CN113087757A - Preparation of type B cardiac glycoside with 5 alpha structure and application of type B cardiac glycoside in resisting liver cancer - Google Patents
Preparation of type B cardiac glycoside with 5 alpha structure and application of type B cardiac glycoside in resisting liver cancer Download PDFInfo
- Publication number
- CN113087757A CN113087757A CN202110269244.XA CN202110269244A CN113087757A CN 113087757 A CN113087757 A CN 113087757A CN 202110269244 A CN202110269244 A CN 202110269244A CN 113087757 A CN113087757 A CN 113087757A
- Authority
- CN
- China
- Prior art keywords
- cardiac glycoside
- liver cancer
- butanol
- type
- methanol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229940097217 cardiac glycoside Drugs 0.000 title claims abstract description 22
- 239000002368 cardiac glycoside Substances 0.000 title claims abstract description 22
- 229930002534 steroid glycoside Natural products 0.000 title claims abstract description 22
- 201000007270 liver cancer Diseases 0.000 title claims abstract description 19
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 19
- LPMXVESGRSUGHW-HBYQJFLCSA-N ouabain Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1C[C@@]2(O)CC[C@H]3[C@@]4(O)CC[C@H](C=5COC(=O)C=5)[C@@]4(C)C[C@@H](O)[C@@H]3[C@@]2(CO)[C@H](O)C1 LPMXVESGRSUGHW-HBYQJFLCSA-N 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 31
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000002021 butanolic extract Substances 0.000 claims abstract description 7
- 238000004440 column chromatography Methods 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 6
- 239000000499 gel Substances 0.000 claims abstract description 4
- 210000004024 hepatic stellate cell Anatomy 0.000 claims abstract description 4
- 239000011347 resin Substances 0.000 claims abstract description 4
- 229920005989 resin Polymers 0.000 claims abstract description 4
- 239000000741 silica gel Substances 0.000 claims abstract description 4
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000012071 phase Substances 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 125000002351 beta-D-glucopyranosyloxy group Chemical group 0.000 claims description 2
- 239000002034 butanolic fraction Substances 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000011894 semi-preparative HPLC Methods 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 abstract description 9
- 240000001825 Chimonanthus praecox Species 0.000 abstract description 4
- 239000003560 cancer drug Substances 0.000 abstract description 4
- 235000007519 Chimonanthus praecox Nutrition 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000011161 development Methods 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 238000002953 preparative HPLC Methods 0.000 abstract description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 abstract 2
- 241000693930 Helleborus thibetanus Species 0.000 abstract 1
- 241000218201 Ranunculaceae Species 0.000 abstract 1
- 230000009982 effect on human Effects 0.000 abstract 1
- 229910052742 iron Inorganic materials 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000000843 powder Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- -1 phytoecdysone Chemical class 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000003527 anti-angiogenesis Effects 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
- 230000001644 anti-hepatocarcinoma Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000935235 Fritillaria meleagris Species 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- ZSBXGIUJOOQZMP-UHFFFAOYSA-N Isomatrine Natural products C1CCC2CN3C(=O)CCCC3C3C2N1CCC3 ZSBXGIUJOOQZMP-UHFFFAOYSA-N 0.000 description 1
- ZSBXGIUJOOQZMP-JLNYLFASSA-N Matrine Chemical compound C1CC[C@H]2CN3C(=O)CCC[C@@H]3[C@@H]3[C@H]2N1CCC3 ZSBXGIUJOOQZMP-JLNYLFASSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940109262 curcumin Drugs 0.000 description 1
- 235000012754 curcumin Nutrition 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 201000003146 cystitis Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 238000012826 global research Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229930014456 matrine Natural products 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000005856 steroid saponins Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 208000000143 urethritis Diseases 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
- C07J19/005—Glycosides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a type B cardiac glycoside with a 5 alpha structure, which has the following structure:
Description
Technical Field
The invention belongs to the field of natural medicinal chemistry, and relates to a compound separated and extracted from chopsticks, a preparation method thereof and application thereof in preparing anti-liver cancer medicaments.
Background
According to the statistics of the world health organization, the death cases of the liver cancer account for 8.2 percent of the death cases of the cancer all over the world. In recent years, the incidence and mortality of liver cancer have increased year by year, and liver cancer has become one of the major diseases impeding social development, so the prevention and treatment of liver cancer has become one of the global research hotspots. As one of malignant tumors with high morbidity and mortality, the traditional Chinese medicine composition has the problems of high postoperative recurrence rate, easy metastasis of cancer cells, multi-drug resistance, low body immunity, poor treatment prognosis and the like; currently, an effective treatment means is lacked, so that a novel anti-liver cancer drug which is safe, efficient and low in toxicity is urgently searched. In recent years, more and more researches prove that natural products play an anti-tumor role through a plurality of molecular mechanisms, such as matrine, thunberg fritillary bulb, quercetin, curcumin and the like. The active ingredients of the natural products have the characteristics of preferentially killing cancer cells, small toxic and side effects, difficult generation of drug resistance and the like, and are a hotspot for researching novel anti-liver cancer drugs.
The Chimonanthus praecox is a specific plant in China, is mainly distributed in northwest Sichuan, south Gansu and south Shanxi, and is mainly used for treating diseases such as activating blood circulation to dissipate blood stasis, urethritis, cystitis, sores, furuncles, pyogenic infections and the like. In recent years, the chemical components of the chopsticks mainly comprise steroid saponin, flavone, phytoecdysone, fatty acid and B-type cardiac glycoside, and the chopsticks have good biological activity including anti-angiogenesis activity. Therefore, the separation of the compound with anti-angiogenesis function from the chopsticks plays an important role in the research and development of novel anti-angiogenesis medicines.
Disclosure of Invention
The invention aims to provide an B-type cardiac glycoside compound with a 5 alpha structure, which has the effect of resisting liver cancer.
Another object of the present invention is to provide a method for preparing the aforementioned type b cardiac glycoside.
The invention also aims to solve the problem of application of the type B cardiac glycoside in anti-liver cancer drugs.
The invention relates to a specific technical scheme.
A cardiac glycoside B has the following structural formula:
is named as: 14 beta, 16 beta-dihydroxy-3 beta- (beta-D-glucopyranosyloxy) -5a-bufa-20,22-dienolide
The preparation method of the B cardiac glycoside compound comprises the following steps:
(1) naturally drying the collected rhizome parts of the Chimonanthus praecox plant, crushing, soaking in 95% ethanol, and concentrating the soaking solution under reduced pressure to obtain a concentrated solution;
(2) preparing the concentrated solution into a turbid water solution, extracting with n-butanol, and concentrating the organic phase to obtain an n-butanol extract;
(3) performing D101 macroporous resin column chromatography on the n-butanol extract, eluting with water, 30%, 50% and 95% ethanol respectively, and finally distilling and concentrating respectively to obtain four fractions;
(4) subjecting 50% n-butanol fraction of radix Chimonanthi Praecocis to MCI reversed phase column chromatography, eluting with ethanol: gradient elution with water (35%, 50%, 60%, 80%) to obtain four components Fr.1-Fr.4;
(5) fr.1 was first separated on a normal phase silica column, purified with dichloromethane: gradient eluting with methanol (15:1, 8:1, 5:1, 2:1) to obtain five components Fr.1.1-Fr.1.5;
(6) continuously separating Fr.1.2 in reverse phase chromatographic column by gradient elution with methanol-water eluent to obtain five components Fr.1.2.1-Fr.1.2.5;
(7) fr.1.2.3 purification was repeated further on a gel column (dichloromethane: methanol 1:1) and a silica gel column (dichloromethane: methanol 13:1, 8:1, 5:1), and finally on a semi-preparative hplc methanol: eluting with 68:32 solvent, and detecting at 280 nm; collecting components at 320nm for 6.4min to obtain pure B cardiac glycoside.
In the step (1), the chopsticks are crushed until the particle size diameter is less than 3 mm.
In the step (1), the volume ratio of the chopsticks to 95% ethanol is 1:2-1: 3.
In the step (1), soaking is carried out for four times, and the soaking time is one week each time.
In the step (2), the volume ratio of the concentrated solution to water is 1:3-1: 4.
In the step (2), the volume ratio of the n-butanol to the turbid water solution is 3:1, and the extraction times are 3-4.
In step (7), the flow rate was 3 mL/min.
An application of type B cardiac glycoside with 5 alpha structure in preparing medicine for treating liver cancer is disclosed.
The invention has the advantages and beneficial effects that:
(1) the chopsticks are a single plant peculiar to China and are traditional Chinese herbal medicines;
(2) the B cardiac glycoside compound obtained by the invention has good anti-liver cancer activity and low toxicity to normal cells;
(3) the preparation method of the extract is simple and convenient to operate and low in cost.
Drawings
FIG. 1 is a structural formula of a compound;
FIG. 2 is a graph of hydrogen (600MHz) and carbon (150MHz) spectra (CDOD) data for the compound of FIG. 13)；
FIG. 3 is the activity test of the compound shown in FIG. 1 on liver cancer cell HepG 2;
FIG. 4 is a graph showing the activity of the compound shown in FIG. 1 on rat hepatic stellate cell HSC-T6.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples. The experimental procedures in the examples, unless otherwise specified, were carried out by conventional techniques in the art and the experimental reagents were all purchased commercially.
Example 1 preparation of n-butanol extract of Chimonanthus praecox
The collected rootstock parts of the chopsticks are naturally dried in the air, and 7.5kg of the rootstock parts of the chopsticks are weighed. Pulverizing to particle size of less than 3mm, soaking in 95% ethanol for 7 days for 4 times (15L each time). Removing solvent from all soaking solutions with rotary evaporator to obtain concentrated solution 4L; adding 12L ethyl acetate into the concentrated solution for extraction, extracting for 4 times, and collecting the extracted organic phase (ethyl acetate phase); adding n-butanol into the water phase, extracting for four times to obtain n-butanol phase, and discarding the water phase. The n-butanol extract was concentrated under reduced pressure to constant weight using a rotary evaporator to give a brownish red slurry as a crude extract (650 g).
EXAMPLE 2 preparation of type B cardiac glycoside
Concentrating n-butanol phase of radix Chimonanthi Praecocis to 500mL to obtain dark brown red viscous liquid, performing column chromatography with D101 macroporous resin, performing gradient elution with water, 30%, 50% and 95% ethanol, and distilling and concentrating to obtain four fractions. The n-butanol 50% fraction (65.8g) was subjected to MCI reverse phase column chromatography using ethanol: gradient elution with water (35%, 50%, 60%, 80%) gave four fractions Fr.1-Fr.4. Fr.1 normal phase silica gel column chromatography is first performed with dichloromethane: methanol (15:1, 8:1, 5:1 and 2:1) gradient elution gave five fractions Fr.1.1-Fr.1.5. Separating Fr.1.2(800mg) on reverse phase chromatography column with methanol-water eluent gradient to obtain five components Fr.1.2.1-Fr.1.2.5. Fr.1.2.3 gel column (dichloromethane: methanol 1:1), silica gel column (dichloromethane: methanol 13:1, 8:1, 5:1) further repeated purification, and semi-preparative high performance liquid chromatography (chromatography column YMC-Pack ODS-A10 mm × 250mm, flow rate 3.0mL/min, mobile phase methanol: water 68:32) purification with detection wavelength of 280 nm; collecting components at 320nm for 6.4min to obtain pure B cardiac glycoside.
The compound is white solid, is easily soluble in chloroform, methanol and DMSO, is hardly soluble in water, and has a molecular formula of C30H44O10。
Subjecting the isolated compound to1H NMR、13C NMR analysis, in contrast to literature, determines the structure.1H and13the C NMR data are shown in FIG. 2, and the structure of the isolated compound is shown in FIG. 1.
Example 3 anti-hepatoma Activity of type B cardiac glycoside Compounds
Adding 2.05mm/L glutamine and 10% FBS into liver cancer cell HepG2 and rat hepatic stellate cell HSC-T6RPMI-1640 medium at 37 deg.C in 5% CO2Culturing in a humidified environment. Cells were isolated from the flask with trypsin EDTA. After dilution to 5X 10 with complete medium4after/mL, 100 μ L of cell suspension was added to each well of a 96-well culture plate. Followed by incubation at 37 ℃ in an atmosphere of 5% carbon dioxide for 24 h. Test compounds at the indicated concentrations were added to each well and then incubated for 48 h. Doxorubicin served as a positive control. The attached cells were fixed with cold 50% trichloroacetic acid for 30min and then stained with 0.04% SRB for 30 min. After dissolving the bound SRB in 10mm Tris medium, absorbance at 450nm was measured using a Tecan multi-functional microplate reader.
The anti-hepatocarcinoma activity of type B cardiac glycoside is shown in fig. 3 and 4.
The experimental results show that the compound inhibits the survival rate of HepG2 cells at the concentration of 200 mu M; the cell line has concentration dependence on HSC-T6 cells to inhibit the survival rate, and the inhibition effect is obvious.
Claims (9)
1. A cardiac glycoside B compound with 5 alpha structure is named as:
14 beta, 16 beta-dihydroxy-3 beta- (beta-D-glucopyran-osyloxy) -5a-bufa-20,22-dienolide, the structural formula:
2. the method of claim 1, wherein the method comprises the steps of:
(1) pulverizing rhizome of radix Chimonanthi Praecocis, soaking in 95% ethanol, and concentrating the soaking solution under reduced pressure to obtain concentrated solution;
(2) preparing the concentrated solution into a turbid water solution, extracting with n-butanol, and concentrating the organic phase to obtain an n-butanol extract;
(3) performing D101 macroporous resin column chromatography on the n-butanol extract, eluting with water, 30%, 50% and 95% ethanol respectively, and finally distilling and concentrating respectively to obtain four fractions;
(4) subjecting 50% n-butanol fraction of radix Chimonanthi Praecocis to MCI reversed phase column chromatography, eluting with ethanol: gradient elution with water (35%, 50%, 60%, 80%) to obtain four components Fr.1-Fr.4;
(5) fr.1 was first separated on a normal phase silica column, purified with dichloromethane: gradient eluting with methanol (15:1, 8:1, 5:1, 2:1) to obtain five components Fr.1.1-Fr.1.5;
(6) continuously separating Fr.1.2 in reverse phase chromatographic column by gradient elution with methanol-water eluent to obtain five components Fr.1.2.1-Fr.1.2.5;
(7) fr.1.2.3 purification was repeated further on a gel column (dichloromethane: methanol 1:1) and a silica gel column (dichloromethane: methanol 13:1, 8:1, 5:1), and finally on a semi-preparative hplc methanol: eluting with 68:32 solvent, and detecting at 280 nm; collecting components at 320nm for 6.4min to obtain pure B cardiac glycoside.
3. The method according to claim 2, wherein in the step (1), the chopsticks are crushed to a particle size diameter of less than 3 mm.
4. The preparation method according to claim 2, wherein in the step (1), the volume ratio of the chopsticks to 95% ethanol is 1:2-1: 3.
5. The method according to claim 2, wherein in the step (1), the soaking is performed four times, and each soaking time is one week.
6. The method according to claim 2, wherein in the step (2), the volume ratio of the concentrate to water is 1:3 to 1: 4.
7. The method according to claim 2, wherein in the step (2), the volume ratio of n-butanol to the aqueous turbid solution is 3:1, and the number of extraction times is 3 to 4.
8. Use of the type b cardiac glycoside of claim 1 in the preparation of an anti-liver cancer medicament.
9. The use of claim 8, wherein the cells are human hepatoma cells (HepG2) and rat hepatic stellate cells (HSC-T6).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110269244.XA CN113087757A (en) | 2021-03-12 | 2021-03-12 | Preparation of type B cardiac glycoside with 5 alpha structure and application of type B cardiac glycoside in resisting liver cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110269244.XA CN113087757A (en) | 2021-03-12 | 2021-03-12 | Preparation of type B cardiac glycoside with 5 alpha structure and application of type B cardiac glycoside in resisting liver cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113087757A true CN113087757A (en) | 2021-07-09 |
Family
ID=76667236
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110269244.XA Pending CN113087757A (en) | 2021-03-12 | 2021-03-12 | Preparation of type B cardiac glycoside with 5 alpha structure and application of type B cardiac glycoside in resisting liver cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113087757A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008040548A2 (en) * | 2006-10-04 | 2008-04-10 | Ares Trading S.A. | Treatment for non-alcoholic-steatohepatitis |
| CN103254263A (en) * | 2013-05-31 | 2013-08-21 | 贵州省中国科学院天然产物化学重点实验室 | Cardiac glycoside compound and preparation method as well as application thereof |
| CN104817609A (en) * | 2015-04-01 | 2015-08-05 | 江苏省中医药研究院 | Notoginsenoside compound with liver cancer-resistant activity and its preparation method and use |
| CN106714809A (en) * | 2014-08-20 | 2017-05-24 | 耶鲁大学 | Novel compositions and methods useful for treating or preventing liver diseases or disorders, and promoting weight loss |
| CN109438542A (en) * | 2018-09-04 | 2019-03-08 | 天津科技大学 | A kind of novel cardiac glycosides monomeric compound and its purposes in the preparation of antitumor drugs |
| US20190350923A1 (en) * | 2017-01-31 | 2019-11-21 | Medizinische Hochschule Hannover (Mhh) | Natural compounds and fibrosis |
-
2021
- 2021-03-12 CN CN202110269244.XA patent/CN113087757A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008040548A2 (en) * | 2006-10-04 | 2008-04-10 | Ares Trading S.A. | Treatment for non-alcoholic-steatohepatitis |
| CN103254263A (en) * | 2013-05-31 | 2013-08-21 | 贵州省中国科学院天然产物化学重点实验室 | Cardiac glycoside compound and preparation method as well as application thereof |
| CN106714809A (en) * | 2014-08-20 | 2017-05-24 | 耶鲁大学 | Novel compositions and methods useful for treating or preventing liver diseases or disorders, and promoting weight loss |
| CN104817609A (en) * | 2015-04-01 | 2015-08-05 | 江苏省中医药研究院 | Notoginsenoside compound with liver cancer-resistant activity and its preparation method and use |
| US20190350923A1 (en) * | 2017-01-31 | 2019-11-21 | Medizinische Hochschule Hannover (Mhh) | Natural compounds and fibrosis |
| CN109438542A (en) * | 2018-09-04 | 2019-03-08 | 天津科技大学 | A kind of novel cardiac glycosides monomeric compound and its purposes in the preparation of antitumor drugs |
Non-Patent Citations (3)
| Title |
|---|
| FENG-YING YANG 等: "Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus(Ranunculaceae)", 《BIOCHEMICAL SYSTEMATICS AND ECOLOGY》 * |
| LONG MA 等: "A cardiac glycoside HTF-1 isolated fromHelleborus thibetanusFranch displays potentin vitro anti-cancer activity via caspase-9, MAPK and PI3K-Akt-mTOR pathways", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY 》 * |
| 杨凤英 等: "小桃儿七螺甾烷皂苷及其抗肿瘤细胞活性", 《济南大学学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101863871B (en) | Total glycosides of Rhodiola rosea, medical application and preparation method thereof | |
| EP3412679A1 (en) | Baicalin magnesium, preparation method thereof and application of same | |
| Dang et al. | Antioxidative extracts and phenols isolated from Qinghai–Tibet Plateau medicinal plant Saxifraga tangutica Engl. | |
| CN104370871B (en) | The mouth diphenylene ketone oxide class separated from Swertia punicea Hemsl. and the application of suppression hepatitis B virus | |
| CN101824067A (en) | Barrigenol-type triterpenoid saponins compound, preparation method and application thereof | |
| CN105017345B (en) | The method of four kinds of compounds of extraction and the application simultaneously from selfheal | |
| CN102219686A (en) | Caffeoyl derivative and use of coffeeoyl derivative in preparing drugs against respiratory syncytial viruses | |
| CN113264974B (en) | Preparation of type B cardiac glycoside and anti-angiogenesis application thereof | |
| CN101703669B (en) | Smilax china effective fractions and extraction as well as purification process thereof | |
| CN113087757A (en) | Preparation of type B cardiac glycoside with 5 alpha structure and application of type B cardiac glycoside in resisting liver cancer | |
| CN102190692A (en) | Preparation method and medical purpose of novel dihydro-flavonoid | |
| CN112898357B (en) | Diterpene glycoside novel compound in trollius chinensis bunge and separation and purification method and application thereof | |
| CN106905278B (en) | A method of 5 kinds of separation preparation has antiinflammatory active compound from falcate crazyweed herb | |
| CN105348247A (en) | Isocoumarin compound, and preparation method and application thereof | |
| CN112194704B (en) | Steroid saponin compound and preparation method and application thereof | |
| CN104262316A (en) | Flavonoid compound as well as preparation method and application thereof | |
| CN109091602B (en) | Effective component of semen allii tuberosi, extraction method and application thereof in preparing liver injury protection medicine | |
| CN102389456A (en) | Method for extracting isodon japonica var.galaucocalyx total diterpenoids or Glaucocalyxin A | |
| CN103113196B (en) | Glechoma longituba phenol, and preparation method and application thereof | |
| CN106860624B (en) | Cimicifugae rhizoma extract, two cimicifugae flavone bases, and preparation method and application thereof | |
| CN110522776A (en) | It is a kind of Huang the rose of Sharon in anti-tumor effective component preparation method and application | |
| CN110698444A (en) | Phenylpropanoid compound and preparation method thereof | |
| CN1128138C (en) | Carbazole alkaloid derivative and its prepn and use | |
| CN106860771B (en) | Preparation method of rhizoma gastrodiae refined component, rhizoma gastrodiae refined component and application | |
| CN110251552B (en) | Preparation method of total flavonoids of sauropus spatulifolius |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210709 |