CN115677814A - Pentacyclic triterpenoid compound and preparation method and application thereof - Google Patents
Pentacyclic triterpenoid compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a pentacyclic triterpenoid compound and a preparation method and application thereof, belonging to the field of medicines. Pentacyclic triterpenoid compounds with the following chemical structural formula (I) or (II) and pharmaceutically acceptable salts thereof,
Description
Technical Field
The invention relates to a pentacyclic triterpenoid compound and a preparation method and application thereof, belonging to the field of medicines.
Background
Caragana microphylla lam is mainly distributed in northeast, north China, inner Mongolia, gansu and other places of China. It has effects of promoting blood circulation, removing blood stasis, dispelling pathogenic wind, removing dampness, clearing away heat and toxic materials, and relieving inflammation, and can be used for treating vertigo, headache, rheumatalgia, cough, asthma, and hypertension.
At present, the study on chemical components and pharmacological activity of caragana microphylla is less reported, modern pharmacological studies show that caragana microphylla has an anti-inflammatory effect, and the chemical components mainly comprise flavone and glycosides thereof, triterpene and glycosides thereof, phenylpropanoids, alkaloids and the like.
Disclosure of Invention
The invention aims to provide a series of pentacyclic triterpenoids, a preparation method and a new medical application thereof.
The invention provides pentacyclic triterpenoid compounds with the following chemical structural formula (I) or (II) and pharmaceutically acceptable salts thereof,
the invention also aims to provide a preparation method and application of the pentacyclic triterpenoid with the following chemical structural formula (I) or (II).
A process for the preparation of pentacyclic triterpenoids of the following chemical formula (I) or (II), comprising the following process steps:
(1) Heating and extracting root of Caragana microphylla Lam, and recovering extractive solution to obtain crude extract;
(2) Dissolving the crude extract obtained in the step (1) in water, and extracting with an organic solvent to obtain extracts with different polarities;
(3) Separating the 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, or a mixed solvent of petroleum ether and acetone, or a mixed solvent of chloroform and acetone, or a mixed solvent of dichloromethane and acetone, or a mixed solvent of chloroform and methanol, or a mixed solvent of dichloromethane and methanol;
(4) Separating the flow obtained in the step (3) by ODS column chromatography, and performing gradient elution by using a methanol-water or acetonitrile-water mixed solvent as a mobile phase;
(5) And (4) further separating the methanol-water or acetonitrile-water eluate obtained in the step (4) by HPLC, and performing 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 compound 1 and a compound 2.
Preferably, the extraction method in the step (1) comprises heating reflux extraction with ethanol or methanol, and heating ultrasonic extraction for 2-5 times, wherein the material-liquid ratio is 1:8-1.
Preferably, in the organic solvent extraction in the step (2), the volume ratio of the aqueous phase to the organic phase is 1:1-1:5, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol are respectively used for sequentially extracting for 3-5 times, and the organic solvent is recovered under reduced pressure.
Preferably, in the mixed solvent in the step (3), the volume ratio of petroleum ether to ethyl acetate, petroleum ether to acetone is 100-1:1; dichloromethane and acetone, the volume ratio of chloroform to acetone, dichloromethane to methanol, chloroform to methanol is from 1 to 1:1.
Preferably, in the mixed solvent in the step (4), the volume ratio of methanol to water is 2:8-8:2, and the volume ratio of acetonitrile to water is 1:9-9:1.
Preferably, in the mixed solvent in the step (5), the volume ratio of methanol to water is 2:8-7:3, and the volume ratio of acetonitrile to water is 1:9-6:4.
A pharmaceutical composition comprises pentacyclic triterpenoid with the following chemical structural formula (I) or (II) and pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier,
the invention further provides application of the pentacyclic triterpenoid compound with the chemical structural formula (I) or (II) and the pharmaceutically acceptable salt thereof in preparing the anti-neuritis medicine.
The beneficial effects of the invention are as follows: the invention provides a method for preparing and identifying 2 novel compounds by taking the dry roots of caragana microphylla as a raw material for the first time, systematically evaluates the activity of the caragana microphylla in the aspect of resisting neuritis and clarifies the application of the caragana microphylla in the aspect of developing and treating central nervous system disease medicaments.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
A process for the preparation of pentacyclic triterpenoids of formula (I) or (II), comprising the following process steps:
(1) Heating and refluxing dried roots of Caragana microphylla Lam with 70-95% ethanol or 60-90% methanol, and recovering the extract to obtain a crude extract;
(2) Dissolving the crude extract obtained in the step (1) with water, and sequentially extracting for 3-5 times by using petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:1-1:5 to obtain extracts with different polarities;
(3) Separating the extract obtained in the step (2) by silica gel column chromatography, and eluting by using a mixed solvent of petroleum ether and ethyl acetate 100;
(4) Separating the flow obtained in the step (3) by ODS column chromatography, and performing gradient elution by using a mixed solvent of methanol-water 2:8-8:2 or acetonitrile-water 1:9-9:1 as a mobile phase;
(5) Further separating the methanol and water, acetonitrile and water eluates obtained in the step (4) by HPLC, and carrying out gradient elution by taking a mixed solvent of methanol and water 2:8-7:3 or a mixed solvent of acetonitrile and water 1:9-6:4 as a mobile phase to obtain a compound 1 and a compound 2;
in the technical scheme, the extraction method in the step (1) is heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction for 2-5 times, and the used solvent is 70-95% of ethanol or 60-90% of methanol, preferably 75-85% of ethanol or 65-85% of methanol. The feed-liquid ratio is 1:8-1, 20g/mL, preferably 1.
In the above technical scheme, in the organic solvent extraction method in step (2), the crude extract is dissolved by water, and the organic solvent is sequentially extracted for 3 to 5 times, preferably 5 times, by using petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol according to the volume ratio of the aqueous phase to the organic phase of 1:1 to 1:5, preferably 1:1 to 1:3, and the organic solvent is recovered under reduced pressure.
In the technical scheme, the volume ratio of the petroleum ether and the ethyl acetate or the mixed solvent of the petroleum ether and the acetone in the step (3) is 100-1:1, preferably 100; the volume ratio of the mixed solvent of dichloromethane and acetone, or chloroform and acetone, or dichloromethane and methanol, or chloroform and methanol is 100.
In the technical scheme, the volume ratio of the mixed solvent of methanol and water in the step (4) is 2:8-8:2, preferably 2:8-7:3, and the volume ratio of the mixed solvent of acetonitrile and water is 1:9-9:1, preferably 1:9-6:4.
In the technical scheme, the volume ratio of the mixed solvent of the methanol and the water in the step (5) is 2:8-7:3, preferably 2:8-6:4, and the volume ratio of the mixed solvent of the acetonitrile and the water is 1:9-6:4, preferably 1:9-4:6.
The method adopts LPS-induced abnormally activated BV-2 microglia, takes NO release amount as an index, and preliminarily evaluates the effect of the compound 1-2 on inhibiting excessive activation of the microglia. The results show that the novel compound 2 can dose-dependently inhibit LPS-induced NO release from BV-2 microglia. Therefore, the compound prepared by the invention can be applied to the development of medicines for treating central nervous system diseases.
Example 1
(1) Extracting 1000g of dried root of caragana microphylla with 95% ethanol under heating and refluxing for 3 times (dosage: 10L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Dissolving the 95% ethanol crude extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, extracting each organic phase 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 polarity parts;
(3) In the step (2), the n-butanol extract is separated by silica gel column chromatography, and is sequentially eluted by dichloromethane and methanol mixed solution, wherein the ratio of the solvent to the solvent is 100, 10;
(4) Dichloromethane obtained in the above step (3): methanol 10 to 5:1 fractions were subjected to ODS chromatography, and eluted with a mixed solvent gradient of methanol-water 2;
(5) Separating and preparing the methanol-water (2:8-4:6) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 3mL/min, and the mobile phase is acetonitrile: water =45, to give compound 1 (t) R =22 min) (yield 0.0006 ‰).
(6) Separating and preparing the methanol-water (6:4-8:2) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 3mL/min, and the mobile phase is acetonitrile: water =62, yielding compound 2 (t) R =15 min) (yield 0.0002 ‰).
The structure of the compound 1-2 was identified based on its physicochemical properties and spectral data.
The structural identification data for compound 1 is as follows:
white amorphous powder (methanol). HR-ESI-MS gives the excimer peak m/z 831.4109[ 2 ] M + Na] + (calcd.831.4137for C 42 H 64 O 15 Na) and the molecular formula thereof is presumed to be C 42 H 64 O 15 The unsaturation was calculated to be 11.
1 H NMR(600MHz,pyridine-d 5 ) The high field region gives 6 methyl hydrogen signals: delta H 1.31(3H,s,CH 3 -23),0.76(3H,s,CH 3 -25),0.89(3H,s,CH 3 -26),1.26(3H,s,CH 3 -27),1.23(3H,s,CH 3 -28),1.44(3H,s,CH 3 -30); the low field region gives 2 glycosyl-terminal hydrogen signals: delta H 4.92 (1H, d, J =7.8Hz, H-1 '), 5.49 (1H, d, J =7.4Hz, H-1') and 1 alkene hydrogen signal：δ H 5.31 (1H, br. S, H-12); 1 methoxy hydrogen signal: delta H 3.75(3H,s,6'-OCH 3 ) (ii) a Group 1 continuous oxygen methylene hydrogen signal: delta H 4.32(1H,m,H-24a),3.40(1H,m,H-24b)。 13 C NMR(150MHz,pyridine-d 5 ) Giving 42 carbon signals, including 30 triterpene mother nucleus carbon signals and 12 glycosyl carbon signals. Wherein delta C 215.4 (C-22), 179.6 (C-29), 170.8 (C-6') is the 3 carbonyl carbon signal; delta C 125.0 (C-12), 141.9 (C-13) is a group 1 double bond carbon signal; delta C 105.7 (C-1 '), 105.3 (C-1') is the 2 sugar end group carbon signal; delta C 52.6(6'-OCH 3 ) Is a methoxy carbon signal; delta C 22.9(CH 3 -23),16.1(CH 3 -25),17.1(CH 3 -26),25.8(CH 3 -27),21.4(CH 3 -28),22.1(CH 3 -30) is a 6 methyl carbon signal. According to the 1D NMR data of the compound 1, the combination of literature comparison can infer that the mother nucleus of the compound is an oleanane type pentacyclic triterpene saponin structure, and the structure contains 1 glucuronate and 1 arabinose. The hydrogen-carbon data were further assigned according to information related to HSQC spectra (see Table 1).
In HMBC spectra, delta H 4.92 (H-1') and delta C 90.9(C-3)，δ H 5.49 (H-1') and delta C 79.0 (C-2 ') has remote correlation, and the connection mode and the connection position and the connection sequence of the sugar segment on the triterpene saponin mother nucleus are determined, wherein the sugar segment is connected at the C-3 position of the mother nucleus, and the arabinose segment is connected at the C-2' position of the glucuronate; delta H 3.75(6'-OCH 3 ) And delta C 170.8 (C-6') has a remote correlation, suggesting that the carboxyl groups on the sugar segments form esters. Further, the D-glucuronic acid and the L-arabinose are detected by GC after the sugar part obtained by acid hydrolysis is derivatized, and the D-glucuronic acid and the L-arabinose are respectively identified by combining the chemical shift value and the terminal hydrogen coupling constant of the D-glucuronic acid and the L-arabinose: beta-D-glucuronic acid and alpha-L-arabinose.
NOESY spectrum of Compound 1, δ H 1.23(CH 3 -28),δ H 1.44(CH 3 -30) respectively with δ H 2.56 (H-18) related, suggesting that the carboxyl group is substituted at the C-29 position and the D/E ring isCis-fused; delta H 3.40 (H-3) and δ H 1.31(CH 3 -23) correlation, δ H 4.32 (H-24 a) and δ H 0.76(CH 3 -25) correlation, suggesting that H-3 is in the alpha orientation, the hydroxymethyl substitution is at the C-24 position and in the beta orientation.
Through Sci-finder search, the compound 1 is a novel compound which is not reported in the literature.
TABLE 1 assignment of NMR data for Compound 1
The structural identification data for compound 2 is as follows:
white amorphous powder (methanol). HR-ESI-MS gives the excimer peak m/z 875.4400M + Na] + (calcd.875.4405for C 44 H 68 O 16 Na) and the molecular formula thereof is presumed to be C 44 H 68 O 16 The unsaturation was calculated to be 11.
1 H NMR(600MHz,pyridine-d 5 ) The high field region gives 6 methyl hydrogen signals: delta. For the preparation of a coating H 1.35(3H,s,CH 3 -23),0.74(3H,s,CH 3 -25),0.85(3H,s,CH 3 -26),1.20(3H,s,CH 3 -27),1.18(3H,s,CH 3 -28),1.24(3H,s,CH 3 -30); the low field region gives 2 glycosyl-terminal hydrogen signals: delta H 4.94 (1h, d, j =7.7hz, h-1'), 5.54 (1h, d, j =7.7hz, h-1 ") and 1 alkene hydrogen signal: delta H 5.28 (1H, br. S, H-12); 2 methoxy hydrogen signals: delta H 3.66(3H,s,29-OCH 3 ),3.76(3H,s,6'-OCH 3 ) (ii) a Group 1 continuous oxygen methylene hydrogen signal: delta H 4.31(1H,m,H-24a),3.41(1H,m,H-24b)。 13 C NMR(150MHz,pyridine-d 5 ) Giving 44 carbon signals, including 31 triterpene parent nuclear carbon signals and 13 glycosyl carbon signals. Wherein delta C 214.6 (C-22), 177.1 (C-29), 170.8 (C-6') is the 3 carbonyl carbon signal; delta C 125.2 (C-12), 141.6 (C-13) is a group 1 double bond carbon signal; delta C 105.4 (C-1 '), 105.9 (C-1') is the 2 sugar end group carbon signal; delta C 52.6(29,6'-OCH 3 ) Is a 2 methoxy carbon signal; delta C 23.1(CH 3 -23),16.1(CH 3 -25),17.1(CH 3 -26),25.7(CH 3 -27),21.3(CH 3 -28),21.7(CH 3 -30) is a 6 methyl carbon signal. According to the 1D NMR data of the compound 2, the combination of literature comparison can infer that the mother nucleus of the compound is an oleanane type pentacyclic triterpene saponin structure, and the structure contains 1 glucuronic acid ester and 1 galactose. Further according to H 1 -H 1 COSY, HSQC spectra-related information attributing hydrogen-carbon data (see Table 2).
In HMBC spectra, delta H 4.94 (H-1') and delta C 91.2(C-3)，δ H 5.54 (H-1') and delta C 81.2 (C-2 ') respectively have remote correlation, and the connection mode of the sugar segment and the connection position and the connection sequence of the sugar segment on the triterpene saponin mother nucleus are determined, wherein the sugar segment is connected to the C-3 position of the mother nucleus, and the galactose is connected to the C-2' position of the glucuronic acid ester; delta H 3.66(29-OCH 3 ) And delta C 177.1(C-29)，δ H 3.76(6'-OCH 3 ) And delta C 170.8 (C-6') have a remote correlation, respectively, indicating that the carboxyl groups at the C-29 position and on the sugar moiety are both esterified. Further obtaining sugar part derivatization through acid hydrolysis, detecting D-glucuronic acid and D-galactopyranose through GC, and respectively identifying the D-glucuronic acid and the D-galactopyranose by combining chemical shift values and terminal hydrogen coupling constants as follows: beta-D-glucuronic acid and beta-D-galactopyranose.
NOESY spectrum of the compound, δ H 2.47 (H-18) and δ H 1.24(CH 3 -30) correlation, δ H 1.18(CH 3 -28) and δ H 3.27 (H-21. Beta.) related suggests that the methyl ester substitution is at C-29 and the D/E ring is cis-fused; delta H 3.41 (H-3) and δ H 0.81 (H-5) correlation,. Delta. H 4.31 (H-24 a) and δ H 0.74(CH 3 -25) correlation, suggesting that H-3 is in the alpha orientation, the hydroxymethyl substitution is at the C-24 position and in the beta orientation. The compound is a new unreported compound by a Sci-finder search.
TABLE 1 assignment of NMR data for Compound 2
Example 2
(1) Heating and reflux-extracting 500g of Caragana microphylla with 80% ethanol for 2 times (dosage: 8L), and recovering the 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 aqueous phase to organic phase of 1:1, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) Subjecting the n-butanol extract obtained in the step (2) to silica gel column chromatography, and sequentially eluting with a mixed solvent of petroleum ether and acetone 100, 8, 6;
(4) The petroleum ether obtained in the step (3): subjecting the acetone 100-4:1 fraction to ODS chromatography, and performing gradient elution with a mixed solvent of methanol-water 2;
(5) Separating and preparing the methanol-water (2:8-4:6) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 3mL/min, and the mobile phase is methanol: water =70, yielding compound 1 (t) R =30 min) (yield 0.0007 ‰);
(6) And (3) separating the methanol-water (6:4-8:2) fraction obtained in the step (4) by HPLC-RID chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water =75, yielding compound 2 (t) R =28 min) (yield 0.0002 ‰).
1-2 see example 1.
Example 3
(1) Reflux-extracting 1000g of Caragana microphylla with 70% ethanol under heating for 3 times (dosage: 15L), and recovering the 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 dichloromethane, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1:2, and respectively extracting for 4 times to obtain extracts of different polar parts;
(3) Separating the n-butanol extract obtained in the step (2) by silica gel column chromatography, and sequentially eluting with a chloroform and methanol mixed solvent 100, 1, 7;
(4) Chloroform obtained in the step (3): methanol 10 to 5:1 fractions are subjected to ODS chromatography, eluting with a mixed solvent gradient of methanol-water 2;
(5) And (3) separating the methanol-water (2:8-4:6) fraction obtained in the step (4) by HPLC-RID chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water =64, yielding compound 1 (t) R =18 min) (yield 0.0005 ‰).
(6) And (3) separating the methanol-water (5:5-8:2) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 3mL/min, and taking a 55 R =11 min) (yield 0.0003 ‰).
1-2 see example 1.
Example 4
(1) Heating and reflux-extracting 2000g of Caragana microphylla with 65% methanol for 4 times (dosage: 20L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1:2, and respectively extracting for 4 times to obtain extracts of different polar parts;
(3) Subjecting the n-butanol extract obtained in the step (2) to silica gel column chromatography, and sequentially eluting with a mixed solvent 100 of dichloromethane and acetone, wherein the ratio of the solvent;
(4) Dichloromethane obtained in the above step (3): acetone 100 to 1:1 fraction is subjected to ODS chromatography, eluting with a mixed solvent gradient of methanol-water 1;
(5) The methanol-water (1:9) obtained in the above step (4)4:6) fraction prepared by HPLC-UV chromatography with detection at 210nm, flow rate of 3mL/min, mobile phase of acetonitrile: water =42, yielding compound 1 (t) R =17 min) (yield 0.0006 ‰).
(6) The fraction of methanol-water (5:5 to 9:1) obtained in the step (4) was separated and prepared by HPLC-RID chromatography at a flow rate of 3mL/min, and a mixture solvent of 45 acetonitrile 55 and water was used as a mobile phase to obtain compound 2 (t R =25 min) (yield 0.0003 ‰).
1-2 see example 1.
Example 5
(1) Extracting Caragana microphylla 1500g with 80% methanol under reflux for 3 times (dosage: 20L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, chloroform and n-butanol according to the volume ratio of aqueous phase to organic phase of 1:1, and respectively extracting for 4 times to obtain extracts of different polar parts;
(3) The n-butanol extract obtained in the above step (2) is subjected to silica gel column chromatography, and is sequentially eluted with a mixed solvent of dichloromethane and methanol 100;
(4) Dichloromethane obtained in the above step (3): a methanol 100 to 8:1 stream is subjected to ODS chromatography with a mixed solvent gradient of acetonitrile-water 2;
(5) Separating and preparing the acetonitrile-water (2:8-5:5) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 3mL/min, and the mobile phase is methanol: water =70, yielding compound 1 (t) R =19 min) (yield 0.0007 ‰).
(6) And (3) separating the acetonitrile-water (7:3-8:2) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 3mL/min, and taking a 50 R =25 min) (yield 0.0004 ‰).
1-2 see example 1.
Example 6
(1) Heating and reflux-extracting 3000g of Caragana microphylla with 90% methanol for 2 times (dosage: 20L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol 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 1:2, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) Separating the n-butanol extract obtained in the step (2) by silica gel column chromatography, and sequentially eluting with a mixed solvent 100 of petroleum ether and ethyl acetate, i.e. a mixture of the following solvents, i.e. a ratio of 1, 100;
(4) The petroleum ether obtained in the step (3): an ethyl acetate 100-5:1 stream is subjected to ODS chromatography, eluting with a mixed solvent gradient of acetonitrile-water 1;
(5) Separating the acetonitrile-water (1:9-5:5) fraction obtained in the step (4) by HPLC-UV chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water =61, yielding compound 1 (t) R =27 min) (yield 0.0008 ‰).
(6) Separating the acetonitrile-water (7:3-9:1) fraction obtained in the step (4) by HPLC-RID chromatography at a flow rate of 3mL/min, and using a 56 acetonitrile-water mixture solvent as a mobile phase to obtain a compound 2 (t R ＝30min)。
1-2 see example 1.
Example 7 anti-neuritic Activity of Compounds 1-2 prepared in examples 1-6.
(1) The experimental principle is as follows: the neuroinflammation and the neurodegenerative diseases are inseparable, and microglia plays an important role in the generation and development of the neuroinflammation. As a cell with immune defense, microglia can release a large amount of inflammatory factors such as NO, TNF-alpha, IL-6, IL-1 beta and the like under the condition of over activation, so that tissue damage and apoptosis are caused, and a neurodegenerative process is further caused [2-4] This is also one of the mechanisms that cause neurodegenerative diseases. Therefore, research and development of new therapeutic drugs using microglia as a target point become a new hot spot.
In the experiment, the effect of the monomeric compound separated from the root of the caragana microphylla on inhibiting excessive activation of the microglia is preliminarily evaluated by establishing an in vitro LPS-induced abnormally activated BV-2 microglia screening model and taking NO release amount as an index.
(2) The experimental method comprises the following steps:
(1) culture of mouse microglial cell line BV-2
A cell culture solution containing 10% fetal bovine serum was prepared on the basis of DMEM/F-12 medium. The cells were cultured at a rate of about 2.5X 10 5 cells/mL at 5% CO 2 Culturing in an incubator at 37 ℃ until the third day, wherein the adherent cells account for about 80-90% of the bottom area of the culture bottle, digesting the adherent cells with pancreatin, and passaging to another culture bottle.
(2) Method for preparing medicine
The monomeric compound of caragana microphylla root was dissolved in DMSO to prepare a stock solution with a concentration of 100mM, 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 and 1. Mu.M in this order. The final concentration of DMSO is less than 1 ‰.
(3) Griess method for detecting inhibition effect of compound on LPS (LPS) activated microglia
Taking BV-2 microglia in logarithmic growth phase, and adjusting the cell density to 2.0 x 10 by using fresh DMEM medium containing 10% fetal calf serum 5 cells/mL, seeded in 96-well plates at 100. Mu.L/well at 37 ℃ in 5% CO 2 Culturing in the incubator. And (3) replacing the cells with fresh serum-free culture solution after 24 hours of adherent culture, and simultaneously adding medicine. The 37 monomeric compounds were co-administered with LPS at 100. Mu.M, 30. Mu.M, 10. Mu.M, 1. Mu.M doses. Blank control was also set. The final concentration of LPS in each administration group was 100ng/mL. Continuously culturing for 24h after adding medicine into cells, collecting supernatant, and detecting NO in the supernatant by Griess colorimetric method 2- And (4) content.
(4) MTT method for detecting influence of compound on survival rate of microglia cells
Taking BV-2 microglia cultured in logarithmic growth phase, adjusting cell density to 2.0 × 10 by using fresh DMDM culture medium containing 10% fetal calf serum 5 cells/mL, seeded in 96-well plates at 100. Mu.L/well at 37 ℃ in 5% CO 2 Cultured in an 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. The 37 monomeric compounds were co-administered with LPS at 100. Mu.M, 30. Mu.M, 10. Mu.M, 1. Mu.M doses. Blank control was also set. After the cells with the final concentration of LPS of 100ng/mL in each administration group were added with the drug and cultured for 24 hours, MTT solution, 10. Mu.L/well, was added to the cell fluid, the cells were incubated with 0.25mg/mL MTT at 37 ℃ for 3 hours, the culture fluid was aspirated, and then 150. Mu.L DMSO solution was added to determine the optical density OD value. And (3) processing the data, performing data processing by using corresponding software of a microplate reader, calculating an average value of OD values of 3 holes of each sample, and calculating the cell survival (CV%) by using the average value according to the following formula.
Cell survival% = [ mean value of OD value of sample group/mean value of OD value of blank control group ] × 100%
(5) Statistical method
All data were examined using the SPSS (27.0) 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 uses a Leven test, when p is greater than 0.05, the variances are uniform, dunnett's double-sided T test is used for testing the difference of the mean values among the groups, when p is less than 0.05, the variances are not uniform, dunnett T3 test is used for testing the difference of the mean values among the groups. 6
⑥IC 50 Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate 50 。
(3) The experimental results are as follows:
the results are shown in tables 3 and 4.
TABLE 3 Effect of 1-2 on LPS-activated BV-2 microglia NO release (%) (Mean. + -. SEM)
TABLE 4-2 Effect on LPS activated BV-2 microglia survival (%) (Mean. + -. SEM)
Note: * P<0.05,**P<0.01,***P<0.001 compared to LPS-induced group; ### P<0.001 compared to the control group.
As a result, it was found that the novel compounds 1 and 2 prepared in examples 1 to 6 were able to inhibit LPS-induced NO release from BV-2 microglia, and that the compound 2 was able to significantly inhibit LPS-induced NO release from BV-2 microglia in a dose-dependent manner.
Claims (9)
1. Pentacyclic triterpenoid compounds with the following chemical structural formula (I) or (II) and pharmaceutically acceptable salts thereof,
2. a method for preparing pentacyclic triterpenoid of claim 1, which is characterized in that: the method comprises the following steps:
(1) Heating and extracting caragana microphylla root, and recovering an extracting solution to obtain a crude extract;
(2) Dissolving the crude extract obtained in the step (1) in water, and extracting with an organic solvent to obtain extracts with different polarities;
(3) Separating the 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, or a mixed solvent of petroleum ether and acetone, or a mixed solvent of chloroform and acetone, or a mixed solvent of dichloromethane and acetone, or a mixed solvent of chloroform and methanol, or a mixed solvent of dichloromethane and methanol;
(4) Separating the flow obtained in the step (3) by ODS column chromatography, and performing gradient elution by using a methanol-water or acetonitrile-water mixed solvent as a mobile phase;
(5) And (4) further separating the methanol-water or acetonitrile-water eluate obtained in the step (4) by HPLC, and performing 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 compound 1 and a compound 2.
3. The method of claim 2, wherein: the extraction method in the step (1) comprises heating reflux ethanol or methanol extraction and heating ultrasonic extraction for 2-5 times, wherein the material-liquid ratio is 1:8-1.
4. The method of claim 2, wherein: in the organic solvent extraction in the step (2), the volume ratio of the water phase to the organic phase is 1:1-1:5, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butyl alcohol are respectively used for sequentially extracting for 3-5 times, and the organic solvent is recovered under reduced pressure.
5. The method of claim 2, wherein: in the mixed solvent in the step (3), the volume ratio of petroleum ether to ethyl acetate, petroleum ether to acetone is 100-1:1; dichloromethane and acetone, the volume ratio of chloroform to acetone, dichloromethane to methanol, chloroform to methanol is from 1 to 1:1.
6. The method of claim 2, wherein: in the mixed solvent in the step (4), the volume ratio of methanol to water is 2:8-8:2, and the volume ratio of acetonitrile to water is 1:9-9:1.
7. The method of claim 2, wherein: in the mixed solvent in the step (5), the volume ratio of methanol to water is 2:8-7:3, and the volume ratio of acetonitrile to water is 1:9-6:4.
8. A pharmaceutical composition comprising a compound of claim 1 and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.
9. Use of a compound of claim 1 and pharmaceutically acceptable salts thereof or a pharmaceutical composition of claim 8 in the manufacture of an anti-neuritis agent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113072608A (en) * | 2020-01-03 | 2021-07-06 | 沈阳药科大学 | Triterpenoid saponin compound and application thereof |
| CN113072609A (en) * | 2020-01-03 | 2021-07-06 | 沈阳药科大学 | Triterpenoid compound and preparation method and application thereof |
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| CN113072608A (en) * | 2020-01-03 | 2021-07-06 | 沈阳药科大学 | Triterpenoid saponin compound and application thereof |
| CN113072609A (en) * | 2020-01-03 | 2021-07-06 | 沈阳药科大学 | Triterpenoid compound and preparation method and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| GUI-LIN JIN等: "Triterpenoid Saponins from the Seeds of Caragana microphylla", ARCH PHARM RES, vol. 34, no. 6, pages 869 - 873 * |
| SHUFANG WANG等: "Rapid discovery and identification of anti-inflammatory constituents from traditional Chinese medicine formula by activity index, LC-MS, and NMR", SCIENTIFIC REPORTS, vol. 6, pages 31000 * |
| 张燕;石妍;: "蒙药小叶锦鸡儿的化学成分、药理作用研究进展", 包头医学院学报, no. 08 * |
| 李宜玲;朴惠顺;金光洙;金莉莉;: "小叶锦鸡儿的HPLC指纹图谱", 延边大学医学学报, no. 04 * |
| 杜庆波;蔡红;: "锦鸡儿属植物化学成分及药理活性研究新进展", 宿州学院学报, no. 11 * |
| 贺程;朴惠顺;: "小叶锦鸡儿的化学成分研究(Ⅱ)", 延边大学医学学报, no. 04 * |
| 陈龙;张一冰;王佳佳;许启泰;康文艺;: "锦鸡儿属植物化学成分及药理作用研究进展", 中国医药科学, no. 18 * |
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