Triterpenoid component cyclatraine A in stems and leaves of astragalus membranaceus and application of triterpenoid component cyclatraine A in neuroprotection

Abstract

The invention discloses a triterpenoid component cyclatraine A in stems and leaves of astragalus membranaceus and application of the triterpenoid component cyclatraine A in neuroprotection, and belongs to the field of medicines. According to the method, separation methods such as normal-phase silica gel column chromatography, reversed-phase ODS column chromatography and high-performance liquid chromatography are adopted, chemical components in the ethanol extract of the stems and leaves of astragalus membranaceus are separated and purified, and a new triterpenoid component is successfully found and identified. Meanwhile, through an A beta 25-35 induced damaged PC12 cell model, the protection effect of the new triterpenoid component on nerve cells is researched. Experimental results show that the new triterpenoid components in the stems and leaves of the astragalus membranaceus can remarkably protect PC12 cells from being damaged through an anti-inflammatory and apoptosis inhibiting mechanism. The discovery provides an important scientific basis for further development and utilization of the stems and leaves of astragalus membranaceus, and also provides a new research direction for research and development of neuroprotective drugs.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to a triterpenoid cyclastraine A in the stems and leaves of Astragalus membranaceus and its application in neuroprotection. Background Art

[0002] Astragalus membranaceus is the dried root of the leguminous plant Astragalus membranaceus (Fisch.) Bunge or Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao, and was first recorded in "Shennong Ben Cao Jing". Astragalus membranaceus is known as "the best among all qi-tonifying herbs" and has various pharmacological effects such as enhancing immune function, protecting the nervous system, anti-tumor, protecting the cardiovascular and cerebrovascular systems, protecting internal organs, and regulating body metabolism, and is widely used in the treatment of diseases of the human immune system, metabolic system, and cardiovascular and cerebrovascular systems.

[0003] The stems and leaves of Astragalus membranaceus are the dried stems and leaves of Astragalus membranaceus or Astragalus mongholicus. Their medicinal value was first seen in "Ming Yi Bie Lu", which recorded that "the stems and leaves can treat thirst, muscle spasms, carbuncles, and sores", and there are also relevant records in "Compendium of Materia Medica" and "Qian Jin Yi Fang". During the growth and harvesting of Astragalus membranaceus, a large amount of its stems and leaves will be produced. Research has found that they contain similar chemical compositions and pharmacological activities to the medicinal part (the root of Astragalus membranaceus). However, at present, the utilization rate of the stems and leaves of Astragalus membranaceus is relatively low and they are often discarded, resulting in waste of resources. Summary of the Invention

[0004] The purpose of the present invention is to provide a triterpenoid cyclastraine A in the stems and leaves of Astragalus membranaceus and its application in neuroprotection.

[0005] The present invention first provides a compound, whose structural formula is shown in Formula I:

[0006]

[0007] Furthermore, the present invention provides a preparation method of the above compound, including the following steps:

[0008] (1) Using an ethanol aqueous solution as an extraction solvent to extract the stems and leaves of Astragalus membranaceus to obtain an extraction extract;

[0009] (2) Disperse the extraction extract in water, and then extract successively with petroleum ether and ethyl acetate. Concentrate the ethyl acetate extract to obtain an ethyl acetate extraction part extract;

[0010] (3) Purify the ethyl acetate extraction part extract successively by silica gel column chromatography, ODS column chromatography, and preparative HPLC to obtain the compound.

[0011] In the above preparation method, in step (1), the volume percentage concentration of the ethanol aqueous solution is 50% to 95%, specifically 70%;

[0012] The mass ratio of the astragalus membranaceus stems and leaves to the volume of the ethanol aqueous solution is 1 kg: 5 - 12 L.

[0013] In the above preparation method, in step (1), the extraction temperature is 85 - 95 °C, specifically 90 °C;

[0014] The extraction time is 1 - 5 h, specifically 2 h;

[0015] The number of extractions is 2 - 5 times.

[0016] In the above preparation method, in step (2), the mass ratio of the extracted extract to the volume of water is 1 kg: 8 - 15 L;

[0017] The mass ratio of the extracted extract to the volume of petroleum ether is 1 kg: 8 - 15 L;

[0018] The mass ratio of the extracted extract to the volume of ethyl acetate is 1 kg: 8 - 15 L;

[0019] The number of extractions with petroleum ether or ethyl acetate is 2 - 6 times.

[0020] In the above preparation method, in step (1), after extraction, the extract is concentrated under reduced pressure to obtain the extracted extract;

[0021] In step (2), the ethyl acetate extraction solution is concentrated by reduced pressure concentration.

[0022] For the above preparation method, step (3) specifically includes the following steps: S1. Mix the extract of the ethyl acetate extraction part with methanol, then sample it on silica gel, and perform gradient elution using a silica gel chromatographic column, where the eluents are dichloromethane and methanol with volume ratios of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1 in sequence; elute with 100:0 for 1 column volume, 100:1 for 2 column volumes, 50:1 for 2 column volumes, 20:1 for 3 column volumes, 10:1 for 3 column volumes, 5:1 for 4 column volumes, 3:1 for 4 column volumes, 2:1 for 3 column volumes, 1:1 for 3 column volumes, 0:1 for 1 column volume, and collect each eluate in portions of 1 / 4 of the column volume to obtain 104 eluates in sequence; combine the eluates of the 1st - 4th, 5th - 12th, 13th - 21st, 22nd - 30th, 31st - 36th, 37th - 45th, 46th - 57th, 58th - 66th, 67th - 79th, 80th - 92nd, 93rd - 99th, and 100th - 104th portions with similar chromatographic behaviors to obtain components Fr.1 - 12 in sequence;

[0023] S2. Further separate the Fr.8 component by ODS column chromatography, perform gradient elution with methanol - water as the mobile phase, and the volume percentages are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% methanol in sequence, where 10% elutes for 1 column volume, 20% elutes for 2 column volumes, 30% elutes for 2 column volumes, 40% elutes for 3 column volumes, 50% elutes for 2 column volumes, 60% elutes for 3 column volumes, 70% elutes for 3 column volumes, 80% elutes for 3 column volumes, 90% elutes for 1 column volume, 100% elutes for 1 column volume, and collect each eluate in portions of 1 / 3 of the column volume to obtain 63 eluates in sequence; combine the eluates of the 1st - 6th, 7th - 15th, 16th - 27th, 28th - 35th, 36th - 44th, 45th - 57th, and 58th - 63rd portions with similar chromatographic behaviors to obtain a total of 7 components in sequence, labeled as components Fr.8A - 8G;

[0024] S3. Purify Fr.8E by preparative HPLC, use methanol - water as the mobile phase, and the elution volume ratio is 68:32; collect the eluate and concentrate it to obtain the compound.

[0025] For the above preparation method, in step S1, the mass - to - volume ratio of the extract of the ethyl acetate extraction part to methanol is 300 - 500 g:2 L;

[0026] The mass ratio of the extract of the ethyl acetate part to the silica gel used for sampling is 1:1 - 1.5;

[0027] The silica gel used for sample mixing is 80 - 120 mesh silica gel;

[0028] The silica gel for packing the silica gel column is 200 - 300 mesh silica gel;

[0029] The mass ratio of the silica gel for packing the silica gel column to the extract of the ethyl acetate part is 5:1 - 8:1;

[0030] In steps S1 and S2, 10% sulfuric acid - ethanol is used as the color developing agent, and thin - layer chromatography is used to detect each eluent under ultraviolet light at 254 nm and 365 nm and under daylight to judge the eluents with similar chromatographic behaviors;

[0031] In step S1, the flow rate of the mobile phase is 120 - 150 min;

[0032] In step S2, the flow rate of the mobile phase is 60 - 80 min;

[0033] In step S3, the preparative HPLC is Shimadzu Essentia LC - 16P preparative high - performance liquid chromatograph;

[0034] The flow rate of the mobile phase is 3 - 10 mL / min.

[0035] Finally, the present invention provides the application of the above - mentioned compound in the preparation of drugs for treating and / or preventing neurodegenerative diseases or Alzheimer's disease.

[0036] The application of the above - mentioned compound in the preparation of any one of the following drugs also belongs to the protection scope of the present invention;

[0037] (1) Drugs for treating and / or preventing neuronal injury and / or apoptosis;

[0038] (2) Drugs for protecting nerve cells;

[0039] (3) Drugs for reducing the levels of IL - 6, IL - 1β and TNF - α.

[0040] The present invention uses separation methods such as normal - phase silica gel column chromatography, reverse - phase ODS column chromatography and high - performance liquid chromatography to separate and purify the chemical components in the ethanol extract of the stems and leaves of Astragalus membranaceus, and successfully discovers and identifies 1 new triterpenoid component. At the same time, through the PC12 cell model induced by Aβ 25-35 The protective effect of the new triterpenoid component on nerve cells was studied. The experimental results show that the new triterpenoid component in the stems and leaves of Astragalus membranaceus can significantly protect PC12 cells from damage through the mechanisms of anti - inflammation and inhibition of apoptosis. This discovery provides an important scientific basis for the further development and utilization of the stems and leaves of Astragalus membranaceus, and also provides a new research direction for the research and development of neuroprotective drugs. Description of the Drawings

[0041] Figure 1 HR-ESI-MS spectrum of cyclastraine A.

[0042] Figure 2 For cyclastraine A 1 1H NMR spectrum (600 MHz, CD3OD).

[0043] Figure 3 For cyclastraine A 13 13C NMR spectrum (150 MHz, CD3OD).

[0044] Figure 4 DEPT 135 spectrum of cyclastraine A.

[0045] Figure 5 HSQC spectrum of cyclastraine A.

[0046] Figure 6 HMBC spectrum of cyclastraine A.

[0047] Figure 7 For cyclastraine A 1 H- 1 H COSY spectrum.

[0048] Figure 8 For compound 1 1 H- 1 H COSY (red thick line) and key HMBC (blue arrows) correlations.

[0049] Figure 9 NOESY spectrum of cyclastraine A.

[0050] Figure 10 Key NOESY correlations (pink arrows) of compound 1.

[0051] Figure 11 Effect of cyclastraine A on the viability of PC12 cells ( n = 6); Note: Compared with the blank group, *P < 0.05, **P < 0.01.

[0052] Figure 12 Effect of cyclastraine A on Aβ 25-35 -induced viability of PC12 cells ( n = 6); Note: Compared with the blank group, **P < 0.01; compared with the model group,## P < 0.01.

[0053] Figure 13 Effect of cyclastraine A on Aβ 25-35 Effect on the morphology of Aβ-damaged PC12 cells (200×); among them, Figure 13 A in is the blank group; B is the model group; C is the cyclastraine A group.

[0054] Figure 14 Effect of cyclastraine A on Aβ 25-35 Effect on the levels of IL-6, IL-1β and TNF-α in the supernatant of Aβ-damaged PC12 cells ( n = 6).

[0055] Figure 15 Effect of cyclastraine A on Aβ 25-35 Effect on the expression of IL-6, IL-1β and TNF-α in Aβ-damaged PC12 cells; Note: Compared with the blank group, **P < 0.01; compared with the model group, ## P < 0.01.

[0056] Figure 16 Effect of cyclastraine A on Aβ 25-35 Effect on the apoptosis of Aβ-damaged PC12 cells (Hoechst 33342 staining method, 200×); among them, Figure 16 A in is the blank group; B is the model group; C is the cyclastraine A group.

[0057] Figure 17 Effect of cyclastraine A on Aβ 25-35 Effect on the expression of Caspase 3, Bax and Cyt C in Aβ-damaged PC12 cells; Note: Compared with the blank group, **P < 0.01; compared with the model group, ## P < 0.01.

[0058] Figure 18 Effect of cyclastraine A on Aβ 25-35 Effect on the apoptosis of Aβ-damaged PC12 cells; Note: Compared with the blank group, **P < 0.01. Detailed implementation manners

[0059] The present invention will be further described in detail below in combination with the detailed implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0060] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0061] In the quantitative tests in the following examples, unless otherwise specified, three repeated experiments were set, and the results were averaged.

[0062] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0063] Example 1. Preparation of a new triterpenoid component, cyclastraine A, from the stems and leaves of Astragalus membranaceus

[0064] 1. Experimental instruments and materials

[0065] P850 full-automatic polarimeter (China Hanon Systems Co., Ltd.); Bruker DPX 600 nuclear magnetic resonance spectrometer (Bruker, Germany); Q Exactive Focus LC-MS / MS liquid chromatography-mass spectrometry instrument (Thermo Fisher Scientific, USA); Büchi pump Manager C-610 medium-pressure chromatography instrument (Büchi, Switzerland); Voyager analytical high-performance liquid chromatography instrument (Chromai, China); Shimadzu Essentia LC-16P preparative high-performance liquid chromatography instrument (SHIMADZU, Japan); N-1300 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.); Sharpsil-UC18 analytical chromatography column (4.6 mm × 250 mm, 5 μm); Sharpsil-U C18 preparative chromatography column (21.2 mm × 250 mm, 5 μm); column chromatography silica gel (200 - 300 mesh, 80 - 120 mesh, Qingdao Ocean Chemical Factory); column chromatography ODS (YMC, Japan); chromatographic grade methanol (OCEANPAK, Sweden); other reagents are all of analytical grade (Tianjin Zhiyuan Chemical Reagent Co., Ltd.).

[0066] The stems and leaves of Astragalus membranaceus were collected from Hunyuan County, Datong City, Shanxi Province, China in August 2021, and were dried and stored in a ventilated and cool place after collection. Identified by Professor Pei Xiangping of the Traditional Chinese Medicine Identification Teaching and Research Section, School of Traditional Chinese Medicine and Food Engineering, Shanxi University of Traditional Chinese Medicine, this sample is the dried stems and leaves of Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao, a leguminous plant. The original plant specimen (specimen number: 20210820) is now stored in the Shanxi Modern Traditional Chinese Medicine Engineering Laboratory of Shanxi University of Traditional Chinese Medicine.

[0067] 2. Extraction and separation

[0068] Take 6 kg of dry Astragalus membranaceus stems and leaves, cut them into small sections evenly, and soak the medicinal materials with 8 times the amount of 70% (v / v) ethanol aqueous solution (that is, the material-liquid ratio of Astragalus membranaceus stems and leaves to ethanol solution is 1 kg: 8 L), heat under reflux at 90 °C for 2 h, and repeat the extraction 3 times. Combine the extraction solutions of the 3 times and concentrate under reduced pressure (40 °C) to obtain 1.23 kg of extract. Add 10 L of distilled water to the obtained extract and disperse it evenly, and then perform gradient extraction with different solvents. Use petroleum ether for extraction 4 times at 10 L / time, ethyl acetate for extraction 4 times at 10 L / time, and water-saturated n-butanol for extraction 3 times at 10 L / time, and combine the same kind of extraction solutions. After each extraction solution is concentrated under reduced pressure (among them, the concentration temperature of the ethyl acetate extraction part is 35 °C), the extracts of the corresponding extraction parts are obtained respectively, and the extract of the ethyl acetate part is 394 g.

[0069] Take 350 g of the ethyl acetate extract, dissolve it again with 2 L of methanol, then mix it with 400 g of silica gel of 80 - 120 mesh, and load the column by wet method and sample by dry method onto a silica gel chromatographic column (2.0 kg of silica gel of 200 - 300 mesh is loaded into a glass chromatographic column). Gradient elution is carried out by silica gel column chromatography to further separate and purify chemical components. The eluent is prepared according to the ratio of dichloromethane - methanol (100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1, v / v). The amount of the mobile phase for each elution ratio is 2 - 4 column volumes (flow rate is 120 min / column volume), where 100:0 elutes for 1 column volume, 100:1 elutes for 2 column volumes, 50:1 elutes for 2 column volumes, 20:1 elutes for 3 column volumes, 10:1 elutes for 3 column volumes, 5:1 elutes for 4 column volumes, 3:1 elutes for 4 column volumes, 2:1 elutes for 3 column volumes, 1:1 elutes for 3 column volumes, 0:1 elutes for 1 column volume. Each eluate is collected in portions of 1 / 4 of the column volume, and 104 eluates are obtained in sequence. Using 10% sulfuric acid - ethanol (i.e., the volume ratio of sulfuric acid to ethanol is 1:9) as the developer, each eluate is detected by thin - layer chromatography under ultraviolet light at 254 nm and 365 nm and under daylight. The 1st - 4th, 5th - 12th, 13th - 21st, 22nd - 30th, 31st - 36th, 37th - 45th, 46th - 57th, 58th - 66th, 67th - 79th, 80th - 92nd, 93rd - 99th, and 100th - 104th eluates with similar chromatographic behaviors are combined, and components Fr.1 - 12 are obtained in sequence.Among them, the Fr.8 fraction was further separated by ODS column chromatography, gradient elution was carried out with methanol-water as the mobile phase, and the volume percentages were 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol in sequence. The amount of the mobile phase used for each elution concentration was 1 to 3 column volumes (flow rate: 60 min / column volume), where 10% eluted for 1 column volume, 20% eluted for 2 column volumes, 30% eluted for 2 column volumes, 40% eluted for 3 column volumes, 50% eluted for 2 column volumes, 60% eluted for 3 column volumes, 70% eluted for 3 column volumes, 80% eluted for 3 column volumes, 90% eluted for 1 column volume, and 100% eluted for 1 column volume. Each eluate was collected in portions of 1 / 3 of the column volume, and 63 eluates were obtained in sequence; in the thin-layer chromatography identification, 10% sulfuric acid-ethanol was used as the developer, and each eluate was detected by thin-layer chromatography under ultraviolet light at 254 nm and 365 nm and under daylight. The 1st to 6th, 7th to 15th, 16th to 27th, 28th to 35th, 36th to 44th, 45th to 57th, and 58th to 63rd eluates with similar chromatographic behaviors were combined, and a total of 7 fractions were obtained in sequence, labeled as fractions Fr.8A - 8G; subsequently, Fr.8E was purified by preparative HPLC (Shimadzu Essentia LC-16P preparative high-performance liquid chromatograph), with methanol-water as the mobile phase, the elution volume ratio was 68:32, and the volume flow rate was 6 mL / min. Finally, a methanol-aqueous solution containing compound 1 was obtained, and after concentration under reduced pressure (40 °C), compound 1 (5.63 mg, t R = 25.2 min) was obtained.

[0070] 3. Structure Identification of Compound 1

[0071] Compound 1 is a white amorphous powder. An ion peak with m / z of 533.3483 [M+HCOO] - was detected by HR-ESI-MS in the negative ion mode (calculated value: C 31 H 49 O7, 533.3478) ( Figure 1 ), and in combination with 1 H-NMR ( Figure 2 ) and 13 C-NMR ( Figure 3 ) data, its molecular formula was deduced to be C 30 H 48 O5, and the degree of unsaturation was 7.

[0072] The 1 H-NMR data of compound 1 are shown in Table 1. In the high-field region, δ HCharacteristic signals of the cyclopropane methylene protons appeared at 0.93 (1H, d, J = 4.6 Hz, H-19a) and 0.61 (1H, d, J = 4.6 Hz, H-19b), indicating the presence of a cyclopropane structure. In addition, at δ H Five methyl proton signals were observed at 1.45 (3H, s, H3-18), 1.67 (3H, s, H3-26), 1.00 (3H, s, H3-28), 0.80 (3H, s, H3-29) and 0.71 (3H, s, H3-30). These proton signals were directly connected to δ C 12.5 (C-18), 13.7 (C-26), 25.9 (C-28), 13.8 (C-29) and 20.9 (C-30) respectively in the HSQC spectrum. In addition, at δ H A doublet methyl proton signal appeared at 0.95 (3H, d, J = 6.8 Hz, H3-21), indicating that this methyl group was connected to a tertiary carbon; at δ H Three oxygen-linked methine proton signals were observed at 3.38 (1H, t, J = 2.5 Hz, H-1), 3.69 (1H, dd, J = 10.2, 6.4 Hz, H-3) and 4.34 (1H, m, H-16). Based on the above data, it was speculated that compound 1 was a highly oxidized cycloartane-type triterpenoid compound.

[0073] According to the 13 C-NMR (Table 1), DEPT ( Figure 4 ) and HSQC ( Figure 5 ) spectrum analysis showed that compound 1 had a total of 30 carbon signals, including 1 carbonyl carbon signal (δ C 215.6), 6 quaternary carbon signals (one of which was an olefinic carbon signal, δ C 135.3), 8 tertiary carbon signals (three of which were oxygen-linked tertiary carbon signals, δ C 73.5, 74.3 and 72.2; one olefinic tertiary carbon signal, δ C 127.8), 9 secondary carbon signals (one of which was an oxygen-linked secondary carbon signal, δ C 69.2) and 6 methyl carbon signals. One carbonyl and one double bond contributed 2 degrees of unsaturation, and the remaining 5 degrees of unsaturation indicated that compound 1 had a five-membered ring skeleton. The data of compound 1 above were highly consistent with the known cycloartane-type triterpenoid 9β,19-cyclolanost-24E-ene-1α,3β,16β,27-tetraol, and the only difference was that compound 1 had an additional carbonyl group at the C-12 position. Through HMBC spectrum ( Figure 6 ) analysis, H2-11 (δ H3.00, 2.18) and H3-18 (δ H 1.45) and C-12 (δ C 215.6) are correlated, thus determining the position of the carbonyl group ( Figure 8 ). Combining 1 H- 1 H COSY ( Figure 7 ) and the HMBC spectrum to obtain the key H / C correlations ( Figure 8 ), the planar structure of Compound 1 was constructed as shown in Formula I below.

[0074]

[0075] Among the known cycloartane-type triterpenoids, H3-18, H-19, and H3-29 are all in the β configuration, while H3-28 and H3-30 are both in the α configuration. The relative configurations of H-17, OH-1, OH-3, and OH-16 were further determined to be α, α, β, and β, respectively, by the NOESY experiment of Compound 1. The NOESY spectrum ( Figure 9 ) shows correlation signals between H3-30 and H-17, H-17 and H3-21, H2-19 and H-1, H3-28 and H-3, and H3-30 and H-16 ( Figure 10 ). In addition, the Δ 24 double bond was determined to be in the E configuration by the presence of a NOESY correlation signal between H-24 and H2-27 ( Figure 10 ). Combining the above information, the structure of Compound 1 was determined. After searching the Scifinder database, it was found to be a new compound, which was named cyclastraine A.

[0076] Information on cyclastraine A is as follows:

[0077] Molecular formula: C 30 H 48 O5

[0078] HR-ESI-MS m / z: 533.3483 [M+HCOO] -

[0079] Appearance: White amorphous powder

[0080] Specific rotation: = +65 (c = 0.10, MeOH)

[0081] Table 1 1 1H-NMR (600 MHz, CD3OD) and 13 13C-NMR (150 MHz, CD3OD) data of Compound 1

[0082]

[0083] "o" represents an overlapping peak.

[0084] Example 2. Protective effect of cyclastraine A on Aβ 25-35 Study on the protective effect on the Aβ-induced PC12 cell injury model

[0085] Pheochromocytoma (PC12) cells are a monoclonal cell line isolated from rat adrenal pheochromocytoma. Because of their neuron-like characteristics and ease of culture, this cell line is used as an in vitro model in the fields of nervous system diseases, neurobiology, and neurochemistry research. Aβ 25-35 is an active fragment of β-amyloid (Aβ), which can aggregate to form amyloid fibrils in vivo and in vitro, and then produce neurotoxic effects on cells, leading to neuronal injury or apoptosis. This process is highly related to the pathological characteristics of neurodegenerative diseases such as AD. Therefore, Aβ 25-35 -induced PC12 cell injury model has been widely used in the screening of neuroprotective drugs and the study of their action mechanisms. In this study, through the Aβ 25-35 -induced PC12 cell injury model, the protective effect and potential mechanism of cyclastraine A (Compound 1, Comp 1) on nerve cells were investigated.

[0086] 1. Experimental materials and instruments

[0087] (1) Experimental materials

[0088] RPMI-1640 medium (GA24050182813), Wuhan Sevier Biotechnology Co., Ltd.; fetal bovine serum (SA211), Cyagen Biosciences (Beijing) Co., Ltd.; penicillin-streptomycin mixture (G4003), Wuhan Sevier Biotechnology Co., Ltd.; 0.25% trypsin digestion solution (GA2405006), Wuhan Sevier Biotechnology Co., Ltd.; PBS (GA23110190022), Wuhan Sevier Biotechnology Co., Ltd.; dimethyl sulfoxide (D8371), Beijing Solarbio Science & Technology Co., Ltd.; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, M2128-1G), Sigma-Aldrich; Hoechst 33342 (C0031); Beijing Solarbio Science & Technology Co., Ltd.; Aβ 25-35(Y-0044), Beijing BoaoSen Biotechnology Co., Ltd.; IL-6 ELISA Kit (F3066-A), Fankewi Co., Ltd.; IL-1β ELISA Kit (F2923-B), Fankewi Co., Ltd.; TNF-α ELISA Kit (F3056-A), Fankewi Co., Ltd.; Cell Smear (14mm), LanJieKe Technology Co., Ltd.; Triton X-100 (T8200), Beijing Solarbio Science & Technology Co., Ltd.; Goat Serum (G1208), Wuhan Sevier Biotechnology Co., Ltd.; IL-6 Antibody (0782R), Beijing BoaoSen Biotechnology Co., Ltd.; IL-1β Antibody (6319R), Beijing BoaoSen Biotechnology Co., Ltd.; TNF-α Antibody (10802R), Beijing BoaoSen Biotechnology Co., Ltd.; Caspase 3 Antibody (55032R), Beijing BoaoSen Biotechnology Co., Ltd.; Bax Antibody (33283M), Beijing BoaoSen Biotechnology Co., Ltd.; Cytochrome C Antibody (33193M), Beijing BoaoSen Biotechnology Co., Ltd.; Fluorescent Secondary Antibody (BA1039), Boster Biological Technology Co., Ltd.; DAPI Staining Reagent (G1012), Wuhan Sevier Biotechnology Co., Ltd.; Anti-Fluorescence Quenching Mounting Medium (G1401), Wuhan Sevier Biotechnology Co., Ltd.; Annexin V-FITC / PI Apoptosis Detection Kit (40302ES60), Yeasen Biotechnology (Shanghai) Co., Ltd.

[0089] (2) Experimental Instruments

[0090] Countstar Mira BF Automatic Cell Analyzer, Shanghai Ruijue Biotechnology Co., Ltd.; Axioscope5 Inverted Fluorescence Microscope, Zeiss Technology (Suzhou) Co., Ltd.; HAJ-3-160 Carbon Dioxide Incubator, Shanghai Yuejin Medical Instrument Co., Ltd.; EPOCH Full-Wavelength Microplate Reader, Shanghai SennaJie Biotechnology Co., Ltd.; SW-CJ-2FD Laminar Flow Hood, Suzhou Jinghua Co., Ltd.; AE31 Upright Microscope, Shanghai Optical Instrument Factory; CytoFLEX Flow Cytometer, Beckman Coulter Trading (China) Co., Ltd.

[0091] 2. Experimental Methods

[0092] (1) Cell Culture

[0093] During the process of resuscitating PC12 cells, the operation must be carried out quickly. The specific operation is as follows: First, soak the cells in warm water for 1 - 2 minutes, then quickly add complete medium (RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture), and then centrifuge at a speed of 1200 r / min for 5 minutes. After centrifugation, discard the supernatant, add 4 - 5 mL of complete medium, resuspend the cells in a 25 mL culture flask, and then place it in an incubator at 37°C and 5% CO2 for culture. After the PC12 cells are successfully resuscitated, the medium needs to be changed within 12 - 24 hours, that is, discard the original medium, then rinse twice with PBS buffer, and then add complete medium to complete the medium change step.

[0094] During cell culture, subculture is required once every 1 - 2 days. When the cells grow to the logarithmic growth phase, first discard the original medium, then rinse twice with PBS buffer, then add trypsin for digestion. After digestion is completed, add complete medium to terminate the digestion process, then collect the cell suspension, centrifuge at 1200 r / min for 5 minutes, discard the supernatant, add complete medium to resuspend the cells, and subculture according to a volume ratio of 1:2.

[0095] When the cells are passaged to a certain generation, cryopreservation operation needs to be carried out. When the cells grow to the logarithmic growth phase, first discard the medium, rinse twice with PBS buffer, then add trypsin for digestion, add complete medium to terminate the digestion, collect the cell suspension, centrifuge at 1200 r / min for 5 minutes, discard the supernatant, add 1.5 mL of fetal bovine serum, gently pipette to mix evenly, transfer to a 2.5 mL cryopreservation tube, then add 150 μL of dimethyl sulfoxide (DMSO) and mix well, finally seal with a sealing film, make corresponding marks, put it into a programmable freezing box, and then place it in a -80°C refrigerator for cryopreservation.

[0096] (2) Effect of cyclastraine A on the survival rate of PC12 cells

[0097] Select cells in the logarithmic growth phase, at a concentration of 1×10 5PC12 cells were seeded into 96-well plates at a density of cells / mL, with an inoculation volume of 100 μL per well. At the same time, 6 replicate wells were set up in each group. Then, they were placed in an incubator at 37 °C and 5% CO2 for 24 h. After the culture was completed, the medium was discarded. Subsequently, cyclastraine A solutions with concentrations of 5, 25, 50, and 100 μM (cyclastraine A was dissolved in serum-free RPMI-164 medium) were added to each well in turn, with an addition volume of 100 μL / well. The blank group and the negative group were added with serum-free RPMI-1640 medium. After the addition operation was completed, the treated cells were placed in an incubator at 37 °C and 5% CO2 for continued cultivation for 24 h. After the cultivation was completed, they were taken out. 10 μL of MTT reagent was added to each well, and then the cultivation was continued for 4 h and then the cultivation process was terminated. Then, the supernatant in each well was aspirated, and 100 μL of DMSO was added to each well. They were incubated at 37 °C for 10 min under light-proof conditions. After the incubation was completed, the measurement was carried out at a wavelength of 490 nm, and then the cell survival rate was calculated according to the formula.

[0098] Cell survival rate (%) = (Aexperimental - Anegative) / (Ablank - Anegative) × 100%

[0099] (3) Protective effect of cyclastraine A on Aβ 25-35 -induced damaged PC12 cells

[0100] a. Solution preparation

[0101] Aβ 25-35 solution: Dissolve 5 mg of Aβ 25-35 in 4.717 mL of PBS solution to prepare a solution with a concentration of 1 mM. Subsequently, the prepared solution was placed in an incubator at 37 °C for 7 d to age it and form a condensate. Then, it was aliquoted into small portions with a volume of 86.13 μL or 287.1 μL per tube and stored in a -20 °C refrigerator. Before use, the corresponding aliquot solution was taken out, diluted to 5.742 μM with serum-free RPMI-164 medium, and placed at 4 °C for standby.

[0102] cyclastraine A solution: Weigh 2 mg of cyclastraine A and dissolve it in 2 mL of PBS solution to prepare a 1 mg / mL solution. Before use, it was diluted with serum-free RPMI-164 medium to concentrations of 100, 50, 25, and 5 μM respectively.

[0103] b. Detection of cell survival rate

[0104] Cells in the logarithmic growth phase were selected and seeded at 1×10 5PC12 cells were seeded in 96-well plates at a density of cells / mL, with an inoculation volume of 100 μL per well, and 6 replicates were set for each group. After culturing at 37 °C and 5% CO2 for 24 h until the cells adhered, the culture medium was discarded. Subsequently, the cells were divided into a blank group, a model group, a negative group, and a drug administration group. Among them, 100 μL of serum-free RPMI-164 medium was added to each well in the blank group, the model group, and the negative group; in the drug administration group, cyclastraine A solutions with concentrations of 5, 25, 50, and 100 μM were added respectively, and then intervened at 37 °C for 2 h. After that, 100 μL of serum-free RPMI-164 medium was added to the blank group and the negative group respectively, while 100 μL of Aβ 25-35 solution with a concentration of 5.742 μM was added to the model group and the drug administration group, and then they were cultured together at 37 °C and 5% CO2 for 24 h. After the culture was completed, 10 μL of MTT reagent was added to each well, and then the culture was continued at 37 °C and 5% CO2 for 4 h and then terminated. Subsequently, the supernatant in each well was aspirated, and 100 μL of DMSO was added to each well and incubated at 37 °C for 10 min in the dark. After incubation, the absorbance was measured at a wavelength of 490 nm, and then the cell survival rate was calculated.

[0105] c. Observation of cell morphology

[0106] Cells in the logarithmic growth phase were selected and seeded in 24-well plates at a density of 1×10 5 cells / mL, with an inoculation volume of 0.5 mL of cell suspension per well, and 3 replicates were set for each group. Then they were cultured for 24 h. After the cells adhered, the culture medium was discarded, and the grouping and drug administration method described in item "b" was followed. After that, the cell morphology of each group was carefully observed under an inverted microscope and corresponding records were made.

[0107] d. Detection of the contents of IL-6, IL-1β, and TNF-α in the cell supernatant by ELISA

[0108] Group and administer drugs to the cells according to the method described in item "b". Subsequently, collect the cell culture medium and collect the supernatant by centrifugation for detecting the contents of inflammatory factors IL-6, IL-1β, and TNF-α using an ELISA kit. The specific operation steps are as follows: First, take out the required strips and place them at room temperature for 60 min of equilibration. Then, set up standard wells, blank wells, and sample wells in sequence. After adding standards and samples to the corresponding wells, seal the reaction wells with a sealing film, and then place them in an environment of 37 °C and 5% CO2 for incubation for 30 min. After incubation, add 50 μL of detection antibody labeled with horseradish peroxidase (HRP) to each well, seal the reaction wells with a sealing film again, and continue to incubate at 37 °C and 5% CO2 for 30 min. Then, repeat the plate washing operation 5 times for the strips using the washing solution (add 350 μL to each well each time). Referring to the kit instruction manual, add 50 μL of substrate A and B to each well respectively, and then place them in an environment of 37 °C and dark for incubation for 15 min. After incubation is completed, add 50 μL of stop solution to each well, and then measure the OD values of each well at a wavelength of 450 nm.

[0109] e. Detection of apoptosis of PC12 cells by Hoechst 33342 staining method

[0110] First, place the cell slides in a 24-well plate. Then, select cells in the logarithmic growth phase and inoculate PC12 cells into the 24-well plate at a density of 1×10 5 cells / mL, with the volume of the cell suspension inoculated into each well being 0.5 mL. Set 3 replicates for each group, and then culture them for 24 h. After the cells adhere to the wall, discard the culture medium, and then group and administer drugs according to the method described in item "b". After drug administration, co-culture at 37 °C and 5% CO2 for 24 h, and then discard the supernatant. Then, fix the cultured cells with 4% paraformaldehyde for 10 min. After fixation is completed, remove the fixing solution, and then wash the cells with PBS buffer. After the liquid is completely aspirated, add Hoechst 33342 staining solution with a concentration of 10 μg / mL to each well, with the addition amount being 0.5 mL / well, and then incubate in the dark for 5 min. After incubation is completed, continue to wash with PBS buffer, with each washing duration being 5 min, and wash 3 times in total. Finally, cover the slides with attached cells on the glass slides, carefully observe the cell morphology of each group using a fluorescence microscope, and make corresponding records.

[0111] f. Cellular immunofluorescence (IF)

[0112] The cells were grouped and given drug interventions according to the method described in item "b". After the intervention was completed, the cell slides were washed 3 times with PBS buffer. Subsequently, 4% paraformaldehyde was added at room temperature, and the slides were placed on a shaker to fix the cells for 15 min. After fixation, the supernatant was discarded and the cells were washed 3 times with PBS buffer again. Then, the cells were permeabilized with 0.5% Triton for 20 min. After the treatment was completed, the cell slides were gently and quickly washed 3 times with PBS buffer again, and then goat serum was added to block for 15 min. After that, primary antibodies (IL-6, IL-1β, TNF-α, Caspase 3, Bax, Cyt C) were added and incubated overnight at 4°C. After the incubation was completed, the supernatant was discarded and the cells were washed 3 times with PBS buffer. Subsequently, fluorescent secondary antibodies were added in the dark and incubated at room temperature for 2 h. After the incubation was completed, the cells were also washed 3 times with PBS buffer. Then, the cells were stained with 1×DAPI for 5 min. After the staining was completed, the cells were washed 3 times with PBS buffer. Finally, a fluorescence quencher was added to the glass slide, the cell side of the cell slide was placed face down on the glass slide, and the slide was sealed with an anti-fluorescence quenching mounting medium. Then, it was placed under a microscope for observation.

[0113] g. Flow cytometry was used to detect cell apoptosis

[0114] The cells were grouped and given drug interventions according to the method described in item "b". After the intervention was completed, the supernatant was collected first, and then the cells were digested with trypsin without EDTA. Subsequently, the supernatant was used to terminate the digestion process. After that, the cells were centrifuged at 2000 r / min for 5 min. After centrifugation, the supernatant was discarded. Then, pre-cooled PBS buffer was added to wash the cells. After washing twice, the cell density was adjusted to 1×10 6 cells. The cells were centrifuged again at 2000 r / min for 5 min, and the supernatant generated this time was discarded to obtain a cell pellet. 100 μL of 1×Binding Buffer was added to the cell pellet to prepare a cell suspension. According to the operation instructions of the cell apoptosis kit, 5 μL of Annexin V-FITC and 10 μL of PI solution were added in sequence. After thorough mixing, the mixture was placed in the dark at room temperature for reaction for 10 min. After the reaction was completed, 400 μL of 1×BindingBuffer solution was added and mixed well. The mixture was placed on ice, and finally the cell apoptosis was detected by flow cytometry.

[0115] h. Data analysis

[0116] GraphPad Prism 9 software was used to analyze the statistical data, and all data were expressed as mean ± standard deviation It is expressed in the form of. If the data conforms to the normal distribution, then the one-way analysis of variance method is used for analysis; if the data does not conform to the normal distribution, the non-parametric test method is correspondingly used for analysis and processing.

[0117] 3. Experimental results

[0118] (1) Effect of cyclastraine A on the viability of PC12 cells

[0119] The effect of cyclastraine A on the viability of PC12 cells was evaluated using the MTT assay. As Figure 11 shown, compared with the control group, cyclastraine A had little effect on the viability of PC12 cells in the concentration range of 5, 25, and 50 μM. However, when the concentration was increased to 100 μM, the cell viability decreased significantly, indicating that cyclastraine A had obvious cytotoxicity to PC12 cells at a concentration of 100 μM.

[0120] (2) Effect of cyclastraine A on Aβ 25-35 -induced viability of PC12 cells

[0121] Aβ 25-35 is an active fragment of the Aβ protein, which has neurotoxicity and can mimic the pathological environment in the brains of AD patients. To evaluate the neuroprotective potential of cyclastraine A against Aβ-induced toxicity, we constructed an in vitro model of Aβ 25-35 -induced PC12 cells. As Figure 12 shown, compared with the control group, the viability of PC12 cells decreased significantly (P < 0.01) after Aβ 25-35 induction. Compared with the model group, cyclastraine A significantly increased the viability of PC12 cells at concentrations of 5 μM and 25 μM (P < 0.05). Based on this, 25 μM of cyclastraine A was selected for subsequent experiments to carry out relevant research.

[0122] (3) Effect of cyclastraine A on the morphology of Aβ 25-35 -induced PC12 cells

[0123] Normally, PC12 cells usually show a spindle-shaped or polygonal morphology, with a high cell density and significant neurite outgrowth ( Figure 13 ). After Aβ 25-35After induction, the model group showed very obvious morphological changes, specifically manifested as increased cell gaps, rounded cell bodies, and shortened neurites. After intervention with cyclastraine A, the cell morphology was very similar to that of the control group, indicating that cyclastraine A has significant therapeutic effects.

[0124] (4) Cyclastraine A exerts a protective effect on Aβ 25-35 -induced PC12 cells

[0125] Through ELISA analysis of the cell supernatant, it was found that ( Figure 14 ), compared with the control group, the levels of inflammatory factors IL-6, IL-1β, and TNF-α in the model group were significantly increased (P < 0.01). After administration of cyclastraine A, it was observed that it had a regulatory effect on these inflammatory cytokines. Cyclastraine A could significantly reduce the levels of IL-6, IL-1β, and TNF-α (P < 0.05 or P < 0.01).

[0126] In addition, the levels of IL-6, IL-1β, and TNF-α in cells were detected by IF. As Figure 15 shown, the fluorescence intensities of IL-6, IL-1β, and TNF-α in the model group were significantly higher than those in the control group (P < 0.01). In contrast, after administration of cyclastraine A, the fluorescence intensities were significantly decreased (P < 0.01). This indicates that cyclastraine A can exert a protective effect on Aβ 25-35 -induced PC12 cells by reducing the inflammatory response, and thus may relieve memory impairment.

[0127] (5) Cyclastraine A exerts a protective effect on Aβ 25-35 -induced PC12 cells by inhibiting apoptosis

[0128] Apoptotic cells were evaluated by Hoechst 33342 staining ( Figure 16 ). In the control group, the cell nuclei were round and showed pale blue fluorescence. In contrast, after Aβ 25-35 induction, significant nuclear condensation occurred in the model group, manifested as dense blue fluorescence. After administration of cyclastraine A, the apoptosis of cells was significantly improved, indicating that cyclastraine A has an obvious inhibitory effect on Aβ 25-35 -induced cell apoptosis.

[0129] The effects of cyclastraine A on the levels of intracellular pro-apoptotic factors Caspase 3, Bax, and Cytochrome C were detected using IF, and the results are as Figure 17 shown. Compared with the control group, after treatment with Aβ 25-35 , the expressions of Caspase 3, Bax, and Cytochrome C were significantly upregulated (P<0.01). After administration of cyclastraine A, except for its non-significant effect on the downregulation of Bax, the fluorescence intensities of the remaining related indicators were significantly decreased (P<0.01 or P<0.05). This detection result indicates that cyclastraine A can reduce Aβ 25-35 -induced apoptosis of PC12 cells by inhibiting pro-apoptotic proteins.

[0130] Meanwhile, flow cytometry analysis was performed, and the results are as Figure 18 shown. After administration of cyclastraine A, the apoptosis rate decreased. This indicates that cyclastraine A can effectively inhibit Aβ 25-35 -induced apoptosis of PC12 cells, thereby exerting corresponding protective effects.

Claims

1. A compound, the structural formula of which is shown in Formula I:

2. A method for preparing the compound according to Claim 1, comprising the following steps: (1) Using an ethanol aqueous solution as an extraction solvent to extract the stems and leaves of Astragalus membranaceus, to obtain an extraction extract; (2) Disperse the said extraction extract in water, then successively extract with petroleum ether and ethyl acetate, and concentrate the ethyl acetate extract to obtain an ethyl acetate extraction part extract; (3) Purify the ethyl acetate extraction part extract successively by silica gel column chromatography, ODS column chromatography and preparative HPLC to obtain the said compound.

3. The preparation method according to claim 2, characterized in that: In step (1), the volume percentage concentration of the ethanol aqueous solution is 50%-95%, specifically it can be 70%; The mass ratio of the stems and leaves of Astragalus membranaceus to the volume of the ethanol aqueous solution is 1 kg: 5-12 L.

4. The preparation method according to claim 2 or 3, characterized in that: In step (1), the extraction temperature is 85-95 °C; The extraction time is 1-5 h; The extraction times are 2-5 times.

5. The preparation method according to any one of claims 2-4, characterized in that: In step (2), the mass ratio of the extraction extract to the volume of water is 1 kg: 8-15 L; The mass ratio of the extraction extract to the volume of petroleum ether is 1 kg: 8-15 L; The mass ratio of the extraction extract to the volume of ethyl acetate is 1 kg: 8-15 L; The extraction times of the petroleum ether or ethyl acetate are 2-6 times.

6. The preparation method according to any one of claims 2-5, characterized in that: In step (1), after extraction, the extraction solution is concentrated under reduced pressure to obtain the said extraction extract; In step (2), the ethyl acetate extract is concentrated by reduced pressure concentration.

7. The preparation method according to any one of claims 2-6, characterized in that: Step (3) specifically includes the following steps: S1. Dissolve the ethyl acetate extraction part extract in methanol, then mix it with silica gel, and perform gradient elution by silica gel column chromatography, wherein the eluents are dichloromethane and methanol with volume ratios of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1 in sequence; elute with 100:0 for 1 column volume, 100:1 for 2 column volumes, 50:1 for 2 column volumes, 20:1 for 3 column volumes, 10:1 for 3 column volumes, 5:1 for 4 column volumes, 3:1 for 4 column volumes, 2:1 for 3 column volumes, 1:1 for 3 column volumes, 0:1 for 1 column volume, and collect each eluent in portions of 1 / 4 of the column volume, successively obtaining 104 eluents; Combine the eluents of the 1st-4th, 5th-12th, 13th-21st, 22nd-30th, 31st-36th, 37th-45th, 46th-57th, 58th-66th, 67th-79th, 80th-92nd, 93rd-99th, 100th-104th with similar chromatographic behaviors, and successively obtain components Fr.1-12 in order; S2. Further separate the Fr.8 fraction by ODS column chromatography, perform gradient elution with methanol-water as the mobile phase, and the volume percentages are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol in sequence. Among them, 10% elutes for 1 column volume, 20% elutes for 2 column volumes, 30% elutes for 2 column volumes, 40% elutes for 3 column volumes, 50% elutes for 2 column volumes, 60% elutes for 3 column volumes, 70% elutes for 3 column volumes, 80% elutes for 3 column volumes, 90% elutes for 1 column volume, and 100% elutes for 1 column volume. Collect each eluate in portions of 1 / 3 of the column volume, and obtain 63 eluates in sequence; Combine the eluates of the 1st - 6th, 7th - 15th, 16th - 27th, 28th - 35th, 36th - 44th, 45th - 57th, and 58th - 63rd portions with similar chromatographic behaviors, and obtain 7 fractions in sequence, labeled as fractions Fr.8A - 8G; S3. Purify Fr.8E by preparative HPLC, use methanol-water as the mobile phase, and the elution volume ratio is 68:32; Collect the eluate, concentrate it to obtain the said compound.

8. The preparation method according to claim 7, characterized in that: In step S1, the mass ratio of the ethyl acetate extraction part extract to the volume of methanol is 300 - 500 g:2 L; The mass ratio of the ethyl acetate part extract to the silica gel used for sample mixing is 1:1 - 1.5; The silica gel used for sample mixing is 80 - 120 mesh silica gel; The silica gel for packing the silica gel column is 200 - 300 mesh silica gel; The mass ratio of the silica gel for packing the silica gel column to the ethyl acetate part extract is 5:1 - 8:1; In steps S1 and S2, use 10% sulfuric acid - ethanol as the color developing agent, detect each eluate by thin layer chromatography under ultraviolet light at 254 nm and 365 nm and under daylight, and judge the eluates with similar chromatographic behaviors; In step S1, the flow rate of the mobile phase is 120 - 150 min / column volume; In step S2, the flow rate of the mobile phase is 60 - 80 min / column volume; In step S3, the preparative HPLC is Shimadzu Essentia LC - 16P preparative high performance liquid chromatograph; The flow rate of the mobile phase is 3 - 10 mL / min.

9. Use of the compound according to claim 1 in the preparation of a drug for treating and / or preventing neurodegenerative diseases or Alzheimer's disease.

10. Use of the compound according to claim 1 in the preparation of any one of the following drugs: (1) A drug for treating and / or preventing neuronal injury and / or apoptosis; (2) A drug for protecting nerve cells; (3) A drug for reducing the levels of IL - 6, IL - 1β, and TNF - α.

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