Method for extracting active substances of bungarus multicinctus by simulating gastrointestinal digestion in vitro and extract

Abstract

The invention discloses a method for extracting active substances of bungarus multicinctus by simulating gastrointestinal tract digestion in vitro and an extract. The extraction method comprises the following steps: performing oral cavity simulation digestion on a to-be-digested mixture under the action of simulated saliva to obtain an oral cavity simulation digestion extract; the mixture to be digested comprises bungarus parvus and water, and the mass volume ratio of the bungarus parvus to the water is 1: 10-1: 50 g / mL; performing stomach simulated digestion on the oral cavity simulated digestion extract under the action of simulated gastric juice to obtain a stomach simulated digestion extract; the stomach simulated digestion extract is subjected to intestinal simulated digestion under the action of simulated intestinal juice, and the extract is prepared. According to the method, in-vitro digestion simulation and bungarus multicinctus active ingredient extraction are combined for the first time, and mild, efficient, green and directional extraction of bungarus multicinctus active substances is achieved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, and relates to a method for extracting active substances from *Snakehead sinensis* and the extract by simulating gastrointestinal digestion in vitro. Background Technology

[0002] Bungarus multicinctus is one of the main ingredients in the Uyghur hospital preparation "Xiao Aifei Honey Ointment". Bungarus multicinctus has the effects of dispelling wind, clearing the meridians, and relieving spasms; it can be used for stubborn rheumatism, numbness and contractures, stroke, and mouth and eye ailments. Treatment of conditions such as paralysis, hemiplegia, convulsions, tetanus, leprosy, scrofula, and malignant sores. The traditional preparation process of "Xiao Aifei Honey Ointment" involves grinding all the medicinal ingredients into a fine powder and then adding honey to form an ointment. This method preserves the original components of the medicine to the greatest extent possible. However, the large protein molecules in the white-flowered snake may not be fully released and absorbed due to their complex structure and lack of extraction and purification, which to some extent limits its resource utilization efficiency and further improvement of clinical efficacy.

[0003] The golden-ringed snake (Synthetica granatum), listed in Part I of the Pharmacopoeia of the People's Republic of China, is the dried body of a juvenile silver-ringed snake (Elapidae). It contains not only small molecules such as amino acids, nucleotides, trace elements, and lipids, but is also rich in proteins. However, the complex molecular structure of its proteins makes extraction difficult, hindering its resource development and utilization.

[0004] Currently, in the field of extracting active ingredients from *Syngonium wilfordii*, the closest existing technology typically employs traditional solvent extraction methods, such as using acids, alkalis, and ethanol. Existing methods all utilize the differences in the solubility of different components in solvents to achieve the transfer of active ingredients from the medicinal material to the solvent.

[0005] Liu Meng et al. established optimized extraction processes for alcohol-soluble and alkaline proteins from *Smilax china* (Liu Meng, Xie Chao, Zhang Linsong, et al. Optimization of alcohol-soluble protein extraction process from *Smilax china* using response surface methodology [J]. Grains and Oils, 2023, 36(02):73-77.) and alkaline proteins (Xie Chao, Liu Meng, Song Meiying, et al. Optimization and characterization of alkaline-soluble protein extraction process from *Smilax china* [J]. Chinese Medicinal Herbs, 2023, 46(08):2007-2011.). Mu Xuan et al. optimized the acid extraction process of *Smilax china* protein using response surface methodology (Mu Xuan, Liu Meng, Li Liangying, et al. Optimization of acid extraction process of *Smilax china* protein using response surface methodology [J]. Grains and Oils, 2024, 37(03):68-71+81.). The soluble protein extraction rates of the above extraction methods are below 6%.

[0006] Traditional solvent extraction methods have limited options for solvent selection and use, making it difficult to accurately extract the complex and diverse active ingredients from *Snakehead sinensis*. While ethanol can dissolve some fat-soluble components, it is ineffective at extracting water-soluble polysaccharides and amino acids. Alkaline or acidic extraction can yield more alkali-soluble or acid-soluble components, but the extraction range is limited. Moreover, these methods result in relatively low contact between the solvent and the medicinal material, as well as low mass transfer efficiency, leading to incomplete dissolution of active ingredients and low extraction efficiency.

[0007] Ethanol extraction cannot completely avoid the problem of solvent residue. Residual ethanol may affect subsequent formulation production and use; for example, in the preparation of injectable drugs, residual ethanol may increase the safety risks of the formulation.

[0008] Research on the anti-inflammatory activity of *Syngonium fasciatus* is scarce in existing literature. Most existing efficacy observations focus on *Syngonium fasciatus* herbal wine (whose main components include *Syngonium fasciatus*, *Zaocys dhumnades*, safflower, processed strychnos nux-vomica, and achyranthes bidentata), a mixture of multiple medicinal materials. The complex nature of this research makes it difficult to clearly elucidate and attribute its anti-inflammatory activity to a single active ingredient from *Syngonium fasciatus*. Therefore, the anti-inflammatory mechanism and material basis of *Syngonium fasciatus* as a single medicinal material and its high-purity active substances (especially protein components) remain a crucial gap to be filled in current pharmacological research. Summary of the Invention

[0009] To address the shortcomings of existing technologies, such as low extraction efficiency of active ingredients from *Syngonium wilfordii*, easy inactivation of activity, high risk of solvent residue, and lack of high-purity active substances for pharmacological research, this invention provides a method and extract for extracting active substances from *Syngonium wilfordii* using in vitro simulated gastrointestinal digestion. This invention is the first to combine in vitro digestion simulation with the extraction of active ingredients from *Syngonium wilfordii*, achieving a gentle, efficient, green, and targeted extraction of active substances from *Syngonium wilfordii*.

[0010] Existing conventional chemical methods for extracting the active ingredients, especially active proteins, from *Syngonium wilfordii* suffer from limitations in their techniques (such as high temperature and strong acids / alkalis). These limitations lead to denaturation and inactivation of the active ingredients during the extraction process, fundamentally reducing their bioavailability and final efficacy. Furthermore, current protein extraction processes from *Syngonium wilfordii* generally suffer from low extraction efficiency and a high risk of solvent residue contamination, resulting in low levels of active ingredients (proteins) and numerous impurities in the extracted material, severely hindering its industrial application.

[0011] To address these issues, this invention, for the first time, utilizes a gastrointestinal simulated extraction technique in the extraction method of *Snakehead sinensis*. This technical route achieves a mild, efficient, and environmentally friendly extraction process, significantly improving the extraction rate and resulting in a *Snakehead sinensis* protein extract with high content of active ingredients and low impurities. Therefore, the technical solution provided by this invention lays the foundation for large-scale production potential and reagent-grade high-purity preparation.

[0012] Existing studies on the activity of *Smilax china* (a type of snake) in clinical applications are limited, with no research on its anti-inflammatory effects at the cellular level. Therefore, this invention compares the cellular activity of gastrointestinal digestion extracts, laying the foundation for the industrial development and commercialization of *Smilax china* extraction methods using in vitro digestion simulation. This invention addresses the key issues of unclear material basis and lack of cellular pharmacological evidence regarding the anti-inflammatory effects of *Smilax china*. To this end, this invention conducts the first preliminary cellular-level study on the anti-inflammatory activity of *Smilax china* proteins extracted using the novel method (comparing the activity indicators of existing extracts and the standard lipopolysaccharide (LPS)). This study not only demonstrates the good potential for developing anti-inflammatory effects of *Smilax china* extracts, but more importantly, it provides crucial theoretical and experimental support for the standardization, targeted development, and application of *Smilax china* active proteins in biological reagents, functional foods, or pharmaceuticals.

[0013] Many proteins or peptides identified through conventional extraction, despite possessing strong initial activity, have limited practical efficacy due to their instability in the gastrointestinal environment. To overcome this bottleneck and select peptides with high activity and digestive stability, standardized in vitro digestion simulation models are employed prior to activity screening. This approach ensures that specific protein sequences have the true potential to exert their active effects in vivo. Therefore, this invention is the first to combine in vitro digestion simulation with the extraction of active ingredients from *Smilax china*.

[0014] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0015] This invention provides a method for extracting active substances from *Snakehead sinensis* through in vitro simulated gastrointestinal digestion, comprising the following steps:

[0016] S1. The mixture to be digested is subjected to simulated oral digestion under the action of simulated saliva to obtain an oral simulated digestion extract; wherein, the mixture to be digested includes snakehead and water, and the mass-volume ratio of snakehead and water is 1:10~1:50 g / mL;

[0017] S2. The oral simulated digestion extract undergoes simulated gastric digestion under the action of simulated gastric juice to obtain a simulated gastric digestion extract;

[0018] S3. The gastric simulated digestion extract is subjected to intestinal simulated digestion under the action of simulated intestinal fluid to obtain the extract.

[0019] In this invention, the mass-to-volume ratio of the snake and water can be 1:10 g / mL, 1:20 g / mL, 1:30 g / mL, 1:40 g / mL or 1:50 g / mL, preferably 1:20 to 1:40 g / mL.

[0020] In S1, the method for preparing the mixture to be digested preferably includes the following steps: mixing the powder of *Smilax china* and water.

[0021] The stirring speed can be 500~600 r / min, for example 550 r / min.

[0022] The mixing time can be 10 to 20 minutes, for example, 15 minutes.

[0023] The preparation method of the Golden Coin White Flower Snake powder can be conventional in the art, and may include the following steps: pulverizing the Golden Coin White Flower Snake medicinal material, passing it through a 100-mesh sieve, and obtaining the Golden Coin White Flower Snake powder.

[0024] In this invention, the simulated saliva may include the following components:

[0025] (1) Electrolytes: including KCl, KH2PO4, NaHCO3, MgCl2(H2O)6 and (NH4)2CO3;

[0026] (2) CaCl2;

[0027] (3) Enzyme: α-amylase;

[0028] (4) Water.

[0029] In some preferred embodiments, the electrolyte is prepared as follows: the electrolyte is prepared as an electrolyte stock solution, and the electrolyte stock solution is prepared at a concentration of 1.25 times, that is, 4 parts of electrolyte stock solution are mixed with 1 part of water to obtain the ionic strength required to simulate saliva.

[0030] In some preferred embodiments, the ionic strength in the simulated saliva is as follows: K + 18.8 mmol / L, Na + 13.6 mmol / L, Cl - 19.5 mmol / L, H2PO4 - 3.7 mmol / L, HCO3 - and CO3 - The mixture contained 13.7 mmol / L of Mg. 2+0.15 mmol / L, NH4 + 0.12 mmol / L and Ca 2+ 1.5 mmol / L.

[0031] In some preferred embodiments, the electrolyte concentrations in the simulated saliva are as follows: 15.1 mmol / L KCl solution, 3.7 mmol / L KH2PO4 solution, 13.6 mmol / L NaHCO3 solution, 0.15 mmol / L MgCl2(H2O)6 solution, and 0.06 mmol / L (NH4)2CO3 solution.

[0032] In this invention, the volume ratio of the mixture to be digested to the simulated saliva can be (40~60):(40~60), for example, 50:50.

[0033] In this invention, the activity of the α-amylase in the mixture of the to-be-digested mixture and the simulated saliva can be 70-80 U / mL, for example 75 U / mL.

[0034] In this invention, the concentration of CaCl2 in the mixture of the digestible mixture and the simulated saliva can be 0.7~0.8 mM, for example 0.75 mM.

[0035] In this invention, the pH of the mixture to be digested and the simulated saliva is preferably 7.

[0036] In this invention, the preferred temperature for the oral cavity simulated digestion is 37°C.

[0037] In this invention, the oral cavity simulated digestion time can be 3 to 7 minutes, for example, 5 minutes.

[0038] In this invention, the simulated gastric juice comprises the following components:

[0039] (1) Electrolytes: including KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6 and (NH4)2CO3;

[0040] (2) CaCl2;

[0041] (3) Enzyme: pepsin;

[0042] (4) Water.

[0043] In some preferred embodiments, the ionic strength in the simulated gastric fluid is as follows: K + 7.8 mmol / L, Na + 72.2 mmol / L, Cl - 70.2 mmol / L, H2PO4- 0.9 mmol / L, HCO3 - and CO3 - The mixture of 25.5 mmol / L, Mg 2+ 0.1 mmol / L, NH4 + 1.0 mmol / L and Ca 2+ 0.15 mmol / L.

[0044] In some preferred embodiments, the electrolyte concentrations in the simulated gastric fluid are as follows: 6.9 mmol / L KCl solution, 0.9 mmol / L KH2PO4 solution, 25 mmol / L NaHCO3 solution, 47.2 mmol / L KCl solution, 0.1 mmol / L MgCl2(H2O)6 solution, and 0.5 mmol / L (NH4)2CO3 solution.

[0045] In this invention, the volume ratio of the oral simulated digestive extract to the simulated gastric juice can be (40~60):(40~60), for example, 50:50.

[0046] In this invention, the activity of the pepsin in the mixture of the oral simulated digestion extract and the simulated gastric juice can be 1900~2100 U / mL, for example 2000 U / mL.

[0047] In this invention, the concentration of CaCl2 in the mixture of the oral simulated digestion extract and the simulated gastric juice can be 0.07~0.08 mM, for example 0.075 mM.

[0048] In this invention, the pH of the mixture of the oral simulated digestion extract and the simulated gastric juice is preferably 3.

[0049] In this invention, the temperature for the simulated gastric digestion is preferably 37°C.

[0050] In this invention, the time for simulated gastric digestion can be 1.5 to 2.5 hours, for example, 2 hours.

[0051] In this invention, the simulated intestinal fluid comprises the following components:

[0052] (1) Electrolytes: including KCl, KH2PO4, NaHCO3, NaCl and MgCl2(H2O)6;

[0053] (2) CaCl2;

[0054] (3) Enzyme: trypsin;

[0055] (4) Water.

[0056] In this invention, the simulated intestinal fluid preferably does not contain bile.

[0057] In some preferred embodiments, the ionic strength in the simulated intestinal fluid is as follows: K + 7.6 mmol / L, Na + 123.4 mmol / L, Cl - 55.5 mmol / L, H2PO4 - 0.8 mmol / L, HCO3 - and CO3 - The mixture of 85 mmol / L Mg 2+ 0.33 mmol / L and Ca 2+ 0.6 mmol / L.

[0058] In some preferred embodiments, the electrolyte concentrations in the simulated intestinal fluid are as follows: 6.8 mmol / L KCl solution, 0.8 mmol / L KH2PO4 solution, 85 mmol / L NaHCO3 solution, 38.4 mmol / L KCl solution, and 0.33 mmol / L MgCl2(H2O)6 solution.

[0059] In this invention, the volume ratio of the simulated gastric digestion extract to the simulated intestinal fluid can be (40~60):(40~60), for example, 50:50.

[0060] In this invention, the activity of the trypsin in the mixture of the gastric simulated digestion extract and the simulated intestinal fluid can be 90~110 U / mL, for example 100 U / mL.

[0061] In this invention, the concentration of CaCl2 in the mixture of the gastric simulated digestion extract and the simulated intestinal fluid can be 0.2~0.4 mM, for example 0.3 mM.

[0062] In this invention, the pH of the mixture of the gastric simulated digestion extract and the simulated intestinal fluid is preferably 7.

[0063] In this invention, the temperature for the simulated intestinal digestion is preferably 37°C.

[0064] In this invention, the time for simulated intestinal digestion can be 1.5 to 2.5 hours, for example, 2 hours.

[0065] Preferably, the extract is obtained by performing routine post-processing on the product of the intestinal simulated digestion. The routine post-processing may include terminating digestion, separation and purification, and freeze-drying. The extract contains active substances from *Syngonium podophyllum*.

[0066] The step of terminating digestion can be conventional in the art, such as heating in a 90°C water bath for 10 minutes.

[0067] The separation and purification steps can be conventional in the art, such as centrifugation followed by supernatant collection and dialysis. The centrifugation speed can be 5500 r / min, the centrifugation time can be 15 minutes, the dialysis bag can have a retention capacity of 500 Da, and the dialysis time can be 48 hours.

[0068] The present invention also provides an extract prepared by the method of extracting active substances from snakehead root by in vitro simulated gastrointestinal digestion; the concentration of the extract is preferably 50~1000 μg / mL, for example 50 μg / mL, 100 μg / mL, 300 μg / mL, 500 μg / mL, 800 μg / mL or 1000 μg / mL, more preferably 500~1000 μg / mL.

[0069] In this invention, the protein content in the extract is preferably 3% to 10%, more preferably 6% to 9%, for example 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.

[0070] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0071] The reagents and raw materials used in this invention are all commercially available.

[0072] The positive and progressive effects of this invention are as follows:

[0073] This invention utilizes an in vitro method simulating gastrointestinal digestion to extract active substances from *Snakehead sinensis*, thus obtaining the corresponding extract. This method overcomes the problems of low effective component content, low extraction rate, and numerous impurities found in existing processes. It improves the content and extraction rate of the effective component (protein) while reducing impurities, providing a foundation for large-scale production and reagent-grade high-purity preparation. The active extract obtained through this method provides a reliable material basis for in-depth pharmacological research on the active substances of *Snakehead sinensis*, and offers crucial theoretical and experimental support for its standardization, targeted development, and application in biological reagents, functional foods, or pharmaceuticals. Therefore, it lays a solid scientific foundation for the commercialization and industrial application of the active components of *Snakehead sinensis*.

[0074] The method for extracting active substances from snakehead fern by simulating gastrointestinal digestion in this invention can achieve targeted release of active ingredients under mild conditions; it requires no organic solvents, eliminates the risk of solvent residue, and is green and environmentally friendly.

[0075] For example, in a preferred embodiment, during the simulated gastrointestinal digestion process, by precisely controlling the temperature (37°C), pH value (gastric environment pH=3, intestinal environment pH=7) and enzyme type (such as pepsin, trypsin), specific proteases can break down large protein molecules into bioactive small peptides while avoiding denaturation caused by high temperature.

[0076] In a preferred embodiment, this invention is the first to study the anti-inflammatory activity of an in vitro digestion-simulated extract of *Syngonium fasciatus* at the cellular level, filling the gap in the existing technology of *Syngonium fasciatus* lacking clear pharmacological evidence at the cellular level. By comparing the activity indicators of existing extracts and standards (LPS), it is demonstrated that the extract of *Syngonium fasciatus* has good potential for developing anti-inflammatory effects. Furthermore, by comparing the optimal conditions with other traditional extraction methods, the superiority of this invention is indirectly demonstrated.

[0077] In summary, this invention utilizes an economical and green method to directly extract the active ingredients of *Syngonium fasciatus*, avoiding the destruction of its activity by traditional chemical extraction methods. By simulating the in vivo environment of organisms, it comprehensively surpasses existing technologies in terms of active ingredient retention, extraction efficiency, and environmental friendliness, providing an innovative solution for the efficient utilization of *Syngonium fasciatus*. Attached Figure Description

[0078] Figure 1 This is a comparison of the protein extraction rate and protein content of the extracts obtained in Examples 1-5.

[0079] Figure 2 This is a comparison of the protein extraction rate and protein content of the extracts obtained in Example 3 and Comparative Examples 1-3.

[0080] Figure 3 Cell survival rates under different concentrations of extract solutions in Examples 3 and 1-3.

[0081] Figure 4 The NO content is shown in the extracts obtained in Example 3 and Comparative Examples 1-3. Detailed Implementation

[0082] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0083] The experimental materials, reagents, and instruments used in the following examples and comparative examples are as follows:

[0084] Experimental materials: The raw materials of the golden-flowered snake were purchased from Anhui Zhenlu Breeding Co., Ltd., and were transported and stored at low temperature.

[0085] Experimental reagents: trypsin, α-amylase, and pepsin were purchased from Maclean's (China), andrographolide was purchased from Merck, and all other reagents were analytical grade.

[0086] Experimental instruments: analytical balance (model AE224C, Sunny Optical, China), pH meter (model FE-20, METTLERTOLEDO, Germany), constant temperature shaker (model ZHWY-2102c, Changzhou Jintan Liangyou Co., Ltd., China), low organic matter ultrapure water system (model FDY2002-SUV, Fulham, China), benchtop high-speed centrifuge (model 5430R, Eppendorf, Germany), dialysis bags (molecular weight cutoff 500 Da, Solarbio, China), pre-cooling tank (model PFR-1000, EYELA, Japan), freeze dryer (model FDU2100, EYELA, Japan), and microplate reader (model Spectra Max M5, Meigu Molecular, USA).

[0087] Example 1

[0088] A method for extracting active substances from *Snakehead sinensis* by simulating gastrointestinal digestion in vitro, comprising the following steps:

[0089] 1. Preparation of Powder from the White-flowered Snake (Golden Coin Snake)

[0090] The medicinal material of *Syngonium fasciatus* was pulverized using a pulverizer to make *Syngonium fasciatus* powder, which was then passed through a 100-mesh sieve and stored in a sealed container at -20°C for later use.

[0091] 2. Pre-processing of *Snakehead sinensis* powder before in vitro digestion

[0092] The powder of *Syngonium fasciatus* was mixed with ultrapure water at a mass-to-volume ratio of 1:10 g / mL, and stirred at 550 r / min under magnetic stirring at room temperature for 15 minutes to obtain the mixture to be digested.

[0093] 3. Extracting active substances from *Syntheticus aegyptiacus* by simulating gastrointestinal digestion.

[0094] (1) Preparation of electrolyte reserves for simulated saliva, simulated gastric juice and simulated intestinal juice

[0095] Prepare electrolyte stock solutions for simulated saliva, gastric juice, and intestinal juice according to the formulas in Table 1. These formulas are based on a final volume of 500 mL for the simulated digestive fluids (simulated saliva, gastric juice, or intestinal juice). Prepare a 400 mL system of electrolyte stock solution (1.25 times the original concentration) and store it at -20°C for later use. To obtain the required electrolyte concentration in the simulated digestive fluids, dilute the electrolyte stock solution to a 1:1 concentration when preparing the simulated digestive fluids.

[0096] Table 1. Preparation methods of electrolyte reserve solutions for simulated saliva, gastric juice, and intestinal juice.

[0097]

[0098] (2) Using simulated saliva to simulate oral digestion of the mixture to be digested

[0099] 1) Preparation of simulated saliva: Add α-amylase, CaCl2 and water to the electrolyte stock solution of simulated saliva in Table 1 to prepare a 500 mL system of simulated saliva.

[0100] 2) Mix the mixture to be digested with simulated saliva: Mix the mixture to be digested and simulated saliva obtained in step 2 at a ratio of 50:50 (v / v). The concentration and pH value of each component in the mixture of the mixture to be digested and simulated saliva are shown in Table 2.

[0101] Table 2 Concentration ratio of each component in the simulated digestion system

[0102]

[0103] 3) Oral simulated digestion: The mixture to be digested and the simulated saliva were reacted at 37°C for 5 minutes to obtain an oral simulated digestion extract.

[0104] (3) Using simulated gastric juice to simulate gastric digestion of oral simulated digestion extracts

[0105] 1) Preparation of simulated gastric juice: Add pepsin, CaCl2 and water to the electrolyte reserve solution of simulated gastric juice in Table 1 to prepare a 500 mL system of simulated gastric juice.

[0106] 2) Mixing oral simulated digestion extract with simulated gastric juice: Mix oral simulated digestion extract and simulated gastric juice at a ratio of 50:50 (v / v). The concentration and pH value of each component in the mixed system of oral simulated digestion extract and simulated gastric juice are shown in Table 2.

[0107] 3) Gastric simulated digestion: A mixture of oral simulated digestion extract and simulated gastric juice was reacted at 37°C for 2 hours to obtain gastric simulated digestion extract.

[0108] (4) Using simulated intestinal fluid to perform simulated intestinal digestion on gastric simulated digestion extract.

[0109] 1) Preparation of simulated intestinal fluid: Add trypsin, CaCl2 and water to the electrolyte reserve solution of simulated intestinal fluid in Table 1 to prepare a 500 mL system of simulated intestinal fluid.

[0110] 2) Mixing the gastric simulated digestion extract with simulated intestinal fluid: The gastric simulated digestion extract and simulated intestinal fluid were mixed at a ratio of 50:50 (v / v). The concentrations and pH values ​​of each component in the mixed system of gastric simulated digestion extract and simulated intestinal fluid are shown in Table 2.

[0111] 3) Intestinal simulated digestion: A mixture of gastric simulated digestion extract and simulated intestinal fluid was reacted at 37°C for 2 hours to obtain intestinal simulated digestion extract.

[0112] (5) Post-processing

[0113] The intestinal simulated digestion extract was heated in a 90°C water bath for 10 minutes to terminate digestion. The supernatant was collected by centrifugation at 5500 r / min for 15 minutes and placed in a dialysis bag with a capacity cutoff of 500 Da for dialysis to remove compounds such as salts and polyphenols. Dialysis was performed for 48 hours, with the deionized water on the outside of the dialysis bag being replaced every 4 hours. The dialyzed sample was then dried using a freeze dryer to obtain the extract.

[0114] Example 2

[0115] Example 2 follows the same steps as Example 1, except that in step 2 the mass-to-volume ratio of the powder of the golden snake to ultrapure water is 1:20 g / mL.

[0116] Example 3

[0117] Example 3 follows the same steps as Example 1, except that in step 2 the mass-to-volume ratio of the powder of the golden coin snake to ultrapure water is 1:30 g / mL.

[0118] Example 4

[0119] Example 4 follows the same steps as Example 1, except that in step 2 the mass-to-volume ratio of the powder of the golden snake to ultrapure water is 1:40 g / mL.

[0120] Example 5

[0121] Example 5 follows the same steps as Example 1, except that in step 2 the mass-to-volume ratio of the powder of the golden coin snake to ultrapure water is 1:50 g / mL.

[0122] Comparative Example 1

[0123] Comparative Example 1 used an acidic extraction method. Step 1 of Comparative Example 1 was the same as that of Example 1, and the remaining steps were as follows:

[0124] The *Snakehead sinensis* powder obtained in step 1 of Example 1 was extracted with 0.20 mol / L acetic acid-sodium acetate solution (pH=4.2) at a mass-to-volume ratio of 1:40 g / mL for 6 hours at a temperature of 45°C. After extraction, the mixture was centrifuged at 5500 r / min for 15 minutes, and the supernatant was collected and freeze-dried to obtain the extract.

[0125] Comparative Example 2

[0126] Comparative Example 2 uses an alkaline extraction method. Step 1 of Comparative Example 2 is the same as that of Example 1, and the remaining steps are as follows:

[0127] The *Snakehead sinensis* powder obtained in step 1 of Example 1 was extracted with 0.2 mol / L Tris-HCl buffer (pH 8.0) at a mass-to-volume ratio of 1:40 g / mL for 5 hours at a temperature of 45°C. After extraction, the mixture was centrifuged at 5500 r / min for 15 min, and the supernatant was collected and freeze-dried to obtain the extract.

[0128] Comparative Example 3

[0129] Comparative Example 3 used an ethanol extraction method. Step 1 of Comparative Example 3 was the same as that of Example 1, and the remaining steps were as follows:

[0130] The powder of *Snakehead sinensis* obtained in step 1 of Example 1 was extracted with 40% ethanol (volume fraction) at a mass-to-volume ratio of 1:17 g / mL for 4 hours at a temperature of 40°C. After extraction, the mixture was centrifuged at 5500 rpm for 15 minutes, and the supernatant was collected and freeze-dried to obtain the extract.

[0131] Example 1

[0132] I. Testing Methods

[0133] The protein extraction rate and protein content of the extracts obtained in Examples 1-5 and Comparative Examples 1-3 were tested using the following methods:

[0134] 1. Protein extraction rate

[0135] Protein extraction rate refers to the ratio of the mass of the extract obtained after freeze-drying to the mass of 5.00 g of *Snakehead sinensis* powder, used to express the protein extraction rate of *Snakehead sinensis* using the extraction method. The calculation formula is as follows:

[0136] Extraction rate = (1)

[0137] 2. Protein content

[0138] Protein content was detected using a BCA reagent kit. Standard bovine serum albumin (BSA) was used as the standard, and standard solutions were prepared at concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0 mg / mL. To ensure the protein concentration of the extract solution fell within the middle range of the standard curve, the extract solution concentration was set to 2 mg / mL. Based on the required volume of working solution, working solution A (1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% sodium hydroxide, and 0.95% sodium bicarbonate) was mixed with working solution B (4% copper sulfate) at a ratio of 50:1 (v / v). The mixture was then stored in the dark (prepared immediately before use). 100 μL of the test solution (i.e., the standard solution or the extract solution) and 800 μL of the working solution were added to a 1.5 mL centrifuge tube. Each experiment was repeated three times. After reacting at 37°C for 30 minutes in a benchtop drying oven, 250 μL of the mixed reaction solution was transferred to a 96-well plate. The absorbance was measured at 562 nm using an ELISA reader. A standard curve was plotted with the protein concentration of the standard as the independent variable and the absorbance as the dependent variable. The absorbance of the extract solution was then substituted into the standard curve to obtain the protein concentration (mg / mL) of the extract solution. The protein content of the extract was calculated using formula (2).

[0139] (2)

[0140] Note: C1 is the protein concentration of the sample calculated by substituting the sample into the standard curve, and C0 is the concentration of the sample being tested.

[0141] II. Test Results

[0142] 1. Effect of pretreatment mass-to-volume ratio on protein extraction rate and protein content of extracts obtained by in vitro gastrointestinal digestion simulation extraction method

[0143] Table 3 and Figure 1 The effect of mass-to-volume ratio during pretreatment on the protein extraction rate and protein content of the extract obtained by the in vitro gastrointestinal digestion simulation extraction method is shown in Table 3 and... Figure 1 It can be seen that the protein content and extraction rate are highest when the in vitro digestion pretreatment has a mass-to-volume ratio of 1:30 g / mL. Therefore, 1:30 g / mL is the optimal mass-to-volume ratio in the pretreatment process. The protein extraction rate and protein content of the extracts obtained by the in vitro gastrointestinal digestion simulation extraction method under different mass-to-volume ratios are shown in the table below.

[0144] Table 3. Protein extraction rate and protein content of the extracts obtained in Examples 1-5

[0145]

[0146] 2. Comparison of in vitro digestion with existing extraction methods

[0147] Figure 2 This is a bar chart showing the protein extraction rate and protein content of the extracts obtained in Examples 3 and Comparative Examples 1-3 of the present invention. Regarding the protein extraction rate, the extract obtained in Example 3 had the highest protein extraction rate, significantly higher than that obtained by alkaline extraction, acidic extraction, and alcohol-soluble extraction (p<0.0001). This indicates that the in vitro digestion simulation technology of the present invention can achieve highly efficient dissolution of active ingredients to the maximum extent. Regarding the protein content, the extract obtained in Example 3 had the highest protein content, showing a significant advantage compared to the other three traditional extraction methods (p<0.0001). The data on the protein extraction rate and protein content of the obtained extracts are shown in the table below.

[0148] Table 4. Protein extraction rate and protein content of the extracts obtained in Example 3 and Comparative Examples 1-3

[0149]

[0150] Therefore, the in vitro digestion-simulated extraction method of the present invention can achieve efficient enrichment of target protein components in *Syngonium wilfordii* under mild conditions. This method successfully overcomes the key defects of existing technologies, such as low extraction rate and insufficient protein content, and provides reliable technical support for the preparation of highly active and high-purity functional crude extracts of *Syngonium wilfordii*.

[0151] Example 2

[0152] I. Screening for the optimal experimental concentration using the MTT assay

[0153] The extracts from Example 3 (with the highest protein content) and Comparative Examples 1-3 were selected for cytotoxicity assays.

[0154] Experiment 1

[0155] 1. LPS stimulation of RAW264.7 macrophages to construct an inflammatory cell model.

[0156] RAW264.7 cells were removed from liquid nitrogen and quickly placed in a 37°C water bath. The cryovials were gently shaken to thaw the cryopreservation solution. After thawing, the cells were transferred to centrifuge tubes containing 5 mL of culture medium. The cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature, and the supernatant was discarded. The cells were resuspended in complete culture medium containing 10% fetal bovine serum, seeded into culture dishes, and gently mixed by pipetting. The cells were then cultured at 37°C under saturated humidity (5% CO2). RAW cells in the logarithmic growth phase and in good growth condition were seeded at a concentration of 1 × 10⁻⁶ cells / cell. 5RAW264.7 macrophages were seeded per well in 96-well cell culture plates and cultured at 37°C, 5% CO2, in DMEM medium (containing 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). 100 μL / well of LPS complete medium was added to a final concentration of 1 μg / mL, and stimulation was initiated for 24 hours.

[0157] 2. Material handling

[0158] Cells cultured the previous day in 96-well plates were divided into the following groups: blank control, LPS group (1 μg / mL), LPS + andrographolide (positive control) group (100 μM, 75 μM, 50 μM, 25 μM, 10 μM, 5 μM), and LPS + extract solution prepared in Example 3 treatment group (50 μg / mL, 100 μg / mL, 300 μg / mL, 500 μg / mL, 800 μg / mL, 1000 μg / mL). Except for the blank control group, each group was incubated with the drug for 1 hour before LPS was added. The blank control group was cultured in the corresponding culture medium. Cytotoxicity was assessed after 24 hours of incubation.

[0159] 3. Cytotoxicity assay

[0160] Thaw the MTT kit at room temperature, add 10 μL of MTT solution to each well of a 96-well plate, and incubate for 4 hours in a cell culture incubator. Add 100 μL of Formazan solution to each well of the 96-well plate and mix well. Continue incubation in a cell culture incubator until the formazan is completely dissolved under a regular optical microscope. Transfer the culture medium in the well plate and measure the absorbance at 570 nm using a microplate reader. Calculate the cell viability using formula (3).

[0161] (3)

[0162] Comparative Experiment 1

[0163] The steps of Comparative Experiment 1 were the same as those of Experiment 1, except that the LPS+ extract solution treatment group prepared in Example 3 was replaced with the LPS+ extract solution prepared in Comparative Example 1.

[0164] Comparative Experiment 2

[0165] The steps of Comparative Experiment 2 were the same as those of Experiment 1, except that the LPS+ extract solution treatment group prepared in Example 3 was replaced with the LPS+ extract solution prepared in Comparative Example 2.

[0166] Comparative Experiment 3

[0167] The steps of Comparative Experiment 3 were the same as those of Experiment 1, except that the LPS+ extract solution treatment group prepared in Example 3 was replaced with the LPS+ extract solution prepared in Comparative Example 3.

[0168] II. Test Results

[0169] To avoid false positives, this effective example screened for the optimal concentration of four different extracts. After selecting the optimal safe concentration, the nitric oxide (NO) content was determined. The results are shown in Table 5 and... Figure 3 As shown, for the extraction method in Example 3, the cell viability was closest to 100% when the extract solution concentration was 800 µg / mL. Therefore, a sample with a concentration of 800 µg / mL was ultimately selected for the next step of NO content determination. Similarly, for the extraction methods in Comparative Examples 1, 2, and 3, the extract solution concentration at which the cell viability was closest to 100% was selected for NO content determination.

[0170] The cell viability data for different concentrations of extract solutions in Examples 3 and Comparative Examples 1-3 are shown in the table below.

[0171] Table 5. Cell viability under different concentrations of extract solutions in Examples 3 and Comparative Examples 1-3

[0172]

[0173] Example 3

[0174] Experiment 1

[0175] I. Study on the anti-inflammatory activity of different extracts

[0176] 1. Cell Culture

[0177] Remove RAW cells from liquid nitrogen and quickly place them in a 37°C water bath. Gently shake the cryovial to thaw the cryopreservation solution. After thawing, transfer the cells to a centrifuge tube containing 5 mL of culture medium, centrifuge to collect the cells, centrifuge at 1000 r / min for 5 minutes at room temperature, and discard the supernatant. Resuspend the cells in complete culture medium containing 10% fetal bovine serum, seed them into culture dishes, gently pipette to mix, and culture at 37°C under saturated humidity of 5% CO2. Take 1×10⁶ RAW cells in the logarithmic growth phase and in good growth condition. 5 Cells / well were seeded in a 12-well plate (100 μL of sterile PBS was added to the wells around the cells).

[0178] 2. Material Preparation

[0179] The cells cultured in 12-well plates from the previous day were divided into the following groups: blank group, LPS group (1 μg / mL), LPS + 5 μM andrographolide (positive control group), and LPS + extract solution prepared in Example 3 (800 μg / mL). Except for the blank group, each group was incubated with the drug for 1 hour before LPS was added. The blank group was cultured in the corresponding culture medium. The NO content was measured after 24 hours of incubation.

[0180] 3. NO content determination

[0181] After culturing cells in 12-well plates for 24 hours, the cell supernatant was centrifuged at 3000 r / min for 10 minutes before analysis. Sodium nitrite standards were diluted with DMEM + 10% FBS to prepare sodium nitrite standard solutions at concentrations of 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 100 μM. The above concentration gradient of sodium nitrite standard solutions or sample solutions were added to 96-well plates at 50 μL / well. Subsequently, 50 μL of room temperature Griess Reagent I and 50 μL of room temperature Griess Reagent II were added to each well, and the absorbance was measured at 540 nm. The NO content in the samples was calculated based on the standard curve.

[0182] Comparative Experiment 1

[0183] The steps of Comparative Experiment 1 were the same as those of Experiment 1, except that the LPS+ extract solution prepared in Example 3 was replaced with the LPS+ extract solution prepared in Comparative Example 1 (300 μg / mL).

[0184] Comparative Experiment 2

[0185] The steps of Comparative Experiment 2 were the same as those of Experiment 1, except that the LPS+ extract solution treatment group prepared in Example 3 was replaced with the LPS+ extract solution (800 μg / mL) indicated in Comparative Example 2.

[0186] Comparative Experiment 3

[0187] The steps of Comparative Experiment 3 were the same as those of Experiment 1, except that the LPS+ extract solution treatment group prepared in Example 3 was replaced with the LPS+ extract solution prepared in Comparative Example 3 (300 μg / mL).

[0188] II. Test Results

[0189] This embodiment aims to compare the effects of crude protein extracts from *Smilax china* obtained by different extraction methods on NO release from RAW264.7 macrophages in order to evaluate the anti-inflammatory potential of each extract. Figure 4The NO content in extracts obtained by different extraction methods is determined by... Figure 4 It was found that among the four extracts, the NO content of the extract prepared in Example 3 was significantly lower than that of the other three traditional extracts (acidic extraction, alkaline extraction, and ethanol extraction). The ethanol-extracted extract had the highest NO content, while the extract obtained through in vitro simulated gastrointestinal digestion had a NO content of approximately 11.71 µM, indicating its strongest ability to inhibit NO release. The excellent anti-inflammatory potential demonstrated by the in vitro simulated gastrointestinal digestion extraction method in cell experiments provides a crucial pharmacological basis and data support for its application as a highly efficient and mild active substance extraction technology in the fields of functional foods and pharmaceuticals.

[0190] The NO content data of the extracts obtained in Examples 3 and Comparative Examples 1-3 are shown in the table below.

[0191] Table 6. NO content in the extracts obtained in Example 3 and Comparative Examples 1-3

[0192]

[0193] This result strongly demonstrates the superiority of the in vitro gastrointestinal digestion and extraction method. By simulating the mild enzymatic digestion environment within a living organism, this method can maximize the retention or release of bioactive proteins or peptides, thereby exhibiting stronger anti-inflammatory activity at the cellular level.

Claims

1. A method for extracting active substances of Lichanura muda in vitro by simulating gastrointestinal digestion, characterized in that, It comprises the following steps: S1. The mixture to be digested is subjected to oral simulation digestion under the action of simulated saliva to obtain an oral simulation digestion extract; wherein the mixture to be digested comprises money white flower snake and water, and the mass-volume ratio of the money white flower snake and water is 1:10-1:50 g / mL; S2. The oral simulation digestion extract is subjected to gastric simulation digestion under the action of simulated gastric juice to obtain a gastric simulation digestion extract; S3. The gastric simulation digestion extract is subjected to intestinal simulation digestion under the action of simulated intestinal juice to obtain an extract.

2. The method for extracting active substances of Lichanura muda according to claim 1, wherein the active substances of Lichanura muda are extracted by in-vitro simulation of gastrointestinal digestion. The mass-volume ratio of the money white flower snake and water is 1:10 g / mL, 1:20 g / mL, 1:30 g / mL, 1:40 g / mL or 1:50 g / mL, preferably 1:20-1:40 g / mL.

3. The method for extracting active substances of Lichanura muda according to claim 1, wherein the active substances of Lichanura muda are extracted by in-vitro simulated gastrointestinal digestion. The simulated saliva comprises the following components: (1) Electrolytes: KCl, KH2PO4, NaHCO3, MgCl2(H2O)6 and (NH4)2CO3; (2) CaCl2; (3) Enzyme: α-amylase; (4) Water; Preferably, in the simulated saliva, the concentrations of electrolytes are as follows: 15.1 mmol / L KCl solution, 3.7 mmol / L KH2PO4 solution, 13.6 mmol / L NaHCO3 solution, 0.15 mmol / L MgCl2(H2O)6 solution and 0.06 mmol / L (NH4)2CO3 solution.

4. The method for extracting active substances of money white snake in in-vitro simulated gastrointestinal digestion according to claim 3, characterized in that, It meets one or more of the following conditions: (1) The volume ratio of the mixture to be digested to the simulated saliva is (40-60):(40-60), for example 50:50; (2) In the mixed system of the mixture to be digested and the simulated saliva, the activity of the α-amylase is 70-80 U / mL, for example 75 U / mL; (3) In the mixed system of the mixture to be digested and the simulated saliva, the concentration of CaCl2 is 0.7-0.8 mM, for example 0.75 mM; (4) In the mixed system of the mixture to be digested and the simulated saliva, the pH is 7; (5) The temperature of the oral simulation digestion is 37°C; and, (6) The time of the oral simulation digestion is 3-7 minutes, for example 5 minutes.

5. The method for extracting active substances of money white snake in in-vitro simulated gastrointestinal digestion according to claim 1, characterized in that, The simulated gastric juice comprises the following components: (1) Electrolytes: KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6 and (NH4)2CO3; (2) CaCl2; (3) Enzyme: pepsin; (4) Water; Preferably, in the simulated gastric juice, the concentrations of electrolytes are as follows: 6.9 mmol / L KCl solution, 0.9 mmol / L KH2PO4 solution, 25 mmol / L NaHCO3 solution, 47.2 mmol / L KCl solution, 0.1 mmol / L MgCl2(H2O)6 solution and 0.5 mmol / L (NH4)2CO3 solution.

6. The method for extracting active substances of money white snake in in-vitro simulated gastrointestinal digestion according to claim 5, characterized in that, It meets one or more of the following conditions: (1) the volume ratio of the oral simulated digestion extract to the simulated gastric juice is (40-60):(40-60), for example 50:50; (2) in the mixed system of the oral simulated digestion extract and the simulated gastric juice, the activity of the pepsin is 1900-2100 U / mL, for example 2000 U / mL; (3) in the mixed system of the oral simulated digestion extract and the simulated gastric juice, the concentration of CaCl2 is 0.07-0.08 mM, for example 0.075 mM; (4) in the mixed system of the oral simulated digestion extract and the simulated gastric juice, the pH is 3; (5) the temperature of the gastric simulated digestion is 37°C; and, (6) the time of the gastric simulated digestion is 1.5-2.5 hours, for example 2 hours.

7. The method for extracting active substances of money white snake in in-vitro simulated gastrointestinal digestion according to claim 1, characterized in that, The simulated intestinal juice comprises the following components: (1) electrolytes: including KCl, KH2PO4, NaHCO3, NaCl and MgCl2(H2O)6; (2) CaCl2; (3) enzymes: trypsin; (4) water; Preferably, in the simulated intestinal juice, the concentrations of the electrolytes are as follows: 6.8 mmol / L KCl solution, 0.8 mmol / L KH2PO4 solution, 85 mmol / L NaHCO3 solution, 38.4 mmol / L KCl solution and 0.33 mmol / L MgCl2(H2O)6 solution.

8. The method for extracting active substances of money white snake in in-vitro simulated gastrointestinal digestion according to claim 7, characterized in that, It satisfies one or more of the following conditions: (1) the volume ratio of the gastric simulated digestion extract to the simulated intestinal juice is (40-60):(40-60), for example 50:50; (2) in the mixed system of the gastric simulated digestion extract and the simulated intestinal juice, the activity of the trypsin is 90-110 U / mL, for example 100 U / mL; (3) in the mixed system of the gastric simulated digestion extract and the simulated intestinal juice, the concentration of CaCl2 is 0.2-0.4 mM, for example 0.3 mM; (4) in the mixed system of the gastric simulated digestion extract and the simulated intestinal juice, the pH is 7; (5) the temperature of the intestinal simulated digestion is 37°C; and, (6) the time of the intestinal simulated digestion is 1.5-2.5 hours, for example 2 hours.

9. The method for extracting active substances of money white snake in in-vitro simulated gastrointestinal digestion according to claim 1, characterized in that, In S1, the preparation method of the mixture to be digested preferably comprises the following step: stirring and mixing the powder of Lycusuaurora and water; Preferably, the stirring and mixing is performed at a speed of 500-600 r / min, for example 550 r / min; Preferably, the stirring and mixing is performed for 10-20 minutes, for example 15 minutes; Preferably, the preparation method of the powder of Lycusuaurora comprises the following steps: crushing Lycusuaurora medicinal materials, and passing through a 100-mesh sieve to obtain the powder of Lycusuaurora; and / or, the product of the intestinal simulated digestion is subjected to post-treatment to obtain the extract; preferably, the post-treatment comprises: terminating digestion, separation and purification, freeze-drying; the extract contains active substances of Lycusuaurora; Preferably, the step of terminating digestion comprises heating in a 90°C water bath for 10 minutes. Preferably, the step of separation and purification comprises: taking supernatant after centrifugation, dialysis, the rotation speed of the centrifugation is preferably 5500 r / min, the centrifugation time is preferably 15 minutes, the cut-off of the dialysis bag is preferably 500 Da, and the dialysis time is preferably 48 hours.

10. An extract prepared by a method of extracting active substances of Lichanura muda in vitro by simulating gastrointestinal digestion according to any one of claims 1-9; The concentration of the extract is preferably 50-1000 μg / mL, for example 50 μg / mL, 100 μg / mL, 300 μg / mL, 500 μg / mL, 800 μg / mL or 1000 μg / mL, more preferably 500-1000 μg / mL; The protein content in the extract is preferably 3%-10%, more preferably 6-9%, for example 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.

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