CN109180635A - The tunning and its acetic acid ethyl acetate extract of compound E1011 and the preparation method and application thereof, potato endogenetic fungus - Google Patents
The tunning and its acetic acid ethyl acetate extract of compound E1011 and the preparation method and application thereof, potato endogenetic fungus Download PDFInfo
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Abstract
The present invention relates to a kind of compound E1011 and the preparation method and application thereof, the tunning and its acetic acid ethyl acetate extract of potato endogenetic fungus, belong to chemical field.The chemical structural formula of compound E1011 is
Description
Technical field
The present invention relates to chemical fields, and in particular to a kind of compound E1011 and the preparation method and application thereof, horse
The tunning and its acetic acid ethyl acetate extract of bell potato endogenetic fungus.
Background technique
Potato (Solanum tuberosum L.) is also known as potato, potato, foreign sweet potato etc., belongs to Solanaceae perennial herb and plants
Object, stem tuber can be edible, are the fourth-largest important cereal crops in the whole world, are only second to wheat, paddy and corn.
Chinese medicine thinks potato " sweet natured nontoxic, energy strengthening the spleen and stomach, tonifying qi and regulating middle energizer, relieving spasm to stop pain, tonneau stool.It is right
Weakness of the spleen and the stomach, indigestion, gastrointestinal disharmony, stomach duct and abdomen are had a pain, the patient outcome of inhibited defecation is significant ".Modern study proves that Ma Ling
Potato has special efficacy to indigestion is reconciled, and is the good medicine and high-quality health care product of stomach trouble and cardiac.Also, potato is rich in battalion
It supports, is one of food of anti-aging.
Domestic and foreign scholars achieve certain progress to the chemical component of potato and its application study at present, but also need pair
Potato is furtherd investigate.
Summary of the invention
One of the objects of the present invention is to provide a kind of compound E1011, the chemical structural formula of compound E1011 is
The second object of the present invention is to provide the preparation method of compound E1011 a kind of, and this method is simple, easy to operate,
The higher compound E1011 of purity can effectively be prepared.
The third object of the present invention is to provide a kind of potato endogenetic fungus Xylaria for being prepared as described above method and obtaining
The tunning of curta.
The fourth object of the present invention is to provide a kind of potato endogenetic fungus Xylaria for being prepared as described above method and obtaining
The acetic acid ethyl acetate extract of curta tunning.
The fifth object of the present invention is to provide a kind of above compound E1011, bell potato endogenetic fungus Xylaria curta
The acetic acid ethyl acetate extract of tunning or potato endogenetic fungus Xylaria curta tunning is in treatment leukaemia, liver
Application in cancer or colon cancer.
The present invention solves its technical problem and adopts the following technical solutions to realize:
The present invention proposes a kind of compound E1011, the chemical structural formula of compound E1011 are as follows:
The present invention proposes the preparation method of compound E1011 a kind of, comprising the following steps:
Fermenting potato endogenetic fungus Xylaria curta, extracts tunning with ethyl acetate, to acetic acid second
Ester extract liquor carries out silica gel post separation；To standby chromatographic isolation is suppressed in the 4th elution fraction progress, it is sub- to collect the 4th elution
Component carries out the preparation of high pressure liquid phase, collects the highest chromatographic peak component of content, dry.
Wherein, the condition of silica gel post separation includes: using chloroform and methanol as eluent, is 100:0,50 by concentration gradient:
The ratio of 1:20:1,10:1 and 5:1 are eluted, and five elution fractions are obtained.
The middle condition for suppressing standby chromatographic isolation includes: using first alcohol and water as eluent, is 50:50,60 by concentration gradient:
The ratio of 40:70:30,80:20 and 90:10 are eluted, and five elution subfractions are obtained.
The condition of high pressure liquid phase preparation includes: using acetonitrile and water as eluent, is 70 by the initial concentration of acetonitrile and water:
30, ultimate density is that 85:10 carries out gradient elution 40min.
The invention also provides the hairs of potato endogenetic fungus Xylaria curta that above-mentioned preparation method obtains a kind of
The acetic acid ethyl acetate extract of ferment product and the tunning.
The invention also provides the fermentations of a kind of above compound E1011, potato endogenetic fungus Xylaria curta to produce
Application of the acetic acid ethyl acetate extract of object or tunning in treatment leukaemia, liver cancer or colon cancer.
Compound E1011 provided by the present application and the preparation method and application thereof, potato endogenetic fungus tunning and
The beneficial effect of its acetic acid ethyl acetate extract includes:
The raw material of compound E1011 provided by the present application is natural products, and green safe, toxic side effect is small.On and
Stating compound E1011 is novel substance isolated from potato endogenetic fungus for the first time.Preparation method is simple, easy to operate,
The higher compound E1011 of purity can effectively be prepared.Compound E1011 and during the preparation process obtained potato
The tunning of endogenetic fungus Xylaria curta and the acetic acid ethyl acetate extract of tunning are to leukaemia, liver cancer or colon
Cancer all has certain therapeutic effect.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Below to the tunning of compound E1011 of the application and the preparation method and application thereof, potato endogenetic fungus
And its acetic acid ethyl acetate extract is specifically described.
Compound E1011 provided by the present application is separated from the tunning of potato endogenetic fungus Xylaria curta
It obtains, the chemical structural formula of compound E1011 is
Specifically, potato endogenetic fungus Xylaria curta can be obtained through following manner: using the place of production as Yunnan Province Lincang
City, the potato that kind is cloud potato 505 are raw material, surface sterilization, then by potato root, stem, the leaf fritter point after disinfection
It is not inoculated in added on the solid medium of penicillin and streptomysin mixed liquor, every plate 1-2 block, number consecutively is placed in 22-25 DEG C
It is cultivated in constant incubator.Daily routine observation, when observing each different organization internal of potato to four perimeter of culture medium
Out when macroscopic bacterium colony, according to colonial morphology, color and the time is grown, the apparent single colonie of picking difference carries out purifying training
It supports.
The above-mentioned apparent single colonie of difference includes but are not limited to have the feature that mycelia as white, and bacterium colony center is
Dark brown, it is sparse, at radial growth.
The condition for purifying culture is as follows: on picking colony forward position white hypha to fresh cultured plate, being repeated 3 times, general
The form of light passing microscopically observation mycelia purifies success if form is uniform.
Potato endogenetic fungus Xylaria curta after purifying culture has but is not limited only to following Physiology and biochemistry spy
Sign: being grown in bacterium colony center in PDA culture medium is dark brown, is around the radial mycelia of white, and later period bacterium produces pigment,
Making the radial mycelia of white becomes dark brown.Being grown in bacterium colony mycelia on oat medium is white, sparse, radial,
Produce minute quantity pigment.
Can refer to ground, the preparation method of compound E1011 the following steps are included:
Fermenting potato endogenetic fungus Xylaria curta, extracts tunning with ethyl acetate, to acetic acid second
Ester extract liquor carries out silica gel post separation；To standby chromatographic isolation is suppressed in the 4th elution fraction progress, it is sub- to collect the 4th elution
Component carries out the preparation of high pressure liquid phase, collects the highest chromatographic peak component of content, dry.
In this application, potato endogenetic fungus Xylaria curta first can be inoculated in the seeding tank of 15L the 5- that ferments
7 days, it is then transferred to the fermentation cylinder for fermentation culture of 100L 18-22 days again.It is worth noting that above-mentioned seeding tank and fermentor
Volume be not limited to 15L and 100L, can carry out according to actual needs correspondence expand or shrink.
Optionally, the inoculum concentration of potato endogenetic fungus Xylaria curta can be 8-12vt%, such as
8vt%, 9vt%, 10vt%, 11vt% or 12vt%, or any numerical value between above-mentioned each inoculum concentration point value, such as
8.5vt%, 9.5vt%, 10.5vt% or 11.5vt%.In some preferred embodiments, potato endogenetic fungus
The inoculum concentration of Xylaria curta is 10vt%.
In some specific embodiments, culture medium used in fermentation process contains the grape of 4.8-5.2wt%
Sugar, the pork protein peptone of 0.12-0.18wt%, the yeast powder of 0.45-0.55wt%, 0.045-0.055wt% KH2PO4And
The MgSO of 0.045-0.055wt%4.The culture medium of each nutriment containing above-mentioned content is conducive to potato endogenetic fungus
Xylaria curta growth metabolism obtains target product compound E1011.
In some embodiments of the application, fermentation can be 5.9-6.1 in 24-26 DEG C, pH value and revolving speed is
It is carried out under conditions of 220-280r/min.In some preferred embodiments, fermenting in 25 DEG C, pH value is 6.0 and revolving speed
To be carried out under conditions of 250r/min.
Can be 0.8-1.2vvm with ventilatory capacity in fermentation process, for example, 0.8vvm, 0.9vvm, 1.0vvm, 1.1vvm or
1.2vvm is passed through air into fermentation system, and wherein vvm represents ventilation ratio, i.e. minute ventilation volume and the practical material liquid volume of tank body
Ratio.
It is worth noting that the tunning of the application meaning is without mycelial fermentation liquid.
Before extractive fermentation product, can also first centrifugal filtration contain mycelial fermentation liquid, obtain mycelium and filtrate (hair
Ferment product).Mycelium is impregnated with organic reagent, obtains organic reagent soak.Impregnating number can be 1 time, or repeatedly,
Such as 3 times.
In some preferred embodiments, organic reagent is acetone, with preferable penetrating power, can make Ma Ling
It is not discharged into extracellular secondary metabolite into the cell and to the greatest extent may be used by the membranolysis of potato endogenetic fungus Xylaria curta
The release of energy improves the yield of final compound E1011.
Then organic reagent (acetone) soak and acetic acid ethyl acetate extract are mixed and successively carries out silica gel post separation, middle pressure
Prepare chromatographic isolation and the preparation of high pressure liquid phase.
It is worth noting that before separation, organic reagent (acetone) soak and acetic acid ethyl acetate extract can be concentrated to give
To medicinal extract, medicinal extract and silica gel materials are mixed with the ratio of 1:2.5-3.5 (preferably 1:3) and mix sample, then carry out silicagel column point again
From.
Silica gel post separation is original with the column layer chromatography silicone rubber (200-300 mesh) of the sale of Qingdao Marine Chemical Co., Ltd.
Material.The condition of silica gel post separation includes: using chloroform and methanol as eluent, is 100:0,50:1:20:1,10:1 by concentration gradient
And the ratio of 5:1 is eluted, the elution volume of each gradient is 3L, obtain five elution fractions namely component A, component B,
Component C, component D and component E.
To suppress standby chromatographic isolation in the progress of above-mentioned 4th elution fraction (component D), middle suppress can be used for chromatographic column
Common high voltage bearing reversed silicagel column.The middle condition for suppressing standby chromatographic isolation includes: using first alcohol and water as eluent, by concentration
Gradient is that the ratio of 50:50,60:40:70:30,80:20 and 90:10 are eluted, and elution time amounts to 60min, obtains five
A elution subfraction namely subfraction a, subfraction b, subfraction c, subfraction d and subfraction e.
With above-mentioned 4th elution subfraction, (subfraction d) carries out the preparation of high pressure liquid phase, the condition packet of high pressure liquid phase preparation
It includes: being 70:30 by the initial concentration of acetonitrile and water using acetonitrile and water as eluent, ultimate density is that 85:10 carries out gradient elution
40min collects the highest chromatographic peak component of content, drying to obtain compound E1011.
In addition, present invention also provides a kind of above compound E1011, bell potato endogenetic fungus Xylaria curta fermentations
The application of the acetic acid ethyl acetate extract of product or potato endogenetic fungus Xylaria curta tunning, such as can be used
In treatment leukaemia, liver cancer or colon cancer.
With can refer to, in above compound E1011, bell potato endogenetic fungus Xylaria curta tunning or potato
The acetic acid ethyl acetate extract of raw fungi Xylaria curta tunning can be used for inhibiting leukemia HL-60 or liver cancer SMMC-
The growth of 7721 or colon cancer SW480.
Embodiment 1
It with inoculum concentration is that 10vt% is inoculated in the seeding tank of 15L and ferments 6 by potato endogenetic fungus Xylaria curta
It, is then transferred to the fermentation cylinder for fermentation culture of 100L 20 days again, obtains containing mycelial fermentation liquid.Culture medium contains 5.0wt%
Glucose, the pork protein peptone of 0.15wt%, the yeast powder of 0.5wt%, 0.05wt% KH2PO4And 0.05wt% culture
In base.It ferments and is carried out under conditions of 25 DEG C, pH value are 6.0 and revolving speed is 250r/min, be with ventilatory capacity in fermentation process
1.0vvm is passed through air into fermentation system.
Centrifugal filtration is above-mentioned to contain mycelial fermentation liquid, obtains mycelium and filtrate (tunning).With acetone soak bacterium
Filament 3 times, collect 3 resulting acetone soak liquid.Filtrate (tunning) is extracted 3 times with ethyl acetate, collects 3 gained
Acetic acid ethyl acetate extract.Above-mentioned acetone soak liquid and acetic acid ethyl acetate extract are mixed, medicinal extract is concentrated to get.
Medicinal extract and silica gel materials are mixed with the ratio of 1:3 mixes sample, using chloroform and methanol as eluent, is by concentration gradient
The ratio of 100:0,50:1:20:1,10:1 and 5:1 carry out the elution of silica gel post separation, and the elution volume of each gradient is 3L, obtain
To five elution fractions: component A, component B, component C, component D and component E.
Said components D is collected, is 50:50,60:40:70:30,80:20 by concentration gradient using first alcohol and water as eluent
And suppress standby chromatographic isolation in the ratio progress of 90:10 and elute, elution time amounts to 60min, obtains five elution subfractions:
Subfraction a, subfraction b, subfraction c, subfraction d and subfraction e.
Above-mentioned subfraction d is collected, is 70:30 by the initial concentration of acetonitrile and water using acetonitrile and water as eluent, it is final dense
Degree is that 85:10 carries out gradient elution 40min, collects the highest chromatographic peak component of content, dry, obtains compound E1011.
Embodiment 2
It with inoculum concentration is that 8vt% is inoculated in the seeding tank of 15L and ferments 5 by potato endogenetic fungus Xylaria curta
It, is then transferred to the fermentation cylinder for fermentation culture of 100L 18 days again, obtains containing mycelial fermentation liquid.Culture medium contains 4.8wt%
Glucose, the pork protein peptone of 0.12wt%, the yeast powder of 0.45wt%, 0.045wt% KH2PO4And 0.045wt%
In culture medium.It ferments and is carried out under conditions of 24 DEG C, pH value are 5.9 and revolving speed is 220r/min, with ventilation in fermentation process
Amount is that 0.8vvm is passed through air into fermentation system.
Centrifugal filtration is above-mentioned to contain mycelial fermentation liquid, obtains mycelium and filtrate (tunning).With acetone soak bacterium
Filament 3 times, collect 3 resulting acetone soak liquid.Filtrate (tunning) is extracted 3 times with ethyl acetate, collects 3 gained
Acetic acid ethyl acetate extract.Above-mentioned acetone soak liquid and acetic acid ethyl acetate extract are mixed, medicinal extract is concentrated to get.
Medicinal extract and silica gel materials are mixed with the ratio of 1:2.5 mixes sample, using chloroform and methanol as eluent, is by concentration gradient
The ratio of 100:0,50:1:20:1,10:1 and 5:1 carry out the elution of silica gel post separation, and the elution volume of each gradient is 3L, obtain
To five elution fractions: component A, component B, component C, component D and component E.
Said components D is collected, is 50:50,60:40:70:30,80:20 by concentration gradient using first alcohol and water as eluent
And suppress standby chromatographic isolation in the ratio progress of 90:10 and elute, elution time amounts to 60min, obtains five elution subfractions:
Subfraction a, subfraction b, subfraction c, subfraction d and subfraction e.
Above-mentioned subfraction d is collected, is 70:30 by the initial concentration of acetonitrile and water using acetonitrile and water as eluent, it is final dense
Degree is that 85:10 carries out gradient elution 40min, collects the highest chromatographic peak component of content, dry, obtains compound E1011.
Embodiment 3
It with inoculum concentration is that 12vt% is inoculated in the seeding tank of 15L and ferments 7 by potato endogenetic fungus Xylaria curta
It, is then transferred to the fermentation cylinder for fermentation culture of 100L 22 days again, obtains containing mycelial fermentation liquid.Culture medium contains 5.2wt%
Glucose, the pork protein peptone of 0.18wt%, the yeast powder of 0.55wt%, 0.055wt% KH2PO4And 0.055wt%
In culture medium.It ferments and is carried out under conditions of 26 DEG C, pH value are 6.1 and revolving speed is 280r/min, with ventilation in fermentation process
Amount is that 1.2vvm is passed through air into fermentation system.
Centrifugal filtration is above-mentioned to contain mycelial fermentation liquid, obtains mycelium and filtrate (tunning).With acetone soak bacterium
Filament 3 times, collect 3 resulting acetone soak liquid.Filtrate (tunning) is extracted 3 times with ethyl acetate, collects 3 gained
Acetic acid ethyl acetate extract.Above-mentioned acetone soak liquid and acetic acid ethyl acetate extract are mixed, medicinal extract is concentrated to get.
Medicinal extract and silica gel materials are mixed with the ratio of 1:3.5 mixes sample, using chloroform and methanol as eluent, is by concentration gradient
The ratio of 100:0,50:1:20:1,10:1 and 5:1 carry out the elution of silica gel post separation, and the elution volume of each gradient is 3L, obtain
To five elution fractions: component A, component B, component C, component D and component E.
Said components D is collected, is 50:50,60:40:70:30,80:20 by concentration gradient using first alcohol and water as eluent
And suppress standby chromatographic isolation in the ratio progress of 90:10 and elute, elution time amounts to 60min, obtains five elution subfractions:
Subfraction a, subfraction b, subfraction c, subfraction d and subfraction e.
Above-mentioned subfraction d is collected, is 70:30 by the initial concentration of acetonitrile and water using acetonitrile and water as eluent, it is final dense
Degree is that 85:10 carries out gradient elution 40min, collects the highest chromatographic peak component of content, dry, obtains compound E1011.
Embodiment 4
Potato endogenetic fungus Xylaria curta is inoculated in the seeding tank of 15L with inoculum concentration for 10.5vt% and is sent out
Ferment 6.5 days, it is then transferred to the fermentation cylinder for fermentation culture of 100L 21 days again, obtains containing mycelial fermentation liquid.Culture medium contains
The glucose of 5.1wt%, the pork protein peptone of 0.16wt%, the yeast powder of 0.52wt%, 0.052wt% KH2PO4And
In 0.052wt% culture medium.It ferments and is carried out under conditions of 25.5 DEG C, pH value are 6.0 and revolving speed is 260r/min, fermented
Air is passed through into fermentation system for 1.1vvm with ventilatory capacity in journey.
Centrifugal filtration is above-mentioned to contain mycelial fermentation liquid, obtains mycelium and filtrate (tunning).With acetone soak bacterium
Filament 1 time, collect resulting acetone soak liquid.Filtrate (tunning) is extracted 1 time with ethyl acetate, collects resulting acetic acid
Ethyl ester extract liquor.Above-mentioned acetone soak liquid and acetic acid ethyl acetate extract are mixed, medicinal extract is concentrated to get.
Medicinal extract and silica gel materials are mixed with the ratio of 1:3 mixes sample, using chloroform and methanol as eluent, is by concentration gradient
The ratio of 100:0,50:1:20:1,10:1 and 5:1 carry out the elution of silica gel post separation, and the elution volume of each gradient is 3L, obtain
To five elution fractions: component A, component B, component C, component D and component E.
Said components D is collected, is 50:50,60:40:70:30,80:20 by concentration gradient using first alcohol and water as eluent
And suppress standby chromatographic isolation in the ratio progress of 90:10 and elute, elution time amounts to 60min, obtains five elution subfractions:
Subfraction a, subfraction b, subfraction c, subfraction d and subfraction e.
Above-mentioned subfraction d is collected, is 70:30 by the initial concentration of acetonitrile and water using acetonitrile and water as eluent, it is final dense
Degree is that 85:10 carries out gradient elution 40min, collects the highest chromatographic peak component of content, dry, obtains compound E1011.
Embodiment 5
Potato endogenetic fungus Xylaria curta is inoculated in the seeding tank of 15L with inoculum concentration for 9.5vt% and is sent out
Ferment 5.5 days, it is then transferred to the fermentation cylinder for fermentation culture of 100L 19 days again, obtains containing mycelial fermentation liquid.Culture medium contains
The glucose of 4.9wt%, the pork protein peptone of 0.14wt%, the yeast powder of 0.48wt%, 0.048wt% KH2PO4And
In 0.048wt% culture medium.It ferments and is carried out under conditions of 24.5 DEG C, pH value are 6.0 and revolving speed is 240r/min, fermented
Air is passed through into fermentation system for 0.9vvm with ventilatory capacity in journey.
Filtrate (tunning) is extracted 3 times with ethyl acetate, 3 resulting acetic acid ethyl acetate extracts is collected, is concentrated to give
To medicinal extract.
Medicinal extract and silica gel materials are mixed with the ratio of 1:3 mixes sample, using chloroform and methanol as eluent, is by concentration gradient
The ratio of 100:0,50:1:20:1,10:1 and 5:1 carry out the elution of silica gel post separation, and the elution volume of each gradient is 3L, obtain
To five elution fractions: component A, component B, component C, component D and component E.
Said components D is collected, is 50:50,60:40:70:30,80:20 by concentration gradient using first alcohol and water as eluent
And suppress standby chromatographic isolation in the ratio progress of 90:10 and elute, elution time amounts to 60min, obtains five elution subfractions:
Subfraction a, subfraction b, subfraction c, subfraction d and subfraction e.
Above-mentioned subfraction d is collected, is 70:30 by the initial concentration of acetonitrile and water using acetonitrile and water as eluent, it is final dense
Degree is that 85:10 carries out gradient elution 40min, collects the highest chromatographic peak component of content, dry, obtains compound E1011.
Test example 1
It repeats to implement embodiment 1-5, obtains enough compound E1011.Structure is carried out to resulting compound E1011
Measurement, including the measurement of nuclear magnetic resonance measuring, high resolution mass spectrum (HRESIMS), ultraviolet determination and infrared analysis.
Nuclear magnetic resonance test result is as shown in table 1:
1 compound E1011's of table1H NMR (600MHz) and13C NMR (150MHz) data (CDCl3, δ in ppm, J in
Hz)
| Position | δH | δC |
| 1 | - | 164.9 |
| 2 | 5.84,dd,16.0,1.7 | 121.7 |
| 3 | 6.75,dd,16.0,4.6 | 147.5 |
| 4 | 5.53,m | 72.1 |
| 5 | 2.05,m,1.75,m | 27.1 |
| 6 | 1.75,m,1.54,m | 26.9 |
| 7 | 5.16,m | 69.7 |
| 8 | 1.19,d,6.5 | 17.8 |
| 9 | - | 164.8 |
| 10 | 5.98,dd,15.8,0.75 | 126.1 |
| 11 | 6.55,dd,15.8,7.8 | 141.8 |
| 12 | 5.12,m | 76.8 |
| 13 | 5.16,m | 69.6 |
| 14 | 1.37,d,5.7 | 17.8 |
| 4-OAc | - | 170.2 |
| 2.07 | 21.1 | |
| 12-OAc | - | 170.0 |
| 2.10, | 21.1 |
High resolution mass spectrum measurement result are as follows: the molecular weight of compound E1011 is m/z [M+K]+
(calculated value: C18H24O8, 368.14).
Ultraviolet (UV) measurement result are as follows: the main ultraviolet data (MeOH) of compound E1011: 225nm (3.1).
Infrared (IR) measurement result are as follows: the main infrared data of compound E1011: 1635.44,600.12.
In addition, above compound E1011 is colorless oil, specific rotatory power is [α]22 D=14 (c 0.05, CH3OH)。CD
Data (CH3OH): 200nm (- 17.0), 217nm (+31.4).
By said determination, show that the chemical structural formula of compound E1011 obtained in this programme isEach position corresponds respectively in table 1
Test example 2
Using following test method, compound E1011 resulting to embodiment carries out cell activity and inhibits test.It is to be measured thin
Born of the same parents include leukemia HL-60, lung cancer A-549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW480.
(1), the concentration of compound E1011 is 40 μM
1. inoculating cell: it is outstanding to be made into individual cells with the culture solution (DMEM or RMPI1640) containing 10% fetal calf serum
Liquid is inoculated into 96 orifice plates, every 100 μ L of pore volume with 3000-15000, every hole allogenic cell, and cell shifts to an earlier date 12-24 hours and is inoculated with
Culture.
2. compound E1011 solution is added: compound E1011 is dissolved with DMSO, and compound E1011 is at the beginning of 40 μM of concentration
Sieve, every 200 μ L of hole final volume, every kind of cell processing are all provided with 3 multiple holes.
3. colour developing: after 37 DEG C are cultivated 48 hours, attached cell abandons culture solution in hole, and every hole adds 20 μ L of MTS solution and culture
100 μ L of liquid；Suspension cell abandons 100 μ L culture supernatants, and every hole adds the MTS solution of 20 μ L；If 3 blank multiple holes (MTS solution 20
The mixed liquor of 100 μ L of μ L and culture solution), continue to be incubated for 2-4 hours, measures absorbance value after the progress that reacts fully.
4. colorimetric: selection 492nm wavelength, multi-function microplate reader (MULTISKAN FC) read each hole absorbance value, record
As a result, obtaining cell average inhibition after data processing.
5. positive reference compound: experiment is all provided with two positive compounds of cis-platinum (DDP) and taxol (Taxol) every time,
Using concentration as abscissa, cell survival rate is that ordinate draws cell growth curve, using two-point method (Reed and Muench
Method) calculate compound IC50Value.
Its result is as shown in table 2 and table 3.
2 cell inhibitory rate of table
3 IC of table50Value
(2), setting compound E1011 gradient concentration is 40 μM, 8 μM, 1.6 μM, 0.32 μM and 0.064 μM
1. inoculating cell: it is outstanding to be made into individual cells with the culture solution (DMEM or RMPI1640) containing 10% fetal calf serum
Liquid is inoculated into 96 orifice plates, every 100 μ L of pore volume with 3000-15000, every hole allogenic cell, and cell shifts to an earlier date 12-24 hours and is inoculated with
Culture.
2. compound E1011 solution is added: compound E1011 is dissolved with DMSO, and compound E1011 is with 40 μM, 8 μM, 1.6
μM, 0.32 μM and 0.064 μM of concentration secondary screening, 200 μ L of every hole final volume, every kind of cell processing is all provided with 3 multiple holes.
3. colour developing: after 37 DEG C are cultivated 48 hours, attached cell abandons culture solution in hole, and every hole adds 20 μ L of MTS solution and culture
100 μ L of liquid；Suspension cell abandons 100 μ L culture supernatants, and every hole adds the MTS solution of 20 μ L；If 3 blank multiple holes (MTS solution 20
The mixed liquor of 100 μ L of μ L and culture solution), continue to be incubated for 2-4 hours, measures absorbance value after the progress that reacts fully.
4. colorimetric: selection 492nm wavelength, multi-function microplate reader (MULTISKAN FC) read each hole absorbance value, record
As a result, calculating the IC of compound E1011 after data processing50Value.
The results are shown in Table 4 for it.
4 IC of table50Value
Contrast table 3 and table 4 can be seen that compound E1011 to the leukemia HL-60 in above-mentioned five kinds of cells, liver cancer
SMMC-7721 and colon cancer SW480 are significantly inhibited, and wherein to liver cancer SMMC-7721 and colon cancer
The IC of SW480 cell50Value is significantly lower than positive control cis-platinum.The compound E1011 for illustrating that the application obtains can be used in inhibiting
The growth of leukemia HL-60, liver cancer SMMC-7721 and colon cancer SW480, more particularly to liver cancer SMMC-7721 and colon
Cancer SW480 plays preferable inhibitory effect.
In other words, compound E1011 can be used for treating leukaemia, liver cancer or colon cancer, contain above compound E1011
Bell potato endogenetic fungus Xylaria curta tunning and potato endogenetic fungus Xylaria curta tunning
Acetic acid ethyl acetate extract can also be used for treatment leukaemia, liver cancer or colon cancer.
In conclusion the raw material of compound E1011 provided by the present application is natural products, green safe, toxic side effect
It is small.And above compound E1011 is novel substance isolated from potato endogenetic fungus for the first time.Preparation method letter
It is single, easy to operate, it can effectively prepare the higher compound E1011 of purity.Compound E1011 and during the preparation process gained
The acetic acid ethyl acetate extract of the tunning of the potato endogenetic fungus Xylaria curta arrived and tunning to leukaemia,
Liver cancer or colon cancer all have certain therapeutic effect.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of compound E1011, which is characterized in that the chemical structural formula of the compound E1011 is
2. the preparation method of compound E1011 as described in claim 1, which comprises the following steps:
Fermenting potato endogenetic fungus Xylaria curta, extracts tunning with ethyl acetate, extracts to ethyl acetate
Liquid is taken to carry out silica gel post separation；To standby chromatographic isolation is suppressed in the 4th elution fraction progress, the 4th elution subfraction is collected
The preparation of high pressure liquid phase is carried out, the highest chromatographic peak component of content is collected, it is dry；
Wherein, the condition of silica gel post separation includes: using chloroform and methanol as eluent, is 100:0,50:1:20 by concentration gradient:
1, the ratio of 10:1 and 5:1 is eluted, and obtains five elution fractions；
The middle condition for suppressing standby chromatographic isolation includes: using first alcohol and water as eluent, is 50:50,60:40:70 by concentration gradient:
30, the ratio of 80:20 and 90:10 is eluted, and obtains five elution subfractions；
The condition of high pressure liquid phase preparation includes: using acetonitrile and water as eluent, is 70 by the initial concentration of the acetonitrile and water:
30, ultimate density is that 85:10 carries out gradient elution 40min.
3. preparation method according to claim 2, which is characterized in that further include using acetone before extracting the tunning
Mycelium after soaking fermentation obtains acetone soak liquid；Then the acetone soak liquid and the acetic acid ethyl acetate extract are mixed simultaneously
Successively carry out silica gel post separation, middle suppress prepares for chromatographic isolation and high pressure liquid phase.
4. preparation method according to claim 2, which is characterized in that fermentation be in 24-26 DEG C, pH value be 5.9-6.1 with
And revolving speed be 220-280r/min under conditions of carry out.
5. preparation method according to claim 2, which is characterized in that culture medium used in fermentation process contains 4.8-
Glucose, the pork protein peptone of 0.12-0.18wt%, the yeast powder of 0.45-0.55wt%, the 0.045- of 5.2wt%
The KH of 0.055wt%2PO4And the MgSO of 0.045-0.055wt%4。
6. preparation method according to claim 5, which is characterized in that the potato endogenetic fungus Xylaria curta
Inoculum concentration in the medium is 8-12vt%.
7. preparation method according to claim 2, which is characterized in that logical for 0.8-1.2vvm with ventilatory capacity in fermentation process
Enter air.
8. a kind of potato endogenetic fungus Xylaria curta obtained such as the described in any item preparation methods of claim 2-7
The tunning.
9. a kind of acetic acid ethyl acetate extract obtained such as the described in any item preparation methods of claim 2-7.
10. compound E1011 as described in claim 1, tunning as claimed in claim 8 or such as claim 9 institute
Application of the acetic acid ethyl acetate extract stated in treatment leukaemia, liver cancer or colon cancer.
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| CN111808115A (en) * | 2020-07-23 | 2020-10-23 | 中南民族大学 | Fermented organic matter with anti-pathogenic activity of potato endophyte, preparation process and application thereof, and fermented product |
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| CN111808115A (en) * | 2020-07-23 | 2020-10-23 | 中南民族大学 | Fermented organic matter with anti-pathogenic activity of potato endophyte, preparation process and application thereof, and fermented product |
| CN111808115B (en) * | 2020-07-23 | 2021-04-23 | 中南民族大学 | Fermented organic matter with anti-pathogenic activity of potato endophyte, preparation process and application thereof, and fermented product |
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