CN101574384A - Production method for preparing anti-cancer medicament preparation and health care food by taking fibrous ginseng root as raw material - Google Patents

Abstract

The invention discloses a production method for preparing anti-cancer drug preparations and health food from ginseng root. It is found that the ginseng root is superfine powdered and then extracted according to the method provided by the present invention. Two components of ginsenoside and ginseng polysaccharide can be obtained at the same time. Experimental studies have proved that the ingredients extracted from the superfine powder of ginseng have obvious inhibitory effect on human cancer cells, and also have obvious inhibitory effect on transplanted cancer in mice. These results indicate that the components extracted from ginseng by ultrafine powder can be used to prepare anticancer drug preparations and health food. The ingredients extracted from the superfine powder of ginseng can be prepared into pills, tablets, granules, capsules, injections and any clinically acceptable anticancer drug preparations for clinical use to prevent and treat tumors through traditional production processes , can also be made into health food for preventing various tumors.

Description

With Leptoradix Ginseng is feedstock production anti-cancer drug preparation and production method of health food-Yizhibao

Technical field

The present invention relates to prepare anti-cancer drug preparation and production method of health food-Yizhibao with the Leptoradix Ginseng.

Background technology

Global annual cancer new cases are 1,000 ten thousand at present, and because of the cancer mortality number is 7,000,000, existing patient's case has 2,200 ten thousand.World Health Organization's calendar year 2001 report, world's cancer morbidity and mortality rate have risen 22% than the nineteen ninety, also will rise about 50% in 20 years from now on.In the conference of 18 world anticancer disease alliances, the research report that World Health Organization (WHO) delivers shows: global cancer condition will be serious day by day, the number of new tumor patient will be increased to 1,500 ten thousand by present every year 1000 ten thousand in 20 years from now on, because of the number of cancer mortality also will increase to 1,000 ten thousand by annual 6000000.To the year two thousand twenty along with world population reaches 8,000,000,000, to have 1,500 ten thousand New Development cases of cancers every year, wherein death toll will reach 1,000 ten thousand, survival number 3,000 ten thousand (the year two thousand twenty Data Source: Chinese cancer prevention and control planning outline (2004-2010)), wherein the overwhelming majority will occur in developing country.Therefore, treatment for cancer becomes numerous scientific and technical personnel's urgent problem.

Chemotherapy, operative treatment, radiotherapy are three big means of cancer Comprehensive Treatment.Since the forties in 20th century, first kind of chemotherapeutics chlormethine found, the research and development of chemotherapeutics have started one and another climax in the whole world, the other drug relevant with oncotherapy constantly poured into clinical, and the status of chemotherapy in the malignant tumor Comprehensive Treatment also becomes more and more important.Global in recent years antitumor drug market development is rapid, global cancer therapy drug gross sales amount was 28,200,000,000 dollars in 2005, account for 4.6% of global medicine total value, increased by 25% than 2004, estimate will to keep in 5 years from now on the growth rate of market of 15.5%-25%, and Chinese antitumor drug sales volume is also with annual 15% to 17% speed increment, domestic only hospital clinical in 2005 medication is just above 25,000,000,000 yuan, annual rate of growth surpasses 30%, and market is considerable.Yet because present stage is used for that clinical chemotherapeutics is generally relatively poor to the tumor cell selectivity, tumor cell drug resistance, toxicity is big, safety range is little, becomes increase dosage, improves the major obstacle of curative effect.Therefore it is efficient to seek low toxicity from different approach, and special cancer therapy drug is the task of top priority of tumor pharmacother.From plant, marine organisms, seek the antitumor drug of high-efficiency low-toxicity, become an important component part of antitumor drug research.Because this class medicament sources is in plant, anticancer effect is remarkable, and the mechanism of action uniqueness demonstrates specificity highly to some tumor, and the serious toxicity effect of antimetabolite as none, the discovery of paclitaxel and derivant thereof are exactly a lively example.It is not toxicity problem that its application can not be popularized so far, but the medicine source is in short supply.Developed at present the plant amedica kind of patent medicine and had only a few kinds such as paclitaxel, vinca, cephalotaxin, indirubin yet, and a large amount of plant amedica is not exploited.Some plant amedica also exists curative effect not high, and toxicity is big, for example, and bone marrow depression, gastrointestinal reaction, immunosuppressant, alopecia, Liver and kidney toxicity etc.Therefore, be necessary further in plant, to seek new, that the source is abundant, selectivity height, antineoplastic component that toxicity is low, provide new effective antitumour medicine for clinical.

Radix Ginseng (Panax ginseng C.A.Mey.) is araliaceae ginseng plant's root and rhizome, is the traditional rare medicinal herbs of China, in Korea S, and the Far East Area countries such as Japan, it should have long history is also arranged.Put down in writing according to China's classic of TCM: its slightly warm in nature, sweet and slightly bitter taste is returned spleen, lung, heart channel has strongly invigorating primordial QI, invigorating the spleen to benefit the lung, promoting the production of body fluid to quench thirst, the effect of the Fructus Alpiniae Oxyphyllae of calming the nerves.The effective ingredient that extracts in Radix Ginseng that studies show that in recent years can be regulated the central nervous system, improves the excitement and the process of inhibition of brain, makes it to tend to balance; Can improve mental and blue-collar ability, increase work efficiency, and the antifatigue effect is arranged; Promote the utilization of brain, can improve ability of learning and memory energy matter, can be by increasing myocardial contraction, decreased heart rate increases cardiac output and coronary flow and improves cardiac function, can be to resisting myocardial ischemia and arrhythmia.The obvious functions of blood sugar effect, enhancing body's immunological function; Raising is to the defensive ability/resistance ability of destructive stimulus, but the stress ability of enhancing body and adaptability; Antitumor action and antioxidation also have antiviral, shock, effects such as fat-reducing.

Radix Ginseng mainly contains saponin, polysaccharide, and volatile oil, organic acid, multiclass compositions such as trace element, wherein saponin part has been separated so far and has been obtained the ginsenoside Ro, Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, compositions such as Rg3 and Rh.And pharmacology and biochemical test prove: the ginsenoside has the physiologically active identical with Radix Ginseng, is the main effective ingredient of Radix Ginseng.Show that with the in vitro tests result ginsenoside all has the obvious suppression effect to growth of tumor such as mastocytoma, leukemia, osteosarcoma, malignant lymphoma, melanoma, glioma teratoma, thyroid carcinoma, rectal cancer, pulmonary carcinoma, hepatocarcinoma, esophageal carcinoma, cancer of biliary duct, breast carcinoma, gastric cancer, adenocarcinoma of the uterine cervix, ovarian cancers in the body, can significantly reduce the tumor number, dwindle the tumor body, inducing tumor cell is transferred the transfer die, break up and suppress tumor cell, diffusion etc.

Another kind of effective ingredient is a polysaccharose substance.In recent years, a large amount of pharmacology and clinical research data show that ginseng polysaccharide's chemical compound is a kind of immunomodulator, its energy activate immunity receptor, and the enhancing human body immunity function has antitumor, antiviral, infection, the anti-ageing multiple function of waiting for a long time.And its toxic and side effects is little, and is safe, particularly shown its advantage in the auxiliary treatment day by day in treatment for cancer.

Ginseng polysaccharide's antitumor action mechanism: the direct repression of 1 pair of tumor cell (1) changes the growth characteristics of tumor cell membrane, (2) influence the ultrastructure of tumor cell, (3) influence the signal transduction of tumor cell, (4) inducing apoptosis of tumour cell and the differentiation of inducing that suppresses tumor cell, (5) nucleic acid and the protein synthesis of inhibition tumor cell, (6) suppress angiogenesis, (7) influence oncogene.2 enhancing human body immunity functions, the antitumor action of raising body.Some polysaccharide does not have direct repression to tumor cell, brings into play its antitumor action [20] by enhancing body's immunological function.The immune cancer suppressing action of polysaccharide is many target spots, the a plurality of links that almost spread all over nonspecific immunity and specific immune response, mainly comprise (1) activating macrophage, (2) activated lymphocyte, (3) promote cytokine secretion, (4) complement activation, (5) participate in regulating nerve-endocrine-immunity (NIM) network.3 antioxidation, the effect of removing free radical, 4 mutation.

Ginseng polysaccharide's activity is relevant with molecular weight, dissolubility, viscosity, substituent group, primary structure and higher structure, if the spiral stereochemical structure is destroyed, then ginseng polysaccharide's activity reduces greatly.In addition, the size of ginseng polysaccharide's anti-tumor activity is also relevant with factors such as approach and extracting method with dosage, time.Usually ginseng polysaccharide and radiotherapy, chemotherapy, cytokine, tumor-killing effector lymphocyte's use in conjunction can significantly strengthen ginseng polysaccharide's cancer suppressing action, the bone marrow depression that the using dosage of reduction cytokine and radiotherapy or chemotherapy cause.

The ginseng polysaccharide is as another important activity composition of Chinese medicine Radix Ginseng, and it all has comparatively sure pharmacotoxicological effect to body's immunological function, hemopoietic function and at aspects such as antitumor and blood sugar lowering.From pharmacology analysis and clinical practice situation, it kills and wounds and inhibitory action tumor cell, can activate phagocyte, T lymphocyte, bone-marrow-derived lymphocyte, and enhancing antibody forms and complement activation, and its cytotoxic factor mainly comprises tumor necrosis factor and interferon.Tumor necrosis factor can be brought into play antitumor action by number of ways in vivo: the direct killing tumor cell; Act on the tumor blood circulation, cause tumor tissues blood for minimizing, thereby influence growth of tumor; Activate the antitumor action of body immune system.Multinomial experiment simultaneously shows, the ginseng polysaccharide can efficiently press down tumor killing cell, can significantly improve the animal economy immunologic function again, has immunologic enhancement, mainly be by stimulating maturation, differentiation and the breeding of the various immunologically competent cells of body, body immune system is restored balance, and the resistance of performance body self removes to eliminate, engulf cancerous cell.The ginseng polysaccharide also has the efficacy enhancing and toxicity reducing effect to chemotherapy and radiation, and effect in increasing leukocyte is obvious, has embodied the marrow-strengthening the body resistance effect of China's Chinese medicine.Though the research to the ginseng polysaccharide is started late, obtained faster progress, especially Japan and Korea S scholar are to the separating of GPS, extraction and purification, and the relation between chemical constitution and the pharmacological action has been carried out comparatively deep research.But domestic research still belongs to the starting stage, and research means is also relatively backward, and also there is big difference in some aspect.It is worthy of note, the ginseng polysaccharide of separate sources, the polysaccharide that the different extracting method of same plant obtains, even the biologic activity difference of ginseng polysaccharide's different component is very big, this is one of laboratory research reason of presenting difference as a result.Though separate different with the means of purification, the structure of the polysaccharide that obtains in addition active can be different, along with the raising of analysis means and the people further research to the ginseng polysaccharide, the ginseng polysaccharide can become another antineoplastic effective composition behind the ginsenoside.

The also not research of antitumaous effect that ginsenoside and ginseng polysaccharide are share simultaneously at present both at home and abroad, this project discovers that the Leptoradix Ginseng is behind superfine powder, extract, can obtain ginsenoside and ginseng polysaccharide simultaneously, studies have shown that by experiment composition that the Leptoradix Ginseng extracts through superfine powder has the effect of obvious suppression human cancer cell, also has tangible tumor-inhibiting action to mice zoografting cancer.These presentation of results the Leptoradix Ginseng can prepare anti-cancer drug preparation and health food through the composition that superfine powder extracts, promote the well-being of mankind.

Summary of the invention

A kind of only to be that the Chinese medicine Leptoradix Ginseng is the anti-cancer drug preparation and the production method of health food-Yizhibao of composition of raw materials simply, this method mainly comprises the extraction and the preparation process of Leptoradix Ginseng's superfine powder effective ingredient.The extraction of Leptoradix Ginseng's superfine powder effective ingredient: get the Leptoradix Ginseng, pulverize, carry out micronization, add 15 times of 20 ℃ ~ 25 ℃ distilled water immersions 30 minutes, filter, keep filtrate (filtrate 1) with the Baily pulverizing mill; Filtering residue adds 15 times of 20 ℃ ~ 25 ℃ distilled water immersions 30 minutes again, filters, and keeps filtrate (filtrate 2); Filtering residue adds 10 times of distilled water, transfers PH to 1 with hydrochloric acid; Reacting by heating 2 hours is put coldly, and hydro-oxidation sodium is transferred PH to 7; Filter, get filtrate (filtrate 3).Merging filtrate 1,2 and 3 concentrates, and (is equivalent to 1g crude drug/ml) to 100%.

Leptoradix Ginseng's superfine powder effective ingredient with above-mentioned technology extraction, can be by the production technology of traditional Chinese medicine preparation, be prepared into pill, tablet, granule, capsule, injection and any clinical preparation of accepting respectively, for clinical some tumor of preventing or treating of being used to, also can be used as the prevention that anti-cancer health-care food is used for tumor.State Leptoradix Ginseng's superfine powder effective ingredient that technology is extracted, also can be prepared into pill, tablet, granule, capsule, injection and any clinical acceptable anti-cancer drug preparation, supply clinical being used for to prevent to treat some tumor by modern crafts.

The specific embodiment:

Embodiment one

1 materials and methods

1.1 the extraction of Leptoradix Ginseng's superfine powder effective ingredient

Get the Leptoradix Ginseng, pulverize, carry out micronization (200 orders are following), add 15 times of 20 ℃ ~ 25 ℃ distilled water immersions 30 minutes, filter, keep filtrate (filtrate 1) with the Baily pulverizing mill; Filtering residue adds 15 times of 20 ℃ ~ 25 ℃ distilled water immersions 30 minutes again, filters, and keeps filtrate (filtrate 2); Filtering residue adds 10 times of distilled water, transfers PH to 1 with hydrochloric acid; Heat 92 ℃ of reactions 2 hours, put coldly, hydro-oxidation sodium is transferred PH to 7; Filter, get filtrate (filtrate 3).Merging filtrate 1,2 and 3 concentrates, and (is equivalent to 1g crude drug/ml) to 100%.

1.2 the mensuration of Leptoradix Ginseng's superfine powder effective component extracting HPLC collection of illustrative plates

Instrument: high performance liquid chromatograph: the Agulent1100 high performance liquid chromatograph comprises Agulent1100 series quaternary gradient pump, Agulent1100 manual injector, Agulent1100 series diode array detector, Agulent1100 work station;

1/10000 electric r analytical balance Shenyang dragon rises electric r weighing apparatus company limited

Filter Dalian Yi Lite scientific instrument company limited

Vacuum air pump U.S. Waters company

KQ32000E medical supersonic washer Jiangsu Kunshan Ultrasonic Instruments Co., Ltd.

Ginsenoside Rg1's reference substance (lot number 119703-200424) Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides

Ultra-pure water is from the Millipore system.

Reagent: chemical plant, acetonitrile (chromatograph alcohol) Shandong king Industrial Co., Ltd. city; Phosphoric acid (chromatograph alcohol) Guangzhou Chemical Reagent Factory; Methanol (chromatograph alcohol) Shantou brilliance chemical reagent work.

(200mm * 4.6mm 5um), is a mobile phase with acetonitrile-0.5% phosphate aqueous solution gradient elution to the selection Hhypersil ODS C18 chromatographic column of chromatographic condition, flow velocity: 1.0mL/min; Column temperature: 30 ℃; Sample is carried out eluting.Investigate suitable detection wavelength with the DAD detector simultaneously.

The preparation mobile phase agents useful for same of test solution faces with preceding and filters with solvent filter.

The need testing solution preparation is extracted with reference to the extraction process of front, repeats to extract 3 times, and extracting solution 20 times of microporous filter membrane of dilution (0.22um) get final product sample introduction after filtering.

The preparation precision of reference substance solution takes by weighing ginsenoside's standard substance 0.015g, is settled to 1mL.

1.3 measuring content of ginsenoside

Ginsenoside's linear relationship is investigated and be the results are shown in Table 1:

Table 1: ginsenoside's linear relationship is investigated the result

Y＝1263.36804x-1.96910 r＝0.9998

1.4 Leptoradix Ginseng's superfine powder extracts the assay of Rg1 in the composition

Sample thief 5ul detects by the condition of 3.1.1, and testing result and ginsenoside's standard substance Rg1 linear relationship collection of illustrative plates compare, and obtain following data: sample 50mg/ml, peak area (n=5) 147.63,147.34,147.89,148.78,147.14.

Calculate the content that Leptoradix Ginseng's superfine powder extracts Rg1 in the composition according to ginsenoside's standard substance Rg1 linear relationship chart: to extract the content of ginsenoside Rg1 in the composition be 2.48mg to Leptoradix Ginseng's superfine powder in obtaining (being equivalent to the 1g crude drug) in every ml extracting solution.

1.5 Leptoradix Ginseng's superfine powder effective component extracting high-efficient liquid phase chromatogram spectrum

The high-efficient liquid phase chromatogram spectrum of ginsenoside's standard substance Rg1 is seen Figure of description Fig. 1 (the high-efficient liquid phase chromatogram spectrum of ginsenoside's standard substance Rg1); Leptoradix Ginseng's superfine powder effective component extracting high-efficient liquid phase chromatogram spectrum is seen Figure of description Fig. 2 (Leptoradix Ginseng's superfine powder effective component extracting high-efficient liquid phase chromatogram spectrum).

1.6 ginseng polysaccharide's quantitative analysis

1. the sulphuric acid phynol method is measured neutral sugar content

The drafting of a standard curve

Precision take by weighing be dried to constant weight glucose 0.1g in the volumetric flask of 100ml, dissolved in distilled water, and be added into scale.Get this solution 1ml again and be diluted to 10ml.According to the form below is prepared the glucose titer of each concentration.

Precision is measured above each standard solution 2ml in stoppered test tube, establishes blank simultaneously, adds 6% phenol solution 1ml, mixing, add concentrated sulphuric acid 5ml along test tube wall again, leave standstill 5min, put boiling water bath 15min, lucifuge is cooled to room temperature, and with 1cm optical path colorimetric pool, the 490nm place measures absorbance.With method working sample absorbance, and neutral sugar content in the calculation sample.

(μ g/ml) is abscissa with concentration of glucose, is the A490 vertical coordinate, and the drawing standard curve is obtained regression equation according to the result and is:

Regression equation: y=0.00680x-0.0153 r=0.9997

x＝(y+0.00153)÷0.0068

Leptoradix Ginseng's superfine powder extracts ginseng polysaccharide's Determination on content in the composition: the method for reference mark curve is measured, sample thief 0.1g crude drug/ml, and absorbance (n=3) 0.486,0.506,0.448,

Calculate content: ginseng polysaccharide's neutral sugar content is 708.15ug/ml in (being equivalent to the 1g crude drug) in every ml extracting solution.

2. sulphuric acid-anthrone colorimetric method for determining neutral sugar content

The drafting of a standard curve

Precision take by weighing be dried to constant weight glucose 0.1g in the volumetric flask of 100ml, dissolved in distilled water, and be added into scale.Get this solution 1ml again and be diluted to 10ml.According to the form below is prepared the glucose titer of each concentration.

The B sample determination

Draw sugared liquid 1.0ml to be measured in doing clean test tube, in psychrolusia, slowly add 5ml 1% anthrone-sulfuric acid solution along test tube wall, mixing, 96 ℃ of water-bath colour developing 3min take out cooling rapidly in ice bath, mixing, the room temperature lucifuge is placed 15min. and is surveyed absorbance with 0.5cm optical path colorimetric pool in 620nm, establishes blank simultaneously.

(μ g/ml) is abscissa with concentration of glucose, is A 620 vertical coordinates, and the drawing standard curve is obtained regression equation according to the result and is:

Regression equation: y=0.0113x+0.0283 r=0.9966

x＝(y-0.0283)/0.0113

Leptoradix Ginseng's superfine powder extracts ginseng polysaccharide's Determination on content in the composition: the method for reference mark curve is measured, and gets 0.1g crude drug/ml, absorbance (n=3) 0.898,0.878,0.900; Calculate content: in (being equivalent to the 1g crude drug) in every ml extracting solution among the ginseng polysaccharide content of neutral sugar be 763.45ug/ml.

In sum: in (being equivalent to the 1g crude drug) in every ml extracting solution among the ginseng polysaccharide content of neutral sugar be (708.15+763.45)/2=735.83ug/ml.

1.7 discuss

Leptoradix Ginseng's superfine powder extraction process can obtain ginsenoside Rg1 and ginseng polysaccharide's two class material cancer-resisting substances simultaneously, and Radix Ginseng is the dry root of araliaceae ginseng plant's Radix Ginseng, concocts the processing method difference according to it, is divided into Radix Ginseng again, Radix Ginseng Rubra and sugared Radix Ginseng.Radix Ginseng has many-sided pharmacology and biological activity, contain multiple category and get chemical constituent, as saponins, polysaccharide, the polypeptide class, fatty acid, aminoacid, polyacetylene alcohols etc., its main active is ginsenoside and ginseng polysaccharide, get main root at Radix Ginseng, fibrous root, reed head, stem and leaf, alabastrum, specific examples of such components is all contained at positions such as fruit, and the composition that current technology of testing the employing superfine powder obtains is determined to extract composition with HPLC and has been contained the ginsenoside and determined that with sulphuric acid phynol method and sulphuric acid-anthrone colorimetry the extraction composition contains ginseng polysaccharide's material.

Experimental result shows, Leptoradix Ginseng's superfine powder that this research technology is extracted extracts composition, contains ginsenoside Rg1 2.48mg, ginseng polysaccharide 0.74mg in (being equivalent to the 1g crude drug) in every ml extracting solution.Leptoradix Ginseng's superfine powder effective component extracting high-efficient liquid phase chromatogram spectrum is seen Figure of description Fig. 2.Leptoradix Ginseng's superfine powder extracts composition can be prepared into pill, tablet, granule, capsule, injection respectively by traditional production technology.

2. Leptoradix Ginseng's superfine powder extracts the extracorporeal anti-tumor function research of composition to human cancer cell

In order to investigate the anti tumor activity in vitro that Leptoradix Ginseng's superfine powder extracts composition, we have detected Leptoradix Ginseng's superfine powder with mtt assay and have extracted suppression ratio and the IC50 of composition to people's hepatocarcinoma SK-HEP-1 cell, human promyelocytic leukemia cell HL-60, the low differentiation of people nasopharyngeal carcinoma cell CNE-2Z; And be model with the low differentiation of people nasopharyngeal carcinoma cell CNE-2Z, by flow cytometry, morphological observation, the mensuration of cell culture fluid SOD and NO, observer's Leptoradix Ginseng superfine powder effective component extracting is to the influence of tumor cell.

2.1 experiment material

Trial drug and preparation: Leptoradix Ginseng's superfine powder effective component extracting is extracted in Guangdong Medical College's development centre laboratory by me, be yellow solution, contain ginsenoside Rg1 2.48mg in (being equivalent to the 1g crude drug) in every ml extracting solution, ginseng polysaccharide 0.74mg, in 4 ℃ of preservations, use physiological saline solution during experiment, be made into 1gml-1, high temperature sterilize, packing.-20 ℃ frozen standby.When handling cell, the concentration of normal saline≤1% (V/V), verified this concentration cell growth of preliminary experiment does not have influence.Used positive drug is 5-Fu (the general company limited in the rising sun East Sea, 5-fluorouracil Shanghai) in the experiment.

The strain of test tumor:

SK-HEP-1 (human liver cancer cell) draws the medical college Experimental Animal Center from Zhongshan University

HL-60 (human promyelocytic leukemia) draws the medical college Experimental Animal Center from Zhongshan University

CNE-2Z (people low differentiation nasopharyngeal carcinoma) draws from the above tumor strain of Zhongshan University's medical college Experimental Animal Center by the preservation of going down to posterity of this seminar.

2.2 experimental technique

2.2.1 Leptoradix Ginseng's superfine powder extracts composition the antitumaous effect of the low differentiation of people nasopharyngeal carcinoma cell CNE-2Z is observed

The low differentiation of people nasopharyngeal carcinoma cell CNE-2Z cell culture: it is 10% deactivation calf serum that cell grows in volume fraction, in the RPMI-1640 complete medium of 100uml-1 penicillin and 100ugml-1 streptomycin, in 37 ℃, cultivate in the 5%CO2 saturated humidity incubator.Go down to posterity once with about 1: 3 ratio every other day.Cell disperses to go down to posterity and gather in the crops through pancreatin, and the trophophase cell of taking the logarithm experimentizes.

The low differentiation of people nasopharyngeal carcinoma cell CNE-2Z growth and proliferation of cell suppresses experiment: MTT tests with reference to Hansen[47] etc. method.The low differentiation of the people of exponential phase nasopharyngeal carcinoma cell CNE-2Z cell is added 96 orifice plates with the concentration 100 μ l of 1 * 105ml-1, after treating cell attachment, renew culture medium (90 μ l), the medication group adds the medicine of 10 μ l variable concentrations respectively, and each concentration is established 8 multiple holes, establish blank (10 μ l1640 culture medium) simultaneously, reagent contrast (1%NS), positive control (5-FU) is cultivated 24h, 48h, 72h more respectively after dosing, every hole adds 5mgml-1MTT 20 μ l, continues to cultivate 4h.Clappers, every hole adds 150 μ lDMSO lysates, and treating to dissolve fully the back is to detect wavelength with 570nm with microplate reader, and 630nm is a reference wavelength, measures absorbance A.Experiment repeats 3 times.According to formula: growth and proliferation of cell suppression ratio %=(absorbance of 1-processed group/matched group absorbance) * 100% calculates suppression ratio, and draws out concentration-suppression ratio curve, calculates IC50 by improvement Karber formula.

2.2.2 Leptoradix Ginseng's superfine powder extracts composition the antitumaous effect of human promyelocytic leukemia cell HL-60 is observed

HL-60 (human promyelocytic leukemia) cultivates: the cancerous cell suspension culture is put 37 ℃ in the RPMI-1640 complete medium that contains 10% deactivation calf serum, 100uml-1 penicillin and 100ugml-1 streptomycin, cultivates in the 5%CO2 saturated humidity incubator.Go down to posterity and gather in the crops, the trophophase cell of taking the logarithm experimentizes.

Leptoradix Ginseng's superfine powder extracts the tumor-inhibiting action of composition to the strain of HL-60 tumor: this test method is basic identical with said method, difference is that it is 1 * 107/l living cells suspension 90ul that every hole adds concentration, add immediately and contain the RPMI-1640 complete medium 10ul/ hole that variable concentrations Leptoradix Ginseng superfine powder extracts composition, each concentration is established 8 parallel holes, cultivate 24h respectively, 48h, behind the 72h, plate of each taking-up, need not inhale culture medium after adding 5mg/lMTT 20ul, directly add DMSO150ul, place in the shaking table and jolt 15 minutes, treat that it dissolves fully after, be the detection wavelength with 570nm with microplate reader, 630nm is a reference wavelength, measures absorbance A.Experiment repeats 3 times.Tumour inhibiting rate and IC50 computational methods are with the CNE-2Z cell.

2.2.3 Leptoradix Ginseng's superfine powder extracts composition the antitumaous effect of people's hepatocarcinoma SK-HEP-1 cell is observed

People's hepatocarcinoma SK-HEP-1 cell culture: it is 10% deactivation calf serum that cell grows in volume fraction, in the RPMI-1640 complete medium of 100uml-1 penicillin and 100ugml-1 streptomycin, in 37 ℃, cultivates in the 5%CO2 saturated humidity incubator.Go down to posterity once with about 1: 3 ratio every other day.Cell disperses to go down to posterity and gather in the crops through pancreatin, and the trophophase cell of taking the logarithm experimentizes.People's hepatocarcinoma SK-HEP-1 cell growing multiplication suppresses experiment: experimental technique, tumour inhibiting rate and IC50 computational methods are with the CNE-2Z cell.。

2.3 experimental result

2.3.1 Leptoradix Ginseng's superfine powder extracts the antitumaous effect of composition to the low differentiation of people nasopharyngeal carcinoma cell CNE-2Z

Leptoradix Ginseng's superfine powder extracts composition the low antitumaous effect that breaks up nasopharyngeal carcinoma cell CNE-2Z of people be the results are shown in Table 2.3.1:

Table 2.3.1: Leptoradix Ginseng's superfine powder extracts composition to the tumour inhibiting rate % of nasopharyngeal carcinoma CNE-2Z tumor strain (x ± s)

From table 2.3.1 as seen, Leptoradix Ginseng's superfine powder extraction composition has dosage dependence characteristics to the tumour inhibiting rate of nasopharyngeal carcinoma CNE-2Z tumor strain, compare with 5-FU, Leptoradix Ginseng's effective component extracting of 10mg/ml and 5mg/ml is at 24h, tumour inhibiting rate in the time of 48h is all than the tumour inhibiting rate height of 5-FU, but in 72h both tumour inhibiting rates just near, the early stage tumor-inhibiting action that this prompter's Leptoradix Ginseng extracts composition is significantly in the tumor-inhibiting action in its later stage.

2.3.2 Leptoradix Ginseng's superfine powder extracts the antitumaous effect of composition to human promyelocytic leukemia cell HL-60

Leptoradix Ginseng's superfine powder extraction composition the results are shown in Table 2.3.2 to the antitumaous effect of human promyelocytic leukemia cell HL-60:

Table 2.3.2: Leptoradix Ginseng's superfine powder extracts composition to the tumour inhibiting rate % of HL-60 tumor strain (x ± s)

From table 2.3.2 as seen, Leptoradix Ginseng's superfine powder extraction composition has dosage dependence characteristics to the tumour inhibiting rate of HL-60 tumor strain, compare with 5-FU, the tumour inhibiting rate of Leptoradix Ginseng's effective component extracting of high concentration (10mg/ml) is similar, to similar, increase and to increase the influence of administration time when drug level is greater than the influence of tumour inhibiting rate tumour inhibiting rate to the influence of human nasopharyngeal carcinoma CNE-2Z tumor strain.

2.3.3 Leptoradix Ginseng's superfine powder extracts the antitumaous effect of composition to people's hepatocarcinoma SK-HEP-1 cell

Leptoradix Ginseng's superfine powder extraction composition the results are shown in Table 2.3.3 to people's antitumaous effect;

Table 2.3.3: Leptoradix Ginseng's superfine powder extracts composition to the tumour inhibiting rate % of hepatocarcinoma tumor strain (x ± s)

From table 2.3.3 as seen, Leptoradix Ginseng's superfine powder extraction composition has dosage and time-dependent characteristics to the tumour inhibiting rate of people's hepatocarcinoma SK-HEP-1 cell, compare with 5-FU, at 24h, all (tumour inhibiting rate of Leptoradix Ginseng's effective component extracting of 2.5mg/ml ~ 10mg/ml) is low, and this prompter's hepatocarcinoma SK-HEP-1 cell more has sensitivity to Leptoradix Ginseng's effective component extracting than 5-FU than middle and high concentration for tumour inhibiting rate in the time of 48h, 72h.

2.3 discuss

Adopt people's hepatocarcinoma SK-HEP-1 cell, human leukemia HL-60 cell, the low differentiation of people nasopharyngeal carcinoma CNE-2Z cell to investigate the extracorporeal anti-tumor function of Leptoradix Ginseng's superfine powder effective component extracting from this research, and inquire into the antitumor mechanism of Leptoradix Ginseng's superfine powder effective component extracting with the low differentiation of people nasopharyngeal carcinoma CNE-2Z cell.Discover that Leptoradix Ginseng's superfine powder extracts composition, the low differentiation of people nasopharyngeal carcinoma CNE-2Z cell, people's hepatocarcinoma SK-HEP-1 cell, human leukemia HL-60 cell are all had tumor-inhibiting action, the IC50 value of effect 24h is respectively 2.82,5.28,1.55mgml-1 the IC50 value of effect 48h is respectively 1.44,2.82,1.83mgml-1, the IC50 value of effect 72h is respectively 1.77,2.97,1.45mgml-1.(shown in Fig3.1,3.2,3.3), wherein the inhibitory action to HL-60 is stronger, may be relevant with lymphsystem tumor sensitivity height.Rose in 1985, the U.S. has just begun the previous work of " the special cancer therapy drug of disease is found ".The U.S. state-run institute of oncology (NCI) adopts external drug screening method, by deriving from nearly 60 cell strains of 8 kinds of different tumors (comprising lung, colon, brain, leukemia, ovary, kidney, carcinoma of prostate and breast carcinoma), screen and estimate cancer therapy drug, in the hope of finding new antitumor mechanism.Along with the variation of social living environment, hepatocarcinoma has become the highest cancer of case fatality rate in the global range.And nasopharyngeal carcinoma is the tumor occurred frequently of China's southern area.Therefore this research adopt people's hepatocarcinoma SK-HEP-1 cell, human leukemia HL-60 cell, people low break up nasopharyngeal carcinoma CNE-2Z cell investigate Leptoradix Ginseng's superfine powder effective component extracting extracorporeal anti-tumor function be significant.

Cancer is the serious disease that threatens human health, the human second dead cause of disease, and its sickness rate is ascendant trend year by year.Along with the variation of social living environment, hepatocarcinoma has become the highest cancer of case fatality rate in the global range.Its incidence rate case fatality rate is still rising year by year.At present common antitumor drug is desirable not enough to the curative effect of solid tumor, and it is not high also to exist curative effect, and toxicity is serious, and is serious to normal tissue injury, influences shortcomings such as patient's life quality.In recent years, a large amount of pharmacology and clinical research data show that the main effective ingredient ginsenoside who extracts and ginseng polysaccharide's energy enhancing human body immunity function have antitumor, antiviral, infection, the anti-ageing multiple function of waiting for a long time from Stem and leaf of Radix Ginseng, alabastrum.And its toxic and side effects is little, and is safe, particularly shown its advantage in the auxiliary treatment day by day in treatment for cancer.This experiment shows that the composition that extracts from Leptoradix Ginseng's superfine powder is external an anti-tumor activity, may be a kind of active anticancer height, the water soluble ingredient that toxicity is little, and the application and development prospect is better.Leptoradix Ginseng's superfine powder effective ingredient, can be by the production technology of traditional Chinese medicine preparation, be prepared into pill, tablet, granule, capsule, injection and any clinical preparation of accepting, for clinical some tumor of preventing or treating of being used to, also can be used as the prevention that anti-cancer health-care food is used for tumor.

Claims (3)

1, a kind of anti-cancer drug preparation and production method of health food-Yizhibao, the prescription that it is characterized in that this production method are only to be Chinese medicine Leptoradix Ginseng simply.

2, a kind of anti-cancer drug preparation as claimed in claim 1 and production method of health food-Yizhibao, the Leptoradix Ginseng's superfine powder that it is characterized in that the preparation of this production method extracts composition by following prepared: get the Leptoradix Ginseng, pulverize, carry out micronization with the Baily pulverizing mill, add 15 times of 20 ℃ ~ 25 ℃ distilled water immersions 30 minutes, filter, keep filtrate; Filtering residue adds 15 times of 20 ℃ ~ 25 ℃ distilled water immersions 30 minutes again, filters, and keeps filtrate; Filtering residue adds 10 times of distilled water, transfers PH to 1 with hydrochloric acid; Reacting by heating 2 hours is put coldly, and hydro-oxidation sodium is transferred PH to 7; Filter, get filtrate, merge three times filtrate, concentrate.

3,, it is characterized in that Leptoradix Ginseng's superfine powder extraction composition of this production method preparation can be prepared into pill, tablet, granule, capsule, injection respectively by traditional production technology as claim 1 and 2 described a kind of anti-cancer drug preparation and production method of health food-Yizhibao.

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