CN1279928C - Extracts and use of Leimo - Google Patents
Extracts and use of Leimo Download PDFInfo
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- CN1279928C CN1279928C CN 03140855 CN03140855A CN1279928C CN 1279928 C CN1279928 C CN 1279928C CN 03140855 CN03140855 CN 03140855 CN 03140855 A CN03140855 A CN 03140855A CN 1279928 C CN1279928 C CN 1279928C
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- Medicines Containing Plant Substances (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention discloses an extract of Leimo and an application thereof. The present invention aims at providing an extract of Leimo and a medicine for curing cancer with the extract of Leimo as an active ingredient. The extract of Leimo is prepared through the following method: Leimo is stirred and ground, added with 0.6 to 0.8% of NaCl aqueous solution or water with the quantity 8 to 12 times of the volume of the Leimo, centrifugated to obtain supernatant fluid, or filtered through a net with 120 to 200 meshes to obtain filter liquor, namely the extract of Leimo. The present invention relates to also provides a medicine for curing cancer with the extract of Leimo as an active ingredient. The present invention has conspicuous curative effect.
Description
Technical field
The present invention relates to a kind of fungal extract and application thereof, particularly a kind of Leucopaxillus giganteus (Sow ex Franch.) singer. extract and the application in preparation treatment cancer drug thereof.
Background technology
At present, malignant tumor has become one of human main cause of death.The annual emerging tumor patient about 1,000,000, dead about 800,000 of China.Particularly leukemia is in rising trend in recent years, the life and health of harm humans seriously, and also the patient is child and teenager mostly.Be the second largest cause of death that the children malignant tumors of representative has become China 5-15 year child at present with the leukemia, annual on average have a child's cases with leukemia below 15 years old about 20,000.Scientist can't find out so far and cause leukemic reason.Therefore the control of malignant tumor becomes the key subjects that modern medicine needs to be resolved hurrily.Many scientists and medical worker have carried out big quantity research, obtain much can killing and suppressing the medicine of tumor cell, but these medicines of while are also to normal cell generation hazardness to a certain degree.
At the beginning of the seventies, whether people begin one's study can make tumor cell break up to normal cell by inducing differentiation, thereby reaches treatment malignant tumor ground purpose.The differentiation therapy of inducing of tumor cell is better than traditional chemotherapy and radiation, and this therapy is not a killing tumor cell, but under the effect of differentiating inducer, inducing tumor cell broke up to the maturation period, recovers the normal or approaching normal phenotype and the function of cell.Current, seeking tumor cell differentiation revulsant has become new research field.
Kind of a chemical compound can be used as tumor cell differentiation revulsant surplus having now found that ten.Wherein studying maximum is retinoid compounds (RA) and assorted polar compound, as dimethyl sulfoxine (DMSO), hexa-methylene bis-dimethylsilyl-acetamide (HMBA), butanoic acid etc.According to reported in literature dimethyl sulfoxine inductivity is 80%, is to generally acknowledge best derivant.China at first used all-trans-retinoic acid (ATRA) induction-differential therapy acute promyelocytic leukemia in 1986, had obtained good result.Rui Jin hospital of Shanghai Second Emdical University Shanghai hematology's institute and discovering of Harbin Medical University can be used As
2O
3Treatment acute promyelocytic leukemia, its mechanism mainly are to promote the acute progranulocyte apoptosis.Though ATRA and As
2O
3On the treatment acute promyelocytic leukemia, obtained success, retinoic acid, HMBA have entered clinical stage, but these medicines all belong to the pure chemistry medicament, side effect is very big, and exist limitation at aspects such as adapting to state of an illness scope, dose-dependent toxicity, can't be widely used in clinical cancer therapy.
China utilizes our traditional advantage as a Chinese medicine big country, has done a lot of researchs at the Chinese medicine anticancer aspect.Find a lot of class Chinese herbal medicine can anticancer growth, and toxicity is lower.At present developed some and can treat the Chinese herbal medicine of cancer, as Ganoderma spore powder, krestin, lentinan and the TIANXIAN WAN of having gone on the market etc.But because these medicinal herb components complexity, its effective ingredient and mechanism are not also studied clear, and curative effect is not remarkable, is not suitable for leukemia and hepatocarcinoma.
Leucopaxillus giganteus (Sow ex Franch.) singer. is the Tricholoma mongolicum Imai of a kind of natural gill fungi whitish eye mushroom section Tricholoma, and Latin is called Leucopaxillusgiganteus; Its sporophore is grown on the grassland summer and autumn, and the about 5-30cm of sporophore cap is common in the grassland, Siklingelei, the Inner Mongol in autumn.Application to Leucopaxillus giganteus (Sow ex Franch.) singer. at present still is in the development phase, and its all kinds of chemical constituents that contain are not studied in great detail record, and prescription generally is used for sending out infantile measles, treatment flu, treating pulmonery tuberculosis bacillus etc.
The innovation and creation content
The purpose of this invention is to provide a kind of Leucopaxillus giganteus (Sow ex Franch.) singer. extract that can be utilized effectively.
A kind of Leucopaxillus giganteus (Sow ex Franch.) singer. extract obtains by following method:
Leucopaxillus giganteus (Sow ex Franch.) singer. is blended, add NaCl aqueous solution or the water of its 8-12 times of volume 0.6-0.8%, centrifugal, get supernatant or, get filtrate with 120-200 order net filtration, be the Leucopaxillus giganteus (Sow ex Franch.) singer. extract.
Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention can also can extract from the Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium of artificial culture from wild Leucopaxillus giganteus (Sow ex Franch.) singer. sporophore.
Described Leucopaxillus giganteus (Sow ex Franch.) singer. is generally selected dry Leucopaxillus giganteus (Sow ex Franch.) singer. for use, for the effective ingredient that makes Leucopaxillus giganteus (Sow ex Franch.) singer. is fully discharged, dry Leucopaxillus giganteus (Sow ex Franch.) singer. sporophore pulverizing machine should be crushed to the 70-90 order.
The volume of described NaCl aqueous solution or water is 10 times of Leucopaxillus giganteus (Sow ex Franch.) singer. volume preferably, the concentration of described NaCl aqueous solution preferably 0.7%.
Described centrifugal be 4000 to change 20 minutes.
Described Leucopaxillus giganteus (Sow ex Franch.) singer. extract shows that through electrophoresis and full gloss analysis of spectrum its effective ingredient is mainly GL-PP and small-molecule peptide, wherein also contains clitocybine (Clitocybin).
Another object of the present invention provides a kind of medicine for the treatment of cancer.
The medicine of treatment cancer provided by the present invention, its active component are above-mentioned Leucopaxillus giganteus (Sow ex Franch.) singer. extracts.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention can be made various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream, is preferably oral liquid.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The consumption of said medicine is generally extract stock solution 0.2ml/kg/day, also should add pharmaceutically acceptable carrier, diluent, absorption enhancer, flavouring agent, sweeting agent etc.Be generally the course of treatment 45-60 days.
Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention contains DIF, can break up to normal cell by inducing cancer cell under certain concentration; Through the cell in vitro experimental verification, this extract not only can kill the leukaemia but also the human normal cell not had any lethal effect, has no side effect; Compare with present common both at home and abroad similar medicine, of the present inventionly induce differentiation rate all to be higher than other to induce differentiation agent, the inventor under equal conditions makes of conventional method and induces the differentiation test relatively, dimethyl sulfoxine DMSO induces the differentiation denier, the retinoic acid of having gone up at present clinical trial only about 30%, and Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention induces differentiation rate to reach more than 40%, and consumption is less, is a kind of cancer therapy drug that has very much exploitation to be worth.
The specific embodiment
Embodiment 1, cultivation Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium
Get fresh Leucopaxillus giganteus (Sow ex Franch.) singer. sporophore, under aseptic condition, go clean surface irregularities, use 70% alcohol disinfecting, with aseptic dissecting knife sporophore is cut away half then, extract the piece of tissue of the about Semen Glycines size of mid portion of sporophore stem and cap junction, insert in the test tube culture medium, the culturing base prescription is: sucrose 20g, Rhizoma Solani tuber osi 200g, KH
2PO
43g, MgSO
40.5g, vitamin B
12mg, agar 10g, water 1000ml, pH6.1 (pH6.0-6.2 all can), 29 ℃ of (28 ℃-30 ℃ all can) constant temperature culture 15 days (10-20 days all can) grow dense Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium of former generation in media surface; This Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium of former generation being inserted goes down to posterity on another and the culture medium of above-mentioned culture medium with component again cultivated for 3 generations (1-4 generation all can), the Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium strain that obtains taming;
The domestication Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium strain that above-mentioned switching is obtained inserts through sterilization (1.5kg/cm
2Under the highly compressed condition sterilization 30 minutes) fluid medium (sucrose 20g, analysis for soybean powder 10g, Semen Maydis powder 10g, KH
2PO
43g, MgSO
40.5g, vitamin B
12mg, yeast powder 1g, CaCO
31g, water 1000ml) in, and on shaking table 29 ℃ of (28 ℃-30 ℃ all can) constant temperature culture 3 days (4-5 days all can), speed is 120 rev/mins, insert fermentor cultivation then, its inoculum concentration is 10v%, in 29 ℃ of (28 ℃-30 ℃ all can) constant temperature culture 4 days (3-5 days all can), and after filtration, obtain the Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium.
Embodiment 2, preparation Leucopaxillus giganteus (Sow ex Franch.) singer. extract
Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium 10g (weight in wet base) with embodiment 1 obtains rubs to pulpous state, adds 100ml 0.7%NaCl aqueous solution, stirs 10 minutes, and 4000 left the heart 20 minutes, got supernatant, obtained 60ml Leucopaxillus giganteus (Sow ex Franch.) singer. extract.
Embodiment 3, preparation Leucopaxillus giganteus (Sow ex Franch.) singer. extract
Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium 10g (weight in wet base) with embodiment 1 obtains rubs to pulpous state, adds the 100ml pure water, stirs 20 minutes, with 200 order nylon net filters, gets filtrate, obtains 60ml Leucopaxillus giganteus (Sow ex Franch.) singer. extract.
Embodiment 4, preparation Leucopaxillus giganteus (Sow ex Franch.) singer. extract
With the Leucopaxillus giganteus (Sow ex Franch.) singer. sporophore 10g that collects, drying is crushed to 80 orders, adds 100ml 0.75%NaCl aqueous solution, stirs 30 minutes, and 4000 left the heart 20 minutes, got supernatant, obtained 80ml Leucopaxillus giganteus (Sow ex Franch.) singer. extract.
Embodiment 5, preparation Leucopaxillus giganteus (Sow ex Franch.) singer. extract
With the Leucopaxillus giganteus (Sow ex Franch.) singer. sporophore 10g that collects, drying is crushed to 90 orders, adds the 100ml pure water, stirs 30 minutes, with 150 order nylon net filters, obtains 80ml Leucopaxillus giganteus (Sow ex Franch.) singer. extract.
Embodiment 6, acute toxicity test experience
With body weight is that 20 30 mices that restrain are divided into three groups: first group 10 are matched group, with the common Mus food nursing of animal feeding center, Beijing preparation; Second group 10,, press 5g mycelium (weight in wet base)/d.Kg and feed with containing the mycelial Mus food of Leucopaxillus giganteus (Sow ex Franch.) singer.; The 3rd group 10,, give mouse stomach with Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention (being diluted to 10wt%) with 0.7%NaCl or water.Other daily raising condition is identical.
The Leucopaxillus giganteus (Sow ex Franch.) singer. extract is pressed 5ml/d.Kg and is irritated stomach.
Feed, irritated stomach 30 days, weigh, first group of mice average weight is 31.2g, and second group of mice average weight is 32.8g, and the 3rd group of mice average weight is 32.9g.
Feed the average weight of mice of Leucopaxillus giganteus (Sow ex Franch.) singer. mycelium and Leucopaxillus giganteus (Sow ex Franch.) singer. extract a little more than matched group; And do not have any untoward reaction, thus the result as can be known, the Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention preparation does not have toxicity.
Embodiment 7, Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention are to the inhibition of hepatoma carcinoma cell
The S180 ascites cells that is provided with Chinese Academy of Sciences animal carries out inoculation experiments to mice.Used mice is available from the public Mus in the Kunming at animal feeding center, Beijing, and body weight is 20 grams.30 mices are divided into five groups, 5 every group, fed 30 days for the first～two group, fed 60 days for the three～five group.
With the common Mus food of animal feeding center, Beijing preparation, or, give mouse stomach with Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention (being diluted to 10wt%) with 0.7%NaCl or water.
The first winding kind cancerous cell uses common Mus food to feed, after 30 days dead 4;
The second winding kind cancerous cell is fed the mice that is inoculated with common Mus food earlier, and mice produces ascites after the week, and abdominal part obviously rises greatly compared with the control, and touching abdominal part with hands has the ascites sensation; The Leucopaxillus giganteus (Sow ex Franch.) singer. extract that begins 0.2 milliliter/day of every usefulness this moment is irritated stomach, all survivals after 30 days, and abdominal part diminishes;
The 3rd winding kind cancerous cell uses common Mus food to feed, and is all dead after 60 days;
The 4th winding kind cancerous cell, and inoculating the Leucopaxillus giganteus (Sow ex Franch.) singer. extract filling stomach of 0.2 milliliter/day of cancerous cell while every usefulness, all survivals after 60 days, no ascites occurs;
The 5th winding kind cancerous cell is fed the mice that is inoculated with common Mus food earlier, and mice produces ascites after the week, and abdominal part obviously rises greatly compared with the control, and touching abdominal part with hands has the ascites sensation; Begin 0.1 milliliter/day of every usefulness/Leucopaxillus giganteus (Sow ex Franch.) singer. extract only this moment and irritate stomach, all survivals after 60 days, abdominal part diminishes.
The medicine feed group is dissected liver observe, no abnormal, and the cancerous cell suppression ratio reaches 95%, illustrate that the Leucopaxillus giganteus (Sow ex Franch.) singer. extract that the present invention prepares has inhibitory action significantly to cancerous cell, but normal cell is had no side effect.
With first and second, 20 mices of four and five groups of survivals are divided into two groups, carry out physical ability experiment and anoxia enduring is tested with normal mouse.Physical ability experiment: the afterbody of mice is bundled 5 grammes per square metre things with iron wire, the mice of heavy burden just can be sunk, the breathing of constantly floating again.Anoxia enduring experiment: the mice of carrying out mark is inserted in the hermetic container, and extracting air gradually makes only surplus little air in the container then.Physical ability experiment and anoxia enduring experimental result show, 20 mices of survival aspect physical ability and anoxia enduring with normal mouse and zero difference, illustrate that the Leucopaxillus giganteus (Sow ex Franch.) singer. extract can cure cancer, make the mice recovery normally.
Grouping experiment is the result show, and is identical with the Leucopaxillus giganteus (Sow ex Franch.) singer. extract effect that natural sporophore obtains from the artificial hyphostoma with NaCl aqueous solution or water.
Embodiment 8, Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention are to the inhibitory action of leukaemia cancer cell
Human erythroleukemia cell's strain K562 is the malignant cell of human hematopoietic system, and K562 leukaemia system is derived from U.S. ATCC.The K562 that this experiment is used is that consonance medical university cell biological chamber stores, and with 1640 culture medium that contain 10% calf serum routinely condition of culture cultivate.
Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention is diluted to 1%, 0.5%, 0.1% solution with 0.7%NaCl or sterilized water, human erythroleukemia K562 cell is suppressed the growth checking.With 3 * 10
4/ ml K562 cell is inoculated into 96 orifice plates respectively, by one group in 8 holes, is divided into 12 groups, adds 1%, 0.5%, 0.1% Leucopaxillus giganteus (Sow ex Franch.) singer. extract 200 microlitres respectively, cultivate after 1,3,5 day, and the cell that counts respectively, and draw growth curve.Experimental result shows, 1%, 0.5% extract of 200 microlitres all can 100% kills 1000～5000 cancerous cell, and 0.1% extract can be induced the differentiation cancerous cell, and inductivity is about more than 40%.
Embodiment 9, Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention are to the influence of immune organ thymus, spleen weight
Thymus and spleen are intravital main immune organs.Thymus is primary lymphatic organ, and the hematopoietic stem cell of migration enters primordium of thymus, becomes T lymphocyte (T cell) at this differentiation and proliferation, and is relevant with cellular immunization.Spleen is secondary lymphocyte, immunologically competent cell is divided a word with a hyphen at the end of a line in this, and alloantigen stimulates further proliferation and differentiation and maturation in the immunologic process because of being subjected to herein, T lymphocyte and bone-marrow-derived lymphocyte (B cell) are arranged in the spleen, also have macrophage, with humoral immunization, cellular immunization substantial connection is arranged all, some immunosuppressant such as cyclophosphamide, cortisone etc., all can make thymus, the obvious atrophy of spleen, immunostimulant such as some immune polysaccharides then make thymus or spleen weightening finish.
Leucopaxillus giganteus (Sow ex Franch.) singer. extract of the present invention is diluted to 1% with 0.7%NaCl, give mouse stomach, young Mus body weight 11-15g, adult rats body weight 18-22g, dosage children Mus is 0.1g/kg/d, adult rats is 0.5g/kg/d, gastric infusion 7 days, every day 1 time, after after the last administration 24 hours, put to death, extraction thymus and spleen are weighed, and are thymus or spleen index with thymus or spleen weight (mg) with likening to of body weight (g).The result is as shown in table 1:
Table 1, extract are to mouse immune organ weight's influence
| Medicine | Number of mice | Body weight (g) | Thymus index | Spleen index |
| Contrast | 10 | 10-15 | 4.1±0.43 | 5.23±0.55 |
| Extract | 10 | 10-15 | 4.9±0.22 | 6.12±0.75 |
| Contrast | 10 | 18-22 | 2.8±0.33 | 3.23±0.51 |
| Extract | 10 | 18-22 | 2.9±0.25 | 3.22±0.45 |
From the data of table 1 as can be seen, give mouse stomach, can obviously increase mouse thymus, spleen weight with extract of the present invention.Therefore infer that extract of the present invention has potentiation to the immunologic function of animal.
Claims (8)
1, a kind of Leucopaxillus giganteus (Sow ex Franch.) singer. extract with cancer suppressing action, obtain by following method: Leucopaxillus giganteus (Sow ex Franch.) singer. is blended, the NaCl aqueous solution or the water that add its 8-12 times of volume 0.6-0.8%, stir, centrifugal, get supernatant or with 120-200 order net filtration, get filtrate, being effective ingredient is the Leucopaxillus giganteus (Sow ex Franch.) singer. extract of GL-PP, small-molecule peptide and clitocybine.
2, Leucopaxillus giganteus (Sow ex Franch.) singer. extract according to claim 1, it is characterized in that: described Leucopaxillus giganteus (Sow ex Franch.) singer. is dry Leucopaxillus giganteus (Sow ex Franch.) singer., and is crushed to the 70-90 order.
3, Leucopaxillus giganteus (Sow ex Franch.) singer. extract according to claim 1 is characterized in that: the volume of described NaCl aqueous solution or water is 10 times of Leucopaxillus giganteus (Sow ex Franch.) singer. volume.
4, Leucopaxillus giganteus (Sow ex Franch.) singer. extract according to claim 1 is characterized in that: described centrifugal be 4000 rev/mins, centrifugal 20 minutes.
5, according to claim 1 or 2 or 3 or 4 described Leucopaxillus giganteus (Sow ex Franch.) singer. extracts, it is characterized in that: the concentration of described NaCl aqueous solution is 0.7%.
6, a kind of medicine for the treatment of cancer, its active component are the described Leucopaxillus giganteus (Sow ex Franch.) singer. extract with cancer suppressing action of claim 1.
7, the medicine of treatment cancer according to claim 6 is characterized in that: described medicament is an oral liquid.
8, according to the medicine of claim 6 or 7 described treatment cancers, it is characterized in that: also be added with pharmaceutically acceptable carrier in the described medicine.
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