Background technology
EGCG, NVP-XAA 723, is that extraction is refined and obtained from green tea.The main component of Green Tea Polyphenols is catechin.Wherein comprise again l-Epicatechol (EC)/NVP-XAA 723 (EGCG), L-Epicatechin gallate (ECG) etc., and EGCG accounts for 50% of catechin total amount.Research shows that EGCG has the function of stronger oxidation-resistance and preventing cancer and cardiovascular disorder, and can improve the susceptibility of cancer cells to chemotherapy, reduces the toxicity of chemotherapy to heart simultaneously.EGCG has Green Tea Extract DNA infringement simultaneously, radioprotective and ultraviolet ray, stop oil peroxidation, reduce the content of low density cholesterol, extremely-low density cholesterol and triglyceride level in serum, the signal transmission that interfere with cancer cells is survived required, suppresses the carcinogenic substance in diet, the vigor that jointly stops some carcinogenic substance with other enzymes in intestines, liver and lung and antioxidant action, remove free radical, resist the impact of pollution, Exposure to Sunlight and smoking, control skin aging and the effect such as wrinkling.Polyphenolic compound EGCG in green tea and senile dementia correlation research (Chinese pharmacology communication the 24 volume second phase in 2007) have confirmed that a kind of polyphenolic compound EGCG in green tea can prevent that A beta-amyloyd spot from gathering, by diet, supplement, will be the early effective ways of senile dementia of control.According to documents and materials, show, EGCG is in treatment cancer simultaneously, and there is stronger pharmacological action DNA plerosis, the aspect such as antitumor, can strengthen immune function, suppress the growth of liver fat and cholesterol, suppress the growth of tumour, dysentery injury, streptococcus aureus are also had to extremely strong restraining effect.EGCG is mainly used in the products such as foodstuff additive, healthcare products at present.
Because its chemical structure of the polyphenols extracting from green tea is all extremely similar to character, all there is strong polarity, general separation method is all difficult to be separated.The method of directly extracting at present epi-nutgall catechin gallic acid ester monomer from tea-polyphenol mainly contains column chromatography, high-speed counter-current partition chromatography, ionic reaction method, acetylation method etc.Chinese patent application CN1136024C discloses a kind of adverse current chromatogram method for separating and preparing of catechin, this separation method is first to prepare with ethyl acetate: ethanol: the solvent system that water forms stationary phase, moving phase carries out NVP-XAA 723, nutgall catechin gallic acid ester obtains separated, the purity of the purification thing obtaining can arrive 98%, but the amount of countercurrent chromatography flash liberation sample is little at present, separation cycle is long, is difficult to be applied to industrial production.Chinese patent CN102311419A discloses the process for refining and purifying of a kind of high-content EGCG, this method of purification is mainly to utilize ethyl acetate and sherwood oil by 1:1 mixed solution sample dissolution, with ethyl acetate-sherwood oil-acetic acid, be mixed in proportion as elutriant, raw material is through upper prop, wash-out, concentrated, crystallization, dry and obtain, the product content obtaining is greater than 98%, yield is greater than 80%, but the method preparation lysate and elutriant complex operation, the content of the product obtaining and yield effect do not reach maximization.
Hanfangchin A (having another name called Tetrrine) derives from the root of menispermaceous plants Radix stephaniae tetrandrae (Stephania tetrandra S.Moore), studies have found that hanfangchin A is a good slow channel blocking agent.Clinically be mainly used in treating early stage mild hypertension.The papillary muscles of report hanfangchin A to animal such as the gold language of the Manchus, exempt from the cardiovascular aspects such as atrium and active vein system and all present different pharmacological actions.
Senile dementia, claim again alzheimer's disease (Alzhimer ' s Disease, AD), it is a kind of degenerative disease of central nervous system, its clinical manifestation is the continuous deterioration of cognitive and memory capability, and carrying out property of activity of daily living goes down and occurs various schizophrenia symptoms and behavior disorder.Along with advancing of disease, patient can lose self care ability gradually, brings white elephant to patient's individual family and society.The pathogenesis of senile dementia is still indefinite at present, about the pathogenetic main flow theory of senile dementia, is amyloid beta cascade hypothesis.This hypothesis thinks that the abnormal degraded of amyloid beta sample precursor (APP) generates excessive aβ protein, and in brain the core of formation of deposits senile plaque, it can activate microglia, reaction causes inflammation, lesion wire plastochondria causes energy metabolism impairment, generate polyoxy freely, caused the oxidative stress infringement of body, activating cells apoptosis pathway.On the other hand, A β can also activated protein kinase, impels Protein tau Abnormal Phosphorylation, and damage cholinergic neuron causes the pathology of central cholinergic system.These pathological changes promote again generation and the abnormal stacking of A β, produce the cascade scale effect of positive regeeration, finally cause neuronic minimizing and mediator to discharge abnormal.Research EGCG to the experiment of curing senile dementia in, applicant finds unexpectedly, the successful when pharmaceutical composition that EGCG and hanfangchin A form is used for the treatment of senile dementia, when EGCG and hanfangchin A are combined for the treatment of senile dementia, two kinds of medicines can embody the generation process that significant synergy both can delay senile dementia, show significant result for the treatment of simultaneously, strengthen patient's reminiscence ability, slow down the misery of patients of senile dementia.
Summary of the invention
Low in order to solve in prior art EGCG purification content, the defect of the low existence of yield and in order to overcome the defect that current curing senile dementia medicine effect is indefinite, poisonous side effect of medicine is large.First object of the present invention is to provide the method for purification of a kind of EGCG, and it is raw material (low levels EGCG refers to that the content of EGCG is 40-50%) that the method be take the tea-polyphenol of low levels EGCG, and its step is as follows:
(1) dress post: adopting dry method that silica gel is loaded on to diameter is 5cm, and height is in the glass chromatography column of 80cm;
(2) dissolve: after the tea-polyphenol of the alcohol immersion low levels EGCG with 70%, pour into and in silicagel column, carry out chromatography;
(3) wash-out: then add eluent to carry out wash-out;
(4) collect: use thin-layer chromatography to collect the EGCG wash-out section liquid of each wash-out section liquid;
(5) concentrated: EGCG to be collected after liquid merges and concentrated, obtain EGCG medicinal extract;
(6) crystallization: EGCG medicinal extract is placed in to Erlenmeyer flask, adds 75% ethanol, then, at 65 ℃ of heating in water bath, heat while stirring, dissolve 1 h, place the crystallization of spending the night, filter, collect crystal;
(7) dry: crystal to be placed in to drying under reduced pressure under 70 ℃ of conditions, after being dried, can to obtain EGCG powder.
Through contriver, constantly study, the ethanol that the optimum extraction solvent of finding tea-polyphenol and EGCG is 70%, when the weight ratio of the addition of the tea-polyphenol of the addition of ethanol and low levels EGCG is 3 ~ 4:1, its lixiviate amount reaches maximization.Wherein chromatographic silica gel is to adopt 70~90 order chromatographic silica gels, and contriver is through a large amount of experiment discoveries, and when the weight ratio of the addition of the tea-polyphenol of the addition of silica gel and low levels EGCG is 1:5 ~ 1:7, its separating effect is best; Described eluent is that the volume ratio of ethyl acetate and sherwood oil is 3:5 ~ 5:7 mixed solvent; Suddenly the weight ratio of the addition of the addition of the ethanol in (6) and the tea-polyphenol of low levels EGCG is 1:7 ~ 9 o'clock, and its crystallization effect is best.
Compared with prior art, the invention has the advantages that: the method for purification of EGCG of the present invention is simple to operate, drying under reduced pressure temperature is low, reach energy-conservation effect, solvating agent and elutriant consumption low, play that cost is low, the effect of environmental protection; And the EGCG powder yield that the present invention obtains is high, purity is high, yield >=90%, purity >=99%.
Second object of the present invention is to provide a kind of pharmaceutical composition for the treatment of senile dementia, the EGCG obtaining according to above-mentioned pure method is used for preparing prevention or treatment senile dementia pharmaceutical composition, this pharmaceutical composition comprises EGCG and hanfangchin A, the ratio of weight and number of the own first element of EGCG and Chinese prescription is 1-5:1-5, and wherein the ratio of weight and number of the own first element of EGCG and Chinese prescription is preferably 3:2.Pharmaceutical composition of the present invention is mainly due to its restraining effect to the gathering of A β to the therapeutic action of senile dementia, and it adopts the machine-processed senile dementia coupling of different treatments can obtain beyond thought treatment or preventive effect from other.Experiment showed, that drug combination not only can strengthen the result for the treatment of of medicine, and can reduce adverse drug reaction and the resistance of body to medicine.Based on this, the invention provides a kind of pharmaceutical composition that contains EGCG and hanfangchin A for the treatment of senile dementia.Applicant finds, when this pharmaceutical composition is used for the treatment of senile dementia, two kinds of medicines can embody significant synergy (referring to the embodiment of the present invention 7 and embodiment 8), not only can delay the generation process of senile dementia, more embody significant result for the treatment of, strengthen patient's reminiscence ability, can alleviate the misery of patients of senile dementia.
Embodiment
Adopt specific embodiment to further illustrate content of the present invention below
Following content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Embodiment 1, EGCG purifying technique of the present invention are as follows:
(1) dress post: adopting dry method that the 70 order silica gel of 100 g are loaded on to diameter is 5cm, and height is in the glass chromatography column of 80cm;
(2) dissolve: add to pour into after 70% the alcohol immersion 20 g tea-polyphenol of 60ml and in silicagel column, carry out chromatography;
(3) wash-out: then adding volume ratio is that ethyl acetate-sherwood oil mixed solvent of 3:5 carries out wash-out;
(4) collect: use thin-layer chromatography to collect the EGCG wash-out section liquid of each wash-out section liquid;
(5) concentrated: EGCG to be collected after liquid merges and concentrated, obtain EGCG medicinal extract;
(6) crystallization: the Erlenmeyer flask that EGCG medicinal extract is placed in, add 75% ethanol 140ml, then, at 65 ℃ of heating in water bath, heat while stirring, dissolve 1 h, place the crystallization of spending the night, filter, collect crystal;
(7) dry: crystal is placed in to drying under reduced pressure under 70 ℃ of conditions, after being dried, can obtains 18.4 g EGCG powder, yield reaches 92%, detects its purity reach 99.3% through HPLC.
Embodiment 2, EGCG purifying technique of the present invention are as follows:
(1) dress post: adopting dry method that the 70 order silica gel of 120 g are loaded on to diameter is 5cm, and height is in the glass chromatography column of 80cm;
(2) dissolve: add to pour into after 70% the alcohol immersion 20 g tea-polyphenol of 70ml and in silicagel column, carry out chromatography;
(3) wash-out: then adding volume ratio is that ethyl acetate-sherwood oil mixed solvent of 4:6 carries out wash-out;
(4) collect: use thin-layer chromatography to collect the EGCG wash-out section liquid of each wash-out section liquid;
(5) concentrated: EGCG to be collected after liquid merges and concentrated, obtain EGCG medicinal extract;
(6) crystallization: the Erlenmeyer flask that EGCG medicinal extract is placed in, add 75% ethanol 160ml, then, at 65 ℃ of heating in water bath, heat while stirring, dissolve 1h, place the crystallization of spending the night, filter, collect crystal;
(7) dry: crystal is placed in to drying under reduced pressure under 70 ℃ of conditions, after being dried, can obtains 19 g EGCG powder, yield reaches 95%, detects its purity reach 99.6% through HPLC.
Embodiment 3, EGCG purifying technique of the present invention are as follows:
(1) dress post: adopting dry method that the 70 order silica gel of 140 g are loaded on to diameter is 5cm, and height is in the glass chromatography column of 80cm;
(2) dissolve: add to pour into after 70% the alcohol immersion 20 g tea-polyphenol of 80ml and in silicagel column, carry out chromatography;
(3) wash-out: then adding volume ratio is that ethyl acetate-sherwood oil mixed solvent of 5:7 carries out wash-out;
(4) collect: use thin-layer chromatography to collect the EGCG wash-out section liquid of each wash-out section liquid;
(5) concentrated: EGCG to be collected after liquid merges and concentrated, obtain EGCG medicinal extract;
(6) crystallization: the Erlenmeyer flask that EGCG medicinal extract is placed in, add 75% ethanol 180ml, then, at 65 ℃ of heating in water bath, heat while stirring, dissolve 1 h, place the crystallization of spending the night, filter, collect crystal;
(7) dry: crystal is placed in to drying under reduced pressure under 70 ℃ of conditions, after being dried, can obtains 18.6 g EGCG powder, yield reaches 93%, detects its purity reach 99.4% through HPLC.
Contrast experiment's example one, the refining purifying technique of a kind of high-content EGCG:
Contrast experiment's example is the embodiment mono-to embodiment five of Chinese patent CN102311419A together.
Embodiment mono-to five by embodiments of the invention 1-3 and Chinese patent CN102311419A can draw, the EGCG powder that EGCG method of purification of the present invention draws, yield and purity are all high than the yield of Chinese patent CN102311419A and purity, and the method for purification of EGCG of the present invention is simple to operate, drying under reduced pressure temperature is low, reach energy-conservation effect, solvating agent and elutriant consumption low, cost is low, environmental protection; Yield is greater than 90%, and purity is greater than 99%, is conducive to industrialized production.
Embodiment 4, a kind of pharmaceutical composition for the treatment of senile dementia:
Preparation technology:
The EGCG, hanfangchin A, dextrin and the low-substituted hydroxypropyl cellulose that take recipe quantity mix.60% ethanol of separately getting sufficient quantity, is incorporated in mixed powder, mixes rear softwood processed, by 18 mesh sieves, granulates, and 60 ℃ following dry.After completing after dry, use 22 mesh sieves carry out whole grain, sift out the fine powder in dry granular, mix, and then be mixed evenly with dry particle with the Magnesium Stearate sieving, and compressing tablet, obtains.
Embodiment 5, a kind of EGCG medicine for the treatment of senile dementia:
Preparation technology is with embodiment 4.
Embodiment 6, a kind of hanfangchin A medicine for the treatment of senile dementia:
Preparation technology is with embodiment 4.
Embodiment 7, EGCG pharmaceutical composition of the present invention are to A β
25-35due to the therapeutic action of rat Senlie dementia model
Also do not find at present AD uniqueness, animal model that generally acknowledge, desirable.Existing AD animal model has 2 classes: damaging animal model and transgenic animal model.Damaging animal model mainly comprises physics, chemistry, organism damage and autoimmunization damage model.Transgenic animal model mainly refers to single transgene or many transgenic animal models of the related genes such as the precursor sample albumen (APP) relevant with AD, lipophorin (Apo) E4, senilism albumen 1 (PSI), senilism albumen 2 (psII).Transgenic models is the focus of studying at present, but modeling process is complicated, somewhat expensive, and deficient in stability goes down to posterity.One of neuropathological feature that AD patient is main is senile plaque (sP) deposition, and A β is the main component in senile plaque.All oneself confirms the neurotoxic effect of A β to the inside and outside test of body, can cause neuronal damage and cognitive function decline, be the key factor of AD formation and development, and the AD rat model that hippocampal injection A sets up is observed the multiple pathological changes such as Abnormal Phosphorylation (being neurofibrillary tangles) of neuronal damage, A β deposition, Protein tau.
1 materials and methods
1.1 experiment material
Laboratory animal is male SD rat, and in 9~12 weeks ages of mouse, body weight 260-280 g, is provided by The 2nd Army Medical College Experimental Animal Center.A β
25-35purchased from U.S. Sigma company, Morris water maze is purchased from Shanghai Ji Liang company.
The foundation of 1.2 rat Senlie dementia models and evaluation
1.2.1 intracerebral ventricle injection A β
25-35the preparation of solution: by A β
25-35be dissolved in stroke-physiological saline solution, making A β concentration is 10 mmol/L, puts in 37 ℃ of thermostat containers, to hatch 3 d and carry out aging.
1.2.2 the making of animal model: raise under standard environment, be divided at random 2 groups: control group and model group, 10 every group.2 groups of there was no significant differences in mouse age and body weight.Animal gives adaptability and feeds after 1 week, by 2% vetanarcol intraperitoneal anesthesia for rat (40~50 mg/kg weight), be fixed on stereo brain orienting instrument, cut off crown portion hair, after iodine tincture disinfection, cut skin, with reference to < < rat brain stereotaxic atlas > >, select right side tricorn for injection target area, in bregma 1.0 mm backward, the other 1.7 mm places that open of center line, with three-edged needle, bore and open skull, expose endocranium, use again microsyringe with the speed of 15 μ m/s from vertical inserting needle 4.0 mm in brain surface, by 10 mmol/L A β
25-35solution 5 μ l slowly inject, and after let the acupuncture needle remain at a certain point 2 min, slowly remove pin, sew up the incision.Control group injects equal-volume stroke-physiological saline solution.
1.2.3 Morris water maze study of behaviour is measured: 2 groups of rats are respectively within postoperative the 10th day, starting to carry out the test of Morris water maze.Test procedure is orientation navigation test: last 5 d, front 2 d are the training adaptation phase, rear 3 d
Record achievement, if rat is found platform in 1 min, record its actual escape latency; If do not find yet platform in 1 min, by experimenter, to be drawn upper mounting plate and stopped 20 S, escape latency is recorded as 1 min.
1.2.4 the evaluation of animal model
By following table, can be found out, the escape latency of model group just obviously extends (P < 0.05 or P < 0.01) from the control group that starts for the 1st day of experimental record, and escape latency no significant difference between escape latency between model group 3 days and control group 3 days, show that the Senlie dementia model that adopts the method to set up is reliably accurate, can be for the evaluating drug effect of curing senile dementia medicine.
2 animal models and grouping administration
According to above-mentioned modeling method modeling, and control group, normal group are set, 20 every group, under standard environment, raise.It is as described below that each organizes administering mode:
Normal group: gavage gives the physiological saline of same volume;
Control group: gavage gives the physiological saline of same volume;
Model group: gavage gives the physiological saline of same volume;
EGCG group: gavage gives the physiological saline of same volume containing 15 mg/kg EGCG;
Hanfangchin A group: gavage gives the physiological saline of same volume containing 0.5 mg/kg hanfangchin A;
Compound group: gavage gives the physiological saline of same volume containing 15 mg/kg EGCG and 0.5 mg/kg hanfangchin A pharmaceutical composition;
Above-mentioned administration group respectively at modeling after administration after 10 days, within the 11st day, be recorded as the 1st day, be administered once every day, observe drinking water for animals and diet situation every day, respectively at administration the 1st day, administration the 5th day, administration the 10th day, administration the 15th day, the escape latency of rat is measured in administration for 20 days with Morris water maze study of behaviour measuring method.Measured for the last time rear execution rat.It is as shown in the table that each organizes treated rats in Morris water maze performance study of behaviour measurement result.
Each administration group of table 1 is to A β
25-35due to the result for the treatment of of rat Senlie dementia model
As can be seen from Table 1: each administration group escape latency thing significant difference of the 1st day, but along with the prolongation of administration time, the escape latency difference of each administration group rat strengthens, and wherein EGCG and compound group all have positive therapeutic action.Be embodied in:
1) the rat escape latency of EGCG group is compared and is had significant difference with model group, all significantly shortens the escape latency of rat, and wherein pharmacological agent is after 15 days, and the escape latency of its escape latency and positive drug has significant difference.
2) the rat escape latency of compound group is compared and is had significant difference with model group, and the escape latency of its rat all obviously shortens, wherein compound medicine treatment is after 15 days, and its escape latency is compared and had significant difference with single therapy group escape latency.This shows that EGCG is to A β
25-35due to therapeutic action and the hanfangchin A of rat Senlie dementia model its therapeutic action is not repelled, to senile dementia rat model, there is synergy in two kinds of medicines.
3) each group of compound single medicine group corresponding with it compared and had significant difference, this shows that EGCG of the present invention can exist with the combination of hanfangchin A significant synergy, not only on medication effect, significantly strengthen, more can improve the motion function of patients of senile dementia, and effect rapidly, accelerates patient's recovery from illness process.
The embodiment of the present invention 7 or show that the mechanism that EGCG acts on senile dementia do not conflict with the medicine that other drug acts on senile dementia, it can combine use, and can obtain the synergy in treatment.