Composition, preparation method of pseudo-ginseng extract and application of pseudo-ginseng extract

Abstract

The invention relates to the field of medicines, in particular to a composition, a preparation method of a pseudo-ginseng extract and application of the pseudo-ginseng extract. The composition is prepared from notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re, and the weight ratio of the notoginsenoside R1 to the ginsenoside Rg1 to the ginsenoside Re is (2.05 to 2.5) to (7.5 to 9.4) to 1. The invention also provides application of the composition in preparation of drugs for preventing and / or treating cardiovascular and cerebrovascular diseases. The preparation method of the pseudo-ginseng extract provided by the invention comprises the following steps: cleaning pseudo-ginseng, removing impurities, pretreating, crushing, mixing with ethanol, soaking, percolating, collecting percolate, concentrating, and filtering to obtain the pseudo-ginseng extract. The composition provided by the invention can be used for effectively treating cardiovascular and cerebrovascular diseases, and can generate a better treatment effect compared with the existing composition adopting panaxatriol saponin.

Description

[0001] This divisional application is based on the patent application filed with the National Intellectual Property Administration on September 8, 2020, with the application number 202010934338.X, the invention title "Composition, Preparation Method of Notoginseng Extract, and Application of Notoginseng Extract", and the applicant is Chengdu Huashen Technology Group Co., Ltd. Technical Field

[0002] The present invention relates to the field of drugs, and particularly to a composition, a preparation method of notoginseng extract, and an application of notoginseng extract. Background Art

[0003] Notoginseng (Panax notoginseng (Burk.) F.H. Chen), also known as Tianqi, Ginseng Notoginseng, Tian Sanqi, Shanqi, Jinbuchuan, or Xuecan, etc., is a perennial herb of the genus Panax in the Araliaceae family. It is named Notoginseng because it is dug after sowing for three to seven years and each plant has three petioles, and each petiole bears seven leaves. Its stems, leaves, and flowers can all be used as medicine, and it is a precious traditional Chinese medicine unique to China. Notoginsenoside Rg1, Re, and R1 are the saponin extracts of the single traditional Chinese medicine notoginseng (also known as Tianqi, Ginseng Notoginseng), which are the effective parts for promoting blood circulation and removing blood stasis in notoginseng, containing all the triterpene saponins in notoginseng. The main components are ginsenoside Rg1, Re, notoginsenoside R1, and a small amount of Rb1, Rd, etc.

[0004] Existing research mainly focuses on the extraction or preparation of total notoginsenosides. Total notoginsenosides belong to dammarane-type tetracyclic triterpenoid saponins, and can be further divided into protopanaxadiol-type and notoginsenoside Rg1, Re, and R1 according to their structural characteristics. Due to the different structures of these two types of saponins, their biological activities are also very different.

[0005] However, currently, it is difficult for drug preparations based on this notoginsenoside Rg1, Re, and R1 to exert better targeted efficacy for specific diseases. Therefore, it is of great practical significance to isolate effective parts containing different components from notoginseng in order to provide drugs more suitable for specific diseases. Summary of the Invention

[0006] In view of this, the present invention provides a composition, a preparation method of notoginseng extract, and an application of notoginseng extract. The composition or notoginseng extract provided by the present invention can effectively treat cardiovascular and cerebrovascular diseases, and can produce better treatment effects compared with the existing ones using notoginsenoside Rg1, Re, and R1.

[0007] In order to achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides a composition, the active ingredient of which is notoginsenoside triol, and the notoginsenoside triol includes notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re. The weight ratio of the notoginsenoside R1, the ginsenoside Rg1 and the ginsenoside Re is: (2.05 - 2.5):(7.5 - 9.4):1.

[0009] The beneficial effect of the present invention is that the ratio of the three components of the above-mentioned notoginsenoside triol can greatly improve the therapeutic efficacy in the treatment of cardiovascular and cerebrovascular diseases. Compared with the existing notoginsenoside triol compositions, the ratio of the present invention enables better synergistic effects among the components, with better medicinal effects, enabling patients to recover health faster.

[0010] In some specific embodiments of the present invention, the weight ratio of the notoginsenoside R1, the ginsenoside Rg1 and the ginsenoside Re is: (2.075 - 2.4):(8.125 - 9.25):1.

[0011] The beneficial effect of the above specific embodiment of the present invention is that the preferred ratio of the present invention enables the medicinal effect to be further enhanced, can better treat patients, shortens the treatment cycle of patients, and has strong social and economic benefits.

[0012] In some specific embodiments of the present invention, the weight ratio of the notoginsenoside R1, the ginsenoside Rg1 and the ginsenoside Re is: (2.1 - 2.3):(8.7 - 9.0):1.

[0013] The beneficial effect of the above specific embodiment of the present invention is that the preferred ratio of the present invention enables the medicinal effect to be further enhanced, can better treat patients, and further shortens the treatment time.

[0014] On the other hand, the present invention also provides the use of the described composition in the preparation of a drug for preventing and / or treating cardiovascular and cerebrovascular diseases.

[0015] The beneficial effect of the present invention is that the above composition has pharmacological effects such as reducing blood viscosity, enabling it to have good applications in cardiovascular and cerebrovascular diseases.

[0016] In some specific embodiments of the present invention, the composition is used for reducing blood viscosity, antiplatelet aggregation and / or improving microcirculation; or

[0017] The composition stimulates the estrogen receptor α and / or the glucocorticoid receptor to produce activity.

[0018] The beneficial effects of the above specific embodiments of the present invention are that through in-depth pharmacological research, the present invention discovers that the composition stimulates the estrogen receptor α and glucocorticoid receptor to produce activity. Thereby, it can play functions such as antiplatelet aggregation, and can be better applied in cardiovascular and cerebrovascular diseases.

[0019] In addition, the present invention also provides a preparation method of the Panax notoginseng extract, comprising the following steps:

[0020] Step 1: Take the Panax notoginseng, wash and remove impurities, then mix it with an alkali solution, soak it for the first time for 1 - 2 hours, wash it, and then mix it with carbendazim, soak it for the second time for 20 - 30 minutes to obtain pretreated Panax notoginseng;

[0021] Step 2: Take the pretreated Panax notoginseng obtained in Step 1 and crush it to obtain Panax notoginseng powder;

[0022] Step 3: Take 62 - 69 wt% ethanol and mix it with the Panax notoginseng powder obtained in Step 2, soak for 13 - 22 hours to obtain the soaked Panax notoginseng powder;

[0023] Step 4: Take the soaked Panax notoginseng powder and percolate it with 62 - 69 wt% ethanol, collect the percolate, concentrate and filter it to obtain the Panax notoginseng extract.

[0024] The beneficial effects of the present invention are that in Step 1, after soaking with saturated calcium hydroxide and carbendazim, some impurities such as dencichine in the Panax notoginseng are removed to obtain pretreated Panax notoginseng. Then, through the crushing in Step 2 and the addition of ethanol in Step 3, and then ethanol percolation, etc., some impurities such as notoginsenoside diol are removed. The obtained Panax notoginseng extract contains notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re, which form notoginsenoside triol. And notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re have the corresponding ratios announced above, ensuring the efficacy of the obtained notoginsenoside triol. This preparation method has a simple process and can remove a large amount of impurities in the Panax notoginseng, obtaining notoginsenoside triol with a relatively high purity.

[0025] In some specific embodiments of the present invention, the temperature of the first soaking or the second soaking in Step 1 is 20 - 45 °C;

[0026] The alkali solution in Step 1 includes saturated calcium hydroxide solution or saturated sodium hydroxide solution;

[0027] The time of the first soaking in Step 1 is 1.5 hours, and the time of the second soaking is 25 minutes;

[0028] The particle size of the Panax notoginseng powder in Step 2 is 2 - 10 mesh; in Step 3, the weight ratio of ethanol to the Panax notoginseng powder is (3 - 9):1.

[0029] The beneficial effects of the present invention are that the specific soaking temperature makes the whole preparation process more stable, and can ensure the more stable preparation of notoginsenoside.

[0030] In some specific embodiments of the present invention, in step 4, the mass ratio of ethanol to the soaked notoginseng powder is (2-7):1; the concentration condition is carried out under reduced pressure concentration under the conditions of a vacuum degree of 0.04-0.1 Mpa and a temperature of 51-57 °C; the filtration is carried out using a styrene-type macroporous resin column.

[0031] The beneficial effects of the present invention are that the above specific conditions make the ratio of notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re in the obtained notoginsenoside better, making the therapeutic effect of the composition better and the medicinal effect stronger.

[0032] The present invention also provides a notoginseng extract prepared by the above preparation method, including notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re, and the weight ratio of notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re is: (2.05-2.5):(7.5-9.4):1; preferably, the weight ratio of notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re is: (2.075-2.4):(8.125-9.25):1; more preferably, the weight ratio of notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re is: (2.1-2.3):(8.7-9.0):1.

[0033] On the basis of the above research, the present invention also provides the application of the above-mentioned notoginseng extract in the preparation of drugs for preventing and / or treating cardiovascular and cerebrovascular diseases.

[0034] More importantly, the present invention also provides a drug for preventing and / or treating cardiovascular and cerebrovascular diseases, including the above composition or the above notoginseng extract and pharmaceutically acceptable excipients.

[0035] The beneficial effects of the present invention are that the present invention provides a preparation method of a composition, a notoginseng extract and the application of the notoginseng extract, and the composition and the notoginseng extract can produce better therapeutic effects compared with the existing ones using notoginsenoside. Detailed implementation mode

[0036] The present invention discloses a composition, a preparation method of notoginseng extract, and an application of notoginseng extract. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred preparation examples. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0037] The raw materials and reagents used in the composition, the preparation method of notoginseng extract, and the application of notoginseng extract provided by the present invention can all be purchased from the market.

[0038] The following further elaborates the present invention in conjunction with examples:

[0039] Preparation Example 1

[0040] A preparation method of notoginsenoside triol includes the following steps:

[0041] S1. Take 150 kg of notoginseng (origin: Wenshan Prefecture, Yunnan Province), wash and remove impurities, then add it to 200 kg of saturated aqueous solution of calcium hydroxide, soak it at 35 °C for 1.5 h and then take it out. Then rinse the notoginseng with sterile water, and then soak the notoginseng in carbendazim at room temperature for 25 min and then take it out to obtain the treated notoginseng.

[0042] S2. Crush the treated notoginseng in step S1 with a universal crusher (purchased from Shanghai Tianhe Pharmaceutical Machinery Factory, model GF-300) to obtain notoginseng powder, and the particle size of the notoginseng powder is 2-10 mesh.

[0043] S3. Soak the notoginseng powder in step S2 with 62 wt% ethanol for 19 h, and then fill the notoginseng powder into a percolation device to obtain the to-be-percolated notoginseng powder; the volume ratio of ethanol to the notoginseng powder is 3:1.

[0044] S4. Add 62 wt% ethanol to the percolation device in step S3 and carry out percolation, collect the percolate, then carry out vacuum concentration under the conditions of 0.08 Mpa and 54 °C, and then filter the solution after vacuum concentration with a styrene-type macroporous resin column to obtain the filtrate, namely notoginsenoside triol. The volume ratio of ethanol to the notoginseng powder is 2:1. The obtained notoginsenoside triol is collectively referred to as PTS (the same below), and the weight of PTS obtained is 7 kg.

[0045] Preparation Example 2

[0046] The difference from Preparation Example 1 is that in step S3, the volume ratio of ethanol to the notoginseng powder is 5:1. In step S4, ethanol with a concentration of 65 wt% is added, and the volume ratio of ethanol to the notoginseng powder is 5:1. The weight of the obtained PTS is 7.1 kg.

[0047] Preparation Example 3

[0048] The difference from Preparation Example 1 is that in step S3, the volume ratio of ethanol to the notoginseng powder is 9:1. In step S4, ethanol with a concentration of 65 wt% is added, and the volume ratio of ethanol to the notoginseng powder is 7:1. The weight of the obtained PTS is 6.9 kg.

[0049] Preparation Example 4

[0050] The difference from Preparation Example 1 is that in step S3, 65 wt% ethanol is used. In step S4, ethanol with a concentration of 65 wt% is added. The weight of the obtained PTS is 7.2 kg.

[0051] Preparation Example 5

[0052] The difference from Preparation Example 1 is that in step S3, 69 wt% ethanol is used. In step S4, 69 wt% ethanol is added. The obtained notoginseng powder to be percolated is dissolved in sterile water and centrifuged at 1000 - 2000 r / min for 3 - 4 min, and the supernatant is taken and filtered through a quartz sand column to obtain the filtrate. The weight of the obtained PTS is 7.3 kg.

[0053] Preparation Example 6

[0054] The difference from Preparation Example 2 is that in step S3, 65 wt% ethanol is used. The weight of the obtained PTS is 7.25 kg.

[0055] Preparation Example 7

[0056] The difference from Preparation Example 2 is that in step S3, 69 wt% ethanol is used. The weight of the obtained PTS is 6.95 kg.

[0057] Preparation Example 8

[0058] The difference from Preparation Example 3 is that in step S3, 65 wt% ethanol is used. The weight of the obtained PTS is 6.92 kg.

[0059] Preparation Example 9

[0060] The difference from Preparation Example 3 is that in step S3, 69 wt% ethanol is used. In step S4, 69 wt% ethanol is added. The weight of the obtained PTS is 6.91 kg.

[0061] Preparation of Comparative Example 1

[0062] It is different from Preparation Example 1 in that the pretreatment step is cancelled. The weight of the obtained PTS is 6 kg.

[0063] Preparation of Comparative Example 2

[0064] It is different from Preparation Example 2 in that the pretreatment step is cancelled. The weight of the obtained PTS is 5.9 kg.

[0065] Preparation of Comparative Example 3

[0066] It is different from Preparation Example 3 in that the pretreatment step is cancelled. The weight of the obtained PTS is 5.96 kg.

[0067] Preparation of Comparative Example 4

[0068] It is different from Preparation Example 4 in that the pretreatment step is cancelled. The weight of the obtained PTS is 6.08 kg.

[0069] Preparation of Comparative Example 5

[0070] It is different from Preparation Example 5 in that the pretreatment step is cancelled. The weight of the obtained PTS is 6.31 kg.

[0071] Preparation of Comparative Example 6

[0072] It is different from Preparation Example 6 in that the pretreatment step is cancelled. The weight of the obtained PTS is 6.09 kg.

[0073] Preparation of Comparative Example 7

[0074] It is different from Preparation Example 7 in that the pretreatment step is cancelled. The weight of the obtained PTS is 6.01 kg.

[0075] Demonstration Example 1

[0076] In order to know the contents of notoginsenoside R1, ginsenoside Rg1, and ginsenoside Re in the notoginsenosides obtained from Preparation Example 1 to Preparation Example 9, Preparation Comparative Example 1, and Preparation Comparative Example 7 above, Preparation Example 1 to Preparation Example 9, Preparation Comparative Example 1, and Preparation Comparative Example 7 were specifically measured by high performance liquid chromatography as follows:

[0077] Chromatographic conditions and system suitability test:

[0078] Using octadecylsilyl silica gel as the filler; using acetonitrile - water (19.5:80.5) as the mobile phase; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the ginsenoside Rg1 peak should be not less than 4000, the resolution between ginsenoside Rg1 and notoginsenoside R1 should be not less than 1.5, and the resolution between ginsenoside Rg1 and ginsenoside Re should be not less than 1.3.

[0079] Preparation of reference substance solution: Take appropriate amounts of ginsenoside Rg1 reference substance, ginsenoside Re reference substance, and notoginsenoside R1 reference substance, weigh accurately, dissolve with the mobile phase and dilute to a mixed solution containing 2.5 mg of ginsenoside Rg1, 0.4 mg of ginsenoside Re, and 0.8 mg of notoginsenoside R1 in each 1 ml, shake well, and it is ready.

[0080] Preparation of test solution: Take 0.13 g of the test samples from Preparation Examples 1 to 9, Preparation Comparative Example 1, and Preparation Comparative Example 7 respectively, weigh accurately, place in a 25 - ml volumetric flask, add 20 ml of the mobile phase, ultrasonically treat (power 160 W, frequency 40 KHZ) for 30 minutes, let it cool, dilute to the scale with the mobile phase, shake well, filter, and take the subsequent filtrate, and it is ready.

[0081] Determination method: Accurately pipette 10 μl each of the reference substance solution and the test solutions from Preparation Examples 1 to 9, Preparation Comparative Example 1, and Preparation Comparative Example 7 into the liquid chromatograph for determination, and the contents of ginsenoside Rg1, ginsenoside Re, and notoginsenoside R1 can be obtained. The statistics are shown in Table 1 below:

[0082] Table 1

[0083]

[0084]

[0085] From the contents of each component in the above PTS, it can be known that the preparation method of the present invention can extract the corresponding contents of ginsenoside Rg1, ginsenoside Re, and notoginsenoside R1 required by the present invention from Panax notoginseng. The pretreatment therein plays an important role in impurity removal and purification. In Preparation Example 5, the obtained notoginseng powder to be percolated is dissolved with sterile water and centrifuged at 1000 - 2000 r / min for 3 - 4 min, and the supernatant is filtered through a quartz sand column to obtain the filtrate, which can further separate and purify Panax notoginseng to obtain notoginsenoside suitable for the ratio of ginsenoside Rg1, ginsenoside Re, and notoginsenoside R1.

[0086] Meanwhile, purchase notoginsenoside R1, ginsenoside Rg1, and ginsenoside Re respectively from "Standard Substance Network", weigh accurately, and form 16 groups of compositions. The components and ratios of the 16 groups of compositions are shown in Table 2 below:

[0087] Table 2

[0088] Number <![CDATA[Quality of Ginsenoside Rg1, Ginsenoside Re and Notoginsenoside R1]]> Composition 1 <![CDATA[Notoginsenoside R1: 17.425 g; Ginsenoside Rg1: 63.75 g; Ginsenoside Re: 8.5 g]]> Composition 2 <![CDATA[Notoginsenoside R1: 18 g; Ginsenoside Rg1: 67.68 g; Ginsenoside Re: 7.2 g]]> Composition 3 <![CDATA[Notoginsenoside R1: 16.767 g; Ginsenoside Rg1: 64.8 g; Ginsenoside Re: 8.1 g]]> Composition 4 <![CDATA[Notoginsenoside R1: 17.64 g; Ginsenoside Rg1: 73.08 g; Ginsenoside Re: 8.4 g]]> Composition 5 <![CDATA[Notoginsenoside R1: 18.04 g; Ginsenoside Rg1: 72.98 g; Ginsenoside Re: 8.2 g]]> Composition 6 <![CDATA[Notoginsenoside R1: 18.4 g; Ginsenoside Rg1: 72 g; Ginsenoside Re: 8 g]]> Composition 7 <![CDATA[Notoginsenoside R1: 17.845 g; Ginsenoside Rg1: 69.875 g; Ginsenoside Re: 8.6 g]]> Composition 8 <![CDATA[Notoginsenoside R1: 18.24 g; Ginsenoside Rg1: 70.3 g; Ginsenoside Re: 7.6 g]]> Composition 9 <![CDATA[Notoginsenoside R1: 19.505 g; Ginsenoside Rg1: 70.55 g; Ginsenoside Re: 8.3 g]]> Composition 10 <![CDATA[Notoginsenoside R1: 14.676 g; Ginsenoside Rg1: 72.744 g; Ginsenoside Re: 9.036 g]]> Composition 11 <![CDATA[Notoginsenoside R1: 17.556 g; Ginsenoside Rg1: 67.452 g; Ginsenoside Re: 9.24 g]]> Composition 12 <![CDATA[Notoginsenoside R1: 18.928 g; Ginsenoside Rg1: 69.16 g; Ginsenoside Re: 7.28 g]]> Composition 13 <![CDATA[Notoginsenoside R1: 14.058 g; Ginsenoside Rg1: 73.414 g; Ginsenoside Re: 7.81 g]]> Composition 14 <![CDATA[Notoginsenoside R1: 22.308 g; Ginsenoside Rg1: 63.492 g; Ginsenoside Re: 8.58 g]]> Composition 15 <![CDATA[Notoginsenoside R1: 14.96 g; Ginsenoside Rg1: 71.06 g; Ginsenoside Re: 7.48 g]]> Composition 16 <![CDATA[Notoginsenoside R1: 13.869 g; Ginsenoside Rg1: 73.968 g; Ginsenoside Re: 7.705 g]]>

[0089] In order to know the effects, safety and curative effects of the notoginsenoside triol saponins prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 in the treatment of cardiovascular and cerebrovascular diseases, the following experiments were specifically conducted:

[0090] 100 g of the dried PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were respectively taken, dissolved in ethanol with a concentration of 50 wt%, 100 g of starch was respectively added, mixed evenly, granulated by a one-step granulator, and filled into 16 groups of experimental capsules, with 1000 capsules in each group. At the same time, Compositions 1 to 16 were taken, dissolved in ethanol with a concentration of 50 wt% respectively, 100 g of starch was respectively added, mixed evenly, granulated by a one-step granulator, and filled into 16 groups of composition standard capsules. The experimental group capsules and the composition standard capsules were tested in the following manner.

[0091] Effect Example 1. Influence on platelet aggregation function and release function

[0092] Using the turbidimetry method and a PAM-3 type dual-channel platelet aggregometer (purchased from Danyang Radio Factory, Jiangsu Province), in vivo experimental evaluation was carried out on the function of inhibiting platelet aggregation of the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 of the present invention. 320 healthy male SD rats were taken and evenly divided into 16 experimental groups. Each experimental group was further evenly divided into three dose groups of large (150 mg / kg), medium (75 mg / kg), and small (37.5 mg / kg) and one aspirin positive control group (300 mg / kg). The PTS powders prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were respectively formulated into solutions with physiological saline and corresponded to the 16 experimental groups one by one. Each experimental group was administered the prepared PTS solution by duodenal injection according to the predetermined doses (large dose group, medium dose group, and small dose group). After administration once, blood was taken from the abdominal aorta 1 hour later, rich platelet plasma was prepared, and an inducer adenosine diphosphate (ADP) with a concentration of 3×10 -6 mol / L was added. The experimental results showed that the PTS prepared in Preparation Examples 1 to 9 could significantly inhibit the platelet aggregation of rats induced by ADP. The PTS prepared in Preparation Comparative Examples 1 to 7 could also inhibit the platelet aggregation of rats induced by ADP, and the effect was enhanced with the increase of the dose. The intensity of the effect of inhibiting platelet aggregation caused by ADP was basically equivalent to that of the control group aspirin.

[0093] Using the turbidimetry method and a PAM-3 type dual-channel platelet aggregometer (purchased from Danyang Radio Factory, Jiangsu Province), an in vitro experimental evaluation was carried out on the function of the composition of the present invention in inhibiting platelet aggregation. First, the PTS powders prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were respectively dissolved in physiological saline to form solutions with a concentration of 1 mg / ml. Sixteen healthy male rabbits were taken, and blood was drawn from the common carotid artery. After preparing rich platelet plasma, the 16 rabbits were respectively corresponding to the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 one by one and diluted with an equal volume of the prepared PTS solution. Then, an inducer ADP with a concentration of 3×10 -6 mol / L was added to the above-diluted plasma to induce platelet aggregation. The experimental results proved that Preparation Examples 1 to 9 all had an obvious effect of inhibiting rabbit platelet aggregation induced by ADP. The PTS prepared in Preparation Comparative Examples 1 to 7 also had an effect of inhibiting rabbit platelet aggregation induced by ADP, and this effect was dose-dependent. Therefore, it was proved that the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 had a definite pharmacodynamic effect of inhibiting platelet aggregation.

[0094] The above experiments showed that the composition of notoginsenoside Rg1, notoginsenoside Re and notoginsenoside R1, nototriol saponin, had a therapeutic effect on platelet aggregation function and release function and could be used to treat cardiovascular and cerebrovascular diseases.

[0095] Effect Example 2. Acute toxicity test

[0096] Eight hundred healthy NIH mice weighing 18 - 22 g were selected and evenly divided into 16 large groups. Each large group was randomly divided into 5 small groups, with 10 mice in each small group, including 5 pairs of male and female mice. The dosing doses of the 5 pairs of male and female mice in each small group were 4444.44 mg / kg, 4000.00 mg / kg, 3600.00 mg / kg, 3240.00 mg / kg, and 2916.00 mg / kg respectively, and the dose ratio was 1∶0.9. The PTS powders prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were formulated into PTS solutions with sterilized physiological saline and corresponded to the 16 large groups one by one. Each small group of mice was intravenously injected with the prepared PTS solution according to the predetermined dose. The LD50 (drug median lethal dose: the dose at which the drug causes half of the experimental animals to die in the acute toxicity experiment) was calculated according to the experimental results as shown in Table 3 below. Before death, there were transient convulsions, startles, and respiratory depression followed by death.

[0097] Table 3

[0098]

[0099] Since the qualified range of LD 50 is 3261.8 mg / kg to 3590.6 mg / kg, Preparation Examples 1 to 9 of the present invention are all within the qualified range. Thus, it can be proved that the composition of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 of the present invention is reliable in acute toxicity, indicating the safety of the pharmaceutical composition of the present invention. Moreover, the safety of Preparation Comparative Examples 1 to 7 of the present invention is also reliable.

[0100] In order to verify that in the PTS prepared from Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, it is indeed the notoginsenoside composed of notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re that has sufficient safety, the safety of Compositions 1 to 16 purchased from the market was verified. The verification method was the same as the test conditions and methods corresponding in Table 3 above. The specific results are shown in Table 4 below:

[0101] Table 4

[0102]

[0103] From the data in Table 4, it can be known that the safety of Compositions 1 to 16 is determined, and it can further confirm the safety of the PTS prepared from Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7.

[0104] Effect Example 3. Long-term toxicity test

[0105] 64 healthy male SD rats were taken and evenly divided into 16 experimental groups. The PTS powders prepared from Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were respectively made into concentrated suspensions with normal saline and corresponded to the 16 experimental groups one by one. The rats were gavaged with the drug at a dose of 2 g / kg body weight per day using a gavage needle for a total of 90 days. After the test ended, the physiological conditions of the rats were evaluated. It was found that the test rats in the 16 experimental groups had normal diet, normal weight gain, no obvious influence on hematological and blood biochemical indexes, and no obvious lesions were seen in the histological examination of each organ. This shows that the pharmaceutical composition of the present invention has long-term safety.

[0106] Effect Example 4. Influence on experimental cerebral ischemia in gerbils

[0107] 480 gerbils were taken and evenly divided into 16 large groups. Each large group was randomly divided into 1 normal group, 1 ischemic control group, 1 positive drug control group and 2 experimental groups, with 6 in each group. Among them:

[0108] The normal group was raised normally without giving any drugs, and the other groups adopted an experimental model of unilateral common carotid artery ligation in gerbils to form infarction in one cerebral hemisphere.

[0109] The sham operation control group was intraperitoneally injected with normal saline (150 mg / kg) once a day for 10 consecutive days.

[0110] The positive control drug group was intragastrically administered with nimodipine at 60 mg / kg once a day for 10 consecutive days.

[0111] The experimental groups of 16 large groups were intraperitoneally injected with the physiological saline solution of PTS (concentration: 50 mg / ml) prepared in Preparation Example 1 to Preparation Example 9 and Preparation Comparative Example 1 to Preparation Comparative Example 7. Among them: in the experimental groups of each large group, 3 gerbils were administered at a high dose group (150 mg / kg), and another 3 gerbils were at a low dose group (75 mg / kg), once a day for 10 consecutive days.

[0112] One hour after the last administration of all mice, they were anesthetized with ether, and the effects of PTS prepared in Preparation Example 1 to Preparation Example 9 and Preparation Comparative Example 1 to Preparation Comparative Example 7 on cerebral dysfunction and potassium and sodium ions in brain tissue caused by local cerebral ischemia were observed. The animals were behaviorally scored according to the method of Butterfield et al. (Selected Materials of the National Symposium on Coronary Heart Disease in 1973, page 261, People's Medical Publishing House, 1974). The results showed that PTS prepared in Preparation Example 1 to Preparation Example 9 significantly improved the experimental cerebral ischemia behavior of gerbils, and the positive control drug nimodipine also had an obvious effect. PTS prepared in Preparation Comparative Example 1 to Preparation Comparative Example 7 improved the experimental cerebral ischemia behavior of gerbils, and the positive control drug nimodipine also had an effect. The contents of potassium and sodium ions in brain tissue were determined by flame emission spectrophotometry. The results showed that the sodium content in the ischemic hemisphere of the sham operation control group gerbils increased and the potassium content decreased. In the high-dose PTS (150 mg / kg) group, there were no obvious changes in potassium and sodium contents. In the low-dose PTS (75 mg / kg) group, except that the potassium content decreased significantly, the sodium content had no significant change. Nimodipine also had such an antagonistic effect.

[0113] In summary, it is shown that PTS prepared in Preparation Example 1 to Preparation Example 9 and Preparation Comparative Example 1 to Preparation Comparative Example 7 has a certain protective effect on experimental cerebral ischemia in gerbils.

[0114] In order to prove the curative effects in Preparation Example 1 to Preparation Example 9 and Preparation Comparative Example 1 to Preparation Comparative Example 7, the following experiment was specifically conducted:

[0115] Effect Example 5. Influence on experimental thrombosis in rats

[0116] 960 healthy male SD rats with a body weight of over 300 g were evenly divided into 16 large groups. Each large group was divided into 2 control groups and 10 experimental groups, with 5 rats in each control group and experimental group. The PTS powders prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were dissolved in physiological saline to form solutions corresponding to the 16 large groups one by one, and then the PTS solutions were all administered via the duodenum.

[0117] Among them: the administration doses of the 10 experimental groups in each large group were 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 50 mg / kg, 100 mg / kg, and 200 mg / kg respectively, and the administration was carried out once.

[0118] The 2 control groups in each large group were a negative control group and a positive control group respectively. The duodenal administration dose of the negative control group was an equal volume of distilled water as the maximum dose experimental group, that is, 200 mg / kg. The positive drug control group was given aspirin (300 mg / kg) by gavage, and the administration was carried out once.

[0119] One hour after administration, surgery was performed using the arteriovenous bypass method to establish blood circulation. The statistical results of the administration doses of the prepared PTS that could significantly inhibit experimental thrombosis in rats are shown in Table 5 below: Among them: B indicates that it could not significantly inhibit experimental thrombosis in rats, and A indicates that it could significantly inhibit experimental thrombosis in rats.

[0120] Table 5

[0121]

[0122]

[0123] As the dose increased, the effect was enhanced. From the data in Table 5, it can be seen that the pharmacodynamic effect in Preparation Example 5 was the best. Only the PTS in Preparation Example 5 could significantly inhibit experimental thrombosis in rats at 20 mg / kg, and the pharmacodynamic effect was the strongest. In Preparation Example 4 and Preparation Example 6, experimental thrombosis in rats could be significantly inhibited at 25 mg / kg. In Preparation Example 7, Preparation Example 8, and Preparation Example 9, experimental thrombosis in rats could be significantly inhibited at 30 mg / kg. In Preparation Examples 1 to 3, experimental thrombosis in rats could be significantly inhibited at 35 mg / kg. The worst pharmacodynamic effect was in Preparation Comparative Example 1 and Preparation Comparative Example 7, where experimental thrombosis in rats could be significantly inhibited at 50 mg / kg. Therefore, the pharmacodynamic effect of the Panax notoginseng extract prepared in the present invention is much stronger than that of Preparation Comparative Examples 1 to 7.

[0124] Meanwhile, in order to prove that in the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, it is indeed the notoginsenoside R1, ginsenoside Rg1, and ginsenoside Re that constitute the notoginsenoside triol saponins that produce the medicinal effects. Therefore, 960 healthy male SD rats with a body weight of more than 300 g were taken and evenly divided into 16 large groups. Each large group was divided into 2 control groups and 10 experimental groups, with 5 rats in each control group and experimental group. The PTS powder prepared from Composition 1 to Composition 16 was formulated into a solution with physiological saline and corresponded to the 16 large groups one by one. Then, the PTS solution prepared by duodenal administration was used, and the remaining administration conditions and verification methods were the same as the corresponding test conditions and methods in Table 5. The specific results obtained are shown in Table 6 below.

[0125] Table 6

[0126]

[0127]

[0128] It can be seen from the data in Table 6 that Composition 1 to Composition 16 can produce medicinal effects very similar to those of the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, which once again proves that it is the notoginsenoside triol saponins in the components prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 that produce the medicinal effects.

[0129] Effect Example 6. Protective effect on cerebral infarction caused by middle cerebral artery occlusion in rats

[0130] (1) Preventive administration

[0131] 400 male Wistar rats were taken and randomly and evenly divided into 16 large groups. Each large group was randomly divided into an ischemic control group, a positive drug control group, a high-dose (200 mg / kg / day) experimental group, a medium-dose (100 mg / kg / day) experimental group, and a low-dose (50 mg / kg / day) experimental group, with 5 rats in each group. Among them:

[0132] The ischemic control group was intragastrically administered 4 ml / kg / day of distilled water.

[0133] The positive control drug group was intragastrically administered nimodipine at 50 mg / kg / day.

[0134] The PTS powder prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 was respectively formulated into a suspension with physiological saline. The animals in the high, medium, and low-dose experimental groups in the 16 large groups were intragastrically administered according to the predetermined dose, and the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 corresponded to the 16 large groups one by one.

[0135] Each large group was continuously administered for 7 days. Two hours after the last administration, the rats in all large groups were intraperitoneally injected with chloral hydrate at 350 mg / kg to be anesthetized, and the middle cerebral artery of the rats was cauterized to cause cerebral infarction. Through observation, statistics and calculation, it was obtained that the average reduction rate of the percentage of cerebral infarction tissue in the nimodipine group of the positive control drug group was 56.3%, and the average reduction rate of the percentage of cerebral infarction tissue in the ischemic control group was 5.2%. Observe the effects of PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 on cerebral dysfunction and infarction range caused by local cerebral ischemia. Statistically calculate the reduction rate (%) of the percentage of cerebral infarction tissue caused by blocking the middle cerebral artery of the rats. The results are shown in Table 7 below: Among them

[0136] Table 7

[0137]

[0138]

[0139] The data in Table 7 show that: PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 by continuous intragastric administration for 7 days (200, 100, 50 mg / kg / day) has obvious curative effects on cerebral infarction caused by blocking the middle cerebral artery of the rats. The pharmacodynamic effect is similar to that of nimodipine, and the high- and medium-dose experimental groups can significantly improve their behavioral disorders. And in terms of the specific curative effect, the effect of Preparation Example 5 is the best. The average reduction rate of the percentage of cerebral infarction tissue in the low-dose experimental group is 69.9%, the average reduction rate of the percentage of cerebral infarction tissue in the medium-dose experimental group is 79.1%, and the average reduction rate of the percentage of cerebral infarction tissue in the high-dose experimental group is 86.4%. The curative effects of other groups decrease in turn: Preparation Example 4 and Preparation Example 6 > ( > indicates that the pharmacodynamic effect is superior to) Preparation Examples 7 to 9 > ( > indicates that the pharmacodynamic effect is superior to) Preparation Examples 1 to 3 > ( > indicates that the pharmacodynamic effect is superior to) Preparation Comparative Examples 1 to 7.

[0140] At the same time, in order to prove that in the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, it is indeed the notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re in the notoginsenoside triol saponins that produce the pharmacodynamic effect, 400 male Wistar rats were taken and evenly divided into 16 large groups. Each large group was randomly divided into an ischemic control group, a positive drug control group, a high-dose (200 mg / kg / day) experimental group, a medium-dose (100 mg / kg / day) experimental group, and a low-dose (50 mg / kg / day) experimental group, with 5 rats in each group. The PTS powder prepared from Compositions 1 to 16 was formulated into a suspension with physiological saline and corresponded to the 16 large groups one by one. The remaining administration conditions and verification methods were the same as the corresponding test conditions and methods in Table 7. The specific obtained results are shown in Table 8 below.

[0141] Table 8

[0142]

[0143] It was observed, statistically calculated, that the average percentage reduction rate of cerebral infarction tissue of nimodipine in the positive control drug group was 56.4%, and that in the ischemic control group was 5.21%, which was basically the same as the data measured by the method corresponding to Table 7. It can be seen from the data in Table 8 that Compositions 1 to 16 can produce similar pharmacological effects to the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, which once again proves that the pharmacological effects are produced by notoginsenoside triol in the components prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7.

[0144] (2) Therapeutic administration

[0145] Four hundred male Wistar rats were randomly and evenly divided into 16 large groups, and each large group was randomly divided into an ischemic control group, a positive drug control group, a high-dose (200 mg / kg / day) experimental group, a medium-dose (100 mg / kg / day) experimental group, and a low-dose (50 mg / kg / day) experimental group, with 5 rats in each group. Among them:

[0146] The ischemic control group was intragastrically administered 4 ml / kg / day of distilled water;

[0147] The positive control drug group was intragastrically administered 50 mg / kg / day of nimodipine.

[0148] All the rats in each large group were anesthetized by intraperitoneal injection of chloral hydrate at 350 mg / kg, and the middle cerebral artery of the rats was cauterized to cause cerebral infarction. Then, the PTS powders prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 were made into suspensions with normal saline, and all the experimental animals were intragastrically administered according to the predetermined doses. Moreover, the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 corresponded one by one to the 16 large groups;

[0149] Each large group was continuously administered for 6 days. Two hours after the last administration, it was observed, statistically calculated, that the average percentage reduction rate of cerebral infarction tissue of nimodipine in the positive control drug group was 38.3%, and that in the ischemic control group was 2.9%. Observe the effects of the PTS powders prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 on cerebral dysfunction and infarction range caused by local cerebral ischemia. Statistically calculate the percentage reduction rate (%) of the infarction tissue of cerebral infarction caused by blocking the middle cerebral artery of the rats. The results are shown in Table 9 below.

[0150] Table 9

[0151]

[0152]

[0153] In summary, it is shown that the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 has a protective effect on local cerebral ischemia in rats. Continuous intragastric administration for 7 days (200, 100, 50 mg / kg / day) of the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7 has an obvious curative effect on cerebral infarction caused by blocking the middle cerebral artery of rats. The pharmacodynamic effect is similar to that of nimodipine, and the high- and medium-dose experimental groups can significantly improve their behavioral disorders. In terms of specific curative effects, the effect of Preparation Example 5 is the best. The average reduction rate of the percentage of cerebral infarction tissue in the low-dose experimental group is 50.2%, the average reduction rate of the percentage of cerebral infarction tissue in the medium-dose experimental group is 65.1%, and the average reduction rate of the percentage of cerebral infarction tissue in the high-dose experimental group is 78.2%. The pharmacodynamic effects of other groups gradually decrease: Preparation Example 4 and Preparation Example 6 > (">" indicates better pharmacodynamic effect than) Preparation Examples 7 to 9 > (">" indicates better pharmacodynamic effect than) Preparation Examples 1 to 3 > (">" indicates better pharmacodynamic effect than) Preparation Comparative Examples 1 to 7. Therefore, the Panax notoginseng extract prepared in the present invention can also significantly improve the behavioral disorders of cerebral infarction rats.

[0154] At the same time, in order to prove that among the PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, it is indeed the notoginsenoside R1, ginsenoside Rg1, and ginsenoside Re in the notoginsenoside triol saponins of Panax notoginseng that produce the pharmacodynamic effects, 400 male Wistar rats were taken and evenly divided into 16 large groups. Each large group was randomly divided into an ischemic control group, a positive drug control group, a high-dose (200 mg / kg / day) experimental group, a medium-dose (100 mg / kg / day) experimental group, and a low-dose (50 mg / kg / day) experimental group, with 5 rats in each group. The PTS powder prepared from Compositions 1 to 16 was formulated into a suspension with physiological saline and corresponded to the 16 large groups one by one. The remaining administration conditions and verification methods were the same as the corresponding test conditions and methods in Table 9. The specific results obtained are shown in Table 10 below.

[0155] Table 10

[0156]

[0157]

[0158] Observation and statistical calculation showed that the average percentage reduction rate of cerebral infarction tissue of nimodipine in the positive control drug group was 38.2%, and the average percentage reduction rate of cerebral infarction tissue in the ischemic control group was 2.89%, which was basically the same as the data measured by the method corresponding to Table 9. From the data in Table 10, it can be seen that Compositions 1 to 16 can produce pharmacodynamic effects very similar to those of PTS prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7, further proving that the pharmacodynamic effects are produced by notoginsenoside triol in the components prepared in Preparation Examples 1 to 9 and Preparation Comparative Examples 1 to 7.

[0159] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing Panax notoginseng extract, characterized in that, It includes the following steps: Step 1: Take Panax notoginseng, wash and remove impurities, then mix it with an alkali solution, soak it for 1 - 2 hours in the first soaking, wash it, and then mix it with carbendazim and soak it for 20 - 30 minutes in the second soaking to obtain pretreated Panax notoginseng; Step 2: Take the pretreated Panax notoginseng obtained in Step 1 and crush it to obtain Panax notoginseng powder; Step 3: Take 62 - 69wt% ethanol and mix it with the Panax notoginseng powder prepared in Step 2 and soak it for 13 - 22 hours to obtain the soaked Panax notoginseng powder; Step 4: Take the soaked Panax notoginseng powder in Step 3 and percolate it with 62 - 69wt% ethanol, then collect the percolate, concentrate it, and then filter it to obtain the Panax notoginseng extract.

2. The preparation method of the notoginseng extract according to claim 1, characterized in that, The temperature of the first soaking or the second soaking in Step 1 is 20 - 45°C; The time of the first soaking in Step 1 is 1.5 hours, and the time of the second soaking is 25 minutes; The alkali solution in Step 1 includes a saturated calcium hydroxide solution or a saturated sodium hydroxide solution; The particle size of the Panax notoginseng powder in Step 2 is 2 - 10 mesh; The weight ratio of ethanol to the Panax notoginseng powder in Step 3 is (3 - 9):1; The mass ratio of ethanol to the soaked Panax notoginseng powder in Step 4 is (2 - 7):1; The concentration condition is carried out under reduced pressure concentration at a vacuum degree of 0.04 - 0.1 Mpa and a temperature of 51 - 57°C; The filtration is carried out using a styrene-type macroporous resin column.

3. The notoginseng extract prepared by the method for preparing a notoginseng extract according to claim 1 or 2, characterized in that, It includes notoginsenoside R1, ginsenoside Rg1 and ginsenoside Re, and the weight ratio of the notoginsenoside R1, the ginsenoside Rg1 and the ginsenoside Re is: (2.05 - 2.5):(7.5 - 9.4):1; preferably, the weight ratio of the notoginsenoside R1, the ginsenoside Rg1 and the ginsenoside Re is: (2.075 ～ 2.4):(8.125 ～ 9.25):1; more preferably, the weight ratio of the notoginsenoside R1, the ginsenoside Rg1 and the ginsenoside Re is: (2.1 - 2.3):(8.7 - 9.0):

1.

4. Use of the Panax notoginseng extract according to claim 3 in the preparation of a drug for preventing and / or treating cardiovascular and cerebrovascular diseases.

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