CN117024507A - Kansui-type triterpene compound with anti-tumor effect and preparation method thereof - Google Patents
Kansui-type triterpene compound with anti-tumor effect and preparation method thereof Download PDFInfo
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- -1 triterpene compound Chemical class 0.000 title claims abstract description 29
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 6
- 208000029742 colonic neoplasm Diseases 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 44
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 238000004440 column chromatography Methods 0.000 claims description 20
- 238000004809 thin layer chromatography Methods 0.000 claims description 20
- 239000003480 eluent Substances 0.000 claims description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 10
- GLYLMXARZJNUEY-UHFFFAOYSA-N dichloromethane;methanol;hydrate Chemical compound O.OC.ClCCl GLYLMXARZJNUEY-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- DQFAAIWCCHDCLO-UHFFFAOYSA-N acetonitrile;formic acid;hydrate Chemical compound O.CC#N.OC=O DQFAAIWCCHDCLO-UHFFFAOYSA-N 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 5
- 238000010262 high-speed countercurrent chromatography Methods 0.000 claims description 4
- 239000000178 monomer Substances 0.000 claims description 4
- 238000010898 silica gel chromatography Methods 0.000 claims description 4
- 210000001944 turbinate Anatomy 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 241000242759 Actiniaria Species 0.000 abstract 1
- 238000001228 spectrum Methods 0.000 description 13
- 238000005481 NMR spectroscopy Methods 0.000 description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 3
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- 239000007787 solid Substances 0.000 description 3
- 238000004611 spectroscopical analysis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000732800 Cymbidium Species 0.000 description 2
- 241000158728 Meliaceae Species 0.000 description 2
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- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
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- 150000002630 limonoids Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 244000247747 Coptis groenlandica Species 0.000 description 1
- 235000002991 Coptis groenlandica Nutrition 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 241000007383 Magnolia sinica Species 0.000 description 1
- 244000141359 Malus pumila Species 0.000 description 1
- 240000007997 Munronia pinnata Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000405414 Rehmannia Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241000723873 Tobacco mosaic virus Species 0.000 description 1
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001887 anti-feedant effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
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- 239000006285 cell suspension Substances 0.000 description 1
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- 238000001142 circular dichroism spectrum Methods 0.000 description 1
- 238000004185 countercurrent chromatography Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 125000000567 diterpene group Chemical group 0.000 description 1
- 238000005516 engineering process Methods 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
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
-
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
- C07C59/46—Unsaturated compounds containing hydroxy or O-metal groups containing rings other than six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
- C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
- C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
- C07C2603/16—Benz[e]indenes; Hydrogenated benz[e]indenes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
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Abstract
The invention relates to an kansui-type triterpene compound with an anti-tumor effect and a preparation method thereof, wherein the kansui-type triterpene compound has one structure of Munrepene A, munrepene B and Munrepene C, and the kansui-type triterpene compounds Munrepene A-C show a specific inhibition effect on colon cancer cells, which is obviously superior to the inhibition effect on other types of tumor cells, thus showing that the kansui-type triterpene compound extracted from the rhizoma anemones Aristolochiae is hopeful to be developed into a new preparation for treating colon cancer.
Description
Technical Field
The invention relates to the field of natural medicines, in particular to an kansui type triterpene compound with an anti-tumor effect and a preparation method thereof.
Background
About 15 plants of the genus rehmannia in the natural world are distributed in the regions of Spirachica, india, peninsula, indonesia and Philippines, of which 8and 1 varieties are distributed in China. Previous studies on the chemical composition of this class of plants have successfully isolated a range of structurally diverse compounds including limonoids, triterpenes, flavonoids, lignin, sterols, sesquiterpenes, diterpenes and the like. Some of the compounds have remarkable biological activities such as anti-inflammatory effect, tumor inhibiting effect, tobacco mosaic virus resisting effect and antifeedant effect.
The plant cymbidium (Munronia pinnata (wall.) W.Theob) (synnyms: M.henyi Harms, M.pumila light, and M.sinica Diels) belongs to one of the members of the family Meliaceae, a perennial herb plant that grows in the temperate zone of south China. The subject group has previously isolated a number of novel limonoids from the plant cymbidium.
At present, no research on the components containing the kansui type triterpene compounds in the dwarf gyroscope is reported, and no record is made on the fact that the kansui type triterpene compounds in the dwarf gyroscope have anticancer activity.
Disclosure of Invention
In order to solve the technical problems, the invention provides an kansui-type triterpene compound with an anti-tumor effect and a preparation method thereof.
The invention provides an kansuine type triterpene compound with an anti-tumor effect, which has one structure of Munrepene A, munrepene B and Munrepene C, wherein the Munrepene A is shown as a formula (I), the Munrepene B is shown as a formula (II), and the Munrepene C is shown as a formula (III):
a method for preparing an anti-tumor kansuine type triterpene compound, wherein the anti-tumor kansuine type triterpene compound is Munrepene A, and the preparation method of the Munrepene A comprises the following steps:
s1, adding 95% ethanol into dried dwarf gyroscope powder according to a feed liquid ratio of 1 kg:10-15L, soaking and extracting for 2-4 times at room temperature, merging filtrate for 1 time every 3 days, filtering and concentrating to obtain extract, sequentially extracting the extract by petroleum ether, ethyl acetate and water with equal volumes respectively, subjecting a water layer to macroporous resin column chromatography with the weight of 45-55 times of the water layer, sequentially eluting by 2.0-3.0L of 20% -80% ethanol respectively, and collecting 40% ethanol part eluent to obtain Fr3;
s2, performing silica gel column chromatography by taking Fr3 with the weight of 100 times of that of the Fr3, performing gradient elution by adopting dichloromethane-methanol with the volume ratio of 80:20-100:0 as a mobile phase, collecting 1 test tube every 10-15 ml, and observing and combining eluent containing the same components by using a TLC (thin-layer chromatography) plate every test tube to obtain 20 fractions of Fr 3.1-Fr 3.20;
s3, taking Fr3.3, purifying by adopting a C18 high performance liquid chromatography column and acetonitrile-water-formic acid with a volume ratio of 18:82:0.1 as a mobile phase, and keeping for 20-25 min to obtain Munrepene A.
A method for preparing an anti-tumor kansuine type triterpene compound, wherein the anti-tumor kansuine type triterpene compound is Munrepene B, and the preparation method of the Munrepene B comprises the following steps:
s1, adding 95% ethanol into dried dwarf turbinate powder according to a feed liquid ratio of 1 kg:10-15L, soaking and extracting for 2-4 times at room temperature, 1 time every 3 days, merging filtrate, filtering and concentrating to obtain extract, sequentially extracting the extract by petroleum ether, ethyl acetate and water with equal volumes respectively, sequentially eluting a water layer by using macroporous resin column chromatography with the weight of 45-55 times of the water layer, sequentially eluting by using 2.0-3.0L of 20-80% ethanol, collecting an 80% ethanol eluting part, sequentially eluting by using gel column chromatography with the weight of 100 times of the water layer again, and continuously eluting by using 100% methanol for 1.0-2.0L to obtain Fr2;
s2, performing C18 column chromatography with the weight of 100 times of Fr2, performing gradient elution with methanol-water at the volume ratio of 30:70-50:50 of 1.0-1.5L, collecting 1 test tube every 10-15 ml, and observing and combining the eluents containing the same components by TLC (thin layer chromatography) plates in each test tube to obtain 7 fractions of Fr 2.1-Fr 2.7;
s3, subjecting Fr2.1 to silica gel column chromatography with the weight of 100 times of the Fr2.1, eluting with methylene dichloride-methanol at the volume ratio of 4:1, collecting 1 test tube per 10-15 mL, and observing and combining the eluents containing the same components by TLC (thin layer chromatography) plates to obtain 5 components such as Fr 2.1.1-Fr 2.1.5;
s4, taking Fr2.1.2, adopting a high-speed countercurrent chromatography method, adopting 1.5-2.5L of dichloromethane-methanol-water as a mobile phase, reversely pushing the solution, collecting 1 test tube per 20-25 ml of mobile phase, and combining the same components through a TLC (thin layer chromatography) point plate to obtain 6 components of Fr 2.1.2.1-Fr 2.1.2.6 and the like, wherein the component Fr2.1.2.3 is a monomer compound, namely Munrepene B.
A method for preparing an anti-tumor kansuine type triterpene compound, wherein the anti-tumor kansuine type triterpene compound is Munrepene C, and the preparation method of the Munrepene C comprises the following steps:
s1, adding 95% ethanol into dried dwarf turbinate powder according to a feed liquid ratio of 1 kg:10-15L, soaking and extracting for 2-4 times at room temperature, 1 time every 3 days, merging filtrate, filtering and concentrating to obtain extract, sequentially extracting the extract by petroleum ether, ethyl acetate and water with equal volumes respectively, sequentially eluting a water layer by using macroporous resin column chromatography with the weight of 45-55 times of the water layer, sequentially eluting by using 2.0-3.0L of 20-80% ethanol, collecting an 80% ethanol eluting part, sequentially eluting by using gel column chromatography with the weight of 100 times of the water layer again, and continuously eluting by using 100% methanol for 1.0-2.0L to obtain Fr2;
s2, performing C18 column chromatography with the weight of 100 times of Fr2, performing gradient elution with methanol-water at the volume ratio of 30:70-50:50 of 1.0-1.5L, collecting 1 test tube every 10-15 ml, and observing and combining the eluents containing the same components by TLC (thin layer chromatography) plates in each test tube to obtain 7 fractions of Fr 2.1-Fr 2.7;
s3, taking Fr2.3, adopting 550-650 mL of dichloromethane-methanol-water as a mobile phase in a volume ratio of 2:2:1, collecting 1 test tube every 10-15 mL, and observing and combining eluent containing the same components by a TLC (thin layer chromatography) plate in each test tube to obtain 3 fractions of Fr 2.3.1-Fr 2.3.3; taking Fr2.3.3, preparing a high performance liquid chromatography, using a C18 preparation column, adopting acetonitrile-water-formic acid as a mobile phase in a volume ratio of 23:77:0.1, and preparing and purifying for 10-15 min to obtain Munrepene C.
The kansui alkane type triterpene compound has the following characteristics and advantages:
1. the kansuine type triterpene compounds Munrepene A-C extracted from the short-tuo have specific inhibition effect on colon cancer cells, are obviously superior to inhibition effect on other types of tumor cells, and show that the kansuine type triterpene compounds extracted from the short-tuo are hopefully developed into novel drugs for treating colon cancer.
2. The preparation method is convenient to operate, easy for mass production and stable in quality; the raw materials of the invention are abundant in China and have proper price, so that the large-scale production of the invention has no high cost limit.
Drawings
FIG. 1 is a 2D NMR correlation and plan view block diagram of Munrepene A and Munrepene B.
FIG. 2 is a 2D NMR correlation and plane configuration diagram of Munrepene C.
FIG. 3 is a diagram showing the structure of an X-ray crystal of Munrepene A.
FIG. 4 is a perspective view of the ROESY spectrum of Munrepene A.
FIG. 5 is a perspective view of the ROESY spectrum of Munrepene B.
FIG. 6 is a perspective view of the ROESY spectrum of Munrepene C.
FIG. 7 is a schematic diagram of a skeleton rotation model of Munrepene A, wherein a) is C-17-C-20, b) is C-20-C-22, C) is C-22-C-23, and d) is C-23-C-24.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of kansuixane triterpene Compounds Munrepene A to F
The plant of the dwarf tuo is collected from the Guangxi Jinxi city in 2021 at 7 months, is identified by Li Dianpeng professor as pinna (wall.) w.Theob, the genus Coptis, the family Meliaceae. The sample was stored in the plant specimen room (21-GX-001) of the natural product chemical research center of Guangxi plant institute of China academy of sciences.
S1, adding 95% ethanol into dried short tourbillon powder (25 kg), soaking and extracting for 3 times at room temperature, 250L each time, 1 time every 3 days, merging filtrate, filtering and concentrating to obtain extract, sequentially extracting the extract (1.71 kg) respectively by petroleum ether, ethyl acetate and water with equal volumes in sequence, subjecting a water layer (560 g) to macroporous resin column chromatography (D-101) with 50 times of the weight of the extract, sequentially eluting by 2.5L of 20%, 40% and 80% ethanol respectively, and collecting 40% ethanol partial eluent to obtain Fr3 (34 g). The 80% ethanol eluted fraction (86 g) was collected and subjected to LH-20 gel column chromatography again in an amount 100 times its weight, followed by eluting with 100% methanol for 1.5L to give Fr2 (15 g).
S2. Fr2 was subjected to column chromatography with YMC 50. Mu.mC 18 in an amount 100 times the weight thereof, and gradient elution was carried out with methanol-water at a volume ratio of 30:70,40:60,50:50 each with 1L, 1 test tube was collected per 10ml, and the same component-containing eluents were pooled by TLC plate observation per test tube to obtain 7 total fractions of Fr2.1 to Fr2.7 (numbered 1, 2, 3 … … according to the order of the effluent components, and so forth, the same applies hereinafter), wherein Fr2.1 was 1.46g, fr2.3 was 1.27g, fr2.4 was 1.39g, and Fr2.7 was 0.9g.
S3, taking Fr2.1 (1.46 g) and carrying out silica gel H column chromatography with the weight of 100 times of the Fr2.1, eluting with dichloromethane-methanol in a volume ratio of 4:1, collecting 1 test tube per 10mL, and combining eluents containing the same components by observing each test tube through a TLC (thin layer chromatography) plate to obtain Fr 2.1.1-Fr 2.1.5 and other 5 components. Fr2.1.2 (163 mg) was prepared by high-speed countercurrent chromatography using 2L of dichloromethane-methanol-water in a volume ratio of 2:2:1 as flow reverse push, 1 tube was collected per 20ml of mobile phase, and the same components were combined by TLC plate to obtain 6 components, fr 2.1.2.1-Fr 2.1.2.6, wherein component Fr2.1.2.3 was a monomer compound, i.e., munrepene B (2) (68 mg).
S4, taking Fr2.3 (1.27 g) and adopting 600mL of dichloromethane-methanol-water as a mobile phase in a volume ratio of 2:2:1 by a high-speed countercurrent chromatography method, collecting 1 test tube per 10mL, and observing and combining eluent containing the same components by a TLC (thin layer chromatography) point plate per test tube to obtain 3 fractions of Fr 2.3.1-Fr 2.3.3. Fr2.3.3 was purified by preparative high performance liquid chromatography using Agilent ZORBAX SB-C18 column using acetonitrile-water-formic acid at a volume ratio of 23:77:0.1 as mobile phase to give Munrepene C (3) (26 mg, tR=10.1 min).
S5, taking Fr2.4 (1.39 g), eluting with 2L of dichloromethane-methanol-water by taking the volume ratio of 2:2:1 as a mobile phase, collecting each 20ml of mobile phase as 1 pipe, and combining the same components by a TLC (thin layer chromatography) plate to obtain 6 components such as Fr 2.4.1-Fr 2.4.6; fr2.4.4 (131 mg) was then subjected to preparative high performance liquid chromatography using Agilent ZORBAX SB-C18 preparative column eluting with acetonitrile-water-formic acid at a volume ratio of 23:77:0.1 as mobile phase to give Munrepene E (5) (10 mg, tR=12.2 min) and Munrepene F (6) (15 mg, tR=14.7 min), respectively.
S6, taking Fr2.7 (0.9 g), eluting by adopting 1.5L of dichloromethane-methanol-water as a mobile phase in a volume ratio of 2:2:1, collecting each 20ml of mobile phase as 1 pipe, and combining the same components by a TLC (thin layer chromatography) plate to obtain 5 components such as Fr 2.7.1-Fr 2.7.5, wherein the component Fr2.7.3 is a monomer compound, namely Munrepene D (4) (184 mg).
S7. Fr3 (30 g) was subjected to silica gel H column chromatography in an amount 100 times the weight of the mixture, and methylene chloride-methanol was used in a volume ratio of 80:20, 90:10 Gradient elution is carried out by taking 1.5L of each 100:0 as mobile phase, 1 test tube is collected every 10ml, and 20 fractions from Fr3.1 to Fr3.20 are obtained after each test tube is observed by a TLC (thin layer chromatography) plate and combined to contain the same component eluent; fr3.3 (1.49 g) was purified by preparative HPLC using a ChromCore 120-C18 HPLC column with acetonitrile-water-formic acid at a volume ratio of 18:82:0.1 as mobile phase to give Munrepene A (1) (321 mg, tR=21.3 min).
Identification of kansuine triterpene compounds Munrepene A-F
Jasco P-1020 polarimeter for measuring optical rotation. TENSOR27 type Fourier transform Infrared spectrometer (Therom Fisher, USA) IR spectroscopy of KBr tabletsAnd (5) analyzing. CD spectra were recorded on a J-810 type CD spectrometer. Mass spectra were determined using an LC/MS-IT-TOF mass spectrometer. Nuclear magnetic resonance spectra were recorded with a bruck AVANCE III-HD 500 spectrometer, internal standard TMS. CCC was performed on TBE-300C system (Tauto biotechnology, shanghai, china). HPLC analysis used Agilent 1260 Infinicity II LC (Agilent Technologies, U.S.) using Agilent Poroshell cb-C18 (4mm,4.6mm x 150mm,Agilent, U.S.) ChromCore 120-C18 (5mm,10mm x 250mm,NanoChrom, china) and Agilent ZORBAX cb-C18 (5mm,9.4mm x 250mm,Agilent, U.S.). Column chromatography was performed on silica gel (200-300 mesh, qingdao ocean chemical plant, china) and MCI gel (Mitsubishi chemical corporation, japan). TLC analysis was performed using pre-coated silica gel 60f254 plates (Merck Millipore, germany with 10% H 2 SO 4 The silica gel plate sprayed in ethanol was heated, and spots were observed.
Munrepene A: a colorless amorphous solid having optical activity; molecular formula C 32 H 50 O 12 ；[α] D 20.1 +4.98(c0.10,MeOH)；HRESIMS:m/z 608.2496([M+Na] + Delta+2.4 mmu; IR:1748and 1689cm-1, carbonyl function absorbance peak. The nuclear magnetic resonance hydrogen spectrum is shown in table 1. The nuclear magnetic resonance carbon spectrum is shown in Table 2. The 2D NMR correlation and plane structure are shown in fig. 1, the X-ray crystal structure is shown in fig. 3, the ROESY spectrum is shown in fig. 4, and the skeleton rotation model is shown in fig. 7.
Munrepene B: a colorless amorphous solid having optical activity; molecular formula C 32 H 50 O 12 ；[α] D 20.1 -5.00(c0.10,MeOH)；HRESIMS:m/z 608.2493([M+Na]+,Δ+2.1mmu；IR：1748and 1689cm -1 Carbonyl function absorbance peak. The nuclear magnetic resonance hydrogen spectrum is shown in table 1. The nuclear magnetic resonance carbon spectrum is shown in Table 2. The 2D NMR correlation and plane structure are shown in fig. 1, and the ROESY spectrum is shown in fig. 5.
Munrepene C: a colorless amorphous solid having optical activity; molecular formula C 32 H 52 O 13 ；[α] D 20.0 ＝-50.51(c0.10,MeOH)；HRESIMS:m/z643.3216([M-H]-, Δ11.9 mmu); the nuclear magnetic resonance hydrogen spectrum is shown in table 1. The nuclear magnetic resonance carbon spectrum is shown in Table 2. 2D NMRThe correlation and plane structure are shown in FIG. 2, and the ROESY spectrum is shown in FIG. 6.
Cytotoxicity test of kansuine triterpene Compounds Munrepene A-F
The cytotoxicity of the compounds Munrepene A-F on the human non-small cell lung cancer cell strain A549, the human colon cancer cell strain HCT and the mouse mononuclear macrophage leukemia cell strain RAW264.7, the liver cancer cell HepG2, the triple negative breast cancer cell MCF7 and the human ductal breast cancer cell MDAMB was detected by adopting a CCK-8 method, and 100 mu L of cell suspension (2×10 ^5 Individual cells/mL) were inoculated into 96-well microtiter plates and after incubation for 24h, the compounds were added. Compounds Munrepene A-F (5,10,20,40,80,160. Mu.M) were added at various concentrations to the dishes, the blank was added with an equal volume of DMSO, 3 wells were repeated for each group, and incubation was performed for 24h, with 10. Mu.L of CCK-8 reagent added to each well. After 1h, the absorbance at 450nm was measured with a microplate reader, and the cell viability was calculated.
TABLE 1 Nuclear magnetic resonance Hydrogen Spectrometry 1 H NMR data for munropenes A-D in CD 3 OD)
TABLE 2 Nuclear magnetic resonance carbon Spectrum [ (ll) 13 C NMR data for munropenes A-D in CD 3 OD)
TABLE 3 Nuclear magnetic resonance carbon Spectrometry and Hydrogen Spectrometry 1 H and 13 C NMRdata for munropenesE-F in CD 3 OD)
TABLE 4 cytotoxicity (IC) 50 (μM))
| Compounds | HCT116 | A549 | HepG2 | MCF7 | MDAMB |
| Munropene A | 19.13 | >160 | >160 | >160 | >160 |
| Munropene B | 40.90 | >160 | >160 | >160 | >160 |
| Munropene C | >160 | >160 | >160 | >160 | >160 |
| Munropene D | 17.66 | >160 | >160 | >160 | >160 |
| Munropene E | 57.90 | >160 | >160 | >160 | >160 |
| Munropene F | 32.62 | >160 | >160 | >160 | >160 |
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. An kansuine type triterpene compound with an anti-tumor effect is characterized in that the kansuine type triterpene compound has one structure of Munrepene A, munrepene B and Munrepene C, wherein the Munrepene A is shown as a formula (I), the Munrepene B is shown as a formula (II), and the Munrepene C is shown as a formula (III):
2. the kansuine type triterpene compound with anti-tumor effect according to claim 1, wherein the preparation method of the Munropene a comprises the following steps: s1, adding 95% ethanol into dried dwarf gyroscope powder according to a feed liquid ratio of 1 kg:10-15L, soaking and extracting for 2-4 times at room temperature, merging filtrate for 1 time every 3 days, filtering and concentrating to obtain extract, sequentially extracting the extract by petroleum ether, ethyl acetate and water with equal volumes respectively, subjecting a water layer to macroporous resin column chromatography with the weight of 45-55 times of the water layer, sequentially eluting by 2.0-3.0L of 20% -80% ethanol respectively, and collecting 40% ethanol part eluent to obtain Fr3;
s2, performing silica gel column chromatography by taking Fr3 with the weight of 100 times of that of the Fr3, performing gradient elution by adopting dichloromethane-methanol with the volume ratio of 80:20-100:0 as a mobile phase, collecting 1 test tube every 10-15 ml, and observing and combining eluent containing the same components by using a TLC (thin-layer chromatography) plate every test tube to obtain 20 fractions of Fr 3.1-Fr 3.20;
s3, taking Fr3.3, purifying by adopting a C18 high performance liquid chromatography column and acetonitrile-water-formic acid with a volume ratio of 18:82:0.1 as a mobile phase, and keeping for 20-25 min to obtain Munrepene A.
3. The kansuine type triterpene compound with anti-tumor effect according to claim 1, wherein the preparation method of the Munropene B comprises the following steps: s1, adding 95% ethanol into dried dwarf turbinate powder according to a feed liquid ratio of 1 kg:10-15L, soaking and extracting for 2-4 times at room temperature, 1 time every 3 days, merging filtrate, filtering and concentrating to obtain extract, sequentially extracting the extract by petroleum ether, ethyl acetate and water with equal volumes respectively, sequentially eluting a water layer by using macroporous resin column chromatography with the weight of 45-55 times of the water layer, sequentially eluting by using 2.0-3.0L of 20-80% ethanol, collecting an 80% ethanol eluting part, sequentially eluting by using gel column chromatography with the weight of 100 times of the water layer again, and continuously eluting by using 100% methanol for 1.0-2.0L to obtain Fr2;
s2, performing C18 column chromatography with the weight of 100 times of Fr2, performing gradient elution with methanol-water at the volume ratio of 30:70-50:50 of 1.0-1.5L, collecting 1 test tube every 10-15 ml, and observing and combining the eluents containing the same components by TLC (thin layer chromatography) plates in each test tube to obtain 7 fractions of Fr 2.1-Fr 2.7;
s3, subjecting Fr2.1 to silica gel column chromatography with the weight of 100 times of the Fr2.1, eluting with methylene dichloride-methanol at the volume ratio of 4:1, collecting 1 test tube per 10-15 mL, and observing and combining the eluents containing the same components by TLC (thin layer chromatography) plates to obtain 5 components such as Fr 2.1.1-Fr 2.1.5;
s4, taking Fr2.1.2, adopting a high-speed countercurrent chromatography method, adopting 1.5-2.5L of dichloromethane-methanol-water as a mobile phase, reversely pushing the solution, collecting 1 test tube per 20-25 ml of mobile phase, and combining the same components through a TLC (thin layer chromatography) point plate to obtain 6 components of Fr 2.1.2.1-Fr 2.1.2.6 and the like, wherein the component Fr2.1.2.3 is a monomer compound, namely Munrepene B.
4. The kansuine type triterpene compound with anti-tumor effect according to claim 1, wherein the preparation method of the Munropene C comprises the following steps: s1, adding 95% ethanol into dried dwarf turbinate powder according to a feed liquid ratio of 1 kg:10-15L, soaking and extracting for 2-4 times at room temperature, 1 time every 3 days, merging filtrate, filtering and concentrating to obtain extract, sequentially extracting the extract by petroleum ether, ethyl acetate and water with equal volumes respectively, sequentially eluting a water layer by using macroporous resin column chromatography with the weight of 45-55 times of the water layer, sequentially eluting by using 2.0-3.0L of 20-80% ethanol, collecting an 80% ethanol eluting part, sequentially eluting by using gel column chromatography with the weight of 100 times of the water layer again, and continuously eluting by using 100% methanol for 1.0-2.0L to obtain Fr2;
s2, performing C18 column chromatography with the weight of 100 times of Fr2, performing gradient elution with methanol-water at the volume ratio of 30:70-50:50 of 1.0-1.5L, collecting 1 test tube every 10-15 ml, and observing and combining the eluents containing the same components by TLC (thin layer chromatography) plates in each test tube to obtain 7 fractions of Fr 2.1-Fr 2.7;
s3, taking Fr2.3, adopting 550-650 mL of dichloromethane-methanol-water as a mobile phase in a volume ratio of 2:2:1, collecting 1 test tube every 10-15 mL, and observing and combining eluent containing the same components by a TLC (thin layer chromatography) plate in each test tube to obtain 3 fractions of Fr 2.3.1-Fr 2.3.3; taking Fr2.3.3, preparing a high performance liquid chromatography, using a C18 preparation column, adopting acetonitrile-water-formic acid as a mobile phase in a volume ratio of 23:77:0.1, and preparing and purifying for 10-15 min to obtain Munrepene C.
5. The use of an anti-tumor kansui type triterpene compound according to any one of claims 1 to 4 for preparing a medicament for treating colon cancer.
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