Traditional Chinese medicine composition for treating ischemic diseases of lower limbs and preparation method of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for treating ischemic diseases of lower limbs and also provides a preparation method of the traditional Chinese medicine composition, the synergistic combination effect of the two medicines is exerted, the curative effect of a single medicine is obviously improved, and the anticoagulant effect is far greater than that of the independent administration of caltrop; the sum of the action intensity of two independent administration is achieved, and the in-vitro anticoagulation effect and the effect of improving the ischemic blood flow of the lower limbs can be remarkably improved; blood vessel growth factors are remarkably increased, the activity of creatine kinase and lactic dehydrogenase is reduced, and tissue damage is relieved; glutathione peroxidase is promoted, malondialdehyde and active oxygen are inhibited, and the anti-oxidation effect is achieved; hypoxia and mitochondrial functions can be improved by inhibiting hypoxia-inducible factors (hypoxia-inducible factors)-1 alpha and mitochondrial compounds 3; the anti-inflammatory effect can be achieved by inhibiting CRP, IL-1beta and TNF-alpha; the rat lower limb ischemia can be obviously improved and treated.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine preparations, in particular to a traditional Chinese medicine composition for treating lower limb ischemic diseases, and also provides a preparation method of the medicine. Background Art

[0002] Ischemic diseases are diseases caused by various factors that lead to insufficient blood supply to local tissues or organs, resulting in hypoxia, metabolic disorders, and dysfunction. They can affect multiple organs, including the heart, brain, kidneys, and intestines. Their core mechanisms include vascular stenosis / occlusion, thromboembolism, hemodynamic abnormalities, and increased blood viscosity. They mainly include coronary heart disease, cerebral stroke, and peripheral arterial disease. Peripheral arterial disease is primarily caused by lower limb atherosclerosis. CLI refers to ischemic lower limb disease (CLI) caused by arterial stenosis or occlusion in the corresponding blood supply area of the lower limbs, resulting in ischemia and hypoxia, leading to intermittent claudication, rest pain, ulcers, and gangrene. Currently, traditional surgery or endovascular treatment is the preferred method of clinical treatment, but approximately 30% of patients are still not suitable for surgery or endovascular treatment due to severe complications. The limited time window for thrombolysis and thrombectomy limits the effectiveness of thrombolysis. While anticoagulants reduce the risk of bleeding, there is still a residual risk of ischemic events. Statins, while able to stabilize plaques, do not significantly improve blood flow or alleviate ischemic symptoms. High doses can easily cause liver damage and poor patient compliance, impacting efficacy. Furthermore, the effective time window for fibrin-targeted thrombolytics is extremely short, limiting their clinical applicability. Currently, there is an urgent need for drugs that can effectively improve ischemic symptoms for the treatment of ischemic diseases such as lower limb ischemia.

[0003] Traditional Chinese medicine emphasizes improving local blood circulation through multiple components and targets, while also addressing overall Qi and blood flow to regulate lower limb ischemia. Its primary characteristic is the synergistic effect of multiple components and targets. Therefore, it is crucial to clarify how to rationally integrate these components to achieve a cascade-amplified efficacy. This invention aims to identify drugs that can effectively treat ischemia in the body for the treatment of ischemic diseases such as lower limb ischemia. Summary of the Invention

[0004] The present invention provides a traditional Chinese medicine composition for treating lower limb ischemic diseases. The composition utilizes a combination of extracts of two crude drugs, Tribulus terrestris and Astragalus membranaceus, to prepare a medicine for treating lower limb ischemic diseases through the synergistic effect of the two drugs, specifically and significantly enhancing anticoagulant activity and improving ischemic symptoms.

[0005] The present invention exerts a significant synergistic effect in the treatment of lower limb ischemic diseases. Its mechanism is to reduce ischemic body damage by promoting angiogenesis, anti-oxidative stress, improving energy metabolism, maintaining mitochondrial function and anti-inflammatory effects, thereby improving ischemic symptoms and achieving therapeutic effects.

[0006] The Chinese medicine composition for treating lower limb ischemic disease of the present invention is implemented by the following technical solution: The traditional Chinese medicine composition is prepared by uniformly mixing the alcohol extract or water extract of the crude drug Tribulus terrestris and the crude drug Astragalus membranaceus at a mass ratio of 1-10:1-10.

[0007] Among them, the optimal ratio of Tribulus terrestris and Astragalus membranaceus is 2~10:2~10; The most preferred mass ratio of Tribulus terrestris and Astragalus membranaceus is 10:7; 1. The tribulus terrestris alcohol extract of the present invention is extracted by the following steps: Step 1: Weigh the amount of Tribulus terrestris and flatten it; Step 2: Add 8 times the amount of 50% ethanol, soak for 16 hours, heat under reflux for 2 hours, and filter to collect the filtrate; Step 3: Add 6 times the amount of 50% ethanol to the residue in step 2, heat and reflux for 2 hours, and filter and collect the filtrate; Step 4: Add 6 times the amount of 50% ethanol to the residue in step 3, heat and reflux for 2 hours, and filter and collect the filtrate; Step 5: Combine the filtrates collected in steps 2, 3 and 4, concentrate and dry to obtain the tribulus terrestris alcohol extract.

[0008] 2. The astragalus alcohol extract is extracted by the following steps: Step 1: Weigh the amount of Astragalus slices in the formula and add 8 times the amount of 70% ethanol, soak for 16 hours, heat under reflux for 1.5 hours, and filter to collect the filtrate; Step 2: Add 6 times the amount of 70% ethanol to the residue in step 1, heat and reflux for 1.5 hours, and filter and collect the filtrate; Step 3: Add 6 times the amount of 70% ethanol to the residue in step 2, heat and reflux for 1.5 hours, and filter and collect the filtrate; Step 4: Combine the filtrates collected in steps 1, 2 and 3, concentrate and dry to obtain the astragalus alcohol extract.

[0009] 3. The water extract of Tribulus terrestris of the present invention is extracted by the following steps: Step 1: Weigh the amount of Tribulus terrestris and flatten it; Step 2: Add 8 times the amount of water, soak for 16 hours, boil for 2 hours, and filter to collect the filtrate; Step 3: Add 6 times the amount of water to the residue from step 2, boil for 2 hours, and filter to collect the filtrate; Step 4: Combine the filtrates collected in steps 2 and 3, concentrate and dry to obtain the water extract of Tribulus terrestris.

[0010] 4. The astragalus water extract is extracted by the following steps: Step 1: Weigh the amount of Astragalus slices in the formula, add 8 times the amount of water, soak for 16 hours, decoct for 1.5 hours, and filter to collect the filtrate; Step 2: Add 6 times the amount of water to the residue in step 1, boil for 1.5 hours, and filter to collect the filtrate; Step 3: Combine the filtrates collected in steps 1 and 2, concentrate and dry to obtain the astragalus water extract.

[0011] 5. The crude drug of Tribulus terrestris prepared in the above steps and the crude drug alcohol extract or water extract of Astragalus membranaceus are uniformly mixed according to the crude drug ratio to obtain the Chinese medicine composition of the present invention.

[0012] The yield of the Tribulus terrestris extract is 5-15% of the raw medicinal slices, and the yield of the Astragalus membranaceus extract is 25-40% of the raw medicinal slices.

[0013] The Chinese medicine composition of the present invention can significantly increase anticoagulant activity in vitro; in vivo, it can reduce damage to ischemic sites by promoting angiogenesis, resisting oxidative stress, improving energy metabolism, and maintaining mitochondrial function, thereby improving ischemic symptoms and treating ischemic diseases.

[0014] As a preferred embodiment of the present invention, the Chinese medicine composition is prepared by respectively extracting the crude drug Tribulus terrestris and the crude drug Astragalus membranaceus in a mass ratio of 10:7 and uniformly mixing the extracts. Alternatively, the two crude drugs can be extracted simultaneously to obtain a mixed extract.

[0015] The positive effects of the present invention are:

[0016] The organic combination of Tribulus terrestris extract and Astragalus membranaceus extract can bring into play the synergistic effect of the two drugs, significantly improving the efficacy of the single drugs. The anticoagulant effect is much greater than the anticoagulant effect of Tribulus terrestris alone, reaching 1.38-6.14 times the sum of the two single-dose effects. It can significantly increase the in vitro anticoagulant effect and improve the blood flow effect of lower limb ischemia (see Experimental Examples 1 and 3); significantly increase the activity of vascular endothelial growth factor (VEGF), reduce the activity of creatine kinase (CK) and lactate dehydrogenase (LDH), and reduce tissue damage; by promoting glutathione Glycines peroxidase (GPX) inhibits malondialdehyde (MDA) and reactive oxygen species (ROS) to play an antioxidant role; it can improve hypoxia and mitochondrial function by inhibiting the hypoxia-inducible factor hypoxia-inducible factor-1α (HIF-1α) and mitochondrial complex 3; it can exert an anti-inflammatory effect by inhibiting CRP, IL-1β and TNF-α; the efficacy of different doses of the composition in regulating these indicators is greater than the sum of the efficacy of the two alone, reaching 1.16-5.66 times the sum of the two individual administration strengths, indicating that the composition can significantly improve and treat lower limb ischemia in rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The in vitro anticoagulation time of Tribulus terrestris combined with different proportions of Astragalus membranaceus (A: Tribulus terrestris (raw drug dosage 240 mg / ml) combined with different proportions of Astragalus membranaceus; B: Tribulus terrestris (raw drug dosage 320 mg / ml) combined with different proportions of Astragalus membranaceus); Figure 2 The anti-thrombotic effect of Tribulus terrestris combined with different proportions of Astragalus membranaceus in vivo; Figure 3 The effect of HJ composition on blood flow and VEGF in ischemic area of rats with lower limb ischemia; Figure 4 Effects of the HJ composition on CK in rats with lower limb ischemia (A: CK test values of each group, B: sum of individual pharmacodynamics and the pharmacodynamics of the HJ composition, where the pharmacodynamics is the difference between the test values of the model group and the drug-treated group, and the sum of individual pharmacodynamics is the sum of the pharmacodynamics of Tribulus terrestris and Astragalus membranaceus alone); Figure 5 Effects of the HJ composition on LDH in rats with lower limb ischemia (A: LDH test values of each group, B: sum of individual efficacy and efficacy of the HJ composition, where the efficacy is the difference between the test values of the model group and the drug-treated group, and the sum of individual efficacy is the sum of the efficacy of Tribulus terrestris alone and Astragalus alone); Figure 6 Effects of the HJ composition on GPX in rats with lower limb ischemia (A: GPX test values of each group, B: sum of individual efficacy and efficacy of the HJ composition, where the efficacy is the difference between the test values of the model group and the drug-treated group, and the sum of individual efficacy is the sum of the efficacy of Tribulus terrestris alone and Astragalus alone); Figure 7 Effects of the HJ composition on MDA in rats with lower limb ischemia (A: MDA test values of each group, B: sum of individual efficacy and efficacy of the HJ composition, where the efficacy is the difference between the test values of the model group and the drug-treated group, and the sum of individual efficacy is the sum of the efficacy of Tribulus terrestris alone and Astragalus alone); Figure 8 Effects of the HJ composition on ROS in rats with lower limb ischemia (A: ROS detection value of each group, B: The sum of the individual pharmacodynamics and the efficacy of the HJ composition, where the pharmacodynamics is the difference between the detection values of the model group and the drug-treated group, and the sum of the individual pharmacodynamics is the sum of the pharmacodynamics of Tribulus terrestris and Astragalus membranaceus alone); Figure 9 Effects of the HJ composition on HIF-1α in rats with lower limb ischemia (A: HIF-1α detection value of each group, B: the sum of the individual efficacy and the efficacy of the HJ composition, where the efficacy is the difference between the detection values of the model group and the drug-treated group, and the sum of the individual efficacy is the sum of the efficacy of Tribulus terrestris alone and Astragalus alone); Figure 10Effects of the HJ composition on mitochondrial complex 3 in rats with lower limb ischemia (A: detection value of mitochondrial complex 3 in each group, B: sum of individual efficacy and efficacy of the HJ composition, where the efficacy is the difference between the detection values of the model group and the drug-treated group, and the sum of individual efficacy is the sum of the efficacy of Tribulus terrestris alone and Astragalus alone); Figure 11 The effect of the optimal ratio of HJ composition on CRP in rats with lower limb ischemia; Figure 12 The effect of the optimal ratio of HJ combination on IL-1β in rats with lower limb ischemia; Figure 13 The effect of the optimal ratio composition of HJ on TNF-α in rats with lower limb ischemia. DETAILED DESCRIPTION

[0018] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention. Example 1

[0019] The present invention provides a traditional Chinese medicine composition for treating ischemic diseases. The traditional Chinese medicine composition is prepared by respectively extracting Tribulus terrestris and Astragalus membranaceus, and uniformly mixing the extracts at a mass ratio of 1-10:1-10 between the crude drug Tribulus terrestris and the crude drug Astragalus membranaceus, wherein: The tribulus terrestris alcohol extract is extracted by the following steps: Step 1: Weigh 1500 g of flattened Tribulus terrestris; Step 2: Add 8 times the amount of 50% ethanol, soak for 16 hours, heat under reflux for 2 hours, and filter to collect the filtrate; Step 3: Add 6 times the amount of 50% ethanol to the residue in step 2, heat and reflux for 2 hours, and filter and collect the filtrate; Step 4: Add 6 times the amount of 50% ethanol to the residue in step 3, heat and reflux for 2 hours, and filter and collect the filtrate; Step 5: Combine the filtrates collected in steps 2, 3 and 4, concentrate and dry to obtain the tribulus terrestris alcohol extract.

[0020] The astragalus alcohol extract is extracted by the following steps: Step 1: Weigh 1800 g of Astragalus slices, add 8 times the amount of 70% ethanol, soak for 16 hours, heat under reflux for 1.5 hours, and filter to collect the filtrate; Step 2: Add 6 times the amount of 70% ethanol to the residue in step 1, heat and reflux for 1.5 hours, and filter and collect the filtrate; Step 3: Add 6 times the amount of 70% ethanol to the residue in step 2, heat and reflux for 1.5 hours, and filter and collect the filtrate; Step 4: combining the filtrates collected in steps 1, 2, and 3, concentrating and drying to obtain an astragalus alcohol extract; The water extract of Tribulus terrestris is extracted by the following steps: Step 1: Weigh 1500 g of flattened Tribulus terrestris; Step 2: Add 8 times the amount of water, soak for 16 hours, boil for 2 hours, and filter to collect the filtrate; Step 3: Add 6 times the amount of water to the residue from step 2, boil for 2 hours, and filter to collect the filtrate; Step 4: Combine the filtrates collected in steps 2 and 3, concentrate and dry to obtain the water extract of Tribulus terrestris.

[0021] The astragalus water extract is extracted by the following steps: Step 1: Weigh 1800 g of Astragalus slices and add 8 times the amount of water, soak for 16 hours, decoct for 1.5 hours, and filter to collect the filtrate; Step 2: Add 6 times the amount of water to the residue in step 1, boil for 1.5 hours, and filter to collect the filtrate; Step 3, combining the filtrates collected in steps 1 and 2, concentrating and drying to obtain an astragalus water extract; The yield of the Tribulus terrestris extract is 5-15% of the raw medicinal slices, and the yield of the Astragalus membranaceus extract is 25-40% of the raw medicinal slices. Example 2

[0022] The steps for preparing a Chinese medicine composition by combining the crude drug of Tribulus terrestris at a concentration of 320 mg / ml and the crude drug of Astragalus membranaceus at a ratio of 1:0.5, 0.6, 0.7, 0.8, 0.9, 1 are as follows: Step 1: Weigh 6 portions of 96 mg of the Tribulus terrestris extract from Example 1, equivalent to 640 mg of the crude drug Tribulus terrestris, and place them in 6 2 mL volumetric flasks respectively; Step 2, weighing 124.8, 149.76, 174.72, 199.68, 224.64, and 249.6 mg of the astragalus extract of Example 1, equivalent to 320, 384, 448, 512, 576, and 640 mg of the astragalus crude drug, respectively; Step 3, adding different masses of the astragalus extracts weighed in step 2 into the six volumetric flasks in step 1 respectively; Step 4, add pure water to the volumetric flask of step 3, dilute to 2mL, mix evenly to obtain a compatibility drug with a concentration of 320mg / ml of Tribulus terrestris and a ratio of 1:0.5, 0.6, 0.7, 0.8, 0.9, 1; The steps for preparing the medicine are as follows: Step 1: Weigh 6 portions of 72 mg of the Tribulus terrestris extract from Example 1 (equivalent to 480 mg of the crude drug Tribulus terrestris) and place them in 6 2 mL volumetric flasks respectively; Step 2, weighing 93.6, 112.32, 131.04, 149.76, 168.48, and 187.2 mg of the astragalus extract of Example 1, equivalent to 240, 288, 336, 384, 432, and 480 mg of the astragalus raw drug, respectively; Step 3, adding different masses of the astragalus extracts weighed in step 2 into the six volumetric flasks in step 1 respectively; Step 4, add pure water to the volumetric flask of step 3, dilute to 2 mL, mix evenly to obtain a compatibility drug with a concentration of 240 mg / ml of Tribulus terrestris and a ratio of 1:0.5, 0.6, 0.7, 0.8, 0.9, 1 of Astragalus membranaceus. Example 3

[0023] The dosage for mice was 200 mg / kg of Tribulus terrestris extract (raw drug amount 1.34 g / kg) and the raw drug of Astragalus membranaceus at a ratio of 1:0.5, 0.6, and 0.7. The steps for preparing the Chinese medicine composition were as follows: Step 1: Weigh 2 g of the Tribulus terrestris extract from Example 1, equivalent to 13.34 g of the Tribulus terrestris crude drug, into three 100 ml graduated cylinders respectively; Step 2: Weigh 2.60, 3.12, and 3.64 g of the Astragalus extract from Example 1, respectively, equivalent to 6.67, 8.00, and 9.34 g of the Astragalus crude drug; Step 3, adding different masses of the astragalus extract weighed in step 2 into the three measuring cylinders in step 1 respectively; Step 4: Add pure water to the graduated cylinder of step 3 to the 100 mL mark, mix well, and obtain a dosage for mice of 200 mg / kg of Tribulus terrestris extract (crude drug amount 1.34 g / kg) and 1:0.5, 0.6, and 0.7 of Astragalus membranaceus crude drug. Example 4

[0024] The steps for preparing high, medium and low doses of Tribulus terrestris and Astragalus membranaceus combination (HJ) are as follows: 36 g of the Tribulus terrestris extract and 28.08 g of the Astragalus membranaceus extract (240 g of the Tribulus terrestris crude drug) of Example 1 and 72 g of the Astragalus membranaceus crude drug were weighed respectively, placed in a 1000 mL graduated cylinder, and pure water was added to the 900 mL mark. The mixture was ultrasonically mixed to obtain the HJ high-dose drug. Weigh 27 g of the Tribulus terrestris extract and 21.06 g of the Astragalus membranaceus extract (180 g of the Tribulus terrestris crude drug) in Example 1 and 54 g of the Astragalus membranaceus crude drug in a 1000 mL graduated cylinder. Add pure water to the 900 mL mark and mix thoroughly by ultrasonic mixing to obtain the HJ medium-dose drug. 18 g of the Tribulus terrestris extract and 14.04 g of the Astragalus membranaceus extract (120 g of the Tribulus terrestris crude drug) were weighed in a 1000 mL graduated cylinder. Pure water was added to the 900 mL mark, and the mixture was ultrasonically mixed to obtain a low-dose HJ drug. Example 5

[0025] The steps for preparing the high, medium and low doses of the optimal ratio combination of Tribulus terrestris and Astragalus membranaceus (HJ) are as follows: 25.04 g of the Tribulus terrestris extract and 45.10 g of the Astragalus membranaceus extract (166 g of the Tribulus terrestris crude drug) of Example 1 were weighed and placed in a 1000 mL graduated cylinder. Pure water was added to the 600 mL mark and the mixture was ultrasonically mixed to obtain the HJ high-dose drug. Weigh 12.52 g of the Tribulus terrestris extract and 22.55 g of the Astragalus membranaceus extract (83.0 g of the Tribulus terrestris crude drug) in Example 1 and 58.1 g of the Astragalus membranaceus crude drug in a 1000 mL graduated cylinder. Add pure water to the 600 mL mark and mix thoroughly by ultrasonication to obtain the HJ medium-dose drug. 6.26 g of the Tribulus terrestris extract and 11.27 g of the Astragalus membranaceus extract (41 g of the Tribulus terrestris crude drug) of Example 1 were weighed and placed in a 1000 mL graduated cylinder. Pure water was added to the 600 mL mark, and ultrasonic mixing was performed to obtain the HJ low-dose drug.

[0026] The synergistic effect and therapeutic effect of the present invention are demonstrated by the following experimental examples: Experimental Example 1

[0027] In vitro coagulation assays provide key insights into the treatment and clinical management of lower limb ischemia by assessing coagulation factor activity, fibrin formation, and anticoagulant function. The thrombin time (TT) is an important indicator for evaluating fibrinogen function and the presence of anticoagulants. A prolonged TT indicates a decrease in plasma fibrinogen concentration or activity, which may be due to factors such as fibrinogen deficiency or deficiencies in coagulation factors II, V, VII, and X. In vitro assessment of drug effects on the TT is an important method for evaluating their efficacy in lower limb ischemia. This method offers the advantage of rapid screening for drug effects on the terminal stages of coagulation, making it suitable for high-throughput screening of anticoagulant drugs. This study evaluated the anticoagulant efficacy of Tribulus terrestris, Astragalus membranaceus, and their combination by in vitro testing of the TT, thereby assessing the efficacy of this traditional Chinese medicine formula in treating lower limb ischemia.

[0028] 1. Experimental Materials 1.1 Experimental Animals One SD male rat, weighing 180–220 g, SPF grade, was purchased from Liaoning Changsheng Biotechnology Co., Ltd. The laboratory animal quality certificate number is 210726250100317075, and the laboratory animal production license number is SCXK (Liaoning) 2020-0001.

[0029] 1.2 Drugs and reagents Tribulus terrestris extract: Jilin Academy of Traditional Chinese Medicine (Batch No.: XSJ202501091); Astragalus extract: Jilin Academy of Traditional Chinese Medicine (Batch No.: XSH202501091); Heparin: Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Batch No.: obc10650); Thrombin time kit, Shanghai Sun Biotechnology Co., Ltd. (Batch No.: 122353).

[0030] 2 Experimental methods 2.1 Sample ratio design In vitro anticoagulation experiments were performed with a crude drug concentration of 320 mg / ml of Tribulus terrestris and a crude drug ratio of 1:0.5, 0.6, 0.7, 0.8, 0.9, and 1 of Astragalus membranaceus, and a crude drug concentration of 240 mg / ml of Tribulus terrestris and a crude drug ratio of 1:0.5, 0.6, 0.7, 0.8, 0.9, and 1 of Astragalus membranaceus (Example 2). The anticoagulation effects of Tribulus terrestris alone, Astragalus membranaceus alone, and a combination of Tribulus terrestris and Astragalus membranaceus at the same concentration were also investigated.

[0031] 2.2 TT measurement Rats were anesthetized with 1.25% tribromoethanol injected intraperitoneally. The abdominal skin and muscles were cut open, and the abdominal aorta was isolated. Blood was collected from the abdominal aorta with a needle. After collection, the tube (containing sodium citrate anticoagulant) was gently inverted three times to mix thoroughly. The tube was centrifuged at 3500 rpm for 10 minutes, and the upper plasma layer was collected for later use. A blank control group received normal saline. For the other groups, 100 μl of each sample was added to a coagulation test cup. 100 μl of plasma was added and mixed for 3 minutes. Then, 100 μl of TT reagent was added and the test was started simultaneously. Three replicates were performed.

[0032] 3 Experimental results The experimental results show that the corresponding compatibility ratio of Astragalus alone has no anticoagulant effect. Although Tribulus terrestris can prolong the coagulation time, its anticoagulant effect is far inferior to the combination of the two. Figure 1 As shown, the anticoagulant effects of the compositions of Tribulus terrestris crude drug concentrations of 240 and 320 mg / ml combined with Astragalus membranaceus crude drug ratios of 1:0.5, 0.6, 0.7, 0.8, 0.9, 1 were far greater than the anticoagulant effect of Tribulus terrestris administered alone, reaching 1.38-6.14 times the sum of the two individual administration intensities, indicating that Astragalus membranaceus can significantly enhance the anticoagulant effect of Tribulus terrestris, and the combination of the two Chinese medicines can significantly increase the anticoagulant activity.

[0033] Experimental Example 2: Effect of the composition on collagen-epinephrine-induced thrombosis in mice

[0034] Thrombosis is a key pathological mechanism of lower limb ischemia, primarily caused by vascular endothelial damage, blood stasis, and a hypercoagulable state. Collagen-epinephrine combined induction is a commonly used method for mouse thrombosis models, simulating the pathological process of lower limb ischemia by synergistically activating platelets and vasoconstriction. This experiment induced thrombosis in mice using collagen-epinephrine after administering Tribulus terrestris, Astragalus membranaceus, and a combination of the two. The anti-thrombotic effect of the traditional Chinese medicine formula was evaluated by observing the number of mice that died or experienced hemiplegia that did not recover within 15 minutes, thereby evaluating the efficacy of the formula against lower limb ischemia.

[0035] 1. Experimental Materials 1.1 Experimental animals 108 KM male mice weighing 17–20 g, SPF grade, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. Laboratory Animal Quality Certificate No.: 210726240101928316, Laboratory Animal Production License No.: SCXK(Liao)2020-0001.

[0036] 1.2 Drugs and reagents Drugs: Dengzhan Shengmai Capsules (National Medical Approval No.: HJ20170088), Tribulus Terrestris Extract (Batch No.: J20240727), and Astragalus Extract (Batch No.: H20240727). All extracts were produced in-house by the Jilin Academy of Traditional Chinese Medicine. Collagen: Solebo (Cat. No. C8062), epinephrine (Batch No.: 221208).

[0037] 2 Experimental methods 2.1 Experimental Grouping 108 KM male mice weighing 17-20 g were randomly divided into 9 groups, with 12 mice in each group, namely the control group, the positive drug group (Dengzhan Shengmai group), the Tribulus terrestris single group, three Tribulus terrestris and Astragalus herbal drug compatibility groups (1:0.5, 1:0.6 and 1:0.7) and three Astragalus herbal drug single groups with corresponding Astragalus herbal drug concentrations. The Tribulus terrestris dosage was 200 mg / kg of extract, and the Astragalus herbal drug dosage was converted by the crude drug ratio (Example 3).

[0038] 2.2 Induction of thrombosis Each group of animals was gavage-administered once daily (dose of 0.2 ml / 10 g) for 14 days according to the grouping. One hour after the last administration, a mixed inducer of collagen (225 μg / mouse) and epinephrine (9 μg / mouse) was injected through the tail vein. The number of mice that died or did not recover from hemiplegia within 15 minutes after the injection was observed.

[0039] 3. Experimental Results The experimental results showed that the combination of Tribulus terrestris and Astragalus membranaceus could significantly inhibit the collagen-adrenaline-induced thrombosis in mice ( Figure 2 ), compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone, the antithrombotic effect of the combination in different proportions was greater than the sum of the efficacy of the two alone, reaching 2.51-3.33 times the sum of the efficacy of the two alone ( Figure 2 ). This indicates that the combination of Tribulus terrestris and Astragalus membranaceus can significantly enhance the anti-thrombotic efficacy compared with single administration.

[0040] Experimental Example 3: Therapeutic effect of the composition on rats with lower limb ischemia

[0041] The lower limb ischemia rat model is an animal model for evaluating the primary efficacy of drugs for lower limb ischemia. It simulates the pathological state of human lower limb ischemia by surgically ligating the femoral artery or inducing thromboembolism. This model allows for systematic evaluation of drug effects on lower limb ischemia blood flow recovery and collateral angiogenesis, as well as monitoring drug efficacy in response to vascular endothelial growth factor (VEGF), muscle tissue damage (CK, LDH), hypoxia response (HIF-1α), and oxidative stress (GPX, MDA, and ROS). This model provides key experimental evidence for the mechanism research and clinical translation of Traditional Chinese Medicine (TCM) compounds.

[0042] 1. Experimental Materials 1.1 Experimental Animals Eighty male SD rats, weighing 180–200 g, SPF grade, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The laboratory animal quality certificate number is 210726250100317075, and the laboratory animal production license number is SCXK (Liaoning) 2020-0001.

[0043] 1.2 Drugs and reagents Dengzhan Shengmai Capsule: Yunnan Biovalley Pharmaceutical Co., Ltd.; Tribulus terrestris extract: Jilin Academy of Traditional Chinese Medicine (Batch No.: XSJ202501091); Astragalus extract: Jilin Academy of Traditional Chinese Medicine (Batch No.: XSH202501091); Tribromoethanol: Nanjing Aibei Biotechnology Co., Ltd.

[0044] 2 Experimental methods 2.1 Experimental Grouping Eighty male SD rats were randomly divided into eight groups of ten rats each: a control group, a model group, a positive drug Dengzhan Shengmai Capsule group (Dengzhan Shengmai group), a medium-dose Tribulus terrestris group, a medium-dose Astragalus membranaceus group, and high-, medium-, and low-dose HJ groups (Example 4). The oral administration materials and the doses of the Tribulus terrestris and Astragalus membranaceus combinations are shown in Table 1.

[0045]

[0046] 2.2 Modeling, drug administration, and blood flow detection The model was established by anesthetizing rats with an intraperitoneal injection of 1.25% tribromoethanol. The rats were then immobilized in the supine position. A 1.5 cm transverse incision was made on the lateral abdomen and the surface of the inguinal ligament. The arteries were ligated at both ends, severed, and their branches removed. The skin was then sutured. Laser speckle flowmetry was used to monitor blood flow in the lower limb muscles and foot. Significant reduction in blood flow was considered a successful model. Animals with intraoperative vascular rupture or those failing this criterion were excluded. Rats were then placed on a 37°C electric blanket until awake. In the control group, only the femoral artery was exposed, without ligating the artery and its branches. Postoperatively, iodine solution was sprayed on the suture site. Drugs were administered orally starting 1 hour before surgery on the first day and continued for 14 consecutive days. After 14 days of administration, blood flow was measured using a laser speckle flowmetry device, and blood was collected from the abdominal aorta to measure VEGF, CK, LDH, HIF-1α, GPX, MDA, and ROS.

[0047] 3. Experimental results: 3.1 Establishment of rat lower limb ischemia model After the modeling was completed, the success of the modeling was judged based on the blood flow ratio of the lower limb muscles and feet before and after the modeling. The experimental results are shown in Table 2. Compared with before the modeling, the blood flow after the modeling was significantly decreased, indicating that the lower limb ischemia model was successfully established.

[0048]

[0049] 3.2 Effects on blood flow and VEGF in ischemic areas of rats with lower limb ischemia The experimental results showed that compared with the model group, Tribulus terrestris and Astragalus membranaceus alone had no effect on the blood flow of rats with lower limb ischemia, while the HJ combination could significantly increase the blood flow of the hind limbs of rats with lower limb ischemia ( Figure 3 A); Compared with the model group, Tribulus terrestris and Astragalus membranaceus alone had no effect on the VEGF content in the gastrocnemius muscle of rats with lower limb ischemia, while the HJ combination could significantly increase the VEGF content in the gastrocnemius muscle of rats with lower limb ischemia ( Figure 3 B), indicating that the composition can synergistically treat rats with lower limb ischemia.

[0050] 3.3 Effects on CK in rats with lower limb ischemia The experimental results showed that compared with the model group, Tribulus terrestris and Astragalus membranaceus alone had no effect on the CK activity in the serum of rats with lower limb ischemia, while the HJ combination could significantly reduce the CK activity in the serum of rats with lower limb ischemia ( Figure 4 A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone (the sum of the difference between the test value of the model group and the test value of the Tribulus terrestris and Astragalus membranaceus alone), the efficacy of the combination at different doses in reducing CK activity (the difference between the test value of the model group and the test value of the combination at different doses) was greater than the sum of the efficacy of the two drugs alone, and reached 3.23-5.66 times the sum of the two individual efficacy ( Figure 4 B).

[0051] 3.4 Effects on LDH in rats with lower limb ischemia The experimental results showed that compared with the model group, Tribulus terrestris and Astragalus membranaceus alone had no effect on the LDH activity in the serum of rats with lower limb ischemia, while the HJ combination could significantly reduce the LDH activity in the serum of rats with lower limb ischemia ( Figure 5 A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone (same as 3.3), the efficacy of the combination at different doses in reducing LDH activity was greater than the sum of the efficacy of the two alone, reaching 1.51-2.46 times the sum of the efficacy of the two alone ( Figure 5 B).

[0052] 3.5 Effects of oxidative stress on rats with lower limb ischemia The experimental results showed that Tribulus terrestris and Astragalus membranaceus alone had no effect on GPX in the serum of rats with lower limb ischemia, while the HJ combination could significantly increase the activity of GPX in the serum of rats with lower limb ischemia ( Figure 6 A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone, the efficacy of the combination at different doses in increasing GPX activity was greater than the sum of the efficacy of the two alone, reaching 1.16-2.14 times the sum of the efficacy of the two alone ( Figure 6 B).

[0053] The experimental results showed that Tribulus terrestris and Astragalus membranaceus alone had no effect on the MDA content in the serum of rats with lower limb ischemia, while the HJ combination could significantly reduce the MDA content in the serum of rats with lower limb ischemia ( Figure 7 A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone, the efficacy of the combination at different doses in reducing MDA content was greater than the sum of the efficacy of the two alone, reaching 2.02-2.35 times the sum of the efficacy of the two alone ( Figure 7 B).

[0054] The experimental results showed that the HJ composition could significantly reduce the ROS content in the serum of rats with lower limb ischemia ( Figure 8 A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone, the efficacy of the combination at different doses in reducing ROS content was greater than the sum of the efficacy of the two alone, reaching 1.19 times the sum of the efficacy of the two alone ( Figure 8 B).

[0055] Effects of 3.6 on HIF-1α and mitochondrial complex 3 in rats with lower limb ischemia The experimental results showed that Astragalus alone had no effect on HIF-1α in rats with lower limb ischemia, and Tribulus terrestris alone had no significant effect. The HJ combination could significantly reduce the HIF-1α content in rats with lower limb ischemia ( Figure 9A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone, the efficacy of the combination at different doses in reducing HIF-1α content was greater than the sum of the efficacy of the two alone, reaching 1.55-2.65 times the sum of the efficacy of the two alone ( Figure 9 B).

[0056] The experimental results showed that Astragalus alone had no effect on the activity of mitochondrial complex 3 in rats with lower limb ischemia, and Tribulus terrestris alone had no obvious effect. The HJ combination could significantly reduce the activity of mitochondrial complex 3 in rats with lower limb ischemia ( Figure 10 A); Compared with the sum of the efficacy of Tribulus terrestris and Astragalus membranaceus alone, the efficacy of the combination at different doses in reducing mitochondrial complex 3 activity was greater than the sum of the efficacy of the two alone, reaching 1.18-2.2 times the sum of the efficacy of the two alone ( Figure 10 B).

[0057] Experimental Example 4: Therapeutic effect of the optimal ratio composition on rats with lower limb ischemia

[0058] 1. Experimental Materials 1.1 Experimental Animals Sixty male Sprague-Dawley rats, weighing 180–200 g, SPF grade. Laboratory Animal Quality Certificate: 110324251102450351, Production License Number: SCXK(Beijing)2024-0001. Animals were purchased from Spefox (Beijing) Biotechnology Co., Ltd., quality tested by Suzhou Xishan Biotechnology Co., Ltd., Laboratory Unit Use License Number: SYXK(Ji)2022-0004.

[0059] 1.2 Drugs and reagents Dengzhan Shengmai Capsule: Yunnan Biovalley Pharmaceutical Co., Ltd.; Tribulus terrestris extract: Jilin Academy of Traditional Chinese Medicine (Batch No.: XSJ202501091); Astragalus extract: Jilin Academy of Traditional Chinese Medicine (Batch No.: XSH202501091); Tribromoethanol: Nanjing Aibei Biotechnology Co., Ltd.

[0060] 2 Experimental methods 2.1 Experimental Grouping Sixty male SD rats were randomly divided into six groups of 10 rats each: a control group, a model group, a positive drug Dengzhan Shengmai Capsule group (Dengzhan Shengmai group), and HJ optimal ratio high, medium, and low dose groups (Example 5). The rat gavage material and the dose of Tribulus terrestris combined with Astragalus are shown in Table 3.

[0061]

[0062] 2.2 Modeling, drug administration, and blood flow detection The model was established by anesthetizing rats with an intraperitoneal injection of 1.25% tribromoethanol. The rats were then immobilized in the supine position. A 1.5 cm transverse incision was made on the lateral abdomen and surface of the inguinal ligament. The arterial vessels were ligated at both ends, severed, and their branches removed. The skin was then sutured. Laser speckle blood flowmetry was used to monitor blood flow in the lower limb muscles and foot. Significant reduction in blood flow indicated successful model establishment. Animals with intraoperative vascular rupture or those failing this criterion were excluded. Rats were then placed on a 37°C electric blanket until awake. In the control group, only the femoral artery was exposed, without ligating the artery and its branches. Postoperatively, the suture sites of the rats were sprayed with iodine solution. Drugs were administered orally starting 1 hour before surgery and continued for 14 consecutive days. After 14 days of administration, inflammatory markers such as CRP, IL-1β, and TNF-α in the gastrocnemius muscle were measured.

[0063] 3. Experimental results: 3.1 Establishment of rat lower limb ischemia model After the modeling was completed, the success of the modeling was judged based on the blood flow ratio of the lower limb muscles and feet before and after the modeling. The experimental results are shown in Table 4. Compared with before the modeling, the blood flow after the modeling was significantly decreased, indicating that the lower limb ischemia model was successfully established.

[0064]

[0065] 3.2 Effects on CRP in rats with lower limb ischemia The experimental results showed that compared with the control group, the CRP content in the gastrocnemius muscle of the model group rats increased significantly, and the HJ optimal ratio composition could significantly reduce the CRP content in the gastrocnemius muscle of the rats with lower limb ischemia in a dose-dependent manner ( Figure 11 ), indicating that the optimal ratio of HJ composition can alleviate the inflammatory response in rats with lower limb ischemia and play a therapeutic role.

[0066] 3.3 Effects of IL-1β on rats with lower limb ischemia The experimental results showed that compared with the control group, the IL-1β content in the gastrocnemius muscle of the model group rats increased significantly, and the HJ optimal ratio composition could significantly reduce the IL-1β content in the gastrocnemius muscle of the rats with lower limb ischemia ( Figure 12 ), indicating that the optimal ratio of HJ composition reduces the IL-1β content in the gastrocnemius muscle of rats with lower limb ischemia and exerts an anti-inflammatory effect.

[0067] 3.4 Effects of TNF-α on rats with lower limb ischemia The experimental results showed that compared with the control group, the TNF-α content in the gastrocnemius muscle of the model group rats increased significantly, and the HJ optimal ratio composition could significantly reduce the TNF-α content in the gastrocnemius muscle of the rats with lower limb ischemia in a dose-dependent manner ( Figure 13 ), indicating that the optimal ratio of HJ composition reduces the TNF-α content in the gastrocnemius muscle of rats with lower limb ischemia and exerts an anti-inflammatory effect.

Claims

1. A Chinese medicine composition for treating lower limb ischemic disease, characterized in that: The traditional Chinese medicine composition is prepared by uniformly mixing the crude drug Tribulus terrestris and the crude drug alcohol extract or water extract of Astragalus membranaceus at a mass ratio of 1-10:1-10.

2. A Chinese medicine composition for treating lower limb ischemic disease according to claim 1, characterized in that: The preferred mass ratio of the crude drug of Tribulus terrestris to the crude drug of Astragalus membranaceus is 2-10:2-10.

3. A Chinese medicine composition for treating lower limb ischemic disease according to claim 1, characterized in that: The most preferred mass ratio of the crude drug of Tribulus terrestris to the crude drug of Astragalus membranaceus is 10:

7.

4. A method for preparing a Chinese medicine composition for treating lower limb ischemic disease as claimed in claim 1, 2 or 3, comprising the following steps: 1) The tribulus terrestris alcohol extract is extracted by the following steps: Step 1: Weigh the amount of Tribulus terrestris and flatten it; Step 2: Add 8 times the amount of 50% ethanol, soak for 16 hours, heat under reflux for 2 hours, and filter to collect the filtrate; Step 3: Add 6 times the amount of 50% ethanol to the residue in step 2, heat and reflux for 2 hours, and filter and collect the filtrate; Step 4: Add 6 times the amount of 50% ethanol to the residue in step 3, heat and reflux for 2 hours, and filter and collect the filtrate; Step 5, combining the filtrates collected in steps 2, 3, and 4, concentrating and drying to obtain a tribulus terrestris alcohol extract; 2) The astragalus alcohol extract is extracted by the following steps: Step 1: Weigh the amount of Astragalus slices in the formula, add 8 times the amount of 70% ethanol, soak for 16 hours, heat under reflux for 1.5 hours, and filter to collect the filtrate; Step 2: Add 6 times the amount of 70% ethanol to the residue in step 1, heat and reflux for 1.5 hours, and filter and collect the filtrate; Step 3: Add 6 times the amount of 70% ethanol to the residue in step 2, heat and reflux for 1.5 hours, and filter and collect the filtrate; Step 4: combining the filtrates collected in steps 1, 2, and 3, concentrating and drying to obtain an astragalus alcohol extract; 3) The Tribulus terrestris crude drug and the Astragalus crude drug extract prepared in the above steps are uniformly mixed according to the crude drug ratio.

5. A method for preparing a Chinese medicine composition for treating lower limb ischemic disease as claimed in claim 1, 2 or 3, comprising the following steps: 1) The water extract of Tribulus terrestris is extracted by the following steps: Step 1: Weigh the amount of Tribulus terrestris and flatten it; Step 2: Add 8 times the amount of water, soak for 16 hours, boil for 2 hours, and filter to collect the filtrate; Step 3: Add 6 times the amount of water to the residue from step 2, boil for 2 hours, and filter to collect the filtrate; Step 4: combining the filtrates collected in steps 2 and 3, concentrating and drying to obtain a water extract of Tribulus terrestris; 2) The astragalus water extract is extracted by the following steps: Step 1: Weigh the amount of Astragalus slices in the formula, add 8 times the amount of water, soak for 16 hours, decoct for 1.5 hours, and filter to collect the filtrate; Step 2: Add 6 times the amount of water to the residue in step 1, boil for 1.5 hours, and filter to collect the filtrate; Step 3, combining the filtrates collected in steps 1 and 2, concentrating and drying to obtain an astragalus water extract; 3) The Tribulus terrestris crude drug and the Astragalus crude drug extract prepared in the above steps are uniformly mixed according to the crude drug ratio.

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