CN115894499A - Novel natural sulfur-containing alkaloid and preparation method and application thereof - Google Patents
Novel natural sulfur-containing alkaloid and preparation method and application thereof Download PDFInfo
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 42
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 title claims abstract description 9
- 229910052717 sulfur Inorganic materials 0.000 title claims abstract description 9
- 239000011593 sulfur Substances 0.000 title claims abstract description 9
- 235000019504 cigarettes Nutrition 0.000 claims abstract description 100
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims abstract description 84
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a novel natural sulfur-containing alkaloid, a preparation method and application thereof, wherein the alkaloid 4-acetoxy maca thiourea D is a novel natural alkaloid with a tetrahydroimidazo tetrahydropyrrole framework, and the compound is prepared by taking dry roots or fresh roots of maca as a raw material through the routes of stewing, processing, extracting, separating, purifying, structure identifying and the like. Having a molecular formula of C 15 H 16 N 2 O 3 S, the molecular weight is 304, the Chinese name is 4-acetoxy maca thiourea D, and the structural formula is as follows:
. The 4-acetoxy maca thiourea D prepared by the invention is added to acetate fiber tows to prepare functional filter sticks for cigarettes, so that the functional filter sticks can be remarkably reducedTotal particulate matter, nicotine, tar and benzo [ a ] in cigarette mainstream smoke]Content of pyrene. The novel alkaloid disclosed by the invention is derived from traditional medicine and food dual-purpose plants, and the raw materials are simple and easy to obtain, and are safe and reliable. The invention can obviously reduce the release of harmful substances in the cigarette, has good harmony between the cigarette smoke and the original taste, and has potential research value and application value.
Description
Technical Field
The invention belongs to the technical field of natural organic compounds and tobacco additives in reducing the release of harmful substances in cigarettes, and particularly relates to a novel natural sulfur-containing alkaloid 4-acetoxy-lepidium meyenii thiourea D in medicinal plant lepidium meyenii, a preparation method thereof and application thereof in removing harmful substances in cigarettes.
Background
The cigarette smoke components are extremely complex, a large amount of compounds can be generated in the combustion process of tobacco, and the 13 compounds comprise 44 main cigarette smoke harmful components in total. The first is nicotine, which stays mostly in the lungs after entering the human body with smoke, and only a small part enters the blood, and reaches the brain in a very short time, thus having an inhibitory effect on sympathetic nerves. The second is carbon monoxide, and researches show that the symptoms of human body such as hypoxic poisoning, cardiovascular disorder and the like are greatly related to the carbon monoxide, and the excessive carbon monoxide can even cause organic lesions. The tar is a mixture with extremely complex components, consists of various hydrocarbons, hydrocarbon oxides, sulfides, nitrides and the like, contains aroma substances special for cigarettes, carcinogens or carcinogens extremely harmful to human bodies, such as polycyclic aromatic hydrocarbons such as benzo [ a ] pyrene and the like, aromatic amines, nitrosamines, chlorohydrocarbons and the like.
The selective reduction of harmful components in the smoke and the maintenance of the original quality of the cigarette become a major topic for tobacco practitioners. Wherein the active ingredients in the medicinal plants are used as additives to selectively reduce the harmful ingredients in the cigarette smokeIs an effective strategy. Maca is a medicinal plant of 1 to 2 years old in Lepidium of Brassicaceae (Latin name isLepidium meyenii Walp), originally produced south american andes mountainous area (altitude 3500 to 4500), is a medicinal plant and edible material commonly used in local places, and is in prime reputation of "peru ginseng". The invention discovers a novel sulfur-containing alkaloid 4-acetoxyl maca thiourea D with a tetrahydroimidazo tetrahydropyrrole mother nucleus from the plant, and prompts that the component can be one of main active components of maca serving as a medicine and food dual-purpose plant. The compound 4-acetoxy maca thiourea D has the thiocarbonyl at the 2-position, the oxycarbonyl at the 4-position andNthe special groups help to adsorb harmful substances in cigarettes, and the tobacco additive is a medicinal plant tobacco additive with potential application prospect.
Disclosure of Invention
The first object of the present invention is to provide a novel alkaloid; the second purpose is to provide the preparation method of the alkaloid; the third purpose is to provide the application of the alkaloid.
The first object of the present invention is achieved by a novel alkaloid having a tetrahydroimidazotetrahydropyrrole skeleton, which is a compound derived from Lepidium meyenii Walp (Latin name:Lepidium meyenii walp.) dried or fresh roots as raw materials, and is prepared by the routes of cooking, processing, solvent extraction, separation and purification, structure identification and the like; the molecular formula of the compound is C 15 H 16 N 2 O 3 S, the molecular weight is 304, the Chinese name of the compound is 4-acetoxy maca thiourea D, the English name is 4-OAc macathiohydantoin D, and the systematic name is 2-benzyl-1-oxo-3-thioxotetrahydro-1H- pyrrolo[1,2-c]imidazole-7 a (5H) -yl acetate, having the chemical formula:
the second purpose of the invention is realized by taking the roots of Lepidium meyenii Walp of Lepidium of Cruciferae as a raw material, and carrying out the steps of sample cooking and processing, solvent extraction, MCI decolorization, normal phase column chromatography segmentation, preparation and semi-preparation high performance liquid chromatography enrichment, wherein the specific steps (1) to (6):
(1) Steaming and boiling: lepidium meyenii walp of Lepidium (Lepidium meyenii Walp.)Lepidium meyenii) And (2) drying or steaming the fresh roots at a high temperature of 100 to 150 ℃ by a wet method, naturally airing the sample, crushing, and sieving by a sieve with 10 to 100 meshes to obtain a material a.
(2) Solvent extraction: carrying out cold soaking on the material a by using an organic solvent with the weight being 2-10 times of that of the material a, carrying out ultrasonic extraction for 2-5 times, carrying out 1-3 h each time, filtering to obtain a filtrate, and carrying out reduced pressure distillation and concentration to obtain a maca extract b;
(3) And (3) extraction: adding deionized water with the weight 2-4 times of that of the extract b into the extract b, stirring to obtain a suspension, extracting for 2-5 times by using an organic solvent which has the same volume with the suspension and is immiscible with water, combining extraction phases of the organic solvent, and concentrating under reduced pressure to obtain an extract c;
(4) MCI decoloration: dissolving the extract c in a methanol-water mixed solvent with the weight 2-5 times that of the extract c, purifying by using MCI column chromatography, eluting by using a methanol water solution with the volume percentage concentration of 20-30% to remove impurities, eluting by using a methanol water solution with the volume percentage concentration of 80-90% and mixing the eluates, and concentrating under reduced pressure to obtain an extract d;
(5) Positive phase column chromatography segmentation: dissolving the extract D with an organic solvent which is 2-3 times of the weight of the extract D, stirring a sample with silica gel which is 80-100 meshes or 200-300 meshes and is 1-3 times of the weight of the extract D, purifying by using normal-phase column chromatography, loading the column with 100-200 meshes of silica gel which is 5-10 times of the weight of the extract D, performing gradient elution by using a mixed organic solvent with a volume ratio of 1;
(6) Preparation and semi-preparation high performance liquid chromatography enrichment: and (3) preparing the high performance liquid chromatography for the mixture e, eluting by using a methanol solution with the volume percentage concentration of 40-90% or an acetonitrile solution with the volume percentage concentration of 30-80% to obtain a crude product of the 4-acetoxy maca thiourea D, and separating and purifying by using a semi-preparative high performance liquid chromatography to obtain a pure product of the 4-acetoxy maca thiourea D.
The alkaloid provided by the invention is a novel alkaloid compound discovered from nature for the first time, and the compound is characterized by methods such as nuclear magnetic resonance, mass spectrum, ultraviolet and infrared chromatography and the like, and is determined to be named as 2-benzyl-1-oxo-3-thioxotetrahydro-1H-pyrrolo[1,2-c]imidazole-7 a (5H) -yl acetate having the formula:
the third purpose of the invention is realized by the following steps:
(1) Fully dissolving a prepared target compound 4-acetoxy maca thiourea D (20 to 40 mg) into 1 mL of glyceryl triacetate to prepare a solution of 20 to 40 mg/mL, immersing a filter tip into the solution until all the solution is absorbed by an acetate fiber filter tip, naturally drying the filter tip in the shade, and then filling the filter tip into cigarette tipping paper in an original shape to obtain the functional filter stick capable of reducing the release amount of harmful substances, wherein the filter stick comprises three different specifications of a fine cigarette, a medium cigarette and a conventional cigarette;
(2) And respectively measuring the contents of total particulate matters (T.P.M.), moisture and nicotine in the main stream smoke of the cigarette according to industrial standards YC/T2, YC/T8 and YC/T30, and removing the moisture and nicotine contents from the total particulate matters to obtain the tar content. Compared with a control cigarette, after the alkaloid of the invention with three concentrations (20, 30 and 40 mg) is added, the contents of total particulate matters (T.P.M.) of main stream smoke of the cigarette, nicotine, tar and benzo [ a ] pyrene in the smoke of the cigarettes with three different specifications are all obviously reduced, wherein the reduction rate of the total particulate matters (T.P.M.) is between 4.36 and 13.75 percent, the reduction rate of the nicotine is between 3.17 and 16.67 percent, the reduction rate of the tar is between 4.53 and 16.86 percent, and the reduction rate of the benzo [ a ] pyrene is between 3.39 and 14.11 percent. The above results illustrate that: the alkaloid and the functional filter stick thereof have the obvious effects of intercepting or reducing four types of harmful substances (total particulate matter (T.P.M.), nicotine, tar and benzo [ a ] pyrene) in the mainstream smoke of cigarettes, and the interception capability of the alkaloid is obviously enhanced along with the increase of the concentration of the alkaloid added, and cigarettes of different specifications are slightly different;
(3) Toxicology evaluation and sensory evaluation showed that: the cigarette added with the alkaloid has no toxicity in the aspect of obvious cell inhibition; meanwhile, after the alkaloid of the invention is added, the difference of the sensory evaluation results of the contrast is very small, the main difference is that the taste is slightly cool, the irritation and the aftertaste are slightly improved, and no obvious negative effect is caused.
The invention has the following outstanding advantages: (1) The target compound 4-acetoxy maca thiourea D is derived from safe and healthy medicinal and edible plants, is safe and nontoxic, can effectively intercept harmful substances in cigarette smoke by adding a natural compound with an adsorption function into a cigarette filter stick, is technically easier to realize, and has low cost. (2) The compound and the functional filter stick thereof have the obvious effect of intercepting harmful substances in main stream smoke of cigarettes, and the interception capability of the compound is obviously enhanced along with the increase of the concentration of the compound added. (3) After the compound is added into a filter tip, the cigarette filter tip is safe and non-toxic, cigarette smoke and the original taste of cigarettes are good in harmony, and the cigarette filter tip has a slight cool taste when being eaten and has a slight improvement effect on irritation and aftertaste.
Drawings
FIG. 1 shows the NMR spectrum of 4-acetoxy-maca thiourea D ( 1 H NMR）；
FIG. 2 is a nuclear magnetic resonance DEPT spectrum (DEPT) of 4-acetoxy maca thiourea D.
Detailed Description
The present invention is further described with reference to the following examples and drawings, but the present invention is not limited thereto in any way, and any changes or modifications based on the teaching of the present invention are within the scope of the present invention.
1. The alkaloid is a new alkaloid with a tetrahydroimidazo-tetrahydropyrrole skeleton, and the compound is a compound prepared from Lepidium meyenii Walp (Latin name:Lepidium meyenii walp.) dried or fresh root of the species Melaleuca alternifolia as raw material, by steaming and processingSolvent extraction, separation and purification, structure identification and the like; the molecular formula of the compound is C 15 H 16 N 2 O 3 S, the molecular weight is 304, the compound is named as 4-acetoxy maca thiourea D in Chinese and 4-OAc macathiohydatoin D in English, and the systematic name is 2-benzyl-1-oxo-3-thioxotetrahydro-1H-pyrrolo[1,2-c]Imidazol-7a (5H) -yl acetate having the chemical formula:
2. the preparation method of the alkaloid is characterized in that roots of Lepidium meyenii Walp of Lepidium of Cruciferae are used as raw materials, and a sample is prepared by the steps of stewing, processing, solvent extraction, MCI (methanol-to-ethanol) decolorization, normal phase column chromatography segmentation, preparation and semi-preparation high performance liquid chromatography enrichment, and specifically comprises the following steps:
(1) Steaming and boiling and processing: lepidium meyenii walp of Lepidium (Lepidium meyenii Walp.)Lepidium meyenii) And (3) drying or steaming the fresh roots at a high temperature of 100-150 ℃ by a wet method, naturally airing the sample, crushing, and sieving with a sieve of 10-100 meshes to obtain the material a.
(2) Solvent extraction: and (3) carrying out cold soaking on the material a in an organic solvent with the weight being 2-10 times of that of the material a, carrying out ultrasonic extraction for 2-5 times, wherein each time lasts for 1-3 hours, filtering to obtain a filtrate, and carrying out reduced pressure distillation and concentration to obtain a maca extract b. The organic solvent is 50-100% of acetone, 80-100% of ethanol, 80-100% of ethyl acetate or 80-100% of methanol;
(3) And (3) extraction: adding deionized water with the weight 2-4 times of that of the extract b into the extract b, stirring to obtain a suspension, extracting for 2-5 times by using an organic solvent which has the same volume with the suspension and is immiscible with water, combining extraction phases of the organic solvent, and concentrating under reduced pressure to obtain an extract c. The organic solvent is diethyl ether, chloroform, dichloromethane, ethyl acetate, n-butanol, isopropanol, cyclohexane or petroleum ether.
(4) MCI decoloration: dissolving the extract c in a methanol-water mixed solvent with the weight 2-5 times that of the extract c, purifying by using MCI column chromatography, eluting by using a methanol water solution with the volume percentage concentration of 20-30% to remove impurities, eluting by using a methanol water solution with the volume percentage concentration of 80-90% and mixing the eluates, and concentrating under reduced pressure to obtain an extract d.
(5) Positive phase column chromatography segmentation: dissolving the extract D with an organic solvent which is 2-3 times of the weight of the extract D, stirring a sample with silica gel which is 80-100 meshes or 200-300 meshes and is 1-3 times of the weight of the extract D, purifying by using normal phase column chromatography, loading the column with 100-200 meshes of silica gel which is 5-10 times of the weight of the extract D, performing gradient elution by using a mixed organic solvent with a volume ratio of 1. The mixed organic solvent is chloroform-acetone, and the volume ratio is generally 10.
(6) Preparation and semi-preparation high performance liquid chromatography enrichment: and (3) preparing the high performance liquid chromatography for the mixture e, eluting by using a methanol solution with the volume percentage concentration of 40-90% or an acetonitrile solution with the volume percentage concentration of 30-80% to obtain a crude product of the 4-acetoxy maca thiourea D, and separating and purifying by using a semi-preparative high performance liquid chromatography to obtain a pure product of the 4-acetoxy maca thiourea D. The preparation of the high performance liquid chromatography enrichment is characterized in that 40-90% of methanol aqueous solution or 30-80% of acetonitrile aqueous solution in volume percentage concentration is used as a mobile phase, the flow rate is 10-14mL/min, a Zorbax PrepHT GF reversed-phase preparation column of 5 mu m and 21.2 x 250mm is used as a stationary phase, the detection wavelength of an ultraviolet detector is 202nm and 254nm, the sample size is 10-1000 mu L each time, chromatographic peaks within the range of 10-25min are collected, and after the same steps are accumulated for multiple times, a rotary evaporator is used for evaporating to dryness to obtain the 4-acetoxyl maca thiourea D crude product; the semi-preparative high performance liquid chromatography purification is to use methanol aqueous solution with volume percentage concentration of 40-90% or acetonitrile aqueous solution with volume percentage concentration of 30-80% as a mobile phase, the flow rate is 1-4 mL/min, and Zorbax SB-C with the flow rate of 10 mu m and the flow rate of 9.4 x 250mm is used as a mobile phase 18 Using a reversed phase semi-preparative column as a stationary phase, detecting wavelengths of a DAD detector are 203nm, 220nm, 254nm, 265nm and 306nm, sampling volume of each time is 1-100 mu L, collecting chromatographic peaks within the range of 10-25min, accumulating for multiple times by the same steps, and evaporating to dryness by using a rotary evaporator to obtain the final product4-acetoxy-group pure maca thiourea D.
3. The preparation method of the filter stick capable of reducing the release amount of the harmful substances comprises the following specific steps: weighing 20-40 mg of a compound 4-acetoxy maca thiourea D, fully dissolving the compound 4-acetoxy maca thiourea D into 1 mL of triacetin, preparing the 4-acetoxy maca thiourea D into 20-40 mg/mL solution, immersing a filter tip into the solution until all the solution is absorbed by an acetate fiber filter tip, naturally drying the filter tip in the shade, and then filling the filter tip into cigarette tipping paper in an original shape to obtain the functional filter stick capable of reducing the release amount of harmful substances, wherein the filter stick comprises three different specifications of a thin cigarette, a medium cigarette and a conventional cigarette.
4. And (3) measuring the content of total particulate matters (T.P.M.) in the mainstream smoke of the cigarette. The method specifically comprises the following steps: the cigarette added with the compound is used for analyzing the smoke of the cigarette, and the cigarette without the compound is used as a reference. The content determination calculation formula is as follows:
total particulate matter (t.p.m.) content = [ (M) 2 - M 1 ) / N] × 1000
M 1 Is the weight (unit: gram) of the Cambridge filter sheet smoke catcher before smoking
M 2 The weight of the Cambridge filter smoke catcher after smoking (unit: g)
N is the number of smoking cigarettes (unit: count)
Total particulate matter (t.p.m.) is the weight of total particulate matter (in mg/count)
The reduction rate of total particulate matter (T.P.M.) is calculated by comparing with a control sample, and after the compound is added into filter sticks with different specifications, compared with a control cigarette, the reduction rate of the total particulate matter (T.P.M.) in the mainstream smoke of the cigarette is 4.36 to 13.75 percent, so that the compound and the functional filter stick thereof have a remarkable effect of intercepting the total particulate matter (T.P.M.) in the mainstream smoke of the cigarette, and the interception capability of the compound and the functional filter stick thereof is remarkably enhanced along with the increase of the concentration of the compound added.
5. And measuring the content of nicotine and tar in the mainstream smoke of the cigarette. The method comprises the following specific steps: the cigarette added with the compound is subjected to cigarette smoke analysis, content measurement is carried out according to Chinese tobacco industry standards YC/T2, YC/T8 and YC/T30, the contents of total particulate matters (T.P.M.), moisture and nicotine in the main stream smoke of the cigarette are respectively measured, and the content of the tar is obtained by removing the moisture and the nicotine from the total particulate matters. The reduction rates of water, nicotine and tar are calculated by comparing with a control sample, and compared with a control cigarette, the reduction rate of nicotine in the main stream smoke of the cigarette is between 3.17 and 16.67 percent and the reduction rate of tar is between 4.53 and 16.86 percent after the compound is added into filter rods with different specifications. The above results illustrate that: the alkaloid and the functional filter stick thereof have the obvious effect of intercepting or reducing nicotine and tar in main stream smoke of cigarettes, the interception capability of the alkaloid is obviously enhanced along with the increase of the concentration of the alkaloid, and cigarettes with different specifications have slight difference.
6. And measuring the content of benzo [ a ] pyrene in the mainstream smoke of the cigarette. The method specifically comprises the following steps: the cigarette added with the compound is subjected to cigarette smoke analysis, and the determination of the content of benzo [ a ] pyrene in the mainstream smoke of the cigarette is carried out according to national standard GBT 27525-2011 of the people's republic of China. The reduction rate of water, nicotine and tar is calculated by comparing with a control sample, and after the compound is added into filter rods with different specifications, compared with a control cigarette, the reduction rate of benzo [ a ] pyrene in the main stream smoke of the cigarette is 3.39 to 14.11 percent. The results show that the compound and the functional filter stick thereof have obvious effect of intercepting benzo [ a ] pyrene in the mainstream smoke of cigarettes, the interception capability of the compound is obviously enhanced along with the increase of the concentration of the compound, and cigarettes with different specifications are slightly different.
7. Toxicology assessment and sensory evaluation. The results of three in vitro toxicology tests, namely toxicology evaluation, ames test and in vitro micronucleus test, on the cigarette smoke condensate added with the compound of the invention in the filter stick show that: statistical analysis is carried out on the cell inhibition rate of the total particulate matters in the smoke of the cigarettes of the tested sample and the control sample by adopting one-way ANOVA of SPSS software, and in three toxicological experiments, the total particulate matters in the smoke of the cigarettes added with the compound have no statistical difference (P is more than 0.05). 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 alkaloid has no toxicity in the aspect of obvious cell inhibition; meanwhile, after the alkaloid is added, the difference of the result of sensory evaluation is very small, the main difference is that the taste is slightly cool, the irritation and aftertaste are slightly improved, and no obvious negative effect is caused.
The invention is further illustrated by the following specific examples:
example 1
Taking fresh Lepidium meyenii Walp (Lepidium meyenii Walp) of LepidiumLepidium meyenii Walp.) is wrapped by medical gauze, then the wrapped roots are put into an autoclave and steamed for 2 hours by adding water at 110 ℃, and a sample is naturally aired to obtain a material a. Coarse pulverizing to 50 mesh, cold soaking with 70% acetone water solution, ultrasonic extracting for 3 times (60 min each time), vacuum filtering the extractive solution, mixing filtrates, vacuum concentrating with rotary evaporator to obtain acetone-free suspension, standing, filtering to remove precipitate, and concentrating to obtain 5kg extract b. And (3) adding 10kg of water into the extract b, extracting for 5 times by using chloroform with the same volume as the water phase, combining the extract phases, and concentrating under reduced pressure to obtain 250g of extract c. And (3) filling the extract c into a column by using MCI, adding 500g of a mixed solvent of 80% methanol and water into the extract c for dissolving, loading the mixture into the column after sample mixing, eluting by using a 90% methanol water solution for 4-5L, collecting eluent, and concentrating under reduced pressure to obtain 200g of extract d. Adding 450g of acetone into the extract d for dissolution, then adding 300g of 80-mesh silica gel for sample stirring, loading the mixture into a column after sample stirring, loading the mixture into a 100-mesh silica gel 2000g of a column, performing gradient elution by using a chloroform-acetone mixed organic solvent with the volume ratio of 10Five fractions, of which the fourth fraction, fraction 2. And taking 50% methanol as a mobile phase, the flow rate of the mixture e is 12mL/min, taking a Zorbax PrepHT GF (5 mu m,21.2 multiplied by 250 mm) reverse phase preparation column as a stationary phase, the detection wavelength of an ultraviolet detector is 203nm and 306nm, the sample size of each sample is 100 mu L, collecting chromatographic peaks in the range of 10 to 20min, accumulating for multiple times by the same steps, and evaporating to dryness by using a rotary evaporator to obtain 1200mg of the crude 4-acetoxy maca thiourea D product. Separating and purifying the 4-acetoxy maca thiourea D crude product by semi-preparative high performance liquid chromatography, taking 85% methanol aqueous solution as a mobile phase, the flow rate is 3mL/min, and using Zorbax SB-C 18 And (10 mu m,9.4 multiplied by 250 mm) reverse phase semi-preparative column is a stationary phase, the detection wavelength of the DAD detector is 203nm, 220nm, 254nm, 265nm and 306nm, the sample amount of each time is 35 mu L, chromatographic peaks of 12.5min are collected, after the same steps are accumulated for multiple times, a rotary evaporator is used for evaporating to dryness, and the pure 4-acetoxy maca thiourea D product 850mg with the purity of 95% is obtained.
Example 2
Taking the 4-acetoxy maca thiourea D prepared in the example 1 as a colorless oily compound, identifying the chemical structure of the compound by nuclear magnetic resonance and combining identification technologies such as ultraviolet spectrum, infrared spectrum, convention and high-resolution mass spectrum, and the specific data are as follows:
(1) Data of ultraviolet spectrum (solvent is methanol):λ max (log ε): 210 (2.11), 255 (3.05) and 278 (3.23).
(2) Infrared spectroscopy (potassium bromide pellet) data: v is max ：3430，2944，2850，1732，1428，1333，1120 cm -1 。
(3) Hresms data: the excimer ion peak of the compound of the invention is M/z 327 [ M + Na ]] + (ii) a HRESIMS (positive ion mode) M/z 327.0768 [ M + Na ]] + (Calcd. 327.0774, C 15 H 16 N 2 NaO 3 S + )。
（4） 1 H NMR（CDCl 3 400 MHz) and 13 C NMR（CDCl 3 100 MHz) data: 1 H NMR (CDCl 3 ，100MHz)：δ H 7.26−7.45 (m，5H，H-3a−7a)，4.94−5.06, (d，2H，J = 12.0，H 2 -1a)，3.94−4.04 (m，2H，H 2 -7)，2.32−2.36 (m，2H，H 2 -6)，1.75−2.27 (m，2H，H 2 -5)，2.09 (s，3H，H 3 -2a')； 13 C NMR（CDCl 3 ，100MHz）：δ C 186.8 (s，C-1)，169.1 (s，C-3)，93.6 (s，C-4)，32.0 (t，C-5)，24.7 (t，C-6)，49.0 (t，C-7)，45.7 (t，C-1a)，135.3 (s，C-2a)，128.7 (d，C-3a)，128.4 (d，C-4a)，127.7 (d，C-5a)，128.4 (d，C-6a)，128.7 (d，C-7a)，168.8 (s，C-1a')，20.6 (q，C-2a')。
the alkaloid structure analysis process of the invention comprises the following steps: the high resolution mass spectrum of the 4-acetoxy maca thiourea D gives an excimer ion peak of M/z 327.0768 [ M + Na ]] + (Calcd. 327.0774, C 15 H 16 N 2 NaO 3 S + ) In combination with 1 The H and DEPT NMR spectra (FIGS. 1 and 2) give 4-acetoxy-maca thiourea D of formula C 15 H 16 N 2 O 3 S, the unsaturation degree is 9. DEPT NMR spectra (FIG. 2) and 1 the H NMR spectrum (fig. 1) shows 15 carbon signals and 16 hydrogen signals. These signals typically include a benzyl group consisting of a methylene group and an acetyl groupδ H 4.94−5.06, (d，2H，J = 12.0，H 2 -1 a) and a monosubstituted phenyl ringδ H 7.26-7.45 (m, 5H, H-3a-7 a), and the signal of acetyl isδ H 2.09 (s，3H，H 3 -2 a'); accordingly, they occupy a signal of 9 carbon atoms in the carbon spectrumδ C 45.7 (t, C-1 a), 135.3 (s, C-2 a), 128.7 (d, C-3 a), 128.4 (d, C-4 a), 127.7 (d, C-5 a), 128.4 (d, C-6 a) and 128.7 (d, C-7 a) are benzyl signals,δ C 168.8 (s, C-1a ') and 20.6 (q, C-2 a') are acetyl signals. The other 6 carbon atoms being part of the backbone, including the 3 typical methylene signalsδ C 32.0 (t, C-5), 24.7 (t, C-6) and 49.0 (t, C-7), 1 Quaternary carbon Signal with two heteroatoms attachedδ C 93.6 (s，C-4), 2 ester group signalsδ C δ C 186.8 (s, C-1) and 169.1 (s, C-3) with corresponding hydrogen spectra data:δ H 3.94−4.04 (m，2H，H 2 -7)，2.32−2.36 (m，2H，H 2 -6) and 1.75-2.27 (m, 2H 2 -5). In combination with two-dimensional NMR, it was determined that the 6 carbon atoms and heteroatoms form two heterocycles, where C-1, N-2, C-3, C-4 and N-8 form a 1-thiocarbonyl-3-carbonyl-tetrahydroimidazolidine ring fragment and C-4, C-5, C-6, C-7 and N-8 form a tetrahydropyrrole fragment, these two fragments forming a rare 1-thiocarbonyl-3-carbonyl-tetrahydroimidazotetrahydropyrrole core, where C-1 thiocarbonylation and C-3 oxycarbonylation. This is very similar to the data reported in the literature for maca thiourea D (macadamia chemontoin D), except that 4-acetoxy maca thiourea D has one more acetyl signal, and only 4-OH in combination with maca thiourea D can link acetyl, suggesting that the acetoxy group of this compound is linked to C-4. These projections are by key H in two-dimensional HMBC spectra 3 2a 'is related to C-1a', H 2 -5 is related to C-3 and C-4, H 2 -7 is related to C-1, H 2 The correlation of-1 a with C-1 and C-3, etc. was confirmed. Therefore, the plane structure of the alkaloid is determined, the structure is not reported in the literature, and the alkaloid is a new natural product, namely 4-acetoxy maca thiourea D in Chinese, 4-OAc macaohydantoin D in English, and the systematic name is as follows: 2-benzyl-1-oxo-3-thioxotetrahydro-1H-pyrrolo [1,2-c ]]High performance liquid chromatography type chromatographic analysis of imidazole-7 a (5H) -yl acetate equipped with chiral column shows that the compound is a pair of racemates, and the structural formula is as follows:
example 3
Example 1 preparation of 4-acetoxy maca thiourea D, preparation of three solutions of 20, 30 and 40 mg/mL with glyceryl triacetate, dipping the filter in the above solutions until all the solutions are absorbed by the acetate filter, and filling the filter in cigarette tipping paper as it is after natural drying in the shade to obtainThe filter stick with the function of reducing the release amount of harmful substances comprises three different specifications of a fine cigarette, a medium cigarette and a conventional cigarette. The cigarette smoke analysis is carried out on the cigarette added with the compound of the invention in the filter stick, and the cigarette smoke analysis refers to Chinese tobacco industry standards (YC/T2, YC/T8 and YC/T30) and national standards of the people's republic of China (GBT 27525-2011) to respectively carry out the analysis on nicotine, tar and benzo [ a ]]The content of pyrene was measured. The content of the total particulate matter (T.P.M.) in the smoke is determined according to the following calculation formula: total particulate matter (t.p.m.) content = [ (M) 2 - M 1 ) / N]X 1000, wherein M 1 The weight (unit: gram) of the Cambridge filter sheet smoke catcher before smoking, M 2 The weight of the Cambridge filter disc smoke catcher after smoking (unit: g), N is the number of cigarettes smoked (unit: one), and the weight of the total particulate matter (T.P.M.) is the weight of the total particulate matter (unit mg/one). On the basis of this, cigarettes to which the compounds of the present invention were not added were used as a control, and the reduction rate of each harmful component was calculated by comparison with the control sample, and the results are shown in tables-1 to-3. The results show that after the compounds (20 mg, 30 mg and 40 mg) of the invention are added into filter sticks (conventional, medium and fine cigarettes) with three different specifications, compared with a control cigarette, the total particulate matter (T.P.M.), nicotine, tar and benzo [ a ] in the mainstream smoke of the cigarette]The content of pyrene is obviously reduced. After the compound of the invention with three concentrations is added, the reduction rate of total particulate matters (T.P.M.) in the smoke of cigarettes with three different specifications is 4.36 to 13.75 percent, the reduction rate of nicotine is 3.17 to 16.67 percent, the reduction rate of tar is 4.53 to 16.86 percent, and benzo [ a ] is]The reduction rate of pyrene was 3.39 to 14.11%. The above results illustrate that: the alkaloid and the functional filter stick thereof have the obvious effect of intercepting or reducing four types of harmful substances in main stream smoke of cigarettes, including total particulate matters (T.P.M.), nicotine, tar and benzo [ a ]]Pyrene, and the retention capacity of the pyrene is obviously enhanced along with the increase of the concentration of the alkaloid added in the invention, and cigarettes of different specifications are slightly different.
TABLE-1 determination of harmful substance content in conventional cigarette by adding the inventive compound
TABLE-2 determination of harmful substance content in cigarette
TABLE-3 determination of harmful substance content in fine cigarette by adding the compound of the invention
Example 4
The preparation method comprises the steps of preparing 4-acetoxyl maca thiourea D in the embodiment 1, preparing 20-40 mg/mL solution by using glyceryl triacetate, immersing a filter tip into the solution until all the solution is absorbed by an acetate fiber filter tip, naturally drying the filter tip in the shade, and then filling the filter tip into cigarette tipping paper in an original shape to obtain the functional filter tip capable of reducing the release amount of harmful substances, wherein the filter tip comprises three different specifications of a thin cigarette, a medium cigarette and a conventional cigarette. And carrying out toxicological evaluation and sensory evaluation research on the filter sticks with nine different specifications and different additive amounts. Three in vitro toxicology tests are carried out on the cigarette smoke condensate with the compound added in the filter stick, including toxicology evaluation, ames test and in vitro micronucleus test, and the results 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. On this basis, 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 cigarette added with the alkaloid has no toxicity in the aspect of obvious cell inhibition; meanwhile, after the alkaloid is added, the difference of the result of sensory evaluation is very small, the main difference is that the taste is slightly cool, the irritation and aftertaste are slightly improved, and no obvious negative effect is caused.
Claims (8)
1. A new natural sulfur-containing alkaloid is characterized in that the alkaloid is a new alkaloid with a tetrahydroimidazo tetrahydropyrrole skeleton, and is prepared by taking dried or fresh roots of Lepidium meyenii Walp of Lepidium of Cruciferae as a raw material through the routes of cooking, processing, extraction, separation, purification, structural identification and the like; the molecular formula of the compound is C 15 H 16 N 2 O 3 The molecular weight of S is 304, the Chinese name is 4-acetoxy maca thiourea D, the English name is 4-OAc macathiohydantoin D, and the chemical structural formula is as follows:
2. the preparation method of the novel natural sulfur-containing alkaloid according to claim 1, which is characterized in that the novel natural sulfur-containing alkaloid is prepared by taking roots of Lepidium meyenii Walp of Lepidium of Cruciferae as a raw material through the steps of sample cooking and processing, solvent extraction, MCI (methanol-glucose oxidase) decoloration, normal phase column chromatography segmentation, preparation and semi-preparative high performance liquid chromatography enrichment, and specifically comprises the following steps:
(1) Steaming and boiling and processing: lepidium meyenii walp of Lepidium (Lepidium meyenii Walp.)Lepidium meyenii) Drying or steaming fresh roots at a high temperature of 100-150 ℃ by a wet method, naturally airing a sample, crushing, and sieving with a sieve of 10-100 meshes to obtain a material a;
(2) Solvent extraction: carrying out cold soaking on the material a by using an organic solvent with the weight being 2-10 times of that of the material a, carrying out ultrasonic extraction for 2-5 times, carrying out 1-3 h each time, filtering to obtain a filtrate, and carrying out reduced pressure distillation and concentration to obtain a maca extract b;
(3) And (3) extraction: adding deionized water with the weight 2-4 times of that of the extract b into the extract b, stirring to obtain a suspension, extracting for 2-5 times by using an organic solvent which has the same volume with the suspension and is immiscible with water, combining extraction phases of the organic solvent, and concentrating under reduced pressure to obtain an extract c;
(4) MCI decoloration: dissolving the extract c by using a methanol-water mixed solvent with the weight 2 to 5 times that of the extract c, purifying by using MCI column chromatography, eluting by using a methanol water solution with the volume percentage concentration of 20-30% to remove impurities, eluting by using a methanol water solution with the volume percentage concentration of 80-90% and mixing the eluates, and concentrating under reduced pressure to obtain an extract d;
(5) Positive phase column chromatography segmentation: dissolving the extract D with an organic solvent which is 2-3 times of the weight of the extract D, stirring the mixture with silica gel which is 1-3 times of the weight of the extract D and is 80-100 meshes or 200-300 meshes, purifying the mixture by using normal phase column chromatography, filling the mixture into a column with silica gel which is 5-10 times of the weight of the extract D and is 100-200 meshes, performing gradient elution by using a mixed organic solvent with a volume ratio of 1;
(6) Preparation and semi-preparation high performance liquid chromatography enrichment: and (3) eluting the mixture e by using a methanol solution with the volume percentage concentration of 40-90% or an acetonitrile solution with the volume percentage concentration of 30-80% through preparing a high performance liquid chromatography to obtain a crude product of the 4-acetoxy maca thiourea D, and separating and purifying the crude product by using a semi-preparative high performance liquid chromatography to obtain a pure product of the 4-acetoxy maca thiourea D.
3. The preparation method according to claim 2, wherein the organic solvent in step (2) is 50-100% acetone, 80-100% ethanol, 80-100% ethyl acetate or 80-100% methanol; the organic solvent in the step (3) is chloroform, dichloromethane, ethyl acetate, n-butanol, isopropanol, cyclohexane or petroleum ether.
4. The preparation method according to claim 2, wherein the mixed organic solvent in step (5) is chloroform-acetone, and the volume ratio is 10.
5. The method of claim 2, wherein the preparative HPLC enrichment in step (6) is a 40-90% by volume aqueous solution of methanol or 30-80% by volume acetonitrileThe method comprises the following steps of (1) taking an aqueous solution as a mobile phase, taking a Zorbax PrepHT GF reversed phase preparation column with the flow rate of 10-14mL/min and the size of 21.2 x 250mm as a stationary phase, taking the detection wavelength of an ultraviolet detector as 202nm and 254nm, taking the sample size of 10-1000 mu L each time, collecting chromatographic peaks in the range of 10-25min, accumulating for multiple times by the same steps, and evaporating to dryness by using a rotary evaporator to obtain a 4-acetoxyl thiosemicarbazide D crude product; the semi-preparative high performance liquid chromatography purification is to use methanol aqueous solution with volume percentage concentration of 40-90% or acetonitrile aqueous solution with volume percentage concentration of 30-80% as a mobile phase, the flow rate is 1-4 mL/min, and Zorbax SB-C with the flow rate of 10 mu m and the flow rate of 9.4 x 250mm is used as a mobile phase 18 And (3) taking the reversed-phase semi-preparative column as a stationary phase, detecting wavelengths of the DAD detector as 203nm, 220nm, 254nm, 265nm and 306nm, sampling volume of 1-100 mu L each time, collecting chromatographic peaks within the range of 10-25min, accumulating for multiple times by the same steps, and evaporating by using a rotary evaporator to dryness to obtain the pure 4-acetoxy maca thiourea D.
6. The application of the novel natural sulfur-containing alkaloid 4-acetoxy maca thiourea D according to any one of claims 1 to 5 in removing harmful substances from cigarettes.
7. The application of the invention as claimed in claim 6, wherein the 4-acetoxy maca thiourea D prepared by the invention is added on acetate fiber tows to prepare the functional filter stick of the cigarette.
8. The application of claim 7, wherein the specific method for preparing the cigarette functional filter stick is as follows: weighing 20-40 mg of the alkaloid 4-acetoxy maca thiourea D disclosed by the invention, fully dissolving the alkaloid 4-acetoxy maca thiourea D into 1 mL of glyceryl triacetate, preparing the 4-acetoxy maca thiourea D into a solution of 20-40 mg/mL, immersing the filter tip into the solution until all the solution is absorbed by an acetate fiber filter tip, naturally drying the filter tip in the shade, and then filling the filter tip into cigarette tipping paper in an original shape to obtain the functional filter stick capable of reducing the release amount of harmful substances, wherein the filter stick comprises three different specifications of a thin cigarette, a medium cigarette and a conventional cigarette.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107556314A (en) * | 2017-09-30 | 2018-01-09 | 云南民族大学 | A kind of sulfur-bearing alkaloid and preparation method and application |
| CN107629060A (en) * | 2017-09-30 | 2018-01-26 | 云南民族大学 | A kind of sulfur-bearing alkaloid compound and preparation method and application |
| CN111134359A (en) * | 2019-12-25 | 2020-05-12 | 云南中烟工业有限责任公司 | Application of natural nitrogen-sulfur heterocyclic compound in reducing heavy metals in cigarette smoke |
| CN114621224A (en) * | 2022-03-23 | 2022-06-14 | 云南省农业科学院质量标准与检测技术研究所 | Maca alkaloid and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107556314A (en) * | 2017-09-30 | 2018-01-09 | 云南民族大学 | A kind of sulfur-bearing alkaloid and preparation method and application |
| CN107629060A (en) * | 2017-09-30 | 2018-01-26 | 云南民族大学 | A kind of sulfur-bearing alkaloid compound and preparation method and application |
| CN111134359A (en) * | 2019-12-25 | 2020-05-12 | 云南中烟工业有限责任公司 | Application of natural nitrogen-sulfur heterocyclic compound in reducing heavy metals in cigarette smoke |
| CN114621224A (en) * | 2022-03-23 | 2022-06-14 | 云南省农业科学院质量标准与检测技术研究所 | Maca alkaloid and preparation method and application thereof |
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