Ilex cornuta leaf extract as well as preparation method and application thereof

Abstract

The invention provides a folium ilicis cornutae extract as well as a preparation method and application thereof. The core component of the holly leaf extract comprises polyphenol compounds, the total polyphenol content is 10-500mg / g, and the polyphenol components comprise neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C and isochlorogenic acid A according to a ratio of (95-100): (300-450): (70-109): (120-256): (20-46): (21-156): (24-43): (27-61). The preparation method of the Chinese holly leaf extract comprises the whole processes of raw material pretreatment, multi-solvent synergistic extraction, multi-stage double-effect filtration and impurity removal, deep concentration and drying, parameters of all links are accurately controlled, the method is high in extraction rate, and the obtained Chinese holly leaf extract can effectively reduce the uric acid level and prevent and treat hyperuricemia.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly relates to an extract of Ilex cornuta Lindl. leaves, a preparation method thereof, and an application thereof. Background Art

[0002] Hyperuricemia is a common metabolic disease. In recent years, its prevalence has been increasing year by year and showing a trend of getting younger, and it has become the fourth most common basic disease after hypertension, hyperlipidemia, and diabetes. Hyperuricemia is associated with a variety of diseases and is an independent risk factor for diseases such as hypertension, cardiovascular diseases, diabetes, and kidney diseases, and has gradually become one of the common diseases threatening national health, imposing a serious burden on the public health system of our country.

[0003] At present, the treatment of hyperuricemia is mainly achieved by reducing the uric acid level. The drugs used for anti-hyperuricemia are mainly divided into three categories: xanthine oxidase inhibitors, urate oxidase, and uricosuric drugs. Although these drugs can effectively reduce the uric acid level in the body of hyperuricemia patients, long-term use will cause a series of adverse reactions such as liver damage, renal function abnormalities, and gastrointestinal reactions.

[0004] Traditional Chinese medicine has a long history in the treatment of hyperuricemia, and there are relevant records in "Synopsis of the Golden Chamber". Modern research also shows that a variety of traditional Chinese medicines can play an anti-hyperuricemia role by reducing uric acid, anti-inflammatory, antioxidant, and protecting the kidneys, and its curative effect is obvious; at the same time, the treatment approach of traditional Chinese medicine also provides guarantees for its safety and effectiveness. Therefore, using traditional Chinese medicine to treat hyperuricemia is a choice worthy of consideration. The leaves of Ilex cornuta Lindl. are the dried leaves of the plant Ilex cornuta Lindl. in the family Aquifoliaceae. They are bitter in taste and cool in nature, and belong to the liver and kidney meridians, and have the effects of clearing heat and nourishing yin, tonifying the kidney, and calming the liver. At present, there is no report on the treatment of hyperuricemia with the polyphenol components of Ilex cornuta Lindl. leaves. Summary of the Invention

[0005] The object of the present invention is to provide an extract of Ilex cornuta Lindl. leaves, a preparation method thereof, and an application thereof in the prevention and treatment of hyperuricemia. By clarifying that the core component of the extract of Ilex cornuta Lindl. leaves is polyphenols and contains a variety of components in specific proportions, an optimized preparation process including multiple fine steps such as extraction, filtration, concentration, and drying is provided, and an anti-hyperuricemia drug is prepared using this extract to achieve effective prevention and treatment of hyperuricemia and provide a better treatment option for patients.

[0006] An Ilex cornuta Lindl. leaf extract has clear core polyphenol components, a specific total polyphenol content, and precisely defined proportions of each component, laying a key material foundation for subsequent precise preparation and playing a unique medicinal effect in the prevention and treatment of hyperuricemia, with great value in medical innovation and application. The Ilex cornuta Lindl. leaf extract provided by the present invention has core components including polyphenolic compounds, and the total polyphenol content is 10 - 500 mg / g; the polyphenolic components include: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and the proportions of each component are neochlorogenic acid:chlorogenic acid:cryptochlorogenic acid:caffeic acid:isoquercitrin:isochlorogenic acid B:isochlorogenic acid C:isochlorogenic acid A = 95 - 100:300 - 450:70 - 109:120 - 256:20 - 46:21 - 156:24 - 43:27 - 61.

[0007] The total polyphenol content range of 10 - 500 mg / g and the precise component proportions constitute the core quality indicators of the extract, ensuring the constant quality of each batch of the extract and guaranteeing the consistency and safety of the medicinal effect. The polyphenolic components with precise proportions act synergistically. Neochlorogenic acid, chlorogenic acid, etc. are combined in a specific proportion, closely and precisely acting on liver xanthine oxidase. Chlorogenic acid can be embedded in the key region of the enzyme active site, and neochlorogenic acid stabilizes the conformation of the enzyme protein. The two act synergistically to inhibit its activity by more than 30%, precisely reducing uric acid production from the source, far exceeding the effect of a single component, and precisely exerting force for the treatment of hyperuricemia. At the same time, in kidney protection, caffeic acid, isoquercitrin, etc. build a strong antioxidant network according to the proportion. The efficiency of caffeic acid in scavenging free radicals is improved, and the ability of isoquercitrin to repair damaged cell membranes is enhanced. They synergistically reduce the concentration of kidney inflammatory factors, strengthen the antioxidant defense while anti - inflammatory, and multi - dimensionally and precisely protect the kidneys from hyperuric acid damage, maintain the homeostasis of the kidney cell microenvironment, and improve the integrity of kidney physiological functions.

[0008] The present invention provides a preparation method of an Ilex cornuta Lindl. leaf extract. By reasonably selecting solvents and extraction conditions, rigorously filtering, precisely concentrating, and using various drying means, it ensures the precise extraction and stable preservation of active ingredients, laying a solid foundation for the application of the Ilex cornuta Lindl. leaf extract in multiple fields such as the prevention and treatment of hyperuricemia.

[0009] The preparation method of an Ilex cornuta Lindl. leaf extract provided by the present invention ensures the efficient and precise extraction of active ingredients from Ilex cornuta Lindl. leaves, and through processes such as filtration, concentration, and drying, finally obtains a high - quality and highly active extract finished product, laying a foundation for subsequent applications in fields such as the prevention and treatment of hyperuricemia. Specifically, it includes the following steps: S1 Extraction step: Obtain the active ingredients (such as polyphenolic compounds) from the raw material of Ilex cornuta leaves. Using the medicinal materials or decoction pieces of Ilex cornuta leaves as raw materials, they are crushed and screened through a 20-80 mesh sieve to increase the specific surface area of the raw materials, enabling the solvent to fully contact the medicinal materials and improving the extraction efficiency. Add a solvent with a solid-liquid ratio of 5-40 times water or 20-80% (v / v) ethanol. Ethanol can enhance the dissolution ability of polyphenolic compounds and improve the extraction rate. At 50-100 °C, use one of the ultrasonic extraction method, reflux extraction method, microwave extraction method, or decoction method; among them, the ultrasonic extraction method utilizes the cavitation effect of ultrasonic waves to accelerate the contact between the solvent and the medicinal materials, improving the extraction speed and efficiency; the reflux extraction method enables the solvent to be recycled for extraction through heating reflux, which is suitable for large-scale production; the microwave extraction method utilizes the thermal effect and electromagnetic field effect of microwaves to quickly extract the active ingredients; the decoction method is a traditional extraction method, and the active ingredients are dissolved out by heating and boiling. Control the temperature and time to ensure the full dissolution of the active ingredients, while avoiding the degradation of ingredients caused by high temperature and long-term treatment. Continuously extract the active ingredients for 20-120 minutes, and the number of extractions is one of one, two, or three times. Multiple extractions can improve the extraction rate and ensure that the active ingredients are extracted as much as possible to obtain the extract.

[0010] S2 Filtration process: Remove solid impurities and macromolecular impurities in the extract to improve the purity of the extract. The extract is centrifuged at a centrifugal force of 1000-10000×g for 5-30 minutes to separate the solid impurities by centrifugal force and obtain a clear supernatant. Use one or more filtration techniques such as gravity filtration, pressure filtration, microfiltration, ultrafiltration, nanofiltration, or reverse osmosis for deep impurity removal. Among them, gravity filtration uses a filter paper or filter cloth with a pore size of 10-50 μm to remove larger solid particles; pressure filtration / microfiltration uses a microporous membrane with a pore size of 0.45-1 μm to remove fine particles and some microorganisms; the membrane pore size of ultrafiltration is 1-20 nm, which can remove macromolecular impurities such as proteins and polysaccharides; the pore size of nanofiltration is 0.1-100 nm to further remove small molecular impurities; the pore size of reverse osmosis is 0.1-1 nm, which can remove dissolved impurities and further purify the extract. Double-effect filtration ensures the removal of impurities and obtains the filtrate; through the dual effects of centrifugation and filtration, it ensures the thorough removal of impurities in the extract and provides high-quality raw materials for subsequent concentration and drying.

[0011] In the S3 concentration step, the volume of the extract is reduced and the concentration of the active ingredients is increased to facilitate subsequent drying. Using pressurized or atmospheric or vacuum concentration techniques, or combined with membrane concentration techniques, the filtrate is concentrated to 0.1 - 3.0 g of crude drug / ml to obtain a concentrated solution. Select the appropriate concentration method according to different requirements. Among them, pressurized concentration is carried out at a pressure of 0.2 - 2.0 MPa and a temperature of 50 - 80 °C, which is suitable for rapid concentration; vacuum concentration is carried out at a pressure of -0.10 to -0.05 MPa and a temperature of 50 - 70 °C, which can avoid the destruction of active ingredients by high temperature; atmospheric concentration is carried out at 80 - 100 °C, which is suitable for large-scale production. Membrane technology is based on the characteristics of molecular size, charge, etc. for separation, and can accurately separate the target components from solvents or other impurities, and can achieve a higher concentration multiple at a lower operating pressure, greatly improving the concentration efficiency. The membrane concentration process is usually carried out at room temperature, avoiding the destruction or denaturation of heat-sensitive components that may be caused by traditional concentration methods such as heating and evaporation. The filtrate is concentrated to 0.1 - 3.0 g of crude drug / ml to obtain a concentrated solution, ensuring that the concentration of the active ingredients in the concentrated solution reaches the range required for subsequent drying.

[0012] S4 Drying, converting the concentrated solution into a dry solid extract for easy storage and subsequent preparation. Use one of atmospheric drying, vacuum drying, spray drying, and freeze drying. Among them, atmospheric drying is carried out at 60 - 100 °C, which is suitable for large-scale production; vacuum drying is carried out at a pressure of 0.01 - 0.1 MPa and a temperature of 40 - 80 °C, which can reduce the loss of heat-sensitive components; spray drying is carried out by rapid drying at high temperature, which is suitable for large-scale production to obtain a powdery extract; freeze drying is carried out by low-temperature freezing and vacuum sublimation, which can retain the activity and quality of the active ingredients to the greatest extent, and is suitable for high-value-added products. The extract is dried to a moisture content of 3 - 15% to convert the concentrated solution into a stable extract form, locking in the activity and quality of the components to obtain the finished product of Ilex cornuta Lindl. leaf extract.

[0013] In the preparation method of the Ilex cornuta Lindl. leaf extract of the present invention, in the S1 extraction step, the extraction method uses ultrasonic extraction to strengthen cell disruption and component dissolution through ultrasonic cavitation and mechanical vibration at a specific frequency and power, which is energy-efficient and can protect the active ingredients by cooling; microwave extraction uses microwave thermal effect and non-thermal effect to accelerate molecular movement and break cell walls to promote the dissolution of active ingredients at a specific temperature and frequency period, with high speed and high yield; reflux extraction uses the synergistic effect of solvents and high-temperature reflux to break intermolecular chemical bonds, enhance dissolution efficiency, improve extraction efficiency and prevent oxidation and deterioration; decoction method is carried out by step-by-step boiling and filtering, with traditional warm and hot complementary extraction and dissolution, a combination of various operation methods and accurate parameters, and all-round deep extraction of polyphenols and other components, adapting to the complexity of raw material components to ensure the medicinal quality and efficacy of the extract. Specifically, it includes the following parameters: Ultrasonic extraction method accelerates the contact between the solvent and the medicinal materials through the cavitation effect of ultrasonic waves, thereby improving the extraction efficiency. When ultrasonic waves propagate in a liquid, the cavitation effect will occur, that is, tiny bubbles are formed in the liquid and burst instantly, generating local high temperature and high pressure. This effect can break the cell walls of the medicinal materials, enabling the active ingredients to be quickly dissolved into the solvent. After the raw materials of Ilex cornuta Lindl. leaves are mixed with the solvent, ultrasonic treatment is carried out at 50 - 80 °C for 20 - 120 minutes, with an ultrasonic frequency of 20 - 40 kHz and a power of 250 - 500 W; it has high extraction efficiency, short time, is suitable for extracting thermosensitive components, avoids high-temperature degradation, has simple operation, and is suitable for production.

[0014] Microwave extraction method utilizes the thermal effect and electromagnetic field action of microwaves to quickly extract active ingredients. Microwaves can cause polar molecules in the solvent and the medicinal materials to vibrate rapidly, generating heat, and accelerating the penetration of the solvent and the dissolution of components. The electromagnetic field action of microwaves can further break the cell walls and improve the extraction efficiency. After the raw materials of Ilex cornuta Lindl. leaves are mixed with the solvent, microwave treatment is carried out at 60 - 80 °C for 20 - 120 minutes, with a microwave power of 200 - 600 W and a frequency of 20 - 50 MHz; it has a fast extraction speed, high efficiency, high energy utilization efficiency, is suitable for large-scale production, and can achieve rapid temperature rise and shorten the extraction time.

[0015] Reflux extraction method improves the extraction rate by heating and refluxing to make the solvent circulate and extract multiple times. After the medicinal materials are mixed with the solvent, they are heated to boiling, and the solvent vapor is cooled by a condenser and then refluxed into the extraction tank, repeating this process. Through multiple cycles, it ensures sufficient contact between the solvent and the medicinal materials and improves the extraction rate. After the raw materials of Ilex cornuta Lindl. leaves are mixed with the solvent, reflux is carried out at 60 - 100 °C for 20 - 120 minutes; it has a high extraction rate, is suitable for large-scale production, has simple operation, and has low equipment requirements.

[0016] Decoction method makes the active ingredients dissolve out from the medicinal materials through the traditional heating and boiling method. After the medicinal materials are mixed with the solvent, they are pre-soaked for 10 - 60 minutes to allow the solvent to fully penetrate the medicinal materials. After boiling vigorously, it is turned to a low heat for decoction, and the active ingredients are dissolved out through the action of heat. After the raw materials of Ilex cornuta Lindl. leaves are mixed with the solvent, they are pre-soaked for 10 - 60 minutes, and after boiling vigorously, it is turned to a low heat for decoction for 20 - 120 minutes; it is a traditional method, has simple operation, low cost, is suitable for large-scale production, especially under the condition of limited resources.

[0017] In the preparation method of the Ilex cornuta Lindl. leaf extract of the present invention, the S2 filtration process includes two key steps, namely S21 centrifugation and S22 filtration, which jointly ensure the purification of the extract and the removal of impurities. Specifically, it includes the following steps: S21 Centrifugation separates solid impurities and suspended particles in the extract by centrifugal force to obtain a clear supernatant. Using the high centrifugal force generated by a centrifuge, the solid particles and suspended matter in the extract precipitate to the bottom of the centrifuge tube, thus achieving solid-liquid separation. The extract is placed in a centrifuge and centrifuged at 2000 - 10000×g for 5 - 30 minutes to obtain a clear supernatant, removing most of the solid impurities and suspended matter, and providing a purer liquid for the subsequent filtration step.

[0018] S22 Filtration further removes soluble impurities, microorganisms, macromolecular impurities, etc. in the supernatant to ensure the purity and quality of the extract. One or more filtration techniques among gravity filtration, pressure filtration, microfiltration, ultrafiltration, nanofiltration or reverse osmosis are used to filter the centrifuged supernatant. Among them, gravity filtration utilizes the gravity effect to make the liquid pass through filter paper or filter cloth to remove larger solid particles and suspended matter; pressure filtration / microfiltration, under negative pressure, the liquid passes through a microporous membrane with a pore size of 0.45 - 1μm to remove fine particles, bacteria and some microorganisms; ultrafiltration, under a pressure of 0.1 - 0.5MPa, the liquid passes through an ultrafiltration membrane with a pore size of 1 - 20nm to remove macromolecular impurities (such as proteins, polysaccharides, etc.), further purifying the extract, used to remove macromolecular impurities and improve the purity of the extract, suitable for the extraction of small molecule active ingredients such as polyphenols; nanofiltration filters through a nanofiltration membrane with a pore size of 0.1 - 100nm to remove small molecule impurities, some salts and pigments, used for deep purification to further improve the quality of the extract; reverse osmosis, under a high pressure of 1.0 - 5.0MPa, the liquid passes through a reverse osmosis membrane with a pore size of 0.1 - 1nm to remove soluble small molecule impurities, salts and most of the impurities in water; used to prepare a high-purity extract. Deep impurity removal is carried out to obtain a filtrate, ensuring that the extract reaches a high purity before entering the concentration step, providing high-quality raw materials for subsequent concentration and drying, and thus ensuring the quality and activity of the final extract.

[0019] In the preparation method of the Ilex cornuta Lindl. extract of the present invention, the filtration techniques in the S22 filtration step, through reasonable selection and combined use, can achieve multi-level removal from coarse impurities to soluble small molecule impurities, ensuring the purity and quality of the extract. Specifically, it includes the following parameters: Gravity filtration removes larger solid particles and suspended matter. Utilizing the gravity effect, the liquid passes through filter paper or filter cloth with a pore size of 10 - 50μm. Solid particles larger than the pore size will be intercepted, while the liquid and soluble components pass through the filter paper to obtain a filtrate. The operation is simple and the cost is low, suitable for preliminary purification in large-scale production.

[0020] Suction filtration / microfiltration removes fine particles and microorganisms. Under negative pressure, the liquid passes through a microporous membrane with a pore size of 0.45 - 1 μm, effectively retaining fine particles and microorganisms (such as bacteria), but allowing the liquid and soluble components to pass through, obtaining a filtrate. The operation is rapid and suitable for removing impurities with medium particle sizes.

[0021] Ultrafiltration removes macromolecular impurities such as proteins and polysaccharides. Under a pressure of 0.1 - 0.5 MPa, the liquid passes through an ultrafiltration membrane with a pore size of 1 - 20 nm, retaining macromolecular substances, but allowing small molecule components and solvents to pass through, obtaining a filtrate, improving the purity of the extract, and reducing the burden of subsequent processing.

[0022] Nanofiltration removes small molecule impurities, some salts, and pigments. The liquid passes through a nanofiltration membrane with a pore size of 0.1 - 100 nm, retaining small molecule impurities, some salts, and pigments, but allowing water and small molecule solvents to pass through, obtaining a filtrate, improving the purity and transparency of the extract.

[0023] Reverse osmosis removes soluble small molecule impurities, salts, and most impurities in water. Under a high pressure of 1.0 - 5.0 MPa, the liquid passes through a reverse osmosis membrane with a pore size of 0.1 - 1 nm, retaining almost all soluble small molecule impurities and salts, but allowing water molecules to pass through, obtaining a filtrate, improving the purity of the extract, and being suitable for the production of high - value - added products.

[0024] In the preparation method of the Ilex cornuta Lindl. leaf extract of the present invention, the membrane concentration and concentration steps in the S3 concentration link act together to ensure that the active ingredients of the extract are fully concentrated, while avoiding over - concentration of ineffective ingredients, providing a high - quality concentrated solution for the preparation of the final extract. Specifically, it includes the following steps: S31 Membrane concentration initially removes the solvent in the filtrate to achieve initial concentration of the active ingredients. Using a membrane concentration device, under an operating pressure of 0.1 - 0.5 MPa, the liquid passes through an ultrafiltration membrane with a pore size of 1 - 20 nm. The solvent (such as water or ethanol) and small molecule components can pass through the membrane, while the macromolecular active ingredients are retained, thus achieving initial concentration, increasing the concentration of the active ingredients and reducing the solvent content.

[0025] S32 Concentration further concentrates the extract to obtain a highly concentrated solution. According to different requirements, pressure concentration, vacuum concentration, or atmospheric pressure concentration can be selected. Among them, pressure concentration is carried out under an operating pressure of 0.2 - 2.0 MPa and a temperature of 50 - 80 °C, suitable for rapid concentration; vacuum concentration is carried out under an operating pressure of - 0.10 to - 0.05 MPa and a temperature of 50 - 70 °C, which can avoid the destruction of active ingredients by high temperature; atmospheric pressure concentration is carried out at an operating temperature of 80 - 100 °C, suitable for large - scale production, obtaining a concentrated solution of 0.1 - 2.0 g of crude drug / ml, ensuring that the concentration of active ingredients in the concentrated solution reaches the range required for subsequent drying.

[0026] In the preparation method of the Ilex cornuta Lindl. leaf extract of the present invention, the concentration method in the S32 concentration step depends on the nature of the extract, the thermal stability of the components, and the production requirements. By reasonably selecting the concentration method and parameters, the active ingredients of the extract can be maximally retained and protected during the concentration process. Specifically, the following parameters are included: Pressurized concentration increases the evaporation rate of the solvent by increasing the operating pressure, thereby shortening the concentration time. Appropriate temperature can ensure rapid evaporation of the solvent while avoiding the destruction of heat-sensitive components by high temperature. The concentration speed is fast and suitable for large-scale production. By adjusting the pressure and temperature, the concentration process can be precisely controlled. At an operating pressure of 0.2 - 2.0 MPa and a temperature of 50 - 80°C, for 1 - 3 hours, the extract is concentrated to a concentrated solution of 0.1 - 2.0 g of crude drug / ml.

[0027] Vacuum concentration reduces the boiling point of the solvent by reducing the system pressure, thereby achieving evaporation at a lower temperature. The low temperature can effectively protect heat-sensitive components and avoid degradation or denaturation due to high temperature; it is suitable for treating heat-sensitive components and avoiding the destruction of active ingredients by high temperature. At the same time, the lower temperature and pressure can reduce energy consumption. At an operating pressure of -0.10 to -0.05 MPa and a temperature of 50 - 70°C, for 2 - 6 hours, the extract is concentrated to a concentrated solution of 0.1 - 2.0 g of crude drug / ml.

[0028] Atmospheric pressure concentration directly heats the extract to evaporate the solvent under atmospheric pressure. A temperature of 80 - 100°C can ensure rapid evaporation of the solvent, but the time needs to be controlled to avoid over-concentration or component degradation; the operation is simple, the equipment requirements are low, and it is suitable for large-scale production. The evaporation rate of the solvent is fast under atmospheric pressure, suitable for treating a large volume of extract. At an operating temperature of 80 - 100°C, for 3 - 8 hours, the extract is concentrated to a concentrated solution of 0.1 - 2.0 g of crude drug / ml.

[0029] In the preparation method of the Ilex cornuta Lindl. leaf extract of the present invention, the S4 drying process covers multiple methods such as atmospheric pressure, vacuum, spray, and freeze drying. By reasonably selecting the drying method and parameters, the extract can be maximally retained and protected during the drying process, and the Ilex cornuta Lindl. leaf extract is dried to a moisture content of 3 - 15%. Specifically, the following parameters are included: Atmospheric pressure drying uses hot air to directly contact the extract to evaporate the moisture. The operation is simple and the cost is low, suitable for large-scale production; the drying temperature is 60 - 100°C, and it is dried to a moisture content of 3 - 15%.

[0030] Under vacuum drying conditions, the boiling point of water decreases, allowing it to evaporate at a lower temperature. This low-temperature drying method is suitable for protecting heat-sensitive components and features high drying efficiency, making it suitable for rapid drying. The vacuum is set to a pressure of 0.01 - 0.1 MPa, and the drying temperature is 40 - 80 °C.

[0031] Spray drying involves spraying the extract into fine droplets, increasing the surface area and enabling rapid water evaporation. It has a fast drying speed, is suitable for large-scale production, and can yield a uniform powdered product. The inlet air temperature is 180 - 250 °C, the outlet air temperature is 80 - 100 °C, and the feeding speed is 10 - 30 ml / minute.

[0032] Freeze drying rapidly cools the water to an ice crystal state, and then directly sublimes it from the solid state to the gaseous state under low pressure. This low-temperature drying method effectively protects heat-sensitive components, and the dried extract maintains good physical structure and biological activity. Rapidly cool to -40 to -20 °C for pre-freezing to turn the water into ice crystals, and maintain this low temperature for 2 - 4 hours. Evacuate to a low-pressure environment of 0.01 - 0.1 Pa and raise the temperature to -10 to 25 °C for sublimation for 5 - 36 hours.

[0033] The present invention provides the application of such Ilex cornuta Lindl. leaf extract in the prevention and treatment of hyperuricemia, specifically including the application of the Ilex cornuta Lindl. leaf extract in the preparation of anti-hyperuricemia drugs. Polyphenolic compounds (such as neochlorogenic acid, chlorogenic acid, etc.) in the Ilex cornuta Lindl. leaf extract can inhibit the activity of xanthine oxidase. Xanthine oxidase is the key enzyme for uric acid production. After its activity is inhibited, the production amount of uric acid will be significantly reduced. Neochlorogenic acid and chlorogenic acid can embed into the active site of the enzyme, thereby inhibiting its catalytic action and reducing the production of uric acid. Components such as caffeic acid and isoquercitrin in the Ilex cornuta Lindl. leaf extract have significant antioxidant capacity, can scavenge free radicals in the body, and reduce oxidative stress damage. The antioxidant effect helps to alleviate the kidney inflammatory response caused by hyperuricemia. Isoquercitrin can repair damaged cell membranes and reduce the concentration of inflammatory factors, thereby protecting kidney cells from the damage of high uric acid. Hyperuricemia often leads to kidney damage, while the polyphenolic components in the Ilex cornuta Lindl. leaf extract can maintain the microenvironment homeostasis of kidney cells through antioxidant and anti-inflammatory effects. Caffeic acid and isoquercitrin can act synergistically to reduce the concentration of kidney inflammatory factors and reduce renal tubular damage, thereby protecting kidney function. The components in the Ilex cornuta Lindl. leaf extract can increase uric acid excretion or inhibit uric acid reabsorption by regulating uric acid transporters in the kidney (such as URAT1, OAT1 / 3, etc.), and promote the excretion of uric acid from the kidney, thereby reducing the blood uric acid level. Hyperuricemia often coexists with metabolic syndrome (such as obesity, hypertension, hyperlipidemia, etc.). The polyphenolic components in the Ilex cornuta Lindl. leaf extract can improve these related symptoms by regulating metabolic pathways, and can also indirectly reduce the uric acid level by regulating the intestinal flora or affecting lipid metabolism. Therefore, the Ilex cornuta Lindl. leaf extract can be used as the basic raw material for developing new anti-hyperuricemia drugs. Clinically, this extract can be used as an adjuvant therapy drug and used in combination with existing uric acid-lowering drugs to improve the treatment effect and reduce side effects.

[0034] In summary, the present invention has the following beneficial effects: 1. The core polyphenolic components of the Ilex cornuta Lindl. leaf extract provided by the present invention are clear, the proportion of each component is accurate, and neochlorogenic acid, chlorogenic acid, etc. act synergistically to provide a precise pharmacodynamic mechanism for the prevention and treatment of hyperuricemia; 2. The preparation method of the Ilex cornuta Lindl. leaf extract provided by the present invention is different from the traditional preparation process, covering four links of extraction, filtration, concentration and drying. Each link has clear parameter control to ensure the quality of the extract and the stability of active ingredients; A variety of modern extraction technologies such as ultrasonic extraction and microwave extraction are adopted. Compared with the traditional method, it has higher extraction efficiency and lower energy consumption, and at the same time avoids the damage of high temperature to thermosensitive components; Through multi-stage filtration (gravity filtration, ultrafiltration, nanofiltration, etc.) and membrane concentration technology, deep purification of the extract solution is realized, effectively removing impurities and improving the purity and safety of the extract; 3. Different from the traditional preparation method of Ilex cornuta Lindl. leaf extract, the present invention has made a technical improvement. In the filtration process, the present invention adopts a variety of advanced membrane separation technologies (such as ultrafiltration, nanofiltration, reverse osmosis, etc.) to accurately remove impurities with different particle sizes, ensuring the high purity and high quality of the extract; according to the properties of different components and product requirements, the present invention provides a variety of drying methods such as atmospheric drying, vacuum drying, spray drying and freeze drying to ensure that the active ingredients are retained to the greatest extent during the drying process of the extract; through the combination of membrane concentration and a variety of concentration methods (pressure, vacuum, atmospheric pressure), efficient concentration of the extraction solution is achieved, while avoiding component degradation and improving the yield and quality of the extract. 4. The extract of the present invention shows significant pharmacological activities in the prevention and treatment of hyperuricemia, can effectively reduce uric acid levels, protect kidney function, and has potential clinical application value; through precise component control and optimized preparation process, the extract of the present invention has significant advantages in terms of safety, and at the same time, the synergistic action mechanism of its polyphenol components makes it show higher effectiveness in reducing uric acid; the application of the Ilex cornuta Lindl. leaf extract provided by the present invention in the prevention and treatment of hyperuricemia opens up a new path for the treatment of hyperuricemia with natural drugs, fills the blank of polyphenol treatment of Ilex cornuta Lindl. leaf, provides a safe and effective alternative scheme, reduces the adverse reactions of western medicine on the liver, kidneys and gastrointestinal tract, and promotes the modern innovation of traditional Chinese medicine and expands the application scope of traditional Chinese medicine in metabolic diseases. Description of the Drawings

[0035] Figure 1 It is a schematic diagram of the ultraviolet absorption spectra of the test solution of Ilex cornuta Lindl. leaf and the reference solution of gallic acid. Figure 2 It is the HPLC chromatogram of the test solution of Ilex cornuta Lindl. leaf and the mixed reference solution of 8 polyphenol components. In the figure, A1 is the test solution of Ilex cornuta Lindl. leaf, B1 is the reference solution of gallic acid, A2 is the test sample of Ilex cornuta Lindl. leaf, and B2 is the mixed reference sample. In the figure: 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Cryptochlorogenic acid; 4. Caffeic acid; 5. Isoquercitrin; 6. Isochlorogenic acid B; 7. Isochlorogenic acid A; 8. Isochlorogenic acid. Detailed Embodiments

[0036] This specific embodiment is only an explanation of the present invention, and it does not limit the present invention. After reading this specification, those skilled in the art can make modifications without creative contributions to this embodiment as needed, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

[0037] Example 1

[0038] Weigh 100 g of Ilex cornuta Lindl. ex Paxt. leaf powder, add 5 times the amount of 40% ethanol, and perform ultrasonic extraction at 50 °C for 30 minutes with a power of 500 w. After extraction, make up the weight. After centrifuging the extract at 5000×g for 10 minutes to obtain the supernatant, perform double filtration and concentration by gravity filtration and suction filtration through a 0.45 μm microporous membrane. After membrane concentration at 0.2 MPa with a filtration flow rate of 10 L / min, concentrate under normal pressure at 90 °C for 4 hours to obtain an Ilex cornuta Lindl. ex Paxt. leaf extract solution of 0.5 g crude drug / mL. Dry the Ilex cornuta Lindl. ex Paxt. leaf extract solution at 90 °C under normal pressure to obtain the finished product of Ilex cornuta Lindl. ex Paxt. leaf extract, with a yield of 12.32%. After testing, the moisture content of the finished product is 8%.

[0039] Weigh 100 mg of Ilex cornuta Lindl. ex Paxt. leaf extract, dissolve it in 10 mL of 40% ethanol to prepare a 10 mg / mL extract solution. Precisely transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Use the colorimetric method to detect that the total polyphenol mass content in Ilex cornuta Lindl. ex Paxt. leaf is 22.84 mg / g. As Figure 1 shown, the ultraviolet absorption spectra of the test solution (A1) of Ilex cornuta Lindl. ex Paxt. leaf and the reference solution (B1) of gallic acid both show characteristic absorption peaks at 275 nm, and the absorbance of the test solution has a good fitting degree with the standard curve (R2 > 0.99), indicating that the total polyphenol quantification method is reliable, and the ultraviolet response characteristics of polyphenol components in the extract are consistent with those of the reference standard, further supporting the accuracy of the total polyphenol content detection results. Take 1 mL of the extract solution and pass it through a 0.22 μm microporous membrane, and use HPLC to determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A. As Figure 2 shown, the HPLC chromatograms of the test solution (A2) of Ilex cornuta Lindl. ex Paxt. leaf and the mixed reference solution (B2) show that the resolution of each target component (peaks 1 - 8) is good, the retention time is consistent with that of the reference standard, and the peak shape is symmetrical without tailing, indicating that this method can accurately determine the proportion of polyphenol components in the extract. Specifically, peak 1 is neochlorogenic acid (retention time 6.2 min), peak 2 is chlorogenic acid (retention time 8.5 min), peak 3 is cryptochlorogenic acid (retention time 10.1 min), corresponding to caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in sequence. By calculating the peak area integration, the mass content ratio of each component is neochlorogenic acid:chlorogenic acid:cryptochlorogenic acid:caffeic acid:isoquercitrin:isochlorogenic acid B:isochlorogenic acid C:isochlorogenic acid A = 100:443.63:72.55:208.38:41.77:126.96:42.78:55.92, which meets the proportion limit range of each component of the Ilex cornuta Lindl. ex Paxt. leaf extract of the present invention, verifying the stability of the component proportion of the extract.

[0040] Example 2

[0041] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 20 times the amount of 40% ethanol, perform ultrasonic extraction at 60 °C for 30 minutes with a power of 500 w, and extract 2 times. After the extraction is completed, make up the weight. Centrifuge the extract at 4000×g for 12 minutes to obtain the supernatant, filter and concentrate it through a 10 nm ultrafiltration membrane under a pressure of 0.3 Mpa. After membrane concentration at 0.3 MPa with a filtration flow rate of 7 L / min, perform vacuum concentration at -0.05 MPa at 60 °C for 4 hours to obtain an Ilex cornuta Lindl. leaf extract with a concentration of 0.5 g crude drug / mL. Vacuum dry the Ilex cornuta Lindl. leaf extract at 40 - 80 °C under a pressure of 0.05 MPa to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 13.68%, and the moisture content is 7%.

[0042] Weigh 100 mg of Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 40% ethanol to prepare a 10 mg / mL extract solution. Precisely transfer 5 μL to a 5 mL centrifuge tube, make up to 1 mL with ultrapure water, and use the colorimetric method to detect the total polyphenol mass content of Ilex cornuta Lindl. leaf as 33.28 mg / g. Take 1 mL of the extract solution and pass it through a 0.22 μm microporous filter membrane, and use HPLC method to determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A. The mass content ratios of each component are neochlorogenic acid:chlorogenic acid:cryptochlorogenic acid:caffeic acid:isoquercitrin:isochlorogenic acid B:isochlorogenic acid C:isochlorogenic acid A = 100:446.81:88.43:214.06:42.61:124.84:40.04:56.31.

[0043] Example 3

[0044] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 40 times the amount of 40% ethanol, perform ultrasonic extraction at 70 °C for 30 minutes with a power of 500 w, and extract 3 times. After the extraction is completed, make up the weight. Centrifuge the extract at 6000×g for 15 minutes to obtain the supernatant, filter it through a nanofiltration membrane with a pore size of 10 nm. After membrane concentration at 0.5 MPa with a filtration flow rate of 10 L / min, perform pressure concentration at 65 °C under a pressure of 0.5 MPa for 2 hours to obtain an Ilex cornuta Lindl. leaf extract with a concentration of 0.1 g crude drug / mL. Spray dry the Ilex cornuta Lindl. leaf extract under the conditions of an inlet air temperature of 200 °C and an outlet air temperature of 90 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 14.93%, and the moisture content is 10%. Weigh 100 mg of Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 40% ethanol to prepare a 10 mg / mL extract solution. Precisely transfer 5 μL to a 5 mL centrifuge tube, make up to 1 mL with ultrapure water, and use the colorimetric method to detect the total polyphenol mass content of Ilex cornuta Lindl. leaf as 33.28 mg / g.

[0045] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous filter membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100: 438.97: 84.88: 209.67: 40.74: 127.20: 42.58: 57.76.

[0046] Example 4

[0047] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 40 times the amount of 20% ethanol, and perform ultrasonic extraction at 80 °C for 90 minutes with a power of 500 w for three times. After extraction, make up the weight. After centrifuging the extract at 9000×g for 10 minutes to obtain the supernatant, filter it through a 0.5 μm microporous filter membrane, and concentrate it through a membrane under 0.5 MPa with a filtration flow rate of 8 L / min. Then concentrate it under normal pressure at 85 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract solution of 0.5 g of crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract solution at 70 °C under normal pressure to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 10.84%, and the moisture content is 6%. Weigh 100 mg of the Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 20% ethanol to prepare a 10 mg / mL extract solution. Accurately transfer 5 μL to a 5 mL centrifuge tube and make up to 1 mL with ultrapure water. Detect the total polyphenol mass content of the Ilex cornuta Lindl. leaf by colorimetry to be 62.25 mg / g.

[0048] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous filter membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100: 426.15: 81.70: 180.75: 21.27: 47.56: 24.16: 27.09.

[0049] Example 5

[0050] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 40 times the amount of 40% ethanol, and perform ultrasonic extraction at 80 °C for 30 minutes with a power of 500 W. After extraction, make up the weight. After centrifuging the extract at 3000×g for 25 minutes to obtain the supernatant, filter it through a reverse osmosis membrane with a pore size of 0.2 nm under a pressure of 3.0 MPa. After membrane concentration at 0.1 MPa with a filtration flow rate of 5 L / min, perform normal-pressure concentration at 90 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract of 0.1 g crude drug / mL. Rapidly cool the Ilex cornuta Lindl. leaf extract to -30 °C for pre-freezing to make the water in a crystalline state. After maintaining this low temperature for 3 hours, evacuate to a low-pressure environment of 0.05 Pa and raise the temperature to 10 °C for sublimation for 24 hours to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 12.38%, and the moisture content of the finished product is 4%. Weigh 100 mg of Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 40% ethanol to prepare a 10 mg / mL extract solution. Accurately pipette 5 μL into a 5 mL centrifuge tube and make up to 1 mL with ultrapure water. Use the colorimetric method to detect that the total polyphenol mass content of Ilex cornuta Lindl. leaf is 82.13 mg / g.

[0051] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous filter membrane. Use the HPLC method to determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100:449.64:79.26:214.62:44.59:121.47:40.38:57.48.

[0052] Example 6

[0053] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 10 times the amount of 60% ethanol, and perform ultrasonic extraction at 80 °C for 90 minutes with a power of 500 W. After extraction, make up the weight. After centrifuging the extract at 1000×g for 30 minutes to obtain the supernatant, filter it through a microporous filter membrane with a pore size of 0.5 μm by suction filtration. After membrane concentration at 0.2 MPa with a filtration flow rate of 15 L / min, perform pressurized concentration at 2.0 MPa at 80 °C for 1 hour to obtain an Ilex cornuta Lindl. leaf extract of 1.0 g crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract under a pressure of 0.1 MPa at 80 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 15.13%, and the moisture content of the finished product is 13%. Weigh 100 mg of Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 60% ethanol to prepare a 10 mg / mL extract solution. Accurately pipette 5 μL into a 5 mL centrifuge tube and make up to 1 mL with ultrapure water. Use the colorimetric method to detect that the total polyphenol mass content of Ilex cornuta Lindl. leaf is 54.93 mg / g.

[0054] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100:446:89.76:234.80:45.28:145.46:42.92:60.68.

[0055] Example 7

[0056] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 20 times the amount of 60% ethanol, and perform ultrasonic extraction at 80 °C for 30 minutes with a power of 500 w. After the extraction is completed, make up the weight. After centrifuging the extract at 9000×g for 5 minutes to obtain the supernatant, filter it under the pressure of 0.2 Mpa through an ultrafiltration membrane with a pore size of 20 nm. After membrane concentration at 0.2 MPa and a filtration flow rate of 10 L / min, perform vacuum concentration at -0.05 MPa at 50 °C for 5 hours to obtain an Ilex cornuta Lindl. leaf extract of 0.1 g crude drug / mL. Spray-dry the Ilex cornuta Lindl. leaf extract at an inlet air temperature of 220 °C and an outlet air temperature of 95 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After testing, the yield of the finished product is 14.92%, and the moisture content of the finished product is 11%. Weigh 100 mg of the Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 60% ethanol, prepare a 10 mg / mL extract solution, accurately transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Use the colorimetric method to detect the total polyphenol mass content of the Ilex cornuta Lindl. leaf extract as 57.35 mg / g.

[0057] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100:433.92:81.82:212.63:41.19:136.51:36.65:60.06.

[0058] Example 8

[0059] Weigh 100 g of Ilex cornuta Lindl. leaf powder, add 40 times the amount of 80% ethanol, and perform ultrasonic extraction at 80 °C for 60 minutes with a power of 500 w. After extraction, make up the weight. After centrifuging the extract at 5000×g for 15 minutes to obtain the supernatant, filter it through a reverse osmosis membrane with a pore size of 0.1 nm under a pressure of 1.0 MPa. After membrane concentration at 0.5 MPa with a filtration flow rate of 5 L / min, perform atmospheric pressure concentration at 90 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract with 0.1 g of crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract at atmospheric pressure at 85 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 15.35%, and the moisture content of the finished product is 12%. Weigh 100 mg of Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 80% ethanol to prepare a 10 mg / mL extract solution. Precisely transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Use the colorimetric method to detect the total polyphenol mass content of Ilex cornuta Lindl. leaf as 71.58 mg / g. Dry the Ilex cornuta Lindl. leaf extract at atmospheric pressure at 85 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the moisture content of the finished product is 12%.

[0060] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous filter membrane. Use HPLC method to determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A. The mass content ratio of each component is neochlorogenic acid:chlorogenic acid:cryptochlorogenic acid:caffeic acid:isoquercitrin:isochlorogenic acid B:isochlorogenic acid C:isochlorogenic acid A = 100:429.21:79.09:255.29:40.03:155.42:40.36:60.40.

[0061] Example 9

[0062] Weigh 100 g of dried Ilex cornuta Lindl. leaves, soak them in 12 times the amount of water for 30 minutes, bring to a boil over high heat and then turn to low heat and decoct for 30 minutes. After centrifugal filtration, add 10 times the amount of water to the residue, bring to a boil over high heat and then turn to low heat and decoct for 30 minutes, and perform centrifugal filtration. Combine the filtrates, and after membrane concentration at 0.1 MPa with a filtration flow rate of 5 L / min, perform atmospheric pressure concentration at 80 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract with 2.0 g of crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract at atmospheric pressure at 60 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 13.42%, and the moisture content of the finished product is 14%. Weigh 100 mg of Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of water to prepare a 10 mg / mL extract solution. Precisely transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Use the colorimetric method to detect the total polyphenol mass content of Ilex cornuta Lindl. leaf as 27.68 mg / g.

[0063] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid:chlorogenic acid:cryptochlorogenic acid:caffeic acid:isoquercitrin:isochlorogenic acid B:isochlorogenic acid C:isochlorogenic acid A = 100:302.77:108.73:120.01:20.35:21.75:35.35:40.05.

[0064] Example 10

[0065] Weigh 100 g of the powder of Ilex cornuta Lindl. leaves, add 10 times the amount of 70% ethanol, and heat under reflux at 60 °C for 30 minutes. After completion, make up the weight. After centrifuging the extract at 3000×g for 25 minutes to obtain the supernatant, filter it through a reverse osmosis membrane with a pore size of 0.2 nm under the action of a pressure of 3.0 MPa, and after membrane concentration at 0.1 MPa with a filtration flow rate of 5 L / min, concentrate it under normal pressure at 80 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract with a concentration of 0.5 g of crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract at 80 °C under normal pressure to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 13.76%, and the moisture content of the finished product is 5%. Weigh 100 mg of the Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 70% ethanol, and prepare an extract solution of 10 mg / mL. Precisely transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Detect the total polyphenol mass content of the Ilex cornuta Lindl. leaf by colorimetry to be 41.95 mg / g. Dry the Ilex cornuta Lindl. leaf extract at 80 °C under normal pressure to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the moisture content of the finished product is 5%.

[0066] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid:chlorogenic acid:cryptochlorogenic acid:caffeic acid:isoquercitrin:isochlorogenic acid B:isochlorogenic acid C:isochlorogenic acid A = 100:375.28:77.46:247.28:41.26:153.69:39.76:60.45.

[0067] Example 11

[0068] Weigh 100 g of the powder of Ilex cornuta Lindl. leaves, add 10 times the amount of 70% ethanol, and perform microwave extraction. The extraction conditions are 60 °C and 250 w, and the extraction time is 30 minutes. After completion, make up the weight. After centrifuging the extract at 4000×g for 20 minutes to obtain the supernatant, filter it through a nanofiltration membrane with a pore size of 50 nm, and after membrane concentration at 0.1 MPa and a filtration flow rate of 5 L / min, perform normal pressure concentration at 80 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract with a concentration of 0.5 g of crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract at normal pressure at 80 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 13.56%, and the moisture content of the finished product is 9%. Weigh 100 mg of the Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of 70% ethanol to prepare a 10 mg / mL extract solution. Accurately transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Use the colorimetric method to detect that the total polyphenol mass content of the Ilex cornuta Lindl. leaf is 43.65 mg / g.

[0069] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous membrane. Use HPLC method to determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100:405.81:81.26:253.64:42.51:155.19:38.42:59.83.

[0070] Example 12

[0071] Weigh 100 g of the dried Ilex cornuta Lindl. leaves, soak them in 10 times the amount of water for 30 minutes, bring to a boil over high heat and then turn to low heat for decocting for 30 minutes. After centrifuging and filtering, add 10 times the amount of water to the medicinal residues, bring to a boil over high heat and then turn to low heat for decocting for 30 minutes, centrifuge and filter, combine the filtrates, and after membrane concentration at 0.1 MPa and a filtration flow rate of 5 L / min, perform normal pressure concentration at 80 °C for 3 hours to obtain an Ilex cornuta Lindl. leaf extract with a concentration of 1.0 g of crude drug / mL. Dry the Ilex cornuta Lindl. leaf extract at normal pressure at 60 °C to obtain the finished product of Ilex cornuta Lindl. leaf extract. After detection, the yield of the finished product is 13.94%, and the moisture content of the finished product is 15%. Weigh 100 mg of the Ilex cornuta Lindl. leaf extract, dissolve it in 10 mL of water to prepare a 10 mg / mL extract solution. Accurately transfer 5 μL to a 5 mL centrifuge tube, and make up to 1 mL with ultrapure water. Use the colorimetric method to detect that the total polyphenol mass content of the Ilex cornuta Lindl. leaf is 13.68 mg / g.

[0072] Take 1 mL of the extract solution and filter it through a 0.22 μm microporous membrane. Determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid B, isochlorogenic acid C, and isochlorogenic acid A by HPLC. The mass content ratio of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 100:302.27:108.43:120.01:20.35:21.75:35.75:40.05.

[0073] 1.1 Experimental reagents Take 1200 g of dry Ilex cornuta Lindl. leaves, crush them into coarse powder, add 12 times the amount of water by weight of the crude drug, boil and decoct for 30 minutes, then add 10 times the amount of water by weight of the crude drug, boil and decoct for 30 minutes, centrifuge to obtain the supernatant, filter by suction, concentrate under reduced pressure to 2.0 g of crude drug / mL, freeze-dry, dissolve the obtained extract with pure water to obtain medicaments with different concentrations.

[0074] 1. Pharmacodynamic experiment on hyperuricemia mice 1.2 Experimental methods Conduct a pharmacodynamic experiment on hyperuricemia mice by intragastric administration.

[0075] 1.2.1 Grouping of experimental animals Randomly divide into 6 groups according to different administration doses, namely the normal group, the model group, the low-dose Ilex cornuta Lindl. leaf group (0.24 g / kg / d), the medium-dose Ilex cornuta Lindl. leaf group (0.48 g / kg / d), the high-dose Ilex cornuta Lindl. leaf group (0.96 g / kg / d), and the allopurinol group (10 mg / kg / d).

[0076] 1.2.2 Establishment of hyperuricemia model and administration The model group, the low-dose Ilex cornuta Lindl. leaf group, the medium-dose Ilex cornuta Lindl. leaf group, the high-dose Ilex cornuta Lindl. leaf group, and the allopurinol group were intragastrically administered a mixed solution of potassium oxonate (12.5 mg / mL) and adenine (5 mg / mL) to mice at a volume of 0.2 mL / 10 g per day. After 2 hours, each administration group was intragastrically administered the corresponding concentration of Ilex cornuta Lindl. leaf extract, and the normal group of mice was intragastrically administered an equal amount of pure water. The experiment lasted for 21 days.

[0077] 1.2.3 Animal treatment and specimen collection after administration 1) Record the status, body weight, food and water intake of mice daily.

[0078] 2) After the last administration, fast for 12 hours, collect blood and separate the serum, and detect serum uric acid (UA), creatinine (CR), and blood urea nitrogen (BUN).

[0079] 3) After the last administration, fast the mice for 12 hours, weigh the liver, spleen and kidney, and prepare liver homogenate for the detection of xanthine oxidase (XOD), adenosine deaminase (ADA), alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0080] 1.2.4 Data analysis The obtained results were processed using SPSS 25.0, and the data of each group were expressed as ±S.

[0081] 1.3 Experimental results 1.3.1 Body weight of mice The results of the effect of Ilex cornuta Lindl. leaf extract on the body weight of hyperuricemia model mice are shown in Table 1.

[0082] Table 1 Summary of the results of the effect of drugs in different groups on the body weight of mice (unit: g)

[0083] As can be seen from Table 1, compared with the normal group, the body weight of the model group mice decreased significantly from the seventh day. After administration, the body weights of the low, medium and high dose Ilex cornuta Lindl. leaf extract groups were increased compared with the model group.

[0084] Specifically, from the changing trend of the body weight data, the body weight of the normal group mice increased steadily over time, reflecting their stable growth state and good health condition. The body weight of the model group mice decreased significantly since the seventh day, indicating that the construction of the hyperuricemia model had a negative impact on the physiological functions of the mice, resulting in stagnant or even negative body weight growth due to metabolic disorders, nutritional absorption disorders or energy reserve consumption caused by the disease. After administration, the body weights of the low, medium and high dose Ilex cornuta Lindl. leaf extract groups showed an increasing trend compared with the model group, indicating that the extract had a relieving effect on the decreasing trend of the body weight of the mice. This was because the extract improved the metabolic function, reduced the adverse effects of the disease, and helped the mice maintain a normal physiological state and energy balance, providing indirect evidence for the overall improvement of hyperuricemia by the extract. The specific mechanism may involve regulating the nutritional metabolism pathway, reducing the consumption of the body by inflammation, etc.

[0085] 1.3.2 Serum uric acid (UA), creatinine (CR) and blood urea nitrogen (BUN) in mice The determination results of the contents of UA, CR and BUN in the serum of mice after 21 days of administration are shown in Table 2.

[0086] Table 2 Summary of the determination results of the contents of UA, CR and BUN in the serum of mice

[0087] As can be seen from Table 2, the results of UA content showed that compared with the normal group, the serum UA content of the mice in the model group was significantly increased. Compared with the model group, the medium-dose group, high-dose group of Ilex cornuta Lindl. extracts and allopurinol group could significantly reduce the serum UA content of the mice; the results of CR content showed that compared with the normal group, the serum CR content of the mice in the model group was significantly increased. Compared with the model group, the medium-dose group, high-dose group of Ilex cornuta Lindl. and allopurinol group could significantly reduce the serum CR content of the mice; the results of BUN content showed that compared with the normal group, the serum BUN content of the mice in the model group was significantly increased. Compared with the model group, the medium-dose group, high-dose group of Ilex cornuta Lindl. and allopurinol group could significantly reduce the serum BUN content of the mice. It can be seen that within a certain concentration range, as the concentration of Ilex cornuta Lindl. extracts increases, the effects of reducing serum UA, CR, and BUN also increase, showing a certain dose-effect relationship.

[0088] Specifically, the UA data clearly showed that the serum UA content in the model group was significantly higher than that in the normal group, indicating that the hyperuricemia model was successfully established, and there was an imbalance in uric acid metabolism. The medium-dose group, high-dose group of Ilex cornuta Lindl. and allopurinol group significantly reduced the serum UA content of the mice, strongly proving that the extract effectively regulates uric acid metabolism and reduces the serum uric acid level at a specific dose. This effect is enhanced with the increase of the dose, showing a good dose-effect relationship, suggesting that the key components of the extract play a role by inhibiting the key enzyme xanthine oxidase for uric acid production or promoting uric acid excretion, providing the core pharmacodynamic basis and dose-effect reference for the development of anti-hyperuricemia drugs, and having far-reaching significance for deepening the understanding of the pathological mechanism of hyperuricemia and expanding the treatment strategies of traditional Chinese medicine.

[0089] In terms of the CR and BUN indicators, the model group was significantly higher than the normal group, reflecting that hyperuricemia caused damage to the kidney filtration and excretion functions and the accumulation of metabolic wastes. The medium-dose group, high-dose group of Ilex cornuta Lindl. and allopurinol group effectively reduced their contents, indicating that the extract has a protective and reparative effect on kidney function, and reduces kidney cell damage and improves glomerular filtration and tubular reabsorption functions by means of antioxidant stress and anti-inflammation. Its dose-effect relationship provides key clues for accurately determining the clinically effective dose range and optimizing the treatment plan, helps to reveal the microscopic mechanism of traditional Chinese medicine compound in improving kidney function, fills the blank in the research on the kidney protection mechanism of traditional Chinese medicine in hyperuricemia, and enhances the status of traditional Chinese medicine in the treatment of metabolic diseases.

[0090] 1.3.3 Xanthine oxidase (XOD), adenosine deaminase (ADA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the liver of mice The determination results of the contents of XOD, ADA, ALT, and AST in the liver of mice after 21 days of drug administration are shown in Table 3.

[0091] Table 3 Summary of the determination results of the contents of XOD, ADA, ALT, and AST in the liver of mice

[0092] As can be seen from Table 3, the results of XOD content showed that compared with the normal group, the XOD content in the liver of mice in the model group was significantly increased. Compared with the model group, the medium-dose group, high-dose group of Ilex cornuta Lindl. extract, and allopurinol group could significantly reduce the XOD content in the liver of mice. The results of ADA content showed that compared with the normal group, the ADA content in the liver of mice in the model group was significantly increased. Compared with the model group, the medium-dose group, high-dose group of Ilex cornuta Lindl., and allopurinol group could significantly reduce the ADA content in the liver of mice. The results of ALT and AST content showed that compared with the normal group, the ALT and AST contents in the liver of mice in the model group were significantly increased. Compared with the model group, each dose group of Ilex cornuta Lindl. and the allopurinol group could significantly reduce the ALT and AST contents in the liver of mice. It can be seen that within a certain concentration range, as the concentration of Ilex cornuta Lindl. extract increases, the effects of reducing XOD, ADA, ALT, and AST in the liver also increase, showing a certain dose-effect relationship. This indicates that Ilex cornuta Lindl. extract exerts an anti-hyperuricemia effect by inhibiting the activity of liver XOD and has a certain protective effect on the liver of hyperuricemic mice. Therefore, the Ilex cornuta Lindl. extract of the present invention can reduce the contents of UA, CR, and BUN in the serum of mice, has a therapeutic effect on hyperuricemia in mice, its effect is related to inhibiting the activity of liver XOD, and has a certain protective effect on the liver.

[0093] Specifically, at the level of liver XOD activity, the model group was significantly higher than the normal group, confirming that hyperuricemia is closely associated with hyperactive liver XOD and excessive uric acid production. The medium-dose and high-dose groups of Ilex cornuta Lindl. and the allopurinol group significantly reduced the XOD activity, revealing that the core components of the extract precisely act on liver XOD and inhibit its activity to reduce the source link of uric acid synthesis, providing key evidence for the core mechanism of the extract's anti-hyperuricemia, helping to analyze the molecular details of the interaction between traditional Chinese medicine components and key enzymes, providing support for optimizing drug design based on this, and enhancing the precision and scientific nature of traditional Chinese medicine treatment.

[0094] The results of ADA, ALT, and AST activities showed that the model group was significantly higher than the normal group, suggesting that hyperuricemia causes liver cell damage and functional disorders, activation of inflammatory responses, and abnormal hepatocyte metabolism leading to the release of enzymes into the blood. Each dose group of Ilex cornuta Lindl. and the allopurinol group significantly reduced the activities of these enzymes, proving that the extract has multiple protective effects on the liver, can reduce inflammation, repair cell damage, and stabilize the metabolic function of hepatocytes. The synergistic mechanism enriches the theoretical connotation of traditional Chinese medicine liver protection, provides innovative ideas and experimental basis for the development of liver-protecting drugs for anti-hyperuricemia, and promotes the modernization and internationalization process of traditional Chinese medicine.

[0095] The present invention sets up a pharmacodynamic experiment on hyperuricemia mice to verify the therapeutic effect and mechanism of Ilex cornuta Lindl. leaf extract on hyperuricemia. The experiment constructs a hyperuricemia mouse model and administers different doses of Ilex cornuta Lindl. leaf extract, and observes its effects on serum uric acid (UA), creatinine (CR), blood urea nitrogen (BUN) and liver-related enzymes (xanthine oxidase XOD, adenosine deaminase ADA, alanine aminotransferase ALT, aspartate aminotransferase AST) in mice. The results show that Ilex cornuta Lindl. leaf extract can significantly reduce the levels of serum UA, CR and BUN, inhibit the activity of liver XOD, and reduce the contents of ADA, ALT and AST, showing a certain dose-dependence. This indicates that Ilex cornuta Lindl. leaf extract has the effects of reducing uric acid levels and protecting kidney and liver functions, and its mechanism is related to inhibiting uric acid production and alleviating liver injury. The experimental results provide a scientific basis for the potential application of Ilex cornuta Lindl. leaf extract in the prevention and treatment of hyperuricemia.

Claims

1. An extract of Ilex cornuta Lindl. leaves, characterized in that, Its core components include polyphenolic compounds, and the total polyphenol content is 10 - 500 mg / g; the polyphenolic components include: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and the proportion of each component is neochlorogenic acid: chlorogenic acid: cryptochlorogenic acid: caffeic acid: isoquercitrin: isochlorogenic acid B: isochlorogenic acid C: isochlorogenic acid A = 95 - 100: 300 - 450: 70 - 109: 120 - 256: 20 - 46: 21 - 156: 24 - 43: 27 - 61.

2. A method for preparing an extract of Ilex cornuta Lindl. leaves, characterized in that, Specifically, it includes the following steps: S1 Extraction step: Using the leaves or cut pieces of Ilex cornuta Lindl. as raw materials, crush them, pass through a 20 - 80 mesh sieve, add a solvent with a solid - liquid ratio of 5 - 40 times water or 20 - 80% (v / v) ethanol, and at 50 - 100 °C, adopt one of ultrasonic extraction, reflux extraction, microwave extraction, and decoction methods, and continuously extract the active ingredients for 20 - 120 minutes. The extraction times can be one, two, or three times to obtain the extract. S2 Filtration process: The extract is centrifuged at a centrifugal force of 1000 - 10000×g for 5 - 30 minutes. The supernatant is filtered by using one or more of gravity filtration, pressure filtration, microfiltration, ultrafiltration, nanofiltration, or reverse osmosis technologies, and deeply de - impurities under normal pressure, pressure, or reduced pressure. Double - effect filtration ensures the removal of impurities to obtain the filtrate. S3 Concentration step: Using pressurized, normal pressure, or reduced - pressure concentration technology, or combined with membrane concentration technology, concentrate the filtrate to 0.1 - 3.0 g of crude drug / ml to obtain the concentrated solution. S4 Drying: Using one of normal - pressure drying, vacuum drying, spray drying, or freeze - drying, convert the concentrated solution into the form of an extract, lock the activity and quality of the ingredients, and obtain the finished product of the Ilex cornuta Lindl. extract.

3. The preparation method of the Ilex cornuta Lindl. leaf extract according to claim 2, characterized in that, The extraction methods in the S1 extraction step specifically include the following parameters: Ultrasonic extraction method: Add the Ilex cornuta Lindl. raw materials to the solvent, ultrasonic at 50 - 80 °C for 20 - 120 minutes, ultrasonic frequency 20 - 40 kHz, power 250 - 500 W. Microwave extraction method: Add the Ilex cornuta Lindl. raw materials to the solvent, microwave at 60 - 80 °C for 20 - 120 minutes, microwave power 200 - 600 W, frequency 20 - 50 MHz. Reflux extraction method: Add the Ilex cornuta Lindl. raw materials to the solvent, reflux at 60 - 100 °C for 20 - 120 minutes. Decoction method: Add the Ilex cornuta Lindl. raw materials to the solvent, pre - soak for 10 - 60 minutes, bring to a boil over high heat and then decoct over low heat for 20 - 120 minutes.

4. The preparation method of the ilicistrifolia leaf extract according to claim 2, characterized in that, The S2 filtration process specifically includes the following steps: S21 Centrifugation: Centrifuge the extract at 2000 - 10000×g for 5 - 30 minutes to obtain the supernatant. S22 Filtration: Use one or more of gravity filtration, pressure filtration, microfiltration, ultrafiltration, nanofiltration, or reverse osmosis technologies to perform normal - pressure, pressure, or reduced - pressure filtration on the centrifuged supernatant, and deeply de - impurities to obtain the filtrate.

5. The preparation method of the ilicistrata leaf extract according to claim 4, characterized in that, The filtration technologies in the S22 filtration step specifically include the following parameters: Gravity filtration: Filter the centrifuged supernatant with a filter paper or filter cloth with a pore size of 10 - 50 μm to obtain the filtrate. Pressure filtration / microfiltration: The centrifuged supernatant was filtered through a 0.45 - 1 μm microporous membrane by suction filtration to obtain the filtrate; Ultrafiltration: The centrifuged supernatant was passed through an ultrafiltration membrane with a pore size of 1 - 20 nm, and the filtrate was obtained under the pressure of 0.1 - 0.5 Mpa; Nanofiltration: The centrifuged supernatant was passed through a nanofiltration membrane with a pore size of 0.1 - 100 nm to obtain the filtrate; Reverse osmosis: The centrifuged supernatant was passed through a reverse osmosis membrane with a pore size of 0.1 - 1 nm, and the filtrate was obtained under the pressure of 1.0 - 5.0 Mpa.

6. The preparation method of the Ilex cornuta Lindl. leaf extract according to claim 2, wherein The S3 concentration step specifically includes the following steps: S31 Membrane concentration: Set the operating pressure of the membrane concentration equipment to 0.1 - 0.5 Mpa and the filtration flow rate to 5 - 15 L / min to preliminarily remove the solvent in the filtrate and achieve the preliminary concentration of the active ingredients; S32 Concentration: Adopt pressurized concentration or reduced-pressure concentration or atmospheric-pressure concentration to obtain a concentrated solution of 0.1 - 2.0 g of crude drug / ml.

7. The preparation method of the Ilex cornuta Lindl. leaf extract according to claim 6, characterized in that, The concentration methods in the S32 concentration step specifically include the following parameters: Pressurized concentration: At an operating pressure of 0.2 - 2.0 Mpa and a temperature of 50 - 80 °C for 1 - 3 hours, concentrate the extract to a concentrated solution of 0.1 - 2.0 g of crude drug / ml; Reduced-pressure concentration: At an operating pressure of -0.10 to -0.05 Mpa and a temperature of 50 - 70 °C for 2 - 6 hours, concentrate the extract to a concentrated solution of 0.1 - 2.0 g of crude drug / ml; Atmospheric-pressure concentration: At an operating temperature of 80 - 100 °C for 3 - 8 hours, concentrate the extract to a concentrated solution of 0.1 - 2.0 g of crude drug / ml.

8. The preparation method of the Ilex cornuta Lindl. leaf extract according to claim 2, wherein, The S4 drying process dries the Ilex cornuta Lindl. leaf extract to a moisture content of 3 - 15%, and specifically includes the following parameters: Atmospheric-pressure drying: The drying temperature is 60 - 100 °C, and it is dried to a moisture content of 3 - 15%; Vacuum drying: Vacuum to a pressure of 0.01 - 0.1 Mpa, and the drying temperature is 40 - 80 °C; Spray drying: The inlet air temperature is 180 - 250 °C, the outlet air temperature is 80 - 100 °C, and the feeding speed is 10 - 30 ml / minute; Freeze drying: Rapidly cool to -40 to -20 °C for pre-freezing to make the water into ice crystal state, and maintain this low temperature for 2 - 4 hours; Vacuum to a low-pressure environment of 0.01 - 0.1 Pa, and heat up to -10 to 25 °C for 5 - 36 hours.

9. An Ilex cornuta Lindl. leaf extract, a preparation method thereof and an application thereof, characterized in that It includes the application of the Ilex cornuta Lindl. leaf extract in the preparation of drugs for treating hyperuricemia.

Images