CN110551066B - Isoquinoline alkaloid compound extracted from tobacco rhizome and application thereof - Google Patents

Isoquinoline alkaloid compound extracted from tobacco rhizome and application thereof Download PDF

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CN110551066B
CN110551066B CN201910825546.3A CN201910825546A CN110551066B CN 110551066 B CN110551066 B CN 110551066B CN 201910825546 A CN201910825546 A CN 201910825546A CN 110551066 B CN110551066 B CN 110551066B
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compound
cigarette
heavy metal
isoquinoline alkaloid
extracting
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CN110551066A (en
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曾婉俐
高茜
向海英
杨光宇
李雪梅
李晶
黄海涛
王晋
许�永
宋春满
孔维松
刘欣
胡秋芬
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China Tobacco Yunnan Industrial Co Ltd
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    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/24—Treatment of tobacco products or tobacco substitutes by extraction; Tobacco extracts
    • A24B15/241—Extraction of specific substances
    • A24B15/243—Nicotine
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances
    • A24B15/302—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances by natural substances obtained from animals or plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
    • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
    • C07D217/16—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals substituted by oxygen atoms

Abstract

The invention discloses an isoquinoline alkaloid compound extracted from tobacco roots and stems and application thereof. The compound is obtained by taking tobacco roots and stems as raw materials and separating and purifying the raw materials through ethyl acetate extraction, silica gel column chromatography and high performance liquid chromatography. Molecular formula C16H17NO2Having the following structural formula:
Figure DDA0002188911610000011
is named as: 1- (6-hydroxy-7-isopentenyl-isoquinolin-1-yl) ethanone. The compound has a functional group with a heavy metal chelating function, can generate a stable chelate with heavy metal elements, can obviously reduce the content of the heavy metal elements in cigarette smoke when being added into a cigarette filter, has no obvious negative effect on the cigarette smoke, has good harmony with the original taste of the cigarette, has slightly bitter cool taste and sweet taste when being put into the mouth, and also has an improvement effect on the irritation and aftertaste of cigarette smoking. The invention has the advantages of easily obtained production raw materials, capability of changing waste into valuable, low cost, safe and nontoxic compound, simple preparation process and large-scale popularization and application conditions.

Description

Isoquinoline alkaloid compound extracted from tobacco rhizome and application thereof
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to an isoquinoline alkaloid compound extracted and separated from tobacco roots and stems for the first time. The compound has a functional group with a heavy metal chelating function, and can generate a stable chelate with a free heavy metal element, so the invention also relates to the application of the compound in reducing the heavy metal content in cigarette smoke.
Background
Tobacco is the plant with the most complex chemical components in the world, secondary metabolites are very abundant, and through decades of researches, more than 3000 monomeric chemical substances are identified from tobacco at present, and many components are not identified yet. Besides being mainly used for cigarette smoking, the tobacco can also extract a plurality of chemical components with useful values, and guide compounds with development and utilization values are found from the chemical components. The tobacco roots are waste materials in the tobacco leaf production process, but the tobacco roots are also rich in a large amount of components with comprehensive utilization values, such as: contains a large amount of plant fibers, and the tobacco stems can be pressed into fiber boards or made into active carbon and the like; the tobacco stalk also contains considerable pectin, pectinase and resin, and the pectin is a chemical product used in a plurality of industries on a large scale. The tobacco rhizome and low-grade tobacco leaf can be used for extracting nicotine, and the nicotine has the functions of sterilization and hemostasis, and can prevent and treat crop pests and livestock skin parasites. Especially, in recent years, researchers at home and abroad discover a plurality of secondary metabolites with antibacterial, antioxidant, antiviral and other activities from tobacco roots and stalks, and the comprehensive utilization value of the tobacco roots and stalks is remarkably improved.
Heavy metal elements such As As, Pb, Cd, Ni, Cr and the like in the mainstream smoke of the cigarettes are harmful substances in the Hoffman list. Studies have shown that the blood cadmium concentration of smoking men is significantly higher than that of non-smokers. Therefore, the control of trace heavy metals of the cigarettes is an important problem related to smoking safety, and the research on how to reduce the content of heavy metals in the smoking process of the cigarettes has important scientific significance and application value.
According to the invention, through the research on chemical components of tobacco roots and stems, a novel isoquinoline alkaloid compound is obtained through extraction and separation, and the compound has not been reported so far. It is worth mentioning that the compound has a group with a heavy metal element chelating function, can generate a stable five-membered ring chelate with the heavy metal element, can effectively intercept the heavy metal element in the mainstream smoke of the cigarette, and has the effect of remarkably reducing the content of the heavy metal element in the smoke of the cigarette.
Disclosure of Invention
The invention aims to provide a novel isoquinoline alkaloid compound.
Another object of the present invention is to provide a method for preparing said isoquinoline alkaloid compounds.
The invention also aims to provide application of the isoquinoline alkaloid compound in reducing the content of heavy metal elements in the mainstream smoke of cigarettes.
The purpose of the invention is realized by the following technical scheme.
All percentages used in the present invention are mass percentages unless otherwise indicated.
An isoquinoline alkaloid compound with molecular formula of C16H17NO2Having the following structural formula:
Figure GDA0003413768960000021
the compound was named: 1- (6-hydroxy-7-isopentenyl-isoquinolin-1-yl) ethanone, with the english name: 1- (6-hydroxy-7-phenyl-isoquinolin-1-yl) ethanone.
A method for preparing said isoquinoline alkaloid compounds comprising the steps of:
(1) extracting the extractum: drying tobacco roots in the sun, crushing the tobacco roots to 35-60 meshes, placing the crushed sample in a glass reaction kettle, performing reflux extraction for 2 times by using 95% ethanol, combining extracting solutions obtained in the two times, concentrating the extracting solutions to a small volume, diluting the extracting solutions by using 3% tartaric acid solution, and extracting the diluted extracting solutions by using ethyl acetate for 2 times; saturating the water phase with sodium carbonate, extracting with ethyl acetate for 2 times, mixing the extracted ethyl acetate phases, and concentrating under reduced pressure to obtain alkaloid extract;
(2) silica gel column chromatography: dissolving the extract obtained in the step (1) by using acetone or methanol in an amount which is 1.5-3 times the weight of the extract, and then mixing the sample by using 80-100 mesh silica gel in an amount which is 0.8-1.6 times the weight of the extract; the silica gel filled in the column is 160-200 meshes, and the using amount of the silica gel is 3-8 times of the weight of the extract; performing gradient elution with chloroform-acetone mixed solvent at volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions;
(3) high performance liquid chromatography separation: and (3) performing high performance liquid chromatography separation and purification on chloroform-acetone eluent with the ratio of 8: 2: and (2) taking 58% methanol aqueous solution as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the thickness of 5 mu m as a stationary phase, setting the flow rate at 20mL/min, the detection wavelength at 338nm by an ultraviolet detector, feeding 0.5-1.0 mL each time, collecting a chromatographic peak for 34.2min, accumulating for multiple times, and evaporating to dryness to obtain the crude isoquinoline alkaloid compound.
And (3) dissolving the crude compound obtained after the separation and purification by the high performance liquid chromatography in pure methanol, and separating by a sephadex column chromatography with the pure methanol as a mobile phase to obtain a yellow jelly, namely a pure compound.
Structural identification of the compounds:
the compound is yellow jelly, and HRESI-MS shows that the peak of the quasi-molecular ion is 278.1152[ M + Na ]]+Is combined with1H and13c NMR spectrum to confirm the molecular formula as C16H17NO2Is a alkaloid compound, and the unsaturation degree of the compound is 9. The infrared spectrum shows that the compound has hydroxyl (3412 cm)-1) Carbonyl group (1665 cm)-1) And aromatic rings (1612, 1568 and 1453cm-1) The signals, the absorption maxima of the UV spectra at 220, 264, 299 and 338nm also confirm the presence of aromatic ring structures in the compounds. Process for preparing compounds1H、13C-NMR and DEPT spectra (Table-1) showed that it contained 16 carbons and 17 hydrogens. The method comprises the following steps: a 1,6, 7-substituted isoquinoline nucleus (C-1 to C-10; H-3, H-4, H-5, and H-8), an isopentenyl group (-CH)2CH＝C(CH3)2，C-1″～C-5″，H2-1″、H-2″、H3-4 "and H3-5') and an acetyl group (-CO-CH)3(ii) a C-1 'and C-2'; h3-2'), and one phenolic hydroxy group (δ)H10.80 s). The isoquinoline nucleus can exist through H-3 and C-1, C-4, C-10; h-4 and C-3, C-5, C-9, C-10; h-5 and C-4, C-9, C-10; and HMBC correlation of H-8 and C-1, C-9, C-10 (FIG. 3). The presence of isopentenyl can be determined by H2Of-1 "and H-21H-1H COSY related, and H2-1 "and C-2", C-3 ", H-2" and C-1 ", C-3", C-4 ", C-5", H3-4 "and C-3", C-2 ", C-5", H3HMBC correlation of-5 "with C-3", C-2 ", C-4" confirmed that the presence of acetyl groups was confirmed by H3HMBC correlation of-2 'and C-1' was confirmed.
After the parent nucleus and key substituted fragments of the compound are determined, the positions of isopentenyl, acetyl and phenolic hydroxyl groups can be further determined by HMBC correlation. By H2HMBC correlation of-1 ' with C-6, C-7, C-8, H-2 ' with C-7, and H-8 with C-1 ', confirmed the prenyl substitution at the C-7 position. By H3HMBC at-2' and C-1 correlate and the substitution of the acetyl group at the C-1 position can be confirmed. By phenolic hydroxy hydrogen (. delta.)H10.80) and HMBC at C-5, C-6, C-7, it was confirmed that the phenolic hydroxyl group was substituted at the C-6 position. The structure of the compound of the present invention was confirmed so far, and the compound was named: 1- (6-hydroxy-7-isopentenyl-isoquinolin-1-yl) ethanone.
Watch-1. Process for preparing compounds1H and13c NMR data (solvent CDCl3, 500 and 125MHz)
Figure GDA0003413768960000041
Infrared, ultraviolet and mass spectral data of compounds: UV (methanol), lambdamax(log ε)220(4.12), 264(3.53), 299(3.22),338 (341); IR (potassium bromide pellet): v ismax 3412、3050、2954、1665、1612、1568、1453、1386、1243、1160、1056、936、794cm-1；1H and13c NMR data (500 and 125MHz, (CDCl)3) See Table-1; ESIMS M/z 278[ M + Na ] in positive ion mode]+(ii) a Positive ion mode HRESIMS M/z 278.1152[ M + Na ]]+(calculation value C)16H17NNaO2，278.1157)。
The isoquinoline alkaloid compound is applied to reducing the content of heavy metal elements in the mainstream smoke of cigarettes.
1. Determination of the stability constant of the heavy metal complexes of the compounds:
the nitrogen and carbonyl oxygen of isoquinoline in the compound can coordinate with heavy metal atoms to form a stable five-membered ring chelate. The chelate stability constants of the compounds of the invention were determined using the classical method developed by Scogs and Miniquad. The results show that: the compound and four heavy metal elements with high toxicity, namely arsenic, lead, cadmium and mercury, can generate stable chelate in a neutral medium (the pH value is 6.2-7.9), and the stability constant of the chelate (lgK)MY) Respectively as follows: 18.2, 22.1, 26.4 and 19.6.
2. The adding method of the compound in the cigarette filter and the analysis of the reduction effect of the heavy metal elements in the smoke are as follows:
the isoquinoline alkaloid compounds are prepared into a solution of 0.5mg/mL by using glycerol triacetate. In the process of forming the cigarette filter, the heavy metal element is uniformly sprayed on the filter tow according to 5-8% of the weight of the filter tow to prepare a filter stick, and then the filter stick and the cigarette are rolled to prepare the cigarette for measuring the heavy metal element in the smoke of the cigarette.
Mainstream smoke analysis is carried out on the cigarettes with the compounds of the invention added in the filter sticks, smoke heavy metal element determination is carried out by referring to Chinese tobacco industry standards (YC/T389-2011 and YCT 404-2011), the same kind of cigarettes without the compounds of the invention are used as a reference, and the reduction rate of the heavy metal elements is calculated by comparing with a reference sample.
The results show that: the heavy metal release amount of the cigarette smoke with 6 different specifications added with the compound of the invention is respectively as follows: 0.385-0.547 mu g/20 arsenic, 2.58-3.67 mu g/20 lead, 3.22-4.67 mu g/20 cadmium and 0.148-0.213 mu g/20 mercury, wherein the heavy metal release amount of the smoke of the control cigarettes without the compound is respectively as follows: 0.527-0.785 mu g/20 of arsenic, 4.72-6.58 mu g/20 of lead, 5.61-8.86 mu g/20 of cadmium and 0.217-0.366 mu g/20 of mercury, wherein the reduction rates of the four heavy metal elements are respectively as follows: 26.9 to 33.2 percent of arsenic, 44.2 to 45.5 percent of lead, 43.6 to 47.8 percent of cadmium and 31.8 to 42.3 percent of mercury; has the effect of remarkably reducing the content of heavy metal elements in the mainstream smoke of the cigarettes.
3. Toxicology assessment and sensory evaluation
Toxicology analysis was performed on the cigarette smoke condensate with the compound of the invention added to the filter stick. The results of three in vitro toxicology tests, namely a cytotoxicity test, an Ames test and an in vitro micronucleus test, show that: statistical analysis is carried out on the cell inhibition rate of the total particulate matter of the smoke of the cigarettes of the tested sample and the control sample by using one-way ANOVA of SPSS software, and in three toxicology experiments, the total particulate matter of the smoke of the cigarettes added with the compound has no statistical difference (P is more than 0.05) with the total particulate matter of the smoke of the cigarettes of the control sample.
Sensory evaluation was also performed on cigarettes to which the compounds of the present invention were added, and the same cigarettes to which the compounds were not added were used as controls. The evaluation and analysis result shows that: the cigarette added with the compound has bitter and cool taste and sweet aftertaste, has good harmony with the original taste of the cigarette after the compound is added, has no obvious negative effect, and has the effect of improving the irritation and aftertaste of cigarette smoking.
Compared with the prior art, the invention has the following advantages:
(1) the isoquinoline alkaloid compound is obtained by first extracting and separating from tobacco roots and stems, has a functional group with a heavy metal chelating function, and can generate a stable chelate with heavy metal elements; the cigarette filter tip is added to the cigarette filter tip, so that the content of heavy metal elements in cigarette smoke can be obviously reduced, the cigarette smoke is not obviously influenced negatively, the harmony with the original taste of the cigarette is good, the cigarette filter tip has slight bitter cool taste and sweet taste in the mouth, and the cigarette filter tip also has the effect of improving the irritation and aftertaste of cigarette smoking.
(2) The tobacco roots and stems are waste in the tobacco production process, the tobacco is widely planted in Yunnan, and a large number of roots and stems are discarded in the tobacco production process every year, so that the production raw materials are easy to obtain, waste can be changed into valuable, the cost is low, and the industrial application is easy to realize.
(3) The compound is safe and nontoxic, has simple preparation process and has the condition of large-scale popularization and application.
Drawings
FIG. 1 is the nuclear magnetic resonance carbon spectrum of the isoquinoline alkaloid compounds of the invention (13C NMR)；
FIG. 2 shows the NMR spectra of isoquinoline alkaloids of the invention1H NMR)；
FIG. 3 is a key HMBC correlation diagram for isoquinoline alkaloid compounds of the present invention.
Detailed Description
The present invention is further described in detail with reference to the drawings and examples, which are not intended to limit the technical scope of the present invention, and all changes and equivalents which are made based on the teachings of the present invention should fall within the protective scope of the present invention.
The tobacco described in the examples is not limited by region or variety, and the present invention can be realized.
Example 1
The tobacco root stem is from Yunnan Kunming, and the variety is safflower Dajinyuan. Taking 4.6kg of dried tobacco roots, crushing the dried tobacco roots to 35-60 meshes, putting the crushed tobacco roots into a 20L glass reaction kettle, adding 6-10L of 95% ethanol, performing reflux extraction for 35-60 min, and filtering an extracting solution; and adding 6-10L of 95% ethanol into the filter residue again, performing reflux extraction for 35-60 min, filtering out an extracting solution, combining the two extracting solutions, and concentrating to a small volume. Then diluting the mixture with 4-6L of 3% tartaric acid solution, and extracting the diluted mixture with 4-6L of ethyl acetate for 2 times. And after extraction, saturating the water phase with sodium carbonate, extracting for 2 times with 4-6L of ethyl acetate, combining the extracted ethyl acetate phases, and concentrating under reduced pressure to obtain 28.6g of alkaloid part extract. Adding 45g of acetone into the extract for dissolving, then adding 38g of 100-mesh silica gel for sample mixing, and filling 86g of 200-mesh silica gel into a column after sample mixing; gradient elution is carried out by chloroform-acetone mixed organic solvent with volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1 respectively, gradient eluent is collected and concentrated, TLC monitoring is carried out, the same parts are combined to obtain 6 parts A-F, wherein, the collected part C (8:2) of a sample is further processed by using a Zorbax PrepHT GF reversed phase preparation column with the size of 21.2 multiplied by 250mm and the size of 5 mu m as a stationary phase, 58% methanol as a mobile phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 338nm, each sample injection is 0.6mL, 34.2min chromatographic peaks are collected, and the crude isoquinoline alkaloid compound is obtained after multiple accumulation and evaporation to dryness. And dissolving the crude compound by using pure methanol again, separating by using sephadex column chromatography by using the pure methanol as a mobile phase, collecting the outflow part of the alkaloid compound, evaporating the solvent to obtain yellow jelly, and evaporating the solvent to obtain the pure compound.
Example 2
The tobacco root stem is from Yunnan Dali and the variety is Yunyan-85. Pulverizing the sample to about 40 mesh, weighing 6.4kg of the pulverized sample, placing in a 20L glass reaction kettle, adding 95% ethanol 6L, reflux extracting for 50min, and filtering to obtain extractive solution; adding 95% ethanol 6L into the residue, reflux extracting for 50min, filtering to obtain extractive solution, mixing the two extractive solutions, and concentrating to small volume. Then diluted with 6L of 3% tartaric acid solution and extracted 2 times with 6L of ethyl acetate. After extraction, the water phase is saturated with sodium carbonate, extracted for 2 times with 6L ethyl acetate again, the extracted ethyl acetate phases are combined, and concentrated under reduced pressure to obtain 38.4g of alkaloid part extract. Adding 80g of acetone into the extract for dissolving, then adding 45g of 100-mesh silica gel for sample mixing, and filling 120g of 180-mesh silica gel into a column after sample mixing; gradient elution is carried out by chloroform-acetone mixed organic solvents with volume ratios of 20:1,9:1,8:2,7:3,6:4 and 1:1 respectively, gradient eluent is collected and concentrated, TLC monitoring is carried out, the same parts are combined to obtain 6 parts A-F, wherein, the collected part C (8:2) of a sample is further processed by using a Zorbax PrepHT GF reversed-phase preparation column with the size of 21.2 multiplied by 250mm and the size of 5 mu m as a stationary phase, 58% methanol as a mobile phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 338nm, each sample injection is 0.8mL, 34.2min chromatographic peaks are collected, and the crude isoquinoline alkaloid compound is obtained after multiple accumulation and evaporation to dryness. And dissolving the crude compound by using pure methanol again, separating by using sephadex column chromatography by using the pure methanol as a mobile phase, collecting the outflow part of the alkaloid compound, and evaporating the solvent to dryness to obtain the pure compound.
Example 3
Example 1 the structure of the isoquinoline alkaloid compounds prepared is identified by the following method:
the compound is yellow jelly, and HRESI-MS shows that the peak of the quasi-molecular ion is 278.1152[ M + Na ]]+Is combined with1H and13c NMR spectrum to confirm the molecular formula as C16H17NO2Is a alkaloid compound, and the unsaturation degree of the compound is 9. The infrared spectrum shows that the compound has hydroxyl (3412 cm)-1) Carbonyl group (1665 cm)-1) And aromatic rings (1612, 1568 and 1453cm-1) The signals, the absorption maxima of the UV spectra at 220, 264, 299 and 338nm also confirm the presence of aromatic ring structures in the compounds. Process for preparing compounds1H、13C-NMR and DEPT spectra (Table-1) showed that it contained 16 carbons and 17 hydrogens. The method comprises the following steps: a 1,6, 7-substituted isoquinoline nucleus (C-1 to C-10; H-3, H-4, H-5, and H-8), an isopentenyl group (-CH)2CH＝C(CH3)2，C-1″～C-5″，H2-1″、H-2″、H3-4 "and H3-5') and an acetyl group (-CO-CH)3(ii) a C-1 'and C-2'; h3-2'), and one phenolic hydroxy group (δ)H10.80 s). The isoquinoline nucleus can exist through H-3 and C-1, C-4, C-10; h-4 and C-3, C-5, C-9, C-10; h-5 and C-4, C-9, C-10; and HMBC correlation of H-8 and C-1, C-9, C-10 (FIG. 3). The presence of isopentenyl can be determined by H2Of-1 "and H-21H-1H COSY related, and H2-1 "and C-2", C-3 ", H-2" and C-1 ", C-3", C-4 ", C-5", H3-4 "and C-3", C-2 ", C-5", H3HMBC correlation of-5 "with C-3", C-2 ", C-4" confirmed that the presence of acetyl groups was confirmed by H3HMBC correlation of-2 'and C-1' was confirmed.
After the parent nucleus and key substituted fragments of the compound are determined, the positions of isopentenyl, acetyl and phenolic hydroxyl groups can be further determined by HMBC correlation. By H2HMBC correlation of-1 ' with C-6, C-7, C-8, H-2 ' with C-7, and H-8 with C-1 ', confirmed the prenyl substitution at the C-7 position. By H3HMBC at-2' and C-1 correlate and the substitution of the acetyl group at the C-1 position can be confirmed. By phenolic hydroxy hydrogen (. delta.)H10.80) and HMBC at C-5, C-6, C-7, it was confirmed that the phenolic hydroxyl group was substituted at the C-6 position. The structure of the compound of the present invention was confirmed so far, and the compound was named: 1- (6-hydroxy-7-isopentenyl-isoquinolin-1-yl) ethanone.
Infrared, ultraviolet and mass spectral data of compounds: UV (methanol), lambdamax(log ε)220(4.12), 264(3.53), 299(3.22),338 (341); IR (potassium bromide pellet): v ismax 3412、3050、2954、1665、1612、1568、1453、1386、1243、1160、1056、936、794cm-1；1H and13c NMR data (500 and 125MHz, (CDCl)3) See Table-1; ESIMS M/z 278[ M + Na ] in positive ion mode]+(ii) a Positive ion mode HRESIMS M/z 278.1152[ M + Na ]]+(calculation value C)16H17NNaO2，278.1157)。
Example 4
The compound prepared in example 2 was taken as a yellow gum. The compound prepared in example 2 was confirmed to be 1- (6-hydroxy-7-isopentenyl-isoquinolin-1-yl) ethanone, which is the isoquinoline alkaloid compound, in the same manner as in example 3.
Example 5
The nitrogen and carbonyl oxygen of isoquinoline in the compound can coordinate with heavy metal atoms to form a stable five-membered ring chelate. So the classic developed by Scogs and Miniquad is adoptedThe method determines the chelate stability constant of the compounds of the invention. The results show that: the compound and four heavy metal elements with high toxicity, namely arsenic, lead, cadmium and mercury, can generate stable chelate in a neutral medium (the pH value is 6.2-7.9), and the stability constant of the chelate (lgK)MY) Respectively as follows: 18.2, 22.1, 26.4 and 19.6.
Example 6
The isoquinoline alkaloid compounds are prepared into a solution of 0.5mg/mL by using glycerol triacetate. In the process of forming the cigarette filter, the heavy metal element is uniformly sprayed on the filter tow according to 5-8% of the weight of the filter tow to prepare a filter stick, and then the filter stick and the cigarette are rolled to prepare the cigarette for measuring the heavy metal element in the smoke of the cigarette.
Mainstream smoke analysis is carried out on the cigarettes with the compounds of the invention added in the filter sticks, smoke heavy metal element determination is carried out by referring to Chinese tobacco industry standards (YC/T389-2011 and YCT 404-2011), the same kind of cigarettes without the compounds of the invention are used as a reference, and the reduction rate of the heavy metal elements is calculated by comparing with a reference sample.
The results show that: the heavy metal release amount of the cigarette smoke with 6 different specifications added with the compound of the invention is respectively as follows: 0.385-0.547 mu g/20 arsenic, 2.58-3.67 mu g/20 lead, 3.22-4.67 mu g/20 cadmium and 0.148-0.213 mu g/20 mercury, wherein the heavy metal release amount of the smoke of the control cigarettes without the compound is respectively as follows: 0.527-0.785 mu g/20 of arsenic, 4.72-6.58 mu g/20 of lead, 5.61-8.86 mu g/20 of cadmium and 0.217-0.366 mu g/20 of mercury, wherein the reduction rates of the four heavy metal elements are respectively as follows: 26.9 to 33.2 percent of arsenic, 44.2 to 45.5 percent of lead, 43.6 to 47.8 percent of cadmium and 31.8 to 42.3 percent of mercury; has the effect of remarkably reducing the content of heavy metal elements in the mainstream smoke of the cigarettes.
Example 7
Toxicology analysis was performed on the cigarette smoke condensate with the compound of the invention added to the filter stick. The results of three in vitro toxicology tests, namely a cytotoxicity test, an Ames test and an in vitro micronucleus test, show that: statistical analysis is carried out on the cell inhibition rate of the total particulate matter of the smoke of the cigarettes of the tested sample and the control sample by using one-way ANOVA of SPSS software, and in three toxicology experiments, the total particulate matter of the smoke of the cigarettes added with the compound has no statistical difference (P is more than 0.05) with the total particulate matter of the smoke of the cigarettes of the control sample.
Sensory evaluation was also performed on cigarettes to which the compounds of the present invention were added, and the same cigarettes to which the compounds were not added were used as controls. The evaluation and analysis result shows that: the cigarette added with the compound has bitter and cool taste and sweet aftertaste, has good harmony with the original taste of the cigarette after the compound is added, has no obvious negative effect, and has the effect of improving the irritation and aftertaste of cigarette smoking.

Claims (5)

1. An isoquinoline alkaloid compound with molecular formula of C16H17NO2Having the following structural formula:
Figure FDA0002188911580000011
the compound was named: 1- (6-hydroxy-7-isopentenyl-isoquinolin-1-yl) ethanone, with the english name: 1- (6-hydroxy-7-phenyl-isoquinolin-1-yl) ethanone.
2. A process for the preparation of isoquinoline alkaloid compounds as claimed in claim 1 comprising the steps of:
(1) extracting the extractum: drying tobacco roots in the sun, crushing the tobacco roots to 35-60 meshes, placing the crushed sample in a glass reaction kettle, performing reflux extraction for 2 times by using 95% ethanol, combining extracting solutions obtained in the two times, concentrating the extracting solutions to a small volume, diluting the extracting solutions by using 3% tartaric acid solution, and extracting the diluted extracting solutions by using ethyl acetate for 2 times; saturating the water phase with sodium carbonate, extracting with ethyl acetate for 2 times, mixing the extracted ethyl acetate phases, and concentrating under reduced pressure to obtain alkaloid extract;
(2) silica gel column chromatography: dissolving the extract obtained in the step (1) by using acetone or methanol in an amount which is 1.5-3 times the weight of the extract, and then mixing the sample by using 80-100 mesh silica gel in an amount which is 0.8-1.6 times the weight of the extract; the silica gel filled in the column is 160-200 meshes, and the using amount of the silica gel is 3-8 times of the weight of the extract; performing gradient elution with chloroform-acetone mixed solvent at volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions;
(3) high performance liquid chromatography separation: and (3) performing high performance liquid chromatography separation and purification on chloroform-acetone eluent with the ratio of 8: 2: and (2) taking 58% methanol aqueous solution as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the thickness of 5 mu m as a stationary phase, setting the flow rate at 20mL/min, the detection wavelength at 338nm by an ultraviolet detector, feeding 0.5-1.0 mL each time, collecting a chromatographic peak for 34.2min, accumulating for multiple times, and evaporating to dryness to obtain the crude isoquinoline alkaloid compound.
3. The method of claim 2, wherein: and (3) dissolving the crude compound obtained after the separation and purification by the high performance liquid chromatography in pure methanol, and separating by a sephadex column chromatography with the pure methanol as a mobile phase to obtain a yellow jelly, namely a pure compound.
4. The use of isoquinoline alkaloid compounds of claim 1 in reducing the content of heavy metal elements in the mainstream smoke of a cigarette.
5. Use according to claim 4, characterized in that: preparing the isoquinoline alkaloid compounds into 0.5mg/mL solution by glycerol triacetate, uniformly spraying the solution on filter tow according to 5-8% of the weight of the filter tow, and then preparing the filter rod for cigarettes by a conventional process.
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