CN102068584A - Hydrophobic steroid saponin extract of peltate yam rhizome as well as preparation method and use thereof - Google Patents
Hydrophobic steroid saponin extract of peltate yam rhizome as well as preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a hydrophobic steroid saponin extract of peltate yam rhizome as well as a preparation method and use thereof. In the invention, in vitro anti-cancer drug experimental study proves that the hydrophobic steroid saponin extract of the peltate yam rhizome has strong growth inhibition activity on cell lines of colon cancer, lung cancer, breast cancer, cervical carcinoma, ovarian cancer, larynx cancer and the like. The extract of peltate yam rhizome has the advantages of wide resource, simple extraction and separation process of saponin, and wide prospect in cancer treatment, thus developing a new anticancer use of a hydrophobic dioscin component in the peltate yam rhizome.
Description
Technical field
The present invention relates to a kind of HUANGJIANG hydrophobicity steroidal saponin extract, belong to drug world.
Background technology
Tumor is the principal disease of harm humans health and lives.The whole world has 5,000,000 people to die from malignant tumor every year approximately.In recent years, because the deterioration of environment, and modern's operating pressure, transmission of disease, the sickness rate of malignant tumor is in rising trend, and becomes the second largest deadly cause of disease after cardiovascular disease.Therefore, the annual lot of manpower and material resources that drops in the whole world is used to research and develop the novel drugs of preventing curing cancers.In recent years, the materia medica expert has also carried out a lot of researchs to Chinese medicine except the research Western medicine, the mechanism that malignant tumor forms is also inquired into, and the invention of advanced technologies such as combinatorial chemistry, genetic engineering and application have been quickened the malignant tumor medicine development process; Particularly be that lead compound searching new drug has vast potential for future development at present with the active skull cap components, assessed the anti-tumor activity of kind of higher plant surplus in the of 40,000 up to now.According to the foreign data introduction, hundreds of kind PTS has been developed in the whole world, wherein has much to belong to natural plant extracts, in recent years, the screening widely that research worker is carried out from Chinese medicine, found a large amount of anticancer plants, as Ramulus et folium taxi cuspidatae, Semen Ginkgo, Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae), Ganoderma etc., in above-mentioned plant, people are more to the research of Ramulus et folium taxi cuspidatae, Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae), a lot of plants are owing to poor growth, and natural resources shortage is difficult to meet the need of market.
Kind surplus China's yam has 80, wherein belong to rhizomatic and have 17 kinds, 1 subspecies and 1 mutation, contain sapogenin in the yam, it is the main medicine source of extracting saponin, sapogenin is one of primary raw material of synthesizing steroid hormone drug and contraceptive, the water-solubility saponin preparation is effective to treatment coronary heart disease, can reduce angina pectoris, regulate metabolism, domestic is raw material with yam not of the same race, extract its total steroid saponin as the man plant produced of the existing number of hypolipidemic clinically, the new drug " peltate leaf perhexiline " of wherein using Rhizoma Dioscoreae Zingiberensis (D.zingibernsis) to succeed in developing, clinical treatment coronary heart disease for raw material, angina pectoris has certain curative effect; " DIAOXINXUE KANG " of having gone on the market also is a kind of medicine of blood fat reducing, this medicine is to be the mixture of the dioscin of raw material extraction with Dioscorea panthaica Prain et Burkill (Dioscorea panthaiapraia et Burkill) rhizome, comprising monoglycosides, bioside etc., be used for the treatment of coronary heart disease, angina pectoris, breathe hard, symptom such as cardiopalmus, uncomfortable in chest, chest pain, total effective rate reaches more than 90%.
Rhizoma Dioscoreae Zingiberensis Dioscoreazingiberensis C.H.Wrigh, have another name called HUANGJIANG, be yam, popular name " duration and degree of heating root ", another name claims Rhizoma Polygonati Cirrhifolii, monkey joint lotus etc., be that perennial herbaceous stem twines liana, its root stock likeness in form Rhizoma Zingiberis Recens, the section endoplasm is crocus, so claim HUANGJIANG, be the distinctive Rhizoma Dioscoreae type of China, its active substance diosgenin (being commonly called as Saponin) is present in the rhizome with the form of saponin, have treatment coronary heart disease, regulate metabolism, lipidemia, effect such as anticancer, be the important source material of synthetic hormone class medicine.HUANGJIANG is one of best medicinal plants of extracting on the medical industry Saponin, modern Saponin can only be extracted from minority plants such as HUANGJIANG, valley dragon, Dioscorea panthaica Prain et Burkill, the Saponin of wherein extracting from HUANGJIANG accounts for more than 70% of total amount, and Saponin is " mother of hormone ", and the method for extracting diosgenin has direct acid-hydrolysis method, fermentation-acid-hydrolysis method, enzymolysis-acid-hydrolysis method, pre-separation cellulose starch method, supercritical extraction etc.Most of enterprise adopts direct acid-hydrolysis method in producing at present, and the shortcoming of this method is that acid and extractant consumption are big, and the waste water and dregs of generation is many, and side-product starch and cellulose can't reclaim etc.The contained dioscin of HUANGJIANG is not only the important intermediate of synthesizing steroid hormonal medicaments, but also has biological activitys such as antibacterial, parasite killing, antiinflammatory, is the raw material that present domestic bio-pharmaceuticals producer is badly in need of.
At present less at the report of HUANGJIANG hydrophobicity steroidal saponin, there is not HUANGJIANG hydrophobicity steroidal saponin extract to be used for the treatment of the pertinent literature report of cancer.
Summary of the invention
Technical scheme of the present invention has provided a kind of HUANGJIANG hydrophobicity steroidal saponin extract, and another technical scheme of the present invention has provided the purposes of this HUANGJIANG hydrophobicity steroidal saponin extract.
The invention provides a kind of HUANGJIANG hydrophobicity steroidal saponin extract, contain Trillium tschonoskii Maxim saponin 0-1.021mg, diosgenin cellobioside 8-30.342mg, triangle leaf saponin 16.461-93.947mg, gracillin 0-8.456mg in every g extract.
Further preferably, contain Trillium tschonoskii Maxim saponin 0.891mg, diosgenin cellobioside 28.178mg, triangle leaf saponin 83.974mg, gracillin 1.450mg in the every g extract.
It is to be prepared from by following steps:
A, with medicinal plants HUANGJIANG Dioscorea zingiberensis C.H.Wrigh tuber oven dry, pulverize, add 70-100%v/v ethanol lixiviate 10-24 hour, solid-liquid separation;
The solid phase of b, isolated by filtration added the 70-100%v/v alcohol reflux 2-5 hour, and 3-5 time, filtered while hot;
After c, the liquid phase that the liquid phase of a step lixiviate and the hot reflux of b step are extracted merged, distilling under reduced pressure was reclaimed ethanol and is distinguished the flavor of to there being ethanol substantially, gets brown extractum;
D, brown extractum is dissolved in water, at the centrifugal 15-20 of 4000-6000r/min minute, water phase separated and solid phase;
E, with the solid phase lyophilization that d obtains, promptly get HUANGJIANG hydrophobicity steroidal saponin extract.
Wherein, the HPLC finger printing of hydrophobicity steroidal saponin extract of the present invention as shown in Figure 1; Chromatographic condition: Tianjin, island chromatograph LC-20; Chromatographic column: Shim-pack VP-ODS C
18(250 * 4.6mmi.d.5um), pre-column (5 μ m, 10 * 4.6mm i.d.); Binary eluent gradient eluting, water (A) and acetonitrile (B), 0-5min, 20%-30%B; 5-35min, 30%-40%B; 35-40min, 30%-40%B; 40-60min, 60%-85%B; 60-75min, 85%-100%B; 75-80min, 100%B; Flow velocity: 1ml/min; Column temperature: 30 ℃; Detect wavelength: λ=204nm; Sample size: 20 μ l.
The invention provides a kind of method for preparing described HUANGJIANG hydrophobicity steroidal saponin extract, it comprises the steps:
A, with medicinal plants HUANGJIANG Dioscorea zingiberensis C.H.Wrigh tuber oven dry, pulverize, add 70-100%v/v ethanol lixiviate 10-24 hour, solid-liquid separation;
The solid phase of b, isolated by filtration added the 70-100%v/v alcohol reflux 2-5 hour, and 3-5 time, filtered while hot;
After c, the liquid phase that the liquid phase of a step lixiviate and the hot reflux of b step are extracted merged, distilling under reduced pressure was reclaimed ethanol and is distinguished the flavor of to there being ethanol substantially, gets brown extractum;
D, brown extractum is dissolved in water, at the centrifugal 15-20 of 4000-6000r/min minute, water phase separated and solid phase;
E, with the solid phase lyophilization that d obtains, promptly get HUANGJIANG hydrophobicity steroidal saponin extract.
The invention provides the purposes of described HUANGJIANG hydrophobicity steroidal saponin extract in the preparation cancer therapy drug.
Further preferably, described medicine is the medicine of treatment colon cancer, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer, laryngeal carcinoma.
The present invention also provides a kind of anticancer pharmaceutical composition, and it is that HUANGJIANG hydrophobicity steroidal saponin extract by effective dose is an active component, adds the medicament that acceptable accessories or complementary composition are prepared from.
Wherein, described medicament is oral formulations or ejection preparation.
Medicine of the present invention is used for the treatment of various cancers, as colon cancer, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer, laryngeal carcinoma, efficacy stability, have efficient active anticancer, and directly extract the hydrophobicity steroidal saponin from HUANGJIANG, method is easy, cost is low, steady quality, controllability are strong, are suitable for industrialized great production, provide a kind of new medication to select for clinical.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Description of drawings
The HPLC finger printing of the hydrophobicity saponin of Fig. 1 the present invention preparation
Fig. 2 HUANGJIANG hydrophobicity steroidal saponin to growth of tumour cell propagation influence (annotate: HUANGJIANG steroidal saponin drug level is 10mg/L among the last figure, 1, control group, i.e. negative control group; 2, HUANGJIANG hydrophobicity steroidal saponin)
Fig. 3 HUANGJIANG hydrophobicity steroidal saponin and diosgenin anti-human ovarian carcinoma cell (SKOV3) drug effect comparison diagram.
Fig. 4 HUANGJIANG hydrophobicity of the present invention steroidal saponin extracts the drug effect of resistive connection intestinal cancer in the object
Fig. 5 HUANGJIANG hydrophobicity of the present invention steroidal saponin extracts the drug effect of anti-pulmonary carcinoma in the object
Fig. 6 HUANGJIANG hydrophobicity of the present invention steroidal saponin extracts the drug effect of anti-hepatocarcinoma in the object
Fig. 7 HUANGJIANG hydrophobicity of the present invention steroidal saponin extracts the drug effect of anti-cervical cancer in the object
The comparative experiments of Fig. 8 HUANGJIANG hydrophobicity of the present invention steroidal saponin extract survival rate life cycle
The specific embodiment
The preparation of embodiment 1 medicine of the present invention
After fresh Rhizoma Dioscoreae Zingiberensis tuber cleans up, be cut into bulk, 50 ℃ of oven dry are pulverized.Take by weighing about 600g HUANGJIANG powder in the 5000ml beaker, add about 2000ml soak with ethanol and spend the night, sucking filtration separates solid phase and liquid phase, and solid phase changes in the 5L Backflow bottle, adds 1200ml-2000ml ethanol, 78 ℃ of backflow 2h-5h, and filtered while hot repeats reflux, extract, 3 times.Filtrate and soak are merged, distilling under reduced pressure is to there not being the ethanol flavor substantially, reclaim ethanol, the 6-10 that presses extractum weight again is the about 2000ml water of water ultrasound wave dissolving solubility saponin extraordinarily, suspension centrifugal (4000-6000r/min, 10-20min), fractionate aqueous solutions and precipitation, to precipitate-65 ℃ of frozen drying 36h again, promptly get hydrophobicity total steroidal saponin 40g.
Rhizoma Dioscoreae Zingiberensis hydrophobicity total steroidal saponin 16g, at first adopt normal phase silica gel column chromatography to separate, eluant is mixed solvent chloroform-methanol (V/V), gradient elution, gradient is 100: 10 (600ml)-90: 10 (1000ml)-80: 20 (800ml)-70: 30 (500ml), TLC follows the tracks of detection, the colour developing of 10% sulphuric acid ethanol, merge the Rf value same composition, collect 4 different components, after gel chromatography SephadexLH20, eluant chloroform-methanol (8: 1) (volume ratio), get the monomer steroidal saponin behind the recrystallization repeatedly again, detect purity all greater than 98% through HPLC, through high resolution mass spec HR-ESI-MS, infrared spectrum IR, nuclear magnetic resoance spectrum, comprise 1H-NMR, 13C-NMR, 135DEPT, 1H-1HCOSY, HMBC, HMQC, spectrographic techniques such as NOECOSY identify that its structure is respectively: Trillium tschonoskii Maxim saponin (12mg), diosgenin cellobioside (68mg), triangle leaf saponin (128mg), gracillin (50mg);
Trillium tschonoskii Maxim saponin Trillin (C
33H
52O
8, 576): (glycosyl part contains a part glucose to 3-O-β-D-pyranose-diosgenin for 3-O-β-D-glucopyranosyl-diosgenin), belong to the spirostane saponin, C3 position link glycosyl part;
Diosgenin cellobioside Diosgenin diglucoside (C
39H
62O
13738): 3-O-β-D-pyranose-[(1 → 4)-β-D-Glucopyranose .]-diosgenin (3-O-β-D-glucopyranosyl (1 → 4)-β-D-glucopyranoside-diosgenin), belong to the spirostane saponin, C3 position link glycosyl part, glycosyl part contains two molecule glucoses, and the C3 position of inboard glucose is connected with a part glucose;
Triangle leaf saponin D eltonin (C
45H
72O
17848): 3-O-β-D-pyranose-[(1 → 2)-α-L-pyrans rhamnose]-[(1 → 4)-β-D-Glucopyranose .]-diosgenin (3-O-β-D-glucopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-glucopyranoside-diosgenin), belong to the spirostane saponin, C3 position link glycosyl part, glycosyl part contains two molecule glucoses and a part rhamnose, and the C2 of inboard glucose and C4 position are connected with a part rhamnose and a part glucose respectively.
Gracillin Gracillin (C
45H
72O
17, 848): diosgenin-3-O-[β-D-glucopyanosyl-(1 → 3)]-O-[α-L-pyrans rhamnose (1 → 2)]-O-β-D-glucopyranoside (3-O-β-D-glucopyranosyl (1 → 3)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-glucopyranoside-diosgenin)
By mass spectrum (HR-ESI-MS), infrared spectrum (IR), one-dimensional nuclear magnetic resonance (1D-NMR) and its structure of two dimensional NMR (2D-NMR) Analysis and Identification.HUANGJIANG hydrophobicity total steroidal saponin component is got a plurality of monomer saponins through normal phase silica gel column chromatography and gel chromatography separation and recrystallization.Utilization high resolution mass spec (HR-ESI-MS), infrared spectrum (IR), one-dimensional nuclear magnetic resonance (1D-NMR) and two dimensional NMR methods such as (2D-NMR) are identified active ingredient monomer saponin structure.In the normal phase column chromatography that is adopted, can select normal pressure or pressurized silica gel column chromatography for use, the optional column chromatography of used column packing is with silica gel or thin layer chromatography silica gel, eluant can be selected benzene/acetone for use, dichloromethane/acetone, chloroform/acetone, benzene/methanol, methylene chloride, chloroform/methanol, ethyl acetate/methanol, benzene/acetone, chloroform/methanol, one or more of solvent in methylene chloride/water or the ethyl acetate/methanol/water etc., can isocratic elution during eluting, also can gradient elution, whole separation process TLC tracking and monitoring.
Embodiment 2 medicine HPLC finger printing of the present invention
Chromatographic condition: Tianjin, island chromatograph LC-20; Chromatographic column: Shim-pack VP-ODS C
18(250 * 4.6mm i.d.5um), pre-column (5 μ m, 10 * 4.6mm i.d.); Binary eluent gradient eluting, water (A) and acetonitrile (B), 0-5min, 20%-30%B; 5-35min, 30%-40%B; 35-40min, 30%-40%B; 40-60min, 60%-85%B; 60-75min, 85%-100%B; 75-80min, 100%B; Flow velocity: 1ml/min; Column temperature: 30 ℃; Detect wavelength: λ=204nm; Sample size: 20 μ l.Finger printing is seen Fig. 1, wherein: 1. diosgenin, 2. Trillium tschonoskii Maxim saponin, 3. diosgenin cellobioside, 4. gracillin, 5. dioscin, 6. triangle leaf saponin, 8. former very thin saponin, 9. protodioscin, 10. former triangle leaf saponin, 11 Dioscorea parvifcora Ting saponin
The mensuration of each saponin content (mg/g) in the table 1 Rhizoma Dioscoreae Zingiberensis hydrophobicity total saponins
aComputing formula: saponin content=(cv)/m
0Wherein: c: the concentration of representative sample liquid (μ g/ml), measure three times and average; V: the cumulative volume of representative sample liquid (ml); m
0: the quality (mg) of representing the hydrophobicity total saponins;
bDo not detect;
cBe lower than minimum detection by quantitative limit (LOD).
By medicine of the present invention is measured, it is as follows that each organizes the content range of chemical compound, sees Table 2:
Each components contents scope in the table 2 HUANGJIANG hydrophobicity total extractives of steroid saponin
aComputing formula: saponin content=(cv)/m
0Wherein: c: the concentration of representative sample liquid (ug/ml), measure three times and average; V: the cumulative volume of representative sample liquid (ml); m
0: the quality (mg) of representing the hydrophobicity total saponins
Below prove beneficial effect of the present invention by pharmacodynamics test.
Adopt mtt assay to detect HUANGJIANG hydrophobicity steroidal saponin extract the malignant cell growth in vitro is suppressed activity research, method and result are as follows:
Test example 1 anti tumor activity in vitro experimental technique
1 cell strain and cultivation
With colon cancer C26, pulmonary carcinoma A549, breast carcinoma MCF-7, cervical cancer HeLa and oophoroma tumor Sk-OV-3, laryngeal carcinoma Hep-2 cell culture is in 10% inactivated fetal bovine serum, the 100U/ml penicillin, among the RPMI1640 or DMEM culture medium of 100 μ g/ml streptomycins, cultivate under 37 ℃ of 10%CO2 incubators and the saturated humidity condition, went down to posterity once in 3-4 days.
2 medicine antitumor cytolytic activities
Every hole adds 200 μ l and (contains 2-3 * 10 in 96 well culture plates
5Individual/the ml colon cancer, pulmonary carcinoma, breast carcinoma, cervical cancer, laryngeal carcinoma and ovarian cancer cell) contain the RPMI1640 of 10%FBS or the cell suspension of DMEM culture medium, put 37 ℃ of 10%CO
2After cultivating 12h in the incubator, be subjected to reagent to establish 6 dosage groups, every group of 5 parallel holes, matched group then adds the culture fluid of isoconcentration equal-volume solvent, repeats 3 experiments.96 well culture plates are put 37 ℃ of 10%CO
2After cultivating 48h in the incubator, abandoning supernatant adds the serum-free medium of the freshly prepared 0.5mg/ml of the containing MTT in 200 μ l/ holes, and 37 ℃ are continued to cultivate 4h.Discard the stillness of night, add 150 μ l DMSO, behind the concussion mixing, on microplate reader, be 570nm with the wavelength, reference wavelength is that 490nm measures the OD value, calculates suppression ratio, and formula is: inhibitory rate of cell growth=(matched group OD value-experimental group OD value)/matched group OD value * 100%.And by inhibitory rate of cell growth and corresponding concentration, by computed in software IC
50Value (half suppression ratio), IC
50(inhibitory rate of cell growth is 50% drug level)
Two, experimental result: mtt assay is measured variable concentrations HUANGJIANG hydrophobicity steroidal saponin influences (see figure 2) for the tumor cell line growth activity
Table 3 HUANGJIANG hydrophobicity steroidal saponin suppresses active to tumor cell extracorporeal growth
Three, conclusion
Mtt assay is the common method of anti tumor activity in vitro, the primary dcreening operation experiment of Chang Zuowei anti-tumor activity medicine.The experiment proved that HUANGJIANG hydrophobicity spirostane steroidal soap total glycosides is to colon cancer, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer, cell lines such as laryngeal carcinoma have obvious suppression cell line growth activity.
Test example 2 HUANGJIANG hydrophobicity steroidal saponin extracts of the present invention and diosgenin anti-human ovarian carcinoma cell (SKOV3) drug effect are relatively
With the DMSO dissolving, the reuse culture medium dilutes it for concentration is 5,10 respectively respectively for diosgenin and HUANGJIANG hydrophobicity steroidal saponin extract, and 20mg/L guarantees that simultaneously DMSO concentration is lower than 0.01% (V/V).Every hole adds 200ul and (contains 1 * 10 in 96 well culture plates
5Individual/ml oophoroma tumor cell) contain the cell suspension of the RPMI1640 culture medium of 10%FBS, put 37 ℃ of 10%CO
2After cultivating 12h in the incubator, adding concentration respectively is 5,10, and 20mg/L diosgenin and HUANGJIANG hydrophobicity total steroidal saponin are organized in contrast to add the 0.01%DMSO culture medium, repeat 3 experiments.96 well culture plates are put 37 ℃ of 10%CO
2After cultivating 48h in the incubator, abandoning supernatant adds the freshly prepared serum-free medium that contains 0.5mg/ml MTT in 200ul/ hole, and 37 ℃ are continued to cultivate 4h.Discarding the stillness of night, add 150ul DMSO, behind the concussion mixing, is 570nm with the wavelength on microplate reader, and reference wavelength is that 490nm measures OD value, calculating cell growth vigor, and formula is: cell growth vigor=experimental group OD value/matched group OD value * 100%.
HUANGJIANG hydrophobicity steroidal saponin extract and diosgenin anti-human ovarian carcinoma cell (SKOV3) drug effect are relatively seen Fig. 3.
As shown in Figure 3, with under the isoconcentration, the cell proliferation rate of HUANGJIANG hydrophobicity steroidal saponin extract of the present invention is starkly lower than diosgenin, proves that HUANGJIANG hydrophobicity steroidal saponin extract of the present invention has obvious suppression cell line growth activity.
The 3 HUANGJIANG hydrophobicity steroidal saponin extract anti-tumor in vivo experiments of the present invention of test example
1. the foundation of animal model
(1) the colon cancer C26 cell that will be in exponential phase is made into cell suspension, and cell concentration is 10
7/ ml.
Get 19～23g male BALB/C mice in (6--8 age in week), sterilized near the right hind place in the back with alcohol swab, subcutaneous injection C26 cell suspension 50ul, tumor can grow after about 7 days.
(2) people's pulmonary carcinoma LL2, hepatocarcinoma H22, the s that will be in exponential phase is made into cell suspension, and cell concentration is respectively 10
7/ ml.
Get 18～20g male nude mouse in (6--8 age in week), sterilized near the right hind place in the back with alcohol swab, subcutaneous pulmonary carcinoma LL2, hepatocarcinoma H22, the s suspension 50ul of injecting respectively, tumor can grow after about 7 days.
2. experiment grouping and treatment
Inoculation one week of back has become the animal model of tumor to be divided into 4 groups respectively at random with all kinds of after the inoculation, 10 every group, irritates the stomach treatment.
First group is matched group (CN).When other groups were treated, this group poured into the normal saline that is dissolved with DMSO (＜0.1%) of equal volume.
The back is the administration group for three groups, and medicine (by the HUANGJIANG hydrophobicity steroidal saponin extract of embodiment 1 preparation) is dissolved in and contains in DMSO (＜0.1%) normal saline, and administration concentration is 10mg/kg, 20mg/kg, 40mg/kg.
3. detection index
Weighing mice body weight before the administration for the first time is by the body weight administration.Later one all weighings twice, the record body weight change.
Measure the tumor line of apsides before the administration for the first time, biweekly later on, draw tumor growth curve behind the calculating gross tumor volume.Calculate gross tumor volume with length/minor axis by following formula:
Volume (mm
3Long (mm) * wide by 2 (mm2) in)=1/2
Take off neck execution model mouse, the complete tumor of peeling off after treating for 3 weeks.
Get two mices for every group and strip tumor and do SABC, HE dyeing, western blot etc. does drug mechanism research.See Fig. 4, Fig. 5, Fig. 6, Fig. 7.
Above result shows, HUANGJIANG hydrophobicity steroidal saponin extract 20mg/kg, 30mg/kg, 40mg/kg be all to colon cancer in the body, pulmonary carcinoma, and hepatocarcinoma and cervical cancer all have inhibitory action, and increase tumor-inhibiting action with concentration and strengthen.
4 HUANGJIANG hydrophobicity steroidal saponin extract of the present invention experiments life cycle of test example
1. the foundation of animal model
The C26 cell that will be in exponential phase is made into cell suspension, and cell concentration is 10
7/ ml.Get 19～23g male BALB/C mice in (6--8 age in week), sterilized near the right hind place in the back with alcohol swab, subcutaneous injection C26 cell suspension 0.1ml, tumor can grow after about 7 days.
2. experiment grouping
Inoculation one week of back will become the animal model of tumor to be divided into 4 groups at random after the inoculation, 10 every group, irritates stomach and treats for three weeks.
First group is matched group (CN).When other groups were treated, this group poured into the normal saline that is dissolved with DMSO (＜0.1%) of equal volume.
The back is the administration group for three groups, and medicine (the HUANGJIANG hydrophobicity steroidal saponin extract of embodiment 1 preparation) is dissolved in and contains in DMSO (＜0.1%) normal saline, and administration concentration is 10mg/kg, 20mg/kg, 40mg/kg.
Observe the existence situation of a mouse in per three days after treatment is finished, continue to observe 60 days, draw curve life cycle, see Fig. 8.
The mean survival time (MST) that finished each group of experiment by 65 days, be respectively 31.3d, 41.2d, and 48.9d, 55.1d has obvious prolongation the life cycle of administration group mice.
In sum, HUANGJIANG hydrophobicity steroidal saponin extract of the present invention is used for the treatment of various cancers, as colon cancer, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer, laryngeal carcinoma, efficacy stability, has efficient active anticancer, drug effect obviously is better than the diosgenin of Isodose, can obviously prolong the life cycle of tumor patient, and directly extract the hydrophobicity steroidal saponin from HUANGJIANG, method is easy, and cost is low, steady quality, controllability are strong, be suitable for industrialized great production, steady quality, controllability are strong, provide a kind of new medication to select for clinical.
Claims (9)
1. a HUANGJIANG hydrophobicity steroidal saponin extract is characterized in that: contain Trillium tschonoskii Maxim saponin 0-1.021mg, diosgenin cellobioside 8-30.342mg, triangle leaf saponin 16.461-93.947mg, gracillin 0-8.456mg in every g extract.
2. HUANGJIANG hydrophobicity steroidal saponin extract according to claim 1 is characterized in that: contain Trillium tschonoskii Maxim saponin 0.891mg, diosgenin cellobioside 28.178mg, triangle leaf saponin 83.974mg, gracillin 1.450mg in every g extract.
3. HUANGJIANG hydrophobicity steroidal saponin extract according to claim 1 and 2, it is characterized in that: it is to be prepared from by following steps:
A, with medicinal plants HUANGJIANG Dioscorea zingiberensis C.H.Wrigh tuber oven dry, pulverize, add 70-100%v/v ethanol lixiviate 10-24 hour, solid-liquid separation;
The solid phase of b, isolated by filtration added the 70-100%v/v alcohol reflux 2-5 hour, 3-5 time, filtered;
C, the liquid phase of the liquid phase of a step lixiviate and b step reflux, extract, merged after, distilling under reduced pressure is reclaimed ethanol and is distinguished the flavor of to there being ethanol substantially, gets brown extractum;
D, brown extractum is dissolved in water, at the centrifugal 15-20 of 4000-6000r/min minute, water phase separated and solid phase;
E, with the solid phase lyophilization that d obtains, promptly get HUANGJIANG hydrophobicity steroidal saponin extract.
4. according to any described HUANGJIANG hydrophobicity steroidal saponin extract of claim 1-3, it is characterized in that: the HPLC finger printing of this extract as shown in Figure 1; Chromatographic condition: Tianjin, island chromatograph LC-20; Chromatographic column: Shim-pack VP-ODS C
18(250 * 4.6mm i.d.5um), pre-column (5 μ m, 10 * 4.6mm i.d.); Binary eluent gradient eluting, water (A) and acetonitrile (B), 0-5min, 20%-30%B; 5-35min, 30%-40%B; 35-40min, 30%-40%B; 40-60min, 60%-85%B; 60-75min, 85%-100%B; 75-80min, 100%B; Flow velocity: 1ml/min; Column temperature: 30 ℃; Detect wavelength: λ=204nm; Sample size: 20 μ l.
5. method for preparing any described HUANGJIANG hydrophobicity steroidal saponin extract of claim 1-4, it is characterized in that: it comprises the steps:
A, with medicinal plants HUANGJIANG Dioscorea zingiberensis C.H.Wrigh tuber oven dry, pulverize, add 70-100%v/v ethanol lixiviate 10-24 hour, solid-liquid separation;
The solid phase of b, isolated by filtration added the 70-100%v/v alcohol reflux 2-5 hour, and 3-5 time, filtered while hot;
C, the liquid phase of the liquid phase of a step lixiviate and b step reflux, extract, merged after, distilling under reduced pressure is reclaimed ethanol and is distinguished the flavor of to there being ethanol substantially, gets brown extractum;
D, brown extractum is dissolved in water, at the centrifugal 15-20 of 4000-6000r/min minute, water phase separated and solid phase;
E, with the solid phase lyophilization that d obtains, promptly get HUANGJIANG hydrophobicity steroidal saponin extract.
6. any described HUANGJIANG hydrophobicity steroidal saponin extract of claim 1-4 is in the purposes of preparation in the cancer therapy drug.
7. purposes according to claim 6 is characterized in that: described medicine is the medicine of treatment colon cancer, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer or laryngeal carcinoma.
8. anticancer pharmaceutical composition is characterized in that: it is to be active component by any described HUANGJIANG hydrophobicity steroidal saponin extract of the claim 1-4 of effective dose, adds the medicament that acceptable accessories or complementary composition are prepared from.
9. pharmaceutical composition according to claim 8 is characterized in that: described medicament is oral formulations or ejection preparation.
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| CN108295085A (en) * | 2018-01-30 | 2018-07-20 | 中山大学附属第医院 | Application of the protodioscin in preparing anti-drug resistance bone and flesh tumor medicine |
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