CN107573314B - Isoamyl flavonoid compound in rose waste residue and preparation method and application thereof - Google Patents
Isoamyl flavonoid compound in rose waste residue and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an isoamyl flavonoid compound in rose waste residue, a preparation method and application thereof, belonging to the technical field of phytochemistry. The isoamyl flavonoid compound is separated from waste residue after the production of the rose essential oil, and is named as 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-alkene) -flavone with the molecular formula of C22H20O5The structural formula is shown as the formula (I):
formula (I); the rose essential oil is prepared by taking waste residues after rose essential oil production as raw materials and performing the steps of extract extraction, organic solvent extraction, MCI (methanol-to-ethanol) decoloration, silica gel column chromatography and high performance liquid chromatography separation. The compound of the invention is added into cigarette filters, can alleviate the stimulation of smoke to throats, stimulate salivary secretion, has sweet aftertaste effect in oral cavities, improves the aftertaste of the oral cavities of cigarettes, covers miscellaneous gas and improves the smoking quality of the cigarettes.
Description
Technical Field
The invention belongs to the technical field of phytochemistry, relates to an isoamyl flavonoid compound in rose waste residue, and a preparation method and application thereof, and particularly relates to an isoamyl flavonoid compound extracted from waste residue after rose essential oil production for the first time. The compound is added into cigarette filter tip, and can alleviate irritation of smoke gas to throat, stimulate salivary secretion, and has sweet aftertaste in oral cavity, so as to improve aftertaste of cigarette oral cavity, cover offensive odor, and improve smoking quality of cigarette.
Background
Damascus rose (academic name)Rosa DamascenaThe Damask rose is commonly named as Damask rose), also named as jujuan rose, belongs to the rose in the rosaceous family, belongs to the classical garden, and is a bushy plant which is native to syria and is widely planted in france from the beginning of the 14 th century, and the Damask rose is a main rose variety planted in bulgaria, and has the characteristics of petal opening, light color of petal edge and silk-satin texture; pure and fine floral fragranceThe rose is pressed to have fragrance, and becomes the top grade of the rose for oil, so the rose is widely planted to extract the rose essential oil. Rose essential oil is the most expensive essential oil in the world and is called "after essential oil". Besides being widely applied to foods and cosmetics, the rose essential oil is also widely applied to cigarette flavoring; the cigarette added with the rose essential oil has the characteristic of composite fragrance of the tobacco and the rose, is rich and sweet in taste, fresh and light in smoke flavor, and can bring elegant and fragrant unique enjoyment to consumers.
The rose essential oil is prepared by taking fresh rose flowers picked in the morning as a raw material through a distillation or supercritical extraction method, the yield of the rose essential oil is extremely low, about five tons of heavy flowers can only extract two pounds of rose oil, and the waste residue generated by the essential oil is also rich in a large amount of chemical components with development and utilization values, such as quercitrin, bitter substances, tannin, fatty oil, organic acid (gallic acid), haematochrome, uranidin, β -carotene and the like.
Research shows that besides conventional chemical components such as protein, amino acid, sugar, phosphorus, calcium, potassium and the like, the waste residue after the rose essential oil is produced also contains a large amount of secondary metabolites, and the types of the compounds comprise flavone, coumarin, tannin, furan, flavone, phenylpropanoid, terpenoids, alkaloid and the like. The compounds have various biological activities and have remarkable effects in multiple aspects such as antibiosis, antiphlogosis, anti-mutation, blood pressure reduction, heat clearing and detoxifying, sedation, diuresis, antioxidation, anticancer, cancer prevention, lipase inhibition and the like.
The flavonoids generally refer to a series of compounds in which two benzene rings (A-and B-rings) having phenolic hydroxyl groups are connected with each other through a central three-carbon atom, and the basic nucleus of the compounds is 2-phenylchromone. The structure of the flavonoid compound is often connected with functional groups such as phenolic hydroxyl, methoxyl, methyl, isopentenyl and the like. In addition, it is often combined with a sugar to form a glycoside. The flavone has various effects, is a strong antioxidant, has oxidation prevention capability more than ten times of that of vitamin E, and can prevent cell degeneration, aging and cancer. The flavone has effects of improving blood circulation, reducing cholesterol, and improving cardiovascular and cerebrovascular diseases. In addition, the flavonoids have outstanding effect of improving the taste, some flavonoids have very special taste performance, and have natural sweet taste after being taken. The flavone contained in the olive is the main reason for returning the sweet taste, and the higher the flavone content is, the more obvious the returning sweet taste is, and the more mellow the smell is. The flavonoid compound is separated from the rose essential oil production waste residue, and when the flavonoid compound is added into a cigarette filter, the irritation of smoke on the throat can be alleviated, the salivary secretion can be stimulated, the oral cavity has the sweet aftertaste effect, the aftertaste of the oral cavity is improved, the offensive odor can be covered, and the smoking quality of cigarettes is improved.
Disclosure of Invention
The invention aims to provide an isoamyl flavonoid compound in rose waste residue; the second purpose is to provide a preparation method of the isoamyl flavonoid compound; the third purpose is to provide the application of the isoamyl flavonoid compound in flavoring the cigarette filter, the compound can relieve the stimulation of smoke to the throat, stimulate salivary secretion, endow the effect of sweet aftertaste to the oral cavity, improve the aftertaste of the oral cavity, cover up offensive odor and improve the smoking quality of the cigarette.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the first purpose of the invention is realized by that the isoamyl flavonoid compound in the rose waste residue is separated from the waste residue after the rose essential oil is produced, and the compound is light yellow jelly and is named as: 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone, english name: 8, 2' -dimethoxy-7- (3-methyl-2-oxobout-3-enyl) -flavanone with molecular formula C22H20O5The structural formula is shown as the formula (I):
The second purpose of the invention is realized by adopting the preparation method of the isoamyl flavonoid compound, which is prepared by taking waste residue after the production of the rose essential oil as a raw material and carrying out the steps of extract extraction, organic solvent extraction, MCI decoloration, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps:
A. extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced for 2-5 times by using a solvent, wherein the mass of the extraction solvent used each time is 2-6 times of that of the waste residue powder, the extraction time is 30-60 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitates are separated out, standing for 20-60 minutes, filtering out the precipitates, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 1-2 times that of the extract a into the extract a, extracting for 3-5 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding a 95% methanol aqueous solution with volume concentration which is 3-5 times of the weight of the extract b into the extract b, loading the extract b on an MCI (methanol-to-oil) column after the extract b is completely dissolved, eluting by using a 90-95% methanol aqueous solution, merging the eluents, and then concentrating the merged eluents under reduced pressure to obtain an extract c;
D. silica gel column chromatography: performing silica gel column chromatography on the extract c, wherein the silica gel filled in the column is 160-200 meshes, and the weight of the silica gel is 6-10 times that of the extract c; performing gradient elution by using a chloroform-acetone mixed organic solvent with a volume ratio of 1: 0-0: 1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC spot plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: and (3) separating and purifying a part obtained by eluting with a chloroform-acetone mixed organic solvent with a volume ratio of 9:1 by using high performance liquid chromatography to obtain the isoamyl flavonoid compound.
Preferably, the solvent in step a is an acetone aqueous solution with a volume concentration of 70-100%, an ethanol aqueous solution with a volume concentration of 90-100%, or a methanol aqueous solution with a volume concentration of 90-100%.
Further, it is preferable that the organic solvent of the step B is dichloromethane, chloroform, ethyl acetate, diethyl ether or petroleum ether.
Further, preferably, in the step D, before the extract c is subjected to silica gel column chromatography, acetone or methanol with the weight 1.5-3 times that of the extract c is used for dissolving the extract c, then 80-100 mesh silica gel with the weight 0.8-1.2 times that of the extract c is used for mixing samples, and then the samples are loaded.
Further, in the step D, it is preferable that the chloroform-acetone mixed organic solvent used in the gradient elution has a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 in this order; after each gradient eluted to the point where the TLC spot plate was no longer spotted, the next gradient elution was changed.
Further, it is preferable that the separation and purification by high performance liquid chromatography in the step E is performed at a flow rate of 20ml/min at a flow rate of 21.2' 250mm, 5 with an aqueous methanol solution having a volume concentration of 68% as a mobile phasemm Zorbax PrepHT GF reversed-phase preparation column is a fixed phase, the detection wavelength of an ultraviolet detector is 354nm, 50-200 mL of sample is fed each time, a chromatographic peak of 30.5 min is collected, and the mixture is evaporated to dryness after multiple accumulation to obtain the compound. Those skilled in the art will appreciate that the parameters of the HPLC separation and purification are not limited thereto.
The invention has no limit to the granularity of the waste residue powder.
The structure of the prepared compound was identified by the following method:
the compounds of the invention are pale yellow gums;
HRESI-MS shows that the peak of the excimer ion is 363.1202 [ M + Na ]]+(calculated 363.1208), combined1The molecular formula of the compound is C determined by HNMR and DEPT spectra22H20O5The unsaturation degree was 13.
The infrared spectrum shows carbonyl groups (1680 and 1665 cm)-1) And aromatic rings (1610, 1537 and 1469 cm)-1) The resonance absorption peak of (1). The maximum absorption of the ultraviolet spectrum at 210 nm, 258 nm and 360 nm also indicates that aromatic ring structures possibly exist in the compound.
Process for preparing compounds1H and13c NMR spectra (see Table 1, FIG. 1 and FIG. 2)The catalyst contains 22 carbon atoms and 20 hydrogen atoms, and comprises 1,2,3, 4-tetra-substituted benzene ring (C-5-C-10, H-5 and H-6), 1, 2-di-substituted benzene ring (C-1 '-C-6', H-3 '-H-6'), and 1α,βUnsaturated carbonyl (C-2, C-3, C-4, H-3), 1 3-methyl-2-oxobutyl-3-ene structural fragment (C-1'' to C-5 ''; H)2-1''，H2-4'' and H3-5 "), two methoxy groups (d C61.5 q and 56.1 q,d H3.84 s and 3.81 s). According to the typical 2 benzene rings,α,βUnsaturated carbonyl and double bond signals, which are presumed to be flavonoids. The compound was further confirmed to have a flavonoid structure based on HMBC correlation (see FIG. 3) between H-3 and C-2, C-4, C-10, C-1', H-5 and C-4, C-9, C-10, and H-6' and C-2.
TABLE 1 preparation of the compounds of the invention1H NMR and13c NMR data (CCl)3)
After the parent compound is identified, the remaining substituents, 3-methyl-2-oxobutyl-3-ene and methoxy, can be considered as substituents on the flavone. Two methoxyhydrogens (A), (B) can be observed in the HMBC spectrum of the compound (as shown in FIG. 3)d H3.84 and 3.81) in connection with HMBC at C-8 and C-2', it can be speculated that two methoxy groups are substituted at the C-8 and C-2' positions, respectively; according to H2-1'' (d H4.61) and C-6, C-7, C-8 and H-6: (d H6.72) and HMBC at C-1 ″, it was confirmed that the substitution of 3-methyl-2-oxobutyl-3-ene is at the C-7 position. In addition the proton signal on the benzene ring is typical [ H-5,d H7.43 (d) 8.2；H-6,d H6.72 (d) 8.2，H-3′d H6.93 (d) 7.6；H-4′，d H7.32 (t) 7.6；H-5′，d H6.88 (t) 7.6；H-6′，d H7.89 (d)7.6]it was also confirmed that the flavone compound of the present invention has a 6, 8-disubstituted ring B and a 2' -monosubstituted ring C.
To this end, the structure of the compound was determined and the compound was named: 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone.
Infrared, ultraviolet and mass spectral data of compounds: UV (methanol)λ max(logε) 360 (3.69), 258(3.87), 210 (4.32) nm, IR (Potassium bromide pressed):ν max2967、2854、1680、1665、1610、1537、1469、1356、1139、1055 cm–1;1H and13c NMR data (500 and 125 MHz) in Table-1, positive ion ESIMSm/z387 positive ion mode HRESIMSm/z387.1215 [M+Na]+(calculation value C)22H20O5，387.1208).
The third object of the present invention is achieved by:
the isoamyl flavonoid compound in the rose waste residue is used for preparing the cigarette filter additive. The compound of the invention can alleviate the stimulation of smoke gas to the throat and stimulate the saliva secretion in the cigarette smoking, and has the effects of improving the aftertaste of the oral cavity and covering up the miscellaneous gas.
The present invention contemplates that glyceryl triacetate is the most commonly used plasticizer for cigarette filter formation, and that the compounds of the present invention are soluble in glyceryl triacetate, through which they can be added to the filter during cigarette filter formation.
The cigarette for adding cigarette is Hongta mountain classic, and the flavone compound is prepared into 0.5 mg/mL solution by using triacetyl glycerine. Uniformly spraying the mixture on filter tow in an amount of 5-8% of the weight of the filter tow to prepare a filter stick, then making the filter stick into a cigarette by conventional cigarette rolling, performing sensory evaluation, and using the same cigarette without the compound as a reference. The evaluation and analysis result shows that: compared with the contrast, the compound of the invention is added into the cigarette filter, can alleviate the stimulation of smoke to the throat, stimulate the secretion of saliva, has sweet aftertaste effect in the oral cavity, improves the aftertaste of the oral cavity, covers the offensive odor and improves the smoking quality of the cigarette.
Compared with the prior art, the invention has the beneficial effects that:
the compound is obtained by separating waste residues after the rose essential oil is produced, and has very wide raw material sources and low cost; the compound can comprehensively utilize waste resources in production, and has important practical significance for improving the added value of rose processing.
The compound has simple structure, can be produced by artificial synthesis, is easy to realize, and provides a new additive for the tobacco industry. The compound is dissolved in the glycerol triacetate, and can be added into a filter tip through the glycerol triacetate in the forming process of the cigarette filter tip, so that no additional process is added, and the application in the cigarette is easy to realize.
Compared with the contrast, the compound of the invention is added into the cigarette filter, can alleviate the stimulation of smoke to the throat, stimulate the secretion of saliva, has sweet aftertaste effect in the oral cavity, improves the aftertaste of the oral cavity, covers the offensive odor and improves the smoking quality of the cigarette.
Drawings
FIG. 1 nuclear magnetic resonance carbon spectrum of isoamyl flavonoid of the present invention: (13C NMR）；
FIG. 2 shows the NMR spectra of isoamyl flavonoids of the present invention: (1H NMR）；
FIG. 3 is a key HMBC correlation chart of the isoamyl flavonoid of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
The isoamyl flavonoid compound in the rose waste residue is obtained by separating the waste residue after the rose essential oil is produced, and is named as: 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone, english name: 8, 2' -dimethoxy-7- (3-methyl-2-oxobout-3-enyl) -flavanone with molecular formula C22H20O5The structural formula is shown as the formula (I):
The compound was a pale yellow gum, designated: the compound was named: 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone, english name: 8, 2' -dimethoxy-7- (3-methyl-2-oxobout-3-enyl) -flavanone.
The preparation method of the isoamyl flavonoid compound takes waste residue after the production of rose essential oil as a raw material, and is prepared by the steps of extract extraction, organic solvent extraction, MCI decoloration, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps:
A. extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced for 2-5 times by using a solvent, wherein the mass of the extraction solvent used each time is 2-6 times of that of the waste residue powder, the extraction time is 30-60 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitates are separated out, standing for 20-60 minutes, filtering out the precipitates, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 1-2 times that of the extract a into the extract a, extracting for 3-5 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding a 95% methanol aqueous solution with volume concentration which is 3-5 times of the weight of the extract b into the extract b, loading the extract b on an MCI (methanol-to-oil) column after the extract b is completely dissolved, eluting by using a 90-95% methanol aqueous solution, merging the eluents, and then concentrating the merged eluents under reduced pressure to obtain an extract c;
D. silica gel column chromatography: performing silica gel column chromatography on the extract c, wherein the silica gel filled in the column is 160-200 meshes, and the weight of the silica gel is 6-10 times that of the extract c; performing gradient elution by using a chloroform-acetone mixed organic solvent with a volume ratio of 1: 0-0: 1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC spot plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: and (3) separating and purifying a part obtained by eluting with a chloroform-acetone mixed organic solvent with a volume ratio of 9:1 by using high performance liquid chromatography to obtain the isoamyl flavonoid compound.
The solvent in the step A is an acetone aqueous solution with volume concentration of 70-100%, an ethanol aqueous solution with volume concentration of 90-100% or a methanol aqueous solution with volume concentration of 90-100%.
And the organic solvent in the step B is dichloromethane, chloroform, ethyl acetate, diethyl ether or petroleum ether.
And D, dissolving the extract c in acetone or methanol which is 1.5-3 times of the weight of the extract c before the extract c is subjected to silica gel column chromatography, mixing the sample with 80-100 meshes of silica gel which is 0.8-1.2 times of the weight of the extract c, and then loading the sample.
In the step D, in the gradient elution, the volume ratio of the chloroform to the acetone mixed organic solvent is 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 in sequence; after each gradient eluted to the point where the TLC spot plate was no longer spotted, the next gradient elution was changed.
And E, separating and purifying the high performance liquid chromatography in the step E by taking a methanol water solution with volume concentration of 68% as a mobile phase, wherein the flow rate is 15-25 ml/min, and the separation and purification are carried out at 21.2' 250mm, 5mAnd m Zorbax PrepHT GF reversed-phase preparation column is a stationary phase, the detection wavelength of an ultraviolet detector is 354nm, 50-200 mL of sample is fed every time, a chromatographic peak of 30.5 min is collected, and the chromatographic peak is evaporated to dryness after multiple accumulation.
The compound of the invention is added into a cigarette filter, can alleviate the stimulation of smoke to the throat, stimulate the salivary secretion, has sweet aftertaste effect in the oral cavity, improves the aftertaste of the oral cavity, covers the miscellaneous gas and improves the smoking quality of the cigarette.
Example 1
The waste residue sample after producing the rose essential oil is from Yuxi yunnan, and the variety is Damascus rose.
A. Extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced with a solvent for 2 times, wherein the mass of the extraction solvent used each time is 2 times of that of the waste residue powder, the extraction time is 30 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitate is separated out, standing for 20 minutes, filtering out the precipitate, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 1 time that of the extract a into the extract a, extracting for 3 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding a 95% methanol aqueous solution with volume concentration which is 3 times of the weight of the extract b into the extract b, loading the extract b on an MCI column after the extract b is completely dissolved, eluting by using a 90% methanol aqueous solution, merging the eluents, and then concentrating the merged eluents under reduced pressure to obtain an extract c;
D. silica gel column chromatography: dissolving the extract c by using acetone or methanol with the weight 1.5 times that of the extract c, then mixing the sample by using 80-mesh silica gel with the weight 0.8 times that of the extract c, then carrying out column chromatography on the sample, wherein the silica gel filled in the column is 160 meshes, and the weight of the used silica gel is 6 times that of the extract c; performing gradient elution by using a mixed organic solvent of chloroform and acetone in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC point plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: separating and purifying a part eluted by a chloroform-acetone mixed organic solvent with a volume ratio of 9:1 by using a high performance liquid chromatography, wherein the specific method for separating and purifying the high performance liquid chromatography is as follows: using 68% methanol water solution as mobile phase, flowing at 20ml/min, and 21.2' 250mm, 5 ″mm Zorbax PrepHT GF reversed phase preparation column is a fixed phase, the detection wavelength of an ultraviolet detector is 354nm, 50mL of sample is injected each time, 30.5 min chromatographic peak is collected, and after multiple accumulation, the isopentyl flavonoid compound is obtained by evaporation to dryness.
Wherein, the solvent in the step A is acetone aqueous solution with the volume concentration of 70 percent. And the organic solvent in the step B is chloroform.
Example 2
The waste residue sample after producing the rose essential oil is from Yuxi yunnan, and the variety is Damascus rose.
A. Extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced with a solvent for 5 times, wherein the mass of the extraction solvent used each time is 6 times of that of the waste residue powder, the extraction time is 60 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitate is separated out, standing for 60 minutes, filtering out the precipitate, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 2 times that of the extract a into the extract a, extracting for 5 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding a 95% methanol aqueous solution with volume concentration which is 5 times of the weight of the extract b into the extract b, loading the extract b on an MCI column after the extract b is completely dissolved, eluting by using the 95% methanol aqueous solution, merging the eluents, and then concentrating the merged eluents under reduced pressure to obtain an extract c;
D. silica gel column chromatography: dissolving the extract c by using acetone or methanol with the weight 3 times that of the extract c, then mixing the sample by using 100-mesh silica gel with the weight 1.2 times that of the extract c, then carrying out column chromatography on the sample, wherein the silica gel filled in the column is 200 meshes, and the weight of the silica gel is 10 times that of the extract c; performing gradient elution by using a mixed organic solvent of chloroform and acetone in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC point plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: separating and purifying a part eluted by a chloroform-acetone mixed organic solvent with a volume ratio of 9:1 by using a high performance liquid chromatography, wherein the specific method for separating and purifying the high performance liquid chromatography is as follows: using 68% methanol water solution as mobile phase, flowing at 20ml/min, and 21.2' 250mm, 5 ″mm Zorbax PrepHT GF reversed phase preparation column as fixed phase, and ultraviolet detector for detectionAnd (3) sampling 200mL of the isopentyl flavonoid compound at a wavelength of 354nm every time, collecting chromatographic peaks for 30.5 min, accumulating for multiple times, and evaporating to dryness to obtain the isopentyl flavonoid compound.
Wherein, the solvent in the step A is acetone. The organic solvent in the step B is dichloromethane.
Example 3
The waste residue sample after producing the rose essential oil is from Yuxi yunnan, and the variety is Damascus rose.
A. Extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced with a solvent for 3 times, wherein the mass of the extraction solvent used each time is 3 times of that of the waste residue powder, the extraction time is 40 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitate is separated out, standing for 40 minutes, filtering out the precipitate, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 1.5 times of the weight of the extract a into the extract a, extracting for 4 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding a 95% methanol aqueous solution with volume concentration 4 times of the weight of the extract b into the extract b, loading the extract b on an MCI column after the extract b is completely dissolved, eluting by using a 93% methanol aqueous solution, merging the eluents, and then concentrating the merged eluents under reduced pressure to obtain an extract c;
D. silica gel column chromatography: dissolving the extract c with acetone or methanol 2 times the weight of the extract c, mixing with 90-mesh silica gel 1 times the weight of the extract c, loading the sample, performing column chromatography, wherein the silica gel filled in the column is 180 meshes, and the weight of the silica gel is 8 times the weight of the extract c; performing gradient elution by using a mixed organic solvent of chloroform and acetone in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC point plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: chloroform-acetone mixed organic solvent with volume ratio of 9:1 is adoptedSeparating and purifying the eluted part by high performance liquid chromatography, wherein the specific method for separating and purifying the high performance liquid chromatography is as follows: using 68% methanol water solution as mobile phase, flowing at 20ml/min, and 21.2' 250mm, 5 ″mm Zorbax PrepHT GF reversed phase preparation column is a fixed phase, the detection wavelength of an ultraviolet detector is 354nm, 100mL of sample is injected each time, a chromatographic peak of 30.5 min is collected, and after multiple accumulation, the isoamyl flavonoid compound is obtained by evaporation to dryness.
Wherein, the solvent in the step A is ethanol. And the organic solvent in the step B is ethyl acetate.
In the embodiment, 4.4kg of waste residue powder is adopted to obtain 120g of extract a, 80g of extract b, 62g of extract c, and 18 g of part eluted by chloroform-acetone mixed organic solvent with the volume ratio of 9: 1.
Example 4
The waste residue sample after producing the rose essential oil comes from Xinjiang Hotan and the variety is Damascus rose.
A. Extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced with a solvent for 4 times, wherein the mass of the extraction solvent used each time is 4 times of that of the waste residue powder, the extraction time is 50 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitate is separated out, standing for 40 minutes, filtering out the precipitate, and then concentrating the obtained filtrate under reduced pressure into an extract a;
B. organic solvent extraction: adding water with the weight 1.2 times of the weight of the extract a into the extract a, extracting for 4 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding 95% methanol aqueous solution 4.5 times the weight of the extract b, loading the extract b on MCI column after the extract b is completely dissolved, eluting with 92% methanol aqueous solution, mixing eluates, and concentrating the mixed eluates under reduced pressure to obtain extract c;
D. silica gel column chromatography: dissolving the extract c by acetone or methanol with the weight 2.5 times that of the extract c, mixing the sample by 80-mesh silica gel with the weight 0.9 time that of the extract c, loading the sample to perform column chromatography, wherein the silica gel filled in the column is 160 meshes, and the weight of the silica gel is 8 times that of the extract c; performing gradient elution by using a mixed organic solvent of chloroform and acetone in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC point plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: separating and purifying a part eluted by a chloroform-acetone mixed organic solvent with a volume ratio of 9:1 by using a high performance liquid chromatography, wherein the specific method for separating and purifying the high performance liquid chromatography is as follows: using 68% methanol water solution as mobile phase, flowing at 20ml/min, and 21.2' 250mm, 5 ″mm Zorbax PrepHT GF reversed phase preparation column is a fixed phase, the detection wavelength of an ultraviolet detector is 354nm, each sample injection is 80mL, 30.5 min chromatographic peak is collected, and after multiple accumulation, the isopentyl flavonoid compound is obtained by evaporation to dryness.
Wherein, the solvent in the step A is methanol. And the organic solvent in the step B is diethyl ether.
In the embodiment, 10kg of waste residue powder is adopted to obtain 350g of extract a, 210g of extract b, 150g of extract c and 55g of part eluted by chloroform-acetone mixed organic solvent with the volume ratio of 9: 1.
Example 5
The waste residue sample after producing the rose essential oil comes from Xinjiang Hotan and the variety is Damascus rose.
A. Extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced with a solvent for 4 times, wherein the mass of the extraction solvent used each time is 5 times of that of the waste residue powder, the extraction time is 35 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitate is separated out, standing for 55 minutes, filtering out the precipitate, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 1.6 times of the weight of the extract a into the extract a, extracting for 4 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding 95% methanol aqueous solution 4.5 times the weight of the extract b, loading the extract b on MCI column after the extract b is completely dissolved, eluting with 94% methanol aqueous solution, mixing eluates, and concentrating the mixed eluates under reduced pressure to obtain extract c;
D. silica gel column chromatography: dissolving the extract c by acetone or methanol with the weight 2.4 times that of the extract c, mixing the sample by 100-mesh silica gel with the weight 1 times that of the extract c, loading the sample to perform column chromatography, wherein the silica gel filled in the column is 200 meshes, and the weight of the silica gel is 7 times that of the extract c; performing gradient elution by using a mixed organic solvent of chloroform and acetone in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC point plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: separating and purifying a part eluted by a chloroform-acetone mixed organic solvent with a volume ratio of 9:1 by using a high performance liquid chromatography, wherein the specific method for separating and purifying the high performance liquid chromatography is as follows: using 68% methanol water solution as mobile phase, flowing at 20ml/min, and 21.2' 250mm, 5 ″mm Zorbax PrepHT GF reversed phase preparation column is a fixed phase, the detection wavelength of an ultraviolet detector is 354nm, 120mL of sample is injected each time, 30.5 min chromatographic peak is collected, and after multiple accumulation, the isopentyl flavonoid compound is obtained by evaporation to dryness.
Wherein, the solvent in the step A is a methanol water solution with the volume concentration of 95 percent. And the organic solvent in the step B is petroleum ether.
Example 6
The structure of the isoamyl flavonoid prepared by the method of example 1 was determined by the following method:
the compounds of the invention are pale yellow gums; HRESI-MS shows that the peak of the excimer ion is 363.1202 [ M + Na ]]+(calculated 363.1208), combined1H NMR and DEPT spectra confirm that the molecular formula is C22H20O5The unsaturation degree was 13.
In infrared spectrum showsCarbonyl (1680 and 1665 cm)-1) And aromatic rings (1610, 1537 and 1469 cm)-1) The resonance absorption peak of (1). The maximum absorption of the ultraviolet spectrum at 210 nm, 258 nm and 360 nm also indicates that aromatic ring structures possibly exist in the compound.
Process for preparing compounds1H and13c NMR spectra (see Table 1, FIG. 1 and FIG. 2) show that they contain 22 carbons and 20 hydrogens, including 1,2,3, 4-tetra-substituted benzene ring (C-5-C-10, H-5, H-6), 1, 2-di-substituted benzene ring (C-1 '-C-6', H-3 '-H-6'), and 1α,βUnsaturated carbonyl (C-2, C-3, C-4, H-3), 1 3-methyl-2-oxobutyl-3-ene structural fragment (C-1'' to C-5 ''; H)2-1''，H2-4'' and H3-5 "), two methoxy groups (d C61.5 q and 56.1 q,d H3.84 s and 3.81 s). According to the typical 2 benzene rings,α,βUnsaturated carbonyl and double bond signals, which are presumed to be flavonoids. The compound was further confirmed to have a flavonoid structure based on HMBC correlation (see FIG. 3) between H-3 and C-2, C-4, C-10, C-1', H-5 and C-4, C-9, C-10, and H-6' and C-2.
After the parent compound is identified, the remaining substituents, 3-methyl-2-oxobutyl-3-ene and methoxy, can be considered as substituents on the flavone. Two methoxyhydrogens (A), (B) can be observed in the HMBC spectrum of the compound (as shown in FIG. 3)d H3.84 and 3.81) in connection with HMBC at C-8 and C-2', it can be speculated that two methoxy groups are substituted at the C-8 and C-2' positions, respectively; according to H2-1'' (d H4.61) and C-6, C-7, C-8 and H-6: (d H6.72) and HMBC at C-1 ″, it was confirmed that the substitution of 3-methyl-2-oxobutyl-3-ene is at the C-7 position. In addition the proton signal on the benzene ring is typical [ H-5,d H7.43 (d) 8.2；H-6,d H6.72 (d) 8.2，H-3′d H6.93 (d) 7.6；H-4′，d H7.32 (t) 7.6；H-5′，d H6.88 (t) 7.6；H-6′，d H7.89 (d)7.6]it was also confirmed that the flavone compound of the present invention has a 6, 8-disubstituted ring B and a 2' -monosubstituted ring C.
To this end, the structure of the compound was determined and the compound was named: 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone.
Example 7
The compounds prepared in examples 2-5 were taken as pale yellow gums. The determination method was the same as in example 6, and it was confirmed that the compound prepared in examples 2 to 5 was 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone, which is the aforementioned isoamyl flavonoid.
Example 8
The isoamyl flavonoid compound prepared in any one of the embodiments 1 to 5 is used for carrying out the addition effect test of the cigarette filter, and the test conditions are as follows:
the added cigarette is a cigarette 'Hongtashan classic' of Hongta group, and the isoamyl flavonoid compound is prepared into a solution of 0.1 mg/mL by using triacetyl glycerine. Uniformly spraying the compound on the filter tow according to 8 percent of the weight of the filter tow to prepare a filter stick, then preparing the filter stick into a cigarette through conventional cigarette rolling, carrying out sensory evaluation, and taking the same cigarette without the compound as a reference. The evaluation and analysis result shows that: can alleviate the stimulation of smoke gas to the throat, stimulate the secretion of saliva, and has sweet aftertaste effect in the oral cavity, so that the aftertaste of the oral cavity is improved, the miscellaneous gas can be covered, and the smoking quality of the cigarette is improved.
Example 9
The isoamyl flavonoid compound prepared in any one of the embodiments 1 to 5 is used for carrying out the addition effect test of the cigarette filter:
adding cigarette "soft red plum" of Hongta group, and using triacetin to prepare 0.5 mg/mL solution of the isoamyl flavonoid compound. Uniformly spraying the mixture on filter tow according to 5% of the weight of the filter tow to prepare a filter stick, then preparing the filter stick into a cigarette through conventional cigarette rolling, carrying out sensory evaluation, and taking the same cigarette without the compound as a reference. The evaluation and analysis result shows that: can alleviate the stimulation of smoke gas to the throat, stimulate the secretion of saliva, and has sweet aftertaste effect in the oral cavity, so that the aftertaste of the oral cavity is improved, the miscellaneous gas can be covered, and the smoking quality of the cigarette is improved.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. An isoamyl flavonoid compound in rose waste residue is characterized in that the isoamyl flavonoid compound is named as: 8, 2' -dimethoxy-7- (3-methyl-2-oxobutyl-3-ene) -flavone, english name: 8, 2' -dimethoxy-7- (3-methyl-2-oxobout-3-enyl) -flavanone with molecular formula C22H20O5The structural formula is shown as the formula (I):
2. The method for preparing isoamyl flavonoid compounds in rose waste residue according to claim 1, wherein the waste residue after the production of rose essential oil is used as a raw material, and the steps of extract extraction, organic solvent extraction, MCI decoloration, silica gel column chromatography and high performance liquid chromatography are carried out to obtain the rose residue, and the method specifically comprises the following steps:
A. extracting the extractum: ultrasonically extracting the waste residue powder after the rose essential oil is produced for 2-5 times by using a solvent, wherein the mass of the extraction solvent used each time is 2-6 times of that of the waste residue powder, the extraction time is 30-60 minutes each time, combining the extracting solutions, filtering, concentrating the filtrate under reduced pressure until just precipitates are separated out, standing for 20-60 minutes, filtering out the precipitates, and then concentrating the obtained filtrate under reduced pressure to obtain an extract a;
B. organic solvent extraction: adding water with the weight 1-2 times that of the extract a into the extract a, extracting for 3-5 times by using an organic solvent, combining organic solvent extraction phases, and then concentrating the combined organic solvent extraction phases under reduced pressure to obtain an extract b, wherein the volume of the organic solvent used in each time is the same as that of the water;
C. MCI decoloration: adding a 95% methanol aqueous solution with volume concentration which is 3-5 times of the weight of the extract b into the extract b, loading the extract b on an MCI (methanol-to-oil) column after the extract b is completely dissolved, eluting by using a 90-95% methanol aqueous solution, merging the eluents, and then concentrating the merged eluents under reduced pressure to obtain an extract c;
D. silica gel column chromatography: performing silica gel column chromatography on the extract c, wherein the silica gel filled in the column is 160-200 meshes, and the weight of the silica gel is 6-10 times that of the extract c; performing gradient elution by using a chloroform-acetone mixed organic solvent with a volume ratio of 1: 0-0: 1, and replacing the next gradient elution after each gradient elution is performed until no point exists on a TLC spot plate; collecting gradient eluates of each gradient, concentrating, monitoring by TLC, and mixing the same fractions;
E. high performance liquid chromatography separation: separating and purifying the part eluted by chloroform-acetone mixed organic solvent with the volume ratio of 9:1 by high performance liquid chromatography to obtain the isoamyl flavonoid compound;
in the step D, in the gradient elution, the volume ratio of the chloroform to the acetone mixed organic solvent is 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 in sequence; after each gradient eluted to the point where the TLC spot plate was no longer spotted, the next gradient elution was changed.
3. The method for preparing isoamyl flavonoid compounds in rose waste residue according to claim 2, wherein the solvent in step a is an aqueous solution of acetone with a volume concentration of 70-100%, an aqueous solution of ethanol with a volume concentration of 90-100%, or an aqueous solution of methanol with a volume concentration of 90-100%.
4. The method for preparing isoamyl flavonoid compounds in rose waste residue according to claim 2, wherein the organic solvent in step B is dichloromethane, chloroform, ethyl acetate, diethyl ether or petroleum ether.
5. The method for preparing isoamyl flavonoid compounds in rose waste residue according to claim 2, wherein in the step D, before silica gel column chromatography, the extract c is dissolved by acetone or methanol which is 1.5-3 times of the weight of the extract c, then the sample is mixed by 80-100 meshes of silica gel which is 0.8-1.2 times of the weight of the extract c, and then the sample is loaded.
6. The method for preparing isoamyl flavonoid compounds in rose waste residue according to claim 2, wherein the high performance liquid chromatography separation and purification of step E is performed by taking a 68% methanol aqueous solution as a mobile phase, taking a flow rate of 20ml/min, taking a Zorbax PrepHT GF reversed-phase preparation column of 21.2 x 250mm and 5 μm as a stationary phase, using an ultraviolet detector to detect the wavelength of 354nm, feeding 50-200 μ L of sample each time, collecting 30.5 min of chromatographic peak, and evaporating to dryness after multiple accumulation.
7. The use of the isoamyl flavonoid compounds contained in the rose dregs of claim 1 as an additive for preparing cigarette filters.
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| CN107118194A (en) * | 2017-06-02 | 2017-09-01 | 云南中烟工业有限责任公司 | A kind of isoflavonoid that can improve cigarette smoking throat comfortableness and preparation method and application |
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| 山楂黄酮萃取工艺优化及在卷烟中的应用;李小斌 等;《食品工业》;20161231;第37卷(第10期);第104-108页 * |
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