CN113121561A - Sulfur-containing diene compound and preparation method and application thereof - Google Patents
Sulfur-containing diene compound and preparation method and application thereof Download PDFInfo
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- CN113121561A CN113121561A CN202110366990.0A CN202110366990A CN113121561A CN 113121561 A CN113121561 A CN 113121561A CN 202110366990 A CN202110366990 A CN 202110366990A CN 113121561 A CN113121561 A CN 113121561A
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- 229910052717 sulfur Inorganic materials 0.000 title claims abstract description 43
- 239000011593 sulfur Substances 0.000 title claims abstract description 43
- -1 diene compound Chemical class 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000002386 leaching Methods 0.000 claims abstract description 18
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- 238000010828 elution Methods 0.000 claims description 24
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- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a sulfur-containing diene compound and a preparation method and application thereof; the molecular formula of the sulfur-containing diene compound is C17H16O5S; the extraction method of the compound comprises the following steps: leaching the rubus hirsutus in an organic solvent to obtain a leaching solution, concentrating the leaching solution, and adding water to prepare an aqueous suspension; extracting the water suspension, and concentrating the extract to obtain an extract; separating and purifying the extract to obtain the sulfur-containing diene compound; the invention separates and identifies a sulfur-containing diene compound with a novel structure from rubus hirsutus, and the compound has the pharmacological activity of resisting tumor and can be used for preparing anti-tumor drugs or functional foods.
Description
Technical Field
The invention belongs to the technical field of active ingredients of medicines, and particularly relates to a sulfur-containing diene compound and a preparation method and application thereof.
Background
The Rubus hirsutus (Rubus hirsutus Thunb.) is a Rubus L plant of Rubus of Rosaceae (Rosaceae), also called Eugenia japonica and Blastus fortunei, is perennial deciduous shrub, has strong tillering capacity, wide distribution and large resource amount; the Tephrodis Te; at present, the research on the rubus hirsutus domesticus and abroad is relatively few particularly aiming at the separation and identification of the monomer components and the pharmacological activity of the monomer components, and the monomer components are not related to the anti-tumor pharmacological activity of the rubus hirsutus, so the available range of the rubus hirsutus as a medicament still needs to be further expanded.
Disclosure of Invention
The invention aims to provide a sulfur-containing diene compound, a preparation method and application thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a sulfur-containing diene compound, wherein the sulfur-containing diene hirsutupyran A has a structural formula:
the preparation method of the sulfur-containing diene compound comprises the following steps:
(1) placing the rubus hirsutus in an organic solvent, and leaching by a cold leaching method or a hot reflux extraction method to obtain a leaching solution;
(2) concentrating the leaching liquor, adding water to prepare water suspension, extracting the water suspension, and concentrating the extract to obtain an extract;
the cold soaking method comprises the steps of putting the rubus hirsutus into a sealed container, adding a certain volume of organic solvent according to a formula, and soaking for about 2 weeks; during the period, the rubus hirsutus is shaken frequently, and after the active ingredients in the rubus hirsutus are fully dissolved into the organic solvent, the leaching liquor can be obtained by filtration;
the hot reflux extraction method is to use an organic solvent for heating extraction, and a reflux heating device is needed to avoid solvent volatilization loss; the hot reflux extraction method is more complicated than the cold leaching method, but the extraction efficiency is high;
(3) and separating and purifying the extract by adopting normal-phase silica gel column chromatography to obtain the sulfur-containing diene compound.
Before the step (1), the method also comprises a crushing step of crushing the rubus hirsutus.
In the step (1), the ratio of the weight (kg) of the rubus hirsutus to the volume (L) of the organic solvent is 1: 1-2: 1.
The organic solvent is one or more of methanol, ethanol, chloroform, ethyl acetate and acetone.
Preferably, the organic solvent is methanol, ethanol or a mixture thereof; the polarity of methanol and ethanol is relatively high, and more active ingredients with medium and low polarity can be obtained by leaching with methanol or ethanol.
During extraction, a solvent which is not soluble with the organic solvent adopted during extraction is selected; when leaching with methanol, extraction is carried out with ethyl acetate; when the ethanol is used for leaching, chloroform is used for extraction; in addition, in order to improve the extraction yield, preferably, the leaching liquor is concentrated and then water is added to prepare water suspension, and then the water suspension is extracted.
The eluent in the step (3) is a mixed solution of petroleum ether and ethyl acetate; carrying out gradient elution on petroleum ether and ethyl acetate according to the volume ratio of 9:1, 7:1, 4:1, 3:1, 1:3 and 1:9 in sequence, collecting fractions eluted when the volume ratio of the petroleum ether to the ethyl acetate is 7:1, and carrying out rotary evaporation on the fractions to obtain the sulfur-containing diene compound; the petroleum ether and the ethyl acetate have lower polarity, and can carry out full chromatography on the target compound to remove all or most impurities; compared with other organic solvents, the petroleum ether and the ethyl acetate have lower toxicity and are beneficial to the health of researchers.
Before the fraction is subjected to rotary evaporation to dryness, the method also comprises the steps of high performance liquid chromatography separation and recrystallization which are sequentially carried out on the eluted fraction.
Before the high performance liquid chromatography separation step, the method also comprises a reverse silica gel column chromatography step, wherein an eluant used in the reverse silica gel column chromatography step is a mixed solution of methanol and water; sequentially carrying out gradient elution on methanol and water according to the volume ratio of 9:1, 7:1, 4:1, 1:3 and 1:9, carrying out gradient elution on 1L of each gradient elution, detecting each fraction by TLC, merging, concentrating fractions with the elution ratio of 7:1, and recrystallizing with acetone to obtain the target compound.
The application of the sulfur-containing diene compound is used for preparing antitumor medicines or functional foods.
The beneficial technical effects of the invention compared with the prior art are that,
(1) the invention extracts and separates the sulfur-containing diene compound with a novel structure from the rubus hirsutus by utilizing the polarity difference of the sulfur-containing diene substances, and the method has the advantages of simple and convenient operation, high extraction yield and high product purity, and is suitable for large-scale production.
(2) The sulfur-containing diene compound provided by the invention has wide pharmacological activity and can be used for preparing antitumor drugs or antitumor functional foods.
Drawings
FIG. 1 shows the preparation of sulfur-containing diene compound hirsutupyran A1H-NMR spectrum (500 MHz).
FIG. 2 is a diagram of sulfur-containing diene compound hirsutupyran A13C-NMR spectrum (125 MHz).
FIG. 3 is a structural formula of sulfur-containing diene hirsutupyran A.
FIG. 4 is a graph of hydrogen dependence and remote carbon-hydrogen dependence of sulfur-containing diene compound hirsutupyran A.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
The preparation of the sulfur-containing diene compound comprises the following steps:
example 1
Pulverizing 10kg of fructus Tephrodii Tephrodis, extracting with 10L of methanol for 2 weeks, concentrating the methanol extractive solution, suspending with 1L of distilled water, extracting the water suspension with 1L of ethyl acetate for 3 times, and concentrating the ethyl acetate extractive solution to obtain 260g of extract; mixing with silica gel (100 mesh, 100g), performing normal phase silica gel column chromatography (200 mesh, 300 mesh, 1 kg; silica gel column size L500 mm,
) Carrying out gradient elution by mixed solution of petroleum ether and ethyl acetate in volume ratio of 9:1, 7:1, 4:1, 3:1, 1:3 and 1:9 in sequence, wherein each gradient elution is 5L; detecting the fraction by TLC, collecting fractions with an elution ratio of 7:1 (petroleum ether: ethyl acetate), mixing, concentrating to obtain a crude product of 300mg with a purity of 60% and a yield of 3%, and recrystallizing with acetone (at room temperature) to obtain a target compound of 100mg with a purity of 99% and a yield of 1%.
Example 2
Crushing 10kg of rubus hirsutus, extracting with 5L of ethanol under reflux, concentrating the ethanol extract, suspending with 1L of distilled water, extracting the water suspension with 1L of chloroform for 3 times, and concentrating the chloroform extract to obtain 234g of extract; mixing with silica gel (100 mesh, 100g), performing normal phase silica gel column chromatography (200 mesh, 300 mesh, 1 kg; silica gel column size L500 mm,
) Carrying out gradient elution by mixed solution of petroleum ether and ethyl acetate in volume ratio of 9:1, 7:1, 4:1, 3:1, 1:3 and 1:9 in sequence, wherein each gradient elution is 5L; detecting the fraction by TLC, collecting fractions with an elution ratio of 7:1 (petroleum ether: ethyl acetate), merging, concentrating to obtain a crude product of 260mg with a purity of 65% and a yield of 2.6%, and recrystallizing with acetone (at room temperature) to obtain a target compound of 120mg with a purity of 99% and a yield of 1.2%.
Example 3
Pulverizing 10kg of pulverized fructus Tephrodii Tephrodis, extracting with 5L of mixed solution of methanol and ethanol, and collecting methanolConcentrating ethanol leaching solution to obtain extract 366g, mixing with 100g diatomaceous earth, hot soaking with 5L chloroform for 3 times, performing normal phase silica gel column chromatography (200 meshes 300 mesh, 1 kg; silica gel column size L500 mm,
) Carrying out gradient elution by mixed solution of petroleum ether and ethyl acetate in volume ratio of 9:1, 7:1, 4:1, 3:1, 1:3 and 1:9 in sequence, wherein each gradient elution is 5L; detecting the fraction by TLC, collecting fractions with an elution ratio of 10:1 (chloroform: methanol), mixing, concentrating to obtain crude product 350mg with purity of 60% and yield of 3.5%, and recrystallizing with acetone/n-hexane (volume ratio of 2:3) (room temperature) to obtain target compound 210mg with purity of 99% and yield of 2.1%.
Example 4
Extracting 10kg of pulverized fructus Tephrodii Tephrodis with 5L of mixed solution of methanol and ethanol, concentrating the extractive solution to obtain extract 350g, mixing with 100g of diatomaceous earth, hot-soaking with 5L of chloroform for 3 times, performing normal phase silica gel column chromatography (200 meshes 300, 1 kg; silica gel column size L500 mm),
) Gradient elution is sequentially carried out by mixed solution of chloroform and methanol with volume ratio of 9:1, 7:1, 4:1, 3:1, 1:3 and 1:9, and each gradient elution is 5L; detecting fractions by TLC, collecting fractions with elution ratio of 4:1, mixing, and concentrating; and (3) carrying out reverse phase silica gel column chromatography on the concentrated fractions, sequentially eluting with mixed liquor of methanol and water in volume ratios of 9:1, 7:1, 4:1, 1:3 and 1:9, carrying out gradient elution for 1L, detecting each fraction by TLC, combining and concentrating fractions with an elution ratio of 7:5:1 (petroleum ether: chloroform: methanol) to obtain a crude product of 400mg, wherein the purity is 65 percent, the yield is 4 percent, and recrystallizing with acetone to obtain a target compound of 340mg, the purity is 99 percent and the purity is 3.4 percent.
Concentrating the target fraction 7:5:1 (petroleum ether: chloroform: methanol) obtained by normal phase silica gel column chromatography, and purifying by high performance liquid chromatography; detecting with high performance liquid chromatography at wavelength of 210nm, eluting with 20-100% methanol sequentially, collecting eluate with retention time of 45-48min, mixing eluates, concentrating, and recrystallizing with acetone to obtain target compound with yield of 1%.
Structural identification of sulfur-containing diene compounds:
performing purity identification on the target compound obtained in the examples 1-4 by adopting HPLC, and performing structure identification by adopting mass spectrum and nuclear magnetic resonance technology, wherein nuclear magnetic resonance is measured by using a Bruker AVANCE DRX-500 NMR Sectrometer, and TMS is used as an internal standard; high resolution mass spectra fticrrms were measured using a Bruker Apex Spectrometer; ESI-MS electrospray ionization mass spectrometry with Bruker Esquire 3000plusSpectrometer assay.
Of the compound1H-NMR spectrum and13the C-NMR spectra are shown in FIG. 1 and FIG. 2, respectively, and the NMR data are shown in Table 1; the compound structure was resolved by hydrogen-hydrogen correlation and remote hydrocarbon correlation maps (fig. 4).
NMR data for the Compounds of Table 1
| Position of | δC | δH |
| 2 | 144.9 | |
| 3 | 120.8 | 6.78 |
| 3a | 150.6 | |
| 4 | 115.2 | 6.92 |
| 4a | 121.5 | |
| 5 | 151.5 | |
| 6 | 171.8 | |
| 8 | 82.8 | |
| 8a | 136.7 | |
| 8b | 128.0 | |
| 9 | 126.0 | 5.56 |
| 10 | 134.6 | 6.21 |
| 11 | 132.2 | 5.96 |
| 12 | 135.7 | 5.67 |
| 13 | 37.1 | 2.30 |
| 16 | 62.4 | 3.87 |
| 15 | 60.7 | 4.08 |
ESI-TOF-MS shows molecular ion peak m/z333, and the NMR spectrum and analysis of the compound are combined with mass spectrum data to show that the compound is a novel sulfur-containing diene compound which is named as hirsutupyran A and has a molecular formula of C17H16O5S, the structure is shown in figure 3.
The application of the sulfur-containing diene compound is used for preparing antitumor medicines or functional foods.
And (3) analyzing the anti-tumor activity of the sulfur-containing diene compound:
hela cells were cultured in RP-MI 1640 medium containing 10% calf serum, 100IU/mL penicillin and 100g/mL streptomycin, 1 change every 3 days, and 1 passage every 5 days. The cells were all placed at 37 ℃. Taking logarithmic growth phase cells, diluting to 5 × 10 with RPMI 1640 medium4Single cell suspension/mL, seeded in 96-well cell culture plates, 3 wells per concentration, for eachWells 180. mu.L. After the culture box is placed for incubation for 12h, 20 mu L of test solution with different concentrations is added into each hole of the drug group, a blank control group (the RPMI 1640 culture medium with the same volume is used for replacing the tested drug) is arranged in parallel, and the total culture time is 48 h; adding 50 mu L of 1mg/mL MTT solution into each hole, continuously culturing for 4h, completely sucking supernatant, adding 150 mu L of dimethyl sulfoxide (DMSO) into each hole, and fully dissolving the MTT reduction product; measuring optical density (D) of each drug group and blank group at 492nm on a microplate reader, and calculating the inhibition rate (IR,%) and half Inhibition Concentration (IC) of the drug on tumor cell growth according to a formula50) And the drug effect is preliminarily evaluated.
IR (%) - (1-mean D value of dosing group/mean D value of control group) × 100%.
As is clear from the results of the experiments, the IC of the compound50The compound has better anti-tumor effect as shown by 11 mu M (Hela cells).
The anti-tumor medicine is prepared by taking the sulfur-containing diene compound as a main active ingredient and adding pharmaceutically acceptable auxiliary materials, and can be prepared into preparations according to preparation methods recorded in pharmaceutics; the preparation can be injection, infusion solution, powder for injection, granule, tablet, granule, powder, oral liquid, sugar-coated tablet, film-coated tablet, enteric-coated tablet, buccal agent, granule, pill, unguent, pellet, spray, dripping pill, disintegrant, orally disintegrating tablet, pellet, etc.
The preparation method of the sulfur-containing diene compound dripping pill preparation comprises the following steps:
0.5g of sulfur-containing diene compound R-3-methyl-6, 8-dihydroxy-7-methyl-3, 4-dihydro sulfur-containing diene-1-ketone is taken to be uniformly mixed with 10.5g of polyethylene glycol-6000, heated and melted, the mixture is moved into dripping pill drop irrigation after melting, the medicine drops are dropped into liquid paraffin with the temperature of 6-8 ℃, oil is removed, and 300 dropping pills are prepared.
The preparation method of the sulfur-containing diene compound freeze-dried powder injection comprises the following steps:
taking 0.5g of sulfur-containing diene compound R-3-methyl-6, 8-dihydroxy-7-methyl-3, 4-dihydro sulfur-containing diene-1-ketone, 4.5g of glucose, 0.9g of sodium thiosulfate and 1000mL of distilled water, uniformly mixing the components, freeze-drying, and subpackaging 400 to obtain the sulfur-containing diene compound.
The anti-tumor functional food is prepared by taking the sulfur-containing diene compound as a main active ingredient and adding acceptable auxiliary materials.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (9)
1. A sulfur-containing diene compound, wherein the sulfur-containing diene compound has a structural formula:
2. the preparation method of the sulfur-containing diene compound is characterized by comprising the following steps:
(1) placing the rubus hirsutus in an organic solvent, and leaching by a cold leaching method or a hot reflux extraction method to obtain a leaching solution;
(2) concentrating the leaching liquor, adding water to prepare water suspension, extracting the water suspension, and concentrating the extract to obtain an extract;
(3) and separating and purifying the extract by adopting normal-phase silica gel column chromatography to obtain the sulfur-containing diene compound.
3. The process for preparing sulfur-containing diene compounds according to claim 2, wherein the process further comprises a step of pulverizing the Tephrodis prior to the step (1).
4. The method of claim 3, wherein in step (1), the weight ratio of the Tephrodis hirsutus to the volume of the organic solvent is 1: 1-2: 1.
5. The method according to claim 4, wherein the organic solvent is one or more selected from methanol, ethanol, chloroform, ethyl acetate and acetone.
6. The process according to claim 5, wherein the eluent in step (3) is a mixture of petroleum ether and ethyl acetate; and (3) carrying out gradient elution on petroleum ether and ethyl acetate according to the volume ratio of 9:1, 7:1, 4:1, 3:1, 1:3 and 1:9 in sequence, collecting fractions eluted when the volume ratio of the petroleum ether to the ethyl acetate is 7:1, and carrying out rotary evaporation on the fractions to obtain the sulfur-containing diene compound.
7. The process according to claim 6, wherein the process further comprises the steps of sequentially subjecting the eluted fractions to HPLC separation and recrystallization before subjecting the fractions to rotary evaporation.
8. The process according to claim 7, wherein the process further comprises a reverse silica gel column chromatography step before the HPLC separation step, wherein the eluent used in the reverse silica gel column chromatography step is a mixture of methanol and water; sequentially carrying out gradient elution on methanol and water according to the volume ratio of 9:1, 7:1, 4:1, 1:3 and 1:9, carrying out gradient elution on 1L of each gradient elution, detecting each fraction by TLC, merging, concentrating fractions with the elution ratio of 7:1, and recrystallizing with acetone to obtain the target compound.
9. The use of the sulfur-containing diene compound according to claim 1, wherein the sulfur-containing diene compound is used for preparing an antitumor drug or functional food.
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