CN116283874A - Rhododendron dauricum hetero-terpenoid, extraction method and application - Google Patents
Rhododendron dauricum hetero-terpenoid, extraction method and application Download PDFInfo
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- CN116283874A CN116283874A CN202211696752.7A CN202211696752A CN116283874A CN 116283874 A CN116283874 A CN 116283874A CN 202211696752 A CN202211696752 A CN 202211696752A CN 116283874 A CN116283874 A CN 116283874A
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- 238000000605 extraction Methods 0.000 title claims abstract description 12
- 241000681946 Rhododendron dauricum Species 0.000 title description 2
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- 150000001875 compounds Chemical class 0.000 claims abstract description 40
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- 238000004440 column chromatography Methods 0.000 claims abstract description 16
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 14
- -1 diterpenoid compound Chemical class 0.000 claims abstract description 14
- 239000002024 ethyl acetate extract Substances 0.000 claims abstract description 12
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- 238000002360 preparation method Methods 0.000 claims description 32
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/80—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
- C07C49/24—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups
- C07C49/245—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups containing six-membered aromatic rings
- C07C49/248—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups containing six-membered aromatic rings having unsaturation outside the aromatic rings
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Plant Pathology (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- General Chemical & Material Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention discloses a rhododendron flower diterpenoid compound, an extraction method and application thereof, wherein the invention utilizes a natural medicinal chemistry research method to obtain a total extract by crushing the overground part of rhododendron flower, and carrying out heat reflux extraction in alcohols; extracting the total extract with ethyl acetate, and repeatedly separating the ethyl acetate extract phase by column chromatography to obtain six hetero-terpenoids. The compound has good antibacterial activity to plant bacteria such as kiwifruit canker and bacillus cereus, and also has good antibacterial activity to pathogenic bacteria such as staphylococcus aureus, and is safe to environment, people, livestock and other beneficial organisms.
Description
Technical Field
The invention belongs to the field of natural pharmaceutical chemistry, and particularly relates to a rhododendron flower hetero-terpene compound, an extraction method and application.
Background
The Rhododendron (R.capitatum Maxim) is a Rhododendron (Rhododendron) plant of the Rhododendron family (Ericaceae), is mainly distributed in the whole country except Ningxia and Xinjiang, and is mainly used for activating blood circulation to relieve pain, treating rheumatism and the like. The report in literature indicates that the plant has rich chemical components, mainly including sesquiterpene, hetero-terpene, diterpene, triterpene, alkaloid, glycoside and other compounds, and Chromane hetero-terpene as main characteristic components of rhododendron, and has wide pharmacological activity, such as antitumor, antiviral, antiinflammatory, PTP1B inhibiting activity, cytotoxicity and the like. However, few reports are reported on the azalea heteroterpenes, and no report on the antibacterial activity of the heteroterpenes is found, and further research on functional active ingredients is needed so as to better develop the medicinal value.
Disclosure of Invention
Aiming at the defects and the shortcomings of the prior art, the invention aims to provide a rhododendron flower hetero-terpene compound, an extraction method and application.
In order to achieve the above purpose, the present application adopts the following technical scheme:
a rhododendron flower hetero terpene compound comprises at least one of structures shown in formulas 1-6;
optionally, comprises pulverizing aerial parts of Rhododendron pulvis, and extracting under reflux in alcohol to obtain total extract; extracting the total extract with ethyl acetate, and repeatedly subjecting the obtained ethyl acetate extract phase to column chromatography.
Alternatively, the alcohol is selected from methanol and ethanol.
Optionally, the preparation method specifically includes:
drying aerial parts of rhododendron cephali in shade, pulverizing to 30 meshes, heating and reflux-extracting in methanol for 3 times to obtain an extracting solution, concentrating the extracting solution under reduced pressure to recover methanol to obtain a crude extract, and extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract phase;
dissolving an ethyl acetate extract phase by using chloroform or acetone, then carrying out column chromatography segmentation and rough separation by using silica gel, and carrying out gradient elution by using a chloroform/acetone system, wherein the volume ratio of the gradient elution of the chloroform/acetone system is sequentially 1:0, 9:1, 8:2, 7:3, 1:1 and 0:1; taking sulfuric acid ethanol solution as a color developing agent, and combining similar fractions according to TLC color development to obtain 8 components EA 1-EA 6;
after the EA2 component is subjected to silica gel column chromatography, carrying out gradient elution by using a petroleum ether/ethyl acetate system, wherein the elution concentration of the petroleum ether/ethyl acetate system is sequentially 10:1, 5:1, 4:1 and 2:1 in volume ratio, and 9 components 1A-1I are obtained according to the polarity;
separating component 1B by RP-C18 reverse phase chromatographic column, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compounds 1 and 2;
separating the component 1A sequentially by RP-C18 reverse chromatographic column, forward silica gel chromatographic column, RP-C18 reverse chromatographic column chromatography and HPLC to obtain compound 3;
separating the component 1A by RP-C18 reverse chromatographic column chromatography, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compound 4;
separating component 1C by RP-C18 reverse chromatographic column, forward silica gel chromatographic column and HPLC to obtain compound 5;
carrying out forward silica gel column chromatography on the component EA1, carrying out coarse separation on the component EA1 by using a petroleum ether-ethyl acetate system, and obtaining 10 components 34A-34J according to the polarity, wherein the volume ratio of the petroleum ether-ethyl acetate system is sequentially 1:0, 25:1, 15:1, 10:1, 8:1, 4:1 and 2:1;
separating the component 34G by a forward silica gel chromatographic column, a Sephadx-LH20 gel chromatographic column and HPLC after preparing a liquid phase by the RP-C18 semi-preparation, and obtaining the compound 6, wherein the solvent condition used by the RP-C18 semi-preparation liquid phase is methanol with volume concentration of 20%, 40%, 60%, 80% and 100% in sequence.
The rhododendron flower hetero terpene compound or the pharmaceutically acceptable salt thereof is applied to preparation of antibacterial agents.
Optionally, the antibacterial agent is used for preventing and treating diseases caused by kiwi fruit canker, bacillus cereus, staphylococcus aureus and/or methicillin-resistant staphylococcus aureus.
Optionally, the antibacterial agent preparation is prepared into a pharmaceutically acceptable preparation by adding pharmaceutically acceptable auxiliary materials according to a conventional process, and the pharmaceutically acceptable preparation is a solid preparation or a liquid preparation.
Optionally, the solid formulation comprises a granule, a capsule, a tablet or a pill.
Optionally, the liquid formulation comprises an injectable formulation.
In the antibacterial agent preparation, the content of the rhododendron cephaloporphin compound or the pharmaceutically acceptable salt thereof is 2-8% by mass percent.
Compared with the prior art, the invention has the beneficial technical effects that:
the six hetero-terpene compounds are firstly extracted and separated from the aerial parts of the rhododendron, and the specific preparation method of the compounds has important practical significance and broad market prospect for the development of hetero-terpene components and the comprehensive development and utilization of rhododendron plant resources. Experiments prove that the compound has good antibacterial activity on plant bacteria such as kiwifruit canker and bacillus cereus, also has good antibacterial activity on pathogenic bacteria such as staphylococcus aureus, and is safe to environment, people, livestock and other beneficial organisms. The antibacterial agent formulation prepared by using the compound of the present invention may be administered orally, nasally, rectally or parenterally, and may be formulated into conventional solid formulations, liquid formulations or solutions for injection, etc., preferably tablets, capsules and injections.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a specific isolation scheme for compounds of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The compounds of the present disclosure may be obtained by a variety of techniques, such as chemical synthesis; as another example, from other rhododendrons, the extraction process may be by any of a variety of known techniques, such as: supercritical fluid extraction, thermal reflux extraction with solvent (ethanol, methanol, acetone, etc.), percolation with solvent (ethanol, methanol, acetone, etc.), etc.
Unless otherwise indicated, the structural formulae described herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers). Thus, individual stereochemical isomers or mixtures of enantiomers, diastereomers or geometric isomers of the compounds of the invention are all within the scope of the invention.
The invention provides rhododendron cephali diterpenoid compounds, which have any one or more of structures shown in formulas 1-6;
the preparation method of the rhododendron cephalopod hetero terpene compound comprises the steps of crushing overground parts of rhododendron cephalopod, and carrying out hot reflux extraction in alcohols to obtain total extract; extracting the total extract with ethyl acetate, and repeatedly subjecting the obtained ethyl acetate extract phase to column chromatography.
Still further, the alcohol is selected from methanol and ethanol.
Furthermore, the extraction and preparation method specifically comprises the following steps: drying aerial parts of rhododendron cephali in shade, pulverizing to 30 meshes, heating and refluxing in methanol to obtain an extracting solution, concentrating the extracting solution under reduced pressure to recover methanol to obtain a crude extract, and extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract phase;
dissolving an ethyl acetate extract phase by using chloroform or acetone, then carrying out column chromatography segmentation and rough separation by using silica gel, and carrying out gradient elution by using a chloroform/acetone system, wherein the volume ratio of the gradient elution of the chloroform/acetone system is sequentially 1:0, 9:1, 8:2, 7:3, 1:1 and 0:1; taking sulfuric acid ethanol solution as a color developing agent, and combining similar fractions according to TLC color development to obtain 6 components EA 1-EA 6;
after the EA2 component is subjected to silica gel column chromatography, carrying out gradient elution by using a petroleum ether/ethyl acetate system, wherein the elution concentration of the petroleum ether/ethyl acetate system is sequentially 10:1, 5:1, 4:1 and 2:1 in volume ratio, and 9 components 1A-1I are obtained according to the polarity;
separating component 1B by RP-C18 reverse phase chromatographic column, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compounds 1 and 2;
separating the component 1A sequentially by RP-C18 reverse chromatographic column, forward silica gel chromatographic column, RP-C18 reverse chromatographic column chromatography and HPLC to obtain compound 3;
separating the component 1A by RP-C18 reverse chromatographic column chromatography, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compound 4;
separating component 1C by RP-C18 reverse chromatographic column, forward silica gel chromatographic column and HPLC to obtain compound 5;
carrying out forward silica gel column chromatography on the component EA1, carrying out coarse separation on the component EA1 by using a petroleum ether-ethyl acetate system, and obtaining 10 components 34A-34J according to the polarity, wherein the volume ratio of the petroleum ether-ethyl acetate system is sequentially 1:0, 25:1, 15:1, 10:1, 8:1, 4:1 and 2:1;
separating the component 34G by a forward silica gel chromatographic column, a Sephadx-LH20 gel chromatographic column and HPLC after preparing a liquid phase by the RP-C18 semi-preparation, and obtaining the compound 6, wherein the solvent condition used by the RP-C18 semi-preparation liquid phase is methanol with volume concentration of 20%, 40%, 60%, 80% and 100% in sequence.
The invention also discloses application of the rhododendron flower hetero terpene compound or pharmaceutically acceptable salt thereof in preparing an antibacterial agent preparation.
Furthermore, the antibacterial agent preparation is used for preventing and controlling kiwifruit canker, bacillus cereus and staphylococcus aureus.
Furthermore, the antibacterial agent preparation is prepared into a pharmaceutically acceptable preparation by adding pharmaceutically acceptable auxiliary materials according to a conventional process, and the pharmaceutically acceptable preparation is a solid preparation or a liquid preparation.
Further, the solid preparation comprises granules, capsules, tablets and pills; the liquid formulation includes an injectable formulation.
Furthermore, in the antibacterial agent preparation, the content of the rhododendron cephalopoietini hetero terpene compound or the pharmaceutically acceptable salt thereof is 2-8% by mass percent.
The aerial parts of the rhododendron capitatum used in the present disclosure were collected in the Qinling region 5 months in 2018, and obtained by natural drying.
In the invention, 6 components are obtained by combining similar fractions according to TLC color development, the components 1A-1I are obtained by gradient elution through a petroleum ether/ethyl acetate system, each component is named according to the polarity-magnitude outflow sequence, namely, the outflow is performed first with small polarity, the outflow is performed after the polarity is large, and the similar components are combined according to the TLC color development condition.
The biological activity measurement result and the physiological and biochemical experiment result in the embodiment of the invention prove that the rhododendron flower hetero-terpenoid has antibacterial activity. Those skilled in the art will appreciate that biological activity assays and physiological and biochemical experiments establish the general utility of rhododendron hetercerpenes as antibacterial agents.
In order to better understand the essence of the invention, the technical contents of the invention will be described in detail with examples, but the invention is not limited to these examples.
Example 1:
the embodiment discloses a rhododendron flower hetero terpene compound which has any one of structures shown in formulas 1-6 or a combination thereof:
the physical and spectroscopic data for compounds 1-6 are as follows:
compound 1: pale yellow oil;
(c＝0.036g/100ml,MeOH)；CD(MeOH)λ max (Δε):221(+2.38),204(-3.54)；UV(MeOH)λmax(logε)228(0.29)nm；IR(KBr)ν max 3319,3051,2970,2920,1625,1446,1073cm -1 ；HR-ESI-MS m/z365.2195[M+Na] + [calcd for C 22 H 30 O 3 Na,365.2093]； 1 H and 13 the C NMR data are shown in the Table.
Compound 2: pale brown oily;
(c＝0.036g/100ml,MeOH)；CD(MeOH)λ max (Δε):223(+3.07),299(-1.99)；UV(MeOH)λmax(logε)228(0.31)nm；IR(KBr)ν max 3342,3051,2960,2928,1709,1625,1453cm -1 ；HR-ESI-MS m/z381.2144[M+Na] + (calcd for C 22 H 30 O 4 Na,381.2041)； 1 H and 13 the C NMR data are shown in the Table.
Compound 3: brown oily;
(c＝0.040g/100ml,MeOH)；CD(MeOH)λ max (Δε):279(+3.41),207(-4.63)；UV(MeOH)λmax(logε)230(0.45)nm；IR(KBr)ν max 3278,2974,2928,1625,1451,1067cm -1 ；HR-ESI-MS m/z379.2351[M+Na] + (calcd for C 23 H 32 O 3 Na,379.2228)； 1 H and 13 the C NMR data are shown in the Table.
Compound 4: pale yellow oil;
(c＝0.030g/100ml,MeOH)；CD(MeOH)λ max (Δε):275(+1.10),313(-0.30)；UV(MeOH)λmax(logε)228(0.40)nm；IR(KBr)ν max ,3047,2958,2923,1713,1619,1445,1070cm -1 . HR-ESI-MS m/z342.2195[M+H] + (calcd for C 22 H 31 O 3 ,343.2264)； 1 H and 13 the C NMR data are shown in the Table.
Compound 5: pale yellow oil;
(c＝0.033g/100ml,MeOH)；CD(MeOH)λ max (Δε):280(+3.73),205(-6.27)；UV(MeOH)λ max (logε)230(0.53)nm；IR(KBr)ν max 3316,2966,2925,1625,1447,1087cm -1 ；HR-ESI-MS m/z365.2195[M+Na] + (calcd for C 22 H 30 O 3 Na,365.2093)； 1 H and 13 the C NMR data are shown in the Table.
Compound 6: brown oily;
(c＝0.036g/100ml,MeOH)；UV(MeOH)λ max (logε)209(1.10)nm；IR(KBr)ν max 3405,2957,2923,1697,1426,1043cm -1 ；HR-ESI-MS m/z367.2351[M+Na] + (calcd for C 22 H 32 O 3 Na,367.2242) 1 H and 13 the C NMR data are shown in the Table.
1 H NMR(400MHz,J in Hz)spectroscopic data for 1–4.
1 H NMR(400 MHz,J in Hz)spectroscopic data for 5–6.
13 C NMR(400 MHz,CD 3 COCD 3 )Spectroscopic Data for 1–6.
Note that: the nuclear magnetic data are all measured by a Bruker DRX-400MHz nuclear magnetic resonance instrument.
Example 2:
as shown in fig. 1, the embodiment discloses an extraction method of rhododendron flower diterpenoid compounds, which specifically comprises the following steps:
drying aerial parts of rhododendron cephali in shade, pulverizing to 30 meshes, heating and refluxing in methanol to obtain an extracting solution, concentrating the extracting solution under reduced pressure to recover methanol to obtain a crude extract, and extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract phase;
dissolving an ethyl acetate extract phase by using chloroform or acetone, then carrying out column chromatography segmentation and rough separation by using silica gel, and carrying out gradient elution by using a chloroform/acetone system, wherein the volume ratio of the gradient elution of the chloroform/acetone system is sequentially 1:0, 9:1, 8:2, 7:3, 1:1 and 0:1; taking sulfuric acid ethanol solution as a color developing agent, and combining similar fractions according to TLC color development to obtain 6 components EA 1-EA 6;
after the EA2 component is subjected to silica gel column chromatography, carrying out gradient elution by using a petroleum ether/ethyl acetate system, wherein the elution concentration of the petroleum ether/ethyl acetate system is sequentially 10:1, 5:1, 4:1 and 2:1 in volume ratio, and 9 components 1A-1I are obtained according to the polarity;
separating component 1B by RP-C18 reverse phase chromatographic column, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compounds 1 and 2;
separating the component 1A sequentially by RP-C18 reverse chromatographic column, forward silica gel chromatographic column, RP-C18 reverse chromatographic column chromatography and HPLC to obtain compound 3;
separating the component 1A by RP-C18 reverse chromatographic column chromatography, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compound 4;
separating component 1C by RP-C18 reverse chromatographic column, forward silica gel chromatographic column and HPLC to obtain compound 5;
carrying out forward silica gel column chromatography on the component EA1, carrying out coarse separation on the component EA1 by using a petroleum ether-ethyl acetate system, and obtaining 10 components 34A-34J according to the polarity, wherein the volume ratio of the petroleum ether-ethyl acetate system is sequentially 1:0, 25:1, 15:1, 10:1, 8:1, 4:1 and 2:1;
separating the component 34G by a forward silica gel chromatographic column, a Sephadx-LH20 gel chromatographic column and HPLC after preparing a liquid phase by the RP-C18 semi-preparation, and obtaining the compound 6, wherein the solvent condition used by the RP-C18 semi-preparation liquid phase is methanol with volume concentration of 20%, 40%, 60%, 80% and 100% in sequence.
Example 3:
antibacterial activity detection of the compound obtained by the preparation method disclosed in example 2:
1. test strain: actinidia canker (Pseudomonas syringae pv. Actinidae, psa), bacillus subtilis (Bacillus subtilis), bacillus cereus (Bacillus cereus), staphylococcus aureus (Staphylococcus aureus) and Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus). The strains are all preserved by glycerol tubes, taken out from a refrigerator at the temperature of minus 80 ℃ and are continuously activated twice in fresh sterile LB culture medium in advance for use.
2. The activity test method comprises the following steps: bacteria were inoculated in LB medium for activation, and were shake-cultured on a shaker at 37℃and 170rpm/min to logarithmic growth phase. Diluting the bacterial liquid to 2 x 10-degree by LB culture medium 6 CFU/mL, the cultured bacterial liquid is added into a sterile 96-well plate, and 100 mu l of each well is obtained. Samples and controls were diluted to 200 μm with LB medium. Test compounds are respectively dissolved in DMSO to prepare mother solutions of 200mM, the mother solutions are diluted to 200 mu M by LB culture medium, 100 mu L of diluted sample solution is added into each well of a 96-well plate, and after standing culture is carried out for 12-14 hours in a 37 ℃ incubator, the growth condition of the thalli is observed. Three groups of parallel samples were established, LB medium containing 0.5% DMSO was used as negative control, gentamicin and ciprofloxacin, which are common antibacterial agents, were used as positive control, and a blank control group was set. And (3) combining the primary screening result, and further carrying out rescreening on a series of concentration gradients (1.56-100 mu M) of the test compound with the antibacterial activity. Bacteria grow in 96-well plate LB culture medium, white sediment can be generated after a period of time due to large quantity, the holes with bacteria growth are turbid, sediment appears at the bottom, and the concentration of the compound corresponding to the holes without sediment growth is the minimum antibacterial concentration. Finally, the absorbance value corresponding to the wavelength of 600nm is tested by using an enzyme-labeled instrument to verify the accuracy of the MIC value (minimum inhibitory concentration). The activity data are shown in the table.
Data on bacteriostatic activity of Compounds 1-6
The data in the table show that compounds 1-6 show different degrees of bacteriostasis against kiwi fruit canker pathogen, bacillus cereus, staphylococcus aureus and methicillin-resistant staphylococcus aureus. Among them, the bacteriostatic effect of compounds 2 and 3 is most remarkable, especially for bacillus cereus and staphylococcus aureus, the MIC value is 6.25 μm, and the inhibitory activity for kiwi fruit canker is moderate, and the MIC value is 25 and 12.5 μm, respectively.
Meanwhile, compound 6 also showed remarkable antibacterial activity against Bacillus cereus, and the MIC value was 6.25. Mu.M. According to the results, the compounds 2, 3 and 6 have obvious antibacterial effect and can be used as antibacterial agents for preventing and treating the bacterial infection.
Example 4:
this example discloses a tablet comprising any of the compounds disclosed in example 1, further comprising lactose, starch and magnesium stearate.
The preparation method comprises the following steps: mixing the compound, lactose and starch, uniformly wetting with propylene glycol, sieving the wetted mixture, drying, sieving again, adding magnesium stearate, tabletting the mixture, each tablet weighing 250mg, and the compound content being 10mg.
The obtained tablet can be used as antibacterial agent for treating bacterial infection. The daily dose may be from 0.01 to 10mg/kg body weight, preferably from 0.1 to 5mg/kg body weight, and may be administered in one or more administrations.
Example 5:
the embodiment discloses an ampoule agent, which is obtained by mixing one or more of the compounds disclosed in the embodiment 1 with propylene glycol, wherein the content of the rhododendron flower diterpenoid compounds is 2% -8%;
the preparation method comprises the following steps: any one of the compounds obtained in examples 1-2 was dissolved in 3 ml of propylene glycol, filtered, and the resulting solution was filled into an ampoule under aseptic conditions.
The obtained ampoule agent can be used as antibacterial agent for treating pathogen infection. The daily dose may be from 0.01 to 10mg/kg body weight, preferably from 0.1 to 5mg/kg body weight, and may be administered in one or more administrations.
Example 6:
this example discloses a capsule comprising 10mg of the compound disclosed in example 1, 187mg of lactose, 3mg of magnesium stearate;
the preparation method comprises the following steps: the compound was mixed with adjuvants, sieved, mixed homogeneously, and the obtained mixture was filled into hard gelatin capsules each weighing 200mg and the active ingredient content was 10mg. The daily dose may be from 0.01 to 10mg/kg body weight, preferably from 0.1 to 5mg/kg body weight, and may be administered in one or more administrations.
The capsule can be used as antibacterial agent for treating germ infection.
The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. The rhododendron flower hetero-terpenoid is characterized by comprising at least one of structures shown in formulas 1-6;
2. the method for preparing the rhododendron flower diterpene compound of claim 1, which is characterized by comprising the steps of crushing overground parts of rhododendron flower, and carrying out heat reflux extraction in alcohols to obtain total extract; extracting the total extract with ethyl acetate, and repeatedly subjecting the obtained ethyl acetate extract phase to column chromatography.
3. The method for preparing the rhododendron hetercerpenes according to claim 2, wherein the alcohol is selected from methanol and ethanol.
4. The method for preparing the rhododendron flower diterpenoid compound according to claim 2, which is characterized by comprising the following steps:
drying aerial parts of rhododendron cephali in shade, pulverizing to 30 meshes, heating and reflux-extracting in methanol for 3 times to obtain an extracting solution, concentrating the extracting solution under reduced pressure to recover methanol to obtain a crude extract, and extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract phase;
dissolving an ethyl acetate extract phase by using chloroform or acetone, then carrying out column chromatography segmentation and rough separation by using silica gel, and carrying out gradient elution by using a chloroform/acetone system, wherein the volume ratio of the gradient elution of the chloroform/acetone system is sequentially 1:0, 9:1, 8:2, 7:3, 1:1 and 0:1; taking sulfuric acid ethanol solution as a color developing agent, and combining similar fractions according to TLC color development to obtain 8 components EA 1-EA 6;
after the EA2 component is subjected to silica gel column chromatography, carrying out gradient elution by using a petroleum ether/ethyl acetate system, wherein the elution concentration of the petroleum ether/ethyl acetate system is sequentially 10:1, 5:1, 4:1 and 2:1 in volume ratio, and 9 components 1A-1I are obtained according to the polarity;
separating component 1B by RP-C18 reverse phase chromatographic column, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compounds 1 and 2;
separating the component 1A sequentially by RP-C18 reverse chromatographic column, forward silica gel chromatographic column, RP-C18 reverse chromatographic column chromatography and HPLC to obtain compound 3;
separating the component 1A by RP-C18 reverse chromatographic column chromatography, forward silica gel chromatographic column, sephadx-LH20 gel chromatographic column and HPLC to obtain compound 4;
separating component 1C by RP-C18 reverse chromatographic column, forward silica gel chromatographic column and HPLC to obtain compound 5;
carrying out forward silica gel column chromatography on the component EA1, carrying out coarse separation on the component EA1 by using a petroleum ether-ethyl acetate system, and obtaining 10 components 34A-34J according to the polarity, wherein the volume ratio of the petroleum ether-ethyl acetate system is sequentially 1:0, 25:1, 15:1, 10:1, 8:1, 4:1 and 2:1;
separating the component 34G by a forward silica gel chromatographic column, a Sephadx-LH20 gel chromatographic column and HPLC after preparing a liquid phase by the RP-C18 semi-preparation, and obtaining the compound 6, wherein the solvent condition used by the RP-C18 semi-preparation liquid phase is methanol with volume concentration of 20%, 40%, 60%, 80% and 100% in sequence.
5. Use of the rhododendron flower diterpenoid compound or the pharmaceutically acceptable salt thereof as claimed in claim 1 for preparing an antibacterial agent.
6. The use according to claim 5, wherein the antimicrobial agent is used for controlling diseases caused by kiwifruit canker, bacillus cereus, staphylococcus aureus and/or methicillin-resistant staphylococcus aureus.
7. The use according to claim 5, wherein the antimicrobial agent formulation is a pharmaceutically acceptable formulation prepared by adding pharmaceutically acceptable excipients according to conventional techniques, and the pharmaceutically acceptable formulation is a solid formulation or a liquid formulation.
8. The use according to claim 7, wherein the solid formulation comprises granules, capsules, tablets or pills.
9. The use of claim 7, wherein the liquid formulation comprises an injectable formulation.
10. The use according to claim 7, wherein the content of rhododendron hetercerpene or a pharmaceutically acceptable salt thereof in the antimicrobial formulation is 2-8% by mass.
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