CN114853841A - Nepetalactone as well as preparation method and application thereof - Google Patents
Nepetalactone as well as preparation method and application thereof Download PDFInfo
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- CN114853841A CN114853841A CN202210637029.5A CN202210637029A CN114853841A CN 114853841 A CN114853841 A CN 114853841A CN 202210637029 A CN202210637029 A CN 202210637029A CN 114853841 A CN114853841 A CN 114853841A
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- vangueractone
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- ZDKZHVNKFOXMND-UHFFFAOYSA-N cis-Nepetalactone Natural products O=C1OC=C(C)C2C1C(C)CC2 ZDKZHVNKFOXMND-UHFFFAOYSA-N 0.000 title abstract description 5
- ZDKZHVNKFOXMND-NBEYISGCSA-N cis-trans-nepetalactone Chemical compound O=C1OC=C(C)[C@@H]2[C@H]1[C@@H](C)CC2 ZDKZHVNKFOXMND-NBEYISGCSA-N 0.000 title abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 15
- 208000032612 Glial tumor Diseases 0.000 claims abstract description 14
- 206010018338 Glioma Diseases 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 70
- 238000010828 elution Methods 0.000 claims description 60
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 44
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 239000000284 extract Substances 0.000 claims description 22
- 241000951473 Schizonepeta Species 0.000 claims description 17
- 241000951376 Schizonepeta tenuifolia Species 0.000 claims description 14
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000011347 resin Substances 0.000 claims description 14
- 229920005989 resin Polymers 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 8
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 7
- 238000007873 sieving Methods 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 230000001348 anti-glioma Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
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- 230000005764 inhibitory process Effects 0.000 abstract description 4
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 235000010679 Nepeta cataria Nutrition 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 description 3
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- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010011224 Cough Diseases 0.000 description 2
- 241000207923 Lamiaceae Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
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- 208000006673 asthma Diseases 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- -1 catnip lactone Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005100 correlation spectroscopy Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 2
- 150000002596 lactones Chemical group 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
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- 238000004611 spectroscopical analysis Methods 0.000 description 2
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- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001612423 Nepeta bracteata Species 0.000 description 1
- 240000009215 Nepeta cataria Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000612118 Samolus valerandi Species 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
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- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
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- 230000003247 decreasing effect Effects 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 150000003431 steroids Chemical class 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the technical field of separation and purification of overground parts of bract nepeta, and discloses bract nepetalactone and a preparation method and application thereof. The Vangueractone disclosed by the invention has a strong in-vitro inhibition effect on drug-resistant glioma, so that the Vangueractone can be applied to preparation of drugs for preventing glioma.
Description
Technical Field
The invention relates to the technical field of separation and purification of overground parts of bract catnip, and discloses a bract catnip lactone and a preparation method and application thereof, wherein the bract catnip lactone is Vangueractone for short.
Background
Nepeta Bracteata Bunge Benth, a Nepeta plant of the genus Nepeta of the family Labiatae (Labiatae), also known as "ancestor" Uygur "and using dried whole plant as a medicine. Research shows that the whole herb contains components such as flavonoid, triterpenes, alkanes, steroids and volatile oil. Has effects in relieving cough and asthma, clearing away heat, and promoting diuresis, and can be used for treating tracheitis, cough and asthma, common cold, fever, and dysuresia. Although modern pharmacological studies have shown that extracts thereof have significant anticancer activity, there have been fewer related studies to characterize the chemical composition thereof. The ursolic acid type triterpenoid contained in the compound has better anti-cancer activity in the anti-tumor aspect, and has the advantages of small adverse reaction, low price and wide source, thereby gaining the attention of wide Chinese and foreign researchers. Therefore, the triterpenoid monomer compound of the schizonepeta bracteata is developed and utilized, the potential medicinal value of the schizonepeta bracteata is further excavated, the structure and the physicochemical properties of the monomer compound of the schizonepeta bracteata are determined and characterized, and the application has important significance for developing and utilizing the schizonepeta bracteata.
Disclosure of Invention
The invention provides a kind of bract nepetalactone and its preparation method and application, and overcomes the deficiency of the prior art, the invention discloses Vangueractone for the first time, the Vangueractone of the invention has stronger in vitro inhibition to drug-resistant glioma, thus making Vangueractone can be used for preparing the application of preventing glioma drug.
One of the technical schemes of the invention is realized by the following measures: a Vanguerolactone having the chemical formula:
the following is a further optimization or/and improvement of one of the above-mentioned technical solutions of the invention:
the Vangueractone was obtained as follows: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part and performing methanol-water gradient elution, performing high performance liquid chromatography on the 3 rd fraction and performing methanol-water gradient elution, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
In the first step, every 6kg of the aerial parts of the schizonepeta cataria are added with distilled water in an amount which is 10 times of that of the aerial parts of the schizonepeta cataria, decocted and heated and refluxed for extraction for 2 times.
In the fourth step, the eluent for gradient elution by the high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 85: 15.
the second technical scheme of the invention is realized by the following measures: a preparation method of Vangueractone is characterized by comprising the following steps: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part to perform methanol-water gradient elution, performing high performance liquid chromatography methanol-water gradient elution on the 3 rd fraction, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
The following is further optimization or/and improvement of the second technical scheme of the invention:
in the first step, every 6kg of the aerial parts of the schizonepeta cataria are added with distilled water in an amount which is 10 times of that of the aerial parts of the schizonepeta cataria, decocted and heated and refluxed for extraction for 2 times.
In the fourth step, the eluent for gradient elution of the high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 85: 15.
the third technical scheme of the invention is realized by the following measures: an application of Vangueractone in preparing the medicine for preventing glioma.
The fourth technical scheme of the invention is realized by the following measures: an application of Vangueractone in preparing anti-glioma medicine.
The fifth technical scheme of the invention is realized by the following measures: an application of Vangueractone in preparing health products for preventing and treating glioma.
The invention discloses Vangueractone with an ursolic acid type triterpene structural skeleton containing a hexa-membered lactone ring for the first time, and the Vangueractone has a strong in-vitro inhibition effect on drug-resistant glioma, so that the Vangueractone can be applied to preparation of drugs for preventing and treating glioma.
Drawings
FIG. 1 is a chemical structural diagram of Vangaueractone of the present invention.
FIG. 2 is an HR-MS map of Vangueractone of the present invention.
FIG. 3 is a drawing of Vangueractone of the present invention 1 H-NMR spectrum.
FIG. 4 shows Vangueractone of the present invention 13 C-APT spectrum.
FIG. 5 is H of Vangueractone of the present invention 1 -H 1 COSY spectrogram.
FIG. 6 is an HSQC spectrum of Vangueractone of the present invention.
FIG. 7 is an HMBC spectrum of Vangueractone of the present invention.
FIG. 8 is a NOESY spectrum of Vangueractone of the present invention.
FIG. 9 is a bar graph showing the toxic effect of Vangueractone on RAW264.7 cells.
FIG. 10 is a graph showing the effect of Vangaueractone of the present invention on TNF-. alpha.levels in LPS-induced RAW264.7 cells.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments may be determined according to the technical solutions and practical situations of the present invention. The various chemical reagents and chemical articles mentioned in the invention are all the chemical reagents and chemical articles which are well known and commonly used in the prior art, unless otherwise specified; the percentages in the invention are mass weight percentages unless otherwise specified; the solution in the invention is an aqueous solution of water as a solvent unless otherwise specified, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution; the normal temperature and room temperature in the present invention generally mean a temperature of 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to the following examples:
example 1: the Vangueractone has a chemical structural formula as follows:
example 2: as an optimization of the above embodiment, it was obtained as follows: a preparation method of Vangueractone is characterized by comprising the following steps: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part and performing methanol-water gradient elution, performing high performance liquid chromatography on the 3 rd fraction and performing methanol-water gradient elution, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
Example 5: the preparation method of Vangueractone is carried out according to the following method: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part and performing methanol-water gradient elution, performing high performance liquid chromatography on the 3 rd fraction and performing methanol-water gradient elution, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
Example 3: as optimization of the above example, in the first step, every 6kg of the aerial parts of Nepeta cataria are decocted with 10 times of distilled water and heated under reflux for 2 times.
Example 4: as an optimization of the above embodiment, in the fourth step, the eluent for gradient elution by high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 85: 15.
example 6: the Vangueractone is applied to preparing the glioma prevention medicine.
Example 7: the Vangueractone is applied to preparing anti-glioma drugs.
Example 8: the Vangueractone is applied to the preparation of health care products for preventing and treating glioma.
Example 9: the Vanguerolactone was obtained as follows: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part and performing methanol-water gradient elution, performing high performance liquid chromatography on the 3 rd fraction and performing methanol-water gradient elution, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
NMR Hydrogen Spectroscopy of Vangueractone obtained in example 9 of the present invention ( 1 H-NMR) and nuclear magnetic resonance carbon Spectroscopy (C 13 C-APT), HR-MS spectrum, 1H-NMR spectrum, 13 C-APT spectrum, H 1 -H 1 The COSY spectrogram, HSQC spectrogram, HMBC spectrogram and NOESY spectrogram are shown in figures 2 to 8.
Fig. 3 and 4 were subjected to map analysis with reference to fig. 2, 5, 6, 7, and 8, and the peaks in fig. 3 and 4 were assigned, and the peak assignments in fig. 3 and 4 are shown in table 1. As can be seen from the data in fig. 3, fig. 4 and table 1, the chemical structural formula of Vanguerolactone of the present invention is shown in fig. 1, and Vanguerolactone of the present invention is amorphous powder and is easily soluble in chloroform and methanol.
The Vangueractone is subjected to in vitro cytotoxicity experiments and anti-inflammatory pharmacodynamic experiments, and the in vitro cytotoxicity experiments utilize an MTT colorimetric method. The level of inflammatory factor TNF-alpha in the cell supernatant was determined by ELISA.
Taking Vangueractone as an experimental group, indomethacin (anti-inflammatory drug) as a control group, simultaneously setting a blank group, taking RAW264.7 cells in a logarithmic growth phase, blowing and collecting the cells into a centrifuge tube by a pipette gun, diluting and counting, calculating the cell density in a cell suspension, preparing a 2 x 105/mL cell suspension by using a complete culture medium, inoculating the cell suspension into a 96-well plate, placing 100 mu L/well, and culturing in an incubator. After 12h, discarding the supernatant in the wells, adding 100 μ L Vangerolactone culture medium or blank culture medium with concentration of 200, 100, 50, 25, 12.5, 6.25 μ g/mL respectively, namely an administration group and a blank control group, setting 3 multiple wells in each group, placing in an incubator for 24h, discarding the supernatant, adding 100 μ L DMEM culture medium 20 μ L MTT solution (5mg/mL) in each well, continuing to culture for 4h, discarding the supernatant, adding 150 μ L dimethyl sulfoxide (DMSO) in each well, shaking on a shaker for 10min, fully dissolving the purple crystals, and detecting the optical density D (λ) value of each well at 490nm of a microplate reader.
Collecting RAW264.7 cells in logarithmic growth phase, collecting in a centrifuge tube by blowing with a pipette, diluting, counting, calculating cell density in cell suspension, and preparing into 1 × 10 with complete culture medium 6 The cell suspension was inoculated in a 96-well plate at 100. mu.L/well and cultured in an incubator. After 12h, the supernatant in the wells was discarded, and 200. mu.L of LVanguerolactone medium containing 1. mu.g/mL of LPS, 4. mu.g/mL of aspirin medium containing 1. mu.g/mL of LPS, 1. mu.g/mL of LPS and a medium, i.e., an administration group, a positive control group, a model group and a blank control group, each group having 3 multiple wells, were placed in an incubator and cultured for 24h, and then the level of TNF-. alpha.secreted from the cells was measured using the kit.
The bar chart of the toxic effect of Vanguerolactone on RAW264.7 cells is shown in figure 9. Figure 9 shows that Vanguerolactone has a strong anti-inflammatory effect on Lipopolysaccharide (LPS) -stimulated RAW264.7 cells.
The effect of Vanguerolactone on LPS-induced TNF- α levels in RAW264.7 cells of the invention is shown in figure 10. As can be seen from FIG. 10, TNF-. alpha.secretion was significantly reduced in each administration group as compared with the normal group and the model group.
In conclusion, the Vangueractone containing the ursolic acid type triterpene structural skeleton with the hexa-membered lactone ring is disclosed for the first time, and has a strong in-vitro inhibition effect on drug-resistant glioma, so that the Vangueractone can be applied to preparation of drugs for preventing glioma.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.
TABLE 1
Claims (10)
1. A Vanguerolactone characterized by the chemical structural formula:
2. the Vanguerolactone according to claim 1, characterized in that it is obtained according to the following method: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part and performing methanol-water gradient elution, performing high performance liquid chromatography on the 3 rd fraction and performing methanol-water gradient elution, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
3. The Vangueractone according to claim 2, wherein in the first step, every 6kg of the aerial parts of Schizonepeta cataria are decocted with 10 times of distilled water.
4. The Vangueractone of claim 2 or claim 3, wherein in the fourth step, the eluent from the HPLC gradient is a mixture of methanol and water, wherein the volume ratio of methanol to water is 85: 15.
5. A method for preparing Vanguerolactone according to claim 1, characterized by the following steps: firstly, smashing and sieving overground part of schizonepeta tenuifolia, decocting and extracting for 2 times by using distilled water, wherein each time lasts for 1-3 hours, combining decoction liquid of each time, and recovering and concentrating under reduced pressure to obtain overground part water decoction of the schizonepeta tenuifolia; secondly, subjecting the overground part water decoction of the schizonepeta cataria to D101 macroporous adsorption resin, performing gradient elution by using ethanol and water, and concentrating 30 percent of ethanol elution parts into extractum; thirdly, the 30 percent ethanol elution part extract is taken to pass through AB-8 macroporous resin, ethanol-water gradient elution is carried out, and the 10 percent ethanol elution part is taken to be concentrated into extract; and step four, performing ODS reversed phase chromatography on the extract of the 10% ethanol elution part and performing methanol-water gradient elution, performing high performance liquid chromatography on the 3 rd fraction and performing methanol-water gradient elution, purifying and separating, collecting and concentrating the eluate, and obtaining Vangueractone at the 22.3 th minute.
6. The method of producing Vangueractone according to claim 5, wherein in the first step, every 6kg of the aerial parts of Schizonepeta cataria are decocted in distilled water in an amount of 10 times.
7. The process according to claim 5 or 6, wherein the eluent for gradient elution by HPLC is a mixture of methanol and water, wherein the volume ratio of methanol to water is 85: 15.
8. use of Vangueractone according to claim 1, 2, 3 or 4 for the preparation of a medicament for the prevention of glioma.
9. Use of Vangueractone according to claim 1, 2, 3 or 4 for the preparation of an anti-glioma medicament.
10. Use of Vangueractone according to claim 1, 2, 3 or 4 as a health product for the prevention and treatment of glioma.
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| KONGUL SAFAK, ESRA等: "Cytotoxic Effects of Some Nepeta Species against Breast Cancer Cell Lines and Their Associated Phytochemical Properties", 《PLANTS》 * |
| WANG, CHEN等: "Bioactive pentacyclic triterpenoids from the leaves of Cleistocalyx operculatus", 《JOURNAL OF NATURAL PRODUCTS》 * |
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