A kind of water-soluble ampelopsin polymerTechnical field
The invention belongs to field of medicaments, in particular to one kind for treating tumour, suppressing bacterium infection, liver protection shieldLiver, hypoglycemic, reducing blood lipid water-soluble ampelopsin polymeric articles.
Background technology
Ampelopsin（AMP）Also known as dihydromyricetin, dihydromyricetin, Ampelopstin etc., it is a kind of polyphenol hydroxylFlavanonol, belongs to flavone compound（See below formula）., there is rhizome of Chinese monkshood porcelain ampelopsis, Fujian white in its aboundresources, wide material sourcesIn the plants such as tea, vine tea, its extraction and analytical method is simple, toxicity is low, bioactivity is strong, with anti-oxidant, analgesia, cough-relieving, suppressionMany pharmacological activity such as bacterium, liver protecting, hypoglycemic, reducing blood lipid, enhancing body immunity, antitumor.At present, porcelain ampelopsisThe study hotspot of element, to the In-vitro Inhibitory Effect of tumour cell, but under study for action, has in the exploitation of its antitumor activityScholar find ampelopsin body in anticancer effect it is undesirable, analysis reason may have it is following some：1. solubility property is poor；2. stabilizationProperty is poor；3. belong to flavonoid drugs, there is the shortcomings of action target spot is more, selectivity is not strong, pharmacodynamics effect is weaker.
Ampelopsin structural formula
（The entitled AMP of chemistry, molecular formula is C15H12O8）
For ampelopsin above mentioned problem, this seminar selects high molecular polymer：Gamma-polyglutamic acid（γ-PGA) and α-poly-Glutamic acid（α-PGA) connection ampelopsin, γ-PGA are a kind of natural polymers, are to pass through alpha-amido by glutamic acid repeating unitsWhat the amido link formed between γ-carboxyl was formed by connecting.It is a kind of biodegradable, nontoxic, without immunogenicity medicineCarrier.Active side chain carboxyl group higher on its strand, it is easy to and some cancer therapy drugs form polymer drug and are combinedBody, with good pharmacological profile, can be such that medicine is easily accumulated in tumor tissues by EPR effects.Therefore, γ-PGA areThe preferable pharmaceutical macromolecular material in vivo of one class.α-polyglutamic acid（α-PGA) it is Synthetic artifact, its synthetic method and characteristicRefer to patent document [14], the similar γ-PGA of its application pharmaceutically.To promote ampelopsin this natural materials earlyClinic is applied to, this seminar intends using the c-terminus that ampelopsin is connected to macromolecule carrier PGA.By the method toIn the case where ampelopsin biological agent is retained, the advantage of macromolecular compound is played, reach raising solubility, increase stabilizationProperty, biological half-life in extension body, strengthen the purpose of drug effect, the anti-cancer agent for meeting clinical demand is developed, it is cancerTreat the new prospect of developing.
Bibliography
[1] Wu Xiaoming, He Yanping, Li Song water solubility cephalotoxinum polyaminoacid esters or its salt [P] .CN：1288010A,2001-03-21.
[2] Yan Le, Cao little Hong, Wang Chunling, wait the preparation of gamma-polyglutamic acid-D- galactolipins esterification derivative-CDDP complexAnd its hepatic targeting analysis [J] Chinese biological engineering magazines, 2009,29 (5):11-16.
[3] Primary Study [D] the Guangdong of the synthesis, physicochemical property and its pharmacological activity of positive happy woods .PEG-AMP esters：Middle mountain is bigLearn, 2010.
[4] research and utilization present situation [J] Chinese patent drugs of Zhang Yousheng, Ning Zhengxiang, Hu Yanyong flavone compound dihydromyricetins,2002,24(12): 970-973.
[5]Qi S M, Xin Y Q, Guo Y T. Ampelopsin reduces endotoxic inflammationvia repressing ROS-mediated activation of PI3K/Akt/NF-κB signaling pathways[J]. International Immunopharmacology, 2012, 12, (1) : 278–287.
[6] Liu Deyu, Luo Man, Xie Bingfen, wait the antitumor action of ampelopsins to study [J] cancers, 2001,20 (12):1372 - 1375.
[7]Seong H J, Wan Joo C, Yong J C, et al. Cyto-toxic constituents fromthe bark of Salix hulteni[J]. Archives of Pharmacal Research, 2008, 31(8):978-982.
[8]G. Pasut, F.M. Veronese. Polymer–drug conjugation, recent achievementsand general strategies [J]. Prog. Polym. Sci., 2007,31:933-961.
[9] a kind of water-soluble anticancer compound-polyamino acid toxol esters of the bright of Wu Xiao [P]:CN 1164533 A.
[10] preparation [J] Chinese medicines of Liu Deyu ampelopsins micro-capsule, 2003,26 (5):355-357.
[11] Dong Qianqian, Chen Li peak the dihydromyricetins pharmacological research progress Central-South journals of [J], 2005,3 (5):295-298.
[12] foundation of all outstanding person's maleimide polyglutamic acid aspartic acid targeting peptides polymer drug carrier systems and application[D] Shanghai：East China Normal University, 2013.
[13] imperial court is consoled, what dogface, Xu Hong, waits the relative molecular mass of biosynthesis polyglutamic acids to characterize [J], Nanjing industryCollege journal, 2003（25）6：6-10.
[14] Zhao Jun, Ji Tongqin, Sun Min, the water-soluble paclitaxel polymer [P] with tumor-targeting that waits a kind of:CN103012778B。
The content of the invention
It is an object of the present invention to provide a kind of for treating tumour, antibacterial, liver protecting, hypoglycemic, reducing blood lipidWater-soluble poly glutamic acid-ampelopsin ester polymer, the hydroxyl of ampelopsin and polyglutamic acid free carboxyl group is anti-by esterificationLinkage should be formed, obtains being connected with the polyglutamic acid derivative of ampelopsin.
The invention provides a kind of polyglutamic acid derivative or its pharmaceutically acceptable salt as shown in Formulas I or Formula II,It is characterized in that：Containing with selected from poly- l- glutamic acid, poly- d- glutamic acid, poly- d-l- glutamic acid water-soluble polymer be conjugatedMedicine composition, in each repeat unit R be selected from hydroxyl or ampelopsin class group；
Formulas I (n=38~390)
(n=38~390) Formula II
One in hydroxyl, ampelopsin class group of R in each repeat unit；
Wherein ampelopsin class group sees below formula III,
Formula III.
The molecular weight of described polyglutamic acid is 5000~50,000 dalton.
N is integer, and cause the polyglutamic acid derivative molecular weight be 6000~600,000 dalton, preferablyIt is 30,000~60,000 dalton；
Described water-soluble polymer and ampelopsin 7 selected from poly- l- glutamic acid, poly- d- glutamic acid or poly- d-l- glutamic acidPosition is hydroxyl conjugated.
Composition of the present invention, it is comprising selected from poly- l- glutamic acid, poly- d- glutamic acid or poly- d-l- glutamic acid7 and the 3 ' disubstituted conjugates of position hydroxyl of water-soluble polymer and ampelopsin.
Composition of the present invention, it is comprising selected from poly- l- glutamic acid, poly- d- glutamic acid or poly- d-l- glutamic acid7 and the 5 ' disubstituted conjugation of position hydroxyl of water-soluble polymer and ampelopsin.
Composition of the present invention, said composition is dispersed in pharmaceutical carrier solution.
Composition of the present invention is used to prepare treatment patient cancer, bacterium infection, high fat of blood, hyperglycaemia, liver diseaseApplied in the medicine of disease.
Wherein described cancer is breast cancer, liver cancer, lung cancer, prostate cancer, oophoroma, malignant mela noma, stomach cancer, knotIntestinal cancer, incidence cancer or leukaemia.
Pharmaceutical composition of the present invention, wherein described conjugate contains ampelopsin is up to 32.3%.
Pharmaceutical composition of the invention, wherein described pharmaceutical composition contains 5%~50% ampelopsin.
The mass percent that ampelopsin group accounts for the polyglutamic acid derivative in the polyglutamic acid derivative is 5%~50%, preferably 20~35%；
Polyglutamic acid derivative shown in Formulas I or Formula II has three class repeat units：
1) free glutaminic acid residue, i.e. R are hydroxyl；
2) it is connected with the glutaminic acid residue of porcelain ampelopsis chlorins compound；Preferably ampelopsin passes through 7 hydroxyls and polyglutamicThe carboxyl of acid forms ester bond and is connected on the polymer long-chain of polyglutamic acid.
Method another object of the present invention is to provide above-mentioned polyglutamic acid derivative is prepared.It is used in preparation processSmall molecule polyglutamic acid can be prepared according to the method described in embodiment in the present invention, it is also possible to referred to according to prior artIt is prepared by the method in bibliography [12].
The English of the reagent used by synthesizing in the present invention is write a Chinese character in simplified form and is defined as follows：
AMP：Ampelopsin
PGA：Polyglutamic acid
DCC：Dicyclohexylcarbodiimide
EDC ：1- ethyls-（3- dimethylaminopropyls）Carbodiimide hydrochloride
NHS ：N- hydroxysuccinimides
DMAP：4- dimethylamino pyridines
DMSO：Dimethyl sulfoxide (DMSO)
Heretofore described polyglutamic acid derivative can be prepared in accordance with the following methods：
Polyglutamic acid reacts with ampelopsin under condensation reagent and catalyst action, prepares above-mentioned target polyglutamic acid and derivesThing.Reaction condensation reagent used is EDC and NHS or DCC, and catalyst is DMAP.Solvent dimethyl sulfoxide used, reaction is completedAfterwards, synthetic product separating and extracting solvent for use is ether in reaction solution.Preferred preparation method is that ampelopsin is dissolved in into twoIn first sulfoxide, DCC, DMAP stirring half an hour are added.Under strong agitation, the diformazan that solution is slowly dropped into polyglutamic acid is sub-In the solution of sulfone, reacted 24 hours at 40 DEG C.Reaction solution adds ether, and concussion, standing separation takes the subnatant of extract and separateBody, nitrogen protection lower rapid filtration under suction, drip washing, vacuum freezedrying obtains polyglutamic acid ampelopsin ester (PGA-AMP).
In the above method, condensation reagent used is DCC or EDC and/or NHS, and catalyst is DMAP.Reaction dissolvent is poleProperty aprotic solvent, selected from dimethyl sulfoxide, DMF, 1-METHYLPYRROLIDONE, preferably dimethyl sulfoxide.Separate extractionSolvent for use is taken for ether.
A further object of the present invention there is provided the Pharmaceutical composition containing above-mentioned polyglutamic acid derivative.
In Pharmaceutical composition containing above-mentioned polyglutamic acid derivative containing solvent, decentralized medium, coating, antibacterial agent andAny component such as antifungal agent and isotonic agent.The purposes that these media and reagent are used for pharmaceutically active substance is art technologyPersonnel are known.Any commonly employed medium or reagent are in addition to incompatible with the polyglutamic acid derivative, it should can be used for treatment groupIn compound.Also the active component of supplement can be added in composition.
A further object of the present invention there is provided purposes of the above-mentioned polyglutamic acid derivative in pharmacy.
Polyglutamic acid derivative in the present invention can be used for preparing the medicine for the treatment of cancer.Described cancer is mammary glandCancer, liver cancer, lung cancer, prostate cancer, oophoroma, malignant mela noma, stomach cancer, colon cancer, incidence cancer or leukaemia.PreferablyLiver cancer, breast cancer, lung cancer.
Polyglutamic acid derivative in the present invention can be used for preparing treatment bacterium infection, high fat of blood, hyperglycaemia, liver diseaseThe purposes of the medicine of disease.
The beneficial effects of the present invention are, ampelopsin is connected by polyglutamic acid main chain, retaining ampelopsinIn the case of biological agent, the advantage of macromolecular compound is played, reach raising solubility, increase stability, it is raw in extension bodyThing half-life period, improve the purpose of curative effect.
Polyglutamic acid-ampelopsin ester (PGA-AMP) composition prepared by the present invention, good water solubility, curative effect is improved.Can be withFor the preparation of antineoplastic.
Polyglutamic acid-porcelain ampelopsis cellulose ester compositions (PGA-AMP) prepared by the present invention, good water solubility, curative effect is improved.Can be withFor bacterium infection, high fat of blood, hyperglycaemia, the medicine of liver diseases preparation.
Brief description of the drawings
Fig. 1 is the uv-visible absorption spectra of γ-PGA-AMP.
Fig. 2 is the uv-visible absorption spectra of AMP.
Fig. 3 is the infared spectrum of γ-PGA-AMP.
Fig. 4 is the infared spectrum of ampelopsin.
Fig. 5 is PGA-AMP administration groups, ampelopsin (AMP) control group, solvent control group to human breast carcinoma MCF-7 tumoursThe action diagram of cell.
Fig. 6 is PGA-AMP administration groups, ampelopsin (AMP) control group, solvent control group thin to human liver cancer HepG-2 tumoursThe action diagram of born of the same parents.
Fig. 7 is PGA-AMP administration groups, ampelopsin (AMP) control group, solvent control group thin to human lung cancer A549 tumoursThe action diagram of born of the same parents.
Specific embodiment
The following examples are to prove the preferred embodiment of the present invention.Those of ordinary skill in the art should understandTechnology disclosed in example below be the inventors discovered that, the technology that can play a role very well in the present invention is implemented, becauseIt can be considered as a part preferred embodiment by this.But, according to the present invention, those of ordinary skill in the art it is also to be understood thatRoad, on the premise of present disclosure and scope is not departed from, can carry out various modifications to disclosed particular embodiment,But still can obtain same or analogous result.
Embodiment 1：The preparation of small molecule gamma-polyglutamic acid sodium salt
Take the macromolecule gamma-polyglutamic acid of 100g biosynthesis（γ-PGA, molecular weight is about 2,000,000, and synthetic method is with reference to existingThere is technical literature [12]）Water dissolves are distilled with 500ml, 3 times of absolute ethyl alcohols of volume are added, standing obtains γ-PGA precipitations, usesWater is dissolved again, ultrafiltration, removes insoluble matter, and freeze-drying is carried out to sediment, obtains white chunks γ-PGA.
Take the above-mentioned γ-PGA of 10g and be made into 2% with distilled water（The PGA of 2g adds water to 100ml）γ-PGA the aqueous solution, uses hydrochloric acidAdjust PH to 2-3 or so, HTHP（121℃、0.1MPa）Effect 20min, immediately ice bath cooling, PH to 7-8 is adjusted with NaOH, is enteredRow freeze-drying, obtains white flocculence small molecule γ-PGA sodium salts.γ-PGA sodium salt distilled water is made into 2% γ-PGA sodium saltsThe aqueous solution, 2.0 or so are recalled to using HCl by PH, using distilled water dialysis 48h.After freeze-drying being carried out to the solution after dialysis,Obtain white flock small molecule γ-PGA salt.
Embodiment 2：The synthesis of γ-PGA-AMP
Take ampelopsin（0.59g, about 1.8mmol) it is dissolved in 20ml dimethyl sulfoxide (DMSO)s, stirring and dissolving, takes two hexamethylenes at room temperatureBase carbodiimide (DCC) 0.37g（About 1.8mmol), 0.1gDMAP is added, add and contain γ-PGA（1.24g, aboutDimethyl sulfoxide solution 180ml 9.37mmol), is placed in heat-collecting magnetic stirring device, 40 DEG C, after reaction carries out 3h, is supervised using TLCCourse of reaction is surveyed, reaction carries out 24h, and monitoring result display ampelopsin is completely converted into polymer conjugate.In reaction solution10 times of ether washings are added, is shaken, 4 DEG C stand overnight, and solution is divided into two-layer, extraction, collect lower floor's pale yellow solution.By γ-PGA-AMP reaction solutions carry out freeze-drying, obtain yellow powder product 1.02g, yield 55.7%.
Polyglutamic acid ampelopsin is dissolved in polyglutamic acid AMP-Na is obtained in 0.5mol/L sodium bicarbonate solutionsSalt.Dialysis is carried out to the aqueous solution of polyglutamic acid ampelopsin sodium salt with distilled water, to remove the small impurity of molecular weight and residueSodium acid carbonate.Dialysis thing freeze-drying is obtained into white powder.
With UV absorbance detection method measure the ampelopsin in this polyglutamic acid ampelopsin sodium salt containing 32.3%(w/w)。
With similarity method increase ampelopsin used and the ratio of polyglutamic acid, the polyglutamic acid snake of generation can be improvedAmpelopsin ratio in grape element sodium salt, in synthesized polymer, ampelopsin amount is up to 50%（w/w）.
The structural characterization data of synthetic product：
The UV-Vis scans spectral data of polyglutamic acid ampelopsin ester（See accompanying drawing 1）：Maximum absorption band is located at 293.66nm,With the uv absorption spectra of AMP（See accompanying drawing 2）Compare, maximum absorption band red shift about 2nm on phenyl ring.In Fig. 1 between 200-270nmShow multiple absworption peaks of the carboxyl double bond of polyglutamic acid.
The infrared scan spectral data of polyglutamic acid ampelopsin ester：IR (KBr, cm-1)
Fig. 3 is the infrared spectrogram of γ-PGA-AMP, by infrared spectrum it can be seen that ν-CO-O-：1090cm-1、1249cm-1, carboxylic acidνC=O：1646cm-1, ester bond νC=O：1728cm-1；νC-H：2921cm-1、975cm-1、1352cm-1、1315cm-1.The lower Fig. 4 snakes of contrastThe infared spectrum of grape element, 3630cm-1The absworption peak for locating ampelopsin free hydroxyl group has disappeared, 1650cm-1- C=O the bases at placeAbsorb because the change of substituted radical is moved to 1646cm-1.These are all proved on free hydroxyl group and γ-PGA on ampelopsinCarboxyl there is esterification.
The hydrogen nuclear magnetic resonance spectrum data of polyglutamic acid ampelopsin ester：
1H NMR (500MHz, D2O)：δ=4.9 (-CH2CO-), signal peak δ=4.4 (C on the big ring of ampelopsin2- H), 4.1(C3- H), 5.6（C3-OH）5.65 and 5.9 (C6- H), 5.9 (C8- H), 6.4 (C2’,6’- H), 8.0 (NH) and 8.9(C3’,5’- OH), 8.3 (C4’- OH).Signal peak on polyglutamic acid chain：δ=1.7-2.0 (aCH2), 2.2 (bCH2), 4.0（cCH2）
Embodiment 3：Polyglutamic acid ampelopsin ester vitro cytotoxicity test
Cell line and culture：Human breast cancer cell line Bcap-37, human liver cancer cell HepG-2, human lung cancer cell A549 be incubated at containingPercentage by volume is 10% inactivation NBCS, 1x105U•L-1Penicillin and 100mg L-1Streptomysin RPMI-In 1640 culture mediums, 37 DEG C, CO are positioned over2Content is the 5% interior culture of incubator.Liquid or passage are changed in centrifugation in 2~3 days, and it is right to takeNumber growth period cell is tested.
The nutrient solution of ampelopsin serum-free is configured to concentration for 200 μ g mL-1Solution, 0.22 μm of filtering with microporous membraneIt is degerming, it is instant to match somebody with somebody.γ-PGA-AMP are also directly to be dissolved to be configured to 200 μ g mL with the nutrient solution of serum-free-1Solution experiments instituteConcentration is needed, 0.22 μm of filtering with microporous membrane is degerming, instant to match somebody with somebody.Dosing final concentration is（200、100、50、25、12.5μg•ml-1）.It is thin with the nutrient solution culture human breast cancer cell line Bcap-37s of RPMI medium 1640 containing 10% inactivation calf serum, human liver cancerBorn of the same parents HepG-2, human lung cancer cell A549, cell are inoculated in 96 well culture plates, per hole number of cells about 5 × 103It is individual, it is wet in 37 DEG CThe CO of degree 5%2Incubated overnight in gas.Cell culture is allowed under the same conditions afterwards in the above-mentioned acute drug solution of 200 μ L24h。
Cell inhibitory rate is detected with mtt assay, by PBS solution (the 20 μ l, 5 mg ml of MTT-1) be added in each hole,Cultivate 4 hours under the same conditions.At room temperature, plate is placed on ELIASA and reads data after concussion, be 497 per hole exciting lightnm.It is blank well without the hole of refinement born of the same parents only to add culture medium 200uL, other hole readings subtract the blank well numerical value, medicineTo the computational methods such as following formula of inhibition of cancer cell rate：
In formula：A：Tested sample group absorbance values；A0：Solvent control group absorbance values.
γ-PGA-AMP are shown in Table 1 to cytotoxicity result.To people when measuring γ-PGA-AMP adding consistencies for 200 μ g/mlBreast cancer cell MCF-7, human liver cancer cell HepG-2, the inhibiting rate of human lung cancer cell A549 is respectively 78.82%, 76.14%,68.84%.γ-PGA-AMP are calculated to human breast cancer cell line Bcap-37, human liver cancer cell HepG-2 and typeⅡ pneumocyte threePlant the IC of cell50It is respectively 42.2,30.6 and 54.7 μM.
γ-PGA-AMP have to human breast cancer cell line Bcap-37, human liver cancer cell HepG-2, human lung cancer cell A549's cellObvious lethal effect and as drug concentration is incremented by, kills cell ability enhancing, and sensitive concentration is 5 ~ 20 μ g/ml.γ-PGA-AMP, ampelopsin group, solvent control group compare the toxic action of tumour cell sees Fig. 5-7.
Various concentrations γ-the PGA-AMP of table 1. are to MCF-7, HepG-2 and A549 cytotoxic effect
Tab1. Effect of different concentration ofγ-PGA-AMP on the inhibitionratio
rate of cancer cell