CN107034253A - A kind of method that use Yunnan rough gentian endogenetic fungus carries out bioconversion - Google Patents
A kind of method that use Yunnan rough gentian endogenetic fungus carries out bioconversion Download PDFInfo
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Abstract
The present invention relates to endogenetic fungus, and the method for utilizing the Viability compound of endogenetic fungus bioconversion gentiamarin is isolated and purified by Yunnan rough gentian plant, belong to biological technical field, endogenetic fungus is stable can long term storage, biotransformation is simple, quick, and mild condition is pollution-free；Conversion and separating resulting stabilization, controllable, high conversion rate.Significant is produced to the biotechnology for expanding medicine resource.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of use Yunnan rough gentian endogenetic fungus converts the side of gentiamarin
Method, and successful conversion, isolated two compositions with liver-protecting activity.
Background technology
Rough gentian（Gentianae Radix et Rhizoma）, it is gentianaceae plant Gentiana manshuricaGentiana manshurica Kitag, rough gentianG. scabra Bge., G. trifloraG. triflora Or Yunnan rough gentian Pall.G. rigescens Franch. drying root and rhizome.First recorded in《Sheng Nong's herbal classic》, middle product are classified as, are the conventional of tcm clinical practice
Chinese medicine, bitter in taste, cold, Return liver gallbladder channel, can heat-clearing and damp-drying drug, purging the liver of pathogenic fire courage fire, for jaundice with damp-heat pathogen, swelling of vulva pruritus vulvae, under band, eczema scabies
Itch, irascibility hot eyes, Hiccough and deaf, hypochondriac pain bitter taste, persistent erection, convulsion.Wherein, Yunnan rough gentian alias Gentiana rigescens, southern rough gentian, indigo plant
Hua Gen, felwort, snow mountain eel grass etc., are Yunnan genunie medicinal materials, Yunnan Yi nationality medicine principal item is numerous enterprises production andrographis paniculata again
The important source material of piece, Longdanxiegan piece etc., is the good medicine of hepatic cholagogic.
Main active in rough gentian is gentiamarin（gentiopicroside）, Swertiamarin
（swertiamarin）, sweroside（sweroside）Deng, wherein, gentiamarin is as the quality control index of rough gentian
Composition, be《Chinese Pharmacopoeia》（Version in 2015）Recorded.Modern pharmacology experiment finds that these active components are respectively provided with significant guarantor
Liver choleretic effect.Gentiamarin can substantially protect CCl4Caused hepatic injury, reduces the impaired release of paddy the third ammonia transaminase,
Gentiamarin can also substantially increase the concentration of bile flow and bilirubin simultaneously, with notable liver protection, cholagogic, anti-inflammatory, analgesia
Deng pharmacological action.Such compound is pro-drug, and after gut flora or liver metabolism, pharmacophore is produced in vivo, because
And there is obvious drug effect in whole animal level（Activity）, in vitro in the level of cell or enzyme, biology is not shown but
Activity.
Endogenetic fungus（endophytic fungi）, refer to that those a certain periods in its history of life live in plant group
In knitting, the fungi of obvious Disease symptoms is not caused to host plant.According to " endosymbiotic theory " theory, endogenetic fungus and host
There is the exchange of inhereditary material in long-term evolutionary process, with host plant coevolution.The research of nearly twenty or thirty year is found, interior
Raw fungi is almost present in all plants studied, with abundant species diversity, participates in coordinate plant growth, is to plant
The natural resources of thing biological control of diseases；Secondary metabolite enriches, and can produce and the same or analogous composition of host, research
Have a extensive future.Thus speculate, the endogenetic fungus in the rough gentian of Yunnan can assist host to synthesize analog necessary to some host metabolisms
Or precursor compound, it is the resources domain with great potential.
Microorganism conversion, is to carry out structural modification and transformation to xenobiotic substrates by microbial cell or enzyme system, and is obtained
The biochemical reactions of valuable product.Microorganism conversion is the treasure-house of the huge new native compound structure of acquisition, can be with
Effectively protect medicine resource, low-carbon environment-friendly.
In view of endogenetic fungus and host's coevolution, may take part in the synthesis of secondary metabolite, host's synthesis is assisted
There is abundant enzyme system in analog necessary to some host metabolisms or precursor substance, and endogenetic fungus, its progress is utilized
Bioconversion can obtain valuable derivative, and we convert gentiamarin using Yunnan rough gentian endogenetic fungus, and obtain 1 pair
Active metabolite.
The content of the invention
The technical problem to be solved in the present invention be use endogenetic fungus successful conversion gentiamarin,
The present invention relates to be following 1 pair of active metabolite by gentiamarin conversion in substrate：
5α-(hydroxymethyl)-6β-methyl-1H,3H-5,6-dihydropyrano[3,4-c]pyran-1(3H)-
one（Compound A）；
With 5β-(hydroxymethyl)-6β-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one（Change
Compound B）Method.
The application for the composition for treating hepatic injury is being prepared the present invention relates to compound A.
The application for the composition for treating hepatic injury is being prepared the present invention relates to compound B.
The application of oxidation resistant composition is being prepared the present invention relates to compound A.
The application of oxidation resistant composition is being prepared the present invention relates to compound B.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
One plant of Yunnan rough gentian endogenetic fungus and its method for converting gentiamarin：It is characterized in that：Turn for endogenetic fungus direct fermentation
It is prepared by change method, including culture, fermentation, the addition of substrate and the product of endogenetic fungus.
The endogenetic fungus is one plant of endogenetic fungus 1-10 isolated from the rough gentian of Yunnan, is accredited as through 18S rDNAFusarium oxysporum；
The bioconversion fermentations liquid is PDA liquid fermentation mediums：Potato 200g, glucose 5g, 1000ml is settled to after boiling,
pH7.0；
The method that rough gentian endogenetic fungus in Yunnan of the present invention converts gentiamarin, is comprised the following specific steps that：
A) separation of Yunnan rough gentian endogenetic fungus
1. from the root of Yunnan rough gentian Fresh Plants, stem, leaf, spend middle separation endogenetic fungus：Washing, 75% is respectively adopted in Fresh Plants
The mode of alcohol-pickled and 5% sodium hypochlorite immersion is sterilized, and is inoculated in PDA solid mediums, is trained in 28 DEG C of constant incubators
Support, until growing fungus colony；
2. control group is set up in separation process, the plant disinfected is contacted in being rolled on blank cultures, and culture dish is same
Sample is placed in insulating box and cultivated, and bacterium colony generation has been seen whether, to ensure that endogenetic fungus is separated successfully；
3. isolated endogenetic fungus is purified repeatedly.
B) screening of bioconversion activity：It is bitter that its host's active component rough gentian is converted using isolated endogenetic fungus
Glycosides, and set up only fermentation simultaneously and be added without substrate, and only add without bacterium the control group of substrate.
1. ferment：Bacterial strain is fermented respectively, shaking table condition is 150rpm, 28 DEG C；
2. substrate is added：Fermentation two days, when mycelial growth is vigorous in fermentation flask, adds gentiamarin and continues shaking table culture；
3. fermentation is terminated：Continue to co-culture 7 days, after substrate is converted substantially, add double amount methanol terminating reaction, and carry out Q-
TOF-MS is detected.
C) bioconversion amplification test
Obtained aimed strain progress bulk fermentation will be screened, and increase the input amount of substrate.
D) converted product is separated
1. the sample that bioconversion is completed is filtered, obtains filtered solution, it is anti-in three times with isometric water-saturated n-butanol
Multiple extraction, combining extraction liquid is concentrated under reduced pressure and volatilizes n-butanol.
2. it is residue obtained dissolved with methanol after, cross MCI posts（60 cm, 50g of MCI gel）Chromatographic purifying, eluant, eluent according to
Secondary use water, MeOH-H2O（50/50,80/20, v/v）Gradient elution.
3. MeOH-H is collected2O（50/50,80/20, v/v）Eluent, is recovered under reduced pressure solvent.
Residue methanol redissolves, inverted high performance liquid preparative chromatography, obtains product.
E) product analysis and Structural Identification
Compound A's and compound B1H（400 MHz）、13C（100 MHz）NMR is as follows：
Structural Identification is：
Compound A compounds B
Separate the preferred process of the method for converted product：
A) preferred process of MCI posts purifying：
Eluant, eluent uses water, MeOH-H successively2O（10/90th, 50/50,90/10, v/v）Gradient elution（Each 2-3 times of column volume）；
Eluant, eluent uses water, MeOH-H successively2O（20/80th, 50/50,80/20, v/v）Gradient elution（Each 2-3 times of column volume）；
Detect, found through water, MeOH-H through HPLC2O（50/50,80/20, v/v）Elution（Each 2-3 times of column volume）, target production
Thing is concentrated mainly in this two parts eluent, therefore uses this elution requirement.
B) preferred process prepared by RP-HPLC：
Mobile phase A is pure water, and Mobile phase B is acetonitrile, and gradient condition is set to：
A：B=11%：89%, the time is 30min
A：B=8%：92%, the time is 30min
Through investigating, gradient is A：B=8%：92%, the time is 30min, is relatively adapted to compound A/B preparation.
Compound A and compound B retention times in anti-phase preparation liquid phase are respectively about 15min and 18min.
The present invention has the advantages that：
1) endogenetic fungus is easy to culture, and fermentation process is easily controllable.2) conversion process is pollution-free, controllable, safe and environment-friendly.3) turn
Rate is up to 1-2.5%.
The preparation method of liver protection compound of the present invention, is that a large amount of higher liver protection compounds of purity that prepare are used to grind
Study carefully its physiologically active and mechanism of action provides premise, with application prospect.
Specific embodiment
The one plant of endogenetic fungus isolated from the rough gentian of Yunnan of embodiment 1Fusarium oxysporum SchlSide
Method
Wild Yunnan rough gentian intact plant is gathered from Lincang, is transported in 48 hours in Shanghai Univ. of Traditional Chinese Medicine's laboratory progress
Raw fungi separation.
Yunnan rough gentian endogenetic fungus separation：Whole plant is disposed into soil, carefully cleaned up, is divided into root, stem, leaf, flower
Position, rinses 1 hour under flowing running water, (20~30s) → aseptic water washing is rinsed with 75% ethanol in aseptic operating platform
4 times → 5%NaClO solution rinse 5min) → aseptic water washing 4 times → 75% ethanol rinsing (20~30s) → aseptic water washing
4 times.Aseptic filter paper piece suck dry moisture, is cut into the mm of the mm of 5 mm × 5 × 1 fritter, is inoculated on PDA solid mediums, each
4 pieces are inoculated with culture dish, 3~30 d of culture in 28 DEG C of constant incubators are put, it was observed that on culture medium out of each plant tissue block
Portion to surrounding grow mycelia when, using Tip Splitting picking method fungi be transferred on new culture medium continue cultivate, switching, directly
To Economical Purification.
Control group is set in separation process, is rolled using the tissue block through surface sterilization on blank cultures, makes tissue
Each surface of block touches culture dish, is cultivated together with the culture dish being inoculated with, and does not grow bacterium colony on control culture dish, illustrates surface
Sterilization is thorough.
The culture medium that separation, purifying are used is PDA solid medium：Potato 200g, glucose 20g, agar 18g, after boiling
It is settled to 1000ml, pH7.0.
By above step, the isolated bacterial strain from the fresh leaf of Yunnan rough gentianFusarium oxysporum Schl。
The method that the Yunnan rough gentian endogenetic fungus of embodiment 2 converts gentiamarin
Bacterial strain 1-10 is inoculated with aseptic operating platform into PDA liquid fermentation mediums, cultivated in 28 DEG C, 150rpm.Treat that 48 is small
When fungal spore is abundant in Shi Hou, conical flask, substrate gentiamarin is added, 28 DEG C, 150rpm cultures is continued at.
Gentiamarin addition is 100mg/L.
PDA liquid fermentation mediums：Potato 200g, glucose 5g, are settled to 1000ml, pH7.0 after boiling.250ml tapers
It is bottled enter 100ml culture mediums.
The method of the Q-TOF-MS of embodiment 3 detections
Use Waters ACQUITYTM Synapt G2 systems.
Chromatographic process
Chromatographic column uses 1.8 μm of Waters HSS T3,2.1*50mm, 45 DEG C of column temperature.
Mobile phase A is the 0.1% FA aqueous solution（v/v）,
Mobile phase B is acetonitrile, and flow velocity is 0.4 ml/min.
Using gradient elution, method is as follows：
| Time（min） | 0 | 5 | 9 | 13 | 14 | 16 | 17 |
| A% | 98 | 92 | 82 | 72 | 10 | 10 | 98 |
| B% | 2 | 8 | 18 | 28 | 90 | 90 | 2 |
Mass spectrometry method
Capillary voltage is 1.5kV；Sampling taper hole voltage is 45V；Extraction taper hole voltage is 4V；Ion source temperature is 120 DEG C；It is dry
Pathogenic dryness temperature is 400 DEG C, and flow velocity is 800L/h；Taper hole gas velocity is 50L/h.
Compound A and compound the B retention time in UPLC are respectively about 6.9min and 7.1min.
The method of the bioconversion amplification test of embodiment 4
By bacterial strainFusarium oxysporum SchlIt is inoculated with aseptic operating platform into PDA liquid fermentation mediums, in 28
DEG C, 150rpm culture.After after 48 hours, when fungal spore is abundant in conical flask, substrate gentiamarin is added, 28 are continued at
DEG C, 150rpm cultivate 7 days.
PDA liquid fermentation mediums：Potato 800g, glucose 20g, are settled to 4000ml, pH7.0 after boiling.250ml tapers
It is bottled enter 100ml culture mediums.
40 bottles of fermentation cultures altogether, ferment and add gentiamarin substrate 1000mg respectively.
The method that embodiment 5 separates converted product
Extraction：By the nutrient solution filtering in conical flask, merge nutrient solution, nutrient solution extracted with the water-saturated n-butanol of triplication,
Merge organic phase, organic phase is recovered under reduced pressure, residue is obtained.
MCI posts are purified：By residue（800 mg）With water and methanol dissolving, ultrasonic, upper MCI posts（60 cm, filler containing MCI is about
50g）.Methanol elutes 2-3 column volume, and water, MeOH-H are then used successively2O（50/50,80/20, v/v）Elution（Each 2-3 times of post
Volume）, collect MeOH-H2O（50/50,80/20, v/v）Eluent, LC analysis result identicals component is merged, depressurized back
Receive.
It is prepared by RP-HPLC：Residue sample methanol is redissolved, filtered, be prepared by upper RPLC.
Chromatographic column：YMC-PA is exported, ODS-A, 10 μm, 250*20mm.
Mobile phase：A is pure water,
B is acetonitrile,
Gradient：A：B=8%：92%,
Time is 30min,
Compound A and compound B monomer compound are prepared, is brown ceramic powder, respectively 25mg, 10mg.
Compound A and compound B retention times in anti-phase preparation liquid phase are respectively about 15min and 18min.
The liver-protecting activity of embodiment 6
1. protective effect of the compound to the human liver cell damage of hydrogen peroxide-induced
1.1 experiment materials, medicine and instrument
Material：Normal liver cell system (HL-7702) is purchased from Chinese Academy of Sciences's Shanghai cell bank.With containing 10% hyclone
RPMI-1640 medium cultures.Every milliliter of culture medium contains 100 units of Penicillin and 100 unit streptomysins, and all cells are equal
Containing 5%CO237 DEG C of incubators in cultivate, per 3-4 days pass on once；CCK-8 kits.
Instrument：Flow cytometer, full-automatic ELIASA, CO2 incubators, 96 well culture plates.
1.2 given the test agent：Compound A, compound B
1.3 CCK-8 methods evaluate the vigor of liver cell
Take the logarithm growth period cell, after 0.25% Trypsin Induced, suspended again with the DMEM solution containing 10%FBS, carried out
Cell count, will be diluted to the suspension inoculation containing 5 × 106/L HL-7702 cell in 96 well culture plates, the inoculation per hole
5000, treat that cell grows up to subgrade, you can for testing, selection grows up to the cell of exponential phase, changes original fluid, plus
Enter each metabolite of various concentrations, 37 DEG C, 50 mL/L CO2Under the conditions of cultivate 24 h, then add content be 2.0 mM
H2O2, 100 μ L/ holes cultivate 1 h, cause H2O2Damage model.Add CCK-8 and be incubated 2 h, with enzyme linked immunological instrument in wavelength 450
OD values are surveyed at nm.Each drug concentration repeats to survey 5 times.
Cell Viability %=(medicine group A_450)/(ModeA_450) × 100%
Protection result of 1.4 compounds to damage liver cell
（1）In experimentation, 3 concentration of compound have been done respectively（5、10、20 uM）Bioactivity, compound A is in 20 uM
When have protective effect to the HL-7702 cells of damage, compared with model group, with significant difference（P＜ 0.01）；Compound B
There is protective effect to the HL-7702 cells of damage in 5 uM, compared with model group, with significant difference（P＜ 0.01）.
The suppression for the Hepatic Stellate Cell Activation that compound is induced TGF-β 1
2.1 experiment materials, medicine and instrument
Material：Human liver microsome proteins system (LX-2) is purchased from Chinese Academy of Sciences's Shanghai cell bank；Every milliliter of culture medium is single containing 100
Position penicillin and 100 unit streptomysins, all cells are containing 5%CO237 DEG C of incubators in cultivate, per 3-4 days pass on once.
Medicine：Compound A, compound B
Instrument：Flow cytometer, full-automatic ELIASA, CO2Incubator, 96 well culture plates.
2.2 mtt assay evaluate the propagation of HSCs (LX-2 cells)
Take the logarithm growth period cell, after 0.25% Trypsin Induced, suspended again with the DMEM solution containing 10%FBS, carried out
Cell count, will be diluted to the suspension inoculation containing 5 × 106/L LX-2 cell in 96 well culture plates, the inoculation 4000 per hole
It is individual, treat that cell grows up to subgrade, you can for testing, selection grows up to the cell of exponential phase, changes original fluid, Ran Houjia
Enter each metabolite of various concentrations while adding TGF-β 1 2.5 ng/mL, 37oC, 50 mL/L CO2Under the conditions of cultivate 24
H, after incubation terminates, adds 1000 rpm/min after MTT solution, 4 h and centrifuges 15 min, abandon supernatant, it is molten to add DMSO per hole
The μ L min of constant temperature 20 at 37 DEG C of liquid 100, OD values are surveyed in enzyme linked immunological instrument at the nm of wavelength 490.Each drug concentration is repeated
Survey 5 times.
Inhibition of Cell Viability % =(【"ModeA" 】_ " 490 " "-medicine group " A_490)/("
Mode" "A" _"490" )×100%
Suppression of 2.3 compounds to activated hepatic stellate cells
Compound A has certain inhibitory action when concentration is 10 μ g/mL to the HSCs of activation, compared with model group, tool
There is significant difference（P＜ 0.01）；Compound B has certain suppression to make the HSCs of activation when concentration is 20 μ g/mL
With compared with model group, with significant difference（P＜ 0.01）.
Conclusion：Two products A and B, which are shown, protective effect to the HL-7702 cells of damage, and starlike to the liver of activation
The inhibitory action of cell, and their substrate does not have inhibitory action to the HSCs of activation.
The preparation of the compound A of embodiment 7 tablet
Prescription：Compound A1mg, starch 94mg, magnesium stearate 5mg.
Preparation technology：Compound A is taken to sieve, plus starch, magnesium stearate are well mixed, and particle is made, and dry, tabletting, i.e.,
.
The preparation of the compound B of embodiment 8 tablet
Prescription：Compound B-11 mg, starch 94mg, magnesium stearate 5mg.
Preparation technology：Compound B is taken to sieve, plus starch, magnesium stearate are well mixed, and particle is made, and dry, tabletting, i.e.,
.
Finally be necessary described herein be：The above is served only for being described in further detail technical scheme,
It is not intended that limiting the scope of the invention, those skilled in the art made according to the above of the present invention one
A little nonessential modifications and adaptations belong to protection scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Univ. of Traditional Chinese Medicine
<120>A kind of method that use Yunnan rough gentian endogenetic fungus carries out bioconversion
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1085
<212> DNA
<213> Fusarium oxysporum
<400> 1
taccccgacc tgcgaatggc tcattatata agttatcgtt tatttgaaaa aggatactac 60
ttggataacc gtggtaattc tagagctaat acatgctaaa aatcccgact tcggaaggga 120
tgtatttatt agattaaaaa ccaatgccct tcggggctca ctggtgattc atgataactc 180
ctcgaatcgc atggccttgt gccggcgatg gttcattcaa atttcttccc tatcaacttt 240
cgatgtttgg gtattggcca aacatggttg caacgggtaa cggagggtta gggctcgacc 300
ccggagaagg agcctgagaa acggctacta catccaagga aggcagcagg cgcgcaaatt 360
acccaatccc gacacgggga ggtagtgaca ataaatactg ttttagggct cttttgggtc 420
ttgtaattgg aatgagtaca atttaaatcc cttaacgagg aacaattgga gggcaagtct 480
ggtgccagca gccgcggtaa ttccagctcc aatagcgtat attaaagttg ttgtggttaa 540
aaagctcgta gttgaacctt gggcctggct ggccggtccg cctcaccgcg tgtactggtc 600
cggccgggcc tttccctctg tggaacccca tgcccttcac tgggtgtggc ggggaaacag 660
gacttttact gtgaaaaaat tagagtgctc caggcaggcc tatgctcgaa tacattagca 720
tggaataata gaataggacg tgtggttcta ttttgttggt ttctaggacc gccgtaatga 780
ttaataggga cagtcggggg catcagtatt caattgtcag aggtgaaaat tcttggattt 840
aatggaaact aactactgcg aaaagccatt tgccaaggat gttttcatta atcaggaacg 900
aaagttaggg gatcgaaaac gatcagatac cgtcgtagtc taaccataaa ctatgccgac 960
tagggatcgg acggtgttaa tttttgaccc gtttcggctc ccttacgaga aaatcaaaat
1020
gccttggctt ccaggggaag ttaggttccg caggctgaaa ccttcatgaa ttgacgaaac
1080
gcact 1085
Claims (6)
1. the method that one plant of Yunnan rough gentian endogenetic fungus converts gentiamarin：It is characterized in that：
It is prepared by culture, fermentation, the addition of substrate and product for endogenetic fungus direct fermentation conversion method, including endogenetic fungus；
The endogenetic fungus is one plant of endogenetic fungus isolated from the rough gentian of Yunnan, is accredited as through 18S rDNAFusarium oxysporumSchl；
The bioconversion fermentations liquid is PDA liquid fermentation mediums：Potato 200g, glucose 5g, 1000ml is settled to after boiling,
pH7.0。
2. the method that one plant of Yunnan rough gentian endogenetic fungus converts gentiamarin：It is characterized in that：
A) separation of Yunnan rough gentian endogenetic fungus；
1. from the root of Yunnan rough gentian Fresh Plants, stem, leaf, spend middle separation endogenetic fungus：Washing, 75% is respectively adopted in Fresh Plants
The mode of alcohol-pickled and 5% sodium hypochlorite immersion is sterilized, and is inoculated in PDA solid mediums, is trained in 28 DEG C of constant incubators
Support, until growing fungus colony；
2. control group is set up in separation process, the plant disinfected is contacted in being rolled on blank cultures, and culture dish is same
Sample is placed in insulating box and cultivated, and bacterium colony generation has been seen whether, to ensure that endogenetic fungus is separated successfully；
3. isolated endogenetic fungus is purified repeatedly；
B) screening of bioconversion activity：Its host's active component gentiamarin is converted using isolated endogenetic fungus, and
Only fermentation is set up simultaneously and substrate is added without, and only adds without bacterium the control group of substrate；
1. ferment：Bacterial strain is fermented respectively, shaking table condition is 150rpm, 28 DEG C；
2. substrate is added：Fermentation two days, when mycelial growth is vigorous in fermentation flask, adds gentiamarin and continues shaking table culture；
3. fermentation is terminated：Continue to co-culture 7 days, after substrate is converted substantially, add double amount methanol terminating reaction, and carry out Q-
TOF-MS is detected；
C) bioconversion amplification test
Obtained aimed strain progress bulk fermentation will be screened, and increase the input amount of substrate；
D) converted product is separated
1. the sample that bioconversion is completed is filtered, obtains filtered solution, it is anti-in three times with isometric water-saturated n-butanol
Multiple extraction, combining extraction liquid is concentrated under reduced pressure and volatilizes n-butanol；
2. it is residue obtained dissolved with methanol after, cross MCI posts, 60 cm, 50g of MCI gel, chromatographic purifying, eluant, eluent uses successively
Water, MeOH-H2O,
50/50,
80/20,
V/v,
Gradient elution；
3. collect
MeOH-H2O
50/50,
80/20,
v/v
Eluent, is recovered under reduced pressure solvent；
Residue methanol redissolves, inverted high performance liquid preparative chromatography, obtains product.
3. compound A is in the application for the composition for preparing treatment hepatic injury.
4. compound B is in the application for the composition for preparing treatment hepatic injury.
5. compound A is preparing the application of oxidation resistant composition.
6. compound B is preparing the application of oxidation resistant composition.
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| CN108611275A (en) * | 2018-05-16 | 2018-10-02 | 四川农业大学 | The antioxidant activity of pale reddish brown jade hairpin endophytic fungal hypha alcohol extract and its application |
| CN110859786A (en) * | 2019-11-14 | 2020-03-06 | 广州伽能生物科技有限公司 | Preparation method and application of anti-irritant factor of discolored gentian |
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| CN103289906A (en) * | 2013-05-23 | 2013-09-11 | 都晓伟 | Gentiana manshurica kitag endophytic fungi and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108611275A (en) * | 2018-05-16 | 2018-10-02 | 四川农业大学 | The antioxidant activity of pale reddish brown jade hairpin endophytic fungal hypha alcohol extract and its application |
| CN110859786A (en) * | 2019-11-14 | 2020-03-06 | 广州伽能生物科技有限公司 | Preparation method and application of anti-irritant factor of discolored gentian |
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