CN105669631B - A kind of potentilla plants extract and the method for therefrom separating four kinds of tanninses compounds - Google Patents
A kind of potentilla plants extract and the method for therefrom separating four kinds of tanninses compounds Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
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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
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
Abstract
The invention discloses a kind of potentilla plants extract, and it contains the OPC B7 not less than 6%w/w or/and 2,3,4, the 6 four O galloyl glucoses not less than 4%w/w.The extract can be used for anti-inflammatory, it is anti-oxidant, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control the disturbance of lower legs due to pathogenic cold and dampness, treatment pruigo or treatment mastitis.Meanwhile PB3, OPC B6, OPC B7 and 2,3 has been prepared in present invention separation also from the extract, four kinds of tanninses compounds of 4,6 four O galloyl glucoses, product purity are all higher than 95%, this method preparation amount is big, is suitable for commercial Application.
Description
Technical field
The present invention relates to natural medicine field, and in particular to from potentilla plants leaflet bush cinqefoil extract, Yi Jicong
Separation prepares PB3, OPC the B6 ,-O- nutgall acyl grapes of OPC B7 and 2,3,4,6- tetra- in the extract
The method of sugar.
Background technology
Now there are some researches show the various plants of Potentilla are medicinal plant, have an anti-inflammatory, and anti-diabetic is antitumor
Deng effect；And tanninses compound is the important component of the platymiscium.Tanninses active material is divided into two classes：That is condensed type tannin
(including OPC) and hydrolysis-type tannin, they have anti-inflammatory, anti-oxidant, promotion wound healing, antibacterial, antiviral etc.
Effect.
Leaflet bush cinqefoil (Potentilla parvifolia Fisch.) is one kind in potentilla plants, is specifically
Rose family Potentilla machaka, also known as littleleaf ciquefoil leaf and flower etc..It is used as medicine as a kind of traditional middle Tibetan medicine with flower, leaf.
《Dictionary of medicinal plant》Described in littleleaf ciquefoil leaf and flower major function：Diuresis disappears water, controls the disturbance of lower legs due to pathogenic cold and dampness, pruigo, mastitis.
At present, it there are no the leaflet bush cinqefoil extract of enrichment tannin and separated from leaflet bush cinqefoil and prepared
Obtain the report of tanninses compound.
The content of the invention
To solve the above problems, the invention provides a kind of potentilla plants leaflet bush cinqefoil extract, it contains not
OPC B7 less than 6%w/w or/and the-O- galloyl glucoses of the 2,3,4,6- not less than 4%w/w tetra-.Above two
Material is to be found first from Potentilla.
Further, the extract also contains the original flower not less than 14%w/w PB3s and not less than 7%w/w
Blue or green plain B6.
PB3 (I), OPC B6 (II), OPC B7 (III) and-O- nutgall acyls Portugals of 2,3,4,6- tetra-
The structure of grape sugar (IV) is as follows：
Further, the extract contains 15~16%w/w PB3,8~9%w/w OPC B6,
7~8%w/w OPC B7 and the 5~6%w/w-O- galloyl glucoses of 2,3,4,6- tetra-.
Further, described 2, in 3,4,6- tetra--O- galloyl glucoses ,-O- the nutgall acyls of α-type 2,3,4,6- tetra-
The mol ratio of glucose and the-O- galloyl glucoses of β-type 2,3,4,6- tetra- is 3:1.
Further, the extract is leaflet bush cinqefoil Potentilla parvifolia Fisch. aerial parts
Extract.The aerial part is all aerial parts, including stem, leaf, flower.
Present invention also offers a kind of method for preparing the leaflet bush cinqefoil extract, it is characterised in that：Including following
Step：
(1) leaflet bush cinqefoil alcohol crude extract is prepared：Leaflet bush cinqefoil, cutting or crushing are taken, alcohol extracting, is concentrated to give leaflet
Bush cinqefoil alcohol crude extract；
(2) ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract is prepared：Take step (1) that alcohol is prepared slightly to carry
Thing, petroleum ether extraction, remaining liq is taken, ethyl acetate extraction, obtains the ethyl acetate extraction portion of leaflet bush cinqefoil alcohol crude extract
Point；
(3) concentrate for being enriched with four kinds of tanninses compounds is prepared：It is prepared into silica gel column chromatography separating step (2)
The ethyl acetate extraction part arrived, with petroleum ether-ethyl acetate volume ratio 100:0~0:100 gradient elutions, collection petroleum ether-
Ethyl acetate volume ratio 1:1~0:100 flow point, concentrates and produces；It is preferred that 0:Under 100 flow point, i.e. neat ethyl acetate
Flow point.
Further, in step (1), the alcohol is 60%-90% ethanol, preferably 70% ethanol；The temperature of the extraction
Degree is 60 DEG C；The number of extraction is 3 times；Every time during extraction, the volume mass ratio of alcohol and leaflet bush cinqefoil is 30:6.5L/Kg；
Further, in step (2), the extraction times of the petroleum ether are 2~10 times, when extracting every time, are slightly carried with alcohol
The volume ratio of thing is 1:8；The extraction times of the ethyl acetate are 2~10 times, when extracting every time, the volume ratio with alcohol crude extract
For 1:10.
Further, in step (3), the gradient elution is successively using the volume ratio of petroleum ether-ethyl acetate as 100：
1、50：1、10：1、1：1、2：3 eluent and ethyl acetate eluent (the i.e. volume ratio 0 of petroleum ether-ethyl acetate:100) enter
Capable gradient elution.
Further, in step (3), the volume ratio of the petroleum ether-ethyl acetate is 100：1、50：1、10：1、1：1、
2：3 eluent and ethyl acetate eluent are followed successively by 3 with the volume mass ratio of leaflet bush cinqefoil：6.5、4：6.5、6：6.5、
10：6.5、10.5：6.5 and 11.5：6.5L/Kg.
Further, foregoing leaflet bush cinqefoil extract is prepared by foregoing method.
Present invention also offers from foregoing leaflet bush cinqefoil extract separation prepare four kinds of tanninses compounds method,
Four kinds of tanninses compounds are PB3, OPC the B6 ,-O- nutgall acyls of OPC B7 and 2,3,4,6- tetra-
Glucose, comprise the following steps：
Separated using high-speed countercurrent chromatography and prepare four kinds of tanninses compounds：
(a) take foregoing leaflet bush cinqefoil extract to be dissolved in sample introduction solvent, sample introduction solution is prepared；
(b) sample introduction solution is injected into high-speed counter-current chromatograph, PB3, OPC B6, former cyanine are collected in detection
Plain B7 and 2, the flow point of 3,4,6- tetra--O- galloyl glucoses, solvent is removed, produces PB3, OPC B6, original
The product of-O- the galloyl glucoses of anthocyanidin B7 and 2,3,4,6- tetra-；
The stationary phase of the high-speed countercurrent chromatography is the upper phase of two phase solvent system, and mobile phase is two phase solvent system
Lower phase；The two phase solvent system is by n-hexane-ethyl acetate-methanol-water=(0.8~1.2):(10.8~11.2):(1~
1.4):(10.8~11.2) (v/v) is formed, and preferable ratio is 1:11:1.2:11(v/v).
Further, the sample introduction solvent is according to 1 by the upper and lower phase:The solvent of 1 volume ratio composition；It is described
The volume mass of sample introduction solvent and extract ratio is 1:15~1:30mL/mg.
Further, the rotating speed of separating pipe is 900 ± 50rpm in the high-speed counter-current chromatograph.
Further, the temperature setting of separating pipe is 30 ± 5 DEG C in the high-speed counter-current chromatograph.
Further, the flow velocity of the mobile phase is 1.2 ± 0.3mL/min.
Further, the wavelength of the detection is 270 ± 5nm.
Present invention also offers a kind of separation to prepare PB3, OPC B6, OPC B7 and 2,3,4,6-
The method of four-O- galloyl glucoses, comprises the following steps：
Take containing PB3, OPC B6, OPC B7 and 2,3,4,6- tetra--O- galloyl glucoses
Solution or extract, carried out according to foregoing high-speed countercurrent chromatography.
Present invention also offers the extract prepare anti-inflammatory, it is anti-oxidant, promote wound healing, antibacterial, antiviral, sharp
Urine, consumer edema, the disturbance of lower legs due to pathogenic cold and dampness is controlled, treat pruigo or treats the purposes in mastitis medicament.
Present invention also offers a kind of pharmaceutical composition, and it is that described leaflet bush cinqefoil extract is active component, is added
On preparation that pharmaceutically conventional auxiliary material or complementary composition is prepared.
Present invention firstly discovers that Potentilla containing OPC B7 Yu the-O- galloyl glucoses of 2,3,4,6- tetra-
Plant extracts.
Leaflet bush cinqefoil extract prepared by the present invention, is enriched with a variety of tannins, the OPC in the extract
B3 content is up to 15~16%w/w；OPC B6 content is up to 8~9%w/w；OPC B7 content is up to 7~
8%w/w；The content of-O- the galloyl glucoses of 2,3,4,6- tetra- is up to 5~6%w/w.The extract can be used for anti-inflammatory, antioxygen
Change, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control the disturbance of lower legs due to pathogenic cold and dampness, treatment pruigo or treatment mastitis.
Meanwhile method provided by the invention, the former cyanine of high-purity has been prepared in separation from leaflet bush cinqefoil first
Plain B3, OPC B6, OPC B7 and 2,3,4,6- tetra--O- galloyl glucoses, their purity are followed successively by
98.3%, 97.2%, 99.0% and 95.2%.Aforementioned four tanninses compound is to be sent out first from leaflet bush cinqefoil plant
It is existing, and OPC B7 and 2,3,4,6- tetra--O- galloyl glucoses greatly extend to be found first from Potentilla
The ranges of choice of these compound plant materials.
Also, high-speed countercurrent chromatography (the high-speed couter-current that the present invention uses
Chromatography, HSCCC), it is a kind of liquid liquid partition chromatography technology without using solid state support body or carrier, which overcomes
Solid phase carrier the shortcomings of caused loss, denaturation, has to the absorption of sample and realizes efficient preparative separation in a short time
The advantages of.This method has the advantages of reproducible, product purity is high, preparation amount is big, suitable for containing above-mentioned four kinds from various
High-purity monomer is prepared in the natural products of compound, is with a wide range of applications.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The embodiment of form by the following examples, the above of the present invention is remake further specifically
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 is dicyandiamide solution n-hexane-ethyl acetate-methanol-water=1:11:1.2:When 11, leaflet bush cinqefoil extract
High speed adverse current chromatogram figure.
In Fig. 2, (a) is the high-efficient liquid phase chromatogram of leaflet bush cinqefoil extract of the present invention, and (b)~(e) is followed successively by chemical combination
The high-efficient liquid phase chromatogram of thing I~IV.
Fig. 3 is PB3 (I), OPC B6 (II), OPC B7 (III) and-O- nutgalls of 2,3,4,6- tetra-
The ultraviolet spectra of acyl glucose (IV).
Fig. 4 is the petrochina ether of embodiment 2：Ethyl acetate=1:The high-efficient liquid phase chromatogram of 1 (v/v) eluent part.
Fig. 5 is the petroleum ether of embodiment 2：Ethyl acetate=2:The high-efficient liquid phase chromatogram of 3 (v/v) eluent part.
Fig. 6 is dicyandiamide solution n-hexane-ethyl acetate-methanol-water=1:10:1:When 10, leaflet bush cinqefoil extract
High speed adverse current chromatogram figure.
Embodiment
Medicinal material used in the present invention is the drying overground part of leaflet bush cinqefoil (Potentilla parvifolia Fisch.)
Point, adopted in August, 2014 in Haibei Prefecture.Plant species by Northwest Plateau-organisms Research Inst. of Chinese Academy of Sciences Zhang Faqi
Doctor identifies that plant specimen is stored in Qinghai-Tibet biological sample shop, and sample numbering is 252558.
Experiment uses reagent：Analyze pure petroleum ether, ethyl acetate, n-hexane, methanol (the limited public affairs of Shanghai fuzz chemical reagent
Department), chromatogram methanol (Chemical Reagent Co., Ltd., Sinopharm Group).
Instrument：TBE-300B high-speed counter-current chromatographs (Shanghai Tauto Biotechnology Co., Ltd.), equipped with TBP-
5002 constant flow pumps and TBD-2000UV detectors (the same field in Shanghai)；(the rich doctor's health laboratory apparatus in Beijing has HX-1050 constant temperature circulators
Limit company) and N2000 chromatographic work stations (Zhejiang University's intelligence reaches information engineering company).
Excellent general ultrapure water machine (Sichuan UP Hyperpure Technology Co., Ltd.).
Yi Lite P230 high performance liquid chromatographs, equipped with P230 double pump systems, manual sampler (Rheodyne 7725i),
Column oven (PH-530), PDAD (DAD230), Dubhe-C18Analytical column (4.6mm × 250mm, 10 μm) and
EC2000DAD single channels chromatographic work station (Dalian Yilite Analytical Instrument Co., Ltd).
NMR is the NMR systems of Varian INOVA 600 (Varian, Palo Alto, CA, USA).
Embodiment 1
1. the preparation of leaflet bush cinqefoil extract of the present invention
Leaflet bush cinqefoil dry aerial parts (6.5kg) scissors is taken to shred the segment for being about 1.5cm, using 70% ethanol,
With boiling machine (temperature is 60 DEG C) heating extraction 3 times (each 30L), merging is concentrated to give leaflet bush cinqefoil alcohol crude extract, depressurized
4L is concentrated to, then successively with petroleum ether (every time with 5000mL, extracting 10 times), ethyl acetate (each 4000mL, extracting 10 times)
Extraction.
Ethyl acetate extraction part is concentrated, normal phase silicagel column is crossed, successively with petroleum ether-ethyl acetate 100：1
(3000mL)、50：1(4000mL)、10：1(6000mL)、1：1(10000mL)、2：3 (10500mL) and ethyl acetate
(11500mL) elution, the elution fraction of ethyl acetate eluent is taken, the removing solvent that is concentrated under reduced pressure obtains dry solid
Powder, i.e. leaflet bush cinqefoil extract 9.8g.
2. high speed adverse current chromatogram isolates and purifies four kinds of tanninses compounds in leaflet bush cinqefoil extract
A. solvent system is prepared
According to n-hexane-ethyl acetate-methanol-water=1:11:1.2:11 volume ratio is prepared solvent system and leaked in liquid separation
In bucket, stratification after abundant shake, upper phase solvent and lower phase solvent are collected respectively, respectively ultrasonic 20min, the above is mutually fixation
Phase, lower phase are mobile phase；
B. it is pumped into institute by 30mL/min of flow velocity into the separating pipe (temperature setting is 30 DEG C) of high-speed counter-current chromatograph device
Stationary phase is stated, after phase to be fixed is full of separating pipe, main frame is rotated forward and keeps rotating speed to balance 30min for 900rpm, with
2.0mL/min flow pump enter mobile phase to the separating pipe port of export have mobile phase overflow and chromatographic work station baseline it is steady；
Obtained leaflet bush cinqefoil extract in 120mg steps 1 is taken, 8mL is dissolved in and mixes up and down in liquid (upper and lower equal quantities), by sample introduction
Valve injection, while the flow velocity that the mobile phase is pumped into is adjusted to 1.2mL/min.
The high speed adverse current chromatogram figure (Detection wavelength 270nm) of leaflet bush cinqefoil extract is as shown in Figure 1.
Flow point " I ", " II ", " III " and " IV " is received according to the spectrogram, retention time difference is as follows：“Ⅰ”：94-106min,
“Ⅱ”：211-223min, " III "：243-245min, " IV "：248-258min.
Received cut is spin-dried for (60 DEG C) with Rotary Evaporators, chemical compounds I~IV is obtained and is followed successively by 18.8mg, 9.8mg,
9.1mg and 6.9mg；In the leaflet bush cinqefoil extract that i.e. step 1 is prepared, the chemical compounds I containing 15.7%w/w, 8.2%
The compounds Ⅳ of w/w compound ii, 7.6%w/w compound III and 5.8%w/w.
3rd, the structural confirmation and purity detecting of flow point " I ", " II ", " III " and " IV "
Purity detecting (peak area normalization method) is carried out to each flow point using HPLC methods, chromatographic condition is as follows：
Dubhe-C18Analytical column (4.6mm × 250mm, 10 μm), mobile phase：A- methanol, B- water；Gradient elution program：
25%~40% methanol (0-20min), flow velocity 1.0mL/min, 30 DEG C, Detection wavelength 270nm of column temperature, sample size are 5 μ L.
As a result as shown in Fig. 2 wherein (a) is sample A to be separated high-efficient liquid phase chromatogram, (b)~(e) is followed successively by chemical combination
The high-efficient liquid phase chromatogram of thing I~IV.
It is in single chromatographic peak to measure flow point " I "~" IV ", and its purity is more than 95%.Their purity is followed successively by
98.3%, 97.2%, 99.0% and 95.2%.
Through structural confirmation, compound " I "~" IV " is respectively PB3, OPC B6, OPC B7 and 2,
- O- the galloyl glucoses of 3,4,6- tetra-.
The ultraviolet spectra of compound " I "~" IV " is as shown in Figure 3.
The nuclear magnetic data of compound " I "~" IV " is as follows：
PB3 (I), light brown red powder；1H NMR(600MHz,CD3OD)δ:6.73 (1H, d, J=1.8Hz),
6.67 (1H, d, J=2.4Hz), 6.66 (1H, d, J=8.0Hz), 6.58 (1H, d, J=1.8Hz), 6.46 (1H, dd, J=
), 1.8,8.4Hz 6.24 (1H, dd, J=1.8,8.4Hz), 6.06 (1H, s), 5.88 (1H, d, J=2.4Hz), 5.78 (1H, d,
), J=1.8Hz 4.53 (1H, d, J=7.2Hz), 4.40 (1H, d, J=7.8Hz), 4.34 (1H, t, J=9.0Hz), 4.25
(1H, d, J=9.6Hz), 3.79 (1H, dd, J=7.2,13.2Hz), 2.75 (1H, dd, J=6.0,16.8Hz), 2.48 (1H,
Dd, J=7.8,16.2Hz)；13C NMR(150MHz,CD3OD)δ:158.6,157.1(×2),155.8,155.6,154.9,
146.1,145.8,145.6,145.5,132.6,131.8,120.6,119.9,116.4,116.2,116.0,115.5,
108.2,107.2,102.2,97.3,96.8,96.0,83.9,82.4,73.7,68.9,38.6,28.8.
OPC B6 (II), light brown red powder；1H NMR(600MHz,CD3OD)δ:6.95 (1H, d, J=8.4Hz),
6.81 (1H, d, J=10.8Hz), 6.66 (1H, d, J=8.0Hz), 6.58 (1H, d, J=1.8Hz), 6.46 (1H, dd, J=
), 1.8,8.4Hz 6.24 (1H, dd, J=1.8,8.4Hz), 6.06 (1H, s), 5.88 (1H, d, J=2.4Hz), 5.78 (1H, d,
), J=1.8Hz 4.53 (1H, d, J=7.2Hz), 4.40 (1H, d, J=7.8Hz), 4.34 (1H, t, J=9.0Hz), 4.25
(1H, d, J=9.6Hz), 3.79 (1H, dd, J=7.2,13.2Hz), 2.75 (1H, dd, J=6.0,16.8Hz), 2.48 (1H,
Dd, J=7.8,16.2Hz)；13C NMR(150MHz,CD3OD)δ:158.7,158.2,158.0,157.4,156.0.154.7,
146.4,146.2(×2),146.1,132.2,132.0,121.0,119.9,116.1(×2),115.9,115.2,105.4,
102.1,97.7,97.3,96.4,96.2,82.6,82.4,74.2,68.9,38.6,29.3.
OPC B7 (III), light brown red powder；1H-NMR(600MHz,CD3OD)δ:4.90(1H,br s,H-2),
4.00(1H,br s,H-3),4.55(1H,br s,H-4),6.05(1H,br s,H-6),5.99(1H,br s,H-8),6.88
(1H, br s, H-2'), 6.75 (1H, d, J=7.8Hz, H-4'), 6.68 (1H, d, J=7.8Hz, H-6'), 4.57 (1H, d, J
=7.8Hz, H-2 "), 3.96 (1H, m, H-3 "), 2.46 (1H, d, J=7.8Hz, H-4 "), 5.99 (1H, br s, H-8 "),
2.75 (1H, d, J=13.2Hz, H-10 "), 6.84 (1H, br s, H-2 " '), 6.72 (1H, d, J=7.8Hz, H-4 " '), 6.71
(1H, d, J=4.2Hz, H-6 " ')；13C-NMR(150MHz,CD3OD)δ:77.3(C-2),72.7(C-3),37.7(C-4),
154.9(C-5),96.8(C-6),156.0(C-7),96.1(C-8),159.3(C-9),101.4(C-10),132.3(C-1'),
115.2(C-2'),146.2(C-3'),145.7(C-4'),115.9(C-5'),119.2(C-6'),82.6(C-2”),68.8
(C-3”),28.6(C-4”),155.5(C-5”),96.8(C-6”),158.0(C-7”),96.1(C-8”),159.4(C-9”),
101.4(C-10”),132.2(C-1”'),115.2(C-2”'),146.2(C-3”'),145.9(C-4”'),116.1(C-
5”'),120.0(C-6”').
2,3,4,6- tetra--O- galloyl glucoses (IV), light brown red powder, 3:The rotational isomer of 1 mixing.
Main isomers：- O- the galloyl glucoses of α-type 2,3,4,6- tetra-,1H NMR(600MHz,CD3OD)δ:
5.51 (d, J=3.5Hz, H-1), 5.14 (dd, J=9.9,3.5Hz, H-2), 6.00 (t, J=9.9Hz, H-3), 5.52 (t, J
=9.9Hz, H-4), 4.55 (ddd, J=9.9,4.5,2.0Hz, H-5), 4.32 (dd, J=12.2,4.5Hz, H-6a), 4.43
(dd, J=12.2,2.0Hz, H-6b)；6.90,6.97,7.01,7.11(each s,galloyl-2',6').13C NMR
(150MHz,CD3OD)δ:63.7(C-6),68.8(C-4),70.6(C-2),71.6(C-5),73.4(C-3),91.5(C-1)；
110.3,110.3,110.4,110.4(galloyl-2',6'),120.4,120.5,120.8,121.2(galloyl-1'),
139.9,140.0,140.2,140.3(galloyl-4'),146.3,146.4,146.4,146.5(galloyl-3',5'),
167.1,167.5,167.7,168.1(galloyl-7').
Secondary isomers：- O- the galloyl glucoses of β-type 2,3,4,6- tetra-,1H NMR(150MHz,CD3OD)δ:
5.07 (d, J=7.9Hz, H-1), 5.23 (dd, J=9.7,7.9Hz, H-2), 5.74 (t, J=9.7Hz, H-3), 5.51 (t, J
=9.7Hz, H-4), 4.20 (ddd, J=9.7,4.6,2.0Hz, H-5), 4.31 (dd, J=12.3,4.6Hz, H-6a), 4.49
(dd, J=12.3,2.0Hz, H-6b)；6.87,6.95,6.99,7.10(each s,galloyl-2',6').13C NMR datas
It is identical with the above-mentioned-O- galloyl glucoses of α-type 2,3,4,6- tetra-.
The present invention separation of embodiment 2 prepares the screening of process conditions in four kinds of tanninses compound methods
Inventor is mainly to being obtained the solvent system in elution fraction and high speed adverse current chromatogram by ethyl acetate extraction part
System is screened.
(1) screening of eluant component
Elution fraction 1:Petroleum ether：Ethyl acetate=1:1 (v/v) eluent part.According to efficient liquid phase in embodiment 1
Chromatographic condition determines：Difference is gradient elution program：10%-70% methanol (0~45min), Detection wavelength 210nm.As a result
As shown in Figure 4.
Elution fraction 2:Petroleum ether：Ethyl acetate=2:3 (v/v) eluent part.According to efficient liquid phase in embodiment 1
Chromatographic condition determines：Difference is gradient elution program：25%-50% methanol (0~25min), Detection wavelength 250nm.As a result
As shown in Figure 5.
Compared with ethyl acetate elution fraction used in the embodiment 1 of display in Fig. 2 (a), above two elution fraction
Main peak is less, and baseline is uneven, and impurity is more, it is not easy to reach baseline separation, therefore the acetic acid second selected in preferred embodiment 1
Ester elution fraction.
(2) screening of solvent system：
Distribution coefficient (K) is the ratio that solute is distributed after two-phase solvent balance between two-phase, is high speed adverse current chromatogram system
Unite the key factor of selection.Defining K value is surveyed by the high performance liquid chromatography peak area ratio of distribution samples in two-phase solvent.According to
Document, in order that good separating effect, K values will be between 0.5-2.The solvent system selected in the present invention is n-hexane-acetic acid second
The different proportion of ester-methanol-water, the dicyandiamide solution K values of different proportion are shown in Table 1.
K value of the target compound I-IV of table 1 in different solvents system
As a result show, be 1 in dicyandiamide solution ratio:10:1:10、1:10:0.8:10 and 1:11:1.2:When 11, four mesh
The K values of compound are marked all in OK range.
However, when dicyandiamide solution is 1:10:1:When 10, it is applied to HSCCC separation process, as a result as described in Figure 6,
As a result show, its effect is unsatisfactory.
When dicyandiamide solution ratio is 1:10:0.8:10 when, compound I and III α values (α=K2/K1,K2>K1) close
1, the two compounds can be caused inseparable.
Therefore, the result in table 1 and embodiment 1, final choice solvent system be n-hexane-ethyl acetate-methanol-
Water volume ratio is 1:11:1.2:11, under this dicyandiamide solution, during HSCCC, the retention rate of stationary phase is 62.7%.
In summary, present invention firstly discovers that containing OPC B7 and 2,3,4,6- tetra--O- galloyl glucoses
Potentilla plants extract.
Leaflet bush cinqefoil extract prepared by the present invention, is enriched with a variety of tannins, the OPC in the extract
B3 content is up to 15~16%w/w；OPC B6 content is up to 8~9%w/w；OPC B7 content is up to 7~
8%w/w；The content of-O- the galloyl glucoses of 2,3,4,6- tetra- is up to 5~6%w/w.The extract can be used for anti-inflammatory, antioxygen
Change, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control the disturbance of lower legs due to pathogenic cold and dampness, treatment pruigo or treatment mastitis.
Meanwhile PB3, OPC B6, former flower has been prepared in the present invention separation from leaflet bush cinqefoil first
Blue or green plain B7 and 2, four kinds of tanninses compounds of 3,4,6- tetra--O- galloyl glucoses, product purity are all higher than the 95%, party
Method preparation amount is big, is suitable for commercial Application.
Claims (12)
- A kind of 1. method for preparing potentilla plants leaflet bush cinqefoil extract, it is characterised in that：Comprise the following steps：(1) leaflet bush cinqefoil alcohol crude extract is prepared：Leaflet bush cinqefoil, cutting or crushing, alcohol extracting are taken, is concentrated to give leaflet gold dew Plum alcohol crude extract；(2) ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract is prepared：Take step (1) that alcohol crude extract, stone is prepared Oily ether extraction, remaining liq is taken, ethyl acetate extraction, obtains the ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract；(3) concentrate for being enriched with four kinds of tanninses compounds is prepared：It is prepared with silica gel column chromatography separating step (2) Ethyl acetate extraction part, with petroleum ether-ethyl acetate volume ratio 100:0~0:100 gradient elutions, collect petroleum ether-acetic acid Ethyl ester volume ratio 1:1~0:100 flow point, concentrates and produces；Four kinds of tanninses compounds are PB3, and OPC B6, OPC B7 and 2,3,4,6- tetra--O-, which, not to be eaten Sub- acyl glucose.
- 2. according to the method for claim 1, it is characterised in that：In step (3), the Fraction collection petroleum ether-acetic acid second Ester volume ratio is 0:100.
- 3. according to the method for claim 2, it is characterised in that：In step (1), the alcohol is 60%-90% ethanol；The temperature of the extraction is 60 DEG C；The number of extraction is 3 times；Often During secondary extraction, the volume mass ratio of alcohol and leaflet bush cinqefoil is 30:6.5L/Kg；In step (2), the extraction times of the petroleum ether are 2~10 times, and when extracting every time, the volume ratio with alcohol crude extract is 1: 8；The extraction times of the ethyl acetate are 2~10 times, and when extracting every time, the volume ratio with alcohol crude extract is 1:10；In step (3), the gradient elution is successively using the volume ratio of petroleum ether-ethyl acetate as 100：1、50：1、10：1、1： 1、2：The gradient elution that 3 eluent and ethyl acetate eluent is carried out；3 are followed successively by with the volume mass ratio of leaflet bush cinqefoil： 6.5、4：6.5、6：6.5、10：6.5、10.5：6.5 and 11.5：6.5L/Kg.
- 4. according to the method for claim 3, it is characterised in that：The concentration of the ethanol is 70%.
- 5. according to the method described in claim 1-4 any one, it is characterised in that：The extract contains not less than 6%w/w OPC B7 or/and-O- galloyl glucoses of 2,3,4,6- tetra- not less than 4%w/w.
- 6. according to the method for claim 5, it is characterised in that：The extract also contains and is not less than 14%w/w original cyanine The plain B3 and OPC B6 not less than 7%w/w.
- 7. according to the method for claim 6, it is characterised in that：The extract contains 15~16%w/w OPC B3,8~9%w/w OPC B6,7~8%w/w OPC B7 and 5~6%w/w 2,3,4,6- tetra--O- are not eaten Sub- acyl glucose.
- 8. according to the method for claim 5, it is characterised in that：In described 2,3,4,6- tetra--O- galloyl glucoses, α- The mol ratio of-O- the galloyl glucoses of type 2,3,4,6- tetra- and the-O- galloyl glucoses of β-type 2,3,4,6- tetra- is 3:1.
- 9. the method according to claim 6 or 7, it is characterised in that：- O- the galloyl glucoses of 2,3,4,6- tetra- In, the mol ratio of-O- galloyl glucoses of α-type 2,3,4,6- tetra- and the-O- galloyl glucoses of β-type 2,3,4,6- tetra- is 3:1。
- 10. a kind of separate the method for preparing four kinds of tanninses compounds, it is characterised in that：Four kinds of tanninses compounds are original Anthocyanidin B3, OPC B6, OPC B7 and 2,3,4,6- tetra--O- galloyl glucoses, comprise the following steps：Separated using high-speed countercurrent chromatography and prepare four kinds of tanninses compounds：(a) the leaflet bush cinqefoil extract for taking the method described in claim 1-9 any one to prepare, is dissolved in sample introduction solvent, Sample introduction solution is prepared；(b) sample introduction solution is injected into high-speed counter-current chromatograph, PB3, OPC B6, OPC B7 are collected in detection With 2, the flow point of 3,4,6- tetra--O- galloyl glucoses, solvent is removed, produces PB3, OPC B6, former cyanine The product of the plain-O- galloyl glucoses of B7 and 2,3,4,6- tetra-；The stationary phase of the high-speed countercurrent chromatography is the upper phase of two phase solvent system, and mobile phase is under two phase solvent system Phase；The two phase solvent system is (0.8~1.2) by n-hexane-ethyl acetate-methanol-water volume ratio:(10.8~11.2): (1~1.4):(10.8~11.2) form.
- 11. according to the method for claim 10, it is characterised in that：The volume of the n-hexane-ethyl acetate-methanol-water Than for 1:11:1.2:11.
- 12. the method according to claim 10 or 11, it is characterised in that：The sample introduction solvent is by the upper and lower phase According to 1:The solvent of 1 volume ratio composition；The volume mass ratio of the sample introduction solvent and extract is 1:15~1:30mL/mg； The rotating speed of separating pipe is 900 ± 50rpm in the high-speed counter-current chromatograph, the temperature of separating pipe in the high-speed counter-current chromatograph 30 ± 5 DEG C are arranged to, the flow velocity of the mobile phase is 1.2 ± 0.3mL/min, and the wavelength of the detection is 270 ± 5nm.
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