CN106674311B - A kind of benzofuran glycosides compound and its preparation method and application - Google Patents
A kind of benzofuran glycosides compound and its preparation method and application Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C07H1/08—Separation; Purification from natural products
Abstract
The present invention relates to a kind of benzofuran glycosides compounds and its preparation method and application, take the dry root of Xingan's Radix Angelicae Pubescentis to carry out refluxing extraction and extraction, obtain extracting n-butyl alcohol layer；Extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, carries out gradient elution, by effluent volume than merging respectively for the fraction of 10:1 and 8:1, solvent is removed, obtains sample a1 and b1；Sample a1 and b1 are splined on reversed-phase silica gel chromatography column respectively, using methanol-water system as eluent, carry out gradient elution, by effluent volume than the fraction merging for 100:0, remove solvent, obtain sample a2 and b2；Sample a2 and b2 are splined on high performance liquid chromatography separation column respectively, carry out isocratic elution with mobile phase, obtain benzofuran glycosides compound.The compound of the present invention has good anti-inflammatory effect, is expected to develop into new anti-inflammatory drug or be used to prepare with the health food for preventing and treating inflammation.
Description
[technical field]
The invention belongs to medicine and field of health care food, and in particular to a kind of benzofuran glycosides compound and its preparation side
Method and application.
[background technique]
Inflammation is a kind of defense reaction of the body for stimulation, is to have the living tissue of vascular system to damage factor institute
The defense reaction of generation.It is broadly divided into acute inflammation and two kinds of chronic inflammation.The substance for participating in inflammatory reaction has histamine, 5- hydroxyl color
The amine substances such as amine, the polypeptides such as bradykinin, kallidin, fibrin lyase, kallikrein protein enzyme etc..
A variety of diseases such as inflammation and artery sclerosis, heart disease, apoplexy, cancer, diabetes are closely related, are to cause a variety of diseases
The key factor of disease.Therefore, have the characteristics that the constantly research and development of the anti-inflammatory drug of high-efficiency low-toxicity are particularly significant.
The plant of nature and the secondary metabolite of microorganism are always the important source of new drug discovery, because natural produce
Object has important structure diversity and diverse biological activities, and the structure of many compounds is unpredictable in advance and is difficult to use
Synthetic method obtains.Although more and more new drugs are chemical drugs now, but still have phase with the development of synthetic pharmacochemistry
When the new drug of ratio is directly or indirectly from natural products.Therefore, natural products is still that new drug development in the process can not
An or scarce important compound treasure-house.
Xingan's Radix Angelicae Pubescentis (Heracleum dissectum) is distributed mainly on the Northeast, is that composite family ox radix saposhnikoviae belongs to perennial
Herbaceous plant, Hubei Province language are known as Kan Kula, the wild plant of dual-purpose of drug and food Kan Kula important as the Oroqen, spring and summer season its
Tender cauline leaf is used as delicious vegetables by locals and eats, and root is as traditional Hubei Province race medicinal material for dispelling wind and eliminating dampness, analgesic
Antidiarrheal etc..Therefore, Kan Kula is considered as jewellery by the Olunchuns, just stews the reception of robe meat with Kan Kula when only honored guest arrives
Guest.But up to the present few to the research of its chemical component, also parmacodynamics-less activity research is reported.
[summary of the invention]
It is an object of the invention to overcome problems of the prior art, provide a kind of benzofuran glycosides compound and
A kind of new benzofuran glycosides compound can be made, for anti-inflammatory in preparation method and application.
In order to achieve the above object, the present invention adopts the following technical scheme: its molecular formula is C23H30O13Or C22H28O13。
Further, general structure are as follows:
Wherein, R is-H or-CH3。
Preparation method of the present invention, comprising the following steps:
1) take Xingan's Radix Angelicae Pubescentis dry root carry out refluxing extraction several times, extracting solution is concentrated under reduced pressure, obtain total medicinal extract or
Concentrate；
2) total medicinal extract is suspended in water, obtains medicinal extract liquid, medicinal extract liquid or concentrate after ethyl acetate extraction several times
Organic layer is isolated, combining extraction liquid after remaining water layer passes through extracting n-butyl alcohol several times is removed under reduced pressure n-butanol and obtains positive fourth
Alcohol extract layer；
3) extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, by volume (100:
0) gradient elution~(0:100) is carried out, efflux is detected, by effluent volume than the fraction merging for 10:1, is removed
Solvent obtains sample a1；By effluent volume than the fraction merging for 8:1, solvent is removed, sample b1 is obtained；
4) sample a1 is splined on reversed-phase silica gel chromatography column, using methanol-water system as eluent, by volume (0:
100) gradient elution~(100:0) is carried out, by effluent volume than the fraction merging for 100:0, solvent is removed, obtains sample
a2；
Sample b1 is splined on reversed-phase silica gel chromatography column, using methanol-water system as eluent, by volume (0:100)
~(100:0) carries out gradient elution, by effluent volume than the fraction merging for 70:30, removes solvent, obtains sample b2；
5) sample a2 and sample b2 are splined on high performance liquid chromatography separation column respectively, carry out isocratic elution with mobile phase,
Obtain benzofuran glycosides compound.
Further, the ethyl alcohol in the refluxing extraction of step 1) using methanol, water or volume fraction 10~95%, which is used as, mentions
Agent is taken, the dry root of Xingan's Radix Angelicae Pubescentis and the mass volume ratio of extractant are 1kg:(1~8) L；When extractant is methanol or volume point
When the ethyl alcohol of number 10~95%, solvent recovery in obtained extracting solution is obtained into total medicinal extract；When extractant is water, by extracting solution
Volume concentration to 1/10 to ten/6ths, obtain concentrate.
Further, extraction time is 1~6 time, every time 1~4 hour in step 1).
Further, the volume ratio of total medicinal extract and water is 1:1~1:5 in step 2).
Further, medicinal extract liquid or concentrate are directly over ethyl acetate extraction in step 2)；Or first successively through petroleum
After ether and chloroform extraction, extracted using ethyl acetate；Extraction is equal-volume extraction every time；Every kind of solvent extracts 1~6 respectively
It is secondary.
Further, every 300~800mL collects a fraction after gradient elution in step 3)；Gradient elution in step 4)
Every 200~500mL collects a fraction afterwards；It is to carry out TLC detection to efflux in step 3) and step 4)；Step 5) is medium
The flow velocity of degree elution is 3~6mL/min.
Further, mobile phase is -0.5% acetic acid water system of -0.5% acetic acid water system of methanol or acetonitrile in step 5)；
When carrying out isocratic elution to sample a2, the volume ratio of methanol, water and acetic acid is 45 in -0.5% acetic acid water system of methanol that uses:
55:0.5, the volume ratio of acetonitrile, water and acetic acid is 42:58:0.5 in -0.5% acetic acid water system of acetonitrile used, obtained benzene
And furans glycosides compound molecular formula is C23H30O13；When carrying out isocratic elution to sample a2, -0.5% acetic acid water of methanol of use
The volume ratio of methanol, water and acetic acid is 35:65:0.5 in system, acetonitrile in -0.5% acetic acid water system of acetonitrile of use, water and
The volume ratio of acetic acid is 31:69:0.5, and obtained benzofuran glycosides compound molecular formula is C22H28O13。
The application of benzofuran glycosides compound aspect in preparing anti-inflammatory drug and/or health care product as described above.
Compared with prior art, the beneficial effects of the present invention are:
New benzofuran glycosides compound in the present invention has anti-inflammatory effect, tests prove that, to mouse
RAW264.7 small cell has good anti-inflammatory effect, which has the characteristics that efficient, low toxicity, is expected to develop into new resist
Scorching drug, or be used to prepare with the health food for preventing and treating inflammation.
It since the polarity of the compound in the present invention is larger, is difficult to purify to obtain with general method, such as with general anti-
The phase chromatographic column compound can not retain, and flow out with mobile phase, cannot achieve the purpose that separate with other compounds, this hair
It is bright middle using hydrophilic Interaction Chromatography that benzofuran glycosides compound is isolated, and purity is higher (> 98.5%), is separation
Purify the effective means that big polar small molecule aglycon connects multiple saccharide compounds.
[Detailed description of the invention]
Fig. 1 is the compounds of this invention 11H-NMR map；
Fig. 2 is the compounds of this invention 113C-NMR map；
Fig. 3 is the DEPT map of the compounds of this invention 1；
Fig. 4 is the compounds of this invention 11H-1H COSY map；
Fig. 5 is the HMQC map of the compounds of this invention 1；
Fig. 6 is the HMBC map of the compounds of this invention 1；
Fig. 7 is the compounds of this invention 21H-NMR map；
Fig. 8 is the HMQC map of the compounds of this invention 2；
Fig. 9 is the compounds of this invention 21H-1H COSY map.
[specific embodiment]
The present invention is further illustrated combined with specific embodiments below, and however, it is not limited to this.
The structural formula of the new benzofuran glycosides compound of the present invention is as follows:
Wherein, the R in structural formula are as follows:-H or-CH3。
The preparation method of the new benzofuran glycosides compound of the present invention, comprising the following steps:
1) the Xingan's Radix Angelicae Pubescentis dry root for taking certain mass (kg) is 1~8 times of Xingan's Radix Angelicae Pubescentis dry root quality amount with volume
Methanol, ethyl alcohol or water (L) heating and refluxing extraction 1~6 time near respective boiling point that volume fraction is 10~95%, every time 1~4
Hour, when extractant is the ethyl alcohol of methanol or volume fraction 10~95%, removing solvent is recovered under reduced pressure in combined extract, obtains
Total medicinal extract, when extractant is water, combined extract and by its volume concentration to 1/10 to ten/6ths is concentrated
Liquid；
2) total medicinal extract is suspended in water, the volume ratio of total medicinal extract and water is 1:1~1:5, obtain medicinal extract liquid, medicinal extract liquid or
Concentrate is successively extracted with isometric organic solvent respectively, wherein with petroleum ether to medicinal extract liquid or concentration when extracting for the first time
Liquid carries out equal-volume extraction, and extraction is to isolate last organic layer extracted every time later, by remaining water layer with having
Solvent is extracted next time in equal volume；Every kind of solvent extracts 1~6 time, combining extraction liquid, and normal pressure or vacuum distillation remove
Organic solvent respectively obtains each extract layer and water layer.Organic solvent includes petroleum ether, chloroform, ethyl acetate and n-butanol etc., and
Extracting order is solvent first small with polarity, then the organic solvent big with polarity, petroleum ether and chloroform can save.
3) extracting n-butyl alcohol layer is taken, by using separation methods such as column chromatographic purifyings, obtains benzofuran glycosides of the invention
Class compound.
Column chromatography includes following three phases:
First stage: extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, by volume
Gradient elution is carried out than (100:0)~(0:100), every 300~800ml collects a fraction, TLC detection is carried out to efflux,
Merge identical fraction, 30 fractions are obtained, is respectively designated as FrB1-FrB30, normal pressure or evaporated under reduced pressure solvent, takes therein
FrB20 fraction and FrB24 fraction, i.e. effluent volume than be respectively 10:1 and 8:1 fraction, as first time cross post part；
Second stage: first time is crossed into the FrB20 fraction and FrB24 fraction in post part, is splined on reverse phase silica gel respectively
Chromatographic column, using methanol-water system as eluent, the gradient elution of (0:100)~(100:0) progress by volume, every 200~
500mL collects a fraction, carries out TLC detection to efflux, merges identical fraction, obtain 4 Arius part (FrB20.1-
FrB20.4) and 2 Arius parts (FrB24.1-FrB24.2), solvent is removed under reduced pressure, obtained second and crosses post part；
Phase III: post part FrB20.4 fraction will be crossed second, i.e., effluent volume is than the fraction for 100:0；
FrB24.1 fraction, i.e. effluent volume are splined on high performance liquid chromatography separation column than the fraction for 70:30 respectively, isolated
Benzofuran glycosides compound.Wherein, high performance liquid chromatography separation column is the Megres C18 column of Jiangsu Chinese nation, high-efficient liquid phase color
Spectrum separation is with differential refraction detector, using -0.5% acetic acid water system of methanol or -0.5% acetic acid water system of acetonitrile as flowing
Phase carries out isocratic elution by 3~6mL/min.
The volume ratio of methanol, water and acetic acid is respectively 45:55:0.5 and 35:65 in -0.5% acetic acid water system of methanol:
0.5；The volume ratio of acetonitrile, water and acetic acid is respectively 42:58:0.5 and 31:69:0.5 in -0.5% acetic acid water system of acetonitrile.
New benzofuran glycosides compound in the present invention can be applied in preparing anti-inflammatory drug and/or health care product.
Embodiment 1
1. benzofuran glycosides compound isolates and purifies
1) Kan Kula dry root 6kg is taken, with methanol heating and refluxing extraction 3 times of 30L, every time 2 hours, at this point, and methanol
Volume be 5 times of quality of Xingan's Radix Angelicae Pubescentis dry root, merge methanol extract liquid and solvent be recovered under reduced pressure, obtain total medicinal extract；
2) total medicinal extract is suspended in 3 times of volume water, with petroleum ether equal-volume extraction 4 times, then successively through chloroform, second
Acetoacetic ester and n-butanol equal-volume extraction, every kind of organic solvent extract 4 times, after organic layer normal pressure or vacuum distillation are removed solvent
Respectively obtain petroleum ether layer, chloroform layer, ethyl acetate layer, n-butanol layer and remaining water layer.
3) extracting n-butyl alcohol layer 100g is taken, silica gel column chromatography is splined on, using chloroform-methanol system as eluent, by body
Product is that (0:100)~(100:0) carries out gradient elution than (v/v), and every 500mL collects primary, air-distillation recycling design, TLC
Inspection, which is known, merges identical fraction, and 30 fractions are obtained, are respectively designated as FrB1-FrB30, take FrB20 fraction therein and FrB24
Fraction, i.e. effluent volume than be respectively 10:1 and 8:1 fraction, as first time cross post part.
Wherein, the inverted silica gel column chromatography of FrB20 fraction uses methanol-water system as eluent, by volume (v/v)
For 0:100~100:0 gradient elution, every 200mL is collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction, obtains 4
A Arius part, is respectively designated as FrB20.1-FrB20.4.Then FrB20.4 fraction, i.e. effluent volume are than the stream for 100:0
Part, with half preparative high-performance liquid chromatographic instrument, using Megres C18 chromatographic column, using methanol and 0.5% acetic acid water as flowing
Equality elutes (45:55), and flow velocity 3mL/min obtains benzofuran glycosides compound 1 (retention time 27min).
The inverted silica gel column chromatography of FrB24 fraction, with methanol-water gradient elution (30:70,45:55,70:30,100:0,
V/v), every 200mL is collected primary, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction, is obtained 2 Arius parts and is respectively designated as
FrB24.1-FrB24.2.Then FrB24.1 fraction, i.e. effluent volume are than the fraction for 70:30, with half preparation efficient liquid phase
Chromatograph, using Megres C18 chromatographic column, using methanol and 0.5% acetic acid water as mobile phase isocratic elution (35:65), stream
Speed is 3mL/min, obtains benzofuran glycosides compound 2 (retention time 20min).
4) present invention is identified by physicochemical constant and Modern spectroscopy technological means (HR-ESI-MS, 1D-NMR, 2D-NMR)
The structure of compound, compound 1 are 6-methoxycarbonylethyl-benzofuran-5-O- β-D-xylopyranosyl
(1 → 2)-β-D-glucopyranoside, compound 2 are 6-hydroxycarbonylethyl-benzofuran-5-O- β-
D-xylopyranosyl(1→2)-β-D-glucopyranoside.The Structural Identification process of compound 1 and 2 is as described below.
2. the Structural Identification of new benzofuran glycosides compound
(1) Structural Identification of noval chemical compound 1
Compound 1 is yellow oil, and HR-ESIMS shows that quasi-molecular ion peak is [M+Na]+at m/z
537.1584 (calcd 537.1579), it is thus determined that molecular formula is C23H30O13.Infrared spectroscopy shows hydroxyl group absorption band
(3403cm-1), alkyl absorption band (2933cm-1), carbonyl absorption band (1718cm-1) etc..Fig. 1's1In H NMR, four hydrogen matter
Subsignal δH7.64 (1H, d, J=2.2Hz), 7.18 (1H, m), 7.11 (1H, d, J=8.4Hz), 7.19 (1H, m) show point
There are disubstituted benzofuran structures in son, and two substituent groups are in the substitution of ortho position two.Fig. 2's13In C NMR, in addition to one
Outer (the δ of a carbonylC174.8), there are also eight fragrant carbon signals, including four methines, four quaternary carbons, the neighbours with aforementioned supposition
Position disubstituted benzofuran structure fragment is consistent.It is composed in conjunction with the DEPT of Fig. 3, Fig. 4's1H-1The HMQC of H COSY spectrum and Fig. 5, two groups
Multiplet proton signal δH3.30 (1H, m), 3.01 (1H, m), δH2.72 (1H, m), 2.65 (1H, m) and their corresponding carbon
Signal (δC25.2 and δC34.9) show the methylene being connected directly in molecule there are two.In the HMBC spectrum of Fig. 6, this two
A methylene and a carbonyl carbon (δC174.8) related, and this carbonyl carbon is connected (δ with a methoxyl groupH3.68 δC
50.6).?1H and13In C H NMR spectroscopy, two anomeric proton signal δH5.09 (1H, d, J=7.6Hz) and 4.79 (1H, d, J
=7.3Hz) and their corresponding carbon signal (δC101.8, δC104.5) show in molecule to be used after sour water solution there are two sugared unit
It is D-Glucose and D- xylose that GC, which analyzes and identifies both sugar, and the end group configuration of the two sugar is beta comfiguration, because of their end
The coupling constant of matrix is 7.6Hz and 7.3Hz.The position of substitution and the order of connection of substituent group and sugar pass through integration analysis1H–1H-COSY, HMQC and HMBC are determined.
Therefore, compound identification are as follows: 6-methoxycarbonylethyl-benzofuran-5-O- β-D-
Xylopyranosyl (1 → 2)-β-D-glucopyranoside, i.e. 6- dion e-benzofuran -5-O- β-D-
Xylopyranose-(1 → 2)-β-D- glucopyranoside, it is one and has no the noval chemical compound reported in the literature with new aglycon.
Its structure sees below formula:
Nuclear magnetic data is shown in Table 1.
(2) Structural Identification of noval chemical compound 2
Compound 2 is yellow oil, and HR-ESIMS shows that quasi-molecular ion peak is [M+Na]+at m/z
523.1427 (calcd 523.1422) determine that molecular formula is C22H28O13.The hydrogen of its Fig. 7 is composed and the carbon modal data and chemical combination of Fig. 8
Object 1 is quite similar, has only lacked a methoxyl group signal in hydrogen spectrum, therefore speculate that compound 2 is acid rather than the structure of methyl esters,
This is consistent with molecular weight.Integration analysis Fig. 9's1H–1H-COSY, HMQC and HMBC spectrum determine that compound 2 is 6-
hydroxycarbonylethyl-benzofuran-5-O-β-D-xylopyranosyl(1→2)-β-D-
Glucopyranoside, i.e. 6- dion e-benzofuran -5-O- β-D- xylopyranose-(1 → 2)-β-D- pyrans
Glucoside is one and has no noval chemical compound reported in the literature.2 structural formula of compound is as follows:
Compound 1 and 2 in 1 present invention of table1H NMR and13C NMR data
Pharmacological activity detection is further done to the compound 1 and 2 of identification separated in the present invention below.
3. extracorporeal anti-inflammatory is tested
Experimental method:
By RAW264.7 cell inoculation in 96 orifice plates, LPS (lipopolysaccharides) is added afterwards for 24 hours in culture.Then different component is other
The sample (the compounds of this invention 1 and 2, positive drug Indomethacin) of various concentration is added, blank group is added isometric
vehicle.50 μ L cell culture fluids mix isometric Griess reagent I and are added in above-mentioned 96 orifice plate, are subsequently added into Griess examination
Agent II.Microplate reader measures OD value of each group sample under 546nm wavelength, calculates NO using following equation and generates inhibiting rate, is used in combination
SPSS software calculates the half-inhibitory concentration (IC of tested sample50Value, μM).
NO inhibiting rate (%)=[1-(sample sets/blank group)] × 100%.
Experimental result:
The cell RAW264.7 of 2 the compounds of this invention 1 and 2 pair LPS of table stimulation generates the inhibiting effect of NO
Interpretation of result: table 2 statistics indicate that, the present invention in compound 1 and 2 have good extracorporeal anti-inflammatory effect, it is right
Lipopolysaccharides LPS, which stimulates mouse RAW264.7 cell to generate NO, has good inhibiting effect, half-inhibitory concentration IC50Respectively
28.6 ± 1.6 μM, 32.9 ± 2.1 μM are superior to 39.5 ± 3.2 μM of positive control medicine Indomethacin.Illustrate in the present invention
Benzofuran glycosides compound 1 and 2 have preferable anti-inflammatory effect.
Embodiment 2
1) Kan Kula dry root 10kg is taken, with 70% ethyl alcohol heating and refluxing extraction 3 times of 40L, 2 hours every time, merging was mentioned
It takes liquid and solvent is recovered under reduced pressure, obtain total medicinal extract；
2) total medicinal extract is suspended in 4 times of amount volume of water, after being first extracted with ethyl acetate 3 times, then it is isometric with n-butanol
Extraction 4 times, respectively obtains ethyl acetate layer and n-butanol layer after organic solvent is removed under reduced pressure.
3) extracting n-butyl alcohol layer 200g is taken, silica gel column chromatography is splined on, using chloroform-methanol system as eluent, by body
Product is that 100:0~0:100 carries out gradient elution than (v/v), and every 800mL collects primary, air-distillation recycling design, and TLC inspection is known
Merge identical fraction, 30 fractions are obtained, are respectively designated as FrB1-FrB30, FrB20 fraction therein and FrB24 is taken to flow
Part, i.e., effluent volume than be respectively 10:1 and 8:1 fraction, as first time cross post part.
Wherein, the inverted silica gel column chromatography of FrB20 fraction uses methanol-water system as eluent, by volume (v/v)
Gradient elution is carried out for 100:0~0:100, every 400mL is collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction,
4 Arius parts are obtained, FrB20.1-FrB20.4 is respectively designated as.Then FrB20.4 fraction, i.e. effluent volume ratio are 100%
Methanol-eluted fractions, with half preparative high-performance liquid chromatographic instrument, using Megres C18 chromatographic column, with acetonitrile and 0.5% acetic acid
Water is as mobile phase isocratic elution (42:58), flow velocity 3mL/min, and obtaining benzofuran glycosides compound 1, (retention time is
25min)。
The inverted silica gel column chromatography of FrB24 fraction is that 100:0~0:100 carries out gradient with methanol-water volume ratio (v/v)
Elution, every 400mL are collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction, and obtaining 2 Arius parts is respectively
FrB24.1-FrB24.2.Then FrB24.1 fraction, i.e. effluent volume are than the fraction for 70:30, with half preparation efficient liquid phase
Chromatograph, using Megres C18 chromatographic column, using acetonitrile and 0.5% acetic acid water as mobile phase isocratic elution (31:69), stream
Speed is 3mL/min, obtains benzofuran glycosides compound 2 (retention time 18min).
Embodiment 3
1) Kan Kula dry root 10kg is taken, with water heating and refluxing extraction 1 time of 30L, 4 hours every time, combined extract was simultaneously
Solvent is recovered under reduced pressure, obtains total medicinal extract；
2) total medicinal extract is suspended in 2 times of amount volume of water, after being first extracted with ethyl acetate 3 times, then it is isometric with n-butanol
Extraction 6 times, respectively obtains ethyl acetate layer and n-butanol layer after organic solvent is removed under reduced pressure.
3) extracting n-butyl alcohol layer 200g is taken, silica gel column chromatography is splined on, using chloroform-methanol system as eluent, by body
Product is that 100:0~0:100 carries out gradient elution than (v/v), and every 800mL collects primary, air-distillation recycling design, and TLC inspection is known
Merge identical fraction, 30 fractions are obtained, are respectively designated as FrB1-FrB30, FrB20 fraction therein and FrB24 is taken to flow
Part, i.e., effluent volume than be respectively 10:1 and 8:1 fraction, as first time cross post part.
Wherein, the inverted silica gel column chromatography of FrB20 fraction uses methanol-water system as eluent, by volume (v/v)
Gradient elution is carried out for 100:0~0:100, every 500mL is collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction,
4 Arius parts are obtained, FrB20.1-FrB20.4 is respectively designated as.Then FrB20.4 fraction, i.e. effluent volume ratio are 100%
Methanol-eluted fractions, with half preparative high-performance liquid chromatographic instrument, using Megres C18 chromatographic column, with acetonitrile and 0.5% acetic acid
Water is as mobile phase isocratic elution (42:58), flow velocity 3mL/min, and obtaining benzofuran glycosides compound 1, (retention time is
25min)。
The inverted silica gel column chromatography of FrB24 fraction is by volume (v/v) that 100:0~0:100 carries out ladder with methanol-water
Degree elution, every 400mL are collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction, and obtaining 2 Arius parts is respectively
FrB24.1-FrB24.2.Then FrB24.1 fraction, i.e. effluent volume are than the fraction for 70:30, with half preparation efficient liquid phase
Chromatograph, using Megres C18 chromatographic column, using acetonitrile and 0.5% acetic acid water as mobile phase isocratic elution (31:69), stream
Speed is 3mL/min, obtains benzofuran glycosides compound 2 (retention time 18min).
Embodiment 4
1) Kan Kula dry root 10kg is taken, with 10% ethyl alcohol heating and refluxing extraction 6 times of 80L, 1 hour every time, merging was mentioned
It takes liquid and solvent is recovered under reduced pressure, obtain total medicinal extract；
2) total medicinal extract is suspended in 5 times of amount volume of water, after first using petroleum ether extraction 2 times, then successively uses chloroform, acetic acid second
Ester and n-butanol equal-volume extraction 1 time, respectively obtain ethyl acetate layer and n-butanol layer after organic solvent is removed under reduced pressure.
3) extracting n-butyl alcohol layer 200g is taken, silica gel column chromatography is splined on, using chloroform-methanol system as eluent, by body
Product is that 100:0~0:100 carries out gradient elution than (v/v), and every 300mL collects primary, air-distillation recycling design, and TLC inspection is known
Merge identical fraction, 30 fractions are obtained, are respectively designated as FrB1-FrB30, FrB20 fraction therein and FrB24 is taken to flow
Part, i.e., effluent volume than be respectively 10:1 and 8:1 fraction, as first time cross post part.
Wherein, the inverted silica gel column chromatography of FrB20 fraction uses methanol-water system as eluent, by volume (v/v)
Gradient elution is carried out for 100:0~0:100, every 300mL is collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction,
4 Arius parts are obtained, FrB20.1-FrB20.4 is respectively designated as.Then FrB20.4 fraction, i.e. effluent volume ratio are 100%
Methanol-eluted fractions, with half preparative high-performance liquid chromatographic instrument, using Megres C18 chromatographic column, with acetonitrile and 0.5% acetic acid
Water is as mobile phase isocratic elution (42:58), flow velocity 3mL/min, and obtaining benzofuran glycosides compound 1, (retention time is
25min)。
The inverted silica gel column chromatography of FrB24 fraction is by volume (v/v) that 100:0~0:100 carries out ladder with methanol-water
Degree elution, every 400mL are collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction, and obtaining 2 Arius parts is respectively
FrB24.1-FrB24.2.Then FrB24.1 fraction, i.e. effluent volume are than the fraction for 70:30, with half preparation efficient liquid phase
Chromatograph, using Megres C18 chromatographic column, using acetonitrile and 0.5% acetic acid water as mobile phase isocratic elution (31:69), stream
Speed is 3mL/min, obtains benzofuran glycosides compound 2 (retention time 18min).
Embodiment 5
1) Kan Kula dry root 10kg is taken, with 95% ethyl alcohol heating and refluxing extraction 4 times of 35L, 3 hours every time, merging was mentioned
It takes liquid and solvent is recovered under reduced pressure, obtain total medicinal extract；
2) total medicinal extract is suspended in 1 times of amount volume of water, after first using petroleum ether extraction 1 time, then successively uses chloroform, acetic acid second
Ester and n-butanol equal-volume extraction 2 times, respectively obtain ethyl acetate layer and n-butanol layer after organic solvent is removed under reduced pressure.
3) extracting n-butyl alcohol layer 200g is taken, silica gel column chromatography is splined on, using chloroform-methanol system as eluent, by body
Product is that 100:0~0:100 carries out gradient elution than (v/v), and every 600mL collects primary, air-distillation recycling design, and TLC inspection is known
Merge identical fraction, 30 fractions are obtained, are respectively designated as FrB1-FrB30, FrB20 fraction therein and FrB24 is taken to flow
Part, i.e., effluent volume than be respectively 10:1 and 8:1 fraction, as first time cross post part.
Wherein, the inverted silica gel column chromatography of FrB20 fraction uses methanol-water system as eluent, by volume (v/v)
Gradient elution is carried out for 100:0~0:100, every 200mL is collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction,
4 Arius parts are obtained, FrB20.1-FrB20.4 is respectively designated as.Then FrB20.4 fraction, i.e. effluent volume ratio are 100%
Methanol-eluted fractions, with half preparative high-performance liquid chromatographic instrument, using Megres C18 chromatographic column, with methanol and 0.5% acetic acid
Water is as mobile phase isocratic elution (45:55), flow velocity 3mL/min, and obtaining benzofuran glycosides compound 1, (retention time is
27min)。
The inverted silica gel column chromatography of FrB24 fraction is by volume (v/v) that 100:0~0:100 carries out ladder with methanol-water
Degree elution, every 400mL are collected once, and solvent is removed under reduced pressure, and TLC inspection, which is known, merges identical fraction, and obtaining 2 Arius parts is respectively
FrB24.1-FrB24.2.Then FrB24.1 fraction, i.e. effluent volume are than the fraction for 70:30, with half preparation efficient liquid phase
Chromatograph, using Megres C18 chromatographic column, using methanol and 0.5% acetic acid water as mobile phase isocratic elution (35:65), stream
Speed is 3mL/min, obtains benzofuran glycosides compound 2 (retention time 20min).
In conjunction with the above experiment and its experimental result, show that the benzofuran glycosides compound in the present invention has good body
Outer anti-inflammatory effect, being expected to exploitation is new anti-inflammatory drug；Or it is used to prepare the health food for preventing and treating inflammation.
Comparative example 1
Changing the mobile phase in step 5) the identical mobile phase of other mobile phases or other ratios into, (or chromatographic column is replaced
Change common reverse phase silica gel chromatographic column into), other steps and condition are same as Example 1.It was found that benzofuran glycosides can not be isolated
Class compound.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. benzofuran glycosides compound, it is characterised in that: its molecular formula is C23H30O13Or C22H28O13；
Its general structure are as follows:
Wherein, R is-H or-CH3。
2. the preparation method of benzofuran glycosides compound as described in claim 1, it is characterised in that: the following steps are included:
1) it takes the dry root of Xingan's Radix Angelicae Pubescentis to carry out refluxing extraction several times, extracting solution is concentrated under reduced pressure, total medicinal extract or concentration are obtained
Liquid；
2) total medicinal extract is suspended in water, obtains medicinal extract liquid, medicinal extract liquid or concentrate and is separated after ethyl acetate extraction several times
Organic layer out, combining extraction liquid after remaining water layer passes through extracting n-butyl alcohol several times are removed under reduced pressure n-butanol and obtain n-butanol extraction
Take layer；
3) extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, by volume (100:0)~
(0:100) carries out gradient elution, detects to efflux, by effluent volume than the fraction merging for 10:1, removes solvent,
Obtain sample a1；By effluent volume than the fraction merging for 8:1, solvent is removed, sample b1 is obtained；
4) sample a1 is splined on reversed-phase silica gel chromatography column, using methanol-water system as eluent, by volume (0:100)~
(100:0) carries out gradient elution, by effluent volume than the fraction merging for 100:0, removes solvent, obtains sample a2；
Sample b1 is splined on reversed-phase silica gel chromatography column, using methanol-water system as eluent, by volume (0:100)~
(100:0) carries out gradient elution, by effluent volume than the fraction merging for 70:30, removes solvent, obtains sample b2；
5) sample a2 and sample b2 are splined on high performance liquid chromatography separation column respectively, isocratic elution is carried out with mobile phase, obtains
Benzofuran glycosides compound；
Ethyl alcohol in the refluxing extraction of step 1) using methanol, water or volume fraction 10~95% is as extractant, Xingan's Radix Angelicae Pubescentis
Dry root and extractant mass volume ratio be 1kg:(1~8) L；When extractant is methanol or volume fraction 10~95%
When ethyl alcohol, solvent recovery in obtained extracting solution is obtained into total medicinal extract；When extractant is water, extremely by the volume concentration of extracting solution
1/10 to ten/6ths, obtain concentrate；
Medicinal extract liquid or concentrate are directly over ethyl acetate extraction in step 2)；Or it is first successively extracted through petroleum ether and chloroform
Afterwards, it is extracted using ethyl acetate；Extraction is equal-volume extraction every time；Every kind of solvent extracts 1~6 time respectively；
Mobile phase is -0.5% acetic acid water system of -0.5% acetic acid water system of methanol or acetonitrile in step 5)；Sample a2 is carried out etc.
When degree elution, in -0.5% acetic acid water system of methanol that uses the volume ratio of methanol, water and acetic acid for 45:55:0.5, use
The volume ratio of acetonitrile, water and acetic acid is 42:58:0.5, obtained benzofuran glycoside chemical combination in -0.5% acetic acid water system of acetonitrile
Object molecular formula is C23H30O13；When carrying out isocratic elution to sample a2, methanol, water in -0.5% acetic acid water system of methanol of use
Be 35:65:0.5 with the volume ratio of acetic acid, in -0.5% acetic acid water system of acetonitrile used the volume ratio of acetonitrile, water and acetic acid for
31:69:0.5, obtained benzofuran glycosides compound molecular formula are C22H28O13。
3. the preparation method of benzofuran glycosides compound according to claim 2, it is characterised in that: extracted in step 1)
Number is 1~6 time, every time 1~4 hour.
4. the preparation method of benzofuran glycosides compound according to claim 2, it is characterised in that: always soaked in step 2)
The volume ratio of cream and water is 1:1~1:5.
5. the preparation method of benzofuran glycosides compound according to claim 2, it is characterised in that: gradient in step 3)
Every 300~800mL collects a fraction after elution；Every 200~500mL collects a fraction after gradient elution in step 4)；Step
It is rapid 3) and step 4) in efflux be carry out TLC detection；The flow velocity of isocratic elution is 3~6mL/min in step 5).
6. benzofuran glycosides compound as described in claim 1 in preparing anti-inflammatory drug and/or health care product aspect answer
With.
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| CN1633432A (en) * | 2002-04-08 | 2005-06-29 | 科学与工业研究委员会 | Novel glucopyranoside, process for isolation thereof, pharmaceutical composition containing same and use thereof |
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| CN101502507A (en) * | 2008-02-04 | 2009-08-12 | 成都地奥九泓制药厂 | Egonol type benzofuran and novel use in pharmacy of glycosides thereof |
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| CN1830976A (en) * | 2005-03-08 | 2006-09-13 | 成都地奥制药集团有限公司 | Extractive of total benzofuran of benzoin plants, prepn. method and use thereof |
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