CN105017375A - Anticoagulation blackberry seed effective component and extraction and separation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant extraction, and in particular relates to an anticoagulant active ingredient of blackberry seeds, an extraction and separation method and application thereof. The present invention uses a special method to separate and obtain 6 compounds from the ethyl acetate part and n-butanol part of blackberry seeds, and investigates their anticoagulant effects in vitro. The results show that compounds 1~6 all have good anticoagulant effects. effect, they can be used alone or in combination as anticoagulants.

Description

An anticoagulant blackberry seed active ingredient and its extraction and separation method and application

technical field

The present invention belongs to the technical field of plant extraction, and in particular relates to an anticoagulant blackberry seed active ingredient and its extraction and separation method and application.

Background technique

Blackberry Rubus spp. Blackberry is an aggregate fruit plant of the Rosaceae Rubus genus, not used for medicine, and is one of the four emerging small fruit trees in the world. Native to North America, its sweet and sour fruit has high nutritional value and medicinal value. In addition to fresh food, most of it is made into quick-frozen fruit, fruit juice, fruit wine and jam. Literature search found that research on blackberry mainly focused on fruit volatile oil, fatty acid composition of blackberry seed oil, and content analysis of flavonoids, anthocyanins and vitamin E in blackberry leaves and seeds.

Blackberry seeds are by-products of processed products such as blackberry wine and blackberry juice. They contain about 27.14% of functional oils, of which the total amount of unsaturated fatty acids is as high as 93.55%. In addition, there are essential amino acids for the human body. It plays an important role in diabetes, immune regulation, growth promotion and intellectual development.

The essence of blood coagulation is the process of converting water-soluble fibrinogen into water-insoluble solid fibrin. It is the generation of prothrombin activator under the endogenous or (and) exogenous coagulation pathway. Thrombin is generated under the action of thrombin, and finally fibrinogen is converted into fibrin under the action of thrombin. PT mainly reflects the activity of coagulation factors I, II, V, VII, and X in the extrinsic coagulation pathway; APTT mainly reflects the status of the endogenous coagulation system, which is different from the activities of endogenous coagulation factors such as VIII, X, XI, and XII. Related; TT value is an important indicator mainly reflecting the degree of fibrinogen into fibrin; FIB mainly reflects the content of fibrinogen.

Thrombosis is the basic pathological process of many diseases such as myocardial infarction, ischemic stroke, deep vein thrombosis, etc. It seriously endangers human health. Its formation is related to platelet aggregation, adhesion, endogenous and exogenous It is closely related to the blood coagulation system and the formation of fibrin. According to the survey report of the World Health Organization, the death caused by thrombosis disease due to atherosclerosis has been ranked first. Arterial thrombosis is due to atherosclerosis, plaque instability and plaque rupture, causing platelets in the blood to aggregate, activating the blood coagulation system in the body, and finally leading to thrombus formation. Once blocked, tissue ischemia and necrosis will occur, resulting in serious consequences. In the acute or special state of arterial thrombotic disease, it is necessary to use anticoagulant drugs in order to achieve better antithrombotic effect.

Invention content

The object of the present invention is to provide an anticoagulant blackberry seed active ingredient, and simultaneously provide an extraction and separation method and application of the anticoagulant blackberry seed active ingredient.

To achieve the above object, the present invention adopts the following technical solutions:

A method for extracting and separating the active ingredients of anticoagulant blackberry seeds, comprising the following steps: 1) using blackberry seeds as raw materials, after the blackberry seeds are crushed, leaching with petroleum ether (leaching 3-4 times, 3 days each time), petroleum After the ether extracts are combined and evaporated to dry petroleum ether, add 70% ethanol for extraction (leaching 3 times, each time for 3 days). The ethanol extracts are combined, filtered, and concentrated to obtain the total ethanol extract. Ethyl, n-butanol extraction, solvent recovery, petroleum ether, ethyl acetate and n-butanol parts;

2) The ethyl acetate part was subjected to 200-300 mesh silica gel column chromatography, dichloromethane-methanol gradient elution, the ratio was 100:0-1:1, combined by TLC thin-layer chromatography to obtain 6 components, according to the obtained The polarity of the components is marked as components 1-6 in order from small to large (the system used for polarity determination is petroleum ether-ethyl acetate), and component 5 is subjected to repeated silica gel H column chromatography and Sephadex LH-20 column chromatography Obtain compound 1 ;

3) After adding 70% ethanol to the n-butanol part to dissolve, D-101 macroporous resin was used to absorb and load the sample, let stand overnight, and then followed by water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and absolute ethanol Gradient elution, each eluent was eluted with 5 column volumes, 2000 mL each time, after recovering the eluent, the corresponding eluted fraction was obtained;

The fraction eluted with 80% ethanol was subjected to silica gel H column chromatography to obtain 2 components, which were marked as components 1-2 according to the polarity of the obtained components from small to large (the system used for polarity determination was dichloromethane-methanol) , component 1 obtains compound 2 through Sephadex LH-20 gel column chromatography, and component 2 obtains compound 3 through Sephadex LH-20 gel column chromatography and repeated silica gel H column chromatography;

The part eluted with 60% ethanol was first subjected to 200-300 mesh silica gel column chromatography, and then combined by silica gel H column chromatography and TLC thin layer chromatography to obtain 2 components, which were marked as groups according to the polarity of the obtained components from small to large. Divide 1-2 (used system is dichloromethane-methanol during polarity judgment), component 1 obtains compound 4 through repeated silica gel H column chromatography, Sephadex LH-20 gel column chromatography; Component 2 obtains compound 4 through repeated Sephadex LH- 20 gel column chromatography, silica gel H column chromatography, and the obtained sample obtains compound 5 through reversed-phase liquid phase preparative chromatography;

The 40% ethanol eluted part was first subjected to 200-300 mesh silica gel column chromatography, followed by repeated silica gel H column chromatography and Sephadex LH-20 gel column chromatography to obtain compound 6 ;

Compounds 1, 2, 3, 4, 5, and 6 are the active ingredients of anticoagulant blackberry seeds.

 The active ingredients of anticoagulant blackberry seeds obtained according to the above extraction and separation methods.

Application of active ingredients of anticoagulant blackberry seeds in the preparation of anticoagulant drugs.

Compounds 1, 2, 3, 4, 5, 6 are 1 α , 2 α , 3 β , 19 α -tetrahydroxy-12-en-28-ursolic acid (compound 1 ), pollinate acid (compound 2 ), 9( Z )-octadecenoic acid amide (compound 3 ), glucoside R1 (compound 4 ), pampic acid-28- O - β -D-glucopyranose ester (compound 5 ), 3,5 ,7,2',5'-pentahydroxyflavan (compound 6 ), its structure is as follows:

.

The present invention uses a special method to separate and obtain 6 compounds from the ethyl acetate part and n-butanol part of blackberry seeds, and investigates their anticoagulant effects in vitro. The results show that compounds 1~6 all have good anticoagulant effects. effect, and the anticoagulant effect of compounds 4~6 is better than that of breviscapine. It can be seen that they can be used alone or in combination as anticoagulants.

Description of drawings

Figure 1 is a diagram of the coagulation mechanism during the effect experiment.

Detailed ways

Example 1

The blackberry seeds are purchased from the blackberry planting base in Fengqiu County, Henan Province, and are Rubus spp. Blackberry seeds of the plant Rubus spp. of the family Rosaceae.

The ethanol concentration in this article is the volume concentration.

  Extraction and separation methods for active compounds:

(1) After the blackberry seeds are crushed, extract them with petroleum ether for 4 times, each time for 3 days. After evaporating the petroleum ether, add 70% ethanol and extract them for 3 times, each time for 3 days. The extracts are combined, filtered, and concentrated Obtain the total extract of ethanol, sequentially extract with petroleum ether, ethyl acetate, n-butanol, reclaim the solvent, obtain the petroleum ether part, ethyl acetate part and n-butanol part;

(2) The ethyl acetate part was subjected to 200-300 mesh silica gel column chromatography, dichloromethane-methanol gradient elution (volume ratio 100:0-1:1), combined with TLC thin-layer chromatography to obtain 6 components, According to the polarity of the obtained components from small to large, they are marked as components 1-6 (the system used for polarity determination is petroleum ether-ethyl acetate), and component 5 is subjected to repeated silica gel H column chromatography, Sephadex LH-20 Column chromatography etc. obtain compound 1 ;

(3) After adding 70% ethanol to the n-butanol part to dissolve, the D-101 macroporous resin was adsorbed and loaded, and left to stand overnight, followed by water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and anhydrous Ethanol gradient elution, each eluent was eluted with 5 column volumes, each time 2000 mL, after recovering the eluent, the corresponding eluted fraction was obtained;

The fraction eluted with 80% ethanol was subjected to silica gel H column chromatography to obtain 2 components, which were marked as components 1-2 according to the polarity of the obtained components from small to large (the system used for polarity determination was dichloromethane-methanol) , component 1 obtains compound 2 through Sephadex LH-20 gel column chromatography; Component 2 obtains compound 3 through Sephadex LH-20 gel column chromatography and repeated silica gel H column chromatography again;

The part eluted with 60% ethanol was first subjected to 200-300 mesh silica gel column chromatography, and then combined by silica gel H column chromatography and TLC thin layer chromatography to obtain 2 components, which were marked as groups according to the polarity of the obtained components from small to large. Divide 1-2 (used system is dichloromethane-methanol during polarity judgment), component 1 obtains compound 4 through repeated silica gel H column chromatography, Sephadex LH-20 gel column chromatography; Component 2 obtains compound 4 through repeated Sephadex LH- 20 gel column chromatography, silica gel H column chromatography, and the obtained sample is carried out to Gilson preparative liquid phase to obtain compound 5 ;

The fraction eluted with 40% ethanol was first subjected to 200-300 mesh silica gel column chromatography, followed by repeated silica gel H column chromatography and Sephadex LH-20 gel column chromatography to obtain compound 6 .

Compounds 1, 2, 3, 4, 5, 6 are 1 α , 2 α , 3 β , 19 α -tetrahydroxy-12-en-28-ursolic acid ( 1 ), pollinate ( 2 ), 9( Z )-octadecenoic acid amide ( 3 ), steafoside R1 ( 4 ), pampic acid-28- O - β -D-glucopyranose ester ( 5 ), 3,5,7,2',5'-Pentahydroxyflavan ( 6 ), the structure was identified by various spectroscopy techniques as follows:

1 α ,2 α ,3 β ,19 α -tetrahydroxy-12-en-28-ursolic acid ( 1 ) : white powder, C ₃₀ H ₄₈ O ₆ , EI-MS m/z : 504[M] ^＋ . ¹ H-NMR (CD ₃ OD,400 MHZ) δ : 0.85 (3H, s, 23-CH ₃ ), 0.93 (3H, s, 24-CH ₃ ), 0.97 (3H, d, J =4.0 HZ, 30 -CH ₃ ), 1.02 (3H, s, 25-CH ₃ ), 1.06 (3H, s, 26-CH ₃ ), 1.24 (3H, s, 29-CH ₃ ), 1.40 (3H, s, 27-CH ₃ ). ¹³ C-NMR (CD ₃ OD,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).

Potentate acid ( 2 ) : white powder, C ₃₀ H ₄₈ O ₅ , EI-MS m/z : 488[M] ^＋ . ¹ H-NMR (CD ₃ OD,400 MHZ) δ : 0.79 (3H, s, CH ₃ -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-3 α ), 3.62 (1H, d, J =12 HZ, H-2 β ), 2.5 (1H, s, H-18), 5.29 (1H, s, H-12). ¹³ C-NMR (CD ₃ OD,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 amide (3): white powder, C ₁₈ H ₃₅ NO, EI-MS m/z : 281[M] ⁺ . ¹ H-NMR(CDCl ₃ -CD ₃ OD,400 MHZ) δ : 5.30 (2H, m, -CH=CH-), 2.16 (2H, t, J =8 HZ, -COCH ₂ ), 1.98 (4H, m, 8,11-CH ₂ ), 1.57 (2H, t, J =8 HZ, 3-CH ₂ ), 1.27 (20H, s, 4～7, 12～17-CH ₂ ), 0.85 (3H, t , J =8 HZ, 18-CH ₃ ). ¹³ C-NMR(CDCl ₃ -CD ₃ OD,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).

Steferoside R ₁ ( 4 ) : Colorless needle crystals, C ₃₆ H ₅₆ O ₁₂ , mp 72.0～73.0°C, EI-MS m/z : 680[M] ⁺ . ¹ H-NMR(C ₅ D ₅ N,400 MHZ) δ : 1.14, 1.19, 1.37, 1.61, 1.70 (3H×5, s, CH ₃ -), 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'). ¹³ C-NMR(C ₅ D ₅ N,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').

Plmectic acid-28- O - β -D-glucopyranose ester ( 5): white powder, C ₃₆ H ₅₈ O ₉ , EI-MS m/z : 650[M] ^＋ . ¹ H-NMR (CD ₃ OD,400 MHZ) δ : 0.75 (3H, s, 24-CH ₃ ), 0.79 (3H, s, 26-CH ₃ ), 0.95 (3H, s, CH ₃ -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). ¹³ C-NMR(CD ₃ OD,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'-pentahydroxyflavan (6): red solid, C _I5 H ₁₄ O ₆ , EI-MS m/z : 290[M] ^＋ . ¹ H-NMR (C ₅ D ₅ N,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'). ¹³ C-NMR(C ₅ D ₅ N ,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 ').

Anticoagulant effect test of compound:

Method: Four tests of plasma coagulation in vitro in rabbits (see Figure 1 for the diagram of the coagulation mechanism)

Instruments and materials: TGL-16gR high-speed desktop refrigerated centrifuge (Shanghai Anting Scientific Instrument Factory); LRH-150 biochemical incubator (Shanghai Yiheng Technology Co., Ltd.); sodium chloride injection (Hebei Tiancheng Pharmaceutical Co., Ltd., A14091701); 0.109 mol/L sodium citrate solution (self-made), vitamin K ₁ injection (Tianjin Pharmaceutical Group Xinzheng Co., Ltd., 1403112); breviscapine for injection (Hunan Hengsheng Pharmaceutical Co., Ltd., 20110202); coagulation Proenzyme Time (PT) Assay Kit (105241); Activated Partial Thrombin Time (APTT) Assay Kit (112175); Thrombin Time (TT) Assay Kit (121132); Fibrinogen (FIB) Content Determination Reagent Boxes (1320701) were produced by Shanghai Sun Biotechnology Co., Ltd.

Experimental animals: Rex rabbit, male, weighing 2.0-2.5 kg, provided by the Institute of Traditional Chinese Medicine, School of Pharmacy, Henan University (Yidongzi No. 14-2-6).

Preparation of sample solution: Dissolve compounds 1-6 (1 mg) in 80 μL of solvent to prepare 12.5 mg/mL solution. Dissolve 8 mg of breviscapine in 240 μL of solvent to obtain a 33.33 mg/mL solution, and dissolve vitamin K ₁ injection in a solvent to obtain a solution with a vitamin K ₁ concentration of 12.5 mg/mL. The solvent is: DMSO:1,2-propanediol=3:2 (volume ratio).

experimental method:

(1) Detection method for impact on APTT

Plasma preparation: 3.6 mL of blood was collected from the ear vein of rabbits, placed in a 4 mL centrifuge tube containing 400 μL of 0.109 mol/L sodium citrate, mixed gently by inversion, centrifuged at 3000 rpm for 15 min, and the supernatant was taken spare.

Add 20 μL of sample solution to the test cup, then add 100 μL of plasma and 100 μL of APTT reagent pre-warmed at 37 °C, incubate at 37 °C for 5 min, then add 100 μL of 0.025 mol/L CaCl ₂ solution pre-warmed at 37 °C, record the coagulation time.

(2) Detection method for impact on PT

The plasma preparation method is the same as (1). Add 20 μL of sample solution to the test cup, then add 100 μL of plasma, incubate at 37 °C for 3 minutes, add 200 μL of PT reagent pre-warmed at 37 °C, and record the coagulation time, which is PT value.

(3) Detection method for impact on TT

The plasma preparation method is the same as (1). Add 40 μL sample solution and 200 μL plasma to the test cup, add 200 μL TT reagent after incubation for 3 minutes, and record the clotting time, which is the TT value.

(4) Detection method for impact on FIB

The plasma preparation method is the same as (1), and the standard curve is determined according to the reagent instructions. Take 200 μL plasma and 100 μL sample, and then add 700 μL buffer. After mixing, take 200 μL of diluted plasma, pre-warm at 37°C for 3 min, add 100 μL of thrombin solution, measure the clotting time, and record the content of fibrinogen.

Data processing: The results are expressed by the arithmetic mean and standard deviation, and the numerical statistics are compared by SPSS19.0 software one-way ANOVA (One-Way ANOVA) to compare the significant differences. The measurement results are shown in Table 1.

Table 1 Effects of blackberry seed compounds on the coagulation time of rabbits ( ± s )

Note: Compared with the blank group: ^△ P >0.05, ^△△△ P <0.001.

Compared with the breviscapine group: 0.001< ^** P <0.01, ^*** P <0.001.

Compared with vitamin K ₁ group: ^### P <0.001.

Compared with the blank group, the anticoagulant effect of compounds 1~6 has a very significant difference ( P <0.001), and the anticoagulant effect of compound 4~6 is better than that of breviscapine ( P <0.001), which shows that the present invention is obtained from blackberry The six compounds isolated from the seeds all have good anticoagulant effects, and the anticoagulant effects of compounds 4-6 are better than breviscapine, and compounds 1-6 can be used alone or in combination as anticoagulants.

Claims (3)

1. A method for extracting and separating the effective components of anticoagulant blackberry seeds, characterized in that it comprises the following steps: 1) taking blackberry seeds as raw material, after the blackberry seeds are broken, extracting with petroleum ether, merging the petroleum ether extracts, After evaporating the petroleum ether, add 70% ethanol for extraction, the ethanol extracts are combined, filtered, and concentrated to obtain the total ethanol extract, which is sequentially extracted with the solvent petroleum ether, ethyl acetate, and n-butanol, and the solvent is recovered to obtain the petroleum ether fraction , ethyl acetate site and n-butanol site; 2) The ethyl acetate part was subjected to 200-300 mesh silica gel column chromatography, dichloromethane-methanol gradient elution, the ratio was 100:0-1:1, combined by TLC thin-layer chromatography to obtain 6 components, according to the obtained Component polarity is marked as component 1-6 successively from small to large, wherein component 5 obtains compound 1 through repeated silica gel H column chromatography, Sephadex LH-20 column chromatography again; 3) After adding 70% ethanol to the n-butanol part to dissolve, D-101 macroporous resin was used to absorb and load the sample, let stand overnight, and then followed by water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and absolute ethanol Gradient elution, each eluent was eluted with 5 column volumes, 2000 mL each time, after recovering the eluent, the corresponding eluted fraction was obtained; The fraction eluted with 80% ethanol was subjected to silica gel H column chromatography to obtain 2 components, which were marked as components 1-2 according to the polarity of the obtained components from small to large, and component 1 was subjected to Sephadex LH-20 gel column chromatography, Compound 2 was obtained, and component 2 was subjected to Sephadex LH-20 gel column chromatography and repeated silica gel H column chromatography to obtain compound 3 ; The part eluted with 60% ethanol was first subjected to 200-300 mesh silica gel column chromatography, and then combined by silica gel H column chromatography and TLC thin layer chromatography to obtain 2 components, which were marked as groups according to the polarity of the obtained components from small to large. Divide 1-2, component 1 obtains compound 4 through repeated silica gel H column chromatography, Sephadex LH-20 gel column chromatography; Reversed-phase liquid phase preparative chromatography obtains compound 5 ; The 40% ethanol eluted part was first subjected to 200-300 mesh silica gel column chromatography, followed by repeated silica gel H column chromatography and Sephadex LH-20 gel column chromatography to obtain compound 6 ; Compounds 1, 2, 3, 4, 5, and 6 are the active ingredients of anticoagulant blackberry seeds. 2. utilize the anticoagulant blackberry seed active ingredient that claim 1 extracts and separates the method to obtain. 3. the application of the anticoagulant blackberry seed active ingredient described in claim 2 in the preparation of anticoagulant medicine.