Anticoagulant active part of massa medicata fermentata fujianensis as well as preparation

Abstract

The invention discloses an anticoagulant active site of massa medicata fermentata fujianensis as well as a preparation method and application thereof, and belongs to the field of biological medicines. The massa medicata fermentata fujianensis is used for preparing anticoagulant drugs for the first time. The method comprises the following steps: soaking a fermented mass raw material with methanol, performing reflux extraction, and recovering a solvent under reduced pressure to obtain a total extract; and separating the total extract by adopting macroporous adsorption resin column chromatography to obtain a water part, a 30% methanol part, a 50% methanol part and a pure methanol part. Platelet aggregation detection results show that all the parts can inhibit ADP-induced platelet aggregation, and the anticoagulant effect of the 30% methanol part is most prominent and is similar to that of a positive drug. According to the preparation method of the anticoagulant active part of the massa medicata fermentata fujianensis, the anticoagulant active part in the massa medicata fermentata fujianensis can be effectively separated and enriched, and the obtained anticoagulant active part can be applied to preparation of anticoagulant drugs and has good industrial application prospects and clinical value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the anticoagulant active fraction of gentian root, its preparation method, and its application. Background Technology

[0002] Jianqu is a fermented koji made from a mixture of various medicinal herbs, including Polygonum hydropiper, Artemisia annua, Xanthium sibiricum, bitter almond, red adzuki bean, and malt, along with wheat bran and flour. It has a long history of clinical use. Jianqu is warm in nature, pungent and sweet in taste, and enters the spleen and stomach meridians. It has the effects of relieving exterior symptoms and harmonizing the middle jiao (digestive system). Clinically, it is mainly used to treat headaches due to cold or heat, food stagnation in the middle jiao, vomiting, and abdominal distension. Modern pharmacological research shows that Jianqu has biological activities such as promoting fat / starch decomposition, regulating gastrointestinal motility, protecting the gastrointestinal mucosa, and possessing anti-inflammatory and antibacterial properties.

[0003] Currently, the main monoclonal drugs used clinically for anticoagulation include vitamin K antagonists (such as warfarin), heparin derivatives (such as heparin), thrombin inhibitors (such as hirudin), and factor Xa inhibitors (such as rivaroxaban). These drugs have safety concerns, including a narrow therapeutic window, a high risk of thrombocytopenia, and a high risk of bleeding. No research has been reported on the use of Jianqu extract for anticoagulation. Therefore, there is an urgent need to develop the anticoagulant function of Jianqu extract. Summary of the Invention

[0004] The purpose of this invention is to provide the anticoagulant active fraction of Jianqu, its preparation method and application, in order to solve the problems existing in the prior art. The method for preparing the anticoagulant active fraction of Jianqu provided by this invention can effectively separate and enrich the anticoagulant active fraction in Jianqu. The obtained anticoagulant active fraction can be used to prepare anticoagulant drugs, and has good industrial application prospects and clinical value.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of Jianqu in the preparation of anticoagulant drugs.

[0007] Furthermore, the anticoagulant is an inhibitor of platelet aggregation.

[0008] This invention also provides a method for preparing the aqueous fraction of Jianqu extract, comprising the following steps:

[0009] Take Jianqu (a type of fermented bamboo), mix and soak it with methanol solution, reflux extract, concentrate under reduced pressure to obtain total Jianqu extract;

[0010] The total extract of Jianqu was mixed with water, dissolved, filtered, and the filtrate was collected.

[0011] The filtrate was subjected to chromatographic separation, eluted with water, and the eluent was collected, which is the aqueous fraction of the Jianqu extract.

[0012] Furthermore, the volume fraction of the methanol solution is 80%;

[0013] The mass ratio of the methyl methacrylate to the methanol solution is 1:5;

[0014] The reflux extraction was performed three times, with each extraction lasting 2.5 hours.

[0015] The present invention also provides an aqueous fraction of Jianqu extract obtained according to the above preparation method.

[0016] The present invention also provides a method for preparing the 30% methanol fraction of Jianqu extract, comprising the following steps: taking Jianqu, mixing and soaking it with a methanol solution with a volume fraction of 80%, reflux extraction, and concentration under reduced pressure to obtain the total Jianqu extract;

[0017] The total extract of Jianqu was mixed with water, dissolved, filtered, and the filtrate was collected.

[0018] The filtrate was subjected to chromatographic separation, and eluted sequentially with water and a 30% (v / v) methanol solution. The methanol eluent was collected, which is the 30% methanol fraction of the Jianqu extract.

[0019] The present invention also provides a 30% methanol fraction of Jianqu extract obtained according to the above preparation method.

[0020] This invention also provides the application of the aqueous fraction of the above-mentioned Jianqu extract or the 30% methanol fraction of the above-mentioned Jianqu extract in the preparation of anticoagulant drugs, wherein the preparation method of the total Jianqu extract includes the following steps:

[0021] Take Jianqu (a type of medicinal herb), mix and soak it in methanol solution, reflux to extract, and concentrate under reduced pressure to obtain the total extract of Jianqu.

[0022] Furthermore, the anticoagulant is an inhibitor of platelet aggregation.

[0023] The present invention also provides an anticoagulant drug, wherein the drug has the aqueous fraction of the above-mentioned Jianqu extract and / or the 30% methanol fraction of the above-mentioned Jianqu extract as the main active ingredients.

[0024] The present invention discloses the following technical effects:

[0025] This invention is the first to use Jianqu (a type of medicinal herb) in the preparation of anticoagulant drugs. The invention involves soaking Jianqu raw materials in methanol and then reflux extraction to obtain a total extract. Macroporous adsorption resin column chromatography is used to separate the fractions in the extract, yielding aqueous, 30% methanol, 50% methanol, and pure methanol fractions. Platelet aggregation detection results show that all fractions can inhibit ADP-induced platelet aggregation, with the 30% methanol fraction exhibiting the most prominent anticoagulant effect, similar to that of positive control drugs. The method for preparing the anticoagulant active fraction of Jianqu provided by this invention can effectively separate and enrich the anticoagulant active fraction in Jianqu. The obtained anticoagulant active fraction can be used to prepare anticoagulant drugs, showing good prospects for industrial application and clinical value. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The image shows the HPLC analysis of the total extract of Jianqu (a type of medicinal herb); where 1 is caffeic acid, 2 is glycyrrhizin, 3 is ferulic acid, 4 is quercetin, 5 is naringin, 6 is hesperidin, 7 is neohesperidin, 8 is artemisinin, 9 is luteolin, 10 is norihesperidin, and 11 is tangeretin.

[0028] Figure 2 HPLC chromatogram of the Jianqu River fraction;

[0029] Figure 3 HPLC chromatogram of the 30% methanol fraction in Jianqu (a type of Chinese liquor);

[0030] Figure 4 HPLC chromatogram of the 50% methanol fraction in Jianqu (a type of Chinese liquor);

[0031] Figure 5 HPLC chromatogram of the 100% methanol fraction in Jianqu;

[0032] Figure 6 To construct a statistical graph of the inhibition rate of ADP-induced platelet aggregation at different polar sites;

[0033] Figure 7 Chromatogram of the component identification of 30% methanol fraction of Jianqu; where 1 is caffeic acid, 2 is glycyrrhizin, 5 is naringin, 6 is hesperidin, 7 is neohesperidin, and 12 is rosmarinic acid. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Some of the experimental materials involved in the embodiments of this invention:

[0040] 1. Samples and reagents: Jianqu (production batch number: 20230814) was purchased from Sichuan Qianyuan Traditional Chinese Medicine Pieces Co., Ltd.; Ticagrelor and sodium citrate were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; adenosine diphosphate (ADP) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; methanol (analytical grade) and acetonitrile (analytical grade) were purchased from Shanghai Titan Technology Co., Ltd.; and formic acid (analytical grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0041] 2. Reference standards: Caffeic acid, ferulic acid, quercetin, naringin, hesperidin, neohesperidin, artemisinin lactone, luteolin, nonotrimonin, tangeretin, and rosmarinic acid were purchased from the National Institutes for Food and Drug Control, and glycyrrhizin was purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0042] 3. Instruments: LC2050 high performance liquid chromatograph (quaternary low-pressure gradient pump, column temperature control chamber, DAD detector) was purchased from Shimadzu Corporation, Japan; HX502T electronic balance was purchased from Cixi Tiandong Weighing Instrument Factory; AUW220D 0.0001 g electronic balance was purchased from Shimadzu Corporation, Japan; EYELA OSB-2100 rotary evaporator was purchased from Tokyo Rikagi K.K. Co., Ltd.; AG400 semi-automatic platelet aggregation analyzer was purchased from Terexon; XS-500i fully automatic five-part differential hematology analyzer was purchased from Sysmex Corporation, Japan.

[0043] 4. Experimental animals: 10 male SD rats were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0044] Example 1: Identification of components in the total extract of Jianqu (a type of medicinal herb).

[0045] 1. Extraction of medicinal materials: Take 2g of Jianqu (batch 20230814) into a stoppered Erlenmeyer flask, add 20mL of water, heat and reflux twice for 1h each time, combine the extracts, filter the residue, dry the filtrate under reduced pressure, and redissolve it in water to prepare a total extract solution with a concentration of 40mg / mL.

[0046] 2. HPLC detection

[0047] Preparation of reference solution: Accurately weigh 2 mg each of caffeic acid, glycyrrhizin, ferulic acid, quercetin, naringin, hesperidin, neohesperidin, artemisinin, luteolin, nosocomioterpenoid, tangeretin, and rosmarinic acid, dissolve them in methanol solution to prepare a 2 mg / mL reference solution.

[0048] Liquid chromatography conditions: Column: Kromasil C8; 0.1% formic acid aqueous solution as mobile phase A; 0.1% formic acid acetonitrile solution as mobile phase B; gradient elution: 0-75 min, 2%-65% B, 76-80 min, 65%-2% B, 81-90 min, 2% B; flow rate: 1.0 mL / min; detection wavelength: 300 nm; column temperature: 35 ℃; injection volume: 10 μL.

[0049] 3. Composition identification

[0050] The results are as follows Figure 1As shown. By comparing the chromatographic peak retention time and ultraviolet absorption characteristics with those of the reference standard, 11 major components in the total extract of Jianqu were identified, including caffeic acid, glycyrrhizin, ferulic acid, quercetin, naringin, hesperidin, neohesperidin, artemisinin lactone, luteolin, nonotrimonin, and tangeretin.

[0051] Example 2: Enrichment of Different Polar Components from Jianqu Herbs

[0052] 1. Extraction of medicinal materials

[0053] Take 5 kg of Jianqu medicinal material, add 5 times the mass of methanol (80% by volume), soak at 55℃ for 6 hours, reflux extract 3 times, 2.5 hours each time, combine the extracts, concentrate under reduced pressure at 50℃ to obtain the total extract.

[0054] 2. Accumulation at different polarity sites

[0055] The total extract (0.82 kg) was dissolved in water, filtered, and the solution was separated by D101 macroporous adsorption resin column chromatography. Elution was performed sequentially with water (6 L), 30% methanol (4 L), 50% methanol (3 L), and 100% methanol (2 L). The eluents were collected and named the water fraction, 30% methanol fraction, 50% methanol fraction, and 100% methanol (pure methanol) fraction, respectively. The solvent in each eluent was recovered under reduced pressure at 50 °C, and the eluent was concentrated to a thick paste.

[0056] 3. Yield of each part

[0057] After separation by D101 macroporous adsorption resin column chromatography, the enrichment of different polar fractions of Jianqu was achieved. The yields of each polar fraction enriched fraction are as follows: water fraction thick paste weight 0.34 kg, yield 46.57%; 30% methanol fraction thick paste weight 0.14 kg, yield 19.18%; 50% methanol fraction thick paste weight 0.17 kg, yield 23.29%; 100% methanol fraction thick paste weight 0.08 kg, yield 10.96%.

[0058] Example 3: Liquid Chromatography Detection of Different Polarity Parts of the Formulation

[0059] 1. Preparation of the test solution

[0060] Take 400 mg of the thick extract from each part, redissolve the thick extract from the aqueous part with deionized water, and redissolve the thick extract from the other parts with 50% methanol solution to prepare a solution with a concentration of 40 mg / mL. Centrifuge at 12000 r / min for 10 min and collect the supernatant, which is the test solution.

[0061] 2. Liquid phase conditions

[0062] Chromatographic column: Kromasil C8; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution; gradient elution: 0-75 min, 2%-65% B, 76-80 min, 65%-2% B, 81-90 min, 2% B; flow rate: 1 mL / min; detection wavelength: 300 nm; column temperature: 35 ℃; injection volume: 10 μL.

[0063] 3. Test Results

[0064] like Figures 2-5 As shown, the different fractions obtained by chromatography using D101 macroporous adsorption resin column showed significant differences in polarity. The chromatographic peaks of the water fraction were mainly distributed between 2 min and 20 min, the chromatographic peaks of the 30% methanol fraction were mainly concentrated between 12 min and 35 min, the chromatographic peaks of the 50% methanol fraction were mainly concentrated between 20 min and 40 min, and the chromatographic peaks of the 100% methanol fraction were mainly concentrated between 30 min and 75 min.

[0065] Example 4 Evaluation of the anticoagulant activity of total extract of *Jianqu* and its different polar fractions

[0066] 1. Preparation of the test solution

[0067] Take the total extract, water fraction, 30% methanol fraction, 50% methanol fraction and 100% methanol fraction respectively, freeze dry, take 200mg of each and add 1mL of physiological saline to make a 200mg / mL solution, centrifuge at 12000r / min for 10min, take the supernatant to obtain the test solution of each fraction.

[0068] 2. Platelet isolation and preparation

[0069] Sodium citrate solution: Weigh 3.8g of sodium citrate into a 100mL volumetric flask, dissolve it in purified water, and make up to volume to obtain a 38g / L sodium citrate solution.

[0070] After anesthetizing normal SD rats, blood was collected from the abdominal aorta and placed in a 38 g / L sodium citrate anticoagulant blood collection tube (the tube was pre-lubricated with 38 g / L sodium citrate to prevent blood clotting). The blood was centrifuged at 1200 r / min for 10 min, and the supernatant was separated to obtain platelet-rich plasma (PRP). After carefully aspirating the PRP, the remaining blood was centrifuged at 4000 r / min for 10 min, and the supernatant was aspirated to obtain platelet-poor plasma (PPP), which was then used. After platelet counting with PRP, the platelet count was adjusted to 400,000 / μL with PPP. Subsequent experiments used PRP with a platelet count adjusted to 400,000 / μL.

[0071] 3. Preparation of positive reagent and solvent

[0072] Preparation of the positive control solution: Weigh 4.5 mg of ticagrelor powder and add 172.2 μL of dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. Take 4 μL of the stock solution and dilute with 28 μL of DMSO to obtain a 6.25 mM stock solution. Take 4 μL of the stock solution and add 996 μL of platelet-rich plasma (PRP) to obtain a final concentration of 25 μM ticagrelor.

[0073] 4. Platelet aggregation rate detection

[0074] 4.1 Dosing

[0075] Zeroing the test cup: Add 270 μL of PPP, then add 30 μL of physiological saline (without adding a stir bar).

[0076] Blank group test cup: Add 270μL of PRP, then add 30μL of physiological saline (with stir bar).

[0077] Positive drug and test sample group test cups: Add 270 μL of PRP, then add 30 μL of positive drug solution or test sample solution (with stir bar), and incubate in the incubation chamber for 4 min.

[0078] 4.2 Maximum platelet aggregation rate

[0079] Place the sample in a blank test cup and wait for the light flux value to stabilize. Then, add 6 mL of adenosine diphosphate inducer to the bottom of the cup and record the maximum platelet aggregation rate of the blank group within 5 minutes.

[0080] 4.3 Detection of positive reagents and test samples

[0081] Place the platelet sample into the test cup, add 6 μL of adenosine diphosphate inducer to the bottom of the cup, and record the platelet aggregation rate data within 5 minutes. Each group has 3 parallel samples.

[0082] 5. Results

[0083] The results are as follows Figure 6 As shown. The anticoagulant activity was evaluated by the inhibition of ADP-induced platelet aggregation by the total extract and each fraction. The platelet aggregation rates of each sample under ADP induction were as follows: saline 54.84±6.78%, positive control drug -11.35±1.27%, total extract of Jianqu 4.96±6.05%, water fraction 2.37±1.93%, 30% methanol fraction 0.40±0.13%, 50% methanol fraction 3.48±0.33%, and methanol fraction 10.30±4.8%.

[0084] If the inhibition rate of physiological saline against ADP-induced platelet aggregation is considered to be 0, and the inhibition rate of positive control drug against ADP-induced platelet aggregation is considered to be 100%, then the total extract of Jianqu (a traditional Chinese medicine) showed an inhibition rate of 75.36% against ADP-induced platelet aggregation, the aqueous fraction showed an inhibition rate of 79.27%, the 30% methanol fraction showed an inhibition rate of 82.25%, the 50% methanol fraction showed an inhibition rate of 77.59%, and the methanol fraction showed an inhibition rate of 67.29%.

[0085] The results showed that the total extract of Jianqu and its various polar fractions all inhibited ADP-induced platelet aggregation. Among them, the 30% methanol fraction had the strongest inhibitory effect on ADP-induced platelet aggregation (P<0.05), followed by the water fraction, while the 100% methanol fraction had the weakest anticoagulant effect.

[0086] Example 5: Identification of the principal component in the 30% methanol fraction

[0087] 1. Prepare a 30% methanol fractional solution using the sample preparation method described in Example 3.

[0088] 2. Prepare the reference solution using the reference preparation method described in Example 1.

[0089] 3. Using the HPLC detection method described in Example 3, the polar region with the strongest inhibitory effect on ADP-induced platelet aggregation was identified.

[0090] 4. Test results: such as Figure 7 As shown. The main components of the 30% methanol fraction are caffeic acid and hesperidin, and it also contains glycyrrhizin, naringin, neohesperidin and rosmarinic acid.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of Jianqu in the preparation of anticoagulant drugs.

2. The application as described in claim 1, characterized in that, The anticoagulant is used to inhibit platelet aggregation.

3. A method for preparing the aqueous fraction of Jianqu extract, characterized in that, Includes the following steps: Take Jianqu (a type of fermented bamboo), mix and soak it with methanol solution, reflux extract, concentrate under reduced pressure to obtain total Jianqu extract; The total extract of Jianqu was mixed with water, dissolved, filtered, and the filtrate was collected. The filtrate was subjected to chromatographic separation, eluted with water, and the eluent was collected, which is the aqueous fraction of the Jianqu extract.

4. The preparation method according to claim 3, characterized in that, The volume fraction of the methanol solution is 80%; The mass ratio of the methyl methacrylate to the methanol solution is 1:5; The reflux extraction was performed three times, with each extraction lasting 2.5 hours.

5. An aqueous fraction of Jianqu extract obtained by the preparation method according to claim 3 or 4.

6. A method for preparing the 30% methanol fraction of Jianqu extract, characterized in that, Includes the following steps: Take Jianqu (a type of medicinal herb), mix and soak it with an 80% methanol solution, reflux to extract, concentrate under reduced pressure to obtain the total extract of Jianqu; The total extract of Jianqu was mixed with water, dissolved, filtered, and the filtrate was collected. The filtrate was subjected to chromatographic separation, and eluted sequentially with water and a 30% (v / v) methanol solution. The methanol eluent was collected, which is the 30% methanol fraction of the Jianqu extract.

7. A 30% methanol fraction of Jianqu extract obtained by the preparation method according to claim 6.

8. The use of the aqueous fraction of Jianqu extract as described in claim 5, or the 30% methanol fraction of Jianqu extract as described in claim 7, or the total Jianqu extract in the preparation of an anticoagulant drug, characterized in that, The preparation method of the total extract of *Jianqu* includes the following steps: Take Jianqu (a type of medicinal herb), mix and soak it in methanol solution, reflux to extract, and concentrate under reduced pressure to obtain the total extract of Jianqu.

9. The application as described in claim 8, characterized in that, The anticoagulant is used to inhibit platelet aggregation.

10. An anticoagulant drug, characterized in that, The drug uses the aqueous fraction of Jianqu extract as described in claim 5 and / or the 30% methanol fraction of Jianqu extract as described in claim 7 as its main active ingredients.

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