Schisandra chinensis leaf reduced triterpene compound and application thereof

Abstract

The invention relates to a Chinese magnoliavine leaf reduced triterpenoid compound and application thereof. The schisandra chinensis leaf nortriterpenoid compound is extracted from schisandra chinensis leaves, has an excellent neuroprotective effect, and is expected to be used for preventing and treating neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to nortriterpenoid compounds from Schisandrae Chinensis leaves and applications thereof. Background Art

[0002] Schisandra chinensis (Turcz.) Baill. is a deciduous woody vine of the genus Schisandra in the family Magnoliaceae. It prefers slightly acidic humus soil and has poor drought tolerance. Young branches are reddish brown, old branches are gray-brown, often wrinkled, and flaky. The whole plant is nearly glabrous, with ridges at the tip. The petiole is 1-4 cm long. The leaves are membranous, broadly elliptical, obovate or nearly circular, 5-10 cm long, 3-5 cm wide. The apex is acute, the base is cuneate, and there are small teeth on the edge. The flowers are mostly unisexual and dioecious, occasionally on the same plant, solitary or clustered in the leaf axils, the perianth segments are pink-white or pink, the male flower pedicels are 5-25 mm long, and the stamens are nearly obovate; the female flower pedicels are 17-38 mm long, and the pistils are nearly ovate. The aggregated small berries are 1.5-8.5 cm long, the small berries are red, nearly spherical or obovate. The flesh smells slightly sour and has a special aroma after being broken. The mature fruit is dark red and contains 1-2 seeds. The seeds are light brown, kidney-shaped, 4-5mm long, 2.5-3mm wide, with a smooth seed coat and a distinct U-shaped recessed hilum. The flowering period is from May to July, and the fruiting period is from July to October. As one of the major traditional Chinese medicinal materials, the fruit is often used as medicine. It has the effects of astringing, strengthening qi and promoting body fluid, nourishing the kidney and calming the mind, and calming the nerves. It can be used to treat lung deficiency, cough, dry mouth, spontaneous sweating, night sweats, nocturnal emission, insomnia, dreaminess, forgetfulness, and fatigue.

[0003] Schisandra leaves contain lignans, flavonoids and phenolic acids. Wu Yanan, Wen Sihua, Wu Xu, Zhao Chongbo, Liu Hang, Sun Jing, etc. Comparison of 7 components in Schisandra fruits and leaves (Northwest Journal of Pharmacy, 2022, Vol. 37, No. 4, pp. 15-19); Jin Yinping, Qu Zhengyi, Cui Lili, Pu Xiangmin, Guo Jing, Wang Yingping, etc. Determination of the content and antioxidant activity of flavonoids and phenolic acids in different parts of Schisandra (Chinese Journal of Experimental Traditional Chinese Medicine, 2018, Vol. 24, No. 19, pp. 79-84). However, there are few reports on the systematic separation, identification and application of triterpenoid compounds contained in Schisandra leaves. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides Schisandrae Chinensis Leaf Nortriterpenoid Compounds 1-9 or pharmaceutically acceptable salts, prodrugs, stereoisomers, solvates thereof, and uses thereof for neuroprotection.

[0005] In one aspect, the present invention provides the following Schisandrae Chinensis Leaf Nortriterpenoid Compounds 1-9, or pharmaceutically acceptable salts, prodrugs, stereoisomers, solvates thereof:

[0006]

[0007] In the present invention, the prodrug refers to a compound that is converted into an active compound as a result of a reaction in vivo with an enzyme, gastric acid, etc. As prodrugs, 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.

[0008] In the present invention, the solvate refers to a compound form that forms a complex in a solid or liquid state by coordination with solvent molecules.

[0009] The present invention also provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, or solvate thereof.

[0010] The present invention also provides a pharmaceutical composition of the compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, or solvate thereof, or use of the pharmaceutical composition in preparing a drug, wherein the drug is used for protecting nerves.

[0011] Preferably, the drug is used to protect SH-SY5Y cells.

[0012] The present invention also provides a pharmaceutical composition of the compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, or solvate thereof, or use of the pharmaceutical composition in preparing a drug for preventing or treating a neurodegenerative disease.

[0013] Preferably, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

[0014] Preferably, the pharmaceutical composition or medicine of the present invention further comprises a pharmaceutically acceptable carrier or adjuvant.

[0015] As pharmaceutically acceptable carriers or adjuvants, any carrier or adjuvant known to be suitable for preparing a specific drug or pharmaceutical composition can be used. Examples include, but are not limited to, solvents, excipients, dispersants, emulsifiers, solubilizers, gel formers, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, bonding agents, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizers, lubricants, coating agents, propellants, tonicity regulators, surfactants, colorants, flavoring agents, sweeteners and dyes. Particularly adjuvants suitable for the type of desired preparation and desired mode of administration are used.

[0016] 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 polyvinyl pyrrolidone, low-substituted hydroxypropyl methylcellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, starch Powder paste, glucose syrup, sucrose syrup, fructose syrup, sorbitol, gelatin paste, acacia paste, tragacanth paste, microcrystalline cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, calcium carboxymethylcellulose, polymethacrylate, alginic acid, sodium alginate, polyethylene glycol, colloidal magnesium aluminum silicate, stearic acid, calcium stearate, magnesium stearate, zinc stearate, talc, glyceryl monostearate, glyceryl palmitostearate, magnesium lauryl sulfate, polyethylene glycol, sodium stearyl fumarate.

[0017] The pharmaceutical composition or drug is presented in a unit dosage form, such as tablets, pills, capsules, powders, granules, suspensions, oral liquids, aerosols or liquid sprays, drops; for oral parenteral, intranasal, sublingual or rectal administration or for inhalation or spray administration. Preferably, the pharmaceutical dosage form is an oral preparation, more preferably tablets, pills, capsules, granules, suspensions, powders, oral liquids.

[0018] The pharmaceutical composition or drug of the present invention can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, making sugar-coated pills, grinding, emulsifying, freeze-drying, etc. Pharmaceutically, the content of the active ingredient in the pharmaceutical composition of the present invention should be in the range of 0.05% to 90% by weight, preferably 0.1% to 50% by weight of the whole composition.

[0019] For adult patients, the compound of the present invention can be administered orally or parenterally in a dose of 0.001 to 500 mg once a day or several times a day. It should be noted that the dose can be appropriately increased or decreased depending on the type of disease to be treated, the patient's age, weight, symptoms, etc.

[0020] The present invention also provides a method for preparing compound 1-9, which comprises extracting Schisandrae Chinensis leaves with a solvent to obtain a total extract of Schisandrae Chinensis, and then separating compound 1-9 from the total extract of Schisandrae Chinensis.

[0021] Preferably, the solvent extraction comprises heating extraction with an organic solvent.

[0022] The organic solvent is selected from at least one of methanol and ethanol, preferably 60-95% ethanol, more preferably 70-85% ethanol; the solvent can be heated to reflux; the mass of the solvent during extraction is 5-20 times the mass of Schisandrae Chinensis leaves; the number of extractions can be 1-5 times, preferably 2-4 times.

[0023] After extraction, the extracts were filtered and combined, and the solvent was evaporated under reduced pressure to obtain the total extract of Schisandra chinensis.

[0024] Preferably, the isolating compounds 1-9 from the total extract of Schisandra chinensis comprises separating the total extract of Schisandra chinensis through a macroporous resin, and then separating the compounds through a silica gel column, MCI column chromatography, ODS column chromatography, gel column chromatography, and preparative HPLC to obtain compounds 1-9.

[0025] The macroporous adsorption resin separation comprises loading the total extract of Schisandra chinensis onto a macroporous adsorption resin column, performing gradient elution with water-ethanol (1:0-5:95, v / v), and collecting 95% ethanol elution components.

[0026] Preferably, the macroporous adsorption resin includes HPD-100 macroporous adsorption resin, AB-8 macroporous adsorption resin, and X-5 macroporous adsorption resin.

[0027] Preferably, the gradient elution with water-ethanol includes eluting with water, 15-25% ethanol, 35-45% ethanol, and 95% ethanol in sequence; more preferably, eluting with water, 20% ethanol, 40% ethanol, and 95% ethanol in sequence. During elution, the amount of each eluent is 1-6BV, which can be 1BV, 2BV, 3BV, 4BV, 5BV or 6BV.

[0028] Preferably, the silica gel column separation comprises loading the 95% ethanol elution component onto the silica gel column, performing gradient elution with dichloromethane-methanol (1:0-0:1, v / v), identifying the eluate by thin layer analysis, and combining the eluate to obtain 8 fractions: Fr.A-H.

[0029] In one embodiment, Fr.C is separated by MCI column chromatography (the mobile phase is a methanol-water system, and the volume ratio of methanol to water is 8:2) and then by ODS column chromatography (the mobile phase is a methanol-water system, and the volume ratio of methanol to water is 3:7-9:1) to obtain Fr.C1-C8, wherein Fr.C4 is separated by Sephadex LH-20 gel column chromatography (the mobile phase is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol is 1:1) to obtain Fr.C4-1-8, and Fr.C4-4 is separated by preparative HPLC (methanol / water = 62%, 5 mL / min) to obtain compound 2 (t R =29.3min,5.2mg).

[0030] In one embodiment, Fr.D is separated by MCI column chromatography (the mobile phase is a methanol-water system, and the volume ratio of methanol to water is 8:2) and then by ODS column chromatography (the mobile phase is a methanol-water system, and the volume ratio of methanol to water is 3:7-9:1) to obtain Fr.D1~D10, wherein Fr.D4 is separated by Sephadex LH-20 gel column chromatography (the mobile phase is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol is 1:1) to obtain Fr.D4-1~7, Fr.D4-4 is separated by silica gel column chromatography (the mobile phase is a petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone is 5:1-0:1) to obtain Fr.D4-4-1~18, and Fr.D4-4-7 is separated by preparative HPLC (methanol / water=60%, 5mL / min) to obtain compound 1 (t R =27.2min, 2.5mg), compound 4 (t R =30.7min, 4.0mg), compound 9 (t R =34.6min, 6.2mg); Fr.D4-4-9 was separated by preparative HPLC (methanol / water=60%, 5mL / min) to give compound 3 (t R =26.4min, 3.8mg); Fr.D4-4-10 was separated by preparative HPLC (methanol / water=60%, 5mL / min) to give compound 6 (t R =27.0min, 7.2mg); Fr.D4-4-11 was separated by preparative HPLC (methanol / water=58%, 5mL / min) to give compound 5 (t R =28.2min, 3.2mg), compound 7 (t R =32.0min,8.1mg).

[0031] Preferably, Fr.D6 is separated by Sephadex LH-20 gel column chromatography (the mobile phase is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol is 1:1) to obtain Fr.D6-1 to 8, and Fr.D6-5 is separated by silica gel column chromatography (the mobile phase is a petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone is 5:1-0:1) to obtain Fr.D6-5-1 to 12. Fr.D6-5-6 is separated by preparative HPLC (methanol / water = 62%, 5 mL / min) to obtain compound 8 (t R =33.5min, 4.3mg); Fr.D6-5-7 was separated by preparative HPLC (methanol / water=60%, 5mL / min) to give compound 9 (t R =34.6min,6.2mg).

[0032] In the present invention, the percentage of ethanol refers to the volume fraction.

[0033] Beneficial Effects

[0034] The invention provides a schisandra leaf nortriterpenoid compound, which is extracted from the schisandra leaf and has an excellent neuroprotective effect and is expected to be used for the prevention and treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Flow chart of the isolation of compounds 1-9 from Schisandrae Chinensis leaves;

[0036] Figure 2 :HMBC of compound 1, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0037] Figure 3 : ECD spectrum of compound 1;

[0038] Figure 4 :HMBC of compound 2, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0039] Figure 5 : Crystallographic structure of compound 2;

[0040] Figure 6 :HMBC of compound 3, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0041] Figure 7 : Crystallographic structure of compound 3;

[0042] Figure 8 :HMBC of compound 4, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0043] Fig. 9 :HMBC of compound 5, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0044] Fig.10 :HMBC of compound 6, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0045] Fig.11 : Crystallographic structure of compound 6;

[0046] Fig.12 :HMBC of compound 7, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0047] Fig.13 :HMBC of compound 8, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0048] Fig.14 : ECD spectrum of compound 8;

[0049] Fig.15 : HMBC of compound 9, 1 H- 1 Key related signals in H COSY and NOESY spectra;

[0050] Fig.16 : ECD spectrum of compound 9. DETAILED DESCRIPTION

[0051] The present invention is described in more detail hereinafter to facilitate understanding of the present invention.

[0052] The experimental methods in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0053] Experimental materials and instruments

[0054] Experimental Materials

[0055]

[0056] Experimental instruments

[0057]

[0058] Extraction and separation

[0059] 50 kg of dried Schisandra leaves were extracted with 10 times the mass of 80% ethanol under heating and reflux for 3 times, each time for 2 hours, filtered, the filtrate was combined, and the solvent was recovered under reduced pressure to obtain 10.15 kg of total extract (with an extract yield of 20.3%). -1Column chromatography was performed with a flow rate of 1.5447 W / m2. The eluents were successively water (2BV), 20% ethanol (2BV), 40% ethanol (2BV), and 95% ethanol (4BV). The corresponding eluates were collected and concentrated under reduced pressure to obtain 2.74 kg of water elution component, 0.91 kg of 20% ethanol elution component, 1.82 kg of 40% ethanol elution component and 1.64 kg of 95% ethanol elution component.

[0060] Take the 95% ethanol elution component (0.55kg) and pass it through silica gel column chromatography, eluting with dichloromethane-methanol (1:0~0:1, v / v) gradient, and identify the eluent by thin layer analysis. Combine the bottles to get 8 fractions: Fr.A~H. Comprehensively use silica gel, MCI, ODS column chromatography and preparative HPLC separation methods to systematically separate Fr.B~D. The specific separation process is detailed in Figure 1 , the steps are as follows:

[0061] Fr.C was separated by MCI column chromatography (the mobile phase was a methanol-water system, the volume ratio of methanol to water was 8:2) and then by ODS column chromatography (the mobile phase was a methanol-water system, the volume ratio of methanol to water was 3:7-9:1) to obtain Fr.C1-C8, among which Fr.C4 was separated by Sephadex LH-20 gel column chromatography (the mobile phase was a dichloromethane-methanol system, the volume ratio of dichloromethane to methanol was 1:1) to obtain Fr.C4-1-8. Fr.C4-4 was separated by preparative HPLC (methanol / water = 62%, 5mL / min) to obtain compound 2 (t R =29.3min,5.2mg).

[0062] Fr.D was separated by MCI column chromatography (the mobile phase was a methanol-water system, and the volume ratio of methanol to water was 8:2) and then by ODS column chromatography (the mobile phase was a methanol-water system, and the volume ratio of methanol to water was 3:7-9:1) to obtain Fr.D1~D10, among which Fr.D4 was separated by Sephadex LH-20 gel column chromatography (the mobile phase was a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol was 1:1) to obtain Fr.D4-1~7. Fr.D4-4 was separated by silica gel column chromatography (the mobile phase was a petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone was 5:1-0:1) to obtain Fr.D4-4-1~18. Fr.D4-4-7 was separated by preparative HPLC (methanol / water = 60%, 5mL / min) to obtain compound 1 (t R =27.2min, 2.5mg), compound 4 (t R =30.7min, 4.0mg), compound 9 (t R =34.6min, 6.2mg); Fr.D4-4-9 was separated by preparative HPLC (methanol / water=60%, 5mL / min) to give compound 3 (tR =26.4min, 3.8mg); Fr.D4-4-10 was separated by preparative HPLC (methanol / water=60%, 5mL / min) to give compound 6 (t R =27.0min, 7.2mg); Fr.D4-4-11 was separated by preparative HPLC (methanol / water=58%, 5mL / min) to give compound 5 (t R =28.2min, 3.2mg), compound 7 (t R =32.0min,8.1mg);

[0063] Fr.D6 was separated by Sephadex LH-20 gel column chromatography (the mobile phase was dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol was 1:1) to obtain Fr.D6-1 to 8. Fr.D6-5 was separated by silica gel column chromatography (the mobile phase was petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone was 5:1-0:1) to obtain Fr.D6-5-1 to 12. Fr.D6-5-6 was separated by preparative HPLC (methanol / water = 62%, 5 mL / min) to obtain compound 8 (t R =33.5min, 4.3mg); Fr.D6-5-7 was separated by preparative HPLC (methanol / water=60%, 5mL / min) to give compound 9 (t R =34.6min,6.2mg).

[0064] Through the determination of physicochemical constants and various spectral data analysis (UV, NMR, MS, etc.), the structures of 9 triterpenoid compounds were identified, as shown below:

[0065]

[0066] The H-NMR spectrum and C-NMR spectrum of compound 1-9 are shown in Table 1-3:

[0067] Table 1. Compounds 1-3 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (C 5 D 5 N,δin ppm,JinHz)

[0068]

[0069]

[0070] Table 2. Compounds 4-6 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (C 5 D5 N,δin ppm,JinHz)

[0071]

[0072]

[0073] Table 3. Compounds 7-9 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (C 5 D 5 N,δin ppm,JinHz)

[0074]

[0075]

[0076] Structural identification of compound 1

[0077] Compound 1 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+NH 4 ] + Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 36 O 10 , and its unsaturation is calculated to be 12.

[0078] Compound 1 1 H-NMR (600MHz, C 5 D 5 In the N) spectrum, an olefin proton signal can be clearly observed at δ7.52 (1H, brs); δ4.22 (1H, d, J = 5.4), 5.30 (1H, brs) are oxymethyl proton signals. In the high field region, δ1.77 (3H, s), 1.34 (3H, d, J = 7.0), 1.68 (3H, s), 1.15 (3H, s) show four methyl proton signals; at δ2.62 (1H, d, J = 18.1), 2.89 (1H, dd, J = 18.1, 5.3) and 1.92 (1H, ABd, J = 15.8), 2.33 (1H, ABd, J = 15.8), typical methylene proton signals in two groups of triterpenoids can be observed.

[0079] Compound 1 13 C-NMR (150MHz, C 5 D5 N) spectrum showed 29 carbon signals, which included 1 ketone carbonyl at δ223.4, 2 ester carbonyl carbon signals at δ175.4 and 174.3, and 1 double bond carbon signal at δ149.5 and 129.8, excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 1 contains eight rings. At the same time, the compound also has four methyl carbon signals at δ27.8, 14.9, 10.4, and 16.8; six methylene carbon signals (one oxidized); eight methine carbon signals (two oxidized) and six quaternary carbon signals (five oxidized). The above information indicates that compound 1 is a highly oxidized nortriterpenoid compound.

[0080] The 1D-NMR data of compound 1 were compared with those of the known compound lancifodilactone I reported in literature 1, and it was found that the two compounds had the same AC, G, and H rings, and the 1 H- 1 The relevant signals in the H COSY and HMBC spectra were verified. However, by analyzing the 1D-NMR and 2D-NMR data of the two, it was found that some structural data had huge differences. The keto carbonyl group belonging to C-14 in lancifodilactone I disappeared, providing 1 unsaturation, and the methylene group belonging to C-12 became a methyne group, suggesting that the oxidative rearrangement of the eight-membered ring changed in compound 1. The two five-membered rings D and E in compound 1 were fused (5 / 5[12,14]), and C-9 and C-15 formed an oxygen bridge structure. Combined with DEPT-135, it can be seen that C-12 is a methyne group and C-14 is a quaternary carbon. According to the relevant signals of the HMBC spectrum, H-8 and C-15, H-12 and C-9, C-14 and C-15, H-16 and C-15, CH 3 -18 with C-12, C-13 and C-16, H 2 -19 is related to C-8 and C-9, H-22 is related to C-15, and 1 H- 1 In the H COSY spectrum, H 2 -11 / H-12, H-16 / H-22 related signals, it was found that compound 1 has a rare five-membered D and E ring, and through the fusion of the carbon-carbon single bond between C-12 and C-14. At the same time, the chemical shift of C-14 and C-15 and the remaining unsaturation, combined with the chemical formula given by high-resolution mass spectrometry, we can conclude that C-14 and C-15 form an epoxy structure. 13 C-NMR and DEPT spectra show that C-30 is an oxidized methylene. According to the HMBC spectrum related signals, H 2-30 and C-4, C-5 and C-29 have related signals, which can confirm that C-30 is substituted by hydroxyl. So far, the planar structure of compound 1 has been determined, and the eight-membered D ring structure has undergone a skeleton rearrangement to form a new skeleton triterpenoid with two five-membered D and E rings fused together. This skeleton type of triterpenoid is isolated from nature for the first time.

[0081] In the NOESY spectrum of compound 1, H-5 / H-8 / H 2 -30, CH 3 -21 / H-22, CH 3 -29 / H-1, CH 3 The related signals of H-18 / H-12 / H-16 / H-20, and H-16 / H-23 indicate that H-5, H-8, CH 3 -21, H-22 and H 2 -30 is α-configuration; on the contrary, H-1, H-12, H-16, CH 3 -18, H-20, H-23 and CH 3 -29 is β configuration. Using quantum chemical calculation, we calculated its ECD spectrum and finally determined 23S.

[0082] The structure of compound 1 was determined by comprehensive analysis of its 2D-NMR spectrum and detailed attribution of its 1D-NMR data, and it was named schinensilactone G.

[0083] Structural identification of compound 2

[0084] Compound 2 is a colorless crystal, soluble in methanol. HR-ESI-MS shows [M-H+H 2 O] - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 34 O 12 , and its unsaturation is calculated to be 13.

[0085] Compound 2 1 H-NMR (600MHz, C 5 D 5In the 1H N) spectrum, δ3.97 (1H, t-like, J = 6.7), 5.29 (1H, brs), 5.06 (1H, brs) are oxymethyl proton signals. In the high field region, δ0.96 (3H, s), 1.16 (3H, d, J = 7.0), 2.15 (3H, s), 1.33 (3H, s), 1.30 (3H, s) show five methyl proton signals; at δ3.18 (H, ABd, J = 17.9), 3.22 (H, ABd, J = 17.9) and 2.51 (1H, ABd, J = 16.4), 2.69 (1H, ABd, J = 16.4), typical AB type methylene proton signals in triterpenoids can be observed.

[0086] Compound 2 13 C-NMR (150MHz, C 5 D 5 N) spectrum showed 29 carbon signals, which included 2 ketone carbonyl signals at δ208.3 and 220.1, and 2 ester carbonyl carbon signals at δ173.0 and 177.6, excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 1 contains nine rings. At the same time, the compound also has five methyl carbon signals at δ26.5, 14.6, 17.8, 24.8, 29.3; five methylene carbon signals at δ43.2, 27.9, 36.5, 31.9, 37.6; seven methine carbon signals (three oxidized) and eight quaternary carbon signals (seven oxidized). The above information indicates that compound 2 is a highly oxidized nortriterpenoid compound.

[0087] The 1D-NMR spectrum data of compound 2 was compared with the known compound schindilactone G reported in reference 2. The chemical shifts of the two compounds were found to be the main difference between them. The chemical shift of C-25 in the H ring changed and moved downfield to δ77.1, which was speculated to be hydroxyl substituted. Combined with DEPT-135, it was known that C-25 was a quaternary carbon. Further analysis of the HMBC related spectrum showed that H-23 and C-25, C-26 and CH 3 -27 has related signals with C-25 and C-26, indicating that C-25 of compound 2 is substituted by hydroxyl. Thus, the planar structure of compound 2 is determined.

[0088] In the NOESY spectrum of compound 2, H-5 / H-7 / CH 3 -30, CH 3 -18 / H-16 / H-22, H-16 / CH 3 The related signals of H-21 and H-20 / H-23 / H-24 indicate that H-5, H-7, H-20, H-23, H-24 and CH3 -30 is in the α configuration; on the contrary, H-16, CH 3 -18, CH 3 -21, H-22 and CH 3 -29 is β configuration. There are multiple chiral carbons in this structure. To determine the absolute configuration, we tried to grow a single crystal and finally obtained it in methanol solvent of compound 2 at 23°C. By Ga KαX-single crystal diffraction [orthorhombic, space group P4 1 (no.76), Z=4, T=193.00K, μ(GaKα)=0.601mm -1 ,Dcalc=1.412g / cm 3 ,44112reflections measured(5.626°≤2θ≤121.09°),6311unique(R int =0.0505,R sigma =0.0338)which were used in all calculations.The finalR 1 was 0.0355(I>2σ(I))andwR 2 was 0.0836 (all data). Flack: -0.02 (7)] and its absolute configurations were determined to be 1R, 5S, 7S, 8S, 9S, 10R, 13S, 15S, 16S, 20S, 22S, 23R, 24S and 25R.

[0089] The 2D-NMR spectrum of compound 2 was comprehensively analyzed, and its 1D-NMR data were attributed in detail. Combined with the results of single crystal diffraction, the structure of compound 2 was determined and named schinensilactone H.

[0090] Structural identification of compound 3

[0091] Compound 3 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [MH] at m / z 559.2192 (calcd.for559.2185). - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 36 O 11 , and its unsaturation is calculated to be 12.

[0092] Compound 3 1H-NMR (600MHz, C 5 D 5 In the 1H, 4.79 (1H, brs), 4.59 (1H, brs), and 4.64 (1H, brs) spectra, oxymethyl proton signals are at δ4.79 (1H, brs), 4.59 (1H, brs), and 4.64 (1H, brs). In the high field region, 5 methyl proton signals are shown at δ0.91 (3H, s), 1.21 (3H, d, J = 6.9), 1.35 (3H, d, J = 7.2), 1.44 (3H, s), and 1.33 (3H, s); the typical AB type methylene proton signals in triterpenoids can be observed at δ2.93 (1H, ABd, J = 16.3) and 3.71 (1H, ABd, J = 16.3).

[0093] Compound 3 13 C-NMR (150MHz, C 5 D 5 N) spectrum showed 29 carbon signals, which included 2 ketone carbonyl signals at δ209.2 and 222.1, and 2 ester carbonyl carbon signals at δ172.7 and 178.0, excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 3 contains eight rings. At the same time, the compound also has five methyl carbon signals at δ26.8, 14.4, 8.1, 25.7, 30.9; four methylene carbon signals at δ31.4, 37.5, 31.8, 48.6; 10 methine carbon signals (3 oxidized) and six quaternary carbon signals (5 oxidized). The above information indicates that compound 3 is a highly oxidized schisanartane-type nortriterpenoid compound.

[0094] In the HMBC correlation spectrum of compound 3, H-2 is related to C-1 and C-10, H-5 is related to C-4, and H 2 -6 and C-10, CH 3 -29 has related signals with C-4 and C-5, and binds 1 H- 1 In the H COSY spectrum, H-2 / H-7 / H 2 -6 / H-5 related signals, it was found that compound 3 has a rare six-membered A ring. At the same time, H-16 and C-13 and C-15, CH 3 -18 with C-12, C-13, C-16 and C-17, CH 3 -21 with C-17, H-22 with C-24, H-24 with C-23 and C-26, CH 3 -27 and C-26 HMBC correlation spectra, and 1 H- 1In the HCOSY spectrum, the correlation signals of H-11 / H-12, H-16 / H-22, H-21 / H-20 / H-22 / H-23 and H-24 / H-25 / H-27 confirmed the existence of the DH loop. 2 The HMBC spectrum correlation signals of H-19 and C-1, C-9 and C-10 confirm that the compound has a five-membered C ring. The HMBC spectrum correlation signals of H-2 / C-3, combined with the chemical shifts and remaining unsaturation of C-1 (δ90.6), C-2 (δ51.7), C-3 (δ174.7) and C-10 (δ100.2), indicate that the B ring is a five-membered lactone ring. In addition, H-8 and C-2, H 2 -19 and C-1 and C-10 HMBC correlation signals, thus inferring the fusion of ring B and ring C through the carbon-carbon single bond between C-1 and C-10. The above information shows that compound 3 has the same planar structure as the known compound chinorlactoneA reported in reference 3.

[0095] Compound 3 differs from chinorlactone A in that the chemical shift values ​​at C-2 and C-7 are significantly different, and we speculate that it is a diastereomer of chinorlactone A. Combined with the NOESY spectrum, CH 3 -18 / H-16 / H-22, H-22 / CH 3 -21, H-23, H-20 / H-23 / H-24 and H-23 / H-25 related signals indicate that H-20, H-23, H-24 and H-25 are in α configuration; on the contrary, H-16, CH 3 -21, H-22, and CH 3 -27 is a β configuration. There are multiple chiral carbons in this structure. To determine the absolute configuration, we tried to grow a single crystal and finally obtained it in methanol solvent of compound 3 at 23°C. By Cu KαX-single crystal diffraction [tetragonal, space group P4 3 2 1 2(no.96), Z=8, T=170.00K, μ(GuKα)=0.763mm -1 ,Dcalc=1.189g / cm 3 ,30752reflections measured(8.118°≤2θ≤149.174°),6258unique(R int =0.0742,R sigma=0.0476)which were used in all calculations.The finalR 1 was0.0429(I>2σ(I))and wR 2 was0.1196(all data).Flack:0.13(8)] and determined that their absolute configurations were 1R, 2S, 5S, 7R, 8R, 9S, 10R, 13S, 15S, 16S, 20S, 22S, 23R, 24R and 25S.

[0096] The 2D-NMR spectrum of compound 3 was comprehensively analyzed, and its 1D-NMR data were attributed in detail. Combined with the results of single crystal diffraction, the structure of compound 3 was determined and named schinensilactone I.

[0097] Structural identification of compound 4

[0098] Compound 4 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+FA-H] at m / z 587.2142 (calcd.for587.2134). - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 34 O 10 , and its unsaturation is calculated to be 13.

[0099] Compound 4 1 H-NMR (600MHz, C 5 D 5 In the 1H, 2H) spectrum, an olefin proton signal at δ6.19 (1H, s) can be clearly observed; δ4.76 (1H, brs), 4.72 (1H, brs), and 4.81 (1H, brs) are oxymethyl proton signals. In the high field region, δ1.20 (3H, s), 1.26 (3H, d, J = 7.0), 1.37 (3H, d, J = 7.1), 1.58 (3H, s), and 1.66 (3H, s) show five methyl proton signals; at δ1.58 (1H, ABd, J = 13.8) and 2.53 (1H, ABd, J = 13.8), the typical AB type methylene proton signals in triterpenoids can be observed.

[0100] Compound 4 13 C-NMR (150MHz, C 5 D 5N) spectrum showed 29 carbon signals, which included 1 ketone carbonyl at δ221.0; 3 ester carbonyl carbon signals at δ172.7, 173.6, 177.8; a group of double bond carbon signals at δ178.4, 117.6; excluding the above sp 2 The five unsaturations occupied by the hybridized carbon signals indicate that compound 1 contains eight rings. At the same time, the compound also has five methyl carbon signals at δ27.2, 14.3, 8.1, 25.0, 31.1; four methylene carbon signals at δ30.3, 35.9, 31.7, 42.4; nine methine carbon signals (three oxidized) and five quaternary carbon signals (four oxidized). The above information indicates that compound 5 is a highly oxidized nortriterpenoid compound.

[0101] Comparison of the NMR spectrum data of compound 4 with the known compound schinensilactone A reported in reference 4 revealed that the two have the same parent nucleus structure. Further comparison of their NMR data revealed that there was a certain difference in the chemical shift of C-20-22, which suggested that the structure may have undergone a configuration change. In the NOESY spectrum, H-5 / H-14 / CH 3 -30, CH 3 -30 / H-24, H-20 / H-23, H-23 / H-25, CH 3 -18 / H-16 / H-22 and H-22 / CH 3 The related signals of H-21 indicate that H-14, H-20, H-23, H-24, H-25 and CH 3 -30 is in the α configuration; on the contrary, H-16, CH 3 -21, H-22 and CH 3 -27 is the β configuration.

[0102] The structure of compound 4 was determined by comprehensive analysis of its 2D-NMR spectrum and detailed attribution of its 1D-NMR data, and it was named schinensilactone J.

[0103] Structural identification of compound 5

[0104] Compound 5 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M-H+H 2 O] - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 36 O 11, and its unsaturation is calculated to be 12.

[0105] Compound 5 1 H-NMR (600MHz, C 5 D 5 In the 1H, 4H (N) spectrum, δ5.39 (1H, brs), 4.71 (1H, brs), and 4.82 (1H, brs) are oxymethyl proton signals. In the high field region, δ1.21 (3H, s), 1.29 (3H, d, J = 6.8), 1.35 (3H, d, J = 7.0), 1.51 (3H, s), and 1.69 (3H, s) show five methyl proton signals; at δ3.13 (1H, ABd, J = 18.1) and 3.30 (1H, ABd, J = 18.1), typical AB-type methylene proton signals in triterpenoids can be observed.

[0106] Compound 5 13 C-NMR (150MHz, C 5 D 5 N) spectrum showed 29 carbon signals, which included 1 ketone carbonyl at δ222.6, 3 ester carbonyl carbon signals at δ174.7, 175.5, and 178.2, and excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 1 contains eight rings. At the same time, the compound also has five methyl carbon signals at δ27.3, 14.4, 8.0, 24.9, 31.1; five methylene carbon signals at δ47.0, 31.6, 36.9, 31.9, 44.5; nine methine carbon signals (three oxidized) and six quaternary carbon signals (four oxidized). The above information indicates that compound 4 is a highly oxidized nortriterpenoid compound.

[0107] The NMR spectrum data of compound 5 was compared with the known compound schinensilactoneA reported in literature 4. It was found that the two have the same parent core structure. The main difference is that the chemical shifts of C-1 and C-2 in compound schinensilactoneA have changed significantly. Combined with DEPT-135, it can be seen that the double bond sp 2 The hybridization becomes an oxidized quaternary carbon (C-1) and a methylene group (C-2). Further analysis of the HMBC correlation spectrum shows that H 2 -2 has related signals with C-1, C-3 and C-10, and H-8 has related signals with C-1. This indicates that C-1 of compound 4 is substituted by hydroxyl and C-2 is methylene. Further comparison of its NMR data revealed that there was a certain difference in the chemical shift of C-20-22, which suggested that the structure may have undergone a configuration change. In the NOESY spectrum, H-5 / H-14 / CH3 -30, CH 3 -30 / H-24, H-20 / H-23, H-23 / H-25, CH 3 -18 / H-16 / H-22 and H-22 / CH 3 The related signals of H-21 indicate that H-14, H-20, H-23, H-24, H-25 and CH 3 -30 is in the α configuration; on the contrary, H-16, CH 3 -21, H-22 and CH 3 -27 is the β configuration.

[0108] The structure of compound 5 was determined by comprehensive analysis of its 2D-NMR spectrum and detailed attribution of its 1D-NMR data, and it was named schinensilactone K.

[0109] Structural identification of compound 6

[0110] Compound 6 is a colorless crystal, soluble in methanol. HR-ESI-MS shows [MH] at m / z 559.2195 (calcd.for559.2185). - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 36 O 11 , and its unsaturation is calculated to be 12.

[0111] Compound 6 1 H-NMR (600MHz, C 5 D 5 In the 1H, 2H (N) spectrum, δ4.39 (1H, s) and 4.73 (1H, s) are oxymethyl proton signals. In the high field region, δ0.92 (3H, s), 1.15 (3H, d, J = 7.2), 1.54 (3H, d, J = 7.5), 1.08 (3H, s), and 1.21 (3H, s) show five methyl proton signals; at δ2.61 (1H, ABd, J = 16.0) and 2.73 (1H, ABd, J = 16.0), typical AB type methylene proton signals in triterpenoids can be observed.

[0112] Compound 6 13 C-NMR (150MHz, C 5 D 5N) spectrum showed 29 carbon signals, which included 2 ketone carbonyl signals at δ214.8 and 219.4, and 2 ester carbonyl carbon signals at δ176.8 and 178.5, excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 6 contains eight rings. At the same time, the compound also has five methyl carbon signals at δ26.1, 18.2, 17.2, 21.0, 27.8; six methylene carbon signals at δ19.2, 32.2, 35.8, 31.6, 43.4, 37.9; seven methine carbon signals (two oxidized) and seven quaternary carbon signals (six oxidized). The above information indicates that compound 6 is a highly oxidized nortriterpenoid compound.

[0113] The 1D-NMR spectrum data of compound 6 was compared with the known compound schigrandilactoneA reported in literature 5. The chemical shifts of the two compounds were found to be mainly different in that compound schigrandilactoneA lacked one oxidized methylene and methylene, and had one more oxidized methine and methyl. Combined with DEPT-135, it was known that C-2 was methine and C-30 was methyl. Further analysis of the HMBC correlation spectrum showed that H-2 was closely related to C-1, C-10 and CH 3 -30 has related signals with C-4, C-5, and C-29, indicating that C-2 of compound 6 is substituted by a hydroxyl group and C-30 is a methyl group. Thus, the planar structure of compound 6 has been determined.

[0114] In the NOESY spectrum of compound 6, H-5 / CH 3 -30, CH 3 -29 / H-1, CH 3 -18 / H-16 / H-22, H-22 / CH 3 -21 and H-20 / CH 3 The related signals of H-5, H-20, CH 3 -27 and CH 3 -30 is in the α configuration; on the contrary, H-16, CH 3 -18, CH 3 -21, H-22, H-25 and CH 3 -29 is β-configuration. There are multiple chiral carbons in this structure. To determine the absolute configuration, we tried to grow a single crystal and finally obtained compound 6 in methanol solvent at 23°C. By Cu KαX-single crystal diffraction [orthorhombic, space group P2 1 (no.4), Z=4, T=193.00K, μ(CuKα)=0.917mm-1 ,Dcalc=1.364g / cm 3 ,45999reflections measured(7.892°≤2θ≤159.188°),12158unique(R int =0.0642,R sigma =0.0519)which were used in all calculations.The final R 1 was0.1127(I>2σ(I))and wR 2 was 0.3095 (all data). Flack: 0.11 (5)] and determined that their absolute configurations were 1R, 2R, 5S, 8S, 9S, 10R, 13S, 15R, 16S, 20S, 22S, 23R and 25S.

[0115] The 2D-NMR spectrum of compound 6 was comprehensively analyzed, and its 1D-NMR data were attributed in detail. Combined with the results of single crystal diffraction, the structure of compound 6 was determined and named schinensilactone L.

[0116] Structural identification of compound 7

[0117] Compound 7 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [MH] at m / z 559.2192 (calcd.for559.2185). - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 36 O 11 , and its unsaturation is calculated to be 12.

[0118] Compound 7 1 H-NMR (600MHz, C 5 D 5 In the 1H, 2H (N) spectrum, δ4.42 (1H, s) and 4.76 (1H, s) are oxymethyl proton signals. In the high field region, δ0.95 (3H, s), 1.19 (3H, d, J = 6.9), 1.26 (3H, d, J = 7.1), 1.10 (3H, s), and 1.23 (3H, s) show five methyl proton signals; at δ2.65 (1H, ABd, J = 16.0) and 2.77 (1H, ABd, J = 16.0), typical AB-type methylene proton signals in triterpenoids can be observed.

[0119] Compound 713 C-NMR (150MHz, C 5 D 5 N) spectrum showed 29 carbon signals, which included 2 ketone carbonyl signals at δ214.7 and 219.5, and 2 ester carbonyl carbon signals at δ176.9 and 178.3, excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 6 contains eight rings. At the same time, the compound also has five methyl carbon signals at δ26.0, 18.3, 14.5, 21.0, 27.9; six methylene carbon signals at δ19.3, 32.2, 35.7, 31.6, 43.4, 39.6; seven methine carbon signals (two oxidized) and seven quaternary carbon signals (six oxidized). The above information indicates that compound 7 is a highly oxidized nortriterpenoid compound.

[0120] The 1D-NMR spectrum data of compound 7 was compared with that of compound 6, and it was found that the spectra of the two were very similar. Further comparison of their NMR data revealed that there were slight differences in the chemical shifts of C-24, C-25 and C-27, which suggested that the configuration of C-25 in the structure might have changed. 3 -30, CH 3 -29 / H-1, CH 3 -18 / H-16 / H-22, H-22 / CH 3 -2, CH 3 -21 / H-24β and H-24β / CH 3 The related signals of H-5, H-20, H-25 and CH 3 -30 is in the α configuration; on the contrary, H-16, CH 3 -18, CH 3 -21, H-22, CH 3 -75 and CH 3 -29 is the β configuration.

[0121] The structure of compound 7 was determined by comprehensive analysis of its 2D-NMR spectrum and detailed attribution of its 1D-NMR data, and it was named schinensilactone M.

[0122] Structural identification of compound 8

[0123] Compound 8 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+FA-H] at m / z 591.2456 (calcd.for591.2447). - Ion peak, combined 1 H-NMR, 13C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 38 O 10 , and its unsaturation is calculated to be 11.

[0124] Compound 8 1 H-NMR (600MHz, C 5 D 5 In the spectrum of 1H, an olefin proton signal at δ7.15 (1H, s) can be clearly observed; δ5.12 (1H, dd, J = 5.4, 1.4), 4.51 (1H, d, J = 7.5), 5.16 (1H, m) are oxymethyl proton signals. In the high field region, δ1.17 (3H, s), 1.81 (3H, brs), 1.80 (3H, brs), 1.30 (3H, s), 1.23 (3H, s) show five methyl proton signals. 13 C-NMR (150MHz, C 5 D 5 N) spectrum, one ketone carbonyl signal at δ215.8; two ester carbonyl carbon signals at δ175.1, 174.8; one double bond carbon signal at δ148.8, 130.1; excluding the above sp 2 The four unsaturations occupied by the hybridized carbon signals indicate that compound 8 contains seven rings. At the same time, the compound also has five methyl carbon signals at δ28.4, 17.6, 10.6, 21.3, 27.7; five methylene carbon signals at δ35.8, 21.9, 21.9, 29.8, 26.2; nine methine carbon signals (four oxidized) and five quaternary carbon signals (four oxidized). The above information indicates that compound 8 is a highly oxidized nortriterpenoid compound.

[0125] The NMR spectrum data of compound 8 was compared with the known compound preschisanartanin X reported in literature 6, and it was found that the two had the same parent core structure. Further comparison of their NMR data revealed that compound 8 had one less set of acetoxy carbon signals than preschisanartanin X and the chemical shift of C-22 changed only slightly. Combined with DEPT-135, it was known that C-22 was a methine. According to the HMBC related signals, CH 3 -21 and C-22 have related signals and bind simultaneously 1 H- 1 In the H COSY spectrum, the H-17 / H-20 / H-22 / H-23 related signals further confirmed that C-22 was substituted by a hydroxyl group. Thus, the planar structure of compound 8 was determined.

[0126] In the NOESY spectrum of compound 8, H-5 / H-8 / CH3 -30, CH 3 -29 / H-1 and CH 3 The correlation signals of H-18 / H-16 indicate that H-5, H-8 and CH 3 -30 is α-configuration; on the contrary, H-1, H-16, CH 3 -18 and CH 3 -29 is the β configuration. 3 J H-16 / H-17 (9.2 Hz) suggests that they are in the same plane, and the absolute configuration of C-17 is R (see reference 7). At the same time, quantum chemical calculations were used to calculate the ECD spectrum of compound 8, and finally 20R, 22R, and 23S were determined.

[0127] The structure of compound 8 was determined by comprehensive analysis of its 2D-NMR spectrum and detailed attribution of its 1D-NMR data, and it was named schinensilactone N.

[0128] Structural identification of compound 9

[0129] Compound 9 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+FA-H] at m / z 573.2350 (calcd.for573.2341). - Ion peak, combined 1 H-NMR, 13 C-NMR and DEPT spectra, etc., inferred that its molecular formula is C 29 H 36 O 9 , and its unsaturation is calculated to be 12.

[0130] Compound 9 1 H-NMR (600MHz, C 5 D 5 In the 1H, 2H, 3H, 4H, 5H, s) spectrum, two olefin proton signals can be clearly observed at δ5.78 (1H, d, J = 10.4), 7.76 (1H, brs); δ5.10 (1H, d, J = 5.3), 3.87 (1H, d, J = 6.2) are oxymethyl proton signals. In the high field region, δ1.00 (3H, s), 1.59 (3H, d, J = 6.7), 1.96 (3H, brs), 1.31 (3H, s), 1.21 (3H, s) show five methyl proton signals. At δ2.92 (1H, d, J = 18.2), 3.03 (1H, dd, J = 18.2, 5.3), a group of typical methylene proton signals in triterpenoids can be observed.

[0131] exist 13 C-NMR (150MHz, C5 D 5 N) spectrum shows 29 carbon signals. Combining DEPT-135 and HSQC spectra, we can clearly observe a ketone carbonyl at δ215.6; two ester carbonyl carbon signals at δ176.3 and 171.1; two groups of double bond carbon signals at δ119.8, 148.1, 134.8, and 130.7. 2 The five unsaturations occupied by the hybridized carbon signals indicate that compound 9 contains seven rings. At the same time, the compound also has five methyl carbon signals at δ28.1, 24.1, 10.6, 22.0, 28.5; five methylene carbon signals at δ35.4, 23.6, 27.6, 23.9, 25.7; seven methine carbon signals (two oxidized) and five quaternary carbon signals (four oxidized). The above information indicates that compound 9 is a highly oxidized nortriterpenoid compound.

[0132] The NMR spectrum data of compound 9 was compared with the known compound pre-schisanartanin reported in literature 7, and it was found that the two have the same parent core structure. Further comparison of their NMR data revealed that pre-schisanartanin has one less set of acetoxy carbon signals and one more set of double bond carbon signals. Combined with DEPT-135, it can be seen that C-22 is a methine and C-23 is a quaternary carbon. According to the HMBC related signals, CH 3 -21 and C-22 and H-22 and C-23 have related signals, and they bind 1 H- 1 In the HCOSY spectrum, the H-20 / H-22 correlation signal further confirmed that C-22 and C-23 are double bond sp 2 Hybridization. So far, the planar structure of compound 9 has been determined.

[0133] In the NOESY spectrum of compound 9, H-5 / H-8 / CH 3 -30, CH 3 -29 / H-1, H-1 / H-19 and CH 3 The correlation signals of H-18 / H-16 indicate that H-5, H-8 and CH 3 -30 is α-configuration; on the contrary, H-1, H-16, CH 3 -18 and CH 3 -29 is β-configuration. In the NOESY spectrum, no H-22 / H-24 related signals were found, but H-20 / H-24 related signals appeared, confirming that the double bond configuration of C-22 / C-23 is E-configuration. 3 J H-16 / H-17(8.9 Hz) suggests that they are in the same plane and the absolute configuration of C-17 is R (see reference 7). At the same time, quantum chemical calculations were used to calculate the ECD spectrum of compound 9, and 20R was finally determined.

[0134] The structure of compound 9 was determined by comprehensive analysis of its 2D-NMR spectrum and detailed attribution of its 1D-NMR data, and it was named schinensilactone O.

[0135] Document 1: Xiao WL, Huang SX, Zhang L, et al. Nortriterpenoids from Schisandralancifolia[J]. Journal of natural products, 2006, 69(4): 650-653.

[0136] Document 2: Huang SX, Han QB, Lei C, et al. Isolation and characterization of miscellaneous terpenoids of Schisandra chinensis[J]. Tetrahedron, 2008, 64(19): 4260-4267.

[0137] Document 3: Yang YC, Zhu SY, Liu LQ, et al.Chinorlactone A:aschinortriterpenoid with a6 / 5 / 8 / 5-fused carbocyclic core from the stems andleaves ofSchisandra chinensis[J].Organic Chemistry Frontiers, 2022,9(7):1917-1923.

[0138] Literature 4: Liu Y, Liu GZ, Li XM, et al. Anti-proliferative Properties of Schinensilactone A, A Schinortriterpenoid with 7,8-Seco-1,8-cyclo Scaffold against Caco-2 by Inducing Cell Apoptosis from the Leaves of Schisandra chinensis[J]. Chinese Journal of Chemistry, 2022, 40(11):1331-1336.

[0139] Literature 5: Xiao WL, Gong YQ, Wang RR, et al. Bioactive nortriterpenoids from Schisandra grandiflora[J]. Journal of natural products, 2009, 72(9):1678-1681.

[0140] Literature 9: Kun HU, Xing-Ren LI, Tang JW, et al. Structural determination of eleven new preschisanartane-type schinortriterpenoids from two Schisandra species and structural revision of preschisanartanin J using NMR computation method[J]. Chinese journal of natural medicines, 2019, 17(12):970-981.

[0141] Literature 7: Huang SX, Li RT, Liu JP, et al. Isolation and characterization of biogenetically related highly oxygenated nortriterpenoids from Schisandra chinensis[J]. Organic Letters, 2007, 9(11):2079-2082.

[0142] Example 2: In vitro screening of neuroprotective activity of Schisandrae Chinensis leaf nortriterpenoids

[0143] Experimental materials and instruments

[0144] instrument

[0145]

[0146] Material

[0147]

[0148]

[0149] Preparation of test solution

[0150] Test compounds: Weigh an appropriate amount (0.5 mg) of the above-isolated nortriterpenoid compounds from Schisandrae Chinensis leaves, dissolve them into a mother solution with a small amount of DMSO, filter and sterilize with a 0.22 μm microporous filter membrane, and store at 4°C for later use. Dilute with DMEM medium to the required concentration before use.

[0151] Experimental methods

[0152] 1. Cell Culture

[0153] SH-SY5Y cells were revived by conventional methods and cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin / streptomycin / amphotericin B (37°C, 5% CO 2 ), and the cells were passaged once every 2-3 days by repeated pipetting with the culture medium. All cells used in the experiment were in the logarithmic growth phase.

[0154] 2. Cell protection experiment

[0155] (1) SH-SY5Y cells were seeded in 96-well plates (1×10 4 cells / well) and cultured overnight.

[0156] (2) The cells were divided into normal control group and H 2 O 2 770μmol / L (model group), H 2 O 2 770μmol / L+N-acetylcysteine ​​(NAC) 0.5mmol / L (positive control group), H 2 O 2 770μmol / L+various concentrations of compounds and blank control group.

[0157] Inoculate overnight, dilute each drug group with complete medium (2-fold dilution), and make solvent control (DMSO) at the same time. Discard the original medium in the well plate, add the drug to be tested, 100 μL per well, culture for 22 hours, discard the original medium, add 10 μL CCK-8 solution to each well, 37°C, 5% CO 2 After culturing at constant temperature for 2 h, the OD value of the sample was measured using an enzyme reader at a wavelength of 450 nm.

[0158] Cell survival rate % = (experimental group - blank group) OD / (control group - blank group) OD × 100%

[0159] Statistical analysis

[0160] The experimental data are The data were expressed as follows. GraphPad Prism Version 8.0 (La Jolla, CA, USA) software was used to perform one-way analysis of variance (ANOVA) analysis for comparison among the groups.

[0161] Experimental Results

[0162] Table 4 Effect of Compounds 1-9 on H at 50 μmol / L 2 O 2 Induced neuroprotection in SH-SY5Y cells

[0163]

[0164] ** P<0.01, * P<0.05vs.Model(55.24±3.36%)

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, or solvate thereof, wherein the compound is selected from the group consisting of compounds 1-9 shown below:

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

3. Use of the pharmaceutical composition of the compound according to claim 1 or its pharmaceutically acceptable salt, prodrug, stereoisomer, solvate or the pharmaceutical composition according to claim 2 in the preparation of a drug for protecting nerves.

4. The use according to claim 3, characterized in that The drug is used for protecting SH-SY5Y cells.

5. Use of a pharmaceutical composition of the compound of claim 1 or a pharmaceutically acceptable salt, prodrug, stereoisomer, solvate thereof or a pharmaceutical composition of claim 2 in the preparation of a medicament for preventing or treating a neurodegenerative disease.

6. The use according to claim 5, characterized in that The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

7. A method for preparing the compound according to claim 1, characterized in that: The Schisandra leaves are subjected to solvent extraction to obtain the Schisandra total extract, and then compounds 1-9 are separated from the Schisandra total extract.

8. The method according to claim 7, characterized in that The solvent extraction comprises heating extraction with an organic solvent; the organic solvent is selected from at least one of methanol and ethanol; after the extraction, the extract is filtered and combined, and the solvent is evaporated under reduced pressure to obtain the total extract of Schisandra chinensis.

9. The method according to claim 7 or 8, characterized in that: The method of separating compounds 1-9 from the total extract of Schisandra chinensis comprises separating the total extract of Schisandra chinensis by macroporous resin, and then separating the total extract of Schisandra chinensis by silica gel column chromatography, MCI column chromatography, ODS column chromatography, gel column chromatography, and preparative HPLC to obtain compounds 1-9.

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