CN103585237A - Application of Berberis fruit extract in preparation of anticancer drugs - Google Patents
Application of Berberis fruit extract in preparation of anticancer drugs Download PDFInfo
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- CN103585237A CN103585237A CN201310525980.2A CN201310525980A CN103585237A CN 103585237 A CN103585237 A CN 103585237A CN 201310525980 A CN201310525980 A CN 201310525980A CN 103585237 A CN103585237 A CN 103585237A
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- Prior art keywords
- radix berberidis
- berberidis amurensis
- berry extract
- amurensis berry
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Abstract
The invention discloses new application of a Berberis fruit extract in preparation of anticancer drugs. The extract is prepared by: taking Berberis fruits as raw materials, conducting extraction with 30%-90% ethanol, and carrying out macroporous adsorption resin purification twice to obtain the extract containing a Berberis fruit extract A and a Berberis fruit extract B. The Berberis fruit extract provided by the invention is of great significance in preparation of anticancer drugs, especially anti-liver cancer, lung cancer and gastric cancer drugs.
Description
Technical field
The invention belongs to biomedicine field, relate to the application of a kind of Chinese crude drug extract in preparing cancer therapy drug, specifically relate to the application of Radix Berberidis Amurensis berry extract in preparing cancer therapy drug.
Background technology
According in February, 2010 Ministry of Public Health statistics, annual global cancer mortality number reaches 1,000 ten thousand.At present the whole world 1/4th human mortalities' reason is due to cancer, and prediction goes down by current trend development, to the year two thousand fifty by the died that has 1/2nd, be owing to having suffered from cancer.The pernicious cancer morbidity of China, with annual 2.5-5% speed increment, has become serious harm human life's arch-criminal.Higher with sickness rate such as pulmonary carcinoma, gastric cancer, hepatocarcinoma in the middle of various types of cancers.
It is essential that the formation that blood vessel is normal structure organ betides function maintenance, and it is closely related with all diseases that human development's imbalance and Different types of etiopathogenises cause.Tumor neovascularization plays very important effect in tumorigenesis, transfer and recurrence.A series of research shows, as long as effectively suppress tumor neovascularization, just can suppress tumor and generate, shift and recurrence.In addition, the diseases such as diabetes and rheumatic arthritis have blood vessel hyperplasia, and its aberrant angiogenesis and tumor vessel are closely similar.Therefore, angiogenic inhibitor also can be used for treating the vascular proliferative diseases such as diabetes and rheumatic arthritis.In recent years, people deepen continuously to the research of tumor-blood-vessel growth mechanism, and the medicine that many antineoplastic vasculars generate is developed in succession as Avastin, Angiostatin etc., applies clinically.This class medicine not only can be for the treatment of most of solid tumors, can also be for the prevention of tumor and the treatment of Malignancy, simultaneously to the disease of other and associated angiogenesis as prevention and the treatment of diabetic renal papillary necrosis, rheumatic arthritis, psoriasis, hemangioma, atherosclerosis etc., all there is certain theory and realistic meaning.
Tumor cell in tumor can be divided into two classes.One class is common tumor cell, and a class is tumor stem cell.Common tumor cell has quick division, to the sense of antitumor susceptibility, there is no the features such as self updating ability.Therefore common tumor cell can be dead after the certain algebraically of division.Tumor stem cell has following features: generally remains static, and splitting status not, insensitive to antineoplastic agent, there is self updating ability, there is the ability of unlimited breeding.In chemotherapy of tumors, a large amount of tumor cells is killed (because responsive to chemotherapeutic), yet tumor stem cell can survive (because insensitive to chemotherapeutic).The recurrence of tumor is because chemotherapy of tumors medicine is invalid to tumor stem cell.Looking for the medicine that can effectively treat tumor stem cell is a current direction.Can the key that eradicate tumor do not lie in kill common tumor cell, and is to eradicate tumor stem cell.
The history of Chinese medicine cancer is of long standing and well established, and existing Ramulus et folium taxi cuspidatae, Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae), arsenicum, green tea, Ganoderma, Radix Ginseng, Herba Catharanthi Rosei, Radix Asparagi, Herba Scutellariae Barbatae, Radix Semiaquilegiae etc., for clinical anticancer, have been obtained some good effects so far.For a long time, people apply Traditional Thinking, theory and method research and development anticancer herbal drug, although obtained progress, produce little effect.Chinese herbal medicine becomes first-selected cancer therapy drug not yet at home and abroad so far, and the anticancer therapeutic assistant officer of existing clinical Chinese herbal medicine needs further to improve.Therefore, research and development Chinese herbal medicine cancer therapy drug is the task of top priority of the pernicious cancer for the treatment of at present, and the chemical nature of illustrating the low molecule tumour-inhibitory substance of endogenous is the key problem of this area research.
Radix Berberidis Amurensis is really the dry mature fruit of Berberidaceae plant Berberis nummularia Bge. Berberis nummularia Bge..Record in < < Drug Standard of Ministry of Public Health of the Peoples Republic of China Uigurs medicine fascicle > > in 1998.Its property is flat, have stomach invigorating and in, promoting the production of body fluid to quench thirst, the effect of heat-clearing and toxic substances removing.For dyspepsia, under dysentery is rushed down, thirsty, aphtha, pharyngitis.For China, hide the common drug of the minority areas such as illiteracy.But the basic research of relevant Radix Berberidis Amurensis fruit is still very limited, and the follow-up promotion and application of this medical material are restricted.Modern study shows, Radix Berberidis Amurensis really contains the alkaloids compositions such as berberine, jateorhizine, palmatine, and the anthocyan composition such as pelargonidin, enidin.Other chemical compositions are not quite clear; Pharmacological research has no report.
Domestic patent search result, has no Radix Berberidis Amurensis fruit Patents.
Above-mentioned document and patent etc., there is not yet Radix Berberidis Amurensis fruit or Radix Berberidis Amurensis berry extract for the preparation of the report of cancer therapy drug.
Summary of the invention
The application of a kind of Radix Berberidis Amurensis berry extract that the object of the present invention is to provide Radix Berberidis Amurensis berry extract in preparing cancer therapy drug.
The present invention is achieved through the following technical solutions:
The present invention Radix Berberidis Amurensis used is really the dry mature fruit of Berberidaceae plant Berberis nummularia Bge. Berberis nummularia Bge..
The application of Radix Berberidis Amurensis berry extract of the present invention in preparing cancer therapy drug, the preparation method of described Radix Berberidis Amurensis berry extract is:
(1) Radix Berberidis Amurensis fruit, with concentration 30%-90% ethanol, as solvent, extract temperature 50 C-95 ℃, extraction time is 1-3 time, each extraction time is 1-4 hour, each solvent load is 6-15 times of Radix Berberidis Amurensis fruit weight, filters merge extractive liquid,, reclaim ethanol, be concentrated into relative density d=1.10-1.18, filter, obtain medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, pass through nonpolar macroporous adsorption resin, first wash with water, water elution liquid directly passes through polar macroporous adsorption resin, use again the alcoholic solution eluting nonpolar macroporous adsorption resin of concentration 50%-95%, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting polar macroporous adsorption resin of concentration 50%-95% again, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A, Radix Berberidis Amurensis berry extract B, wherein one or both mix by a certain percentage, obtain Radix Berberidis Amurensis berry extract of the present invention.
The preparation method of Radix Berberidis Amurensis berry extract is:
(1) Radix Berberidis Amurensis fruit, with concentration 30%-90% ethanol, as solvent, extract temperature 50 C-95 ℃, extraction time is 1-3 time, each extraction time is 1-4 hour, each solvent load is 6-15 times of Radix Berberidis Amurensis fruit weight, filters merge extractive liquid,, reclaim ethanol, be concentrated into relative density d=1.10-1.18, filter, obtain medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, pass through nonpolar macroporous adsorption resin, first wash with water, water elution liquid directly passes through polar macroporous adsorption resin, use again the alcoholic solution eluting nonpolar macroporous adsorption resin of concentration 50%-95%, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting polar macroporous adsorption resin of concentration 50%-95% again, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
The preparation method of Radix Berberidis Amurensis berry extract is:
(1) Radix Berberidis Amurensis fruit, as solvent, extracts temperature 70 C with concentration 70% ethanol, and extraction time is 2 times, and each extraction time is 2.5 hours, and each solvent load is 12 times of Radix Berberidis Amurensis fruit weight; Filter, merge extractive liquid,, reclaims ethanol, is concentrated into relative density d=1.15, filters, and obtains medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, by D101 nonpolar macroporous adsorption resin, first wash with water, water elution liquid is directly by LSA-40 polar macroporous adsorption resin, use again the alcoholic solution eluting D101 nonpolar macroporous adsorption resin of concentration 70%, collect 70% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting LSA-40 polar macroporous adsorption resin of concentration 80% again, collect 80% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
In Radix Berberidis Amurensis berry extract of the present invention, mainly contain: berberine, jateorhizine, palmatine, magnoflorine, pelargonidin, enidin.
Radix Berberidis Amurensis berry extract preparation method characteristic of the present invention is: the nonpolar macroporous adsorption resin adopting is D101 macroporous adsorbent resin, AB-8 macroporous adsorbent resin; The polar macroporous adsorption resin adopting is LSA-40 macroporous adsorbent resin, HPD600 macroporous adsorbent resin.
Radix Berberidis Amurensis berry extract of the present invention, by the various adjuvants that add pharmaceutics to allow, makes the peroral dosage forms such as tablet on pharmaceutics, granule, capsule.
Cancer therapy drug of the present invention is anti-lung-cancer medicament, medicines resistant to liver cancer, anti-gastric cancer medicine.Radix Berberidis Amurensis berry extract of the present invention, for the preparation of cancer therapy drug, is especially prepared anti-pulmonary carcinoma, anti-hepatocarcinoma, anti-gastric cancer medicine.
Radix Berberidis Amurensis berry extract A of the present invention, Radix Berberidis Amurensis berry extract B be for the preparation of cancer therapy drug, especially anti-pulmonary carcinoma, anti-hepatocarcinoma, anti-gastric cancer medicine.
The cancer therapy drug that Radix Berberidis Amurensis berry extract of the present invention and chemical drugs or Chinese medicine or natural drug form.
The cancer therapy drug that Radix Berberidis Amurensis berry extract A, Radix Berberidis Amurensis berry extract B of the present invention and chemical drugs or Chinese medicine or natural drug form.
The present invention's exploratory development first be take the dry mature fruit of Berberidaceae plant Berberis nummularia Bge. Berberis nummularia Bge. and as raw material extracts, is prepared anticancer extract.This experimentation shows, Radix Berberidis Amurensis berry extract is high to the tumour inhibiting rate of little S180 sarcoma of rats, has as seen strong anticancer effect, and we further study its impact on angiogenesis on this basis.Blood capillary number while evaluating the objective criterion of cancer angiogenesis degree in cancerous tissue, this experiment is by experiment in mice transplantability cancer model body, the dyeing of tumor body blood capillary labelling, microvessel density shows, each extract of Radix Berberidis Amurensis fruit all can suppress the generation of cancer blood vessel, with matched group comparison, there is significant difference.The present invention measures the exercising result demonstration that Radix Berberidis Amurensis berry extract kills and wounds cancerous cell (HepG2, GLC, MFC), and its main manifestations is the cytotoxicity feature of concentration dependent form, and, along with drug dose increases, inhibitory action strengthens gradually.
The specific embodiment
Below by specific experiment example and embodiment, to Radix Berberidis Amurensis berry extract, the application in preparing cancer therapy drug is described further, but is not limited to the present invention.
Embodiment 1: Radix Berberidis Amurensis berry extract and monomeric compound preparation
(1) Radix Berberidis Amurensis fruit 15kg, as solvent, extracts temperature 70 C with concentration 70% ethanol, and extraction time is 2 times, and each extraction time is 2.5 hours, and each solvent load is 12 times of Radix Berberidis Amurensis fruit weight; Filter, merge extractive liquid,, reclaims ethanol, is concentrated into relative density d=1.15, filters, and obtains medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, by D101 nonpolar macroporous adsorption resin, first wash with water, water elution liquid is directly by LSA-40 polar macroporous adsorption resin, use again the alcoholic solution eluting D101 nonpolar macroporous adsorption resin of concentration 70%, collect 70% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting LSA-40 polar macroporous adsorption resin of concentration 80% again, collect 80% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
Radix Berberidis Amurensis berry extract carries out silica gel column chromatography, Preparative TLC chromatograph and Sephadex LH mono-2O column chromatography, obtains respectively sloughing off berberine, jateorhizine, palmatine, magnoflorine, pelargonidin, enidin.The chemical constitution of each compound is all through wave spectrum means confirmations such as mass spectrum and nuclear magnetic resonance, NMR above, and purity detects and is all greater than 98% through high performance liquid chromatography.
Embodiment 2: Radix Berberidis Amurensis berry extract and monomeric compound preparation
(1) Radix Berberidis Amurensis fruit 20kg, as solvent, extracts 95 ℃ of temperature with concentration 30% ethanol, and extraction time is 1 time, and each extraction time is 4 hours, and each solvent load is 15 times of Radix Berberidis Amurensis fruit weight; Filter, merge extractive liquid,, reclaims ethanol, is concentrated into relative density d=1.18, filters, and obtains medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, by AB-8 nonpolar macroporous adsorption resin, first wash with water, water elution liquid is directly by HPD600 polar macroporous adsorption resin, use again the alcoholic solution eluting AB-8 nonpolar macroporous adsorption resin of concentration 50%, collect 50% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting HPD600 polar macroporous adsorption resin of concentration 50% again, collect 50% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
Radix Berberidis Amurensis berry extract carries out silica gel column chromatography, Preparative TLC chromatograph and Sephadex LH mono-2O column chromatography, obtains respectively sloughing off berberine, jateorhizine, palmatine, magnoflorine, pelargonidin, enidin.The chemical constitution of each compound is all through wave spectrum means confirmations such as mass spectrum and nuclear magnetic resonance, NMR above, and purity detects and is all greater than 98% through high performance liquid chromatography.
Embodiment 3: Radix Berberidis Amurensis berry extract and monomeric compound preparation
(1) Radix Berberidis Amurensis fruit 30kg, as solvent, extracts temperature 50 C with concentration 90% ethanol, and extraction time is 3 times, and each extraction time is 1 hour, and each solvent load is 6 times of Radix Berberidis Amurensis fruit weight; Filter, merge extractive liquid,, reclaims ethanol, is concentrated into relative density d=1.10, filters, and obtains medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, by D101 nonpolar macroporous adsorption resin, first wash with water, water elution liquid is directly by HPD600 polar macroporous adsorption resin, use again the alcoholic solution eluting D101 nonpolar macroporous adsorption resin of concentration 95%, collect 95% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting HPD600 polar macroporous adsorption resin of concentration 95% again, collect 95% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
Radix Berberidis Amurensis berry extract carries out silica gel column chromatography, Preparative TLC chromatograph and Sephadex LH mono-2O column chromatography, obtains respectively sloughing off berberine, jateorhizine, palmatine, magnoflorine, pelargonidin, enidin.The chemical constitution of each compound is all through wave spectrum means confirmations such as mass spectrum and nuclear magnetic resonance, NMR above, and purity detects and is all greater than 98% through high performance liquid chromatography.
Embodiment 4: the preparation of Radix Berberidis Amurensis berry extract tablet
Get embodiment 1 Radix Berberidis Amurensis berry extract 320g, add starch 50g, mix, granulate, dry, sieve, add microcrystalline Cellulose 23g, magnesium stearate 2.0g, mixes, and is pressed into 1000, obtains Radix Berberidis Amurensis berry extract tablet.
Embodiment 5: the preparation of Radix Berberidis Amurensis berry extract capsule
Get embodiment 2 Radix Berberidis Amurensis berry extract 245g, add starch 60g, mix, granulate, dry, granulate, sieves, and encapsulated 1000, obtains Radix Berberidis Amurensis berry extract capsule.
Embodiment 6: the preparation of Radix Berberidis Amurensis berry extract granule
Get embodiment 3 Radix Berberidis Amurensis berry extract 265g, add dextrin 50g, starch 50g, mixes, granulate, and granulate,, obtain Radix Berberidis Amurensis berry extract granule.
Experimental example 1: Radix Berberidis Amurensis berry extract is to mice transplantability S
180sarcoma vascularization inhibitory action
Method: murine sarcoma S
180tumor strain, the abdominal cavity inoculation of going down to posterity.Until ascites, grow when vigorous, extract ascites out, cell counting, adjustment cell concentration is 2*10
7individual cells/ml, at mice oxter sc S
180sarcoma cell, every inoculation 0.2ml.
Laboratory animal and tumor strain: kunming mice, male and female half and half, in age in 6-8 week, body weight (20 ± 2) g ，You Tongji Medical College, Huazhong Science and Technology Univ. Experimental Animal Center provides.Murine sarcoma S
180by Wuhan University's Chinese Typical Representative culture collection center, provided.
Medicine: Radix Berberidis Amurensis berry extract, Radix Berberidis Amurensis berry extract A, Radix Berberidis Amurensis berry extract B are for to prepare gained by embodiment 1 preparation method, and lot number is respectively: 20110506,20110507,20110508, be configured to respectively 0.1g/ml normal saline solution.
Treatment and grouping: 120 mices were divided into 5 groups in inoculation the same day at random: blank group, Radix Berberidis Amurensis berry extract group, Radix Berberidis Amurensis berry extract A group, Radix Berberidis Amurensis berry extract B group.Radix Berberidis Amurensis berry extract group, Radix Berberidis Amurensis berry extract A group, Radix Berberidis Amurensis berry extract B group, give respectively injection 25ml/kg.Matched group only gives normal saline 0.2ml/.After treatment 10d, put to death mice, peel off tumor body and weigh, by formula, calculate tumour inhibiting rate: tumour inhibiting rate=(the average tumor weight of the average tumor weight-experimental group of matched group) average tumor heavy * 100% of/matched group.
Blood capillary dyeing: SABC SABC method dyeing blood vessel, using miniature blood vessel staining kit (the relevant Factor VIII of primary antibodie behaviour) is Wuhan doctor's moral biotech firm product.Each group of Radix Berberidis Amurensis berry extract and matched group tumor body cut specimen after peeling off, fixing, embedding, section.Section after dyed, endotheliocyte is brown to be dyed, and blood vessel is yellowish-brown, is easy to identification.The mensuration of MVD is undertaken by the method for the reports such as Bosari, first under low power field, chooses the abundantest region of cancer blood capillary, i.e. and " focus ", then be dyed to brown blood capillary number at 400 times of field range countings, get the meansigma methods of 3 numerical value as MVD value.The endotheliocyte that any palm fibre is dyed or endotheliocyte bunch, as long as separate with the blood capillary of closing on, cancerous cell or other connective tissues, be just considered as a blood vessel, although lumen of vessels often can see, conduct judges the standard of blood vessel.For fear of the interference to counting compared with trunk, to tube wall, there is thicker smooth muscle to hold or the blood vessel of tube chamber >8 erythrocyte area will not be counted.
VEGF, bFGF SABC: VEGF, bFGF immunologic combined detection reagent kit (instant) are purchased from Wuhan doctor's moral biotech firm.Positive cell is brown color, immunohistochemical staining scoring is undertaken by the method for Rahman etc., the standards of grading that are VEGF, bFGF are: according to the ratio of cytochrome (dyeing scope) and staining power, dyeing scope (positive cell ratio) is divided into 0-4 level, and feminine gender is 0 minute; Positive cell 1%-25% is 1 minute; Positive cell 26%-50% is 2 minutes; Positive cell 51%-75% is 3 minutes; Positive cell 76%-100% is 1 minute.Staining power is divided into 0-3 level; Feminine gender is 0 minute; The weak positive is 1 minute; Moderate strength is 2 minutes; Strong positive is 3 minutes, and score adds up to last scoring.
Statistical method: use SPSS statistical software, adopt t check.
Result:
(1) reaction and the tumour inhibiting rate of mice with tumor to treatment measure
Latter the 6th day of inoculation, each organizes the existing Semen Glycines size of mice with tumor oxter transplanted tumor, and mice is active, and diet is normal.The 8th day posterior tuberosity bulk-growth of control group mice accelerated, and part mice torpescence, takes food poor; It is active that mice is respectively organized in treatment, and diet is normal, and hair luster is normal, and tumor bulk-growth is slow.Each Radix Berberidis Amurensis berry extract group is to mice S
180sarcoma all has inhibitory action.In Table 1
Table 1 Radix Berberidis Amurensis berry extract is to mice S
180the inhibitory action of sarcoma (n=10).
(2) Radix Berberidis Amurensis berry extract is to S
180the impact of tumor body microvessel density
Section after the dyeing of SABC SABC method, vascular endothelial cell is dyed by brown, and the visible blood capillary of matched group is distributed widely in cancerous tissue interstitial, and experimental group blood capillary is relatively rare, and P<0.05, in Table 2.
Table 2 Radix Berberidis Amurensis berry extract is to mice S
180transplant the impact (n=10) of intratumoral microvascular density
Note: compare * P<0.05 with matched group, * * compares P<0.01 with matched group.
(3) Radix Berberidis Amurensis berry extract is to S
180the impact that tumor body VEGF, bFGF express
Showed by immune group result, VEGF, bFGF albumen are at S
180in sarcoma tissue, be high expressed, be mainly expressed in the cytoplasm of cell.Radix Berberidis Amurensis berry extract has obvious inhibitory action to the expression of VEGF, bFGF, the results are shown in Table 3.
Table 3 Radix Berberidis Amurensis berry extract is to mice S
180the impact (n=10) that tumor body VEGF, bFGF express
Note: * compares P<0.05 with matched group, and * * compares P<0.01 with matched group.
This experimentation shows, Radix Berberidis Amurensis berry extract is to mice S
180the tumour inhibiting rate of sarcoma is 49.2%, has as seen strong anticancer effect, and we further study its impact on angiogenesis on this basis.Blood capillary number while evaluating the objective criterion of cancer angiogenesis degree in cancerous tissue, this experiment is by experiment in mice transplantability cancer model body, the dyeing of tumor body blood capillary labelling, microvessel density shows, Radix Berberidis Amurensis berry extract all can suppress the generation of cancer blood vessel, with matched group comparison, there is significant difference.Vascularization, sprouts and generates new blood capillary from existing blood vessel, and this multi-step process depends on vascularization and promotes the coordination of the factor and inhibitive factor to produce.Research shows, VEGF, bFGF all have expression in multiple cancer, and VEGF, bFGF play an important role in the angiogenesis of multiple cancer.
Experimental example 2: the external anticancer test of Radix Berberidis Amurensis berry extract.
1. modeling method: get one bottle of well-grown attached cell, with the trypsinization 5min of 0.1%-0.2%.After centrifugal, get collecting cell, with containing the culture fluid washed cell of 10% calf serum 1 time, make single cell dispersion suspension, be adjusted to every milliliter containing 60 cells.Get 5ml and inject 60min plastic culture dish, confluent cultures ware surface, is uniformly distributed cell, and every ware, containing 300-500 cell, is put 37 ℃ of 5%CO by culture dish
2in incubator, spend the night.Under inverted microscope, the adherent distribution situation of observation of cell, if adherent good, be evenly distributed, discard culture fluid and can add the different reagents (general treat reagent with the culture fluid preparation containing 10% calf serum) for the treatment of, if treat, reagent is Chinese medicine crude preparation by using, as water decoction, alcohol extract etc., generally directly do not add at present crude preparation by using in Tissue Culture Dish, but add not commensurability Contained Serum, can improve like this credibility of experimental result.Feed Chinese medicine crude preparation by using both to blood supply animal (generally with rat or rabbit), after a period of time, got blood, separation of serum (the another chapter introduction of concrete grammar).After medicine and cytosis certain hour (2-4h), remove pastille culture fluid.With after fresh medium rinsing, add without medicine serum free culture system liquid cultivation 10-12d and can clone numeration.Discard culture fluid, use again Han ' s liquid washing 1-2 time, the fixative that adds new preparation, be methanol ice vinegar liquid (3:1) 3-5ml, fixedly 10min, discards fixative, adds the 10%Giemsa dyeing liquor about 20min that dyes after dry, under 20x anatomic microscope, numeration is containing 50 above colony numbers (clone).
2, the external anticancer experimentation of Radix Berberidis Amurensis berry extract.
Method: modeling as stated above.Stomach cancer cell MFC (Shanghai cell research is introduced) before cell culture: human lung adenocarcinoma cell GLC (Shanghai cell research is introduced), HepG2 cell lines (Shanghai cell research is introduced), Mus.Containing (including 0.1% penicillin, streptomycin) in RPM1640 (U.S. Gibco) culture fluid of 10% calf serum, cultivating 37 ℃ of 4%CO
2standby in the incubator of saturated humidity.
Medicine: positive control cisplatin (PDD) is the product of Qilu Pharmaceutical Factory, 5-fluorouracil (5-FU) is the product of Tianjin people pharmaceutical factory, fresh preparation during use, its concentration PDD is 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 5-FU is 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, Radix Berberidis Amurensis berry extract, Radix Berberidis Amurensis berry extract A, Radix Berberidis Amurensis berry extract B are for to prepare gained by embodiment 1 preparation method, lot number is respectively: 20110506,20110507,20110508, being is 50 μ g/ml, 25 μ g/ml, 12 μ g/ml.
Medicine inhibition test: cytotoxicity test adopts improvement mtt assay, makes 3*10
5/ ml cell suspension, inserts in 96 well culture plates, every hole 100 μ L.Cancerous cell negative control group (with physiologic saline for substitute), positive controls and Radix Berberidis Amurensis berry extract group, Radix Berberidis Amurensis berry extract A group, Radix Berberidis Amurensis berry extract B group are established in experiment.Variable concentrations sample is added in respectively in 96 orifice plates.3 parallel holes of every kind of dosage.After drug effect 48h, abandon the every hole of supernatant and add MTT 10 μ l, hatch 4h for 37 ℃, after abandoning supernatant, every hole adds dimethyl sulfoxide (DMSO) 100 μ l, micro oscillator concussion 5min, elisa reading instrument is surveyed OD value (wavelength 570 μ m) automatically, calculates the suppression ratio of medicine to cancerous cell.The average OD value * 100% that average OD value/control wells that average OD value-dosing group that cancerous cell kill rate=control wells is measured is measured is measured.
The analysis that morphocytology changes: collect the cell of Radix Berberidis Amurensis berry extract, PDD processing, observe and take pictures with phase contrast microscope.
Apoptotic side is fixed: cultured cell is mixed with 1*10
5/ ml cell suspension, be seeded in 24 well culture plates that are placed with coverslip, if after normal cancerous cell negative control and PDD (10 μ g/ml), 5-FU (25 μ g/ml) and Radix Berberidis Amurensis berry extract 12 μ g/ml effect 48h, with PBS liquid, wash 2 times, by 10% neutral formalin, fix, PBS washes 3 times, after hydrogen peroxide treatment, 37 ℃ of the buffer of labelling are hatched, and PBS washes 3 times, add again biotinylated dUTP labelling, hatch for 37 ℃, PBS washes 3 times, and 0.5%DAB substrate reactions colour developing haematoxylin is redyed, conventional mounting, microscopy.
Statistical method: every kind of medicine is done parallel assay 3 holes, asks its x ± s, adopts the variance analysis of SAS software, q check, each drug test group and negative control group comparison.
2. result
(1) impact of each group of Radix Berberidis Amurensis berry extract on cancerous cell: 5-FU, PDD, Radix Berberidis Amurensis berry extract medicine group, increase to the lethality of cancerous cell with drug level, be that suppression ratio increases (P<0.01) gradually, have utmost point significant difference, in Table 4.
Table 4 MTT method is measured the impact of each group of Radix Berberidis Amurensis berry extract on cancerous cell.
(3) antitumaous effect of Radix Berberidis Amurensis berry extract: the action principle of MIT method is that MIT can be by the mitochondrial mitochondrial dehydrogenase of living cells (as succinate dehydrogenase and diaphorase), be reduced into indigo Jia Za (Fornazan), dead cell or erythrocyte are without this ability, thereby available colorimetry is inferred survival and the propagation degree of cell.The method is simple to operate, quick, responsive, and in recent years not only for the experiment of cancer therapy drug extracorporeal sensitivity, national cancer institute is method as routine screening chemotherapeutics with it.We adopt mtt assay screening PTS, have shortened experimental period, and repeatability better.
The present invention measures the exercising result demonstration that Radix Berberidis Amurensis berry extract kills and wounds cancerous cell (HepG2, GLC, MFC), and its main manifestations is the cytotoxicity feature of concentration dependent form.Along with drug dose increases, inhibitory action strengthens (in Table 4) gradually.Kill capability and the 5-FU of Radix Berberidis Amurensis berry extract are more approaching, slightly inferior to PDD.The demonstration of apoptosis result, Radix Berberidis Amurensis berry extract is cancer cell specific induction of apoptosis effectively, and apoptotic index is more consistent with positive drug PDD, 5-FU.
Claims (10)
1. the application of Radix Berberidis Amurensis berry extract in preparing cancer therapy drug, the preparation method of described Radix Berberidis Amurensis berry extract is:
(1) Radix Berberidis Amurensis fruit, with concentration 30%-90% ethanol, as solvent, extract temperature 50 C-95 ℃, extraction time is 1-3 time, each extraction time is 1-4 hour, each solvent load is 6-15 times of Radix Berberidis Amurensis fruit weight, filters merge extractive liquid,, reclaim ethanol, be concentrated into relative density d=1.10-1.18, filter, obtain medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, pass through nonpolar macroporous adsorption resin, first wash with water, water elution liquid directly passes through polar macroporous adsorption resin, use again the alcoholic solution eluting nonpolar macroporous adsorption resin of concentration 50%-95%, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting polar macroporous adsorption resin of concentration 50%-95% again, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A, Radix Berberidis Amurensis berry extract B, wherein one or both mix by a certain percentage, obtain Radix Berberidis Amurensis berry extract of the present invention.
2. the preparation method of Radix Berberidis Amurensis berry extract is according to claim 1:
(1) Radix Berberidis Amurensis fruit, with concentration 30%-90% ethanol, as solvent, extract temperature 50 C-95 ℃, extraction time is 1-3 time, each extraction time is 1-4 hour, each solvent load is 6-15 times of Radix Berberidis Amurensis fruit weight, filters merge extractive liquid,, reclaim ethanol, be concentrated into relative density d=1.10-1.18, filter, obtain medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, pass through nonpolar macroporous adsorption resin, first wash with water, water elution liquid directly passes through polar macroporous adsorption resin, use again the alcoholic solution eluting nonpolar macroporous adsorption resin of concentration 50%-95%, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting polar macroporous adsorption resin of concentration 50%-95% again, collect different concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
3. the preparation method of Radix Berberidis Amurensis berry extract is according to claim 1:
(1) Radix Berberidis Amurensis fruit, as solvent, extracts temperature 70 C with concentration 70% ethanol, and extraction time is 2 times, and each extraction time is 2.5 hours, and each solvent load is 12 times of Radix Berberidis Amurensis fruit weight; Filter, merge extractive liquid,, reclaims ethanol, is concentrated into relative density d=1.15, filters, and obtains medicinal liquid A;
(2) medicinal liquid A step (1) being obtained, by D101 nonpolar macroporous adsorption resin, first wash with water, water elution liquid is directly by LSA-40 polar macroporous adsorption resin, use again the alcoholic solution eluting D101 nonpolar macroporous adsorption resin of concentration 70%, collect 70% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract A; Use the alcoholic solution eluting LSA-40 polar macroporous adsorption resin of concentration 80% again, collect 80% concentration ethanol eluent, concentrate drying, obtains Radix Berberidis Amurensis berry extract B;
(3) above-mentioned Radix Berberidis Amurensis berry extract A and Radix Berberidis Amurensis berry extract B mix, and obtain Radix Berberidis Amurensis berry extract of the present invention.
4. according to claim 1, claim 2, Radix Berberidis Amurensis berry extract claimed in claim 3, it is characterized in that mainly containing: berberine, jateorhizine, palmatine, magnoflorine, pelargonidin, enidin.
5. according to the preparation method of claim 1, Radix Berberidis Amurensis berry extract claimed in claim 2, it is characterized in that: the nonpolar macroporous adsorption resin adopting is D101 macroporous adsorbent resin, AB-8 macroporous adsorbent resin; The polar macroporous adsorption resin adopting is LSA-40 macroporous adsorbent resin, HPD600 macroporous adsorbent resin.
6. according to claim 1, claim 2, Radix Berberidis Amurensis berry extract claimed in claim 3, by the various adjuvants that add pharmaceutics to allow, make the peroral dosage forms such as tablet on pharmaceutics, granule, capsule.
7. cancer therapy drug is anti-lung-cancer medicament, medicines resistant to liver cancer, anti-gastric cancer medicine according to claim 1.
8. the cancer therapy drug that Radix Berberidis Amurensis berry extract according to claim 1 and chemical drugs or Chinese medicine or natural drug form.
9. Radix Berberidis Amurensis berry extract A according to claim 1, the application of Radix Berberidis Amurensis berry extract B in preparing cancer therapy drug.
10. the cancer therapy drug that Radix Berberidis Amurensis berry extract A according to claim 1, Radix Berberidis Amurensis berry extract B and chemical drugs or Chinese medicine or natural drug form.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103948627A (en) * | 2014-04-27 | 2014-07-30 | 王小艳 | Pharmaceutical composition used for treating small cell carcinoma |
| CN103948627B (en) * | 2014-04-27 | 2016-11-30 | 青岛市中心医院 | For treating the pharmaceutical composition of small cell carcinoma |
| CN106692141A (en) * | 2015-11-13 | 2017-05-24 | 天津中医药大学 | Applications of berberine in preparing drugs used for treating stomach cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101757074B (en) * | 2009-11-13 | 2012-03-21 | 山东轻工业学院 | Chewable tablet prepared from berberis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103948627A (en) * | 2014-04-27 | 2014-07-30 | 王小艳 | Pharmaceutical composition used for treating small cell carcinoma |
| CN103948627B (en) * | 2014-04-27 | 2016-11-30 | 青岛市中心医院 | For treating the pharmaceutical composition of small cell carcinoma |
| CN106692141A (en) * | 2015-11-13 | 2017-05-24 | 天津中医药大学 | Applications of berberine in preparing drugs used for treating stomach cancer |
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