CN1158082C - Pharmaceutical usage of diosgenin - Google Patents
Pharmaceutical usage of diosgenin Download PDFInfo
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- CN1158082C CN1158082C CNB011293179A CN01129317A CN1158082C CN 1158082 C CN1158082 C CN 1158082C CN B011293179 A CNB011293179 A CN B011293179A CN 01129317 A CN01129317 A CN 01129317A CN 1158082 C CN1158082 C CN 1158082C
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- diosgenin
- dio
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Abstract
The present invention discloses a new medicinal application of diosgnin in preparing medicine for resisting tumors. The rumors can be sarcoma-180, ascites type hepatic tumors, mouse cervical cancer and Ehrlich ascites cancer.
Description
The present invention relates to the pharmaceutic usage of diosgenin, is the pharmaceutic usage of diosgenin in the preparation antitumor drug specifically.
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.
The object of the invention provides the pharmaceutic usage of diosgenin, i.e. the pharmaceutic usage of diosgenin in the preparation antitumor drug.
Diosgenin is a kind of known material with pharmaceutically active, therefore, can it be prepared into any medicament applicatory clinically according to the formulation method of routine, for example, and tablet, capsule, injection, oral liquid, granule etc.
In the middle of the process of the above-mentioned preparation of preparation, diosgenin can be mixed use with any excipient that is prepared into clinical medicament that is applicable to, be prepared into pharmaceutical preparation.
Because the application of diosgenin in the preparation antitumor drug is inventor's discovery first; therefore; no matter be to use diosgenin to make medicament separately as active component; still utilize the anti-tumor activity of diosgenin and other active component to be used and make medicament, all within the protection domain of this patent.
The anti-tumor activity of diosgenin is confirmed by following pharmacological evaluation.
The extracorporeal anti-tumor function of experimental example one 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% anti-cattle element (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 RPMI RPMI-1640,3 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 death of neoplastic cells rate (%)
ug/ml L929 HeLa
1 0 0
5 7.5 12.3
10 15.0 20.5
20 25.8 37.3
50 45.2 50.5
100 82.4 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 1h with RnaseA and E.C. 3.4.21.64 altogether in 37 ℃ respectively, add 20 μ 1NaCl (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 DNA Ladder 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 20-100Llg/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.
The whole antitumor action of experimental example two diosgenins
Experiment purpose: observe diosgenin (Diosgenin, whole antitumor action Dio).
Method: medicine shown in the observation is to 4 kinds of mice transplanted tumors [S-180 (S-180), hepatoma ascitic type (HepA), mouse cervical cancer-14 (U
14) ehrlich carcinoma (EAC)] during the armpit skin just connecing, administration every day was 1 time in inoculation 10 days, calculates each administration group struma suppression ratio.Result: (1) Dio 200,100 and 50mg.kg
-1Ig is to S
180Have the obvious suppression effect, tumour inhibiting rate is respectively 50.6%, 42.9% and 35.9%; Dio 10050 and 25mg.kg
-1Ip is to S
180All have the obvious suppression effect, 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 is to U
14Also have the obvious suppression effect, tumour inhibiting rate is respectively 44.6%34.7% and 28.3% "; Dio100 and 50mg.kg
-1Ip is to U
14Have the obvious suppression effect, 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 1-4.
Table 1 Dio is to mice (S
180) the heavy influence of 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 with matched group,
*P＜0.05;
*P＜0.01
The influence that table 2 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
Table 3 Dio is to mice U
14The influence that tumor weighs (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 4 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) 26.9±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;
Embodiment:
Prescription
Diosgenin 250 gram starch 2500 grams
Make 1000 capsules altogether, each includes 250 milligrams of principal agents
Method for making
Starch is carried out drying earlier, crosses 120 mesh sieves,, cross twice 120 mesh sieves then with the diosgenin mix homogeneously, abundant mixing, inspect qualified after, branch is packed in the capsulae vacuus, promptly.
Claims (6)
1, the application of diosgenin in preparation treatment sarcoma medicine.
2, the application of diosgenin in preparation treatment liver tumor medicine.
3, the application of diosgenin in preparation treatment cervical cancer medicine.
4, the application of diosgenin in preparation treatment S-180 medicine.
5, the application of diosgenin in preparation treatment ascitic type hepatoma medicine.
6, the application of diosgenin in preparation treatment mouse cervical cancer medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011293179A CN1158082C (en) | 2001-06-11 | 2001-06-11 | Pharmaceutical usage of diosgenin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011293179A CN1158082C (en) | 2001-06-11 | 2001-06-11 | Pharmaceutical usage of diosgenin |
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| CN1390551A CN1390551A (en) | 2003-01-15 |
| CN1158082C true CN1158082C (en) | 2004-07-21 |
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| CNB011293179A Expired - Fee Related CN1158082C (en) | 2001-06-11 | 2001-06-11 | Pharmaceutical usage of diosgenin |
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| CN114560904B (en) * | 2022-01-28 | 2024-01-09 | 首都医科大学附属北京安定医院 | IL-15 inhibitor Diospgenin, screening method and application thereof |
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Owner name: XIUZHENG PHARMACEUTICAL GROUP CO., LTD. Free format text: FORMER OWNER: TIANYAO SCIENCE AND TECHNOLOGY CO LTD, JILIN Effective date: 20070706 |
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Granted publication date: 20040721 Termination date: 20160611 |
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