z-15), contortarine AN-oxide (Z-16), hunterlanine U (Z-17), hunterlanine V (Z-18), hunterizeyline B (Z-19), hunterzeynine A (Z-20), hunterzeynine B (Z-21).

3. A Hunteria zeylanica alkaloid composition, characterized in that: at least one of the Hunteria zeylanica alkaloids of claim 1 or 2.

4. A method for preparing a composition of an alkaloid from the tree of olive, characterized in that comprising the following steps: The dried Hunteria zeylanica branches and leaves are ground into medicinal powder, and then subjected to percolation extraction. The pH value of the extract is adjusted to be acidic, and then the extract is subjected to organic solvent extraction. The obtained organic layer is concentrated under reduced pressure to obtain extract, which is dissolved in mixed solvent and then subjected to three-phase extraction with pure water. The obtained water layer is concentrated, and finally subjected to chromatography column chromatography with mixed solvent elution, and the first eluted part is collected as the Hunteria zeylanica alkaloid composition containing bisindole alkaloids.

5. The preparation method of the Hunteria zeylanica alkaloid composition according to claim 4, characterized in that: the extraction solvent is at least one of water, acidic water, anhydrous ethanol, or an ethanol aqueous solution with any concentration; the solution used in the percolation extraction is a 1-2% hydrochloric acid aqueous solution; the pH value is adjusted to 1-2; the organic solvent is chloroform or dichloromethane; the mixed solvent is a solvent obtained by mixing CH3OH and CH2Cl2 at a volume ratio of 1:2-5; the chromatography column is a Sephadex LH-20 chromatography column.

6. Use of the Hunteria alkaloid of any one of claims 1-2 or the Hunteria alkaloid composition of claim 3 in the preparation of a medicament for protecting nerve cells.

7. Use of the Hunteria alkaloid of any one of claims 1-2 or the Hunteria alkaloid composition of claim 3 in the preparation of a medicament for treating neurodegenerative diseases.

8. Use of the Hunteria alkaloid of any one of claims 1-2 or the Hunteria alkaloid composition of claim 3 in the preparation of an anti-epilepsy medicament.

9. The use according to any one of claims 6-8, characterized in that: the medicament comprises the bisindole alkaloid compound and derivatives thereof, and / or pharmaceutically acceptable salts thereof, and / or stereoisomers thereof, and / or prodrug molecules thereof.

10. The use according to any one of claims 6-8, characterized in that: the medicament comprises one or more pharmaceutically acceptable carriers and / or excipients; the excipients include any one or a combination of at least two of carriers, wetting agents, disintegrating agents, emulsifying agents, co-solvents, solubilizing agents, osmotic pressure adjusting agents, surfactants, coating materials, coloring agents, pH adjusting agents, antioxidants, bacteriostatic agents, or buffers; the dosage form of the medicament includes any one of tablets, capsules, dripping pills, granules, injections, powder injections, or aerosols.

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