CN103800351A - Pharmaceutical application of akebin E - Google Patents
Pharmaceutical application of akebin E Download PDFInfo
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- CN103800351A CN103800351A CN201410042091.5A CN201410042091A CN103800351A CN 103800351 A CN103800351 A CN 103800351A CN 201410042091 A CN201410042091 A CN 201410042091A CN 103800351 A CN103800351 A CN 103800351A
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Abstract
The invention discloses a pharmaceutical application of akebin E. The pharmaceutical application is to prepare anti-tumor drugs with akebin E as one of the active ingredients or a sole active ingredient. Study results show that akebin E can inhibit malignant proliferation of liver cancer cells, lung cancer cells, breast cancer cells, stomach cancer cells and other tumor cells by inducing severe endoplasmic reticulum stress, and can be widely used in preparing pharmaceutical preparations for treating primary liver cancer, lung cancer, breast cancer, stomach cancer and other malignant tumors. When akebin E is used in combination with other therapeutic drugs or chemotherapeutic drugs, the drug resistance of tumor cells is decreased due to endoplasmic reticulum stress, and cell apoptosis or death is accelerated, so that dose reduction and efficacy enhancement are achieved, the toxic and adverse effects of drugs are reduced, the treatment opportunity of patients is increased, and the effective pharmaceutical treatment period is prolonged. Akebin E has wide and important application prospects and development values.
Description
Technical field
The present invention relates to the medicinal usage of akebin E, specifically, relate to akebin E in the purposes of preparing in anti-tumor medicinal preparation, belong to medical technical field.The invention still further relates to the application of akebin E as inducing cell er stress reagent, belong to biochemistry and cell biological molecular techniques field.
Background technology
The malignant tumor such as hepatocarcinoma, gastric cancer, pulmonary carcinoma, breast carcinoma are the major diseases of contemporary serious harm human health and life.These malignant tumor patients are losing after surgical indication, often rely on the composite treatments such as chemotherapy.Composite treatment take chemotherapy as representative often, because of its toxic and side effects that severe inhibition such as patient's hemopoietic system, immune system, digestive system are caused, has limited the treatment of its continuous and effective.If can search out the antitumor drug that is different from cellulotoxic chemotherapeutics action pathway, for example inducing cell endoplasmic reticulum seriously stress medicine, chemotherapy Comprehensive Treatment alone or in combination, the consumption of chemotherapeutics will be expected to reduce, heighten the effect of a treatment, thereby extend patient treatment cycle and life-span, improve its life quality, thereby produce good society and economic benefit.
Fructus Akebiae is plants of Lardizabalaceae Caulis Akebiae (Akebia quinata(Thunb.) Decne.), threeleaf akebia (Akebia trifoliate(Thunb.) Koidz.), Caulis Akebiae (Akebia trifoliate(Thunb.) Koidz.var.australis((Diels)) Rehd.) and dry almost ripe fruit, contain akebin (Akebia saponin) A, B, C, D, E, F and its sapogenin of G(and be helexin) etc. tens kinds of compositions.Fructus Akebiae is traditional Chinese medical science conventional Chinese medicine, and its nature and flavor bitter cold, returns liver, gallbladder, stomach, urinary bladder channel, has the effects such as depressed liver-energy dispersing and QI regulating, stomach function regulating, promoting blood circulation and stopping pain, hard masses softening and resolving, diuresis.Up to now, the Fructus Akebiae literature contents such as treatise, patent, scientific and technological achievement of being correlated with are main mainly with the form of one of compound recipe composition medicine in clinical practice, and single medicinal material research is rare.
Fructus Akebiae seed is the seed of peeling off out from Fructus Akebiae, accounts for 1/3 of dry fruit weight.The ethanol extraction that only limits to the inventor's disclosed Fructus Akebiae seed in Chinese patent application CN201310140809.X about independent effect of Fructus Akebiae seed in prior art can be used as the pharmaceutical preparation for the preparation for the treatment of primary hepatocarcinoma of one of active component or unique active component.
Akebin E(Akebia saponin E), its chemistry is by name: 3-O-β-D-xylopyranose-(1 → 2)-α-L-arabopyranose helexin-28-O-B-D-Glucopyranose .-(1 → 6)-Glucopyranose., has following chemical structural formula:
Research shows: in Fructus Akebiae, the content of akebin E is only 0.74%, and the content of the akebin E in Fructus Akebiae seed is about 1%; Up to now, there is not yet correlational study report about the medicinal usage of akebin E.
Summary of the invention
The object of this invention is to provide the medicinal usage of a kind of akebin E.
The medicinal usage of akebin E of the present invention, refer to using akebin E as one of active component or unique active component for the preparation for the treatment of anti-tumor drug preparation.
Furtherly, the medicinal usage of akebin E of the present invention, refers to using akebin E as one of active component or unique active component is treated anti-tumor drug preparation for the preparation of comprising by induction er stress approach.
Described tumor comprises the Several Kinds of Malignancies such as hepatocarcinoma, gastric cancer, pulmonary carcinoma, breast carcinoma.
Furtherly, in described pharmaceutical preparation, can comprise akebin E and other treatment anti-tumor drug of effective dose.
Furtherly, described akebin E also comprises based on akebin E structure conformational characteristic and carries out analog and the derivant that molecular modification or structure of modification obtain.
Furtherly, described akebin E can derive from the extract of chemosynthesis and Fructus Akebiae seed.
Furtherly, extracted the method for obtaining akebin E by Fructus Akebiae seed, comprise the steps:
A) with the ethanol water of 60vol%～95vol%, Fructus Akebiae seed is pulverized to coarse powder and carry out reflux, extract, 2～3 times, collect extracting solution, filtration, decompression recycling ethanol, obtain the Fructus Akebiae seed ethanol extract containing akebin E;
B) good to visual flow of solution containing the Fructus Akebiae seed ethanol extract of akebin E with deionized water dilution, without thickness state, then respectively extract 2～3 times with petroleum ether, ethyl acetate, the n-butyl alcohol of 1:1 volume successively, collect n-butanol extraction phase, reclaim under reduced pressure n-butyl alcohol, obtains the Fructus Akebiae seed n-butanol extraction position containing akebin E;
C) will contain D101 macroporous resin adsorption post on the Fructus Akebiae seed n-butanol extraction position of akebin E, the ethanol water of water～95vol% carries out gradient elution successively; Get the eluent of the ethanol water of 50vol%, carry out decompression recycling ethanol, dry, obtain the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E;
D) the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E is mixed after sample with dissolve with methanol and silica white, reclaim under reduced pressure methanol, dry, upper silica gel column chromatography, with dichloromethane and methanol by volume content be that 100%:0～0:100% carries out gradient elution; Get the 3rd eluting part, carry out concentrating under reduced pressure, dry, obtain the Fructus Akebiae seed silicagel column adsorption chromatography separated part containing akebin E;
E) will be containing ODS reversed-phase silica gel chromatography post on the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E, carry out gradient elution with methanol aqueous solution～absolute methanol of 5vol%; Get the 1st eluting part, carry out reclaim under reduced pressure methanol, dry, obtain akebin E.
Pharmaceutical preparation of the present invention can various route of administration give patient, includes but not limited to oral, transdermal, muscle, subcutaneous and intravenous injection.
Above-mentioned pharmaceutical preparation can be any pharmaceutically useful dosage form, comprising: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch etc.; Preferred oral dosage form, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.Described peroral dosage form can contain conventional excipient, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.Suitable filler comprises cellulose, mannitol, lactose and other similar filler; Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate; Suitable lubricant comprises, for example magnesium stearate; The acceptable wetting agent of suitable medicine comprises sodium lauryl sulphate.
Result of study of the present invention shows: akebin E has inducing cell significantly and occur the pharmacodynamic properties of serious er stress; And the place of the endoplasmic reticulum synthetic processing that is intracellular protein, its generation stress cause cell serious " dyspepsia ", function to fall sharply, and degree severe patient can direct cell death inducing or necrosis; Therefore, akebin E can be by inducing serious er stress to suppress the malignant proliferation of the kinds of tumor cells such as hepatocarcinoma, pulmonary carcinoma, breast carcinoma, gastric cancer, thereby can be widely used in, in the preparation of the pharmaceutical preparation for the treatment of the Several Kinds of Malignancies such as primary hepatocarcinoma, pulmonary carcinoma, breast carcinoma, gastric cancer, thering is medical value; In the time that akebin E and other medicine or chemotherapeutics are used in combination, tumor cell will decline because er stress makes its drug-resistant capability, accelerate its apoptosis or death, can realize decrement potentiation, reduce the toxic and side effects of medicine, will increase patient treatment chance and extend the cycle of active drug treatment, thering is extensive and important application prospect and be worth with exploitation.
Accompanying drawing explanation
Fig. 1 is the Fructus Akebiae seed ethanol extract and the each inhibitory action comparison diagram of medicine to SMMC7721 hepatoma carcinoma cell malignant proliferation that contrast containing akebin E, wherein: a is ordinary cells group, b is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 0.94mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, d is that Drug level is the amycin group of 3.33 μ g/mL, e is that Drug level is the amycin group of 6.67 μ g/mL, f is the adriamycin medication group of the Fructus Akebiae seed ethanol extract+3.33 μ g/mL containing akebin E of 0.94mg crude drug/mL.
Fig. 2 be containing the Fructus Akebiae seed ethanol extract of akebin E with contrast medicine and partly measures the impact (adopting the form picture of inverted microscope (400 times) observation) that drug combination group is learned SMMC7721 Morphology of Hepatocellular Carcinoma after 72 hours in administration, wherein: scheming a is the hepatoma carcinoma cell of cellar culture; Figure b is that administration concentration is the hepatoma carcinoma cell of the amycin effect of 6.67 μ g/mL; Figure c is that administration concentration is the hepatoma carcinoma cell of the Fructus Akebiae seed ethanol extract effect containing akebin E of 1.88mg crude drug/mL; Figure d be 0.94mg crude drug/mL containing the hepatoma carcinoma cell after the amycin effect of the Fructus Akebiae seed ethanol extract+3.33 μ g/mL of akebin E.
Fig. 3 is the Fructus Akebiae seed ethanol extract and the each inhibitory action comparison diagram of medicine to A549 lung carcinoma cell malignant proliferation that contrast containing akebin E, wherein: a is common cultivation A549 lung carcinoma cell group, b is that Drug level is the amycin group of 6 μ g/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.25mg crude drug/mL, d is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, e is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 2.5mg crude drug/mL, f is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 3.75mg crude drug/mL.
Fig. 4 is Fructus Akebiae seed ethanol extract and the inhibitory action comparison diagram of chemotherapeutics coupling to A549 lung carcinoma cell malignant proliferation containing akebin E, wherein: a is common cultivation A549 lung carcinoma cell group, b is that Drug level is the amycin group of 6 μ g/mL, c Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, and d is that Drug level is the drug combination group of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E of 0.94mg crude drug/mL.
Fig. 5 is containing the Morphology Effects to A549 lung carcinoma cell after the Fructus Akebiae seed ethanol extract medication 72h of akebin E (the form picture that adopts inverted microscope (400 times) to observe).Wherein: figure a is the lung carcinoma cell of cellar culture; Figure b is the lung carcinoma cell after the amycin effect of 6 μ g/mL; Figure c is the lung carcinoma cell after the Fructus Akebiae seed ethanol extract effect of akebin E that contains of 1.88mg crude drug/mL; Figure d be 0.94mg crude drug/mL containing the lung carcinoma cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
Fig. 6 is Fructus Akebiae seed ethanol extract containing the akebin E inhibitory action comparison diagram to MCF7 breast cancer cell malignant proliferation.Wherein: a is common cultivation MCF7 breast cancer cell group, b is that Drug level is the amycin group of 6 μ g/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.25mg crude drug/mL, d is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, e is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 2.5mg crude drug/mL, and f is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 3.75mg crude drug/mL.
Fig. 7 is Fructus Akebiae seed ethanol extract and the inhibitory action comparison diagram of chemotherapeutics coupling to MCF7 breast cancer cell malignant proliferation containing akebin E.Wherein: a is common cultivation MCF3 breast cancer cell group, b is that Drug level is the amycin group of 6 μ g/mL, c Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, and d is that Drug level is the drug combination group of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E of 0.94mg crude drug/mL.
Fig. 8 is containing the Morphology Effects to MCF7 breast cancer cell after the Fructus Akebiae seed ethanol extract medication 72h of akebin E (the form picture that adopts inverted microscope (400 times) to observe).Wherein: figure a is the breast cancer cell of cellar culture; Figure b is the breast cancer cell after the amycin effect of 6 μ g/mL; Figure c is the breast cancer cell after the Fructus Akebiae seed ethanol extract effect of akebin E that contains of 1.88mg crude drug/mL; Figure d be Drug level be 0.94mg crude drug/mL containing the breast cancer cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
Fig. 9 is Fructus Akebiae seed ethanol extract containing the akebin E inhibitory action comparison diagram to SGC7901 stomach cancer cell malignant proliferation.Wherein: a is the stomach cancer cell group of common cultivation, b is that Drug level is the amycin group of 6 μ g/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.25mg crude drug/mL, d is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, e is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 2.5mg crude drug/mL, and f is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 3.75mg crude drug/mL.
Figure 10 is Fructus Akebiae seed ethanol extract and the inhibitory action comparison diagram of chemotherapeutics coupling to SGC7901 stomach cancer cell malignant proliferation containing akebin E.Wherein: a is common cultivation stomach cancer cell group, b is that Drug level is the amycin group of 6 μ g/mL, c Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, and d is that Drug level is the drug combination group of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E of 0.94mg crude drug/mL.
Figure 11 is containing the Morphology Effects to SGC7901 gastric cancer after the Fructus Akebiae seed ethanol extract medication 72h of akebin E (the form picture that adopts inverted microscope (400 times) to observe).Wherein: figure a is the stomach cancer cell of cellar culture; Figure b is the stomach cancer cell after the amycin effect of 6 μ g/mL; Figure c is the stomach cancer cell after the Fructus Akebiae seed ethanol extract effect of akebin E that contains of 1.88mg crude drug/mL; D figure be Drug level be 0.94mg crude drug/mL containing the stomach cancer cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
Figure 12 is the inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed n-butanol extraction position of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 0.625mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 1.25mg crude drug/mL, and d is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 1.88mg crude drug/mL.
Figure 13 is the inhibitory action comparison diagram to HepG2 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed n-butanol extraction position of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is 0.45mg/mL G418 group, c is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 0.47mg crude drug/mL, and d is that Drug level is the drug combination group of 0.24mg crude drug/mL containing Fructus Akebiae seed n-butanol extraction position group+0.225mg/mL G418 of akebin E.
Figure 14 is the inhibitory action comparison diagram to Huh7 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed n-butanol extraction position of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is 0.45mg/mL G418 group, c is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 0.625mg crude drug/mL, and d is that Drug level is the drug combination group of 0.31mg crude drug/mL containing Fructus Akebiae seed n-butanol extraction position group+0.225mg/mL G418 of akebin E.
Figure 15 is the inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is the Fructus Akebiae seed macroporous resin adsorption was separated part hyte containing akebin E of 0.75mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed macroporous resin adsorption was separated part hyte containing akebin E of 1mg crude drug/mL, and d is that Drug level is the Fructus Akebiae seed macroporous resin adsorption was separated part hyte containing akebin E of 2mg crude drug/mL.
Figure 16 is the inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte containing akebin E of 2mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte containing akebin E of 4mg crude drug/mL, and d is that Drug level is the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte containing akebin E of 8mg crude drug/mL.
Figure 17 is the akebin E of the various dose inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation.In figure: a is ordinary cells matched group, b is that Drug level is the akebin E group of 30 μ M, and c is that Drug level is the akebin E group of 60 μ M, and d is that Drug level is the akebin E group of 120 μ M.
Figure 18 is the high-efficient liquid phase analysis figure of akebin E.
Figure 19 is ultra high efficiency liquid phase and the level Four bar time-of-flight mass spectrometry instrument analysis chart of akebin E.
Figure 20 is the carbon-13 nmr spectra figure of akebin E.
Figure 21 is the carbon spectrum data of akebin E.
The specific embodiment
Below in conjunction with specific embodiments and the drawings, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
Embodiment 1: preparation is containing the Fructus Akebiae seed ethanol extract of akebin E
Fructus Akebiae seed is ground into coarse powder, soaks 2h, heating and refluxing extraction 2～3 times with the ethanol water of the 60vol%～95vol% of 8～10 times of amounts, each 2h, filters merging filtrate, decompression recycling ethanol is until extracting solution without alcohol taste, obtains the Fructus Akebiae seed ethanol extract containing akebin E.
Embodiment 2: the effect experiment that contains the Fructus Akebiae seed ethanol extract of akebin E
Adopt distilled water to dissolve and contain the Fructus Akebiae seed ethanol extract of akebin E and regulate pH to 7.0, then add distilled water and be settled to 1g crude drug/mL; The sterilizing of cell culture condition sucking filtration, subpackage be stored in-80 ℃ for subsequent use; Tumor cell is carried out to 96 orifice plate paving wares; Spend the night after cell attachment, carry out administration; After administration 24,48,72h, adopt inverted microscope to detect respectively and take pictures, mtt assay detects tumor cell proliferation rate of growth, and draws amount effect curve.
Effect experiment one, the inhibitory action containing the Fructus Akebiae seed ethanol extract of akebin E to SMMC7721 hepatoma carcinoma cell malignant proliferation:
Fig. 1 is the Fructus Akebiae seed ethanol extract and the each inhibitory action MTT result of medicine to SMMC7721 hepatoma carcinoma cell malignant proliferation that contrast containing akebin E, wherein: a is ordinary cells group, b is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 0.94mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, d is that Drug level is the amycin group of 3.33 μ g/mL, e is that Drug level is the amycin group of 6.67 μ g/mL, f is the adriamycin medication group of the Fructus Akebiae seed ethanol extract+3.33 μ g/mL containing akebin E of 0.94mg crude drug/mL.
As seen from Figure 1: the Fructus Akebiae seed ethanol extract containing akebin E of amycin+0.94mg crude drug/mL of the 3.33 μ g/mL that dosage reduces by half separately, the inhibitory action of hepatoma carcinoma cell is all obviously better than to the independent role of the amycin of 3.33 μ g/mL and the Fructus Akebiae seed ethanol extract containing akebin E of 0.94mg/mL, the amycin group that approaches 6.67 μ g/mL, presents the effect of decrement potentiation.
Fig. 2 be containing the Fructus Akebiae seed ethanol extract of akebin E with contrast medicine and partly measures the impact (adopting the form picture of inverted microscope (400 times) observation) that drug combination group is learned SMMC7721 Morphology of Hepatocellular Carcinoma after 72 hours in administration, wherein: scheming a is the hepatoma carcinoma cell of cellar culture; Figure b is that administration concentration is the hepatoma carcinoma cell of the amycin effect of 6.67 μ g/mL; Figure c is that administration concentration is the hepatoma carcinoma cell of the Fructus Akebiae seed ethanol extract effect containing akebin E of 1.88mg crude drug/mL; Figure d be Drug level be 0.94mg crude drug/mL containing the hepatoma carcinoma cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
As seen from Figure 2: hepatoma carcinoma cell is containing after the Fructus Akebiae seed ethanol extract effect of akebin E of 1.88mg crude drug/mL in administration concentration, (figure c) to occur serious er stress; After the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E that is 0.94mg crude drug/mL through Drug level, hepatoma carcinoma cell has typical er stress, residual a large amount of cavitys in cell, and see cell pseudopodium and apoptosis phenomenon, the hepatoma carcinoma cell quantity of survival obviously reduces, and the effect with obvious decrement potentiation (is schemed d).
Effect experiment two, the inhibitory action containing the Fructus Akebiae seed ethanol extract of akebin E to A549 lung carcinoma cell malignant proliferation:
Fig. 3 is the Fructus Akebiae seed ethanol extract and the each inhibitory action MTT result of medicine to A549 lung carcinoma cell malignant proliferation that contrast containing akebin E, wherein: a is common cultivation A549 lung carcinoma cell group, b is that Drug level is the amycin group of 6 μ g/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.25mg crude drug/mL, d is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, e is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 2.5mg crude drug/mL, f is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 3.75mg crude drug/mL.
As seen from Figure 3: the Fructus Akebiae seed ethanol extract medication containing akebin E has obvious dose-effect relationship to the inhibited proliferation of lung carcinoma cell, the Fructus Akebiae seed ethanol extract medication containing akebin E of 1.25mg crude drug/mL, has inhibited proliferation significantly to lung carcinoma cell; Be better than the amycin of 6 μ g/mL as 1.88mg crude drug/mL containing its inhibited proliferation after the Fructus Akebiae seed ethanol extract medication of akebin E.
Fig. 4 is Fructus Akebiae seed ethanol extract and the inhibitory action MTT result of chemotherapeutics coupling to A549 lung carcinoma cell malignant proliferation containing akebin E, wherein: a is common cultivation A549 lung carcinoma cell group, b is that Drug level is the amycin group of 6 μ g/mL, c Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, and d is that Drug level is the drug combination group of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E of 0.94mg crude drug/mL.
As seen from Figure 4: containing the Fructus Akebiae seed ethanol extract of akebin E and amycin separately half amount combination (0.94mg crude drug/mL contains Fructus Akebiae seed ethanol extract+amycin 3 μ g/mL of akebin E) medication lung carcinoma cell is had to obvious inhibited proliferation; And the inhibited proliferation after its half amount combination approaches containing the Fructus Akebiae seed ethanol extract enough (1.88mg crude drug/mL) of akebin E and the effect of amycin enough (6 μ g/mL), can realize the synergism of decrement potentiation.
Fig. 5 is containing the Morphology Effects to A549 lung carcinoma cell after the Fructus Akebiae seed ethanol extract medication 72h of akebin E (the form picture that adopts inverted microscope (400 times) to observe).Wherein: figure a is the lung carcinoma cell of cellar culture; Figure b is the lung carcinoma cell after the amycin effect of 6 μ g/mL; Figure c is the lung carcinoma cell after the Fructus Akebiae seed ethanol extract effect of akebin E that contains of 1.88mg crude drug/mL; Figure d be Drug level be 0.94mg crude drug/mL containing the lung carcinoma cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
As seen from Figure 5: 1.88mg crude drug/mL contains after the Fructus Akebiae seed ethanol extract medication of akebin E, and lung carcinoma cell number reduces sharply, and inside generally occurs serious er stress, (figure c) to show as a large amount of cavitys; After amycin 6 μ g/mL medications, lung carcinoma cell number reduces sharply (schemes b); Fructus Akebiae seed ethanol extract and amycin containing akebin E half are measured after combination (0.94mg crude drug/mL is containing Fructus Akebiae seed ethanol extract+amycin 3 μ g/mL of akebin E) medication separately, the same number of lung carcinoma cell reduces sharply, and (figure d) to remain the feature that still has er stress in cell.
Effect experiment three, the inhibitory action containing the Fructus Akebiae seed ethanol extract of akebin E to MCF7 breast cancer cell malignant proliferation:
Fig. 6 is Fructus Akebiae seed ethanol extract containing the akebin E inhibitory action MTT result to MCF7 breast cancer cell malignant proliferation.Wherein: a is common cultivation breast cancer cell group, b is that Drug level is the amycin group of 6 μ g/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.25mg crude drug/mL, d is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, e is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 2.5mg crude drug/mL, and f is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 3.75mg crude drug/mL.
As seen from Figure 6, Fructus Akebiae seed ethanol extract medication containing akebin E has obvious dose-effect relationship to the inhibited proliferation of breast cancer cell, 1.88, the Fructus Akebiae seed ethanol extract medication containing akebin E of 2.5mg crude drug/mL, has inhibited proliferation significantly to breast cancer cell; Be better than the amycin (b) of 6 μ g/mL as 3.75mg crude drug/mL far away containing its inhibited proliferation after Fructus Akebiae seed ethanol extract (f) medication of akebin E.
Fig. 7 is Fructus Akebiae seed ethanol extract and the inhibitory action MTT result of chemotherapeutics coupling to MCF7 breast cancer cell malignant proliferation containing akebin E.Wherein: a is common cultivation MCF3 breast cancer cell group, b is that Drug level is the amycin group of 6 μ g/mL, c Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, and d is that Drug level is the drug combination group of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E of 0.94mg crude drug/mL.
As seen from Figure 7, Fructus Akebiae seed ethanol extract and amycin containing akebin E half are measured after combination (d) medication separately, breast cancer cell is had to obvious inhibited proliferation, effect is obviously better than the Fructus Akebiae seed ethanol extract enough (c) containing akebin E, and approach the effect of amycin enough (b), presented obvious decrement potentiation.
Fig. 8 is containing the Morphology Effects to MCF7 breast cancer cell after the Fructus Akebiae seed ethanol extract medication 72h of akebin E (the form picture that adopts inverted microscope (400 times) to observe).Wherein: figure a is the breast cancer cell of cellar culture; Figure b is the breast cancer cell after the amycin effect of 6 μ g/mL; Figure c is the breast cancer cell after the Fructus Akebiae seed ethanol extract effect of akebin E that contains of 1.88mg crude drug/mL; Figure d be Drug level be 0.94mg crude drug/mL containing the breast cancer cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
As seen from Figure 8, after the ethanol extraction medication of Fructus Akebiae seed 1.88 crude drugs/mL, there is serious er stress in breast cancer cell, and in cell, free bubbling out now (schemed c); After amycin 6 μ g/mL medications, breast cancer cell number reduces sharply, and iuntercellular connects to dissociate (schemes b); Containing the Fructus Akebiae seed ethanol extract of akebin E and amycin half amount combination separately, (figure is d) after medication, and breast cancer cell number reduces sharply, and iuntercellular connection is dissociated; Remain cavitation phenomena in cell.
Effect experiment four, the inhibitory action containing the Fructus Akebiae seed ethanol extract of akebin E to SGC7901 stomach cancer cell malignant proliferation:
Fig. 9 is Fructus Akebiae seed ethanol extract containing the akebin E inhibitory action MTT result to SGC7901 stomach cancer cell malignant proliferation.Wherein: a is the stomach cancer cell group of common cultivation, b is that Drug level is the amycin group of 6 μ g/mL, c is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.25mg crude drug/mL, d is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, e is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 2.5mg crude drug/mL, and f is that Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 3.75mg crude drug/mL.
As seen from Figure 9, Fructus Akebiae seed ethanol extract medication containing akebin E has obvious dose-effect relationship to the inhibited proliferation of stomach cancer cell, the Fructus Akebiae seed ethanol extract medication containing akebin E of 1.25mg crude drug/mL, has inhibited proliferation significantly to stomach cancer cell; Be better than the amycin (b) of positive control drug 6 μ g/mL as 2.5mg crude drug/mL containing its inhibited proliferation after Fructus Akebiae seed ethanol extract (e) medication of akebin E.
Figure 10 is Fructus Akebiae seed ethanol extract and the inhibitory action MTT result of chemotherapeutics coupling to SGC7901 stomach cancer cell malignant proliferation containing akebin E.Wherein: a is common cultivation stomach cancer cell group, b is that Drug level is the amycin group of 6 μ g/mL, c Drug level is the Fructus Akebiae seed ethanol extract group containing akebin E of 1.88mg crude drug/mL, and d is that Drug level is the drug combination group of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin containing akebin E of 0.94mg crude drug/mL.
As seen from Figure 10, containing the Fructus Akebiae seed ethanol extract of akebin E and amycin separately half amount combine (d) medication stomach cancer cell had to obvious inhibited proliferation, approach containing the Fructus Akebiae seed ethanol extract enough (c) of akebin E and the effect of amycin enough (b), can present the synergism of obvious decrement potentiation.
Figure 11 is containing the Morphology Effects to SGC7901 gastric cancer after the Fructus Akebiae seed ethanol extract medication 72h of akebin E (the form picture that adopts inverted microscope (400 times) to observe).Wherein: figure a is the stomach cancer cell of cellar culture; Figure b is the stomach cancer cell after the amycin effect of 6 μ g/mL; Figure c is the stomach cancer cell after the Fructus Akebiae seed ethanol extract effect of akebin E that contains of 1.88mg crude drug/mL; D figure be Drug level be 0.94mg crude drug/mL containing the stomach cancer cell after the drug combination group effect of the Fructus Akebiae seed ethanol extract+3 μ g/mL amycin of akebin E.
As seen from Figure 11,1.88mg crude drug/mL contains after the Fructus Akebiae seed ethanol extract medication of akebin E, and serious er stress has appearred in stomach cancer cell mostly, and (figure c) in endochylema, to be covered with cavity; After amycin 6 μ g/mL medications, stomach cancer cell swelling, decreased number, (figure is b) in indivedual cells, also to occur cavity; Fructus Akebiae seed ethanol extract and amycin containing akebin E are partly measured after composite reagent separately, and the metamorphosis that stomach cancer cell occurs being similar to after the enough medications of amycin (is schemed d).
Embodiment 3: preparation is containing the Fructus Akebiae seed n-butanol extraction position of akebin E
Good to visual flow of solution containing the Fructus Akebiae seed ethanol extract of akebin E with deionized water dilution, without thickness state, then use the petroleum ether extraction 3 times of 1:1 volume, gained water extracts 3 times by the ethyl acetate of 1:1 volume again, gained water is used the n-butanol extraction 3 times of 1:1 volume again, collect n-butanol extraction phase, reclaim under reduced pressure n-butyl alcohol, until n-butyl alcohol is waved to the greatest extent, obtains the Fructus Akebiae seed n-butanol extraction position containing akebin E.
Embodiment 4: the effect experiment that contains the Fructus Akebiae seed n-butanol extraction position of akebin E
Adopt distilled water to dissolve and contain the Fructus Akebiae seed n-butanol extraction position of akebin E and regulate pH to 7.0, then add distilled water and be settled to 1g crude drug/mL; The sterilizing of cell culture condition sucking filtration, subpackage be stored in-80 ℃ for subsequent use; Tumor cell is carried out to 96 orifice plate paving wares; Spend the night after cell attachment, carry out administration; After administration 24,48,72h, adopt inverted microscope to detect and take pictures respectively.
Effect experiment one, the inhibitory action containing the Fructus Akebiae seed n-butanol extraction position of akebin E to SMMC7721 hepatoma carcinoma cell malignant proliferation:
Figure 12 is the inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed n-butanol extraction position of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 0.625mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 1.25mg crude drug/mL, and d is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 1.88mg crude drug/mL.
As seen from Figure 12: Drug level is causing hepatoma carcinoma cell all cells containing the Fructus Akebiae seed n-butanol extraction position of akebin E serious er stress occurring of 0.625mg crude drug/mL, is covered with cavity in endochylema; While reaching 1.25mg crude drug/mL containing the Drug level at the Fructus Akebiae seed n-butanol extraction position of akebin E, hepatoma carcinoma cell starts to occur cell pyknosis, de-endochylema.
Effect experiment two, the inhibitory action containing the Fructus Akebiae seed n-butanol extraction position of akebin E to HepG2 hepatoma carcinoma cell malignant proliferation:
Figure 13 is the inhibitory action comparison diagram to HepG2 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed n-butanol extraction position of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is 0.45mg/mL G418 group, c is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 0.47mg crude drug/mL, and d is that Drug level is the drug combination group of 0.24mg crude drug/mL containing Fructus Akebiae seed n-butanol extraction position group+0.225mg/mL G418 of akebin E.
As seen from Figure 13: Drug level is causing hepatoma carcinoma cell all cells containing the Fructus Akebiae seed n-butanol extraction position of akebin E serious er stress occurring of 0.47mg crude drug/mL, is covered with cavity in endochylema; Containing Fructus Akebiae seed n-butanol extraction position and the G418 half amount combination separately of akebin E, there is obvious pyknosis, necrosis and apoptosis in hepatoma carcinoma cell, and part cell is visible serious er stress still.
Effect experiment three, the inhibitory action containing the Fructus Akebiae seed n-butanol extraction position of akebin E to Huh7 hepatoma carcinoma cell malignant proliferation:
Figure 14 is the inhibitory action comparison diagram to Huh7 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed n-butanol extraction position of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is 0.45mg/mL G418 group, c is that Drug level is the Fructus Akebiae seed n-butanol extraction position group containing akebin E of 0.625mg crude drug/mL, and d is that Drug level is the drug combination group of 0.31mg crude drug/mL containing Fructus Akebiae seed n-butanol extraction position group+0.225mg/mL G418 of akebin E.
As seen from Figure 14: Drug level is causing hepatoma carcinoma cell all cells containing the Fructus Akebiae seed n-butanol extraction position of akebin E serious er stress occurring of 0.625mg crude drug/mL, is covered with cavity in endochylema; Containing Fructus Akebiae seed n-butanol extraction position and the G418 half amount combination separately of akebin E, there is obvious pyknosis, necrosis and apoptosis in hepatoma carcinoma cell, and part cell is visible serious er stress still.
Embodiment 5: preparation is containing the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E
To contain D101 macroporous resin adsorption post on the Fructus Akebiae seed n-butanol extraction position of akebin E (column volume and crude drug calculate in 5:1 ratio), the ethanol water of water～95vol% carries out gradient elution successively; Get the eluent of the ethanol water of 50vol%, reclaim under reduced pressure, until ethanol is waved to the greatest extent, obtains the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E.
Embodiment 6: the effect experiment that contains the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E
Adopt distilled water to dissolve and contain the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E and regulate pH to 7.0, then add distilled water and be settled to 1g crude drug/mL; The sterilizing of cell culture condition sucking filtration, subpackage be stored in-80 ℃ for subsequent use; Tumor cell is carried out to 96 orifice plate paving wares; Spend the night after cell attachment, carry out administration; After administration 24,48,72h, adopt inverted microscope to detect and take pictures respectively.
Effect experiment one, the inhibitory action containing the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E to SMMC7721 hepatoma carcinoma cell malignant proliferation:
Figure 15 is the inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is the Fructus Akebiae seed macroporous resin adsorption was separated part hyte containing akebin E of 0.75mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed macroporous resin adsorption was separated part hyte containing akebin E of 1mg crude drug/mL, and d is that Drug level is the Fructus Akebiae seed macroporous resin adsorption was separated part hyte containing akebin E of 2mg crude drug/mL.
As seen from Figure 15, Drug level be 0.75mg crude drug/mL containing the causing hepatoma carcinoma cell part cell containing the Fructus Akebiae seed macroporous resin adsorption was separated part of akebin E serious er stress phenomenon occurred of akebin E, in endochylema, there is cavity; In the time that the Drug level of the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E containing akebin E reaches 1mg crude drug/mL, nearly all cell has all produced serious er stress.In the time that the Drug level of the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E containing akebin E reaches 2mg/mL, hepatoma carcinoma cell starts to occur pyknosis, death.
Embodiment 7: preparation is containing the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E
Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E is mixed after sample (mass ratio of Fructus Akebiae seed macroporous resin adsorption was separated part and silica white is 1:3) with dissolve with methanol and silica white (100-200), reclaim under reduced pressure methanol, dry, upper silica gel (200-300) chromatographic column (mass ratio of silica gel and Fructus Akebiae seed macroporous resin adsorption was separated part is 20:1), with dichloromethane and methanol by volume content be that 100%:0～0:100% carries out gradient elution; Get the 3rd eluting part, reclaim under reduced pressure methanol, dry, obtains the Fructus Akebiae seed silicagel column adsorption chromatography separated part containing akebin E.
Embodiment 8: the effect experiment that contains the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E
Adopt distilled water to dissolve and contain the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E and regulate pH to 7.0, then add distilled water and be settled to 1g crude drug/mL; The sterilizing of cell culture condition sucking filtration, subpackage be stored in-80 ℃ for subsequent use; Tumor cell is carried out to 96 orifice plate paving wares; Spend the night after cell attachment, carry out administration; After administration 24,48,72h, adopt inverted microscope to detect and take pictures respectively.
Effect experiment one, the inhibitory action containing the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E to SMMC7721 hepatoma carcinoma cell malignant proliferation:
Figure 16 is the inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation containing the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E of various dose.In figure: a is ordinary cells matched group, b is that Drug level is the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte containing akebin E of 2mg crude drug/mL, c is that Drug level is the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte containing akebin E of 4mg crude drug/mL, and d is that Drug level is the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte containing akebin E of 8mg crude drug/mL.
From the comparing result of Figure 16, Drug level is causing hepatoma carcinoma cell part cell containing the Fructus Akebiae seed silicagel column adsorption chromatography separated part hyte of akebin E serious er stress phenomenon having occurred of 2mg crude drug/mL, occurs cavity in endochylema; When Drug level reaches 4mg crude drug/mL, there is serious er stress phenomenon in nearly all cell; When Drug level reaches 8mg crude drug/mL, there are pyknosis, the phenomena of mortality in cell.
Embodiment 9: prepare akebin E
Will be containing ODS reversed-phase silica gel chromatography post (mass ratio of ODS reverse phase silica gel and Fructus Akebiae seed silicagel column adsorption chromatography separated part is 40:1) on the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E, carry out gradient elution with methanol aqueous solution～absolute methanol of 5vol%; Get the 1st eluting part, reclaim under reduced pressure methanol, dry, obtains akebin E.
Embodiment 10: the effect experiment of akebin E
Adopt distilled water to dissolve akebin E and also regulate pH to 7.0, then add distilled water and be settled to 1g crude drug/mL(molecular weight and be converted to μ M after clear and definite); The sterilizing of cell culture condition sucking filtration, subpackage be stored in-80 ℃ for subsequent use; Tumor cell is carried out to 96 orifice plate paving wares; Spend the night after cell attachment, carry out administration; After administration 24,48,72h, adopt inverted microscope to detect and take pictures respectively.
The inhibitory action of effect experiment one akebin E to SMMC7721 hepatoma carcinoma cell malignant proliferation:
Figure 17 is the akebin E of the various dose inhibitory action comparison diagram to SMMC7721 hepatoma carcinoma cell malignant proliferation.In figure: a is ordinary cells matched group, b is that Drug level is the akebin E group of 30 μ M, and c is that Drug level is the akebin E group of 60 μ M, and d is that Drug level is the akebin E group of 120 μ M.
From the comparing result of Figure 17, Drug level is that the akebin E of 30 μ M produces serious er stress phenomenon to part cell in hepatoma carcinoma cell, occurs cavity in endochylema; When Drug level reaches 60 μ M, nearly all cell produces serious er stress phenomenon, and has part cell generation pyknosis.
Adopt Agilent1260HPLC to carry out high performance liquid chromatography detection, chromatographic column: Kromasil ODS4.6 × 250,5 μ m, evaporating temperature: 70 ℃, atomization temperature: 40 ℃, Gas:1.00, mobile phase is acetonitrile, gradient elution: 0～3min5%, 3～18min35%～75%, detects and determines that this position is single chemical composition through high performance liquid chromatography.
Figure 18 is the high-efficient liquid phase analysis figure of akebin E.
Adopt the analyses of Ultra Performance Liquid Chromatography and level Four bar time-of-flight mass spectrometry instrument: in Ultra Performance Liquid Chromatography method, mobile phase A is 0.1% formic acid, and Mobile phase B is acetonitrile; Chromatographic column temperature is 45 ℃, and sample room temperature is 10 ℃, and flow velocity is 0.4mL/min, and sampling volume is 5 μ l.Mass spectral analysis process adopts electric spray ion source negative ion mode to detect; Mass spectrum parameter under negative ion mode is as follows: capillary voltage is 2000V, and taper hole voltage is 40V, and ion source temperature is 120 ℃, desolventizing temperature degree is 400 ℃, desolventizing throughput is 800l/h, and gas curtain throughput is 50l/h, level Four bar sweep limits m/z100-1500.The molecular weight of specifying this part is 1060.Supposition is that Hederagenin is connected with two pentoses (xylose or arabinose) and two glucoses.Molecular formula: C
52h
84o
22.
Figure 19 is ultra high efficiency liquid phase and the level Four bar time-of-flight mass spectrometry instrument analysis chart of akebin E.
Adopt BRUKER AVANCE III (400MHZ) nuclear magnetic resonance spectrometer to measure, mobile phase is pyridine, through comparing, this compound is akebin E, i.e. 3-O-β-D-xylopyranose-(1 → 2)-α-L-arabopyranose helexin-28-O-β-D-Glucopyranose .-(1 → 6)-Glucopyranose..
Figure 20 is the carbon-13 nmr spectra figure of akebin E.
Figure 21 is the carbon spectrum data of akebin E.
In Figure 21,13C NMR spectrum shows 52 carbon signals altogether, comprises 30 aglycon carbon signals.Wherein aglycon carbon signal and Caulis Hederae Sinensis aglycon are basically identical.In 13C NMR spectrum, there are 4 sugared signals, in conjunction with NMR data and UPLC analysis result, proved that it is respectively L-arabinose, D-xylose and 2 D-Glucoses.Through above-mentioned analysis, and and disclosed document [Ma Shuancheng, Chen Dechang, Zhao Shujie. Chinese yam is known sub chemical constitution study (IV). Chinese herbal medicine, 1995,26 (3): 122-124] data in compare, and prove that its structure is 3-O-β-D-xylopyranose-(1 → 2)-α-L-arabopyranose helexin-28-O-B-D-Glucopyranose .-(1 → 6)-Glucopyranose..
Visible in sum: 1) akebin E has inducing cell significantly and occur the pharmacodynamic properties of serious er stress; And the place of the endoplasmic reticulum synthetic processing that is intracellular protein, its generation stress cause cell serious " dyspepsia ", function to fall sharply, and degree severe patient can direct cell death inducing or necrosis; Therefore, can akebin E as one of active component or unique active component for the preparation for the treatment of anti-tumor drug preparation, by inducing serious er stress to suppress the malignant proliferation of the kinds of tumor cells such as hepatocarcinoma, pulmonary carcinoma, breast carcinoma, gastric cancer, there is medical value; 2) in the time that akebin E and other medicine or chemotherapeutics are used in combination, tumor cell will decline because er stress makes its drug-resistant capability, accelerate its apoptosis or death, can realize decrement potentiation, reduce the toxic and side effects of medicine, will increase patient treatment chance and extend the cycle of active drug treatment, thering is extensive and important application prospect and be worth with exploitation; 3) in view of akebin E has the optionally serious er stress of inducing cell, therefore can be used as experiment reagent, be widely used in the experiment of Celluar and Molecular Biology.
Finally be necessary described herein: above embodiment is only for being described in more detail technical scheme of the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (6)
1. a medicinal usage of akebin E, is characterized in that: using akebin E as one of active component or unique active component for the preparation for the treatment of anti-tumor drug preparation.
2. the medicinal usage of akebin E as claimed in claim 1, is characterized in that: using akebin E as one of active component or unique active component for the preparation of comprising by induction er stress approach treatment anti-tumor drug preparation.
3. the medicinal usage of akebin E as claimed in claim 1 or 2, is characterized in that: described tumor at least comprises hepatocarcinoma, gastric cancer, pulmonary carcinoma, breast carcinoma.
4. the medicinal usage of akebin E as claimed in claim 1 or 2, is characterized in that: described akebin E comprises analog and the derivant based on akebin E structure conformational characteristic.
5. the medicinal usage of akebin E as claimed in claim 1 or 2, is characterized in that: described akebin E derives from the extract of chemosynthesis or Fructus Akebiae seed.
6. the medicinal usage of akebin E as claimed in claim 5, is characterized in that, is extracted the method for obtaining akebin E by Fructus Akebiae seed, comprises the steps:
A) with the ethanol water of 60vol%～95vol%, Fructus Akebiae seed is pulverized to coarse powder and carry out reflux, extract, 2～3 times, collect extracting solution, filtration, decompression recycling ethanol, obtain the Fructus Akebiae seed ethanol extract containing akebin E;
B) good to visual flow of solution containing the Fructus Akebiae seed ethanol extract of akebin E with deionized water dilution, without thickness state, then respectively extract 2～3 times with petroleum ether, ethyl acetate, the n-butyl alcohol of 1:1 volume successively, collect n-butanol extraction phase, reclaim under reduced pressure n-butyl alcohol, obtains the Fructus Akebiae seed n-butanol extraction position containing akebin E;
C) will contain D101 macroporous resin adsorption post on the Fructus Akebiae seed n-butanol extraction position of akebin E, the ethanol water of water～95vol% carries out gradient elution successively; Get the eluent of the ethanol water of 50vol%, carry out decompression recycling ethanol, dry, obtain the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E;
D) the Fructus Akebiae seed macroporous resin adsorption was separated part containing akebin E is mixed after sample with dissolve with methanol and silica white, reclaim under reduced pressure methanol, dry, upper silica gel column chromatography, with dichloromethane and methanol by volume content be that 100%:0～0:100% carries out gradient elution; Get the 3rd eluting part, carry out concentrating under reduced pressure, dry, obtain the Fructus Akebiae seed silicagel column adsorption chromatography separated part containing akebin E;
E) will be containing ODS reversed-phase silica gel chromatography post on the Fructus Akebiae seed silicagel column adsorption chromatography separated part of akebin E, carry out gradient elution with methanol aqueous solution～absolute methanol of 5vol%; Get the 1st eluting part, carry out reclaim under reduced pressure methanol, dry, obtain akebin E.
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| CN109966330A (en) * | 2019-05-27 | 2019-07-05 | 江西省药品检验检测研究院 | Active component, the preparation method and application for having diuresis are extracted from threeleaf akebia |
| CN110078783A (en) * | 2019-05-30 | 2019-08-02 | 江西省药品检验检测研究院 | Caulis akebiae saponin(e H and preparation method thereof |
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| JEONG SI等: "Apoptosis-inducing effect of akebia saponin D from the roots of Dispsacus asper Wall in U937 cells", 《ARCH PHARM RES》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109966330A (en) * | 2019-05-27 | 2019-07-05 | 江西省药品检验检测研究院 | Active component, the preparation method and application for having diuresis are extracted from threeleaf akebia |
| CN110078783A (en) * | 2019-05-30 | 2019-08-02 | 江西省药品检验检测研究院 | Caulis akebiae saponin(e H and preparation method thereof |
| CN110078783B (en) * | 2019-05-30 | 2021-07-23 | 江西省药品检验检测研究院 | Akebia saponin H and preparation method thereof |
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Application publication date: 20140521 |