CN110483536B - Tricyclic isoquinoline alkaloid compound extracted from sabina chinensis and application thereof - Google Patents

Tricyclic isoquinoline alkaloid compound extracted from sabina chinensis and application thereof Download PDF

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CN110483536B
CN110483536B CN201910825539.3A CN201910825539A CN110483536B CN 110483536 B CN110483536 B CN 110483536B CN 201910825539 A CN201910825539 A CN 201910825539A CN 110483536 B CN110483536 B CN 110483536B
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compound
tricyclic
heavy metal
cigarette
extracting
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CN110483536A (en
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高茜
曾婉俐
向海英
杨光宇
孔维松
李雪梅
李晶
刘欣
宋春满
黄海涛
王晋
许�永
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
    • A24D—CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES FOR CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
    • A24D3/00—Tobacco smoke filters, e.g. filter-tips, filtering inserts; Filters specially adapted for simulated smoking devices; Mouthpieces for cigars or cigarettes
    • A24D3/02—Manufacture of tobacco smoke filters
    • A24D3/0204—Preliminary operations before the filter rod forming process, e.g. crimping, blooming
    • A24D3/0212—Applying additives to filter materials
    • A24D3/022—Applying additives to filter materials with liquid additives, e.g. application of plasticisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered

Abstract

The invention discloses a tricyclic isoquinoline alkaloid compound extracted from sabia japonica and application thereof. The compound is obtained by taking branches and leaves of the sabia japonica 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 C17H17NO3Having the following structural formula:
Figure DDA0003430456220000011
the compound has a functional group with a heavy metal chelating function, and can generate a stable chelate with a heavy metal element; the cigarette filter tip has the advantages of obvious effect of reducing heavy metal elements in cigarette smoke, no obvious negative effect on the cigarette smoke, good harmony with the original taste of the cigarette, and slight improvement effect on the irritation and aftertaste of cigarette smoking. The invention has the advantages of wide distribution of production raw materials, large biological yield, simple compound preparation process, low production cost and easy realization of industrialized application.

Description

Tricyclic isoquinoline alkaloid compound extracted from sabina chinensis and application thereof
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to a tricyclic isoquinoline alkaloid compound which is obtained by first extracting and separating branches and leaves of a plant of Cassia of Leguminosae. 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
The name of the iron knife wood (Cassia siamea Lam.) is hard and hard to be inserted. Evergreen arbor of Cassia of Leguminosae, about 10 m in height; the bark is grey, smooth near light and slightly longitudinally split; the twigs have ribs, so that the quilt is thinned and softened. The leaf length is 20-30 cm; the leaf axis and the petiole have no gland and are slightly soft; the lobules are in pairs. The raceme grows from the leaf axil at the top end of the branch and is arranged into an umbrella house inflorescence shape; the shape of bract line; the sepals are approximately round; the petals are yellow and wide and inverted egg-shaped. Flat pods, thickened edges; 10-20 seeds. The flowering phase is 10-11 months; fruit stage 12 months-next year 1 month. The seeds have a long cultivation history in China, and are widely planted in Fujian, Taiwan, Guangdong, Hainan, Guangxi, Yunnan and West, wherein the salary forest cultivation history of Yunnan Jinghong is long. Because the wood is hard and compact, is resistant to water and humidity, is not polluted by insects and oysters, and is a first-class furniture raw material. The old tree is black and has beautiful texture, and can be used as musical instrument decoration. Because of its rapid growth, strong sprouting power, easily-flammable branches and trunks, strong fire, it can be extensively cultivated in Yunnan as firewood forest, and cut down by adopting head-shaped operation, and the cut-down trees can be up to 3-4 m in two years, and can be cut down once every four years. Scholars at home and abroad have conducted some researches on the sabdariffa, and the main reported compound types are as follows: chromone, flavonoid, anthraquinone, terpenes, steroids, alkaloids, etc.
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, a novel tricyclic isoquinoline alkaloid compound is obtained by researching chemical components of the sabina chinensis bunge through extraction and separation, and the compound has no related report so far. It is worth mentioning that the compound has a group with 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 tricyclic isoquinoline alkaloid compound.
Another object of the present invention is to provide a method for preparing the tricyclic isoquinoline alkaloid compounds.
The invention also aims to provide application of the tricyclic 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.
Tricyclic isoquinoline alkaloid compound with molecular formula of C17H17NO3Having the following structural formula:
Figure GDA0003430456210000021
。
a method of preparing the tricyclic isoquinoline alkaloid compounds, comprising the steps of:
(1) extracting the extractum: drying branches and leaves of the sabia japonica in the sun, crushing the branches and leaves to 35-60 meshes, putting the crushed sample into a glass reaction kettle, performing reflux extraction for 2 times by using 95% ethanol, combining the two extracting solutions, concentrating the extracting solution to a small volume, diluting the extracting solution by using a 3% tartaric acid solution, and extracting the diluted solution 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 by using a chloroform-acetone mixed solvent with the volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
(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: taking a 62% methanol aqueous solution as a mobile phase, a Zorbax PrepHT GF reversed-phase preparation column with the thickness of 21.2 multiplied by 250mm and the thickness of 5 mu m as a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 346nm, the sample injection amount is 0.5-1.0 mL each time, the chromatographic peak of 32.9min is collected, and the tricyclic isoquinoline alkaloid compound crude product is obtained after multiple accumulation and evaporation to dryness.
And (3) dissolving the crude compound obtained after the separation and purification by the high pressure liquid chromatography in pure methanol, and separating by using 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 306.1112[ M + Na ]]+Is combined with1H and13c NMR spectrum to confirm the molecular formula as C17H17NO3Is a alkaloid compound, and the unsaturation degree of the compound is 10. The infrared spectrum of the compound showed that the compound had a hydroxyl group (3386 cm)-1) Carbonyl group (1665 cm)-1) And aromatic rings (1612, 1568 and 1440 cm)-1) The signal, the absorption maxima of the UV spectrum at 225, 273, 310 and 346nm also confirms the presence of aromatic ring structures in the compounds. Process for preparing compounds1H、13C-NMR and DEPT spectra (Table-1) showed that it contained 17 carbons and 17 hydrogens. The method comprises the following steps: a 1,6, 7-substituted isoquinoline core (C-1 to C-10; H-3, H-4, H-5, and H-8), a synthosphane ring (-CH ═ CH-C (CH)3)2-O-, C-1 'to C-5', H-1 ', H-2' and H6-4 ", 5"), a 3-hydroxypropan-1-one group (-CO-CH)2CH2-OH；C-1′～C-3′；H2-2' and H2-3'). 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 harmonic dimethylchromene ring can be present by H-1 'and C-2', C-3 ', H-2' and C-1 ', C-3', C-4 ', 5', and H6HMBC correlation of-4 ", 5" with C-2 ", C-3" was confirmed; the 3-hydroxypropan-1-one group may be present via H2-2' and H2-3' of1H-1H COSY related, and H22 ' and C-1 ', C-3 ', H3HMBC correlation of-3 ' and C-1 ', C-2 ' was confirmed. Further analysis of its HMBC correlation spectrum, based on H-1' and C-5, C-6, C-7; h-2 ' and C-7, and H-8 and C-1 ' are related to HMBC, it is speculated that the gem-dimethyl chromene ring is substituted at the C-6 and C-7 positions of the phenyl ring of the isoquinoline, and the carbon at the C-1 ' position is attached at the C-7 position of the phenyl ring of the isoquinoline. The parent structure of the compound can be determined to be isoquinoline tricyclic alkaloid with gem-dimethyl chromene ring. After the parent nucleus of the compound is determined, the remaining 3-hydroxypropan-1-one group may be defined as a substituent. By H2HMBC at-2' and C-1 correlate and a 3-hydroxypropan-1-keto substitution at C-1 can be determined. The structure of the compounds of the present invention has thus far been confirmed.
TABLE-1 preparation of the compounds1H and13c nuclear magnetic resonance data (solvent CDCl)3500 and 125MHz)
Figure GDA0003430456210000041
Infrared, ultraviolet and mass spectral data of compounds: UV (methanol), lambdamax(logε)225(4.26)、273(3.68)、310(3.43), 346 (3.62); IR (potassium bromide pellet): v ismax 3386、3065、2948、1665、1612、1568、1440、1352、1244、1163、1057、857、760cm-1；1H and13c NMR data (500 and 125MHz, (CDCl)3) See Table-1; positive ion mode ESIMS M/z 306[ M + Na ]]+(ii) a Positive ion mode HRESIMS M/z 306.1112[ M + Na ]]+(calculation value C)17H17NNaO3，306.1106)。
The tricyclic isoquinoline alkaloid compound disclosed by the invention is applied to reduction of the heavy metal element content 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 (lg) of the chelate isKMY) Respectively as follows: 19.6, 23.4, 25.8 and 20.2.
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 tricyclic isoquinoline alkaloid compound is 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: 0.367 to 0.552 microgram/20 arsenic, 2.48 to 3.46 microgram/20 lead, 3.15 to 4.38 microgram/20 cadmium and 0.136 to 0.205 microgram/20 mercury, and the heavy metal release amount of the smoke of the control cigarettes without the compound is respectively as follows: 0.533-0.792 microgram/20 arsenic, 4.82-6.77 microgram/20 lead, 5.70-8.92 microgram/20 cadmium and 0.224-0.375 microgram/20 mercury, wherein the reduction rates of the four heavy metal elements are respectively as follows: 26.2 to 34.1 percent of arsenic, 44.8 to 47.2 percent of lead, 44.0 to 49.1 percent of cadmium and 32.4 to 43.5 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 difference between the sensory evaluation results of the cigarettes added with the compound is very small, and the main difference is that the cigarettes slightly bring back sweet feeling to the mouth, the harmony with the original taste of the cigarettes is good, and no obvious negative effect exists; but also has slight improvement effect on the irritation and aftertaste of cigarette smoking.
Compared with the prior art, the invention has the following advantages:
(1) the invention extracts and separates a tricyclic isoquinoline alkaloid compound from cassia siamea which belongs to leguminosae for the first time, and the compound has a functional group with heavy metal chelating function and can generate stable chelate with heavy metal elements; the cigarette filter tip has the advantages of obvious effect of reducing heavy metal elements in cigarette smoke, no obvious negative effect on the cigarette smoke, good harmony with the original taste of the cigarette, and slight improvement effect on the irritation and aftertaste of cigarette smoking.
(2) The raw materials of the cycas revoluta are widely distributed, a large amount of cycas revoluta are cultivated in Yunnan to be used as salary woods, head-shaped operation is adopted for cutting, the trees can be cut to be 3-4 meters in two years after cutting, generally, the trees can be cut once every four years, the biological yield is very high, the raw materials are easy to obtain, 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 a nuclear magnetic resonance carbon spectrum of a tricyclic isoquinoline alkaloid compound of the present invention: (13C NMR)；
FIG. 2 shows the NMR spectra of tricyclic isoquinoline alkaloids of the invention1H NMR)；
FIG. 3 is key HMBC and key HMBC derivatives of tricyclic isoquinoline alkaloids of the present invention1H-1HCOSY correlation graph.
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 method for manufacturing the steel cutting wood is not limited by regions and varieties, and can be realized.
Example 1
The samples of the kefir were taken from the Yunnan Xishuangbanna. Taking 5.2kg of dried ramulus et folium Sambuci Buergerianae and leaves, pulverizing to about 35-60 meshes, placing in a 20L glass reaction kettle, adding 6-10L of 95% ethanol, reflux-extracting for 35-60 min, and filtering to obtain extractive 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 33.5g of alkaloid part extract. Adding 60g of acetone into the extract for dissolving, then adding 45g of 100-mesh silica gel for sample mixing, and filling 120g 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 collected sample C (8:2) is further treated 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, 62% methanol as a mobile phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 346nm, each sample injection is 0.8mL, a chromatographic peak with the size of 32.9min is collected, and the sample is evaporated to dryness after multiple accumulation to obtain the tricyclic isoquinoline alkaloid compound crude product. Dissolving the crude compound with pure methanol, separating by Sephadex column chromatography with pure methanol as mobile phase, collecting the effluent of alkaloid compounds, evaporating solvent to obtain yellow jelly, and evaporating solvent to obtain pure compound.
Example 2
The ramulus Et folium Picrasmae is taken from Yun nan Yuan Jiang. Pulverizing the sample to 45 mesh, weighing 7.2kg of the pulverized sample, placing in a 20L glass reaction kettle, adding 10L of 95% ethanol, reflux extracting for 50min, and filtering to obtain extractive solution; adding 10L of 95% ethanol 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 8L of 3% tartaric acid solution and extracted 2 times with 8L of ethyl acetate. After extraction, the water phase is saturated with sodium carbonate, and is extracted with 8L ethyl acetate for 2 times, and the extracted ethyl acetate phases are combined and concentrated under reduced pressure to obtain 45.6g of alkaloid part extract. Adding 80g of acetone into the extract for dissolving, then adding 60g of 100-mesh silica gel for sample mixing, and filling 200g 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 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, 62 percent methanol as a mobile phase with the flow rate of 20mL/min, the detection wavelength of an ultraviolet detector is 346nm, 1.0mL is injected each time, chromatographic peaks with the size of 32.9min are collected, and the crude product of the tricyclic isoquinoline alkaloid compound is obtained after multiple accumulation and evaporation to dryness. Dissolving the crude compound with pure methanol, separating with Sephadex column chromatography with pure methanol as mobile phase, collecting the outflow part of the alkaloid compounds, and evaporating the solvent to obtain the pure compound.
Example 3
Example 1 the structure of the tricyclic isoquinoline alkaloid compounds prepared by the following method is identified:
the compound is yellow jelly, and HRESI-MS shows that the peak of the quasi-molecular ion is 306.1112[ M + Na ]]+Is combined with1H and13c NMR spectrum to confirm the molecular formula as C17H17NO3Is a alkaloid compound, and the unsaturation degree of the compound is 10. The infrared spectrum of the compound showed that the compound had a hydroxyl group (3386 cm)-1) Carbonyl group (1665 cm)-1) And aromatic rings (1612, 1568 and 1440 cm)-1) The signal, the absorption maxima of the UV spectrum at 225, 273, 310 and 346nm also confirms the presence of aromatic ring structures in the compounds. Process for preparing compounds1H、13C-NMR and DEPT spectra (Table-1) showed that it contained 17 carbons and 17 hydrogens. The method comprises the following steps: a 1,6, 7-substituted isoquinoline core (C-1 to C-10; H-3, H-4, H-5, and H-8), a synthosphane ring (-CH ═ CH-C (CH)3)2-O-, C-1 'to C-5', H-1 ', H-2' and H6-4 ", 5"), a 3-hydroxypropan-1-one group (-CO-CH)2CH2-OH；C-1′～C-3′；H2-2' and H2-3'). 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 harmonic dimethylchromene ring can be present by H-1 'and C-2', C-3 ', H-2' and C-1 ', C-3', C-4 ', 5', and H6HMBC correlation of-4 ", 5" with C-2 ", C-3" was confirmed; the 3-hydroxypropan-1-one group may be present via H2-2' and H2-3' of1H-1H COSY related, and H22 ' and C-1 ', C-3 ', H3HMBC correlation of-3 ' and C-1 ', C-2 ' was confirmed. Further analysis of HMBC correlationSpectra according to H-1' and C-5, C-6, C-7; h-2 ' and C-7, and H-8 and C-1 ' are related to HMBC, it is speculated that the gem-dimethyl chromene ring is substituted at the C-6 and C-7 positions of the phenyl ring of the isoquinoline, and the carbon at the C-1 ' position is attached at the C-7 position of the phenyl ring of the isoquinoline. The parent structure of the compound can be determined to be isoquinoline tricyclic alkaloid with gem-dimethyl chromene ring. After the parent nucleus of the compound is determined, the remaining 3-hydroxypropan-1-one group may be defined as a substituent. By H2HMBC at-2' and C-1 correlate and a 3-hydroxypropan-1-keto substitution at C-1 can be determined. The structure of the compounds of the present invention has thus far been confirmed.
Example 4
The compound prepared in example 2 was taken as a yellow gum. The compound prepared in example 2 was confirmed to be a tricyclic isoquinoline alkaloid compound, sabcomeline alkaloid-S, 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. The chelate stability constants of the compounds of the invention were determined in the present invention 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 (lg) of the chelate isKMY) Respectively as follows: 19.6, 23.4, 25.8 and 20.2.
Example 6
The tricyclic isoquinoline alkaloid compound is 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), and the 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 the reference samples with the cigarettes.
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: 0.367 to 0.552 microgram/20 arsenic, 2.48 to 3.46 microgram/20 lead, 3.15 to 4.38 microgram/20 cadmium and 0.136 to 0.205 microgram/20 mercury, and the heavy metal release amount of the smoke of the control cigarettes without the compound is respectively as follows: 0.533-0.792 microgram/20 arsenic, 4.82-6.77 microgram/20 lead, 5.70-8.92 microgram/20 cadmium and 0.224-0.375 microgram/20 mercury, wherein the reduction rates of the four heavy metal elements are respectively as follows: 26.2 to 34.1 percent of arsenic, 44.8 to 47.2 percent of lead, 44.0 to 49.1 percent of cadmium and 32.4 to 43.5 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.
The cigarettes to which the compounds of the present invention were added were subjected to sensory evaluation, and the same cigarettes to which the compounds were not added were used as controls. The evaluation and analysis result shows that: the difference between the sensory evaluation results of the cigarettes added with the compound is very small, and the main difference is that the cigarettes slightly bring back sweet feeling to the mouth, the harmony with the original taste of the cigarettes is good, and no obvious negative effect exists; but also has slight improvement effect on the irritation and aftertaste of cigarette smoking.

Claims (5)

1. Tricyclic isoquinoline alkaloid compound with molecular formula of C17H17NO3Having the following structural formula:
Figure FDA0003430456200000011
。
2. a process for the preparation of tricyclic isoquinoline alkaloid compounds according to claim 1 comprising the steps of:
(1) extracting the extractum: drying branches and leaves of the sabia japonica in the sun, crushing the branches and leaves to 35-60 meshes, putting the crushed sample into a glass reaction kettle, performing reflux extraction for 2 times by using 95% ethanol, combining the two extracting solutions, concentrating the extracting solution to a small volume, diluting the extracting solution by using a 3% tartaric acid solution, and extracting the diluted solution 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 by using a chloroform-acetone mixed solvent with the volume ratio of 20:1,9:1,8:2,7:3,6:4 and 1:1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
(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: taking a 62% methanol aqueous solution as a mobile phase, a Zorbax PrepHT GF reversed-phase preparation column with the thickness of 21.2 multiplied by 250mm and the thickness of 5 mu m as a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 346nm, the sample injection amount is 0.5-1.0 mL each time, the chromatographic peak of 32.9min is collected, and the tricyclic isoquinoline alkaloid compound crude product is obtained after multiple accumulation and evaporation to dryness.
3. The method of claim 2, wherein: and (3) dissolving the crude compound obtained after the separation and purification by the high pressure liquid chromatography in pure methanol, and separating by using 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 tricyclic isoquinoline alkaloid compounds of claim 1 to reduce the content of heavy metal elements in the mainstream smoke of a cigarette.
5. Use according to claim 4, characterized in that: preparing the tricyclic isoquinoline alkaloid compound into 0.5mg/mL solution by using glycerol triacetate, uniformly spraying the solution onto filter tow according to 5-8% of the weight of the filter tow, and then manufacturing the filter rod for cigarettes by using a conventional process.
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US5162335A (en) * 1986-05-21 1992-11-10 Duphar International Research B.V. Di- and tetrahydroisoquinoline derivatives
CN106117138A (en) * 2016-06-29 2016-11-16 云南中烟工业有限责任公司 A kind of isoquinoline alkaloids alkaloid compound, its preparation method and application with antibacterial activity

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