CN105175259B - Mulberry monomer compounds, preparation method and application thereof - Google Patents

Mulberry monomer compounds, preparation method and application thereof Download PDF

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CN105175259B
CN105175259B CN201510514623.5A CN201510514623A CN105175259B CN 105175259 B CN105175259 B CN 105175259B CN 201510514623 A CN201510514623 A CN 201510514623A CN 105175259 B CN105175259 B CN 105175259B
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fructus mori
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methanol
extract
monomeric compound
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CN105175259A (en
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苟德明
康康
吴志琴
王立岩
李荔
黄炼
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Shenzhen University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment

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Abstract

The invention discloses mulberry monomer compounds, a preparation method and applications thereof. The mulberry monomer compounds are prepared by the following steps: extracting mulberry dichloromethane extract from mulberries by an acidification ethanol method and an organic solvent extraction method, and then processing the mulberry dichloromethane extract by column chromatography and HPLC (high performance liquid chromatography) equipment to obtain the mulberry monomer compounds in one step. The experiment results show that two extracted mulberry monomer compounds are both belonged to the functional grease and both have a prominent anti-inflammation activity in cell level. The provided preparation method is simple, and can be applied to the industrial massive production. The provided mulberry monomer compounds can be used to develop anti-inflammation healthcare products.

Description

A kind of Fructus Mori monomeric compound and its preparation method and application
Technical field
The invention belongs to technical field of plant extraction, it is more particularly related to a kind of Fructus Mori monomeric compound and Its preparation method and application.
Background technology
Inflammation is the adaptation response caused by pathogenic bacterium, destructive stimuluses or physical damnification, is body to infection or organizes A kind of defensive measure produced by damaging.In inflammation early period of origination, this defense mechanism is beneficial, and inflammatory cell discharges phase The inflammatory factor answered, carries out a series of defense reaction to damage factor, such as dilute and kill etc., at the same time, discharged Inflammatory factor participate in the reparation and healing at body injury position；And when inflammatory reaction is excessively violent or lasting, body is beneficial Inflammation will change, and induce various inflammation related diseases in turn, or even develop into chronic or persistency disease.By Chronic inflammatory reaction produce a large amount of inflammatory mediators be proved to take part in the pathological process of numerous disease, including cardiovascular disease, Rheumatoid arthritiss, cancer, lupus erythematosus, diabetes and tatanic cervical spondylitiss etc..There are many factors induce inflammation, such as Infection, physico-chemical stresses stimulation, toxin etc., wherein cause pathogeny imcrobe infection is a modal class, such as lipopolysaccharide (lipopolysaccharides,LPS).LPS is one of constituent of gram-negative bacteria cell wall, is only entered on antibacterial Just it is released when row breeding or death, which causes inflammatory extremely strong.When being made body sustain damage by gram positive bacterial infection When, substantial amounts of LPS is released, subsequently with cell membrane on Toll-like receptor 4 (Toll like receptor 4, TLR4) With reference to, and then interact with host cell, by the transmission of intracellular signaling pathway by stimulus signal Cascaded amplification, finally draw Play a series of inflammatory reaction.LPS causes the synthesis and release of substantial amounts of bioactie agent after stimulating body, such as：Prostatitis The inflammatory mediators such as parathyrine (prostaglandin, PGs), nitric oxide (nitric oxide, NO), and Interleukin -1β (interleukin-1 β, IL-l β), interleukin-6 (interleukin-6, IL-6), tumor necrosis factor α (tumor Necrosis factor α, TNF-α) etc. inflammatory cytokine, make body show various inflammatory reactions, when serious, can cause complete It is body inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), septic shock, many Organ failure (multiple organ failure, MOF) or even death.Inflammatory cytokine (IL-l β, the IL- of unconventionality expression 6, TNF-α etc.), COX-2 (Cyclooxygenase-2, COX-2) and nuclear Factor-Kappa B (Nuclear factor- κ B, NF- κ B) be inflammation canceration on molecular level inducement.Therefore, it is desirable to the related disease that preferably prevention or treatment inflammation cause Disease, it is necessary to suppress the generation of inflammatory mediator or block its biological function, and screen Anti-Inflammatory Actives and study its effect Mechanism, then can become the important means of the new prevention and treatment cancer of exploitation.
Functional grease refers to the oils and fatss with certain special physiological function, although at present to functional grease also without very The definition of authority, but biodiesel, by the unofficial a subset for classifying as functional food, they are typically used to conduct Supplement beyond traditional food, but they possess the advantage beyond basal nutrient function, with good Physiological Benefit and Reduce the effects such as the risk of chronic disease, such as pre- preventing obesity, blood fat reducing, prevention senile dementia.
Functional grease relates generally to following several：(1) omega-fatty acid：Including alpha-linolenic acid (alpha-linolenic Acid, ALA), eicosapentaenoic acid (eicosapentaenoic acid, EPA) and docosahexenoic acid (docosahexaenoic acid, DHA), (2) ω -6 fatty acids：Including gamma-Linolenic acid (gamma linolenic acid, GLA) and linoleic acid (linoleic acid, LA), (3) conjugated linoleic acid (conjugated linoleic acid, CLA), (4) Medium-chain Triglycerides (medium chain triglyceride, MCTs) and (6) plant sterol (phytosterols).
Mammal cannot voluntarily synthesize ω -3 and omega 6 polyunsaturated fatty acid (polyunsaturated fatty Acids, PUFAs), it is necessary to absorb from food.These PUFAs are the important composition compositions of cell membrane.Fatty acid in diet Content can affect the cell membrane fat acid composition of normal and tumor tissues, and this may affect the attribute of film, such as permeability or fat The work of the specified proteins such as matter packaging, gene expression, transcription factor activity, Protein kinase C (protein kinase C, PKC) Property and signal transduction.Numerous studies show that omega-fatty acid and GLA can be used to preventing and treating numerous diseases, such as acne, different Position property eczema, psoriasises, cardiovascular disease, allergic dermatitises etc..
Fructus Mori (mulberry) as can nourishing YIN and supplementing blood, fluid dryness integration of edible and medicinal herbs based food, containing many active matters, Including in addition to flavonol, anthocyanin, phenolic acid, aminoacid, also substantial amounts of trace element and mineral.Fructus Mori are used as the medicinal beginning It is loaded in Tang《Newly Revised Canon of Materia Medica》, its effect is all on the books in many Chinese medicine works, such as《Compendium of Materia Medica》、《Bencao Shiyi》、《With breath Occupy diet spectrum》Deng.Folium Mori, Radix Mori skin and Ramulus Mori are used for treating heating, hepatoprotective, improving eyesight, diuresis and drop blood in Traditional Chinese Medicine Pressure etc..Also there are some researches show sorosis, Radix Mori skin and Ramulus Mori be widely used in treat jaundice, hepatitis, cancer, diabetes, diarrhoea, Hypertension etc..Become the agricultural product for enjoying " being both food and medicine " this laudatory title early in Fructus Mori in 1993, with very high Edible and medical value, be the quality raw materials of development functionality food.
The extract of mulberry, most common is to be extracted as many with Folium Mori as material, and effective-component extract of Foliun-Mori grinds Study carefully report and patent application is very more, and by the use of fruit Fructus Mori as the few of material, not yet have Fructus Mori dichloromethane at present In extract, effective content of anti inflammation is extracted or the research of Fructus Mori monomeric compound is reported.
The content of the invention
In view of this, in order to overcome above-mentioned problems of the prior art, the invention provides a kind of Fructus Mori singulation Compound and its preparation method and application.
In order to realize foregoing invention purpose, technical scheme below is this invention takes：
A kind of preparation method of Fructus Mori monomeric compound, comprises the following steps：
(1), fresh Fructus Mori are blended, the ethanol extracted overnight being acidified with hydrochloric acid under normal temperature and pressure, collects supernatant extraction Liquid, repeats the step 2～3 time, and decompression recycling ethanol, concentrated extract, rotation are evaporated to quality and no longer change, and obtain Fructus Mori Crude extract；Will be Fructus Mori crude extract soluble in water, add isopyknic water saturation dichloromethane and extracted, collect lower floor's dichloro Methane extract, repeats the step 2～3 time, and recovered under reduced pressure dichloromethane, concentrated extract, rotation are evaporated to quality and no longer change Become, obtain Fructus Mori dichloromethane extract；
(2), by macroporous resin column on Fructus Mori dichloromethane extract, with the cyclohexane-ethyl acetate of 3 times of post retention volumes Mixed solvent carry out system gradient elution, it is after gradient elution terminates, colourless to eluent with methanol-eluted fractions, collect eluent, Jing after silica gel column chromatography and TLC analyses, saccharide, the mixture of fatty Halogenated-hydrocarbons is obtained；
(3), the mixture of the ester type compound that fractions get off carries out hydroxypropyl polydextran gel column chromatography, with etc. The mixed solvent of concentration chloroform-methanol affords the live part of ester type compound, subsequently carries out reversed-phase silica gel column chromatography, uses Methanol solution carries out gradient elution, is concentrated to give concentrate；Normal-phase silica gel column chromatography is carried out again, with the volume ratio containing 1% acetic acid For 97:The mixed solvent of 3 cyclohexane-acetone carries out Isocratic clution, obtains the enriched substance of compound, subsequently to the enriched substance Using high performance liquid chromatography device, purification is carried out in methanol-water solution, you can obtain the high-purity monomer of ethyl linolenate Compound；
(4), the mixture of the fatty acid compound for eluting step (2) carries out reversed-phase silica gel column chromatography, uses first Alcoholic solution carries out gradient elution, is concentrated to give the enriched substance of compound, subsequently adopts high performance liquid chromatography device to the enriched substance, Purification is carried out in methanol-water solution, you can obtain linoleic high-purity monomer compound.
Wherein in some embodiments, the volume ratio of mixed solvent cyclohexane described in step (2) and ethyl acetate according to It is secondary to be：100:0、99:1、98:2、97:3、96:4、95:5、9:1、8:2、7:3、6:4 and 0:100, the temperature of gradient elution is 25 ± 5 DEG C, eluting pressure is 100KPa.
Wherein in some embodiments, described in step (3), in methanol solution, methanol is followed successively by 8 with the volume ratio of water:2 Hes 10:0, the temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa；The preparation parameter of HPLC is：0-40min, 90- 100%；40-45min, 100-100%；45-50min, 90-90%.
Wherein in some embodiments, described in step (4), in methanol solution, methanol is followed successively by 8 with the volume ratio of water:2 Hes 10:0, the temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa；The preparation parameter of HPLC is：0-30min, 80- 100%；30-35min, 100-100%；35-40min, 80-80%.
Wherein in some embodiments, the aperture of macroporous resin described in step (2) is 200-300 mesh.
Wherein in some embodiments, hydroxypropyl polydextran gel described in step (2) is Sephadex LH-20 molecules Sieve resin.
Wherein in some embodiments, reverse silica gel described in step (2) and (3) is the reverse silica gel of 75C18-OPN.
Wherein in some embodiments, described in step (2), the aperture of positive silica gel is 300-400 mesh.
Wherein in some embodiments, the ethanol of the acidifying of hydrochloric acid described in step (1) is the 80% of 0.1% hydrochloric acid acidifying Ethanol.
Wherein in some embodiments, the preparation method of the ethanol of the acidifying of hydrochloric acid described in step (1) is：First prepare dense eventually The ethanol solution for 80% is spent, then is acidified with hydrochloric acid so that hydrochloric acid final concentration of 0.1%.
Wherein in some embodiments, described in step (1), extraction conditionss are：Extraction temperature is 25 ± 5 DEG C, extracting pressure For 100KPa.
Wherein in some embodiments, it is 50 ± 5 DEG C to rotate the temperature being evaporated described in step (1).
Present invention also offers the Fructus Mori monomeric compound that above-mentioned preparation method is prepared.
Present invention also offers application of the above-mentioned Fructus Mori monomeric compound in the health product for preparing prevention enteritis.
Compared with prior art, the invention has the advantages that：
The present invention adopts acidic ethanol method and organic solvent extractionprocess to extract from Fructus Mori and obtain Fructus Mori dichloromethane extraction Thing, and column chromatography and high performance liquid chromatography device is utilized, further prepare corresponding Fructus Mori monomeric compound.Jing experiments are ground Study carefully and show, two for extracting Fructus Mori monomeric compound belongs to biodiesel, on a cellular level with significant antiinflammatory Activity.The preparation method of Fructus Mori monomeric compound provided by the present invention is simple, is adapted to large-scale industrial production, is expected to out Send out into new anti-inflammatory health product.
Description of the drawings
Preparation method flow charts of the Fig. 1 for Fructus Mori monomeric compound in the embodiment of the present invention 1, wherein MB-E1-B2-d1-4： That is MB-DCM-D, gamma-ethyl linolenate；MB-E1-F2-5：That is MB-DCM-F, linoleic acid.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, below in conjunction with accompanying drawing and it is embodied as Example, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only to explain this It is bright, it is not intended to limit the present invention.
A kind of preparation method of 1 Fructus Mori monomeric compound of embodiment
Fig. 1 is referred to, the preparation method of the Fructus Mori monomeric compound of the embodiment, including step in detail below：
(1), fresh Fructus Mori 3.5Kg, cleans and dries naturally excessive moisture, adds 80% ethanol of 0.1% hydrochloric acid acidifying Solution, after homogenate, 25 ± 5 DEG C, react under the conditions of 100KPa, sucking filtration after 24h, collects filtrate, this step in triplicate, by three times Filtrate mixing, 50 ± 5 DEG C of decompression recycling ethanols, concentration filtrate to small size, the filtrate after concentration is transferred in small container, 50 ± 5 DEG C of rotations are evaporated to concentrate quality and keep constant, and weighing obtains primary extract 412.67g.
(2) 370.13g primary extracts, are taken, is dissolved completely with water, added isopyknic water saturated dichloromethane and enter OK, turn upside down mixing, 25 ± 5 DEG C, stand to demixing of solvents under the conditions of 100KPa, collect lower floor's dichloromethane extract, this Step in triplicate, the extract of three times is mixed, 50 ± 5 DEG C of recovered under reduced pressure dichloromethane, and concentration filtrate, will be dense to small size Extract after contracting is transferred in small container, and 50 ± 5 DEG C are evaporated to extract quality and keep constant, and weighing obtains dichloromethane and carries Take thing 6.54g.
(3), (the friend's silica gel exploitation of Yantai river is limited for macroporous resin column on the dichloromethane extract for extracting step (2) Company produces, 200-300 mesh), with the mixed solvent of the cyclohexane-ethyl acetate of 3 times of post retention volumes (hexamethylene in mixed solvent Alkane is followed successively by with the volume ratio of ethyl acetate：100:0、99:1、98:2、97:3、96:4、95:5、9:1、8:2、7:3、6:4 and 0: 100) system gradient elution (temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa) is carried out, gradient elution terminates Afterwards, it is colourless to eluent with methanol-eluted fractions, eluent is collected, Jing after silica gel column chromatography and TLC analyses, merges similar according to polarity Mixture, according to polarity size distribution, obtains the mixture of A-G class materials, wherein B classes (mixture of ester type compound) 0.97g, F class (mixture of fats compound) 1.49g.
(4) B classes material is passed through into hydroxypropyl sephadex column (Sephadex LH-20 pillars, Pharmacia companies, Production), chloroform：Methanol=5:5 eluent is separated, and obtains 2 classes according to the sulphuric acid of thin layer chromatography sample spot colour developing result The live part MB-E1-B-2 material 609mg of material, wherein ester type compound；C18- of the F classes material by 28cm × 3.5cm OPN reversed-phase columns (Nacalai Tesque companies), methanol：Water=8:2 and pure methanol carry out the gradient elution (temperature of gradient elution For 25 ± 5 DEG C, eluting pressure is 100KPa), judge to obtain 3 class materials, wherein MB-E1-F-2 according to the color of sulphuric acid developer Material 139.9mg.(after B classes material is separated with F class materials, screening can develop the color and content is prevailing in material composition Material, is respectively designated as MB-E1-B-2 and MB-E1-F-2).
(5), further by the C18-OPN reversed-phase columns of 28cm × 3.5cm, methanol：Water=8:2 and pure methanol carry out gradient Eluting (temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa) separates MB-E1-B-2 class materials, big according to polarity Little distribution obtains 9 class materials, wherein d materials 48.7mg, then normal phase silicagel column (the Qingdao Haiyang chemical industry by 288.4cm × 6mm The 300-400 mesh of field production), use hexamethylene：Acetone=97:The mixed solvent Isocratic clution of 3 (1% acetic acids) is separated, 3 class materials, wherein MB-E1-B2-d1 materials 36mg are obtained according to the sulphuric acid of thin layer chromatography sample spot colour developing result.
(6), finally by the YMC Pack Pro C18 pillars of 250 × 10mml, carry out in methanol-water solution pure Change, with reference to high performance liquid chromatography separation MB-E1-B2-d1, (preparation parameter of HPLC is：0-40min, 90-100%；40- 45min, 100-100%；The percent concentration of 45-50min, 100-90%, i.e. methanol was gradually risen with the time：0-40min, 90% rises to 100%；40-45min, 100%；45-50min, 100% is down to 90%, when methanol concentration is 98%, isolates The compound) and MB-E1-F-2 (preparation parameter of HPLC is：0-30min, 80-100%；30-35min, 100-100%；35- The percent concentration of 40min, 100-80%, i.e. methanol was gradually risen with the time：0-30min, 80% rises to 100%；30- 35min, 100%；35-40min, 100% is down to 80%, when methanol concentration is 91.5%, isolates the compound) after, obtain To two monomeric compounds, respectively MB-E1-B2-d1-4 (17mg) and MB-E1-F2-5 (56.7mg), using MS,1H-NMR 、13The Modern spectroscopy such as C-NMR technology is measured to Fructus Mori active monomer compound, according to the parsing to returned data, it is determined that single Body compound is respectively MB-DCM-D (i.e. gamma-ethyl linolenate) and MB-DCM-F (i.e. linoleic acid).
The identification of A, MB-E1-B2-d1-4
Colourless oil liquid, GC-MS is in 292.2 [M-CH of m/z2] provide molecule peak.Prompting compound molecular weight be 306.2.Binding compounds1H-NMR and13C-NMR data (table 1) determine that the molecular formula of compound is C20H34O2, degree of unsaturation is 4。
Table 1MB-E1-B2-d1-4's1H-NMR and13C-NMR data (400,100MHz, DMSO-d6, TMS, δ ppm)
a:Overlapped signal
1H-NMR (400MHz, DMSO-d6)δ：5.32 (6H, m, H-9,10,12,13,15,16), 4.02 (2H, q, J= 7.1Hz ,-OCH2CH3), 2.76 (4H, dd, J=5.8Hz, H-11, H-14), 2.24 (2H, t, J=7.3Hz, H-2), 2.03 (4H, m, H-8, H-17), 1.50 (2H, t, 7.0Hz, H-3), 1.30 (8H, m, H-4,5,6,7), 1.16 (3H, t, J= 7.0Hz ,-OCH2CH3), 0.92 (3H, t, J=7.5Hz, H-18).
13C-NMR (100MHz, DMSO-d6)δ：172.7 (C-1), 131.4 (C-16), 129.8 (C-9), 127.8 (C-12, C-13), 127.5 (C-10), 126.9 (C-15), 59.5 (- OCH2-), 33.4 (C-2), 28.9 (C-6), 28.4 (C-7, C-5), 28.3 (C-4), 26.5 (C-8), 25.1 (C-14), 25.0 (C-11), 24.3 (C-3), 19.9 (C-17), 14.1 (- OCH2CH3), 14.0(C-18)。
Above-mentioned NMR data is consistent with the gamma-ethyl linolenate of document, and identification of M B-E1-B2-d1-4 (i.e. MB-DCM-D) is Gamma-ethyl linolenate, structural formula are as follows.
Gamma-ethyl linolenate is the derivant of GLA, is the ethyl ester compound of GLA, with more steady than the more preferable antioxidation of GLA It is qualitative, therefore more conducively storage and transport.Gamma-ethyl linolenate has same physiological action with GLA, and GLA must as human body ω -6 series fatty acids for needing, GLA is by the middle generation generated after △ 6- desaturases (rate-limiting enzyme) metabolism LA in animal body Thank to product, GLA is existed in certain plants.Then gamma-Linolenic acid is extended to dihomo-gamma-linolenic acid (dihomo gamma linolenic acid,DGLA).DGLA has 2 metabolic pathways, is likely to become cell membrane phospholipid or goes to satisfy by △ 5- With obtain arachidonic acid (arachidonic acid, AA) after ferment treatment, AA further can aoxidize various in cell Between quasi-eicosane acid compound critically important in signal transduction.GLA has fibrosis, antiinflammatory and an anti-tumor activity, but Mechanism of action in chronic hepatitiss is but still indefinite.The research such as Park finds that GLA has protection hepatocyte and treats chronic liver The potentiality of disease, GLA is by blocking the activation of Smad3/4 and p38 signal paths so as to suppress transforming growth factor-beta 1 (Transforming growth factor beta 1, the plasminog en-activating thing inhibitor that 1) TGF-β mediates The expression of (Plasminogen activator inhibitor 1, PAI-1).In fatty acid metabolism approach, the participation of ethanol Ethyl linolenate can be synthesized in interior various fatty-acid ethyl ester compounds (fatty acid ethyl esters, FAEE). The research such as Li finds ethyl linolenate by increasing the expression of cyclin E (cyclin E), strengthening the cyclin E/ cycles The mitosiss of the activity of protein dependent kinase 2 (cyclin-dependent kinase 2, CDK2) and then promotion cell, Additionally, ethyl linolenate can increase the activity of ERK and JNK, play an important role in the gene expression that activation AP-1 is relied on.
The identification of B, MB-E1-F2-5
Pale yellow oily liquid.GC-MS is in 294.0 [M+CH of m/z2] provide molecule peak.Prompting compound molecular weight be 280.0.Binding compounds1H-NMR and13C-NMR data (table 2) determine that the molecular formula of compound is C18H32O2, degree of unsaturation is 3。
Table 2MB-E1-F2-5's1H-NMR and13C-NMR data (400,100MHz, DMSO-d6, TMS, δ ppm)
a:Overlapped signal
1H-NMR (400MHz, DMSO-d6)δ：5.32 (4H, m, J=1.2Hz, H-9,10,12,13), 2.73 (2H, dd, J =6.1Hz, H-11), 2.16 (2H, t, J=7.3Hz, H-2), 2.01 (4H, m, J=7.2Hz, H-8, H-14), 1.47 (2H, t, J=7.0Hz, H-3), 1.29 (14H, m, H-4,5,6,7,15,16,17), 0.85 (3H, t, J=7.0Hz, H-18).
13C-NMR (100MHz, DMSO-d6)δ：174.4 (C-1), 129.6,127.7 (C-9,10,12,13), 33.7 (C- 2), 30.8 (C-6) 28.9 (C-16), 28.6 (C-7), 28.5 (C-4,5,15), 26.5 (C-8, C-14), 25.1 (C-11), 24.5 (C-3), 21.9 (C-17), 13.8 (C-18).Above-mentioned NMR data is consistent with the linoleic acid of document, identification of M B-E1-F2-5 (i.e. MB-DCM-F) is linoleic acid, and structural formula is as follows.
Linoleic acid is also a class essential fatty acid, and human body is only capable of synthesizing very small amount and is insufficient for itself needing or can not Synthesis, has functions that the prevention of cardiovascular such as blood fat reducing, blood pressure lowering, vessel softening disease or reduces cardiovascular morbidity.Human body The operating of cholesterol and metabolism need linoleic help, if lacking linoleic acid in vivo, cholesterol will be full because of combining And fatty acid, so as to hinder its transhipment and metabolism, cholesterol is just deposited on blood vessel wall so that vessel wall thickening, hinders blood Circulation, and then develop into cardiovascular and cerebrovascular disease.As linoleic acid is prevented from deposition of the cholesterol in blood vessel wall, quilt Referred to as " blood vessel scavenger ", the health product of manufacture prevention of cardiovascular disease are widely used in.
In recent years, CLA is one kind of PUFA, is that a class contains the ten of cis or trans conjugated double bond at specific carbon atom Eight carbon dienoic acids, are the general designations of linoleic one group of position and geometric isomer.CLA's generally has many conformations, including the 9th with 11, the 10th and 12 or the 11st and 13, natural CLA is mainly cis-9, trans-11CLA.It is present in milk product and anti- The biological characteristicses that the CLA of hay animal meat has prevention and reduces disease harm because of which, have caused sizable concern. CLA has, therefore has in the food industry wide Wealthy application prospect.
Impact of the 1 Fructus Mori monomeric compound of test example to RAW264.7 macrophage toxicities and vigor and to LPS induce RAW264.7 Macrophage Inflamatories medium and inflammatory factor generate impact
Using 2,4 dinitrophenyl hydrazine colorimetric determination Fructus Mori monomeric compound to macrophage lactic acid dehydrogenase The impact that (1actatedehydrogenase, LDH) discharges, determines Fructus Mori monomeric compound using MTS methods and macrophage is increased The impact grown.
Using NO contents in Griess methods detection RAW264.7 macrophage medium supernatant, Fructus Mori monomer chemical combination is investigated The RAW264.7 cells that thing is induced to LPS produce the suppression ratio of NO.The results are shown in Table 1.
Impact of the 1 Fructus Mori active monomer compound of table to NO growing amounts
MB (Mulberry, Fructus Mori), DCM (dichloromethane extracts, dichloromethane extract), MB- DCM-D：Gamma-ethyl linolenate, MB-DCM-F：Linoleic acid
1 result of table shows that two Fructus Mori monomeric compounds are not affected in 25-100 μ g/ml concentration ranges The normal proliferative of RAW264.7 cells, that is, do not observe inhibitory action, illustrates the equal no cytotoxicity of two Fructus Mori monomeric compounds.
The detection of NO growing amounts finds that when cell is not affected by environmental stimuli, cell does not express NO substantially, in LPS (1 μ g/ Ml after) stimulating 24h, macrophage discharges substantial amounts of NO (26.32 ± 0.28 μM), shows that LPS stimulates RAW264.7 macrophages thin After born of the same parents, can a large amount of inflammatory mediator NO of inducing cell release.Compared with simple LPS treatment groups, while LPS process, addition monomer enters Row is processed, and when concentration for the treatment of is 25 μ g/ml, 50 μ g/ml and 100 μ g/ml, the corresponding NO growing amounts of MB-DCM-D are respectively 15.67 ± 2.38 μM, 13.27 ± 2.54 μM and 10.46 ± 2.05 μM, the corresponding NO growing amounts of MB-DCM-F be respectively 8.58 ± 1.29 μM, 5.71 ± 0.35 μM and 3.93 ± 0.78 μM, illustrate that two Fructus Mori monomeric compounds can effectively suppress LPS to lure The great expression of the NO for having guided, and with good dosage effect.
The impact of the RAW264.7 macrophages iNOS expression that 2 Fructus Mori monomeric compound of test example is induced to LPS
The expression of iNOS is detected using immunofluorescence staining, further clear and definite Fructus Mori monomeric compound is lured to LPS The inhibitory action for leading RAW264.7 macrophages generation NO is played a role by adjusting iNOS expression.
Immunofluorescence dyeing result shows, when cell is not affected by stimulating, the intracellular expression for not observing iNOS albumen, After LPS stimulates cell, the expression of iNOS albumen is significantly improved, while after adding low concentration monomeric compound to process, two lists Body can significantly reduce the expression of iNOS albumen.
The RAW264.7 macrophage p65 that 3 Fructus Mori monomeric compound of test example is induced to LPS enter the impact of core
The nuclear translocation situation of p65 albumen in RAW264.7 macrophages is detected using immunofluorescence staining, Fructus Mori are studied Monomeric compound enters the inhibitory action of core to p65 albumen.
Immunofluorescence dyeing result shows that, when cell is in quiescent condition, p65 albumen is blocked in Cytoplasm；When After LPS stimulates cell, NF- κ B signal paths are activated, and p65 albumen is quickly entered in nucleus, now nucleus and Cytoplasm In contain p65 albumen；Giving LPS after two kinds of monomeric compound pretreatment 12h again stimulates cell, heavy-stained in nucleus P65 albumen has been reduced, or even the cell p65 albumen having is blocked in Cytoplasm completely, after wherein MB-DCM-F process, is removed P65 albumen is blocked outside Cytoplasm, and intracytoplasmic p65 expressing quantities have also declined.
It is aobvious that the result of above test example 1～3 shows that the Fructus Mori monomeric compound of present invention preparation has on a cellular level The antiphlogistic effects of work.
Embodiment described above only expresses embodiments of the present invention, and its description is more concrete and detailed, but can not Therefore it is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, Without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the protection model of the present invention Enclose.Therefore, the protection domain of patent of the present invention should be defined by claims.

Claims (9)

1. a kind of preparation method of Fructus Mori monomeric compound, it is characterised in that comprise the following steps：
(1), fresh Fructus Mori are blended, the ethanol extracted overnight being acidified with hydrochloric acid under normal temperature and pressure, collects supernatant extract, weight The multiple step 2～3 time, decompression recycling ethanol, concentrated extract, rotation are evaporated to quality and no longer change, and obtain Fructus Mori crude extract； Will be Fructus Mori crude extract soluble in water, add isopyknic water saturation dichloromethane and extracted, collect lower floor's dichloromethane extraction Liquid is taken, repeats the step 2～3 time, recovered under reduced pressure dichloromethane, concentrated extract, rotation are evaporated to quality and no longer change, obtain Fructus Mori dichloromethane extract；
(2) it is, by macroporous resin column on Fructus Mori dichloromethane extract, mixed with the cyclohexane-ethyl acetate of 3 times of post retention volumes Bonding solvent carries out system gradient elution, after gradient elution terminates, colourless to eluent with methanol-eluted fractions, collects eluent, Jing silicon After plastic column chromatography and TLC analyses, saccharide, the mixture of fatty Halogenated-hydrocarbons is obtained；
(3), the mixture of the ester type compound that fractions get off carries out hydroxypropyl polydextran gel column chromatography, uses isoconcentration The mixed solvent of chloroform-methanol affords the live part of ester type compound, subsequently carries out reversed-phase silica gel column chromatography, uses methanol Solution carries out gradient elution, is concentrated to give concentrate；Normal-phase silica gel column chromatography is carried out again, is 97 with the volume ratio containing 1% acetic acid: The mixed solvent of 3 cyclohexane-acetone carries out Isocratic clution, obtains the enriched substance of compound, and subsequently the enriched substance is adopted High performance liquid chromatography device, carries out purification in methanol-water solution, you can obtain the high-purity monomer chemical combination of ethyl linolenate Thing；
(4), the mixture of the fatty acid compound for eluting step (2) carries out reversed-phase silica gel column chromatography, molten with methanol Liquid carries out gradient elution, is concentrated to give the enriched substance of compound, subsequently adopts high performance liquid chromatography device to the enriched substance, in first Purification is carried out in alcohol-water system, you can obtain linoleic high-purity monomer compound.
2. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that in step (2), it is described mixed Bonding solvent cyclohexane is followed successively by with the volume ratio of ethyl acetate：100:0、99:1、98:2、97:3、96:4、95:5、9:1、8: 2、7:3、6:4 and 0:100, the temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa.
3. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that in step (3), the first In alcoholic solution, methanol is followed successively by 8 with the volume ratio of water:2 and 10:0, the temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa；The preparation parameter of HPLC is：0-40min, 90-100%；40-45min, 100-100%；45-50min, 100- 90%.
4. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that in step (4), the first In alcoholic solution, methanol is followed successively by 8 with the volume ratio of water:2 and 10:0, the temperature of gradient elution is 25 ± 5 DEG C, and eluting pressure is 100KPa；The preparation parameter of HPLC is：0-30min, 80-100%；30-35min, 100-100%；35-40min, 100- 80%.
5. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that big described in step (2) The aperture of hole resin is 200-300 mesh.
6. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that hydroxyl described in step (2) Propyl group polydextran gel is Sephadex LH-20 molecular sieve resins；The aperture of the positive silica gel is 300-400 mesh.
7. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that salt described in step (1) The ethanol of acid acidifying is 80% ethanol of 0.1% hydrochloric acid acidifying, and its preparation method is：Final concentration of 80% ethanol is prepared first Solution, then be acidified with hydrochloric acid so that hydrochloric acid final concentration of 0.1%.
8. the preparation method of Fructus Mori monomeric compound according to claim 1, it is characterised in that extract described in step (1) The condition of taking is：Extraction temperature is 25 ± 5 DEG C, and extracting pressure is 100KPa；The temperature being evaporated that rotates is 50 ± 5 DEG C.
9. the Fructus Mori monomeric compound that the preparation method described in any one of claim 1～8 is prepared is preparing prevention enteritis Health product in application.
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