CN117229269A - Flavonoid compound with antibacterial activity and preparation method and application thereof - Google Patents
Flavonoid compound with antibacterial activity and preparation method and application thereof Download PDFInfo
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- CN117229269A CN117229269A CN202311143453.5A CN202311143453A CN117229269A CN 117229269 A CN117229269 A CN 117229269A CN 202311143453 A CN202311143453 A CN 202311143453A CN 117229269 A CN117229269 A CN 117229269A
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- -1 Flavonoid compound Chemical class 0.000 title claims abstract description 60
- 229930003935 flavonoid Natural products 0.000 title claims abstract description 50
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 50
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000000284 extract Substances 0.000 claims abstract description 52
- 235000019504 cigarettes Nutrition 0.000 claims abstract description 24
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 23
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 19
- ZZLCFHIKESPLTH-UHFFFAOYSA-N 4-Methylbiphenyl Chemical compound C1=CC(C)=CC=C1C1=CC=CC=C1 ZZLCFHIKESPLTH-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 16
- 239000002994 raw material Substances 0.000 claims abstract description 15
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 33
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 29
- 239000000741 silica gel Substances 0.000 claims description 23
- 229910002027 silica gel Inorganic materials 0.000 claims description 23
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- 238000000034 method Methods 0.000 claims description 12
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- 238000011068 loading method Methods 0.000 claims description 10
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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
- ZPSJGADGUYYRKE-UHFFFAOYSA-N 2H-pyran-2-one Chemical group O=C1C=CC=CO1 ZPSJGADGUYYRKE-UHFFFAOYSA-N 0.000 description 2
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- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical group C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
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- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 125000001844 prenyl group Chemical group [H]C([*])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
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- 244000025352 Artocarpus heterophyllus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001070941 Castanea Species 0.000 description 1
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a flavonoid compound with antibacterial activity, a preparation method and application thereof, which are prepared by taking fine branches of melaleuca acuminata as raw materials through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and are named as follows: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavone, english name: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavane, having the followingThe structure is as follows:. The antibacterial active flavonoid compound is prepared by taking fine branches of honey as raw materials, extracting the extract, performing silica gel column chromatography and performing high-pressure liquid chromatography separation. The activity test shows that the isopentenyl flavonoid compound has a good antibacterial effect and a sweet returning effect. The compound is used for the cigarette tipping paper, can eliminate or reduce the possibility of the growth and reproduction of harmful bacteria in the cigarette tipping paper, and can improve the taste of the cigarette tipping paper.
Description
Technical Field
The invention belongs to the technical field of natural product chemistry, and particularly relates to a flavonoid compound extracted and separated from a jackfruit plant acuminate melaleuca of the Moraceae for the first time and application of the flavonoid compound in cigarette tipping paper.
Background
The Jianmila is native to Malaysia and Mexico, and cultivated in Hainan, fujian, guangxi and Yunnan China. The Jianmila is a typical tropical fruit tree, is a warm and moist environment, and has the best growth and development in soil with good fertility and drainage under the altitude of 1000 meters, and has weak cold resistance. The ripe pulp of the Shandong Mila has thick and sweet taste and aromatic flavor, is rich in nutrition, has better flavor, can be eaten fresh, and has soft pulp; can also be processed into jam, confection, fruit juice, jelly, and can. The roasted or boiled seeds are edible and have a taste such as chestnut, and are rich in proteins, fats, carbohydrates, etc. In addition, modern medical research proves that the acuminate honey has the functions of anti-edema, antibacterial, anti-inflammatory and the like. After eating the Jianmila, the hydrolysis of fibrin in human body can be enhanced, and fibrin and blood clot blocked in tissues and blood vessels can be dissolved, so that local blood and body fluid circulation is promoted, inflammation and oedema are absorbed and resolved, and the Xinmamila has certain auxiliary treatment effect on cerebral thrombosis and other diseases caused by thrombosis.
The main active ingredient of the acuminate is isopentenyl flavone. The isopentenyl flavone is a branch in a flavonoid compound and is characterized in that an isopentenyl side chain exists on the framework of the flavonoid compound. The isopentenyl flavone has wide biological activity, and has obvious curative effects in the aspects of promoting stem cell differentiation, resisting bacteria, resisting inflammation, regulating immunity, protecting cardiovascular system, improving metabolic diseases, improving osteoporosis, protecting nerves, resisting tumor, resisting aging, promoting reproduction and the like. There is a significant correlation between the chemical structure and the biological activity of isopentenyl flavones. Therefore, more isopentenyl flavonoids are found, and the method is particularly important for researching the structure-activity relationship of isopentenyl flavonoids. The invention separates a new isopentenyl flavonoid compound from the acuminate, and the compound has no report. It is worth mentioning that the compound has remarkable antibacterial activity and also has the effect of returning to the sweet. The antibacterial tipping paper can inhibit harmful microorganisms in the tipping paper and improve the taste of the cigarettes; in addition, the compounds of the present invention form 4-methyl-1 for the prenyl side chains first found in natural productsH-flavonoid compounds of the pyrrole ring, the structure of which has a high novelty.
Disclosure of Invention
The first object of the present invention is to provide a flavonoid compound having antibacterial activity; the second aim is to provide a preparation method of the flavonoid compound with antibacterial activity; a third object is to provide the use of said flavonoid compounds with antibacterial activity.
The first object of the invention is achieved by using medicinal plant JianmalaArtocarpus champedenThe (Lour.) Spren fine branch is used as raw material, and is prepared by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. The compounds were named: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavone, english name: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavane, having the following structure:
。
the second purpose of the invention is realized by taking medicinal plant acuminate fine branches as raw materials, and preparing the acuminate fine branches by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and the method specifically comprises the following steps:
A. pretreatment: fine-drawing branches of a medicinal plant of raw materials and crushing to obtain a material a;
B. extracting extract: adding an organic extraction solvent with the mass which is 2-6 times that of the material a into the material a, soaking and extracting for 2-5 times at normal temperature, wherein the extraction time is 12-20 h each time, combining the extracting solutions, and filtering to obtain a sample extracting solution b;
C. MCI decolorization: decolorizing the sample extract with MCI column, collecting effluent and concentrating under reduced pressure to obtain extract c;
D. silica gel column chromatography:
1) Adding 200-250 mesh silica gel with the weight 3-10 times of that of the extract c into the extract c, loading the mixture into a column, performing gradient elution by using chloroform-methanol solution with the volume ratio of 20:1-1:1, monitoring by TLC, and combining the same parts;
2) Adding 200-250 mesh silica gel with the weight 3-10 times of the extract into eluent obtained by eluting chloroform-methanol solution with the volume ratio of 9:1, loading the eluent into a column, performing gradient elution by using chloroform-acetone solution with the volume ratio of 1:0-1:2, monitoring by TLC, and merging the same parts;
E. high performance liquid chromatography separation: eluting with chloroform-acetone solution at ratio of 7:3, and separating and purifying by high pressure liquid chromatography to obtain isopentenyl flavonoid compound with antibacterial activity.
The structure of the isopentenyl flavonoid compound prepared by the steps can be identified by the following method:
high resolution mass spectrometry (HRESI-MS) analysis was performed first, and HRESI-MS showed an excimer ion peak of 370.1053[ M+Na ]] + (calculated 370.1050), combined with nuclear magnetic resonance 1 H NMR、 13 C and DEPT spectra confirm that its molecular formula is C 21 H 17 NO 4 The unsaturation was 14.
The compounds show hydroxyl (3418), carbonyl (1668) and aromatic rings (1616, 1549, 1457 cm) in the infrared spectrum -1 ) Is a resonance absorption peak of (2). The maximum absorption at 376, 275 and 215nm of the UV spectrum also indicates the possible presence of aromatic ring structures in the compounds.
Of compounds 1 H and 13 the C NMR data (as in Table 1, FIGS. 1 and 2) shows that it contains 21 carbons and 17 hydrogens, including 1,2,3, 5-tetrasubstituted benzene ring (C-5~C-10, H-5, H-7), 1, 4-disubstituted benzene ring (C-1 '-C-6', H) 2 -2',6 and H 2 -3', 5'), and 1α,β-unsaturated carbonyl (C-2, C-3, C-4, H-3), a (4 '' -methylpyrrolidin-2-yl) fragment (C-2 '' -C-6 '', H-3'', H-5'', H 3 -6 "and-NH), one methoxy group (delta) C 56.2 q and delta H 3.83 s) and a phenolic hydroxyl group (delta) H 10.22 s). Based on typical 2 benzene rings, α, β -unsaturated carbonyl and double bond signals. In addition, the unsaturation degree of two benzene rings is 8;1 alpha, beta-unsaturated carbonyl group has unsaturation degree of 2; the azole ring unsaturation is 3 and there should be one ring in the compound. Further analysis of nuclear magnetic resonance data shows the presence of two oxidized aromatic aprotic carbons (C-9 and C-2) in the compound, which indicates that C-9 and C-2 are linked through an oxygen atom to form a pyrone ring, and it is presumed that Compound 1 is a flavonoid. The flavonoid structure of the compound can be further confirmed according to the HMBC correlation (as shown in FIG. 3) of H-3 and C-4, C-10, C-1', H-5 and C-4, C-9, C-10, and H-2' and C-2.
TABLE 1 Compounds of the invention 1 H NMR 13 C NMR data (CDCl) 3 )
After the parent compounds are identified, the remaining substituents, such as hydroxy and methoxy, can be considered substituents on the flavone. The presence of (3-methylpyrrolidin-2-yl) structural fragments can also be taken from H-3'' and C-2'', C-4'', C-5'', C-6''; h-5'' and C-2'', C-3'', C-4'', C-6''; -NH andthe HMBC correlation of C-2'', C-3'', C-4'', C-5'', and H-6'', and C-3'', C-4'', C-5'', was confirmed. At the same time, it was found by further analysis that the methoxy proton signal (δ H 3.83 s) and C-8 indicate that the methoxy group is located at the C-8 position. The HMBC correlation of H-5, H-7 and C-2 ", and the correlation of-NH, H-3' and C-6 indicate that the (3-methylpyrrolidin-2-yl) structural fragment is attached at the C-6 position. Protons of phenolic hydroxyl groups (delta) H 10.22 HMBC related to C-4', C-2',6' indicate that the phenolic hydroxyl group is substituted at the C-8 position. Thus far, the structure of compound 1 was determined. The compound was named: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavone, english name: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl)-flavone。
Through literature query, the compound of the invention forms 4-methyl-1 for the isopentenyl group which is first found in natural productsH-flavonoid compounds of pyrrole ring, the compound structure of which has higher novelty.
Infrared, ultraviolet and mass spectral data for the compounds: ultraviolet spectrum (methanol),λ max (logε) 376 (3.72), 275 (3.93), 215 (4.26) nm; infrared spectrum (potassium bromide tablet):ν max 3418,3360、3049、2987、2842、1668、1616、1549、1457、1362、1254、1170、1062、870cm -1 ； 1 h and 13 c NMR data (500 and 125MHz, (C) 5 D 5 N) is shown in Table-1; positive ion mode ESIMSm/z370[M+Na] + The method comprises the steps of carrying out a first treatment on the surface of the Positive ion mode HRESIMSm/z370.1053[M+Na] + (calculated 370.1050, C) 21 H 17 NNaO 4 )。
The third purpose of the invention, the application of the isopentenyl flavonoid compound as an antibacterial agent and the application in preparing antibacterial tipping paper for cigarettes are realized in the following way:
an in vitro antibacterial activity experiment of the compound of the invention was performed by an agar diffusion method. Firstly, uniformly coating a tested bacterium on a flat plate of a common agar culture medium (beef extract, peptone, sodium chloride, serum and agar), dissolving a compound to be tested (isopentenyl flavonoid compound with 10mL of DMSO, diluting with water to form a 50 mug/mL solution), placing the soaked tablet (with the diameter of 5 mm) on the culture medium with the bacterium, placing the culture medium into an incubator, incubating at 25 ℃ for 24-72h, and observing the size of a bacteriostasis zone. The results show that: the compound has strong activity on staphylococcus aureus, escherichia coli, bacillus subtilis, proteus and the like; the inhibition rate exceeds 95%.
The use of the compounds of the present invention for antimicrobial tipping paper was further tested. The compound is prepared at 50µThe concentration of g/mL is added to the cigarette tipping paper; according to the detection method of the sanitary standard for disposable sanitary products GB15979-2002 of the people's republic of China, the tipping paper for cigarettes added with the compound is used for detecting the total number of bacteria, coliform, staphylococcus aureus, pseudomonas aeruginosa, hemolytic streptococcus and fungi. The results show that the total colony count of tipping paper added with the compound of the invention is obviously reduced, the compound has obvious inhibition effect on several tested bacteria, and the inhibition rate on escherichia coli, staphylococcus aureus and the like is all over 93.8 percent. The compound of the invention is added into the cigarette tipping paper, and has obvious inhibition effect on harmful bacteria. The tipping paper is directly contacted with the oral cavity, so that the compound can be used in the cigarette tipping paper to avoid microbial contamination of cigarettes in the smoking and transferring processes, and the sanitation and safety of the cigarettes are effectively improved. In addition, the compound has the effect of returning sweet, has good compatibility with the characteristic flavor of tobacco, and has good durability; has the effect of improving the taste of the tipping paper of the cigarettes.
The invention has the advantages that:
(1) The compound is obtained by separating the isopentenyl flavone from medicinal plant point melaleuca alternifolia, the biological yield of the point melaleuca alternifolia branches is high, the raw material sources are very wide, the cost is low, and the separation and preparation of the compound are easy to realize.
(2) The preparation method of the invention adopts solvent extraction and then combines conventional column chromatography with high performance liquid chromatography, the operation flow of compound preparation is simple, the purity of the obtained compound is high, and the subsequent industrialized production is easy to realize.
(3) The compound disclosed by the invention is nontoxic to animals, safe to use, and good in antibacterial activity, and the antibacterial rate on escherichia coli, staphylococcus aureus and the like is all over 93%; the microbial agent is applied to the cigarette tipping paper and can inhibit the microorganism polluted by the cigarette tipping paper. The cigarette tipping paper is directly contacted with the oral cavity, and the compound can avoid microbial contamination of the cigarettes in the smoking and transferring process, thereby effectively improving the sanitation and safety of the cigarettes.
(4) The compound has the effect of returning sweet, good compatibility with the characteristic fragrance of tobacco, and good durability; has the effect of improving the taste of the tipping paper of the cigarettes.
(5) The compounds of the invention form 4-methyl-1 for the prenyl side chains first found in natural productsHThe structure of the flavonoid compound with the pyrrole ring has higher novelty and higher scientific significance for perfecting the structure-activity relationship of the flavonoid compound.
Drawings
FIG. 1 nuclear magnetic resonance carbon spectrum of the antibacterial active flavonoid compound of the invention 13 C NMR）；
FIG. 2 shows nuclear magnetic resonance hydrogen spectrum of the antibacterial active flavonoid compound 1 H NMR）；
FIG. 3 is a diagram showing the correlation of key HMBC of an antimicrobial flavonoid of the present invention.
Detailed Description
The invention is further described below with reference to examples and figures, but is not limited in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The invention relates to an isopentenyl flavonoid compound with antibacterial activity, which is prepared from medicinal plant acuminatumArtocarpus champeden(Lour.) Spren) fine branch is used as raw material, and is prepared by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and is named as: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavone, english name: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavane, whichThe structure is as follows:
the flavonoid compound with antibacterial activity is prepared by taking medicinal plant fine branches of melaleuca alternifolia as a raw material through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps:
A. pretreatment: fine-drawing branches of a medicinal plant of Yunnan nationality with honey, and crushing to obtain a material a;
B. extracting extract: adding an organic extraction solvent with the mass which is 2-6 times that of the material a into the material a, soaking and extracting for 2-5 times at normal temperature, wherein the extraction time is 12-20 h each time, combining the extracting solutions, and filtering to obtain a sample extracting solution b;
C. MCI decolorization: decolorizing the sample extract with MCI column, collecting effluent and concentrating under reduced pressure to obtain extract c;
D. silica gel column chromatography:
1) Adding 200-250 mesh silica gel with the weight 3-10 times of that of the extract c into the extract c, loading the mixture into a column, performing gradient elution by using chloroform-methanol solution with the volume ratio of 20:1-1:1, monitoring by TLC, and combining the same parts;
2) Adding 200-250 mesh silica gel with the weight 3-10 times of the extract into eluent obtained by eluting chloroform-methanol solution with the volume ratio of 9:1, loading the eluent into a column, performing gradient elution by using chloroform-acetone solution with the volume ratio of 1:0-1:2, monitoring by TLC, and merging the same parts;
E. high performance liquid chromatography separation: eluting with chloroform-acetone solution at ratio of 7:3, and separating and purifying by high pressure liquid chromatography to obtain flavonoid compound with antibacterial activity.
The organic extraction solvent in the step B is 70-100% of methanol aqueous solution, 70-100% of ethanol aqueous solution or 70-100% of acetone aqueous solution.
And D, before the column is put on the column, the step D is carried out, namely, an organic solvent which is 1.5-3 times of the weight of the material c is used for dissolving, and then 80-100 meshes of silica gel which is 0.8-2.0 times of the weight of the material c is added for sample mixing.
The organic solvent is pure methanol or pure acetone.
The volume ratio of the chloroform-methanol solution in the step D1) is 20:1,9:1,8:2,7:3,6:4 and 1:1.
And E, in the step of high performance liquid chromatography separation and purification, a methanol aqueous solution with the volume concentration of 50-65% is taken as a mobile phase, the flow rate is 12mL/min, a Zorbax PrepHT GF reversed phase preparation column with the flow rate of 2.12X 250mm and 5 mu m is taken as a stationary phase, the detection wavelength of an ultraviolet detector is 376nm, 0.5-1.0 mL of sample is injected each time, chromatographic peaks of 30-42 min are collected, and the target antibacterial active flavonoid compound is obtained after accumulation for multiple times and evaporation.
The application of the flavonoid compound with the isopentenyl group in the invention is the application of the flavonoid compound with the antibacterial activity in the preparation of cosmetic additives.
The preparation method comprises the following specific operations:
A. sample extraction and purification: sun-drying the fine branches of the spine honey, crushing to 20-50 meshes, extracting with a solvent for 2-5 times, wherein each time the solvent is 2-6 times of the raw material, the extracting time is 12-20 hours, filtering out precipitate to obtain a sample extracting solution, and concentrating under reduced pressure to obtain an extract. The ethanol extract prepared was partitioned between ethyl acetate and aqueous solution. The ethyl acetate layer was concentrated under reduced pressure to obtain a crude extract. Filtering the obtained crude extract, decolorizing with MCI column, collecting effluent, concentrating under reduced pressure to obtain extract, and separating with column chromatography.
B. Primary silica gel column chromatography: diluting the obtained extract with 1.5-3 times of acetone or methanol, and then mixing the extract with 0.8-2.0 times of silica gel with the weight of the extract, wherein the mixed silica gel is 100-150 meshes. Coarsely separating the mixed sample by silica gel column chromatography, wherein the weight of column-packed silica gel is 200-250 meshes, and the weight of the silica gel is 3-10 times of the weight of the extract; and (3) carrying out gradient elution by using a mixed organic solvent of chloroform and methanol in a volume ratio of 20:1-1:1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 6 components (A-F).
C. Again silica gel column chromatography: component B (chloroform-methanol 9:1 fraction) from step B was further subjected to silica gel column chromatography: the column-packed silica gel is 200-250 meshes, the weight of the silica gel is 3-10 times of the weight of the extract, chloroform and acetone mixed organic solvent with the volume ratio of 1:0-1:2 are used for gradient elution, and gradient eluent of each gradient is collected and concentrated.
D. High performance liquid chromatography separation: and (3) separating and purifying the component C (part obtained by eluting the chloroform-acetone 7:3 mixed organic solvent) in the step C by adopting high performance liquid chromatography to obtain the flavonoid compound.
Further, it is preferable that the solvent in the step a is an aqueous acetone solution with a volume concentration of 70 to 100%, an aqueous ethanol solution with a volume concentration of 70 to 100%, or an aqueous methanol solution with a volume concentration of 70 to 100%.
Further, preferably, before the extract in the step C is subjected to silica gel column chromatography, acetone or methanol with the weight being 1.5-3 times of that of the extract is used for diluting, then 80-100 mesh silica gel with the weight being 0.8-2.0 times of that of the extract is used for sample mixing, and then sample loading is performed.
Further, it is preferable that in the step C, the volume ratio of chloroform and methanol mixed organic solvent used in the gradient elution is 20:1,9:1,8:2,7:3,6:4 and 1:1 in this order. (each time eluting until no components flow out, i.e. no components remain in the distillation flask after evaporating the solvent, then changing to the next gradient).
Further, preferably, the high performance liquid chromatography separation and purification in the step E uses a methanol aqueous solution with the volume concentration of 50-65% as a mobile phase, the flow rate is 12mL/min, a Zorbax PrepHT GF reversed phase preparation column with the flow rate of 2.12 multiplied by 250mm and 5 μm is used as a stationary phase, the detection wavelength of an ultraviolet detector is 376nm, 0.5-1.0 mL of sample is injected each time, chromatographic peaks with the volume concentration of 30-42 min are collected, and the chromatographic peaks are accumulated for multiple times and evaporated to dryness to obtain a pure compound.
The medicinal plant acuminate is not limited by regions and varieties, can realize the invention,
the invention is further illustrated by the following materials of the Jianmla from different producing areas in Yunnan:
example 1
The embodiment provides a preparation method of the antibacterial active flavonoid compound, which comprises the following steps: extracting extract, performing silica gel column chromatography and high performance liquid chromatography, wherein medicinal plant Jianmila is used as a raw material, and the specific operation is as follows:
the medicinal plant point honey-drawn is produced from the mouth of Yunnan Honghe river, and the raw material is a sample obtained by crushing or cutting medicinal plant point honey-drawn branches, and soaking and extracting for 4 times with 70% methanol aqueous solution for 15 hours each time. Mixing the extractive solutions, decolorizing with MCI column, and concentrating the eluate under reduced pressure to obtain extract; dissolving the extract with methanol 2 with the mass 2 times of the extract, adding 90-mesh silica gel, stirring, using 220-mesh silica gel column, and loading the mixture after stirring; gradient eluting with chloroform-methanol eluents with volume ratios of 20:1,9:1,8:2,7:3,6:4 and 1:1 respectively, collecting gradient eluents, concentrating, monitoring by TLC, combining the same parts to obtain 6 parts A-F, wherein 218g of the collected sample B (9:1) part is filled with 220 mesh silica gel with 8 times of extract, gradient eluting with chloroform-acetone eluents with volume ratios of 1:0,1:1 and 1:2 respectively, collecting gradient eluents, concentrating, monitoring by TLC, combining the same parts to obtain 3 parts, wherein 24.6g of 7:3 part, and using 56% methanol as mobile phase with flow rate of 12ml/min and 2.12×250mm, 5gµm Zorbax PrepHT GF reversed phase preparation column is used as stationary phase, the detection wavelength of an ultraviolet detector is 376nm, 0.8mL is injected each time, the chromatographic peak of 33.5min is collected, accumulated for multiple times and evaporated to dryness, and the antibacterial active isopentenyl flavonoid compound is obtained.
The structure of the prepared isopentenyl flavonoid compound with antibacterial activity is identified by the following method:
high resolution mass spectrometry (HRESI-MS) analysis was performed first, and HRESI-MS showed an excimer ion peak of 370.1053[ M+Na ]] + (calculated 370.1050), combined with nuclear magnetic resonance 1 H NMR、 13 C and DEPT spectra confirm that its molecular formula is C 21 H 17 NO 4 The unsaturation was 14.
The compounds show hydroxyl (3418), carbonyl (1668) and aromatic rings (1616, 1549, 1457 cm) in the infrared spectrum -1 ) Is a resonance absorption peak of (2). The maximum absorption at 376, 275 and 215nm of the UV spectrum also indicates the possible presence of aromatic ring structures in the compounds.
Of compounds 1 H and 13 the C NMR data (as in Table 1, FIGS. 1 and 2) shows that it contains 21 carbons and 17 hydrogens, including 1,2,3, 5-tetrasubstituted benzene ring (C-5~C-10, H-5, H-7), 1, 4-disubstituted benzene ring (C-1 '-C-6', H) 2 -2',6 and H 2 -3', 5'), and 1α,β-unsaturated carbonyl (C-2, C-3, C-4, H-3), a (4 '' -methylpyrrolidin-2-yl) fragment (C-2 '' -C-6 '', H-3'', H-5'', H 3 -6 "and-NH), one methoxy group (delta) C 56.2 q and delta H 3.83 s) and a phenolic hydroxyl group (delta) H 10.22 s). Based on typical 2 benzene rings, α, β -unsaturated carbonyl and double bond signals. In addition, the unsaturation degree of two benzene rings is 8;1 alpha, beta-unsaturated carbonyl group has unsaturation degree of 2; the azole ring unsaturation is 3 and there should be one ring in the compound. Further analysis of nuclear magnetic resonance data shows the presence of two oxidized aromatic aprotic carbons (C-9 and C-2) in the compound, which indicates that C-9 and C-2 are linked through an oxygen atom to form a pyrone ring, and it is presumed that Compound 1 is a flavonoid. The flavonoid structure of the compound can be further confirmed according to the HMBC correlation (as shown in FIG. 3) of H-3 and C-4, C-10, C-1', H-5 and C-4, C-9, C-10, and H-2' and C-2.
After the parent compounds are identified, the remaining substituents, such as hydroxy and methoxy, can be considered substituents on the flavone. The presence of (3-methylpyrrolidin-2-yl) structural fragments can also be taken from H-3'' and C-2'', C-4'', C-5'', C-6''; h-5'' and C-2'', C-3'', C-4'', C-6''; the HMBC correlation of-NH and C-2'', C-3'', C-4'', C-5'', and H-6'', and C-3'', C-4'', C-5'', is demonstrated. At the same time, it was found by further analysis that the methoxy proton signal (δ H 3.83 s) and C-8 indicate that the methoxy group is located at the C-8 position. The HMBC correlation of H-5, H-7 and C-2 ", and the correlation of-NH, H-3' and C-6 indicate that the (3-methylpyrrolidin-2-yl) structural fragment is attached at the C-6 position. Protons of phenolic hydroxyl groups (delta) H 10.22 HMBC related to C-4', C-2',6' indicate that the phenolic hydroxyl group is substituted at the C-8 position. Thus far, the structure of compound 1 was determined. The compound was named: 4' -hydroxy-8-methoxy-6- (4-methyl-1)HPyrrole-2-dioctaneRadical) -flavone, english name: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl)-flavone。
Example 2
The embodiment provides a preparation method of the antibacterial active flavonoid compound, which comprises the following steps: extracting extract, performing silica gel column chromatography and high performance liquid chromatography, wherein medicinal plant Jianmila is used as a raw material, and the specific operation is as follows:
the medicinal plant point honey-drawn is produced from Yuxi Yuanjiang of Yunnan, and the raw material is a sample obtained by crushing or cutting medicinal plant point honey-drawn branches, soaking and extracting for 5 times with an ethanol aqueous solution with the mass concentration of 80% and 12 hours each time. Mixing the extractive solutions, decolorizing with MCI column, and concentrating the eluate under reduced pressure to obtain extract; dissolving the extract with acetone 3 times of the extract, adding 100 mesh silica gel, stirring, using 250 mesh silica gel column, stirring, and loading onto column; gradient eluting with chloroform-methanol eluents with volume ratios of 20:1,9:1,8:2,7:3,6:4 and 1:1 respectively, collecting gradient eluents, concentrating, monitoring by TLC, combining the same parts to obtain 6 parts A-F, wherein 262g of the collected sample B (9:1) part is added into a 250 mesh silica gel column with 10 times of extract, gradient eluting with chloroform-acetone eluents with volume ratios of 1:0,1:1 and 1:2 respectively, collecting gradient eluents, concentrating, monitoring by TLC, combining the same parts to obtain 3 parts, wherein 21.5g of 7:3 part, and using 56% methanol as mobile phase with flow rate of 12ml/min and 2.12×250mm, 5gµm Zorbax PrepHT GF reversed phase preparation column is used as a stationary phase, the detection wavelength of an ultraviolet detector is 376nm, 0.5-1.0 mL of sample is injected each time, chromatographic peaks of 33.5min are collected, accumulated for multiple times, and evaporated to dryness, so that the antibacterial active flavonoid compound is obtained. The identification method is the same as that of example 1, and the molecular formula of the result is C 21 H 17 NO 4 。
Example 3
The embodiment provides a preparation method of the antibacterial active flavonoid compound, which comprises the following steps: extracting extract, performing silica gel column chromatography and high performance liquid chromatography, wherein medicinal plant Jianmila is used as a raw material, and the specific operation is as follows:
the medicinal plant acutangular melasma is produced from Xishuangbanna of Yunnan, and is prepared by soaking and extracting medicinal plant acutangular melasma (sample obtained by pulverizing fine branches or cutting into sections) with 80% acetone aqueous solution for 2 times for 12 hr each time, decolorizing the combined extractive solution with MCI column, and concentrating the eluate under reduced pressure to obtain extract; dissolving the extract with 1.5 times of methanol, adding 80 mesh silica gel, stirring, loading on 200 mesh silica gel column, gradient eluting with chloroform-methanol eluents with volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1 respectively, collecting gradient eluents, concentrating, TLC monitoring, mixing the same parts to obtain 6 parts A-F, wherein 210g of the collected sample B (9:1) part, adding 3 times of the extract of 200 mesh silica gel column, gradient eluting with chloroform-acetone eluents with volume ratio of 1:0,1:1 and 1:2 respectively, collecting gradient eluents, concentrating, TLC monitoring, mixing the same parts to obtain 3 parts, wherein 19.5g of 7:3 parts, and 56% methanol as mobile phase with flow rate of 12ml/min,2.12×250mm, 5gµm Zorbax PrepHT GF reversed phase preparation column is used as a stationary phase, the detection wavelength of an ultraviolet detector is 376nm, 0.5-1.0 mL of sample is injected each time, chromatographic peaks of 33.5min are collected, accumulated for multiple times, and evaporated to dryness, so that the antibacterial active flavonoid compound is obtained. The identification method is the same as that of example 1, and the molecular formula of the result is C 21 H 17 NO 4 。
Example 4
Any of the compounds prepared in examples 1-3 was taken as a yellow gum. The measurement method was the same as in example 1, and the compounds prepared in examples 1 to 3 were confirmed to be the described 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavones.
Example 5
An antibacterial activity test was performed on any of the isopentenyl flavonoids prepared in examples 1 to 4, with the following test conditions and results:
the in vitro antibacterial experiment is carried out by an agar diffusion method, firstly, the test bacteria are uniformly coated on a flat plate of a common agar culture medium (beef extract, peptone, sodium chloride, serum and agar), then, a compound to be tested (benzopyran lactone compound is dissolved by 10mL of DMSO, and diluted into 50 mug/mL of solution by adding water), a soaked tablet (with the diameter of 5 mm) is placed on the culture medium with bacteria, and the culture medium is placed in an incubator, and after incubation is carried out at 25 ℃ for 24-72 hours, the size of a bacteriostasis zone is observed. The results show that: the compound has strong activity on staphylococcus aureus, escherichia coli, bacillus subtilis, proteus and the like; the inhibition rate exceeds 95%.
Example 6
Safety evaluation was carried out on any of the benzopyran lactones prepared in examples 1 to 4, and the test conditions were as follows: the compound of the invention has low toxicity to animals and safe use as proved by a mouse bone marrow micronucleus experiment, an Ames experiment and a TK gene mutation experiment.
An in vitro antibacterial activity experiment of the compound of the invention was performed by an agar diffusion method. Firstly, uniformly coating a tested bacterium on a flat plate of a common agar culture medium (beef extract, peptone, sodium chloride, serum and agar), dissolving a compound to be tested (isopentenyl flavonoid compound with 10mL of DMSO, diluting with water to form a 50 mug/mL solution), placing the soaked tablet (with the diameter of 5 mm) on the culture medium with the bacterium, placing the culture medium into an incubator, incubating at 25 ℃ for 24-72h, and observing the size of a bacteriostasis zone. The results show that: the compound has strong activity on staphylococcus aureus, escherichia coli, bacillus subtilis, proteus and the like; the inhibition rate exceeds 95%.
The compound is added to the cigarette tipping paper at the concentration of 50 mug/mL; according to the detection method of the sanitary standard for disposable sanitary products GB15979-2002 of the people's republic of China, the tipping paper for cigarettes added with the compound is taken, and the total number of bacteria, coliform, staphylococcus aureus, pseudomonas aeruginosa, hemolytic streptococcus and fungi are detected at the size of 2.0 multiplied by 3.0 mm. The results show that the total colony count of tipping paper added with the compound of the invention is obviously reduced, the compound has obvious inhibition effect on several tested bacteria, and the inhibition rate on escherichia coli, staphylococcus aureus and the like is all over 93.8 percent. The compound of the invention is added into the cigarette tipping paper, and has obvious inhibition effect on harmful bacteria. In addition, the compound has the effect of returning sweet, good compatibility of the taste and the characteristic fragrance of tobacco, and good durability; has the effect of improving the taste of the tipping paper of the cigarettes.
Claims (9)
1. The flavonoid compound with antibacterial activity is characterized by being prepared from fine branches of Rumex madaio by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and is named as: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavone, english name: 4' -hydroxy-8-methoxy-6- (4-methyl-1)H-pyrrol-2-yl) -flavane, having the following structure:
。
2. the flavonoid compound with antibacterial activity according to claim 1, which is characterized by being prepared from the medicinal plant of Yunnan nationality, namely, fine branch of Armillariella prinoides, through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps:
A. pretreatment: the method comprises the steps of (1) finely drawing branches of a medicinal sharp honey of a raw material Yunnan nationality, crushing or cutting into sections to obtain a material a;
B. extracting extract: adding an organic extraction solvent with the mass which is 2-6 times that of the material a into the material a, soaking and extracting for 2-5 times at normal temperature, wherein the extraction time is 12-20 h each time, combining the extracting solutions, and filtering to obtain a sample extracting solution b;
C. MCI decolorization: decolorizing the sample extract with MCI column, collecting effluent and concentrating under reduced pressure to obtain extract c;
D. silica gel column chromatography:
1) Adding 200-250 mesh silica gel with the weight 3-10 times of that of the extract c into the extract c, loading the mixture into a column, performing gradient elution by using chloroform-methanol solution with the volume ratio of 20:1-1:1, monitoring by TLC, and combining the same parts;
2) Concentrating eluent obtained by eluting chloroform-methanol solution in a volume ratio of 9:1 under reduced pressure to obtain a component d, loading the component d into a column with 200-250 meshes, wherein the weight of the component d is 3-10 times that of the component d, performing gradient elution by using chloroform-acetone solution in a volume ratio of 1:0-1:2, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: will be described as 7:3 eluting with chloroform-acetone solution, and separating and purifying by high pressure liquid chromatography to obtain flavonoid compound with antibacterial activity.
3. The preparation method of claim 2, wherein the organic extraction solvent in the step B is 70% -100% by mass of aqueous methanol solution, 70% -100% by mass of aqueous ethanol solution or 70% -100% by mass of aqueous acetone solution.
4. The preparation method of claim 2, wherein the step D is characterized in that the step D further comprises the step of mixing samples by adding 80-100 mesh silica gel which is 0.8-2.0 times the weight of the material c after dissolving the mixture by using an organic solvent which is 1.5-3 times the weight of the material c.
5. The method according to claim 4, wherein the organic solvent is pure methanol or pure acetone.
6. The method according to claim 2, wherein the chloroform-methanol solution in step D1) has a volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1.
7. The preparation method of claim 2, wherein in the step E, the high performance liquid chromatography separation and purification is carried out by taking 50-65% methanol aqueous solution as a mobile phase, taking a flow rate of 12mL/min, taking a Zorbax PrepHT GF reversed phase preparation column of 2.12 x 250mm and 5 μm as a stationary phase, detecting the wavelength by an ultraviolet detector to 376, sampling 0.5-1.0 mL each time, collecting chromatographic peaks of 30-42 min, accumulating for multiple times, and evaporating to dryness to obtain the target antibacterial active flavonoid compound.
8. The use of a flavonoid compound with antibacterial activity according to claim 1, characterized in that the use of the flavonoid compound with antibacterial activity in the preparation of an antibacterial agent.
9. The use of the flavonoid compound with antibacterial activity according to claim 1, which is characterized in that the flavonoid compound with antibacterial activity is used for preparing antibacterial tipping paper of cigarettes.
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