CN117143100B - Analgesic compound with Nav1.2 inhibition effect in radix Arnafae, and preparation and application thereof - Google Patents
Analgesic compound with Nav1.2 inhibition effect in radix Arnafae, and preparation and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention relates to a preparation method of a tetramino 6/6/6/5/7/5 octacycloalkaloid compound with a unique 8,14,18,24-tetraazaoctacyclo [21.2.2 1,4.1.02,21.03,18.05,17.09, 16.011,15 ] icosahederane ring system and an anti-inflammatory and analgesic application thereof, wherein the tetramino 6/6/6/5/7/5 octacycloalkaloid compound is extracted and extracted from roots (ananape roots) of Roman pyrethrum (Anacyclus pyrethrum (L.) DC.) by adopting an organic solvent, and then separated by two to three methods of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography to obtain a tetramino 6/6/6/5/7/5 octacycloalkaloid monomer compound with 4 new frameworks, and the structural identification is carried out by methods such as high resolution mass spectrum, nuclear magnetic resonance spectrum and the like. The invention also provides Nav1.2 inhibition and NO inhibition of these compounds.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to analgesic/anti-inflammatory compounds (a compound of formula 1 and a compound of formula 2) with Nav1.2 inhibition and NO inhibition in the root of Arna, and preparation and application thereof.
Background
Pain is a body's defense against disease and is the fifth largest vital sign. However, persistent and severe pain can have an impact on the mental health of the patient, causing anxiety, sadness, and reduced immunity, leading to a range of consequences, most severe leading to death or disability. However, traditional analgesics such as opioids and nonsteroidal drugs have side effects of drug resistance, addiction and gastrointestinal bleeding.
Ion channels can regulate the passage of intracellular and extracellular ions to regulate the voltage potential across the membrane. The ion current generates an electrical pulse which in turn causes adjacent voltage sensitive channels to open continuously, resulting in a spontaneous electrical signal. By blocking ion channels, inhibiting sustained rhythmic potentials, signal transduction can be prevented, thereby reducing pain response. Currently, various types of ion channel inhibitors have been reported, including VGSCs, VGCCs, VGPCs and TRPs. Sodium channels are responsible for the generation and propagation of action potentials and are the fundamental elements of all excitable cells (including nerve and muscle cells, etc.) that produce electrical signals. In humans, there are nine subtypes of sodium channels, designated Nav1.1-Nav1.9. For example, lidocaine reduces the response to pain by inhibiting nav1.7 and nav1.9. Therefore, it is very important to find new ion channel inhibitors with analgesic potential.
The search for lead compounds with significant pharmacodynamic activity from traditional medicinal plants is a hotspot in drug research, and therefore, the discovery of novel ion channel inhibitors and compounds with anti-inflammatory effects from medicinal plants is an effective way to develop novel analgesic/anti-inflammatory drugs.
The Arna root is dried root of Roman pyrethrum (Anacyclus pyrethrum (L.) DC) belonging to Compositae, and has effects in refreshing brain, inducing resuscitation, treating hemiplegia, vitiligo, cornu Naemorhedi, headache, relieving cough, eliminating phlegm, and exhausting air.
Disclosure of Invention
The inventor has conducted intensive studies to first separate analgesic/anti-inflammatory compounds (a compound of formula 1 and a compound of formula 2) having Nav1.2 inhibitory action and NO inhibitory action from the root of Arna, and these compounds are brand-new skeleton compounds, are highly conjugated tetra-amino 6/6/6/5/7/5 octa-ring alkaloids, and have been identified in their structures, and through cell experiments, they have been verified to have Nav1.2 inhibitory action and NO inhibitory action, and can be used for preparing analgesic and/or anti-inflammatory drugs.
Accordingly, the present invention provides the following:
1. a compound selected from the group consisting of:
7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-bridged aza-o [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (compound of formula 1); and
7 A-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (a compound of formula 2).
2. The compound according to 1 above or an isomer thereof, wherein the isomer is an enantiomer.
3. A compound according to 1 or 2 above, or an isomer thereof, selected from the group consisting of:
(4 aR,7aR,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methano-aza [4,5,6-de ] pyrrolo [ 3', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aS,7aS,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aR,7aR,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [ 3', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione; and
(4 AS,7aS,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [ 3', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinolin-1,8,11 (9H) -trione.
4. A compound according to any one of claims 1 to 3 above, or an isomer thereof, having a structural formula selected from the group consisting of:
5. A method of extracting a compound according to any one of 1 to 4 above or an isomer thereof from the root of ananape, comprising the steps of:
a. Drying and pulverizing radix Arnebiae, taking 50-95% (v/v) ethanol water solution, methanol or chloroform as solvent (weight ratio of medicinal material (Kg) to solvent (L) 1:1.5-1:4), extracting by cold soaking, percolating, heating and refluxing or ultrasonic extracting, concentrating under reduced pressure to recover solvent to obtain extract;
b. C, suspending the total extract in the step a by water, dispersing the total extract in an acid water layer obtained by using acid such as hydrochloric acid with the concentration of 1-5% or sulfuric acid with the concentration of 1-5%, extracting the acid water layer by using dichloromethane to remove non-alkaloid, adjusting the pH value to 10-12 by using alkali such as NaHCO 3、Na2CO3, ammonia water or NaOH, extracting the acid water layer by using an organic solvent such as dichloromethane, ethyl acetate or n-butanol, concentrating the acid water layer under reduced pressure, and recovering the organic solvent to obtain the total alkaloid;
c. separating the total alkaloids from step b by silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or isomer thereof.
6. The method according to above 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography and reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, a compound of formula 1 or formula 2 is obtained by reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:0 to 3:1, petroleum ether and acetone in a volume ratio of 500:1 to 3:1, methanol and acetone in a volume ratio of 50:1 to 0:1, or methanol and water in a volume ratio of 1:9 to 1:0, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 20 to 100% (v/v) or an aqueous acetonitrile solution in a volume ratio of 20 to 100% (v/v), and the isocratic eluent used in the semi-preparative high performance liquid chromatography is gradient of normal phase of 99 to 99 v/v.
7. The method according to above 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed with reverse phase silica gel or reverse phase MCI column chromatography, and further, semi-preparative high performance liquid chromatography is performed to obtain the compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution of 20 to 100% (v/v) or an aqueous acetonitrile solution of 20 to 100% (v/v), and the isocratic or gradient elution eluent used in the semi-preparative high performance liquid chromatography is n-hexane/EtOH in a volume ratio of 99 to 50% (v/v).
8. The method according to above 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, sephadex LH-20 column chromatography, gradient elution with reverse phase silica gel or reverse phase MCI column chromatography, and further semi-preparative high performance liquid chromatography is performed to obtain a compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:1 to 0:1, dichloromethane and methanol or chloroform and methanol, the eluent used in the reverse phase silica gel LH-20 column chromatography is an aqueous methanol solution of 20 to 100% (v/v) or an aqueous acetonitrile solution of 20 to 100% (v/v), and the eluent used in the semi-preparative high performance liquid chromatography is a gradient of 99 to 99% (v/v) hexane.
9. The method according to any one of the preceding claims 5 to 8, wherein in step c the silica gel column chromatography is normal pressure or pressure column chromatography and/or the packing used is forward silica gel or reverse phase silica gel.
10. The use of a compound according to any one of 1 to 4 above or an isomer thereof for the preparation of an analgesic or anti-inflammatory drug, preferably, the compound or isomer thereof exerts an analgesic effect by inhibiting nav1.2 and an anti-inflammatory effect by inhibiting NO.
Detailed Description
The invention aims to provide a compound with Nav1.2 inhibition and/or anti-inflammatory effect, a separation preparation method thereof and application value in preparing analgesic drugs.
According to a first aspect of the present invention, there is provided a compound having nav1.2 inhibitory effect and/or having anti-inflammatory effect, the compound having the structural formula shown in the following figure;
Wherein:
compound (+) -1 is: (4 aR,7aR,11aS,14 aR) -7 a-acetyl
-2, 4A,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
Compound (-) -1 is: (4 aS,7aS,11aR,14 aS) -7 a-acetyl
-2, 4A,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
The compound (+) -2 is: (4 aR,7aR,11aR,14 aS) -7 a-acetyl
-2, 4A,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
compound (-) -2 is: (4 aS,7aS,11aS,14 aR) -7 a-acetyl
-2, 4A,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinolin-1,8,11 (9H) -trione.
The extraction and separation method of the alkaloid compounds comprises the following steps:
a. Drying and pulverizing radix Arnebiae, taking 50-95% (v/v) ethanol water solution, methanol or chloroform as solvent (weight ratio of medicinal material (Kg) to solvent (L) 1:1.5-1:4), extracting by cold soaking, percolating, heating and refluxing or ultrasonic extracting, concentrating under reduced pressure to recover solvent to obtain extract;
b. C, suspending the total extract in the step a by water, dispersing the total extract in an acid water layer obtained by using acid such as hydrochloric acid with the concentration of 1-5% or sulfuric acid with the concentration of 1-5%, extracting the acid water layer by using dichloromethane to remove non-alkaloid, adjusting the pH value to 10-12 by using alkali such as NaHCO 3、Na2CO3, ammonia water or NaOH, extracting the acid water layer by using an organic solvent such as dichloromethane, ethyl acetate or n-butanol, concentrating the acid water layer under reduced pressure, and recovering the organic solvent to obtain the total alkaloid;
c. separating the total alkaloids from step b by silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or isomer thereof.
Two of the separation modes are as follows:
The volume ratio of the normal phase silica gel column chromatography eluent is 100:0-3:1 or 500:1-3:1 dichloromethane-methanol, and performing reversed phase silica gel or MCI column chromatography or semi-preparative high performance liquid chromatography to obtain compound of formula 1 or 2;
three separation modes:
the normal phase silica gel column chromatography eluent is petroleum ether-ethyl acetate, methylene dichloride-methanol or chloroform-methanol with the volume ratio of 100:1-0:1, reverse phase silica gel or MCI column chromatography is carried out, methanol aqueous solution with the volume ratio of 20-100% (v/v) or acetonitrile aqueous solution with the volume ratio of 20-100% (v/v) is adopted for gradient elution, semi-preparation high performance liquid chromatography is adopted, and normal hexane/EtOH with the volume ratio of 99-50% (v/v) is adopted as the eluent, so that the compound of the formula 1 or 2 is obtained.
Four separation modes:
The normal phase silica gel column chromatography eluent is prepared by gradient eluting petroleum ether-ethyl acetate, methylene dichloride-methanol or chloroform-methanol with the volume ratio of 100:1-0:1, subjecting to Sephadex LH-20 column chromatography, subjecting to isocratic eluting with methanol, subjecting to reverse phase silica gel or MCI column chromatography, subjecting to gradient eluting with methanol aqueous solution with the volume ratio of 20-100% (v/v) or acetonitrile aqueous solution with the volume ratio of 20-100% (v/v), subjecting to semi-preparative high performance liquid chromatography, and subjecting to normal hexane/EtOH with the volume ratio of 99-50% (v/v) as eluent to obtain the compound of formula 1 or 2.
The preparation method of the alkaloid compound in the root of the Arna is characterized in that the silica gel column chromatography is normal pressure or pressurized column chromatography, the filler is forward silica gel or reverse phase silica gel, and the volume ratio of dichloromethane and methanol is 500:1-3:1; the volume ratio is 100:0-3:1 petroleum ether/ethyl acetate; the volume ratio is 5:2 petroleum ether/acetone elution; or methanol water with the volume ratio of 1:9-1:0 is used as an eluent, and isocratic or gradient elution is adopted.
The preparation method of the alkaloid compound in the root of the argan in the step c is characterized in that the eluent of the sephadex LH-20 column chromatography is methanol and isocratic elution is adopted.
The preparation method of the alkaloid compound in the root of the Arna in the step c is characterized in that the preparation high performance liquid chromatography adopts normal hexane/EtOH with the volume ratio of 99-50% (v/v) as an eluent, and adopts isocratic or gradient elution.
According to another aspect of the present invention, there is provided the use of the compound having nav1.2 inhibiting effect for the preparation of an analgesic drug.
The structures of compound 1 and compound 2 prepared in the examples were determined by combining various spectroscopic analysis methods (high resolution mass spectrum, ultraviolet spectrum, infrared spectrum, and nuclear magnetic resonance spectrum), quantum chemical calculation methods (13 C-NMR dp4+ probability analysis and ECD), and the like for comprehensive analysis. Wherein the relative configuration of compound 1 and compound 2 is determined by X-ray single crystal diffraction, as shown in fig. 1 and 4.
Compound 1 (ANACYPHRETHINE A): yellow needle-like crystals; optical rotation value [ alpha ] 25D 1584 (c 0.08, methanol, (+) -1); [ alpha ] 25D-1584 (c 0.1, methanol, (-) -1); ultraviolet (methanol) lambda max (log epsilon) 427 (4.15) nm,267 (3.96) nm; infrared (KBr) max 3297,2961,2924,1704,1623,and 1448cm -1;ECD(c 3.12×10-4 M, methanol) λ max (Δε216 (-11.39), 253 (-2.36), 289 (-13.16), 417 (17.07) nm, enantiomer compound (+) -1; ECD (c 3.12×10 -4 M, methanol) λ max (Δε) 217 (9.25), 253 (0.05), 290 (13.67), 416 (-21.25) nm, enantiomer compound (-) -1; high resolution mass spectra m/z 641.4051[ M+H ] + (calculated C 39H53O4N4 +, 641.4066), 1 H and 13 C NMR spectra data are shown in tables 1 and 2.
Compound 2 (ANACYPHRETHINE A): yellow needle-like crystals; optical rotation value [ alpha ] 25D-530 (c 0.03, methanol, (-) -2); [ alpha ] 25D 530 (c 0.02, methanol, (+) -2); ultraviolet (methanol) lambda max (log epsilon) 475 (3.90) nm,281 (3.74) nm; infrared (KBr) max 3316,2963,2926,2857,1699,1615,1445,and 1194cm -1;ECD(c 3.12×10-4 M, methanol) λ max (Δε) 219 (-23.50), 265 (17.79), 300 (-35.20), 381 (25.45), 481 (-9.33), enantiomer compound (-) -2; ECD (c 3.12×10 -4 M, methanol) λ max (Δε) 220 (30.48), 264 (-25.61), 301 (46.37), 380 (-34.83), 480 (10.83), enantiomer compound (+) -2; high resolution mass spectra m/z 641.4049[ m+h ] + (calculated for C 39H53O4N4 +, 641.4066), 1 H and 13 CNMR spectra data are shown in tables 1 and 2.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) The compounds 1-2 provided by the invention are brand new framework compounds. Compounds 1 and 2 are two unprecedented pairs of novel skeletal enantiomers of highly conjugated tetra-amino 6/6/6/5/7/5 octacyclic alkaloids, with a unique skeletal structure of the 8,14,18,24-tetraazaoctacyclo [21.2.2 1,4.1.02,21.03,18.05,17.09,16.011,15 ] twenty-nine alkane ring system, with 4 discrete chiral stereocenters.
(2) The compound 1-2 provided by the invention has Nav1.2 inhibitory activity and NO inhibitory activity, and the compound 2 has micromole-level Nav1.2 inhibitory activity and potential Nav1.2 inhibitory activity.
(3) According to the invention, through computer molecule docking, the active results of a compound and Nav1.2 are combined, residues of Gln332, phe38, asn361, asp334, tyr362, asp949, trp948, pro921, trp923, tyr1429 and Met1425 in the Nav1.2 polypeptide chain are summarized as active binding sites of the Nav1.2 polypeptide chain, and a theoretical basis is provided for subsequent development of efficient Nav1.2 inhibitors.
The invention will now be described in further detail with reference to the drawings and examples, which are not intended to limit the scope of the invention. Modifications and substitutions of the method, steps, conditions, etc. of the present invention without departing from the spirit and nature of the present invention are intended to be within the scope of the present invention.
Drawings
FIG. 1 is an X-ray single crystal diffraction pattern of Compound 1;
FIG. 2 is an X-ray single crystal diffraction pattern of compound (+) -1;
FIG. 3 is an X-ray single crystal diffraction pattern of compound (-) -1;
FIG. 4 is an X-ray single crystal diffraction pattern of compound 2;
FIG. 5 is a graph of molecular docking and molecular dynamics modeling results for compound (+) -2;
FIG. 6 is 1 H NMR of compound 1;
FIG. 7 is a 13 C NMR chart of compound 1;
FIG. 8 is 1 H NMR of compound 2;
FIG. 9 is a 13 C NMR chart of compound 2.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The following are specific examples:
example 1
Crushing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC, 15.0 kg), ultrasonically extracting with chloroform (30L), concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 5% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH to 10 with saturated aqueous NaHCO 3 solution under stirring in ice-water bath to obtain alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 2
Crushing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC, 15.0 kg), extracting with 95% ethanol (40L) by percolation, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 5% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and regulating pH to 10 with aqueous ammonia solution under stirring to obtain alkalinized solution. The alkalization liquid is fully extracted by ethyl acetate, and the ethyl acetate extracts are combined and dried to obtain the total alkaloid.
Example 3
Crushing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC, 15.0 kg), extracting with 50% ethanol (40L) by percolation, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 2% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH to 10 with Na 2CO3 aqueous solution under stirring to obtain alkalized solution. And fully extracting the alkalization liquid with n-butanol, combining n-butanol extract and drying to obtain total alkaloids.
Example 4
Crushing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC, 15.0 kg), reflux extracting with 75% ethanol (45L), concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 1% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH with aqueous NaOH solution to 12 under stirring to obtain alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 5
Crushing dried radix Arnafae (Anacyclus pyrethrum (L.) DC, 15.0 kg), cold leaching with 22.5L methanol at normal temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 5% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH to 10 with saturated NaHCO 3 aqueous solution under stirring to obtain alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 6
Crushing dried radix Arnafae (Anacyclus pyrethrum (L.) DC, 15.0 kg), cold leaching with 22.5L methanol at normal temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 1% sulfuric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH to 10 with saturated NaHCO 3 aqueous solution under stirring to obtain alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 7
Crushing dried radix Arnafae (Anacyclus pyrethrum (L.) DC, 15.0 kg), cold leaching with 22.5L methanol at normal temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 2% sulfuric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH to 10 with saturated NaHCO 3 aqueous solution under stirring to obtain alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 8
Crushing dried radix Arnafae (Anacyclus pyrethrum (L.) DC, 15.0 kg), cold leaching with 22.5L methanol at normal temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract with water, acidifying with 5% sulfuric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adjusting pH to 10 with saturated NaHCO 3 aqueous solution under stirring to obtain alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 9
Mixing the total alkaloids with 100-200 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with dichloromethane/methanol (100:0-3:1, V/V), and combining the same components to obtain 6 components Fr.A-Fr.F with polarity from small to large; wherein the 1 st component Fr.A (159.0 g) is mixed with 100-200 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (100:0-3:1, V/V) is carried out, the same components are combined, and 6 components Fr.A1-Fr.A6 with the polarity from small to large are obtained; wherein the 2 nd component Fr.A2 (76.1 g) is subjected to reversed-phase MCI column chromatography, methanol/water gradient elution (20:80-100:0, V/V) is adopted, and the same components are combined to obtain 7 subfractions Fr.A21-Fr.A27 with the polarity from large to small; wherein the component Fr.A25 (10.0 g) is mixed with 200-300 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (10:1-3:1, V/V) is carried out, the same components are combined, and 3 components Fr.A251-Fr.A253 with the polarity from small to large are obtained; mixing the components Fr.A253 (9.0 g) with 200-300 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with petroleum ether/ethyl acetate (10:1-3:1, V/V), and combining the same components to obtain 6 components Fr.A2531-Fr.A2533 with the polarity from small to large; Wherein the component Fr.A2533 (7.8 g) is subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain 3 subfractions Fr.A25331-Fr.A25333 with molecular weights from large to small; subjecting the component Fr.A25333 (6.1 g) to reverse phase C18 silica gel column chromatography, eluting with methanol/water gradient (20:80-100:0, V/V), and mixing the same components to obtain 6 subfractions Fr.A253331-Fr.A253336 with polarity from large to small; the fraction Fr.A253336 (231.2 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 5 subfractions Fr.A2533361-Fr.A2533365 with polarity from small to large; Component fr.a2533363 (71.1 mg) was recrystallized from methanol to give compound 1 (i.e. compound of formula 1) (ANACYPHRETHINES A,30.3mg, 0.000202%); the racemate of compound 1 was subjected to chiral resolution by means of a chiral chromatography column (DAICEL CORPORATION CHIRALPAK ID μm 10×250mm; solvent: n-Hexane/etoh=90:10; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give compound (+) -1 (14.0 mg, t R =12.3 min) and compound (-) -1 (15.4 mg, t R =18.0 min). the fraction Fr.A253335 (214.3 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 6 subfractions Fr.A2533351-Fr.A2533356 with polarity from small to large; component fr.a2533353 (61.3 mg) was subjected to normal phase silica gel column chromatography eluting with petroleum ether/acetone (5:2, V/V) to give compound of formula 2 (i.e. compound of formula 2) (ANACYPHRETHINES B,20.1mg, 0.000134%); the racemate of compound 2 was subjected to chiral chromatography (DAICEL CORPORATION CHIRALPAK ID μm 10×250mm; Solvent: n-Hexane/etoh=60:40; column temperature: 25 ℃; flow rate: 3ml/min; detection wavelength: chiral resolution at 360nm gave compound (+) -2 (7.5 mg, t R =17.0 min) and compound (-) -2 (7.3 mg, t R =12.4 min).
Example 10
Mixing the total alkaloids with 100-200 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with dichloromethane/methanol (100:0-3:1, V/V), and combining the same components to obtain 6 components Fr.A-Fr.F with polarity from small to large; wherein the 1 st component Fr.A (159.0 g) is mixed with 100-200 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (100:0-3:1, V/V) is carried out, the same components are combined, and 6 components Fr.A1-Fr.A6 with the polarity from small to large are obtained; wherein the 2 nd component Fr.A2 (76.1 g) is subjected to reversed-phase MCI column chromatography, methanol/water gradient elution (20:80-100:0, V/V) is adopted, and the same components are combined to obtain 7 subfractions Fr.A21-Fr.A27 with the polarity from large to small; wherein the component Fr.A25 (10.0 g) is mixed with 200-300 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (10:1-3:1, V/V) is carried out, the same components are combined, and 3 components Fr.A251-Fr.A253 with the polarity from small to large are obtained; mixing the components Fr.A253 (9.0 g) with 200-300 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with petroleum ether/ethyl acetate (10:1-3:1, V/V), and combining the same components to obtain 6 components Fr.A2531-Fr.A2533 with the polarity from small to large; wherein the component Fr.A2533 (7.8 g) is subjected to MCI column chromatography and eluted with 20-100% acetonitrile water to obtain 3 subfractions Fr.A25331-Fr.A25333 with molecular weights from large to small; subjecting the component Fr.A25333 (6.1 g) to reverse phase C18 silica gel column chromatography, eluting with methanol/water gradient (20:80-100:0, V/V), and mixing the same components to obtain 6 subfractions Fr.A253331-Fr.A253336 with polarity from large to small; the fraction Fr.A253336 (231.2 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 5 subfractions Fr.A2533361-Fr.A2533365 with polarity from small to large; Component fr.a2533363 (71.1 mg) was recrystallized from methanol to give compound 1 (i.e. compound of formula 1) (ANACYPHRETHINES A,30.3mg, 0.000202%); the racemate of compound 1 was subjected to chiral resolution by means of a chiral chromatography column (DAICEL CORPORATION CHIRALPAK ID μm 10×250mm; solvent: n-Hexane/etoh=90:10; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give compound (+) -1 (14.0 mg, t R =12.3 min) and compound (-) -1 (15.4 mg, t R =18.0 min). the fraction Fr.A253335 (214.3 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 6 subfractions Fr.A2533351-Fr.A2533356 with polarity from small to large; component fr.a2533353 (61.3 mg) was subjected to normal phase silica gel column chromatography eluting with petroleum ether/acetone (5:2, V/V) to give compound of formula 2 (i.e. compound of formula 2) (ANACYPHRETHINES B,20.1mg, 0.000134%); the racemate of compound 2 was subjected to chiral chromatography (DAICEL CORPORATION CHIRALPAK ID μm 10×250mm; Solvent: n-Hexane/etoh=60:40; column temperature: 25 ℃; flow rate: 3ml/min; detection wavelength: chiral resolution at 360nm gave compound (+) -2 (7.5 mg, t R =17.0 min) and compound (-) -2 (7.3 mg, t R =12.4 min).
Example 11
Mixing the total alkaloids with 100-200 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with dichloromethane/methanol (100:0-3:1, V/V), and combining the same components to obtain 6 components Fr.A-Fr.F with polarity from small to large; wherein component 1, fr.a. (159.0 g), was subjected to repeated silica gel column chromatography using a volume ratio of 100:0-3:1, petroleum ether-ethyl acetate, 500:1-3:1 methylene chloride-methanol or petroleum ether/acetone with a volume ratio of 50:1-0:1 as eluent to obtain a compound 1 (i.e. a compound of formula 1) (ANACYPHRETHINES A,30.3mg, 0.000202%) and a compound 2 (i.e. a compound of formula 2) (ANACYPHRETHINES B,20.1mg, 0.000134%); the racemate of compound 1 was subjected to chiral resolution by means of a chiral chromatography column (DAICEL CORPORATION CHIRALPAK ID μm 10X 250mm; solvent: n-Hexane/EtOH=99:1-50-50; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give compound (+) -1 (14.0 mg, t R =12.3 min) and compound (-) -1 (15.4 mg, t R =18.0 min). The racemate of compound 2 was subjected to chiral resolution by a chiral chromatography column (DAICEL CORPORATION CHIRALPAK ID μm 10X 250mm; solvent: n-Hexane/EtOH=99:1-50-50; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give compound (+) -2 (7.5 mg, t R =17.0 min) and compound (-) -2 (7.3 mg, t R =12.4 min).
Example 12
The Nav1.2 inhibitory activity of compound 1 and compound 2 was evaluated by patch clamp electrophysiology experiments. Experiments were performed using HEK293T cells (ATCC cell bank) at 37 ℃ in a 5% co 2 incubator; the culture medium contains 90% DMEM+10% Fetal Bovine Serum (FBS), and after digestion with 0.25% pancreatin when the cell density reaches 80-90%, subculture or hole plating is performed. After 24h plating, the cells were transfected with Lipo2000 transfection kit (Thermo Fisher, shanghai, china) at a ratio of pcDNA3.1-SCN2A (NaV1.2 GenBank accession No. NM-001040142) plasmid (Huada Gene, beijing, china) to pcDNA3.1-EGFP plasmid (Huada Gene, beijing, china) of 9:1 (4000 ng total), and electrophysiological experiments were performed 18h or more after transfection.
The current clamp recording experiments used an Axon patch 700B patch clamp amplifier (Axon Instruments, molecular Devices, usa), digital to analog converter Digidata 1440A (Axon Instruments, molecular Devices, usa), signal acquisition using pClamp 10.0 software (Molecular Devices, usa), filtering at 2kHz, sampling frequency at 10kHz. The patch clamp electrode is prepared by drawing a horizontal electrode drawing instrument P-97 (Sutter Instrument, U.S.) through a multi-step procedure, and the resistance of the filled electrode internal liquid is measured to be 3-5MΩ. The recording process is carried out at room temperature (23-25 ℃). The perfusion system is self-made at the speed of about 2mL/min; the drug delivery system was BPS-8 (ALA SCIENTIFIC Instruments, USA). All electrophysiological data were processed using a Clampfit 10.4 (Molecular Device, usa) and then analyzed using GRAPHPAD PRISM (GraphPad Software, usa). The initial concentration of the monomer compound was 40. Mu.M, and the inhibition ratio was shown in Table 3.
TABLE 3 Nav1.2 inhibitory Activity of Compounds 1-2
[a] Positive drug: TTX (tetrodotoxin, jiangsu Kangte bioengineering limited) [b] each experiment was repeated 3 times.
Conclusion: the compound (+) -2 has remarkable inhibition effect on Nav1.2, wherein the compound (+) -2 has the inhibition activity of Nav1.2 with a micromole level, and the IC 50 value is 23.94+/-2.70 mu M.
Example 13
NO inhibitory activity of the compounds of formula 1 (compound 1) and 2 (compound 2):
1. Cell culture:
BV2 cells (purchased from north-nano biology BeNa Culture Collection, BNCC) were cultured in duchenne's medium (dulbecco's modified eagle medium, DMEM) high sugar medium (purchased from Hyclone, usa) containing 10% Fetal Bovine Serum (FBS) (purchased from Giboco, usa), 1% penicillin and streptomycin, in an incubator at 37 ℃ and 5% co 2;
2. test of the effect of the compounds of the invention on cell viability:
Dissolving the compound with dimethyl sulfoxide (DMSO), taking BV2 cells in a logarithmic growth phase and in a good growth state, inoculating the BV2 cells into a 96-well plate at 5X 10 3 cells/well, adding the compound of the formula I with different concentrations of 12.5, 25, 50 and 100 mu M into an experimental group, adding the dimethyl sulfoxide (DMSO) into a control group, culturing for 24 hours, adding 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt (CCK-8 reagent) into each well, measuring the absorbance at 450nm by using an enzyme-labeled instrument, and calculating the cell survival rate, wherein the experimental results are shown in Table 4;
TABLE 4 cell viability of Compounds 1-2
3. Nitric Oxide (NO) content determination:
The intracellular NO release of BV2 was tested by the Griess method (Arias-NEGRETE ET al., analytical biochemistry328.1 (2004): 14-21.) and after incubation for 2h with samples of different concentrations 25, 50 and 100. Mu.M, 1. Mu.g/mL Lipopolysaccharide (LPS, sigma, L4391) was added for a total incubation of 22h, and after the incubation was completed, the cell supernatant was collected and the nitric oxide content in the cell supernatant was determined by the Griess method; before measurement, GRIESS REAGENT I and II (Nitric Oxide Assay kit, beyotine, S0021M) are taken out, the temperature is restored to the room temperature, a complete culture medium is used for diluting a standard substance (1-100 mu M), the concentration of the standard substance can be 0,1,2,5, 10, 20, 40, 60 and 100 mu M, 50 mu L/hole is adopted, the standard substance and a collected culture solution supernatant are added into a 96-well plate, 50 mu L of GRIESS REAGENT I and 50 mu LGRIESS REAGENT II which are restored to the room temperature are sequentially added into each hole, vibration and uniform mixing are carried out for 5min, absorbance is measured at 540nm, a standard curve is made, and the NO content in the culture solution supernatant is calculated according to the standard curve; the initial concentration of the monomer compound was 40. Mu.M, and the inhibition ratio was shown in Table 5.
TABLE 5 NO inhibitory Activity of Compounds 1-2
[a] Positive drug: andrographis paniculata Nees (Andrograholide, AG, HY-N0191) [b] each experiment was repeated 3 times.
Conclusion: the compounds 1-2 have certain inhibition effect on NO release, wherein the IC 50 values of the compounds (+) -1 and (-) -1 are 27.63+/-3.753 and 37.35 +/-0.807 respectively. The compounds (+) -1 and (-) -1 have remarkable inhibiting effect on the release of NO.
Example 13
This example uses Autodock 4.2.6 molecular docking software (THE SCRIPPS RESEARCH Institute, USA) to study the mode of action of compound 2 with Nav1.2 (pdb ID:6J 8E). Dynamic conformational changes and movement trajectories of the compound bound to the nav1.2 protein were reflected by molecular dynamics simulation. Nav1.2 consists of a polypeptide chain that is folded into four homologous repeats.
Conclusion: the docking results show that (+) -2 binds to the top residue of the active pocket and occupies the pores of the Nav1.2 channel with lower affinity (-9.126 kcal/mol), consistent with previous binding experiments. A detailed analysis of the interaction of the Nav1.2 channel active site with (+) -2 showed that the C-10 carbonyl group in (+) -2 forms a hydrogen bond with the Nav1.2 channel residue Asn333 as a hydrogen bond donor. In addition, (+) -2 interacts with residues Gln332, phe385, asn361, asp334, tyr362, asp949, trp948, pro921, trp923, tyr1429 and Met1425, which are possible sites of action for Nav1.2, providing a theoretical basis for subsequent development of potent Nav1.2 inhibitors.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (17)
1. A compound selected from the group consisting of:
7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-bridged aza-o [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (compound of formula 1); and
7 A-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4A,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (a compound of formula 2).
2. The compound of claim 1, or an isomer thereof, wherein the isomer is an enantiomer.
3. The compound according to claim 1 or 2, or an isomer thereof, selected from the group consisting of:
(4 aR,7aR,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methano-aza [4,5,6-de ] pyrrolo [ 3', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aS,7aS,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aR,7aR,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [ 3', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione; and
(4 AS,7aS,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinolin-1,8,11 (9H) -trione.
4. The compound according to claim 1 or 2, or an isomer thereof, having a structural formula selected from the group consisting of:
5. A method of extracting a compound according to any one of claims 1 to 4, or an isomer thereof, from the root of ananape, comprising the steps of:
a. Drying and pulverizing the root of Arna, taking ethanol water solution, methanol or chloroform with volume fraction of 50-95% (v/v) as solvent, wherein the ratio of the root of Arna (Kg) to the solvent (L) is 1:1.5-1:4, extracting by cold soaking, percolating, heating and refluxing or ultrasonic extracting, concentrating under reduced pressure, and recovering solvent to obtain extract;
b. C, suspending the total extract in the step a by water, dispersing the total extract in an acid water layer obtained by using acid such as hydrochloric acid with the concentration of 1-5% or sulfuric acid with the concentration of 1-5%, extracting the acid water layer by using dichloromethane to remove non-alkaloid, adjusting the pH value to 10-12 by using alkali such as NaHCO 3、Na2CO3, ammonia water or NaOH, extracting the acid water layer by using an organic solvent such as dichloromethane, ethyl acetate or n-butanol, concentrating the acid water layer under reduced pressure, and recovering the organic solvent to obtain the total alkaloid;
c. separating the total alkaloids from step b by silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or isomer thereof.
6. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography and reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography.
7. The method of claim 6, wherein in step c, after gradient or isocratic elution using normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography is used to obtain the compound of formula 1 or formula 2.
8. The method of claim 7, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:0 to 3:1, methylene chloride and methanol in a volume ratio of 500:1 to 3:1, petroleum ether and acetone in a volume ratio of 50:1 to 0:1, or methanol and water in a volume ratio of 1:9-1:0, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 20-100% (v/v) or an aqueous acetonitrile solution in a volume ratio of 20-100% (v/v), and the eluent used in the semi-preparative high performance liquid chromatography is n-hexane/EtOH in a volume ratio of 99-50% (v/v).
9. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography.
10. The method of claim 9, wherein after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed using reverse phase silica gel or reverse phase MCI column chromatography, followed by semi-preparative high performance liquid chromatography to obtain the compound of formula 1 or formula 2.
11. The method according to claim 10, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, methylene chloride and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 20 to 100% (v/v) or an aqueous acetonitrile solution in a volume ratio of 20 to 100% (v/v), and the eluent used in the semi-preparative high performance liquid chromatography for isocratic or gradient elution is n-hexane/EtOH in a volume ratio of 99 to 50% (v/v).
12. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography.
13. The method of claim 12, wherein the compound of formula 1 or formula 2 is obtained by subjecting to sephadex LH-20 column chromatography, gradient elution with reverse phase silica gel or reverse phase MCI column chromatography, and semi-preparative high performance liquid chromatography after gradient or isocratic elution using normal phase silica gel column chromatography.
14. The method according to claim 13, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, methylene chloride and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the sephadex LH-20 column chromatography is eluted with a methanol gradient or isocratic, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 20-100% (v/v) or an aqueous acetonitrile solution in a volume ratio of 20-100% (v/v), and the isocratic or gradient eluting eluent used in the semi-preparative high performance liquid chromatography is n-hexane/EtOH in a volume ratio of 99-50% (v/v).
15. The method according to any one of claims 5 to 14, wherein in step c the silica gel column chromatography is atmospheric or pressurized column chromatography and/or the packing used is forward silica gel or reverse phase silica gel.
16. Use of a compound according to any one of claims 1 to 4, or an isomer thereof, in the manufacture of an analgesic or anti-inflammatory medicament.
17. The use according to claim 16, wherein the compound or isomer thereof is analgesic by inhibition of nav1.2 and anti-inflammatory by inhibition of NO.
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