CN105669631A - Potentilla plant extract and method of separating four tannin compounds therefrom - Google Patents
Potentilla plant extract and method of separating four tannin compounds therefrom Download PDFInfo
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Abstract
The invention discloses a potentilla plant extract which includes not less than 6% w/w of procyanidine B7 and not less than 4% w/w of 2,3,4,6-tetra-O-galloylglucose. The extract is used for resisting inflammation and oxidization, promoting wound healing, resisting bacteria and virus, promoting urine, eliminating edema, treating dermatophytosis due to cold and dampness and treating prurigo and mastitis. In the invention, four tannin compounds, including procyanidine B3, procyanidine B6, the procyanidine B7 and the 2,3,4,6-tetra-O-galloylglucose, are separated and prepared from the potentilla plant extract, product purities being higher than 95%. The method has large preparation quantity and is suitable for industrial application.
Description
Technical field
The present invention relates to natural medicine field, it is specifically related to from potentilla plants leaflet bush cinqefoil extract, and the method for PB3, pycnogenols B6, pycnogenols B7 and 2,3,4,6-tetra--O-Nutgalls acyl glucose is prepared in separation from this extract.
Background technology
Now there are some researches show, the various plants of potentilla is medicinal plant, has anti-inflammation, anti-diabetic, and antitumor grade acts on; And tannins compound is the important component of this platymiscium. Tannins active substance is divided into two classes: i.e. condensed type tannin (comprising pycnogenols) and hydrolysis-type tannin, they have anti-inflammation, anti-oxidant, promote wound healing, the effect such as antibacterial, antiviral.
Leaflet bush cinqefoil (PotentillaparvifoliaFisch.) is the one in potentilla plants, and specifically Rosaceae potentilla machaka, has another name called leaf or flower of Littleleaf cinquefoil etc. It, as a kind of traditional middle Tibetan medicine, is used as medicine with flower, leaf. " Chinese medicine voluminous dictionary " is recorded leaf or flower of Littleleaf cinquefoil function cure mainly: diuresis disappears water, controls cold-dampness beriberi, pruigo, mazoitis.
At present, there are no the leaflet bush cinqefoil extract of enrichment tannin and from leaflet bush cinqefoil separation prepare the report of tannins compound.
Summary of the invention
For solving the problem, the present invention provides a kind of potentilla plants leaflet bush cinqefoil extract, and it contains the pycnogenols B7 being not less than 6%w/w or/and be not less than the 2,3,4,6-tetra--O-Nutgalls acyl glucose of 4%w/w. Above-mentioned two kinds of materials are and find from potentilla first.
Further, described extract is also containing being not less than 14%w/w PB3 and be not less than the pycnogenols B6 of 7%w/w.
The structure of PB3 (I), pycnogenols B6 (II), pycnogenols B7 (III) and 2,3,4,6-tetra--O-Nutgalls acyl glucose (IV) is as follows:
Further, described extract contains the PB3 of 15～16%w/w, the 2,3,4,6-tetra--O-Nutgalls acyl glucose of the pycnogenols B7 and 5～6%w/w of the pycnogenols B6,7～8%w/w of 8～9%w/w.
Further, in described 2,3,4,6-tetra--O-Nutgalls acyl glucose, the mol ratio of α-type 2,3,4,6-tetra--O-Nutgalls acyl glucose and β-type 2,3,4,6-tetra--O-Nutgalls acyl glucose is 3:1.
Further, described extract is the extract of leaflet bush cinqefoil PotentillaparvifoliaFisch. over-ground part. Described over-ground part is all over-ground parts, comprises stem, leaf, flower.
Present invention also offers a kind of method preparing described leaflet bush cinqefoil extract, it is characterised in that: comprise the following steps:
(1) preparing leaflet bush cinqefoil alcohol crude extract: get leaflet bush cinqefoil, cutting or pulverizing, ethanol-extracted, concentrates and obtains leaflet bush cinqefoil alcohol crude extract;
(2) ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract is prepared: get step (1) and prepare alcohol crude extract, petroleum ether extraction, get remaining liq, extraction into ethyl acetate, obtain the ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract;
(3) enriched material of four kinds of tannins compounds described in enrichment is prepared: the ethyl acetate extraction part prepared with silica gel column chromatography separating step (2), with petroleum ether-ethyl acetate volume ratio 100:0～0:100 gradient elution, collect the flow point of petroleum ether-ethyl acetate volume ratio 1:1～0:100, concentrate and get final product; The flow point of preferred 0:100, i.e. flow point under pure ethyl acetate wash-out.
Further, in step (1), described alcohol is the ethanol of 60%-90%, it is preferable that 70% ethanol; The temperature of described extraction is 60 DEG C; The number of times extracted is 3 times; When extracting, alcohol is 30:6.5L/Kg with the volume mass ratio of leaflet bush cinqefoil every time;
Further, in step (2), the extraction times of described sherwood oil is 2～10 times, when extracting, is 1:8 with the volume ratio of alcohol crude extract every time; The extraction times of described ethyl acetate is 2～10 times, when extracting, is 1:10 with the volume ratio of alcohol crude extract every time.
Further, in step (3), described gradient elution is successively taking the volume ratio of petroleum ether-ethyl acetate as the gradient elution that the elutriant of 100:1,50:1,10:1,1:1,2:3 and eluent ethyl acetate liquid (i.e. the volume ratio 0:100 of petroleum ether-ethyl acetate) carry out.
Further, in step (3), to be the elutriant of 100:1,50:1,10:1,1:1,2:3 and eluent ethyl acetate liquid be followed successively by 3:6.5,4:6.5,6:6.5,10:6.5,10.5:6.5 and 11.5:6.5L/Kg with the volume mass ratio of leaflet bush cinqefoil to the volume ratio of described petroleum ether-ethyl acetate.
Further, aforementioned leaflet bush cinqefoil extract is prepared by aforesaid method.
Present invention also offers the method for separation preparation four kinds of tannins compounds from aforementioned leaflet bush cinqefoil extract, described four kinds of tannins compounds are PB3, pycnogenols B6, pycnogenols B7 and 2,3,4,6-tetra--O-Nutgalls acyl glucose, comprises the following steps:
Utilize high-speed countercurrent chromatography separation preparation four kinds of tannins compounds:
A () is got aforementioned leaflet bush cinqefoil extract and is dissolved in into, in sample solvent, preparing into sample solution;
B () will enter sample solution and inject high-speed counter-current chromatograph, PB3, pycnogenols B6, pycnogenols B7 and 2 are collected in detection, 3, the flow point of 4,6-tetra--O-Nutgalls acyl glucose, except desolventizing, obtain PB3, pycnogenols B6, pycnogenols B7 and 2, the product of 3,4,6-tetra--O-Nutgalls acyl glucose;
The stationary phase of described high-speed countercurrent chromatography is the upper phase of two phase solvent system, and moving phase is the lower phase of two phase solvent system; Described 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) forms, it is preferable that ratio be 1:11:1.2:11 (v/v).
Further, the solvent that sample solvent is made up of according to the volume ratio of 1:1 described upper and lower phase is entered described in; Described enter sample solvent and extract volume mass than being 1:15～1:30mL/mg.
Further, in described high-speed counter-current chromatograph, the rotating speed of separator tube is 900 ± 50rpm.
Further, in described high-speed counter-current chromatograph, the temperature of separator tube is set to 30 ± 5 DEG C.
Further, the flow velocity of described moving phase is 1.2 ± 0.3mL/min.
Further, the wavelength of described detection is 270 ± 5nm.
Present invention also offers a kind of method that PB3, pycnogenols B6, pycnogenols B7 and 2,3,4,6-tetra--O-Nutgalls acyl glucose are prepared in separation, comprise the following steps:
Get the solution containing PB3, pycnogenols B6, pycnogenols B7 and 2,3,4,6-tetra--O-Nutgalls acyl glucose or extract, carry out according to aforesaid high-speed countercurrent chromatography.
Present invention also offers described extract the anti-inflammation of preparation, anti-oxidant, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control cold-dampness beriberi, purposes in treatment pruigo or treatment mastitis medicament.
Present invention also offers a kind of pharmaceutical composition, the leaflet bush cinqefoil extract that it is described is activeconstituents, adds the preparation that pharmaceutically conventional auxiliary material or complementary composition are prepared from.
The potentilla plants extract of Late Cambrian of the present invention containing pycnogenols B7 and 2,3,4,6-four-O-Nutgalls acyl glucose.
Leaflet bush cinqefoil extract prepared by the present invention, the multiple tannin of enrichment, the content of the PB3 in this extract is up to 15～16%w/w; The content of pycnogenols B6 is up to 8～9%w/w; The content of pycnogenols B7 is up to 7～8%w/w; The content of 2,3,4,6-tetra--O-Nutgalls acyl glucose is up to 5～6%w/w. This extract can be used for anti-inflammation, anti-oxidant, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control cold-dampness beriberi, treatment pruigo or treatment mazoitis.
Meanwhile, method provided by the invention, from leaflet bush cinqefoil, separation has prepared PB3, pycnogenols B6, the pycnogenols B7 and 2 of high purity first, 3,4,6-tetra--O-Nutgalls acyl glucose, their purity is followed successively by 98.3%, 97.2%, 99.0% and 95.2%. Above-mentioned four tannins compounds are find from leaflet bush cinqefoil plant first, and pycnogenols B7 and 2,3,4,6-four-O-Nutgalls acyl glucose is find from potentilla first, greatly extends the range of choice of these compound plant materials.
And, high-speed countercurrent chromatography (the high-speedcouter-currentchromatography that the present invention adopts, HSCCC), it it is a kind of liquid liquid distribution chromatography technology not using solid-state supporter or carrier, which overcome the shortcomings such as loss that the absorption of sample produces by solid phase carrier, sex change, there is the advantage realizing efficient preparation property separation within the short period of time. The method has the advantage that repeatability is good, product purity height, preparation amount are big, is applicable to from the various natural product containing above-mentioned four kinds of compounds and prepares high-purity monomer, is with a wide range of applications.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, do not departing under the above-mentioned basic fundamental thought prerequisite of the present invention, it is also possible to make the amendment of other various ways, replacement or change.
The embodiment of form by the following examples, is described in further detail the foregoing of the present invention again.But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to following example. All technology realized based on foregoing of the present invention all belong to the scope of the present invention.
Accompanying drawing explanation
When Fig. 1 is solvent system n-hexane-ethyl acetate-methanol-water=1:11:1.2:11, the high speed adverse current chromatogram figure of leaflet bush cinqefoil extract.
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 the high-efficient liquid phase chromatogram of chemical compounds I～IV.
Fig. 3 is the UV spectrum of PB3 (I), pycnogenols B6 (II), pycnogenols B7 (III) and 2,3,4,6-tetra--O-Nutgalls acyl glucose (IV).
Fig. 4 is embodiment 2 PetroChina Company Limited. ether: the high-efficient liquid phase chromatogram of the elutriant part of ethyl acetate=1:1 (v/v).
Fig. 5 is embodiment 2 sherwood oil: the high-efficient liquid phase chromatogram of the elutriant part of ethyl acetate=2:3 (v/v).
When Fig. 6 is solvent system n-hexane-ethyl acetate-methanol-water=1:10:1:10, the high speed adverse current chromatogram figure of leaflet bush cinqefoil extract.
Embodiment
The present invention's medicinal material used is the dry aerial parts of leaflet bush cinqefoil (PotentillaparvifoliaFisch.), adopts in Qinghai Province's Oenothera littaralis in August, 2014. Plant species by Chinese Academy of Sciences's northwest plateau biological study doctor Zhang Faqi qualification, herbarium be stored in Qinghai-Tibet Platean biological sample shop, sample is numbered 252558.
Experiment uses reagent: analytical pure sherwood oil, ethyl acetate, normal hexane, methyl alcohol (Shanghai Zhan Yun chemical reagent company limited), chromatogram methyl alcohol (Chemical Reagent Co., Ltd., Sinopharm Group).
Instrument: TBE-300B high-speed counter-current chromatograph (Shanghai is with field biotechnology limited-liability company), is furnished with TBP-5002 constant flow pump and TBD-2000UV detector (same field, Shanghai); HX-1050 constant temperature circulator (Beijing Bo Yikang laboratory apparatus company limited) and N2000 chromatographic working station (Zhi Da information engineering company of Zhejiang University).
Excellent general ultrapure water machine (the excellent general ultrapure Science and Technology Ltd. in Sichuan).
Yi Lite P230 high performance liquid chromatograph, is furnished with P230 double pump system, manual sampling thief (Rheodyne7725i), column oven (PH-530), diode-array detector (DAD230), Dubhe-C18Analytical column (4.6mm × 250mm, 10 μm) and EC2000DAD single passage chromatographic working station (Dalian Yilite Analytical Instrument Co., Ltd).
Nuclear magnetic resonance analyser is VarianINOVA600 NMR system (Varian, PaloAlto, CA, USA).
Embodiment 1
1. the preparation of leaflet bush cinqefoil extract of the present invention
Get leaflet bush cinqefoil dry aerial parts (6.5kg) scissors and shred the segment into about 1.5cm, adopt 70% ethanol, 3 times (each 30L) is extracted with boiling machine (temperature is 60 DEG C) heating, merge to concentrate and obtain leaflet bush cinqefoil alcohol crude extract, concentrating under reduced pressure is to 4L, again successively with sherwood oil (every time with 5000mL, extract 10 times), ethyl acetate (each 4000mL extracts 10 times) extraction.
Ethyl acetate extraction part is concentrated, cross normal phase silicagel column, 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, get the elution fraction of eluent ethyl acetate liquid, concentrating under reduced pressure removes desolventizing and obtains dry pressed powder, i.e. leaflet bush cinqefoil extract 9.8g.
2. four kinds of tannins compounds in high speed adverse current chromatogram separation and purification leaflet bush cinqefoil extract
A. solvent systems is prepared
Solvent systems is prepared in separating funnel according to the volume ratio of n-hexane-ethyl acetate-methanol-water=1:11:1.2:11, fully stratification after shake, collects upper phase solvent and lower phase solvent, respectively ultrasonic 20min respectively, above phase is stationary phase, and lower phase is moving phase;
B. it is that 30mL/min pump enters described stationary phase taking flow velocity in the separator tube (temperature is set to 30 DEG C) of high-speed counter-current chromatograph device, after phase to be fixed is full of separator tube, make main frame just to rotate and keep rotating speed be 900rpm balance 30min, with the flow pump of 2.0mL/min enter moving phase to described separator tube exit end have moving phase overflow and chromatographic working station baseline steady; Get in 120mg step 1 obtained leaflet bush cinqefoil extract, it is dissolved in 8mL up and down in phase mixed solution (upper and lower equal quantities), enters sample by sampling valve, the flow velocity that described moving phase pump enters is adjusted to 1.2mL/min simultaneously.
The high speed adverse current chromatogram figure (determined wavelength is 270nm) of leaflet bush cinqefoil extract is as shown in Figure 1.
Receiving flow point " I ", " II ", " III " and " IV " according to this spectrogram, retention time is as follows respectively: " I ": 94-106min, " II ": 211-223min, " III ": 243-245min, " IV ": 248-258min.
Point it is spin-dried for receiving to evaporate (60 DEG C) with Rotary Evaporators, obtains chemical compounds I～IV and be followed successively by 18.8mg, 9.8mg, 9.1mg and 6.9mg; Namely in the leaflet bush cinqefoil extract that step 1 prepares, the chemical compounds I containing 15.7%w/w, the compound IV of the compound ii of 8.2%w/w, the compound III of 7.6%w/w and 5.8%w/w.
3, the structural confirmation of flow point " I ", " II ", " III " and " IV " and purity detecting
Adopting HPLC method that each flow point carries out purity detecting (peak area normalization method), chromatographic condition is as follows:
Dubhe-C18Analytical column (4.6mm × 250mm, 10 μm), moving phase: A-methyl alcohol, B-water; Gradient elution program: 25%～40% methyl alcohol (0-20min), flow velocity 1.0mL/min, post temperature 30 DEG C, determined wavelength 270nm, sample size is 5 μ L.
As shown in Figure 2, wherein (a) is the high-efficient liquid phase chromatogram of sample A to be separated to result, and (b)～(e) is followed successively by the high-efficient liquid phase chromatogram of chemical compounds I～IV.
Recording flow point " I "～" IV " all in single chromatographic peak, its purity is all 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, pycnogenols B6, pycnogenols B7 and 2,3,4,6-tetra--O-Nutgalls acyl glucose.
The UV spectrum 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;1HNMR(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),13CNMR(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.
Pycnogenols B6 (II), light brown red powder;1HNMR(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),13CNMR(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.
Pycnogenols B7 (III), light brown red powder;1H-NMR(600MHz,CD3OD) δ: 4.90 (1H, brs, H-2), 4.00 (1H, brs, H-3), 4.55 (1H, brs, H-4), 6.05 (1H, brs, H-6), 5.99 (1H, brs, H-8), 6.88 (1H, brs, 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, brs, H-8 "), 2.75 (1H, d, J=13.2Hz, H-10 "), 6.84 (1H, brs, 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-Nutgalls acyl glucose (IV), light brown red powder, the rotational isomer of 3:1 mixing.
Main isomer: α-type 2,3,4,6-tetra--O-Nutgalls acyl glucose,1HNMR(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 (eachs, galloyl-2', 6').13CNMR(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 isomer: β-type 2,3,4,6-tetra--O-Nutgalls acyl glucose,1HNMR(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 (eachs, galloyl-2', 6').13CNMR data are identical with above-mentioned α-type 2,3,4,6-tetra--O-Nutgalls acyl glucose.
Embodiment 2 the present invention is separated the screening of processing condition in preparation four kinds of tannins compound methods
The solvent systems obtained by ethyl acetate extraction part in elution fraction and high speed adverse current chromatogram has mainly been screened by contriver.
(1) screening of elutriant component
Elution fraction 1: sherwood oil: the elutriant part of ethyl acetate=1:1 (v/v). Measure according to high-efficient liquid phase chromatogram condition in embodiment 1: difference is gradient elution program: 10%-70% methyl alcohol (0～45min), determined wavelength 210nm. Result is as shown in Figure 4.
Elution fraction 2: sherwood oil: the elutriant part of ethyl acetate=2:3 (v/v). Measure according to high-efficient liquid phase chromatogram condition in embodiment 1: difference is gradient elution program: 25%-50% methyl alcohol (0～25min), determined wavelength 250nm. Result is as shown in Figure 5.
Compared with eluent ethyl acetate part used in the embodiment 1 of display in Fig. 2 (a), above-mentioned two kinds of elution fraction main peaks are less, and baseline is uneven, and impurity is more, it is not easy to reach baseline separation, it is preferred to the eluent ethyl acetate part selected in embodiment 1.
(2) screening of solvent systems:
Partition ratio (K) is the ratio of solute distribution between two-phase after two-phase solvent balance, is the key factor of high speed adverse current chromatogram Systematic selection.Defining K value is surveyed by the high performance liquid chromatography peak area ratio of the sample that distributes in two-phase solvent. According to document, in order to make good separating effect, K value will between 0.5-2. The solvent systems selected in the present invention is the different ratios of n-hexane-ethyl acetate-methanol-water, and the solvent system K value of different ratios is in table 1.
The K value of table 1 target compound I-IV in different solvents system
Result shows, when solvent system ratio is 1:10:1:10,1:10:0.8:10 and 1:11:1.2:11, the K value of four target compounds is all in OK range.
But, when solvent system is 1:10:1:10, it being applied to the sepn process of HSCCC, as described in Figure 6, result shows result, and its effect is unsatisfactory.
When solvent system ratio is 1:10:0.8:10, the α value (α=K of Compound I and III2/K1,K2>K1) close to 1, these two compounds can be caused inseparable.
Therefore, according to the result in table 1 and embodiment 1, final selection solvent systems is n-hexane-ethyl acetate-methanol-water volume ratio is 1:11:1.2:11, and under this solvent system, in HSCCC process, the retention rate of stationary phase is 62.7%.
In sum, the potentilla plants extract of Late Cambrian of the present invention containing pycnogenols B7 and 2,3,4,6-four-O-Nutgalls acyl glucose.
Leaflet bush cinqefoil extract prepared by the present invention, the multiple tannin of enrichment, the content of the PB3 in this extract is up to 15～16%w/w; The content of pycnogenols B6 is up to 8～9%w/w; The content of pycnogenols B7 is up to 7～8%w/w; The content of 2,3,4,6-tetra--O-Nutgalls acyl glucose is up to 5～6%w/w. This extract can be used for anti-inflammation, anti-oxidant, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control cold-dampness beriberi, treatment pruigo or treatment mazoitis.
Meanwhile, the present invention is separated from leaflet bush cinqefoil first and has prepared PB3, pycnogenols B6, pycnogenols B7 and 2,3,4, four kinds of tannins compounds of 6-tetra--O-Nutgalls acyl glucose, product purity is all greater than 95%, the method preparation amount is big, is suitable for industrial application.
Claims (10)
1. a potentilla plants leaflet bush cinqefoil extract, it is characterised in that: it contains the pycnogenols B7 being not less than 6%w/w or/and be not less than the 2,3,4,6-tetra--O-Nutgalls acyl glucose of 4%w/w.
2. extract according to claim 1, it is characterised in that: described extract is also containing being not less than 14%w/w PB3 and be not less than the pycnogenols B6 of 7%w/w.
3. extract according to claim 2, it is characterised in that: described extract contains the PB3 of 15～16%w/w, the pycnogenols B6 of 8～9%w/w, the 2 of the pycnogenols B7 and 5～6%w/w of 7～8%w/w, 3,4,6-tetra--O-Nutgalls acyl glucose;
Preferably, in described 2,3,4,6-tetra--O-Nutgalls acyl glucose, the mol ratio of α-type 2,3,4,6-tetra--O-Nutgalls acyl glucose and β-type 2,3,4,6-tetra--O-Nutgalls acyl glucose is 3:1.
4. prepare the method for the described leaflet bush cinqefoil extract of the arbitrary item of claim 1-3 for one kind, it is characterised in that: comprise the following steps:
(1) preparing leaflet bush cinqefoil alcohol crude extract: get leaflet bush cinqefoil, cutting or pulverizing, ethanol-extracted, concentrates and obtains leaflet bush cinqefoil alcohol crude extract;
(2) ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract is prepared: get step (1) and prepare alcohol crude extract, petroleum ether extraction, get remaining liq, extraction into ethyl acetate, obtain the ethyl acetate extraction part of leaflet bush cinqefoil alcohol crude extract;
(3) enriched material of four kinds of tannins compounds described in enrichment is prepared: the ethyl acetate extraction part prepared with silica gel column chromatography separating step (2), with petroleum ether-ethyl acetate volume ratio 100:0～0:100 gradient elution, collect the flow point of petroleum ether-ethyl acetate volume ratio 1:1～0:100, concentrate and get final product; The flow point of preferred 0:100.
5. method according to claim 4, it is characterised in that:
In step (1), described alcohol is the ethanol of 60%-90%, it is preferable that 70% ethanol; The temperature of described extraction is 60 DEG C; The number of times extracted is 3 times; When extracting, alcohol is 30:6.5L/Kg with the volume mass ratio of leaflet bush cinqefoil every time;
In step (2), the extraction times of described sherwood oil is 2～10 times, when extracting, is 1:8 with the volume ratio of alcohol crude extract every time; The extraction times of described ethyl acetate is 2～10 times, when extracting, is 1:10 with the volume ratio of alcohol crude extract every time;
In step (3), described gradient elution is successively taking the volume ratio of petroleum ether-ethyl acetate as the gradient elution that the elutriant of 100:1,50:1,10:1,1:1,2:3 and eluent ethyl acetate liquid carry out; It is followed successively by 3:6.5,4:6.5,6:6.5,10:6.5,10.5:6.5 and 11.5:6.5L/Kg with the volume mass of leaflet bush cinqefoil ratio.
6. want the extract described in the arbitrary item of 1-4 according to right, it is characterised in that: described extract is the leaflet bush cinqefoil extract prepared by the method described in claim 4 or 5.
7. one kind is separated the method for preparation four kinds of tannins compounds, it is characterised in that: described four kinds of tannins compounds are PB3, pycnogenols B6, pycnogenols B7 and 2,3,4,6-tetra--O-Nutgalls acyl glucose, comprise the following steps:
Utilize high-speed countercurrent chromatography separation preparation four kinds of tannins compounds:
A () gets leaflet bush cinqefoil extract according to claim 6, be dissolved in into, in sample solvent, preparing into sample solution;
B () will enter sample solution and inject high-speed counter-current chromatograph, PB3, pycnogenols B6, pycnogenols B7 and 2 are collected in detection, 3, the flow point of 4,6-tetra--O-Nutgalls acyl glucose, except desolventizing, obtain PB3, pycnogenols B6, pycnogenols B7 and 2, the product of 3,4,6-tetra--O-Nutgalls acyl glucose;
The stationary phase of described high-speed countercurrent chromatography is the upper phase of two phase solvent system, and moving phase is the lower phase of two phase solvent system; Described 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) forms, it is preferable that ratio be 1:11:1.2:11 (v/v).
8. method according to claim 7, it is characterised in that: described in enter the solvent that sample solvent is made up of according to the volume ratio of 1:1 described upper and lower phase; Described enter sample solvent and extract volume mass than being 1:15～1:30mL/mg; In described high-speed counter-current chromatograph, the rotating speed of separator tube is 900 ± 50rpm, and in described high-speed counter-current chromatograph, the temperature of separator tube is set to 30 ± 5 DEG C, and the flow velocity of described moving phase is 1.2 ± 0.3mL/min, and the wavelength of described detection is 270 ± 5nm.
9. claim 1-3 or the 6 described extracts of arbitrary item the anti-inflammation of preparation, anti-oxidant, promote wound healing, antibacterial, antiviral, diuresis, consumer edema, control cold-dampness beriberi, purposes in treatment pruigo or treatment mastitis medicament.
10. a pharmaceutical composition, it is characterised in that: it is taking the leaflet bush cinqefoil extract described in the arbitrary item of claim 1-3 or 6 as activeconstituents, adds the preparation that pharmaceutically conventional auxiliary material or complementary composition are prepared from.
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| CN109549938A (en) * | 2018-10-22 | 2019-04-02 | 烟台大学 | Purposes of the procyanidin compounds in the product for preventing and/or treating insulin resistance |
| WO2021042922A1 (en) * | 2019-09-02 | 2021-03-11 | 广东省农业科学院茶叶研究所 | Preparation method for tetragalloylglucose |
| JP2022543794A (en) * | 2019-09-02 | 2022-10-14 | 広東省農業科学院茶叶研究所 | Method for producing tetragalloyl glucose |
| JP7305870B2 (en) | 2019-09-02 | 2023-07-10 | 広東省農業科学院茶叶研究所 | Method for producing tetragalloyl glucose |
| CN114740100A (en) * | 2022-03-10 | 2022-07-12 | 西北农林科技大学 | LC-MS/MS detection method of gallotannin in plant |
| CN114740100B (en) * | 2022-03-10 | 2024-03-01 | 西北农林科技大学 | LC-MS/MS detection method for gallotannins in plants |
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