CN105017375B - Anticoagulation blackberry seed effective component and extraction and separation method and application thereof - Google Patents
Anticoagulation blackberry seed effective component and extraction and separation method and application thereof Download PDFInfo
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- CN105017375B CN105017375B CN201510499998.9A CN201510499998A CN105017375B CN 105017375 B CN105017375 B CN 105017375B CN 201510499998 A CN201510499998 A CN 201510499998A CN 105017375 B CN105017375 B CN 105017375B
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- C07C233/02—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
- C07C233/09—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to carbon atoms of an acyclic unsaturated carbon skeleton
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- 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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Abstract
The invention belongs to the technical field of plant extraction, and particularly relates to an anticoagulation blackberry seed effective component and an extraction and separation method and application thereof. Six compounds are obtained from the ethyl acetate portion and the n-butanol portion of blackberry seeds through a special method in a separation mode, the in-vitro anticoagulation effect of the six compounds is investigated, the result shows that the compounds from the first to the sixth all have a good anticoagulation effect, and the six compounds can be independently used or combined to serve as anticoagulants.
Description
Technical field
The invention belongs to technical field of plant extraction, and in particular to a kind of anticoagulation blackberry seed active ingredient and its extraction point
From method, application.
Background technology
Blackberry, blueberryRubusSpp.Blackberry is rose family rubus polymerization fruit plant, does not make medicinal, is in the world
Emerging4Plant one of fruitlet fruit trees.Original North America, its fruit sweet and sour taste, with very high nutritive value and medical value,
Also major part makes the food such as quick-frozen fruit, fruit juice, fruit wine and jam in addition to eating raw.Literature search finds mainly to collect blackberry, blueberry research
In in fruit volatile oil, blackberry seed oil fatty acid composition and blackberry leaf, seed flavones, anthocyanin and vitaminEContent analysis
Aspect.
Blackberry seed is the accessory substance of the converted products such as blackberry fruit wine, blueberry juice, containing about 27.14% functional grease, its
Middle unrighted acid total amount is up to 93.55%, in addition with amino acid needed by human, to antiatherosclerosis, anti-sugar
The aspects such as urine disease, immunological regulation, promotion growth and intelligence development have important effect.
The essence of blood coagulation is the process for being changed into undissolved solid fabric albumen in water in water miscible fibrinogen,
Be in endogenous or（With）Thrombokinase is produced under exogenous cruor pathway, blood coagulation is produced in the presence of clotting factor
Enzyme, finally makes fibrinogen be changed into fibrin in the presence of fibrin ferment.What PT mainly reflected is extrinsic coagulation way
The activity of factor I, II, V, VII, X in footpath；APTT mainly reflects intrinsic coagulation system situation, with VIII, X, XI, XII
It is relevant Deng intrinsic coagulation factor active；TT values are mainly to reflect that fibrinogen is changed into the important finger of fibrin degree
Mark；FIB mainly reflects the content of fibrinogen.
Thrombosis is the basic pathology process of numerous disease such as dead, DVT of myocardial infarction, ischemic soldier etc., it
Severely compromise the health of the mankind, its formation and hematoblastic aggregation, adhesion, endogenous and extrinsic coagulation system and
It is fibrinous to form closely related.According to the survey report of the World Health Organization, due to thrombus caused by atherosclerotic
The death that disease causes has been discharged to first.Arterial thrombus is that patch is unstable to there occurs spot due to atherosclerotic
The rupture of block, assembles the blood platelet in blood, have activated internal blood coagulation system, finally results in thrombosis.Once
Ischemic and the necrosis of tissue will occur after blocking, serious consequence is produced.In the acute or special of arterial thrombotic disease
Under state, need auxiliary to use anticoagulation medicine, can be only achieved more preferable antithrombotic effect.
The content of the invention
It is an object of the invention to provide a kind of anticoagulation blackberry seed active ingredient, while providing a kind of anticoagulation blackberry seed
The purification methods and uses of active ingredient.
For achieving the above object, the present invention is employed the following technical solutions：
A kind of extraction separation method of anticoagulation blackberry seed active ingredient, comprises the following steps：1）With blackberry seed as raw material,
After blackberry seed is broken, extracted with petroleum ether（Extraction 3-4 time, each 3d）, petroleum ether leaching liquor merge volatilize after petroleum ether, add
70 % alcohol steeps（Extraction 3 times, each 3d）, alcohol extract merges, filters, being concentrated to give the total medicinal extract of ethanol, and solvent is used successively
Petroleum ether, ethyl acetate, extracting n-butyl alcohol, recycling design obtains petroleum ether part, ethyl acetate extract and n-butanol portion；
2）Ethyl acetate extract carries out 200 ~ 300 mesh silica gel column chromatographies, methylene chloride-methanol gradient elution, and ratio is
100:0~1:1, with TLC thin-layer chromatography combining data detections, 6 components are obtained, mark successively according to obtained component polarity is ascending
For component 1-6（System used is for petroleum ether-ethyl acetate when polarity judges）, wherein component 5 again Jing repeatedly silica gel H column chromatography,
Sephadex LH-20 column chromatographys obtain compound 1；
3）N-butanol portion add 70 % ethanol dissolving after, D-101 macroporous resin adsorption loadings stand overnight, after successively
With water, 20 % ethanol, 40 % ethanol, 60 % ethanol, 80 % ethanol and absolute ethyl alcohol gradient elution, every kind of elution 5
Column volume, every time 2000 mL, after reclaiming eluent, obtains corresponding elution fraction；
80% ethanol elution part Jing silica gel H column chromatographys, obtain 2 components, ascending successively according to obtained component polarity
It is labeled as component 1-2（System used is for methylene chloride-methanol when polarity judges）, the Jing Sephadex LH-20 gel columns of component 1
Chromatogram, obtains compound 2, the Jing Sephadex LH-20 gel column chromatographies of component 2 and silica gel H column chromatography repeatedly, obtains compound
3；
60 % ethanol elutions parts first carry out 200～300 mesh silica gel column chromatographies, by silica gel H column chromatography, TLC thin layer colors
Spectrum combining data detection, obtains 2 components, and according to obtained component polarity is ascending component 1-2 is labeled as successively（Institute when polarity judges
It is methylene chloride-methanol with system）, the Jing of component 1 silica gel H column chromatography, Sephadex LH-20 gel column chromatographies repeatedly are changed
Compound 4；The Jing of component 2 Sephadex LH-20 gel column chromatographies, silica gel H column chromatography repeatedly, the afterwards inverted liquid phase system of gained sample
Standby chromatogram obtains compound 5；
40 % ethanol elutions parts first carry out 200～300 mesh silica gel column chromatographies, by silica gel H column chromatography repeatedly,
Sephadex LH-20 gel column chromatographies, obtain compound 6；
Compound 1,2,3,4,5,6 is anticoagulation blackberry seed active ingredient.
The anticoagulation blackberry seed active ingredient obtained according to above extraction separation method.
Application of the anticoagulation blackberry seed active ingredient in terms of anticoagulation medicine is prepared.
Compound 1,2,3,4,5,6 is successively 1α,2α,3β,19α- tetrahydroxy -12- alkene -28- ursolic acids (compound 1),
Tormentic acid (compound 2), 9 (Z)-octadecenic acid acid amides (compound 3), sweetleaf glycosides R1 (compound 4), pomolic acid -28-O-β- D- glucopyranosyl esters (compound 5), 3,5,7,2', 5'- penta hydroxy group flavane (compound 6), its structure is as follows respectively：
The present invention isolated 6 changes from the ethyl acetate extract and n-butanol portion of blackberry seed using special method
Compound, and to the investigation of its external anticoagulant effect, as a result show, compound 1 ~ 6 is respectively provided with good anticoagulant effect, and changes
The anticoagulant effect of compound 4 ~ 6 is better than Breviscapinun, it is seen then that they can be used singly or in combination to anticoagulant.
Description of the drawings
Clotting mechanism figure when Fig. 1 is effect experimental.
Specific embodiment
Embodiment 1
Blackberry seed is purchased from Henan Province Fengqiu County blackberry, blueberry planting base, is rose family rubus plant blackberry, blueberryRubusSpp.Blackberry seeds.
Concentration of alcohol herein is volumetric concentration.
The extraction separation method of reactive compound：
(1) after blackberry seed is broken, first extracted 4 times with petroleum ether, every time 3 d, after volatilizing petroleum ether, adds the leaching of 70 % ethanol
3 times are carried, every time 3 d, leaching liquor merges, filters, being concentrated to give the total medicinal extract of ethanol, successively with petroleum ether, ethyl acetate, n-butanol extraction
Take, recycling design, obtain petroleum ether part, ethyl acetate extract and n-butanol portion；
(2) ethyl acetate extract carries out 200 ~ 300 mesh silica gel column chromatographies, methylene chloride-methanol gradient elution (volume ratio
Example 100:0~1:1), with TLC thin-layer chromatography combining data detections, 6 components are obtained, is marked successively according to obtained component polarity is ascending
It is designated as component 1-6（System used is for petroleum ether-ethyl acetate when polarity judges）, wherein Jing silica gel H post colors repeatedly again of component 5
Spectrum, Sephadex LH-20 column chromatographys etc. obtain compound 1；
(3) n-butanol portion add 70 % ethanol dissolving after, D-101 macroporous resin adsorption loadings stand overnight, after successively
With water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and absolute ethyl alcohol gradient elution, 5 cylinders of every kind of elution
Product, every time 2000 mL, after reclaiming eluent, obtains corresponding elution fraction；
80 % ethanol elutions part Jing silica gel H column chromatographys, obtain 2 components, according to obtained component polarity it is ascending according to
It is secondary to be labeled as component 1-2（System used is for methylene chloride-methanol when polarity judges）, the Jing Sephadex LH-20 gels of component 1
Column chromatography, obtains compound 2；Component 2 Jing Sephadex LH-20 gel column chromatographies and silica gel H column chromatography repeatedly again, are changed
Compound 3；
60 % ethanol elutions parts first carry out 200～300 mesh silica gel column chromatographies, by silica gel H column chromatography, TLC thin layer colors
Spectrum combining data detection, obtains 2 components, and according to obtained component polarity is ascending component 1-2 is labeled as successively（Institute when polarity judges
It is methylene chloride-methanol with system）, the Jing of component 1 silica gel H column chromatography, Sephadex LH-20 gel column chromatographies repeatedly are changed
Compound 4；The Jing of component 2 Sephadex LH-20 gel column chromatographies, silica gel H column chromatography repeatedly, afterwards gained sample carry out the gloomy system of gill
Standby liquid phase obtains compound 5；
40 % ethanol elutions parts first carry out 200～300 mesh silica gel column chromatographies, by silica gel H column chromatography repeatedly,
Sephadex LH-20 gel column chromatographies, obtain compound 6.
Compound 1,2,3,4,5,6 is successively 1α,2α,3β,19α- tetrahydroxy -12- alkene -28- ursolic acids (1), potentilla chinensis
Sour (2), 9 (Z)-octadecenic acid acid amides (3), sweetleaf glycosides R1 (4), pomolic acid -28-O-β- D- glucopyranosyl esters (5), 3,
5,7,2', 5'- penta hydroxy group flavane (6), it is as follows with various spectroscopy techniques identification structure：
1α,2α,3β,19α- tetrahydroxy -12- alkene -28- ursolic acids (1):White powder, C30H48O6, EI-MSm/z: 504
[M]+。1H-NMR (CD3OD,400 MHZ) δ: 0.85 (3H, s, 23-CH3), 0.93 (3H, s, 24-CH3),
0.97 (3H, d, J=4.0 HZ, 30-CH3), 1.02 (3H, s, 25-CH3), 1.06 (3H, s, 26-CH3),
1.24 (3H, s, 29-CH3), 1.40 (3H, s, 27-CH3). 13C-NMR (CD3OD,100 MHZ)δ: 73.6 (C-
1), 80.7 (C-2), 81.3 (C-3), 39.0 (C-4), 55.0 (C-5), 17.8 (C-6), 34.2 (C-7),
44.4 (C-8), 49.6 (C-9), 38.9 (C-10), 27.1 (C-11), 130.7 (C-12), 138.9 (C-13),
42.6 (C-14), 30.7 (C-15), 28.3 (C-16), 49.6 (C-17), 55.0 (C-18), 73.6 (C-19),
43.1 (C-20), 27.1 (C-21), 38.9 (C-22), 29.7 (C-23), 22.4 (C-24), 12.9 (C-25),
16.6 (C-26), 24.9 (C-27), 182.4 (C-28), 19.4 (C-29), 27.3 (C-30)。
Tormentic acid (2):White powder, C30H48O5, EI-MSm/z: 488[M]+。1H-NMR (CD3OD,400 MHZ)δ: 0.79 (3H, s, CH3-29), 0.80 (3H, s), 1.01 (3H, s), 1.01 (3H, s), 1.18 (3H,
s), 1.33 (3H, s), 0.93 (3H, d, J=8 HZ, H-30), 2.92 (1H, d, J=12 HZ, H-3α),
3.62 (1H, d, J=12 HZ, H-2β ), 2.5 (1H, s, H-18), 5.29 (1H, s, H-12). 13C-NMR
(CD3OD,100 MHZ)δ: 48.1 (C-1), 69.6 (C-2), 84.6 (C-3), 41.2 (C-4), 56.8 (C-5),
19.8 (C-6), 34.2 (C-7), 40.6 (C-8), 48.2 (C-9), 39.3 (C-10), 24.9 (C-11),
129.4 (C-12), 140.2 (C-13), 42.7 (C-14), 29.7 (C-15), 26.7 (C-16), 48.3 (C-
17), 55.2 (C-18), 73.7 (C-19), 43.1 (C-20), 27.4 (C-21), 39.1 (C-22), 29.4
(C-23), 17.6 (C-24), 17.1 (C-25), 17.5 (C-26), 24.8 (C-27), 182.3 (C-28),
27.2 (C-29), 16.6 (C-30)。
9(Z)-octadecenic acid acid amides (3):White powder, C18H35NO, EI-MSm/z: 281[M]+。1H-NMR(CDCl3-
CD3OD,400 MHZ) δ: 5.30 (2H, m, -CH=CH-), 2.16 (2H, t, J=8 HZ, -COCH2), 1.98
(4H, m, 8,11-CH2), 1.57 (2H, t, J=8 HZ, 3-CH2), 1.27 (20H, s, 4～7,12～17-
CH2), 0.85 (3H, t, J=8 HZ, 18-CH3). 13C-NMR(CDCl3-CD3OD,400 MHZ) δ: 179.11 (C-
1), 36.5 (C-2), 26.70 (C-3), 30.15 (C-4), 30.17 (C-5), 30.43 (C-6), 30.65 (C-
7), 28.00 (C-8), 130.71 (C-9), 130.71 (C-10), 28.00 (C-11), 30.52 (C-12),
30.40 (C-13), 30.3 (C-14), 30.22 (C-15), 32.84 (C-16), 23.53 (C-17), 14.42
(C-18)。
Sweetleaf glycosides R1(4):Colorless needle crystals, C36H56O12, 72.0～73.0 DEG C of mp, EI-MSm/z: 680[M]+。1H-NMR(C5D5N,400 MHZ) δ: 1.14, 1.19, 1.37, 1.61, 1.70 (3H×5, s, CH3-), 1.04
(3H, d, J=8 HZ, H-30), 2.90 (1H, s, H-18), 4.20~4.49 (6H, glu-2', 3', 4', 5',
6'-H), 5.53 (1H, brs, H-12), 6.28 (1H, d, J=8 HZ, H-1'). 13C-NMR(C5D5N,100
MHZ) δ: 48.66 (C-1), 68.75 (C-2), 81.08 (C-3), 54.47 (C-4), 52.36 (C-5),
21.56 (C-6), 33.36 (C-7), 40.78 (C-8), 48.25 (C-9), 37.74 (C-10), 24.26 (C-
11), 128.26 (C-12), 139.34 (C-13), 42.19 (C-14), 29.21 (C-15), 26.14 (C-16),
48.32 (C-17), 54.85 (C-18), 72.74 (C-19), 42.19 (C-20), 26.75 (C-21), 38.66
(C-22), 180.15 (C-23), 13.49 (C-24), 17.45 (C-25), 17.52 (C-26), 24.56 (C-
27), 177.04 (C-28), 27.05 (C-29), 16.73 (C-30), 95.90 (C-1'), 74.10 (C-2'),
78.98 (C-3'), 71.37 (C-4'), 79.26 (C-5'), 62.48 (C-6')。
Pomolic acid -28-O-β- D- glucopyranosyl esters (5):White powder, C36H58O9, EI-MSm/z: 650[M]+。1H-NMR (CD3OD,400 MHZ) δ: 0.75 (3H, s, 24-CH3), 0.79 (3H, s, 26-CH3), 0.95
(3H, s, CH3-25), 1.21 (3H, s, CH3-29), 1.32 (3H, s, CH3-27), 0.94 (3H, d, J=
4.0 Hz, Me-30), 2.53 (1H, s, H-18), 5.32 (1H, s, H-12), 3.66～3.83 (2H, dd,J
=12 HZ, 14.4 HZ). 13C-NMR(CD3OD,100 MHZ) δ: 39.7 (C-1), 28.0 (C-2), 79.8 (C-
3), 39.7 (C-4), 56.3 (C-5), 21.2 (C-6), 34.2 (C-7), 41.3 (C-8), 49.8 (C-9),
38.1 (C-10), 24.7 (C-11), 129.5 (C-12), 139.4 (C-13), 42.8 (C-14), 30.7 (C-
15), 26.8 (C-16), 48.2 (C-17), 54.8 (C-18), 73.8 (C-19), 42.9 (C-20), 27.1
(C-21), 38.3 (C-22), 28.2 (C-23), 16.8 (C-24), 15.3 (C-25), 17.8 (C-26), 24.7
(C-27), 178.7 (C-28), 27.1 (C-29), 15.3 (C-30), 95.7 (C-1'), 73.8 (C-2'),
78.1 (C-3'), 71.1 (C-4'), 78.5 (C-5'), 62.4 (C-6')。
3,5,7,2', 5'- penta hydroxy group flavane (6):Red solid, CI5H14O6, EI-MSm/z: 290[M]+。1H-NMR
(C5D5N,400 MHZ) δ: 3.41 (1H, dd, J= 16.0, 4.0 HZ, H-4), 3.54 (1H, dd, J= 16.0,
4.0 HZ, H-4), 5.37 (1H, brs, H-2), 4.73 (brs, H-3), 6.67 (d, J= 4.0 HZ, H-6),
6.70 (d, J= 4.0 HZ, H-8), 7.28 (d, J= 8.0 HZ, H-3'), 7.35 (d, J= 8.0 HZ, H-
4'), 7.91 (s, H-6'). 13C-NMR(C5D5N,100 MHZ) δ: 80.14 (C-2), 67.04 (C-3), 29.70
(C-4), 100.29 (C-4a), 158.7 (C-5), 96.8 (C-8), 157.7 (C-8a), 132.2 (C-1'),
146.9 (C-2'), 116.4 (C-3'), 119.5 (C-4'), 147.0 (C-5'), 116.2 (C-6')。
The anticoagulant effect experiment of compound：
Method：Four detections of the external blood plasma blood coagulation of rabbit（Clotting mechanism figure is shown in Fig. 1）
Instrument and material：TGL-16gR high speed desktop refrigerated centrifuges（Anting Scientific Instrument Factory, Shanghai）；LRH-150 gives birth to
Change incubator (the permanent Science and Technology Ltd. in Shanghai one)；Sodium chloride injection（Hebei Tiancheng Pharmaceutical Co., Ltd.,
A14091701）；0.109 mol/L liquor sodii citratises（Self-control）, vitamin K1Parenteral solution (Tianjin medicine company group Xinzheng share
Co., Ltd, 1403112)；Breviscapine (Hu'nan Hengsheng Pharmaceutical Co., Ltd., 20110202)；Prothrombin time
（PT）Determine kit（105241）；Activated partial thromboplastin time（APTT）Determine kit（112175）；Thrombin time
（TT）Determine kit（121132）；Fibrinogen（FIB）Assay kit（1320701）It is biological by the Shanghai sun
Technology Co., Ltd. produces.
Animal used as test：Beaver rabbit, male, the kg of body weight 2.0～2.5 provides (doctor by institute of Chinese materia medica of pharmaceutical college of He'nan University
Dynamic word 14-2-6).
Sample solution is prepared：Compound 1-6 is taken respectively（1 mg）With 80 μ L solvents dissolving 12.5 mg/mL solution of system.Take
The mg of Breviscapinun 8 is obtained 33.33 mg/mL solution, vitamin K with the dissolving of 240 μ L solvents1Parenteral solution is obtained dimension with solvent dissolving
Raw element K1The solution of the mg/mL of concentration 12.5.Solvent is：DMSO:1,2- propane diols=3:2（Volume ratio）.
Experimental technique：
(1) detection method that APTT is affected
The preparation of blood plasma：Rabbit auricular vein takes the mL of blood 3.6, is placed in containing the μ L's of 0.109 mol/L sodium citrates 400
In 4 mL centrifuge tubes, gently overturn and mix, 3000 rpm are centrifuged 15 min, take supernatant liquor standby.
20 μ L sample solution are added in test cup, the APTT reagents 100 of 100 μ L blood plasma and 37 DEG C of pre-temperatures are added
μ L, add 0.025 mol/L CaCl of 37 DEG C of pre-temperatures after 37 DEG C of 5 min of incubation2The μ L of solution 100, record setting time.
(2) detection method that PT is affected
Blood plasma preparation method adds the sample solution of 20 μ L with (1) in test cup, adds the blood plasma of 100 μ L, and 37
The μ L of PT reagents 200 of 37 DEG C of pre-temperatures are added after DEG C 3 min of incubation, setting time, as PT values is recorded.
(3) detection method that TT is affected
Blood plasma preparation method adds the blood plasma of 40 μ L samples solution and 200 μ L with (1) in test cup, is incubated 3min
The μ L of TT reagents 200 are added afterwards, record setting time, as TT values.
(4) detection method that FIB is affected
Blood plasma preparation method calibrates directrix curve with (1) according to reagent specification, takes 200 μ L blood plasma and 100 μ L samples,
It is subsequently adding 700 μ L buffer solutions.After mixing, the μ L of blood plasma 200, the 37 DEG C of min of pre-temperature 3 after dilution are taken, add thrombin solution
100 μ L, determine setting time, record the content of fibrinogen.
Data processing：As a result represented using arithmetic mean of instantaneous value and standard deviation, numerical statistic adopts SPSS19.0 software Dan Yin
Plain method of analysis of variance (One-Way ANOVA) compares its significant difference.Measurement result is shown in Table 1.
Note：Compared with blank group：△ P>0.05, △△△ P＜ 0.001.
Compared with Breviscapinun group： 0.001<** P＜ 0.01,*** P<0.001.
With vitamin K1Group is compared： ### P＜ 0.001.
Compared with blank group, the anticoagulant effect of compound 1 ~ 6 has pole significant difference（P＜ 0.001）, wherein compound 4 ~ 6
Anticoagulant effect it is better than Breviscapinun（P＜ 0.001）, illustrate that six isolated from blackberry seed compounds of the present invention are equal
With good anticoagulant effect, and the anticoagulant effect of compound 4 ~ 6 is better than Breviscapinun, compound 1 ~ 6 can individually or
Combine for anticoagulant.
Claims (1)
1. a kind of extraction separation method of anticoagulation blackberry seed active ingredient, it is characterised in that comprise the following steps：
1）With blackberry seed as raw material, after blackberry seed is broken, extracted with petroleum ether, petroleum ether leaching liquor merges, volatilizes after petroleum ether,
70 % alcohol steeps, alcohol extract are added to merge, filter, being concentrated to give the total medicinal extract of ethanol, successively with solvent petroleum ether, acetic acid second
Ester, extracting n-butyl alcohol, recycling design obtains petroleum ether part, ethyl acetate extract and n-butanol portion；
2）Ethyl acetate extract carries out 200 ~ 300 mesh silica gel column chromatographies, and methylene chloride-methanol gradient elution, ratio is 100:0~
1:1, with TLC thin-layer chromatography combining data detections, 6 components are obtained, component is labeled as successively according to obtained component polarity is ascending
1-6, wherein component 5 again Jing repeatedly silica gel H column chromatography, Sephadex LH-20 column chromatographys obtain compound 1；
3）N-butanol portion add 70 % ethanol dissolving after, D-101 macroporous resin adsorption loadings stand overnight, after successively with water,
20 % ethanol, 40 % ethanol, 60 % ethanol, 80 % ethanol and absolute ethyl alcohol gradient elution, 5 cylinders of every kind of elution
Product, every time 2000 mL, after reclaiming eluent, obtains corresponding elution fraction；
80% ethanol elution part Jing silica gel H column chromatographys, obtain 2 components, mark successively according to obtained component polarity is ascending
For component 1-2, the Jing Sephadex LH-20 gel column chromatographies of component 1, compound 2, the Jing Sephadex LH-20 of component 2 are obtained
Gel column chromatography and silica gel H column chromatography repeatedly, obtain compound 3；
60 % ethanol elutions parts first carry out 200～300 mesh silica gel column chromatographies, by silica gel H column chromatography, the inspection of TLC thin-layer chromatographys
Survey and merge, obtain 2 components, according to obtained component polarity is ascending component 1-2 is labeled as successively, the Jing of component 1 silica gel Hs repeatedly
Column chromatography, Sephadex LH-20 gel column chromatographies, obtain compound 4；The Jing of component 2 Sephadex LH-20 gel columns colors repeatedly
Spectrum, silica gel H column chromatography, the inverted liquid phantom preparing chromatogram of gained sample obtains compound 5；
40 % ethanol elutions parts first carry out 200～300 mesh silica gel column chromatographies, by silica gel H column chromatography, Sephadex repeatedly
LH-20 gel column chromatographies, obtain compound 6；
Compound 1,2,3,4,5,6 is anticoagulation blackberry seed active ingredient, and compound 1,2,3,4,5,6 is successively 1α,2α,3β,19α- tetrahydroxy -12- alkene -28- ursolic acids, tormentic acid, 9 (Z)-octadecenic acid acid amides, sweetleaf glycosides R1, pomolic acid -28-O-β- D- glucopyranosyl esters, 3,5,7,2', 5'- penta hydroxy group flavane, its structure is as follows respectively：
。
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