CN116554258A - Lonicera macranthoides triterpenoid saponin compound and preparation method and application thereof - Google Patents
Lonicera macranthoides triterpenoid saponin compound and preparation method and application thereof Download PDFInfo
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- CN116554258A CN116554258A CN202310599087.8A CN202310599087A CN116554258A CN 116554258 A CN116554258 A CN 116554258A CN 202310599087 A CN202310599087 A CN 202310599087A CN 116554258 A CN116554258 A CN 116554258A
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- lonicera macranthoides
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- 241001170076 Lonicera macranthoides Species 0.000 title claims abstract description 42
- -1 triterpenoid saponin compound Chemical class 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims description 13
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 9
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 7
- 239000002994 raw material Substances 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 68
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- 238000000605 extraction Methods 0.000 claims description 18
- 239000003480 eluent Substances 0.000 claims description 12
- 229930182490 saponin Natural products 0.000 claims description 12
- 235000017709 saponins Nutrition 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 11
- 239000011347 resin Substances 0.000 claims description 11
- 229920005989 resin Polymers 0.000 claims description 11
- 150000007949 saponins Chemical class 0.000 claims description 11
- 150000001875 compounds Chemical class 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 239000003463 adsorbent Substances 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 238000001179 sorption measurement Methods 0.000 claims description 4
- 238000003809 water extraction Methods 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical group O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 238000002386 leaching Methods 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 201000010989 colorectal carcinoma Diseases 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 6
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- 229930014626 natural product Natural products 0.000 abstract description 2
- 150000002611 lead compounds Chemical class 0.000 abstract 1
- 238000012216 screening Methods 0.000 abstract 1
- 229940125904 compound 1 Drugs 0.000 description 17
- 229910052739 hydrogen Inorganic materials 0.000 description 11
- 239000012071 phase Substances 0.000 description 9
- 239000002253 acid Substances 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 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 description 4
- 239000000047 product Substances 0.000 description 4
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 3
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 3
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229960002949 fluorouracil Drugs 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 150000002772 monosaccharides Chemical group 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000208828 Caprifoliaceae Species 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 201000000297 Erysipelas Diseases 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 241000245240 Lonicera Species 0.000 description 1
- 241001570521 Lonicera periclymenum Species 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- BMWPBKOFJSHJAW-UHFFFAOYSA-N Saponin B Natural products CC1(C)CCC2(CCC3(C)C(=CCC4C5(C)CCC(OC6OC(CO)C(O)C(OC7OC(CO)C(O)C(O)C7O)C6=O)C(C)(C)C5CCC34C)C2C1)C(=O)O BMWPBKOFJSHJAW-UHFFFAOYSA-N 0.000 description 1
- ABRVLXLNVJHDRQ-UHFFFAOYSA-N [2-pyridin-3-yl-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound FC(C1=CC(=CC(=N1)C=1C=NC=CC=1)CN)(F)F ABRVLXLNVJHDRQ-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 150000001336 alkenes Chemical group 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 125000000089 arabinosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)CO1)* 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229940096919 glycogen Drugs 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
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- JUJWROOIHBZHMG-RALIUCGRSA-N pyridine-d5 Chemical compound [2H]C1=NC([2H])=C([2H])C([2H])=C1[2H] JUJWROOIHBZHMG-RALIUCGRSA-N 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000004044 tetrasaccharides Chemical group 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229930182493 triterpene saponin Natural products 0.000 description 1
- 150000008130 triterpenoid saponins Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the field of natural pharmaceutical chemistry, in particular to a novel triterpenoid saponin compound which is obtained by taking dried flowers of lonicera macranthoides as raw materials and extracting and separating the dried flowers by a natural product chemical method, has a strong inhibition effect on Hela, HTC-116, B16 and MDA-MB-468 tumor cells, and can provide a novel lead compound for screening anti-tumor drugs.
Description
Technical Field
The invention relates to the field of natural products, in particular to a lonicera macranthoides triterpenoid saponin compound and a preparation method and application thereof.
Background
The lonicera macranthoides (Lonicera macranthoides hand-Mazz.) is vine of lonicera of Caprifoliaceae, is a special species of China, has the effects of clearing heat and detoxicating and dispelling wind and heat, and can be used for treating carbuncle and furuncle, pharyngitis, erysipelas, heat toxin bloody dysentery, wind-heat type common cold and warm disease fever. Triterpenoid saponins are characteristic components of lonicera macranthoides and are mainly present in lonicera macranthoides flower buds (Li Jin et al, northern pharmacy, 2014, 11 (02): 71-73.). A plurality of saponin compounds in the lonicera macranthoides have antitumor activity, such as lonicera macranthoides secondary saponin B which has obvious in-vitro and in-vivo antitumor activity (Wang, J et al food chem. Toxicol.47,1716-1721; shan, Y., et al Nutr. Cancer.68, 280-289). Therefore, the analysis of the antitumor active ingredients of the lonicera macranthoides is further excavated, and a new anticancer drug for preventing and treating tumor growth is developed.
Disclosure of Invention
The invention aims to provide a lonicera macranthoides triterpenoid saponin compound, a preparation method thereof and application thereof in anti-tumor drugs.
In order to achieve the purpose of the invention, the technical scheme is as follows:
a triterpene saponin compound of lonicera macranthoides, the chemical name of which is 3-O-beta-D-glucopyranosyl- (1- & gt 4) -beta-D-glucopyranosyl- (1- & gt 3) -alpha-L-rhamnopyranosyl- (1- & gt 2) -alpha-L-arabinopyranosyl-23-hydroxy-ol ean-18-ene-28-oic acid, has the following chemical structural formula:
the compound is prepared by the following method, which comprises the following steps:
s1, preparing total saponins of lonicera macranthoides by taking dried flowers of lonicera macranthoides as raw materials, and then performing reverse phase silica gel column chromatography, wherein an elution system is ethanol-water;
s2, guiding the product obtained in the step S1 by taking liquid quality as a guide, and preparing the compound by using gel column chromatography and preparation liquid chromatography, wherein an elution system is methanol-water.
Further, in S1, ethanol is taken as an eluting system: the water ratio is 60:40 is subjected to step S2.
Further, in S2, a gel column eluent system methanol is taken: the elution fraction with the water ratio of 35:65 enters liquid chromatography, and the eluent system methanol is taken: the eluting fraction, having a water ratio of 40:60, gives the compound.
In a specific embodiment, the reverse phase silica gel column chromatography is selected from the group consisting of C18 reverse phase column and gel column chromatography is selected from the group consisting of Sephadex LH-20.
Further, the preparation method of the lonicera macranthoides total saponins comprises the following steps: using Lonicera macranthoides Lonicera macranthoides hand-Mazz dried flower as raw material, and using water, alcohol or mixture of alcohol and water to obtain extractive solution by reflux method, water extraction method or cold leaching method; subjecting the extractive solution to macroporous adsorbent resin column chromatography, eluting with ethanol-water.
Further, macroporous adsorption resin column chromatography is used, and ethanol in an elution system is taken: the water ratio is 70:30-75:15 to obtain the lonicera macranthoides total saponins.
Further, when alcohol or an alcohol-water mixture is used for extraction, the method further comprises concentrating the extract before the extract enters the macroporous adsorption resin column for chromatographic separation.
Further, the extraction temperature of the extracting solution is 25-100 ℃ at room temperature, the extraction times are 2-3 times, and the extraction time is 2-7 d each time.
The alcohol is small molecular alcohol, preferably methanol or ethanol. In one embodiment, when an alcohol-water mixture is used, a mixture of ethanol and water is preferred, with a volume ratio of 70% ethanol to 95% ethanol. In one embodiment, when using a reflux method or a water extraction method, the extraction temperature is 80 to 100 ℃ each time and the extraction time is 2 to 3 hours each time. In another embodiment, when cold extraction is used, the extraction temperature is room temperature each time and the extraction time is 7d each time.
Preferably, the macroporous adsorbent resin is selected from any one or more of D101, AB-8 and HP-20. More preferably, the macroporous adsorbent resin is selected from D101.
The invention also provides application of the compound in preparing antitumor drugs. Further, the tumor is cervical cancer, colorectal cancer, melanoma or breast cancer. The antitumor drug comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises the compound.
The beneficial effects of the invention are as follows: the lonicera macranthoides triterpenoid saponins compound discovered for the first time can inhibit the growth of tumor cells in vitro by measuring the tumor cell inhibition activity, has good effect of measuring the cell inhibition activity of Hela, HTC-116, B16 and MDA-MB-468 tumor cells, and has the potential of developing antitumor drugs.
Drawings
Fig. 1 is a key HMBC signal diagram of the lonicera macranthoides triterpenoid saponins compound provided by the invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto, and various substitutions and modifications can be made by those skilled in the art without departing from the technical spirit of the present invention, and are intended to be included in the scope of the present invention.
Example 1 preparation and identification of Compound 1 of interest
Reflux-extracting dried flos Lonicerae with 70% ethanol water solution at 80deg.C for 3 times (each for 2 hr), mixing extractive solutions, and vacuum filtering with Buchner funnel to obtain filtrate;
then, the filtrate is distilled in a rotary way to obtain 96.4g of brown gelatinous crude extract without alcohol taste;
separating the crude extract with D101 macroporous resin column, eluting with ethanol: the water ratio is 0:100,30:70,70:30,95:5, 4 main fractions (Fr.I-Fr.IV) are obtained, wherein Fr.III is total saponins of lonicera macranthoides;
taking ethanol of an elution system: the water ratio was 70:30 (34.2 g) was separated on a C18 reverse phase column, eluting with ethanol: the water ratio is 30:70,40:60,60:40,80:20 in sequence, and four subfractions Fr.III-A-D are obtained;
then taking liquid as a guide, and taking Fr.III-A eluting system ethanol: the water ratio is 60:40 The eluted fraction (16.4 g) was separated with Sephadex LH-20 (eluent system: methanol: water ratio: 35:65) and preparation liquid phase (eluent system: methanol: water ratio: 40:60, respectively) to finally obtain the objective compound 1 (27 mg) provided by the present invention.
The structure of compound 1 of interest was identified using a combination of various spectroscopic techniques (MS, NMR, UV, IR).
The target compound 1 is white powder, is easily dissolved in water, methanol-water, is difficultly dissolved in small polar solvents such as ethyl acetate and the like, and has optical rotation of [ alpha ]]20D-28.91 (c 0.11, methanol), TCL plate developed with vanillin-concentrated sulfuric acid and heated to a bluish purple color suggesting that compound 1 may be a saponated compound. HR-ESI-MS showed an m/z of 1097.5529[ M+Na ]] + (calculated value: 1097.5503), the molecular formula thereof can be determined to be C 53 H 86 O 22 The unsaturation was calculated to be 11.
Target Compound 1 1 In the H-NMR spectrum, delta is shown H 1.09(3H,s,H-24)、δ H 0.86(3H,s,H-25)、δ H 1.01(3H,s,H-26)、δ H 0.88(3H,s,H-27)、δ H 1.11 (3H, s, H3-29) and delta H 1.03 (3H, s, H3-30) is 6 methyl hydrogen signals. 13 In the C-NMR spectrum, delta is shown C 13.9(C-24)、δ C 17.4(C-25)、δ C 16.3(C-26)、δ C 15.2(C-27)、δ C 30.8 (C-29) and delta C 29.3 (C-30) is the 6 corresponding methyl carbon signals. In addition, according to delta C 81.2 (C-3) and delta C 179.3 The chemical shift of (C-28) can confirm that the sugar chain is linked to C-3 of the aglycone. In HMBC spectra, delta H 5.24 Olefin protons and delta of (H, s, H-19) C 41.6(C-13)、δ C 48.5(C-17)、δ C 32.4(C-20)、δ C 34.2(C-21)、δ C 30.8 (C-29) 5 carbon atoms, while delta C 139.1 (C-18) and delta H 2.68 (H, d, H-13) are related, indicating that the double bond is located at C-18 and C-19. Further analysis of 2D-NMR (COSY, ROESY, HSQC and HMBC) revealed that the aglycone moiety of target compound 1 was 23-hydroxylean-18-ene-28-oic acid.
The monosaccharide residue at the C-3 position of Compound 1 of interest is at delta H 5.24(H,d,H-1′)、δ H 6.27(H,s,H-1′)、δ H 5.43 (H, d, H-1') and delta H 5.18 (H, d, H-1') and delta, respectively C 104.8(C-1′)、δ C 101.4(C-1′)、δ C 106.7(C-1′)、δ C 104.9 (C-1 "") indicating that it contains 4 monosaccharides. The trimethylsilyl derivative of the target compound 1 after acid hydrolysis was subjected to gas chromatography-mass spectrometry analysis with raw sugar, and the result showed that the sugar moiety consisted of L-Ara, L-Rha and D-Glc (ratio: 1:1:2). The beta-terminal configuration of the glucose unit is represented by J 1,2 Coupling constants (7.8-8.0 Hz), the alpha-terminal configuration of the arabinose units was determined by J 1,2 The coupling constant (6.27 Hz) determines the alpha-terminal configuration of the rhamnose unit by chemical shift delta C 69.6 (C-5') determination. The order of the four sugar chains at the glycogen C-3 was deduced from the following HMBC correlation: delta of Ara H 5.24 (H, d, H-1') aglycone delta C 81.2 (C-3) correlation, delta of Rha H 6.27 Delta of (H, s, H-1') and Ara C 75.3 (C-2') correlation, delta of GlcI H 5.43 (H, d, H-1') and delta of Rha C 83.5 (C-3') correlation, delta of GlcII H 5.18 Delta of (H, d, H-1 "") and GlcI C 81.1 (C-4') correlation. The above nuclear magnetic data indicate that the sugar moiety of target compound 1 is identical to the tetrasaccharide moiety of macroside B. All will be analyzed by HSQC and HMBC spectra 1 H and 13 and C, attributing nuclear magnetic signals. Finally, the product is identified as 3-O-beta-D-glucopyranosyl- (1.fwdarw.4) -beta-D-glucopyranosyl- (1.fwdarw.3) -alpha-L-rhamnopyranosyl- (1.fwdarw.2) -alpha-L-arabinopyranosyl-23-hydroxy-olean-18-ene-28-oic acid (1). The hydrogen spectrum and carbon spectrum data of the specific target compound 1 are shown in table 1.
TABLE 1 Hydrogen Spectrum and carbon Spectrum data for the target Compound 1 provided by the invention
Table 1 1 H and 13 C NMR spectral data of 1
Data were measured at 600MHz for 1 H and 150MHz for 13 C in Pyridine-d 5 ,δin ppm,J inHz.
LC-MS determines the purity of the target compound 1, LC chromatographic conditions are: column Agilent Poroshell SB-AQ C18 (3.0X100 mm,2.7 um); column temperature is 30 ℃; the mobile phase is 0.1% formic acid solution (A) -methanol (B), gradient elution (0-3 min, 10-45% B, 3-12 min, 45-60% B, 12-18 min, 60-95% B, 18-20 min,95% B) and the flow rate is 0.3 mL.min -1 The method comprises the steps of carrying out a first treatment on the surface of the The sample loading was 5. Mu.L. The mass spectrum conditions are as follows: adopting ESI ion source, ionization mode is negative ion mode, capillary voltage is 3.5kV, drying gas temperature is 350 ℃, and cracking voltage is: the fragmentation voltage is 0-16min,135V;16-30min,175V;30-38min,210V, the second-level voltage is: 0-30min,30V,30-38min,40V, and N as atomizing gas 2 The scanning range of the sub-ions is m/z 100-1700, the atomizing air pressure is 50psi, and the drying air flow rate is high10.0L·min -1 The Skimmer voltage is 65V. 3-O-beta-D-glucopyranosyl- (1.fwdarw.4) -beta-D-glucopyranosyl- (1.fwdarw.3) -alpha-L-rhamnofuranosyl- (1.fwdarw.2) -alpha-L-arabin opyranosyl-23-hydroxy-olean-18-ene-28-oic acid (1) has a retention time of 31.24min and a purity of 98.74% calculated by area normalization.
Example 2 preparation of target Compound 1 Using Water extraction
Taking 1kg of dried flowers of lonicera macranthoides, and extracting with water for three times, wherein the water consumption is 10L each time, the extraction time is 2h each time, and the extraction temperature is 100 ℃ each time;
the obtained extract is adsorbed by macroporous resin HP-20, and the ethanol is eluted: the water ratio is 0:100,30:70,70:30,95:5 in sequence, and the eluent of 70% ethanol is taken for decompression and solvent recovery to obtain 47g of total saponins of lonicera macranthoides;
performing C-18 reverse phase silica gel column chromatography on the obtained total saponins of lonicera macranthoides, and performing mobile phase ethanol: the water ratio is 30:70,40:60,60:40,80:20, 0:100 in sequence;
taking a third fraction of ethanol: the water ratio is 60:40 eluting the fraction, using liquid as guide, separating the fraction by using sephadex LH-20 (eluent system is methanol: water ratio is 35:65) and preparing liquid phase (eluent system is methanol: water ratio is 40:60), obtaining the 3-O-beta-D-glucopyranosyl- (1- > 4) -beta-D-glucopyranosyl- (1- > 3) -alpha-L-rhamnofuranyl- (1- > 2) -alpha-L-arabinopyranosyl-23-hydroxy-ol-18-ene-28-oic acid (1) 12mg, the yield is 0.0012%, and the purity of the product measured by LC-MS is 98.2%.
Example 3: preparation of target Compound 1 Using methanol Cold extraction
Taking 1Kg of dried honeysuckle flower, extracting with methanol for three times, wherein the dosage of methanol is 20 liters each time, the extraction time is 7 days each time, the extraction temperature is room temperature each time, and concentrating the extract into extractum without alcohol smell (dry weight is 210 g);
dissolving the extract with 10 times of water, and filtering with filter paper to remove water insoluble substance;
the obtained filtrate is adsorbed by macroporous resin AB-8, and the ethanol of the elution system is: the water ratio was 0:100,15:75, 75:15, ethanol is taken: the water ratio was 75:15, concentrating the eluent to obtain 54g of lonicera macranthoides total saponins;
performing C-18 reverse phase silica gel column chromatography on the obtained total saponins of lonicera macranthoides, and performing mobile phase ethanol: the water ratio is 30:70,40:60,60:40,80:20, 0:100 in sequence;
taking a third fraction of ethanol: the water is 60:40 eluting the fraction, taking liquid as guide, separating the fraction by using sephadex LH-20 (the eluent system is methanol with the water ratio of 35:65) and preparing a liquid phase (the eluent system is methanol with the water ratio of 40:60), and obtaining the 3-O-beta-D-glucopyranosyl- (1- & gt 4) -beta-D-glucopyranosyl- (1- & gt 3) -alpha-L-rhamnopyranosyl- (1- & gt 2) -alpha-L-arabinopyranosyl-23-hydroxy-ol-18-ene-28-oic acid (1) 13mg, wherein the yield is 0.0013%, and the purity of the product measured by LC-MS is 98.5%.
EXAMPLE 4 determination of tumor cell inhibitory Activity
In this example, MTT method was used to test the activity of the target compound 1 in vitro and 5-fluorouracil, an anticancer drug, was used as a positive control. The tumor cells selected are Hela, HTC-116, B16 and MDA-MB-468. The specific method comprises the following steps:
the tumor cells of Hela, HTC-116, B16 and MDA-MB-468 in logarithmic growth phase were inoculated into 96-well culture plates, respectively, at a density of 5000 cells per 100. Mu.L of each well, and treated with the target compound 1 at a concentration of 10, 5, 2.5, 1.25, 0.625, 0.3125. Mu. Mol/L, at 37℃with 5% CO 2 Is incubated in an incubator. After 72h, 10 μLMTT (5 mg/mL, PBS) was added to each well, and after further incubation in the incubator for 4h, centrifugation was continued at 1000rpm for 5min, taking care to discard the supernatant. 100. Mu.L of DMSO was added to each well, and the wells were shaken for 10min, and the absorbance of each well was measured with an ELISA reader at a wavelength of 570nm and the cell growth inhibition was calculated. The cell growth inhibition rate was calculated as follows:
cell growth inhibition (%) = (1-experimental group OD value/cell control group OD value) ×100
The positive control was 5-fluorouracil at a concentration of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125. Mu. Mol/L. Calculation of IC using DPS software 50 Values. The measurement results are shown in the table2。
TABLE 2 tumor cell inhibitory Activity of target Compound 1 against Hela, HTC-B16 and MDA-MB-468 (IC 50 ,μM)
Claims (10)
1. A lonicera macranthoides triterpenoid saponin compound is characterized in that: the lonicera macranthoides triterpenoid saponin compound has the following chemical structural formula:
2. a preparation method of a lonicera macranthoides triterpenoid saponin compound is characterized by comprising the following steps of: the method comprises the following steps:
s1, preparing total saponins of lonicera macranthoides by using dried flowers of lonicera macranthoides as a raw material, and then performing reverse phase silica gel column chromatography by using ethanol-water as an elution system;
s2, guiding the product obtained in the step S1 by taking liquid quality as a guide, and preparing the compound by using gel column chromatography and preparation liquid chromatography, wherein an elution system is methanol-water.
3. The method for preparing the lonicera macranthoides triterpenoid saponin compound according to claim 2, which is characterized in that: s1, taking ethanol in an elution system: the water ratio is 60:40 is subjected to step S2.
4. The method for preparing the lonicera macranthoides triterpenoid saponin compound according to claim 2, which is characterized in that: s2, taking a gel column eluent system methanol: the elution fraction with the water ratio of 35:65 enters liquid chromatography, and the eluent system methanol is taken: the eluting fraction, having a water ratio of 40:60, gives the compound.
5. The method for preparing the lonicera macranthoides triterpenoid saponin compound according to claim 2, which is characterized in that: the preparation method of the lonicera macranthoides total saponins comprises the following steps: using dried flowers of lonicera macranthoides as raw materials, and using water, alcohol or a mixture of alcohol and water to obtain an extracting solution by a reflux method, a water extraction method or a cold leaching method; subjecting the extractive solution to macroporous adsorbent resin column chromatography, eluting with ethanol-water.
6. The method for preparing the lonicera macranthoides triterpenoid saponin compound according to claim 5, which is characterized in that: macroporous adsorption resin column chromatography is adopted, and ethanol in an eluting system is taken: the eluting fraction with the water ratio of 70:30-75:15 is used for obtaining the lonicera macranthoides total saponins.
7. The method for preparing the lonicera macranthoides triterpenoid saponin compound according to claim 5, which is characterized in that: when alcohol or alcohol-water mixture is used for extraction, the method further comprises concentrating the extract before the extract enters the macroporous adsorption resin column for chromatographic separation.
8. The method for preparing the lonicera macranthoides triterpenoid saponin compound according to claim 5, which is characterized in that: the extraction temperature of the extracting solution is between room temperature and 100 ℃, the extraction times are between 2 and 3, and the extraction time is between 2 and 7 days each time.
9. An application of Lonicera macranthoides triterpenoid saponin compound in preparing antitumor drug is provided.
10. The use of a lonicera macranthoides triterpenoid saponin compound in preparing an antitumor drug according to claim 9, characterized in that: the tumor is cervical cancer, carcinoma of large intestine, melanoma or breast cancer.
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