Platycladus orientalis leaf extract and preparation method thereof

Abstract

The present invention relates to the technical field of the biopharmaceutical industry, and specifically to a Platycladus orientalis leaf extract and a preparation method thereof, comprising the following raw materials: a modified Platycladus orientalis leaf extract, a modified total saponin of Panax notoginseng, Panax notoginseng, fibrin, chitosan, iron oxide, glycerol, and a modified spinach solution. In the present invention, the modified Platycladus orientalis leaf extract, the modified total saponin of Panax notoginseng, and the modified spinach solution work together through a synergistic effect. The modified Platycladus orientalis leaf extract contains a relatively high amount of volatile oil, which can stimulate platelet aggregation and promote the release of coagulation factors. The modified total saponin of Panax notoginseng can inhibit excessive platelet activation and aggregation, preventing thrombosis. The modified Platycladus orientalis leaf extract and the modified total saponin of Panax notoginseng can jointly promote platelet aggregation and the release of coagulation factors to accelerate blood coagulation. The modified spinach solution promotes the synthesis and release of coagulation factors by providing vitamin K, thereby accelerating blood coagulation. The three form a dynamic equilibrium system, which promotes blood coagulation through a synergistic effect, achieving a hemostatic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine industry, and in particular to a Platycladus orientalis leaf extract and a preparation method thereof. Background Art

[0002] Blood coagulants play a crucial role in the biomedical field. They are not only crucial for ensuring the integrity of blood samples during collection, storage, and transportation, but also play an irreplaceable role in surgical procedures, the treatment of bleeding disorders, and the study of coagulation mechanisms. Common ingredients of currently available blood coagulants include heparin salts, potassium EDTA, and sodium citrate. These ingredients are mostly organic compounds, and long-term use can cause side effects in patients, including gastrointestinal reactions, allergic reactions, bleeding tendencies, and potential damage to specific organs or systems. In addition to the use of traditional blood coagulants, advances in medical technology have led to the emergence of new anticoagulant drugs and monitoring devices. These new drugs and devices show great potential in improving anticoagulant efficacy and reducing side effects, but they also face the challenge of high costs. The high cost of treatment may limit access to treatment for some patients, particularly in resource-constrained areas and low-income populations. Therefore, the exploration of safe, effective, affordable, and less-side-effect alternative anticoagulants is crucial for improving the treatment and prevention of thrombotic diseases. Platycladus orientalis leaves, as a plant of the genus Platycladus in the Cupressaceae family, have attracted much attention due to their rich medicinal active ingredients. The chemical composition of Platycladus orientalis leaves is complex, and studies have shown that they contain multiple types of medicinal active ingredients, among which flavonoids and volatile oils are relatively high. Both traditional Chinese medicine and modern medical research have confirmed that Platycladus orientalis leaves have significant therapeutic effects in the treatment of various diseases. For example, they have antibacterial, anti-tumor, hemostatic, hair growth promoting, anti-inflammatory and other pharmacological effects. The potential medicinal value of Platycladus orientalis leaves and their auxiliary effects on various diseases in clinical practice are becoming increasingly apparent. Therefore, further in-depth research on the pharmacological mechanism of Platycladus orientalis leaves and the development of new anticoagulants based on Platycladus orientalis leaves will not only help solve the side effects of long-term use of traditional anticoagulants, but may also provide patients with more economical and effective treatment options, which will be conducive to promoting the development of the biomedical field. Summary of the Invention

[0003] (1) Technical problems to be solved

[0004] The purpose of the present invention is to provide a Platycladus orientalis leaf extract and a preparation method thereof, to accurately extract components that have a significant promoting effect on blood coagulation, and by optimizing the main components of the blood coagulant, combining the research content of modern medicine, and extracting and using the natural components of traditional Chinese medicine plants, not only the safety and effectiveness of the blood coagulant are dually improved, but also the production cost is reduced, providing a higher quality and more efficient blood coagulant solution for the biomedicine field, and finally obtaining a Platycladus orientalis leaf extract.

[0005] (2) Technical solution

[0006] To achieve the above object, the present invention provides a Platycladus orientalis leaf extract, comprising the following raw materials in parts by weight: 20-30 parts of modified Panax notoginseng total saponins, 5-15 parts of Panax notoginseng, 10-30 parts of fibrin, 5-15 parts of chitosan, 5-15 parts of iron oxide, 2-6 parts of glycerol and 20-40 parts of modified spinach liquid;

[0007] The Platycladus orientalis leaf extract further comprises:

[0008] modified Platycladus orientalis leaf extract;

[0009] The mass ratio of the modified Platycladus orientalis leaf extract to the modified spinach extract is 1:(3-5);

[0010] The modified Platycladus orientalis leaves are prepared by extracting volatile oil through ultrasonic and acetone extraction methods.

[0011] Furthermore, the preparation method of the modified Platycladus orientalis leaf extract comprises:

[0012] S11. Weigh 100-150 g of Platycladus orientalis leaf sample, wash the Platycladus orientalis leaf with distilled water, and then dry it at 30-50 ° C. After drying, grind the dried Platycladus orientalis leaf with a grinder and pass it through an 80-90 mesh sieve to obtain Platycladus orientalis leaf particles;

[0013] S12. The Platycladus orientalis leaf particles were cleaned with an ultrasonic cleaner for 1 to 2 hours, and then sealed and immersed in 99 to 99.5% acetone for 2 to 3 hours to obtain a first soaking solution;

[0014] S13. The first soaking liquid is subjected to steam distillation for 3 to 5 hours to extract volatile oil to obtain a first extract, and then the first extract is extracted with 2 to 5 ml of petroleum ether. After extraction 3 to 5 times, a modified Platycladus orientalis leaf extract is obtained.

[0015] Furthermore, the preparation method of the modified Panax notoginseng total saponins comprises:

[0016] S21. Wash and chop 200-220 g of Panax notoginseng root, then add it to 1000-1200 mL of deionized water and heat it to 90-100 ° C for 1-2 h to obtain a first mixed solution;

[0017] S22. Add 10-20 mL of sodium hydroxide to the cooled first mixed solution to adjust the pH to 9-10, and then extract in a water bath for 2-4 hours at a temperature of 60-80°C to obtain a second mixed solution.

[0018] S23. After the extraction is completed, 10 to 20 mL of dilute nitric acid is added dropwise to the second mixed solution, the pH value is adjusted to about 3 to 5, and then placed in a freeze dryer and stirred at -5 to 0 ° C. After stirring, the solid residue is removed by filtration to obtain high-purity Panax notoginseng saponins;

[0019] S24. 10 to 15 g of lysine was dissolved in 99.0 to 99.9% dimethylformamide in a first container, 10 to 15 g of anhydride was dissolved in 99.0 to 99.9% dimethylformamide in a second container, the liquid in the second container was slowly added dropwise to the first container, followed by magnetic stirring at 35 to 45 ° C, a stirring speed of 300 to 400 rpm, and a stirring time of 30 to 40 min to obtain an activated lysine solution;

[0020] S25. The activated lysine solution was slowly added dropwise to the high-purity Panax notoginseng total saponins, and the coupling reaction was carried out at 10 to 15 ° C., followed by magnetic stirring at room temperature at a stirring speed of 500 to 700 rpm for 2 to 3 h. During the stirring process, 5 to 10 g of microbial transglutaminase was added to obtain a first modified solution;

[0021] S26. Filter the first modified liquid, and then centrifuge it with 2-5 ml of methanol. After centrifugation 3-5 times, the modified Panax notoginseng total saponins are obtained.

[0022] Furthermore, the modified spinach liquid preparation method comprises:

[0023] S31. Wash fresh radish tops and spinach leaves separately, then chop into 1-3 cm segments. Weigh 100-150 g of each radish top and spinach segments.

[0024] S32. The weighed radish and spinach segments were placed together in 1000-1500 mL of deionized water and boiled in a water bath at 80-90°C for 15-20 min. The filtrate was then filtered and collected, labeled as the first filtrate.

[0025] S33. Take 30-400 mL of a mixed solvent of acetone:n-hexane (mass ratio 1:1), add the first filtrate while stirring at room temperature, then let it stand for 30-40 minutes, then extract the first filtrate, collect the organic phase, and obtain a modified spinach liquid.

[0026] Furthermore, the modified spinach liquid is rich in vitamin K, and the mass ratio of the modified spinach liquid to the modified Panax notoginseng total saponins is 2:(5-7).

[0027] Furthermore, the mass ratio of the fibrin to the modified Panax notoginseng total saponins is (10-20): (5-10).

[0028] Furthermore, the mass ratio of the chitosan to the modified notoginseng total saponins is (1-3): (4-6).

[0029] Furthermore, the mass ratio of the iron oxide to the modified Panax notoginseng total saponins is 3:(2-4).

[0030] On the other hand, based on the same inventive concept, the present invention also provides a method for preparing a Platycladus orientalis leaf extract, which is applied to the Platycladus orientalis leaf extract, comprising the following steps:

[0031] S41 deionized water was added to the container, followed by adding glycerol, Panax notoginseng and modified spinach liquid and stirring, while heating in a water bath at a temperature of 50 to 65 ° C until fully dissolved to obtain a first mixed solution;

[0032] S42. The first mixed solution was filtered to obtain a second mixed solution, and the modified notoginseng total saponins and modified Platycladus orientalis leaf extract were slowly added to the second mixed solution while stirring at a stirring speed of 200 to 500 rpm for 1 to 2 h to obtain a third mixed solution;

[0033] S43. The third mixed solution is subjected to steam distillation. After the distillation is completed, fibrin, chitosan, and iron oxide are added in sequence, followed by magnetic stirring at a speed of 300 to 700 rpm for 5 to 6 hours. After the stirring is completed, a Platycladus orientalis leaf extract is obtained.

[0034] The mechanism of action of the above raw material components is as follows:

[0035] Platycladus orientalis leaves, also known as cypress leaves, bush cypress leaves, and cypress leaves, are the dried young branches and leaves of Platycladus orientalis, a plant of the Cupressaceae family. Cultivated throughout China, they are an excellent landscape tree species. According to Traditional Chinese Medicine (TCM), Platycladus orientalis leaves have a delicate fragrance, a bitter and slightly pungent taste, and possess the properties of cooling blood and stopping bleeding, dispelling wind and dampness, resolving phlegm and relieving coughs, reducing swelling and toxins, and promoting hair growth and darkening. Platycladus orientalis leaves have a complex chemical composition, primarily consisting of anise ketone, camphor, the cypress biflavonoid apigenin, quercetin, kaempferol-7-O-glucoside, pinene, caryophyllene, palmitic acid, stearic acid, lauric acid, and myristic acid. They possess antibacterial and anti-inflammatory properties, vasodilator properties, antidandruff and anti-hair loss properties, and hair growth. Ancient texts document the hemostatic properties of quercetin in Platycladus orientalis leaves, and modern medical research has shown that Platycladus orientalis charcoal also possesses hemostatic properties. The main component of the volatile oil in Platycladus orientalis leaves is α-ene, which has extremely strong medicinal value. It can work with other substances to promote blood coagulation, reduce bleeding, and has strong antibacterial and anti-inflammatory effects.

[0036] Modified Panax notoginseng total saponins are the active medicinal ingredients extracted from Panax notoginseng. They typically appear as a brownish-yellow to tan powder with a slightly bitter taste. Their main components include various ginsenosides, over twenty saponin active substances, seventeen trace elements, proteins, and a rich source of vitamins. Modified Panax notoginseng total saponins can improve blood circulation and eliminate blood stasis. They also have anti-fatigue and anti-aging effects, and enhance immunity. They can also be used to treat hemoptysis, hematemesis, epistaxis, bloody stools, metrorrhagia, and traumatic bleeding. They also have an adjunctive therapeutic effect on chest and abdominal pain, swelling and pain from falls, and other conditions.

[0037] Modified spinach juice is a kind of 2+ , vitamin C, iron and other nutrients in spinach. Among them, vitamin K plays an important role in the blood coagulation process. Vitamin K is a cofactor required for the synthesis of blood coagulation factors, especially coagulation factor II (heparin). Vitamin K deficiency can lead to bleeding tendency and cause coagulation factors to be unable to activate normally. In addition, the modified spinach liquid is rich in antioxidants that help inhibit the formation of free radicals and oxidative stress. The antioxidant effect can maintain vascular health, reduce the damage of oxidative stress to the blood system, and at the same time can inhibit inflammatory responses and reduce vascular inflammation and fibrosis.

[0038] Fibrin, a protein generally composed of collagen and elastin, is insoluble in water and appears as fibers or thin rods with a regular molecular structure. Fibrin plays an important role in maintaining blood vessel integrity, preventing blood loss, and promoting wound healing.

[0039] Chitosan is a naturally occurring polysaccharide secreted by insects, typically the Asian bloodworm. Chitosan is primarily composed of two types of polysaccharides: araconic acid and its isomers. Chitosan has low toxicity and can be used as a drug carrier. Furthermore, chitosan exhibits good blood compatibility and biodegradability, promoting platelet aggregation and coagulation factor activation. It also promotes cell proliferation and differentiation, facilitating tissue repair and angiogenesis. In thrombus dressings, chitosan can promote wound healing and tissue repair, reducing traumatic and post-operative damage.

[0040] Iron oxide can influence the activity of the coagulation factor system and promote the coagulation process. It also has an antioxidant effect during blood coagulation. Iron oxide can bind to oxidative stress substances and inhibit the formation of free radicals, thereby protecting platelets and the coagulation factor system from oxidative damage, helping to maintain the health and normal function of the blood system.

[0041] Glycerol, also known as propylene glycol, is a colorless, odorless, sweet, viscous liquid. It can be extracted from vegetable oils (such as soybean oil and palm oil) and animal oils (such as beef fat), primarily in the form of triglycerides. Glycerol's antioxidant properties protect the blood system from oxidative damage and maintain the normal function of the coagulation system. Glycerol also has antibacterial properties, preventing and treating infections, particularly in traumatic and postoperative settings.

[0042] (3) Beneficial effects

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The modified Platycladus orientalis leaf extract, modified Panax notoginseng total saponins, and modified spinach liquid work through a synergistic effect. The modified Platycladus orientalis leaf extract and modified Panax notoginseng total saponins jointly promote platelet aggregation and the release of coagulation factors to accelerate blood coagulation. The modified spinach liquid promotes the synthesis and release of coagulation factors, accelerates blood coagulation, and achieves a hemostatic effect.

[0045] 2. The modified Platycladus orientalis leaf extract contains a large amount of volatile oil, which stimulates platelet aggregation by binding to receptors on the platelet surface. It can also promote the release of coagulation factors and further accelerate the coagulation process.

[0046] 3. Modified Panax notoginseng saponins inhibit platelet activation and aggregation by increasing the cyclic adenosine monophosphate (cAMP) content in platelets. As an important second messenger molecule, cAMP plays a key regulatory role in platelets. Modified Panax notoginseng saponins can inhibit platelet aggregation through multiple pathways, including inhibiting the production of thromboxane A2 and improving endothelial function.

[0047] 4. Modified spinach liquid contains various nutrients, among which vitamin K is a coenzyme required for the synthesis of coagulation factors 2, 7, 9, and 10. These coagulation factors participate in the synthesis of coagulation factors during the coagulation process, forming a fibrin network to achieve the purpose of hemostasis. In addition, vitamin K can regulate the release of coagulation factors, effectively promoting the synthesis and release of coagulation factors and accelerating blood coagulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the SEM image of the volatile oil in the Platycladus orientalis leaves in Example 1 of the present invention. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0050] The experimental equipment and preparations of the embodiments described below are as follows: steam distillation apparatus, KO5200DA type numerically controlled ultrasonic cleaner, electronic balance (Sartorius, Germany), magnetic stirrer (Meiyingpu, Shanghai), high-speed centrifuge (Guangzhou Jidi), freeze dryer (Shanghai Pudong Freeze Drying), high-speed pulverizer (Shandong Tianfang Machinery), pH meter (Shanghai Yidian), oscillator (Shanghai Analyzer), rotary evaporator (Rikakikai, Japan), SB-1000 water bath (Rikakikai, Japan), scanning electron microscope (Zeiss, Germany); chemicals and reagents were purchased from Sigma-Aldrich.

[0051] Example 1: This example discloses a Platycladus orientalis leaf extract, comprising the following raw materials in parts by weight: 20 parts of modified Panax notoginseng total saponins, 5 parts of Panax notoginseng, 10 parts of fibrin, 5 parts of chitosan, 5 parts of iron oxide, 2 parts of glycerol and 20 parts of modified spinach liquid, the mass ratio of the modified Platycladus orientalis leaf extract to the modified spinach liquid is 1:3; the volatile oil of the modified Platycladus orientalis leaf is extracted by ultrasonic and acetone extraction.

[0052] In the Platycladus orientalis leaf extract, the modified Platycladus orientalis leaf extract, the modified Panax notoginseng total saponins, and the modified spinach extract work synergistically. The volatile oil component in the modified Platycladus orientalis leaf extract is the key to rapid hemostasis. It directly acts on the receptors on the surface of the platelet membrane, activating the signal transduction pathway inside the platelets, prompting the platelets to transition from a resting state to an activated state. Activated platelets release active substances such as ADP and 5-HT, which can further stimulate the aggregation of more platelets. At the same time, activated platelets also promote the release of coagulation factor V and coagulation factor VIII, thereby accelerating the coagulation cascade reaction. Then, excessive platelet activation and activation can lead to some thrombotic reactions. Therefore, modified Panax notoginseng saponins can moderately inhibit excessive platelet activation, altering platelet morphology by regulating the expression of platelet membrane glycoprotein receptors. This, in turn, reduces the secretion of dense granules and platelet adhesion, thereby lowering platelet concentration. Furthermore, modified Panax notoginseng saponins can lower platelet calcium concentration, maintaining a moderate platelet activation level and overall regulating platelet concentration. Fibrin forms a three-dimensional network structure, providing a scaffold for platelet adhesion and enhancing clot strength. Furthermore, modified Panax notoginseng saponins can balance the procoagulant and anticoagulant systems by regulating the synthesis of coagulation factors, preventing the risk of thrombosis caused by excessive platelet aggregation. Chitosan, with its positive charge and strong water absorption properties, adsorbs blood components and promotes the activation of coagulation factors. Furthermore, vitamin K in the modified spinach extract enhances hemostasis by promoting the synthesis and release of coagulation factors. As an essential cofactor, vitamin K participates in the γ-carboxylation modification of multiple coagulation factors, promoting the synthesis of vitamin K-dependent coagulation factors II, VII, IX, and X. The increase in these active coagulation factors accelerates the conversion of prothrombin to thrombin and promotes fibrin formation, thereby consolidating the hemostatic effect and enhancing coagulation factor activity. Iron oxide forms a mechanical barrier by constricting blood vessels and promoting local tissue coagulation, while also providing metal ions to accelerate the coagulation cascade. Glycerol optimizes overall performance by regulating material viscosity, maintaining water balance, and enhancing the dispersibility of other ingredients. The three components—modified Platycladus orientalis leaf extract, modified Panax notoginseng total saponins, and modified spinach extract—form a mutually restrictive and mutually reinforcing network, ultimately forming a dynamically balanced hemostatic system that rapidly and effectively promotes hemostasis, prevents thrombosis, and ensures the persistence of the hemostatic effect.

[0053] The preparation method of the modified Platycladus orientalis leaf extract comprises:

[0054] S11. Weigh 150g of Platycladus orientalis leaf sample, wash the Platycladus orientalis leaf with distilled water, and then dry it at 30 to 50 ° C. After drying, grind the dried Platycladus orientalis leaf with a grinder and pass it through an 80-90 mesh sieve to obtain Platycladus orientalis leaf particles;

[0055] S12. The Platycladus orientalis leaf particles were cleaned with an ultrasonic cleaner for 1 to 2 hours, and then sealed and immersed in 99 to 99.5% acetone for 2 to 3 hours to obtain a first soaking solution;

[0056] S13. The first soaking liquid is subjected to steam distillation for 3 to 5 hours to extract volatile oil to obtain a first extract, and then the first extract is extracted with 2 to 5 ml of petroleum ether. After extraction 3 to 5 times, a modified Platycladus orientalis leaf extract is obtained.

[0057] The preparation method of the modified Panax notoginseng total saponins comprises:

[0058] S21. Wash and chop 200 g of Panax notoginseng root, then add it to 1000-1200 mL of deionized water and heat it to 90-100 ° C for 1-2 h to obtain a first mixed solution;

[0059] S22. Add 10-20 mL of sodium hydroxide to the cooled first mixed solution to adjust the pH to 9-10, and then extract in a water bath for 2-4 hours at a temperature of 60-80°C to obtain a second mixed solution.

[0060] S23. After the extraction is completed, 10 to 20 mL of dilute nitric acid is added dropwise to the second mixed solution, the pH value is adjusted to about 3 to 5, and then placed in a freeze dryer and stirred at -5 to 0 ° C. After stirring, the solid residue is removed by filtration to obtain high-purity Panax notoginseng saponins;

[0061] S24. 10 to 15 g of lysine was dissolved in 99.0 to 99.9% dimethylformamide in a first container, 10 to 15 g of anhydride was dissolved in 99.0 to 99.9% dimethylformamide in a second container, the liquid in the second container was slowly added dropwise to the first container, followed by magnetic stirring at 35 to 45 ° C, a stirring speed of 300 to 400 rpm, and a stirring time of 30 to 40 min to obtain an activated lysine solution;

[0062] S25. The activated lysine solution was slowly added dropwise to the high-purity Panax notoginseng total saponins, and the coupling reaction was carried out at 10 to 15 ° C., followed by magnetic stirring at room temperature at a stirring speed of 500 to 700 rpm for 2 to 3 h. During the stirring process, 5 to 10 g of microbial transglutaminase was added to obtain a first modified solution;

[0063] S26. Filter the first modified liquid, and then centrifuge it with 2-5 ml of methanol. After centrifugation 3-5 times, the modified Panax notoginseng total saponins are obtained.

[0064] The preparation method of the modified spinach liquid comprises:

[0065] S31. Wash fresh radish tops and spinach leaves separately, then chop into 1-3 cm segments. Then weigh 100 g of each radish top segment and spinach segment.

[0066] S32. The weighed radish and spinach segments were placed together in 1000-1500 mL of deionized water and boiled in a water bath at 80-90°C for 15-20 min. The filtrate was then filtered and collected, labeled as the first filtrate.

[0067] S33. Take 30-400 mL of a mixed solvent of acetone:n-hexane (mass ratio 1:1), add the first filtrate while stirring at room temperature, then let it stand for 30-40 minutes, then extract the first filtrate, collect the organic phase, and obtain a modified spinach liquid.

[0068] The mass ratio of the modified spinach liquid to the modified Panax notoginseng total saponins is 2:5.

[0069] The mass ratio of the fibrin to the modified notoginseng total saponins is 10:5.

[0070] The mass ratio of the chitosan to the modified notoginseng total saponins is 1:4.

[0071] The mass ratio of the iron oxide to the modified notoginseng total saponins is 3:2.

[0072] A method for preparing a Platycladus orientalis leaf extract, applied to the Platycladus orientalis leaf extract, comprises the following steps:

[0073] S41 deionized water was added to the container, followed by adding glycerol, Panax notoginseng and modified spinach liquid and stirring, while heating in a water bath at a temperature of 50 to 65 ° C until fully dissolved to obtain a first mixed solution;

[0074] S42. The first mixed solution was filtered to obtain a second mixed solution, and the modified notoginseng total saponins and modified Platycladus orientalis leaf extract were slowly added to the second mixed solution while stirring at a stirring speed of 200 to 500 rpm for 1 to 2 h to obtain a third mixed solution;

[0075] S43. The third mixed solution is subjected to steam distillation. After the distillation is completed, fibrin, chitosan, and iron oxide are added in sequence, followed by magnetic stirring at a speed of 300 to 700 rpm for 5 to 6 hours. After the stirring is completed, a Platycladus orientalis leaf extract is obtained.

[0076] Example 2: This example discloses a Platycladus orientalis leaf extract, comprising the following raw materials in parts by weight: 25 parts of modified Panax notoginseng total saponins, 10 parts of Panax notoginseng, 15 parts of fibrin, 10 parts of chitosan, 10 parts of iron oxide, 4 parts of glycerol and 30 parts of modified spinach liquid, the mass ratio of the modified Platycladus orientalis leaf extract to the modified spinach liquid is 1:4; the volatile oil of the modified Platycladus orientalis leaf is extracted by ultrasonic and acetone extraction.

[0077] The preparation method of the modified Platycladus orientalis leaf extract comprises:

[0078] S11. Weigh 100 g of Platycladus orientalis leaf sample, wash the Platycladus orientalis leaf with distilled water, and then dry it at 30 to 50 ° C. After drying, grind the dried Platycladus orientalis leaf with a grinder and pass it through an 80-90 mesh sieve to obtain Platycladus orientalis leaf particles;

[0079] S12. The Platycladus orientalis leaf particles were cleaned with an ultrasonic cleaner for 1 to 2 hours, and then sealed and immersed in 99 to 99.5% acetone for 2 to 3 hours to obtain a first soaking solution;

[0080] S13. The first soaking liquid is subjected to steam distillation for 3 to 5 hours to extract volatile oil to obtain a first extract, and then the first extract is extracted with 2 to 5 ml of petroleum ether. After extraction 3 to 5 times, a modified Platycladus orientalis leaf extract is obtained.

[0081] The preparation method of the modified Panax notoginseng total saponins comprises:

[0082] S21. Wash and chop 210 g of Panax notoginseng root, then add 1000-1200 mL of deionized water and heat to 90-100 ° C for 1-2 h to obtain a first mixed solution;

[0083] S22. Add 10-20 mL of sodium hydroxide to the cooled first mixed solution to adjust the pH to 9-10, and then extract in a water bath for 2-4 hours at a temperature of 60-80°C to obtain a second mixed solution.

[0084] S23. After the extraction is completed, 10 to 20 mL of dilute nitric acid is added dropwise to the second mixed solution, the pH value is adjusted to about 3 to 5, and then placed in a freeze dryer and stirred at -5 to 0 ° C. After stirring, the solid residue is removed by filtration to obtain high-purity Panax notoginseng saponins;

[0085] S24. 10 to 15 g of lysine was dissolved in 99.0 to 99.9% dimethylformamide in a first container, 10 to 15 g of anhydride was dissolved in 99.0 to 99.9% dimethylformamide in a second container, the liquid in the second container was slowly added dropwise to the first container, followed by magnetic stirring at 35 to 45 ° C, a stirring speed of 300 to 400 rpm, and a stirring time of 30 to 40 min to obtain an activated lysine solution;

[0086] S25. The activated lysine solution was slowly added dropwise to the high-purity Panax notoginseng total saponins, and the coupling reaction was carried out at 10 to 15 ° C., followed by magnetic stirring at room temperature at a stirring speed of 500 to 700 rpm for 2 to 3 h. During the stirring process, 5 to 10 g of microbial transglutaminase was added to obtain a first modified solution;

[0087] S26. Filter the first modified liquid, and then centrifuge it with 2-5 ml of methanol. After centrifugation 3-5 times, the modified Panax notoginseng total saponins are obtained.

[0088] The preparation method of the modified spinach liquid comprises:

[0089] S31. Wash fresh radish tops and spinach leaves separately, then chop into 1-3 cm segments. Then weigh 120 g of each radish top and spinach segments.

[0090] S32. The weighed radish and spinach segments were placed together in 1000-1500 mL of deionized water and boiled in a water bath at 80-90°C for 15-20 min. The filtrate was then filtered and collected, labeled as the first filtrate.

[0091] S33. Take 30-400 mL of a mixed solvent of acetone:n-hexane (mass ratio 1:1), add the first filtrate while stirring at room temperature, then let it stand for 30-40 minutes, then extract the first filtrate, collect the organic phase, and obtain a modified spinach liquid.

[0092] The mass ratio of the modified spinach liquid to the modified Panax notoginseng total saponins is 2:6.

[0093] The mass ratio of the fibrin to the modified Panax notoginseng total saponins is 15:10.

[0094] The mass ratio of the chitosan to the modified notoginseng total saponins is 1:5.

[0095] The mass ratio of the iron oxide to the modified total saponins of Panax notoginseng is 1: 1. The preparation method of the Platycladus orientalis leaf extract in this embodiment is the same as that in the first embodiment.

[0096] Example 3: This example discloses a Platycladus orientalis leaf extract, comprising the following raw materials in parts by weight: 25 parts of modified Panax notoginseng total saponins, 10 parts of Panax notoginseng, 15 parts of fibrin, 10 parts of chitosan, 10 parts of iron oxide, 4 parts of glycerol and 30 parts of modified spinach liquid, the mass ratio of the modified Platycladus orientalis leaf extract to the modified spinach liquid is 1:4; the volatile oil of the modified Platycladus orientalis leaf is extracted by ultrasonic and acetone extraction.

[0097] The preparation method of the modified Platycladus orientalis leaf extract comprises:

[0098] S11. Weigh 120 g of Platycladus orientalis leaf sample, wash the Platycladus orientalis leaf with distilled water, and then dry it at 30 to 50 ° C. After drying, grind the dried Platycladus orientalis leaf with a grinder and pass it through an 80-90 mesh sieve to obtain Platycladus orientalis leaf particles;

[0099] S12. The Platycladus orientalis leaf particles were cleaned with an ultrasonic cleaner for 1 to 2 hours, and then sealed and immersed in 99 to 99.5% acetone for 2 to 3 hours to obtain a first soaking solution;

[0100] S13. The first soaking liquid is subjected to steam distillation for 3 to 5 hours to extract volatile oil to obtain a first extract, and then the first extract is extracted with 2 to 5 ml of petroleum ether. After extraction 3 to 5 times, a modified Platycladus orientalis leaf extract is obtained.

[0101] The preparation method of the modified Panax notoginseng total saponins comprises:

[0102] S21. Wash and chop 220 g of Panax notoginseng root, then add 1000-1200 mL of deionized water and heat to 90-100 ° C for 1-2 h to obtain a first mixed solution;

[0103] S22. Add 10-20 mL of sodium hydroxide to the cooled first mixed solution to adjust the pH to 9-10, and then extract in a water bath for 2-4 hours at a temperature of 60-80°C to obtain a second mixed solution.

[0104] S23. After the extraction is completed, 10 to 20 mL of dilute nitric acid is added dropwise to the second mixed solution, the pH value is adjusted to about 3 to 5, and then placed in a freeze dryer and stirred at -5 to 0 ° C. After stirring, the solid residue is removed by filtration to obtain a modified high-purity total saponins of Panax notoginseng;

[0105] S24. 10 to 15 g of lysine was dissolved in 99.0 to 99.9% dimethylformamide in a first container, 10 to 15 g of anhydride was dissolved in 99.0 to 99.9% dimethylformamide in a second container, the liquid in the second container was slowly added dropwise to the first container, followed by magnetic stirring at 35 to 45 ° C, a stirring speed of 300 to 400 rpm, and a stirring time of 30 to 40 min to obtain an activated lysine solution;

[0106] S25. The activated lysine solution was slowly added dropwise to the high-purity Panax notoginseng total saponins, and the coupling reaction was carried out at 10 to 15 ° C., followed by magnetic stirring at room temperature at a stirring speed of 500 to 700 rpm for 2 to 3 h. During the stirring process, 5 to 10 g of microbial transglutaminase was added to obtain a first modified solution;

[0107] S26. Filter the first modified liquid, and then centrifuge it with 2-5 ml of methanol. After centrifugation 3-5 times, the modified Panax notoginseng total saponins are obtained.

[0108] The preparation method of the modified spinach liquid comprises:

[0109] S31. Wash fresh radish tops and spinach leaves separately, then chop into 1-3 cm segments. Then weigh 150 g of each radish tops and spinach segments.

[0110] S32. The weighed radish and spinach segments were placed together in 1000-1500 mL of deionized water and boiled in a water bath at 80-90°C for 15-20 min. The filtrate was then filtered and collected, labeled as the first filtrate.

[0111] S33. Take 30-400 mL of a mixed solvent of acetone:n-hexane (mass ratio 1:1), add the first filtrate while stirring at room temperature, then let it stand for 30-40 minutes, then extract the first filtrate, collect the organic phase, and obtain a modified spinach liquid.

[0112] The mass ratio of the modified spinach liquid to the modified Panax notoginseng total saponins is 2:7.

[0113] The mass ratio of the fibrin to the modified Panax notoginseng total saponins is 20:10.

[0114] The mass ratio of the chitosan to the modified notoginseng total saponins is 1:4.

[0115] The mass ratio of the iron oxide to the modified total saponins of Panax notoginseng is 3:4. The preparation method of the Platycladus orientalis leaf extract in this embodiment is the same as that in Example 1.

[0116] Example 4: This example discloses a Platycladus orientalis leaf extract, comprising the following raw materials in parts by weight: 30 parts of modified Panax notoginseng total saponins, 10 parts of Panax notoginseng, 15 parts of fibrin, 10 parts of chitosan, 10 parts of iron oxide, 4 parts of glycerol and 30 parts of modified spinach liquid, the mass ratio of the modified Platycladus orientalis leaf extract to the modified spinach liquid is 1:3; the volatile oil of the modified Platycladus orientalis leaf is extracted by ultrasonic and acetone extraction.

[0117] The mass ratio of the modified spinach liquid to the modified Panax notoginseng total saponins is 2:5.

[0118] The mass ratio of the fibrin to the modified notoginseng total saponins is 10:5.

[0119] The mass ratio of the chitosan to the modified notoginseng total saponins is 1:4.

[0120] The mass ratio of the iron oxide to the modified total saponins of Panax notoginseng is 3:2. The preparation method of the Platycladus orientalis leaf extract in this embodiment is the same as that in Example 1.

[0121] Example 5: This example discloses a Platycladus orientalis leaf extract, comprising the following raw materials in parts by weight: 20 parts of modified Panax notoginseng total saponins, 10 parts of Panax notoginseng, 15 parts of fibrin, 10 parts of chitosan, 10 parts of iron oxide, 4 parts of glycerol and 20 parts of modified spinach liquid, the mass ratio of the modified Platycladus orientalis leaf extract to the modified spinach liquid is 1:3; the volatile oil of the modified Platycladus orientalis leaf is extracted by ultrasonic and acetone extraction.

[0122] The mass ratio of the modified spinach liquid to the modified Panax notoginseng total saponins is 2:5.

[0123] The mass ratio of the fibrin to the modified notoginseng total saponins is 10:5.

[0124] The mass ratio of the chitosan to the modified notoginseng total saponins is 1:4.

[0125] The mass ratio of the iron oxide to the modified total saponins of Panax notoginseng is 3:2. The preparation method of the Platycladus orientalis leaf extract in this embodiment is the same as that in Example 1.

[0126] Control group 1: This example differs from Example 1 in that the modified Platycladus orientalis leaf extract is not contained. The preparation method of the Platycladus orientalis leaf extract in this example is the same as that in Example 1.

[0127] Control group 2: This example differs from Example 1 in that the modified notoginseng total saponins are not contained. The preparation method of the Platycladus orientalis leaf extract in this example is the same as that in Example 1.

[0128] Control group 3: This embodiment differs from embodiment 1 in that the modified spinach solution is not included. The preparation method of the Platycladus orientalis leaf extract in this embodiment is the same as that in embodiment 1.

[0129] Control group 4: This example differs from Example 1 in that it does not contain modified spinach liquid and modified Platycladus orientalis leaf extract. The preparation method of Platycladus orientalis leaf extract in this example is the same as that in Example 1.

[0130] Control group 5: This example differs from Example 1 in that it does not contain modified notoginseng total saponins and modified Platycladus orientalis leaf extract. The preparation method of Platycladus orientalis leaf extract in this example is the same as that in Example 1.

[0131] Control group 6: This example differs from Example 1 in that it does not contain modified notoginseng total saponins and modified spinach liquid. The preparation method of the Platycladus orientalis leaf extract in this example is the same as that in Example 1.

[0132] Control group 7: This example differs from Example 1 in that it does not contain modified notoginseng total saponins, modified Platycladus orientalis leaf extract, and modified spinach liquid. The preparation method of Platycladus orientalis leaf extract in this example is the same as that in Example 1.

[0133] Effect evaluation: Analysis of the blood coagulation performance of Platycladus orientalis leaf extract: The Platycladus orientalis leaf extract prepared in each experimental group was randomly selected and divided into 5 parts on average. Blood was taken from the common carotid artery of rabbits. The effect of Platycladus orientalis leaf extract on blood coagulation in vitro was observed using the coagulation plate method with clotting time (CT), activated partial thromboplastin time (APTT), and platelet aggregation rate as indicators. Table 1 shows the statistical results of the factors affecting blood coagulation of Platycladus orientalis leaf extract. It can be seen from Table 1 that there are certain differences in the proportions of Platycladus orientalis leaf extracts in each experimental group. By comparing the clotting time of the Platycladus orientalis leaf extracts prepared in Examples 1 to 5 with that of the control groups 1 to 7, it can be found that, on the whole, the Platycladus orientalis leaf extracts prepared in Examples 1 to 5 have a better effect on blood coagulation, and the average clotting time is lower than that of the control group. In addition, the Platycladus orientalis leaf extract prepared in Example 1 has the best effect on blood coagulation and the shortest clotting time. This shows that the synergistic effect of modified Platycladus orientalis leaf extract, modified Panax notoginseng total saponins and modified spinach liquid plays a great role. Modified Platycladus orientalis leaf extract and modified Panax notoginseng total saponins jointly promote platelet aggregation and the release of coagulation factors to accelerate blood coagulation. Modified spinach liquid promotes the synthesis and release of coagulation factors, accelerates blood coagulation, and achieves a hemostatic effect. Comparing the Platycladus orientalis leaf extract prepared in Example 1 with control groups 1 to 7, it can be found that the coagulation time, activated partial thromboplastin time and platelet aggregation rate are all in an optimal range, among which the coagulation time is the shortest among the entire experimental group and the control group, and blood coagulation is completed in only 30 seconds. The activated partial thromboplastin time is also the shortest among all experimental groups, taking 25 seconds. Among them, the platelet aggregation rate is 10.6%, the highest in the entire experimental group. The normal value of the platelet aggregation rate is generally 9% to 13%. This shows that when the modified Platycladus orientalis leaf extract, modified Panax notoginseng total saponins and modified spinach liquid are added simultaneously during the preparation of the Platycladus orientalis leaf extract, the biomedical value of the Platycladus orientalis leaf extract can be significantly improved.

[0134] Table 1 Statistical results of factors affecting blood coagulation of Platycladus orientalis leaf extract in each experimental group

[0135] Experimental group Coagulation time (s) Activated partial thromboplastin time (s) Platelet aggregation rate (%) Example 1 30 25 10.6 Example 2 33 26 10.3 Example 3 36 26 10.1 Example 4 34 26 10.2 Example 5 35 27 10.4 Control group 1 50 30 9.9 Control group 2 40 30 10.0 Control group 3 49 32 9.2 Control group 4 61 36 9.6 Control group 5 66 33 9.5 Control group 6 63 35 9.0 Control group 7 69 40 9.0

[0136] After the above limited experiments, the Platycladus orientalis leaf extract material of Example 1 of the present invention has significant effects. By optimizing the preparation method of Platycladus orientalis leaf volatile oil, modified Platycladus orientalis leaf extract, modified Panax notoginseng total saponins and modified spinach liquid and other plant ingredients are added to the process of preparing the Platycladus orientalis leaf extract, the medicinal value of Platycladus orientalis leaves is significantly improved, and the effect of Platycladus orientalis leaf extract on blood coagulation is improved. Finally, a high-quality Platycladus orientalis leaf extract is obtained. The obtained Platycladus orientalis leaf extract has great biomedical application potential. The Platycladus orientalis leaf extract raw material can be applied to cosmetics, medicine and other fields to achieve large-scale production, thereby promoting the healthy and orderly development of the industry.

[0137] Finally, it should be noted that although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Platycladus orientalis leaf extract, characterized in that The invention comprises the following raw materials in parts by weight: 20-30 parts of modified total saponins of Panax notoginseng, 5-15 parts of Panax notoginseng, 10-30 parts of fibrin, 5-15 parts of chitosan, 5-15 parts of iron oxide, 2-6 parts of glycerol and 20-40 parts of modified spinach liquid. The Platycladus orientalis leaf extract further comprises: modified Platycladus orientalis leaf extract; The mass ratio of modified Platycladus orientalis leaf extract to modified spinach extract is 1:(3-5); The modified Platycladus orientalis leaves are extracted with volatile oil by ultrasonic and acetone extraction; The preparation method of the modified Platycladus orientalis leaf extract comprises: S11. Weigh 100-150 g of Platycladus orientalis leaf sample, wash the Platycladus orientalis leaf with distilled water, and then dry it at 30-50 ° C. After drying, grind the dried Platycladus orientalis leaf with a grinder and pass it through an 80-90 mesh sieve to obtain Platycladus orientalis leaf particles; S12. The Platycladus orientalis leaf particles were cleaned with an ultrasonic cleaner for 1 to 2 hours, and then placed in 99 to 99.5% acetone for a sealed soak for 2 to 3 hours to obtain a first soaking solution; S13. The first soaking liquid was steam distilled for 3 to 5 hours to extract the volatile oil to obtain a first extract, which was then extracted with 2 to 5 ml of petroleum ether. After extraction 3 to 5 times, a modified Platycladus orientalis leaf extract was obtained; The preparation method of the modified Panax notoginseng total saponins comprises: S21. Wash and mince 200-220 g of Panax notoginseng root, then soak it in 99.0-99.9% dimethylformamide at room temperature for 1-2 hours. After soaking, remove the minced Panax notoginseng root and place it in 1000-1200 mL of deionized water. Heat the mixture to 90-100°C for 1-2 hours to obtain a first mixed solution. S22 was added to the cooled first mixed solution 10 ~ 20mL of sodium hydroxide, adjusted to pH 9 to 10, then placed in a water bath for extraction, the extraction time was 2 to 4h, the extraction temperature was 60 ~ 80 ° C, to give a second mixed solution; S23. After the extraction is completed, 10 to 20 mL of dilute nitric acid was added dropwise to the second mixed solution, the pH value was adjusted to about 3 to 5, and then placed in a freeze dryer and stirred at -5 to 0 ° C. After stirring, the solid residue was removed by filtration to obtain high-purity Panax notoginseng saponins; S24. 10 to 15 g of lysine was dissolved in 99.0 to 99.9% dimethylformamide in a first container, 10 to 15 g of anhydride was dissolved in 99.0 to 99.9% dimethylformamide in a second container, the liquid in the second container was slowly added dropwise to the first container, followed by magnetic stirring at 35 to 45 ° C, a stirring speed of 300 to 400 rpm, a stirring time of 30 to 40 min, to obtain an activated lysine solution; S25 activated lysine solution was slowly added dropwise to high-purity Panax notoginseng saponins, the coupling reaction was carried out at 10~15 ° C, followed by magnetic stirring at room temperature, a stirring speed of 500~700 rpm, stirring time 2~3h, 5~10g of microbial transglutaminase was added during stirring to obtain a first modified solution; S26 The first modified liquid was filtered, and then centrifuged with 2 to 5 ml of methanol, and centrifuged 3 to 5 times to obtain modified Panax notoginseng total saponins; The preparation method of the modified spinach liquid comprises: S31. Wash fresh radish and spinach leaves separately, then chop into 1-3 cm segments. Then weigh 100-150g of radish and spinach segments respectively. S32. The weighed radish sprouts and spinach segments were placed together in 1000~1500 mL of deionized water, boiled in a water bath at 80~90 ° C for 15-20min, and then filtered to collect the filtrate, labeled as the first filtrate; S33. 30 to 400 mL of an acetone:n-hexane mixed solvent was added to the first filtrate while stirring at room temperature, and then allowed to stand for 30 to 40 minutes. The first filtrate was then extracted and the organic phase was collected to obtain a modified spinach solution. The modified spinach liquid is rich in vitamin K and Ga 2+ content.

2. A method for preparing a Platycladus orientalis leaf extract, which is used to prepare the Platycladus orientalis leaf extract according to claim 1, characterized in that: The method comprises the following steps: S41 deionized water was added to the container, followed by the addition of glycerol, Panax notoginseng and modified spinach solution and stirring, while heating in a water bath at a temperature of 50 to 65 ° C until fully dissolved to obtain a first mixed solution; S42 The first mixed solution was filtered to obtain a second mixed solution, and the modified notoginseng total saponins and modified Platycladus orientalis leaf extract were slowly added to the second mixed solution while stirring at a stirring speed of 200 to 500 rpm for 1 to 2h to obtain a third mixed solution; S43. The third mixed solution is subjected to steam distillation. After the distillation is completed, fibrin, chitosan, and iron oxide are added in sequence, followed by magnetic stirring at a speed of 300-700 rpm for 5-6 hours. After the stirring is completed, a Platycladus orientalis leaf extract is obtained.

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