CN104940245B - A kind of application of great Bai bolts bacterium water extract in pharmacy - Google Patents
A kind of application of great Bai bolts bacterium water extract in pharmacy Download PDFInfo
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Abstract
The present invention relates to the New function and purposes of great Bai bolt bacterium water extracts.The material has the function that anti-peptic ulcer, increase WeiDongLi Capsule and then anti-non-ulcer dyspepsia；Great Bai bolt bacterium water extracts have certain inhibitory action in the propagation to the propagation of stomach cancer cell cell, the propagation of the cells of cancer of the esophagus Eca 109 and colorectal cancer LOVO cells, and it has extensive scientific research and mass production edibility in terms of the medication such as digestive system and anti-gastric cancer.
Description
Technical field
The application is related to the purposes of great Bai bolt bacterium water extracts, more particularly to its purposes in pharmacy and health food.
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 separated 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.Research both at home and abroad to great Bai bolt bacterium is few.In recent years, to the research of great Bai bolts bacterium, to show that great Bai bolt fungus extracts have good
Good immunological enhancement and antitumor action.
The content of the invention
The extract of great Bai bolt bacterium is entered using electron ionization mass spectrometry analysis instrument (VGAnalytical 70-250S, 70eV)
Row molecular weight determination, the nuclear magnetic resonance data for the compound extracted are
The great Bai bolt fungus extracts 3- of table 1 hydrogenates loose Siberian cocklebur acid B nuclear magnetic resonance data
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 report such as Francesca Cateni [1] are coincide.
The mass spectrum of acquisition and nuclear magnetic resonance figures modal data are made comparisons with data in literature, identify main compound, 3- hydrogenations
Loose Siberian cocklebur acid B (3 beta-hydroxy wool steroid -8,24- diene -21- acid), the structural formula that 3- hydrogenates loose Siberian cocklebur acid B is 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 component of diabetes.
It is an object of the invention to prepare great Bai bolts mycelia or great Bai bolts fungus extract, 3- to hydrogenate loose Siberian cocklebur acid B, 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, 3- to hydrogenate loose Siberian cocklebur acid B, and should
The application in medicine for preventing and treating non-ulcer dyspepsia.
Another object of the present invention is to prepare great Bai bolts mycelia or great Bai bolts fungus extract, 3- to hydrogenate loose Siberian cocklebur acid B, and should
The application in medicine for preventing and treating stomach cancer.
Another object of the present invention is that preparing great Bai bolts mycelia or great Bai bolts fungus extract, 3- hydrogenates loose Siberian cocklebur acid B, and should
The application in medicine for preventing and treating the cancer of the esophagus.
Another object of the present invention is that preparing great Bai bolts mycelia or great Bai bolts fungus extract, 3- hydrogenates loose Siberian cocklebur acid B, and should
The application in medicine for preventing and treating colorectal cancer.
Another object of the present invention is that preparing great Bai bolts mycelia or great Bai bolts fungus extract, 3- hydrogenates loose Siberian cocklebur acid B, and should
For the application in health food, medicine.
The great Bai bolts mycelia of the present invention or great Bai bolts fungus extract, 3-, which hydrogenate loose Siberian cocklebur acid B, has suppression H+, K+- ATPase lives
Property and anti-ulcer effect.
Brief description of the drawings
Fig. 1 is the structural formula that 3- hydrogenates loose Siberian cocklebur acid B.
Fig. 2 is that 3- hydrogenates loose Siberian cocklebur acid B and H+, K+- ATPase molecular docking results.
Specific embodiment
Great Bai bolt bacterium mycelia, great Bai bolt extracts 3- hydrogenate loose Siberian cocklebur acid B, great Bai bolt water extract, 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) H is suppressed+, K+- ATPase active functions：Take normal kunming mice, body weight 20-25g, non-fasting, cervical dislocation
Put 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.
Take and as far as possible wipe 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, clamp and do not cut off end, washed swill to the greatest extent, in cold PBS with PBS buffer salt solutions
Temporarily preserved in buffer salt solution.By prepare 5% trypsin solution, according to each gastric pouch 4-5ml dosage, 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 shaken once per 2-3min in digestion process, prevent the cell accumulation of digestion.After the completion of digestion, in nothing
Gently blown and beaten with liquid-transfering gun under the conditions of bacterium, cell is tried one's best more come off.Cell and tissue are separated with the screen cloth of 200 mesh.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 the μ l of hole 100.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- hydrogenates loose Siberian cocklebur acid B (40,20 and 10ug/ml), and (added medicine 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, every kind of drug concentration is total to
Three parallel determination samples are counted, use H+, K+- ATPase Activity Assay Kits determine enzymatic activity, calculate inhibiting rate.
The great Bai bolt fungus extracts of table 2 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 male and female half and half.Test group fills daily
Stomach gives relative medicine, totally five days, after administration in the 4th day fasting can't help water, after last dose after 1 hour, gavage aspirin-
Ethanol solution 0.2ml (dissolves aspirin 200mg/kg with 40% ethanol), and mouse is put to death after 4h, and mouse is big along stomach
Crook is cut off, and content is washed away with clear water after upset, and gastric ulcer point is observed under disecting microscope, is scored according to lesion degree.
Rubescent contrafluxion is 1 point, and petechial hemorrhage or erosion are respectively 1 point, and rotten to the corn 1 of wire is 3 points, and ulcer index is used as after total.
Take gastric tissue to be homogenized simultaneously, determine enzyme activity.
The great Bai bolts fungus extract of table 3 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 bolt bacterium have anti-peptic ulcer effect, great Bai bolts extract, 3- hydrogenation pines
Siberian cocklebur acid B is principle active component.
Molecular docking calculates：In the softwares of Auto Dock 4.0, H is built respectively+, K+- ATPase (acceptor) and 3- hydrogenation pines
Siberian cocklebur acid B (part) three-dimensional structure, part and acceptor are docked, runs software, check docking result.Docking result is to dock point
Value is represented with energy △ G are combined, and loose Siberian cocklebur acid B and H are hydrogenated by Fig. 2 3-+, K+- ATPase molecular dockings result understands H+, K+-
ATPase and 3- hydrogenates loose Siberian cocklebur acid B combination energy △ G=-4.75.
2. improve the experiment of WeiDongLi Capsule
The low rat model of WeiDongLi Capsule makes：
After SD rats are bought, adaptability raising 3d, random packet, blank group and model group give 2ml physiology salt 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 during the 7th day gavage
Rat is put to death in vertebra dislocation, splits belly, is ligatured orifice of the stomach and pylorus, is won stomach and small intestine.
Stomach residue rate and Intestinal propulsive rate measure：
Stomach residue rate：Take 0.002g/ml phenol red solution 1ml to add into 10ml 0.1mol/L NaOH solution, shake
Mix.0.2ml is taken to be dissolved in 1ml pure water, as titer.Under wavelength 560nm ultraviolet-uisible spectrophotometer in determine its
Absorbance, as titer OD values.Full stomach is dipped in 0.1mol/L NaOH solution and shredded, fully washing, is shaken up, room temperature
Lower 4000rpm centrifuges 10min, collects supernatant 0.2ml, adds pure water 1ml, is equally determined using ultraviolet-uisible spectrophotometer
Its absorbance under 560nm again, as determines 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 total length.Observe phenol
Red forward position to pylorus distance and measure its length.If phenol red can not be clearly showed that, from intestinal tube is distally cut off, syringe drips
Add 0.1mol/L NaOH, prompt phenol red to be advanced into herein if color is shown in red.Grown with the phenol red propulsion in small intestine
The percentage that degree accounts for small intestine total length calculates Intestinal propulsive rate.
Intestinal propulsive rate (%)=phenol red advance distance/small intestine total length (cm) × 100%.
The great Bai bolts fungus extract of table 4 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, the loose Siberian cocklebur acid B of 3- hydrogenations are that mainly have
Imitate composition.
WeiDongLi Capsule refers to the contraction peristaltic forces of stomach muscle, including the strength and frequency that stomach muscle is shunk.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, poor 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 line：People's gastric epithelial cell GES-1 and gastric carcinoma cells HGC-27 DMEM culture mediums
Culture, cell culture fluid volume fraction containing hyclone 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 used for real
Test.
3- hydrogenates the loose Siberian cocklebur acid B influences to cell survival rate at various ph values：
Single cell suspension is made in growth period cell routine of taking the logarithm digestion, and cell concentration is adjusted to 5 × 10 with nutrient solution4-1
×105Ml or so, 96 orifice plates are inoculated in, per the μ l of hole 100.Cultivate add after 24h 3- hydrogenate loose Siberian cocklebur acid B (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 cultivate 24h, after, 5mg/ml MTT20 μ l are added per hole, 37 DEG C of incubation 4h, discard nutrient solution,
The μ l of DMSO 150 are added per hole, gentle agitation, crystal is fully dissolved, is examined with enzyme-linked immunosorbent assay instrument at wavelength 492nm
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 the softwares of SPSS 13.0 carry out statistical analysis, experimental data withRepresent, with P<0.05 is with statistics
Learn difference.
The medicine of table 5 under different PH to HGC-27 cells propagation influence (N=6)
With blank group than * p ﹤ 0.05, * * p ﹤ 0.01
The medicine of table 6 under normal pH value (PH=7.5) to GES-1 cells propagation influence (N=6)
Test result indicates that great Bai bolts alcohol extract has certain inhibitory action to the propagation of stomach cancer cell cell, in acyclic acidic
Its inhibitory action greatly enhances under border, and low concentration 3- hydrogenates loose Siberian cocklebur acid B (10 μ g/ml, 5 μ g/ml), high concentration great Bai bolt alcohol extracts
Respectively reach 61.4% after (40 μ g/ml) and great Bai bolts water extract (40 μ g/ml) effect 24h to the inhibiting rate of cell, 81.5%,
40% and 32.9%, and this concentration has little to no effect under normal pH environment to people's normal gastric mucosa epithelial cell.
4. anti esophageal cancer Experiment on Function
The in vitro culture of cell line：Human esophagus cancer cell strain Eca-109 is placed in RPMI1640 nutrient solutions and (contains 10% tire
Cow's serum and each 100U/ml of penicillin, streptomysin), in 37 DEG C of saturated humidities, 5%CO2Cultivated in incubator.Treat that cell enters
After exponential phase, through 0.25% Trypsin Induced, collect, be deployed into corresponding cell concentration.
MTT is tested：It is prepared into 1 × 104/ ml Eca-109 cell suspensions, added 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
3- hydrogenates loose 5 experimental groups of Siberian cocklebur acid B, each to test 4 dosage groups of component, be separately added into various concentrations medicine (100,80,
40,20 and 10 μ g/ml), control group adds nutrient solution, continues to cultivate 48h, MTT (5mg/ml) 20 μ l are then added per hole, continue
Cultivate 4h.Nutrient solution is discarded, the μ l of DMSO 150 are added per hole, gentle agitation, are examined with enzyme-linked immunosorbent assay instrument at wavelength 492nm
Survey the trap (OD values) per hole.
The great Bai bolts extract of table 7 to the In-vitro Inhibitory Effects of Eca-109 cells (N=6)
As a result show, great Bai bolts extract has inhibitory action to the propagation of esophageal carcinoma Eca-109 cell line.
5. anti-colorectal cancer Experiment on Function
The in vitro culture of cell line：Human Large Intestine Carcinoma Cells strain LOVO is placed in DMEM nutrient solutions and (contains 10% hyclone
With penicillin, each 100U/ml of streptomysin), cultivated in 37 DEG C of saturated humidities, 5%CO2 incubators.Treat that cell is given birth into logarithm
After long-term, through 0.25% Trypsin Induced, collect, be deployed into corresponding cell concentration.
MTT is tested：It is prepared into 5 × 104/ ml LOVO cell suspensions, added after blowing and beating uniformly in 96 orifice plates, 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-
Hydrogenate loose 3 experimental groups of Siberian cocklebur acid B, it is each to test 4 dosage groups of component, be separately added into various concentrations medicine (100,80,40,
20 and 10 μ g/ml), control group adds nutrient solution, continues to cultivate 48h, MTT (5mg/ml) 20 μ l are then added per hole, continue to train
Support 4h.Nutrient solution is discarded, the μ l of DMSO 150 are added per hole, gentle agitation, are detected with enzyme-linked immunosorbent assay instrument at wavelength 492nm
Trap (OD values) per hole.
The great Bai bolts extract of table 8 to the In-vitro Inhibitory Effects of LOVO cells (N=6)
As a result show, great Bai bolts extract has inhibitory action to the propagation of colorectal cancer LOVO cells.
Claims (3)
- A kind of 1. application of great Bai bolts bacterium water extract in the medicine for suppressing gastric carcinoma cells HGC-27 is prepared.
- A kind of 2. application of great Bai bolts bacterium water extract in the medicine for suppressing human esophagus cancer cell strain Eca-109 is prepared.
- A kind of 3. application of great Bai bolts bacterium water extract in the medicine for suppressing Human Large Intestine Carcinoma Cells strain LOVO is prepared.
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| 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 |
| CN201510328463.5A CN104940245B (en) | 2013-05-14 | 2013-05-14 | A kind of application of great Bai bolts bacterium water extract in pharmacy |
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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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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101245318A (en) * | 2008-02-28 | 2008-08-20 | 三峡大学 | Artificial cultivation method of Basidiomycetes and uses thereof |
| CN102028716A (en) * | 2009-09-28 | 2011-04-27 | 郭志勇 | New application of basidiomycetes or extract thereof (3-trametenloic acid B) |
| CN103316051B (en) * | 2013-05-14 | 2015-07-22 | 三峡大学 | New functions and applications of trametes lactinea(Berk.)Pat. or extractives and 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 |
| 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 |
| CN101933950A (en) * | 2009-07-03 | 2011-01-05 | 启东盖天力药业有限公司 | Application of Trametes robiniophila Murr aqueous extract |
| JP2014001141A (en) * | 2010-09-10 | 2014-01-09 | Kureha Corp | Medicine for tumor treatment and anti-tumorigenic agent |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245318A (en) * | 2008-02-28 | 2008-08-20 | 三峡大学 | Artificial cultivation method of Basidiomycetes and uses thereof |
| CN102028716A (en) * | 2009-09-28 | 2011-04-27 | 郭志勇 | New application of basidiomycetes or extract thereof (3-trametenloic acid B) |
| CN103316051B (en) * | 2013-05-14 | 2015-07-22 | 三峡大学 | New functions and applications of trametes lactinea(Berk.)Pat. or extractives and trametenloic acid B |
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| CN104940244B (en) | 2018-03-02 |
| CN104940245A (en) | 2015-09-30 |
| CN104983750B (en) | 2018-06-26 |
| CN103316051A (en) | 2013-09-25 |
| CN103316051B (en) | 2015-07-22 |
| CN104940244A (en) | 2015-09-30 |
| CN104971069A (en) | 2015-10-14 |
| CN104983750A (en) | 2015-10-21 |
| CN104971069B (en) | 2017-12-19 |
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