Application of notoginsenoside component in preparation of anticoagulant medicine

Abstract

The invention relates to an application of notoginsenoside components in preparation of anticoagulant drugs. The invention relates to the field of anticoagulant drugs, in particular to application of a compound selected from the group consisting of notoginsenoside R2 (Notoginsenoside R2), notoginsenoside Fa (Notoginsenoside Fa), ginsenoside Rf (Ginsenoside Rf), ginsenoside Rh3 (Ginsenoside Rh3), ginsenoside Ra1 (Ginsenoside Ra1), Vietnam ginsenoside R4 (Vinsenoside R4), 20-O-glucosyl ginsenoside Rf (20-O-glucose ginsenoside Rf), 5, 6-dehydroginsenoside Rd (5, 6-dehydroginsenoside Rd) and notoginsenoside R4 (Notoginsenoside R4) in the preparation of anticoagulant drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to the use of notoginsenoside components in the preparation of anticoagulant drugs. Background Art

[0002] Anticoagulants are a class of drugs that prevent blood coagulation by interfering with coagulation factors, and are mainly used for the prevention and treatment of thromboembolic diseases. Commonly used anticoagulants in clinical practice include: parenteral anticoagulants (such as heparin), coumarin anticoagulants (such as warfarin), antiplatelet aggregation drugs (such as aspirin), etc.

[0003] Antiplatelet aggregation is to inhibit platelets from aggregating and adhering to each other to form blood clots, and to avoid the formation of thrombi or embolisms. There are many types of antiplatelet aggregation drugs, mainly including platelet glycoproteins, tirofiban, phosphodiesterase inhibitors, dipyridamole, etc.

[0004] However, currently commonly used anticoagulants and antiplatelet aggregation drugs are all Western medicines, and these drugs will have different side effects while treating diseases.

[0005] Traditional Chinese medicine notoginseng is known as "the magic medicine for hemostasis and the wonderful product for regulating blood circulation", and has traditional effects of dissipating stasis and stopping bleeding, anti-inflammatory and relieving pain, as well as related pharmacological activities such as promoting blood circulation and stopping bleeding. It mainly contains saponin components. Literature reports that there are 14 saponin components in notoginseng with anticoagulant and antiplatelet aggregation effects: ginsenoside Rg3, Rb3, Rg1, Rg2, Re, Ro, Rd, F2, Rb2, Rk1, Rb1, Rh1, Ra3 and notoginsenoside R1. These saponins are mostly components with relatively high content and easy availability in notoginseng. And more than 100 saponin components have been isolated and identified in notoginseng. Therefore, it is necessary to further study and discover saponin components in notoginseng with blood-activating or hemostatic effects. Summary of the Invention

[0006] Based on the textual research of Chinese herbal medicines, literature review, and extensive collection of notoginseng samples, the inventors of the present application systematically studied and compared the chemical components of genuine and non-genuine notoginseng herbs by using HPLC fingerprint, LC-MS-based plant metabolomics method, and LC-MS quantitative analysis method. Combining multiple data analysis methods, the inventors found the differences in chemical components between genuine and non-genuine notoginseng herbs, and selected 9 components related to the genuineness of notoginseng for in vitro anticoagulant and antiplatelet aggregation activity experiments. During the research process, the inventors found that these 9 saponins related to the genuineness of notoginseng and with relatively low content in notoginseng have good anticoagulant effects, and also found a special saponin with a dual regulatory effect of both promoting blood circulation and stopping bleeding. Further, the inventors of the present application also found that specific saponins have antiplatelet aggregation effects.

[0007] The technical solution of the present application is as follows:

[0008] On the one hand, the present application provides the use of any one of the following 9 compounds newly discovered to have anticoagulant effects in the preparation of anticoagulant drugs: Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd, and Notoginsenoside R4.

[0009] On the other hand, the present application provides the use of Notoginsenoside R4 in the preparation of drugs with dual regulatory effects of promoting blood circulation and / or stopping bleeding.

[0010] On still another hand, the present application provides the use of the compounds selected from the following in the preparation of antiplatelet aggregation drugs: Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd.

[0011] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0012] The compounds of the present invention, when prepared into anticoagulant drugs, antiplatelet aggregation drugs, or drugs for promoting blood circulation and stopping bleeding, have low side effects and good curative effects, and are suitable for large-scale promotion.

[0013] The present invention first reports that nine components, namely Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd, and Notoginsenoside R4, have anticoagulant effects in vitro; and first reports that eight components, namely Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, and 5,6-didehydroginsenoside Rd, have antiplatelet aggregation effects in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Effects of the solvent control group and the blank control group on APTT, PT, TT, and FIB; APTT: activated partial thromboplastin time; PT: prothrombin time; TT: thrombin time; FIB: fibrinogen content; n = 3;

[0015] Figure 2 : Effects of nine components at different concentrations on APTT; APTT: activated partial thromboplastin time; N-R2: Notoginsenoside R2, G-Rf: Ginsenoside Rf, G-Rh3: Ginsenoside Rh3, G-Ra1: Ginsenoside Ra1, Vina-R4: Vinaginsenoside R4, 20-O-glu Rf: 20-O-glucoginsenoside Rf, 5,6-di Rd: 5,6-didehydroginsenoside Rd, N-Fa: Notoginsenoside Fa, N-R4: Notoginsenoside R4; compared with the control, ***p < 0.001, **p < 0.01, *p < 0.05; n = 3;

[0016] Figure 3: Effects of 9 components at different concentrations on PT. PT: Prothrombin time; N-R2: Notoginsenoside R2, G-Rf: Ginsenoside Rf, G-Rh3: Ginsenoside Rh3, G-Ra1: Ginsenoside Ra1, Vina-R4: Vinaginsenoside R4, 20-O-glu Rf: 20-O-glucoginsenoside Rf, 5,6-di Rd: 5,6-didehydroginsenoside Rd, N-Fa: Notoginsenoside Fa, N-R4: Notoginsenoside R4; Compared with the control, ***p < 0.001, **p < 0.01, *p < 0.05; n = 3;

[0017] Figure 4 : Effects of 9 components at different concentrations on TT. TT: Thrombin time; N-R2: Notoginsenoside R2, G-Rf: Ginsenoside Rf, G-Rh3: Ginsenoside Rh3, G-Ra1: Ginsenoside Ra1, Vina-R4: Vinaginsenoside R4, 20-O-glu Rf: 20-O-glucoginsenoside Rf, 5,6-di Rd: 5,6-didehydroginsenoside Rd, N-Fa: Notoginsenoside Fa, N-R4: Notoginsenoside R4; Compared with the control, *p < 0.05; n = 3;

[0018] Figure 5 : Effects of 9 components at different concentrations on FIB (fibrinogen content). FIB: Fibrinogen content; N-R2: Notoginsenoside R2, G-Rf: Ginsenoside Rf, G-Rh3: Ginsenoside Rh3, G-Ra1: Ginsenoside Ra1, Vina-R4: Vinaginsenoside R4, 20-O-glu Rf: 20-O-glucoginsenoside Rf, 5,6-di Rd: 5,6-didehydroginsenoside Rd, N-Fa: Notoginsenoside Fa, N-R4: Notoginsenoside R4; Compared with the control, ***p < 0.001; n = 3;

[0019] Figure 6: Effects of the solvent control group and the blank control group on platelet aggregation; n = 3;

[0020] Figure 7 : Effects of 8 components at different concentrations on ADP-induced platelet aggregation in rats; N-R2: Notoginsenoside R2, G-Rf: Ginsenoside Rf, G-Rh3: Ginsenoside Rh3, G-Ra1: Ginsenoside Ra1, Vina-R4: Vinaginsenoside R4, 20-O-glu Rf: 20-O-glucoginsenoside Rf, 5,6-di Rd: 5,6-didehydroginsenoside Rd, N-Fa: Notoginsenoside Fa; compared with the control, ***p < 0.001, **p < 0.01, *p < 0.05. Detailed implementation mode

[0021] The technical solution of the present invention will be further described below in conjunction with the specific implementation mode.

[0022] Example 1: Anticoagulant effect experiment

[0023] The blood coagulation process mainly includes three stages, namely the formation of prothrombin activator, the activation of prothrombin into thrombin, and the conversion of fibrinogen into gel-like fibrin. Thrombin Time (TT), Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), and Fibrinogen content (FIB) are important indicators for monitoring the coagulation system. APTT mainly reflects the status of the intrinsic coagulation system, PT mainly reflects the status of the extrinsic coagulation system, TT mainly reflects the time of fibrinogen conversion into fibrin, and FIB mainly reflects the content of fibrinogen. If a compound has the effect of prolonging TT, APTT, PT and reducing the FIB content, it indicates that it has an anticoagulant effect, otherwise it indicates that it has a coagulant effect. Platelet aggregation is an important factor in thrombus formation, and compounds with blood-activating effects can inhibit platelet aggregation. In this example, the effects of notoginsenoside components at different concentrations on the four coagulation indexes and platelet aggregation were measured to screen the blood-activating effects of each component.

[0024] I. Experimental materials

[0025] (I) Test drugs

[0026] Ginsenoside Rh3 (lot No: MUST-22032505); Vinaginsenoside R4 (lot No: 21051702); Ginsenoside Rf (lot No: 21071411); Notoginsenoside R2 (lot No: 21042812); Ginsenoside Ra1 (lot No: 21050703); Notoginsenoside Fa (lot No: 21103021); 20-O-glucoginsenoside Rf (lot No: 20103012); 5,6-didehydroginsenoside Rd (lot No: MUST-21082805) were purchased from Chengdu Mansite Biotechnology Co., Ltd. Notoginsenoside R4 (lot No: J23GB152507) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0027] The purities of the above 9 saponin components were all ≥98% determined by HPLC area normalization method.

[0028] (II) Instruments

[0029] Electronic analytical balance (Precisa, XB120A, Precisa International Trade (Shanghai) Co., Ltd.); Milli-Q ultrapure water preparation instrument (Millipore, USA); Coagulation analyzer (SMT-120VP, Chengdu Smart Technology Co., Ltd.); Platelet aggregometer (TYXN-96, Shanghai General Mechanical and Electrical Technology Research Institute); Centrifuge (TGL-16gR, Shanghai Anting Scientific Instrument Factory).

[0030] (III) Reagents and Consumables

[0031] Coagulation Four-Item Quantitative Detection Reagent Disk (Chengdu Smart Technology Co., Ltd., lot No: 922304); Adenosine Diphosphate (ADP, Shanghai Jizhi Biochemical Technology Co., Ltd., lot No: A21535DC8); Phosphate Buffered Saline (PBS, Beijing Bairuiji Biotechnology Co., Ltd., lot No: BN20559); Dimethyl Sulfoxide (DMSO, Shanghai Beyotime Biotechnology Co., Ltd., lot No: ST038); Chloral Hydrate (Shanghai Aladdin Biochemical Technology Co., Ltd., lot No: H2121173); Disposable Vacuum Blood Collection Tube (Shandong Chengwu Medical Products Factory, lot No: 20211210); Disposable Sterile Syringe (Wuhan Mingankang Technology Co., Ltd., lot No: 20220107).

[0032] (IV) Experimental Animals

[0033] Male SD rats, SPF grade, weighing 200 - 250 g, a total of 60 rats, were purchased from Chengdu Yakang Biotechnology Co., Ltd. All animal experiments were approved by the Medical Ethics Committee of Peking University, and the review permit number is: LA2016205.

[0034] II. Experimental Methods

[0035] (I) Effects on Coagulation Four-Item Indexes

[0036] 1. Preparation of rat plasma: After male SD rats were purchased, they were adaptively fed for one week, anesthetized by intraperitoneal injection of 7% chloral hydrate (0.5 mL / 100 g), blood was collected from the abdominal aorta, placed in a centrifuge, and centrifuged at 3500 r·min -1 , 4°C for 10 min, and the supernatant was collected for standby.

[0037] 2. Sample preparation: Weigh 9 saponin components respectively, and prepare solutions with mass concentrations of 0.25, 0.50, 1.00, 2.00, and 5.00 mg / mL with PBS containing 5% DMSO respectively, mix well, and wait for measurement.

[0038] 3. Determination of TT, APTT, PT, and FIB: Set up a solvent control group (PBS containing 5% DMSO added to plasma), a blank control group (plasma without DMSO and PBS), and each concentration sample group. Take 50 μL of sample solutions with different mass concentrations and PBS containing 5% DMSO and add them to 50 μL of plasma respectively. After mixing, add them to the coagulation four-item quantitative detection reagent disk respectively, and measure the values of TT, APTT, PT, and FIB in a coagulation analyzer. Three samples were prepared in parallel for each group, and the data of each group were expressed as mean ± SD. The t-test or non-parametric test (Mann-Whitney U test) was used for comparison between groups, and p < 0.05 was considered statistically significant.

[0039] (2) Influence on platelet aggregation

[0040] 1. Preparation of platelet - poor plasma (PPP) and platelet - rich plasma (PRP): Male SD rats were purchased and adaptively fed for one week. They were anesthetized by intraperitoneal injection of 7% chloral hydrate (0.5 mL / 100 g), and blood was collected from the abdominal aorta. The blood was centrifuged at 1000 r·min -1 , 4℃ for 10 min. The supernatant was aspirated and placed in a 5 mL EP tube. The remaining blood was operated once again, and the supernatant was aspirated and placed in the aforementioned EP tube to obtain platelet - rich plasma (PRP); the remaining blood was centrifuged at 3500 r·min -1 , 4℃ for 15 min, and the supernatant was aspirated and placed in another 5 mL EP tube to prepare platelet - poor plasma (PPP).

[0041] 2. Preparation of the inducer: Weigh 3 mg of ADP freeze - dried powder, add 3.5 mL of PBS, and gently shake until completely dissolved to obtain the inducer stock solution. Appropriate amount of the stock solution was aspirated and diluted with PBS at a ratio of 1:3 to obtain the inducer working solution.

[0042] 3. Sample preparation: Weigh 9 saponin components respectively, dissolve them with appropriate amount of DMSO, and then add a certain amount of PRP respectively and mix well to obtain medicated PRP containing 2.5% DMSO with concentrations of 0.25, 0.50, 1.00, 2.00, 5.00 mg / mL respectively.

[0043] 4. Determination of platelet aggregation rate: The platelet aggregometer was turned on and pre - heated for 30 min in advance. After the pre - heating was completed, first use 200 μL of the PPP sample to zero the transmittance. Set up a solvent control group (PRP containing 2.5% DMSO), a blank control group (PRP without DMSO), and each concentration sample group. Add 200 μL of medicated PRP with different mass concentrations, PRP containing 2.5% DMSO, and PRP without DMSO to each glass colorimetric tube respectively, and then add a stir bar. Place the colorimetric tube in the preparatory channel and pre - heat at 37℃ for 3 min, then put the colorimetric tube into the zero - adjusted detection channel, add 12.5 μL of ADP inducer working solution for detection. Before measuring each group of samples, a group of solvent control samples were measured separately, and the maximum platelet aggregation rate of each sample was recorded. And calculate the inhibition rate of the compound on platelet aggregation according to the following formula: Inhibition rate = (maximum aggregation rate of the blank group - maximum aggregation rate of the medicated group) / maximum aggregation rate of the blank group × 100%. Three samples were prepared in parallel for each group, and the data of each group were expressed as mean±SD. The comparison between groups was performed by t - test or non - parametric test (Mann - Whitney U test), and p < 0.05 was considered statistically significant.

[0044] III. Experimental Results

[0045] (I) Effects of 9 chemical components on four coagulation indexes

[0046] There was no significant difference in the effects on the four coagulation indexes between the solvent control group (PBS containing 5% DMSO added to plasma) and the blank control group (plasma without DMSO and PBS) ( Figure 1 ), indicating that the solvent used to dissolve the 9 compounds had no effect on the four coagulation indexes.

[0047] The measurement results showed that the 9 components had different degrees of effects on the four coagulation indexes. Their effects on the four coagulation indexes of APTT (activated partial thromboplastin time), PT (prothrombin time), TT (thrombin time), and FIB (fibrinogen content) at concentrations of 0.25, 0.50, 1.00, 2.00, and 5.00 mg / mL are shown in Table 1 and Figures 2-5 as follows.

[0048] Table 1: Effects of 9 components on APTT, PT, TT, and FIB (mean±SD, n = 3)

[0049]

[0050]

[0051] Note: APTT: Activated partial thromboplastin time; PT: Prothrombin time; TT: Thrombin time; FIB: Fibrinogen content; Control is the solvent control. Compared with Control, ***p<0.001, **p<0.01, *p<0.05

[0052] Continued Table 1: Effects of 9 components on APTT, PT, TT, and FIB (mean±SD, n = 3)

[0053]

[0054]

[0055] Note: APTT: Activated partial thromboplastin time; PT: Prothrombin time; TT: Thrombin time; FIB: Fibrinogen content; Control is the solvent control. Compared with Control, ***p<0.001, **p<0.01, *p<0.05

[0056] The results showed that Notoginsenoside R2 could significantly prolong APTT at the concentrations of 1.00 and 5.00 mg / mL, Ginsenoside Rf at the concentrations of 0.50, 1.00, 2.00 and 5.00 mg / mL, Ginsenoside Rh3 at the concentrations of 1.00 and 5.00 mg / mL, Ginsenoside Ra1 at the concentrations of 1.00, 2.00 and 5.00 mg / mL, Vinaginsenoside R4 at the concentrations of 0.50 and 5.00 mg / mL, 20-O-glucoginsenoside Rf at the concentrations of 0.25, 1.00 and 5.00 mg / mL, 5,6-didehydroginsenoside Rd at the concentrations of 0.50, 2.00 and 5.00 mg / mL, Notoginsenoside Fa at the concentrations of 1.00, 2.00 and 5.00 mg / mL, and Notoginsenoside R4 at the concentrations of 0.25, 0.50 and 2.00 mg / mL. This indicated that the above 9 compounds could significantly prolong the activated partial thromboplastin time at the corresponding concentrations, showing anticoagulant effects.

[0057] The 9 compounds could significantly prolong PT and significantly reduce the plasma FIB content at the concentrations of 0.25, 0.50, 1.00, 2.00 and 5.00 mg / mL. That is, the above 9 compounds could significantly prolong the prothrombin time and significantly reduce the plasma fibrinogen content at the corresponding concentrations, showing anticoagulant effects.

[0058] Among the 9 compounds, only Notoginsenoside R4 showed a significant effect of shortening TT at the concentrations of 0.25, 1.00 and 2.00 mg / mL. That is, this compound could significantly shorten the thrombin time, showing a coagulation effect, while the other 8 components had no significant effect on TT, suggesting that Notoginsenoside R4 had a dual regulatory effect of promoting blood circulation and stopping bleeding.

[0059] In summary, 9 components including Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd and Notoginsenoside R4 all had in vitro anticoagulant effects.

[0060] (2) Effects of 8 differential chemical components on platelet aggregation

[0061] There was no significant difference in the effect on platelet aggregation between the solvent control group (PRP containing 2.5% DMSO) and the blank control group (PRP without DMSO) Figure 6 , indicating that DMSO at this concentration has no inhibitory effect on platelet aggregation.

[0062] After measuring the maximum platelet aggregation rate of 8 components at different concentrations, the inhibition rates of 8 components at different concentrations on ADP-induced platelet aggregation in rats were calculated (as shown in Table 2 and Figure 7 as follows). The results showed that NotoginsenosideR2, Ginsenoside Rf, Ginsenoside Rh3, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd at concentrations of 0.25, 0.50, 1.00, 2.00 and 5.00 mg / mL, GinsenosideRa1 at a concentration of 0.25 mg / mL, and Notoginsenoside Fa at concentrations of 0.25 and 2.00 mg / mL could all significantly inhibit ADP-induced platelet aggregation in rats.

[0063] In summary, 8 components such as Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, and 5,6-didehydroginsenoside Rd all have the effect of anti-platelet aggregation in vitro.

[0064] Table 2: Effects of 8 components on ADP-induced platelet aggregation in rats (n = 3)

[0065]

[0066]

[0067] Control is the solvent control. Compared with Control, ***p < 0.001, **p < 0.01, *p < 0.05

[0068] Continued Table 2: Effects of 8 components on ADP-induced platelet aggregation in rats (n = 3)

[0069]

[0070]

[0071] Control was the solvent control. Compared with Control, ***p < 0.001, **p < 0.01, *p < 0.05.

Claims

1. Use of a compound selected from the following in the preparation of an anticoagulant drug: Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd, and Notoginsenoside R4.

2. Use of Notoginsenoside R4 in the preparation of a drug with a dual regulatory effect of activating blood circulation and / or stopping bleeding.

3. Use of a compound selected from the following in the preparation of an antiplatelet aggregation drug: Notoginsenoside R2, Notoginsenoside Fa, Ginsenoside Rf, Ginsenoside Rh3, Ginsenoside Ra1, Vinaginsenoside R4, 20-O-glucoginsenoside Rf, 5,6-didehydroginsenoside Rd.

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