CN103860602A - Application of phellinus igniarius extract in preparation of medicine for treating liver cancer, cervical cancer and breast cancer - Google Patents

Abstract

The invention discloses the application of Phellinus phellidis extract in the preparation of medicines for treating liver cancer, cervical cancer and breast cancer. The preparation method of the Phellinus Phellinus extract is as follows: (1) after the fruiting body of Phellinus Phellinus is pulverized, reflux extraction with 95% ethanol for three times, and the three extracts are combined to obtain the total extract; (2) the total extract is concentrated to an appropriate amount after recovering ethanol , add distilled water to make a suspension, and then sequentially extract with petroleum ether, ethyl acetate, and n-butanol; (3) Distill the ethyl acetate extract under reduced pressure, recover the solvent, and concentrate to dryness to obtain the Phellinus Phellinus extract. The Phellinus Phellinus extract obtained by separating and extracting Phellinus Phellinus fruiting bodies in the present invention can be effectively used to prepare medicines for treating liver cancer, cervical cancer, and breast cancer, and the extraction process is simple and easy to operate, and can effectively extract various Active ingredients, high extraction efficiency, and stable product quality are of great significance to the comprehensive utilization and industrialization of Phellinus japonica resources.

Description

Phellinus linteus extract is in the application of preparing in Hepatoma therapy, cervical cancer, breast cancer medicines

Technical field

The present invention relates to a kind of phellinus linteus extract in the application of preparing in Hepatoma therapy, cervical cancer, breast cancer medicines, belong to medical technical field.

Background technology

Hepatocarcinoma, cervical cancer, breast carcinoma are current three kinds of more common malignant tumor, not only have a strong impact on patient's quality of life, and bring heavy economy and mental burden to family and society, also be the global Social Events of puzzlement, the treatment of cancer and prevention are one of the most urgent problem of global range class all the time.At present, chemotherapy is the Main Means of resisting tumor, although there is good curative effect, but often cause the side reaction such as bone marrow depression, immunologic hypofunction, make patient be difficult to adhere to treatment, and the drug resistance that chemotherapeutics occurs in therapeutic process become one of difficult problem in current clinical treatment.In recent years, global antitumor drug market is rapid growth situation, and according to U.S. FDA statistical data, global cancer therapy drug market sale total value, by 24,000,000,000 dollars in 2004, is increased sharply to 39,600,000,000 dollars of 2007.Although the whole world all constantly has new type antineoplastic medicine to come out every year, but the mankind still do not have a kind of effectively means beat cancer so far, constantly find new cancer species simultaneously, and generation and the enhancing of resist/drug resistance of tumor, make the needs to finding novel effective antitumor medicine seem particularly urgent.Make a general survey of the cancer therapy drug that the whole world was developed during 1981 to 2002, approximately 60% directly or indirectly comes from natural product, fully shows that nature is the most important source that the mankind obtain antitumor drug.China's natural resources of Chinese medicinal materials is abundant, and from traditional medicine resource, screening searching anticancer natural active site is feasible.

Phellinus igniarius (L. ex Fr.) Quel., base was the sporophore of Polyporaceae phellinus originally, Latin formal name used at school: phellinus igniarius(L.ex Fr.) Quel.Have another name called: phellinus igniarius, sporophore stockless, the flat hemispherical of cap or the shape of a hoof.Modern study confirms that Phellinus igniarius (L. ex Fr.) Quel. contains the effective ingredient such as flavone, polysaccharide, has many-sided pharmacologically active such as immunomodulating, antitumor, and therefore, it is feasible from Phellinus igniarius (L. ex Fr.) Quel., finding highly active antitumor active site.

Summary of the invention

The object of this invention is to provide a kind of phellinus linteus extract in the application of preparing in Hepatoma therapy, cervical cancer, breast cancer medicines.

For achieving the above object, the technical solution used in the present invention is as follows:

Phellinus linteus extract is in the application of preparing in Hepatoma therapy medicine.

The application of phellinus linteus extract in preparation treatment cervical cancer medicine.

The application of phellinus linteus extract in preparation treatment breast cancer medicines.

Described pharmaceutical dosage form can be the dosage forms such as tablet, granule, pill, capsule, oral liquid, syrup, freeze-dried powder, liquid drugs injection.

The preparation method of described phellinus linteus extract is as follows:

(1) after Phellinus igniarius (L. ex Fr.) Quel. sporophore is pulverized, with 95% alcohol reflux three times, extract 2-3 hour, extraction temperature is 75-85 ℃, merges three extracting solution and obtains total extracting solution at every turn;

(2) total extracting solution reclaims after ethanol, is concentrated in right amount, and adding distil water is made suspension, then use petroleum ether successively, ethyl acetate, n-butanol extraction;

(3) by acetic acid ethyl acetate extract distilling under reduced pressure, reclaim solvent, be concentrated into dryly, obtain phellinus linteus extract.

Beneficial effect of the present invention:

The present invention's phellinus linteus extract that separation and Extraction obtains from Phellinus igniarius (L. ex Fr.) Quel. sporophore; can be effectively for the preparation of the medicine of Hepatoma therapy, cervical cancer, breast carcinoma; and extraction process is simple; easy to operate; can effectively extract plurality of active ingredients; extraction efficiency is high, constant product quality, and comprehensive utilization and industrialized development to Phellinus igniarius (L. ex Fr.) Quel. resource are significant.

Accompanying drawing explanation

Fig. 1 is that phellinus linteus extract suppresses active microgram to hepatoma Hep G 2 cells in-vitro multiplication; Wherein, a, matched group, b, positive drug group, c, experimental group (200 μ g/ml).

Fig. 2 is that phellinus linteus extract suppresses active microgram to cervical cancer HeLa in-vitro multiplication; Wherein, a, matched group, b, positive drug group, c, experimental group (200 μ g/ml).

Fig. 3 is that phellinus linteus extract suppresses active microgram to breast carcinoma MCF-7 in-vitro multiplication; Wherein, a, matched group, b, positive drug group, c, experimental group (150 μ g/ml), d, experimental group (200 μ g/ml).

Fig. 4 is the impact analysis figure of phellinus linteus extract to hepatocarcinoma tumor bearing nude mice serum NO content; Wherein, * P<0.05, * * P<0.01vs. model group; #P<0.05, ##P<0.01vs. normal group.

Fig. 5 is the impact analysis figure of phellinus linteus extract to hepatocarcinoma tumor bearing nude mice NK cell killing activity; Wherein, * P<0.05, * * P<0.01vs. model group.

Fig. 6 is the impact of phellinus linteus extract on hepatocarcinoma tumor bearing nude mice ConA induction splenic lymphocytes; Wherein, * P<0.05, * * P<0.01vs. model group; ##P<0.01vs. normal group.

Fig. 7 is the impact analysis figure of phellinus linteus extract to cervical cancer tumor bearing nude mice serum NO content; Wherein, * P<0.05, * * P<0.01vs. model group; #P<0.05, ##P<0.01vs. normal group.

Fig. 8 is the impact analysis figure of phellinus linteus extract to cervical cancer tumor bearing nude mice NK cell killing activity; Wherein, * P<0.05, * * P<0.01vs. model group.

Fig. 9 is the impact analysis figure of phellinus linteus extract to cervical cancer tumor bearing nude mice ConA induction splenic lymphocytes; Wherein, * P<0.05, * * P<0.01vs. model group; ##P<0.01vs. normal group.

Figure 10 is the impact analysis figure of phellinus linteus extract to breast carcinoma tumor bearing nude mice serum NO content; Wherein, * P<0.05, * * P<0.01vs. model group; #P<0.05, ##P<0.01vs. normal group.

Figure 11 is the impact analysis figure of phellinus linteus extract to breast carcinoma tumor bearing nude mice NK cell killing activity; Wherein, * P<0.05, * * P<0.01vs. model group.

Figure 12 is the impact analysis figure of phellinus linteus extract to breast carcinoma tumor bearing nude mice ConA induction splenic lymphocytes; Wherein, * P<0.05, * * P<0.01vs. model group; ##P<0.01vs. normal group.

The specific embodiment

Enumerate embodiment below preparation method of the present invention is described, but preparation method of the present invention is not limited to this.

Embodiment

A preparation method for phellinus linteus extract, comprises the following steps:

(1) get Phellinus igniarius (L. ex Fr.) Quel. sporophore 10kg, pulverize, with 95% alcohol reflux three times (6 times of amounts, 5 times of amounts, 4 times of amounts), extract 2 hours at every turn, extracting temperature is 80 ℃, merges three extracting solution and obtains total extracting solution;

(2) total extracting solution reclaims after ethanol, is concentrated into 1.5g crude drug/ml, and adding distil water is made suspension, then use petroleum ether successively, ethyl acetate, n-butanol extraction;

(3) by the acetic acid ethyl acetate extract distilling under reduced pressure obtaining, reclaim solvent, be concentrated into dryly, obtain phellinus linteus extract 462g.

Below by the anti-tumor activity of evidence phellinus linteus extract of the present invention.

Cellulotoxic experiment:

One, experiment material:

1, cell strain

Hepatoma cell strain HepG2; Cervical cancer cell strain HeLa; Breast cancer cell line mcf-7; Pharmaceutical college of Medical University Of Anhui provides.

2, medicine and reagent

Phellinus igniarius (L. ex Fr.) Quel. ethanol extraction, ethyl acetate extract, n-butanol extract;

DMEM cultivates powder: purchased from Gibco company of the U.S.;

Calf serum, hyclone: purchased from Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biomaterial institute.Be placed in-20 ℃ of Refrigerator stores, 4 ℃ of refrigerator overnight are melted, and after 56 ℃ of water-bath 30min deactivation, use;

Fluorouracil: purchased from Qilu Pharmaceutical Co., Ltd.;

Penicillin: purchased from HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory;

Streptomycin: purchased from south, south China, Shenzhen pharmaceutical Co. Ltd;

Dimethyl sulfoxide (DMSO): purchased from Sigma company;

Tetrazolium bromide (MTT): purchased from Sigma company;

50mm ²tissue Culture Flask, 96 orifice plates: purchased from Corning Incorporated

3, main solution preparation

DMEM cell culture fluid: DMEM powder one wraps, Sodium Pyruvate 0.11g, glutamine 0.58g, sodium bicarbonate 2.0g, adds tri-distilled water to be settled to 1000ml, and 0.22 μ m filtration sterilization is placed in-20 ℃ after packing and saves backup.37 ℃ of water-baths are melted before use, after add 10 ⁵iU/L penicillin, 100mg/L streptomycin, 10% calf serum (10% hyclone is used for cultivating breast cancer cell).

Phosphate buffer (PBS): 8.0g NaCl, 0.2g KCl, 2.9g Na ₂hPO ₄12H ₂o, 0.2g KH ₂pO ₄;

Be dissolved in appropriate tri-distilled water, regulate pH value to 7.2, be settled to 1L, filter subpackage, after autoclaving ,-20 ℃ save backup.

MTT solution: it is in 7.4 PBS solution that MTT powder is dissolved in PH, concentration 5mg/ml, lucifuge, ultrasonic dissolution assisting, filtration sterilization, now with the current, 4 ℃ of preservations.

4, instrument and equipment

4 ℃ ,-20 ℃ of cryogenic refrigerators: SANYO GS company;

MDF-U40865 type-80 ℃ ultra cold storage freezer: SANYO GS company;

SB25-12DTD type ultrasonic washing unit: Ningbo Xin Zhi company;

DHG-9243BS-III type electric heating constant-temperature blowing drying box: Shanghai new talent;

DK-8D tri-hole electric heating constant temperature tanks: Cologne, Hefei company;

3-16K type refrigerated centrifuger: Sigma company;

TGL High speed refrigerated centrifuge: Changsha Xiang Zhi centrifugal apparatus company limited;

2306-Z type CO ₂incubator: Shellab company of the U.S.;

Vertical pressure steam sterilizer:

SW-CJ-IF type clean work station: Suzhou purifies company's product;

Rios83olPE ultrapure water machine: Millipore Corp. of U.S. product;

MULISKAN MK3 microplate reader: Thermo forma Instrument Ltd.;

Pipettor: French PIPETMA company;

XD-202 inverted microscope: Cologne, Hefei;

Two, experimental technique

2.1 cell culture

This tests hepatoma carcinoma cell HepG2 used, the thin Hela of cervical cancer, breast cancer cell MCF-7.Be the cell strain of adherent growth, except the hyclone DMEM culture fluid containing 10% for breast cancer cell, all the other are the calf serum DMEM culture fluid that contains 10% for two kinds of cell strains, and adds the cultivating system of penicillin 10IU/mL and streptomycin 100 μ g/mL to cultivate.Culture environment is 37 ℃, 5% CO ₂incubator, changes culture fluid once in every 2 days, had digestive transfer culture when cell covers with culture bottle, and (cell category difference, the ratio that goes down to posterity difference), gets in the cell of exponential phase and tests.

2.2MTT method detects cell proliferation situation

Succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet Jie Jing formazan (Formazan) and be deposited in cell, and dead cell is without this function.Formazan in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value by microplate reader at 492nm wavelength place, and within the scope of certain cell number, the amount that MTT crystallization forms is directly proportional to cell number.According to the absorbance A recording, judge living cells quantity, A value is larger, and cytoactive is stronger.

Hepatocarcinoma, cervical cancer, breast cancer cell is with 5 × 10 ⁵the concentration of individual/ml is inoculated in 96 well culture plates, and every hole inoculum concentration is 100 μ L.After cell attachment, be divided into blank group, solvent control group (1%DMSO), positive control 5-Fu group, establishes 5 multiple hole groups for every group, and every hole cumulative volume is 200 μ L, after 48h, 72h, discard liquid in hole, clean 2 just with PBS, add afterwards 180 μ L serum-free mediums, and add MTT (concentration is 5mg/mL) 20 μ L, continue to cultivate after 4h at incubator, abandon supernatant, every hole adds the DMSO of 150 μ L, and concussion shakes up, and first a ceremonial jade-ladle, used in libation granule is fully dissolved.On enzyme-linked immunosorbent assay instrument, with wavelength 492nm, the zeroing of reagent matched group, surveys absorbance (A ₄₉₂) value, repeat 3 times, get its meansigma methods.By SPSS17.0 calculating cell IC50 value, calculate according to the following formula inhibitory rate of cell growth:

Inhibitory rate of cell growth=((matched group average A ₄₉₂value-blank group average A ₄₉₂value)-experimental group average A ₄₉₂value-blank group average A ₄₉₂value)/matched group average A ₄₉₂value-blank group average A ₄₉₂value) × 100%.

Three, experimental result

The inhibitory action of table 1 phellinus linteus extract to hepatoma Hep G 2 cells

Group	Dosage (μ g/ml)	OD value	Suppression ratio %	Survival rate %
					Blank group	DMEM	0.161±0.005	?	?
Matched group	1%DMSO	0.956±0.012	?	?
					Positive drug	50	0.190±0.082	96.3	3.7
Experimental group	25	0.888±0.022	8.6	91.4
					?	37.5	0.856±0.026	12.6	87.4
?	50	0.601±0.018	44.7	55.3
					?	75	0.345±0.033	76.9	23.1
?	100	0.313±0.039	80.9	19.1
					?	150	0.223±0.016	92.2	7.8
?	200	0.201±0.013	95.0	5.0

The inhibitory action of table 2 phellinus linteus extract to Cervical Cancer HeLa Cells

Group	Dosage (μ g/ml)	OD value	Suppression ratio %	Survival rate %
					Blank group	DMEM	0.186±0.02	?	?
Matched group	1%DMSO	1.113±0.114	?	?
					Positive drug	50	0.247±0.012	93.4	6.6
Experimental group	25	0.952±0.062	17.3	82.7
					?	50	0.756±0.037	38.5	61.5
?	75	0.531±0.082	62.7	37.3
					?	100	0.419±0.041	74.8	25.2
?	150	0.248±0.062	93.4	6. w
					?	200	0.191±0.047	95.0	5.0

The inhibitory action of table 3 phellinus linteus extract to MCF-7 Breast Cancer Cell

Group

Dosage (μ g/ml)

OD value

Suppression ratio %

Survival rate %

Blank group	DMEM	0.152±0.027	?	?
					Matched group	1%DMSO	0.969±0.034	?	?
Positive drug	25	0.187±0.081	95.7	4.3
					Experimental group	25	0.952±0.051	2.1	97.9
?	37.5	0.781±0.012	23.0	77.0
					?	50	0.642±0.047	40.0	60.0
?	75	0.445±0.036	64.1	35.9
					?	100	0.368±0.071	73.6	26.4
?	125	0.327±0.055	78.6	21.4
					?	150	0.254±0.038	91.2	8.8
?	200	0.213±0.046	92.5	7.5

Testing result shows, ethyl acetate extract has remarkable inhibitory action to hepatoma Hep G 2 cells, Cervical Cancer HeLa Cells, MCF-7 Breast Cancer Cell, and within the scope of certain drug level, along with the increase of drug level, suppression ratio increases gradually, action effect strengthens, and in the time that drug level reaches 75 μ g/ml, shows stronger inhibitory action.

Animal model test:

One, the therapeutical effect of phellinus linteus extract to hepatocellular carcinoma in nude mice transplanted tumor

1, experiment material

1.1 laboratory animals and cell strain

BALB/C _(nu/nu)nude mice, male, body weight 15-20g, in age in 4-5 week, buys in Fukang biotech inc of China, Beijing, raises in zoopery center, Anhui Province SPF level Animal House, and first adaptability is raised 7 days; Laboratory temperature is controlled at 20-25 ℃, and humidity, at 50%-60%, is freely drunk water and feed.

The Shanghai cell bank in the Chinese Academy of Sciences is bought in HepG-2 cell line strain, by the DMEM culture medium that contains 10% hyclone, and the CO that is 5% at 37 ℃, volume fraction ₂, complete cellar culture under saturated humidity condition, the trophophase cell of taking the logarithm is tested.

1.2 experimental drug

Phellinus linteus extract, is made by oneself by the Li Ning of Medical University Of Anhui professor laboratory.

5-fluorouracil is provided by Tianjin Jin Yao aminoacid company limited;

DMEM cultivates powder, dimethyl sulfoxide (DMSO), and tetramethyl azo azoles indigo plant (MTT), dimethyl diaminophenazine chloride is sigma company of U.S. product;

Cell pyrolysis liquid: acetic acid and dehydrated alcohol are 1:1 configuration by volume;

Interleukin-22 (IIL-2), necrocytosis factor-alpha (TNF-α) ELISA test kit are bought the company in RD;

NO test kit is bought in Nanjing and is built up Bioengineering Research Institute.

All the other reagent are domestic AR rank.

1.3 preparations of going down to posterity tumor tissue

HepG2 hepatoma carcinoma cell, under microscope, observation of cell is round, sharp outline, some visible cell core, cell grows to more than 90%, collecting cell after digestion, the blue dyeing of Placenta Hominis survival rate >95%, regulating cell concentration with PBS is 1 × 10 ⁷/ ml; 0.2ml/ is only injected in the right axillary fossa blank space of nude mice, and after cell becomes tumor, nude mice, hepatocarcinoma tumor body interior generation 3 times are put to death in dislocation.After 3 generations, tumor tissue carries out subcutaneous transplantation as tumor source.

1.4 grouping and administrations

In the time that diameter of tumor is 0.3-0.5cm, 90 become tumor nude mice to be divided at random 6 groups, every group 15, be divided into model group (be left intact, allow its ad lib), positive drug group (fluorouracil, 5-Fu, 20mg/kg/d), administration group low dose group (50mg/kg/d), dosage group (100mg/kg/d) in administration group, administration group high dose group (200mg/kg/d); Successive administration 10 days.After drug withdrawal 24h, nude mice is put to death in cervical vertebra dislocation, gets tissue on super-clean bench, detects index.Within during administration every 2 days, record long and short footpath and the body weight of tumor.

1.5 transplanted tumor bodies are weighed and cubing

Gastric infusion 10 days, last administration finishes latter second day eyeball and gets blood, and nude mice is put to death in dislocation, after 75% soak with ethanol, cuts off axillary fossa place skin, peels off tumor body, removes the nonneoplastic tissue of tumor surface, claims tumor weight; Every 2 days major diameters with vernier caliper measurement tumor body (a), minor axis (b) during administration.

The calculating of 1.6 relative tumor suppression appreciation rates

The general status of close observation growth of xenografted situation and nude mice after nude inoculation tumor cell, measure major diameter (a), the minor axis (b) of nude mice body weight and transplanted tumor every 1 day, calculate gross tumor volume (V), relative tumour volume (RTV) and relative tumor appreciation rate (T/C) according to formula below:

V=a × b ²/ 2; RTV=V/V ₀(V ₀for administration pre-neoplastic volume, V is for putting to death pre-neoplastic volume);

T/C=treatment group RTV/ model group RTV × 100%;

The mensuration of 1.7 tumor-bearing mice index and spleen index

Cervical vertebra dislocation is put to death after nude mice, peels off spleen, weighs.Formula is index and spleen index=spleen weight (mg)/nude mice body weight (g);

The mensuration of 1.8ConA induction splenocyte increment reaction

Cervical vertebra dislocation is put to death after nude mice, peels off spleen, is placed in aseptic plate and crosses 4 layer of 200 order nylon mesh screen and use syringe nook closing member to grind, with the PBS flushing of pre-cooling, collect PBS in centrifuge tube, centrifugal 2000rpm, 10min, removes upper strata liquid afterwards, adds erythrocyte cracked liquid, be mixed even, static 5min, recentrifuge, 2000rpm, 10min, removes supernatant liquid.Trypan Blue cell counting, living cells >95%, regulating splenocyte concentration with RPMI-1640 culture fluid is 1 × 10 ⁷individual/ml, being inoculated in every porocyte suspension in 96 orifice plates is 10 μ l, then adds the ConA(5mg/ml of 100 μ l), 5 multiple holes are set, in incubator, cultivate 36h, after cultivation finishes, discard supernatant, every hole adds the MTT(5mg/ml of 20 μ l) and 180 μ l serum-free mediums, continue to cultivate 4h in incubator, centrifugal (2000rpm, 5min), abandons supernatant, add the DMSO in 150 μ l/ holes, in microplate reader, measure absorbance (A) value in 570nm.

1.9 tumor-bearing mice peritoneal macrophages are engulfed the detection of dimethyl diaminophenazine chloride ability

Cervical vertebra dislocation is put to death after mice, is dipped in 1min in 75% alcoholic solution, in super-clean bench, operates.The PBS solution 5ml of lumbar injection pre-cooling, gently rub abdominal part with have gentle hands, with 5ml syringe sucking-off intraperitoneal liquid, in 4 ℃ of centrifugal (12000rpm, 10min) discard supernatant, with PBS cleaning twice, with the hyclone RPMI-1640 culture fluid re-suspended cell that contains 10%, cell counting, and to regulate cell concentration be 1 × 10 ⁷individual/ml, is inoculated in 96 orifice plates, and every hole is 100 μ l, and separately adding complete medium RPMI-1640 is blank group, is placed in cell culture incubator and cultivates 4h, removes and takes attached cell, obtains needed macrophage concentration.

The every hole of macrophage of 96 orifice plates adds 0.075% dimethyl diaminophenazine chloride normal saline solution 100 μ l, continue to cultivate 30min, discard supernatant, with PBS cleaning 3 times, every hole adds cell pyrolysis liquid (acetic acid: dehydrated alcohol=1:1) 150 μ l, room temperature leaves standstill 3h, after cytolysis, measures absorbance (A) value in microplate reader in 570nm place.

1.10 enzyme linked immunosorbent assays (ELISA method) detect IL-1, TNF-alpha content in serum

Nude mice eyeball is got after blood, and room temperature is placed 1h, centrifugal 2000rpm, and 10min, sucts clear liquid, and serum is sub-packed in EP pipe, 50 μ l/ pipes.-80 ℃ of preservations, measure according to the measuring method of ELISA test kit.

The content of 1.11 nitrate reductase method side face nude mice serum NO

Nude mice eyeball is got after blood, and room temperature is placed 1h, centrifugal 2000rpm, and 10min, sucts clear liquid, and serum is sub-packed in EP pipe, 50 μ l/ pipes.-80 ℃ of preservations.The content that detects serum NO with NO test kit, experimental procedure is carried out in strict accordance with test kit description method.

The detection of 1.12NK effector lymphocyte's activity

(1) effector lymphocyte: the conventional aseptic nude mice splenocyte of getting, making cell concentration with the RPMI-1640 culture fluid of 10% hyclone is 1 × 10 ⁷the cell suspension of individual/ml.

(2) preparation of target cell: getting goes down to posterity cultivates the YAC-1 cell of 24-48h, after RPMI-1640 culture fluid washed twice with 10% hyclone, wash once with 0.5%BSA-RPMI-1640 culture fluid, Trypan Blue is confirmed cell survival rate >95%, and adjusting cell concentration is 3 × 10 ⁵individual/ml.

(3) determination of activity: in 96 orifice plates, experimental port adds effector lymphocyte, the each 100 μ l of target cell; Target cell control wells adds target cell, the each 100 μ l of culture fluid; Effector lymphocyte's control wells adds effector lymphocyte, the each 100 μ l of culture fluid, and 3 multiple holes are set.Be placed in incubator and cultivate after 4h, experimental port adds the MTT culture fluid of 10 μ l, is continuing to cultivate 4h.After cultivation finishes, discard supernatant, experimental port respectively adds DMSO, the 0.05mol(pH10.5 of 150 μ l) glycine buffer 20 μ l, put in microplate reader in 570nm place measurement OD value.

NK cytoactive computing formula is as follows:

NK cytoactive (%)=[1-(experimental group OD value-effector lymphocyte organizes OD value)/target cell group OD value] × 100%;

1.13 observation survival time of mice

For observing, the nude mice of life cycle continues to give water and food is raised, and in the time of each group of nude mice exhaustion, puts to death or natural death, records the death time, calculates mean survival time (MST) and increase in life span, and computing formula is as follows:

Increase in life span (%)=(medication group mean survival time-model group mean survival time)/model group mean survival time × 100%

1.14 date processing

Experimental data represents according to mean standard deviation (χ ± S), adopts SPSS17.0 software to carry out statistical data processing, and group is relatively with One-way ANOVA check, and P<0.05 thinks that difference has statistical significance.

2, experimental result

2.1 transplanted human hepatocellular carcinoma models are successfully established

Tumor tissue suspension was injected into axillary fossa after one week, i.e. the kick of visible subcutaneous Semen phaseoli radiati size, and several weeks, visible tumor volume was increasing afterwards.

The body weight change of 2.2 tumor bearing nude mices and the suppression ratio of transplanted tumor

In whole experimentation, without nude mice death, respectively organizing nude mice all can be movable, and drinking-water feed is all normal, and body weight all has increase, not statistically significant (P>0.05) between the body weight of each group.The tumor of administration group weighs with model group and more obviously reduces (P<0.01); phellinus linteus extract (PLEAE) 100mg/kg, 200mg/kg group compares tumor weight with 5-Fu group and all reduces (P<0.05; P<0.01), result is as shown in table 1.

The impact (χ ± S, n=6) of table 1 phellinus linteus extract (PLEAE) on tumor bearing nude mice suppression ratio

* P<0.01, * P<0.05vs. model group; #P<0.05, ##P<0.01vs5-Fu group

2.3 impacts of Phellinus igniarius (L. ex Fr.) Quel. ethyl acetate extract (PLEAE) on transplanted tumor in nude mice change in volume

In experimentation, drinking-water and the feed of respectively organizing nude mice are all normal, without nude mice death.Nude mice after subcutaneous vaccination tumor tissue suspension in body one week, subcutaneous visible tuberosity, after 15 days, diameter of tumor is about 0.3-0.5cm, tumor formation rate 100%.Nude mice by subcutaneous transplanted human hepatocellular carcinoma is nodositas, clear border.Before administration, the change in volume of each group transplanted tumor does not have diversity (P>0.05), during administration, all relative slowing down of tumor growth of the each dosage group of PLEAE and positive drug group, model group is contrary, transplanted tumor is all grown comparatively fast, and ulceration phenomenon has appearred in the tumor body of indivedual nude mices.RTV and model group that administration finishes the each dosage group of rear PLEAE and positive drug group more all have obvious difference (P<0.05, P<0.01).The each dosage group of PLEAE can obviously reduce the volume of transplanted tumor, and has dose dependent, and tumor appreciation rate reduces gradually relatively, and result is as shown in table 2:

The impact (χ ± S, n=6) of table 2 phellinus linteus extract (PLEAE) on transplanted tumor in nude mice change in volume

* P<0.01, * P<0.05vs. model group

The impact of 2.4 phellinus linteus extracts (PLEAE) on tumor bearing nude mice index and spleen index

Relatively not statistically significant of the index and spleen index of PLEAE (50mg/kg) group and 5-Fu class index spleen and model group, index and spleen index and the model group of PLEAE (100,200mg/kg) group tumor bearing nude mice more all raise, and there is statistical significance (P<0.05, P<0.01).Experimental result is as shown in table 3.

The impact (χ ± S, n=6) of table 3PLEAE on the little nude mice index and spleen index of lotus tumor

* P<0.05, * * P<0.01vs. model group

2.5PLEAE engulfs the impact of dimethyl diaminophenazine chloride ability on tumor bearing nude mice peritoneal macrophage

PLEAE(100,200mg/kg) group tumor bearing nude mice the phagocytic activity of peritoneal macrophage and the phagocytic activity of model group compare, phagocytic activity obviously strengthens and has a statistical significance (P<0.05, P<0.01), the phagocytic activity of all the other groups is with the phagocytic activity of the model group not statistically significant of comparing.Shown in experimental result table 4.

Table 4PLEAE engulfs the impact of dimethyl diaminophenazine chloride ability on tumor bearing nude mice peritoneal macrophage

* P<0.05, * * P<0.01vs. model group

The impact of 2.6PLEAE on tumor bearing nude mice serum NO content

The level of the tumor bearing nude mice serum NO of people's HepG-2 cell line strain significantly raises than normal nude mice matched group, and PLEAE can significantly reduce the NO content in tumor bearing nude mice serum, and has certain dosage according to lazy sexual relationship.Experimental result as shown in Figure 4.

The impact of 2.7PLEAE on people's HepG-2 cell line strain tumor bearing nude mice NK cell killing activity

After tumor bearing nude mice successive administration 10 days, NK cell killing activity and the model group of PLEAE group tumor bearing nude mice are relatively significantly increased (P<0.05, P<0.01), and experimental result as shown in Figure 5.

The impact of the tumor bearing nude mice splenic lymphocytes of 2.8PLEAE on ConA induction

With normal group comparison, the model group splenic lymphocytes of ConA induction is starkly lower than normal group (P<0.01), after successive administration 10 days, PLEAE group and 5-Fu group relatively have statistical significance with model group, (P<0.01, P<0.05) PLEAE high dose group can make the tumor bearing nude mice splenic lymphocytes of ConA induction strengthen, and returns to normal level.Experimental result as shown in Figure 6.

The impact of 2.9PLEAE on IL-1, TNF-alpha content in tumor bearing nude mice serum

After tumor bearing nude mice successive administration 10 days, the middle and high dosage group of PLEAE can make the secretion level of IL-1 and TNF-α in nude mice serum improve, with model group comparison and there is statistical significance (P<0.05, P<0.01), it is as shown in the table for experimental result, the expression that can improve nude mice immune factor according to the known PLEAE of data in table 5, table 6, is improved effect to the immunity of nude mice body.

Table 5PLEAE is on secreting the impact (χ ± S, n=6) of IL-1 in tumor bearing nude mice serum

* P<0.05, * * P<0.01vs. model group

The impact (χ ± S, n=6) of table 6PLEAE on TNF secretion-α in tumor bearing nude mice serum

* P<0.05, * * P<0.01vs. model group

2.10PLEAE is on the tumor bearing nude mice impact of life cycle

Raise nude mice to its natural death in SPF level Animal House, record the death time.Positive drug group and PLEAE high dose group can extend the life cycle of tumor bearing nude mice, and relatively have statistical significance (P<0.05, P<0.01) with model group.Record result as shown in table 7.

Table 7PLEAEA is on the tumor bearing nude mice impact of life cycle (χ ± S, n=7)

* P<0.05, * * P<0.01vs. model group

Two, the therapeutical effect of phellinus linteus extract to nude mice cervical cancer transplanted tumor

1, experiment material

1.1 laboratory animals and cell strain

BALB/C _(nu/nu)nude mice, male, body weight 15-20g, in age in 4-5 week, buys in Fukang biotech inc of China, Beijing, raises in zoopery center, Anhui Province SPF level Animal House, and first adaptability is raised 7 days; Laboratory temperature is controlled at 20-25 ℃, and humidity, at 50%-60%, is freely drunk water and feed.

The Shanghai cell bank in the Chinese Academy of Sciences is bought in s strain, by the DMEM culture medium that contains 10% hyclone, and the CO that is 5% at 37 ℃, volume fraction ₂, complete cellar culture in the incubator under saturated humidity condition, the trophophase cell of taking the logarithm is tested.

1.2 experimental drug

Phellinus linteus extract, is made by oneself by the Li Ning of Medical University Of Anhui professor laboratory.

5-fluorouracil is provided by Tianjin Jin Yao aminoacid company limited;

DMEM cultivates powder, dimethyl sulfoxide (DMSO), and tetramethyl azo azoles indigo plant (MTT), dimethyl diaminophenazine chloride is sigma company of U.S. product;

Cell pyrolysis liquid: acetic acid and dehydrated alcohol are 1:1 configuration by volume;

Interleukin-22 (IIL-2), necrocytosis factor-alpha (TNF-α) ELISA test kit are bought the company in RD;

NO test kit is bought in Nanjing and is built up Bioengineering Research Institute.

All the other reagent are domestic AR rank.

1.3 preparations of going down to posterity tumor tissue

S, under microscope, observation of cell is irregular fusiformis, adherent growth, some visible cell core, cell grows to more than 90%, collecting cell after digestion, the blue dyeing of Placenta Hominis survival rate >95%, regulating cell concentration with PBS is 1 × 10 ⁷/ ml; 0.2ml/ is only injected in the right axillary fossa blank space of nude mice, and after cell becomes tumor, nude mice, cervix uteri carcinoma body interior generation 3 times are put to death in dislocation.After 3 generations, tumor tissue carries out subcutaneous transplantation as tumor source.

1.4 grouping and administrations

In the time that diameter of tumor is 0.3-0.5cm, 90 become tumor nude mice to be divided at random 6 groups, every group 15, be divided into model group (be left intact, allow its ad lib), positive drug group (fluorouracil, 5-Fu, 20mg/kg/d), administration group low dose group (50mg/kg/d), dosage group (100mg/kg/d) in administration group, administration group high dose group (200mg/kg/d); Successive administration 10 days.After drug withdrawal 24h, nude mice is put to death in cervical vertebra dislocation, gets tissue on super-clean bench, detects index.Within during administration every 2 days, record long and short footpath and the body weight of tumor.

1.5 transplanted tumor bodies are weighed and cubing

Gastric infusion 10 days, last administration finishes latter second day eyeball and gets blood, and nude mice is put to death in dislocation, after 75% soak with ethanol, cuts off axillary fossa place skin, peels off tumor body, removes the nonneoplastic tissue of tumor surface, claims tumor weight; Every 2 days major diameters with vernier caliper measurement tumor body (a), minor axis (b) during administration.

The calculating of 1.6 relative tumor suppression appreciation rates

The general status of close observation growth of xenografted situation and nude mice after nude inoculation tumor cell, measure major diameter (a), the minor axis (b) of nude mice body weight and transplanted tumor every 1 day, calculate gross tumor volume (V), relative tumour volume (RTV) and relative tumor appreciation rate (T/C) according to formula below:

V=a × b ²/ 2; RTV=V/V ₀(V ₀for administration pre-neoplastic volume, V is for putting to death pre-neoplastic volume);

T/C=treatment group RTV/ model group RTV × 100%;

The mensuration of 1.7 tumor-bearing mice index and spleen index

Cervical vertebra dislocation is put to death after nude mice, peels off spleen, weighs.Formula is index and spleen index=spleen weight (mg)/nude mice body weight (g);

The mensuration of 1.8ConA induction splenocyte increment reaction

Cervical vertebra dislocation is put to death after nude mice, peels off spleen, is placed in aseptic plate and crosses 4 layer of 200 order nylon mesh screen and use syringe nook closing member to grind, with the PBS flushing of pre-cooling, collect PBS in centrifuge tube, centrifugal 2000rpm, 10min, removes upper strata liquid afterwards, adds erythrocyte cracked liquid, be mixed even, static 5min, recentrifuge, 2000rpm, 10min, removes supernatant liquid.Trypan Blue cell counting, living cells >95%, regulating splenocyte concentration with RPMI-1640 culture fluid is 1 × 10 ⁷individual/ml, being inoculated in every porocyte suspension in 96 orifice plates is 10 μ l, then adds the ConA(5mg/ml of 100 μ l), 5 multiple holes are set, in incubator, cultivate 36h, after cultivation finishes, discard supernatant, every hole adds the MTT(5mg/ml of 20 μ l) and 180 μ l serum-free mediums, continue to cultivate 4h in incubator, centrifugal (2000rpm, 5min), abandons supernatant, add the DMSO in 150 μ l/ holes, in microplate reader, measure absorbance (A) value in 570nm.

1.9 tumor-bearing mice peritoneal macrophages are engulfed the detection of dimethyl diaminophenazine chloride ability

Cervical vertebra dislocation is put to death after mice, is dipped in 1min in 75% alcoholic solution, in super-clean bench, operates.The PBS solution 5ml of lumbar injection pre-cooling, gently rub abdominal part with have gentle hands, with 5ml syringe sucking-off intraperitoneal liquid, in 4 ℃ of centrifugal (12000rpm, 10min) discard supernatant, with PBS cleaning twice, with the hyclone RPMI-1640 culture fluid re-suspended cell that contains 10%, cell counting, and to regulate cell concentration be 1 × 10 ⁷individual/ml, is inoculated in 96 orifice plates, and every hole is 100 μ l, and separately adding complete medium RPMI-1640 is blank group, is placed in cell culture incubator and cultivates 4h, removes and takes attached cell, obtains needed macrophage concentration.

The every hole of macrophage of 96 orifice plates adds 0.075% dimethyl diaminophenazine chloride normal saline solution 100 μ l, continue to cultivate 30min, discard supernatant, with PBS cleaning 3 times, every hole adds cell pyrolysis liquid (acetic acid: dehydrated alcohol=1:1) 150 μ l, room temperature leaves standstill 3h, after cytolysis, measures absorbance (A) value in microplate reader in 570nm place.

1.10 enzyme linked immunosorbent assays (ELISA method) detect IL-1, TNF-alpha content in serum

Nude mice eyeball is got after blood, and room temperature is placed 1h, centrifugal 2000rpm, and 10min, sucts clear liquid, and serum is sub-packed in EP pipe, 50 μ l/ pipes.-80 ℃ of preservations, measure according to the measuring method of ELISA test kit.

The content of 1.11 nitrate reductase method side face nude mice serum NO

Nude mice eyeball is got after blood, and room temperature is placed 1h, centrifugal 2000rpm, and 10min, sucts clear liquid, and serum is sub-packed in EP pipe, 50 μ l/ pipes.-80 ℃ of preservations.The content that detects serum NO with NO test kit, experimental procedure is carried out in strict accordance with test kit description method.

The detection of 1.12NK effector lymphocyte's activity

(1) effector lymphocyte: the conventional aseptic nude mice splenocyte of getting, making cell concentration with the RPMI-1640 culture fluid of 10% hyclone is 1 × 10 ⁷the cell suspension of individual/ml.

(2) preparation of target cell: getting goes down to posterity cultivates the YAC-1 cell of 24-48h, after RPMI-1640 culture fluid washed twice with 10% hyclone, wash once with 0.5%BSA-RPMI-1640 culture fluid, Trypan Blue is confirmed cell survival rate >95%, and adjusting cell concentration is 3 × 10 ⁵individual/ml.

(3) determination of activity: in 96 orifice plates, experimental port adds effector lymphocyte, the each 100 μ l of target cell; Target cell control wells adds target cell, the each 100 μ l of culture fluid; Effector lymphocyte's control wells adds effector lymphocyte, the each 100 μ l of culture fluid, and 3 multiple holes are set.Be placed in incubator and cultivate after 4h, experimental port adds the MTT culture fluid of 10 μ l, is continuing to cultivate 4h.After cultivation finishes, discard supernatant, experimental port respectively adds DMSO, the 0.05mol(pH10.5 of 150 μ l) glycine buffer 20 μ l, put in microplate reader in 570nm place measurement OD value.

NK cytoactive computing formula is as follows:

NK cytoactive (%)=[1-(experimental group OD value-effector lymphocyte organizes OD value)/target cell group OD value] × 100%;

1.13 observation survival time of mice

For observing, the nude mice of life cycle continues to give water and food is raised, and in the time of each group of nude mice exhaustion, puts to death or natural death, records the death time, calculates mean survival time (MST) and increase in life span, and computing formula is as follows:

Increase in life span (%)=(medication group mean survival time-model group mean survival time)/model group mean survival time × 100%

1.14 date processing

Experimental data represents according to mean standard deviation (χ ± S), adopts SPSS17.0 software to carry out statistical data processing, and group is relatively with One-way ANOVA check, and P<0.05 thinks that difference has statistical significance.

2, experimental result

2.1 cervical cancer transplanted tumor models are successfully established

Tumor tissue suspension was injected into axillary fossa after one week, i.e. the kick of visible subcutaneous Semen phaseoli radiati size, and several weeks, visible tumor volume was increasing afterwards.

The body weight change of 2.2 tumor bearing nude mices and the suppression ratio of transplanted tumor

In whole experimentation, without nude mice death, respectively organizing nude mice all can be movable, and drinking-water feed is all normal, and body weight all has increase, not statistically significant (P>0.05) between the body weight of each group.The tumor of administration group weighs with model group and more obviously reduces (P<0.01; P<0.05); phellinus linteus extract (PLEAE) 200mg/kg group compares tumor weight with 5-Fu group and all reduces (P<0.01), and experimental result is as shown in table 1.

The impact (χ ± S, n=6) of the suppression ratio of the transplanted tumor of table 1PLEAE on tumor bearing nude mice

* P<0.01, * P<0.05vs. model group; #P<0.05, ##P<0.01vs5-Fu group

The impact of 2.3 phellinus linteus extracts (PLEAE) on transplanted tumor in nude mice change in volume

In experimentation, drinking-water and the feed of respectively organizing nude mice are all normal, without nude mice death.Nude mice after subcutaneous vaccination tumor tissue suspension in body after 10 days, subcutaneous visible tuberosity, after 15 days, diameter of tumor is about 0.3-0.5cm, tumor formation rate 100%.Nude mice by subcutaneous cervical cancer transplanted tumor is nodositas, clear border.Before administration, the change in volume of each group transplanted tumor does not have diversity (P>0.05), during administration, all relative slowing down of tumor growth of the each dosage group of PLEAE and positive drug group, model group is contrary, transplanted tumor is all grown comparatively fast, and ulceration phenomenon has appearred in the tumor body of indivedual nude mices.RTV and model group that administration finishes the each dosage group of rear PLEAE and positive drug group more all have obvious difference (P<0.05, P<0.01).The each dosage group of PLEAE can obviously reduce the volume of transplanted tumor, and has dose dependent, and tumor appreciation rate reduces gradually relatively, and result is as shown in table 2:

The impact (χ ± S, n=6) of table 2 phellinus linteus extract (PLEAE) on transplanted tumor in nude mice change in volume

* P<0.01, * P<0.05vs. model group

The impact of 2.4 phellinus linteus extracts (PLEAE) on tumor bearing nude mice index and spleen index

Relatively not statistically significant of the index and spleen index of PLEAE (50mg/kg) group and 5-Fu class index spleen and model group, index and spleen index and the model group of PLEAE (100,200mg/kg) group tumor bearing nude mice more all raise, and there is statistical significance (P<0.05, P<0.01).Experimental result is as shown in table 3.

The impact (χ ± S, n=6) of table 3PLEAE on the little nude mice index and spleen index of lotus tumor

* P<0.05, * * P<0.01vs. model group

2.5PLEAE engulfs the impact of dimethyl diaminophenazine chloride ability on tumor bearing nude mice peritoneal macrophage

PLEAE(100,200mg/kg) group tumor bearing nude mice the phagocytic activity of peritoneal macrophage and the phagocytic activity of model group compare, phagocytic activity obviously strengthens and has a statistical significance (P<0.05, P<0.01), the phagocytic activity of all the other groups is with the phagocytic activity of the model group not statistically significant of comparing.Shown in experimental result table 4.

The impact (χ ± S, n=6) of table 4.PLEAE on tumor bearing nude mice macrophage phagocytic dimethyl diaminophenazine chloride ability

* P<0.05, * * P<0.01vs. model group

The impact of 2.6PLEAE on tumor bearing nude mice serum NO content

The level of the tumor bearing nude mice serum NO of people's HepG-2 cell line strain significantly raises than normal nude mice matched group, and PLEAE can significantly reduce the NO content in tumor bearing nude mice serum, and has certain dosage according to lazy sexual relationship.Experimental result as shown in Figure 7.

The impact of 2.7PLEAE on human cervical carcinoma Hela cell's strain tumor bearing nude mice NK cell killing activity

After tumor bearing nude mice successive administration 10 days, NK cell killing activity and the model group of the tumor bearing nude mice of PLEAE group, positive drug group are relatively significantly increased (P<0.05, P<0.01), and experimental result as shown in Figure 8.

The impact of the tumor bearing nude mice splenic lymphocytes of 2.8PLEAE on ConA induction

With normal group comparison, the model group splenic lymphocytes of ConA induction is starkly lower than normal group (P<0.01), after successive administration 10 days, PLEAE group and 5-Fu group relatively have statistical significance with model group, (P<0.01, P<0.05) PLEAE high dose group can make the tumor bearing nude mice splenic lymphocytes of ConA induction strengthen, and returns to normal level.Experimental result as shown in Figure 9.

The impact of 1.9PLEAE on IL-1, TNF-alpha content in tumor bearing nude mice serum

After tumor bearing nude mice successive administration 10 days, the middle and high dosage group of PLEAE can make the secretion level of IL-1 and TNF-α in nude mice serum improve, with model group comparison and there is statistical significance (P<0.05, P<0.01), it is as shown in the table for experimental result, the expression that can improve nude mice immune factor according to the known PLEAE of data in table 5, table 6, is improved effect to the immunity of nude mice body.

Table 5.PLEAE is on secreting the impact (χ ± S, n=6) of IL-1 in tumor bearing nude mice serum

* P<0.05, * * P<0.01vs. model group

The impact (χ ± S, n=6) of table 6.PLEAE on TNF secretion-α in tumor bearing nude mice serum

* P<0.05, * * P<0.01vs. model group

1.10PLEAE is on the tumor bearing nude mice impact of life cycle

Raise nude mice to its natural death in SPF level Animal House, record the death time.Positive drug group and PLEAE high dose group can extend the life cycle of tumor bearing nude mice, and relatively have statistical significance (P<0.05, P<0.01) with model group.Record result as shown in table 7.

Table 7.PLEAEA is on the tumor bearing nude mice impact of life cycle (χ ± S, n=7)

* P<0.05, * * P<0.01vs. model group

Three, the therapeutical effect of phellinus linteus extract to human breast cancer in nude mice transplanted tumor

1, experiment material and method

1.1 experiment material

BALB/C _(nu/nu)nude mice, male, body weight 15-20g, in age in 4-5 week, buys in Fukang biotech inc of China, Beijing, raises in zoopery center, Anhui Province SPF level Animal House, and first adaptability is raised 7 days; Laboratory temperature is controlled at 20-25 ℃, and humidity, at 50%-60%, is freely drunk water and feed.

The Shanghai cell bank in the Chinese Academy of Sciences is bought in MCF-7 Human Breast Cancer Cells strain, by the DMEM culture medium of the Gibco powder configuration that contains 10% hyclone, and the CO that is 5% at 37 ℃, volume fraction ₂, complete cellar culture under saturated humidity condition, the trophophase cell of taking the logarithm is tested.

1.2 experimental drug

Phellinus linteus extract (PLEAE) is made by oneself by the Li Ning of Medical University Of Anhui professor laboratory.

5-fluorouracil is provided by Tianjin Jin Yao aminoacid company limited;

DMEM powder is Gibco company;

Dimethyl sulfoxide (DMSO), tetramethyl azo azoles indigo plant (MTT), dimethyl diaminophenazine chloride is sigma company of U.S. product;

Cell pyrolysis liquid: acetic acid and dehydrated alcohol are 1:1 configuration by volume;

Interleukin-22 (IIL-2), necrocytosis factor-alpha (TNF-α) ELISA test kit are bought the company in RD;

NO test kit is bought in Nanjing and is built up Bioengineering Research Institute.

All the other reagent are domestic AR rank.

1.3 preparations of going down to posterity tumor tissue

MCF-7 breast cancer cell, under microscope, observation of cell is polygon, adherent growth, some visible cell core, cell grows to more than 90%, collecting cell after digestion, the blue dyeing of Placenta Hominis survival rate >95%, regulating cell concentration with PBS is 1 × 10 ⁷/ ml; 0.2ml/ is only injected in the right axillary fossa blank space of nude mice, and after cell becomes tumor, nude mice, mammary gland carcinoma body interior generation 3 times are put to death in dislocation.After 3 generations, tumor tissue carries out subcutaneous transplantation as tumor source.

1.4 grouping and administrations

In the time that diameter of tumor is 0.3-0.5cm, 65 become tumor nude mice to be divided at random 5 groups, every group 13, be divided into model group (be left intact, allow its ad lib), positive drug group (fluorouracil, 5-Fu, 20mg/kg/d), administration group low dose group (50mg/kg/d), dosage group (100mg/kg/d) in administration group, administration group high dose group (200mg/kg/d); Successive administration 10 days.After drug withdrawal 24h, nude mice is put to death in cervical vertebra dislocation, gets tissue on super-clean bench, detects index.Within during administration every 2 days, record long and short footpath and the body weight of tumor.

1.5 transplanted tumor bodies are weighed and cubing

Continuous 10 days of gastric infusion, last administration finishes latter second day eyeball and gets blood, and nude mice is put to death in dislocation, after 75% soak with ethanol, cuts off axillary fossa place skin, peels off tumor body, removes the nonneoplastic tissue of tumor surface, claims tumor weight; Every 2 days major diameters with vernier caliper measurement tumor body (a), minor axis (b) during administration.

The calculating of 1.6 relative tumor suppression appreciation rates

The general status of close observation growth of xenografted situation and nude mice after nude inoculation tumor cell, measure major diameter (a), the minor axis (b) of nude mice body weight and transplanted tumor every 1 day, calculate gross tumor volume (V), relative tumour volume (RTV) and relative tumor appreciation rate (T/C) according to formula below:

V=a × b ²/ 2; RTV=V/V ₀(V ₀for administration pre-neoplastic volume, V is for putting to death pre-neoplastic volume);

T/C=treatment group RTV/ model group RTV × 100%;

The mensuration of 1.7 tumor-bearing mice index and spleen index

Cervical vertebra dislocation is put to death after nude mice, peels off spleen, weighs.Formula is index and spleen index=spleen weight (mg)/nude mice body weight (g);

The mensuration of 1.8ConA induction splenocyte increment reaction

Cervical vertebra dislocation is put to death after nude mice, peels off spleen, is placed in aseptic plate and crosses 4 layer of 200 order nylon mesh screen and use syringe nook closing member to grind, with the PBS flushing of pre-cooling, collect PBS in centrifuge tube, centrifugal 2000rpm, 10min, removes upper strata liquid afterwards, adds erythrocyte cracked liquid, be mixed even, static 5min, recentrifuge, 2000rpm, 10min, removes supernatant liquid.Trypan Blue cell counting, living cells >95%, regulating splenocyte concentration with RPMI-1640 culture fluid is 1 × 10 ⁷individual/ml, being inoculated in every porocyte suspension in 96 orifice plates is 10 μ l, then adds the ConA(5mg/ml of 100 μ l), 5 multiple holes are set, in incubator, cultivate 36h, after cultivation finishes, discard supernatant, every hole adds the MTT(5mg/ml of 20 μ l) and 180 μ l serum-free mediums, continue to cultivate 4h in incubator, centrifugal (2000rpm, 5min), abandons supernatant, add the DMSO in 150 μ l/ holes, in microplate reader, measure absorbance (A) value in 570nm.

1.9 tumor-bearing mice peritoneal macrophages are engulfed the detection of dimethyl diaminophenazine chloride ability

Cervical vertebra dislocation is put to death after mice, is dipped in 1min in 75% alcoholic solution, in super-clean bench, operates.The PBS solution 5ml of lumbar injection pre-cooling, gently rub abdominal part with have gentle hands, with 5ml syringe sucking-off intraperitoneal liquid, in 4 ℃ of centrifugal (12000rpm, 10min) discard supernatant, with PBS cleaning twice, with the hyclone RPMI-1640 culture fluid re-suspended cell that contains 10%, cell counting, and to regulate cell concentration be 1 × 10 ⁷individual/ml, is inoculated in 96 orifice plates, and every hole is 100 μ l, and separately adding complete medium RPMI-1640 is blank group, is placed in cell culture incubator and cultivates 4h, removes and takes attached cell, obtains needed macrophage concentration.

The every hole of macrophage of 96 orifice plates adds 0.075% dimethyl diaminophenazine chloride normal saline solution 100 μ l, continue to cultivate 30min, discard supernatant, with PBS cleaning 3 times, every hole adds cell pyrolysis liquid (acetic acid: dehydrated alcohol=1:1) 150 μ l, room temperature leaves standstill 3h, after cytolysis, measures absorbance (A) value in microplate reader in 570nm place.

1.10 enzyme linked immunosorbent assays (ELISA method) detect IL-1, TNF-alpha content in serum

Nude mice eyeball is got after blood, and room temperature is placed 1h, centrifugal 2000rpm, and 10min, sucts clear liquid, and serum is sub-packed in EP pipe, 50 μ l/ pipes.-80 ℃ of preservations, measure according to the measuring method of ELISA test kit.

The content of 1.11 nitrate reductase method side face nude mice serum NO

Nude mice eyeball is got after blood, and room temperature is placed 1h, centrifugal 2000rpm, and 10min, sucts clear liquid, and serum is sub-packed in EP pipe, 50 μ l/ pipes.-80 ℃ of preservations.The content that detects serum NO with NO test kit, experimental procedure is carried out in strict accordance with test kit description method.

The detection of 1.12NK effector lymphocyte's activity

(1) effector lymphocyte: the conventional aseptic nude mice splenocyte of getting, making cell concentration with the RPMI-1640 culture fluid of 10% hyclone is 1 × 10 ⁷the cell suspension of individual/ml.

(2) preparation of target cell: getting goes down to posterity cultivates the YAC-1 cell of 24-48h, after RPMI-1640 culture fluid washed twice with 10% hyclone, wash once with 0.5%BSA-RPMI-1640 culture fluid, Trypan Blue is confirmed cell survival rate >95%, and adjusting cell concentration is 3 × 10 ⁵individual/ml.

(3) determination of activity: in 96 orifice plates, experimental port adds effector lymphocyte, the each 100 μ l of target cell; Target cell control wells adds target cell, the each 100 μ l of culture fluid; Effector lymphocyte's control wells adds effector lymphocyte, the each 100 μ l of culture fluid, and 3 multiple holes are set.Be placed in incubator and cultivate after 4h, experimental port adds the MTT culture fluid of 10 μ l, is continuing to cultivate 4h.After cultivation finishes, discard supernatant, experimental port respectively adds DMSO, the 0.05mol(pH10.5 of 150 μ l) glycine buffer 20 μ l, put in microplate reader in 570nm place measurement OD value.

NK cytoactive computing formula is as follows:

NK cytoactive (%)=[1-(experimental group OD value-effector lymphocyte organizes OD value)/target cell group OD value] × 100%;

1.13 observation survival time of mice

For observing, the nude mice of life cycle continues to give water and food is raised, and in the time of each group of nude mice exhaustion, puts to death or natural death, records the death time, calculates mean survival time (MST) and increase in life span, and computing formula is as follows:

Increase in life span (%)=(medication group mean survival time-model group mean survival time)/model group mean survival time × 100%

1.14 date processing

Experimental data represents according to mean standard deviation (χ ± S), adopts SPSS17.0 software to carry out statistical data processing, and group is relatively with One-way ANOVA check, and P<0.05 thinks that difference has statistical significance.

2, experimental result

2.1 breast cancer transplantable tumor models are successfully established

Tumor tissue suspension was injected into axillary fossa after one week, i.e. the kick of visible subcutaneous Semen phaseoli radiati size, and several weeks, visible tumor volume was increasing afterwards.See Fig. 1:

The body weight change of 2.2 tumor bearing nude mices and the suppression ratio of transplanted tumor

In whole experimentation, without nude mice death, respectively organizing nude mice all can be movable, and drinking-water feed is all normal, and body weight all has increase, but not statistically significant (P>0.05) between the body weight of each group.The tumor of administration group weighs with model group and more obviously reduces (P<0.01; P<0.05); Phellinus igniarius (L. ex Fr.) Quel. ethyl acetate extract (PLEAE) 200mg/kg group compares tumor weight with 5-Fu group and all reduces (P<0.01), and result is as shown in table 1.

The impact (χ ± S, n=6) of the suppression ratio of the transplanted tumor of table 1PLEAE on tumor bearing nude mice

* P<0.01, * P<0.05vs. model group; #P<0.05, ##P<0.01vs5-Fu group

The impact of 2.3 phellinus linteus extracts (PLEAE) on transplanted tumor in nude mice change in volume

In experimentation, drinking-water and the feed of respectively organizing nude mice are all normal, without nude mice death.Nude mice after subcutaneous vaccination tumor tissue suspension in body after 10 days, subcutaneous visible tuberosity, after 15 days, diameter of tumor is about 0.3-0.5cm, tumor formation rate 100%.Nude mice by subcutaneous transplanted human hepatocellular carcinoma is nodositas, clear border.Before administration, the change in volume of each group transplanted tumor does not have diversity (P>0.05), during administration, all relative slowing down of tumor growth of the each dosage group of PLEAE and positive drug group, model group is contrary, transplanted tumor is all grown comparatively fast, and ulceration phenomenon has appearred in the tumor body of indivedual nude mices.RTV and model group that administration finishes the each dosage group of rear PLEAE and positive drug group more all have obvious difference (P<0.05, P<0.01).The each dosage group of PLEAE can obviously reduce the volume of transplanted tumor, and has dose dependent, and tumor appreciation rate reduces gradually relatively, and result is as shown in table 2:

The impact (χ ± S, n=6) of table 2 phellinus linteus extract (PLEAE) on transplanted tumor in nude mice change in volume

* P<0.01, * P<0.05vs. model group

The impact of 2.4 phellinus linteus extracts (PLEAE) on tumor bearing nude mice index and spleen index

Relatively not statistically significant of the index and spleen index of PLEAE (50mg/kg) group and 5-Fu class index spleen and model group, index and spleen index and the model group of PLEAE (100,200mg/kg) group tumor bearing nude mice more all raise, and there is statistical significance (P<0.05, P<0.01).Experimental result is as shown in table 3.

The impact (χ ± S, n=6) of table 3PLEAE on the little nude mice index and spleen index of lotus tumor

* P<0.05, * * P<0.01vs. model group

2.5PLEAE engulfs the impact of dimethyl diaminophenazine chloride ability on tumor bearing nude mice peritoneal macrophage

PLEAE(100,200mg/kg) group tumor bearing nude mice the phagocytic activity of peritoneal macrophage and the phagocytic activity of model group compare, phagocytic activity obviously strengthens and has a statistical significance (P<0.05, P<0.01), the phagocytic activity of all the other groups is with the phagocytic activity of the model group not statistically significant of comparing.Shown in experimental result table 4.

The impact (χ ± S, n=6) of table 4.PLEAE on tumor bearing nude mice macrophage phagocytic dimethyl diaminophenazine chloride ability

* P<0.05, * * P<0.01vs. model group

The impact of 2.6PLEAE on tumor bearing nude mice serum NO content

The level of the tumor bearing nude mice serum NO of MCF-7 Human Breast Cancer Cells strain significantly raises than normal nude mice matched group, and PLEAE can significantly reduce the NO content in tumor bearing nude mice serum, and has certain dosage according to lazy sexual relationship.Experimental result as shown in figure 10.

The impact of 2.7PLEAE on MCF-7 Human Breast Cancer Cells strain tumor bearing nude mice NK cell killing activity

After tumor bearing nude mice successive administration 10 days, NK cell killing activity and the model group of PLEAE group tumor bearing nude mice are relatively significantly increased (P<0.05, P<0.01), and experimental result as shown in figure 11.

The impact of the tumor bearing nude mice splenic lymphocytes of 2.8PLEAE on ConA induction

With normal group comparison, the model group splenic lymphocytes of ConA induction is starkly lower than normal group (P<0.01), after successive administration 10 days, PLEAE group and 5-Fu group relatively have statistical significance with model group, (P<0.01, P<0.05) PLEAE high dose group can make the tumor bearing nude mice splenic lymphocytes of ConA induction strengthen, and returns to normal level.Experimental result as shown in figure 12

The impact of 2.9PLEAE on IL-1, TNF-alpha content in tumor bearing nude mice serum

After tumor bearing nude mice successive administration 10 days, the middle and high dosage group of PLEAE can make the secretion level of IL-1 and TNF-α in nude mice serum improve, with model group comparison and there is statistical significance (P<0.05, P<0.01), it is as shown in the table for experimental result, the expression that can improve nude mice immune factor according to the known PLEAE of data in table 5, table 6, is improved effect to the immunity of nude mice body.

Table 5.PLEAE is on secreting the impact (χ ± S, n=6) of IL-1 in tumor bearing nude mice serum

* P<0.05, * * P<0.01vs. model group

The impact (χ ± S, n=6) of table 6.PLEAE on TNF secretion-α in tumor bearing nude mice serum

* P<0.05, * * P<0.01vs. model group

2.10PLEAE is on the tumor bearing nude mice impact of life cycle

Raise nude mice to its natural death in SPF level Animal House, record the death time.Positive drug group and PLEAE high dose group can extend the life cycle of tumor bearing nude mice, and relatively have statistical significance (P<0.05, P<0.01) with model group.Record result as shown in table 7.

Table 7.PLEAEA is on the tumor bearing nude mice impact of life cycle (χ ± S, n=7)

* P<0.05, * * P<0.01vs. model group.

Claims (5)

1. phellinus linteus extract is in the application of preparing in Hepatoma therapy medicine.

2. the application of phellinus linteus extract in preparation treatment cervical cancer medicine.

3. the application of phellinus linteus extract in preparation treatment breast cancer medicines.

According to the phellinus linteus extract described in claim 1 or 2 or 3 in the application of preparing in Hepatoma therapy, cervical cancer, breast cancer medicines; it is characterized in that, described pharmaceutical dosage form can be the dosage forms such as tablet, granule, pill, capsule, oral liquid, syrup, freeze-dried powder, liquid drugs injection.

According to the phellinus linteus extract described in claim 1 or 2 or 3 in the application of preparing in Hepatoma therapy, cervical cancer, breast cancer medicines, it is characterized in that, the preparation method of described phellinus linteus extract is as follows:

(1) after Phellinus igniarius (L. ex Fr.) Quel. sporophore is pulverized, with 95% alcohol reflux three times, extract 2-3 hour, extraction temperature is 75-85 ℃, merges three extracting solution and obtains total extracting solution at every turn;

(2) total extracting solution reclaims after ethanol, is concentrated in right amount, and adding distil water is made suspension, then use petroleum ether successively, ethyl acetate, n-butanol extraction;

(3) by acetic acid ethyl acetate extract distilling under reduced pressure, reclaim solvent, be concentrated into dryly, obtain phellinus linteus extract.

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