CN107964031A - One kind extracts separated triterpene compound and its methods and applications from rhizoma alismatis - Google Patents
One kind extracts separated triterpene compound and its methods and applications from rhizoma alismatis Download PDFInfo
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Abstract
Separated triterpene compound and its methods and applications are extracted from rhizoma alismatis the invention discloses one kind, its structural formula is such as（Ⅰ）It is shown.The compound is the stem tuber using rhizoma alismatis drying as raw material, extracted through alcohol reflux, ethyl acetate extraction, silica gel column chromatography separation, the extraction of the processing step such as C18 reverse-phase chromatography filler high pressure preparative separations it is isolated, for a kind of new medicinal compound with pharmacological activity, there is good inhibition to staphylococcus aureus, reliable foundation can be provided as the application in antibacterials are prepared, and to prepare novel antibacterial medicine.
Description
Technical field
The invention belongs to technical field of phytochemistry, and in particular to one kind is extracted from rhizoma alismatis separated has pharmacological activity
Triterpene compound and its method and purposes.
Background technology
Rhizoma alismatis is Notes On Alism At Aceae rhizoma alismatisAlisma orientalis （Sam.）Juzep.Dry tuber.Rhizoma alismatis is at me
Traditional Chinese medicines history more than 3000 years existing.The collection of poems before more than 3,000 years《The Book of Songs》, wherein the 3rd section of " preventing gal in Fen " writes：
" that confused song, speech adopt its Mai, that its son is beautiful as beautiful." according to Lu Gong《Poem is dredged》Explain：" speech adopts its Mai, Mai, modern rhizoma alismatis.Its
Leaf such as Asiatic plantain is big, its taste is also similar.Xuzhou, people from Guanling eat it." work such as modern times thorough, Zhao Jun of scholar Zhu Min《Book of Songs annotation of translation》
Will " speech adopts its Mai " and is translated into Modern Chinese " I adopts rhizoma alismatis into basket." written by Chen Zizhan《The Book of Songs is literal translated》, it is translated into and " just adopts that
In be the Mai of rhizoma alismatis." more than the note of scholar, translation at all times, it is exactly rhizoma alismatis all to think Mai.Women just adopts before illustrating more than 3,000 years
Collection oriental waterplantain leaf is eaten.During edible, it is found that it has medical function, just by people as medicine.
The earliest herbal works in China is《Sheng Nong's herbal classic》, it is medicine that it records rhizoma alismatis first, is listed as top grade.After
History tree works has also all recorded rhizoma alismatis, such as the early Medieval China epoch《Mingyi Bielu》、《Bencao jizhu》, the Tang Dynasty
《Tang materia medica》, the Song Dynasty《Bencao Tujing》、《The spare book on Chinese herbal medicine of class is demonstrate,proved through history》、《Amplification on Materia Medica》, the Yuan Dynasty《The Decoction and Material Medica》, the Ming Dynasty
's《Bencao Pinhui Jingyao》、《Compendium of Materia Medica》、《It is brave that book on Chinese herbal medicine multiplies refined half》、《Book on Chinese herbal medicine covers label》, the Qing Dynasty《Herbal true way》、《This warp
Dredge card》、《Book on Chinese herbal medicine must be matched somebody with somebody》、《The new compilation of materia medica》Deng.They be all《Sheng Nong's herbal classic》On the basis of enrich, correct, it is perfect,
And rhizoma alismatis is set to become common Chinese medicine.
Rhizoma alismatis has effects that clearing damp and promoting diuresis, and modern medicine study shows, rhizoma alismatis can reduce serum total cholesterol and trigalloyl
Glycerol content, slows down atherogenesis；Rhizoma alismatis and its preparation, the modern times be additionally operable to treatment auditory vertigo, dyslipidemia,
Seminal emission, fatty liver and diabetes etc..
Modern pharmacy research confirms that rhizoma alismatis stem tuber is containing alisol A, B, C, alisol A monoacetate, alisol B list acetic acid
Ester, alisol C monoacetic ester, epialisol A, rhizoma alismatis Azulene alcohol, rhizoma alismatis Azulene alcohol oxide, 16 'beta '-methoxy alisol B list acetic acid
The various actives such as ester, 16 beta-hydroxy Alisol B 23-monoacetates, sitosterol -3-O- stearyl-β-D- glucopyanosides into
Point, while also containing choline, polysaccharide, and the element such as potassium, calcium, magnesium.It is but most of at present for the further research of rhizoma alismatis medicine
The extracting and developing to various known activity components, purifying etc. are all concentrated on, Patents documents are such as：
CN101724009A provides a kind of extraction and purification process of triterpenoids from oriental waterplantain rhizome, and the present invention is by 5 kinds of macropores
The absorption of resin and the comparison of desorption properties are adsorbed, filters out the optimum resin of triterpenoid in absorption rhizoma alismatis, i.e. HPD-100
Type macroporous absorbent resin.Dissolved before separating technology loading of the present invention using 50% ethanol, make the adsorbance of resin not reduce, this hair
Bright 80% ethanol solution that discloses substantially can be complete as a result, originally triterpenoids from oriental waterplantain rhizome and Alisol B monoacetate elution
Elution solution concentration in invented technology selects 80% ethanol, thus present invention process substantially can triterpenoids from oriental waterplantain rhizome and
Alisol B monoacetate elution is complete.Present invention process has considered triterpenoids from oriental waterplantain rhizome content and 23- acetyl rhizoma alismatis
The content two indices of alcohol B, make extraction and purification process more reasonable, more science.
CN102372759A provides a kind of method of purification for polluting the alisol A small, cost is low, and process is：Pool
Pulverizing medicinal materials, the extraction of 70-90% alcohol refluxs are rushed down, after extracting solution adds activated carbon decolorizing recycling ethanol, concentrate is at 60-80 DEG C
State adds macroporous resin adsorption, and water alcohol gradient elution, is concentrated to give sediment, wash crystallization, crystal is again through high-speed counter-current color
Spectrometer separates, and selects the solvent system of one dichloromethane-acetonitrile of heptane, and product is made.Alisol A, institute are produced using the present invention
Obtain product purity height, high income.
CN102464694A discloses a kind of method for efficiently separating and preparing Alisol B monoacetate.With SCF-CO 2
Total-sterol extract in body extraction rhizoma alismatis, then carries out extract using high speed adverse current chromatogram further to separate and purify；
Finally refined using the method for recrystallization, obtain the Alisol B monoacetate of high-purity.This method step is simple, efficiency
Height, is easy to repeat, and suitable for industrialized production, the Alisol B monoacetate purity of preparation is separated up to more than 99%, in can be used for
Medicine rhizoma alismatis Quality Control reference substance and bulk pharmaceutical chemicals.
CN104497092A provides a kind of method that 23- acetylalisols C is extracted from rhizoma alismatis, mainly solves existing
Complicated in technology, product content is low, and the rate of recovery is low.The method that 23- acetylalisols C is extracted from rhizoma alismatis includes following
Step：1] extract；2] UF membrane；3] clean；4] crystallize.The method that 23- acetylalisols C is extracted from rhizoma alismatis uses buck
Wetting, can remove the impurity molecule of the big polarity in part；Easy to operate, product content is high, and the rate of recovery is high, is suitable for industrial metaplasia
Production.
Since the compound of plant origin is to finding that new drug has huge potentiality, the present inventor lives to rhizoma alismatis
Property component find again and isolated a kind of new medicinal compound when being studied, which is triterpene compound, tool
There is certain antibacterial activity, especially to staphylococcus aureusS.aureusHave with good inhibition.It is as it can be seen that right at present
Contained chemical active ingredient still has the necessity further researched and developed and inquired into rhizoma alismatis.
The content of the invention
Separated triterpene compound is extracted from rhizoma alismatis it is an object of the present invention to providing one kind, by from rhizoma alismatis
The isolated new medicinal compound with pharmacological activity is extracted in medicinal material, has further excavated the new pharmacologic action of rhizoma alismatis,
And provide good reference value to prepare novel antibacterial medicine.
The second object of the present invention in provide it is a kind of from rhizoma alismatis extraction separation triterpene compound method, it be with
Rhizoma alismatis dry tuber is raw material, is extracted through alcohol reflux, ethyl acetate extracts, silica gel column chromatography separates, C18 reverse-phase chromatography fillers
High pressure preparative separation and etc. obtain a kind of new medicinal compound with pharmacological activity, this method operating procedure is simply easy to
Control, it can be ensured that the recovery rate of triterpene compound more than 60%, purity up to more than 99%, and whole production procedure take it is short,
Suitable for industrialized production.
The third object of the present invention is extracting separated triterpene compound in preparation antibacterial in offer one kind from rhizoma alismatis
Application in medicine, reliable foundation is provided to prepare novel antibacterial medicine.
Realize foregoing invention purpose, the technical solution adopted by the present invention is as follows：
One kind extracts separated triterpene compound from rhizoma alismatis, and the triterpene compound has（Ⅰ）Shown structural formula：
The triterpene compound is to extract to be isolated to its chemical name is from the stem tuber of rhizoma alismatis drying：
(1S,3R)-1-((R)-3,3-dimethyloxiran-2-yl)-3-((5R,8S,9S,10S,11S,14R)-11-
hydroxy-4,4,8,10,14-pentamethyl-3-oxo-2,3,4,5,6,7,8,9,10,11,12,14-
Dodecahydro-1H-cyclopenta [a] phenanthren-17-yl) butyl acetate, it is named as certainly：Dehydrogenation 23-
Acetylalisol B.
The triterpene compound is white powder, and 10% sulfuric acid ethanol chromogenic reagent is in aubergine.
The 10% sulfuric acid ethanol is universal visualization agent, its collocation method is：The concentrated sulfuric acid and absolute ethyl alcohol by volume 1:9,
The concentrated sulfuric acid is poured into absolute ethyl alcohol after mixing, is 10% sulfuric acid ethanol color developing agent.
The molecular weight of the triterpene compound is 512, molecular formula C32H48O5。
A kind of method of the extraction separation triterpene compound from rhizoma alismatis, comprises the following steps that：
A. the stem tuber that rhizoma alismatis is dried is crushed, the ethanol heating and refluxing extraction for being 95wt% with concentration, is concentrated under reduced pressure into no alcohol
Afterwards, then add water to carry out decentralized processing concentrated extracting solution and obtain aqueous dispersion；
B. aqueous dispersion is extracted with ethyl acetate, recycling design obtains acetic acid ethyl ester extract and aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with methanol, then using silica gel as carrier, after dry, wet method loading, uses silica gel
Pillar layer separation, collects object chromatographic solution, is concentrated under reduced pressure after doing, is dissolved with ethyl acetate, filters, and after adding petroleum ether, puts
Crystallization is put, obtains crystalline solid；
D. crystalline solid is dissolved with methanol, filtered, filtrate is corresponding with C18 reverse-phase chromatography filler high pressure preparative separations, collection
Chromatographic peak, concentration collection liquid obtain white powder product to dry, i.e.,（Ⅰ）The triterpene compound of shown structural formula.
In step, the dosage of the ethanol is 8~10 times of weight of the rhizoma alismatis dry tuber after crushing.
In step, the refluxing extraction be 3~5 times, every time 2 it is small when.
In step, the concentrated extracting solution by volume 1:5~1:10 extraordinarily water carry out decentralized processing.
In stepb, the aqueous dispersion is extracted 3~5 times with isometric ethyl acetate.
In step C, the silica gel column chromatography separation：A：Petroleum ether B：Ethyl acetate, A:B =5:1~2:1（V/V）For
Mobile phase.
In step D, the C18 reverse-phase chromatographies filler high pressure prepares liquid phase separation：A：Methanol B：Water, A:B =85:15
V/V is mobile phase；Detection wavelength 210nm.
The white powder product that the present invention obtains, 10% sulfuric acid ethanol chromogenic reagent are in aubergine, i.e., the compound is three
Terpenoid.
On this basis, further analysis result is as follows：
（Ⅰ）The electrospray ionization mass spectrometry ESI-MS of the triterpene compound of shown structural formula is shown：535.12 [M+Na of cation
]+, 1047.31 [2M+Na]+；Anion 511.20 [M-H]-, i.e. the compound molecular weight is 512, molecular formula C32H48O5。1H-NMR and13C-NMR data see the table below 1.
1 formula of table（Ⅰ）The triterpene compound nuclear magnetic data of shown structural formula
Pass through1H、13C-NMR and DEPT135o and nuclear-magnetism two dimension HSQC, HMBC, H-HCOSY, NOESY etc. analyze technological means,
It is determined that the compound is：Dehydrogenation Alisol B monoacetate（Triterpene compound）, structural formula is such as（Ⅰ）It is shown.
Through deeper into research show that the triterpene compound has certain antibacterial activity, to Staphylococcus aureus
BacteriumS.aureusThere is good inhibiting effect, can be as the application in antibacterials are prepared.
Advantages of the present invention and beneficial effect are：
1st, it is provided by the invention（Ⅰ）The triterpene compound of shown structural formula is to extract to be isolated from the stem tuber of rhizoma alismatis drying
Arrive, which determines that pharmacological activity is clear and definite, has certain antibacterial activity, to staphylococcus aureus, large intestine bar
Bacterium, hay bacillus etc. have certain inhibitory action.
2nd, modern pharmacology Effect study shows：Triterpene compound has extensive physiological activity, have haemolysis, anticancer,
Anti-inflammatory, antibacterial, it is antiviral, reduce cholesterol, kill mollusk isoreactivity.And the present invention is separated by being extracted from Rhizoma Alismatis
Obtained triterpene compound, is a kind of new medicinal compound, and antibacterial pharmacological testing shows that it is to resistant Staphylococcus grape
The inhibition of coccus is suitable with broad-spectrum antibiotic kanamycin sulfate.Thus the new pharmacologic action of rhizoma alismatis has further been excavated,
There is good reference value for preparing novel antibacterial medicine.
3rd, the present invention is extracted, ethyl acetate extracts, silica gel column chromatography using rhizoma alismatis dry tuber as raw material using alcohol reflux
The modes such as separation, C18 reverse-phase chromatography filler high pressure preparative separations obtain triterpene compound, the compound extraction separation easily,
Be easy to get, method is simply controllable, and whole production procedure take it is short, suitable for industrialized production.
4th, the present invention not only reports the structure of dehydrogenation Alisol B monoacetate first, and according to correlations such as nuclear-magnetism two dimensions
Data determine the noval chemical compound of its relative configuration, and pharmacological research shows that it has staphylococcus aureus and suppresses to imitate well
Fruit, in terms of development of new antibacterials, can develop and utilize as a kind of potential compound.
Embodiment
The present invention is described in further detail with reference to embodiment.But it is necessarily pointed out that following embodiments
It is not intended that limiting the scope of the invention, if what person skilled in art made according to the invention described above content
Some nonessential modifications and adaptations, still fall within protection scope of the present invention.
Embodiment 1
One kind extracts separated triterpene compound from rhizoma alismatis, which is white powder, and 10% sulfuric acid ethanol color developing agent is shown
Color is in aubergine, molecular weight 512, molecular formula C32H48O5, have（Ⅰ）Shown structural formula：
The triterpene compound is to extract to be isolated to concrete technology step is as follows from the stem tuber of rhizoma alismatis drying：
A. the stem tuber that rhizoma alismatis is dried is crushed, the ethanol heating and refluxing extraction for being 95wt% with concentration, is concentrated under reduced pressure into no alcohol
Afterwards, then add water to carry out decentralized processing concentrated extracting solution and obtain aqueous dispersion；
B. aqueous dispersion is extracted with ethyl acetate, recycling design obtains acetic acid ethyl ester extract and aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with methanol, then using silica gel as carrier, after dry, wet method loading, uses silica gel
Pillar layer separation, collects object chromatographic solution, is concentrated under reduced pressure after doing, is dissolved with ethyl acetate, filters, and after adding petroleum ether, puts
Crystallization is put, obtains crystalline solid；
D. crystalline solid is dissolved with methanol, filtered, filtrate is corresponding with C18 reverse-phase chromatography filler high pressure preparative separations, collection
Chromatographic peak, concentration collection liquid obtain white powder product to dry, i.e.,（Ⅰ）The triterpene compound of shown structural formula.
Embodiment 2
A kind of method of the extraction separation triterpene compound from rhizoma alismatis, concrete technology step are as follows：
A. the stem tuber that rhizoma alismatis is dried is crushed, the ethanol heating and refluxing extraction 3 times for being 95wt% with 8 times of concentration of its weight, often
It is secondary 2 it is small when, after being concentrated under reduced pressure into no alcohol, then by concentrated extracting solution by volume 1:5, which add water progress decentralized processing to obtain moisture, dissipates
Body；
B. the isometric ethyl acetate of aqueous dispersion is extracted 3 times, recycling design obtains acetic acid ethyl ester extract and aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with methanol, then using silica gel as carrier, after dry, wet method loading, uses silica gel
Pillar layer separation（A：Petroleum ether B：Ethyl acetate, A:B =5:1 V/V is mobile phase）, object chromatographic solution is collected, decompression is dense
After contracting is dry, dissolved, filtered with ethyl acetate, after adding petroleum ether, placed crystallization, obtain crystalline solid；
D. crystalline solid is dissolved with methanol, filtered, filtrate is with C18 reverse-phase chromatography filler high pressure preparative separations（A：Methanol B：
Water, A:B =85:15 V/V are mobile phase；Detection wavelength 210nm）, collect corresponding chromatographic peak, concentration collection liquid is to dry
To white powder product, i.e.,（Ⅰ）The triterpene compound of shown structural formula.
Embodiment 3
A kind of method of the extraction separation triterpene compound from rhizoma alismatis, concrete technology step are as follows：
A. the stem tuber that rhizoma alismatis is dried is crushed, the ethanol heating and refluxing extraction 5 times for being 95wt% with 10 times of concentration of its weight,
Every time 2 it is small when, after being concentrated under reduced pressure into no alcohol, then by concentrated extracting solution by volume 1:8, which add water to carry out decentralized processing, obtains moisture
Granular media；
B. the isometric ethyl acetate of aqueous dispersion is extracted 5 times, recycling design obtains acetic acid ethyl ester extract and aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with methanol, then using silica gel as carrier, after dry, wet method loading, uses silica gel
Pillar layer separation（A：Petroleum ether B：Ethyl acetate, A:B =2:1 V/V is mobile phase）, object chromatographic solution is collected, decompression is dense
After contracting is dry, dissolved, filtered with ethyl acetate, after adding petroleum ether, placed crystallization, obtain crystalline solid；
D. crystalline solid is dissolved with methanol, filtered, filtrate is with C18 reverse-phase chromatography filler high pressure preparative separations（A：Methanol B：
Water, A:B =85:15 V/V are mobile phase；Detection wavelength 210nm）, collect corresponding chromatographic peak, concentration collection liquid is to dry
To white powder product, i.e.,（Ⅰ）The triterpene compound of shown structural formula.
Embodiment 4
A kind of method of the extraction separation triterpene compound from rhizoma alismatis, concrete technology step are as follows：
A. the stem tuber that rhizoma alismatis is dried is crushed, the ethanol heating and refluxing extraction 4 times for being 95wt% with 9 times of concentration of its weight, often
It is secondary 2 it is small when, after being concentrated under reduced pressure into no alcohol, then by concentrated extracting solution by volume 1:10, which add water progress decentralized processing to obtain moisture, dissipates
Body；
B. the isometric ethyl acetate of aqueous dispersion is extracted 4 times, recycling design obtains acetic acid ethyl ester extract and aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with methanol, then using silica gel as carrier, after dry, wet method loading, uses silica gel
Pillar layer separation（A：Petroleum ether B：Ethyl acetate, A:B =3:1 V/V is mobile phase）, object chromatographic solution is collected, decompression is dense
After contracting is dry, dissolved, filtered with ethyl acetate, after adding petroleum ether, placed crystallization, obtain crystalline solid；
D. crystalline solid is dissolved with methanol, filtered, filtrate is with C18 reverse-phase chromatography filler high pressure preparative separations（A：Methanol B：
Water, A:B =85:15 V/V are mobile phase；Detection wavelength 210nm）, collect corresponding chromatographic peak, concentration collection liquid is to dry
To white powder product, i.e.,（Ⅰ）The triterpene compound of shown structural formula.
The extraction separation of 5 compound of embodiment
A. the stem tuber 10kg for taking rhizoma alismatis to dry, after crushing with 8 times of weight concentrations for 95% ethanol heating and refluxing extraction 3 times, often
It is secondary 2 it is small when, after being concentrated under reduced pressure into no alcohol, concentrated extracting solution 3.5Kg is obtained, by obtained concentrated extracting solution by volume 1:8 extraordinarily
Water carries out decentralized processing；
B. by aqueous dispersion successively with isometric ethyl acetate extract 4 times, recycling design obtain acetic acid ethyl ester extract 1000g and
Aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with 800ml methanol, then using silica gel as carrier, after dry, wet method loading,
Separated with silica gel column chromatography, A：Petroleum ether B：Ethyl acetate, A:B= 2:1 V/V is mobile phase, collects object chromatographic solution, subtracts
After pressure concentration is dry, dissolved with ethyl acetate 60ml, filtering, after adding 300ml petroleum ethers, place crystallization, obtain crystalline solid 3g；
D. crystalline solid is dissolved with methanol, filtered, filtrate is with C18 reverse-phase chromatography filler high pressure preparative separations, A：Methanol B：
Water, A:B= 85:15 V/V are mobile phase；Detection wavelength 210nm, collects corresponding chromatographic peak, and concentration collection liquid is obtained to dry
（Ⅰ）The triterpene off-white powder compound 2.3g of shown structural formula.
Whole about 5 days production procedure used times；
By replacing mobile phase composition, inverse analysis type liquid chromatogram is utilized（RP-HPLC）Product purity is rechecked, measuring result is
99.15%。
The extraction separation of 6 compound of embodiment
A. dry rhizoma alismatis stem tuber 10kg is taken, with the ethanol heating and refluxing extraction 5 times that 10 times of weight concentrations are 95% after crushing, often
It is secondary 2 it is small when, after being concentrated under reduced pressure into no alcohol, concentrated extracting solution 4Kg is obtained, by obtained concentrated extracting solution by volume 1:10 extraordinarily water
Carry out decentralized processing；
B. by aqueous dispersion successively with isometric ethyl acetate extract 3 times, recycling design obtain acetic acid ethyl ester extract 1200g and
Aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with 1000ml methanol, then using silica gel as carrier, after dry, wet method loading,
Separated with silica gel column chromatography, A：Petroleum ether B：Ethyl acetate, A:B= 5:1 V/V is mobile phase, collects object chromatographic solution, subtracts
After pressure concentration is dry, it is concentrated under reduced pressure after doing, is dissolved with ethyl acetate 80ml, filtering, after adding 400ml petroleum ethers, places and crystallize, obtain
To crystalline solid 4g；
D. crystalline solid is dissolved with methanol, filtered, filtrate is with C18 reverse-phase chromatography filler high pressure preparative separations, A：Methanol B：
Water, A:B= 85:15 V/V are mobile phase；Detection wavelength 210nm, collects corresponding chromatographic peak, and concentration collection liquid is obtained to dry
（Ⅰ）The triterpene off-white powder compound 3g of shown structural formula.
Whole about 7 days production procedure used times；
By replacing mobile phase composition, inverse analysis type liquid chromatogram is utilized（RP-HPLC）Product purity is rechecked, measuring result is
99.45%。
The extraction separation of 7 compound of embodiment
A. dry rhizoma alismatis stem tuber 10kg is taken, with the ethanol heating and refluxing extraction 4 times that 9 times of weight concentrations are 95% after crushing, often
It is secondary 2 it is small when, after being concentrated under reduced pressure into no alcohol, concentrated extracting solution 3.2Kg is obtained, by obtained concentrated extracting solution by volume 1:5 extraordinarily
Water carries out decentralized processing；
B. by aqueous dispersion successively with isometric ethyl acetate extract 4 times, recycling design obtain acetic acid ethyl ester extract 1100g and
Aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with 900ml methanol, then using silica gel as carrier, after dry, wet method loading,
Separated with silica gel column chromatography, A：Petroleum ether B：Ethyl acetate, A:B= 3:1 V/V is mobile phase, collects object chromatographic solution, subtracts
After pressure concentration is dry, it is concentrated under reduced pressure after doing, is dissolved with ethyl acetate 70ml, filtering, after adding 350ml petroleum ethers, places and crystallize, obtain
To crystalline solid 3.2g；
D. crystalline solid is dissolved with methanol, filtered, filtrate is with C18 reverse-phase chromatography filler high pressure preparative separations, A：Methanol B：
Water, A:B= 85:15 V/V are mobile phase；Detection wavelength 210nm, collects corresponding chromatographic peak, obtains（Ⅰ）Shown structural formula
Triterpene off-white powder compound 2.4g.
Whole about 6 days production procedure used times.
By replacing mobile phase composition, inverse analysis type liquid chromatogram is utilized（RP-HPLC）Product purity is rechecked, measures knot
Fruit is 99.25%.
8 antifungal activity of embodiment is tested
First, experiment material
1.1 medicine
The extraction of above-described embodiment 5~7 is isolated（Ⅰ）The triterpene off-white powder compound of shown structural formula.
1.2 human body pathogens
Staphylococcus aureus（Staphylococcus aureus）ATCC 51650 is provided by Hainan provincial institute for drug control.
1.3 culture medium
Beef extract-peptone agar medium（NA）：Beef extract 5g, NaCl 7g, peptone 15g, are settled to 1000ml, pH is
7.4~7.6.
2nd, experimental method
Filter paper agar diffusion method measures the antibacterial activity of compound；S.aureusUsing NA culture mediums.
2.1 willS.aureusCertain density bacteria suspension is made（105~107cfu/ml）, take 100 μ l cotton swabs uniform
It is coated on for examination aseptic flat board, tablet containing bacterium is made.
Above-mentioned compound to be determined is configured to lmg/50 μ l solution by 2.2, takes 20 μ l to be added dropwise respectively in sterilizing filter paper piece
On（Φ=6mm）, filter paper is affixed on tablet containing bacterium after solvent volatilization is dry, often processing is repeated 3 times.
2.3 positive control solutions are kanamycin sulfate 0.08mg/ml（10/μl）, cultivate at 37 DEG C, knot observed after 24h
Fruit, measures and records antibacterial circle diameter.
3rd, experimental result
The extraction of embodiment 5~7 is isolated（Ⅰ）Triterpene off-white powder compound, kanamycin sulfate, the first of shown structural formula
Alcohol（Negative control）, they are for staphylococcus aureusS.aureusAntibacterial activity the results detailed in Table 2.
2 antibacterial activity result of table
| Numbering | Title | Concentration（mg/μl） | Staphylococcus aureus antibacterial circle diameter（mm） |
| 1 | Embodiment 5 | 1mg/50μl | 11 |
| 2 | Embodiment 6 | 1mg/50μl | 13 |
| 3 | Embodiment 7 | 1mg/50μl | 12 |
| 4 | Kanamycin sulfate | 1mg/50μl | 31~33 |
| 5 | Methanol | 1mg/50μl | 0 |
Test result indicates that the triterpene compound is to staphylococcus aureusS.aureusThere is certain inhibitory action, can
As the application in antibacterials are prepared.
Claims (10)
1. one kind extracts separated triterpene compound from rhizoma alismatis, it is characterised in that：The triterpene compound has（Ⅰ）Institute
The structural formula shown：
Its chemical name is：
(1S,3R)-1-((R)-3,3-dimethyloxiran-2-yl)-3-((5R,8S,9S,10S,11S,14R)-11-
hydroxy-4,4,8,10,14-pentamethyl-3-oxo-2,3,4,5,6,7,8,9,10,11,12,14-
Dodecahydro-1H-cyclopenta [a] phenanthren-17-yl) butyl acetate, it is named as certainly：Dehydrogenation 23-
Acetylalisol B.
2. separated triterpene compound is extracted from rhizoma alismatis as claimed in claim 1, it is characterised in that：The triterpenes
Compound is white powder, and 10% sulfuric acid ethanol chromogenic reagent is in aubergine.
3. separated triterpene compound is extracted from rhizoma alismatis as claimed in claim 1 or 2, it is characterised in that：The triterpene
The electrospray ionization mass spectrometry ESI-MS of class compound is shown：Cation 535.12 [M+Na]+, 1047.31 [2M+Na]+；Anion
511.20[M-H]-, i.e. the compound molecular weight is 512, molecular formula C32H48O5。
4. prepare the method for the extraction separation triterpene compound from rhizoma alismatis as claimed in claim 1 or 2, it is characterised in that：
Comprise the following steps that：
A. the stem tuber that rhizoma alismatis is dried is crushed, the ethanol heating and refluxing extraction for being 95wt% with concentration, is concentrated under reduced pressure into no alcohol
Afterwards, then add water to carry out decentralized processing concentrated extracting solution and obtain aqueous dispersion；
B. aqueous dispersion is extracted with ethyl acetate, recycling design obtains acetic acid ethyl ester extract and aqueous portion；
C. acetic acid ethyl ester extract is completely dissolved with methanol, then using silica gel as carrier, after dry, wet method loading, uses silica gel
Pillar layer separation, collects object chromatographic solution, is concentrated under reduced pressure after doing, is dissolved with ethyl acetate, filters, and after adding petroleum ether, puts
Crystallization is put, obtains crystalline solid；
D. crystalline solid is dissolved with methanol, filtered, filtrate is corresponding with C18 reverse-phase chromatography filler high pressure preparative separations, collection
Chromatographic peak, concentration collection liquid obtain white powder product to dry, i.e.,（Ⅰ）The triterpene compound of shown structural formula.
5. the method that extraction separates triterpene compound from rhizoma alismatis as claimed in claim 4, it is characterised in that：In step A
In, the dosage of the ethanol is 8~10 times of weight of the rhizoma alismatis dry tuber after crushing；The refluxing extraction is 3~5 times, often
It is secondary 2 it is small when.
6. the method that extraction separates triterpene compound from rhizoma alismatis as claimed in claim 4, it is characterised in that：In step A
In, the concentrated extracting solution by volume 1:5~1:10 extraordinarily water carry out decentralized processing.
7. the method that extraction separates triterpene compound from rhizoma alismatis as claimed in claim 4, it is characterised in that：In step B
In, the aqueous dispersion is extracted 3~5 times with isometric ethyl acetate.
8. the method that extraction separates triterpene compound from rhizoma alismatis as claimed in claim 4, it is characterised in that：In step C
In, the silica gel column chromatography separation：A：Petroleum ether B：Ethyl acetate, A:B =5:1~2:1 volume ratio is mobile phase.
9. the method that extraction separates triterpene compound from rhizoma alismatis as claimed in claim 4, it is characterised in that：In step D
In, the C18 reverse-phase chromatographies filler high pressure prepares liquid phase separation：A：Methanol B：Water, A:B =85:15 V/V are mobile phase；Inspection
Survey wavelength 210nm.
10. a kind of extract separated triterpene compound in antibacterials are prepared from rhizoma alismatis as claimed in claim 1
Using.
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