CN1224392C - Antitumor use of diosgenin - Google Patents
Antitumor use of diosgenin Download PDFInfo
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- CN1224392C CN1224392C CN 02155238 CN02155238A CN1224392C CN 1224392 C CN1224392 C CN 1224392C CN 02155238 CN02155238 CN 02155238 CN 02155238 A CN02155238 A CN 02155238A CN 1224392 C CN1224392 C CN 1224392C
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Abstract
The present invention discloses the new medical purpose of diosgenin obtained from plants, such as dioscorea nipponica, etc., in an anti-tumor aspect. The present invention also discloses a medicinal preparation in the forms of tablets, capsules, drop pills, injections, etc., by using diosgenin as medical preparation raw material, and more specially, the present invention discloses the function of diosgenin in the aspects of treating gastric cancer, mammary cancer, lung cancer, colon cancer, glioma, erythroleukemia, melanoma, cervical cancer, liver cancer, or promyelocytic leukemia.
Description
Technical field
The present invention relates to the pharmaceutic usage of diosgenin, is the pharmaceutic usage of diosgenin in the preparation antitumor drug specifically.
Background technology
Diosgenin is a kind of a kind of ruscogenin that extracts from yam, and the current external synthetic used initiation material of corticosteroid drug has 60% to be diosgenin, and the annual production of Rhizoma Dioscoreae is the 600-800 ton, wherein Mexico's annual production 200-500 ton.Contain the diosgenin in the middle of multiple Rhizoma Dioscoreae, domestic through generaI investigation, it is more to have found that plants such as Dioscorea nipponica, shield leaf, Dioscorea panthaica Prain et Burkill contain the diosgenin amount.For the extraction of diosgenin, the industrial process that all adopts both at home and abroad organic solvent extraction diosgenins such as the direct acid hydrolysis of dioscin tuber dry powder, industrial napthas.For diosgenin application clinically, bibliographical information is less, there is report that diosgenin is used to prepare skin care liquid, with diosgenin, Borneolum Syntheticum, salicylic acid, chloromycetin, glycerol, ethanol compositing formula, be mainly used in dermatosiss such as treatment acne, the excessive dermatitis of matter and tinea versicolor, not seeing so far has bibliographical information that diosgenin is prepared into anti-tumor drug.
Summary of the invention
One of purpose of the present invention has provided the pharmaceutical preparation of diosgenin; Two of purpose of the present invention has provided the antineoplastic new usage of diosgenin.
Pharmaceutical preparation of the present invention contains the diosgenin of 1%-99% and the excipient of 99%-1% (medicine that comprises other adapted), preferably contain the diosgenin of 30%-80% and the pharmaceutical excipient of 70%-20% (comprising the medicine that other compatibility is used), preferably contain the diosgenin of 60%-70% and the excipient of 40%-30% (comprising the medicine that other compatibility is used).
Press practice of pharmacy, dioscin of the present invention can be prepared into the various clinical pharmaceutical dosage form as antitumor drug, comprise the dosage form of oral formulations or parenterai administration.Said oral formulations is selected from any in tablet, capsule, pill, granule, suspensoid, drop pill, the oral liquid; Said non-intestinal is selected from a kind of in the middle of injection, aerosol, suppository or the subcutaneous administration dosage form to dosage form.
Adjuvant in the antitumor drug of the present invention is meant conventional excipient, as solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent etc.The medicine that other compatibility in the antitumor drug of the present invention is used, the diosgenin that refers to effective dose is certain medicine material, again compatibility other allowed the Chinese medicine or the chemical drugs that share.
Diosgenin pharmaceutical preparation of the present invention has antitumor action, is to be confirmed by following experimental example.
Experimental example 1The extracorporeal anti-tumor function of diosgenin
Sample: diosgenin diosgenin
Cell culture:
L929, the HeLa cell strain is from (American Tissue Culture Collection, ATCC, USA), with RPMI 1640 (Gibco) culture fluid, the hyclone of adding 10%, 1% antibiosis Niu Su (10,000U/ml penicillin and 10,00 μ g/ml streptomycins), the 2mM glutamine places 4% incubator to cultivate.
The experiment of cell in vitro poison:
Get the cell that is in exponential phase, attached cell (L929, HeLa) behind trypsinization, be inoculated in respectively 96 well culture plates (1 * 10/ml), after treating that cell is adherent fully. add each concentration sample respectively with the preparation of RPMI1640 culture fluid, 3 multiple holes of every kind of sample, establish 0 contrast and negative control simultaneously, continue to cultivate 48h.Carry out colorimetric determination with mtt assay in 595nm, its meansigma methods is the cell survival optical density, and the result and the matched group of gained compared, and calculates cell mortality under each condition (as shown in the table).
The inductive death of neoplastic cells rate of the diosgenin of table 1. variable concentrations (%)
| Drug level μ g/ml | Death of neoplastic cells rate (%) | |
| L929 | HeLa | |
| 1 5 10 20 50 100 | 0 7.5 15.0 25.8 45.2 82.4 | 0 12.3 20.5 37.3 50.5 98.5 |
Apoptotic morphology research method:
Examine under a microscope cellular morphology.Compare with undressed reward sexual cell, experimental group is carefully embraced (diosgenin administration group) and is had typical apoptosis morphological change, and cell volume dwindles, and becomes circle, pyknosis, has formed apoptotic body the late period that has.
Utilize agarose gel electrophoresis to detect DNA Ladder:
Dna fragmentation extracting and dna gel electrophoresis
(1) experimental technique: collect HeLa cell (about 2 * 10
6Individual), be transferred in the concentrator bowl, centrifugal after, PBS washing, add cell lysis buffer solution (5mM Tris, 20mMEDTA, 0.5%TritonX-100) 37 ℃ of 30min, then in 16,000rpm, 4 ℃ of centrifugal 20min, draw supernatant (containing dna fragmentation) and cultivate lh with RnaseA and E.C. 3.4.21.64 altogether in 37 ℃ respectively, add 20 μ lNaCl (5M), 120 μ l isopropyl alcohols, place-20 ℃ of placements to spend the night, once more the son 16,000rpm, 4 ℃ of centrifugal 15min remove supernatant, and repeated centrifugation once.Remove supernatant fully, add 20TE (Tris-EDTA) buffer, the dissolving DNA fragment, in the voltage factory of 100V, the agarose gel with 2%, electrophoresis 30min in TBE (Tris-borate-EDTA) buffer, make indicator with BPB, the DNALadder molecular weight standard that adds 100bp is simultaneously used 0.5g/mlEB{ethidium bromide as reference) dyeing 15min, the decolouring back is observed under the UV diaphotoscope.
(2) conclusion: agarose gel electrophoresis method for detecting is separation, purification, the segmental typical method of identification of dna, outstanding biochemistry during apoptosis change be chromatin dna the control cracking arranged, activation along with Cobra venom endonuclease, dna degradation becomes the oligomerization nucleosome, the nucleus DNA crack fragment is the oligomerization nucleotides fragment of 180-200 base pair integral multiple, shows as trapezoidal electrophoresis pattern on gel electrophoresis.After diosgenin is handled 48h, HeLa cell suspension is after cracking digests routinely method and extracts DNA, in agarose gel, carry out electrophoresis, bromination second pyridine (EB) dyeing, as seen typical scalariform band (Ladder), and in negative cells, its nuclear is complete, can not produce low-molecular-weight DNA.
Conclusion: under isolated condition, diosgenin is a concentration when being 20-100 μ g/ml, and it has the obvious suppression effect to L929 and HeLa cell.The evidence of dna gel electrophoresis, after diosgenin is handled 48 hours, the HeLa cell suspending liquid is after cracking digestion, after extracting DNA according to a conventional method, electrophoresis in agarose gel, ethidium bromide staining, visible typical scalariform band (Ladder), promptly diosgenin can cause the HeLa natural death of cerebral cells.
Experimental example 2The whole antitumor action of diosgenin
Experiment purpose: observe diosgenin (Diosgenin, whole antitumor action Dio).
Method: during medicine shown in the observation is just connecing 4 kinds of mice transplanted tumors [S-180 (S-180), hepatoma ascitic type (HepA), mouse cervical cancer-14 (U14) ehrlich carcinoma (EAC)] armpit skin, administration every day is 1 time in inoculating 10, calculates each administration group struma suppression ratio.Result: (1) Dio 200,100 and 50mg.kg
-1Ig has the obvious suppression effect to S180, and tumour inhibiting rate is respectively 50.6%, 42.9% and 35.9%; Dio 10050 and 25mg.kg
-1Ip all has the obvious suppression effect to S180, tumour inhibiting rate be respectively 51.3%, 46.8% and 34.6%:(2) Dio 200 and 100mg.kg
-1Ig has the obvious suppression effect to HepA, and tumour inhibiting rate is respectively 41.6% and 30.4%; When its ip dosage is 100 and 50mg.kg
-1The time HepA also had the obvious suppression effect, tumour inhibiting rate is respectively 41.0% and 37.3%; (3) Dio 200,100 and 50mg.kg
-1Ig also has the obvious suppression effect to U14, and tumour inhibiting rate is respectively 44.6%, 34.7% and 28.3%; Dio100 and 50mg.kg
-1Ip has the obvious suppression effect to U14, and tumour inhibiting rate is respectively 45.% and 30.4%; (4) no matter be to irritate stomach, or lumbar injection does not all have inhibitory action (P＞0.05) to EAC. conclusion: diosgenin has obvious antineoplastic.
4 kinds of tumor ascites of animal mice of going down to posterity is all available from the institute of oncology, Jilin Province; Kunming mice, body weight 18-22g is available from Univ. of Farming and Stockbreeding, PLA's Experimental Animal Center (animal quality certification number: 10-5112).
The medicine diosgenin is provided by the Drug Manufacturing Room of Jilin Natural Pharmatech Co., Ltd..The injection cyclophosphamide, specification: 200mg/ props up, lot number: 980101, Hualian Pharmaceutical Co., Ltd., Shanghai's product.
The method experiment mice is divided into contrast (ig distilled water 20ml.kg at random
-1), cyclophosphamide
(ip20mg.kg
-1), Dio (ig 200ml.kg
-1, 100ml.kg
-1And 50ml.kg
-1) and Doi (ip100ml.kg
-1, 50ml.kg
-1And 25ml.kg
-18 groups.Chose transplantation tumor 7 days, tumor growth is good, the abdominal part grand tangible mice of splashing, the skin of abdomen sterilization, thrust the abdominal cavity with disposable sterilized hemostix through stomach wall, extraction ascites is put into aseptic beaker and is diluted to 1: 3 cancerous cell suspension with normal saline, in the right oxter inoculation of every experiment mice 0.2ml
[1]Each treated animal begins administration, every day 1 time, successive administration 10 days in inoculated tumour next day.Administration finishes next day, and dislocation was put to death after animal was weighed, and separated subcutaneous lump and weighed, and respectively organized tumor
Growing state, " t " check between statistical procedures method employing group.
The result
Each treated animal body weight, tumor weight and tumour inhibiting rate statistical result see Table 2.
The influence that table 2 Dio is heavy to mice (S180) tumor (X ± S)
| Group | Dosage mg.kg -1 | Number of animals (n) | The weight of animals (g) | Tumor heavy (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | |||||
| Control group endoxan Dio (ig) Dio (ig) Dio (ig) Dio (ip) Dio (ip) Dio (ip) | - 20 200 100 50 100 50 25 | 10 10 10 10 10 10 10 10 | 18.5±1.51 19.0±1.56 18.7±1.95 18.4±1.78 18.7±1.95 19.1±1.91 18.9±1.97 18.9±1.91 | 24.3±3.06 22.5±2.07 23.3±1.83 22.2±2.57 23.7±3.40 23.2±1.93 24.9±3.14 22.9±3.96 | 1.56±0.70 0.70±0.25** 0.77±0.29** 0.89±0.34* 1.00±0.37* 0.76±0.24** 0.83±0.32** 1.02±0.30* | 55.1 50.6 42.9 35.9 51.3 46.8 34.6 |
Annotate: compare * p＜0.05 with matched group; * p＜0.01
The influence that table 3 Dio is heavy to mice (HepA) tumor (X ± S)
| Group | Dosage mg.kg -1 | Number of animals (n) | The weight of animals (g) | Tumor heavy (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | |||||
| Control group endoxan Dio (ig) Dio (ig) Dio (ig) Dio (ip) Dio (ip) Dio (ip) | - 20 200 100 50 100 50 25 | 9 9 10 10 10 10 10 10 | 18.0±1.41 18.2±1.72 18.0±1.89 18.0±1.33 18.1±1.20 18.1±1.37 18.1±1.20 18.1±1.45 | 26.3±2.96 21.4±3.36 27.1±2.85 2.69±2.18 26.8±2.25 24.2±1.81 26.0±3.09 24.0±3.16 | 1.61±0.38 0.41±0.15*** 0.94±0.34*** 1.12±0.30** 1.21±0.53* 0.95±0.37** 1.01±0.43** 1.17±0.64# | 74.5 41.6 30.4 24.8 41.0 37.3 27.3 |
Annotate: compare #p＞0.05 with matched group; * pp＜0.01; * * p＜0.01
The influence that table 4 Dio is heavy to mice U14 tumor (X ± S)
| Group | Dosage mg.kg -1 | The weight of animals (g) | Tumor heavy (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | ||||
| Control group endoxan Dio (ig) Dio (ig) Dio (ig) Dio (ip) Dio (ip) Dio (ip) | - 20 200 100 50 100 50 25 | 18.3±0.95 18.6±1.08 18.5±0.85 18.5±0.85 18.5±0.85 18.4±0.84 18.4±0.84 18.4±0.84 | 28.9±2.98(9) 25.7±3.40(10) 24.7±4.92(7) 26.4±3.62(8) 26.2±3.90(9) 26.1±3.04(10) 24.0±3.80(10) 26.1±3.14(10) | 1.84±0.40 1.21±0.34** 1.02±0.50** 1.20±0.32** 1.32±0.61* 1.00±0.54** 1.28±0.21** 1.44±0.84# | 34.2 44.6 34.7 28.3 45.7 30.4 21.7 |
Annotate: compare #p＞0.05 with matched group; * p＜0.05; * p＜0.01
Table 5 Dio is to the heavy (X ± S) of mice ehrlich ascites tumor
| Group | Dosage mg.kg -1 | The weight of animals (g) | Tumor heavy (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | ||||
| Control group endoxan Dio (ig) Dio (ig) Dio (ig) Dio (ip) Dio (ip) Dio (ip) | - 20 200 100 50 100 50 25 | 20.4±0.97 20.4±0.97 20.5±0.85 20.4±0.97 20.5±0.85 20.4±0.97 20.4±0.97 20.4±0.97 | 30.3±2.71(10) 26.6±3.89(10) 27.8±3.53(9) 2.69±3.99(10) 29.0±2.18(9) 228.2±1.86(9) 27.1±2.56(10) 30.5±4.72(10) | 1.91±1.10 1.45±0.52# 1.46±0.32# 1.70±0.54# 1.50±0.68# 1.78±0.80# 2.07±0.85# 2.27±1.05# | 24.1 23.6 11.0 21.5 6.8 -8.4 37.3 |
Annotate: compare #p＞0.05 with the photograph group; * p＜0.05;
Experimental example 3:Diosgenin is to transplanting the influence of people's gastric cancer (MGC-803) tumor bearing nude mice tumor weight
1.1 animal BALB/C-nu strain nude mice, SPF level, body weight 16～20g, male and female dual-purpose.No. the 035th, the real moving word of the Liao Dynasty) and laboratory animal portion of Tumour Inst., Chinese Medical Academy (the animal quality certification number: SCXKLL-00-0005) respectively available from laboratory animal portion of Chinese Medical Sciences University (the animal quality certification number:.
1.2 tumor cell people Gastric Cancer MGC-803 tumor cell line is provided by Jilin Province tumour hospital.
1.3 the medicine diosgenin, white powder, purity is more than 95.69%, lot number: 000126, the Drug Manufacturing Room of our unit provide; Cyclophosphamide for injection, specification: 200mg/ props up, lot number: 20010915, Hualian Pharmaceutical Co., Ltd., Shanghai's product.
1.4 conspicuous Li Shi (Heraeus) the HERA D-63450 type CO2 gas incubator of instrument, German product; SB-JB-1B type superclean bench, Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd. product; AB204 one N type list range 100,000/electronics Libra, prunus mume (sieb.) sieb.et zucc. Teller-holder benefit instrument (Shanghai) Co., Ltd. product.
2. whole experimental implementation process of method and nude mice are raised all at no specificity source of disease body (Specific Pathogen-Free; SPF) carry out in the ultra-clean laminar-flow rack under the condition.All (121 ℃, 40min) use the back through autoclaving for mouse cage, feedstuff, drinking water, bedding and padding, experimental drug, experiment equipment.With people's Gastric Cancer MGC-803 tumor cell that the prosthesis unofficial biography are commissioned to train foster, give 5 nude mices earlier, every left side armpit subcutaneous vaccination 0.2mL tumor cell suspension (tumor cell number is 1.1 * 107).After the inoculation, observe routinely and raise.After 20 days, when lump to be inoculated grows to the 1g left and right sides,, strip out lump, reject fibrous capsule, choose well-grown, be the tumor tissue of white powder, flesh of fish shape, place plate sacrifice of animal.Be cut into lmm3 with scalpel) about fritter (the about 40mg of weight), add an amount of normal saline again, as the source of people's gastric cancer (MGC-803) model of nude mice bearing tumor animal inoculation struma.With the tumor piece that cuts with 18
#Puncture needle tubing front end is given 1 of every subcutaneous implantation of left side armpit of accepting the laboratory observation nude mice.Next day, postvaccinal nude mice is divided into contrast (ig distilled water 10ml/kg, every day 1 time) at random; Cyclophosphamide (ig25mg/kg, the next day 1 time); 5 groups of diosgenins (ig400mg/kg, 200mg/kg and 100mg/kg, every day 1 time).And begin administration simultaneously.The administration cycle was 6 weeks.Administration finishes next day, and animal heavy back dislocation is put to death, and separates subcutaneous lump and weighs, and respectively organizes tumor weight.And obtain the tumor suppression percentage rate.For guaranteeing the reliability of experimental result, this experiment repeats 3 batches.
3. result
Each treated animal body weight, tumor weight, tumour inhibiting rate and lump size see Table 6～table 9 respectively.
Table 6, diosgenin are to the influence of people's gastric cancer (MGC-803) tumor bearing nude mice tumor weight (first x ± s)
| Group | Dosage mg/kg | The weight of animals (g) | Tumor piece weight (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | ||||
| Contrast cyclophosphamide diosgenin diosgenin diosgenin | - 25 400 200 100 | 22.0±1.41 21.8±1.47 21.5±1.64 21.5±1.87 21.8±0.75 | 29.2±1.47 29.3±2.06 28.7±2.58 29.0±2.90 29.5±2.34 | 1.22±0.27 0.28±0.18*** 0.45±0.22*** 0.57±0.15*** 0.66±0.24** | 77.1 63.1 53.3 45.9 |
Annotate: compare * * p＜0.01 with matched group; * * p＜0.001.Every group of 6 animals are male.
Table 7, diosgenin are to the influence of people's gastric cancer (MGC-803) tumor bearing nude mice tumor weight (the second crowd of x ± s)
| Group | Dosage mg/kg | The weight of animals (g) | Tumor piece weight (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | ||||
| Contrast cyclophosphamide diosgenin diosgenin diosgenin | - 25 400 200 100 | 20.8±0.98 19.3±1.63 20.7±1.21 21.2±1.60 20.5±1.64 | 26.8±1.60 24.8±1.94 28.0±1.67 26.5±3.02 28.2±1.94 | 1.25±0.27 0.31±0.18*** 0.49±0.30*** 0.63±0.29** 0.74±0.40* | 75.2 60.8 49.6 40.8 |
Annotate: compare * p＜0.05 with matched group; * p＜0.01; * * p＜0.001.Every group of 6 animals are male.
Table 8, diosgenin are to the influence of people's gastric cancer (MGC-803) tumor bearing nude mice tumor weight (the 3rd crowd of x ± s)
| Group | Dosage mg/kg | The weight of animals (g) | Tumor piece weight (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | ||||
| Contrast cyclophosphamide diosgenin diosgenin diosgenin | - 25 400 200 100 | 21.9±2.59 20.9±2.36 22.3±1.37 22.7±1.75 21.1±3.43 | 28.1±1.07 24.5±3.85 26.5±1.22 27.6±1.86 27.5±2.59 | 1.67±0.34 0.36±0.26*** 0.52±0.15*** 0.68±0.27*** 0.89±0.37** | 78.4 68.9 59.3 46.7 |
Annotate: compare * * p＜0.01 with matched group; * * p＜0.001.Every group of 6 animals are male.
(three batches add up to x ± s) in the influence that table 9, diosgenin to people's gastric cancer (MGC-803) tumor bearing nude mice tumor heavily are
| Group | Dosage mg/kg | The weight of animals (g) | Tumor piece weight (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | ||||
| Contrast cyclophosphamide diosgenin diosgenin diosgenin | - 25 400 200 100 | 21.6±1.88 20.8±2.10 21.5±1.51 21.8±1.77 21.2±2.18 | 28.0±1.73 26.1±3.53 27.7±2.02 27.5±2.94 28.3±2.40 | 1.38±0.35 0.32±0.21*** 0.49±0.22*** 0.61±0.24** 0.76±0.34* | 76.8 64.5 55.8 44.9 |
Annotate: compare * p＜0.05 with matched group; * p＜0.01; * * p＜0.001.18 animals of every combination meter, the male and female dual-purpose.
4. conclusion
Three batches of experimental results of comprehensive above-mentioned repetition: no matter be that every batch of experimental result is added up separately, still three batches of experimental results are summed up statistics, diosgenin 400,200 and 100mg/kgig all have the obvious suppression effect to transplanting people's gastric cancer (MGC-803) tumor bearing nude mice tumor growth.And presenting dose-effect relationship preferably, average tumour inhibiting rate can reach 44.47～64.27%.Though its tumour inhibiting rate makes animal occur being not in good state unlike cyclophosphamide not as good as cyclophosphamide, become thin and toxic and side effects such as body weight obviously alleviates.
Experimental example 4:Diosgenin is to transplanting the influence of hepatic ascites (HepA) mice time-to-live
Materials and methods
The animal kunming mice, female, body weight 18～20g, available from laboratory animal portion of Jilin University, the thing quality certification number: 10-1023.Hepatocarcinoma (HepA) the ascites mice of going down to posterity is available from the institute of oncology, Jilin Province.
The medicine diosgenin, specification: purity is more than 98%, lot number: 20000512.The Drug Manufacturing Room of our unit provide; Cyclophosphamide for injection, specification: 200mg/ props up, lot number: 20010915, Hualian Pharmaceutical Co., Ltd., Shanghai's product.
Instrument SB-JB-1B type superclean bench, Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd. product; AB204-N type list range 100,000/electronics Libra, prunus mume (sieb.) sieb.et zucc. Teller one holder benefit instrument (Shanghai) Co., Ltd. product.
Method
[1,2]Experiment mice is divided into contrast ig distilled water 20ml/kg, every day 1 time at random], 5 groups of cyclophosphamide (ig 50mg/kg, the next day 1 time), diosgenin (ig 200mg/kg, 100mg/kg and 50mg/kg, every day 1 time).Choose and transplant the HepA tumor cell line about 7 days, tumor growth is good, the tangible mice of abdominal tympanites.In superclean bench, with the mouse part skin sterilization, thrust the abdominal cavity through stomach wall with disposable sterilized hemostix, extract ascites, put into aseptic beaker and be diluted to 1: 4 cancerous cell suspension with normal saline, examine under a microscope,
Tumor cell number is 1.2 * 107/ml.Give every mouse peritoneal inoculation 0.2ml.Each treated animal is in inoculated tumour random packet next day and begin administration.The natural law that the record animal can survive from the abdominal cavity inoculated tumour.And calculating administration group is with respect to the increase in life span of matched group.For guaranteeing result's accuracy, test repeats 3 batches altogether.
The result
Each treated animal The average survival time natural law of experimental session and increase in life span see Table 10～table 12 respectively.
Table 10, diosgenin are to the influence of intraperitoneal transplantation hepatic ascites (HepA) mice time-to-live (first x ± s)
| Group | Dosage (mg/kg) | Number of animals (only) | Deposit the time (my god) | Increase in life span (%) |
| Rhizoma Dioscoreae soap unit of Rhizoma Dioscoreae soap unit of positive control cyclophosphamide Rhizoma Dioscoreae soap unit | - 50 200 100 50 | 10 10 10 10 10 | 11.0±3.0 27.7±7.6 *** 20.3±4.2 *** 18.0±4.1 *** 16.3±3.3 ** | 151.8 84.5 63.6 50.0 |
Annotate: compare * * p＜0.01 with matched group; * * p＜0.001
Table 11, diosgenin are to the influence of intraperitoneal transplantation hepatic ascites (HepA) mice time-to-live (the second crowd of x ± s)
| Group | Dosage (mg/kg) | Number of animals (only) | Deposit the time (my god) | Increase in life span (%) |
| Rhizoma Dioscoreae soap unit of Rhizoma Dioscoreae soap unit of positive control cyclophosphamide Rhizoma Dioscoreae soap unit | - 50 200 100 50 | 10 10 10 10 10 | 17.3±4.1 28.2±6.5 *** 26.5±3.9 *** 23.3±2.2 *** 19.2±4.1 # | 63.0 53.2 34.7 9.8 |
Annotate: compare * * p＜0.001 with matched group; * p＜0.05
Table 12, diosgenin are to the influence of intraperitoneal transplantation hepatic ascites (HepA) mice time-to-live (the 3rd crowd of x ± s)
| Group | Dosage (mg/kg) | Number of animals (only) | Deposit the time (my god) | Increase in life span (%) |
| Rhizoma Dioscoreae soap unit of Rhizoma Dioscoreae soap unit of positive control cyclophosphamide Rhizoma Dioscoreae soap unit | - 50 200 100 50 | 10 10 10 10 10 | 9.6±2.2 20.2±4.4 *** 29.5±10.6 *** 9.9±4.5 # 8.8±1.4 # | 110.4 207.3 3.1 -8.3 |
Annotate: compare * * * p＜0.001 with matched group; * p＞0.05
Conclusion
Show through repeating three batches of experimental results: diosgenin 200mg/kg ig administration, the time-to-live that can make abdominal cavity inoculation hepatic ascites (HepaA) mice, three crowdes of experiment statistics results were p＜0.001 than the obvious prolongation of matched group; The average life rate elongation is 115%.Diosgenin 100mg/kg, ig administration group in three batches of experimental results, has two batches of animals survived time ratio matched groups obviously to prolong by statistics, is respectively P＜0.001; P＜0.001 and P＞0.05.The average life rate elongation is 33.8%.Dioscin 50mg/kgig administration group in three batches of experimental results, has by statistics that animals survived time ratio matched group obviously prolongs in a collection of experiment, is respectively P＜0.01; P＞0.05 and P＞0,05.The average life rate elongation is 17.2%.Above-mentioned experimental result shows that diosgenin can obviously prolong the time-to-live of abdominal cavity inoculation hepatic ascites (HepA) mice.Particularly high dose (200mg/kg) and middle dosage (100mg/kg) administration group effect are more definite.
Experimental example 5:Diosgenin is to transplanting the influence of Walker carcinosarcoma 256 tumor-bearing rat tumor weights
Materials and methods
Animal Wistar rat, male and female half and half, body weight 105-125g, available from the institute of lab animals, Shenyang City, the animal quality certification number:
Tumor kind rat Walker carcinosarcoma 256 (W256) the ascites rat of going down to posterity is available from institute of Materia Medica,Chinese Academy of Medical Sciences.
Medicine diosgenin, purity are 95.69%, lot number: 000126.The Drug Manufacturing Room of our unit provide; Cyclophosphamide for injection, specification: 200mg/ props up, lot number: 010711, Hualian Pharmaceutical Co., Ltd., Shanghai's product.
Instrument SB-JB-1B type superclean bench, Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd. product; AB204-N type list range 100,000/electronics Libra, prunus mume (sieb.) sieb.et zucc. Teller one holder benefit instrument (Shanghai) Co., Ltd. product.
Method is chosen abdominal part and was gone down to posterity 8 days, the tangible rat of abdominal tympanites, in superclean bench, the skin of abdomen sterilization is thrust the abdominal cavity with disposable sterilized hemostix through stomach wall, extracts ascites, put into aseptic beaker, be diluted to 1: 3 cancerous cell suspension with normal saline, give every experimental rat left side armpit subcutaneous vaccination 0.2ml, tumor cell number is 1 * 107.In inoculating next day, experimental rat is divided into contrast (ig distilled water 10ml/kg, every day 1 time) at random; Cyclophosphamide (ig 25mg/kg, the next day 1 time); 5 groups of diosgenins (ig 100mg/kg, 50mg/kg and 25mg/kg).Begin administration simultaneously, successive administration 14 days.Administration finishes next day, and dislocation was put to death after animal was weighed, and separates subcutaneous lump and weighs, relatively the diversity of tumor weight between each group.And calculate the tumor suppression percentage rate.Institute's column data is added and subtracted standard deviation (x ± s) expression, t-check between statistical procedures method employing group with mean.For guaranteeing result's reliability, this experiment repeats 2 times.
Table 13, Rhizoma Dioscoreae soap unit are to W
256The influence of tumor-bearing rat tumor weight (first x ± s)
| Group | Dosage mg/kg | Animal (only) | The weight of animals (g) | Tumor weight (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | |||||
| Negative control cyclophosphamide diosgenin diosgenin diosgenin | - 25 100 50 25 | 10 10 10 10 10 | 93.2±7.9 93.8±9.5 93.3±10.2 94.6±7.3 94.8±6.7 | 123.4±13.6 104.2±9.6 124.0±9.4 114.5±9.0 124.2±10.8 | 1.70±0.44 0.56±0.21*** 0.75±0.20*** 1.04±0.21*** 1.22±0.22** | 67.1 55.9 38.8 28.2 |
Annotate: compare * * p＜0.01 with matched group; * * p＜0.001
Table 14, Rhizoma Dioscoreae soap unit are to W
256The influence of tumor-bearing rat tumor weight (first x ± s)
| Group | Dosage mg/kg | Animal (only) | The weight of animals (g) | Tumor weight (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | |||||
| Negative control cyclophosphamide diosgenin diosgenin diosgenin | - 25 100 50 25 | 10 10 10 10 10 | 118.1±6.4 117.6±6.5 117.3±6.4 116.3±10.1 115.7±6.7 | 165.5±15.2 149.6±20.2 151.5±12.9 162.8±15.1 169.8±15.5 | 3.02±1.13 0.81±0.34*** 1.01±0.35*** 1.21±0.37*** 1.24±0.29** | 73.2 66.6 59.9 58.9 |
Annotate: compare * * * p＜0.001 with matched group
Conclusion
The integral experiment of this laboratory is the result show: dionin sheet (diosgenin) 100,50 and 25mg.kg
-1Ig is to transplanting W
256The rat tumor weightening finish has the obvious suppression effect.Tumour inhibiting rate can reach 28.2～55.9% (first) and 58.9～66.6% (second batch).Repeat 2 times, obtain same experimental result substantially.Though it presses down tumor not as good as cyclophosphamide, do not resemble and make animal occur being not in good state the cyclophosphamide, become thin and body weight such as obviously alleviates at toxic and side effects.
Experimental example 6:Diosgenin suppresses people's gastric cancer (MGc-803) and Humanmachine tumour (A375-S
2)
The primary action Mechanism Study purpose of growth of tumour cell: utilize flow cytometer to observe diosgenin (Dio) to gastric carcinoma cells (MGC-803) and human melanoma cell (A375-S
2) cell cycle and apoptotic influence, inquire into the mechanism of action that Dio suppresses growth of tumour cell.
1 material
1.1 used gastric carcinoma cells of cell experiment (MGC-803) and human melanoma cell (A375-S2) are provided by the institute of oncology, Jilin Province.
1.2 the sample diosgenin (Diosgenin, Dio), white powder, purity 95% is by this list
The position Drug Manufacturing Room provide.
1.3 reagent and consumptive material DNAREAGENDKIT are U.S. B.D company product; Tissue Culture Dish is available from U.S. CORNING company.
1.4 instrument flow cytometer (the FACSCalibur B.D company U.S.), desk centrifuge (LD5-2A, Beijing)
2 methods
Cell monolayer is seeded in the culture dish of 60mm * 15mm, and cell number is 1 * 10
6Individual, cultivate dosing after 24 hours, the concentration that MGC-803 (gastric carcinoma cells) adds Dio is 0,30 and 60 μ g/mL, A375-S
2The concentration that (human melanoma cell) adds Dio is 0,15 μ g/mL, cultivate 12,24,36 hours respectively after, collects the adherent cell that has suspended, the 1500rmin of reaching
-1Centrifugal 5min, PBS wash once, and 70% ice ethanol-20 ℃ is fixed 18 hours, and PBS washes secondary, and DNAREAGENDKIT dyes, and survey with flow cytometer that cell cycle changes and the influence of pair cell apoptosis.
3 results
3.1 Dio is to the influence of MGC-803 cell cycle and apoptosis
Dio concentration is when 0g/mL, along with prolonged MGC-803 cell cycle (Go/G by 12h-24h-36h action time as a result
1, S, G
2/ M) and apoptosis (Apoptosis) all less than obviously influence; When Dio concentration is increased to 30 or during 60pg/mL, along with the variation of the prolongation cell cycle of action time is still not obvious, but the apoptosis fragment increases (being increased to 15.26% and 18.74% by 5.56% when being increased to 10.60% and 16.95%, 60 μ g/mL by 0.94% during 30 μ g/mL) gradually.Equally, Dio and cytosis time is at 12 hours, and along with Dio concentration is increased to 30 and 60Llg/mL by 0, cell cycle does not change, and the apoptosis fragment has the trend of increase; And being increased to 24 and 36 hours action time, the DNA fragment increase of apoptosis is obvious further, and cell is still not remarkable in the variation in each cycle.
Above-mentioned experimental result shows that Dio can not make the blocking-up of MGC-803 cell cycle in a certain specific period, blocks irrelevant so Dio suppresses the growth of MGC-803 cell with cell cycle.But along with the increase of Dio concentration and the prolongation of action time, apoptotic DNA fragment is regular increase, shows that Dio may induce the MGC-803 apoptosis, by apoptosis-induced inhibition growth of tumour cell.
3.2 Dio is to A375-S
2The influence of cell cycle and apoptosis
The result is at A375-S
2Add 15lug/mL Dio in the cell, along with the prolongation of action time, the DNA fragment of apoptotic cell is increased to 30.50% and 41.93% by 2.39%, and the influence of cell cycle is not obvious.Show that Dio may be by inducing A375-S
2Apoptosis suppresses the growth of tumor cell.
4 conclusions
With flow cytometer the primary action Mechanism Study that Dio suppresses growth of tumour cell is shown that Dio can not make MGC-803 and A375-S
2The cell cycle blocking-up is in a certain specific period, but the energy inducing apoptosis of tumour cell.Conclusion: Dio cell growth inhibiting and cell cycle blocking-up are irrelevant, may suppress growth of tumour cell by cell death inducing.
Experimental example 7:10 kinds of human tumor cell line extracorporeal anti-tumor functions of diosgenin experimental observation
Purpose: observe diosgenin and exsomatize, by measuring IC to the inhibitory action of 10 kinds of representational human body tumour cell strain growths
50Relatively it is to different growth of tumour cell inhibitory action powers.
1 material
1.1 used 10 kinds of cell strain: the MCF-7 of cell experiment (human breast cancer cell), NCI-H446 (human small cell lung carcinoma), MGC-803 (gastric carcinoma cells), Swillc (human colon cancer cell), U251 (people's astroglia oncocyte), SGL-7901 (gastric carcinoma cells), K562 (human erythroleukemia cell), A375-S
2(human melanoma cell), Hela (human cervical carcinoma cell),
BEL-7402 (human liver cancer cell) is all available from the institute of oncology, Jilin Province.
1.2 reagent and consumptive material diosgenin (Diosgenin, Dio, white powder, purity 590%) are provided by the Drug Manufacturing Room of our unit; Benzylpenicillin
Potassium salt is Huabei Pharmaceutic Co., Ltd's product (lot number: 111413); Streptomycin sulfate is produced (lot number: 0010604) by the big pharmaceutical factory of Dalian Metro; HEPES, MTT are SIGMA company product; Glutamine is available from GIBCO company;
The RPMI-1640 culture medium is a HyClone company product; Hyclone (lot number: 011222) Beijing Heng Shengma of unit biotechnology research institute product; 5-fluorouracil (5-FU, lot number: 010114) Shanghai Xudong Hipu Medicine Co., Ltd; Dimethyl sulfoxide (DMSO, lot number: 0-718A01) AMRESCO packing; 96 well culture plates are available from NUNCTM company;
1.3 instrument CO2 gas incubator (HERA D-63450 Germany), inversion micro-(OLYMPUS CK2 Japan), desk centrifuge (LDs-2A Beijing), microplate reader (TECANA-5082 Australia), oscillator (210-A Shenzhen).
2 methods
2.1 the tumor cell culture cell inoculation is containing 10% hyclone, the 2mM glutamine, in the RPMI-1640 culture fluid of 100IU/mL potassium penicillin G and 100 μ g/mL streptomycin sulfates, placing temperature is 37 ℃, gas concentration lwevel is to cultivate in 5% the CO2 gas incubator, passes after 3 generations standby.
2.2MTT method is measured the cell growth with exponential phase cell dissociation, 1000r.min
-1Centrifugal 30min, numeration is with 1 * 10
5Individual/mL density is inoculated in 96 well culture plates, every hole 100 μ L, cultivate after 24 hours, add the Dio (being dissolved among the 0.033%DMSO) that concentration is respectively 0,1.875,3.75,7.5,15,30,60 μ g/mL, establish solvent control group (0.033%DMSO) simultaneously; Positive drug is a 5-fluorouracil, adds in 96 orifice plates with same concentration.After the co-cultivation 44 hours, every hole adds 20.tLMTT (5mg/mL), cultivates 4 hours again, abandons supernatant, and every hole adds 150 μ L dimethyl sulfoxide, vibrates 10 minutes, uses microplate reader and surveys light absorption value in the 492nm place.
1.3 the calculating of suppression ratio and IC
50Statistics
Solvent control A-sample A
Solvent control two cells+culture fluid+0.033%DMSO sample two cells+culture fluid+Dio (5-FU)+0.033%DMSO
1Cso statistical method: adopt method of least square to ask regression equation, ask IC according to regression equation
50
3 results
3.1 Dio and 5-FU hang down the time-effect relationship of differentiation adenocarcinoma of stomach cell strain (MGC-803) effect according to preceding method to the people, at first observe Dio at 24,48,72 hours and 5-FU 24,48,72,96 hours inhibitory action to the MGC-803 cell strain growth, as a result, we select to suppress for Dio in 48 hours the action time of growth of tumour cell.Reason: 1. effect reflected the action intensity of Dio to tumor cell in 48 hours substantially; 2. the incubation time lengthening is easy to microbiological contamination when large quantities of experiment; 3.5-FU as positive control drug, its purpose is not the power (not having comparability between a chemical synthetic drug and a native compound medicine) that comparison and Dio suppress the growth of tumour cell effect in this experiment, but the reliability of verification method.So although 48 hours be not 5-FU the best use of time (optimum reacting time of 5-FU is 72 or 96 hours), this experiment has still been selected 48 hours.
3.2 Dio exsomatizes to the inhibitory action of 10 kinds of human body tumour cells
Experimental result sees Table 15, and Dio is followed successively by A375-S to the action intensity that 10 kinds of human body tumour cells suppress
2＞MCF-7＞Hela＞MGC-803＞SGL-7901＞BEL-7402＞NCi-H446＞K562SMMC-7721＞Swillc＞U
251Dio is to human melanoma cell (A375-S
2), human breast cancer cell (MCF-7), human cervical carcinoma cell (Hela), people low differentiation adenocarcinoma of stomach cell (MGC-803) and gastric carcinoma cells (SGL-7901) act on the strongest, its IC
50All less than 20 μ g/mL.In the table institute's column data only for 5-FU effect 48 hours to 10 kinds of inhibiting IC of human body tumour cell
50Value.
The inhibitory action that table 15, diosgenin (Dio) are grown to 10 kinds of human tumor cell lines
| The tumor cell line kind | IC 50μg | |
| 5-FU | IC 50 | |
| A375-S 2 MCF-7 Hela MGC-803 SGL-7901 BEL-7402 NCL-H446 K562 Swillc U251 | 14.97±0.45 30.60±2.53 39.82±1.24 25.94±1.26 47.68±3.68 29.54±0.57 4.86±0.80 32.09±0.79 61.01±3.19 57.04±4.43 | 7.69±0.05 14.52±0.14 15.24±0.08 15.59±0.20 18.24±0.10 21.29±0.29 25.80±8.86 26.70±0.47 31.12±5.02 33.01±5.42 |
Annotate: be the x ± s of three experimental results in the table
4, conclusion
Potato fragrant plant sapogenin can obviously suppress human melanoma cell (A375-S external
2), human breast cancer cell (MCF-7), human cervical carcinoma cell (Hela), people low differentiation adenocarcinoma of stomach cell (MGC-803) and gastric carcinoma cells (SGL-7901) grow its IC
50All less than 20 μ g/mL.
Experimental example 8:Diosgenin mixes the synthetic influence of people's gastric cancer (1VlGC-803) cell DNA to 3H-TdR
Purpose: observe diosgenin (Dio) to the nucleic acid precursor
3H-TdR mixes the influence of gastric carcinoma cells DNA.
1 material
1.1 cell MGC-803 (gastric carcinoma cells) cell strain is provided by the institute of oncology, Jilin Province.
1.2 (Diosgenin Dio) is white powder to the sample diosgenin, and purity＞90% is provided by the Drug Manufacturing Room of our unit
1.3 reagent 3H-TdR is available from Shanghai Atomic Nucleus Inst., Chinese Academy of Sciences.
1.4 instrument liquid scintillation numeration instrument (WALLAC 1400DSA, the U.S.), water isolation type electro-heating standing-temperature cultivator (WMK-02 type, Hubei)
2 methods
With the exponential phase cell dissociation, centrifugal, numeration is with 1 * 10
5Individual/mL density is inoculated in 96 well culture plates, and every hole 100uL cultivates after 24 hours, add concentration and be respectively 0,1.875,3.75,7.5,15, the Dio of 30lug/mL, establish solvent control (0.033%DMSO) simultaneously, cultivate after 40 hours, every hole adds 3H-TdR logL (37kBq/ hole), cultivates after 8 hours, the careful suction abandoned supernatant, PBS gives a baby a bath on the third day after its birth inferior, and 5% trichloroacetic acid is washed secondary, and ethanol ether (3: 1) is washed once, every hole adds the 100LO.5M sodium hydroxide and spends the night, every hole adds the neutralization of 10 L6M hydrochloric acid, liquid is moved in the filter paper bar dry 5 hours of 60C, add the 3mL scintillation solution, survey the CPM value with liquid scintillation numeration instrument after 30 minutes.
3 results
Dio is right at low concentration 3.75 and 7.5[tg/mL
3H-TdR mixes the MGC-803 cell DNA the obvious suppression effect.When Dio concentration is increased to 15 and during 30iLtg/mL,
3Mixing significantly of H-TdR is suppressed, and incorporation (CPM) has only 1/12 and 1/39 of matched group.We think that the Dio of low concentration suppresses
3H-TdR mixes stomach cancer cell line DNA and suppresses stomach cancer cell propagation and have than confidential relation: but increase to 15 and during 30g/mL when Dio concentration, a large amount of cells are dead, 3H-TdR can not be incorporated into dead cell DNA, so the amount that 3H-TdR mixes DNA can not reflect the inhibitory action (table 16) of Dio to the growth of MGC-803 cell.
Table 16.Dioc mixes the synthetic n=4 of influence of MGC-803 cell DNA hole to 3H-TdR
| Dio/g/mL | 3H-TdR mixes/CP, M |
| 0 1.857 3.75 7.5 15 30 | 64534±1871 64577±2205 55463±3488 ** 33919±3137 *** 5194±966 *** 1631±250 *** |
Compare * * p＜0.01, * * * P＜0.001 with the solvent control group
4 conclusion Dio inhibition stomach cancer cell propagation is synthesized with blocking dna certain relation.
Following embodiment all can realize effect and the object of the invention of above-mentioned experimental example.
Embodiment 1Capsular preparation
Diosgenin 250 gram starch 2500 grams carry out drying earlier with starch, cross 120 mesh sieves, then with the diosgenin mix homogeneously, cross twice 120 mesh sieves, and fully mixing is made 1000 capsules altogether, and every intragranular contains 250 milligrams of diosgenins.Oral, every day 2-3 time, each 1-2 grain.
Embodiment 2The preparation of drop pill
Take by weighing the 300g Macrogol 4000, in water-bath, melt, add diosgenin raw material 100g, stir, in the impouring insulating tube, regulate thermostat, make medicinal liquid under 80-90 ℃, splash in the liquid paraffin that cooled off (temperature ± 4 ℃), after dripping off, to blot paraffin oil on the pill impouring filter paper, add a small amount of Pulvis Talci again, mixing gets 1000 of diosgenin drop pill.Oral, a 2-3 grain, three times on the one, one after each meal.
Embodiment 3The preparation of microencapsule tablet
Take by weighing stearic acid 15g and 21mL respectively as compound recipe capsule material, take by weighing diosgenin 100g as capsule core material, by the spray congealing encystation, capsule directly is 8-100um with compressed air.Then with microcapsule and microcrystalline Cellulose mixing, add the mixed encystation material of 10% ethyl cellulose alcoholic solution, make granule by 18 order nylon mesh,, placed exsiccator interior 24 hours in oven dry below 35 ℃, add 1%-2% magnesium stearate tabletting, get the diosgenin microencapsule tablet, sheet heavily is 0.3g, and is oral, a 1-2 sheet, a twice-daily.
Embodiment 4The preparation of capsule
Diosgenin raw material 1000g, medical starch 1000g, mix homogeneously, the 0# capsule of packing into, every 0.35g, each oral 1-2 grain, twice of every day.
Embodiment 5The preparation of tablet
Diosgenin raw material 1000g, medical starch 500g, mix homogeneously is used an amount of alcohol granulation, through the pelletizing machine granulate, tabletting, every 0.25g, oral, each 1-2 sheet, twice of every day.
Embodiment 6The preparation of granule
Diosgenin raw material 100g, starch 1000g, Icing Sugar 400g, mix homogeneously is used an amount of alcohol granulation, and drying, granulate, packing are promptly.Oral, a 5g, twice on the one.
Embodiment 7The preparation of injection
Diosgenin raw material 10g, propylene glycol 20ml, Polyethylene Glycol-400 50ml, water for injection 300ml mixes heating in water bath 30 minutes, add benzyl alcohol 50ml, reuse water for injection adds to 1000ml, handles 10 minutes in ultrasound wave, heats 30 minutes in the water-bath again, transfer pH value 5.5-6.5, filter clear and bright, embedding, the sterilization promptly.Every 2ml, intramuscular injection, a 2ml, 1-2 time on the one.
Claims (4)
1, the application of diosgenin in preparation treatment gastric cancer medicine.
2, application as claimed in claim 1 is characterized in that described treatment gastric cancer is meant that suppressing the people hangs down differentiation adenocarcinoma of stomach cell or suppress gastric carcinoma cells.
3, application as claimed in claim 1 is characterized in that described treatment gastric cancer is meant that blocking-up stomach cancer cell DNA is synthetic.
4, application as claimed in claim 2 is characterized in that described inhibition people low differentiation adenocarcinoma of stomach cell or suppresses gastric carcinoma cells being meant that blocking-up stomach cancer cell DNA is synthetic.
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| CN101837004B (en) * | 2010-05-28 | 2013-04-17 | 大连医科大学 | Application of dioscin in preparation of liver-protecting pharmaceutical preparation |
| CN102579756B (en) * | 2012-03-14 | 2013-11-06 | 深圳市药品检验所 | Pharmaceutical composition containing ningpo yam rhizome extract and harmane alkaloid, as well as harmane alkaloid type derivatives, and application thereof |
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