CN104983750B - A kind of application of great Bai bolts mycelia in pharmacy - Google Patents
A kind of application of great Bai bolts mycelia in pharmacy Download PDFInfo
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Abstract
The present invention relates to the new functions and purposes of great Bai bolt mycelia.The substance has the function of anti-peptic ulcer, increases WeiDongLi Capsule and then anti-non-ulcer dyspepsia；Great Bai bolts mycelia has certain inhibiting effect in the proliferation to the proliferation of stomach cancer cell cell, the proliferation of 109 cells of cancer of the esophagus Eca and colorectal cancer LOVO cells, it is with extensive scientific research and mass production edible value in terms of the medications such as digestive system and anti-gastric cancer.
Description
Technical field
This application involves the purposes more particularly to its purposes in pharmacy and health food of great Bai bolt mycelia.
Background technology
Great Bai bolts bacterium Trametes lactinea (Berk.) Pat. is a kind of the big of Aphyllophorales Polyporaceae Trametes
Type fungi[1], test and detached with great Bai bolts bacterium from Tu Li Tujias common food Preserved-fish capsicums, great Bai is accredited as through teacher Wang Shaobai
Bolt bacterium.It is few to the research of great Bai bolt bacterium both at home and abroad.In recent years, to great Bai bolts bacterium research shows that great Bai bolts fungus extract is with good
Good immunological enhancement and antitumor action.
Invention content
Using electron ionization mass spectrometry analysis instrument (VGAnalytical 70-250S, 70eV) to the extracts of great Bai bolt bacterium into
Row molecular weight determination, the nuclear magnetic resonance data for the compound extracted are
The nuclear magnetic resonance data of the loose Siberian cocklebur acid B of 1 great Bai bolt fungus extracts 3- of table hydrogenations
It can be seen that molecular ion [M] from the EIMS collection of illustrative plates of compound+454, it is 454 to thereby determine that its molecular weight,
Other fragment peak (439 [M ± CH3]+(100),421[M±CH3±H2O]+(33),393(43),297(48),95(38),69
(43)), the data with the reports such as Francesca Cateni [1] are coincide.
The mass spectrum of acquisition and nuclear magnetic resonance figures modal data with data in literature are made comparisons, identify main compound, 3- hydrogenations
Loose Siberian cocklebur acid B (3 beta-hydroxy wool steroid -8,24- diene -21- acid), the structural formula of the loose Siberian cocklebur acid B of 3- hydrogenations are as shown in Figure 1.
The compound is extracted by great Bai bolts bacterium and is made, it is known that may act on the active constituent of diabetes.
It is an object of the invention to prepare great Bai bolts mycelia or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, and for pre-
Application in anti-and treatment peptic ulcer medicine.
Another object of the present invention is to prepare great Bai bolts mycelia or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, and should
For preventing and treating the application in the medicine of non-ulcer dyspepsia.
Another object of the present invention is to prepare great Bai bolts mycelia or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, and should
For preventing and treating the application in the medicine of gastric cancer.
Another object of the present invention is to prepare great Bai bolts mycelia or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, and should
For preventing and treating the application in the medicine of the cancer of the esophagus.
Another object of the present invention is to prepare great Bai bolts mycelia or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, and should
For preventing and treating the application in the medicine of colorectal cancer.
Another object of the present invention is to prepare great Bai bolts mycelia or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, and should
For the application in health food, drug.
The great Bai bolts mycelia of the present invention or great Bai bolts fungus extract, the loose Siberian cocklebur acid B of 3- hydrogenations, which have, inhibits H+, K+- ATPase lives
Property and anti-ulcer effect.
Description of the drawings
Fig. 1 is the structural formula that 3- hydrogenates loose Siberian cocklebur acid B.
Fig. 2 hydrogenates loose Siberian cocklebur acid B and H for 3-+, K+- ATPase molecular docking results.
Specific embodiment
Great Bai bolt bacterium mycelia, great Bai bolt extracts 3- hydrogenations loose Siberian cocklebur acid B, great Bai bolt water extracts, 95% ethanol extract
Preparation method is shown in the artificial culture method and purposes of patent application great Bai bolt bacterium, Publication No.：200810046969.7.
1. anti-peptic ulcer is tested
(1) inhibit H+, K+- ATPase active functions：Take normal kunming mice, weight 20-25g, non-fasting, cervical dislocation
It puts to death, 5min is sterilized in 75% alcohol, is dissected under aseptic condition, gastric pouch is taken out, is placed in cold PBS buffer salt solutions.
It takes and as possible wipes the coating protein matter on outer membrane and fat etc. off during stomach.Gastric pouch is prolonged into big curved cut off at lower ending opening with scissors
Part, then overturn gastric pouch with tweezers, it clamps and does not cut off end, washed swill to the greatest extent, in cold PBS with PBS buffer salt solutions
It is temporarily preserved in buffer salt solution.By prepare 5% trypsin solution, according to the dosage of each gastric pouch 4-5ml, digested.
Stomach tapetum cell is scraped, is distributed in the cold pancreatin of taking-up.The cold pancreatin for being dispersed with inner membrance confluent monolayer cells is put into 37 DEG C of water-baths
Middle digestion 10min.Gently being shaken per 2-3min in digestion process once, prevents the cell accumulation of digestion.After the completion of digestion, in nothing
It is gently blown and beaten with liquid-transfering gun under the conditions of bacterium, makes cell is more as possible to come off.Cell and tissue are separated with the sieve of 200 mesh.It will
Cell liquid is transferred in 10ml centrifuge tubes, is centrifuged 8min under 1200rpm, is discarded solution, is rapidly added DMEM culture mediums.
Single cell suspension is made in above-mentioned cell, cell concentration is adjusted to 1 × 10 with DMEM culture mediums5/ ml or so, inoculation
In 96 orifice plates, per 100 μ l of hole.Be separately added into Omeprazole (10ug/ml), 95% ethanol extract (80,40 and 20ug/ml),
Great Bai bolts water extract (80,40 and 20ug/ml) 3- hydrogenation pine Siberian cocklebur acid B (40,20 and 10ug/ml), (added drug is small with concentration
After 0.1% DMSO dissolvings plus culture medium dilutes, through 0.22 μm of membrane filtration), after 37 DEG C are incubated 4h, each drug concentration is total to
Three parallel determination samples are counted, use H+, K+- ATPase Activity Assay Kits measure enzymatic activity, calculate inhibiting rate.
2 great Bai bolt fungus extracts of table are to H+, K+- ATPase activity influence (N=6)
With control group than * p ﹤ 0.05, * * p ﹤ 0.01
(2) anti-ulcer effect：Kunming mouse is taken, is randomly divided into group, every group of 10 half male and half females.Test group fills daily
Stomach gives relative medicine, totally five days, is deprived of food but not water after administration in the 4th day, after the last administration after 1 hour, gavage aspirin-
Ethanol solution 0.2ml (dissolves aspirin 200mg/kg with 40% ethyl alcohol), puts to death mouse after 4h, by mouse along greater curvature
Place is cut off, and content is washed away with clear water after overturning, and gastric ulcer point is observed under disecting microscope, is scored according to lesion degree.Office
Rubescent portion's hyperemia is 1 point, and petechial hemorrhage or erosion are respectively 1 point, and linear rotten to the corn 1 is 3 points, as ulcer index after total.Together
When gastric tissue is taken to be homogenized, measure enzyme activity.
3 great Bai bolts fungus extract of table to mouse gastric ulcer influence (N=10)
With model group than * p ﹤ 0.05, * * p ﹤ 0.01
Inside and outside experimental result shows that great Bai bolts bacterium has anti-peptic ulcer effect, great Bai bolts extract, 3- hydrogenation pines
Siberian cocklebur acid B is principle active component.
Molecular docking calculates：In 4.0 softwares of Auto Dock, H is built respectively+, K+- ATPase (receptor) and 3- hydrogenation pines
The three-dimensional structure of Siberian cocklebur acid B (ligand), ligand and receptor are docked, and runs software checks docking result.Docking result is to dock point
Value is represented with energy △ G are combined, by the loose Siberian cocklebur acid B and H of 3- hydrogenations of Fig. 2+, K+- ATPase molecular dockings result understands H+, K+-
The combination energy △ G=-4.75 of the loose Siberian cocklebur acid B of ATPase and 3- hydrogenations.
2. improve the experiment of WeiDongLi Capsule
The low rat model of WeiDongLi Capsule makes：
After SD rats are bought, adaptability raising 3d is random to be grouped, and blank group and model group give the physiology salt of 2ml respectively
Water and glycyrrhiza uralensis (10g/kg), the next day partly measure feed, continue 10 days, make the low rat model of WeiDongLi Capsule, modeling success after,
Model group continues to give licorice solution, Normal group to giving normal saline, and medicine group gives relative medicine, and after
Continue daily gavage Radix Glycyrrhizae solution (10g/kg).Phenol red solution 0.8ml, after gavage terminates 20min, neck are added in during the 7th day gavage
Rat is put to death in vertebra dislocation, splits abdomen, is ligatured cardia and pylorus, is won stomach and small intestine.
Stomach residue rate and Intestinal propulsive rate measure：
Stomach residue rate：0.002g/ml phenol red solution 1ml is taken to add in into the NaOH solution of 10ml 0.1mol/L, is shaken
Mixing.0.2ml is taken to be dissolved in 1ml pure water, as titer.Under wavelength 560nm ultraviolet-uisible spectrophotometer in measure its
Absorbance, as titer OD values.Full stomach is dipped in the NaOH solution of 0.1mol/L and shredded, fully washs, shakes up, room temperature
Lower 4000rpm centrifuges 10min, collects supernatant 0.2ml, adds in pure water 1ml, is equally measured using ultraviolet-uisible spectrophotometer
Its absorbance under 560nm again, as measures liquid OD values.Stomach residue rate is calculated as follows：
Stomach residue rate (%)=measure liquid OD values/titer OD value × 100%.
Intestinal propulsive rate：Pylorus is intercepted to distal end ileocecus part, is laid on dissection plate, measures overall length.Observe phenol
Red forward position to pylorus distance and measure its length.If phenol red cannot be clearly showed that, from intestinal tube is distally cut off, syringe drips
Add 0.1mol/L NaOH, phenol red is prompted to be advanced into herein if color is shown in red.It is grown with the phenol red propulsion in small intestine
The percentage that degree accounts for small intestine overall length calculates Intestinal propulsive rate.
Intestinal propulsive rate (%)=phenol red advance distance/small intestine overall length (cm) × 100%.
4 great Bai bolts fungus extract of table to Stomach residue rate and Intestinal propulsive rate influence (N=8)
With model group than * p ﹤ 0.05, * * p ﹤ 0.01
Experimental result shows that great Bai bolts bacterium can increase WeiDongLi Capsule, and great Bai bolts extract, 3- hydrogenations pine Siberian cocklebur acid B are that mainly have
Imitate ingredient.
WeiDongLi Capsule refers to the contraction peristaltic forces of stomach muscle, the strength and frequency shunk including stomach muscle.Gastric motility disorder
It is the main reason for causing non-ulcer dyspepsia.When there is obstacle in WeiDongLi Capsule, it may occur that indigestion, occur abdominal distension,
The indigestion symptoms such as gas, loss of appetite, sour regurgitation, Nausea and vomiting.Great Bai bolts bacterium can increase WeiDongLi Capsule, be moved available for treatment stomach
Indigestion caused by power deficiency.
3. anti-gastric cancer Experiment on Function
The in vitro culture of cell strain：People's gastric epithelial cell GES-1 and gastric carcinoma cells HGC-27 DMEM culture mediums
Culture, cell culture fluid volume fraction containing fetal calf serum is 10%, containing each 100U/ml of penicillin and streptomycin.All cells are in 37
DEG C, cultivate in 5%CO2 incubators, passed on 0.25% trypsin digestion and cell, growth period cell of taking the logarithm is for real
It tests.
3- hydrogenates pine Siberian cocklebur acid B at various ph values to the influence of cell survival rate：
Single cell suspension is made in growth period cell routine of taking the logarithm digestion, and cell concentration is adjusted to 5 × 10 with culture solution4-1
×105Ml or so is inoculated in 96 orifice plates, per 100 μ l of hole.Culture add in afterwards for 24 hours the loose Siberian cocklebur acid B of 3- hydrogenations (1 μ g/ml, 5 μ g/ml and
10 μ g/ml), great Bai bolt mykols extract (20 μ g/ml and 40 μ g/ml) and great Bai bolt bacterium water extract (20 μ g/ml and 40 μ g/ml),
Blank control group and Normal group, while the different pH value of every group of setting (pH 7.5, pH 6.5 and pH 5.5) separately are set, every group sets
6 multiple holes.37 DEG C are continued to cultivate, and are cultivated for 24 hours, after, 5mg/ml MTT20 μ l are added in per hole, 37 DEG C of incubation 4h discard culture solution,
150 μ l of DMSO are added in per hole, gentle agitation makes crystal fully dissolve, examined at wavelength 492nm with enzyme-linked immunosorbent assay instrument
Survey the trap (OD values) per hole.
Inhibitory rate of cell growth is calculated by following equation：Inhibitory rate of cell growth (%)=1- (dosing group mean OD value/just
Normal control group mean OD value) × 100%.
Using 13.0 softwares of SPSS carry out statistical analysis, experimental data withIt represents, with P<0.05 is with system
Meter learns difference.
5 drug of table under different PH to HGC-27 cell Proliferations influence (N=6)
With blank group than * p ﹤ 0.05, * * p ﹤ 0.01
6 drug of table under normal pH value (PH=7.5) to GES-1 cell Proliferations influence (N=6)
The experimental results showed that great Bai bolts alcohol extract has certain inhibiting effect to the proliferation of stomach cancer cell cell, in acyclic acidic
Its inhibiting effect greatly enhances under border, low concentration 3- hydrogenation pine Siberian cocklebur acid B (10 μ g/ml, 5 μ g/ml), high concentration great Bai bolt alcohol extracts
The effect of (40 μ g/ml) and great Bai bolts water extract (40 μ g/ml) for 24 hours afterwards respectively reaches the inhibiting rate of cell 61.4%, 81.5%,
40% and 32.9%, and this concentration has little effect people's normal gastric mucosa epithelial cell under normal pH environment.
4. anti esophageal cancer Experiment on Function
The in vitro culture of cell strain：Human esophagus cancer cell strain Eca-109 is placed in RPMI1640 culture solutions (containing 10% tire
Cow's serum and each 100U/ml of penicillin, streptomysin), in 37 DEG C of saturated humidities, 5%CO2It is cultivated in incubator.Treat that cell enters
After exponential phase, through 0.25% trypsin digestion, collect, be deployed into corresponding cell concentration.
MTT is tested：It is prepared into 1 × 104The Eca-109 cell suspensions of/ml are added in after blowing and beating uniformly in 96 orifice plates, culture
After 12h, be divided into blank control group, positive group (20 μ g/ml mitomycins) and great Bai bolt mykols extract, great Bai bolt bacterium water extracts and
Loose Siberian cocklebur acid 5 experimental groups of B of 3- hydrogenations, it is each to test 4 dosage groups of component, be separately added into various concentration drug (100,80,
40,20 and 10 μ g/ml), control group adds in culture solution, continues to cultivate 48h, MTT (5mg/ml) 20 μ l are then added in per hole, continue
Cultivate 4h.Culture solution is discarded, 150 μ l of DMSO are added in per hole, gentle agitation is examined with enzyme-linked immunosorbent assay instrument at wavelength 492nm
Survey the trap (OD values) per hole.
7 great Bai bolts extract of table to the In-vitro Inhibitory Effects of Eca-109 cells (N=6)
The result shows that great Bai bolts extract has inhibiting effect to the proliferation of esophageal carcinoma Eca-109 cell line.
5. anti-colorectal cancer Experiment on Function
The in vitro culture of cell strain：Human Large Intestine Carcinoma Cells strain LOVO is placed in DMEM culture solutions (containing 10% fetal calf serum
With penicillin, each 100U/ml of streptomysin), it is cultivated in 37 DEG C of saturated humidities, 5%CO2 incubators.Treat that cell enters logarithm life
After long-term, through 0.25% trypsin digestion, collect, be deployed into corresponding cell concentration.
MTT is tested：It is prepared into 5 × 104The LOVO cell suspensions of/ml add in 96 orifice plates after blowing and beating uniformly, cultivate 12h
Afterwards, it is divided into blank control group, positive group (20 μ g/ml mitomycins) and great Bai bolt mykols extract, great Bai bolt bacterium water extracts and 3-
Loose Siberian cocklebur acid 3 experimental groups of B of hydrogenation, it is each to test 4 dosage groups of component, be separately added into various concentration drug (100,80,40,
20 and 10 μ g/ml), control group adds in culture solution, continues to cultivate 48h, MTT (5mg/ml) 20 μ l are then added in per hole, continue to train
Support 4h.Culture solution is discarded, 150 μ l of DMSO are added in per hole, gentle agitation is detected with enzyme-linked immunosorbent assay instrument at wavelength 492nm
Trap (OD values) per hole.
7 great Bai bolts extract of table to the In-vitro Inhibitory Effects of LOVO cells (N=6)
The result shows that great Bai bolts extract has inhibiting effect to the proliferation of colorectal cancer LOVO cells.
Claims (2)
1. a kind of application of great Bai bolts mycelia in the medicine for preparing prevention and treatment peptic ulcer.
2. a kind of great Bai bolts mycelia is in the medicine of non-ulcer dyspepsia caused by preparation prevention and treatment gastric dynamic dysfunction
Using.
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| CN201510330206.5A CN104983750B (en) | 2013-05-14 | 2013-05-14 | A kind of application of great Bai bolts mycelia in pharmacy |
| CN201310176266.7A CN103316051B (en) | 2013-05-14 | 2013-05-14 | New functions and applications of trametes lactinea(Berk.)Pat. or extractives and trametenloic acid B |
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| CN201510330206.5A Active CN104983750B (en) | 2013-05-14 | 2013-05-14 | A kind of application of great Bai bolts mycelia in pharmacy |
| CN201510328462.0A Active CN104940244B (en) | 2013-05-14 | 2013-05-14 | A kind of application of ethanol extract of great Bai bolts bacterium 95% in pharmacy |
| CN201310176266.7A Active CN103316051B (en) | 2013-05-14 | 2013-05-14 | New functions and applications of trametes lactinea(Berk.)Pat. or extractives and trametenloic acid B |
| CN201510328957.3A Active CN104971069B (en) | 2013-05-14 | 2013-05-14 | A kind of applications of the 3 loose Siberian cocklebur acid B of hydrogenation extracted in great Bai bolts bacterium in pharmacy |
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| CN104940245B (en) * | 2013-05-14 | 2018-03-02 | 三峡大学 | A kind of application of great Bai bolts bacterium water extract in pharmacy |
| CN105949262B (en) * | 2016-05-27 | 2017-05-24 | 三峡大学 | 3-hydrogenated pinicolic acid cyanide ethyl ester medicine and application thereof |
| CN110063960B (en) * | 2019-05-07 | 2021-05-18 | 三峡大学 | Pharmaceutical application of 3-hydrogenated pinolenic acid B cyanomethyl ester |
| CN113384591B (en) * | 2021-06-28 | 2022-09-02 | 三峡大学 | Combined drug of trametes acid and sorafenib and application of combined drug in preparation of antitumor drug |
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| CN102028716A (en) * | 2009-09-28 | 2011-04-27 | 郭志勇 | New application of basidiomycetes or extract thereof (3-trametenloic acid B) |
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| JP4245291B2 (en) * | 2001-12-04 | 2009-03-25 | 正 五井野 | Bioactive composition and method for producing the same |
| CN101439055B (en) * | 2007-11-19 | 2011-08-24 | 启东盖天力药业有限公司 | Use of aqueous extract of Trametes robiniophila Murr. in preparing medicament |
| CN101245318B (en) * | 2008-02-28 | 2013-09-04 | 三峡大学 | Artificial cultivation method of Basidiomycetes and uses thereof |
| JP2009024027A (en) * | 2008-09-16 | 2009-02-05 | Tadashi Goino | Physiologically active composition |
| JP5610415B2 (en) * | 2009-02-10 | 2014-10-22 | 国立大学法人鳥取大学 | Alcohol production method, alcoholic beverage production method, alcohol-containing food production method, and inoculum used therefor |
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| JP2014001141A (en) * | 2010-09-10 | 2014-01-09 | Kureha Corp | Medicine for tumor treatment and anti-tumorigenic agent |
| CN104940245B (en) * | 2013-05-14 | 2018-03-02 | 三峡大学 | A kind of application of great Bai bolts bacterium water extract in pharmacy |
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| Atimicrobial steroids from the fumgus fomitopsis pinicola;Anne Caroline Keller等;《Phytochemistry》;19961231;第41卷(第4期);第1041-1046页 * |
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| CN103316051B (en) | 2015-07-22 |
| CN104940244A (en) | 2015-09-30 |
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