Schisandra chinensis triterpenoid reducing compound as well as preparation and application thereof

Abstract

The invention relates to a schisandra chinensis nortriterpenoid compound which is extracted from schisandra chinensis fruits, has an excellent neuroprotective effect and is expected to be used for preventing and treating neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to schisandrin reduced triterpenoids and preparation and use thereof. BACKGROUND

[0002] Schisandra chinensis (Turcz.) Baill, commonly known as "North Schisandra", is mainly produced in Heilongjiang, Jilin, Liaoning, Inner Mongolia and other places. It was first recorded in "Shennong's Herbal Classic", and was listed as the top product. It has the effects of astringing, tonifying, invigorating, and nourishing the heart and kidney. It is also used for treating various diseases related to the nervous system. As one of the "Dragon Nine Flavors" brand Chinese medicinal materials in Heilongjiang Province, Schisandra chinensis is planted in an area of more than 7,000 mu in Heilongjiang Province, with an annual output of more than 200,000 tons, accounting for more than 40% of the national market share (Zhou X J, Wang Q, Yan B W, et al. Research progress on the effects and mechanisms of Schisandra chinensis on nervous and mental diseases [J]. Chinese Pharmacological Bulletin, 2022, 38: 338-342).

[0003] Schisandra chinensis has various pharmacological effects and biological activities, and its mechanisms of liver protection, anti-tumor, sedation, etc. are relatively systematic and in-depth, and the relative pharmacodynamic material basis is also relatively clear (Zhou X J, Wang Q, Yan B W, et al. Research progress on the effects and mechanisms of Schisandra chinensis on nervous and mental diseases [J]. Chinese Pharmacological Bulletin, 2022, 38: 338-342; Liu Z R. Chemical composition research and neuroprotective activity evaluation of Schisandra chinensis [D]. Northwest A&F University, 2023). However, the research on the anti-nervous system disease of Schisandra chinensis is relatively weak. However, it is worth mentioning that many literatures have reported that Schisandra chinensis has strong biological activities related to anti-nervous system diseases, including neuroprotection, anti-neuroinflammation, inhibition of oxidative stress, etc. Schisandra chinensis can improve the motor balance, dopamine level, dopamine neuron and tyrosine hydroxylase positive cell defects of Parkinson's disease (PD) mice, and is a potential candidate traditional Chinese medicine for improving and preventing PD

[13] Meanwhile, some studies have confirmed that Schisandra chinensis can increase the expression of BDNF in the striatum and hippocampus of PD mice and reduce the activity of GSK-3β, and it is speculated that it may inhibit oxidative stress, neuroinflammation and apoptosis by participating in the BDNF / Nrf6 / NF-κB signaling pathway, thereby playing a role in improving the degenerative changes of DA neurons in 6-OHDA-induced PD mice (Yan T, Mao Q, Zhang X, et al. Schisandra chinensis protects against dopaminergic neuronal oxidative stress, neuroinflammation and apoptosis via the BDNF / Nrf2 / NF-κB pathway in 6-OHDA-induced Parkinson's disease mice [J]. Food Funct. 2021, 12: 4079-4091). Another study shows that Schisandra chinensis can effectively improve the oxidative reaction inside the nerve cells of Alzheimer's disease (AD) mice, promote the expression of Bcl-2 to improve the oxidative damage caused by H2O2, and improve the learning and memory function of AD mice; it can also protect the morphological structure of brain tissue and improve the memory ability of memory disorder mice by reducing the content level of active oxygen in brain tissue and controlling p65.

[0004] The main chemical components of Schisandra chinensis include lignans, terpenes, polysaccharides, and volatile oils. Lignans are considered as the characteristic chemical components of S. chinensis and are one of the main active ingredients. They are often used as the index components for the quality evaluation of S. chinensis and have been reported to have good neuroprotective effects. A total of 86 lignans have been found. Triterpenoids are another main component type of S. chinensis, which have novel structures, high oxidation degree, diverse skeletal rearrangements, and multiple biological activities such as neuroprotection and antioxidant effects. A total of 82 triterpenoids have been isolated from S. chinensis, with a discovery rate of more than 70% and 15 novel skeletons. Polysaccharides and volatile oils of S. chinensis have also been reported to have pharmacological effects such as improving cognitive function, reversing microglial activation, and inhibiting neural apoptosis.Polysaccharide from Schisandra chinensis acts via LRP-1 to reverse microglia activation through suppression of the NF-κB and MAPK signaling[J]. J. Ethnopharmacol. 2020, 256: 112798; Xu M, Zhang X, Ren F, et al. Essential oil of Schisandra chinensis ameliorates cognitive decline in mice by alleviating inflammation[J]. Food Funct. 2019, 10: 5827-5842). In addition, our previous researches have found that lignans from the stems of S. chinensis (Zhang YQ, Liu Y, Wang YQ, et al. Three new schinortriterpenoids from the leaves of Schisandra chinensis (Turcz.) Baill[J]. Natural Product Research, 2023, 39: 2121-2129) can reduce the level of reactive oxygen species and inhibit ROS release to exert neuroprotective effect, and triterpenoids from the leaves of S. chinensis (Liu Y, Guo JT, Wang ZB, et al. Aromatic monoterpenoid glycosides from rattan stems of Schisandra chinensis and their neuroprotective activities[J]. Fitoterapia, 2019, 134: 108-112) can target the regulation of AD mitochondrial autophagy protein MCL-1 and reduce the expression of inflammatory factors to effectively improve the cognitive function of AD mice.

[0005] In summary, S. chinensis has been clinically and pharmacologically proven to have significant neuroprotective effects. As a commonly used kidney-tonifying herb in clinical practice, S. chinensis is sour and salty in flavor, which enters the liver and tonifies the kidney, and is consistent with the therapeutic principle of nervous system diseases. Although previous studies have reported that various types of chemical components of S. chinensis have neuroprotective, anti-neuroinflammatory, and antioxidant activities, there is no research on the neuroprotective activity of triterpenoids from S. chinensis, and the molecular mechanisms and pharmacodynamic material basis have not been systematically elucidated. SUMMARY

[0006] In order to solve the problems in the prior art, the present application provides schisandrid or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof, and a preparation method and use thereof in neuroprotection.

[0007] In one aspect, the present application provides schisandrid 1, 12-14, 18, 21 as shown below, or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof:

[0008]

[0009] In the present application, the prodrug refers to a compound which is converted into an active compound as a result of reaction with enzymes, gastric acid, etc. in the body. As a prodrug, many types of prodrugs are known, for example, esters such as methyl ester, ethyl ester, etc., and suitable prodrugs can be selected from known literature and synthesized by known methods.

[0010] In the present application, the solvate refers to a compound form which forms a complex in a solid or liquid state by coordination with solvent molecules.

[0011] The present application also provides a pharmaceutical composition comprising the schisandrid or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof.

[0012] The present application also provides use of the schisandrid or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof or the pharmaceutical composition in the preparation of a medicament for protecting nerves.

[0013] Preferably, the medicament is for protecting PC12 cells.

[0014] The present application also provides use of the schisandrid or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof or the pharmaceutical composition in the preparation of a medicament for preventing or treating neurodegenerative diseases.

[0015] Preferably, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis.

[0016] Preferably, the pharmaceutical composition or medicament of the present application further comprises a pharmaceutically acceptable carrier or adjuvant.

[0017] As the pharmaceutically acceptable carrier or adjuvant, any carrier or adjuvant known to be suitable for the preparation of a particular drug or pharmaceutical composition can be used. Examples thereof include, but are not limited to, solvents, excipients, dispersing agents, emulsifiers, solubilizers, gel-forming agents, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, binders, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerization agents, lubricants, coating agents, propellants, tonicity-adjusting agents, surfactants, coloring agents, flavoring agents, sweetening agents, and dyes. In particular, adjuvants of a type suitable for the desired formulation and the desired mode of administration are used.

[0018] Further, the pharmaceutically acceptable carrier or adjuvant can be selected from, for example, water, starch, compressible starch, dextrin, sucrose, lactose, fructose, glucose, xylitol, mannitol, microcrystalline cellulose, calcium carbonate, magnesium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, magnesium oxide, aluminum hydroxide, carboxymethyl cellulose calcium, carboxymethyl cellulose, sodium carboxymethyl starch, hydroxypropyl starch, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, starch paste, glucose paste, sucrose paste, fructose paste, sorbitol, gelatin paste, gum arabic paste, gum tragacanth paste, microcrystalline cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose calcium, polymethacrylate, alginic acid, sodium alginate, polyethylene glycol, colloidal magnesium aluminum silicate, stearic acid, calcium stearate, magnesium stearate, zinc stearate, talc, glycerol monostearate, glycerol palmitostearate, magnesium lauryl sulfate, polyethylene glycol, sodium stearyl fumarate.

[0019] The pharmaceutical composition or drug is presented in a unit dosage form such as a tablet, a pill, a capsule, a powder, a granule, a suspension, an oral liquid agent, an aerosol or a liquid spray, a drop; a dosage form for oral, parenteral, intranasal, sublingual or rectal administration in the mouth or for inhalation or jet administration. Preferably, the pharmaceutical dosage form is an oral preparation, more preferably a tablet, a pill, a capsule, a granule, a suspension, a powder, an oral liquid agent.

[0020] The pharmaceutical composition or drug of the present application can be manufactured by methods well known in the art, such as the conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, freeze-drying or lyophilizing processes. The content of the active ingredient in the pharmaceutical composition of the present application should be within the range of 0.05 to 90% by weight, preferably 0.1 to 50% by weight, of the entire composition.

[0021] For adult patients, the compounds of the present application can be administered orally or parenterally in an amount of 0.001 to 500 mg as a single administration, once a day or divided into several times. It should be noted that the administration amount can be appropriately increased or decreased according to the type of disease, the age and weight of the patient, symptoms, etc.

[0022] The present application also provides a method for preparing the schisandrin nor-triterpenoids 1, 12-14, 18, 21, which comprises extracting the fruit of Schisandra chinensis with a solvent to obtain a schisandra extract, and then separating the compounds 1, 12-14, 18, 21 from the schisandra extract.

[0023] Preferably, the solvent extraction comprises heating extraction with an organic solvent.

[0024] Preferably, the solvent extraction comprises heating extraction with an organic solvent.

[0025] After extraction, the extract is filtered and combined, and the solvent is removed by evaporation under reduced pressure to obtain the schisandra extract.

[0026] Preferably, the obtained schisandra extract is dispersed in water, and then extracted with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extract, which is separated by silica gel column chromatography, MCI column chromatography, Sephadex LH-20 chromatography, RP-C 18 chromatography, and semi-preparative chromatography to obtain the compounds 1, 12-14, 18, 21.

[0027] Preferably, the ethyl acetate extract is separated by silica gel column chromatography with dichloromethane-methanol (50:0-0:1, v / v) gradient elution, and the eluate is identified by thin layer chromatography and combined to obtain eight components Fr. I-VIII; Fr. III is separated by MCI column chromatography with 20-40% EtOH (4-6 BV) and 90-95% EtOH (6-10 BV) elution to obtain two sub-components A-B; Fr. B is separated by silica gel chromatography column with petroleum ether-acetone (50:1-0:1, v / v) gradient elution to obtain 10 components Fr. B1-B10; Fr. B7 is further separated by Sephadex LH-20 (MeOH) and RP-C 18 (MeOH:H2O, 10:90-100:0, v / v) to obtain Fr. B7.1-15; Fr. B7.2 is separated by semi-HPLC (MeCN:H2O, 34:66, v / v) to obtain compounds 13 (t R = 17.9 min) and 14 (t R= 37.3 min); Fr. B7.4 was separated by semi-HPLC (MeCN:H2O, 45:55, v / v) to give compound 12 (t R = 35.0 min); Fr. B7.7 was separated by semi-HPLC (MeCN:H2O, 42:58, v / v) to give compound 21 (t R = 32.0 min); Fr. B8 was separated by Sephadex LH-20 (MeOH) and RP-C 18 = 32.0 min); Fr. B8 was separated by Sephadex LH-20 (MeOH) and RP-C R = 35.0 min).

[0028] Fr. IV was subjected to MCI column chromatography, eluted with 20-40% ethanol (4-6 BV) and 90-95% ethanol (6-10 BV) to give two sub-fractions A-B, Fr. B was subjected to silica gel column chromatography, eluted with petroleum ether-acetone (50:1-1:1, v / v) to give eight fractions Fr. B1-B8, Fr. B6 was subjected to Sephadex LH-20 (MeOH) and RP-C 18 = 32.0 min); Fr. B8 was separated by Sephadex LH-20 (MeOH) and RP-C R = 18.0 min).

[0029] In the present application, the percentage of ethanol and the ratio of each solvent refer to volume ratio.

[0030] Beneficial effects: The present application provides Schisandra descending triterpenoids, which are extracted from Schisandra fruit and have excellent neuroprotective effect, and are expected to be used for the prevention and treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Flow chart of separation of Schisandra descending triterpenoids 1, 12-14, 18, 21;

[0032] Figure 2 : Structure of compound 1;

[0033] Figure 3 : HMBC, H- 1 COSY and NOESY correlation signals of compound 1; 1 COSY and NOESY correlation signals of compound 1;

[0034] Figure 4 ECD spectrum of compound 1;

[0035] Figure 5 Crystallographic structure of compound 1;

[0036] Figure 6 Structure of compound 12;

[0037] Figure 7 HMBC, H COSY and NOESY correlation signals of compound 12; 1 H- 1 H COSY and NOESY correlation signals of compound 12;

[0038] Figure 8 ECD spectrum of compound 12;

[0039] Figure 9 Structure of compound 13;

[0040] Figure 10 HMBC, H COSY and NOESY correlation signals of compound 13; 1 H- 1 H COSY and NOESY correlation signals of compound 13;

[0041] Figure 11 ECD spectrum of compound 13;

[0042] Figure 12 Crystallographic structure of compound 13;

[0043] Figure 13 Structure of compound 14;

[0044] Figure 14 HMBC, H COSY and NOESY correlation signals of compound 14; 1 H- 1 H COSY and NOESY correlation signals of compound 14;

[0045] Figure 15 ECD spectrum of compound 14;

[0046] Figure 16 Crystallographic structure of compound 14;

[0047] Figure 17 Structure of compound 18;

[0048] Figure 18 HMBC, H COSY and NOESY correlation signals of compound 18; 1 H- 1 H COSY and NOESY correlation signals of compound 18;

[0049] Figure 19 DP4+ and ECD spectrum of compound 18;

[0050] Figure 20 Structure of compound 21;

[0051] Figure 21 HMBC, COSY and NOESY correlation signals of compound 21; 1 H- 1 H COSY and NOESY correlation signals of compound 21;

[0052] Figure 22 ECD plot of compound 21;

[0053] Figure 23 Cytotoxicity test of compounds 1, 12-14, 18, 21 on PC12 cells (n=3, ).

[0054] Figure 24 Antioxidant activity of compounds 1, 12-14, 18, 21 on H2O2-induced PC12 cells (n=3, ). DETAILED DESCRIPTION

[0055] The present application is described in more details hereinafter to facilitate the understanding of the present application.

[0056] The experimental methods in the following examples are all conventional methods unless otherwise specified. The specific techniques or conditions not mentioned in the examples are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0057] Example 1: Extraction, isolation and structural identification of the schisandrin-type triterpenoids from Schisandra chinensis

[0058] 1.1 Experimental materials and instruments

[0059] 1.1.1 Experimental materials

[0060]

[0061]

[0062] 1.1.2 Experimental instruments

[0063]

[0064] 1.2 Extraction and isolation

[0065] Weigh 45 kg of dried Schisandra chinensis fruits, add 10 times the amount of 70% EtOH, heat and reflux extract 3 times, each for 2 h, filter while hot, concentrate under reduced pressure to obtain 25 kg of Schisandra chinensis extract. Disperse the extract with water, then extract with petroleum ether, ethyl acetate, and n-butanol in sequence. The ethyl acetate extract (1.71 kg) is subjected to silica gel column chromatography, eluted with dichloromethane-methanol (50:0-0:1, v / v) in gradient, and the eluate is subjected to thin layer chromatography, combined, and eight components Fr. I-VIII are obtained. Fr. III (265 g) is subjected to MCI column chromatography, eluted with 30% EtOH (5 BV) and 95% EtOH (8 BV) at 1 BV·h -1 Flow rate, and two sub-components A-B are obtained. Fr. B is subjected to silica gel column chromatography (200-300 mesh), eluted with petroleum ether-acetone (50:1-0:1, v / v) in gradient, and 10 components Fr. B1-B10 are obtained. Figure 1 ) are obtained.

[0066] Fr. B7 is further separated by Sephadex LH-20 (MeOH) and RP-C 18 (MeOH:H2O, 10:90-100:0, v / v) to obtain Fr. B7.1-15. Fr. B7.2 is separated by semi-HPLC (MeCN:H2O, 34:66, v / v) to obtain compound 13 (2.5 mg, t R = 17.9 min) and 14 (2.1 mg, t R = 37.3 min); Fr. B7.4 is separated by semi-HPLC (MeCN:H2O, 45:55, v / v) to obtain compound 12 (2.5 mg, t R = 35.0 min); Fr. B7.7 is separated by semi-HPLC (MeCN:H2O, 42:58, v / v) to obtain compound 21 (3.2 mg, t R = 32.0 min). Fr. B8 is separated by Sephadex LH-20 (MeOH) and RP-C 18 (MeOH:H2O, 10:90-100:0, v / v) to obtain Fr. B8.1-6. Fr. B8.6 is separated by semi-HPLC (MeCN:H2O, 42:58, v / v) to obtain compound 18 (3.5 mg, t R = 35.0 min).

[0067] Fr. IV (200 g) is subjected to MCI column chromatography, eluted with 30% ethanol (5 BV) and 95% ethanol (8 BV) at 1 BV·h -1Elution at a certain flow rate yielded two subfractions, AB. Fr.B was eluted using a silica gel column with a gradient elution of petroleum ether-acetone (50:1–1:1, v / v) to obtain eight fractions, Fr.B1–B8. Fr.B6 was further eluted using Sephadex LH-20 (MeOH) and RP-C. 18 Eight fractions, Fr.B6.1-10, were obtained by separation using (MeOH:H2O, 10:90~100:0, v / v) . Fr.B6.2 was separated by semi-HPLC (MeCN:H2O, 55:45, v / v) to obtain compound 1 (3.2 mg, t R =18.0min).

[0068] 1.3 Identification of the structure of new compounds

[0069] 1.3.1 Structural identification of compound 1

[0070] Compound 1, colorless needle-like crystals. Analyzed by HR-ESI-MS (m / z 589.2277 [M+HCOO)). - The molecular formula can be determined to be C(calcd.589.2290). 29 H 36 O 10 .

[0071] Compound 1 1 Four methyl hydrogen signals can be observed in the H-NMR (600MHz, C5D5N) spectrum [δ] H 0.69 (d, J = 6.7 Hz), 1.21 (s), 1.26 (s), 1.76 (s)], 1 group of methylene oxide hydrogen signals [δ H 3.70(m), 3.76(m)], 1 oxygen-containing methine hydrogen signal [δ H 4.29, (tlike, J = 4.6 Hz)].

[0072] pass 13 Analysis of C-NMR and DEPT spectra revealed 29 carbon signals, including 4 methyl groups, 8 methylene groups (one oxygen-containing carbon), 6 methine groups (one olefin and two oxygen-containing carbons), and 11 quaternary carbons (δ¹²). C 1 sp at 45.9 3 Quaternary carbon, δ C One ketone carbonyl group at 217.3, δ C The two ester groups at positions 175.3 and 171.3, δ C One olefinic carbon at 133.5° and six oxygen-containing carbons). These observations occupy four of the twelve unsaturation levels, indicating that compound 1 is a highly oxidized nortriterpenoid compound with an octyl ring structure. Figure 2).

[0073] Comparison of the 1D-NMR data of compound 1 with schintrilactone C (Jiang Y, Yang GZ, Chen Y, et al. Terpenes from Schisandra sphenanthera [J]. Helv. Chim. Acta, 2011, 94: 491-496) revealed similar structural fragments. 1 H- 1 The cross-peaks of H-1 / H2-2 and H-5 / H2-6 / H2-7 in the H COSY spectrum, and H-1 to C-3 (δ C 175.3) / C-10 (δ C 97.7), H2-7 to C-5 (δ C 50.1), H3-29 to C-4 (δ C 88.3), H2-30 to C-4 (δ C 88.3) / C-5 (δ C 50.1), H2-19 to C-5 (δ C 50.1) / C-8 (δ C 51.9) in the HMBC spectrum indicated the presence of a 5 / 5 / 7 fused ring (Unit 1) fragment. The cross-peaks of H2-11 / H-12 in the H COSY spectrum, and H-12 to C-13, H2-11 to C-13, H3-18 to C-13 in the HMBC spectrum indicated the presence of a quaternary carbon (C-13) in Unit 1. 1 H- 1 The cross-peaks of H2-11 / H-12 in the H COSY spectrum, and H-8 to C-9 / C-15, H2-11 to C-15, H2-16 to C-12 / C-15, H-12 to C-17, H3-18 to C-12 / C-13 / C-16 in the HMBC spectrum indicated the presence of a tricyclo[4.2.1.0 4,8 ]nonane bridge (Unit 2). 1 H- 1 The cross-peaks of H2-20 / H-21 in the H COSY spectrum, and H-20 to C-17 (δ C 112.6) / C-23 (δ C 114.2), H3-22 to C-20 (δ C 41.1) / C-21 (δ C 38.7) / C-23 (δ C 114.2), H-24 to C-23 / C-25 / C-26 / C-27, H3-27 to C-26 in the HMBC spectrum indicated the presence of a spiro[3.3]heptane (Unit 3) fragment Figure 3 ). Thus, the planar structure of compound 1 was determined to be a rare complex tricyclo[4.2.1.04,8 Novel triterpenoid-lowering skeletons of nonane-bridged systems ( Figure 2 This type of skeleton is the first to be isolated from nature.

[0074] Analysis of the NOESY spectrum of compound 1 revealed correlation signals at H-1 / H3-29 and H-12 / H3-18, indicating that H-1, H-12, H-18, and H-29 are in the β configuration, while correlation signals at H3-30 / H-5 / H-8 and H-21 / H-24 indicated that H-5, H-8, H-21, and H3-30 are in the α configuration. Figure 3 The absolute configuration of compound 1 was determined by ECD calculations to be 1R,4R,5S,8R,9S,10R,12R,13R,14R,17S,21S,23R. Figure 4 ).

[0075] Attempts were made to cultivate single crystals of compound 1 under various solvent conditions, ultimately yielding high-quality crystals. From Figure 5 The X-ray single-crystal diffraction analysis results confirmed the accuracy of its structural analysis. It was ultimately named schisaybyterpene A.

[0076] Table 1 Compound 1 1 H-NMR and 13 C-NMR data (δin ppm, J in Hz)

[0077]

[0078]

[0079] Table 2 Crystallographic data of Compound 1

[0080]

[0081]

[0082] 1.3.2 Structural identification of compound 12

[0083] Compound 12, white amorphous powder. Analyzed by HR-ESI-MS (m / z 589.2241 [M+HCOO)). - The molecular formula can be determined to be C(calcd.589.2290). 29 H 36 O 10 .

[0084] Compound 12 1 Five methyl hydrogen signals can be observed in the H-NMR (600MHz, C5D5N) spectrum [δ] H0.94 (3H,s), 1.25 (3H,d,J=6.7Hz), 1.53 (3H,d,J=7.5Hz), 1.02 (3H,s), 1.18 (3H,s)]; 1 oxygen-containing methine hydrogen signal [δ H 4.17 (1H,d,J=6.2Hz)).

[0085] Compound 12 13 C-NMR and DEPT spectra revealed 29 carbon signals, including 5 methyl carbon signals δ C 26.1, 18.1, 17.2, 20.8, 27.9; 7 methylene carbon signals δ C 35.3, 19.2, 32.2, 35.9, 31.7, 42.6, 38.0; 2 ester group carbon signals δ C 175.6, 178.6; two ketone carbonyl carbon signals δ C 214.8, 219.9; in addition, there are 6 methylene carbons (1 of which is an oxygen-containing carbon) and 7 quaternary carbon signals (6 of which are oxygen-containing carbons). Analysis of the data of compound 12 revealed that it is structurally similar to compound schigrandilactones A (Xiao WL,Gong YQ,Wang RR,etal.Bioactive Nortriterpenoids from Schisandra grandiflora[J].J.Nat.Prod,2009,72:1678-1681). Figure 6 ), schigrandilactones A has a hydroxyl substitution at C-29, while compound 12 has a methyl group at C-29. This change can be detected by H3-29 (δ) in the HMBC spectrum. H 1.02) to C-4(δ C The correlation signal between 83.5) was confirmed. Figure 7 Thus, the planar structure of compound 12 was determined.

[0086] In the NOESY spectrum, correlation signals can be observed between H-5 / H3-30, indicating that these protons are of α configuration; conversely, correlation signals can be observed between H-1 / H3-29, H3-18 / H-14 / H-22, and H3-21 / H-22 / H2-24, indicating that these protons are of β configuration. Figure 7 Furthermore, ECD calculations determined the absolute configuration of compound 12 to be 1R,5S,8S,9S,10R,13S,15R,16S,20S,22S,23R,25S. Figure 8The 1D-NMR data of compound 12 were assigned in detail, and it was identified as a new compound by searching the SciFinder database and named schisaterpene J.

[0087] Compound 12 in Table 3 1 H-NMR and 13 C-NMR data (δin ppm, J in Hz)

[0088]

[0089]

[0090] 1.3.3 Structural identification of compound 13

[0091] Compound 13, colorless needle-like crystals. Analyzed by HR-ESI-MS (m / z 635.2408 [M+HCOO]). - The molecular formula can be determined to be C(calcd.635.2345). 30 H 38 O 12 .

[0092] Compound 13 1 Five methyl hydrogen signals can be observed in the H-NMR (600MHz, C5D5N) spectrum [δ] H 0.73 (3H, d, J = 6.1 Hz), 1.64 (3H, s), 0.93 (3H, s), 1.21 (3H, s), 2.04 (3H, s)]; 5 oxygen-containing methine hydrogen signals [δ H 4.85 (1H, brs), 5.57 (1H, d, J = 7.8 Hz), 4.95 (1H, overlap), 3.72 (1H, s), 4.15 (1H, s)]; 1 olefin proton signal δ H 6.94 (1H, s).

[0093] Compound 13 13 C-NMR and DEPT spectra revealed 30 carbon signals, including 5 methyl carbon signals δ C 18.8, 10.3, 21.8, 27.7, 20.8; 5 methylene carbon signals δ C 37.6, 30.3, 37.3, 24.7, 34.4; δ signals of three ester group carbons C 176.1, 171.6, 169.6; Group 1 olefin carbon signal δ C148.7, 131.7; in addition, there are 9 methine carbons (5 of which are oxygen-containing carbons) and 6 oxygen-containing quaternary carbon signals. By analyzing the data of compound 13, it was found that it was similar in structure to compound schinensilactone C (Zhang YQ, Liu Y, Wen B, et al. New triterpenoids and adibenzocyclooctadiene lignan from the leaves of Schisandra chinensis [J]. Phytochem. Lett, 2023, 54: 57-62), the main difference being that compound 13 has an acetyl group at the C-7 position ( Figure 9 ) In the HMBC spectrum, δ H 5.57 to C-1'(δ C 169.6) has a correlation signal, further confirming the above inference ( Figure 10 ) Thus, the planar structure of compound 13 was determined.

[0094] In the NOESY spectrum, there are correlation signals between H-5 / H3-30, indicating that these protons are in the α configuration; on the contrary, there are correlation signals between H-1 / H3-29 / H-19 / H-8 / H-12, H-7 / H-15, indicating that these protons are in the β configuration ( Figure 10 ) By ECD calculation, the absolute configuration of compound 13 was determined to be 1R, 5S, 7R, 8R, 9R, 10S, 12S, 13R, 14S, 15S, 17R, 19R, 20R, 23S ( Figure 11 ) In addition, compound 13 was cultured to obtain high-quality crystals ( Figure 12 ), further verifying the accuracy of its absolute configuration. The 1D-NMR data of compound 13 were assigned in detail, and it was determined to be a new compound by SciFinder database retrieval, named schisaterpene K.

[0095] Table 4 H-NMR and C-NMR data of compound 13 (δ in ppm, J in Hz) 1 H-NMR and C-NMR data of compound 13 (δ in ppm, J in Hz) 13 H-NMR and C-NMR data of compound 13 (δ in ppm, J in Hz)

[0096]

[0097]

[0098] Table 5 Crystallographic data of compound 13

[0099]

[0100] 1.3.4 Structural identification of compound 14

[0101] Compound 14, colorless needle-like crystal. The molecular formula of compound 14 was determined as C - , calcd. 619.2396) by HR-ESI-MS (m / z 619.2424 [M+HCOO] 30 H 38 O 11 .

[0102] The structure of compound 14 1 The 5 methyl hydrogen signals [δ H 0.74 (3H, d, J = 6.7 Hz), 1.72 (3H, s), 0.94 (3H, s), 1.20 (3H, s), 2.03 (3H, s)]; 4 oxygen-containing methine hydrogen signals [δ H 4.20 (1H, d, J = 5.5 Hz), 5.57 (1H, d, J = 8.2 Hz), 4.83 (1H, br d, J = 10.7 Hz), 3.68 (1H, s)]; 1 olefin hydrogen signal δ H 6.97 (1H, s).

[0103] The structure of compound 14 13 The C-NMR and DEPT spectra of compound 14 showed 30 carbon signals, including 5 methyl carbon signals δ C 18.8, 10.4, 21.6, 27.9, 20.8; 6 methylene carbon signals δ C 35.3, 30.8, 42.1, 24.8, 45.9, 34.4; 3 ester group carbon signals δ C 174.8, 171.7, 169.5; 1 set of olefin carbon signals δ C 148.7, 131.8; in addition, there were 8 methine carbons (4 of which were oxygen-containing carbons) and 6 oxygen-containing quaternary carbon signals. By analyzing the data of compound 14, it was found that it was similar to the structure of compound 13, the main difference being that compound 14 had no hydroxyl group at C-19, which was confirmed by the shift of δ C 76.9 to δ C 45.9. Thus, the planar structure of compound 14 was determined. Figure 13

[0104] ​In the NOESY spectrum, a correlation signal can be observed between H-5 / H3-30, indicating that these protons are of α configuration; conversely, a correlation signal can be observed between H3-29 / H-1 and H-7 / H-8 / H-15, indicating that these protons are of β configuration. Figure 14 ECD calculations determined the absolute configuration of compound 14 to be 1R,5S,7R,8R,9S,10R,12S,13R,14S,15S,17R,20R,23S. Figure 15 In addition, compound 14 was cultured into single crystals, ultimately yielding high-quality crystals. Figure 16 This further verified the accuracy of its absolute configuration. The 1D-NMR data of compound 14 were assigned in detail, and a search of the SciFinder database confirmed it as a new compound, named schisaterpene L.

[0105] Compound 14 in Table 6 1 H-NMR and 13 C-NMR data (δin ppm, J in Hz)

[0106]

[0107] Table 7 Crystallographic data of compound 14

[0108]

[0109]

[0110] 1.3.5 Structural identification of compound 18

[0111] Compound 18 is a white amorphous powder. Analyzed by HR-ESI-MS (m / z 659.2790 [M+HCOO)). - The molecular formula can be determined to be C (calcd.659.2709). 33 H 42 O 11 .

[0112] Compound 18 1 Six methyl hydrogen signals can be observed in the H-NMR (600MHz, C5D5N) spectrum [δ]. H 0.86 (3H, d, J = 6.6 Hz), 1.86 (3H, s), 1.01 (3H, s), 1.21 (3H, s), 2.12 (3H, s), 2.04 (3H, brs)]; 6 oxygen-containing methine hydrogen signals [δ H4.24 (1H,brs), 5.66 (1H,brs), 4.03 (1H,brs), 3.75 (1H,s), 3.94 (1H,m), 5.05 (1H,brs)]; 2 olefin proton signals [δ H 7.20(1H,overlap),5.89(1H,brs)].

[0113] Compound 18 13 C-NMR and DEPT spectra revealed 33 carbon signals, including 6 methyl carbon signals δ C 18.8, 10.4, 21.6, 27.9, 20.8; 5 methylene carbon signals δ C 35.2, 30.6, 42.7, 27.1, 45.9; δ signals of three ester group carbons C 174.6, 173.9, 166.1; 2 groups of olefin carbon signals δ C 146.9, 130.8, 127.6, 140.0; in addition, there are 10 methylene carbons (6 of which are oxygen-containing carbons) and 5 oxygen-containing quaternary carbon signals. Analysis of the data of compound 18 revealed that it is structurally similar to compound xuetongdilactones D, except for the substituent at C-12. In xuetongdilactones D (Cao L, Shehla N, Li B, et al. Schinortriterpenoids from Tujiaethnomedicine Xuetong- The stems of Kadsura heteroclita[J]. Phytochemistry, 2020, 169: 112178), the C-12 position is acetyl-substituted, while the C-12 position of compound 18 is hydroxyl-substituted, and the chemical shift of H-12 is from the low field region (δ H 5.30) Displacement to the high field region (δ H 4.03) confirmed this inference. Thus, the planar structure of compound 18 was determined. Figure 17 ).

[0114] In the NOESY spectrum, correlation signals can be observed between H3-30 / H-5, H-20 / H-23, and H-12 / H-20, indicating that 12-OH is in α configuration; conversely, correlation signals can be observed between H-1 / H3-29, H-7 / H-8 / H-15, and H3-21 / H-22, indicating that these protons are in β configuration. Figure 18 NMR calculations and DP4+ analysis indicate that isomer (23R*)-18 is the correct isomer, with a DP4+ probability of 100% and R 2value of 0.9986 Figure 19 In addition, the absolute configuration of compound 18 was determined to be 1R, 5S, 7R, 8R, 9S, 10R, 12S, 13R, 14S, 15S, 17S, 20S, 22R, 23R Figure 19 The 1D-NMR data of compound 18 were assigned in detail and it was determined to be a new compound by SciFinder database search, named schisaterpene N.

[0115] Table 8.1H-NMR data of compound 18 1 H-NMR and 13 C-NMR data (δ in ppm, J in Hz)

[0116]

[0117] 1.3.6 Structure identification of compound 21

[0118] Compound 21, white amorphous powder. The molecular formula of compound 21 was determined to be C - H 29 O9by HR-ESI-MS (m / z 573.2447 [M+HCOO] 36 , calcd. 573.2341).

[0119] The 1D-NMR data of compound 21 were assigned in detail and it was determined to be a new compound by SciFinder database search, named schisaterpene N. 1 H-NMR (600 MHz, C5D5N) spectrum of compound 21 showed 5 methyl hydrogen signals [δ H 1.37 (3H, d, J = 6.8 Hz), 1.85 (3H, s), 1.17 (3H, s), 0.96 (3H, s), 1.23 (3H, s)]; 2 oxygen-containing methine hydrogen signals [δ H 4.24 (1H, d, J = 5.2 Hz), 4.71 (1H, t, J = 6.9 Hz)]; 2 olefinic proton signals [δ H 5.24 (1H, d, J = 10.4 Hz), 7.02 (1H, s)].

[0120] The 1D-NMR data of compound 21 were assigned in detail and it was determined to be a new compound by SciFinder database search, named schisaterpene N. 13 C-NMR and DEPT spectra of compound 21 showed 29 carbon signals, including 5 methyl carbon signals δ C 16.5, 10.5, 18.7, 21.8, 28.6; 6 methylene carbon signals δ C 35.8, 22.0, 24.6, 43.8, 46.4, 41.5; 3 ester carbonyl carbon signals δ C 175.0, 174.0, 170.6; 2 sets of olefinic carbon signals δ C111.8, 149.1, 138.3, 130.3; in addition, there are six methine carbons (two of which are oxygen-containing carbons) and five quaternary carbon signals (four of which are oxygen-containing carbons). Analysis of the data of compound 21 shows that it is similar in structure to compound propinqtrilactones A (Ding WP, Hu K, Liu M, et al. Five new schinortriterpenoids from Schisandra propinqua var. propinqua [J]. Fitoterapia, 2018, 127: 193-200), the main difference being that there is no hydroxyl group substitution at C-6 in compound 21, and C-6 is replaced by δ Figure 20 ). C 65.6 shifted to δ C 22.0, confirming this inference. In addition, in the H- 1 1 H COSY, there are correlation signals between H-5 (δ H 2.37) / H-6 (δ H 1.33) / H-7 (δ H 1.96), and in the HMBC spectrum, there are correlation signals between H-6 (δ H 1.33) and C-8 (δ C 54.9), further confirming the above inference ( Figure 21 ). Thus, the planar structure of compound 21 is determined.

[0121] In the NOESY spectrum, there are correlation signals between H-5 / H3-30, H-8 / H2-11 (δ H 2.23) / H3-28 / H-20, indicating that H-5, H-8, H-20, H3-28, H3-30 are in the α configuration; on the contrary, there are correlation signals between H-1 / H3-29, H-12 / H2-16 (δ H 2.85), H3-21 / H-22, indicating that these protons are in the β configuration; in addition, the correlation signal between H-22 / H-24 confirms that the double bond between C-22 and C-23 is in the Z configuration ( Figure 21 ). Through ECD calculation, the absolute configuration of compound 21 is determined to be 1R, 5S, 8R, 9S, 10R, 12R, 13R, 14S, 17R, 20S ( Figure 22 ). The 1D-NMR data of compound 21 are assigned in detail, and it is determined to be a new compound by SciFinder database retrieval, named schisaterpene M.

[0122] ​Table 9 H-NMR and C-NMR data (δ in ppm, J in Hz) of compound 21 1 H-NMR and C-NMR data (δ in ppm, J in Hz) of compound 21 13 H-NMR and C-NMR data (δ in ppm, J in Hz) of compound 21

[0123]

[0124] Example 2: In vitro neuroprotective activity screening of Schisandra leaf nor-triterpenoid compounds

[0125] 2.1 Experimental materials and instruments

[0126] 2.1.1 Experimental materials

[0127]

[0128] 2.1.2 Experimental instruments

[0129]

[0130] 2.2 Experimental methods

[0131] 2.2.1 Preparation of test compounds

[0132] An appropriate amount of the Schisandra nor-triterpenoid monomer compounds obtained by the above separation was dissolved in a small amount of DMSO to prepare a stock solution, which was stored in a 4°C refrigerator for standby use. Before use, the stock solution was diluted with RPMI-1640 medium to the required concentration.

[0133] 2.2.2 Preparation of H2O2 solution

[0134] 9.09 μL of 880 mM H2O2 solution was taken and diluted with medium to 10 mL to obtain a H2O2 solution with a concentration of 800 μM.

[0135] 2.2.3 Preparation of positive drug

[0136] 5 mg of N-acetyl-cysteine (NAC) was accurately weighed and dissolved in 3.064 mL of DMSO to prepare a stock solution of 10 mM, which was stored at -80°C for standby use. 100 μL was taken and diluted with medium to 1 mL, and vortexed to obtain a positive drug solution with a concentration of 1 mM.

[0137] 2.2.4 Cell culture

[0138] PC12 cells were placed in RPMI-1640 medium (10% fetal bovine serum / RPMI-1640 / penicillin-streptomycin = 10:89:1) and cultured at 37°C, 5% CO2, until the cell density reached about 80-90%, and then subcultured.

[0139] 2.2.5 Cytotoxicity experiment

[0140] The cytotoxicity of compounds (1, 12-14, 18, 21) on PC12 was evaluated by CCK-8 method. Cells were seeded in 96-well plates (5 x 10 3 After 24 h of culture to complete adhesion, 20 μM of compound (1, 12-14, 18, 21) was added to each well, with 3 replicates in each group, and the culture was continued for 24 h. 2 h before the end of the culture, 10 μL of CCK-8 solution was added to each well, followed by placement in a 37 °C, 5% CO2 incubator. After the end of the culture, the absorbance (OD value) of each well of the 96-well plate at 450 nm was measured using a microplate reader, and the experiment was repeated 3 times.

[0141] 2.2.6 Anti-oxidative activity experiment

[0142] Cells were seeded in 96-well plates (5 x 10 3 After 24 h of culture to complete adhesion, 20 μM of compound (1, 12-14, 18, 21) was added to each well, with 3 replicates in each group, and the culture was continued for 24 h. 2 h before the end of the culture, 10 μL of CCK-8 solution was added to each well, followed by placement in a 37 °C, 5% CO2 incubator. After the end of the culture, the absorbance (OD value) of each well of the 96-well plate at 450 nm was measured using a microplate reader, and the experiment was repeated 3 times.

[0143] Cell survival rate (%) = (As-Ab) / (Ac-Ab) x 100% (Ac: control group OD value; As: experimental group OD value; Ab: blank group OD value)

[0144] 2.2.7 Statistical analysis

[0145] The experimental data are represented by SPSS 18.0 statistical software, and one-way ANOVA is used for analysis. The significance of the difference between groups is determined by p<0.05. Data visualization is completed by Graph Pad Prism 10.

[0146] 2.3 Experimental results

[0147] 2.3.1 Analysis of cytotoxicity results

[0148] The isolated triterpenoids were subjected to PC12 cytotoxicity test. The results showed that all compounds did not exhibit cytotoxicity at a concentration of 20 μM Figure 23 ).

[0149] 2.3.2 Analysis of anti-oxidative activity results

[0150] The isolated compounds were tested for in vitro antioxidant activity using a PC12 oxidative stress cell model induced by H2O2, and the results are shown in Table 10. Figure 24 According to the experimental results, when the initial administration concentration was 20 μM, all the compounds had significant differences compared with the model group.

[0151] According to the above screening results, the six compounds with potential antioxidant activity were configured at concentration gradients of 20, 10, 5, 2.5, 1.25, and 0.625 μM, respectively, and antioxidant activity detection was continued. The experimental results (Table 10) showed that compounds 1 (EC 50 = 0.3 ± 0.4 μM), 13 (EC 50 = 1.1 ± 0.1 μM), 14 (EC 50 = 1.2 ± 1.0 μM), and 18 (EC 50 = 1.5 ± 0.7 μM) had significant differences compared with the model group, indicating that they had significant antioxidant activity.

[0152] Table 10 Antioxidant activity of compounds on H2O2-induced PC12 cells

[0153]

[0154] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the method of the present application, several improvements and supplements can also be made, which should also be considered within the protection scope of the present application.

Claims

1. A Schisandra fruit-derived triterpenoid compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof, the compound being selected from the group consisting of compounds 1, 12-14, 18, 21 as shown below:

2. A pharmaceutical composition comprising the Schisandra fruit-derived triterpenoid compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof according to claim 1.

3. Use of the Schisandra fruit-derived triterpenoid compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for protecting nerves.

4. Use according to claim 3, characterized in that, The medicament is for protecting PC12 cells.

5. Use of the Schisandra fruit-derived triterpenoid compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing or treating neurodegenerative diseases.

6. Use according to claim 3, characterized in that, The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis.

7. A method for preparing the Schisandra fruit-derived triterpenoid compound according to claim 1, comprising subjecting Schisandra fruit to solvent extraction to obtain a Schisandra fruit extract, and then separating compounds 1, 12-14, 18, 21 from the Schisandra fruit extract.

8. The method of claim 7, wherein, The solvent extraction comprises heating extraction with an organic solvent; the organic solvent is selected from at least one of methanol and ethanol; after extraction, the extract is filtered and combined, and the solvent is removed by evaporation under reduced pressure to obtain the Schisandra fruit extract.

9. The method of claim 8, wherein, The obtained Schisandra chinensis extract was dispersed in water, and then extracted with petroleum ether and ethyl acetate successively to obtain an ethyl acetate extract. The ethyl acetate extract was separated by silica gel column chromatography, MCI column chromatography, Sephadex LH-20 column chromatography, RP-C 18 chromatography, and semi-preparative chromatography to obtain compounds 1, 12-14, 18, and 21.

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