Beta-carboline-benzisoquinoline alkaloid as well as preparation method and application thereof

Abstract

The invention discloses beta-carboline-benzisoquinoline alkaloid as well as a preparation method and application of the beta-carboline-benzisoquinoline alkaloid. The preparation method comprises the following steps: by taking the stem of the euryale ferox as a raw material, carrying out silica gel column chromatography separation, Sephadex LH-20 gel column separation and semi-preparative liquid chromatography separation on an ethanol part extract to obtain a compound A1, a compound A5 and another three beta-carboline-benzisoquinoline alkaloids A2, A3 and A4, and the compounds A1-A5 have good tumor cell inhibition activity.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicines, and particularly relates to β-carboline-benzisoquinoline alkaloids isolated from Alangium salviifolium, their applications, and a method for isolating β-carboline-benzisoquinoline alkaloids from Alangium salviifolium. Background Art

[0002] Alangium salviifolium ( Alangium salviifolium ), also known as Gesheluo, is a plant of the genus Alangium ( Alangium ) in the family Alangiaceae. In China, it is mainly distributed in the southern coastal areas of Guangdong, Guangxi, and Hainan Province.

[0003] Alangium salviifolium is an excellent medicinal plant, and parts such as leaves, flowers, roots, root bark, stems, and stem bark all have medicinal value. The extract of Alangium salviifolium has potential effects on hypertension, diabetes, epilepsy, cancer, inflammation, ulcers, etc.

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

[0005] Nobuo Yagi (Isolation and biological activity of a novel cadinane-typesesquiterpenoid from the essential oil of Alangium salviifolium , J. OleoSci. 2014, 63(12): 1223-1229.) et al. reported the antioxidant effect of sesquiterpenes in Alangium salviifolium.

[0006] Phanruethai Pailee (Bioactive Cardinane Sesquiterpenes from the Stemsof Alangium salviifolium , Chem- Asian J, 2015, 10: 910-914.) et al. reported the antitumor effect of sesquiterpenes in Alangium salviifolium.

[0007] SHASHIKUMARA (Evaluation of Antidepressant Activity of EthanolicExtract of Alangium SalvifoliumReports such as "Leaves in Swiss Albino Mice, Biomedical&Pharmacology Journal, Vol. 10(1), 427-433, 2017" have reported the antidepressant effect of the ethanol extract of the leaves of *Alangium salviifolium*.

[0008] Md. Nasrullah ("Acetylcholinesterase and Butyrylcholinesterase Enzyme Inhibitory Effect of Alangium salviifolium (L. f.) Wang pericarp Extracts with Their Phytochemical and Antioxidant Values, Journal of Pharmaceutical Research International, 2017, 19(5): 1-11.") and other reports have shown that the pericarp extract of this plant has moderate to strong antioxidant properties, can effectively inhibit acetylcholinesterase and BchE, and has the potential to prevent Alzheimer's disease.

[0009] Preeti Dhruve ("A novel hepatoprotective activity of Alangium salviifolium in mouse model, Drug and Chemical Toxicology, ISSN: 0148-0545") and other reports have reported the hepatoprotective activity of the bark extract of *Alangium salviifolium*.

[0010] The present invention aims to further isolate and study the chemical constituents of *Alangium salviifolium* in order to obtain new alkaloid compounds with antitumor effects. Summary of the Invention

[0011] The object of the present invention is to provide new β-carboline-benzisoquinoline alkaloids and their applications, a method for isolating β-carboline-benzisoquinoline alkaloids from *Alangium salviifolium*, and the applications of the isolated β-carboline-benzisoquinoline alkaloids.

[0012] The technical solution for achieving the first object of the present invention is the β-carboline-benzisoquinoline alkaloids A1 and compound A5 shown in the following formula:

[0013] The technical solution for achieving the second object of the present invention is the application of the compound A1 and compound A5 in the preparation of antitumor drugs.

[0014] Further, the tumors are human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma.

[0015] The technical solution for achieving the third object of the present invention is a method for isolating β-carboline-benzisoquinoline alkaloids from Alstonia scholaris, comprising the following steps: ① After crushing the dried tree stems of Alstonia scholaris, extract them successively with petroleum ether, ethyl acetate, and 95% ethanol, and concentrate the medicinal liquids by vacuum to obtain extracts in different solvents.

[0016] ② Perform silica gel column chromatography separation on the ethanol extract obtained in step ①, and perform gradient elution with petroleum ether / ethyl acetate according to a volume ratio of 100:1 to 0:1. After combining the components with the same polarity, obtain 12 components, Fr.1 to Fr.12.

[0017] ③ Perform silica gel column chromatography separation on the Fr.5 component obtained in step ② again, and perform gradient elution with chloroform / methanol according to a volume ratio of 100∶1 to 1∶100 to obtain 5 components, Fr.5.1 to Fr.5.5.

[0018] ④ Elute the Fr.5.3 - Fr.5.4 components obtained in step ③ through a Sephadex LH-20 gel column with methanol; then separate them by semi-preparative liquid chromatography to obtain compound A1 ( t R = 18 min), A2 ( t R = 23 min), and A5 ( t R = 15 min).

[0019] ⑤ Elute the Fr.5.5 component obtained in step ③ through a Sephadex LH-20 gel column with methanol, and then separate it by semi-preparative liquid chromatography to obtain compound A3 ( t R = 42 min), A4 ( t R = 10 min); The structural formulas of the above compounds A1, A2, A3, A4, and A5 are as follows: .

[0020] Optionally, in the above step ①, extract with petroleum ether 2 - 3 times, each extraction for 2.5 - 3.5 days; extract with ethyl acetate 2 - 3 times, each extraction for 2.5 - 3.5 days; extract with 95% ethanol 2 - 3 times, each extraction for 4.5 - 5.5 days.

[0021] Optionally, in step ①, extract with petroleum ether twice, 3 days each time; extract with ethyl acetate twice, 3 days each time; extract with 95% ethanol twice, 5 days each time.

[0022] In the above step ②, the packing material for silica gel column chromatography is silica gel with a mesh size of 100 - 200; in step ③, the packing material for silica gel column chromatography is silica gel with a mesh size of 200 - 300.

[0023] In the above step ④, the chromatographic column for semi-preparative liquid chromatography is Agilent Eclipse XDB-C 18 , and the mobile phase is CH 3 CN / H 2 O with a volume ratio of 31:69; in step ⑤, the chromatographic column for semi-preparative liquid chromatography is Agilent EclipseXDB-C 18 , and the mobile phase is CH 3 CN / H 2 O with a volume ratio of 45:55.

[0024] The technical solution for achieving the fourth object of the present invention is the application of compound A2, compound A3, and compound A4 prepared according to the above method in the preparation of anti-tumor drugs.

[0025] The tumors are human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma.

[0026] The present invention has positive effects: (1) Two new highly oxidized β-carboline-benzisoquinoline alkaloids A1 and compound A5 are isolated from Alangium salviifolium in the present invention, and these two new compounds A1 and A5 have the activity of inhibiting human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma cells.

[0027] (2) In the present invention, the tree stem of Alangium salviifolium is used as the raw material, and compound A1 and compound A5 are obtained by subjecting the ethanol extract to silica gel column chromatography separation, SephadexLH-20 gel column separation, and semi-preparative liquid chromatography separation.

[0028] In addition to compound A1 and compound A5, the present invention also isolates three other β-carboline-benzisoquinoline alkaloids A2, A3, and A4 from Alangium salviifolium. After experimental verification, compounds A2, A3, and A4 also exhibit outstanding activity in inhibiting human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma cells. Description of the Drawings

[0029] Figure 1 is the 1 H-NMR spectrum (DMSO) of compound A1.

[0030] Figure 2 13C-NMR spectrum (DMSO) of Compound A1 13 13C-NMR spectrum (DMSO) of Compound A1

[0031] Figure 3 DEPT(135°) spectrum (DMSO) of Compound A1

[0032] Figure 4 HSQC spectrum (DMSO) of Compound A1

[0033] Figure 5 HMBC spectrum (DMSO) of Compound A1

[0034] Figure 6 1H- 1 1H- 1 1H COSY spectrum (DMSO) of Compound A1

[0035] Figure 7 NOESY spectrum (DMSO) of Compound A1

[0036] Figure 8 (-)-HR-ESI-MS spectrum of Compound A1

[0037] Figure 9 UV spectrum of Compound A1

[0038] Figure 10 IR spectrum of Compound A1

[0039] Figure 11 1H-NMR spectrum (DMSO) of Compound A5 1 1H-NMR spectrum (DMSO) of Compound A5

[0040] Figure 12 13C-NMR spectrum (DMSO) of Compound A5 13 13C-NMR spectrum (DMSO) of Compound A5

[0041] Figure 13 DEPT(135°) spectrum (DMSO) of Compound A5

[0042] Figure 14 HSQC spectrum (DMSO) of Compound A5

[0043] Figure 15 HMBC spectrum (DMSO) of Compound A5

[0044] Figure 16 1H- 1 1H- 1 1H COSY spectrum (DMSO) of Compound A5

[0045] Figure 17NOESY spectrum of compound A5 (DMSO).

[0046] Figure 18 (-)-HR-ESI-MS spectrum of compound A5

[0047] Figure 19 UV spectrum of compound A5

[0048] Figure 20 IR spectrum of compound A5

[0049] Figure 21 For compound A2 1 1H-NMR spectrum

[0050] Figure 22 For compound A2 13 13C-NMR spectrum

[0051] Figure 23 ORTEP diagram of compound A2 crystal

[0052] Figure 24 For compound A3 1 1H-NMR spectrum

[0053] Figure 25 For compound A3 13 13C-NMR spectrum

[0054] Figure 26 For compound A4 1 1H-NMR spectrum

[0055] Figure 27 For compound A4 13 13C-NMR spectrum

[0056] Figure 28 ECD experimental diagram of compounds A1, A2, and A5 Detailed implementation manners

[0057] The following are some of the multiple possible embodiments of the present invention, aiming to provide a basic understanding of the present invention, and not aiming to identify the key or decisive elements of the present invention or limit the scope to be protected. It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, those of ordinary skill in the art can propose other interchangeable implementation manners. Therefore, the following detailed implementation manners and the accompanying drawings 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 regarded as a limitation or restriction on the technical solution of the present invention.

[0058] (Example 1) Two new β-carboline-benzisoquinoline alkaloids of the present invention were isolated from Alangium salviifolium, namely compound A1 and compound A5. The structural formulas of compound A1 and compound A5 are as follows:

[0059] The structure confirmation spectra of compound A1 are shown in Figures 1 to 10 , and the structure confirmation spectra of compound A5 are shown in Figures 11 to 20 .

[0060] The key 1 H- 1 H COSY and HMBC signals of compounds A1 and A5 are as follows: The key NOESY signals of compounds A1 and A5 are as follows: .

[0061] Compound A1 is a pale yellow powder and is soluble in methanol. The optical rotation value [α] D 20 −24.0 ( c 0.1, CH 3 OH). The molecular formula was determined to be C m / z 446.2788 [M + H] + , theoretical value C 28 H 36 N 3 O 2 , 446.2802) by high-resolution mass spectrometry HRESIMS ( 28 H 35 N 3 O 2 ; The structure was determined based on 1 H, 13 C and two-dimensional nuclear magnetic resonance data. The skeleton type is β -carboline-benzoquinolizidine ( β -carboline-benzoisoquinoline alkaloid), and it was named 9-demethyldeoxytubulosine. The 1 H and 13 C NMR data assignments are shown in Table 1, [600 MHz ( 1 H), 125 MHz ( 13 C), solvent: DMSO- d 6 .

[0062] Compound A5 is a pale yellow powder and is soluble in methanol. The optical rotation value [α] D20 −44.0 ( c 0.1, CH 3 OH). The molecular formula was determined to be C m / z 446.2816 [M + H] + , with a theoretical value of C 28 H 36 N 3 O 2 , 446.2808) by high-resolution electrospray ionization mass spectrometry (HRESIMS). The structure was determined based on 28 H 35 N 3 O 2 ; and two-dimensional nuclear magnetic resonance data. The skeleton type was 1 -carboline-benzoquinolizidine ( 13 -carboline-benzoisoquinoline alkaloid), and it was named 9-demethyldeoxyisotubulosine. The β -carboline-benzoquinolizidine ( β -carboline-benzoisoquinoline alkaloid), and it was named 9-demethyldeoxyisotubulosine. The 1 H and 13 C NMR data assignments are shown in Table 1. [600 MHz ( 1 H), 125 MHz( 13 C), solvent: DMSO- d 6 .

[0063] (Example 2) In this example, the β-carboline-benzoisoquinoline alkaloid described in Example 1 was extracted from Alangium salviifolium, and three other β-carboline-benzoisoquinoline alkaloids A2, A3, and A4 were obtained. The method is as follows: ① After crushing 20 kg of dry Alangium salviifolium tree stems, they were successively extracted with petroleum ether (extracted 2 times, 30 L of solvent each time, denoted as 2 × 30 L, and each extraction for 3 days), ethyl acetate (2 × 30 L, each extraction for 3 days), and 95% ethanol (2 × 30 L, each extraction for 5 days). The extracts of different solvents were obtained by vacuum concentration of the medicinal liquids respectively.

[0064] Optionally, the number of extractions with each solvent and the extraction time for each time can be: petroleum ether extraction 2 - 3 times, each extraction for 2.5 - 3.5 days; ethyl acetate extraction 2 - 3 times, each extraction for 2.5 - 3.5 days; 95% ethanol extraction 2 - 3 times, each extraction for 4.5 - 5.5 days.

[0065] ② Silica gel column chromatography was performed on the ethanol extract obtained in step ① (the packing material was silica gel with a mesh size of 100 - 200), and gradient elution was carried out with petroleum ether / ethyl acetate (100:1 - 0:1, v / v). After combining the fractions with the same polarity, 12 fractions (Fr.1 - Fr.12) were obtained.

[0066] ③ The Fr.5 fraction (1.3 g) obtained in step ② was again separated by silica gel column chromatography (the packing material was silica gel with a mesh size of 200 - 300), and gradient elution was carried out with chloroform / methanol (100∶1 - 1∶100, v / v) to obtain 5 fractions (Fr.5.1 - Fr.5.5).

[0067] ④ The Fr. 5.3 - Fr. 5.4 fractions (680 mg) obtained in step ③ were eluted with methanol through a Sephadex LH - 20 gel column; then, they were further separated by semi - preparative liquid chromatography on an Agilent Eclipse XDB - C 18 (250 mm × 9.4 mm, 5 μm, 2 mL / min, CH 3 CN / H 2 O, 31:69, v / v ) to obtain compound A1 (27.8 mg, t R = 18 min), A2 (35.6 mg, t R = 23 min) and A5 (21.8 mg, t R = 15 min). The ECD experimental spectra of compounds A1, A2, and A5 are shown in Figure 28 .

[0068] ⑤ The Fr. 5.5 fraction (120 mg) obtained in step ③ was eluted with methanol through a Sephadex LH - 20 gel column, and then separated by semi - preparative liquid chromatography on an Agilent Eclipse XDB - C 18 (250 mm × 9.4 mm, 5 μm, 2 mL / min, (CH 3 CN / H 2 O, 45:55, v / v ) to obtain compound A3 (11.8 mg, t R = 42 min), A4 (44.5 mg, t R = 10 min).

[0069] The structures of the isolated compounds A1, A2, A3, A4, and A5 were confirmed. The structure confirmation spectra of compound A1 can be seen in Figures 1 to 10 , and the structure confirmation spectra of compound A2 can be seen in Figures 21 to 23 , the structure confirmation spectra of compound A3 can be seen in Figures 24 to 25 , the structure confirmation spectra of compound A4 can be seen in Figures 26 to 27 , and the structure confirmation spectra of compound A5 can be seen in Figures 11 to 20 ; among them, the spectra of compounds A2, A3, and A4 are consistent with those of the corresponding existing compounds. A1 and A5 are new β-carboline-benzisoquinoline alkaloids. The structural formulas of compounds A1, A2, A3, A4, and A5 are as follows: .

[0070] (Experimental Example) The activities of compounds A1, A2, A3, A4, and A5 were tested in this experimental example.

[0071] (I) Experimental Materials Cells: SGC-7901 (human gastric cancer), Hela (human cervical cancer), K562 (human leukemia), A549 (human lung cancer), BEL-7402 (human liver cancer), HepG2 (human liver cancer), B16 (melanoma cells).

[0072] Cell culture medium: DMEM medium containing 10% fetal bovine serum (FBS), MTT.

[0073] Positive control: doxorubicin hydrochloride.

[0074] (II) Experimental Methods (1) Inoculating cells: Prepare a single cell suspension with a culture medium (DMEM) containing 10% newborn bovine serum, and add 100 μ μL (about 5×10 3 cells per well) of cell suspension into a 96-well plate, and culture for 12 hours until they adhere to the wall.

[0075] (2) Adding drugs: Add 100 μ μL of the sample to be tested. Set 6 wells for addition. Use a concentration of 40 μ μg / ml as the primary screening concentration, and set 5 gradient concentrations for rescreening according to the primary screening results. Set 3 parallel duplicate wells for each concentration.

[0076] (3) Color development: Incubate the 96-well plate after the above drug addition step at 37 °C for 48 hours. Take it out and add 20 μ μL of MTT solution to each well. Continue culturing and then discard the supernatant. Add 150 μ μL of DMSO solution to each well to completely dissolve the crystals.

[0077] (4)Colorimetry: Read the absorbance values of each well with a microplate reader at a wavelength of 490 nm and record the values.

[0078] (5)Data processing: Inhibition rate of cells % = [(OD value of the control group – OD value of the sample group) / OD value of the control group] × 100%, substitute the values to calculate the IC 50 value. The results are as follows: Note: a The values in the table represent the mean ± SD of three parallel experiments.

[0079] b Positive control: doxorubicin hydrochloride.

[0080] From the data in the above table, it can be seen that compounds A1, A2, A3, A4, and A5 have the activity of inhibiting human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma cells; the activity of A1 is better than that of A5.

Claims

1. β-Carboline-benzoisoquinoline alkaloid A1 and compound A5 as shown in the following formula: 。 2. Use of compound A1 and compound A5 as claimed in claim 1 in the preparation of anti-tumor drugs.

3. Use of compound A1 and compound A5 according to claim 2 in the preparation of anti-tumor drugs, characterized in that: The tumors are human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma.

4. A method for separating β-carboline-benzoisoquinoline alkaloids from the genus Tutan tree, characterized in that The following steps are involved: ① After the dried stems of the Tutan tree were crushed, they were extracted with petroleum ether, ethyl acetate, and 95% ethanol in sequence, and the liquid was vacuum concentrated to obtain extracts with different solvents; ② The ethanol extract obtained in step ① was separated by silica gel column chromatography, and gradient eluted with petroleum ether / ethyl acetate at a volume ratio of 100:1 to 0:1, and 12 components Fr.1 to Fr.12 were obtained by combining components with the same polarity; ③ The Fr.5 component obtained in step ② was separated again by silica gel column chromatography, and chloroform / methanol was used for gradient elution at a volume ratio of 100:1 to 1:100 to obtain 5 components Fr.5.1 to Fr. 5.5; ④ The components Fr. 5.3 to Fr. 5.4 obtained in step ③ were passed through a Sephadex LH-20 gel column and eluted with methanol; then, the mixture was separated by semi-preparative liquid chromatography to obtain compound A1 ( t R = 18 min)、A2( t R = 23 min) and A5 ( t R = 15 min); ⑤ The Fr. 5.5 component obtained in step ③ was passed through a Sephadex LH-20 gel column and eluted with methanol, and then separated by semi-preparative liquid chromatography to obtain compound A3 ( t R = 42 min)、A4( t R = 10 min); The structural formulas of the above compounds A1, A2, A3, A4 and A5 are as follows: 。 5. The method for separating β-carboline-benzoisoquinoline alkaloids from the genus Achyranthes bidentata according to claim 4, 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.

6. The method for separating β-carboline-benzoisoquinoline alkaloids from the genus Achyranthes bidentata according to claim 5, 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.

7. The method for separating β-carboline-benzoisoquinoline alkaloids from the genus Achyranthes bidentata according to claim 4, characterized in that: In step ②, the filler of silica gel column chromatography is 100-200 mesh silica gel; in step ③, the filler of silica gel column chromatography is 200-300 mesh silica gel.

8. The method for separating β-carboline-benzoisoquinoline alkaloids from the genus Achyranthes bidentata according to claim 4, characterized in that: In step ④, the chromatographic column for semi-preparative liquid chromatography is Agilent Eclipse XDB-C 18 , the mobile phase was CH3CN / H2O in a volume ratio of 31:69; in step ⑤, the chromatographic column for semi-preparative liquid chromatography was Agilent Eclipse XDB-C 18 , the mobile phase was CH3CN / H2O in a volume ratio of 45:

55.

9. Use of compound A2, compound A3 and compound A4 prepared according to the method of claim 4 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumors are human gastric cancer, human cervical cancer, human leukemia, human liver cancer, human lung cancer, and melanoma.

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