CN102370634A

CN102370634A - Application of epigallocatechin gallate

Info

Publication number: CN102370634A
Application number: CN2011103554316A
Authority: CN
Inventors: 盛军; 郝淑美; 王宣军; 宋爽; 王涵; 于海双; 李春磊; 李冬青; 王腾飞; 黄业伟; 方崇业; 师思; 严亮; 向泽敏; 杜晓翠; 陆洋
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2011-11-10
Filing date: 2011-11-10
Publication date: 2012-03-14
Also published as: WO2013067749A1

Abstract

The invention discloses the effect target of a medicine for treating tumors and indications thereof. The medicine is extracted from tea leaves of which the safety is confirmed by eating for thousands of years. The medicine has a definite chemical structure, namely epigallocatechin gallate with the English name of (-)-Epigallocatechin gallate, EGCG for short. The research of the invention shows that the effect target of the medicine and a preparation thereof in cells is the NOTCH protein of people or animals. The medicine and the preparation thereof have the effect mechanism of suppressing the abnormal activation of an NOTCH signal. The medicine adapts to the treatment of tumors with abnormal NOTCH signals. Because the abnormity of the NOTCH signals nearly occurs in all tumor cells and the medicine and the preparation thereof also have higher safe dose, the medicine and the preparation thereof have extensive and definite market application prospect.

Description

The application of epi-nutgall acid catechin and gallate

Technical field

The present invention relates to the purposes of epi-nutgall acid catechin and gallate, relate in particular to the purposes in pharmaceutical field.

Background technology

Folium Camelliae sinensis is one of most popular in the world beverage; Have antioxidation, defying age, anti-bacteria and anti-virus, antitumor, reduce multiple efficacies such as some disease incidence; Especially in recent years research shows; Drinking tea has definite effect at aspects such as blood sugar lowering, blood fat reducing, anti-curing oncoma and AIDS, makes the health care of Folium Camelliae sinensis cause people's extensive concern.But its concrete mechanism of action is considered to very complicated always and unintelligible, action target spot is indeterminate always, has limited the application of tea on health care, drug development.

The composition of Folium Camelliae sinensis is very complicated, kind surplus the chemical compound of isolation identification has had 500 in the Folium Camelliae sinensis, and kind surplus wherein organic compound has 450, the inorganic matter nutrient also has tens kinds.In the water extract of Folium Camelliae sinensis, catechin accounts for 25%～40%, and epi-nutgall acid catechin and gallate (its abbreviation of * is EGCG, for ease of statement, below adopts abbreviation) accounts for 60% of catechin.EGCG is considered to the main active ingredient in fresh tea leaf and the green tea.

Both at home and abroad the research of Folium Camelliae sinensis effect is also concentrated in the research to EGCG.Through research for many years, it is found that EGCG pair cell, animal have tangible biological effect, especially tumor cell and tumor-bearing mice are had clear and definite GVT.People also attempt to illustrate the molecular mechanism that EGCG causes biological effect; Set forth the dynamic change of variation such as signal transmission in the cell that EGCG causes such as akt, MAPK, AMPK and body, Japanese scholar finds that in 2003 EGCG can be that plain receptor (67LR) specificity of nonconformity that 67,000 ways are paused combines to mediate some cell physiological biochemical reactions with the cell surface molecule amount.But 67LR self and intracellular biochemistry are got in touch as yet and are not illustrated, and the biochemical reactions of the EGCG of its mediation can not be contacted directly with the biologically of body, thereby limited the application of EGCG on drug development.

NOTCH is one type of transmembrane receptor that is positioned on the cell membrane, extensively is present in all known zooblasts.NOTCH mediated cell and intercellular local signal transmission and the reaction of corresponding signal cascade.Think that now NOTCH is the interactional main mechanism of iuntercellular short distance.The NOTCH gene is found in fruit bat the earliest, and the partial function disappearance causes the wing edge to be incised.The NOTCH signal path is the conservative signal transduction pathway of evolution camber, and the function of its regulating cell propagation, differentiation and apoptosis relates to nearly all tissue and organ.NOTCH has important function for neural differentiation and formation.NOTCH is abnormal expression in nearly all mankind and laboratory animal tumor tissues and cell; The NOTCH abnormal activation of tumor tissues in most cases; The NOTCH signal is dependent especially for human acute lymphoblastic leukemia cell; The simple NOTCH signal that suppresses just can make such apoptosis of leukemia, and then cures this type leukemia.The NOTCH inhibitor has been used for the leukemic clinical treatment of this type.Cerebral glioma is also very responsive to the inhibition of NOTCH signal.

Up to now, do not find natural NOTCH inhibitor as yet.But because the NOTCH signal is very important for the growth of body, the NOTCH signal of artificial exploitation transmits inhibitor and upgrades faster for cells such as intestinals that organ has very strong toxicity, has limited their clinical practice.The mankind have four kinds of NOTCH albumen, become NOTCH1, NOTCH2, NOTCH3, NOTCH4 respectively.Human NOTCH has 5 kinds of single-minded parts, is respectively JAG1, JAG2, Dll1, Dll3 and Dll4.The combination of NOTCH receptor and part can cause the activation of NOTCH on the different cells.The combination of part and NOTCH then suppresses the activation of NOTCH on the same cell.Activatory NOTCH regulates transcribing of related gene in nucleus, be the key factor of decision cell fate.NOTCH plays an important role in neural growth course especially.NOTCH can change the cell mode and the consequence that react of signal to external world.The NOTCH of nearly all tumor cell expresses variation has taken place, and they also have significant change to the response mechanism of NOTCH.The intravital oncocyte of some lymphomatosis people particularly, its NOTCH highly expresses and activation, and the activation that only suppresses NOTCH just can make cell get into the apoptosis process, makes oncocyte dead.NOTCH is that the precursor forms of 300KD is synthetic with the molecular weight; Be transported in the intracellular Golgi body after the NOTCH protein translation finishes and handle, NOTCH is by a kind of protease cutting that is called furin in Golgi body, and forming extracellular fragment (about 180kd) and film calmodulin binding domain CaM (is so-called NTM; About 80-120kd); They form a kind of heterodimer of stable bond, are transported on the cell membrane through after a series of modification, become sophisticated NOTCH.Sophisticated NOTCH combines the back to transmit the signal of regulating cell with its part.The inhibitor of NOTCH signal pathway---gamma-secretase inhibitors has been applied to the acute t cell lymphoma of clinical treatment (TALL).

Nature Medicine delivered the NOTCH inhibitor of synthetic in 2009, and the leukemia on the mouse leukemia model is had good inhibition effect.The effect of NOTCH not only is on the tumor treatment, because its participates in each process of life, it all has important effect aspect life process many.The inhibitor of NOTCH and/or conditioner in human diseases treatment and the prevention in can play more effect.

Summary of the invention

The present invention is based on following discovery and accomplishes; Be that the present invention does not directly concern human cell's a lot of biological effects and 67LR through in depth discovering EGCG; The present invention has obtained important breakthrough to this, discovery possibly exist EGCG action target spot and the mechanism of action except that 67LR.

The present invention has disclosed the clear and definite molecular target that EGCG acts on cell first, i.e. the NOTCH receptor on animal cell membrane surface, and EGCG is through the Signal Regulation cell that suppresses NOTCH, the vital movement of body.

The present invention confirms through test:

1.EGCG making the NOTCH albumen of cell surface at short notice degrades rapidly.After adding EGCG, in 0～60 minute, the NOTCH1 of EGCG and surface of cell membrane, 2,3,4 receptor generation specific bond cause the rapid minimizing of surface of cell membrane NOTCH.

2.EGCG suppressed the maturation process of NOTCH, made the NOTCH receptor of surface of cell membrane continue to be in the relative deficiency state.Show as rolling up of the interior NOTCH precursor of tumor cell.

NOTCH4.EGCG can recover after EGCG removes the inhibitory action of NOTCH signal.

3.EGCG can be used for treating tumor, especially acute lymphoma, cerebral glioma to the inhibitory action of NOTCH, diseases such as type 2 diabetes mellitus.

On this basis, the object of the present invention is to provide the new purposes of described EGCG, EGCG promptly is provided the purposes in pharmacy and related field.Simultaneously, EGCG also being provided is the concrete application and the application of action target spot with NOTCH.

In fact, the present invention relates to the application of EGCG in the medicine of preparation treatment or prophylaxis of tumours.

Relating to EGCG is the application of modified compound in the medicine of preparation treatment or prophylaxis of tumours of host compound, and wherein, described modified compound has characteristic same or similar and that NOTCH has an effect.

Relating to EGCG is the application of modified compound in preparation treatment or prevention and NOTCH are associated the medicine of disease unusually of host compound, said disease be characterized as NOTCH abnormal expression or sudden change.

Simultaneously, the invention still further relates to EGCG is that the modified compound of host compound is in preparation treatment or prevention and NOTCH be associated the unusually food of disease or the application in health product or the article of everyday use.

The object of the invention is achieved through following technical proposals.

* except as otherwise noted, the percent that is adopted among the present invention is mass percent.

A. EGCG of the present invention is a kind of native compound, mainly is present in Folium Camelliae sinensis and the extract thereof, and structure is as shown in Figure 2, and title is epigallocatechin gallate (EGCG) ((-)-Epigallocatechin gallate).

Research of the present invention shows; The action target spot of EGCG on cell is the NOTCH protein receptor that is positioned at the eukaryotic cell membrane surface; These receptors comprise NOTCH1, NOTCH2, NOTCH3, the NOTCH4 of people and mammalian cell surface; The N albumen on insect cell surface, the Lin12 and the GLP1 albumen on elegans cell surface.

B. deep research also shows, is chemical compound after the modification of host compound with said chemical compound, and they have same or similarly and the NOTCH characteristic of having an effect, and is used for treating disease such as tumor or is used as preventative preparation.Method of modifying includes but not limited to methylate, phosphorylation, sulphation, acetylation, polymer, formation complex or chelate or the like; Increase or reduce a certain functional group and do not change itself and the interactional character of NOTCH.

C. chemical compound after described chemical compound and the modification thereof, its treatment tumor scope includes but not limited to lymphoma, liver neoplasm, cerebral glioma, digestive tract tumor.It can be used for other and the NOTCH treatment of diseases that is associated unusually.These diseases be characterized as NOTCH abnormal expression or sudden change.

D. with described chemical compound the material medicine and the preparation thereof of main component or important composition.It is characterized in that: contain EGCG or its modification back chemical compound effect and possibility effect thereof NOTCH.The preparation scope includes but not limited to tablet, injection, intravenous injection, spray, lotion, inhalant, granule, dispersant, pill, unguentum, patch.

E. the food and the beverage that contain said chemical compound composition.It is characterized in that EGCG or its modification back chemical compound effect and possibility effect thereof to NOTCH.The scope of food and beverage includes but not limited to general food, beverage, health food, healht-care article, have the food of health care, have the beverage of health care.

F. the daily domestic chemical products such as cosmetics, bath article that contains said chemical compound composition.It is characterized in that EGCG or its modification back chemical compound effect and possibility effect thereof to NOTCH.The scope of daily family product includes but not limited to soap, detergent, facial film, hair shampoo article, various cosmetics, bath gel etc.

G. the domestic pesticide, pesticide, the chemical fertilizer that contain said chemical compound composition.It is characterized in that EGCG or its modification back chemical compound effect and possibility effect thereof to NOTCH.The scope of pesticide, insecticide includes but not limited to household sprays, lotion, and the farmland is with Emulsion, oil preparation, water preparation, suspensoid, powder, seed dressing, various agrochemicals etc.

H. the invention provides the method for preparing of described EGCG, this method adopts following step:

Prepare millet paste with Pu'er raw tea or common green tea, obtain highly purified EGCG through ethyl acetate extraction, column chromatography for separation.

Compared with prior art, the present invention has following obvious improvement:

The present invention discloses action target spot and the indication thereof of EGCG as anti-tumor medicine first.This medicine is from being to extract the safe Folium Camelliae sinensis through thousands of year edible confirmations.This medicine has clear and definite chemical constitution, and this medicine and preparation thereof the action target spot on cell is the NOTCH albumen of humans and animals.The mechanism of action of this medicine and preparation thereof is to suppress the abnormal activation of NOTCH signal.This medicine is adapted to the tumor treatment of NOTCH abnormal signal.The NOTCH abnormal signal occurs in view of nearly all tumor cell, and this medicine and preparation thereof have higher safe dose, make this medicine and preparation thereof have extensive and clear and definite market application foreground.

Description of drawings

Fig. 1 is the amount that EGCG can reduce ripe NOTCH on the cell membrane (is example with NOTCH2).Increase the amount of immaturity NOTCH precursor.The 0th, the situation of NOTCH2 in the cell when not adding EGCG, 1-6 is the situation of the interior NOTCH2 of cell behind the adding EGCG different time.

Fig. 2 is the chemical structural formula of EGCG.

Fig. 3 is the amount that EGCG can reduce ripe NOTCH on human peripheral blood cell's film (is example with NOTCH2).The 0th, intracellular situation when not adding EGCG.1, the 2, the 3rd, intracellular situation when adding behind the EGCG different time.

Fig. 4 is the state diagram of the relative expression quantity of NOTCH target gene.After using EGCG to handle, NOTCH target gene expression amount has tangible reduction with respect to untreated fish group.

To be EGCG can reverse after removing EGCG the effect of NOTCH on human peripheral blood cell's film (is example with NOTCH2) Fig. 5.1, is the situation of the interior NOTCH2 of cell when not adding EGCG; The 2nd, the situation of NOTCH2 in the cell behind the adding EGCG; The 3rd, EGCG is removed after 30 minutes the situation of NOTCH2 in the cell.

Fig. 6 is the design sketch that EGCG can promote people's acute lymphoma cell line Jurkat cell death effectively.

The specific embodiment

In order to make the object of the invention, technical scheme and advantage clearer; Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation; Should be appreciated that specific embodiment described herein only in order to explain the present invention, does not limit protection scope of the present invention.

Embodiment 1

---EGCG is to the effect of Jurkat surface of cell membrane NOTCH receptor

Jurkat is people's acute lymphoma (leukemic a kind of) cell line that can go down to posterity and cultivate.Use contains RPMI 1640 culture medium (GIBCO) of 10% hyclone (Hyclone), 1% mycillin and cultivates the Jurkat cell.With the dissolving of serum-free RPMI 1640 culture medium, concentration is 20mg/ml with EGCG.

The Jurkat cell that goes down to posterity after 24 hours, is added EGCG respectively to different final concentrations with the serum-free medium processing, and contrast adds isopyknic serum-free medium, and each concentration is used 2,000 ten thousand (2*10 ⁷) individual cell experimentizes.Handled 30 minutes.1200 rev/mins of centrifugal 5 minutes collecting cells use full cell pyrolysis liquid cell lysis then, and the collecting cell whole protein is measured protein concentration.

Use 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated cell lysate, the lysate applied sample amount 60ug protein of each processing.100 volts of constant voltage electrophoresis 90 minutes.The albumen that electrophoretic separation finishes is transferred on the nitrocellulose filter through extra electric field.150 milliamperes of constant currents were shifted 150 minutes.Shift the nitrocellulose filter finish after 60 minutes in room temperature with 5% bovine serum albumin closed protein; Use the celluloid film reaction 120 minutes that the anti-people NOTCH1-4 of rabbit antibody (Cell Signal Technology), mouse anti human ubiquitin antibody and sealing finish respectively; Normal saline with phosphate-buffered washs nitrocellulose filter 5 times, each 5 minutes then.After washing finishes, with the goat anti-rabbit antibody (HRP Conjugated goat anti-rabbit IgG) of horse peroxidase labelling and celluloid film reaction 60 minutes.Normal saline with phosphate-buffered after reaction finishes washs nitrocellulose filter 5 times, each 5 minutes.After washing finishes, handle (GE HealthCare) with the ECL chemical luminescence reagent kit with reference to description.Make public with exograph in the darkroom.The result is as shown in the figure.EGCG handles in back 30 minutes, and the proteic amount of NOTCH is compared remarkable minimizing with the control sample that does not carry out any processing on the cell membrane, and the amount of NOTCH precursor rolls up simultaneously.The result sees Fig. 1.

Embodiment 2

Get unit of concentrated human peripheral lymphocyte (PBMC) of fresh separated.After 4 times of serum-free MI1640 culture medium dilutions, personnel selection lymphocyte separation medium separated plasma, leukocyte and erythrocyte.Specific practice is: in disposable 50 milliliters of aseptic apyrogeneity centrifuge tubes, add 20 milliliters of human lymphocyte separating mediums; Concentrated human peripheral lymphocyte after adding dilution on the liquid level of human lymphocyte separating medium is 25 milliliters carefully, avoids the interface of two liquid disturbance to take place to form interface clearly.In room temperature with low speed centrifuge with 2200 rpms centrifugal 25 minutes (about 850g).Not using the brake function of centrifuge to make it nature slowly is decelerated to and stops.With the faint yellow blood plasma sucking-off in upper strata, marked is used as the autologous plasma that uses when leukocyte is cultivated with aseptic pyrogen-free Dispette.The leukocyte intermediate layer sucking-off that certain muddiness is arranged between two-layer liquid level is put in new 50 milliliters of centrifuge tubes, adds 20 milliliters of RPMI 1640 serum-free mediums, with behind the leukocyte mixing 1500 rpms centrifugal 5 minutes, abandon supernatant.The deposition of pipe bottom is isolating human peripheral lymphocyte, repeats with 20 milliliters of serum-free RPMI1640 washed cell depositions once.Add RPMI 1640 culture medium that contain 5% autologous plasma and 1% mycillin then, with automatic cell counter counting cells concentration.

Each is handled sample and uses 1,000 ten thousand human peripheral leucocytes.With containing EGCG (the EGCG working concentration is 0.1-100ug/ml) and 5% autoserous RPMI 1640 culture medium culturing cells after 30 minutes, centrifugal collecting cell.With the cell pyrolysis liquid cell lysis that contains protease inhibitor and inhibitors of phosphatases, be used for immunoblotting behind the protein quantification and detect.Concrete grammar is with reference to embodiment 1.The result sees Fig. 3.

The result shows: EGCG reduces the amount of the ripe NOTCH of human peripheral blood cell's surface of cell membrane significantly.

Embodiment 3

With containing EGCG (the EGCG working concentration is 0.1-100ug/ml) and 5% autoserous RPMI1640 culture medium culturing cell after 5-120 minute, centrifugal collecting cell.Use RNA to extract test kit (QigenInc.) and extract total RNA, transcribing with reverse transcription test kit (TAKARA Inc.) becomes complementary DNA (cDNA).Listed primer sequence carries out Real-time PCR detection NOTCH target gene expression situation in the use sequence table.Showing as the notch target gene expression suppresses.The result sees Fig. 4.

The result shows: EGCG can make the expression of target gene amount of NOTCH reduce, and prompting NOTCH signal has received inhibition.

Embodiment 4

Use the RPMI 1640 culture medium culturing Jurkat cells that contain EGCG (the EGCG working concentration is 0.1-100ug/ml) and 0-10% hyclone after 30 minutes; With cell with centrifugal 5 minutes collecting cells of 1200 rev/mins of kinds; Discard and contain the EGCG culture fluid, with behind the normal saline re-suspended cell of phosphate-buffered once more with cell with centrifugal 5 minutes of 1200 rev/mins of kinds with the residual EGCG of flush away.Spend cultured cells 30 minutes with the RPMI1640 culture medium that does not contain EGCG 37 then.With 1200 rev/mins of centrifugal collecting cells.Do various contrasts simultaneously.With the cell pyrolysis liquid cell lysis that contains protease inhibitor and inhibitors of phosphatases, be used for immunoblotting behind the protein quantification and detect.Concrete grammar is with reference to embodiment 1.The result sees Fig. 5.

The result shows: EGCG reduces the ripe NOTCH of Jurkat cell surface significantly.After removing EGCG30 minute, the amount of the ripe NOTCH of surface of cell membrane has obtained recovery.

Embodiment 5

The JURKAT cell is cultivated according to the method for embodiment 1, added a certain amount of EGCG, the concentration of EGCG is the 0-200 mcg/ml.Measure the survival of cell behind the EGCG processing certain hour.The method of using is mtt assay.The result is as shown in Figure 6, and EGCG has promoted the death of Jurkat cell.

Embodiment 6

Is 1: 10 75 ℃～85 ℃ distilled water immersion with Pu'er raw tea or green tea with weight ratio, and heated and boiled 30 minutes is removed tea grounds.Millet paste is regulated pH=4.0 with citric acid; Adding ethyl acetate that weight is three times in millet paste shakes up the mixed liquor vibration and leaves standstill 10～25 minutes; Treat that the obvious layering of solution can see that the solution on upper strata is the brown ester layer of extraction for the first time that is this moment; Lower floor is water layer and then the extraction that brownish black is extraction for the first time, the ester layer that obtains extracting with the membrane filtration ester metafiltration liquid in 0.45 micron in aperture as the chromatography raw material.

Embodiment 7

The ethyl acetate extraction ester layer that uses embodiment 6 to obtain carries out the chromatogram purification of EGCG as raw material.Use chromatographic column: the ODS filler, diameter 30-40 micron, the chromatographic column specification is 2.8X50cm, detector is a UV-detector, detects wavelength=278nm; Mobile phase V (ethanol): V (water)=15: 85, flow velocity 20ml/min sampling volume 15ml.The chromatograph elution curve of online detection preparative column is collected EGCG according to the situation that goes out the peak.The eluate of collecting 41-70min is collected in together as the product fraction.After lyophilization, get white powder, be required high purity EGCG.

Claims

1.EGCG the application in the medicine of preparation treatment or prophylaxis of tumours.

2. be the application of modified compound in the medicine of preparation treatment or prophylaxis of tumours of host compound with EGCG, wherein, described modified compound has characteristic same or similar and that NOTCH has an effect.

3. be the application of modified compound in preparation treatment or prevention and NOTCH are associated the medicine of disease unusually of host compound with EGCG, said disease be characterized as NOTCH abnormal expression or sudden change.

4. the modified compound that with EGCG is host compound is in preparation treatment or prevention and NOTCH be associated the unusually food of disease or the application in health product or the article of everyday use.