CN103316051B - New functions and applications of trametes lactinea(Berk.)Pat. or extractives and trametenloic acid B - Google Patents
New functions and applications of trametes lactinea(Berk.)Pat. or extractives and trametenloic acid B Download PDFInfo
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- CN103316051B CN103316051B CN201310176266.7A CN201310176266A CN103316051B CN 103316051 B CN103316051 B CN 103316051B CN 201310176266 A CN201310176266 A CN 201310176266A CN 103316051 B CN103316051 B CN 103316051B
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Abstract
The invention relates to new functions and applications of trametes lactinea(Berk.)Pat. or extractives and trametenloic acid B. The substance trametes lactinea(Berk.)Pat has the functions of digestive gastric ulcer resistance, and nonulcerative-indigestion resistance through increasing of gastric motility. The trametes lactinea(Berk.)Pat. or the extractives and the trametenloic acid B have certain inbibitional effects on cell proliferation of gastric cancer cells, esophagus cancer cells Eca-109 and colorectal cancer cells LOVO, and has a wide range of research value and mass production and edible value in dugs for digestive system and gastric cancer resisting and the like.
Description
Technical field
The application relates to the purposes of Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, particularly relates to its purposes in pharmacy and health food.
Background technology
Trametes laclinea Berk Trametes lactinea (Berk.) Pat. is a kind of macro fungi of Aphyllophorales Polyporaceae Trametes
[1], experiment Trametes laclinea Berk from Tu Li Tujia common food Preserved-fish Pepper from, be accredited as Trametes laclinea Berk through teacher Wang Shaobai.Few to the research of Trametes laclinea Berk both at home and abroad.In recent years, Trametes laclinea Berk research is shown that Trametes laclinea Berk extract has good immunological enhancement and antitumor action.
Summary of the invention
Use EIMS instrument (VG Analytical70-250S, the 70eV) extract to Trametes laclinea Berk to carry out molecular weight determination, the nuclear magnetic resonance data obtaining the compound extracted is
The nuclear magnetic resonance data of table 1 Trametes laclinea Berk extract 3-hydrogenation pine Siberian cocklebur acid B
Molecular ion [M] can be seen from the EIMS collection of illustrative plates of compound
+454, determine that its molecular weight is 454 thus, other fragment peak (439 [M ± CH
3]
+(100), 421 [M ± CH
3± H
2o]
+(33), 393 (43), 297 (48), 95 (38), 69 (43)), the data reported with Francesca Cateni [1] etc. are coincide.
The mass spectrum of acquisition and nuclear magnetic resonance map data and data in literature are made comparisons, qualification main compound, 3-hydrogenation pine Siberian cocklebur acid B (3 beta-hydroxy Pilus Caprae seu Ovis steroid-8,24-diene-21-acid), the structural formula of 3-hydrogenation pine Siberian cocklebur acid B as shown in Figure 1.
This compound extracts obtained by Trametes laclinea Berk, known act on diabetes active component.
The object of the invention is to prepare Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, and for the application in the medicine of prevention and therapy peptic ulcer.
Another object of the present invention is to prepare Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, and be applied to the application in the medicine of prevention and therapy non-ucler dyspepsia.
Another object of the present invention is to prepare Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, and be applied to the application in the medicine of prevention and therapy gastric cancer.
Another object of the present invention is to prepare Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, and is applied to the application in the medicine of the prevention and therapy esophageal carcinoma.
Another object of the present invention is to prepare Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, and is applied to the application in the medicine of prevention and therapy colorectal cancer.
Another object of the present invention is to prepare Trametes laclinea Berk silk or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B, and is applied to the application in health food, medicine.
Trametes laclinea Berk silk of the present invention or Trametes laclinea Berk extract, 3-hydrogenation pine Siberian cocklebur acid B have suppression H
+, K
+-ATPase activity and anti-ulcer effect.
Accompanying drawing explanation
Fig. 1 is the structural formula of 3-hydrogenation pine Siberian cocklebur acid B.
Fig. 2 is 3-hydrogenation pine Siberian cocklebur acid B and H
+, K
+-ATPase molecular docking result.
specific embodiment
The preparation method of Trametes laclinea Berk mycelia, DABAI bolt extract 3-hydrogenation pine Siberian cocklebur acid B, DABAI bolt water extract, 95% ethanol extraction is shown in artificial culture method and the purposes of patent application Trametes laclinea Berk, and publication number is: 200810046969.7.
1. anti-peptic ulcer test
(1) H is suppressed
+, K
+-ATPase active function: get normal kunming mice, body weight 20-25g, non-fasting, cervical dislocation is put to death, and sterilize 5min in 75% ethanol, dissects under aseptic condition, is taken out by stomach bag, is placed in cold PBS buffer salt solution.Coating protein matter on adventitia and fat etc. are wiped off when getting stomach as far as possible.With shears stomach bag prolonged and curvedly greatly cut off part from lower ending opening, then with tweezers, stomach bag is overturn, clamp and do not cut off end, with PBS buffer salt solution, food debris is washed to the greatest extent, temporarily preserve in cold PBS buffer salt solution.By prepare 5% trypsin solution, according to the consumption of each stomach bag 4-5ml, digest.Gastric parietal cell is scraped, is distributed in the cold pancreatin of taking-up.The cold pancreatin being dispersed with theca interna cell is put into 37 DEG C of water-baths and digests 10min.In digestion process every 2-3min gently jolting once, prevent digest cell accumulation.After having digested, aseptically blow and beat gently with liquid-transfering gun, make that cell is as far as possible many to come off.With 200 object screen clothes, biological cells and tissues is separated.Proceeded to by Cell sap in 10ml centrifuge tube, under 1200rpm, centrifugal 8min, discards solution, adds rapidly DMEM culture medium.
Above-mentioned cell is made single cell suspension, with DMEM culture medium adjustment cell concentration to 1 × 10
5about/ml, is inoculated in 96 orifice plates, every hole 100 μ l.Add respectively omeprazole (10ug/ml), 95% ethanol extraction (80,40 and 20ug/ml), DABAI bolt water extract (80,40 and 20ug/ml) 3-hydrogenation pine Siberian cocklebur acid B(40,20 and 10ug/ml), (added medicine adds culture medium dilution be all less than the DMSO dissolving of 0.1% by concentration after, through 0.22 μm of membrane filtration), 37 DEG C hatch 4h after, often kind of drug level amounts to three parallel assay samples, uses H
+, K
+-ATPase Activity Assay Kit measures enzymatic activity, calculates suppression ratio.
Table 2 Trametes laclinea Berk extract is to H
+, K
+the impact of-ATPase activity
With matched group than * p ﹤ 0.05, * * p ﹤ 0.01
(2) anti-ulcer effect: get Kunming mouse, in groups, often organizes 10 male and female half and half for random point.Test group gavage every day gives relative medicine, totally five days, after administration in 4th day, water is can't help in fasting, after last administration after 1 hour, gavage aspirin-alcoholic solution 0.2ml(by aspirin 200mg/kg with 40% dissolve with ethanol), put to death mice after 4h, mice is cut off along greater gastric curvature place, wash away content with clear water after upset, under anatomic microscope, observe gastric ulcer point, score according to lesion degree.Contrafluxion is rubescent is 1 point, and petechial hemorrhage or erosion are respectively 1 point, and rotten to the corn 1 of wire is 3 points, as ulcer index after total.Get gastric tissue homogenate simultaneously, measure enzyme and live.
Table 3 Trametes laclinea Berk extract is on the impact of mouse gastric ulcer
With model group than * p ﹤ 0.05, * * p ﹤ 0.01
Experiment in vivo and vitro result shows, and Trametes laclinea Berk has anti-peptic ulcer effect, and DABAI bolt extract, 3-hydrogenation pine Siberian cocklebur acid B are principle active component.
Molecular docking calculates: in Auto Dock4.0 software, build H respectively
+, K
+-ATPase(receptor) and 3-hydrogenation pine Siberian cocklebur acid B (part) three dimensional structure, by part and receptor docking, operating software, checks docking result.Docking result can represent by △ G to dock score value combination, by 3-hydrogenation pine Siberian cocklebur acid B and H of Fig. 2
+, K
+the known H of-ATPase molecular docking result
+, K
+-ATPase and 3-hydrogenation pine Siberian cocklebur acid B in conjunction with energy △ G=-4.75.
2. improve the test of gastric motility
The low rat model of gastric motility makes:
After SD rat is bought, adaptability raises 3d, random packet, blank group and model group give normal saline and the glycyrrhiza uralensis (10g/kg) of 2ml respectively, the next day partly measure feed, continue 10 days, make the low rat model of gastric motility, after modeling success, model group continues to give Radix Glycyrrhizae solution, Normal group to giving normal saline, medicine group gives relative medicine, and continues gavage Radix Glycyrrhizae solution every day (10g/kg).Add phenol red solution 0.8ml during the 7th day gavage, after gavage terminates 20min, cervical dislocation puts to death rat, cuts abdominal part open, ligation cardia and pylorus, wins stomach and small intestinal.
Stomach residue rate and Intestinal propulsive rate measure:
Stomach residue rate: get 0.002g/ml phenol red solution 1ml and be added in the NaOH solution of 10ml0.1mol/L, concussion mixing.Get 0.2ml to be dissolved in 1ml pure water, be titer.Under wavelength 560nm, ultraviolet-uisible spectrophotometer is in its absorbance of mensuration, is titer OD value.Full stomach to be dipped in the NaOH solution of 0.1mol/L and to shred, fully washing, shake up, the centrifugal 10min of 4000rpm under room temperature, collects supernatant 0.2ml, adds pure water 1ml, same use ultraviolet-uisible spectrophotometer measures its absorbance again under 560nm, is and measures liquid OD value.Calculate Stomach residue rate as follows:
Stomach residue rate (%)=mensuration liquid OD value/titer OD value × 100%.
Intestinal propulsive rate: intercept pylorus to far-end ileocecus part, is laid in and dissects on plate, measure total length.Observe phenol red forward position to pylorus distance and measure its length.If can not know display phenol red, then distally cut off intestinal tube, syringe drips 0.1mol/L NaOH, if color is shown in red, prompting phenol red has been advanced into herein.The percent accounting for small intestinal total length with the phenol red propelling length at small intestinal calculates Intestinal propulsive rate.
Intestinal propulsive rate (%)=phenol red advance distance/small intestinal total length (cm) × 100%.
Table 4 Trametes laclinea Berk extract is on the impact of Stomach residue rate and Intestinal propulsive rate
With model group than * p ﹤ 0.05, * * p ﹤ 0.01
Experimental result shows, and Trametes laclinea Berk can increase gastric motility, and DABAI bolt extract, 3-hydrogenation pine Siberian cocklebur acid B are principle active component.
Gastric motility refers to the contraction peristaltic forces of stomach muscle, comprises strength and the frequency of stomach muscle contraction.Gastric motility disorder is the main cause causing non-ucler dyspepsia.When obstacle appears in gastric motility, can dyspepsia be there is, occur the indigestion symptoms such as abdominal distention, belch, inappetence, acid regurgitation, Nausea and vomiting.Trametes laclinea Berk can increase gastric motility, can be used for the dyspepsia that treatment gastric dynamic dysfunction causes.
3. anti-Effect on Gastric Cancer test
The In vitro culture of cell strain: people's gastric epithelial cell GES-1 and gastric carcinoma cells HGC-27 DMEM culture medium culturing, cell culture fluid is all 10% containing hyclone volume fraction, containing each 100U/ml of penicillin and streptomycin.All cells in 37 DEG C, cultivate in 5%CO2 incubator, go down to posterity with the trypsin digestion and cell of 0.25%, trophophase cell of taking the logarithm is for experiment.
B is at various ph values on the impact of cell survival rate for the acid of 3-hydrogenation pine Siberian cocklebur:
Single cell suspension is made in trophophase cell routine of taking the logarithm digestion, with culture fluid adjustment cell concentration to 5 × 10
4-1 × 10
5about ml, is inoculated in 96 orifice plates, every hole 100 μ l.Cultivate after 24h and add 3-hydrogenation pine Siberian cocklebur acid B(1 μ g/ml, 5 μ g/ml and 10 μ g/ml), Trametes laclinea Berk ethanol extract (20 μ g/ml and 40 μ g/ml) and Trametes laclinea Berk water extract (20 μ g/ml and 40 μ g/ml), separately establish blank group and Normal group, often group arranges different pH value (pH7.5 simultaneously, pH6.5 and pH5.5), often group establishes 6 multiple holes.37 DEG C are continued to cultivate, cultivate 24h, after, every hole adds 5mg/ml MTT20 μ l, hatch 4h, discard culture fluid for 37 DEG C, every hole adds DMSO150 μ l, gentle agitation, makes crystal fully dissolve, and detects the trap (OD value) in every hole with enzyme-linked immunosorbent assay instrument at wavelength 492nm place.
By following formulae discovery inhibitory rate of cell growth: inhibitory rate of cell growth (%)=1-(dosing group mean OD value/Normal group mean OD value) × 100%.
Adopt SPSS13.0 software to carry out statistical analysis, experimental data with
represent, with P<0.05 for having significant difference.
Table 5 medicine under different PH on the impact of HGC-27 cell proliferation
With blank group than * p ﹤ 0.05, * * p ﹤ 0.01
The impact of table 6 medicine on GES-1 cell proliferation under normal pH value (PH=7.5)
Experimental result shows, the propagation of DABAI bolt ethanol extract to stomach cancer cell cell has certain inhibitory action, under sour environment, its inhibitory action strengthens greatly, low concentration 3-hydrogenation pine Siberian cocklebur acid B(10 μ g/ml, 5 μ g/ml), high concentration DABAI bolt ethanol extract (40 μ g/ml) and DABAI bolt water extract (40 μ g/ml) reach 61.4%, 81.5%, 40% and 32.9% respectively to the suppression ratio of cell after acting on 24h, and this concentration does not almost affect 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 fluid (containing 10% hyclone and penicillin, each 100U/ml of streptomycin), 37 DEG C of saturated humidities, 5%CO
2cultivate in incubator.Treat that cell enters logarithmic growth after date, through 0.25% trypsinization, collect, be deployed into corresponding cell concentration.
MTT tests: be prepared into 1 × 10
4the Eca-109 cell suspension of/ml, add in 96 orifice plates after piping and druming evenly, after cultivating 12h, be divided into blank group, positive group (20 μ g/ml mitomycin) and Trametes laclinea Berk ethanol extract, Trametes laclinea Berk water extract and 3-hydrogenation pine Siberian cocklebur acid B5 experimental group, each experiment component 4 dosage groups, add the medicine (100 of variable concentrations respectively, 80,40,20 and 10 μ g/ml), matched group adds culture fluid, continues to cultivate 48h, then every hole adds MTT (5mg/ml) 20 μ l, continues to cultivate 4h.Discard culture fluid, every hole adds DMSO150 μ l, gentle agitation, detects the trap (OD value) in every hole with enzyme-linked immunosorbent assay instrument at wavelength 492nm place.
Table 7 DABAI bolt extract is to the In-vitro Inhibitory Effect of Eca-109 cell
Result shows, the propagation of DABAI bolt extract to esophageal carcinoma Eca-109 cell line has inhibitory action.
5. Chinese People's Anti-Japanese Military and Political College's intestinal cancer Experiment on Function
The In vitro culture of cell strain: Human Large Intestine Carcinoma Cells strain LOVO is placed in DMEM culture fluid (containing 10% hyclone and penicillin, each 100U/ml of streptomycin), 37 DEG C of saturated humidities, cultivates in 5%CO2 incubator.Treat that cell enters logarithmic growth after date, through 0.25% trypsinization, collect, be deployed into corresponding cell concentration.
MTT tests: be prepared into 5 × 10
4the LOVO cell suspension of/ml, add in 96 orifice plates after piping and druming evenly, after cultivating 12h, be divided into blank group, positive group (20 μ g/ml mitomycin) and Trametes laclinea Berk ethanol extract, Trametes laclinea Berk water extract and 3-hydrogenation pine Siberian cocklebur acid B3 experimental group, each experiment component 4 dosage groups, add the medicine (100 of variable concentrations respectively, 80,40,20 and 10 μ g/ml), matched group adds culture fluid, continues to cultivate 48h, then every hole adds MTT (5mg/ml) 20 μ l, continues to cultivate 4h.Discard culture fluid, every hole adds DMSO150 μ l, gentle agitation, detects the trap (OD value) in every hole with enzyme-linked immunosorbent assay instrument at wavelength 492nm place.
Table 7 DABAI bolt extract is to the In-vitro Inhibitory Effect of LOVO cell
Result shows, the propagation of DABAI bolt extract to colorectal cancer LOVO cell has inhibitory action.
Claims (6)
1. the application of 3-hydrogenation pine Siberian cocklebur acid B in the medicine preparing prevention and therapy peptic ulcer of extracting in a Trametes laclinea Berk water extract, 95% ethanol extract or Trametes laclinea Berk.
2. the 3-hydrogenation pine Siberian cocklebur acid B extracted in a Trametes laclinea Berk water extract, 95% ethanol extract or Trametes laclinea Berk suppresses H in preparation
+, K
+application in the medicine of-ATPase activity and anti-gastric-ulcer.
3. the application of 3-hydrogenation pine Siberian cocklebur acid B in the medicine preparing prevention and therapy non-ucler dyspepsia of extracting in a Trametes laclinea Berk water extract, 95% ethanol extract or Trametes laclinea Berk.
4. the application of 3-hydrogenation pine Siberian cocklebur acid B in the medicine preparing prevention and therapy gastric cancer of extracting in a Trametes laclinea Berk water extract, 95% ethanol extract or Trametes laclinea Berk.
5. the application of 3-hydrogenation pine Siberian cocklebur acid B in the medicine preparing the prevention and therapy esophageal carcinoma of extracting in a Trametes laclinea Berk water extract, 95% ethanol extract or Trametes laclinea Berk.
6. the application of 3-hydrogenation pine Siberian cocklebur acid B in the medicine preparing prevention and therapy colorectal cancer of extracting in a Trametes laclinea Berk water extract, 95% ethanol extract or Trametes laclinea Berk.
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|---|---|---|---|
| CN201510328463.5A CN104940245B (en) | 2013-05-14 | 2013-05-14 | A kind of application of great Bai bolts bacterium water extract in pharmacy |
| CN201510328957.3A 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 |
| CN201510328462.0A CN104940244B (en) | 2013-05-14 | 2013-05-14 | A kind of application of ethanol extract of great Bai bolts bacterium 95% in pharmacy |
| 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 Division CN104983750B (en) | 2013-05-14 | 2013-05-14 | A kind of application of great Bai bolts mycelia in pharmacy |
| CN201510328463.5A Division CN104940245B (en) | 2013-05-14 | 2013-05-14 | A kind of application of great Bai bolts bacterium water extract in pharmacy |
| CN201510328957.3A Division 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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| CN104940245A (en) * | 2013-05-14 | 2015-09-30 | 三峡大学 | Application of aqueous extract of Trametes lactinea(Berk.)Pat. in pharmacy |
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| 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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| CN104940244A (en) * | 2013-05-14 | 2015-09-30 | 三峡大学 | Application of 95% alcohol extract of Trametes lactinea(Berk.)Pat. in pharmacy |
| CN104971069A (en) * | 2013-05-14 | 2015-10-14 | 三峡大学 | Use of 3-dehydrotrametenolic acid B extracted from Trametes lactinea(Berk.)Pat. in pharmacy |
| CN104971069B (en) * | 2013-05-14 | 2017-12-19 | 三峡大学 | A kind of applications of the 3 loose Siberian cocklebur acid B of hydrogenation extracted in great Bai bolts bacterium in pharmacy |
| 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 |
| CN104940244B (en) * | 2013-05-14 | 2018-03-02 | 三峡大学 | A kind of application of ethanol extract of great Bai bolts bacterium 95% in pharmacy |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104940245B (en) | 2018-03-02 |
| CN104940245A (en) | 2015-09-30 |
| CN104940244B (en) | 2018-03-02 |
| CN104983750A (en) | 2015-10-21 |
| CN104940244A (en) | 2015-09-30 |
| CN103316051A (en) | 2013-09-25 |
| CN104983750B (en) | 2018-06-26 |
| CN104971069A (en) | 2015-10-14 |
| CN104971069B (en) | 2017-12-19 |
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