CN107556314A - A kind of sulfur-bearing alkaloid and preparation method and application - Google Patents
A kind of sulfur-bearing alkaloid and preparation method and application Download PDFInfo
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- CN107556314A CN107556314A CN201710918498.3A CN201710918498A CN107556314A CN 107556314 A CN107556314 A CN 107556314A CN 201710918498 A CN201710918498 A CN 201710918498A CN 107556314 A CN107556314 A CN 107556314A
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Abstract
The invention discloses a kind of sulfur-bearing alkaloid and preparation method and application, described sulfur-bearing alkaloid is the alkaloid with 1,3 diazabicyclo [3.3.1] nonane skeletons, is with Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）Root be prepared for base stock, its molecular formula is C14H16N2O2S, Chinese entitled (±) the maca thiocarbamide C of the compound, entitled (±) the macahydantoin C of English, its chemical structural formula are：.(±) maca thiocarbamide C shows weak inhibitory action to one plant of people source tumor cell line.Alkaloid of the present invention derives from natural medicine-food dual purpose plant, and its structure is novel, and has potential antitumor activity, can have preferable application prospect as the lead compound of development of functional food.
Description
Technical field
The invention belongs to vegetable active chemical composition extractive technique field, and in particular to a kind of sulfur-bearing alkaloid and its preparation
Method and application.
Background technology
(its Latin is entitled for macaLepidium meyenii Walp) (Brassicaceae), it is only to be under the jurisdiction of Cruciferae
1 ~ 2 year raw herbaceous plant of row Lepidium, originates in South America andes region（Height above sea level is more than 3500 meters）, for local conventional characteristic
Resource plant and edible materials, there is the good reputation of " Peru's ginseng " and " South America ginseng ", generally believe its have it is antifatigue and
Improve the effect of both sexes fecundity.Due to maca rich in nutrition content, rich in protein, carbohydrate, aliphatic acid, fiber
Element, vitamin and mineral, thus edible safety food is recommended as by the World Food Programme of the United Nations, China is also in 2011
Maca is included in new resource for food catalogue by May in year.Modern pharmacology activity research shows, maca has and a variety of had to health
The activity of benefit, such as anti-oxidant, strengthen immunity, anticancer, the memory that improves, regulation and control hormone discharge and reduced blood glucose and blood
The effect of fat[3-10], further study showed that, some bioactivity and its Liposoluble of maca have direct relation, such as
Maca ene, macamide, imidazole alkaloid, glucosinolate, sterol and steroids.The researcher of early stage has found that it contains from agate card
There is the natural activity composition that two classes are special：Macamides（macamides）With agate card alkene（macaenes）.Wu H et al. are with conjunction
Into method to the anti-cellulite hydroamidase of part macamide Alkaloid（FAAH）Activity has carried out Primary Study[11],
In addition to characteristic component macamides and Ma Ka alkene, researcher has also obtained a series of novel alkaloids from maca
Compound, and complete the fully synthetic research of multiple molecules.Isolated a kind of novelty in the maca that Zheng et al. produces from Peru
Imidazole alkaloid lepidilines A and B, the structure of such compound determine by X- single crystal diffraction methods, such compound table
Reveal certain antitumor activity, in view of such compound has novel structure and potential pharmacological activity, Wolkenberg
S. E. et al. is catalyzed, through microwave reaction with 2,3- dicarbapentaborane butane and acetaldehyde as reaction initiation material with acetic acid and Ammoniom-Acetate
Obtain the 2 of high yield after 5 minutes, 4,5- tri-substituted imidazoles, by further with benzyl chloride microwave reaction 5 minutes, completing
Lepidiline B's is fully synthetic, and gross production rate is up to 43%.The maca yield in Yunnan accounts for more than the 90% of China at present, accounts for the world
70%, Chinese yunnan Lijing and Shangri-la area are distributed in mostly（Height above sea level is more than 3500 to 2800 meters）, the plant is described as " secret
Shandong ginseng ", and be widely cultivated, annual production is more than 30,000 tons, at the same time, in areas such as Huize, yunnan, Zhaotong, Yuxi, maca
Also it is widely cultivated.However, same characteristic resources plant has larger difference in the different places of production, its composition, China introduces a fine variety
Maca its pharmacology activity research rest essentially within activity rating stage of extract, do not carry out the material base and medicine of system
Composition Study is imitated, its security and validity all urgently illustrate.This method is the correlative study carried out for maca blank field,
An alkaloid compound novel and with potential using value is therefrom found that first.
The content of the invention
The first object of the present invention is to provide a kind of sulfur-bearing alkaloid；Second purpose is to provide described sulfur-bearing biology
The preparation method of alkali；3rd purpose is the application for providing described sulfur-bearing alkaloid.
The first object of the present invention is achieved in that described sulfur-bearing alkaloid is that have 1,3-diazabicyclo
The alkaloid of [3.3.1] nonane skeletons, it is with Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）Root be prepared for base stock, its molecular formula is C14H16N2O2S, the compound
Chinese entitled (±)-maca thiocarbamide C, entitled (±)-macahydantoin C of English, its chemical structural formula are：
。
The second object of the present invention is achieved in that with Cruciferae separate row Vegetable spp characteristic resources plant Maca（Draw
Fourth name：Lepidium meyenii Walp.）Root be base stock, it is extracted, extraction, MCI depigmentations processing, normal phase column color
Spectrometry is drawn section, chromatograph enrichment, chromatogram purification and efficient liquid phase chirality semi-preparative column splitting step and is prepared, and specifically includes：
A, extract：By Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）
Root crush after cross 10 ~ 100 mesh sieves and obtain material a, add the organic solvent of 3 ~ 8 times of material a weight, cold soaking and ultrasonic extraction 2 ~
5 times, every time 1 ~ 3h, extract solution obtains filtrate b through filtering, and distillation and concentration obtains maca medicinal extract c；
B, extract；The water of 1 ~ 3 times of medicinal extract c weight is added into medicinal extract c, stirring obtains suspension, with the isometric and and water with water
Unmixing organic solvent extracts 2 ~ 5 times, merges the extraction phase of organic solvent, is concentrated under reduced pressure to give medicinal extract d；
C, MCI depigmentations are handled：The medicinal extract d Methanol+Waters of 2 ~ 5 times of medicinal extract d weight are dissolved, use MCI post layers
Analysis purifying, eluted with the methanol aqueous solution that concentration expressed in percentage by volume is 80% ~ 90%, merge eluent, be concentrated under reduced pressure to give medicinal extract e；
D, positive column chromatography draws section：Methanol or acetone solution by medicinal extract e with 2 ~ 3 times of medicinal extract e weight, with medicinal extract e weight 1 ~ 3
Times 80 ~ 100 mesh or 200 ~ 300 mesh silica gel mixed sample, purified using positive column chromatography, i.e., with medicinal extract e weight 5 ~
100 ~ 200 mesh silica gel dress post of 10 times of amounts, is 1 with volume ratio:0~0:1 mixed organic solvents gradient elution, collects and concentrates,
Detected through silica gel thin-layer chromatography, merge colour developing and separation identical point, contained (±)-maca thiocarbamide C mixture
f；
E, preparative high-performance liquid chromatographic is enriched with：Using preparative high-performance liquid chromatographic, with the methanol solution of concentration expressed in percentage by volume 40% ~ 90%
Or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% affords (±)-maca thiocarbamide C crude products；
F, half preparative high-performance liquid chromatographic purifies：(±)-maca thiocarbamide C crude products are isolated and purified through half preparative high-performance liquid chromatographic
Obtain (±)-maca thiocarbamide C sterlings；
G, efficient liquid phase chirality semi-preparative column is split：(±)-maca thiocarbamide C sterlings are torn open by efficient liquid phase chirality semi-preparative column
Get (+)-maca thiocarbamide C and (-)-maca thiocarbamide C.
Alkaloid of the present invention has novel 1,3-diazabicyclo [3.3.1] nonane class formation fragments,
This is the new framework types alkaloid found first from nature, by nuclear magnetic resonance, mass spectrum, ultraviolet chromatogram, infrared chromatography,
Optical rotatory dispersion, circular dichroism spectra combination gas chromatographic detection（ECD）The methods of calculating, characterizes, and determines that its structure is 3-benzyl-
5-hydroxy-2-thioxo-1,3-diazabicyclo[3.3.1]nonan-4-
One, its concrete structure are：
On this basis, using (±)-maca thiocarbamide C as base stock, to five plants of tumor cell lines（Early young HL-60 cell lines, lung
Gland cancer A549 cell lines, people marrow neuroblastoma SHSY5Y cell lines, human prostata cancer PC3 cell lines and human breast carcinoma
MCF7 cell lines）Anti tumor activity in vitro evaluation is carried out, as a result shows that (±)-maca thiocarbamide C has to early young HL-60 cell lines
Certain cytotoxic activity, IC50It is worth for 42.11 μM.The compound that result above indicates the present invention is preparing cancer therapy drug, protected
Potential using value in strong product, functional food and food addition.Compound structure of the present invention is novel, possesses certain
Bioactivity, its natural origin is to be selected in the medical and edible dual purpose plant of China's new resource for food catalogue, therefore, can be used as anti-
The lead compound of cancer drug, there is certain researching value and application prospect.
Brief description of the drawings
Fig. 1 is compound (±)-maca thiocarbamide C proton nmr spectra（1H NMR）；
Fig. 2 is compound (±)-maca thiocarbamide C carbon-13 nmr spectra（DEPT）.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but the present invention is not subject in any way
Limitation, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Sulfur-bearing alkaloid of the present invention is the biology for having 1,3-diazabicyclo [3.3.1] nonane skeletons
Alkali, it is with Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）Root be
Base stock is prepared, and its molecular formula is C14H16N2O2S, Chinese entitled (±)-maca thiocarbamide C of the compound, English name
For (±)-macahydantoin C, its chemical structural formula is：
。
The preparation method of sulfur-bearing alkaloid of the present invention, it is with Cruciferae separate row Vegetable spp characteristic resources plant Maca
（Latin name：Lepidium meyenii Walp.）Root be base stock, it is extracted, extraction, MCI depigmentations processing, positive
Column chromatography is drawn section, chromatograph enrichment, chromatogram purification and efficient liquid phase chirality semi-preparative column splitting step and is prepared, specific bag
Include：
A, extract：By Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）
Root crush after cross 10 ~ 100 mesh sieves, obtain material a, add 3 ~ 8 times of material a weight organic solvent cold soaking and ultrasonic extraction 2 ~
5 times, every time 1 ~ 3h, extract solution obtains filtrate b through filtering, and distillation and concentration obtains maca medicinal extract c；
B, extract；The water of 1 ~ 3 times of medicinal extract c weight is added into medicinal extract c, stirring, obtains suspension, with the isometric and and water with water
Unmixing organic solvent extracts 2 ~ 5 times, merges the extraction phase of organic solvent, is concentrated under reduced pressure to give medicinal extract d；
C, MCI depigmentations are handled：The medicinal extract d Methanol+Waters of 2 ~ 5 times of medicinal extract d weight are dissolved, use MCI post layers
Analysis purifying, eluted with the methanol aqueous solution that concentration expressed in percentage by volume is 80% ~ 90%, merge eluent, be concentrated under reduced pressure to give medicinal extract e；
D, positive column chromatography draws section：Methanol or acetone solution by medicinal extract e with 2 ~ 3 times of medicinal extract e weight, with medicinal extract e weight 1 ~ 3
Times 80 ~ 100 mesh or 200 ~ 300 mesh silica gel mixed sample, purified using positive column chromatography, i.e., with medicinal extract e weight 5 ~
100 ~ 200 mesh silica gel dress post of 10 times of amounts, is 1 with volume ratio:0~0:1 mixed organic solvents gradient elution, collects and concentrates,
Detected through silica gel thin-layer chromatography, merge colour developing and separation identical point, contained (±)-maca thiocarbamide C mixture
f；
E, preparative high-performance liquid chromatographic is enriched with：Using preparative high-performance liquid chromatographic, with the methanol solution of concentration expressed in percentage by volume 40% ~ 90%
Or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% affords (±)-maca thiocarbamide C crude products；
F, half preparative high-performance liquid chromatographic purifies：(±)-maca thiocarbamide C crude products are isolated and purified through half preparative high-performance liquid chromatographic
Obtain (±)-maca thiocarbamide C sterlings；
G, efficient liquid phase chirality semi-preparative column is split：(±)-maca thiocarbamide C sterlings are torn open by efficient liquid phase chirality semi-preparative column
Get (+)-maca thiocarbamide C and (-)-maca thiocarbamide C.
The acetone, 80% ~ 100% ethanol, 80% ~ 100% acetic acid second that organic solvent described in step A is 50% ~ 100%
Ester or 80% ~ 100% methanol.
Organic solvent described in step B is chloroform, dichloromethane, ethyl acetate, n-butanol, isopropanol, hexamethylene or
Petroleum ether.
The concentration expressed in percentage by volume of Methanol+Water described in step C is 80%.
Mixed organic solvents described in D steps are chloroform-acetone.
The volume proportion of chloroform and acetone is 10 in described chloroform-acetone:1、9:1、8:2、7:3、6:4 and 5:5.
Preparative high-performance liquid chromatographic enrichment described in E steps, be with the methanol aqueous solution of concentration expressed in percentage by volume 40% ~ 90% or
The acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% is mobile phase, and flow velocity is 10 ~ 14mL/min, with Zorbax PrepHT GF
（5 μm, 21.2 × 250mm）Reverse phase preparative column is stationary phase, and the Detection wavelength of UV-detector is 203nm and 306nm, is entered every time
Sample amount is 10 ~ 1000 μ L, collects the chromatographic peak in the range of 10 ~ 25min, after repeatedly being added up with same steps, uses Rotary Evaporators
It is evaporated, produces described (±)-maca thiocarbamide C crude products.
Half preparative high-performance liquid chromatographic purifying described in F-step is with the methanol aqueous solution of concentration expressed in percentage by volume 40% ~ 90%
Or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% is mobile phase, flow velocity is 1 ~ 4mL/min, with Zorbax SB-C18（10μ
M, 9.4 × 250mm）Reverse phase semi-prep column is stationary phase, the Detection wavelengths of DAD detectors is 203nm, 220nm, 254nm,
265nm and 306nm, each sample size are 1 ~ 100 μ L, collect the chromatographic peak in the range of 10 ~ 25min, are repeatedly tired out with same steps
After adding, it is evaporated with Rotary Evaporators, produces described (±)-maca thiocarbamide C sterlings.
It is water-soluble with the methanol of concentration expressed in percentage by volume 40% ~ 90% that efficient liquid phase chirality semi-preparative column described in G steps, which is split,
Liquid or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% are mobile phase, and flow velocity is 1 ~ 3 mL/min, with Welch Ultimate
Cellu-D (5 μm, 4.6mm × 25cm) reverse phase semi-prep column is stationary phase, the Detection wavelengths of DAD detectors is 203nm,
220nm, 254nm, 265nm and 306nm, each sample size are 1 ~ 100 μ L, the chromatographic peak in the range of 10 ~ 20min are collected, with phase
After repeatedly being added up with step, it is evaporated with Rotary Evaporators, produces optically pure (+)-maca thiocarbamide C and (-)-maca thiocarbamide C is pure
Product.
The application of sulfur-bearing alkaloid of the present invention is that the sulfur-bearing alkaloid is preparing cancer therapy drug, health products and work(
Application in energy food.
Alkaloid of the present invention be with Cruciferae separate row Vegetable spp characteristic resources plant Maca (Lepidium meyenii Walp. rhizome) is base stock, extracted to isolate and purify and Structural Identification means, it was demonstrated that its chemical structural formula
For：
Described alkaloid has novel 1,3-diazabicyclo [3.3.1] nonane basic frameworks, and this is first from certainly
A kind of novel alkaloid compound found in right boundary, is a pair of racemies, its molecular formula is C14H16N2O2S, Chinese name
For (±)-maca thiocarbamide C, entitled (±)-macahydantoin C of English.
The preparation method of described alkaloid, be with Cruciferae separate row Vegetable spp characteristic resources plant Maca (Lepidium meyenii Walp. root) is base stock, through organic solvent cold soaking extraction, organic solvent extraction, MCI depigmentations processing,
The extraction separation skill such as positive column chromatography, preparation and semi-preparative high performance liquid chromatography, the fractionation of efficient liquid phase chirality semi-preparative column
Art is prepared.Concretely comprise the following steps：
(A) organic solvent cold soaking extracts：By Cruciferae separate row Vegetable spp characteristic resources plant Maca (Lepidium meyenii
Walp. rhizome) is crushed to 10 ~ 100 mesh, with organic solvent cold soaking and ultrasonic extraction 2 ~ 5 times, 1 ~ 3 hour every time, gained
Extraction solvent is depressurized with bottle,suction and filtered, and merging filtrate, is evaporated under reduced pressure with Rotary Evaporators, concentrated extracting solution, obtains maca leaching
Cream (a).
(B) organic solvent extracts：The water that weight ratio is 1 ~ 3 times of amount is added into maca medicinal extract (a), is sufficiently stirred into outstanding
Turbid, gained solvent is put into extractor, with isometric with water and extracted 2 ~ 5 times with the unmixing organic solvent of water, merged
Organic solvent extraction phase, is concentrated under reduced pressure, and obtains medicinal extract (b).
(C) MCI depigmentations are handled：Medicinal extract (b) mixed solvent for the first alcohol and water that weight ratio is 2 ~ 5 times of amounts is dissolved,
Purified with MCI column chromatographies, with 80% ~ 90% methanol and water mixed solvent system elutions, merge eluent, be concentrated under reduced pressure, obtain
Medicinal extract (c).
(D) positive column chromatography draws section：By medicinal extract (c) methanol or acetone solution that weight ratio is 2 ~ 3 times of amounts, use
Medicinal extract weighs 1 ~ 3 times of 80 ~ 100 mesh or 200 ~ 300 mesh silica gel mixed samples, then column chromatography eluting with positive（Filling post silica gel is
100 ~ 200 mesh）, dosage is 5 ~ 10 times of amounts, volume ratios 1 of medicinal extract (c) weight:0~0:1 mixed organic solvents gradient elution, ladder
Degree eluent, collection simultaneously concentrate, and are detected through silica gel thin-layer chromatography, merge colour developing and separation identical point, obtain low-purity
Mixture containing (±)-maca thiocarbamide C.
(E) preparative high-performance liquid chromatographic is enriched with：With preparative high-performance liquid chromatographic, with the methanol of volume content 40% ~ 90%
The eluent that (or 30% ~ 80% acetonitrile) aqueous solution affords, is described (±)-maca thiocarbamide C crude products, and purity is
40%~60%.High performance liquid chromatography separation purifying described in the step is with 40% ~ 90% methanol (or 30% ~ 80% acetonitrile)
The aqueous solution is mobile phase, and flow velocity is 10 ~ 14mL/min, with Zorbax PrepHT GF（5 μm, 21.2 × 250mm）Anti-phase preparation
Post is stationary phase, and the Detection wavelength of UV-detector is 203nm and 306nm, and each sample size is 10 ~ 1000 μ L, collection 10 ~
Chromatographic peak in the range of 25min, after repeatedly being added up with same steps, it is evaporated with Rotary Evaporators, produces described (±)-maca
Thiocarbamide C crude products.
(F) half preparative high-performance liquid chromatographic purifies：(±)-maca thiocarbamide C crude products separate through half preparative high-performance liquid chromatographic
Purifying, produces described (±)-maca thiocarbamide C sterlings, and purity is 90% ~ 98%.High performance liquid chromatography separation described in the step
Purifying is that, using 40% ~ 90% methanol (or 30% ~ 80% acetonitrile) aqueous solution as mobile phase, flow velocity is 1 ~ 4mL/min, with
Zorbax SB-C18（10 μm, 9.4 × 250mm）Reverse phase semi-prep column is stationary phase, the Detection wavelengths of DAD detectors is 203nm,
220nm, 254nm, 265nm and 306nm, each sample size are 1 ~ 100 μ L, the chromatographic peak in the range of 10 ~ 25min are collected, with phase
After repeatedly being added up with step, it is evaporated with Rotary Evaporators, produces described (±)-maca thiocarbamide C sterlings.
(G) efficient liquid phase chirality semi-preparative column is split：(±)-maca thiocarbamide C sterlings are through efficient liquid phase chirality semi-preparative column
Split, can further obtain optically pure (+)-maca thiocarbamide C and (-)-maca thiocarbamide C.High-efficient liquid phase color described in the step
It is that flow velocity is 1 ~ 3mL/min using 40% ~ 90% methanol (or 30% ~ 80% acetonitrile) aqueous solution as mobile phase that spectrum, which isolates and purifies,
With Welch Ultimate Cellu-D (5 μm, 4.6mm × 25cm) reverse phase semi-prep column for stationary phase, the inspection of DAD detectors
It is 203nm, 220nm, 254nm, 265nm and 306nm to survey wavelength, and each sample size is 1 ~ 100 μ L, is collected in the range of 10 ~ 20min
Chromatographic peak, with same steps repeatedly add up after, be evaporated with Rotary Evaporators, produce optically pure (+)-maca thiocarbamide C and
(-)-maca thiocarbamide C sterlings.
Wherein, the organic solvent described in A and C experimental procedures be 50% ~ 100% acetone, 80% ~ 100% ethanol, 80% ~
100% ethyl acetate or 80% ~ 100% methanol.Organic solvent described in B and D experimental procedures is chloroform, dichloromethane, acetic acid
Ethyl ester, n-butanol, isopropanol, hexamethylene or petroleum ether equal solvent, its volume proportion are 10:1,9:1,8:2,7:3,6:4,1:
1,1:2 and 0:1 etc..
Application of the described alkaloid in cancer therapy drug health products and functional food, it is using (±)-maca thiocarbamide C as base
This raw material carries out anti tumor activity in vitro evaluation, the results showed that there is the alkaloid certain cell toxicant to live to HL-60 cell lines
Property, IC50It is worth for 42.11 μM.
Cruciferae separate row Vegetable spp characteristic resources plant Maca of the present invention (Lepidium meyenii
Walp.), do not limited by area and kind, can realize the present invention.
So that case is embodied, the present invention will be further described below：
Embodiment 1
Take natural air drying originate from Lijiang, yunnan separate row Vegetable spp plant Maca (Lepidium meyenii Walp. root)
50kg, coarse powder are broken to 50 mesh, and aqueous acetone solution cold soaking and ultrasonic extraction 3 times, each 60min with 70%, extract solution decompression are taken out
Filter, merging filtrate, the suspension of no acetone is concentrated under reduced pressure into Rotary Evaporators；Stand, filter out sediment, be condensed into 10kg leachings
Cream (a)；20kg water is added into medicinal extract (a), is extracted 5 times with the isometric chloroform of water, is merged extraction phase, be concentrated under reduced pressure, obtain
To 500g medicinal extract (b)；Medicinal extract (b) is filled into post with MCI, the mixed solvent of 1000g 80% first alcohol and water is added into medicinal extract (b)
Dissolving, mixes upper prop after sample, is eluted with 90% 8 ~ 10L of methanol aqueous solution, collects eluent, be concentrated under reduced pressure, obtain 400g medicinal extract
(c)；900g acetone solution is added into medicinal extract (c), 80 mesh silica gel 600g is then added and mixes sample, upper prop after sample is mixed, with 200 mesh
Silica gel 4000g fills post, is respectively 10 with volume ratio:1,9:1,8:2,7:3,6:4,5:5 chloroform-acetone mixed organic solvents ladder
Degree elution, gradient eluent, collection are simultaneously concentrated, detected through silica gel thin-layer chromatography, are merged colour developing and separation identical point, are obtained
To 6 parts, wherein, Part II（It is 9 by volume ratio:Obtained by the eluent of 1 chloroform-acetone mixed organic solvents）Obtain sample
Product 35g, positive column chromatography is repeated, be respectively 10 with volume ratio:1,8:1,5:1,2:1,0:1 petroleum ether-acetone mixing
Organic solvent gradient elution, gradient eluent, collection are simultaneously concentrated, detected through silica gel thin-layer chromatography, merge colour developing and separation
Identical point, obtain five parts, wherein Part IV, i.e., 2:1 part is about 24g, then using 50% methanol as mobile phase, stream
Speed is 12mL/min, with Zorbax PrepHT GF（5 μm, 21.2 × 250mm）Reverse phase preparative column is stationary phase, UV-detector
Detection wavelength be 203nm and 306nm, each sample size is 100 μ L, collect 10 ~ 20min in the range of chromatographic peak, with identical
After step repeatedly adds up, it is evaporated with Rotary Evaporators, produces described (±)-maca thiocarbamide C crude products 500mg.(±)-maca sulphur
Urea C crude products isolate and purify through half preparative high-performance liquid chromatographic again, using 85% methanol aqueous solution as mobile phase, flow velocity 3mL/
Min, with Zorbax SB-C18（10 μm, 9.4 × 250mm）Reverse phase semi-prep column is stationary phase, and the Detection wavelength of DAD detectors is
203nm, 220nm, 254nm, 265nm and 306nm, each sample size are 35 μ L, 12.5min chromatographic peak are collected, to be synchronised
It is rapid it is repeatedly cumulative after, be evaporated with Rotary Evaporators, produce described (±)-maca thiocarbamide C sterling 155mg, purity is 90% ~
98%.(±)-maca thiocarbamide C sterlings are split by efficient liquid phase chirality semi-preparative column again, using 55% acetonitrile solution as flowing
Phase, flow velocity 2.5mL/min, it is with Welch Ultimate Cellu-D (5 μm, 4.6mm × 25cm) reverse phase semi-prep column
Stationary phase, the Detection wavelength of DAD detectors is 203nm, 220nm, 254nm, 265nm and 306nm, and each sample size is 25 μ L,
Respectively collect 15.0min and 15.5min in the range of chromatographic peak, obtain optically pure (+)-maca thiocarbamide C (60mg) and (-)-
Maca thiocarbamide C (61mg).
Embodiment 2
Take natural air drying originate from Yunnan Yuxi separate row Vegetable spp plant Maca (Lepidium meyenii Walp. root)
Stem 5kg, coarse powder are broken to 20 mesh, and with 80% methanol ultrasonic extraction 5 times, each 80min, extract solution decompression filters, merging filtrate,
The 1/4 of filtrate volume is concentrated under reduced pressure into Rotary Evaporators；Stand, filter out sediment, concentrate, obtain 1kg medicinal extract (a)；To leaching
2kg water is added in cream (a), is extracted 5 times with the isometric ethyl acetate of water, merges extraction phase, be concentrated under reduced pressure, obtain 50g leachings
Cream (b)；Medicinal extract (b) is filled into post with MCI, the mixed solvent that 100g 80% first alcohol and water is added into medicinal extract (b) dissolves, then
Upper prop, eluted with 80% 3 ~ 5L of methanol-water, collect eluent, be concentrated under reduced pressure, obtain 40g medicinal extract (c)；Added into medicinal extract (c)
100g acetone, dissolving, then adds 80 mesh silica gel 60g and mixes sample, mix upper prop after sample, and post, Ran Houyong are filled with 200 mesh silica gel 400g
Volume ratio is respectively 10:1,9:1,8:2,7:3,6:4,5:5 chloroform-acetone mixed organic solvents gradient elution, gradient elution
Liquid, collection and concentration, are detected through silica gel thin-layer chromatography, are merged colour developing and separation identical point, are obtained 6 parts, wherein,
Part II（It is 9 by volume ratio:Obtained by the eluent of 1 chloroform-acetone mixed organic solvents）Sample 7g is obtained, repeats positive
Column chromatography, it is respectively 10 with volume ratio:1,8:1,5:1,2:1,0:1 petroleum ether-acetone mixed organic solvents gradient elution,
Gradient eluent, collection and concentration, are detected through silica gel thin-layer chromatography, are merged colour developing and separation identical point, are obtained five
Part, wherein Part IV, i.e., 2:1 part is about 2g, then using 40% methanol as mobile phase, flow velocity 12mL/min, with
Zorbax PrepHT GF（5 μm, 21.2 × 250mm）Reverse phase preparative column is stationary phase, and the Detection wavelength of UV-detector is
203nm and 306nm, each sample size are 80 μ L, collect the chromatographic peak in the range of 10 ~ 20min, are repeatedly added up with same steps
Afterwards, it is evaporated with Rotary Evaporators, produces described (±)-maca thiocarbamide C crude products 60mg；(±)-maca thiocarbamide C crude products are passed through again
Half preparative high-performance liquid chromatographic isolates and purifies, using 80% methanol aqueous solution as mobile phase, flow velocity 3mL/min, with Zorbax
SB-C18（10 μm, 9.4 × 250mm）Reverse phase semi-prep column is stationary phase, the Detection wavelengths of DAD detectors is 203nm, 220nm,
254nm, 265nm and 306nm, each sample size are 35 μ L, collect 18min chromatographic peak, after repeatedly being added up with same steps, are used
Rotary Evaporators are evaporated, and produce described (±)-maca thiocarbamide C sterling 30mg, and purity is 90% ~ 98%.(±)-maca thiocarbamide C
Sterling is split by efficient liquid phase chirality semi-preparative column again, using 50% acetonitrile solution as mobile phase, flow velocity 2.5mL/min,
With Welch Ultimate Cellu-D (5 μm, 4.6mm × 25cm) reverse phase semi-prep column for stationary phase, the detection of DAD detectors
Wavelength is 203nm, 220nm, 254nm, 265nm and 306nm, and each sample size is 25 μ L, respectively collect 18.0min and
19.0min chromatographic peak, obtain optically pure (+)-maca thiocarbamide C (10mg) and (-)-maca thiocarbamide C (9mg).
Embodiment 3
(±)-maca thiocarbamide C prepared by Example 1 and 2, is colorless oil compound, with nuclear magnetic resonance, with reference to ultraviolet light
The identification technologies such as spectrum, infrared spectrum, routine and high resolution mass spectrum identify its structure, and specific data are：
（1）Ultraviolet spectra（Solvent is methanol）λ max (logε）：203 (0.51), 265 (0.23), 306 (0.21) nm.
（2）Infrared spectrum（Pressing potassium bromide troche）ν max：3428,2934,1713,1379,1331,1178,1110 cm-1。
（3）HRESIMS shows the [M+CH of the compounds of this invention quasi-molecular ion peak m/z 3313OH+Na]+；HRESIMS
[the M+CH of (positive ion mode) m/z 331.10953OH+Na]+(calculated value 331.1087, C15H20N2O3SNa) ；1H and13C
H NMR spectroscopy（Fig. 1 and Fig. 2）It is C to provide its molecular formula14H16N2O2S。
（4）1H NMR（CDCl3, 400MHz）With13C NMR（CDCl3, 100MHz）Data：1H NMR (CDCl3,
100MHz)：δ H7.33 7.23 (m, 5H, H-3a 7a), 5.57 5.54 (d, J=5.2Hz, 2H, H2- 1a), 4.35 3.33
(m, 2H, H2- 7), 3.53 3.21 (m, 2H, H2- 9), 2.10 1.92 (m, 2H, H2- 5), 1.93 1.75 (m, 2H, H2-6)；13C
NMR（CDCl3, 100MHz）：δ C192.0 (s, C-1), 174.5 (s, C-3), 69.3 (s, C-4), 34.5 (t, C-5), 23.0
(t, C-6), 57.3 (t, C-7), 54.3 (t, C-9), 48.7 (t, C-1a), 136.6 (s, C-2a), 128.0 (d, C-3a),
128.6 (d, C-4a), 127.6 (d, C-5a), 128.65 (d, C-6a), 128.0 (d, C-7a).
Structure elucidation process：(±)-maca thiocarbamide C DEPT H NMR spectroscopies (Fig. 2) and1H H NMR spectroscopies (Fig. 1) show 14
Carbon signal and 16 hydrogen signals.There is a typical benzyl in these signals（benzyl）Substituent signal, be by an Asia
Methylδ H5.57 5.54 (d, J=5.2 Hz, 2H, H2- 1a) and a monosubstituted phenyl ringδ H7.33 7.23 (m, 5H,
H-3a 7a) form.The signal that other 7 carbon atoms belong on skeleton, include four typical methylene signalsδ C 34.5
(t, C-5), 23.0 (t, C-6), 57.3 (t, C-7), and 54.3 (t, C-9), signal of quaternary carbon containing hetero atom 69.3 (s,
C-4), an ester group signal 174.5 (s, C-3) and a thiocarbonyl group signal 192.0 (s, C-1), its corresponding hydrogen modal data
Respectively：4.35 3.33 (m, 2H, H2- 7), 3.53 3.21 (m, 2H, H2- 9), 2.10 1.92 (m, 2H, H2- 5), 1.93
1.75 (m, 2H, H2-6).With reference to two dimensional NMR, determine that this 7 carbon atoms form two heterocycles, wherein C- with hetero atom
1, N-2, C-3, C-4, C-9 and N-8 form a hexahydropyrimidine fragment, and C-4, C-5, C-6, C-7, C-9 and
N-8 then forms piperidine fragments, and the two fragments form rare 1,3-diazabicyclo [3.3.1] nonane
Parent nucleus, wherein, C-1 thiocarbonyl groups, C-3 carbonylations, C-4 hydroxylatings.The compound of two similar skeletons is only reported at present, most
The H that the order of connection of parent nucleus 1,3-diazabicyclo [3.3.1] nonane and benzyl substituent passes through key afterwards2-1a
It is related to C-1/C-3 HMBC to determine.Therefore, the planar structure of the alkaloid is determined, is a new natural products, in
Literary fame is (±)-maca thiocarbamide C, English entitled (±)-macahydantoin C, systematic naming method 3-benzyl-5-
hydroxy-2-thioxo-1,3-diazabicyclo[3.3.1]nonan-4-one.The compound is a pair of racemization isomers,
Its absolute configuration is by chiral resolution, and ECD is calculated and experiment CD value controls determine, wherein, (+)-maca thiocarbamide C is 4S, and
(-)-maca thiocarbamide C is 4R。
Embodiment 4
Maca alkaloid (±)-maca thiocarbamide C prepared by Example 1 or 2, is tied by the method in embodiment 3 respectively
Structure determines, and carries out anti tumor activity in vitro test to raceme (±)-maca thiocarbamide C of gained, test situation is as follows：
Five plants of cell lines are respectively prorubricyte (HL-60) strain, lung carcinoma cell (A549) strain, human neuroblastoma cells
(SHSY5Y) strain, prostate gland cancer cell (PC3) strain and breast cancer cell (MCF7) strain, by Chinese Academy of Sciences's Shanghai drug research
There is provided.
Above cell and various concentrations compound incubation 72 hours, the experiment of every plant of cell is repeated once, and use is real twice
The result tested carries out data processing, using the inhibition level of improvement mtt assay evaluation compound on intracellular propagation, calculates inhibiting rate,
IC is calculated using Logit methods according to inhibiting rate50, the anti tumor activity in vitro of comparative compound.
The proliferation inhibition rate of cell=(the OD values of blank control OD values-medicine feeding hole)/blank control OD value × 100%.
Method is improvement mtt assay, and specific method is：
Take the logarithm the suspension cell in growth period, cell concentration is adjusted to 4 × 104Mol/mL, add 96 well culture plates, 90 μ L/
Hole.Positive control is cis-platinum, uses physiological saline solution.Sample (No. 1 test solution ~ No. 5 examination of 10 μ L various concentrations is separately added into per hole
Liquid).Sample-adding group and control group are all provided with 4 multiple holes, and the dosing that sample-adding group, the high concentration group of positive controls are additionally provided with culture medium is put down
Row hole, every block of plate are equipped with 4 blank control wells (only plus culture medium).The final concentration of sample is respectively 10-2、10-1, 1,10 and
102μ g/mL, corresponding DMSO final concentration are respectively 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%.When sample is at end
Concentration 102During μ g/mL, make negative control with physiological saline by the use of 0.1%DMSO as solvent control, remaining concentration.Positive control
Final concentration of the 10 of medicine cis-platinum-1、1、10µg/mL.Cell is at 37 DEG C, 5% CO2After being incubated 48 hours respectively in incubator, add
MTT (5 mg/ml, Sigma), 10 μ L/ holes.After continuing culture 4 hours, three liquid of addition [10% SDS-5% isobutanols-
0.012mol/L HCL (w/v/v)], 100 μ L/ holes, surveyed after standing overnight with ELIASA under 570 nm, 630 nm dual wavelengths
The OD values in fixed each hole.
Test result indicates that：Through to early young HL-60 cell lines, lung adenocarcinoma A549 cell line, people's marrow neuroblastoma
SHSY5Y cell lines, human prostata cancer PC3 cell lines, the cytotoxic activity experiment of human breast cancer MCF7 cell strain, it is to HL-60
Cell line has certain cytotoxic activity, IC50It is worth for 42.11 μM, it is weaker to other four plants of cytoactives, it is all higher than 80 μM.
Claims (10)
1. a kind of sulfur-bearing alkaloid, it is characterised in that described sulfur-bearing alkaloid is that have 1,3-diazabicyclo [3.3.1]
The alkaloid of nonane skeletons, it is with Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）Root be prepared for base stock, its molecular formula is C14H16N2O2S, the compound it is Chinese entitled
(±)-maca thiocarbamide C, entitled (±)-macahydantoin C of English, its chemical structural formula are：
。
2. the preparation method of the sulfur-bearing alkaloid described in a kind of claim 1, it is characterised in that be with Cruciferae separate row Vegetable spp
Characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）Root be base stock, it is extracted, extraction, MCI
The steps such as section, chromatograph enrichment, chromatogram purification and the fractionation of efficient liquid phase chirality semi-preparative column are drawn in depigmentation processing, positive column chromatography
It is prepared, specifically includes：
A, extract：By Cruciferae separate row Vegetable spp characteristic resources plant Maca（Latin name：Lepidium meyenii Walp.）
Root crush after cross 10 ~ 100 mesh sieves, obtain material a, add 3 ~ 8 times of material a weight organic solvent cold soaking and ultrasonic extraction 2 ~
5 times, every time 1 ~ 3h, extract solution obtains filtrate b through filtering, and distillation and concentration obtains maca medicinal extract c；
B, extract；The water of 1 ~ 3 times of medicinal extract c weight is added into medicinal extract c, stirring obtains suspension, with the isometric and and water with water
Unmixing organic solvent extracts 2 ~ 5 times, merges the extraction phase of organic solvent, is concentrated under reduced pressure to give medicinal extract d；
C, MCI depigmentations are handled：The medicinal extract d Methanol+Waters of 2 ~ 5 times of medicinal extract d weight are dissolved, use MCI post layers
Analysis purifying, eluted with the methanol aqueous solution that concentration expressed in percentage by volume is 80% ~ 90%, merge eluent, be concentrated under reduced pressure to give medicinal extract e；
D, positive column chromatography draws section：Methanol or acetone solution by medicinal extract e with 2 ~ 3 times of medicinal extract e weight, with medicinal extract e weight 1 ~ 3
Times 80 ~ 100 mesh or 200 ~ 300 mesh silica gel mixed sample, purified using positive column chromatography, i.e., with medicinal extract e weight 5 ~
100 ~ 200 mesh silica gel dress post of 10 times of amounts, is 1 with volume ratio:0~0:1 mixed organic solvents gradient elution, collect eluent
And concentrate, detected through silica gel thin-layer chromatography, merge colour developing and separation identical point, contained (±)-maca thiocarbamide C's
Mixture f；
E, preparative high-performance liquid chromatographic is enriched with：Using preparative high-performance liquid chromatographic, with the methanol solution of concentration expressed in percentage by volume 40% ~ 90%
Or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% affords (±)-maca thiocarbamide C crude products；
F, half preparative high-performance liquid chromatographic purifies：(±)-maca thiocarbamide C crude products are isolated and purified through half preparative high-performance liquid chromatographic
Obtain (±)-maca thiocarbamide C sterlings；
G, efficient liquid phase chirality semi-preparative column is split：(±)-maca thiocarbamide C sterlings are torn open by efficient liquid phase chirality semi-preparative column
Get (+)-maca thiocarbamide C and (-)-maca thiocarbamide C.
3. preparation method according to claim 2, it is characterised in that the organic solvent described in step A is 50% ~ 100%
Acetone, 80% ~ 100% ethanol, 80% ~ 100% ethyl acetate or 80% ~ 100% methanol.
4. preparation method according to claim 2, it is characterised in that the organic solvent described in step B is chloroform, dichloro
Methane, ethyl acetate, n-butanol, isopropanol, hexamethylene or petroleum ether.
5. preparation method according to claim 2, it is characterised in that the body of the Methanol+Water described in step C
Product percentage concentration is 80%.
6. preparation method according to claim 2, it is characterised in that mixed organic solvents described in D steps for chloroform-
Acetone.
7. preparation method according to claim 6, it is characterised in that the volume of chloroform and acetone in described chloroform-acetone
Match as 10:1、9:1、8:2、7:3、6:4 and 5:5.
8. preparation method according to claim 2, it is characterised in that the preparative high-performance liquid chromatographic enrichment described in E steps
Be using the methanol aqueous solution of concentration expressed in percentage by volume 40% ~ 90% or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% as mobile phase,
Flow velocity is 10 ~ 14mL/min, with Zorbax PrepHT GF（5 μm, 21.2 × 250mm）Reverse phase preparative column is stationary phase, ultraviolet
The Detection wavelength of detector is 203nm and 306nm, and each sample size is 10 ~ 1000 μ L, collects the chromatogram in the range of 10 ~ 25min
Peak, after repeatedly being added up with same steps, it is evaporated with Rotary Evaporators, produces described (±)-maca thiocarbamide C crude products.
9. preparation method according to claim 2, it is characterised in that half preparative high-performance liquid chromatographic described in F-step is pure
Change is using the methanol aqueous solution of concentration expressed in percentage by volume 40% ~ 90% or the acetonitrile solution of concentration expressed in percentage by volume 30% ~ 80% as flowing
Phase, flow velocity is 1 ~ 4mL/min, with Zorbax SB-C18（10 μm, 9.4 × 250mm）Reverse phase semi-prep column is stationary phase, and DAD is examined
The Detection wavelength for surveying device is 203nm, 220nm, 254nm, 265nm and 306nm, and each sample size is 1 ~ 100 μ L, collection 10 ~
Chromatographic peak in the range of 25min, after repeatedly being added up with same steps, it is evaporated with Rotary Evaporators, produces described (±)-maca
Thiocarbamide C sterlings.
10. the application of the sulfur-bearing alkaloid described in a kind of claim 1, it is characterised in that described sulfur-bearing alkaloid is anti-in preparation
Application in cancer drug, health products and functional food.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN115894499A (en) * | 2022-10-17 | 2023-04-04 | 云南民族大学 | Novel natural sulfur-containing alkaloid and preparation method and application thereof |
-
2017
- 2017-09-30 CN CN201710918498.3A patent/CN107556314A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| MIN ZHOU等: "Biomimetic Synthesis of Macahydantoins A and B from Lepidium meyenii, and Structure Revision of Macahydantoin B as a Class of Thiohydantoin with a 4 Methyl-hexahydropyrrolo[1,2-c]imidazole Skeleton", 《ORG. LETT.》 * |
| MIN ZHOU等: "Supporting Information for Biomimetic Synthesis of Macahydantoins A and B from Lepidium meyenii, and Structure Revision of Macahydantoin B as a Class of Thiohydantoin with a 4 Methyl-hexahydropyrrolo[1,2-c]imidazole Skeleton", 《ORG. LETT.》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN115894499A (en) * | 2022-10-17 | 2023-04-04 | 云南民族大学 | Novel natural sulfur-containing alkaloid and preparation method and application thereof |
| CN115894499B (en) * | 2022-10-17 | 2024-06-14 | 云南民族大学 | Novel natural sulfur-containing alkaloid and preparation method and application thereof |
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