LU503387B1 - Sesquiterpene compound from fructus alpiniae oxyphyllae, and preparation method and application thereof - Google Patents
Sesquiterpene compound from fructus alpiniae oxyphyllae, and preparation method and application thereof Download PDFInfo
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- LU503387B1 LU503387B1 LU503387A LU503387A LU503387B1 LU 503387 B1 LU503387 B1 LU 503387B1 LU 503387 A LU503387 A LU 503387A LU 503387 A LU503387 A LU 503387A LU 503387 B1 LU503387 B1 LU 503387B1
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- compound
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- fructus alpiniae
- alpiniae oxyphyllae
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- 229930004725 sesquiterpene Natural products 0.000 title claims abstract description 40
- -1 Sesquiterpene compound Chemical class 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 150000001875 compounds Chemical class 0.000 claims abstract description 59
- 238000000926 separation method Methods 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims description 58
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 28
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 27
- 239000000741 silica gel Substances 0.000 claims description 27
- 229910002027 silica gel Inorganic materials 0.000 claims description 27
- 229910001868 water Inorganic materials 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 238000011894 semi-preparative HPLC Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- FCSKOFQQCWLGMV-UHFFFAOYSA-N 5-{5-[2-chloro-4-(4,5-dihydro-1,3-oxazol-2-yl)phenoxy]pentyl}-3-methylisoxazole Chemical compound O1N=C(C)C=C1CCCCCOC1=CC=C(C=2OCCN=2)C=C1Cl FCSKOFQQCWLGMV-UHFFFAOYSA-N 0.000 claims description 11
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 10
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 9
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 9
- 239000000469 ethanolic extract Substances 0.000 claims description 9
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 9
- 239000003208 petroleum Substances 0.000 claims description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 14
- 230000000324 neuroprotective effect Effects 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000001681 protective effect Effects 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 3
- 230000004770 neurodegeneration Effects 0.000 abstract description 3
- 208000015122 neurodegenerative disease Diseases 0.000 abstract description 3
- 210000002569 neuron Anatomy 0.000 abstract description 2
- 239000004090 neuroprotective agent Substances 0.000 abstract description 2
- 238000002953 preparative HPLC Methods 0.000 abstract description 2
- 230000003078 antioxidant effect Effects 0.000 abstract 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 21
- 241000572565 Alpinia oxyphylla Species 0.000 description 8
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- NOTFZGFABLVTIG-UHFFFAOYSA-N Cyclohexylethyl acetate Chemical compound CC(=O)OCCC1CCCCC1 NOTFZGFABLVTIG-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 5
- 238000000746 purification Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000005779 cell damage Effects 0.000 description 3
- 208000037887 cell injury Diseases 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 2
- 150000000174 eudesmanes Chemical class 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003505 terpenes Chemical class 0.000 description 2
- 238000010792 warming Methods 0.000 description 2
- MZLKNWMNBXHXMA-UHFFFAOYSA-N 1-phenylheptylbenzene Chemical compound C=1C=CC=CC=1C(CCCCCC)C1=CC=CC=C1 MZLKNWMNBXHXMA-UHFFFAOYSA-N 0.000 description 1
- 241000013298 Alpinia <beetle> Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 241000234299 Zingiberaceae Species 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 235000018927 edible plant Nutrition 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
- 235000021022 fresh fruits Nutrition 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000005227 renal system Anatomy 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 101150005399 sod2 gene Proteins 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 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
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
- C07C35/22—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system
- C07C35/23—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system with hydroxy on a condensed ring system having two rings
- C07C35/36—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system with hydroxy on a condensed ring system having two rings the condensed ring system being a (4.4.0) system, e.g. naphols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
- B01D15/166—Fluid composition conditioning, e.g. gradient
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
- B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/265—Adsorption chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
- B01D15/325—Reversed phase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/613—Unsaturated compounds containing a keto groups being part of a ring polycyclic
- C07C49/617—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system
- C07C49/623—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system having two rings
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C62/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C62/30—Unsaturated compounds
- C07C62/38—Unsaturated compounds containing keto groups
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
- C07D303/02—Compounds containing oxirane rings
- C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
- C07D303/14—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by free hydroxyl radicals
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- C07D—HETEROCYCLIC COMPOUNDS
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- C07D303/02—Compounds containing oxirane rings
- C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
- C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
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- C07C2602/14—All rings being cycloaliphatic
- C07C2602/26—All rings being cycloaliphatic the ring system containing ten carbon atoms
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- C07C2602/14—All rings being cycloaliphatic
- C07C2602/32—All rings being cycloaliphatic the ring system containing at least eleven carbon atoms
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Abstract
The present disclosure relates to the technical field of phytomedicines, and discloses a sesquiterpene compound from Fructus Alpiniae Oxyphyllae, and a preparation method and application thereof. The sesquiterpene compound is any one of sesquiterpene compounds I to VII. The sesquiterpene compound is obtained by semi-preparative high performance liquid chromatography (HPLC) ofalcohol extracts from Fructus Alpiniae Oxyphyllae after repeated Separation, and then through, and the purity is higher. All the sesquiterpene compounds have certain anti-oxidative stress activity, and may be used for preparing neuroprotective drugs. Especially, the compound V has a significant protective effect on HaOa-induced OLN-93 cells, and therefore the compound V has a higher potential in preparation of the neuroprotective medicines for protecting nerve cells from being injured and treating neurodegenerative diseases.
Description
SESQUITERPENE COMPOUND FROM FRUCTUS ALPINIAE OXYPHYLLAE, LU503387
AND PREPARATION METHOD AND APPLICATION THEREOF
[01] The present disclosure relates to the technical field of phytomedicines, in particular to a sesquiterpene compound from Fructus Alpiniae Oxyphyllae, and a preparation method and application thereof.
[02] Fructus Alpiniae Oxyphyllae is a dried ripe fruit of plant Alpinia oxyphylla Mig. of
Alpinia, Zingiberaceae. Alpinia oxyphylla Mig. is mainly distributed in tropical and subtropical provinces, such as Hainan, Guangdong, Guangxi and Yunnan, and it is one of four well-known southern medicinal plants. Alpinia oxyphylla Miq. contains terpene diphenylheptane, flavone, basic groups, nucleosides and other small molecule chemical components, and sesquiterpene chemical components in terpenoids remain dominant.
[03] Alpinia oxyphylla Mig. as Chinese medicinal materials has multiple functions of warming the kidney, consolidating essence, arresting polyuria, warming the spleen, relieving diarrhea, constraining spittle and the like, and is generally used as tablets or decoct herbal medicines for administration. Modern pharmacological research also shows that Alpinia oxyphylla Mig. has rich biological activities, including a nervous system effect, a renal system effect, an anti-tumor effect, anti-inflammatory and antioxidation effects and the like; meanwhile, the clinical applications of prescriptions mainly containing Fructus Alpiniae Oxyphyllae are increasing in terms of invigorating the brain, and a large number of experimental researches on animals have further confirmed that Fructus Alpiniae Oxyphyllae has a good neuroprotective effect. Alpinia oxyphylla Mig. is also made into fresh fruits to eat in folk in Hainan after being soaked in saline water, and then it serves as a medicinal and edible plant resource with high safety. At present, development in Fructus Alpiniae Oxyphyllae products is still minimal, and consequently the application of Fructus Alpiniae Oxyphyllae is limited.
[04] Therefore, a preparation method of a sesquiterpene compound from Fructus Alpiniae
Oxyphyllae is developed to obtain the sesquiterpene compound from Fructus Alpiniae
Oxyphyllae and explore its medicinal effects, which not only benefits further understanding of the composition of the compounds from Fructus Alpiniae Oxyphyllae, but also is of great significance in in-depth development and application of Fructus Alpiniae Oxyphyllae.
[05] The present disclosure is intended to provide a sesquiterpene compound from Fructus
Alpiniae Oxyphyllae, and a preparation method and application thereof to solve the technical problem of less development of existing products related to Fructus Alpiniae Oxyphyllae.
[06] In order to fulfill the above objective, the present disclosure has the following technical solution: a sesquiterpene compound from Fructus Alpiniae Oxyphyllae. The sesquiterpene compound is obtained through purification of alcohol extracts from Fructus Alpiniae
Oxyphyllae. The sesquiterpene compound is any one of compounds I to VII, and chemical structural formulas of the compounds I to VII are shown as follows: 1
CL, LU503387
COLOR à 3 sod 2 40 a. AP Sol LT 4 Hs A D, A 3A le | pe
ST OH il 0 = se T << T = Oo | Ë
Compound IT Ü3R,45,19R)
Compound I (38 458 ,5R, FOR) , Compound TIT (3K4S,185) , Compound IV {65,75S,18#} , _ 9 t4 ; } | = A A = AN A PS 13 AS 0 u in > { Ë La Tt [ 12
OH ; RE | $ Le) ; 2 _ IS ENS Lo I oF OH ia o A RL i i 5 0 6
Compound v{5R, 78,108) , Compound VI {185} , Compound VII {85,12R}
[07] The principle and advantages of the solution are as follows: 7 sesquiterpene compounds provided by the solution are all new compounds extracted from Fructus Alpiniae Oxyphyllae for the first time and having a neuroprotective effect, wherein the compound VII is an unusual rearranged eudesmane sesquiterpene, and has a rare double-ring carbon skeleton formed by linking a six-membered carbon ring with an eight-member carbon ring side by side; the compound VI is eudesmane sesquiterpene undergoing ring opening at C6-C7, separated from a plant for the first time; not only is the type of natural sesquiterpene compounds enriched effectively, but also a theoretical basis is provided for in-depth development of Alpinia oxyphylla Miq. The specific performance is that the compounds I to VII have a better protective effect on H20>-induced OLN-93 injured cells. 50 uM of compound V may increase the viability of the H20>-induced OLN-93 injured cells to 80% or so, which adequately indicates that the compound V has the good neuroprotective effect.
[08] All the compounds prepared in the solution are obtained by semi-preparative HPLC after repeated separation, and the purity is higher (the purity may reach 95% or even higher).
The compounds exhibit a certain neuroprotective effect, and may be used for preparing medicines for treating neurodegenerative diseases, which effectively expands the application range of Fructus Alpiniae Oxyphyllae.
[09] Preferably, a preparation method of the sesquiterpene compound from Fructus Alpiniae
Oxyphyllae. includes the following steps:
[10] ethanol extracts from Fructus Alpiniae Oxyphyllae. are eluted with HP-20 macroporous resins to obtain a total of 4 components F1, F2, F3 and F4; and then, preparation is conducted according to steps in (a) and/or (b) to obtain the sesquiterpene compounds:
[11] (a) the component F3 is eluted by a silica gel column to obtain 10 subcomponents F3 A to F3J in total; then,
[12] the subcomponent F3D is continuously eluted by the silica gel column to obtain 8 subfractions F3D1 to F3D8 in total; the subcomponent F3D3 is separated from an ODS column to obtain 7 subfractions F3D3A to F3D3G in total; the subfraction F3D3D is purified by semi- preparative HPLC to obtain a compound I; and/or, the subfraction F3D4 is separated by semi- 2 preparative HPLC to obtain a compound II and a compound III; and/or, the subfraction F3D5 is separated from the ODS column to obtain 4 subfractions F3D5A to F3D5D in total; the LU503387 subfraction F3D5C is separated by semi-preparative HPLC to obtain a compound VII;
[13] (b) the component F4 is eluted by the silica gel column to obtain 13 subcomponents
F4A to F4M in total; then,
[14] the subcomponent F4E is separated from the ODS column to obtain 6 subfractions
F4E1 to F4F6 in total; the subfraction F4E4 is purified by semi-preparative HPLC to obtain a compound IV and a compound VI; and/or the subcomponent F4E6 is separated from the silica gel column to obtain 8 subfractions F4E6A to F4E6H in total; and the subfraction F4F6D is purified by semi-preparative HPLC to obtain a compound V.
[15] The solution has the beneficial effects that through combined elution of the HP-20 macroporous resins, the silica gel column, the ODS column and the semi-preparative HPLC, a plurality of new compounds with a neuroprotective effect are obtained through separation and purification of alcohol extracts from Fructus Alpiniae Oxyphyllae. which not only facilitates further understanding of chemical composition of Fructus Alpiniae Oxyphyllae, but also provides a new thought for further development of Fructus Alpiniae Oxyphyllae in the preparation of neuroprotective medicines.
[16] Preferably, the conditions for elution with the HP-20 macroporous resins are as follows: ethanol-water serves as an cluant, gradient elution is conducted sequentially according to volume concentration percentages of the ethanol in the eluant, which are 0%, 50%, 70% and 95%, a volume of the eluant used at each gradient is 240 L, elution fractions after each gradient elution are collected respectively, and a total of 4 components F1, F2, F3 and F4 are obtained correspondingly.
[17] The solution has the beneficial effects that, by adopting ethanol solutions with different gradient concentrations, Fructus Alpiniae Oxyphyllae is firstly eluted to obtain 4 components, which facilitates further separation and purification of compounds with different solubilities.
[18] Preferably, the conditions for elution on the silica gel column are as follows: a mixed solvent of any one of cyclohexane and petroleum ether, and ethyl acetate serves as an eluant, and gradient elution is conducted according to different subcomponents/subfractions as per a volume concentration ratio of the cyclohexane or the petroleum ether to the ethyl acetate, which ranges from 1: 0 to 0: 1; a volume of eluant at each elution gradient ranges from 48 L to 50 L, a plurality of elution fractions are collected according to 1/2 to 1/3 of the volume of the eluant used at each elution gradient, and tested, and the elution fractions with consistent results are combined to obtain subfractions.
[19] Preferably, the conditions for elution on the ODS column are as follows: ethanol-water serves as an cluant, and gradient elution is conducted according to different subcomponents/subfractions as per volume concentration percentages of the ethanol to the eluants, which range from 40% to 100%; a volume of eluant at each elution gradient ranges from 350 mL to 450 mL, a plurality of elution fractions are collected according to 1/2 to 1/3 of the volume of the eluant used at each elution gradient, and tested, and the elution fractions with consistent results are combined to obtain subfractions.
[20] Preferably, the eluant used in the semi-preparative HPLC includes a mixed solution of acctonitrile and water, and a mixed solution of methanol and water; wherein a volume ratio of 3 the methanol to the water mixed in the eluant used for separation to obtain the compound II and the compound III is 40: 60. LU503387
[21] Preferably, a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound IV and the compound VI is 45: 55; a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound V is 55: 45.
[22] Preferably, a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound I is 35: 65; a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound VII is 25: 75.
[23] Preferably, extraction of the ethanol extracts from Fructus Alpiniae Oxyphyllae includes steps: Fructus Alpiniae Oxyphyllae is subjected to reflux extraction twice by 60% ethanol in a volume that is 6 times that of Fructus Alpiniae Oxyphyllae, each for 2 h; and the extracting solutions obtained by extraction are combined, and subjected to evaporation under reduced pressure to obtain the ethanol extract powder from Fructus Alpiniae Oxyphyllae.
[24] Preferably, an application of any one of the sesquiterpene compounds from Fructus
Alpiniae Oxyphyllae . in preparation of neuroprotective drugs.
[25] The beneficial effects are as follows: all the compounds I to VII in the solution exhibit a certain neuroprotective effect, including a protective effect on the H:Oz-induced OLN-93 cells; and especially, 50 uM of compound V may increase the viability of the H2O»-induced OLN-93 injured cells to 80% or so, which fully shows that the compound V has a better neuroprotective effect. By this, the compounds prepared from Fructus Alpiniae Oxyphyllae in the solution may be used for preparing neuroprotective medicines to prevent nerve cell injury and treat neurodegenerative diseases.
[26] FIG. 1 to FIG. 7 are UV data spectra of sesquiterpene compounds I to VII from Fructus
Alpiniae Oxyphyllae prepared in embodiments of the present disclosure sequentially.
[27] FIG. 8 to FIG. 14 are graphs showing H>O»-induced cell injury results of sesquiterpene compounds I to VII from Fructus Alpiniae Oxyphyllae prepared in embodiments of the present disclosure sequentially.
[28] The present invention will be further described below in detail with reference to the specific implementations, but the implementations of the present disclosure are not limited thereto. Unless otherwise specially stated, the following technical means used in embodiments are conventional means well-known to those skilled in the art; the used experimental methods are conventional methods; and the used materials, reagents and the like are available commercially.
[29] A sesquiterpene compound from Fructus Alpiniae Oxyphyllae is provided. All sesquiterpene compounds in this solution are separated from fruits (Fructus Alpiniae
Oxyphyllae, purchased from Anguo Shiyuan Trading Co., Ltd.) of A. oxyphylla Mig. from
Hainan as the place of origin. The sesquiterpene compound in this solution is any one of 4 sesquiterpene compounds I to VII, and chemical structural formulas of the compounds I to VII are as follows: LU503387 14
EE ; , =
POSE, AA LA TA 7 ] [ TT 3 ig i Î “ APN ATEN A 3 Bis 3. BG
Soe Se Nec ~~. ANY I SAN +, - pg i } | pond ~~ Se ov” Noe >. | iy ~~ ph 5 4 35 Compound II {3R,4$,15R}
Compound I {35,48,58,18R} , Compound [II (3R,45,195) , Compound IV {65,785,108} ,
N 0 14 - ooo i 3 boo
À = TN A7 TE OS ad a NG 0 oH 2 RE i 3 . 8) 3 x | Sr * PS or OH 1% OF ~~ fog CN 1 ; ] i th 9
Compound V {SR 7X, 10.8) , Compound VI (165) , and Compound VII (88, {2R}
[30] The solution further provides a preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae, which includes the following steps:
[31] (1) Obtainment of ethanol extracts from Fructus Alpiniae Oxyphyllae
[32] Fructus Alpiniae Oxyphyllae is crushed, and is subjected to reflux extraction twice by 60% ethanol in a volume that is 6 times that of Fructus Alpiniae Oxyphyllae, each for 2 h, that is, solid residues after first reflux extraction are re-extracted under the same extraction conditions, extracting solutions obtained in two extractions are combined, and subjected to evaporation under reduced pressure to obtain the ethanol extracts from Fructus Alpiniae
Oxyphyllae.
[33] (2) Separation and purification to obtain compounds I to VII
[34] The ethanol extracts from Fructus Alpiniae Oxyphyllae. are eluted with HP-20 macroporous resins to obtain 4 components F1, F2, F3 and F4 in total; then, preparation is conducted according to steps in (a) and/or (b) to obtain the sesquiterpene compounds I to VII:
[35] (a) the component F3 is eluted on a silica gel column to obtain 10 subcomponents
F3A to F3J in total; then,
[36] the subcomponent F3D is continuously eluted on the silica gel column to obtain 8 subfractions F3D1 to F3DS in total; the subcomponent F3D3 is separated from the ODS column to obtain 7 subfractions F3D3A to F3D3G in total; the subfraction F3D3D is purified by the semi-preparative HPLC to obtain a compound I; and/or, the subfraction F3D4 is separated by semi-preparative HPLC to obtain a compound II and a compound III; and/or, the subfraction
F3DS5 is separated from the ODS column to obtain 4 subfractions F3D5A to F3D5D in total; the subfraction F3D5C is separated by semi-preparative HPLC to obtain a compound VII;
[37] and/or, (b) the component F4 is eluted on the silica gel column to obtain 13 subcomponents F4A to F4M in total; then, LU503387
[38] the subcomponent F4E is separated from the ODS column to obtain 6 subfractions
F4E1 to F4F6 in total; the subfraction F4E4 is purified by semi-preparative HPLC to obtain a compound IV and a compound VI; and/or, the subcomponent F4F6 is eluted on the silica gel column and separated by semi-preparative HPLC sequentially to obtain a compound V.
[39] Wherein, the conditions for elution with the HP-20 macroporous resins are as follows: ethanol-water serves as an eluant, gradient elution is conducted according to volume concentration percentages of the ethanol in the eluant, which are 0%, 50%, 70% and 95% sequentially, a volume of the eluant used at each elution gradient is 240 L, elution fractions after each gradient elution are collected respectively, and a total of 4 components F1, F2, F3 and F4 are obtained correspondingly.
[40] The conditions for elution on the silica gel column are as follows: a mixed solvent of any one of cyclohexane and petroleum ether, and ethyl acetate serves as an eluant, and gradient elution is conducted according to different subcomponents/subfractions as per a volume concentration ratio of the cyclohexane or the petroleum ether to the ethyl acetate, which ranges from 1: 0 to 0: 1; a volume of the eluant at each elution gradient ranges from 48 L to 50 L, a plurality of elution fractions are collected according to 1/2 to 1/3 of the volume of the eluants used at each elution gradient, and tested, and the elution fractions with consistent results are combined to obtain subfractions.
[41] Specifically, during elution of the component F3 on the silica gel column, the eluant includes cyclohexane-ethyl acetate, concentration gradients of the cyclohexane to the ethyl acetate are 98: 2, 95: 5, 9: 1, 85: 15, 8: 2, 7: 3, 6: 4, 1: 1 and 0: 1, and F3 is eluted to obtain 10 subcomponents F3A to F3J in total;
[42] During elution ofthe subcomponent F3D on the silica gel column, the eluant includes cyclohexane-ethyl acetate, concentration gradients of the cyclohexane to the ethyl acetate are 1: 0, 95: 5, 9: 1, 85: 15, 8: 2, 7: 3, 6: 4, 1: 1 and 0: 1, and F3D is eluted to obtain 8 subfractions
F3D1 to F3D8 in total;
[43] during elution of the component F4 on the silica gel column, the eluant includes cyclohexane-ethyl acetate, and concentration gradients of the cyclohexane to the ethyl acetate are 1: 0, 99: 1, 98: 2, 95: 5, 9: 1, 85: 15, 8: 2, 7: 3 and 0: 1; F4 is eluted to obtain 13 subcomponents F4A to F4M in total; and
[44] during elution of the subfraction F4F6 on the silica gel column, the eluant includes petroleum ether-ethyl acetate, and concentration gradients of the petroleum ether to the ethyl acetate are 1: 0, 98: 2, 95: 5, 9: 1, 8: 2, 1: 1 and 0: 1, and F4F6 is eluted to obtain 8 subfractions
F4E6A to FAE6H in total.
[45] The conditions for elution on the ODS column are as follows: ethanol-water serves as an eluant, and gradient elution is conducted according to different subcomponents/subfractions 6 as per volume concentration percentages of the ethanol to the eluants, which range from 40% to 100%; a volume of the eluant at each elution gradient ranges from 350 mL to 450 mL, a LUS03387 plurality of elution fractions are collected according to 1/2 to 1/3 of the volume of the eluant used at each elution gradient, and tested, and the elution fractions with consistent results are combined to obtain subfractions.
[46] Specifically, during elution of the subfraction F3D3 on the ODS column, concentration gradients of the methanol in the used eluant are 40%, 45%, 50%, 55%, 60%, 80% and 100%; F3D3 is eluted to obtain 7 subfractions F3D3A to F3D3G in total;
[47] during elution of the subfraction F3D5 on the ODS column, concentration gradients of the methanol in the used eluant are 40%, 50%, 60%, 70%, 80%, 90% and 100%; F3D5 is eluted to obtain 4 subfractions F3D5A to F3D5D in total;
[48] during elution of the subfraction F4E on the ODS column, concentration gradients of the methanol in the used eluant are 60%, 65%, 70%, 75%, 80%, 90% and 100%; and F4E is eluted to obtain 6 subfractions F4F1 to F4F6 in total.
[49] In this solution, a preparation method of sesquiterpene compounds I to VII is achieved as follows:
[50] 9.8 kg of ethanol extracts extracted from 60 kg of Fructus Alpiniae Oxyphyllae are eluted with HP-20 macroporous resins, wherein an eluant is an ethanol-water solution with concentration gradients of 0%, 50%, 70% and 95%; a total of 4 components F1, F2, F3 and F4 are obtained; then, preparation is conducted according to steps in (a) and/or (b) to obtain the sesquiterpene compounds I to VII, specifically shown as follows:
[51] (a), the component F3 is eluted on a silica gel column with cyclohexane-ethyl acetate with volume ratios of 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1 and 0:1 as the eluant, to obtain subcomponents F3A to F3J in total; and then, the subfractions F3D3, F3D4 and F3DS of the subcomponent F3D are separated from an ODS column and through semi-preparative HPLC under different conditions, to obtain a compound I, a compound II, a compound III and a compound VII respectively.
[52] Specifically, the component F3D (32.8 g) is eluted on the silica gel column with the cyclohexane-ethyl acetate with volume ratios of 1:0, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1 and 0:1 as the eluant, to obtain 8 subfractions F3D1 to F3DS in total.
[53] For reference, a silica gel filler in this solution is specifically silica gel with a mesh number ranging from 200 to 300 (®12x75 cm), and the silica gel is specifically selected from
Silica gel (200 to 300 mesh, Qingdao Haiyang Chemical Co., Ltd., Shandong, China) (for the elution on the silica gel column mentioned below, the silica gel is the same as here, which will not be described below).
[54] The compound I may be prepared as follows: the subfraction F3D3 (8.7 g) is firstly separated from the ODS column with methanol-water with volume percentages of 40%, 45%, 50%, 55%, 60%, 80% and 100% as the eluant, to obtain 7 subfractions F3D3A to F3D3G in 7 total; and F3D3D is subjected to semi-preparative HPLC with 35% acetonitrile-water (a mixed volume ratio of the acetonitrile to the water is 35:65) as the eluant, to obtain the compound I LU503387 (4.1 mg, tz=13.8 min).
[55] For reference, a filler for separation from the ODS column in this solution is specifically: octadecyl silane (ODS) silica gel (12 nm, S-50 um, YMC Ltd., Tokyo, Japan); in a semi- preparative HPLC process, an injection volume is specifically 30 pL, a flow rate is specifically 3 mL/min, and a model of a semi-preparative column is specifically: Phenomenex Gemini C18 column (5 um, phi 10x250 mm; Phenomenex Inc, Los Angeles, USA) (for the ODS column and semi-preparative HPLC mentioned below, instruments used therefor and experimental conditions thereof are the same as here, which will not be described below).
[56] The compound II and the compound III are obtained as follows: the subfraction F3D4 (2.7 g) is subjected to semi-preparative HPLC with 40% methanol-water (a mixed volume ratio of the methanol to the water is 40:60) as the eluant, to obtain the compound II (18.0 mg, tr=15.0 min) and the compound III (6.9 mg, z=15.5 min).
[57] The compound VII is obtained as follows: the subfraction F3D5 (2.5 g) is separated from the ODS column with methanol-water with volume percentages of 40%, 50%, 60%, 70%, 80%, 90% and 100% as the eluant, to obtain 4 subfractions F3D5A to F3D5D in total; and
F3D5C is subjected to semi-preparative HPLC with 25% acetonitrile-water (a mixed volume ratio of the acetonitrile to the water is 25: 75) as the eluant, to obtain the compound VII (4.9 mg, tr =17.5 min).
[58] (b) the component F4 is eluted on the silica gel column with cyclohexane-ethyl acetate with concentration gradients of 1:0, 99:1, 98:2, 95:5, 9:1, 85:15, 8:2, 7:3 and 0:1 as the eluant, to obtain 13 subcomponents F4A to F4M in total; and then, the subfractions F4E4 and F4F6 of the subcomponent F4E are separated from the ODS column and through semi-preparative
HPLC under different conditions, to obtain the compound IV, the compound VI and the compound V respectively.
[539] Specifically, F4E (46.3 g) is separated from the ODS column with methanol-water with volume percentages of 60%, 65%, 70%, 75%, 80%, 90% and 100% as the eluant, to obtain 6 subfractions F4E1 to F4F6 in total; and then,
[60] the compound IV and the compound VI are obtained as follows: semi-preparative
HPLC is conducted on the subfraction F4E4 (870.9 mg) with 45% methanol-water (a mixed volume ratio of the methanol to the water is 45:55) as the eluant, to obtain the compound IV (21.6 mg, tr =37.5 min) and the compound III (87.1 mg, tr =9.0 min).
[61] The compound V is obtained as follows: F4E6 (30.7 g) is separated from the silica gel column with petroleum ether-ethyl acetate with concentration gradients of 1:0, 98:2, 95:5, 9:1, 8:2, 1:1 and 0:1 as the eluant, to obtain 8 subfractions FAE6A to F4E6 in total; and F4E6D is subjected to semi-preparative HPLC with 55% acetonitrile-water (a mixed volume ratio of the acetonitrile to the water is 55: 45), to obtain the compound V (6.3 mg, tr =20.0 min).
[62] Test Example 1: property characterization of the sesquiterpene compounds I to VII prepared in this solution
[63] The sesquiterpene compounds I to VII prepared in this solution were sampled and determined by MRI (including '*C NMR and 'H NMR), mass spectrometry (HR-ESI-MS) and 8 partial physical and chemical methods (UV, IR and the like); wherein MRI test results were shown in Table 1 and Table 2, and the obtained UV spectra were shown in FIG. 1 to FIG. 7 LU503387 respectively. For reference, an instrument used in UV detection in this solution is specifically a JASCOV-550 UV-VIS spectrophotometer.
Table 1 'H NMR and *C NMR data of compounds I to IV
Pos. du (J in Hz) du (J in Hz) du (J in Hz) du (J in Hz) 1.55, ddd 1.52, ddd (13.6, 5.4, 1.58, ddd (13.5, (13.9, 11.7, 5.2) 5.9, 4.6) 1.76 1 [25.60 7.2) 30.0 37.0 34,2 0.95, dd (13.9 1.24, ddd (13.6, 5.2, 1.41, ddd (13.5, 1.74 7.2) 2.1) 10.5, 6.2) 2.12, dddd (15.4, 2.14, dddd (16.5, > | 200 PO oye og) 542116) || lessee | 2.71 0 3 | 2.06, dddd (15.4, | °° | 1.96, dada (15.3, | * on 9 ’ 12.6,5.2, 1.6) 6.2, 4.6, 4.1) es 3 3.28, d (3.1) 3.23,t(1.6) |201.0| 3.36, dt (4.1, 0.9) 6 [171] 696, brs [1278 756 | 3565 2.60, ddd (17.8, 2.63, ddd (17.9, 8 | 210 139 09 342 14.7, 5.6) 19.6 14.9, 5.2) 358 2.06 ’ 225 “ |2.44, ddd (17.8, 4.9, “| 2.39, dddd (17.9, ’ 1.92 2.0) 4.7, 2.0, 0.9) (34 ad 1.86, ddd (14.7, 1.88, ddd (14.9, oa 13.5,4.9) 13.2,4.7) 1.42 9 | 28.8 7.0) 36.6 32.8 34.0 1.74, ddd (13.5, 5.6, 1.67, ddd (13.2, 1.34 1.25, ddd 2.1) 5.2, 2.0) (13.4, 7.0, 0.8) os
Qu] [wel er ol | Tw ww lm me | Tw we iw
Note: * was measured in CDCl; at 150 MHz; ® was measured in CDCl; at 100 MHz; and © was measured in
CD3OD at 100 MHz. 9
Table 2 'H NMR and "*C NMR data of compounds V to VII
LU503387
Pos. 1 41.6 1.45 342 1.48 312 2.31, ddd (13.8, 13.8, 5.0) 1.19 “| 1.32, dt (10.0, 4.2) 1.47, ddd (13.8, 5.0, 3.1) 2 23.5 1.59 18.4 1.63 33.9 2.48, br d (5.0) 2.44, ddd (17.0, 5.0, 3.1) 3 36.7 2.28 31.6 2.00 198.0 ’ 1.95 ’ ’ ’ ee Tee swe [Tei eo] 6 34.4 2.00 175.6 29.2 251 ’ 1.51 ’ ’ 2.26, ddd (13.1, 13.1, 2.5) ’ EEE ee © a 1.69 2.48, ddd (16.0, 8 32.1 2.00 35.8 11.0,4.7) 43.0 2.94 ’ 1.72 12.37, ddd (16.0,11.0, ’ ’ 5.4) 9 38.8 1.40 34.0 1.80 216.1 ’ 1.26 ’ 1.69 ’ 10 | 260 36.0 41.7 | 256. ddd (14.1, 13.6, 3.5) 2.36, ddd (14.1, 4.6, 3.5) 2.66, ddd (14.9, 14.3, 3.5) 11 147.1 40.8 2.59 34.3 1.58, ddd (14.9, 4.7, 3.5) 5.03,s 5.01,s
I
15 | 105.7 4.72, br s 21.8 1.77 11.7 1.70 4.47, br s 05 HS
Note: * was measured in CDCl; at 150 MHz; ® was measured in CDCl; at 100 MHz; and © was measured in
CD+OD at 100 MHz.
[64] The remaining data of the sesquiterpene compounds I to VII from Fructus Alpiniae
Oxyphyllae prepared in the present disclosure was as follows:
[65] Compound I: colorless crystal; {si -109.5 (0.4, CHCl); HR-ESI-MS (positive) m/z 259.1307 [M + Na] (calcd for C:4H200;Na*, 259.1310); UV (MeOH) Amax nm (log €): 202 (3.63), 228 (3.96); IR (KBr) vmax 3471, 2932, 1671, 1441, 1376, 1254, 1232, 1026, 873, 477cm’ !, which is designated as (35,45,5R,10R)-12-nor-eudesma-3,4-epoxy-6-alkene-5-alcohol-11- ketone, namely (3S,4S,5R,10R) -12-nor-eudesma-3,4- epoxy-6-en-5-ol-11-one.
[66] Compound II: yellow oily;f#i-43.3 (0.2, CHClz); HR-ESI-MS (positive) m/z 193.1227
[M + H]" (calcd for C12H1702”, 193.1229); UV (MeOH) 2max nm (log €): 200 (2.65), 241 (3.10);
IR (KBr) vmax 2929, 2861, 1671, 1449, 1239, 1208, 883 cm’, which is designated as LU503387 (3R,45,10R)-11,12,13-trinor-eudesma-3,4-epoxy-5-alkene-7-ketone, namely (3R,45,10R)- 11,12,13-trinor-eudesma-3,4-epoxy-5-en-7-one.
[67] Compound III: yellow oily; ki +53.6 (0.3, CHCl); HR-ESI-MS (positive) m/z 193.1229 [M + H]" (caled for Ci12H17027, 193.1229); UV (MeOH) Amax nm (log €): 200 (3.52), 237 (3.98); IR (KBr) vmax 2932, 2861, 1673, 1448, 1240, 1207, 883cm™!, which is designated as (3R,4S5,105)-11,12,13-trinor-eudesma-3,4-epoxy-5-alkene-7-ketone, namely (3R,4S,108)- 11,12,13-trinor-eudesma -3,4-epoxy-5-en-7-one.
[68] Compound IV: colorless crystal;i«5-203.2 (0.5, CHCl); HR-ESI-MS (positive) m/z 235.1698 [M + HJ" (calcd for C15H2302”, 235.1698); UV (MeOH) 2max (log €): 249 (3.78) nm;
IR (KBr) vmax 2963, 2947, 1666, 1625, 1467, 1441, 1401, 1384, 1327, 1276, 1198, 1139, 1017, 991, 858, 844, 560 cm“, which is designated as (65,75,10R)-eudesma-6,7-epoxy-4-alkene-3- ketone, namely (65,75,10R)-eudesma-6,7-epoxy -4-en-3-one.
[69] Compound V: colorless crystal; ii -86.8 (0.5, CHCI;). HR-ESI-MS (positive) m/z 203.1804 [M + H - HOT (calcd for C:sH23*, 203.1800); UV (MeOH) 2max (log €): 203 (3.69) nm; IR (KBr) vmax 3381, 2947, 2929, 2849, 1648, 1442, 1400, 1384, 1341, 1278, 1221, 1150, 1127, 1052, 994, 974, 905, 896, 889, 616 cm“, which is designated as (5R,75,10S)-eudesma- 4(15),11-diene-7-alcohol, namely (5R,7S,10S)-eudesma-4(15),11-dien-7-ol.
[70] Compound VI: Colorless oily; tx -16.6 (0.5, CHCI;); HR-ESI-MS (positive) m/z 275.1626 [M + Na] (calcd for C:sH240;Na*, 275.1623); UV (MeOH) 2max (log €): 203 (3.48) nm; IR (KBr) vmx 3462, 2934, 1709, 1466, 1383, 1268 cm’, which is designated as alpinoxyphllaone A.
[71] Compound VII: Light yellow oily; is#*-113.5 (0.5, CHCIs); HR-ESI-MS (positive) m/z 235.1706 [M + H]" (caled for C1sH2302”, 235.1698); UV (MeOH) Amax nm (log €): 201 (3.66), 252 (4.08); IR (KBr) vmax 2929, 1705, 1683, 1450, 1345, 1120, 1026, 589 cm“, which is designated as neoxyphyllanene.
[72] Test Example 2: Effects of compounds I to VII on H>O»-induced OLN-93 cell viability
[73] 1. Experimental steps
[74] After trypsinization, cells were pipetted down by a pipette, and a cell density in a
DMEM medium containing 10% FBS was regulated to 8x10* cells/mL; the cells were uniformly inoculated into a 96-well plate at 100 uL/well, and the plate was placed in an incubator for culture for 24 hours after inoculation. Then, the 96-well plate was taken out, and a supernatant was pipetted, and added to a medicine-containing medium prepared from a complete medium; and experiment settings were specifically shown as follows:
[75] (1) blank control group (Group I) + model group: 100 uL of complete medium was added to each well;
[76] (2) administration sample group (including the compounds I to VII): 100 pL of complete medium containing samples with a corresponding concentration and 0.5 ug/ml of LPS were added to cach well; 11
[771 (3) the 96-well plate was placed in a CO» cell incubator for culture for 24 hours after completion of dosing. LU503387
[78] (4) the supernatant was discarded, 100 uL of DMEM medium was added to the blank control group, and a blank medium was added to the model group and the administration group to prepare 100 uL of H:0> solution having a final concentration of 1 mM. The 96-well plate was placed in the CO» cell incubator for culture for 2 hours.
[79] (5) The supernatant was discarded, 100 pL of blank medium and 20 pL of LMTT solution (5 g/L) were added to each well respectively, and the plate was placed in the incubator for incubation for 4 h. The supernatant was pipetted after 4 h, 150 uL of dimethyl sulfoxide was added to each well, and shaken with uniform mixing, and an OD value was determined on an
ELISA analyzer microplate reader at 570 nm for data analysis.
[80] 2. Experimental results
[81] The OLN-93 cells showed dose-dependent protection against H>O»-induced cell injury after being treated by the compounds I to VII, and the results were shown in FIG. 8 to FIG. 14; and compared with the control group, the cell viability of the HO» model group showed a significant decrease, which is statistically significant (p<0.001), and compared with the HO» model group, the viability of the cells treated by the compounds I to VII was improved to different extents, which was statistically significant. The results showed that the protective effects of the compounds I to VII on OLN-93 cells were improved and dose-dependent over the increase of medicinal concentrations within a certain concentration range.
[82] The above descriptions are only the embodiments of the present disclosure, and the specific technical solutions well-known therein and/or characteristics and additional common knowledge in the solution are not described here detailedly. It should be noted that those skilled in the art can also make several modifications and improvements without departing from the technical solution of the present disclosure, and these modifications and improvements should also fall within the protection scope of the present disclosure, and will not affect the implementation effect of the present disclosure and the practicability of the patent. The protection scope of the present application should be based on the content of the claims, and specific implementations and the like in the specification can be used for explaining the content of the claims. 12
Claims (10)
- Oxyphyllae according to claim 2, characterized in that the conditions for elution with the HP-20 macroporous resins are as follows: ethanol-water serves as an eluant, gradient elution LU503387 is conducted sequentially according to volume concentration percentages of the ethanol in the eluant, which are 0%, 50%, 70% and 95%, a volume of the eluant used at each gradient is 240 L, elution fractions after each gradient elution are collected respectively, and a total of 4 components F1, F2, F3 and F4 are obtained correspondingly.4. The preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae according to claim 3, characterized in that the conditions for elution on the silica gel column are as follows: a mixed solvent of any one of cyclohexane and petroleum ether, and ethyl acetate serves as an eluant, and gradient elution is conducted according to different subcomponents/subfractions as per a volume concentration ratio of the cyclohexane or the petroleum ether to the ethyl acetate, which ranges from 1: 0 to 0: 1; a volume of eluant at each elution gradient ranges from 48 ml to 50 ml, a plurality of elution fractions are collected according to 1/2 to 1/3 of the volume of the eluant used at each elution gradient, and tested, and the elution fractions with consistent results are combined to obtain subfractions.5. The preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae according to claim 4, characterized in that the conditions for elution on the ODS column are as follows: ethanol-water serves as an eluant, and gradient elution is conducted according to different subcomponents/subfractions as per volume concentration percentages of the ethanol to the eluant, which range from 40% to 100%; a volume of eluant at each elution gradient ranges from 350 mL to 450 mL, a plurality of elution fractions are collected according to 1/2 to 1/3 of the volume of the eluant used at each elution gradient, and tested, and the elution fractions with consistent results are combined to obtain subfractions.6. The preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae according to claim 5, characterized in that the eluant used in the semi-preparative HPLC includes a mixed solution of acetonitrile and water, and a mixed solution of methanol and water; wherein a volume ratio of the methanol to the water mixed in the eluant used for separation to obtain the compound II and the compound III is 40: 60.7. The preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae according to claim 6, characterized in that a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound IV and the compound VI is 45: 55; and a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound V is 55: 45.8. The preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae according to claim 7, characterized in that a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound I is 35: 65; and a volume ratio of the acetonitrile to the water mixed in the eluant used for separation to obtain the compound VII is 25: 75.9. The preparation method of the sesquiterpene compound from Fructus Alpiniae Oxyphyllae according to claim 8, characterized in that extraction of the ethanol extracts from Fructus Alpiniae Oxyphyllae comprises steps: conducting reflux extraction on Fructus Alpiniae Oxyphyllae twice by 60% ethanol in a volume that is 6 times that of Fructus Alpiniae Oxyphyllae, each for 2 h; and combining the extracting solutions obtained by extraction, and evaporating the extracting solutions under reduced pressure to obtain the ethanol extract from Fructus Alpiniae Oxyphyllae.10. Application of a sesquiterpene compound from Fructus Alpiniae Oxyphyllae 14
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