CN106928287B - A kind of method and its application for extracting phenolic glycoside compound from lepidii,semen - Google Patents
A kind of method and its application for extracting phenolic glycoside compound from lepidii,semen Download PDFInfo
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- CN106928287B CN106928287B CN201710119301.XA CN201710119301A CN106928287B CN 106928287 B CN106928287 B CN 106928287B CN 201710119301 A CN201710119301 A CN 201710119301A CN 106928287 B CN106928287 B CN 106928287B
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- 210000000582 semen Anatomy 0.000 title claims abstract description 41
- 229930182487 phenolic glycoside Natural products 0.000 title claims abstract description 20
- -1 phenolic glycoside compound Chemical class 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 16
- 229930182470 glycoside Natural products 0.000 claims abstract description 68
- 150000002338 glycosides Chemical class 0.000 claims abstract description 68
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 67
- 239000000284 extract Substances 0.000 claims abstract description 23
- 230000002107 myocardial effect Effects 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 282
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 246
- 235000019441 ethanol Nutrition 0.000 claims description 119
- 238000010828 elution Methods 0.000 claims description 52
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 34
- 238000004440 column chromatography Methods 0.000 claims description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 17
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 238000007689 inspection Methods 0.000 claims description 16
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 6
- 235000004204 Foeniculum vulgare Nutrition 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 3
- QBUKAFSEUHGMMX-MTJSOVHGSA-N (5z)-5-[[3-(1-hydroxyethyl)thiophen-2-yl]methylidene]-10-methoxy-2,2,4-trimethyl-1h-chromeno[3,4-f]quinolin-9-ol Chemical compound C1=CC=2NC(C)(C)C=C(C)C=2C2=C1C=1C(OC)=C(O)C=CC=1O\C2=C/C=1SC=CC=1C(C)O QBUKAFSEUHGMMX-MTJSOVHGSA-N 0.000 claims 2
- 240000006927 Foeniculum vulgare Species 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 10
- 230000006698 induction Effects 0.000 abstract description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 7
- 230000006378 damage Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 238000013459 approach Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 23
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 11
- 239000012071 phase Substances 0.000 description 10
- 241000700159 Rattus Species 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 9
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 241000801118 Lepidium Species 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000003795 desorption Methods 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 241000212314 Foeniculum Species 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000001026 1H--1H correlation spectroscopy Methods 0.000 description 2
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001657511 Lepidium apetalum Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 210000004413 cardiac myocyte Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 244000264242 Descurainia sophia Species 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution 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
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000008065 myocardial cell damage Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to the method and its application that phenolic glycoside compound is extracted from lepidii,semen, can effectively excavate the new role of lepidii,semen, and the technical solution solved is the northern new glycosides A of Roripa, molecular formula C18H24O12[M+Na]+M/z 455.1196, the northern new glycosides B of Roripa, molecular formula C18H24O12[M+Na]+M/z 455.1202; the present invention therefrom separates from the water extract of lepidii,semen and identifies two new phenolic glycoside compounds; that is: the northern new glycosides A of the Roripa and new glycosides B of northern Roripa; and two new phenolic glycoside compounds can significantly improve the cell survival rate of the rat myocardial cell H9c2 damage of hydrogen peroxide induction; with myocardial cell protection effect, new approach is provided to prepare myocardial preservation class drug.
Description
Technical field
The present invention relates to field of medicaments, it is especially a kind of from lepidii,semen extract phenolic glycoside compound method and its answer
With.
Background technique
Lepidium seed is parts of generic medicinal plants, first recorded in Shennong's Herbal, is classified as careless subordinate's product, the flavour of a drug are pungent, bitter, and property is big
It is cold, return lung and bladder meridian, have effects that lead off relieving asthma, line water detumescence.2015 editions " Chinese Pharmacopoeia " " lepidium seeds " included have
Point in north and south, wherein the dry mature seed of crucifer Lepidium apetalum Lepidium apetalum Willd., which is practised, claims " north
Lepidium seed ", and the dry mature seed of Descurainia sophia Descurainia sophia (L.) Webbex Prantl. is practised and claims " southern Roripa montana
Son ".Modern pharmacology shows that lepidii,semen mainly has cardiac effect, and its chemical component is mainly flavonoids.And from Roripa montana
New compound is separated in the water extract of son and studies it using the relevant report that yet there are no this respect so far.
Summary of the invention
For above situation, for the defect for solving the prior art, the purpose of the present invention is just to provide a kind of from lepidii,semen
The middle method and its application for extracting phenolic glycoside compound, can effectively excavate the new role of lepidii,semen.
The technical solution that the present invention solves is, from the method for extracting phenolic glycoside compound in lepidii,semen: taking the north after processing
Lepidium seed adds the water of 10 times of weight of lepidii,semen to extract three times, and each 1.5h, extracting solution is concentrated under reduced pressure into medicinal extract shape, concentration
Supernatant centrifugal filtration is concentrated into no alcohol by the ethyl alcohol alcohol precipitation that the volumetric concentration of 2-3 times of weight of medicinal extract lepidii,semen is 80%
Taste, upper DianionHP-20 column (absorption of highly porous styrene/desorption resin), successively with water, 20% ethyl alcohol (v/v),
40% ethyl alcohol (v/v), 60% ethyl alcohol (v/v), 95% ethyl alcohol (v/v) elution, are concentrated under reduced pressure drying for each position, wherein 20% second
Alcohol elution fraction successively uses water, 10% methanol, 20% methanol, 30% methanol, 100% by Toyopearl HW-40 column chromatography
Methanol elution gradient obtains component A1~A5 and (respectively corresponds water, 10% methanol, 20% methanol, 30% methanol, the elution of 100% methanol
It is position, the same below), component A1 through ODS-18 reversed-phase column chromatography, successively with water, 10% methanol, 20% methanol, 30% methanol,
40% methanol, 100% methanol elution gradient obtain B1~B6 component, B1 through Sephadex LH-20 column chromatography, successively with water,
70% methanol elution gradient, the anisaldehyde-concentrated sulfuric acid (it is anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 that it, which is matched) are spraying thin
Layer inspection is known, and merging anisaldehyde-concentrated sulfuric acid colour developing part, upper Sephadex LH-20 column, 100% methanol elute repeatedly again, fennel
Fragrant aldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and coloured moiety is merged, and component B1 is in the above way repeated three times, obtains component C1,
Component C1 successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution again through Toyopearl HW-40 column chromatography,
Component D1-D4, wherein half preparation HPLC separation of component D4, chromatographic column be YMC-Pack ODS-A chromatographic column (specifications and models:
250 × 10mm, 5 μm of partial size, aperture 12nm), mobile phase is acetonitrile: 0.05% trifluoroacetic acid (v/v)=10:90 obtains northern Roripa
The new glycosides A and new glycosides B of northern Roripa；
Wherein, the new glycosides A of northern Roripa, molecular formula C18H24O12[M+Na]+M/z 455.1196 (calculated value 455.1165).Its
Structural formula is as follows:
The northern new glycosides B of Roripa, molecular formula C18H24O12[M+Na]+M/z 455.1202 (calculated value 455.1165), structural formula
It is as follows:
The present invention therefrom separates from the water extract of lepidii,semen and identifies two new phenolic glycoside compounds, it may be assumed that northern Roripa
The new glycosides A and new glycosides B of northern Roripa, and two new phenolic glycoside compounds can significantly improve hydrogen peroxide (H2O2) induction rat myocardial cell
The cell survival rate of H9c2 damage, has myocardial cell protection effect, provides new approach to prepare myocardial preservation class drug.
Detailed description of the invention
Fig. 1 is the new glycosides A of the northern Roripa of the present invention1H-NMR composes (CD3OD)。
Fig. 2 is the new glycosides A of the northern Roripa of the present invention13C-NMR composes (CD3OD)。
Fig. 3 is that the DEPT 135 of the new glycosides A of the northern Roripa of the present invention composes (CD3OD)。
Fig. 4 is the new glycosides A of the northern Roripa of the present invention1H-1H COSY spectrum.
Fig. 5 is the hsqc spectrum of the new glycosides A of the northern Roripa of the present invention.
Fig. 6 is the HMBC spectrum of the new glycosides A of the northern Roripa of the present invention.
Fig. 7 is the NOESY spectrum of the new glycosides A of the northern Roripa of the present invention.
Fig. 8 is the MS spectrum of the new glycosides A of the northern Roripa of the present invention.
Fig. 9 is the IR spectrum of the new glycosides A of the northern Roripa of the present invention.
Figure 10 is the MS spectrum of the new glycosides A of the northern Roripa of the present invention.
Figure 11 is the new glycosides B of the northern Roripa of the present invention1H-NMR composes (CD3OD)。
Figure 12 is the new glycosides B of the northern Roripa of the present invention13C-NMR composes (CD3OD)。
Figure 13 is that the DEPT 135 of the new glycosides B of the northern Roripa of the present invention composes (CD3OD)。
Figure 14 is the new glycosides B of the northern Roripa of the present invention1H-1H COSY spectrum.
Figure 15 is the hsqc spectrum of the new glycosides B of the northern Roripa of the present invention.
Figure 16 is the HMBC spectrum of the new glycosides B of the northern Roripa of the present invention.
Figure 17 is the NOESY spectrum of the new glycosides B of the northern Roripa of the present invention.
Figure 18 is the MS spectrum of the new glycosides B of the northern Roripa of the present invention.
Figure 19 is the IR spectrum of the new glycosides B of the northern Roripa of the present invention.
Figure 20 is the MS spectrum of the new glycosides B of the northern Roripa of the present invention.
Figure 21 is the present invention north new glycosides A of Roripa to H2O2The H9c2 rat myocardial cell survival rate of induction influence (N=
6)。
Figure 22 is the present invention north new glycosides B of Roripa to H2O2The H9c2 rat myocardial cell survival rate of induction influence (N=
6)。
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Embodiment 1
Lepidii,semen 8kg is taken, in 240 DEG C, fries 5.5min, the water of 10 times of weight of lepidii,semen is added to extract three times, every time
1.5h, extracting solution are concentrated under reduced pressure into medicinal extract, the ethyl alcohol alcohol precipitation that the medicinal extract of concentration is 80% with 20L volumetric concentration, by supernatant from
Heart filtering, is concentrated into no alcohol taste, upper Dianion HP-20 column (absorption of highly porous styrene/desorption resin) is successively used
Water, 20% ethyl alcohol (v/v), 40% ethyl alcohol (v/v), 60% ethyl alcohol (v/v), 95% ethyl alcohol (v/v) elution, each position are depressurized dense
Contracting drying, respectively obtain water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution (respectively 361.0g, 71.2g,
89.6g, 67.2g, 28.7g) component, wherein 20% ethanol elution component by Toyopearl HW-40 column chromatography, is successively used
Water, 10% methanol, 20% methanol, 30% methanol, 100% methanol elution gradient obtain component A1~A5, component A1 (25.3g) warp
ODS-18 reversed-phase column chromatography is successively washed with water, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 100% methanol gradient
It is de-, B1~B6 is obtained, component B1 (7.8g) successively uses water, 70% methanol elution gradient, fennel through Sephadex LH-20 column chromatography
Fragrant aldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and anisaldehyde-concentrated sulfuric acid presses anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 proportion closes
And upper Sephadex LH-20 column, 100% methanol elute repeatedly again for anisaldehyde-concentrated sulfuric acid colour developing part, the dense sulphur of anisaldehyde-
The spraying thin layer inspection of acid is known, and merges coloured moiety, component B1 is in the above way repeated three times, obtains component C1 (1.2g), group
Divide C1 again through Toyopearl HW-40 column chromatography, successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution, obtain
Component D1-D4, wherein component D4 (50.3mg) is YMC-Pack ODS-A chromatographic column (rule with half preparation HPLC separation, chromatographic column
Lattice number: 250 × 10mm, 5 μm of partial size, aperture 12nm), mobile phase is -0.05% trifluoroacetic acid (volume ratio 10:90) of acetonitrile,
Obtain new glycosides A (14.0mg, the t of northern RoripaR=80.3min) and new glycosides B (2.9mg, the t of northern RoripaR=62.5min).
Embodiment 2
Lepidii,semen 2kg is taken, in 240 DEG C, fries 5.5min, the water of 10 times of weight of lepidii,semen is added to extract three times, every time
1.5h, extracting solution are concentrated under reduced pressure into medicinal extract, and supernatant is centrifuged by the ethyl alcohol alcohol precipitation that the medicinal extract of concentration is 80% with 5L volumetric concentration
Filtering, is concentrated into no alcohol taste, upper Dianion HP-20 column (absorption of highly porous styrene/desorption resin), successively with water,
20% ethyl alcohol (v/v), 40% ethyl alcohol (v/v), 60% ethyl alcohol (v/v), 95% ethyl alcohol (v/v) elution, each position is concentrated under reduced pressure
It is dry, respectively obtain water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution (respectively 90.1g, 18.0g,
22.5g, 17.0g, 7.3g) component, wherein 20% ethanol elution component by Toyopearl HW-40 column chromatography, is successively used
Water, 10% methanol, 20% methanol, 30% methanol, 100% methanol elution gradient obtain component A1~A5, component A1 (6.2g) warp
ODS-18 reversed-phase column chromatography is successively washed with water, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 100% methanol gradient
It is de-, B1~B6 is obtained, component B1 (1.9g) successively uses water, 70% methanol elution gradient, fennel through Sephadex LH-20 column chromatography
Fragrant aldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and anisaldehyde-concentrated sulfuric acid presses anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 proportion closes
And upper Sephadex LH-20 column, 100% methanol elute repeatedly again for anisaldehyde-concentrated sulfuric acid colour developing part, the dense sulphur of anisaldehyde-
The spraying thin layer inspection of acid is known, and merges coloured moiety, component B1 is in the above way repeated three times, obtains component C1 (300mg), group
Divide C1 again through Toyopearl HW-40 column chromatography, successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution, obtain
Component D1-D4, wherein component D4 (13.0mg) is YMC-Pack ODS-A chromatographic column (rule with half preparation HPLC separation, chromatographic column
Lattice number: 250 × 10mm, 5 μm of partial size, aperture 12nm), mobile phase is -0.05% trifluoroacetic acid of acetonitrile (10:90), obtains north
New glycosides A (3.2mg, the t of RoripaR=80.3min) and new glycosides B (0.6mg, the t of northern RoripaR=62.5min).
Embodiment 3
Lepidii,semen 1kg is taken, in 240 DEG C, fries 5.5min, the water of 10 times of weight of lepidii,semen is added to extract three times, every time
1.5h, extracting solution are concentrated under reduced pressure into medicinal extract, and supernatant is centrifuged by the ethyl alcohol alcohol precipitation that the medicinal extract of concentration is 80% with 3L volumetric concentration
Filtering, is concentrated into no alcohol taste, upper Dianion HP-20 column (absorption of highly porous styrene/desorption resin), successively with water,
20% ethyl alcohol (v/v), 40% ethyl alcohol (v/v), 60% ethyl alcohol (v/v), 95% ethyl alcohol (v/v) elution, each position is concentrated under reduced pressure
It is dry, respectively obtain water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution (respectively 45.0g, 9.0g,
11.3g, 8.5g, 3.6g) component, wherein 20% ethanol elution component (9.0g) is by Toyopearl HW-40 column chromatography, according to
It is secondary to use water, 10% methanol, 20% methanol, 30% methanol, 100% methanol elution gradient, obtain component A1~A5, component A1 (3.2g)
Through ODS-18 reversed-phase column chromatography, water, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 100% methanol gradient are successively used
Elution, obtains B1~B6, and component B1 (0.9g) successively uses water, 70% methanol elution gradient through Sephadex LH-20 column chromatography,
Anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and anisaldehyde-concentrated sulfuric acid presses anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 proportion,
Merging anisaldehyde-concentrated sulfuric acid colour developing part, upper Sephadex LH-20 column, 100% methanol elute repeatedly again, and anisaldehyde-is dense
The inspection of sulfate spray thin layer is known, and coloured moiety is merged, and component B1 is in the above way repeated three times, obtains component C1 (150mg)
Component, C1 successively use water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution again through Toyopearl HW-40 column chromatography,
Component D1-D4 is obtained, wherein component D4 (6.3mg) is YMC-Pack ODS-A chromatographic column (rule with half preparation HPLC separation, chromatographic column
Lattice number: 250 × 10mm, 5 μm of partial size, aperture 12nm), mobile phase is -0.05% trifluoroacetic acid of acetonitrile (10:90), obtains north
New glycosides A (1.8mg, the t of RoripaR=80.0min) and new glycosides B (0.4mg, the t of northern RoripaR=62.0min).
For the effective substance of clear lepidii,semen, the present invention extracts it using water boiling method, and due to
It meets water stickness, does not say so that decocting liquid recovery rate is low, and the ingredient extracted is mostly mucilaginous substance, therefore the present invention is first
First using method is fried, in 240 DEG C, frying 5.5min simply processes it, so that the ingredient in lepidii,semen is easier to dissolve out,
It is therefrom separated from the water extract of lepidii,semen and identifies two new phenolic glycoside compounds, it may be assumed that the northern new glycosides A of Roripa and the new glycosides of northern Roripa
B, experimental result show that the two new phenolic glycoside compounds can significantly improve the rat myocardial cell of hydrogen peroxide (H2O2) induction
The cell survival rate of H9c2 damage, has myocardial cell protection effect.And sufficiently prove through test, correlation test data
It is as follows:
1 instrument and reagent
Nuclear magnetic resonance is used with III 500 Nuclear Magnetic Resonance of BrukerAVANCE (TMS internal standard) (Bruker), infrared spectroscopy
10 Microscope Spectrometer of Nicolet is (Thermo Scientific, USA), high resolution mass spectrum are used
Bruker maxis HD mass spectrometer, ultraviolet spectra Shimadzu UV-2401PC apparatus, efficiently
The serial 2695 efficient liquid phase systems of phase chromatography-use Waters Alliance, are equipped with 2998 type diode array detector,
Empower3 Data Processing in Chromatography Workstation, LC50 type high pressure preparative liquid chromatograph, [match spectrum is sharp to think (north to UV200 type UV detector
Capital) Science and Technology Ltd.], YMC-Pack ODS-A chromatographic column (250 × 10mm.D.S-5mm, 12mm) (YMC Co., Ltd),
It is remaining to have N-1100 type Rotary Evaporators (Shanghai Ai Lang Instrument Ltd), A-1000S type water flow air exhauster (Shanghai Ai Lang instrument
Co., Ltd), N-1111 type freezes water circle device (Shanghai Ai Lang Instrument Ltd), FDU-2110 type freeze drier
(Shanghai Ai Lang Instrument Ltd), DFZ-60508 type vacuum oven (Shanghai Yiheng Scientific Instruments Co., Ltd), AB204-
N a ten thousandth analytical precision balances (METTLER TOLEDO), iMARK type microplate reader (U.S. BIO-RAD), carbon dioxide culture
Case (Shanghai STIK), superclean bench (Su Jing group), the super combined Superpure water machine (SARTORIUS) of Arium 611VF,
It sets microscope (Nikon), PB-10 acidometer (German Sai Duolisi group).
Column chromatographic stuffing Diaion HP-20, MCI Gel CHP-20 (Mitsubishi chemical company), Toyopearl HW-
40 (Japanese TOSOH companies), Sephadex LH-20 (Parmacia Biotech company), column chromatograph used silica gel H (160-
200 mesh) it is that Haiyang Chemical Plant, Qingdao produces, culture dish, 96 well culture plates, cell cryopreservation tube (Corning company), chromatography used
The production of pure reagent position α Cygni friend's fine chemicals Co., Ltd, analytical reagents used are Beijing Chemical Plant and Tianjin third
Learn chemical reagent work's production.
DMEM high sugar culture solution (Gibco), fetal calf serum (Zhejiang Tian Hang Biotechnology Co., Ltd), trypsase
(Gibco), DMSO (Solarbio)；MTT (Biosharp), H2O2Solution (Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd),
Water is ultrapure water, and PBS buffer solution etc. is autogamy.
Lepidii,semen picked up from Nanyang, henan in 2014, was identified as crucifer Lepidium apetalum Lepidium
The dry mature seed of apetalum Willd..
2 Structural Identifications
The northern new glycosides A of Roripa, light yellow crystalline powder (MeOH), molecular formula C18H24O12[M+Na]+m/z 455.1196
(calculated value 455.1165), UV (MeOH) λmax:206.0,230.0,315.0nm；IR(KBr)νmaxcm-1: 3364,2931,
1576,1489,1039.Its structural formula and nuclear magnetic data are as follows:
The northern new glycosides B of Roripa, light yellow crystalline powder (MeOH), molecular formula C18H24O12[M+Na]+m/z 455.1202
(calculated value 455.1165), UV (MeOH) λmax:206.0,238.0,302.0nm；IR(KBr)νmaxcm-1: 3413,2925,
1672,1466,1246,1046.Its structural formula and nuclear magnetic data are as follows:
The new glycosides A's and B of the northern Roripa of table 11H NMR (500MHz) and13C NMR (125MHz) data (in CD3OD)
The 3 north new glycosides A of Roripa and the north new glycosides B of Roripa are to hydrogen peroxide (H2O2) injury rats cardiac muscle cell H9c2 protective effect test
3.1 cell
H9c2 cell strain is purchased from Shenzhen Biowit Biotechnologies Co., Ltd..
3.2 cell culture
H9c2 culture (is contained into penicillin and streptomysin in containing the DMEM culture medium that volume fraction is 10% fetal calf serum
100kU·L-1), it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, equal logarithmic growth phase cell is for testing.
3.3H2O2The foundation of the H9c2 rat myocardial cell damage model of induction
When secondary culture, by the cell of logarithmic growth phase with 5 × 104Density be inoculated in 96 orifice plates, it is complete to cell
After adherent, the DMEM in high glucose culture solution of drug containing is used instead.H2O2It gives containing 200 μM of H2O2The culture solution culture of DMEM in high glucose containing serum
6h。
3.4 experimental group
Every group sets 6 parallel holes, if normal group (NC), H2O2Each monomeric compound administration group of group (M), lepidii,semen.H2O2
Group: it gives containing 200 μM of H2O2The culture solution culture of DMEM in high glucose containing serum 6h；The northern new glycosides A of Roripa and the new glycosides B administration group of northern Roripa: point
It does not give containing 200 μM of H2O2With the culture of DMEM in high glucose containing serum of 1,2,4,8,16,32,64 μM of northern Roripa new glycosides A and the new glycosides B of northern Roripa
Liquid culture 6h.
3.5MTT method detects the north new glycosides A of Roripa and influence of the north new glycosides B of Roripa to H9c2 damaging cells survival rate
Cell inoculation absorbs supernatant, each hole is separately added into containing 1%MTT after 96 orifice plates, adherent rear dosing intervention culture
DMEM in high glucose culture solution 200 μ l, 37 DEG C of incubation 4h of (5g/L) absorb supernatant, and the DMSO of 150 μ L is added, and concussion 10min makes
Crystallization sufficiently dissolution.With OD value at microplate reader detection 490nm wavelength, cell survival rate=(OD experimental group/OD pairs is calculated with formula
According to group) × 100%.Experiment is repeated 3 times, and is averaged.
3.6 statistical method
Experimental data mean ± standard deviationIt indicates, handles data, each comparison among groups with SPSS18.0 statistical software
Using one-way analysis of variance (One-WayANOVA), P < 0.05 indicates significant, and P < 0.01 indicates extremely significant property
Meaning.3.7 result
Result of study shows: compared to the blank group, model group cell viability significantly reduces (P < 0.01)；With model group phase
When than, the northern new glycosides A dosage of Roripa being 1 μM, can extremely significant raising cell viability (P < 0.01), at 2,4,8,16 μM, energy
Enough significantly improve cell viability (P < 0.05)；The northern new glycosides B dosage of Roripa can significantly improve cell at 4,8,16,32 μM
Vigor (P < 0.05).Show that the lepidii,semen monomer north new glycosides A and B of Roripa has certain protective effect to cardiac muscle cell.
The 2 north new glycosides A of Roripa of table is to H2O2The H9c2 rat myocardial cell survival rate of induction influence (N=6)
Note: compared with normal group: P < 0.05 *；P < 0.01 * and H2O2Group compares:#P<0.05；##P<0.01
The 3 north new glycosides B of Roripa of table is to H2O2The H9c2 rat myocardial cell survival rate of induction influence (N=6)
Note: compared with normal group: P < 0.05 *；P < 0.01 * and H2O2Group compares:#P<0.05；##P<0.01
To sum up, the new phenolic glycoside compound of two for separating and identifying from the water extract of lepidii,semen, experimental result are shown
The two new phenolic glycoside compounds can significantly improve hydrogen peroxide (H2O2) induction rat myocardial cell H9c2 damage cell survival
Rate has myocardial cell protection effect, can be effectively used for preparing myocardial preservation class drug, develop the new application of lepidii,semen,
With actual clinical meaning and good application and popularization value.
Claims (7)
1. a kind of phenolic glycoside compound extracted from lepidii,semen, which is characterized in that the northern new glycosides A of Roripa, molecular formula C18H24O12[M
+Na]+M/z 455.1196, structural formula is as follows:
The northern new glycosides B of Roripa, molecular formula C18H24O12[M+Na]+M/z 455.1202, structural formula is as follows:
2. the extracting method of the phenolic glycoside compound described in claim 1 extracted from lepidii,semen, which is characterized in that take processing
Lepidii,semen afterwards adds the water of 10 times of weight of lepidii,semen to extract three times, and each 1.5h, extracting solution is concentrated under reduced pressure into medicinal extract shape,
The ethyl alcohol alcohol precipitation that the volumetric concentration of 2-3 times of weight of medicinal extract lepidii,semen of concentration is 80%, by supernatant centrifugal filtration, concentration
To no alcohol taste, upper Dianion HP-20 column successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution,
Drying is concentrated under reduced pressure in each position, wherein 20% ethanol elution component is by Toyopearl HW-40 column chromatography, successively with water,
10% methanol, 20% methanol, 30% methanol, 100% methanol elution gradient obtain component A1~A5, and component A1 is through ODS-18 reverse phase
Column chromatography successively uses water, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 100% methanol elution gradient, obtains B1
~B6, component B1 successively use water, 70% methanol elution gradient through Sephadex LH-20 column chromatography, and anisaldehyde-concentrated sulfuric acid is spraying
Thin layer inspection is known, and merging anisaldehyde-concentrated sulfuric acid colour developing part, upper Sephadex LH-20 column, 100% methanol elute repeatedly again,
Anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and merges coloured moiety, component B1 is in the above way repeated three times, obtains component
C1, component C1 are successively washed with water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient again through Toyopearl HW-40 column chromatography
It is de-, component D1-D4 is obtained, wherein half preparation HPLC separation of component D4, chromatographic column is YMC-Pack ODS-A chromatographic column, mobile phase
The new glycosides A of northern Roripa and the new glycosides B of northern Roripa are obtained for acetonitrile: 0.05% trifluoroacetic acid=10:90.
3. the extracting method of the phenolic glycoside compound according to claim 2 extracted from lepidii,semen, which is characterized in that take
Lepidii,semen 8kg fries 5.5min in 240 DEG C, and the water of 10 times of weight of lepidii,semen is added to extract three times, each 1.5h, extracting solution
It is concentrated under reduced pressure into medicinal extract, the ethyl alcohol alcohol precipitation that the medicinal extract of concentration is 80% with 20L volumetric concentration, by supernatant centrifugal filtration, concentration
To no alcohol taste, upper Dianion HP-20 column successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution,
Drying is concentrated under reduced pressure in each position, respectively obtains the group of water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution
Point, wherein 20% ethanol elution component is by Toyopearl HW-40 column chromatography, successively with water, 10% methanol, 20% methanol,
30% methanol, 100% methanol elution gradient obtain component A1~A5, component A1 through ODS-18 reversed-phase column chromatography, successively with water,
10% methanol, 20% methanol, 30% methanol, 40% methanol, 100% methanol elution gradient obtain B1~B6, component B1 warp
Sephadex LH-20 column chromatography successively uses water, 70% methanol elution gradient, and anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, fennel
Aldehyde-concentrated sulfuric acid presses anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 proportion, merges anisaldehyde-concentrated sulfuric acid colour developing part again
Secondary upper Sephadex LH-20 column, 100% methanol elute repeatedly, and anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and merge colour developing portion
Point, component B1 is in the above way repeated three times, obtain component C1, component C1 again through Toyopearl HW-40 column chromatography, according to
It is secondary to use water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution, component D1-D4 is obtained, wherein half preparation HPLC of component D4
Separation, chromatographic column are YMC-Pack ODS-A chromatographic column, and mobile phase is -0.05% trifluoroacetic acid of acetonitrile=10:90, obtain northern Roripa
The new glycosides A and new glycosides B of northern Roripa.
4. the extracting method of the phenolic glycoside compound according to claim 2 extracted from lepidii,semen, which is characterized in that take
Lepidii,semen 2kg fries 5.5min in 240 DEG C, and the water of 10 times of weight of lepidii,semen is added to extract three times, each 1.5h, extracting solution
It is concentrated under reduced pressure into medicinal extract, supernatant centrifugal filtration is concentrated by the medicinal extract of concentration with the ethyl alcohol alcohol precipitation that 5L volumetric concentration is 80%
Without alcohol taste, upper Dianion HP-20 column successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution, will
Drying is concentrated under reduced pressure in each position, respectively obtains the component of water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution,
Wherein 20% ethanol elution component successively uses water, 10% methanol, 20% methanol, 30% by Toyopearl HW-40 column chromatography
Methanol, 100% methanol elution gradient obtain component A1~A5, and component A1 successively uses water, 10% first through ODS-18 reversed-phase column chromatography
Alcohol, 20% methanol, 30% methanol, 40% methanol, 100% methanol elution gradient obtain B1~B6, and component B1 is through Sephadex
LH-20 column chromatography successively uses water, 70% methanol elution gradient, and anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, the anisaldehyde-concentrated sulfuric acid
By anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 proportion, merge anisaldehyde-concentrated sulfuric acid colour developing part again on
Sephadex LH-20 column, 100% methanol elute repeatedly, and anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and merge coloured moiety, group
Divide B1 to be in the above way repeated three times, obtains component C1, component C1 through Toyopearl HW-40 column chromatography, is successively used again
Water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution, obtain component D1-D4, wherein half preparation HPLC separation of component D4,
Chromatographic column is YMC-Pack ODS-A chromatographic column, and mobile phase is -0.05% trifluoroacetic acid of acetonitrile=10:90, obtains the new glycosides A of northern Roripa
With the new glycosides B of northern Roripa.
5. the extracting method of the phenolic glycoside compound according to claim 2 extracted from lepidii,semen, which is characterized in that take
Lepidii,semen 1kg fries 5.5min in 240 DEG C, and the water of 10 times of weight of lepidii,semen is added to extract three times, each 1.5h, extracting solution
It is concentrated under reduced pressure into medicinal extract, supernatant centrifugal filtration is concentrated by the medicinal extract of concentration with the ethyl alcohol alcohol precipitation that 3L volumetric concentration is 80%
Without alcohol taste, upper Dianion HP-20 column successively uses water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution, will
Drying is concentrated under reduced pressure in each position, respectively obtains the component of water, 20% ethyl alcohol, 40% ethyl alcohol, 60% ethyl alcohol, 95% ethanol elution,
Wherein 20% ethanol elution component successively uses water, 10% methanol, 20% methanol, 30% by Toyopearl HW-40 column chromatography
Methanol, 100% methanol elution gradient obtain component A1~A5, and component A1 successively uses water, 10% first through ODS-18 reversed-phase column chromatography
Alcohol, 20% methanol, 30% methanol, 40% methanol, 100% methanol elution gradient obtain B1~B6, and component B1 is through Sephadex
LH-20 column chromatography successively uses water, 70% methanol elution gradient, and anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, the anisaldehyde-concentrated sulfuric acid
By anisaldehyde: the concentrated sulfuric acid: 95% ethyl alcohol=1:2:100 proportion, merge anisaldehyde-concentrated sulfuric acid colour developing part again on
Sephadex LH-20 column, 100% methanol elute repeatedly, and anisaldehyde-concentrated sulfuric acid, which is sprayed thin layer inspection, to be known, and merge coloured moiety, group
Divide B1 to be in the above way repeated three times, obtains component C1 component C1 again through Toyopearl HW-40 column chromatography, successively use
Water, 20% ethyl alcohol, 40% ethyl alcohol, 70% ethanol gradient elution, obtain component D1-D4, wherein half preparation HPLC separation of component D4,
Chromatographic column is YMC-Pack ODS-A chromatographic column, and mobile phase is -0.05% trifluoroacetic acid of acetonitrile=10:90, obtains the new glycosides A of northern Roripa
With the new glycosides B of northern Roripa.
6. phenolic glycoside compound described in claim 1 is preparing the application in myocardial preservation drug.
7. the new glycosides A of northern Roripa, the north new glycosides B of Roripa that any one of the claim 2-5 extracting method is extracted are preparing myocardial preservation medicine
Application in object.
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| CN107353315A (en) * | 2017-07-29 | 2017-11-17 | 河南中医药大学 | A kind of method and its application that the new alkali A compounds of northern Roripa are extracted from lepidii,semen |
| CN107446008B (en) * | 2017-09-06 | 2020-10-02 | 河南中医药大学 | Neoside E, and preparation method and application thereof |
| CN107602640A (en) * | 2017-10-18 | 2018-01-19 | 河南中医药大学 | A kind of method and its application that northern Roripa neoflavone C is extracted from lepidii,semen |
| CN107602639B (en) * | 2017-10-18 | 2020-10-02 | 河南中医药大学 | Method for extracting sesquiterpene compound from semen lepidii and application thereof |
| CN108341847B (en) * | 2017-11-29 | 2020-10-02 | 河南中医药大学 | A method for extracting neoline G and neoline H from semen Lepidii, and its preparation method and application |
| CN108191936B (en) * | 2018-01-12 | 2020-10-02 | 河南中医药大学 | A neoline J and K extracted from semen Lepidii, and its preparation method and application |
| CN115671154A (en) * | 2022-11-29 | 2023-02-03 | 浙江大学 | Semen Lepidii flavone component, its extraction method and application in preparing medicine for treating or preventing heart failure |
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