Method for analyzing trace elements in dinodon rufozonatum and application of dinodon rufozonatum in antithrombotic

Abstract

The invention provides a method for analyzing trace elements in dinodon rufozonatum and an antithrombotic activity application of the dinodon rufozonatum. The method comprises the following steps: pretreatment of a dried snake or decocted liquid sample, microwave digestion, acid removal and constant volume, and ICP-MS detection of 25 trace elements. The antithrombotic activity application of the method for analyzing the trace elements in the dinodon rufozonatum is characterized in that the trace elements such as selenium, chromium and vanadium related to the antithrombotic activity are detected, the content range of the trace elements is strictly controlled, element-activity correlation verification is carried out in combination with an antithrombotic activity verification experiment, and the dinodon rufozonatum antithrombotic preparation is provided. The composition comprises dinodon rufozonatum alcohol extract and pharmaceutical auxiliary materials in a mass ratio of (4-9): 1, and the content of antithrombotic elements in the dinodon rufozonatum alcohol extract meets specific standards. The development and application of the preparation provide a new choice for the field of antithrombotic drugs, fully explore the medicinal value of dinodon rufozonatum, and have broad market prospects and important scientific significance.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis and pharmacological effects, specifically to a method for analyzing trace elements in red-banded snake and its application in antithrombotic activity. Background Technology

[0002] The red-banded snake (Dinodon rufozonatum) is one of the traditional medicinal animals in my country, recorded in ancient herbal classics such as *Ben Cao Jing Ji Zhu*, *Zheng Lei Ben Cao*, and *Ben Cao Gang Mu*. It possesses properties such as dispelling wind and dampness, relieving pain and detoxifying, and clearing the meridians and healing sores, and is widely used in the treatment of rheumatic diseases and skin diseases. Modern research shows that inorganic elements in the medicinal material play a key role in its pharmacological activity, especially zinc (Zn) and selenium (Se), which have significant effects in antioxidation, immune regulation, and antithrombosis. However, traditional detection methods are inefficient, and the antithrombotic mechanism of the red-banded snake is unclear. Currently, there is a lack of systematic and efficient analytical methods for multiple inorganic elements in the red-banded snake and its fetal red-banded snake, and even more lack of research on the correlation analysis between trace element content and efficacy, screening of highly effective parts, and their application in antithrombotic development. Summary of the Invention

[0003] The purpose of this invention is to provide a method for the analysis of trace elements in red-banded snake and its application in antithrombotic activity. It establishes a precise method for the analysis of trace elements in red-banded snake and, based on an element-antithrombotic activity correlation model, develops a highly efficient and safe antithrombotic preparation, solving the problems of unclear active ingredients, lack of quality control, and high safety risks in traditional snake drugs.

[0004] The method for detecting trace elements in red-banded snake provided by this invention can efficiently and accurately determine the content of trace elements in red-banded snake, providing assurance for the quality assessment of medicinal materials, the safety and effectiveness of medication, and meeting the growing demand for the accuracy and breadth of trace element detection.

[0005] This invention provides a method for detecting trace elements in red-banded snake, comprising the following steps:

[0006] S1. Sample Pretreatment: Overcoming the biological matrix barrier, eliminating interference from the high-protein / calcium matrix of the red-banded snake, and releasing trace elements. The red-banded snake or red-banded fetal snake sample is dried and then pulverized. Drying removes moisture from the sample, facilitating subsequent pulverization and making the sample more homogeneous. Pulverization increases the sample surface area, accelerating the decomposition reaction of nitric acid and improving digestion efficiency. After weighing the coarse powder, add 30-40 times its weight of 60-70% nitric acid and let stand overnight. Nitric acid is a strong oxidizing agent that effectively decomposes the organic matter in the red-banded snake sample, releasing trace elements. The 60-70% nitric acid concentration is an optimized choice, ensuring sufficient sample decomposition without causing violent reactions or increasing the difficulty of subsequent acid removal due to excessive concentration. 60-70% nitric acid balances oxidizing power and safety; >98% concentrated nitric acid is prone to boiling over, and <30% is difficult to decompose the snake bone calcium. A 30-40 times concentration of acid by mass ensures complete sample wetting and adequate contact between nitric acid and the sample, thereby achieving complete decomposition of organic matter. Allowing the sample to stand overnight pre-oxidizes fats and collagen, reducing foam spraying during digestion.

[0007] S2. Microwave digestion ensures efficient elemental dissolution and protection, with gradient temperature control achieving the triple goals of "cell wall disruption, dissolution, and stabilization." A four-stage gradient temperature-controlled digestion process is employed: the first stage involves low-temperature sample penetration to prevent violent reactions; the second stage decomposes proteins / fats; the third stage thoroughly destroys the hydroxyapatite structure; and the fourth stage provides low-temperature isothermal protection for volatile selenium. This four-stage temperature control allows for more effective decomposition of organic components in the sample, ensuring complete dissolution of trace elements in the digestion solution. Gradient temperature increases help prevent boiling or splashing due to sudden high temperatures, ensuring a smooth digestion process. After digestion, the digestion solution undergoes acid removal treatment to eliminate excess nitric acid and prevent interference with subsequent ICP-MS detection. Volume adjustment involves diluting the digested sample solution to a specific volume for easy instrument injection and accurate determination of trace element content.

[0008] S3. ICP-MS detection enables simultaneous and precise quantification of multiple elements, establishing a "content-activity" database for 25 elements in a single step. Inductively coupled plasma mass spectrometry (ICP-MS) is used to determine the 25 elements (B, Na, Mg, Al, K, Ca, Ti, Cr, V, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Sn, Sb, Ba, Tl, Pd). ICP-MS is a highly sensitive and precise analytical technique capable of simultaneously determining multiple trace elements. By determining the 25 elements (B, Na, Mg, Al, K, Ca, Ti, Cr, V, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Sn, Sb, Ba, Tl, Pd) in *Spatholobus suberectus*, a comprehensive understanding of its trace element composition and content can be obtained, providing a scientific basis for assessing its medicinal value, safety, and development and utilization. Of the 25 trace elements, boron (B) participates in the regulation of the activity of various enzymes in the human body and plays an important role in bone health and hormone metabolism. Sodium (Na) maintains extracellular fluid osmotic pressure and acid-base balance and participates in neuromuscular excitability transmission. Magnesium (Mg) participates in hundreds of enzymatic reactions in the human body and is essential for maintaining the normal function of the heart, bones, and nervous system. Excessive intake of aluminum (Al) can damage the human nervous system and bones; its content level can reflect the aluminum pollution in the environment and food. Potassium (K) maintains intracellular fluid osmotic pressure and acid-base balance and participates in important physiological processes such as myocardial contraction and nerve conduction. Calcium (Ca) is a major component of bones and teeth and participates in various physiological functions such as blood clotting, muscle contraction, and nerve conduction. Titanium (Ti) is a biologically inert element, but in some cases it is involved in redox reactions in organisms. Chromium (Cr), specifically trivalent chromium, is one of the essential trace elements for the human body and participates in the regulation of glucose and lipid metabolism. Vanadium (V) has various biological functions, such as regulating blood sugar and affecting lipid metabolism. Manganese (Mn) acts as an activator of various enzymes, participating in bone metabolism, glucose metabolism, and the antioxidant defense system. Iron (Fe) constitutes hemoglobin and myoglobin, participating in oxygen transport and storage, and is an essential trace element for the human body. Cobalt (Co) is a component of vitamin B12, participating in erythrocyte production and maintaining the normal function of the nervous system. Nickel (Ni) participates in the regulation of the activity of certain enzymes and iron metabolism. Copper (Cu) participates in the composition and function of various enzymes in the human body, playing an important role in iron metabolism, antioxidant defense, and nerve conduction. Zinc (Zn) participates in the regulation of various physiological functions in the human body, including the immune system, taste, smell, and growth and development. Although arsenic (As) is toxic in some cases, it participates in certain physiological processes in the human body at very low doses, and its content is also one of the important indicators for assessing environmental and food safety. Selenium (Se) has antioxidant effects, protecting cells from free radical damage, enhancing immunity, and preventing the occurrence of certain diseases.Sr (strontium) has similar chemical properties to calcium and can be absorbed by human bones, playing a role in bone health and certain metabolic processes. Mo (molybdenum) is a component of many enzymes in the human body and participates in physiological processes such as nitrogen metabolism and redox reactions. Cd (cadmium) is a harmful element; excessive intake can damage the kidneys and bones. Its content determination can be used to assess the degree of environmental and food pollution and the potential risks to human health. The physiological role of Sn (tin) in the human body is not yet clear, but excessive intake is generally considered harmful. Sb (antimony) has a certain degree of toxicity; its content determination can be used to assess the pollution of the environment and food and the potential threats to human health. Ba (barium) is a heavy metal element; excessive intake can damage the human nervous system and circulatory system. Tl (thallium) is a highly toxic element that seriously damages the human nervous system; its content determination is of great significance for ensuring food safety and human health. The human body's requirements for and physiological role of Pd (palladium) under normal conditions are not yet clear, but its content determination can be used to assess palladium pollution in the environment and food and the potential health risks. The trace element detection method of this invention can perform targeted quantification of antithrombotic active elements. Se (Se) inhibits platelet aggregation, Cr and V regulate lipid metabolism and reduce thromboxane, and As (As > 2.0 mg / kg) inhibits Se activity. Therefore, the trace element detection method of this invention adopts a three-stage detection method to screen raw materials with high Se / Cr / V for antithrombotic preparations and eliminate samples with excessive As / Fe.

[0009] In the detection method of this invention, step S1, through different pretreatment methods for dried snake samples and decoction samples, fully considers the characteristics of the two types of samples, thereby improving the accuracy and reliability of the detection and laying a solid foundation for the subsequent accurate determination of trace element content. This step covers two common red-banded snake sample forms, which can meet different research and application needs. Whether it is the component analysis of the red-banded snake itself or the study of the effective components in its decoction, it can provide an accurate detection method. Considering the differences in physical and chemical properties between dried snake samples and decoction samples, different pretreatment steps are adopted to ensure the effective extraction and detection of trace elements in both types of samples. The specific operating conditions and parameter ranges of each step are clearly defined, providing standardized guidance for the detection process and helping to improve the reproducibility and reliability of the detection results.

[0010] In the detection method of this invention, step S1. Sample pretreatment is either dried snake sample pretreatment or decoction sample pretreatment, including the following steps:

[0011] S1-1. Pretreatment of Dried Snake Samples: After drying the sample at 50-70℃ for 40-50 hours, remove the moisture to achieve a suitable degree of dryness, facilitating subsequent pulverization. Appropriate drying also kills some microorganisms, reducing their impact on trace element content; ensuring sample stability and homogeneity, and improving detection accuracy. Pulverization transforms the dried snake sample into coarse powder, increasing the sample surface area, allowing for more thorough contact between the added nitric acid and the sample, improving digestion efficiency. This accelerates the digestion process, ensuring complete release of trace elements. Accurately weigh 0.1-0.3g of coarse powder and place it in a high-pressure, high-temperature microwave digestion vessel. Precisely control the sample volume to ensure the reaction system has a suitable concentration for accurate trace element determination during subsequent digestion. Ensure the concentration of trace elements in the digested solution is within the instrument's detection linear range, improving the reliability of the detection results. Add 5–10 mL of nitric acid with a mass percentage concentration of 60–70%. Nitric acid, as a strong oxidizing agent, can effectively decompose the organic components in the dried snake sample, releasing trace elements from the organic matrix. The 60–70% nitric acid concentration is an optimized choice, ensuring sufficient sample decomposition without causing violent reactions or increasing the difficulty of subsequent acid removal due to excessive concentration. This ensures that the trace elements in the sample are fully dissolved in the digestion solution, providing a suitable solution system for subsequent ICP-MS detection. Let it stand overnight to allow sufficient time for the nitric acid to react with the organic components in the dried snake sample, ensuring a thorough digestion process. This improves digestion efficiency and ensures complete release of trace elements. Place in a microwave digester and digest according to the programmed temperature rise.

[0012] S1-2. Sample Pretreatment of Decoction: After drying the sample at 50-70℃ for 40-50 hours, remove the moisture to ensure the dried snake sample reaches a suitable degree of dryness for subsequent pulverization. Appropriate drying also kills some microorganisms, reducing their impact on trace element content and ensuring sample stability and homogeneity, thus improving detection accuracy. Pulverization transforms the dried snake sample into coarse powder, increasing the sample surface area and allowing for more thorough contact between the added water and the sample, improving the dissolution efficiency of active ingredients during decoction. This accelerates the dissolution of active ingredients and increases the content of trace elements in the decoction. Accurately weigh 1.0-3.0g of coarse powder and place it in an Erlenmeyer flask, precisely controlling the sample volume to ensure a suitable concentration in the reaction system during subsequent decoction, facilitating accurate determination of trace element content. This ensures the concentration of trace elements in the decoction remains within the instrument's detection linear range, improving the reliability of the detection results. Add 50ml of water, seal tightly, and weigh. Water is used as a solvent to extract the effective components, including trace elements, from the dried snake sample. An appropriate amount of water ensures a suitable concentration of the decoction, facilitating subsequent filtration and concentration. This extract provides raw material for determining the trace element content in the decoction. After standing for 0.5–1.5 hours, connect a reflux condenser to fully soak the dried snake sample in water, allowing the effective components to initially dissolve and improving decoction efficiency. This accelerates the dissolution of effective components and increases the content of trace elements in the decoction. Heat to boiling and maintain a gentle boil for 0.5–1.5 hours. Heating allows the effective components in the water to fully dissolve, simulating the extraction process during actual decoction. The 0.5–1.5 hour decoction time is optimized to ensure sufficient dissolution of effective components without causing damage or loss due to excessive time. This extracts the effective components, including trace elements, from the dried snake sample, providing a suitable solution for determining the trace element content in the decoction. After cooling, remove the conical flask, seal it tightly, weigh it, replenish the lost weight with water, and shake well to ensure accurate volume of the decoction and avoid errors caused by water evaporation; ensure accurate concentration of the filtrate for subsequent precise determination of trace element content. Filter the decoction through a dry filter to remove impurities and insoluble components, obtaining a clear filtrate for subsequent concentration and digestion operations; improve the purity of the filtrate and reduce interference from impurities in subsequent detection.

[0013] Accurately measure 20-30 ml of the filtrate and place it in a high-pressure, high-temperature microwave digestion vessel. Precisely control the volume of the filtrate to ensure a suitable concentration in the reaction system during subsequent concentration and digestion processes, facilitating accurate determination of trace element content. Ensure the concentration of trace elements in the concentrated solution is within the instrument's detection linear range, improving the reliability of the results. Evaporate to dryness to remove water from the filtrate, allowing the trace elements to exist in a more concentrated form, facilitating subsequent digestion operations. Increasing the concentration of trace elements in the digestion solution is beneficial for accurate determination of their content. Add 5-10 mL of 60-70% nitric acid. Nitric acid, as a strong oxidizing agent, effectively decomposes the organic components in the decoction, releasing the trace elements from the organic matrix. The 60-70% nitric acid concentration is an optimized choice, ensuring sufficient sample decomposition without causing violent reactions or increasing the difficulty of subsequent acid removal due to excessive concentration. This ensures that the trace elements in the sample are fully dissolved in the digestion solution, providing a suitable solution system for subsequent ICP-MS detection. Place it in a microwave digester and digest it according to the programmed temperature rise.

[0014] The significance and function of the four-stage gradient temperature-controlled microwave digestion process lies in ensuring a stable digestion process by gradually increasing the temperature, while ensuring the complete decomposition of organic components and the complete dissolution of trace elements in the digestion solution, thereby guaranteeing the accuracy and reliability of the detection results. The first stage, with a relatively low temperature, allows the sample to be initially heated, promoting contact and reaction between nitric acid and the sample, while avoiding violent reactions due to excessively high temperatures. The second stage, with an increased temperature, accelerates the decomposition reaction of organic matter in the sample by nitric acid, causing more organic components to be oxidized and decomposed, further releasing trace elements. The third stage, with a short period of high-temperature digestion, ensures the complete decomposition of organic components in the sample, while avoiding excessive heating that could lead to excessive pressure in the digestion vessel or sample loss. The fourth stage, maintaining a lower temperature for a longer period, allows residual nitric acid in the digestion solution to further evaporate and be removed, while simultaneously cooling the digestion solution to facilitate subsequent acid removal operations.

[0015] In the detection method of this invention, step S2. the four-stage gradient temperature-controlled digestion in microwave digestion includes the following operations:

[0016] Phase 1: Heating to 110–130℃ for 4–6 minutes, gradually increasing the temperature from room temperature to 110–130℃, initially heating the sample to promote contact and initiation of the reaction between nitric acid and the sample. At this stage, the temperature is relatively low to prevent boiling or splashing due to sudden high temperatures, while simultaneously allowing the nitric acid to begin decomposing the organic matter in the sample. Maintaining a constant temperature for 4–6 minutes ensures sufficient contact between the nitric acid and the sample, initiating the decomposition of organic components, while avoiding excessively high temperatures that could lead to an overly vigorous reaction, ensuring a stable reaction process.

[0017] The second stage involves heating to 140–160°C for 4–6 minutes, then further increasing the temperature to accelerate the reaction between nitric acid and the organic components in the sample, promoting the decomposition of more organic matter and releasing trace elements. The temperature is then maintained for 8–12 minutes to ensure that most of the organic components in the sample are decomposed, allowing the trace elements to fully dissolve in the digestion solution, while avoiding excessive reaction that could lead to excessive pressure inside the digestion vessel.

[0018] The third stage involves rapidly increasing the temperature to 180–200°C for 4–6 minutes. This high temperature helps to further decompose difficult-to-decompose organic components, ensuring that the organic matter in the sample is completely oxidized. The sample is then held at this temperature for 2–4 minutes. This short period of holding ensures complete digestion of the sample while avoiding excessive pressure or sample loss due to prolonged high temperatures.

[0019] Fourth stage: Maintain a constant temperature of 75-85℃ for 120-180 minutes. The temperature is then lowered to 75-85℃ and maintained for a longer period of time. This helps to remove excess nitric acid from the digestion solution and cools the solution, facilitating subsequent acid removal operations. The lower temperature in this stage helps to reduce the volatilization of nitric acid and ensures the stability of the digestion solution.

[0020] In this step, the four-stage gradient temperature-controlled digestion ensures that the organic components in the sample are fully decomposed through gradual heating, allowing trace elements to completely dissolve in the digestion solution. Controlling the heating rate and isothermal time prevents sample loss or splashing due to violent reactions. High-temperature, short-time digestion ensures complete oxidation of difficult-to-decompose organic components. The final low-temperature, long-time treatment helps remove excess nitric acid, reducing interference with subsequent detection. This optimized digestion procedure ensures accurate trace element content in the digestion solution, providing a reliable sample for ICP-MS detection.

[0021] In the detection method of this invention, step S2, cooling the digestion vessel to room temperature during microwave digestion ensures safe operation and avoids hazards such as burns or splashes during the transfer of the high-temperature digestion solution; it also stabilizes the digestion solution for subsequent transfer and processing. Then, the digestion solution is transferred to an acid removal device and subjected to acid removal at 130–140°C for 15–20 minutes; this removes residual nitric acid from the digestion solution, reducing interference from nitric acid in subsequent detections and preventing chemical reactions or loss of trace elements due to the presence of nitric acid; it improves the accuracy of the detection results, ensuring that the trace element content in the digestion solution truly reflects the actual content in the sample. The temperature range of 130–140°C effectively removes nitric acid without causing loss of trace elements or other adverse reactions due to excessively high temperatures. The 15–20 minutes ensures sufficient removal of nitric acid while avoiding overheating that could lead to changes or loss of trace element concentrations in the digestion solution. Before volume adjustment, wash the digestion vessel walls 3-5 times with ultrapure water, and combine the washings into a 50mL volumetric flask. This ensures that any residual trace elements on the digestion vessel walls are completely collected, improving the accuracy of the detection results and avoiding low results due to trace element loss. This process ensures that the trace elements in the digestion solution are transferred to the volumetric flask as completely as possible, guaranteeing the integrity of the sample. Multiple washes further remove residues from the vessel walls, improving the recovery rate and ensuring complete collection of trace elements. Dilute to the mark with water, shake well, and dilute the digestion solution to a suitable concentration to match the detection linearity range of the inductively coupled plasma mass spectrometer (ICP-MS). This also ensures a uniform distribution of trace elements in the solution, guaranteeing the accuracy and repeatability of the detection results. This provides a suitable concentration of sample solution for ICP-MS detection, improving the sensitivity and reliability of the detection. Shaking well ensures a uniform distribution of trace elements in the solution, avoiding detection errors caused by uneven concentration and ensuring that each injection accurately reflects the actual content of trace elements in the sample.

[0022] This invention provides an application of a trace element analysis method for the antithrombotic activity of *Spodoptera litura*, an application that is highly innovative and practical, offering a completely new perspective and approach to developing the medicinal value of *Spodoptera litura*. The application of this invention's trace element analysis method for the antithrombotic activity of *Spodoptera litura* includes the following:

[0023] I. Elemental Analysis-Driven Screening of Active Substances: Comprehensive analysis of trace elements in *Spodoptera litura* (red-banded snake) can identify bioactive elements, providing a foundation and direction for subsequent screening of substances with antithrombotic activity. Different trace elements participate in various physiological processes in organisms, and some elements are closely related to key aspects of thrombus formation such as blood coagulation and platelet aggregation. For example, copper is a component of many oxidases and participates in the intrinsic coagulation pathway; zinc participates in regulating vascular endothelial cell function, etc. Elemental analysis can quickly identify these potential active elements, thereby narrowing the screening scope and improving screening efficiency.

[0024] II. Element-Activity Correlation Validation: After screening for substances with antithrombotic activity, further validation of the correlation between trace elements and antithrombotic activity can be achieved through a series of in vitro and in vivo experiments. For example, in in vitro experiments, the effects of different concentrations of *Smilax china* extract on platelet aggregation, blood clotting time, and other indicators can be observed, and the role of specific trace elements in these processes can be analyzed in conjunction with the results of elemental analysis. In in vivo experiments, animal models, such as zebrafish thrombosis models, can be used to evaluate the effect of *Smilax china* extract on thrombus formation, and the contribution of trace elements can be explored through appropriate detection methods. This correlation validation helps to deepen the understanding of the material basis and mechanism of action of *Smilax china*'s antithrombotic activity, providing a scientific basis for the development of effective antithrombotic agents.

[0025] III. Development and Application of Antithrombotic Agents: Based on the results of elemental analysis and activity verification, antithrombotic agents using *Spodoptera litura* (red-banded snake) as a raw material can be developed, including determining the dosage form, dosage, and preparation process. For example, different dosage forms such as tablets, capsules, and injections of *Spodoptera litura* extract can be developed to meet different clinical needs. Simultaneously, a reasonable dosage range can be determined based on the content and activity of trace elements to ensure the safety and efficacy of the agent. Furthermore, the quality control standards, stability, and pharmacokinetic properties of the agent can be further studied to ensure its clinical application. This application model, from elemental analysis to activity verification to agent development, can fully explore the medicinal value of *Spodoptera litura*, providing new ideas and resources for the research and development of antithrombotic drugs.

[0026] In this invention, the elemental analysis-driven active substance screening operation is a crucial starting point for realizing the antithrombotic activity of red-banded snake. Through precise detection and quantitative screening, combinations of trace elements with potential antithrombotic activity are identified, providing a clear direction for subsequent research and development of antithrombotic agents.

[0027] In the application of this invention, the elemental analysis-driven screening of active substances includes the following operations: using the method for detecting trace elements in *Spodoptera litura* as described in claim 1, 25 trace elements in *Spodoptera litura* are comprehensively determined, and their content data are obtained. The quantitative screening criteria for antithrombotic elements are: selenium (Se) ≥ 1.5 mg / kg, chromium + vanadium (Cr + V) ≥ 4.5 mg / kg, arsenic (As) ≤ 2.0 mg / kg, and cadmium (Cd) 0.05 mg / kg. Based on previous research and traditional medicinal experience with *Spodoptera litura*, the screening criteria for antithrombotic elements are set as: selenium (Se) content ≥ 1.5 mg / kg. Selenium has antioxidant, anti-inflammatory, and vascular endothelial cell function regulating effects, which are closely related to the prevention of thrombosis. Through its antioxidant effect, selenium can reduce vascular endothelial cell damage and inhibit platelet aggregation, thereby reducing the risk of thrombosis; at the same time, the anti-inflammatory activity of selenium helps to reduce vascular inflammatory response, further maintain vascular health, and reduce the possibility of thrombosis. Therefore, setting a lower limit for selenium content can preliminarily screen for red-banded snake samples with potential antithrombotic activity, providing an important basis for subsequent research and development of antithrombotic agents. Chromium (Cr) and vanadium (V) synergistically regulate lipid metabolism and reduce thromboxane A2 synthesis; arsenic (As) > 2.0 mg / kg antagonizes Se activity and should be discarded directly; cadmium (Cd) ≤ 0.05 mg / kg avoids nephrotoxicity.

[0028] In the application of this invention, the element-activity correlation verification is a crucial step in confirming the relationship between trace elements and antithrombotic activity in *Spodoptera litura*. In the application of this invention, the element-activity correlation verification includes the following operations:

[0029] A1. Construction of a thrombosis model: Transgenic zebrafish embryos Tg (LCR: eGFP) (green fluorescent protein labeled red blood cells) were used, and 1-3 μM phenylhydrazine (PHZ) was added to induce thrombosis. PHZ can oxidize and damage the red blood cell membrane, leading to hemolysis, releasing hemoglobin and cell debris, thereby activating the coagulation system, promoting platelet aggregation and fibrin deposition, and finally forming a thrombus, simulating human thrombosis.

[0030] A2. Grouped administration: Using the method for detecting trace elements in *Spodoptera litura* as described in claim 1, the content of antithrombotic elements (Se, Cr, V) was determined. Based on the content results, a high-element content sample group, a low-element content sample group, and a positive control group were set up. The positive control group may use a drug with known antithrombotic activity, such as cryptotanshinone.

[0031] A3. Dynamic observation: The main vein site of zebrafish is photographed at a speed of 100 frames per second to quantify the number of red blood cells passing through; through high-speed shooting, the flow of red blood cells in blood vessels can be clearly observed and recorded, especially the blood stagnation phenomenon after thrombosis.

[0032] A4. Data Correlation: Compare the red blood cell count and element content (Se / Cr / V) of different groups and calculate the correlation coefficient; calculating the correlation coefficient can quantify the relationship between element content and red blood cell count, thereby making a preliminary judgment on the correlation between element content and antithrombotic activity.

[0033] A5. Statistical Validation: One-way ANOVA was performed using SPSS to confirm that the element content was significantly positively correlated with the antithrombotic activity. One-way ANOVA can test whether the differences between different groups are statistically significant, thus verifying the significant positive correlation between the element content and the antithrombotic activity.

[0034] Through the above steps, the correlation between trace elements and antithrombotic activity in red-banded snake can be scientifically verified, providing experimental evidence for the development of antithrombotic agents.

[0035] In the application of this invention, the development of the antithrombotic agent includes a red-banded snake antithrombotic agent. The development of this agent fully utilizes the detection results of trace elements in the red-banded snake, ensuring the safety and efficacy of the agent. This agent comprises a red-banded snake alcohol extract and pharmaceutical excipients in a mass ratio of 4–9:1. This ratio has been scientifically validated and ensures that the agent maintains stability and safety while possessing good antithrombotic activity. The method for detecting trace elements in *Spodoptera litura* as described in claim 1 was used to determine the antithrombotic elements, wherein the trace elements selenium (Se) ≥ 1.5 mg / kg, arsenic (As) ≤ 2.0 mg / kg, cadmium (Cd) ≤ 0.05 mg / kg, and chromium + vanadium (Cr + V) ≥ 4.5 mg / kg and are positively correlated with antithrombotic activity; selenium is an important antioxidant that can effectively prevent thrombosis; the arsenic content is strictly controlled to ensure the safety of the preparation; similarly, the cadmium content is strictly limited to avoid its harmful effects on the human body; chromium and vanadium are positively correlated with antithrombotic activity, and ensuring their content helps to improve the efficacy of the preparation.

[0036] The preparation method of the red snake ethanol extract includes:

[0037] B1. Dry and pulverize the sample of *Synonymus alatus* or *Synonymus alatus* fetus to increase the surface area, which is beneficial for subsequent decoction extraction. Take the coarse powder and add 50-70% (v / v) dilute ethanol at a material-to-liquid ratio of 1:8-12. The concentration of dilute ethanol is chosen to reduce the dissolution of impurities while extracting the active ingredients. Decoction 1-3 times to ensure that the active ingredients can be fully dissolved. Combine the supernatants for further processing.

[0038] B2. The solvent in the supernatant is removed by vacuum evaporation to obtain the alcohol extract. Vacuum evaporation can be carried out at a lower temperature, effectively preventing the loss of heat-sensitive components. The extract is then dried under vacuum at room temperature to obtain the red snake alcohol extract. This drying method ensures that the product retains its active ingredients while maintaining good stability and flowability.

[0039] The red-banded snake alcohol extract prepared by the above method, combined with strict quality control standards, ensures the safety and efficacy of the antithrombotic preparation in the prevention and treatment of thrombotic diseases. The development of this preparation not only brings new options to the field of antithrombotic drugs but also further explores the modern application value of the red-banded snake, a traditional medicinal resource.

[0040] In summary, the present invention has the following beneficial effects:

[0041] 1. A precise and efficient method for detecting trace elements in *Spodoptera litura* is provided, capable of comprehensively analyzing the content of 25 trace elements, providing a scientific basis for the evaluation of the medicinal value and quality control of *Spodoptera litura*. This method employs steps such as sample pretreatment of dried snake or decoction, microwave digestion, acid removal and volume adjustment, and ICP-MS detection, ensuring the accuracy and reliability of the detection results.

[0042] 2. By using elemental analysis to drive the screening of active substances, the screening criteria for antithrombotic elements are quantified, and the combination of trace elements with potential antithrombotic activity is quickly identified, providing a clear direction for the development of antithrombotic drugs, greatly improving screening efficiency, and saving research and development time and costs.

[0043] 3. A thrombosis model was constructed and the element-activity correlation was verified. Thrombosis was induced by transgenic zebrafish embryos and phenylhydrazine. Combined with the determination of trace element content in red-banded snake and dynamic observation of erythrocytes, the significant positive correlation between trace elements and antithrombotic activity was scientifically verified, and the material basis and mechanism of action of antithrombotic activity of red-banded snake were revealed in depth.

[0044] 4. A safe and effective antithrombotic preparation of *Smilax china* has been developed, comprising *Smilax china* ethanol extract and pharmaceutical excipients in a mass ratio of 4–9:1, with the antithrombotic element content in the *Smilax china* ethanol extract meeting stringent standards. The development of this preparation provides a new option in the field of antithrombotic drugs, fully explores the medicinal value of *Smilax china*, and has broad market prospects and significant scientific importance. Attached Figure Description

[0045] Figure 1 This is a schematic diagram showing the safety test results of the red-banded snake extract;

[0046] Figure 2 This is a schematic diagram of the main vein location;

[0047] Figure 3This is a schematic diagram showing the number of red blood cells at the main vein site in a zebrafish embryo.

[0048] In the attached diagram, csct represents the alcohol extract and csst represents the water extract. Detailed Implementation

[0049] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0050] I. Elemental Analysis-Driven Screening of Active Substances

[0051] 1. Materials

[0052] 1.1 The medicinal materials, including the red-banded snake and the red-banded fetal snake, were mainly collected from the market and provided by breeding farms. They were identified as red-banded snakes. For details of the sample acquisition, please refer to Table 1.

[0053] Table 1 Summary of information on samples of Red-banded Snake and Red-banded Fetal Snake

[0054]

[0055] 1.2 Reagents

[0056] 25-element mixed standard solution: 2000.00 μg / mL calcium (Ca), potassium (K), magnesium (Mg), sodium (Na); 500.00 μg / mL aluminum (Al), iron (Fe); 100.00 μg / mL strontium (Sr); 50.00 μg / mL boron (B), barium (Ba), copper (Cu), manganese (Mn), titanium (Ti), zinc (Zn); 5.00 μg / mL arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), nickel (Ni), lead (Pb), selenium (Se), thallium (Tl), vanadium (V); 0.50 μg / mL molybdenum (Mo), antimony (Sb), tin (Sn) (batch number: P2-MEB) 678634, BF-20190564-01) Inorganic Ventures, USA; Internal standard elemental solutions: (10.00 μg / mL) Germanium (Ge), Indium (In), Bismuth (Bi), Lithium (Li6), Scandium (Sc), Terbium (Tb), Yttrium (Y) (Batch No.: N2-MEB662224) Inorganic Ventures, USA; 65% Nitric Acid (Analytical Grade, Merck & Co., Ltd., Germany); 30% Hydrogen Peroxide (Superior Grade, Nanjing Chemical Reagent Co., Ltd.); Ultrapure Water (In-house).

[0057] 1.3 Preparation of standard solutions: Accurately measure 0.05, 0.10, 0.20, 0.25, 0.50 and 1.00 mL of 25 mixed standard solutions and place them in 50 mL volumetric flasks. Dilute with 10% nitric acid solution to the mark to obtain the standard solutions.

[0058] 1.4 Preparation of internal standard solution

[0059] Accurately measure 1 mL of standard solutions of germanium (Ge), indium (In), bismuth (Bi), lithium (Li), scandium (Sc), terbium (Tb), and yttrium (Y), place them in a 100 mL volumetric flask, and dilute with water to the mark.

[0060] 2. Elemental analysis methods

[0061] 2.1 Sample Pretreatment: After drying the dried snake sample at 60℃ for 48 hours, the sample was crushed; 0.2g of coarse powder (passed through a No. 2 sieve) was accurately weighed and placed in a high-pressure and high-temperature microwave digestion vessel, and 7mL of 65% nitric acid was added sequentially. The sample was left to stand overnight; then it was placed in a microwave digester and digested according to the programmed temperature rise.

[0062] Pretreatment of the decoction sample: Accurately weigh 2.0g of the above-mentioned coarse powder of red-banded snake and red-banded fetal snake, place it in an Erlenmeyer flask, accurately add 50ml of water, seal tightly, weigh, let stand for 1 hour, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 1 hour; after cooling, remove the Erlenmeyer flask, seal tightly, weigh, replenish the lost weight with water, shake well, and filter through a dry filter; accurately measure 25ml of the filtrate, place it in a high-pressure and high-temperature microwave digestion vessel, evaporate to dryness, add 7mL of 65% nitric acid in sequence, place in a microwave digester, and digest according to the programmed temperature rise.

[0063] 2.2 Microwave Digestion

[0064] The microwave digestion procedure in Table 2 is followed by a four-stage gradient temperature-controlled digestion. After digestion, acid removal and volume adjustment are performed. After complete digestion, the digestion vessel is cooled to room temperature, and the digestion solution is transferred to the acid removal device, where acid is removed at 135°C for 15 minutes. Before volume adjustment, the vessel wall is washed three times with ultrapure water, and the washings are combined into a 50mL volumetric flask, diluted to the mark with water, and shaken well.

[0065] Table 2 Microwave Digestion Procedure

[0066]

[0067] 2.3 IPC-MS detection

[0068] IPC-MS employed a fully quantitative analysis mode, using inductively coupled plasma mass spectrometry to determine 25 elements (B, Na, Mg, Al, K, Ca, Ti, Cr, V, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Sn, Sb, Ba, Tl, and Pd). During the analysis, the internal standard sample tube remained immersed in the internal standard solution throughout the process, with the standard solution concentration increasing sequentially. A standard curve was plotted with the measured values ​​on the ordinate and the concentrations on the abscissa. Sample solutions were analyzed sequentially, and the corresponding concentrations were calculated from the standard curve based on the instrument analysis results.

[0069] 3. Results

[0070] 3.1 Data Processing

[0071] Statistical analysis of the raw data was performed using Excel 2010 software, and one-way ANOVA and correlation analysis were performed using SPSS 20.0.

[0072] 3.2 Methodological Validation

[0073] Linear regression was performed with the mass concentration of each element as the x-axis and the instrument response value as the y-axis. The regression equation, linear concentration range, correlation coefficient, and detection limit are shown in Table 3. Table 3 shows that the 25 elements exhibit good linearity within the concentration range, with a correlation coefficient r ≥ 0.9997. The precision of six consecutive injections is between 0.28% and 1.04%, indicating good instrument precision. The repeatability of six analyses of the same sample is between 0.07% and 0.96%, indicating good repeatability. Injections at 0, 2, 4, 8, 12, and 24 hours, with calculated RSD values ​​for each element's content between 0.24% and 0.96%, indicate good stability of each element in the sample solution over 24 hours. Therefore, this method can conveniently, rapidly, and sensitively detect 25 inorganic elements in *Spodoptera litura* and *Spodoptera litura* simultaneously, and is suitable for determining the inorganic element content in these two species.

[0074] Table 3 Methodological Validation of Elemental Analysis

[0075]

[0076] 3.3 Content determination results

[0077] The contents of 25 inorganic elements in the medicinal materials of *Smilax china* and *Smilax china* and their decoctions were analyzed. The contents of each element in *Smilax china* and *Smilax china* and their decoctions were calculated by standard curve based on the IPC-MS response values. The results are shown in Tables 4 and 5.

[0078] Table 4. Results of determination of the content of 25 inorganic elements (mg / kg) in red-banded snake and red-banded fetal snake.

[0079]

[0080]

[0081] Note: "#" indicates that the content exceeds the instrument's detection range.

[0082] Table 5. Results of determination of 25 inorganic elements (mg / kg) in the decoction of *Synonymus alatus* and *Synonymus alatus*.

[0083]

[0084]

[0085] 4. Conclusion

[0086] The data in Tables 4 and 5 show that *Smilax china* and its decoction are rich in various trace elements closely related to antithrombotic activity, such as selenium (Se), with a content ranging from 0.643 to 2.130 mg / kg, and some samples reaching relatively high levels; the contents of chromium (Cr) and vanadium (V) are also considerable, ranging from 1.460 to 6.528 mg / kg and 1.523 to 6.745 mg / kg, respectively. The presence and content range of these elements in *Smilax china* samples indicate that they play an important role in the antithrombotic activity of *Smilax china*. Combined with the screening criteria set in the antithrombotic activity verification experiment (selenium ≥ 1.5 mg / kg, chromium + vanadium ≥ 4.5 mg / kg), *Smilax china* samples that meet the criteria showed significant antithrombotic effects in the experiment, further confirming the correlation between these trace elements and the antithrombotic activity of *Smilax china*. Although the content of trace elements in the decoction is relatively low, it still contains a certain amount of elements such as selenium, chromium, and vanadium, indicating that the decoction process can effectively extract these active ingredients, providing a scientific basis for the development of antithrombotic preparations based on red-banded snake.

[0087] A comprehensive analysis of 25 trace elements in samples from *Syngonium rubrum* and *Syngonium fetus* successfully identified a key combination of trace elements with potential antithrombotic activity: selenium (Se) ≥ 1.5 mg / kg, chromium (Cr) + vanadium (V) ≥ 4.5 mg / kg, while strictly controlling arsenic (As) 2.0 mg / kg and cadmium (Cd) ≤ 0.05 mg / kg. This screening standard was established based on a deep understanding of traditional medicinal experience and modern pharmacological research on *Syngonium rubrum*. The antioxidant and anti-inflammatory properties of selenium, and the regulatory effects of chromium and vanadium on lipid metabolism, are closely related to antithrombotic activity. Experimental data showed that *Syngonium rubrum* samples meeting the above criteria exhibited significant antithrombotic potential, providing a precise direction for subsequent activity verification and formulation development. This not only greatly improved research efficiency but also provided a scientific and efficient method for screening antithrombotic active substances from *Syngonium rubrum*, laying a solid foundation for further exploration of its application in the antithrombotic field.

[0088] II. Element-activity correlation verification

[0089] 1. Experimental Materials

[0090] 1.1 Reagents and Tests

[0091] The red-banded snake medicinal material was purchased from Moganshan Snake Industry Co., Ltd. in Deqing County. It was identified as *Dinodon rufozonatum* (Cantor), belonging to the Colubridae family and the *Dinodon* genus. Other ingredients included: phenylhydrazine (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), cryptotanshinone (110852-201807, China National Institutes for Food and Drug Control), proteinase K (Roche Diagnostics GmbH), methylene blue (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), PTU (1-phenyl-2-thiourea) (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), ethanol, sodium chloride, KCl, potassium chloride, calcium chloride, magnesium sulfate, etc. (Sinopharm Chemical Reagent Co., Ltd.), and Wahaha purified water.

[0092] 1.2 Laboratory Animals

[0093] The experimental zebrafish were transgenic zebrafish Tg (LCR: eGFP) with erythrocytes labeled with green fluorescent protein, and the embryos were cultured in E3 buffer.

[0094] 2. Experimental Methods

[0095] 2.1 Preparation of Red-banded Snake Extract

[0096] Preparation of the water extract of red-banded snake: Weigh the dried coarse powder of red-banded snake, add water at a material-to-liquid ratio of 1:10, decoct, repeat twice, combine the supernatants, remove water by vacuum evaporation to obtain water extract paste, and dry under vacuum at room temperature to constant weight to obtain the water extract of red-banded snake.

[0097] Preparation of red snake alcohol extract: Weigh the dried coarse powder of red snake, add dilute ethanol at a material-to-liquid ratio of 1:10, decoct, repeat twice, combine the supernatants, remove the solvent fraction by vacuum evaporation to obtain alcohol extract paste, and dry under vacuum at room temperature to obtain red snake alcohol extract.

[0098] 2.2 Safety Experiment

[0099] The extracts of *Syngonium fasciatus* were dissolved in water to prepare ethanolic extract (CSCT) solutions of 0.25, 0.5, 1.0, 2.0, and 4.0 μl / ml and aqueous extract (CSST) solutions of 50, 100, 200, 400, and 600 μg / ml.

[0100] Zebrafish embryos were collected in 10cm culture dishes and E3 buffer containing methylene blue was added. After 12 hours, E3 buffer containing 200 μM PTU was added to inhibit melanin production for subsequent observation. When the embryos reached 56 hpf, they were transferred to 24-well plates with 8 embryos per well and 1 ml of liquid per well. If the embryos did not rupture, the egg membrane was removed using proteinase K. Red-banded snake extract solution was added in 10 μl amounts. After 24 hours of administration, the number of dead embryos was counted.

[0101] 2.3 Thrombosis Model Establishment and Drug Intervention: Embryos were collected in 10cm culture dishes and E3 buffer containing methylene blue was added. After 12 hours, E3 buffer containing 200 μM PTU was added to inhibit melanin production for subsequent experimental observation. When the embryos reached 56 hpf, they were transferred to 24-well plates, with 8 embryos per well and 1 ml of liquid. Control, modeling, positive control, and drug treatment groups were established. If the embryos did not rupture, proteinase K was used to remove the membrane. After 24 hours of pre-protection with drug treatment, the embryos were rinsed three times with E3 buffer containing PTU, and then 2 μM phenylhydrazine (PHZ) was added to initiate the model. Subsequent observation experiments were conducted 14 hours after modeling stimulation. The control group was a blank control with no treatment; the modeling group used PHZ for modeling; and the drug treatment group received 10 μl of 1 μg / ml cryptotanshinone for pre-protection for 24 hours, rinsed clean, and then modeled. Based on the safety test results, the drug administration group was given 10 μl of 0.1, 0.2, and 0.25 μl / ml red snake alcohol extract (CSCT) solution and 50, 100, and 200 μg / ml red snake water extract (CSST) solution, respectively, and the procedure was the same as that of the drug administration group.

[0102] 2.4 Dynamic observation of zebrafish erythrocytes

[0103] Transgenic zebrafish Tg (LCR:eGFP) embryos with green fluorescent protein-labeled erythrocytes were anesthetized and transferred to a 24-well plate cover at one well per well. Images were automatically captured using a Leica camera at 100 frames per second. After imaging, the main vein site at the same location in each zebrafish embryo was selected for observation.

[0104] 3. Experimental Results

[0105] 3.1 Security

[0106] After adding red-banded snake extract solution to zebrafish embryo culture medium for 24 hours, embryo mortality was recorded to determine the safe concentration range for red-banded snake. The experimental results are as follows: Figure 1 As shown. The safe concentration of the alcoholic extract of *Syngonium argenteum* is 0.25 μl / ml, and concentrations of 0.5 μl / ml and above can easily lead to the death of all zebrafish embryos; the safe concentration of the aqueous extract of *Syngonium argenteum* is 200 μg / ml, and concentrations of 400 μg / ml and above can easily lead to the death of all zebrafish embryos.

[0107] 3.2 Antithrombotic model

[0108] Zebrafish embryos were divided into a control group, a model group, a positive control group, and a treatment group. The positive control group and the treatment group received pre-protection treatment for 24 hours, followed by modeling and stimulation for 14 hours. The presence of red blood cells through the main vein of the zebrafish embryos was then photographed. The results are as follows: Figure 2As shown in the figure, in the control group, red blood cells in the main vein of zebrafish embryos were clearly visible flowing within the blood vessels; in the model group, red blood cells were visible, but blood flow was stagnant due to thrombosis, indicating that the thrombosis model was successfully established. In both the positive control group and the treated group, red blood cells were clearly visible flowing within the blood vessels, indicating that the extract of *Smilax china* has the same efficacy as cryptotanshinone, possessing antithrombotic effects.

[0109] The experiment used a Leica camera to automatically capture images at 100 frames per second, comparing the number of red blood cells per unit time to determine the intensity of antithrombotic activity. The experimental results are as follows: Figure 3 As shown in the figure, the control group had 138 red blood cells, significantly higher than the model group, while the positive control group had 35 red blood cells, also higher than the model group. The red blood cell counts in the groups with *Smilax china* alcohol extract concentrations of 0.1 μl / ml and 0.25 μl / ml, and the groups with *Smilax china* water extract concentrations of 50 μg / ml and 100 μg / ml, were significantly higher than the positive control group, indicating that *Smilax china* possesses antithrombotic activity similar to cryptotanshinone, but with stronger antithrombotic efficacy.

[0110] 4. Conclusion

[0111] according to Figure 1 , Figure 2 , Figure 3 The analysis results show that the extract of *Smilax china* exhibited good tolerability in safety experiments, without causing significant embryonic death, indicating high safety at the experimental concentration. In the thrombosis model, compared with the model group, the extract of *Smilax china* significantly increased the number of erythrocytes at the main venous site of zebrafish, demonstrating good antithrombotic activity. Its effect was comparable to or even better than that of the positive control drug cryptotanshinone, proving that the extract of *Smilax china* has significant antithrombotic efficacy. These results fully confirm the significant positive correlation between specific trace elements in *Smilax china* and antithrombotic activity, providing a scientific basis for the development of antithrombotic preparations made from *Smilax china*.

[0112] Specifically, in a transgenic zebrafish thrombosis model, experimental results showed that compared with the model group, the number of erythrocytes at the main venous site of zebrafish treated with *Smilax china* extract significantly increased. Furthermore, the ethanol extract of *Smilax china* performed better than the water extract, especially at concentrations of 0.1 μl / ml and 0.25 μl / ml, where its antithrombotic activity even surpassed that of the positive control drug, cryptotanshinone. Correlation coefficient calculations and one-way ANOVA further confirmed a significant positive correlation between the content of trace elements such as selenium, chromium, and vanadium in *Smilax china* and its antithrombotic activity (correlation coefficient r = 0.92, P < 0.05). This result strongly validates the precise association between specific trace elements in *Smilax china* and its antithrombotic activity, providing direct evidence for the scientific validity and rationality of the antithrombotic effect of *Smilax china*, and also providing a crucial basis for the subsequent development of novel antithrombotic drugs.

[0113] III. Development and Application of Antithrombotic Agents

[0114] 1. Preparation of antithrombotic agents.

[0115] Example 1

[0116] Composition: The main component is the alcohol extract of red-chain snake, specifically prepared from a sample of red-chain fetus snake.

[0117] Preparation method:

[0118] Step 1: Take 500g of red-banded fetal snake sample, dry it at 60℃ for 48 hours, and then crush it into coarse powder; take 200g of coarse powder, add 60% dilute ethanol at a material-to-liquid ratio of 1:10, decoct twice, 2 hours each time, and combine the supernatant.

[0119] Step 2: Remove the solvent from the supernatant by vacuum evaporation to obtain an alcohol extract. Dry the extract under vacuum at room temperature to obtain approximately 20g of red snake alcohol extract.

[0120] Step 3: Take 16g of the prepared red snake alcohol extract, add 4g of pharmaceutical excipient starch, mix well; make 100 capsules, each containing 0.16g of red snake alcohol extract and 0.04g of pharmaceutical excipient. The resulting capsules are the red snake antithrombotic preparation.

[0121] Step 4: Using the method for detecting trace elements in red-banded snake of the present invention, the content of antithrombotic elements in the preparation was determined, and the results are as follows: Selenium (Se): 2.0 mg / kg, Arsenic (As): 1.5 mg / kg, Cadmium (Cd): 0.03 mg / kg, Chromium + Vanadium (Cr + V): 5.0 mg / kg; the test results show that the content of antithrombotic elements in the preparation meets the standard.

[0122] Comparative Example 1

[0123] Aspirin formulations contain aspirin, a classic antithrombotic drug that inhibits platelet aggregation by irreversibly inhibiting cyclooxygenase and reducing the production of thromboxane A2.

[0124] Preparation method:

[0125] The aspirin raw material is mixed evenly with an appropriate amount of excipient starch, and then made into a capsule dosage form through conventional pharmaceutical processes. Each capsule contains 0.16g of aspirin raw material and 0.04g of excipient.

[0126] 2. Animal experiments

[0127] 2.1 Laboratory Animals

[0128] SPF-grade ICR mice (male, 18–22 g) were divided into 6 groups (n = 10), including: blank control group (physiological saline), thrombosis model group (physiological saline), positive control group (aspirin 25 mg / kg), low-dose group of red-banded snake preparation (0.8 g / kg, prepared according to Example 1), medium-dose group of red-banded snake preparation (1.6 g / kg), and high-dose group of red-banded snake preparation (3.2 g / kg).

[0129] 2.2 Experimental Methods

[0130] 2.2.1 Dosing regimen

[0131] Administer the medication by gavage for 14 consecutive days, once daily:

[0132] Blank group / model group: equal volume of physiological saline; positive group: aspirin suspension; formulation group: capsule contents (red snake extract + excipients) suspended in physiological saline.

[0133] 2.2.2 Establishment of the thrombosis model

[0134] One hour after the last administration, mice were anesthetized (sodium pentobarbital 40 mg / kg, ip) to establish a carotid artery thrombosis model, including: separating the left common carotid artery, inserting a laser probe to monitor baseline blood flow (T0), covering it with 10% FeCl3 filter paper (1×2 mm) to induce thrombosis, and recording the time when blood flow dropped to 50% T0 (occlusion time, OT).

[0135] 2.3 Sample Collection and Testing

[0136] The collected samples were tested, including for thrombus weight, coagulation function, platelet aggregation rate, serum element levels, and thromboxane B2.

[0137] The collected samples underwent the following tests:

[0138] Thrombus weight: Weigh the thrombus to assess the extent of thrombus formation.

[0139] Coagulation function: measures indicators such as blood clotting time, prothrombin time (PT), and activated partial thromboplastin time (APTT).

[0140] Platelet aggregation rate: Platelet aggregation rate is measured using a platelet aggregometer to assess platelet activity.

[0141] Serum element levels: The levels of antithrombotic elements (Se, Cr, V) in serum were detected by ICP-MS.

[0142] Thromboxane B2 (TXB2): Serum thromboxane B2 levels were detected using enzyme-linked immunosorbent assay (ELISA) to assess related indicators of thrombosis.

[0143] 3. Test Results

[0144] Table 6 Comparison of antithrombotic related indicators in mice from different groups

[0145]

[0146] Note: Compared with the thrombosis model group, ★P<0.05; indicating that there are significant differences between the drug administration groups (positive control group and different doses of red snake preparation group) and the thrombosis model group in terms of thrombus weight, blood clotting time, prothrombin time, activated partial thromboplastin time, platelet aggregation rate, serum trace element level and thromboxane B2, which are statistically significant.

[0147] 4. Conclusion

[0148] Table 6 shows the results of different groups of mice in the detection of antithrombotic related indicators. The specific data analysis is as follows:

[0149] In the thrombus weight test, the model group had the highest thrombus weight, at 3.24±0.51mg, indicating successful thrombus formation. The positive control group had a significantly lower thrombus weight of 1.23±0.24mg, indicating the effectiveness of aspirin. In the red-banded snake preparation group, the thrombus weight gradually decreased with increasing dose, and the high-dose group had the best effect, at 1.08±0.27mg, indicating that the red-banded snake preparation has a significant antithrombotic effect.

[0150] In the blood clotting time recording, the model group had the shortest blood clotting time, at 3.2±0.6 min, indicating a hypercoagulable state. The positive control group had a prolonged blood clotting time of 5.4±0.9 min, reflecting the anticoagulant effect of aspirin. In the red-banded snake preparation group, the blood clotting time was prolonged in all dose groups, with the high-dose group reaching 5.7±1.0 min, indicating that the red-banded snake preparation can effectively prolong blood clotting time and prevent thrombosis.

[0151] In the prothrombin time (PT) and activated partial thromboplastin time (APTT) tests, the PT and APTT in the model group were shortened to 8.4±1.1s and 18.5±2.3s, respectively, indicating that the blood was in a hypercoagulable state. In the positive control group, the PT and APTT were prolonged to 10.2±1.3s and 23.1±2.7s, respectively, indicating that aspirin can improve the hypercoagulable state of the blood. In the red-banded snake preparation group, the PT and APTT of all dose groups were prolonged, with the high-dose group showing the most significant effect, with a PT of 11.4±1.6s and an APTT of 24.5±2.9s, indicating that the red-banded snake preparation can effectively regulate the blood coagulation process.

[0152] In the platelet aggregation rate test, the model group had the highest platelet aggregation rate, which was 78.9±6.3%, indicating that the platelets were highly active. The platelet aggregation rate in the positive control group decreased to 53.2±4.8%, reflecting the inhibitory effect of aspirin on platelet aggregation. The platelet aggregation rate in the red-banded snake preparation group decreased with increasing dose, with the high-dose group at 51.7±4.1%, indicating that the red-banded snake preparation can effectively inhibit platelet aggregation.

[0153] In the serum trace element level records, the contents of selenium (Se), chromium (Cr), and vanadium (V) in the model group were lower than those in the blank control group, at 0.62±0.09 μg / L, 0.15±0.03 μg / L, and 0.10±0.02 μg / L, respectively, due to the consumption of trace elements by thrombosis. In the positive control group, the contents of Se, Cr, and V were 0.78±0.10 μg / L, 0.20±0.03 μg / L, and 0.14±0.02 μg / L, respectively, which were higher than those in the model group but lower than those in the red-banded snake preparation group. In the red-banded snake preparation group, the contents of Se, Cr, and V in each dose group were higher than those in the model group and the positive control group. The high-dose group had Se at 1.18±0.18 μg / L, Cr at 0.36±0.06 μg / L, and V at 0.26±0.04 μg / L, indicating that the red-banded snake preparation can increase the content of antithrombotic trace elements in serum.

[0154] In the thromboxane B2 (TXB2) test, the model group had the highest TXB2 level, at 76.5±9.8 ng / mL, reflecting a high risk of thrombosis. The positive control group had a TXB2 level that decreased to 45.3±6.2 ng / mL, showing the inhibitory effect of aspirin on thrombosis. The TXB2 levels in all dose groups of the red snake preparation group were reduced, with the high-dose group at 39.5±5.7 ng / mL, indicating that the red snake preparation can effectively reduce thrombosis-related indicators.

[0155] Animal experiments have demonstrated that the *Smilax china* preparation of this invention exhibits significant antithrombotic effects, effectively reducing thrombus weight, prolonging blood clotting time, inhibiting platelet aggregation, and regulating coagulation function. Its ability to increase serum Se, Cr, and V levels further confirms the crucial role of trace elements in antithrombotic activity. Furthermore, the *Smilax china* preparation demonstrates good safety, without causing significant abnormalities in physiological indicators. In summary, the *Smilax china* preparation is a safe and effective antithrombotic agent. Its development brings new options to the field of antithrombotic drugs, fully explores the medicinal value of *Smilax china*, and has broad market prospects and significant scientific importance.

Claims

1. A method for detecting trace elements in red-banded snake, characterized in that, Includes the following steps: S1. Sample pretreatment: Dry the sample of red-banded snake or red-banded fetal snake, then crush it. Weigh the coarse powder and add 30 to 40 times its weight of 60 to 70% nitric acid. Let it stand overnight. S2. Microwave digestion: Four-stage gradient temperature-controlled digestion is adopted, followed by acid removal and volume adjustment after digestion; S3. ICP-MS detection: 25 elements (B, Na, Mg, Al, K, Ca, Ti, Cr, V, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Sn, Sb, Ba, Tl, Pd) were determined using inductively coupled plasma mass spectrometry.

2. The detection method according to claim 1, characterized in that, Step S1. Sample pretreatment, which is either pretreatment of dried snake samples or pretreatment of decoction samples, includes the following steps: S1-1. Pretreatment of dried snake samples: After drying the sample at 50-70℃ for 40-50 hours, pulverize it; accurately weigh 0.1-0.3g of coarse powder, place it in a high-pressure and high-temperature microwave digestion vessel, add 5-10mL of nitric acid with a mass percentage concentration of 60-70%, and let it stand overnight; place it in a microwave digester and digest it according to the programmed temperature rise. S1-2. Pretreatment of decoction samples: After drying the sample at 50-70℃ for 40-50 hours, pulverize it; accurately weigh 1.0-3.0g of coarse powder, place it in an Erlenmeyer flask, add 50ml of water, seal tightly, weigh, let stand for 0.5-1.5 hours, connect a reflux condenser, heat to boiling, and maintain a gentle boil for 0.5-1.5 hours; after cooling, remove the Erlenmeyer flask, seal tightly, weigh, replenish the lost weight with water, shake well, and filter through a dry filter; accurately measure 20-30ml of the filtrate, place it in a high-pressure, high-temperature microwave digestion vessel, evaporate to dryness, add 5-10mL of 60-70% nitric acid, place in a microwave digester, and digest according to the programmed temperature rise.

3. The detection method according to claim 1, characterized in that, Step S2. The four-stage gradient temperature-controlled microwave digestion includes the following operations: heating at 110–130℃ for 4–6 minutes, holding at 140–160℃ for 4–6 minutes, holding at 180–200℃ for 4–6 minutes, holding at 180–200℃ for 2–4 minutes, and holding at 75–85℃ for 120–180 minutes.

4. The detection method according to claim 1, characterized in that, Step S2. After the microwave digestion vessel is cooled to room temperature, transfer the digestion solution to the acid removal device and remove the acid at 130-140℃ for 15-20 minutes. Before making up to volume, wash the vessel wall with ultrapure water 3-5 times, combine the washings into a 50mL volumetric flask, dilute with water to the mark, and shake well.

5. An application of a trace element analysis method for red-banded snakes in antithrombotic activity, characterized in that, Includes the following: I. Elemental analysis drives the screening of active substances; II. Element-activity correlation verification; III. Development and application of antithrombotic agents.

6. The application according to claim 5, characterized in that, The elemental analysis-driven screening of active substances includes the following operations: using the method for detecting trace elements in red-banded snake as described in claim 1, 25 trace elements are determined; the quantitative screening criteria for antithrombotic elements are: selenium (Se) ≥ 1.5 mg / kg, chromium + vanadium (Cr + V) ≥ 4.5 mg / kg, arsenic (As) ≤ 2.0 mg / kg, and cadmium (Cd) ≤ 0.05 mg / kg.

7. The application according to claim 5, characterized in that, The element-activity correlation verification includes the following operations: A1. Constructing a thrombosis model: Transgenic zebrafish embryos were used, and 1–3 μM phenylhydrazine (PHZ) was added to induce thrombosis; A2. Grouped administration: The content of antithrombotic elements (Se, Cr, V) in the trace elements in the red-banded snake described in claim 1 was determined by using the method for detecting trace elements in the red-banded snake. A high-element content sample group, a low-element content sample group, and a positive control group were set up. A3. Dynamic observation: The main vein site of zebrafish was photographed at a speed of 100 frames per second to quantify the number of red blood cells passing through; A4. Data Correlation: Compare the red blood cell count and element content (Se / Cr / V) of different groups and calculate the correlation coefficient; A5. Statistical validation: One-way ANOVA using SPSS confirmed that the element content was significantly positively correlated with the antithrombotic activity.

8. The application according to claim 5, characterized in that, The development and application of the antithrombotic agents includes a red-banded snake antithrombotic agent, which comprises red-banded snake alcohol extract and pharmaceutical excipients in a mass ratio of 4 to 9:

1. The antithrombotic elements are determined by the method for detecting trace elements in red-banded snake as described in claim 1, wherein the trace elements selenium (Se) ≥ 1.5 mg / kg, arsenic (As) ≤ 2.0 mg / kg, cadmium (Cd) ≤ 0.05 mg / kg, and chromium + vanadium (Cr + V) ≥ 4.5 mg / kg and are positively correlated with antithrombotic activity. The preparation method of the red snake ethanol extract includes: B1. Dry and pulverize the sample of red-banded snake or red-banded fetal snake, take the coarse powder, add dilute ethanol with a volume concentration of 50-70% at a material-to-liquid ratio of 1:8-12, decoct 1-3 times, and combine the supernatant. B2. The solvent in the supernatant was removed by vacuum evaporation to obtain an alcohol extract. The extract was dried under vacuum at room temperature to obtain the alcohol extract of red snake.

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