Bibenzyl compound and preparation method and application thereof
阅读说明：本技术 联苄类化合物及其制备方法和用途 (Bibenzyl compound and preparation method and application thereof ) 是由 周地 李宁 陈刚 李巍 于 2019-11-21 设计创作，主要内容包括：本发明属于医药技术领域,涉及联苄类化合物及其制备方法和用途,具体涉及3个联苄类化合物及其盐、异构体及其制备方法和在制备预防或治疗神经退行性疾病药物领域中的应用,所述化合物的通式如下：(The invention belongs to the technical field of medicines, relates to bibenzyl compounds, a preparation method and application thereof, and particularly relates to 3 bibenzyl compounds, salts and isomers thereof, a preparation method thereof and application thereof in the field of preparation of medicines for preventing or treating neurodegenerative diseases, wherein the compounds have the following general formula:)
1. The bibenzyl compound and pharmaceutically acceptable salts and isomers thereof have the following structural general formula:
wherein R is1Is hydrogen, C1-C4 alkyl or glucosyl; r2Is hydrogen, hydroxy, C1-C4 alkoxy or a pyrrolidone ring; preferably, R1Is hydrogen, methyl or glucosyl; r2Is hydrogen, hydroxy, methoxy or a pyrrolidone ring.
2. The bibenzyl compound and pharmaceutically acceptable salts and isomers thereof according to claim 1, characterized in that it is one or several of the following structures:
3. the bibenzyl compound and its pharmaceutically acceptable preparation process as claimed in claim 2, including: the method comprises the following steps:
(1) extracting rhizoma Bletillae (Bletilla striata) with ethanol solvent, and recovering extractive solution to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by water, and extracting by an organic solvent to obtain extracts with different polarities;
(3) separating the ethyl acetate extract obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol, and a mixed solvent of dichloromethane and methanol;
(4) separating the 100: 1-100: 25 fractions obtained in the step (3) by ODS column chromatography, and carrying out gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase;
(5) further separating the methanol and water, acetonitrile and water eluates obtained in the step (4) by preparative HPLC-UV, and carrying out gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase to obtain a racemic mixture of bibenzyl compounds 1 and 2 and a compound 3;
(6) and (3) carrying out HPLC chiral resolution on the racemic mixture of the bibenzyl compound obtained in the step (5) to obtain compounds 1 and 2.
4. The method for producing bibenzyls according to claim 3, wherein: the extraction method in the step (1) is heating reflux ethanol extraction or heating ultrasonic extraction for 1-3 times, wherein the volume concentration of ethanol is 70-95%, and the rhizoma bletillae is as follows: the weight-volume ratio of the ethanol is 1: 5-1: 20 g/mL; the organic solvent extraction method in the step (2) comprises the steps of sequentially extracting the water phase and the organic phase for 3-5 times by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of 1:1, and recovering the organic solvent under reduced pressure.
5. The method for producing bibenzyls according to claim 3, wherein: the volume ratio of the mixed solvent of the eluting solvent petroleum ether and the ethyl acetate and the mixed solvent of the petroleum ether and the acetone in the step (3) is 100: 1-1: 1, the volume ratio of a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and methanol, or a mixed solvent of chloroform and methanol is 100: 1-100: 10.
6. the method for producing bibenzyls according to claim 3, wherein: in the step (4), the volume ratio of the methanol-water mixed solvent is 3: 7-9: 1, and the volume ratio of the acetonitrile-water mixed solvent is 1: 9-7: 3.
7. the process for the preparation of bibenzenes according to claim 3, characterized in that: the mixed solvent of methanol and water and the mixed solvent of acetonitrile and water in the step (5) comprise the following components in percentage by volume: 4: 6-9: 1, the volume ratio of the acetonitrile-water mixed solvent is 3: 7-7: 3.
8. the method for producing bibenzyls according to claim 3, wherein: the chiral chromatographic column resolution solvent in the step (6) is a mixed solvent of n-hexane and absolute ethyl alcohol, and the volume ratio of the mixed solvent is 70: 30-95: 5.
9. A pharmaceutical composition comprising the bibenzyl compound and pharmaceutically acceptable salts, isomers and pharmaceutically acceptable carriers thereof according to claim 1 or 2.
10. Use of the bibenzyl compound and pharmaceutically acceptable salts, isomers or pharmaceutical compositions according to claim 1 or 2 in the preparation of medicaments for preventing or treating neurodegenerative diseases.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a novel bibenzyl compound in bletilla striata as well as a preparation method and application thereof.
Background
Rhizoma Bletillae (Bletilla striata) is also known as Glechomae herba, rhizoma Bletilla, etc. Mainly distributed in Guizhou, Sichuan, Yunnan, Hunan, Hubei, Anhui provinces.
The common bletilla pseudobulb is used as a rare and rare Chinese medicinal material, is bitter, sweet, astringent and cool in nature, enters lung, stomach and liver meridians, and is mainly used for treating hemoptysis, hematemesis, traumatic hemorrhage, pyocutaneous disease, pyogenic infection and skin chap. Modern pharmacological activity researches show that the bletilla has obvious effects of resisting tumors, oxidation and bacteria, promoting wound healing and the like, and the chemical components of the bletilla mainly comprise bibenzyls, phenanthrenes, dihydrophenanthrenes, diphenanthrene, terpenoids, polysaccharides and the like.
Disclosure of Invention
The invention aims to provide a series of bibenzyl compounds, a preparation method and a new medical application thereof.
The invention provides bibenzyl compounds, salts and isomers thereof, which have the following structures:
R1is hydrogen, C1-C4 alkyl or glucosyl; r2Is hydrogen, hydroxy, C1-C4 alkoxy or a pyrrolidone ring.
Further, R1Is hydrogen, methyl or glucosyl; r2Is hydrogen, hydroxy, methoxy or a pyrrolidone ring.
The invention specifically discloses the following 3 specific compounds:
the invention also provides a preparation method of the bibenzyl compounds 1-3, which comprises the following steps:
(1) extracting dry tubers of rhizoma bletillae (Bletilla striata) by using 70-95% ethanol, and recovering an extracting solution to obtain a crude extract;
(2) dissolving the crude extract obtained in the step (1) with water, extracting with an organic solvent, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1 to obtain extracts with different polarities;
(3) separating the extract obtained in the step (2) by silica gel column chromatography, and separating the extract by using a mixed solvent of petroleum ether and ethyl acetate 100: 1-1: 1. petroleum ether and acetone mixed solvent 100: 1-1: 1. chloroform-acetone mixed solvent 100: 1-100: 10. dichloromethane and acetone mixed solvent 100: 1-100: 10. chloroform-methanol mixed solvent 100: 1-100: 10. dichloromethane and methanol mixed solvent 100: 1-100: 10 gradient elution;
(4) separating the 100: 1-100: 25 fractions obtained in the step (3) by ODS column chromatography, and carrying out gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase;
(5) and (3) further separating the methanol and water 3: 7-9: 1 and the acetonitrile and water 1: 9-7: 3 eluate obtained in the step (4) by preparative HPLC-UV, and mixing the methanol and water mixed solvent 4: 6-9: 1, or acetonitrile and water 3: 7-7: 3, gradient elution is carried out by using a mixed solvent as a mobile phase to obtain a racemic mixture of bibenzyl compounds 1 and 2 and a compound 3;
(6) and (3) carrying out HPLC chiral resolution on the racemic mixture of the bibenzyl compound obtained in the step (5) to obtain compounds 1 and 2, wherein the solvent is a mixed solvent of n-hexane and absolute ethyl alcohol, and the volume ratio of the mixed solvent is 70: 30-95: 5.
According to the preparation method of the bibenzyl compound 1-3 provided by the invention, the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 1-3 times, and the used solvent is 70-95% of ethanol, preferably 75-95% of ethanol. The medicinal materials are as follows: the weight-volume ratio of the solvent is 1: 5-1: 20g/mL, preferably 1: 10-1: 15.
According to the preparation method of the bibenzyl compounds 1-3, the organic solvent extraction method in the step (2) is adopted, water is adopted to dissolve the crude extract, petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol are respectively used for sequentially extracting for 3-5 times, preferably 5 times according to the volume ratio of the water phase to the organic phase of 1:1, and the organic solvent is recovered under reduced pressure.
According to the preparation method of the bibenzyl compounds 1-3 provided by the invention, in the step (3), the volume ratio of the elution solvent petroleum ether and ethyl acetate mixed solvent to the petroleum ether and acetone mixed solvent is 100: 1-1: 1, preferably 100: 4-10: 1; the volume ratio of the mixed solvent of dichloromethane and acetone, the mixed solvent of chloroform and acetone, the mixed solvent of dichloromethane and methanol, or the mixed solvent of chloroform and methanol is 100: 1-100: 10, preferably 100:1 to 100: 6.
According to the preparation method of the bibenzyl compound 1-3, the volume ratio of the mixed solvent of methanol and water in the step (4) is 3: 7-9: 1, preferably 6: 4-8: 2; the volume ratio of the acetonitrile-water mixed solvent is 1: 9-7: 3, preferably 4:6 to 1: 1.
The invention provides a preparation method of bibenzyl compounds 1-3, which comprises the following steps of (5) preparing a mixed solvent of methanol and water, and a mixed solvent of acetonitrile and water, wherein the volume ratio of the mixed solvent of methanol and water is as follows: 4: 6-9: 1, preferably 6: 4-8: 2; the volume ratio of the acetonitrile-water mixed solvent is 3: 7-7: 3, preferably 4: 6-1: 1.
according to the preparation method of the bibenzyl compounds 1-3, the chiral chromatographic column resolution solvent in the step (6) is a mixed solvent of n-hexane and absolute ethyl alcohol, and the volume ratio of the mixed solvent is 70: 30-95: 5, preferably 75: 25-80: 20.
The invention uses LPS to induce a BV2 microglia overactivation model to evaluate the anti-neuritis activity of the prepared bibenzyl compounds 1-3. The results show that compounds 1, 2 and 3 are able to inhibit LPS-induced release of excess activated BV2 microglia NO, exhibiting moderate-intensity anti-neuritic activity. Therefore, the bibenzyl compound prepared by the invention can be applied to the development of medicines for treating neurodegenerative diseases.
The invention provides a method for preparing and identifying 3 bibenzyl compounds by taking bletilla striata as a raw material for the first time, systematically evaluates the activity of the bibenzyl compounds in the aspect of neuroprotection, and clarifies the application of the bibenzyl compounds in the aspect of developing and treating neurodegenerative diseases.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
(1) Extracting rhizoma Bletillae tuber 500g with 75% ethanol for 1 time (10L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) dissolving the 75% ethanol crude extract obtained in the step (1) with water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol, extracting with each organic solvent for 3 times, wherein the volume ratio of the water phase to the organic phase is 1:1 each time, and obtaining extracts of different polar parts;
(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with a mixed solvent of petroleum ether and ethyl acetate at a ratio of 100:1, 100:3, 100:8 and 100: 10;
(4) the petroleum ether obtained in the step (3): subjecting the ethyl acetate 100: 4-100: 8 flow to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol-water at a ratio of 30:70, 50:50, 70:30, 90: 10;
(5) separating the methanol-water (50: 50-90: 10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 4mL/min, and the mobile phase is methanol: water 75:25 to give a racemic mixture of bibenzyls 1 and 2 (t)R40min) (yield 0.00011%). The racemic mixture of compounds 1 and 2 was further separated by chiral column chromatography using n-hexane: elution with ethanol (75:25) as the mobile phase provided novel compounds 1(9.958min),2(11.304min) (0.00005% yield each).
(6) Separating the methanol-water (50: 50-90: 10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and taking a 60:40 methanol-water mixed solvent as a mobile phase to obtain bibenzyl 3(tR35min) (yield 0.00010%).
The structures of the compounds 1 to 3 were identified based on their physicochemical properties and spectral data.
The structural identification data for compound 1 is as follows:
purple powder (methanol).30.4(c 1.0 MeOH), HR-ESI-MS gave the excimer ion peak [ M + H [ ]]+m/z：328.1539(calcd.328.1543for C19H22NO4) The molecular formula is shown as C19H21NO4。1H NMR(600MHz,CD3OD)：δH7.06(1H, t, J ═ 7.8Hz, H-5'), 6.60(1H, m, H-4'), 6.58(1H, m, H-6'), 6.62(1H, m, H-2') in a set of meta-positionsHydrogen signals of substituted benzene rings, 6.33(1H, d, J ═ 2.4Hz, H-4), 6.23(1H, d, J ═ 2.4Hz, H-6) are hydrogen signals of meta-coupling on benzene rings; deltaH2.74-2.89(4H, m, H-alpha, alpha') is a methylene characteristic hydrogen signal of the bibenzyl compound; deltaH3.75(3H,s,3-OCH3) Is a methoxy hydrogen signal, 5.03(1H, dd, J ═ 9.0,5.0Hz, H-5 "), 2.39(1H, m, H-3" a), 2.50(1H, m, H-3 "b), 2.06(1H, m, H-4" a), 2.32(1H, m, H-4 "b) are 5 aliphatic hydrogen signals, two sets of methylene hydrogen signals are included, one set of pyrrolidone hydrogen signals.13C NMR(150MHz,CD3OD) spectrum gives 19 carbon signals: deltaC143.3(C-1), 120.0(C-2), 161.4(C-3), 99.2(C-4), 159.0(C-5), 109.7(C-6), 144.3(C-1'), 120.9(C-2'), 158.5(C-3'), 114.0(C-4'), 130.4(C-5'), 116.4(C-6') is 12 sp2A hybridized carbon signal; deltaC 55.9(3-OCH3) 39.7 (C-. alpha.'), 36.7 (C-. alpha.) is the aliphatic carbon signal; 181.5(C-2 "), 32.2 (C-3"), 27.4(C-4 "), 53.4 (C-5") constitute a five-membered aza ring-pyrrolidone.
All hydrogen-carbon signals were assigned using HSQC and the position of the substituents was further determined by HMBC spectroscopy. In HMBC spectra, δH5.03 (H-5') and deltaC143.3(C-1), 161.4(C-3) remote correlation, suggesting that pyrrolidone is attached at the C-2 position, δH 3.75(3-OCH3) Remote association with 161.4(C-3) suggests that the methoxy group is attached at the C-3 position, thus determining the structure of the compound. The compound is a new compound which is not reported in the literature through searching and is named as dusuranlansin E1.
TABLE 1 assignment of NMR data for Compounds 1 and 2
The structural identification data for compound 2 is as follows:
purple powder (methanol).30.4(c 1.0 MeOH), HR-ESI-MS gave the excimer ion peak [ M + H [ ]]+m/z：328.1539(calcd.328.1543for C19H22NO4) The molecular formula is shown as C19H21NO4。
As the compounds 1 and 2 are a pair of enantiomers obtained by chiral column resolution, the hydrogen-carbon spectrum data of the compounds are completely consistent (see table 1), and different places are shown on a CD spectrum, wherein the compound 1 has a + cotton effect at 195-220 nm; while compound 2 is a-cotton effect at this wavelength; the compound is a new compound which is not reported in the literature through searching and is named as dusuranlansin E2.
The structural identification data for compound 3 is as follows:
dark yellow powder (methanol). HR-ESI-MS gives the peak of the excimer ion [ M-H ]]-m/z：405.1534(calcd.405.1555for C21H25O8) The molecular formula is shown as C21H26O8，1H NMR(600MHz,DMSO-d6)：δH7.17(1H, t, J ═ 7.8Hz, H-5'), 6.84(1H, m, H-6'), 6.89(1H, m, H-4'), 6.83(1H, m, H-2'), 6.13(1H, m, H-4), 6.21(2H, m, H-2,6) are hydrogen signals on the benzene ring; 4.80(d, J ═ 7.2Hz, H-1') are the sugar terminal hydrogen signals, deltaH2.81-2.88(4H, m, H-alpha, alpha') is a methylene characteristic hydrogen signal of the bibenzyl compound; deltaH 3.65(3H,s,5-OCH3) One methoxy hydrogen signal.13C NMR(150MHz,DMSO-d6) The spectrum gives a 21 carbon signal: deltaC143.4(C-1), 108.2(C-2), 160.3(C-3,5), 98.9(C-4), 104.3(C-6), 143.1(C-1'), 113.5(C-2'), 157.5(C-3'), 116.3(C-4'), 129.0(C-5'), 121.8(C-6') is 12 sp2A hybridized carbon signal; 100.4(C-1 "), 73.3 (C-2"), 76.7(C-3 "), 69.7 (C-4"), 77.0(C-5 "), 60.7 (C-6") are glucose carbon signals, δC 54.7(5-OCH3) 36.7 (C-. alpha.'), 37.2 (C-. alpha.) are signals for aliphatic carbons.
TABLE 2 NMR data assignment for Compound 3
Hydrogen is separated according to HSQC spectrumAnd (3) attributing carbon signals one by one, and determining the connecting positions of the substituent and the sugar fragment by combining HMBC spectrums. Remote correlation of 4.80(H-1 ') with 157.5(C-3') indicates that glucose is attached at the C-3' position, and 3.65 (5-OCH)3) Correlation with 160.3(C-5) indicates that the methoxy group is attached at the C-5 position; the compound is a new compound which is not reported in the literature through search and is named as 3-hydroxy-5-methoxybibenzyl-3' -O-beta-D-glucopyranoside.
Example 2
(1) Reflux-extracting rhizoma Bletillae 1000g with 95% ethanol under heating for 3 times (dosage: 10L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) extracting the ethanol extract obtained in the step (1) by using an organic solvent, and sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1 to obtain extracts of different polar parts;
(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with a mixed solvent of petroleum ether and ethyl acetate at a ratio of 100:2, 100:4, 100:8 and 100: 10;
(4) the petroleum ether obtained in the step (3): subjecting the ethyl acetate 100: 4-100: 7 flow to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol-water at a ratio of 30:70, 60:40, 70:30, 90: 10;
(5) separating the methanol-water (70: 30-90: 10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 4mL/min, and the mobile phase is methanol: water 65:35 to give a racemic mixture of bibenzyls 1 and 2 (t)R40min) (yield 0.00011%). The racemic mixture of compounds 1 and 2 was further separated by chiral column chromatography using n-hexane: elution with ethanol (75:25) as the mobile phase yielded new 1(9.958min),2(11.304min) (0.00005% yield each).
(6) Separating the methanol-water (60: 40-50: 50) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and taking a mixed solvent of 60:40 methanol-water as a mobile phase to obtain bibenzyl 3(tR35min) (yield 0.00011%).
The structure of bibenzenes 1-3 can be identified as described in example 1.
Example 3
(1) Extracting rhizoma Bletillae 800g with 85% ethanol under reflux for 3 times (dosage: 9.6L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1, and respectively extracting for 4 times to obtain extracts of different polar parts;
(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with dichloromethane and acetone mixed solvents of 100:1, 100:3, 100:5 and 100: 7;
(4) dichloromethane obtained in the above step (3): performing ODS chromatography on a 100: 5-100: 7 acetone flow, and performing gradient elution by using a mixed solvent of methanol-water of 30:70, 60:40, 70:30 and 90: 10;
(5) separating the methanol-water (70: 30-90: 10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 4mL/min, and the mobile phase is methanol: water 65:35 to give a racemic mixture of bibenzyls 1 and 2 (t)R36min) (yield 0.00011%). The racemic mixture of compounds 1 and 2 was further separated by chiral column chromatography using n-hexane: elution with ethanol (75:25) as the mobile phase yielded new 1(9.058min),2(11.104min) (0.00005% yield each).
(6) Separating the methanol-water (60: 40-50: 50) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and taking a methanol-water mixed solvent of 55:45 as a mobile phase to obtain bibenzyl 3(tR36min) (yield 0.00011%).
The structure of bibenzenes 1-3 can be identified as described in example 1.
Example 4
(1) Heating and refluxing rhizoma Bletillae 1200g with 95% ethanol for 2 times (dosage: 18L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with dichloromethane and methanol mixed solvents of 100:1, 100:3, 100:5 and 100: 8;
(4) dichloromethane obtained in the above step (3): performing ODS chromatography on a 100: 3-100: 5 flow of methanol, and performing gradient elution by using a mixed solvent of methanol-water of 30:70, 50:50 and 90: 10;
(5) separating the methanol-water (90:10) fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with flow rate of 4mL/min, and using acetonitrile as mobile phase: water 45:55 to give a racemic mixture of bibenzyls 1 and 2 (t)R46min) (yield 0.00010%). The racemic mixture of compounds 1 and 2 was further separated by chiral column chromatography using n-hexane: elution with ethanol (77:23) as the mobile phase yielded new 1(9.008min),2(11.100min) (yield 0.00005% each).
(6) Separating the methanol-water (50:50) fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with flow rate of 4mL/min, and using a 53:47 methanol-water mixed solvent as a mobile phase to obtain bibenzyl 3(tR36min) (yield 0.00013%).
The structure of bibenzenes 1-3 can be identified as described in example 1.
Example 5
(1) Heating and refluxing rhizoma Bletillae 600g with 80% ethanol for 3 times (dosage: 10L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with chloroform and acetone mixed solvents of 100:1, 100:3, 100:5 and 100: 6;
(4) chloroform obtained in the above step (3): performing ODS chromatography on acetone with a flow rate of 100: 5-100: 6, and performing gradient elution by using a mixed solvent of acetonitrile-water with a flow rate of 30:70, 45:55, 60:40, 65: 35;
(5) separating the acetonitrile-water (45:55) fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with flow rate of 4mL/min, and using methanol as mobile phase: water 66:34 to obtainTo racemic mixtures of bibenzyls 1 and 2 (t)R34min) (yield 0.00011%). The racemic mixture of compounds 1 and 2 was further separated by chiral column chromatography using n-hexane: elution with ethanol (78:22) as the mobile phase yielded new 1(9.058min),2(11.104min) (0.00005% yield each).
(6) Separating the acetonitrile-water (30:70) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, and obtaining bibenzyl 3 (t) with a mixed solvent of methanol and water of 55:45 as a mobile phase at a flow rate of 4mL/minR33min) (yield 0.00013%).
The structure of bibenzenes 1-3 can be identified as described in example 1.
Example 6
(1) Reflux-extracting rhizoma Bletillae 300g with 90% ethanol under heating for 3 times (dosage: 3L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with chloroform and methanol mixed solvents of 100:1, 100:3, 100:5 and 100: 7;
(4) chloroform obtained in the above step (3): performing ODS chromatography on 100: 3-100: 5 methanol by using mixed solvent of acetonitrile-water at a ratio of 20:80, 30:70, 50:50, 60: 40;
(5) separating the acetonitrile-water (50:50) fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with flow rate of 4mL/min, and using methanol as mobile phase: water 65:35 to give a racemic mixture of bibenzyls 1 and 2 (t)R36min) (yield 0.00011%). The racemic mixture of compounds 1 and 2 was further separated by chiral column chromatography using n-hexane: elution with ethanol (75:25) as the mobile phase yielded new 1(9.058min),2(11.104min) (0.00005% yield each).
(6) Separating the acetonitrile-water (20:80) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and using a mixed solvent of 30:70 acetonitrile-water as a mobile phase to obtain bibenzyl 3(tR36min) (yield 0.00014%).
The structure of bibenzenes 1-3 can be identified as described in example 1.
EXAMPLE 7 anti-neuritic Activity test of New bibenzenes 1-3 prepared in examples 1-6
(1) The experimental principle is as follows: the chronic inflammatory reaction mediated by the microglia activation is an important link in the generation and development process of neurodegenerative diseases, and the inhibition of the microglia activation can become a new target point for drug discovery. LPS activates microglia to release NO, proinflammatory cytokines, active oxygen and the like. The experiment evaluates the anti-inflammatory activity of the new bibenzyl 1-3 by establishing a screening model for abnormal activation of BV2 microglia through in vitro LPS and taking NO released by activated microglia as an index.
(2) The experimental method comprises the following steps:
culture of mouse microglia line BV2
All glassware and metal instruments (culture bottles, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 121 ℃ for 30min to completely remove the contaminated LPS. A cell culture solution containing 10% fetal calf serum and 50. mu.M 2-mercaptoethanol was prepared on the basis of DMEM medium. Microglia at about 4 × 105cells/ml at 5% CO2And subculturing in a culture bottle at 37 ℃, wherein the adherent cells account for 50-60% of the bottom area of the culture bottle by the third day, digesting the adherent cells by pancreatin, and subculturing to another culture bottle. BV2 thawed in a refrigerator at the ultralow temperature of-80 ℃ is taken as the first generation, and BV2 cells of 3 th to 8 th generations are selected for experiments.
② process for preparing medicine
Test compounds were all in powder form, dissolved in DMSO. A mother liquor was prepared at a concentration of 50mM and stored at-20 ℃. It was diluted with DMEM medium at the time of use to 100. mu.M, 30. mu.M, 10. mu.M, 3. mu.M, and 1. mu.M in this order. The final concentration of DMSO is less than 1 ‰.
③ Griess method for detecting inhibition of compound to LPS activated microglia
Taking BV2 microglia in logarithmic growth phase, and using fresh DMEM medium containing 5% fetal calf serumAdjusting cell density to 3X 105cells/ml, seeded in 96-well plates, 100. mu.l/well, 5% CO at 37 ℃2Culturing in the incubator. And replacing the cells with serum-free fresh culture solution after 24 hours of adherent culture, and simultaneously adding drugs. Each compound was co-administered with LPS at 1, 3, 10, 30, 100. mu.M. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. Continuously culturing for 24h after adding medicine into cells, collecting supernatant, and detecting NO in the supernatant by Griess colorimetric method2-And (4) content.
MTT method for detecting influence of compound on survival rate of microglia cell
Taking BV2 microglia cultured in logarithmic growth phase, adjusting cell density to 3 × 10 by using fresh DMEM medium containing 5% fetal calf serum5cells/ml, seeded in 96-well plates, 100. mu.l/well, 5% CO at 37 ℃2Culturing in the incubator. After the cells are cultured for 24 hours adherent, the cells are changed into fresh culture solution, and meanwhile, the cells are treated by adding medicine. Each compound was co-administered with LPS at 1, 3, 10, 30, 100. mu.M. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. The cells were incubated for 24h after addition of the drug, then MTT solution, 10. mu.l/well, was added to the cell broth, the cells were incubated with 0.25mg/ml MTT at 37 ℃ for 3h, the culture broth was aspirated, then 100. mu.l of DMSO solution was added, and the OD of the optical density was determined. And (3) processing data, namely processing the data by using corresponding software of a microplate reader, calculating an average value of OD values of 6 holes of each sample, and calculating the cell survival rate (CV%) by using the average value according to the following formula.
Percent cell survival%
Fifthly, statistical method
All data were examined using the SPSS (13.0) statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, and the means between groups was analyzed by One-Way ANOVA analysis for homogeneity of variance and by Dunnett's test analysis for comparison between groups. The multiple sample homogeneity of variance test was conducted using a Leven test, where the variances were uniform when p >0.05, the differences in mean among the groups were tested using Dunnett's two-sided T, and the differences in mean among the groups were tested using Dunnett T3 when p <0.05 and the variances were not uniform.
⑥IC50Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate50。
(3) The experimental results are as follows: see Table 3
TABLE 3 results of experiments on inhibitory effect of bibenzyls 1-3 on microglial activation
The significance is as follows:*P<0.05,**P<0.01,***P<0.001 compared to LPS-induced group;###P<0.001 compared to the control group.
The results show that the new bibenzyl compounds 1(100 μ M), 2(100 μ M) and 3(30 μ M, 100 μ M) prepared in examples 1 to 6 can significantly inhibit the release of LPS-induced overactivated BV2 microglia NO.