CN111961013A - Beta-caryophyllene type sesquiterpenoids, separation method and antibacterial application thereof - Google Patents
Beta-caryophyllene type sesquiterpenoids, separation method and antibacterial application thereof Download PDFInfo
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- NPNUFJAVOOONJE-ZIAGYGMSSA-N β-(E)-Caryophyllene Chemical compound C1CC(C)=CCCC(=C)[C@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-ZIAGYGMSSA-N 0.000 title claims abstract description 44
- NPNUFJAVOOONJE-UHFFFAOYSA-N beta-cariophyllene Natural products C1CC(C)=CCCC(=C)C2CC(C)(C)C21 NPNUFJAVOOONJE-UHFFFAOYSA-N 0.000 title claims abstract description 22
- NPNUFJAVOOONJE-UONOGXRCSA-N caryophyllene Natural products C1CC(C)=CCCC(=C)[C@@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-UONOGXRCSA-N 0.000 title claims abstract description 22
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 15
- 238000000926 separation method Methods 0.000 title abstract description 4
- -1 sesquiterpene compound Chemical class 0.000 claims abstract description 12
- 229930004725 sesquiterpene Natural products 0.000 claims abstract description 10
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 3
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- 241000404844 Callicarpa nudiflora Species 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 15
- 238000010828 elution Methods 0.000 claims description 10
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 10
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 125000001033 ether group Chemical group 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
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- 230000002829 reductive effect Effects 0.000 claims description 5
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
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- 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
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
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- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
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- 241001073507 Callicarpa Species 0.000 description 1
- 240000003690 Callicarpa japonica Species 0.000 description 1
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- 241000522190 Desmodium Species 0.000 description 1
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- 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 description 1
- 238000012986 modification Methods 0.000 description 1
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- 150000003648 triterpenes Chemical class 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D301/00—Preparation of oxiranes
- C07D301/32—Separation; Purification
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- 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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- 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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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a beta-caryophyllene type sesquiterpene compound, a separation method and an antibacterial application thereof. Pharmacological experiments show that the compound has better antibacterial activity, and has good application prospect in the aspect of preparing antibacterial drugs.
Description
Technical Field
The invention relates to the technical field of natural phytochemistry, in particular to a beta-caryophyllene type sesquiterpene compound and a separation method and antibacterial application thereof.
Background
Callicarpa nudiflora (Callicarpa nudiflora hook. et Arn.) also known as desmodium, is a plant of Callicarpa of Verbenaceae (Verbenacase), is a medicinal material in provincial province of Hainan, and is also one of common medicinal materials in Li nationality. The beautyberry can be used as medicine for treating suppurative inflammation, traumatic hemorrhage, digestive tract and respiratory tract infection, burn and scald, and has the effects of diminishing inflammation, removing toxic substance, resisting bacteria, stopping bleeding, removing blood stasis, and relieving swelling. The medicine is mainly used for various postoperative hemorrhagic diseases of dermatology, gynecology, ophthalmology and otorhinolaryngology, surgery and the like. Is recorded by newly added Chinese medicine varieties in 'Chinese pharmacopoeia' of 2015 edition.
Researches show that the callicarpa nudiflora mainly contains various chemical components such as flavones (glycosides), phenylpropanoids (glycosides), lignans, triterpenes, diterpenes, iridoids, phenolic acids and the like, and has good biological activity. The cycle (research on the astringent hemostasis and antibacterial action on mycobacterium tuberculosis of Callicarpa nudiflora [ D ]. Master graduate thesis at Hunan university, 2014), the Callicarpa nudiflora can effectively shorten the blood coagulation time of mice; can obviously inhibit inflammatory reaction caused by dimethylbenzene and has the function of astringency and hemostasis; callicarpa nudiflora has antibacterial effect on standard strains of Mycobacterium tuberculosis, clinically isolated non-drug resistant strains and clinically isolated drug resistant strains. However, few sesquiterpenes with antibacterial effect are found in callicarpa nudiflora at present. The invention aims to further separate and research the chemical components of callicarpa nudiflora to obtain a new sesquiterpene compound with an antibacterial effect.
Disclosure of Invention
In view of the disadvantages of the prior art, the present invention provides a novel beta-caryophyllene type sesquiterpene compound having significant antibacterial activity, and discloses a method for separating the compound from callicarpa nudiflora in detail.
The technical scheme of the invention is as follows:
a beta-caryophyllene type sesquiterpenoids compound has a structural formula as follows:
wherein R1, R2, R3, R4, R5 and R6 are selected from hydroxyl, methoxy, methyl or hydrogen, and the number of carbon atoms is 15 and the number of hydrogen atoms is 24. The above compounds can be synthesized on the basis of the compounds of the formula (I) of the present invention.
Preferably, the structural formula of the beta-caryophyllene type sesquiterpenoids is shown as the formula (I):
namely R2, R3 and R6 are CH3R1 and R4 are H, and R5 is OH.
A process for the preparation of a compound of formula (i) as hereinbefore described comprising the steps of:
crushing dried callicarpa nudiflora leaves, extracting with petroleum ether, and concentrating under reduced pressure to obtain petroleum ether part extract;
secondly, subjecting the petroleum ether part extract obtained in the first step to silica gel column chromatography, eluting by adopting a petroleum ether-ethyl acetate solvent system, and collecting fractions; subjecting the fraction to gel column chromatography, and collecting the fraction;
thirdly, carrying out normal phase silica gel column chromatography on the fraction obtained in the second step, wherein an eluent is a petroleum ether-acetone mixed solvent, and collecting the fraction;
fourthly, separating the fraction obtained in the third step by High Performance Liquid Chromatography (HPLC) to obtain the target extract beta-caryophyllene type sesquiterpenoids.
Preferably, the compound of formula (I) is prepared by a process comprising:
crushing dried callicarpa nudiflora leaves, extracting with petroleum ether, and concentrating under reduced pressure to obtain petroleum ether part extract;
performing silica gel column chromatography on the petroleum ether part extract obtained in the step one, performing gradient elution by adopting a petroleum ether-ethyl acetate solvent system, collecting fractions, concentrating each fraction, and combining the same components;
carrying out gel column chromatography on the components eluted in the petroleum ether-ethyl acetate solvent system in a volume ratio of 80:20, eluting 3-6 column volumes with MeOH as an eluent, collecting fractions, and combining the same components to obtain 5 components Fr.1-Fr.5;
subjecting Fr.3 obtained in the step II to normal phase silica gel column chromatography, eluting 3-6 column volumes by using a mixed solvent with a petroleum ether-acetone volume ratio of 10: 1-1: 5, collecting fractions, and combining the same components to obtain 5 components Fr.3A-Fr.3E;
Fr.3B is separated by high performance liquid chromatography HPLC to obtain the target extract beta-caryophyllene type sesquiterpenoids.
More preferably, in the step I, petroleum ether is used for leaching for 2-4 times, each time of extraction is 1-3 hours, and ultrasound is carried out during extraction, and the solution is heated to 35-40 ℃.
More preferably, in the step (II), when the petroleum ether-ethyl acetate solvent is used for gradient elution, the elution gradient is 100:0, 90:10 and 80: 20.
More preferably, in the third step, when gradient elution is performed by using a petroleum ether-acetone mixed solvent, the elution gradient is respectively 10:1, 8:1, 5:1, 1:1 and 1:5, and 3-6 column volumes are eluted, so that 5 components, namely Fr.3A-Fr.3E, are obtained.
More preferably, in the step (iv), the conditions of the High Performance Liquid Chromatography (HPLC) are as follows: the column type is Waters C18(ii) a The specification of the chromatographic column is 9.4 multiplied by 250mm, 4.6 μm; the flow rate is 2 mL/min; the mobile phase is the volume ratio of MeCN to H2O＝68:32。
Experiments prove that the beta-caryophyllene type sesquiterpene compound has obvious antibacterial activity, wherein the bacteria comprise tetragenococcus (Micrococcus tetragenus), Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus), Staphylococcus albus (Staphylococcus albus) or Escherichia coli (Escherichia coli).
The invention has the beneficial effects that:
the invention extracts and obtains a novel beta-caryophyllene type sesquiterpene compound from callicarpa nudiflora, obtains a crystal structure, and determines an absolute configuration, which is rare in the discovery of active ingredients of callicarpa nudiflora.
Pharmacological research shows that the beta-caryophyllene type sesquialter compound has better inhibiting effect on M.teragenus, B.subtilis, S.aureus, S.albus and E.coli, and the MIC range is 2.6-9.5 mu g/mL-1. The compound of the invention has good application prospect in the aspect of preparing antibacterial drugs.
Drawings
FIG. 1: EXAMPLE 1 preparation of Compound I1H-NMR Spectroscopy (MeOD-d)4)；
FIG. 2: EXAMPLE 1 preparation of Compound I13C-NMR Spectroscopy (MeOD-d)4)；
FIG. 3: example 1 DEPT (135 ℃) Spectroscopy (MeOD-d) of Compound I4)；
FIG. 4: EXAMPLE 1 preparation of Compound I1H-1H COSY spectra (MeOD-d)4)；
FIG. 5: EXAMPLE 1 HSQC spectra (MeOD-d) of Compound I4)；
FIG. 6: EXAMPLE 1 HMBC Spectroscopy (MeOD-d) of Compound I4)；
FIG. 7: EXAMPLE 1 NOESY Spectroscopy (MeOD-d) of Compound I4)；
FIG. 8: example 1 HRESIMS spectra of Compound I.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
Example 1
The preparation method of the beta-caryophyllene sesquiterpenoids comprises the following steps:
pulverizing 0.8kg of dried callicarpa nudiflora leaves, leaching for 4 times by using petroleum ether, extracting for 2 hours each time, wherein the solvent consumption is 4.0L each time, and performing ultrasonic extraction while heating the solution to 35-40 ℃. Mixing the extractive solutions, and concentrating under reduced pressure to obtain petroleum ether extract.
Secondly, eluting the petroleum ether part extract (42.5g) obtained in the step I by using a petroleum ether-ethyl acetate solvent system according to the gradient of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100(V/V), collecting fractions per 1000mL, concentrating each fraction, detecting by TLC (thin layer chromatography), and combining the same components;
performing gel column chromatography (Sephadex LH-20) on the components eluted by the petroleum ether-ethyl acetate solvent system at a ratio of 80:20 (V/V), eluting by 3-6 column volumes with MeOH as an eluent, collecting fractions, and combining the same components to obtain 5 components Fr.1-Fr.5;
subjecting Fr.3 obtained in the second step to normal phase silica gel column chromatography, eluting with a petroleum ether-acetone mixed solvent with an elution gradient of 10:1, 8:1, 5:1, 1:1 and 1:5(V/V), eluting for 3-6 column volumes, collecting fractions, and combining the same components to obtain 5 components Fr.3A-Fr.3E;
Fr.3B is separated and prepared by high performance liquid chromatography HPLC, and the chromatographic column is Waters C189.4X 250mm, 4.6 μm, flow rate of 2mL/min, mobile phase MeCN: H2And O is 68: 32. To obtain the target extract beta-caryophyllene type sesquiterpenoids (8.5g), namely compound I. The target extract was a colorless oil in appearance and was readily soluble in chloroform and methanol.
The structural identification data of the target extract beta-caryophyllene type sesquiterpene compound is as follows:
beta-caryophyllene type sesquiterpenoids1H and13the C NMR data are shown in Table 1([400MHz ], (C NMR)1H)，100MHz(13C) The solvent: MeOD-d4])。1H-NMR Spectrum (MeOD-d)4)、13C-NMR Spectrum (MeOD-d)4) DEPT (135 ℃) Spectrum (MeOD-d)4)、1H-1H COSY spectrum (MeOD-d)4) HSQC spectrum (MeOD-d)4) HMBC spectrum (MeOD-d)4) NOESY spectrum (MeOD-d)4) The HRESIMS spectrum is shown in the figure.
TABLE 1 preparation of compound I1H-NMR and13C-NMR data [ (ppm), J (Hz)]
Compound I was treated with HRESI (+) MS (M/z 259.1671[ M + Na ]]+Theoretical value 259.1674) determined to have the molecular formula C15H24O2。
According to1H，13C and two-dimensional nuclear magnetic resonance data determine the structure of the compound, the skeleton type of the compound is beta-caryophyllene type sesquiterpene, and the compound is named as (3S,4R,5S) -5-hydroxycaryophyl-8 (13) -ene 3, 4-epoxide. The structural formula is shown as the formula (I):
the target compound is put into a methanol solvent and slowly volatilized to obtain a crystal, and the crystal structure is shown as a structural formula 1.
Example 2
The difference between this example and example 1 is:
leaching for 2 times by using petroleum ether, wherein the extraction time is 1 hour each time, and the solvent consumption is 3.0L each time.
The structure of the obtained compound was the same as in example 1.
Example 3
The difference between this example and example 1 is:
leaching for 2 times by using petroleum ether, wherein the extraction time is 3 hours each time, and the solvent consumption is 3.0L each time.
The structure of the obtained compound was the same as in example 1.
Test example 1 evaluation of in vitro antibacterial Activity
The compounds of formula (I) of example 1 were evaluated for their antibacterial activity in vitro, using Ciprolixin as a control.
1.1 Experimental materials
Pathogenic bacteria: 5 kinds of human pathogenic bacteria, which are respectively: tetragenococcus (Micrococcus tetragonous), Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus), Staphylococcus albus (Staphylococcus albus), and Escherichia coli (Escherichia coli).
1.2 Experimental methods
The test method comprises the following steps: the method is used for the bacteriostatic activity test of sensitive drugs or new drugs on human pathogenic bacteria by adopting a microdilution method, has simple and convenient operation method, and can be used for a large number of bacteriostatic tests. Inoculating the activated human pathogenic bacteria into a nutrient broth culture medium to prepare a bacterial liquid, and dissolving the drug to be detected into 1mg/mL by using DMSO. Diluting a 96-well plate line by a double-concentration decreasing dilution method, wherein the dilution degree is 1: 500-1: 1000; quantitatively adding the diluted culture solution containing the bacteria into each hole of a 96-hole plate respectively; then adding diluted bacteria solution, reserving two holes as a reference, adding only the culture medium in one hole and adding only the bacteria solution in the other hole, shaking and mixing uniformly after dilution, placing a 96-hole plate in a constant-temperature incubator at 37 ℃ for culturing for 20h, measuring the absorbance at 630nm by using an enzyme-labeling instrument, and measuring the value of the Minimum Inhibitory Concentration (MIC).
The results of the antibacterial activity are shown in Table 2.
TABLE 2 antibacterial Activity of the Compounds
The results show that the compound I shows a certain inhibition effect on tetragenococcus, bacillus subtilis, staphylococcus aureus, staphylococcus albus and escherichia coli, and the compound I can be used as a bacteriostatic drug.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The beta-caryophyllene type sesquiterpenoids are characterized by having the following structural formula:
wherein R1, R2, R3, R4, R5 and R6 are selected from hydroxyl, methoxy, methyl or hydrogen, and the number of carbon atoms is 15 and the number of hydrogen atoms is 24.
2. The beta-caryophyllene type sesquiterpene compound according to claim 1, which has a structural formula shown in formula (I):
3. the method for preparing beta-caryophyllene type sesquiterpenoids according to claim 2, comprising the steps of:
crushing dried callicarpa nudiflora leaves, extracting with petroleum ether, and concentrating under reduced pressure to obtain petroleum ether part extract;
secondly, subjecting the petroleum ether part extract obtained in the first step to silica gel column chromatography, eluting by adopting a petroleum ether-ethyl acetate solvent system, and collecting fractions; subjecting the obtained fraction to gel column chromatography, and collecting the fraction;
step three, the obtained fraction is subjected to normal phase silica gel column chromatography, eluent is petroleum ether-acetone mixed solvent, and the fraction is collected;
fourthly, separating the fraction obtained in the third step by High Performance Liquid Chromatography (HPLC) to obtain the target extract beta-caryophyllene type sesquiterpenoids.
4. The method of claim 3, comprising the steps of:
crushing dried callicarpa nudiflora leaves, extracting with petroleum ether, and concentrating under reduced pressure to obtain petroleum ether part extract;
performing silica gel column chromatography on the petroleum ether part extract obtained in the step one, performing gradient elution by adopting a petroleum ether-ethyl acetate solvent system, collecting fractions, concentrating each fraction, and combining the same components;
carrying out gel column chromatography on the components eluted in the petroleum ether-ethyl acetate solvent system in a volume ratio of 80:20, eluting 3-6 column volumes with MeOH as an eluent, collecting fractions, and combining the same components to obtain 5 components Fr.1-Fr.5;
subjecting Fr.3 obtained in the step II to normal phase silica gel column chromatography, eluting 3-6 column volumes by using a mixed solvent with a petroleum ether-acetone volume ratio of 10: 1-1: 5, collecting fractions, and combining the same components to obtain 5 components Fr.3A-Fr.3E;
Fr.3B is separated by high performance liquid chromatography HPLC to obtain the target extract beta-caryophyllene type sesquiterpenoids.
5. The method of claim 4, wherein: in the step I, petroleum ether is used for leaching for 2-4 times, each time of extraction is 1-3 hours, and ultrasonic treatment is carried out during extraction, and the solution is heated to 35-40 ℃.
6. The preparation method according to claim 4, wherein the gradient elution with the solvent of petroleum ether-ethyl acetate in step (II) is 100:0, 90:10, 80: 20.
7. The preparation method of claim 4, wherein in the third step, when gradient elution is performed with a petroleum ether-acetone mixed solvent, the elution gradient is 10:1, 8:1, 5:1, 1:5, and 3-6 column volumes are eluted, so that 5 components, namely Fr.3A-Fr.3E, are obtained.
8. The preparation method according to claim 4, wherein in the step (iv), the conditions of HPLC are as follows: the column type is Waters C18(ii) a The specification of the chromatographic column is 9.4 multiplied by 250mm, 4.6 μm; the flow rate is 2 mL/min; the mobile phase is the volume ratio of MeCN to H2O＝68:32。
9. The use of the β -caryophyllene sesquiterpene compound of claim 1 in the preparation of antibacterial drugs.
10. The use of claim 9, wherein the bacteria comprise tetragonococcus, bacillus subtilis, staphylococcus aureus, staphylococcus albus or escherichia coli.
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