CN116903578A - Phenolic acid compound in Glechoma hederacea as well as extraction and separation method and application thereof - Google Patents
Phenolic acid compound in Glechoma hederacea as well as extraction and separation method and application thereof Download PDFInfo
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- -1 Phenolic acid compound Chemical class 0.000 title claims abstract description 18
- 238000000605 extraction Methods 0.000 title claims abstract description 16
- 241001136705 Glechoma hederacea Species 0.000 title claims abstract description 14
- 235000011755 Nepeta hederacea Nutrition 0.000 title claims abstract description 14
- 238000000926 separation method Methods 0.000 title claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 20
- 239000003814 drug Substances 0.000 claims abstract description 11
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 7
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 7
- 206010033128 Ovarian cancer Diseases 0.000 claims abstract description 7
- 206010061535 Ovarian neoplasm Diseases 0.000 claims abstract description 7
- 208000005718 Stomach Neoplasms Diseases 0.000 claims abstract description 7
- 208000029742 colonic neoplasm Diseases 0.000 claims abstract description 7
- 206010017758 gastric cancer Diseases 0.000 claims abstract description 7
- 201000007270 liver cancer Diseases 0.000 claims abstract description 7
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 7
- 201000005202 lung cancer Diseases 0.000 claims abstract description 7
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 7
- 201000011549 stomach cancer Diseases 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 5
- 239000003480 eluent Substances 0.000 claims description 29
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 10
- 239000002024 ethyl acetate extract Substances 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- 206010028980 Neoplasm Diseases 0.000 claims description 7
- 239000012141 concentrate Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000004809 thin layer chromatography Methods 0.000 claims description 6
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 5
- 201000011510 cancer Diseases 0.000 claims description 4
- 238000007689 inspection Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000004007 reversed phase HPLC Methods 0.000 claims description 4
- 238000010898 silica gel chromatography Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
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- 239000004480 active ingredient Substances 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 239000002253 acid Substances 0.000 abstract description 26
- 230000000259 anti-tumor effect Effects 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 229940126680 traditional chinese medicines Drugs 0.000 abstract 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 14
- 235000006408 oxalic acid Nutrition 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000002212 electronic circular dichroism spectrum Methods 0.000 description 5
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
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- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 3
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- 230000000144 pharmacologic effect Effects 0.000 description 3
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- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 241000717671 Glechoma longituba Species 0.000 description 2
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 2
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
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- 230000004048 modification Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
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- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010007027 Calculus urinary Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 1
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- BNBQRQQYDMDJAH-UHFFFAOYSA-N benzodioxan Chemical group C1=CC=C2OCCOC2=C1 BNBQRQQYDMDJAH-UHFFFAOYSA-N 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method 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
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 125000005704 oxymethylene group Chemical group [H]C([H])([*:2])O[*:1] 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 208000008281 urolithiasis Diseases 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
- C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
- C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
- C07D319/20—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring with substituents attached to the hetero ring
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application provides phenolic acid compounds in Glechoma hederacea as well as an extraction and separation method and application thereof, belonging to the technical fields of extraction and separation of traditional Chinese medicines, phytochemistry and medicine. The application provides a phenolic acid compound extracted from Glechoma hederacea, the chemical structural formula of which is shown as a formula (I) or a formula (II), wherein the formula (I) is named (+) -Glechoma Qian Caosuan A, the formula (II) is named (-) -Glechoma Qian Caosuan A, and researches show that the compound has anti-tumor activity and the anti-tumor activity of (+) -Glechoma acid A is stronger than that of (-) -Glechoma Qian Caosuan A; meanwhile, the application also provides a simple and rapid extraction and separation method aiming at the pair of compounds. The extraction and separation process is simple in operation and easy to control, and is suitable for industrial production; the enantiomer compound has an anti-tumor effect and has good application prospect in preparing medicines for treating gastric cancer, liver cancer, colon cancer, ovarian cancer and lung cancer.
Description
Technical Field
The application belongs to the technical fields of traditional Chinese medicine extraction and separation, phytochemistry and medicine, and in particular relates to phenolic acid compounds in Glechoma hederacea, and an extraction and separation method and application thereof.
Background
The traditional Chinese medicine Glechoma longituba is a Labiatae plant blood circulation promoting pillGlechomalongituba(Nakai) kupr.); harvesting in spring to autumn, removing impurities, and sun drying; pungent and slightly bitter in flavor, slightly cold in nature, entering liver, kidney and bladder meridians. The function main indications of the Glechoma hederacea are as follows: diuresis inducing and stranguria treating, heat and toxic material clearing away, blood stasis eliminating and swelling eliminating effects; clinically, the traditional Chinese medicine composition is used for treating heat stranguria, urolithiasis, jaundice due to damp-heat, sore, carbuncle, swelling and pain and traumatic injury.
Modern pharmacological researches have proved that Glechoma hederacea has very low acute toxicity, and has the effects of promoting urination and gallbladder, regulating blood lipid, dissolving stone, reducing blood sugar, resisting inflammation and bacteria. The chemical components contained in the Glechomae herba are complex and various, and the main compounds separated from the Glechomae herba are flavonoid, organic acid, terpenoid and the like at present, but no report on phenolic acid enantiomer components is seen.
Disclosure of Invention
Based on the background technology, the application aims to provide a pair of enantiomer phenolic acid compounds extracted and separated from Glechoma hederacea, and the pair of enantiomer phenolic acid compounds have novel structure and pharmacological activity, thereby providing a material basis for the research on the pharmacological action of Glechoma hederacea.
The second purpose of the application is to provide a method for extracting and separating the pair of compounds from Glechoma hederacea, and the whole extraction and separation process is simple to operate, easy to control and suitable for industrial production.
The third purpose of the application is to provide the antitumor effect of the compound and provide a powerful basis for the development of potential antitumor new drugs.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the application provides a phenolic acid compound extracted from Glechoma hederacea, the chemical structural formula of which is shown as a formula (I) or a formula (II), wherein the formula (I) is a dextrorotatory body, the formula (I) is named (+) -Glechoma Qian Caosuan A, the formula (II) is a levorotatory body, the formula (II) is named (-) -Glechoma Qian Caosuan A, and researches show that the compound has anti-tumor activity, and the anti-tumor activity of (+) -Glechoma oxalic acid A is stronger than that of (-) -Glechoma oxalic acid A.
The chemical structural formula of the phenolic acid compound is shown as the following formula (I) or formula (II):
。
the application also provides an extraction and separation method of the phenolic acid compound, which comprises the following steps:
step 1, reflux-extracting Glechoma hederacea by using ethanol solution, and concentrating the extracting solution under reduced pressure to obtain alcohol-free dry extract;
step 2, adding water into the alcohol-free dry extract for suspension, then sequentially extracting with dichloromethane and ethyl acetate, and concentrating the obtained ethyl acetate extract under reduced pressure to obtain ethyl acetate extract concentrate;
step 3, performing silica gel column chromatography on the ethyl acetate extract concentrate obtained in the step 2, performing gradient elution by using chloroform-methanol as an eluent, and performing thin-layer chromatography inspection and merging to obtain 12 fractions;
wherein, the volume ratio of the eluent chloroform-methanol is 100: 1. 25: 1. 20: 1. 17: 1. 15: 1. 12: 1. 10: 1. 7:1, a step of;
step 4, performing silica gel column chromatography on one of the fractions obtained in the step 3, performing gradient elution by using chloroform-methanol as an eluent, and performing thin layer chromatography inspection and merging to obtain 6 fractions;
wherein, the volume ratio of the eluent chloroform-methanol is 8: 1. 7: 1. 6: 1. 5: 1. 4: 1. 3: 1. 2:1, a step of;
step 5, taking one of the fractions obtained in the step 4, and passing through C 18 Middle pressure column layerSeparating, gradient eluting with methanol-water as eluent, and combining by high performance liquid chromatography to obtain 7 fractions;
wherein, the methanol-water volume ratio of the eluent is 20: 80. 30: 70. 40: 60. 50: 50. 60:40, a step of performing a;
step 6, taking one of the fractions obtained in the step 5, preparing a racemate by reversed-phase high-performance preparation liquid chromatography and taking acetonitrile-0.01% trifluoroacetic acid as a mobile phase;
wherein, the volume ratio of acetonitrile-0.01% trifluoroacetic acid of the mobile phase is 32:68, volume flow of 10 mL/min;
and 7, carrying out chiral resolution on the obtained raceme to obtain phenolic acid compounds shown in the formulas (I) and (II).
Preferably, the conditions of the reduced pressure concentration in the step 1 and the step 2 are as follows: the temperature is 50 ℃, the rotating speed is 60 rpm, and the vacuum degree is 40 mbar.
Preferably, the chiral resolution in the step 7 is carried out by using a Lux 5 mu m Cellulose-4 chiral semi-preparative chromatographic column and separating by reverse-phase high performance liquid chromatography.
The present application also provides a pharmaceutical composition comprising as an active ingredient at least one of the above compounds of formula (I), formula (II), and a pharmaceutically acceptable carrier or excipient.
The application also provides application of the phenolic acid compound in preparing a medicament for treating cancer.
Preferably, the cancer is gastric cancer, liver cancer, colon cancer, ovarian cancer or lung cancer.
Compared with the prior art, the application has the beneficial effects that:
1. the application extracts and separates a raceme from Glechomae herba medicinal materials, determines the molecular configuration according to related data such as hydrogen spectrum carbon spectrum and the like, and chiral splits the raceme to obtain a pair of enantiomer compounds (+) -Glechomae oxalic acid A and (-) -Glechomae oxalic acid A.
2. The application takes the Glechomae herba as raw material, and obtains the enantiomer compound through the steps of ethanol reflux extraction, reduced pressure concentration, extraction, chromatographic separation and the like, and the whole extraction and separation process has simple operation and easy control, and is suitable for industrial production.
3. In vitro bioactivity experiments show that the enantiomer compounds (+) -Glechomae oxalic acid A and (-) -Glechomae oxalic acid A have obvious inhibition effects on gastric cancer, liver cancer, colon cancer, ovarian cancer or lung cancer cells, and have good application prospects in preparing medicaments for treating cancers.
Drawings
FIG. 1 is a diagram showing the structure of the compounds (+) -Glechomae acid A and (-) -Glechomae acid A.
Figure 2 is a chiral resolution HPLC chromatogram.
Fig. 3 is a block diagram of hyprhombin E.
Fig. 4 is a high resolution mass spectrum of (+ -) -Glechomaxalic acid A.
FIG. 5 is a UV spectrum of (+ -) -Glechomaxalic acid A.
FIG. 6 shows (+ -) -Glechomaxalic acid A 1 H-NMR spectrum.
FIG. 7 shows (+ -) -Glechomaxalic acid A 13 C-NMR spectrum.
FIG. 8 shows the HSQC spectrum of (+ -) -Glechomazine A.
Fig. 9 is an HMBC spectrum of (+/-) -Glechomaxalic acid a.
FIG. 10 is an ECD spectrum of (+) -Glechomae acid A.
FIG. 11 is an ECD spectrum of (-) -Glechomazine A.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the application, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Example 1 extraction and separation of (+/-) -Glechonic acid A from Glechoma longituba
1. Pulverizing Glechomae herba 20 kg, reflux-extracting with 75% ethanol solution for 3 times and 2 hr each time, mixing extractive solutions, and concentrating under reduced pressure to obtain alcohol-free dry extract. Adding deionized water of which the weight is 20 times that of the alcohol-free dry extract into the alcohol-free dry extract to obtain suspension, sequentially extracting three times by adopting dichloromethane and ethyl acetate respectively, combining ethyl acetate extracts, concentrating the ethyl acetate extracts under reduced pressure to dryness, and obtaining ethyl acetate extract concentrate 210 g.
2. The obtained ethyl acetate extract concentrate is taken and subjected to silica gel column (silica gel granularity is 100-200 meshes) chromatography, and chloroform-methanol (volume ratio of 100:1, 25:1, 20:1, 17:1, 15:1, 12:1, 10:1 and 7:1) is used as eluent for gradient elution (the elution time of 8 gradient volume ratios is 1.5 and h). Collecting eluent, collecting one part of eluent every 1L, numbering the eluent, sequentially inspecting the eluent by thin layer chromatography, and combining similar components to obtain 12 fractions which are marked as F1-F12; wherein fraction F11 of 20.527 g is obtained, and fraction F11 is obtained by combining the 81 st to 86 th eluents.
3. The fraction F11 obtained is chromatographed by a silica gel column (silica gel granularity is 100-200 meshes), and is eluted by chloroform-methanol (volume ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1) as eluent in a gradient way (7 gradient volume ratios are all 1 h). Collecting the eluent, collecting one part of eluent every 500 and mL, numbering the collected eluent sequentially, inspecting the eluent by thin layer chromatography, and combining similar components to obtain 6 fractions which are marked as F11-1-F11-6; wherein 9.352 g fractions F11-5 were obtained, fractions F11-5 were obtained by combining the 26 th to 30 th eluents.
4. Collecting the fraction F11-5, and passing through C 18 Medium pressure column chromatography, eluting with methanol-water (volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40) gradient (5 gradient volume ratio eluting times of 2 h). Collecting the eluent, collecting one part of eluent every 500 and mL, numbering the collected eluent sequentially, inspecting the eluent by high performance liquid chromatography, and combining similar components to obtain 7 fractions which are marked as F11-5-1-F11-5-7; wherein 123.78 mg fraction F11-5-5 is obtained, fraction F11-5-5 is obtained by combining 24-27 parts of eluent.
5. The fraction F11-5-5 is taken to be subjected to reversed phase high performance liquid chromatography, acetonitrile-0.01% trifluoroacetic acid (volume ratio 32:68, flow rate 10 mL/min) is taken as a mobile phase, and (+ -) -even Qian Caosuan A (47.34 mg) is prepared.
EXAMPLE 2 chiral resolution to give a pair of enantiomer compounds
The compound (+) -company Qian Caosuan A18.35 mg and (-) -company Qian Caosuan A16.24 mg was obtained by dissolving 47.34 (+ -) -company Qian Caosuan A obtained in example 1 in methanol, performing reverse phase high performance liquid chromatography, and using a Lux 5 μm chiral semi-preparative chromatographic column (Size: 10.0X1250 mm, P/N: 00G-4491-N0) with acetonitrile-0.01% trifluoroacetic acid (volume ratio: 32:68, flow rate: 2 mL/min) as mobile phase. The structural diagram of the compound (+) -Glechomae oxalic acid A and (-) -Glechomae oxalic acid A is shown in figure 1, and the chiral resolution HPLC chromatogram is shown in figure 2.
Example 3 structural resolution and authentication
The (+ -) -even Qian Caosuan A obtained in example 1 was taken as an off-white powder and dissolved in methanol, and UV-260 was measured by UV-UV Spectrophotometer (MeOH)λ max (logε) 291 (1.96) nm, 323 (1.96) nm; HRESIMS measured by Waters ACQUITY UPLC/Xex G2Q TOFm/z549.1052 [M－H] － (calcd for C 28 H 21 O 12 549.1033) and its molecular formula is C 28 H 22 O 12 。
1 H-NMR spectrum showed 3 1,3, 4-trisubstituted phenyl groups [δ H 7.27 (1H, d,J= 1.8 Hz)，6.93 (1H, d,J=8.4 Hz) and 7.16 (1H, dd,J= 1.8, 8.4 Hz)；6.87 (1H, d,J=1.2 Hz) and 6.76 (2H, overlap); 7.02 (1H, d,J=1.8 Hz), 6.76 (1H, overlap) and 6.93 (1H, dd,J= 1.8, 8.4 Hz)]. Two pairs of trans-oriented olefinic proton hydrogensδ H 7.67 (1H, d,J=16.2 Hz) and 6.46 (1H, d,J= 16.2 Hz)；7.59 (1H, d,J=15.6 Hz) and 6.21 (1H, d,J= 15.6 Hz)]two oxygen-linked methines [δ H 6.53 (1H, d,J=3.0 Hz) and 5.13 (1H, d,J= 3.0 Hz)]one oxymethylene signalδ H 4.71 (1H, s)。
13 The C-NMR spectrum reveals 28 signals including 18 aromatic hydrocarbons, 4 olefin carbons, 3 oxygenated methane carbonsδ C 91.2 76.7 and 61.7) and 3 carboxycarbons [ ]δ C 168.0 166.8 and 171.6).
HMBC spectrum shows H-7%δ H 7.67 And C-2%δ C 117.7)、C-6(δ C 123.7)、C-9(δ C 168.0 A) correlation; h-7'δ H 7.59 C-2%δ C 115.3)、C-6″(δ C 123.5)、C-9″(δ C 166.8 A) correlation; h-7' - (-H)δ H 5.13 C-8' -, Cδ C 91.2)、C-2′(δ C 115.0)、C-6′(δ C 119.8)、C-3 (δ C 144.3 A) correlation; h-8' - (-H)δ H 6.53 C-7' -, Cδ C 76.7)、C-4 (δ C 144.2)、C-9″(δ C 166.8 A) correlation; these data indicate that (+/-) -Glechomazine A has a 1, 4-benzodioxane skeleton, and further shows H-10 @ by HMBCδ H 4.71 And C-9%δ C 168.0)、C-11 (δ C 171.6 Related, indicating that (+/-) -Glechoma acid A is a carboxymethyl ester structure of hyprhombin E (FIG. 3 is a structural diagram of hyprhombin E), due to H-7 'and H-8' (-) -Glechoma acid A is a carboxymethyl ester structure of hyprhombin EJCoupling constant =3.0 Hz) and hyprhombin EJ=1.3 Hz), the two protons are designated as cis-orientation.
The high resolution mass spectrum of (+ -) -Glechomae oxalic acid A is shown in figure 4; the UV spectrum is shown in FIG. 5; 1 H-NMR spectrum, 13 The structure of (+ -) -Glechonic acid A of the present application was confirmed by the C-NMR spectrum, HSQC spectrum, HMBC spectrum, respectively shown in FIGS. 6 to 9, and FIGS. 6 to 9.
Table 1 shows the nuclear magnetic data of (+ -) -Glechonic acid A: 1 H-NMR (600 MHz) 13 C-NMR (150 MHz) at CD 3 In OD.
TABLE 1 (+ -) -Glechonic acid A 1 H-NMR、 13 C-NMR Nuclear magnetic data
The optical rotation of (+ -) -Glechogenic acid A was shown to be 0 as determined by a Perkin-Elmer 341 polarimeter, indicating that (+ -) -Glechogenic acid A is a racemate, was successfully separated into two optically pure enantiomers by HPLC using a Lux 5 μm Cellulose-4 chiral semi-preparative chromatographic column, and the experimental ECD spectra of the enantiomers were measured separately.
The compound (+) -even Qian Caosuan A obtained in example 2 was taken as an off-white powder dissolved in methanol and measured by Perkin-Elmer 341 polarimeter [ alpha ]] 20 D +202.9 (c0.035, meOH); ECD (MeOH) was measured by JASCO J-815 round dichroism spectrometerλ max (∆ε) 218 (3.78) nm, 237 (-1.67) nm, 295 (4.95) nm, 341 (-5.97) nm. The 7'R configuration is similar to that of hyplombin E by its negative cotton effect shown by ECD at 237 nm, with the 7',8' positions being in cis orientation, and thus the absolute configuration of (+) -Glechogenic acid a is designated 7' r,8' r. The ECD spectrum of (+) -Glechomaxalic acid A is shown in FIG. 10.
The compound (-) -even Qian Caosuan A obtained in example 2 was taken as an off-white powder dissolved in methanol and measured by Perkin-Elmer 341 polarimeter [ alpha ]] 20 D -204.3 (c0.046, meOH); ECD (MeOH) was measured by JASCO J-815 round dichroism spectrometerλ max (∆ε) 219 (-3.06) nm, 238 (1.39) nm, 291 (-3.35) nm, 342 (4.58) nm. The 7'S configuration is opposite to (+) -Glechogenic acid A by its positive cotton effect shown by ECD at 238 nm, and the 7',8' position is in cis orientation, therefore the absolute configuration of (-) -Glechogenic acid A is designated 7' S,8' S. The ECD spectrum of (-) -Glechomazine A is shown in FIG. 11.
Example 4 in vitro antitumor Activity test of enantiomer Compounds (+) -Glechomae acid A and (-) -Glechomae acid A
The test principle is as follows: MTT method: in the mitochondria of living cells, there is a dehydrogenase associated with NAPP (nicotinamide adenine dinucleotide phosphate, coenzyme II), and succinic dehydrogenase is capable of reducing exogenously yellow thiazole blue MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) to water-insoluble blue-violet crystalline Formazan (Formazan) and depositing in the cells, where the enzyme disappears and MTT is not reduced. After formazan was dissolved in dimethyl sulfoxide (DMSO), absorbance was measured at 570 nm and 630 nm using an microplate reader, and the optical density value was proportional to the number of living cells.
The cell lines used were: BGC-823 (human gastric cancer cell), bel (human liver cancer cell), HCT-8 (human colon cancer cell), a2780 (human ovarian cancer cell), and a549 (human lung cancer cell).
The test method comprises the following steps: MTT method: taking logarithmic growth cells, digesting, fully blowing into single cell suspension, counting, and diluting to 1×10 5 Inoculating into 96-well culture plate, adding 100 μl of cell suspension into each well, standing at 37deg.C under 5% CO 2 After 24 hours of culture in the incubator of (C), the stock culture was discarded. 8 concentration gradients were designed for each sample, and then 100. Mu.L of medium containing the sample for each concentration gradient was added to the assay wells, each concentration being parallel to 6 wells. 96-well plates were placed at 37℃in 5% CO 2 After 72 hours of incubation in a saturated humidity incubator, 20. Mu.L of freshly prepared serum-free medium containing 5. 5 mg/mL MTT was added to each well and incubation was continued for 4 hours at 37℃and the supernatant removed. 150 μl DMSO is added to each well to dissolve Formazan precipitate, shake for 5 min, and absorbance at 570 nm and 630 nm is measured on an microplate reader to reflect the number of living cells. Drug Inhibition Concentration (IC) was calculated by SPSS software 50 ) Values.
The calculation formula is as follows: tumor cell growth inhibition (%) =1- (assay well assay/control well assay) ×100%.
TABLE 2 liver cancer cell Bel
TABLE 3 gastric cancer cell BGC-823
TABLE 4 colon cancer cell HCT-8
TABLE 5 ovarian cancer cell A2780
TABLE 6 lung cancer cell A549
It can be seen that the enantiomer compounds (+) -Glechomae acid A and (-) -Glechomae acid A have obvious inhibition effects on BGC-823 (human gastric cancer cell), bel (human liver cancer cell), HCT-8 (human colon cancer cell), A2780 (human ovarian cancer cell) and A549 (human lung cancer cell), and the antitumor activity of (+) -Glechomae acid A is stronger than that of (-) -Glechomae Qian Caosuan A, and the two can be used as medicines for treating or researching and treating cancers or tumors.
Finally, it should be emphasized that the foregoing description is merely illustrative of the preferred embodiments of the application, and that various changes and modifications can be made by those skilled in the art without departing from the spirit and principles of the application, and any such modifications, equivalents, improvements, etc. are intended to be included within the scope of the application.
Claims (7)
1. The phenolic acid compound extracted and separated from Glechoma hederacea is characterized in that the chemical structural formula of the phenolic acid compound is shown as the following formula (I) or formula (II):
。
2. the method for extracting and separating phenolic acid compounds as claimed in claim 1, comprising the steps of:
step 1, reflux-extracting Glechoma hederacea by using ethanol solution, and concentrating the extracting solution under reduced pressure to obtain alcohol-free dry extract;
step 2, adding water into the alcohol-free dry extract for suspension, then sequentially extracting with dichloromethane and ethyl acetate, and concentrating the obtained ethyl acetate extract under reduced pressure to obtain ethyl acetate extract concentrate;
step 3, performing silica gel column chromatography on the ethyl acetate extract concentrate obtained in the step 2, performing gradient elution by using chloroform-methanol as an eluent, and performing thin-layer chromatography inspection and merging to obtain 12 fractions;
wherein, the volume ratio of the eluent chloroform-methanol is 100: 1. 25: 1. 20: 1. 17: 1. 15: 1. 12: 1. 10: 1. 7:1, a step of;
step 4, performing silica gel column chromatography on one of the fractions obtained in the step 3, performing gradient elution by using chloroform-methanol as an eluent, and performing thin layer chromatography inspection and merging to obtain 6 fractions;
wherein, the volume ratio of the eluent chloroform-methanol is 8: 1. 7: 1. 6: 1. 5: 1. 4: 1. 3: 1. 2:1, a step of;
step 5, taking one of the fractions obtained in the step 4, and passing through C 18 Performing medium pressure column chromatography, gradient eluting with methanol-water as eluent, and performing high performance liquid chromatography to obtain 7 fractions;
wherein, the methanol-water volume ratio of the eluent is 20: 80. 30: 70. 40: 60. 50: 50. 60:40, a step of performing a;
step 6, taking one of the fractions obtained in the step 5, preparing a racemate by reversed-phase high-performance preparation liquid chromatography and taking acetonitrile-0.01% trifluoroacetic acid as a mobile phase;
wherein, the volume ratio of acetonitrile-0.01% trifluoroacetic acid of the mobile phase is 32:68, volume flow of 10 mL/min;
and 7, carrying out chiral resolution on the obtained raceme to obtain phenolic acid compounds shown in the formulas (I) and (II).
3. The extraction and separation method according to claim 2, wherein the conditions of the reduced pressure concentration in step 1 and step 2 are: the temperature is 50 ℃, the rotating speed is 60 rpm, and the vacuum degree is 40 mbar.
4. The extraction and separation method according to claim 2, wherein the chiral resolution in step 7 is performed by reversed-phase high performance liquid chromatography using a Lux 5 μm chiral semi-preparative chromatographic column.
5. A pharmaceutical composition, characterized in that it comprises as active ingredient at least one of the compounds of formula (I), formula (II) according to claim 1, together with a pharmaceutically acceptable carrier or excipient.
6. Use of phenolic acid compounds according to claim 1 for the preparation of a medicament for the treatment of cancer.
7. The use according to claim 6, wherein the cancer is gastric cancer, liver cancer, colon cancer, ovarian cancer or lung cancer.
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| US4320119A (en) * | 1979-08-23 | 1982-03-16 | Kenyaky Yogyo Kabushiki Kaisha | Extracts of Marsdenia cundurango Reichenbach fil |
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| CN107148409A (en) * | 2014-08-20 | 2017-09-08 | 百时美施贵宝公司 | The bicyclic compound being substituted |
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