Benzopyridine isoquinoline alkaloid extracted from mairei, and extraction method and application of benzopyridine isoquinoline alkaloid

Abstract

The invention discloses four new benzopyridine isoquinoline alkaloids, and discloses an extraction method and application of the benzopyridine isoquinoline alkaloids, the benzopyridine isoquinoline alkaloids are extracted and separated from the bark of the abelmoschus esculentus, and the alkaloids have obvious nerve cell protection activity.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicines, and particularly relates to benzopyridine isoquinoline alkaloids extracted from Alangium salviifolium, an extraction method thereof, and applications thereof. Background Art

[0002] Alangium salviifolium Alangium salviifolium (L.f.) Wangerin is a plant of the genus Alangium in the family Alangiaceae.

[0003] Alangium salviifolium is a medicinal tree species with great development potential. Alkaloids can be extracted from the bark and roots of Alangium salviifolium, sterols and fatty acids can be extracted from the seeds, and steroids and flavonoids can be extracted from the methanol extract of the flowers.

[0004] There are few studies on the chemical constituents of Alangium salviifolium at home and abroad. At present, it is found that its chemical constituents mainly include alkaloids, triterpenes, sesquiterpenes, flavonoids (glycosides), etc., and most of them have good biological activities.

[0005] Tran Manh Hung (Phenolicglycosides from Alangium salviifolium leaves with inhibitory activity on LPS-induced NO, PGE2, and TNF-α production. Bioorganic & Medicinal Chemistry Letters, 2009, 19: 4389–4393.) reported three phenolic glycosides isolated from the leaves of Alangium salviifolium, which have anti-inflammatory activities.

[0006] S.Rajkumar (Isolation chemical characterization and hypoglycemic activity of Alangium Salviifolium Wang bark in alloxan-induced hyperglycemic rats. Indian Journal of Pharmaceutical Science & Research, 2011, 2(6): 1518-1524.) reported two compounds extracted from the bark of Alangium salviifolium, and these two compounds have hypoglycemic activities.

[0007] Md. Nasrullah (Phytochemical screening, antioxidant and anticholinesterase effects of Alangium salvifolium(L.F) Wang, Root extracts. Journal of Medicinal Plants Research 2015, 9(42): 1060-1069.) reported that the extracts of Alangium salviifolium have moderate to strong antioxidant and enzyme inhibitory effects.

[0008] You-Sheng Cai (Octahydro-protoberberine and protoemetine-type alkaloids from the stems of Alangium salviifolium and their cytotoxicity. Journal of Natural Products, 2019, 82:9, 2645-2652.) reported two berberine alkaloids and two protoemetine derivatives isolated from the stems of Alangium salviifolium; and reported that some of these alkaloids have inhibitory effects on three human cancer cells.

[0009] This application further studies the separation of chemical components, structure identification and uses of Alangium salviifolium. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide benzopyridine isoquinoline alkaloids extracted from Alangium salviifolium, and extraction methods and applications thereof.

[0011] The technical solution for achieving the first object of the present invention is benzopyridine isoquinoline alkaloids shown by the following formula, namely Compound 1, Compound 2, Compound 3 and Compound 4, and the structural formulas of each compound are as follows: .

[0012] The technical solution for achieving the second object of the present invention is the application of the above-mentioned Compound 1, Compound 2, Compound 3 and Compound 4 in the preparation of neuroprotective drugs.

[0013] The technical solution for achieving the third object of the present invention is the preparation method of the above-mentioned benzopyridine isoquinoline alkaloids, including the following steps: ① Crush the dry bark of Alangium salviifolium, and then extract it successively with petroleum ether, ethyl acetate, and 95% ethanol. Vacuum concentrate the extracts respectively to obtain crude extracts of different solvents.

[0014] ② Separate the ethanol crude extract by a reduced-pressure chromatography column, and elute it with a petroleum ether-ethyl acetate solvent system according to a volume ratio gradient of 100:1 to 0:1. After combining the components with the same polarity, 29 components Fr.1 ~ Fr.29 are obtained.

[0015] ③Perform ODS column chromatography on the Fr.16 fraction obtained in step ②, with the eluent being MeOH-H 2 O solvent, and perform elution according to the gradient of the volume ratio of MeOH to H 2 O from 3:7 to 1:0. After combining the fractions with the same polarity, 12 fractions Fr.16.1 - Fr.16.12 are obtained.

[0016] ④Perform silica gel column chromatography on the Fr.16.8 fraction obtained in step ③, with the eluent being CHCl 3 -MeOH, and perform elution according to the gradient of the volume ratio of CHCl 3 to MeOH from 15:1 to 5:1. After combining the fractions with the same polarity, 6 fractions Fr.16.8.1 - Fr.16.8.6 are obtained.

[0017] The obtained Fr.16.8.3 fraction is separated by a Sephadex LH-20 chromatography column, and the eluent is CHCl with a volume ratio of 1:1 3 -MeOH; then compound 2 ( t R = 11 min) and compound 1 ( t R = 12 min) are obtained by semi-preparative HPLC method; ⑤Perform silica gel column chromatography on the Fr.16.9 fraction obtained in step ③, with the eluent being CHCl 3 -MeOH, and perform elution according to the gradient of the volume ratio of CHCl 3 to MeOH from 12:1 to 2:1. After combining the fractions with the same polarity, 7 fractions Fr.16.9.1 - Fr.16.9.7 are obtained; The obtained Fr.16.9.3 fraction is further purified by semi-preparative HPLC method to obtain compound 4 ( t R = 29 min) and compound 3 ( t R = 30 min).

[0018] In the above step ①, petroleum ether extraction is carried out 2 - 3 times, 2.5 - 3.5 days each time; ethyl acetate extraction is carried out 2 - 3 times, 2.5 - 3.5 days each time; 95% ethanol extraction is carried out 2 - 3 times, 4.5 - 5.5 days each time.

[0019] Optionally, in step ①, petroleum ether extraction is carried out 2 times, 3 days each time; ethyl acetate extraction is carried out 2 times, 3 days each time; 95% ethanol extraction is carried out 2 times, 5 days each time.

[0020] In step ② above, the packing material of the reduced-pressure chromatography column is silica gel with a mesh size of 200-300.

[0021] In step ④ above, the chromatographic column for semi-preparative HPLC is Agilent Eclipse XDB-C 18 , and the mobile phase is CH 3 CN-H 2 O with a flow rate of 2-3 mL / min.

[0022] In step ⑤ above, the chromatographic column is Agilent Eclipse XDB-C 18 , and the mobile phase is CH 3 CN-H 2 O with a flow rate of 2-3 mL / min.

[0023] The present invention has positive effects: A group of benzopyridine isoquinoline alkaloids are extracted from Alangium salviifolium (L.f.) Wanger., and the benzopyridine isoquinoline alkaloid coded as Compound 1 has obvious neuroprotective activity and has application prospects in the preparation of neuroprotective drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the 1 H NMR spectrum of Compound 1.

[0025] Figure 2 is the 13 C NMR spectrum of Compound 1.

[0026] Figure 3 is the DEPT135 spectrum of Compound 1.

[0027] Figure 4 is the HSQC spectrum of Compound 1.

[0028] Figure 5 is the HMBC spectrum of Compound 1.

[0029] Figure 6 is the 1 H− 1 H COSY spectrum of Compound 1.

[0030] Figure 7 is the NOESY spectrum of Compound 1.

[0031] Figure 8 is the (+)-HR-ESI-MS spectrum of Compound 1.

[0032] Figure 9 is the ultraviolet spectrum of Compound 1.

[0033] Figure 10 is the infrared spectrum of compound 1.

[0034] Figure 11 For compound 2 1 H NMR spectrum.

[0035] Figure 12 For compound 2 13 C NMR spectrum.

[0036] Figure 13 This is the DEPT135 spectrum of compound 2.

[0037] Figure 14 is the HSQC spectrum of compound 2.

[0038] Figure 15 is the HMBC spectrum of compound 2.

[0039] Figure 16 For compound 2 1 H− 1 H COSY spectrum.

[0040] Figure 17 is the NOESY spectrum of compound 2.

[0041] Figure 18 is the (+)-HR-ESI-MS spectrum of compound 2.

[0042] Figure 19 is the UV spectrum of compound 2.

[0043] Figure 20 is the infrared spectrum of compound 2.

[0044] Figure 21 For compound 3 1 H NMR spectrum.

[0045] Figure 22 For compound 3 13 C NMR spectrum.

[0046] Figure 23 This is the DEPT135 spectrum of compound 3.

[0047] Figure 24 is the HSQC spectrum of compound 3.

[0048] Figure 25 is the HMBC spectrum of compound 3.

[0049] Figure 26 For compound 3 1 H− 1 H COSY spectrum.

[0050] Figure 27 is the NOESY spectrum of compound 3.

[0051] Figure 28 is the (+)-HR-ESI-MS spectrum of compound 3.

[0052] Figure 29 is the UV spectrum of compound 3.

[0053] Figure 30 is the infrared spectrum of compound 3.

[0054] Figure 31 For compound 4 1 H NMR spectrum.

[0055] Figure 32 For compound 4 13 C NMR spectrum.

[0056] Figure 33 This is the DEPT135 spectrum of compound 4.

[0057] Figure 34 is the HSQC spectrum of compound 4.

[0058] Figure 35 is the HMBC spectrum of compound 4.

[0059] Figure 36 For compound 4 1 H− 1 H COSY spectrum.

[0060] Figure 37 is the NOESY spectrum of compound 4.

[0061] Figure 38 is the (+)-HR-ESI-MS spectrum of compound 4.

[0062] Figure 39 is the UV spectrum of compound 4.

[0063] Figure 40 is the infrared spectrum of compound 4.

[0064] Figure 41 41-1 is the ECD experimental diagram of compound 1 and compound 2, and 41-2 is the ECD experimental diagram of compound 3 and compound 4.

[0065] Figure 42 This is a crystal image of compound 1-3.

[0066] Figure 43 Compound 1 to H 2 O 2Statistical graph of the induced effects on SH-SY5Y cell survival rate.

[0067] Figure 44 Compound 1 to H 2 O 2 Photographs of induced protection against oxidative damage in SH-SY5Y cells. DETAILED DESCRIPTION

[0068] Introduced below are some of the multiple possible embodiments of the present invention, which are intended to provide a basic understanding of the present invention, and are not intended to confirm the key or decisive elements of the present invention or to limit the scope of protection. It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, a person of ordinary skill in the art can propose other mutually replaceable implementations. Therefore, the following specific embodiments are only exemplary descriptions of the technical solution of the present invention, and should not be regarded as the whole of the present invention or as a limitation or restriction to the technical solution of the present invention.

[0069] (Example 1) The present invention discloses four benzopyridoquinolizine alkaloids extracted from the genus Achyranthes bidentata, namely, compound 1, compound 2, compound 3 and compound 4, and the structural formula of each compound is as follows: .

[0070] The key to compounds 1-4 1 H- 1 The H COSY and HMBC signals are as follows: .

[0071] Among them, the structural confirmation spectrum of compound 1 is shown in Figures 1 to 10 The structural confirmation spectrum of compound 2 is shown in Figures 11 to 20 The structural confirmation spectrum of compound 3 is shown in Figures 21 to 30 The structural confirmation spectrum of compound 4 is shown in Figures 31 to 40 ; The NMR related detection solvent is DMSO. The ECD experimental diagram of compound 1-4 is shown in Figure 41 The crystal diagram of compound 1-3 is shown in Figure 42 .

[0072] Compound 1 (alansaloid A) is a light yellow powder, easily soluble in methanol. The optical rotation value is [α] D 20 + 287.0( c 0.1, DMSO); UV (MeOH) λ max (log ε): 212 (4.62), 281 (414), 364 (3.85) nm; ECD (0.2 mg / mL, MeOH) λ max (∆ ε ): 209 (38.13) nm; high resolution mass spectrometry HRESIMS ( m / z 327.1338 [M+H] + , theoretical value 327.1345) to determine its molecular formula is C 18 H 18 N 2 O 4 ;according to 1 H, 13 Its structure was confirmed by C and 2D NMR data, and its skeleton type was benzopyridine isoquinoline alkaloid, which was named alansaloid A. 1 H and 13 C NMR data assignments are shown in Tables 1 and 2, [600 MHz ( 1 H), 125MHz ( 13 C), solvent: DMSO- d 6 ].

[0073] Compound 2 (alansaloid B) is a light yellow powder with an optical rotation value of [α] D 20 + 206.0 ( c 0.1, DMSO); UV (MeOH) λ max (log ε ): 212 (4.62), 259 (4.23), 361 (4.23) nm; ECD (0.2 mg / mL, MeOH) λ max (∆ ε ): 209 (38.13) nm; high resolution mass spectrometry HRESIMS ( m / z 327.1343 [M + H] + , theoretical value 327.1345) to determine its molecular formula is C 18 H 18 N 2 O 4 ;according to 1 H, 13 Its structure was confirmed by C and 2D NMR data, and its skeleton type was benzopyridine isoquinoline alkaloid, which was named alansaloid B. 1 H and 13C NMR data assignments are shown in Tables 1 and 2, [600 MHz ( 1 H), 125MHz ( 13 C), solvent: DMSO- d 6 ].

[0074] Compound 3 (alansaloid C) is a light yellow powder with an optical rotation value of [α] D 20 + 442.0 ( c 0.1, DMSO); UV (MeOH) λ max (log ε ): 225 (4.85), 278 (4.43), 360 (3.43) nm; ECD (0.2 mg / mL, MeOH) λ max (∆ ε ): 209 (38.13) nm; high resolution mass spectrometry HRESIMS ( m / z 341.1489 [M + H] + , theoretical value 341.1496) to determine its molecular formula is C 19 H 21 N 2 O 4 ;according to 1 H, 13 Its structure was confirmed by C and 2D NMR data, and its skeleton type was benzopyridine isoquinoline alkaloid, so it was named alansaloid C. 1 H and 13 C NMR data assignments are shown in Tables 1 and 2, [600 MHz ( 1 H), 125MHz ( 13 C), solvent: DMSO- d 6 ].

[0075] Compound 4 (alansaloid D) is a light yellow powder with an optical rotation value of [α] D 20 + 332.0 ( c 0.1, DMSO); UV (MeOH) λ max (log ε ): 225 (4.86), 279 (4.44), 363 (3.60) nm; ECD (0.2 mg / mL, MeOH) λ max (∆ε ): 209 (38.13) nm; high resolution mass spectrometry HRESIMS ( m / z 363.1299 [M + H] + , theoretical value 363.1315) to determine its molecular formula is C 19 H 20 N 2 O 4 Na; according to 1 H, 13 Its structure was confirmed by C and 2D NMR data, and its skeleton type was benzopyridine isoquinoline alkaloid, which was named alansaloid D. 1 H and 13 C NMR data assignments are shown in Tables 1 and 2, [600 MHz ( 1 H), 125MHz ( 13 C), solvent: DMSO- d 6 ].

[0076] The above compounds 1 H-NMR (600 M) data are shown in Table 1: .

[0077] The above compounds 13 C-NMR (125 M) data are shown in Table 2: .

[0078] (Example 2) In this example, the benzopyridine isoquinoline alkaloids described in Example 1 were extracted from the spatholobus spatholobus by the following method: ① Crush 20 kg of the bark of the Tutan tree trunk, and then extract it with petroleum ether (extracted twice, each time with a solvent dosage of 30 L, recorded as 2 × 30 L, each extraction for 3 days), ethyl acetate (2 × 30 L, each time for 3 days), and 95% ethanol (2 × 30 L, each time for 5 days). Concentrate the extracts in vacuum to obtain crude extracts of different solvents.

[0079] Optionally, the number of extractions with each of the above solvents and the duration of each extraction can be: 2 to 3 extractions with petroleum ether, 2.5 to 3.5 days each extraction; 2 to 3 extractions with ethyl acetate, 2.5 to 3.5 days each extraction; 2 to 3 extractions with 95% ethanol, 4.5 to 5.5 days each extraction.

[0080] ② The crude ethanol extract (441.0 g) was separated by vacuum chromatography using 200-300 mesh silica gel as filler and gradient elution using a petroleum ether-ethyl acetate solvent system (100:1 to 0:1, v / v). After combining components with the same polarity, 29 components (Fr.1 to Fr.29) were obtained.

[0081] ③ The Fr.16 component (36.02 g) obtained in step ② was separated by ODS column chromatography, and the eluent was MeOH-H 2 O solvent, according to MeOH and H 2 The elution was performed with a gradient of 3:7 to 1:0 of ​​volume ratio of 4-6-nitrogen-2-ol (3:7 to 1:0), and 12 components (Fr.16.1 to Fr.16.12) were obtained after combining the components with the same polarity.

[0082] ④Separate the Fr.16.8 component (1.49 g) obtained in step ③ by silica gel column chromatography, using CHCl as the eluent. 3 -MeOH, according to CHCl 3 The product was eluted with a gradient of 15:1 to 5:1 volume ratio of MeOH, and 6 components (Fr.16.8.1 to Fr.16.8.6) were obtained after combining the components with the same polarity.

[0083] The obtained Fr.16.8.3 component (53.0 mg) was separated by Sephadex LH-20 column chromatography, and the eluent was CHCl (volume ratio 1:1). 3 -MeOH; then compound 2 (8.4 mg, t R = 11 min) and compound 1 (6.6 mg, t R = 12 min). The chromatographic column used for semi-preparative HPLC was Agilent EclipseXDB-C 18 The mobile phase was CH 3 CN-H 2 O, with a flow rate of 2-3 mL / min.

[0084] The structural confirmation spectrum of compound 1 is shown in Figures 1 to 10 The structural confirmation spectrum of compound 2 is shown in Figures 11 to 20 The ECD experimental diagrams of compound 1 and compound 2 are shown in Figure 41-1 ; The crystal images of compound 1 and compound 2 are shown in Figure 42 .

[0085] The structural formulas of compound 1 and compound 2 are as follows: .

[0086] ⑤Separate the Fr.16.9 component (0.85 g) obtained in step ③ by silica gel column chromatography, using CHCl as the eluent. 3 -MeOH, as CHCl 3 The product was eluted with a gradient of 12:1 to 2:1 volume ratio of MeOH, and 7 components (Fr.16.9.1 to Fr.16.9.7) were obtained after combining components with the same polarity.

[0087] The obtained Fr.16.9.3 component was further purified by semi-preparative HPLC to obtain compound 4 (2.1 mg, t R = 29min) and compound 3 (3.7 mg, t R = 30 min); the chromatographic column for semi-preparative HPLC was Agilent EclipseXDB-C 18 The mobile phase was CH 3 CN-H 2 O, with a flow rate of 2-3 mL / min.

[0088] The structural confirmation spectrum of compound 3 is shown in Figures 21 to 30 The structural confirmation spectrum of compound 4 is shown in Figures 31 to 40 ; ECD experimental diagrams of compounds 3 and 4 are shown in Figure 41-2 ; The crystal diagram of compound 3 is shown in Figure 42 .

[0089] The structural formulas of compound 3 and compound 4 are as follows: .

[0090] (Experimental example) This experiment tested the effect of compound 1 on H 2 O 2 Effects of induced SH-SY5Y cell viability.

[0091] In order to study the effects of the above isolates on H 2 O 2 The protective effect of SH-SY5Y cells induced by β-lactamase was detected by CCK-8 method. SH-SY5Y cells were cultured at 6 × 10 5 Cells were seeded at a density of 10 cells / mL in each well of a 96-well plate and incubated at 37°C with 5% CO 2 SH-SY5Y cells were then incubated with 2-fold diluted compounds (vitamin C was used as a positive control) for 3 h. 2 O 2Add to each well of cells. After 6 hours, add 10 μL of CCK8 solution to each well and keep incubating for 0.5-4 hours. The absorbance at 450 nm of each well was measured by Tecan microplate reader, and the cell viability of each group was expressed as a percentage relative to the value of the control group (100%). The results were analyzed using GraphPad Prism software, and the statistical differences between different groups were compared by one-way ANOVA and Dunnett's test.

[0092] Compound 1 for H 2 O 2 The induced SH-SY5Y cell survival rate was shown in Figure 43 , compared with the normal group, ### P <0.001, and H 2 O 2 Group comparison, *** P <0.001.

[0093] Compound 1 for H 2 O 2 The protective effect of inducing oxidative damage in SH-SY5Y cells is shown in Figure 44 , Photos were taken using an Olympus live cell imaging system.

[0094] From the above results, it can be seen that compound 1 of the present invention has obvious protective activity on nerve cells.

Claims

1. The benzopyridine isoquinoline alkaloids shown in the following formula are compound 1, compound 2, compound 3 and compound 4, and the structural formula of each compound is as follows: 。 2. Use of Compound 1, Compound 2, Compound 3 and Compound 4 as claimed in claim 1 in the preparation of neuroprotective drugs.

3. A method for preparing the benzopyridine isoquinoline alkaloid as claimed in claim 1, characterized in that The following steps are involved: ① The bark of the trunk of the Tutan tree was crushed, and then extracted with petroleum ether, ethyl acetate, and 95% ethanol in sequence, and the extracts were vacuum concentrated to obtain crude extracts with different solvents; ② The crude ethanol extract was separated by vacuum chromatography, and a petroleum ether-ethyl acetate solvent system was used for gradient elution at a volume ratio of 100:1 to 0:

1. After combining the components with the same polarity, 29 components Fr.1 to Fr.29 were obtained; ③ The Fr.16 component obtained in step ② was separated by ODS column chromatography, and the eluent was MeOH-H2O solvent, and the eluent was eluted according to the gradient of MeOH and H2O volume ratio of 3:7 to 1:0, and 12 components Fr.16.1 to Fr.16.12 were obtained after combining the components with the same polarity; ④ Separate the Fr.16.8 component obtained in step ③ by silica gel column chromatography, using CHCl3-MeOH as the eluent, and elute according to the gradient of CHCl3 to MeOH volume ratio of 15:1 to 5:1, and combine the components with the same polarity to obtain 6 components Fr.16.8.1~Fr.16.8.6; The obtained Fr.16.8.3 component was separated by Sephadex LH-20 column chromatography, and the eluent was CHCl3-MeOH with a volume ratio of 1:1; then compound 2 ( t R = 11 min) and compound 1 ( t R = 12 min); ⑤ Separate the Fr.16.9 component obtained in step ③ by silica gel column chromatography, using CHCl3-MeOH as the eluent, and elute according to the gradient of CHCl3 to MeOH volume ratio of 12:1 to 2:1, and combine the components with the same polarity to obtain 7 components Fr.16.9.1~Fr.16.9.7; The obtained Fr.16.9.3 component was further purified by semi-preparative HPLC to obtain compound 4 ( t R = 29 min) and compound 3 ( t R = 30 min).

4. The method for preparing the benzopyridine isoquinoline alkaloid according to claim 3, characterized in that: In step ①, petroleum ether is extracted 2 to 3 times, each time for 2.5 to 3.5 days; ethyl acetate is extracted 2 to 3 times, each time for 2.5 to 3.5 days; 95% ethanol is extracted 2 to 3 times, each time for 4.5 to 5.5 days.

5. The method for preparing the benzopyridine isoquinoline alkaloid according to claim 4, characterized in that: In step ①, petroleum ether is extracted twice, each time for 3 days; ethyl acetate is extracted twice, each time for 3 days; 95% ethanol is extracted twice, each time for 5 days.

6. The method for preparing the benzopyridine isoquinoline alkaloid according to claim 3, characterized in that: In step ②, the filler of the vacuum chromatography column is 200-300 mesh silica gel.

7. The method for preparing the benzopyridine isoquinoline alkaloid according to claim 3, characterized in that: In step ④, the chromatographic column for semi-preparative HPLC method is Agilent Eclipse XDB-C 18 , the mobile phase was CH3CN-H2O in a volume ratio of 20:80, and the flow rate was 2-3 mL / min.

8. The method for preparing benzopyridine isoquinoline alkaloids according to claim 3, characterized in that: In step ⑤, the chromatographic column is Agilent Eclipse XDB-C 18 , the mobile phase was CH3CN-H2O in a volume ratio of 20:80, and the flow rate was 2-3 mL / min.

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