CN113754620B - Lignan amide compound in fructus cannabis, and preparation method and application thereof - Google Patents
Lignan amide compound in fructus cannabis, and preparation method and application thereof Download PDFInfo
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- -1 Lignan amide compound Chemical class 0.000 title claims abstract description 27
- 229930013686 lignan Natural products 0.000 title claims abstract description 27
- 235000009408 lignans Nutrition 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title abstract description 18
- 240000004308 marijuana Species 0.000 title 1
- 241000218236 Cannabis Species 0.000 claims abstract description 42
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000000605 extraction Methods 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 10
- 230000004770 neurodegeneration Effects 0.000 claims abstract description 6
- 208000015122 neurodegenerative disease Diseases 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000010828 elution Methods 0.000 claims description 19
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000004440 column chromatography Methods 0.000 claims description 15
- 239000000284 extract Substances 0.000 claims description 15
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 13
- 238000010992 reflux Methods 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical group OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 12
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 11
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 7
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- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 description 3
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/86—Benzo [b] furans; Hydrogenated benzo [b] furans with an oxygen atom directly attached in position 7
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
- C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/22—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
- C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/40—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
- C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
- C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
- C07D319/20—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring with substituents attached to the hetero ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
- C07C2602/04—One of the condensed rings being a six-membered aromatic ring
- C07C2602/10—One of the condensed rings being a six-membered aromatic ring the other ring being six-membered, e.g. tetraline
Abstract
A lignan amide compound in fructus cannabis and a preparation method and application thereof belong to the technical field of traditional Chinese medicine preparation. The structural formula of the lignan amide compound in the fructus cannabis is shown as compounds 1-6 provided in the claims and the specification; the method takes fructus cannabis as a research object, separates and purifies the n-butanol extraction part of the ethanol extract after deoiling to obtain six novel lignan amide compounds, and carries out structure identification and biological activity research on the compounds, so that partial compounds have good neuroprotective activity, namely, have various activities for resisting neurodegenerative diseases, and have potential medical value.
Description
Technical Field
The invention relates to a lignan amide compound in fructus cannabis, a preparation method and application thereof, mainly application in medicaments for treating or preventing neurodegenerative diseases, and belongs to the technical field of traditional Chinese medicine preparation.
Background
Cannabis (Cannabis sativa L.) is also known as Cannabis, flax, pica, hemp, etc., and is a plant of the genus Cannabis (Cannabis) of the subfamily Moraceae (Moraceae) cannabinaceae (subfam. Cannabioideae). Fructus cannabis is a dry mature fruit of fructus cannabis, is a conventional medicinal part of fructus cannabis, is produced in county of Guangxi Bama Yao nationality called by American style of longevity, and is also a medicine and food dual-purpose traditional Chinese medicine which is favored by local residents. Fructus cannabis is used as a medicament in Shennong Ben Cao Jing (Shennong's herbal), is superior in quality, is sweet and flat in nature, enters spleen, stomach and large intestine meridians, has the efficacy of relaxing bowel, and is used for treating blood deficiency, fluid deficiency, constipation due to intestinal dryness and other symptoms. Research shows that the fructus cannabis has certain promotion effect on the health and longevity of local people after long-term eating. According to the report of the literature, the hemp seed extract contains chemical components such as alkaloids, lignan amides, cannabinoids, spiroalkanes, dibenzyls, flavonoids, steroids, fatty acids and the like, and modern pharmacological researches show that the lignan amides in the hemp seed have the activities of resisting tumor, resisting oxidation, reducing blood glucose, resisting anxiety, protecting nerves and the like. However, the component of the cannabis sativa extract is complex, and the existing technology for extracting the lignan amide compound is complicated, the extraction recovery rate is low, and the method is still to be improved, so that the cannabis sativa extract is intensively studied in order to find a new component with neuroprotective activity from the cannabis sativa.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a lignan amide compound in fructus Cannabis, a preparation method and application thereof, wherein the method takes fructus Cannabis (Cannabis sativa L.) as a research object, the n-butyl alcohol extraction part of an ethanol extract after oil removal is separated and purified to obtain six novel lignan amide compounds, and structural identification and biological activity research are carried out on the compounds, so that partial compounds have good neuroprotective activity, namely various activities for resisting neurodegenerative diseases, and have potential medicinal value.
The invention is realized by the following technical scheme:
the structural formula of the lignan amide compound in the fructus cannabis is one or a mixture of several compounds shown as 1-6 provided below;
the invention provides a preparation method of lignan amide compounds in fructus cannabis, which comprises the following steps:
step 1: pulverizing dried fructus Cannabis, adding petroleum ether, and reflux extracting under heating to obtain deoiled fructus Cannabis residue and petroleum ether extract;
adding the deoiled fructus cannabis residue into an ethanol water solution for re-heating reflux extraction to obtain waste residue and ethanol water extract;
distilling the ethanol water extract to remove solvent to obtain total extract;
step 2: adding water into the total extract for suspension to obtain suspension; wherein the solid-to-liquid ratio of the total extract to water is (0.9-1.1) g (9-11) mL;
extracting the suspension with water saturated n-butanol, and obtaining an n-butanol layer (CFE-B) after extraction; wherein, according to the volume ratio, the suspension: water saturated n-butanol= (0.5-1.5): 0.5-1.5;
eluting the n-butanol layer by a liquid chromatography-gradient elution method to obtain 9 fractions named CFE-B1-CFE-B9;
step 3:
performing elution and separation on the CFE-B3 again by adopting a liquid chromatography-gradient elution method to obtain 5 fractions CFE-B31-CFE-B35;
performing elution and separation on the CFE-B4 again by adopting a liquid chromatography-gradient elution method to obtain 9 fractions CFE-B41-CFE-B49;
step 4:
(1) Separating CFE-B33 by ODS column chromatography, and performing gradient elution by using a methanol-water system to obtain CFE-B331-CFE-B339; wherein, in the methanol-water system, the volume percentage of the methanol is 10 to 100 percent;
(2) Separating and purifying CFE-B335 by sephadex LH-20 column chromatography, and performing isocratic elution by a methylene dichloride-methanol system with the volume ratio of 2:1-1:2 to obtain fractions CFE-B3351-CFE-B3353;
separating and purifying CFE-B337 by sephadex LH-20 column chromatography, and isocratically eluting with a dichloromethane-methanol system with a volume ratio of 2:1-1:2 to obtain fractions CFE-B3371-CFE-B3377;
(3) Separating CFE-B3352 by high performance liquid HPLC to obtain compound 2;
separating CFE-B3372 by high performance liquid HPLC to obtain compound 5 and compound 3;
CFE-B3373 was separated by high performance liquid HPLC to give compound 4.
In the step 1, the solid-to-liquid ratio of the fructus cannabis to the petroleum ether is (0.9-1.1) g (9-11) mL; the heating temperature of petroleum ether heating reflux extraction is the boiling point (0-2) DEG C of the solution, the times of reflux extraction are at least 2 times, and the time of each reflux extraction is 1-4 h;
the solid-to-liquid ratio of the fructus cannabis residue to the ethanol water solution is (0.9-1.1) g (9-11) mL; wherein the ethanol water solution is preferably ethanol water solution with the volume percentage of 70-80 percent of ethanol; the heating temperature of the re-heating reflux extraction is the boiling point (0-2) DEG C of the solution, the times of the reflux extraction are at least 2 times, and the time of each reflux extraction is 1-4 h.
In the step 1, the distillation is preferably reduced pressure distillation.
In the step 2, the n-butanol layer is eluted and separated by a liquid chromatography-gradient elution method, a silica gel column chromatography is adopted, the eluent is methylene dichloride-methanol solution, and the gradient concentration is changed to 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1, 85:15,8:2,7:3,6:4,1:1 and 0:100.
In the step 3, the CFE-B3 is eluted again, a silica gel column chromatography column is adopted, the eluent is methylene dichloride-acetone solution, the gradient concentration is determined according to the polarity of the CFE-B3, and the required elution concentration is more specifically 93:7,9:1, 85:15,8:2,7:3,6:4,1:1,0:100 according to silica gel thin layer chromatography.
In the step 3, the CFE-B4 is eluted again, a silica gel column chromatography column is adopted, the eluent is methylene dichloride-acetone solution, the gradient concentration is determined according to the polarity of the CFE-B4, and the required elution concentration is more specifically 93:7,9:1, 85:15,8:2,7:3,6:4,1:1,0:100 according to silica gel thin layer chromatography.
In step 4 (3), the CFE-B3352 is separated by high performance liquid HPLC, and the adopted mobile phase is acetonitrile-water solution with 15-20% of acetonitrile volume percentage concentration.
In step 4 (3), the CFE-B3372 is separated by high performance liquid HPLC, and the adopted mobile phase is acetonitrile-water solution with the volume percentage concentration of 23-27% of acetonitrile.
In step 4 (3), the CFE-B3373 is separated by high performance liquid HPLC, and the adopted mobile phase is acetonitrile-water solution with the volume percentage concentration of acetonitrile of 18-22%.
Further, the preparation method of the lignan amide compound in the fructus cannabis further comprises the following steps:
step 5:
(1) Separating CFE-B43 by ODS column chromatography, and performing gradient elution by using a methanol-water system to obtain CFE-B431-CFE-B439; wherein, in the methanol-water system, the volume percentage of the methanol is 10-100 percent;
(2) Separating and purifying CFE-B436 by gel column chromatography, and performing isocratic elution by a methylene dichloride-methanol system with the volume ratio of 2:1-1:2 to obtain fractions CFE-B4361-CFE-B4365;
(3) Purifying CFE-B4362 by high performance liquid chromatography to obtain a compound 1;
step 6:
(1) Separating CFE-B44 by ODS column chromatography, and performing gradient elution by using a methanol-water system to obtain CFE-B441-CFE-B439; wherein, in the methanol-water system, the volume percentage of the methanol is 10-100 percent;
(2) The CFE-B443 is subjected to one or two high performance liquid chromatography separation or purification to obtain the compound 6.
The gel column in the step (2) of the step 5 is preferably a sephadex LH-20 column.
In step 5 (3), CFE-B4362 was purified by high performance liquid HPLC using an acetonitrile-water solution with a mobile phase of 18% to 22% acetonitrile by volume.
In the step (2) of the step 6, the CFE-B443 is purified by high performance liquid chromatography, and the adopted mobile phase is a methanol-water solution with the volume percentage concentration of 38 to 42 percent of methanol or an acetonitrile-water solution with the volume percentage concentration of 18 to 22 percent of acetonitrile.
The invention relates to a pharmaceutical composition which comprises one or more of the compounds 1-6.
The pharmaceutical composition takes one or more of the compounds 1 to 6 as active ingredients to prepare oral liquid, capsules, tablets, effervescent tablets, granules or one of various known dosage forms.
The pharmaceutical composition can be a medical drug.
The lignan amide compounds in the fructus cannabis of the compounds 3-5 and the application of the pharmaceutical composition taking the lignan amide compounds as active ingredients are used for preparing medicines for treating and/or preventing neurodegenerative diseases.
The neurodegenerative disease is specifically Alzheimer's disease and/or Parkinson's disease.
Compared with the prior art, the lignan amide compound in the fructus cannabis and the preparation method and application thereof have the beneficial effects that: the used reagent has wide sources of raw materials, low cost, suitability for large-scale production, simplicity, easiness in operation, simplicity and convenience in operation, capability of effectively enriching lignan amide compounds, high purity and good stability. The method has wide development and application prospects in the field of medicines.
Drawings
FIG. 1 is a schematic flow chart of a preparation method of lignan amide compounds in fructus cannabis in the embodiment of the invention;
FIG. 2 shows compounds 1 to 6 pairs of H 2 O 2 Inducing the protective effect of PC12 cell injury; wherein (a) is H for compound 3, compound 5 and compound 6 2 O 2 Inducing protection of PC12 cells against damage, (b) Compound 2 against H 2 O 2 Inducing protection of PC12 cells against damage, (c) Compound 1 and Compound 4 against H 2 O 2 Inducing the protective effect of PC12 cell injury.
FIG. 3 shows compounds 1 to 6 pairs of MPPs + Inducing the protective effect of PC12 cell injury; wherein (a) is the MPP of compound 3, compound 5 and compound 6 + Inducing protection of PC12 cells against damage, (b) Compound 2 against MPP + Inducing protection of PC12 cells against damage, (c) Compound 1 and Compound 4 against MPP + Inducing the protective effect of PC12 cell injury.
Detailed Description
The present invention will be described in further detail with reference to examples.
EXAMPLE 1 preparation of n-butanol extract of Cannabis
Dried fructus Cannabis (47.1 kg) was coarsely crushed, and extracted with 10 times of petroleum ether under reflux for 2 times each for 2 hours to obtain deoiled fructus Cannabis residue (36.4 kg). Reflux-heating fructus Cannabis residue with 10 times of 70% ethanol for 2 times each for 2 hr, mixing extractive solutions, and recovering under reduced pressure to obtain total extract (1.8 kg). The extract was suspended with 10 times of water, and extracted 3 times with an equal volume of water-saturated n-butanol to give an n-butanol layer CFE-B (690.7 g).
Example 2
A preparation method of lignan amide compound in fructus Cannabis comprises the following steps:
CFE-B (690.7 g) was first separated by column chromatography on silica gel, and gradient eluted sequentially with a methylene chloride-methanol system (methylene chloride-methanol volume ratio 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1, 85:15,8:2,7:3,6:4,1:1, 0:100), each system washed with 4 retention volumes, 9000mL each volume, giving a total of 9 fractions (CFE-B1-CFE-B9). According to the thin layer chromatography behavior analysis, the volume ratio of dichloromethane to methanol is 97: fraction CFE-B3 (46.6 g) was obtained at 3, at a methylene chloride-methanol volume ratio of 95: fraction CFE-B4 (120.7 g) was obtained at 5. CFE-B3 (46.6 g) and CFE-B4 (120.7 g) were further separated by silica gel column chromatography, eluting with a gradient of dichloromethane-acetone (volume ratio of dichloromethane-acetone 93:7,9:1, 85:15,8:2,7:3,6:4,1:1, 0:100) to give 14 fractions (CFE-B31-CFE-B35 and CFE-B41-CFE-B49). Wherein CFE-B33 is 14.8g, CFE-B43 is 1.7g, and CFE-B44 is 7.2g.
CFE-B33 (14.8 g) was separated by ODS column chromatography, and gradient elution was sequentially performed with methanol-water systems (10%, 20%,30%,40%,50%,60%,70%,100% by volume of methanol-water), each system washed with 4 retention volumes of 400mL each. According to the analysis of the color development behavior of the thin layer chromatography, fraction CFE-B335 was obtained in a 30% by volume methanol-water solution, and fraction CFE-B337 was obtained in a 40% by volume methanol-water solution. CFE-B335 (1.0 g) was further purified by sephadex LH-20 column chromatography, a dichloromethane-methanol system (volume ratio 1:1) was subjected to isocratic elution to give fraction CFE-B3352, CFE-B3352 (624.0 mg) was further separated by preparative High Performance Liquid Chromatography (HPLC), using an ultraviolet detector as detection means, at a wavelength of 280nm, using an acetonitrile-water solution with a volume percentage of 17% as eluent, and performing HPLC preparation at a flow rate of 4mL/min, collecting a chromatographic peak for 26min to give compound 2 (67.5 mg). CFE-B337 (661.7 g) is subjected to further sephadex LH-20 column chromatography separation and purification, a dichloromethane-methanol system (volume ratio of 1:1) is subjected to isocratic elution, so as to obtain fractions CFE-B3372 and CFE-B3373, CFE-B3372 (214.6 mg) is subjected to further separation of a preparation high performance liquid phase (HPLC), an ultraviolet detector is used as a detection means, a wavelength of 210nm, an acetonitrile-water solution with the volume percentage of 25% is used as an eluent is used for HPLC preparation, the flow rate is 4mL/min, a chromatographic peak for 16min is collected to obtain a compound 5 (10.0 mg), and a chromatographic peak for 34min is collected to obtain a compound 3 (10.6 mg). CFE-B3373 (133.1 mg) was further subjected to separation by preparative High Performance Liquid Chromatography (HPLC), using an ultraviolet detector as a detection means, a wavelength of 220nm, and an acetonitrile-water solution with a volume percentage of 25% as an eluent, to prepare by HPLC, a flow rate of 4mL/min, and a chromatographic peak was collected for 20min to give Compound 4 (20.0 mg).
CFE-B43 (1.7 g) was separated by ODS column chromatography, and gradient elution was sequentially carried out with methanol-water systems (10%, 20%,30%,40%,50%,60%,70%,80%,100% by volume of methanol-water), each system washed with 4 retention volumes, each 100mL by volume. Fraction CFE-B436 was obtained in 40% by volume methanol-water solution according to thin layer chromatography chromogenic behavior analysis. Further sephadex LH-20 column chromatography separation and purification were performed on CFE-B436 (61.8 mg), a dichloromethane-methanol system (volume ratio 1:1) was subjected to isocratic elution to obtain fraction CFE-B4362, CFE-B4362 (27.9 mg) was subjected to further separation of preparative High Performance Liquid (HPLC), an ultraviolet detector was used as a detection means, a wavelength of 280nm, and an acetonitrile-water solution with a volume percentage of 20% was used as an eluent to perform HPLC preparation at a flow rate of 4mL/min, and a chromatographic peak of 32min was collected to obtain compound 1 (16.7 mg).
CFE-B44 (7.2 g) was separated by ODS column chromatography, and gradient elution was sequentially carried out with methanol-water systems (10%, 20%,30%,40%,50%,60%,70%,80%,100% by volume of methanol-water), each system washed with 4 retention volumes of 200mL each. Fraction CFE-B443 was obtained in 40% by volume methanol-water solution according to thin layer chromatography chromogenic behavior analysis. CFE-B443 (1.5 g) is prepared by HPLC with an ultraviolet detector as a detection means and a wavelength of 280nm, with a methanol-water solution with a volume percentage of 40% as an eluent, the flow rate is 4mL/min, and a chromatographic peak is collected for 19min, so as to obtain CFE-B4434, CFE-B4434 (242.5 mg) is further prepared, and with an acetonitrile-water solution with a volume percentage of 20% as an eluent, the flow rate is 4mL/min, and a chromatographic peak is collected for 20min, so as to obtain compound 6 (51.2 mg).
Example 3 structural identification of Compounds
Hydrogen spectra of Compounds 1 and 2 1 H-NMR) and carbon Spectrometry 13 C-NMR) is shown in Table 1.
Hydrogen spectra of Compounds 3 and 4 1 H-NMR) and carbon Spectrometry 13 C-NMR) is shown in Table 2.
Hydrogen spectra of Compounds 5 and 6 1 H-NMR) and carbon Spectrometry 13 C-NMR) is shown in Table 3.
TABLE 1 Compounds 1 and 2 1 H-NMR 13 Nuclear magnetic data (CD) of C-NMR 3 OD)
TABLE 2 Compounds 3 and 4 1 H-NMR 13 Nuclear magnetic data (CD) of C-NMR 3 OD)
TABLE 3 Compounds 5 and 6 1 H-NMR 13 Nuclear magnetic data (CD) of C-NMR 3 OD)
The physicochemical properties of the compounds 1 to 6 shown in the present invention are as follows:
compound 1 (cannabisin U)
A yellow powder of the pigment was used,HR-ESI-MS (negative) gives m/z 488.1713[ M-H ]] - (C 28 H 26 NO 7 Calculated as 488.1715) to determine the molecular formula of the compound as C 28 H 27 NO 7 The unsaturation was calculated to be 16.UV spectrum gives lambda max (log ε) 220 (2.88), 280 (2.39) nm.
Compound 2 (cannabisin V)
A pale yellow powder, which is a mixture of a light yellow powder,ESI-MS (positive) gives m/z 476.31[ M+Na ]] + The molecular weight of the compound was indicated to be 453.HR-ESI-MS (positive) gives m/z 476.1685[ M+Na ]] + (C 25 H 27 NO 7 Na, calculated as 476.1680), HR-ESI-MS (negative) gave m/z 452.1709[ M-H ]] - (C 25 H 26 NO 7 Calculated as 52.1715) to determine the molecular formula of the compound as C 25 H 27 NO 7 The unsaturation was calculated to be 13.UV spectrum gives lambda max (log ε) 220 (5.05), 280 (4.59) nm. />
Compound 3 (cannabisin P)
A pale yellow oil-like substance,ESI-MS gives m/z 637.2[ M+Na ]] + ,613.0[M-H] - The molecular weight of the compound was suggested to be 614.HR-ESI-MS gives m/z 613.2189[ M-H ]] - (calcd 613.2186,C 34 H 33 N 2 O 9 ) Determining the molecular formula of the compound as C 34 H 34 N 2 O 9 The unsaturation was calculated to be 19.UV spectrum gives lambda max (log ε) 225 (3.49), 283 (2.99) nm.
Compound 4 (cannabisin X)
A white powder of the pigment is mixed with the pigment,ESI-MS (positive) gave m/z 619.1[ M+Na ]] + The molecular weight of the compound was suggested to be 596.HR-ESI-MS (negative) gives m/z 595.2084[ M-H ]] - (C 34 H 31 N 2 O 8 Calculated as 595.2075) to determine the molecular formula of the compound as C 34 H 32 N 2 O 8 The unsaturation was calculated to be 20.UV spectrum gives lambda max (log ε) 220 (4.03), 280 (3.82) nm.
Compound 5 (cannabisin Y)
White needle crystals (methanol),ESI-MS (positive) gave m/z 306.2[ M+Na] + ESI-MS (negative) gives m/z 282.2[ M-H ]] - The molecular weight of the compound was found to be 283.HR-ESI-MS (positive) gives m/z 284.1281[ M+H ]] + (C 17 H 18 NO 3 Calculated as 284.1287) to determine the molecular formula of the compound as C 17 H 17 NO 3 The unsaturation was calculated to be 9.UV spectrum gives lambda max (log ε) 220 (3.83), 280 (3.78) nm.
Compound 6 (cannabisin Z)
A yellow oil-like substance, which is a mixture of water and water,. HR-ESI-MS (positive) gives m/z 316.1185[ M+H ]] + (C 17 H 18 NO 5 Calculated as 316.1185), the molecular formula of the compound was determined as C 17 H 17 NO 5 The unsaturation was calculated to be 10.UV spectrum gives lambda max (log ε) 220 (3.82), 280 (3.62) nm.
Neuroprotective Activity of the Compounds of example 4
(1) The degree of damage of the compound to PC12 cells is examined, a non-invasive concentration is selected for subsequent experiments, PC12 cells are inoculated in RPMI 1640 (Roswell Park Memorial Institute) medium containing 10% Fetal Bovine Serum (FBS), the cells are placed in a constant temperature incubator at 37 ℃ for culturing for 12 hours, the PC12 cells are acted by the compounds with different concentrations (10 mu M, 25 mu M, 50 mu M and 100 mu M), the survival rate of the PC12 cells is detected after 24 hours, and the compound concentration for further study is selected according to the result.
(2) Investigation of Compounds 1 to 6 vs H 2 O 2 Protection against PC12 cell damage
By H 2 O 2 And (3) inducing PC12 cell oxidative stress and establishing an in vitro nerve cell damage model. PC12 cells (1.0X10) 4 cells/mL, 90. Mu.L/well) was inoculated in 96-well plates and incubated at constant temperature for 12h, 250. Mu. M H was added 2 O 2 After 12h of treatment, different concentrations of compound were then selected and added to the wells, and cell viability was measured after a further incubation of 12 h. The groupings during the experiment were as follows: (1) blank group: only adding the culture medium; (2) negative control group: containing H 2 O 2 (250. Mu.M) of medium; (3) Sample +H 2 O 2 Treatment group:samples of different concentrations +H 2 O 2 (250. Mu.M). After 24h, MTT solution was added to each well, the culture was continued for 4h, the supernatant was aspirated off, 150. Mu.L DMSO was added to each well, and after complete dissolution of the crystals by shaking, the experimental data were read using an ELISA reader at 490 nm.
Cell viability (%) = (a) Sample of –A Blank control )/(A Negative control –A Blank control )×100％
Experimental results: the neuroprotective activity test (FIGS. 2 (a-c)) was performed using MTT assay for compounds 1-6, and the statistical results are shown in Table 4. The compounds 3 to 5 can improve H to a certain extent 2 O 2 The survival rate of PC12 cell damage is induced, suggesting that the PC may have an anti-Alzheimer's disease effect.
Table 4 Compounds 1 to 6 against H 2 O 2 Protection against PC12 cell damage
(3) Monomer Compound pair MPP + Protection against induced PC12 cell damage
PC12 cells were inoculated in RPMI 1640 (Roswell Park Memorial Institute) medium containing 10% Fetal Bovine Serum (FBS) and cultured in a 37℃incubator. The screening concentration of the compound is first selected, followed by MPP + And (3) inducing PC12 cell oxidative stress and establishing an in vitro nerve cell damage model. PC12 cells (1.0X10) 4 cells/mL, 90. Mu.L/well) was inoculated in 96-well plates and incubated at constant temperature for 12h, followed by addition of 500. Mu.M MPP + After 12h of treatment, different concentrations of compound are selected and added into the wells, and the cell viability is detected after culturing for 12 h. The groupings during the experiment were as follows: (1) blank group: only adding the culture medium; (2) negative control group: adding MPP-containing + (500. Mu.M) of medium; (3) Sample +MPP + Treatment group: sample+MPP at different concentrations + (500. Mu.M). After 24h, MTT solution was added to each well and incubation was continued for 4h, the supernatant was aspirated off, 150. Mu.L DMSO was added to each well and shaking was performed thoroughly to crystallizeAfter complete dissolution, the experimental data were read using an enzyme-labeled instrument at 490nm wavelength.
Cell viability (%) = (a) Sample of –A Blank control )/(A Negative control –A Blank control )×100％
Experimental results: the effect of compounds 1 to 6 on the survival rate of PC12 cells was examined by MTT method (FIGS. 3 (a) to (c)), and the statistical results are shown in Table 5. Compounds 3 and 4 are capable of increasing MPP to some extent + The survival rate of PC12 cell damage was induced, suggesting a potential for anti-PD effect.
Table 5 compound vs MPP + Protection against induced PC12 cell damage
The data in Table 5 shows that compounds 3 and 4 are capable of increasing MPP to some extent + The survival rate of PC12 cell injury is induced, suggesting that the PC12 cell injury may have an anti-Parkinson disease effect.
Comparative example
A preparation method of lignan amide compounds in fructus cannabis is the same as in example 1, except that CFE-B3 (46.6 g) is further separated by silica gel column chromatography, and dichloromethane-acetone gradient elution (volume ratio of dichloromethane-acetone is 95:5,9:1, 85:15,8:2,7:3,6:4,1:1, 0:100) is not carried out to obtain eluted products.
Claims (6)
1. The lignan amide compound in the fructus cannabis is characterized in that the lignan amide compound in the fructus cannabis is the following compound 3:
2. the method for preparing lignan amide compound of claim 1, comprising the steps of:
step 1: pulverizing dried fructus Cannabis, adding petroleum ether, heating and reflux extracting at solution boiling point+ (0-2) deg.C to obtain deoiled fructus Cannabis residue and petroleum ether extract;
adding the deoiled fructus cannabis residue into an ethanol water solution for re-heating reflux extraction, wherein the ethanol water solution is 70-80% ethanol water solution by volume percent, and the heating temperature is the solution boiling point+ (0-2) DEG C, so as to obtain waste residue and ethanol water extract;
distilling the ethanol water extract to remove solvent to obtain total extract;
step 2: adding water into the total extract for suspension to obtain suspension; wherein the solid-to-liquid ratio of the total extract to water is (0.9-1.1) g (9-11) mL;
extracting the suspension with water saturated n-butanol, and obtaining an n-butanol layer after extraction; wherein, according to the volume ratio, the suspension: water saturated n-butanol= (0.5-1.5): 0.5-1.5;
eluting the n-butanol layer by a liquid chromatography-gradient elution method, wherein a silica gel column chromatography column is adopted, the eluent is methylene dichloride-methanol solution, the gradient concentration is changed to 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1, 85:15,8:2,7:3,6:4,1:1,0:100, and 9 fractions are respectively named as CFE-B1-CFE-B9;
step 3:
performing elution separation again on the CFE-B3 by adopting a liquid chromatography-gradient elution method, wherein a silica gel column is adopted, the eluent is methylene dichloride-acetone solution, and the gradient concentration changes are 93:7,9:1, 85:15,8:2,7:3,6:4,1:1 and 0:100, so as to obtain 5 fractions CFE-B31-CFE-B35;
step 4:
(1) Separating CFE-B33 by ODS column chromatography, and performing gradient elution by using a methanol-water system to obtain CFE-B331-CFE-B339; wherein, in the methanol-water system, the volume percentage of the methanol is 10 to 100 percent;
(2) Separating and purifying CFE-B337 by sephadex LH-20 column chromatography, and isocratically eluting with a dichloromethane-methanol system with a volume ratio of 2:1-1:2 to obtain fractions CFE-B3371-CFE-B3377;
(3) CFE-B3372 was separated by high performance liquid HPLC to give compound 3.
3. The method for preparing lignan amide compound in fructus cannabis according to claim 2, wherein in the step 1, the solid-to-liquid ratio of fructus cannabis to petroleum ether is (0.9-1.1) g (9-11) mL; heating and reflux-extracting for at least 2 times, wherein the reflux-extracting time is 1-4 h each time;
the solid-to-liquid ratio of the fructus cannabis residue to the ethanol water solution is (0.9-1.1) g (9-11) mL; wherein, the times of reflux extraction of ethanol water solution is at least 2 times, and the time of each reflux extraction is 1-4 h.
4. The method for producing a lignan amide compound of cannabis as claimed in claim 2, wherein in step 4 (3), CFE-B3372 is separated by high performance liquid HPLC using an acetonitrile-water solution having a mobile phase of 23% -27% acetonitrile by volume.
5. A pharmaceutical composition characterized by comprising the compound 3 of claim 1 as an active ingredient, and being formulated into one of oral liquid, capsule, tablet and granule.
6. The use of a lignan amide compound of the cannabis sativa of compound 3 as claimed in claim 1 and a pharmaceutical composition as claimed in claim 5 as an active ingredient for the manufacture of a medicament for the treatment and/or prevention of neurodegenerative diseases.
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