CN105884850A - Production method and use of ergosterol - Google Patents
Production method and use of ergosterol Download PDFInfo
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- CN105884850A CN105884850A CN201610278263.8A CN201610278263A CN105884850A CN 105884850 A CN105884850 A CN 105884850A CN 201610278263 A CN201610278263 A CN 201610278263A CN 105884850 A CN105884850 A CN 105884850A
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- ergosterol
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- silica gel
- eluent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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Abstract
The invention discloses a production method of ergosterol. The production method includes the following steps: adding an extracting solvent to a dried Monascus purpureus-fermented sample, extracting by an extraction process to obtain crude extract, eluting the crude extract by column chromatography to obtain primary purified eluent, subjecting the primary purified elution to silica gel thin-layer chromatography, combining eluents with same silica gel thin-layer chromatography results and performing column chromatography again to obtain secondary purified eluent, removing the solvent from the secondary purified eluent, and recrystallizing through petroleum ether to obtain ergosterol. The preparation of ergosterol from Monascus purpureus as material is provided for the first time, crude extract of the sample is extracted first, and an optimal column chromatography eluent is screened then according to distribution conditions and dissolutions in different solvents, so that ergosterol is isolated and purified effectively. The production method of the ergosterol is simple, easy to implement and low in time consumption, the material is easy to obtain and low in cost, and batch production of ergosterol is achieved.
Description
Technical field
The present invention relates to phytosterin compound extractive technique field, be specifically related to the preparation of a kind of ergosterol
Method and purposes.
Background technology
" ergosterol ", also known as " ergosterol ", is one of steroid.This compound can be used for
Biochemical Research, the same vitamin D2 of partial action.Ergosterol is the important composition portion of microbial cell film
Part, to guaranteeing the integrity of cell membrane, the activity of membrane bound enzyme, the mobility of film, cell viability and
Cellular material transport etc. plays an important role.Produce the intermediate of hormone medicine, can be used to produce can
Pine.There is the effect of vitamin D2.Ergosterol has important physiological action, be food, feedstuff and
Medical industry raw material.
Ergosterol industrialized production, has two approach microorganism biological synthesis and chemosynthesis.Because of steroidal
Compound molecule contains asymmetric center, and synthesis step is many, and difficulty is big, the most extensively carries out microorganism
The research of fermentation synthesis ergosterol.The research of ergosterol biosynthesis pathway at present achieves the biggest
Progress, this will be for carrying by genetic engineering means acquisition high yield ergosterol bacterial strain and antifungal drug design
For theoretical direction.The patent of invention of such as Application No. CN200410040905.8 discloses a kind of Ergota
The preparation method of sterol, by yeast and alkali, carries out saponification again with toluene extracting after sodium chloride solution mixing,
Ergosterol finished product it is vacuum dried to obtain through washing, concentrating and refine.This invention utilizes conventional above-mentioned chemical industry
Supplementary material and vague generalization construction equipment, it is achieved that the industrialized production of ergosterol, production process is the easiest,
Production cost is low, provides raw material guarantee for producing vitamin D2 in enormous quantities.But the preparation side of this invention
The reaction condition needed in method is the gentleest, and the preparation time needed is long, and the bacterial strain of selection is in preparation
During ergosterol, yield is the most not high enough.
As can be seen here, can be for above-mentioned deficiency, it is provided that the preparation method of a kind of ergosterol and purposes,
Make its method simple, easily realize, and raw material is easy to get low cost, it is achieved the batch production of ergosterol, become
For the technical barrier that those skilled in the art are urgently to be resolved hurrily.
Summary of the invention
The present invention is to solve above-mentioned technical problem, it is provided that the preparation method of a kind of ergosterol and purposes,
It includes techniques below scheme:
The preparation method of a kind of ergosterol, including the step using Fermentation Condition of Monascus spp sample to extract as raw material
Suddenly.
Provide the method preparing ergosterol for raw material with monascus first so that it is be different from prior art
In preparation method, and it is simple to have preparation process, effectively realizes the batch production of ergosterol.
Further, comprise the following steps: take dry Fermentation Condition of Monascus spp sample, add Extraction solvent,
Use restricted-access media slightly to be carried product, then use column chromatography eluting slightly to carry product and obtain a purification eluting
Liquid, carries out silica gel thin-layer chromatography to a described purification eluent, by identical silica gel thin-layer chromatography result
Eluent merging again carries out column chromatography and affords secondarily purified eluent, to described secondarily purified eluting
Liquid carries out silica gel thin-layer chromatography, removes solvent also after being merged by the eluent of identical silica gel thin-layer chromatography result
Carry out being recrystallized to give ergosterol by recrystallization solvent.
Infusion process be applicable to viscosity medical material, the medical material of inorganization structure, fresh and be prone to expand medical material,
The extraction of cheap armaticity medical material.And the hot submersion method in infusion process refers to be dissolved in Chinese herbal medicine molten
In agent, useful effect composition is reached 40 DEG C～60 DEG C extracted by water-bath or steam heating.
Further, described Extraction solvent is ethanol, described Fermentation Condition of Monascus spp sample and described extraction
The volume ratio of solvent is 1～3:10～12；Described extraction comprises the following steps: in 12000r min-1
Under the conditions of 60 DEG C extraction 12h.
Further, described ethanol be volume fraction be the ethanol of 90%.
Further, the method for described column chromatography eluting comprises the following steps:
Step one: mix sample: by described slightly carry product or a purification eluent concentrate after the extractum that obtains, adopt
Fully dissolve with solvent, add silica gel and stir to solvent the solid absorption volatilized and separate out on silica gel；
Step 2: dress post: use wet method dress post to obtain silicagel column；
Step 3: loading: after sample step one mixed is poured in silicagel column, adds quartz sand, and
Absorbent cotton is entered at capital end plug；
Step 4: eluting: using eluant to carry out eluting, collect eluent, described eluant wraps respectively
The mixing including the pure solution of petroleum ether, ethyl acetate and methanol and petroleum ether, ethyl acetate and methanol is molten
Agent.
The present invention by column chromatography eluting and screening experiment, obtain slightly carrying in product, petroleum ether polarity is relatively
Little, ethyl acetate is placed in the middle, and methanol polarity is relatively big, therefore selects petroleum ether-ethyl acetate-methanol-eluted fractions.
Further, the solvent in described step one is petroleum ether, the described silica gel of addition and described extractum
Equal-volume；Wet method dress post comprises the following steps: weighs silica gel and is suspended from petroleum ether, is stirred continuously swelling going
Obtain suspension after bubble removing, suspension is at the uniform velocity poured into bottom and is plugged with in the chromatographic column of Cotton Gossypii, open bottom valve and make
Liquid flows out.
Further, described mixed solvent includes that petroleum ether, ethyl acetate and methanol depend on by volume respectively
Secondary for 1:0:0,9:1:0,7:2:1,6:3:1,5:4:1,4:4:2,3:6:1,2:7:1,0:9:1,0:7:3,
The mixed solvent of 0:5:5,0:3:7,0:1:9,0:0:1；The eluant of described each ratio rinses 5 posts
Volume.
Wherein, column volume=(mixing sample silica gel+blank silica gel) × 7/3.
Further, described eluant be the volume ratio of petroleum ether, ethyl acetate and methanol be 9:1:0's
Mixed solution.
When the eluant using above-mentioned volume ratio carries out column chromatography eluting, it is possible to efficiently separate and be purified into Ergota
Sterol.
Further, the described developing solvent in silica gel thin-layer chromatography be volume ratio be the stone of 12～0.5:1
Oil ether and the mixed solvent of ethyl acetate.
When described developing solvent is petroleum ether and the mixed solvent of ethyl acetate that volume ratio is 12～0.5:1,
Chromatographic effect is good, and result is clear, it is possible to obtain each component band.
The purposes of a kind of ergosterol, the medicine of inhibition of lipid peroxidation prepared by described ergosterol
Middle use.
Oxygen free radical reaction and lipid peroxidation play important in the metabolic processes of body
Effect, both are in coordination and dynamic balance state under normal circumstances, maintain internal many Physiology and biochemistries
Reaction and immunoreation.The most this coordination and dynamic equilibrium produce disorderly with imbalance, will cause and one are
Row metabolism is not normal and immunologic function reduces, form oxygen-derived free radicals chain reaction, infringement biomembrane and
Its function, thus formed cell transparency pathological changes, fibrosis, large area cell injury cause into nerve,
Tissue, organ equivalent damage.Lipid peroxidation is just in this reaction.
The process of the ROS oxidative biological film occurred during lipid peroxidation, i.e. ROS is with biomembranous
The macromolecular substances such as the side chain of the polyunsaturated fatty acid that phospholipid, enzyme are relevant with membrane receptor and nucleic acid play lipid
Peroxidization forms lipid peroxidation product (Lipid PerOxide, LPO) such as malonaldehyde
(Malonaldehyde, MDA) and 4-hydroxyl nonenoic acid (4-hydroxynonenal, HNE), so that cell
Mobility and the permeability of film change, and cause the change of cellularity and function eventually.
Ergosterol prepared by the present invention detects through further inhibition of lipid peroxidation, it was demonstrated that Ergota
Sterol can use in the medicine prepare inhibition of lipid peroxidation.
Use technique scheme, including following beneficial effect: present invention firstly provides with Monas cuspurpureus Went
The mould material for raw material preparation with lipoid peroxidization resistant, it is former for providing first with monascus
Ergosterol prepared by material, and monascus ocean product are first slightly carried product and extract by the present invention, basis further
Property distribution situation and the optimal column chromatography eluant of screening of the dissolubility in different solvents so that it is have
Imitate isolated and purified go out ergosterol.The preparation method of the present invention is simple, easily realize, the shortest, and
Raw material is easy to get low cost, it is achieved the batch production of ergosterol.
Accompanying drawing explanation
Fig. 1 is different concentration ethanol extract lipid peroxidation suppression ratio datagram of the present invention；
Fig. 2 is each several part inhibition of lipid peroxidation datagram that the present invention extracts；
Fig. 3 is instant component one, component two, component three thin layer chromatography result figure；
Fig. 4 is instant component three, component four, component five thin layer chromatography result figure；
Fig. 5 is the inhibition of lipid peroxidation datagram of each component that column chromatography of the present invention obtains；
Fig. 6 is the compound thin film analysis chart of isolated of the present invention；
Fig. 7 is the MS collection of illustrative plates of the compounds of this invention H-1；
Fig. 8 is the compounds of this invention H-1's1H-NMR collection of illustrative plates；
Fig. 9 is the compounds of this invention H-1's13C-NMR collection of illustrative plates；
Figure 10 is the compounds of this invention H-1's13The partial enlarged drawing of C-NMR collection of illustrative plates.
Detailed description of the invention
Below by specific embodiment, the present invention is described in further detail.
Embodiment one: the preparation method of a kind of ergosterol, makees including using Fermentation Condition of Monascus spp sample
The step extracted for raw material.
Embodiment two: the purposes of a kind of ergosterol, lipotropism matter mistake prepared by described ergosterol
The medicine of oxidation activity uses.
Embodiment three: the preparation method of a kind of ergosterol, comprises the following steps: take dry red
Aspergillosis fermented sample, adds Extraction solvent, uses restricted-access media slightly to be carried product, then uses
Column chromatography eluting slightly carries product and obtains a purification eluent, and a described purification eluent is carried out silicon
Glue thin layer chromatography, again carries out column chromatography by the eluent merging of identical silica gel thin-layer chromatography result and washes
Take off and obtain secondarily purified eluent, described secondarily purified eluent is carried out silica gel thin-layer chromatography, will
The eluent of identical silica gel thin-layer chromatography result is removed solvent after merging and is carried out by recrystallization solvent
It is recrystallized to give ergosterol.
The purposes of a kind of above-mentioned ergosterol, anti peroxidation of lipid work prepared by described ergosterol
Property medicine in use.
Embodiment four: the preparation method of a kind of ergosterol, comprises the following steps: take dry red
Aspergillosis fermented sample, adds the alcohol solvent of 10 times of volumes, in 12000r min-1Under the conditions of 60 DEG C
Extraction 12h, extracts the product that slightly carried, and then uses column chromatography eluting slightly to carry product and obtains a purification
Eluent, carries out silica gel thin-layer chromatography to a described purification eluent, by identical silica gel thin-layer layer
The eluent merging of analysis result again carries out column chromatography and affords secondarily purified eluent, to described
Secondarily purified eluent carries out silica gel thin-layer chromatography, by the eluent of identical silica gel thin-layer chromatography result
Remove solvent after merging and carry out being recrystallized to give ergosterol by petroleum ether.
The purposes of a kind of above-mentioned ergosterol, anti peroxidation of lipid work prepared by described ergosterol
Property medicine in use.
Embodiment five: the preparation method of a kind of ergosterol, comprises the following steps: take dry red
Aspergillosis fermented sample, adds alcohol solvent, described Fermentation Condition of Monascus spp sample and described Extraction solvent
Volume ratio be 3:10；In 12000r min-1Under the conditions of 60 DEG C extraction 12h, extract slightly carried
Product, then use column chromatography eluting slightly to carry product and obtain a purification eluent, to a described purification
Eluent carries out silica gel thin-layer chromatography, is merged again by the eluent of identical silica gel thin-layer chromatography result
Carry out column chromatography and afford secondarily purified eluent, described secondarily purified eluent is carried out silica gel
Thin layer chromatography, removes solvent and passes through stone after being merged by the eluent of identical silica gel thin-layer chromatography result
Oil ether carries out being recrystallized to give ergosterol.
The method of described column chromatography eluting comprises the following steps:
Step one: mix sample: by described slightly carry product or a purification eluent concentrate after the leaching that obtains
Cream, uses solvent fully to dissolve, and addition silica gel stirring to solvent volatilization and the solid absorption separated out exist
On silica gel；
Step 2: dress post: use wet method dress post to obtain silicagel column；
Step 3: loading: after sample step one mixed is poured in silicagel column, adds quartz
Sand, and enter absorbent cotton at capital end plug；
Step 4: eluting: using eluant to carry out eluting, collect eluent, described eluant divides
Do not include pure solution and petroleum ether, ethyl acetate and the methanol of petroleum ether, ethyl acetate and methanol
Mixed solvent.
The purposes of a kind of above-mentioned ergosterol, anti peroxidation of lipid work prepared by described ergosterol
Property medicine in use.
Embodiment six: the preparation method of a kind of ergosterol, comprises the following steps: take dry red
Aspergillosis fermented sample, adds alcohol solvent, described Fermentation Condition of Monascus spp sample and described Extraction solvent
Volume ratio be 1:12, in 12000r min-1Under the conditions of 60 DEG C extraction 12h, extract slightly carried
Product, then use column chromatography eluting slightly to carry product and obtain a purification eluent, to a described purification
Eluent carries out silica gel thin-layer chromatography, is merged again by the eluent of identical silica gel thin-layer chromatography result
Carry out column chromatography and afford secondarily purified eluent, described secondarily purified eluent is carried out silica gel
Thin layer chromatography, removes solvent and passes through stone after being merged by the eluent of identical silica gel thin-layer chromatography result
Oil ether carries out being recrystallized to give ergosterol.
The method of described column chromatography eluting comprises the following steps:
Step one: mix sample: by described slightly carry product or a purification eluent concentrate after the extractum that obtains,
Fully dissolve with petroleum ether, be placed in plate, stir after adding isopyknic silica gel to solvent volatilization,
Material uniform adsorption is on silica gel, and silica gel particle is loose not to be sticked together；
Step 2: dress post: use wet method dress post, weighs 300g silica gel dress post, the body of described silica gel
The volume ratio of a long-pending post is 1:20, is suspended from 1000ml petroleum ether, is stirred continuously and swelling removes bubble removing
After, by suspension uniform speed slow pour into bottom be plugged with in the chromatographic column of Cotton Gossypii, open bottom valve and make liquid
Flowing out, adsorbent uniformly declines, and suitably vibrates, beneficially aerofluxus, it is to avoid layering；
Step 3: loading: the sample mixed with silica gel is poured slowly in the silicagel column installed, makes
It is layered on post its uniform ground, adds quartz sand afterwards, and entering absorbent cotton at capital end plug, with
Prevent solvent adds the impact for sample；
Step 4: eluting: press as eluant using petroleum ether, ethyl acetate and methanol mixed solvent
Polarity carries out eluting from small to large, collects eluent.
The purposes of a kind of above-mentioned ergosterol, anti peroxidation of lipid work prepared by described ergosterol
Property medicine in use.
Embodiment seven: the preparation method of a kind of ergosterol, comprises the following steps: take dry red
Aspergillosis fermented sample, adds the alcohol solvent that volume fraction is 90% of 10 times of volumes, in 12000r
·min-1Under the conditions of 60 DEG C of extraction 12h, extract and slightly carried product, then use column chromatography eluting slightly to carry
Product obtain a purification eluent, and a described purification eluent is carried out silica gel thin-layer chromatography, will
The eluent merging of identical silica gel thin-layer chromatography result again carries out column chromatography and affords secondarily purified
Eluent, carries out silica gel thin-layer chromatography to described secondarily purified eluent, by identical silica gel thin-layer layer
The eluent of analysis result removes solvent after merging and carries out being recrystallized to give Ergota steroid by petroleum ether
Alcohol.
The method of described column chromatography eluting comprises the following steps:
Step one: mix sample: by described slightly carry product or a purification eluent concentrate after the extractum that obtains,
Fully dissolve with petroleum ether, be placed in plate, stir after adding isopyknic silica gel to solvent volatilization,
Material uniform adsorption is on silica gel, and silica gel particle is loose not to be sticked together；
Step 2: dress post: use wet method dress post, weighs 300g silica gel dress post, the body of described silica gel
The volume ratio of a long-pending post is 3:10, is suspended from 1000ml petroleum ether, is stirred continuously and swelling removes bubble removing
After, by suspension uniform speed slow pour into bottom be plugged with in the chromatographic column of Cotton Gossypii, open bottom valve and make liquid
Flowing out, adsorbent uniformly declines, and suitably vibrates, beneficially aerofluxus, it is to avoid layering；
Step 3: loading: the sample mixed with silica gel is poured slowly in the silicagel column installed, makes
It is layered on post its uniform ground, adds quartz sand afterwards, and entering absorbent cotton at capital end plug, with
Prevent solvent adds the impact for sample；
Step 4: eluting: press as eluant using petroleum ether, ethyl acetate and methanol mixed solvent
Polarity carries out eluting from small to large, collects eluent.Described eluant includes petroleum ether, acetic acid second
The volume ratio of ester and methanol is the mixed solvent of 9:1:0；The eluant of described each ratio rinses 5
Column volume.
The method of described silica gel thin-layer chromatography comprises the following steps:
Step one: point sample: by chromatoplate horizontal positioned, with internal diameter less than 1mm, the mouth of pipe is smooth
The eluting composition of collection is put on the same level line of chromatoplate one end by clean capillary tube one by one, sample
Product spacing is more than 5mm.
Step 2: saturated: pour the developing solvent prepared in advance into chromatography cylinder in right amount, stands after covering tightly
5-10min, makes solvent volatilization gas in chromatography cylinder saturated uniformly.
Step 3: launch: excellent for some chromatoplate is put in chromatography cylinder and launch, make developing solvent soak
Not having thin plate lower end, but be not up to some line-transect, airtight chromatography cylinder launches, when developing solvent is from chromatography
Plate lower end is slowly diffused into forward position, when top 5～10mm, takes out chromatoplate, uses Pencil marks
Position, solvent front.
Step 4: observe, develop the color, qualitative: observe after solvent volatilizes on chromatoplate, GF254
Silica gel plate is placed under uv analyzer directly observing；Some material is unstressed configuration under uv analyzer
Speckle, the most uniformly sprays 10% ethanol solution of sulfuric acid, is baked to colour developing with high temperature rifle, labelling point
Position, measures, and calculates Rf value (Rf value).
Step 5: the selection of developing solvent: the developing solvent of preparation opposed polarity, is carried out sample respectively
Thin layer chromatography, after observing and calculating Rf value, after selecting to launch, component speckle is round and concentrates, no
Be clearly separated with component speckle, Rf value scope be the developing solvent of 0.2-0.8 be optimal.Finally give body
Amass the petroleum ether that ratio is 12:l and petroleum ether and ethyl acetate that ethyl acetate, volume ratio are 1:2,
Through silica gel plate thin layer chromatography analysis, 10% sulphuric acid ethanol colour developing is observed.
The purposes of a kind of above-mentioned ergosterol, anti peroxidation of lipid work prepared by described ergosterol
Property medicine in use.
Embodiment eight: the preparation method of a kind of ergosterol, comprises the following steps: take dry red
Aspergillosis fermented sample, adds the alcohol solvent that volume fraction is 90% of 10 times of volumes, in 12000r
·min-1Under the conditions of 60 DEG C of extraction 12h, extract and slightly carried product, then use column chromatography eluting slightly to carry
Product obtain a purification eluent, and a described purification eluent is carried out silica gel thin-layer chromatography, will
The eluent merging of identical silica gel thin-layer chromatography result again carries out column chromatography and affords secondarily purified
Eluent, carries out silica gel thin-layer chromatography to described secondarily purified eluent, by identical silica gel thin-layer layer
The eluent of analysis result removes solvent after merging and carries out being recrystallized to give Ergota steroid by petroleum ether
Alcohol.
The method of described column chromatography eluting comprises the following steps:
Step one: mix sample: by described slightly carry product or a purification eluent concentrate after the extractum that obtains,
Fully dissolve with petroleum ether, be placed in plate, stir after adding isopyknic silica gel to solvent volatilization,
Material uniform adsorption is on silica gel, and silica gel particle is loose not to be sticked together；
Step 2: dress post: use wet method dress post, weighs 300g silica gel dress post, the body of described silica gel
The volume ratio of a long-pending post is 1:20, is suspended from 1000ml petroleum ether, is stirred continuously and swelling removes bubble removing
After, by suspension uniform speed slow pour into bottom be plugged with in the chromatographic column of Cotton Gossypii, open bottom valve and make liquid
Flowing out, adsorbent uniformly declines, and suitably vibrates, beneficially aerofluxus, it is to avoid layering；
Step 3: loading: the sample mixed with silica gel is poured slowly in the silicagel column installed, makes
It is layered on post its uniform ground, adds quartz sand afterwards, and entering absorbent cotton at capital end plug, with
Prevent solvent adds the impact for sample；
Step 4: eluting: using the mixed solvent of petroleum ether, ethyl acetate and methanol as eluant
Carry out eluting, collect eluent.Described mixed solvent includes petroleum ether, ethyl acetate and methanol
Volume ratio be followed successively by 1:0:0,9:1:0,7:2:1,6:3:1,5:4:1,4:4:2,3:6:1,2:7:1,0:
The mixed solvent of 9:1,0:7:3,0:5:5,0:3:7,0:1:9,0:0:1；The eluting of described each ratio
5 column volumes are rinsed in agent.
The method of described silica gel thin-layer chromatography comprises the following steps:
Step one: point sample: by chromatoplate horizontal positioned, with internal diameter less than 1mm, the mouth of pipe is smooth
The eluting composition of collection is put on the same level line of chromatoplate one end by clean capillary tube one by one, sample
Product spacing is more than 5mm.
Step 2: saturated: pour the developing solvent prepared in advance into chromatography cylinder in right amount, stands after covering tightly
5-10min, makes solvent volatilization gas in chromatography cylinder saturated uniformly.
Step 3: launch: excellent for some chromatoplate is put in chromatography cylinder and launch, make developing solvent soak
Not having thin plate lower end, but be not up to some line-transect, airtight chromatography cylinder launches, when developing solvent is from chromatography
Plate lower end is slowly diffused into forward position, when top 5～10mm, takes out chromatoplate, uses Pencil marks
Position, solvent front.
Step 4: observe, develop the color, qualitative: observe after solvent volatilizes on chromatoplate, GF254
Silica gel plate is placed under uv analyzer directly observing；Some material is unstressed configuration under uv analyzer
Speckle, the most uniformly sprays 10% ethanol solution of sulfuric acid, is baked to colour developing with high temperature rifle, labelling point
Position, measures, and calculates Rf value (Rf value).
Step 5: the selection of developing solvent: the developing solvent of preparation opposed polarity, is carried out sample respectively
Thin layer chromatography, after observing and calculating Rf value, after selecting to launch, component speckle is round and concentrates, no
Be clearly separated with component speckle, Rf value scope be the developing solvent of 0.2-0.8 be optimal.Finally give body
Amass the petroleum ether that ratio is 12:l and petroleum ether and ethyl acetate that ethyl acetate, volume ratio are 1:2,
Through silica gel plate thin layer chromatography analysis, 10% sulphuric acid ethanol colour developing is observed.
The purposes of a kind of above-mentioned ergosterol, anti peroxidation of lipid work prepared by described ergosterol
Property medicine in use.
The lipid peroxidation suppression ratio detection of different solvents:
One, experimental technique:
1, the extraction of product is slightly proposed:
(1) ethyl acetate extraction ethanol extract
Take quantitative undried monascus sample respectively, add 90% ethanol of 10 times of volumes, 60 DEG C
Extraction 12h, 12000r min-1, centrifugal 15min, take supernatant 200ml and be placed in 500mL separatory
In funnel, adding 200mL ethyl acetate, shake well extracts, and stands, treats that solution is layered completely
After, release the extract on upper strata, the lixiviating solution of lower floor extracts 2 times by same method again, adds extraction every time
Agent 200mL, 3 times extractant merges, recovered under reduced pressure extractant at less than 60 DEG C, after being spin-dried for,
Make solvent naturally volatilize, obtain alcohol extraction acetic acid ethyl ester extract.The lixiviating solution of lower floor is less than 60 DEG C
Lower recovered under reduced pressure, after being spin-dried for, makes solvent naturally volatilize, and obtains remaining water after ethyl acetate extraction
Layer.
(2) petroleum ether extracts acetic acid ethyl ester extract further
Take quantitative undried monascus sample respectively, add 10 times of volume 90% ethanol respectively, 60 DEG C
Extraction 12h, 12000r min-1, centrifugal 15min, take supernatant 200ml and be placed in 500mL separatory
In funnel, being separately added into 200mL ethyl acetate, shake well extracts, and stands, treats that solution is complete
After layering, releasing the extract on upper strata, the lixiviating solution of lower floor extracts 2 times by same method again, adds every time
Extractant 200mL, 3 times extractant merges, recovered under reduced pressure extractant at less than 60 DEG C, then adds
Water fully dissolves and is settled to 200ml.
The acetic acid ethyl ester extract fully dissolving constant volume with water is placed in 500mL separatory funnel, point
Not Jia Ru 200mL petroleum ether, shake well extract, stand, after solution is layered completely, release
The extract on upper strata, the lixiviating solution of lower floor extracts 2 times by same method again, adds extractant every time
200mL, 3 times extractant merges, recovered under reduced pressure extractant at less than 60 DEG C, after being spin-dried for,
To petroleum ether extract.The lixiviating solution of lower floor reduces pressure at less than 60 DEG C, after being spin-dried for, obtains oil
Remaining ethyl acetate layer after ether extraction.
2, the preparation of the anti peroxidation of lipid testing sample of extract: weigh the extraction of 0.4mg each several part
Sample, dissolves with distilled water, is settled to 100mL, and this concentration of polymer solution is 8mg/mL.Inhale
Take 8mg/mL solution 25,12.5,6.25,3.13,1.56,0.78,0.39mL, be separately added into
Distilled water is settled to 25mL, the concentration of sample is respectively 0.5,1,2,4,8mg/mL.
3, lipid peroxidation Inhibition test: use egg yolk homogenate (10%, volume fraction) to make
Lipid vehicle for reaction.The testing sample taking the above-mentioned preparation of 0.1mL is homogenized with 0.5mL egg yolk
Liquid, 0.4mL pure water and 50 μ L copperas solution (FeSO4·7H2O, 70mmol/L) mixing,
Hatch 30min for 37 DEG C, be rapidly added 1.5mL acetic acid solution (20%, volume fraction, pH value
3.5) and 1.5mL thiobarbituricacidα-solution (0.8%, mass concentration, use 1.1% dodecyl
Metabisulfite solution is prepared), mix rear 95 DEG C of water-bath 60min at a high speed.Question response mixture is cooled to
Room temperature, adds 5mL n-butyl alcohol, shakes up, and mixture 5000r/min is centrifuged 15min, takes supernatant
Liquid measures its absorbance at 532nm, and pure water does blank.
Computational methods such as following formula:
Lipid peroxidation suppression ratio (%)=(1-experimental group absorbance/blank group absorbance) × 100%
Two, experimental result:
1, testing result is shown in that Fig. 1, result show that water extract inhibition of lipid peroxidation is the highest, secondly
Be 30% ethanol extraction, along with the increase of concentration of alcohol, extract lipid peroxidation suppression ratio by
Gradually reduce, 30% ethanol extraction, 70% ethanol extraction, 90% ethanol extraction and water extract
Suppression ratio difference tool pole significance (p < 0.01), it is notable that water extract and 50% ethanol extract compare difference tool
Property (p < 0.05), until concentration increase to extracting solution during 90% ethanol lipid peroxidation suppression ratio increase,
Difference does not have significance (p > 0.05) compared with 70% ethanol extraction, 90% ethanol extraction, and
Water and 90% concentration of alcohol difference are relatively big, and the active substance composition difference extracted is the biggest.Therefore
Carry out extracting obtained product as extractant using water and ethanol, be respectively provided with anti peroxidation of lipid
Function.
2, the extraction of the product that slightly carry of alcohol extraction:
(1) ethyl acetate extraction ethanol extract
The ethanol extract of monascus sample is obtained by extraction acetic acid ethyl ester extract through ethyl acetate and is labeled as
(ⅴ).After lower floor obtains ethyl acetate extraction, remaining water layer is labeled as ().Such as Fig. 2 institute
Show, often organize stylolitic part, from left to right concentration be followed successively by 8mg/ml, 4mg/ml, 2mg/ml,
1mg/ml, 0.5mg/ml, as seen from Figure 2, partly () is fat during 8mg/ml in concentration
Matter peroxidating suppression ratio has reached 96.05%, and part () is almost without inhibition of lipid peroxidation.
Illustrate that the active substance that alcohol proposes is preferable with ethyl acetate intersolubility, and the active substance extracted is big
Part is the material of middle polarity.
(2) petroleum ether extraction ethyl acetate portion ()
It is labeled as obtaining petroleum ether extraction after () extracts further with petroleum ether by ethyl acetate portion
Substance markers is ().Lower floor after obtaining petroleum ether extraction remaining ethyl acetate layer be labeled as ().
As seen from Figure 2, partly () is that lipid peroxidation suppression ratio during 8mg/ml is in concentration
73.61%, partly () has reached 91.22%.Partly the lipid peroxidation suppression ratio of () is big
In part (), difference tool pole significance (p < 0.01).One is entered through petroleum ether through ethyl acetate layer
After step extraction, major part active substance, still at ethyl acetate layer, also has considerable part to be extracted into stone
Oil ether layer, illustrates that the active substance of alcohol extraction is mainly the material of middle polarity, also has quite a few
The active substance that polarity is less.
The selection of column chromatography eluant:
1, extractum dissolving situation in different solvents: the extractum after alcohol extraction is in different solvents
Dissolving situation: take the alcohol-extracted extract of a little monascus sample respectively through petroleum ether, chloroform, acetic acid second
Ester, n-butyl alcohol, methanol dissolve, and investigate extractum dissolving situation in different solvents, and result shows
Extractum dissolubility in ethyl acetate is preferable.
2, sample separates situation in TLC developing solvent: according to extractum dissolubility in different solvents
Investigation and the investigation of polar contribution situation,
Choose the petroleum ether of opposed polarity proportioning, ethyl acetate and methanol respectively water to be carried and alcohol extraction leaching
Cream carries out thin layer chromatography, after repeatedly thin layer chromatography interpretation of result, in the developing solvent of different ratio
Under ratio, water gets sample product and the thin layer chromatography of alcohol extraction sample, shows that water carries and the thing in alcohol extraction sample
Matter is extremely complex, and opposed polarity Duan Jun has material.But carry with alcohol extraction sample at same polarity bar at water
Part (petroleum ether: ethyl acetate=20:1), Rf value scope is different.
3, the determination of column chromatography eluant: it is more complicated that monascus sample slightly carries product composition, to it
When inhibition of lipid peroxidation material carries out isolated and purified, one-component to be obtained, for avoiding as far as possible
Active substance intersection elutes, so using polarity gradient eluting.According to above-mentioned anti-lipid peroxy
Changing the polar contribution situation of active substance, consider sample dissolubility in a solvent, sample exists
TLC developing solvent separates the selected eluant such as situation, the polarity of solvent.Carrying at water slightly carries in product,
Methanol polarity is relatively big, therefore uses acetate-methanol gradient elution.Slightly carry in product in alcohol extraction, oil
Ether polarity is less, and ethyl acetate is placed in the middle, and methanol polarity is relatively big, therefore selection petroleum ether-ethyl acetate-
Methanol elution gradient.
Slightly put forward product column chromatography purification result first:
Alcohol extraction slightly carries product through silica gel column chromatography, is merged by identical thin layer chromatography elution fraction, obtains group
Point one, component two, component three, component four, component five, component one and component two select petroleum ether-
Ethyl acetate (12:l) as developing solvent, component three, component four and component five select petroleum ether-
Ethyl acetate (1:2) is as developing solvent, and through silica gel plate thin layer chromatography analysis, 10% sulphuric acid ethanol shows
Color is observed, and obtains each component band, and result is shown in Fig. 3 and Fig. 4.
Each component inhibition of lipid peroxidation that column chromatography obtains:
The lipid peroxidation suppression ratio below figure 5 of each component that column chromatography obtains, wherein, component two
With component five without inhibition of lipid peroxidation.Fig. 5 often organizes the most anti-component of columnar data two
Concentration rise successively, the concentration from left to right of the columnar data in VC group is followed successively by 25 μ
Columnar data in g/ml, 50 μ g/ml, 100 μ g/ml, component one and component four groups is from left to right
The concentration of component one and component four is followed successively by 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ
G/ml, the columnar data in component three organize from left to right scattered concentration be followed successively by 6.25 μ g/ml,
12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, result shows, resisting of each component
Lipid peroxidation effect rises, the wherein lipotropism matter mistake of component three along with the increase of each concentration of component
Oxidation effectiveness is the strongest, when concentration reaches 100 μ g/ml, lipid peroxidation suppression ratio up to
90.54%, it is secondly component one, reaches as high as 66.32%.The lipotropism matter mistake of this explanation medicinal fungal substance
Active oxide material major part belongs to the material of middle polarity.
Ethanol extract crosses the secondary column chromatography purification result of post component:
By component one, component three, component four through secondary column chromatography after purification, component one has separated out in vain
Color crystal, remaining component does not all obtain pure compound.And component two presents is yellow, therefore it
The inside is likely in addition to white crystal, is mainly composed of the part of yellow.Component is once silica gel column layer
Analysis, is merged into identical thin layer chromatography elution fraction in penicillin bottle, treats that solvent volatilizes gradually to surplus
During subsequent point, separate out white needle-like crystals in having one bottle, by material in this bottle, entered by petroleum ether
Row recrystallization, obtains compound H-1, and thin layer chromatography result is shown in Fig. 6.
Result shows, from component one 90% from the point of view of the thin layer chromatography of the pure compound of isolated,
Its polarity is the least, and from the point of view of form, its white acicular crystal, density is the lightest.Group at eluting
The eluant dividing one includes that the mixing that volume ratio is 9:1:0 of petroleum ether, ethyl acetate and methanol is molten
Liquid.
Compound H-1 Structural Identification:
1, experiment material, instrument and reagent
Material a: compound of isolated in medicinal fungal substance.
Reagent: methanol is chromatographically pure solvent (Tianjin pass the civil service examinations chemical reagent company limited)
Instrument: gas chromatograph-mass spectrometer (HP-5973 hewlette-packard), liquid phase color
Spectrum GC-MS (HP-1100MSD hewlette-packard), nuclear magnetic resonance chemical analyser (INOVA
400MHz Varian company).
2, experimental technique:
The MS of compound H-1 analyzes: compound is dissolved in petroleum ether solution, carries out EI-MS and divide
Analysis.
The H-NMR of compound H-1 and13C-NMR analyzes: compound is dissolved in CD3Cl solution,
Carry out H-NMR and 13C-NMR analysis of spectrum.
3, result and analysis:
The MS of compound H-1 analyzes:
Compound H-1 is dissolved in petroleum ether solution and carries out MS analysis of spectrum, the spectrogram obtained such as Fig. 7.
EI-MSm/z:396(M+),363(M+-H2O-CH3),337(M+-H2O-CH3-C2H2),271(M+
-C9H17),253(M+-C9H17-H20)。
The NMR of compound analyzes:
It is dissolved in CD3Cl solution, carry out H-NMR and13C-NMR analysis of spectrum, the spectrogram obtained such as figure
Shown in 8～10.
1H-NMR (500MzCDCl) δ: 0.63 (3H, s), 0.83 (3H, d, J=6.0Hz), 0.84 (3H, d,
J=6.8Hz), 0.93 (3H, d, J=6.8Hz), 0.94 (3H, s), 1.03 (3H, d, J=6.5Hz), 3.64 (1H,
M), 5.19 (1H, dd, J=15.5Hz, 6.8HZ), 5.22 (1H, dd, J=15.5Hz, 7.0Hz), 5.39 (1H, d
, J=6.OHz), 5.57 (1H, d, J=6.2Hz).By1H-MR data understand, and have 6 in compound structure
Methyl, is the characteristic signal of sterols material
13C-NMR(400MHz,CD3Cl)δ:12.01(C18),16.24(C19),17.57(C28),19.93(C26
),19.93(C27),21.06(Cll),21.06(C11),22.95(C15),28.27(C16),31.93(C2),33.04(C25)
,36.98(C1O),38.32(C1),39.02(C12),40.42(C2O),40.73(C4),42.77(C13),42.77(C24)
,46.18(C9),54.51(C14),55.65(C17),70.43(C3),116.23(C7),119.54(C6),131.92(C2 3),135.53(C22),139.74(C5),141.35(C8).According to Spectrum Analysis result, comparison document is reported
(Wang Jianping, 2010), authenticating compound is ergosterol, and molecular formula is C28H440, molecular weight is
396.34。
The inhibition of lipid peroxidation detection of ergosterol:
Experimental technique: by the white crystal obtained respectively and yellow crystals, examine as product to be tested
Surveying, described white crystal carries out recrystallization by petroleum ether, obtains ergosterol.White by obtain
Color crystal and yellow crystals carry out inhibition of lipid peroxidation detection.
The preparation of testing sample: weigh 0.4mg ergosterol and yellow crystals respectively, use distilled water
Dissolving, be settled to 100mL, this concentration of polymer solution is 8mg/mL.Draw 8mg/mL solution
25,12.5,6.25,3.13,1.56,0.78,0.39mL, be separately added into distilled water and be settled to
25mL, the concentration of sample is respectively 0.5,1,2,4,8mg/mL.
Lipid peroxidation Inhibition test: use egg yolk homogenate (10%, volume fraction) as anti-
The lipid vehicle answered.Take the testing sample of the above-mentioned preparation of 0.1mL with 0.5mL egg yolk homogenate, 0.4
ML pure water and 50 μ L copperas solution (FeSO4·7H2O, 70mmol/L) mixing, incubate for 37 DEG C
Educate 30min, be rapidly added 1.5mL acetic acid solution (20%, volume fraction, pH value 3.5) and
1.5mL thiobarbituricacidα-solution (0.8%, mass concentration, use 1.1% sodium lauryl sulphate
Solution is prepared), mix rear 95 DEG C of water-bath 60min at a high speed.Question response mixture is cooled to room temperature,
Adding 5mL n-butyl alcohol, shake up, mixture 5000r/min is centrifuged 15min, takes supernatant and measures
Its absorbance at 532nm, pure water does blank.
Experimental result: experimental result is shown in Table 1.
Table 1 ergosterol inhibition of lipid peroxidation detects
| Active substance | Lipid peroxidation suppression ratio (%) |
| Compound H-1 (2 μ g/ml) | 57.42±0.00 |
| Yl moiety (2mg/ml) | 1.2±0.00 |
From the data in table 1, it can be seen that ergosterol makes in the medicine prepare inhibition of lipid peroxidation
With.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, right
For those skilled in the art, the present invention can have various modifications and variations.All in the present invention
Spirit and principle within, any modification, equivalent substitution and improvement etc. made, should be included in
Within protection scope of the present invention.
Claims (10)
1. the preparation method of an ergosterol, it is characterised in that include using Fermentation Condition of Monascus spp
The step that sample extracts as raw material.
The preparation method of a kind of ergosterol the most according to claim 1, including following step
Rapid: to take dry Fermentation Condition of Monascus spp sample, add Extraction solvent, use restricted-access media to obtain slightly
Carry product, then use column chromatography eluting slightly to carry product and obtain a purification eluent, to described the purest
Change eluent and carry out silica gel thin-layer chromatography, the eluent of identical silica gel thin-layer chromatography result is merged again
The secondary column chromatography that carries out affords secondarily purified eluent, and described secondarily purified eluent is carried out silicon
Glue thin layer chromatography, removes solvent and passes through after being merged by the eluent of identical silica gel thin-layer chromatography result
Recrystallization solvent carries out being recrystallized to give ergosterol.
The preparation method of a kind of ergosterol the most according to claim 2, it is characterised in that
Described Extraction solvent is ethanol, and described recrystallization solvent is petroleum ether, described Fermentation Condition of Monascus spp
Sample is 1～3:10～12 with the volume ratio of described Extraction solvent.
The preparation method of a kind of ergosterol the most according to claim 3, it is characterised in that
Described ethanol be volume fraction be the ethanol of 90%；Described extraction comprises the following steps: in
12000r·min-1Under the conditions of 60 DEG C extraction 12h.
The preparation method of a kind of ergosterol the most according to claim 2, it is characterised in that
The method of described column chromatography eluting comprises the following steps:
Step one: mix sample: by described slightly carry product or a purification eluent concentrate after the leaching that obtains
Cream, uses solvent fully to dissolve, and addition silica gel stirring to solvent volatilization and the solid absorption separated out exist
On silica gel；
Step 2: dress post: use wet method dress post to obtain silicagel column；
Step 3: loading: after sample step one mixed is poured in silicagel column, adds quartz
Sand, and enter absorbent cotton at capital end plug；
Step 4: eluting: using eluant to carry out eluting, collect eluent, described eluant divides
Do not include pure solution and petroleum ether, ethyl acetate and the methanol of petroleum ether, ethyl acetate and methanol
Mixed solvent.
The preparation method of a kind of ergosterol the most according to claim 5, it is characterised in that
Solvent in described step one is petroleum ether, the described silica gel of addition and described extractum equal-volume；Wet
Method dress post comprises the following steps: weighs silica gel and is suspended from petroleum ether, is stirred continuously and swelling removes bubble removing
After obtain suspension, suspension is at the uniform velocity poured into bottom be plugged with in the chromatographic column of Cotton Gossypii, open bottom valve and make liquid
Body flows out.
The preparation method of a kind of ergosterol the most according to claim 5, it is characterised in that
Described mixed solvent include respectively petroleum ether, ethyl acetate and methanol be followed successively by by volume 9:1:0,
7:2:1、6:3:1、5:4:1、4:4:2、3:6:1、2:7:1、0:9:1、0:7:3、0:5:5、0:3:7、
The mixed solvent of 0:1:9；Each described eluant rinses 5 column volumes.
The preparation method of a kind of ergosterol the most according to claim 7, it is characterised in that
Described eluant is the mixed solution that volume ratio is 9:1:0 of petroleum ether, ethyl acetate and methanol.
The preparation method of a kind of ergosterol the most according to claim 2, it is characterised in that
The described developing solvent in silica gel thin-layer chromatography be volume ratio be petroleum ether and the acetic acid of 12～0.5:1
The mixed solvent of ethyl ester.
10. the purposes of an ergosterol, it is characterised in that described ergosterol is anti-in preparation
The medicine of lipid peroxidation activity uses.
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| CN107460226B (en) * | 2017-08-29 | 2020-10-23 | 江苏师范大学 | Method for producing ergosterol with high yield by fermenting straw through neurospora crassa |
| CN107536722A (en) * | 2017-09-12 | 2018-01-05 | 广州聚注通用技术研究院有限公司 | Balloonflower root phytosterol and preparation method and application with anti-aging and anti-inflammatory activity |
| CN108558977A (en) * | 2018-05-08 | 2018-09-21 | 刘健 | A method of extracting ergosterol from amylofermentation object |
| CN110669092A (en) * | 2019-10-25 | 2020-01-10 | 宿迁医美科技有限公司 | Method for extracting ergosterol from vinasse |
| CN110669092B (en) * | 2019-10-25 | 2022-04-05 | 宿迁医美科技有限公司 | Method for extracting ergosterol from vinasse |
| CN112390842A (en) * | 2020-12-21 | 2021-02-23 | 天津科技大学 | Preparation method of ergosterol |
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