Houttuynia cordata extract and preparation method thereof

Abstract

The invention discloses a herba houttuyniae extract and a preparation method thereof, and relates to the technical field of Chinese herbal medicine extraction. Comprising the following steps: by taking fresh houttuynia cordata leaves as a unique raw material, carrying out a directional enrichment process, and taking sodium houttuyfonate as a core effective component; the purity of sodium houttuyfonate is not less than 99%, and the extraction rate is not less than 98%; the raw materials are subjected to airflow superfine grinding treatment, and the particle size of the ground raw materials is 1-100 nm; the content of sodium houttuyfonate is reduced by less than or equal to 2% within 12 months of sealed storage of the extract at room temperature. The raw materials are subjected to superfine grinding, so that cells of the raw materials are completely broken, effective components can be fully released, and the extraction time is greatly shortened; the effective component houttuyfonate is adsorbed and eluted by an ion exchange column and then is subjected to a salt forming reaction to generate sodium houttuyfonate, so that the property stability and the purity of the product in the post-treatment process are improved; the whole extraction process is carried out at low temperature, so that the stability and absorptivity of the product are further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Chinese herbal medicine extraction, in particular to a Gynura japonica extract and a preparation method thereof. BACKGROUND

[0002] Gynura japonica is a plant used for both medicine and food, which has been recorded in the Tang Dynasty New Revised Materia Medica. At present, it is widely distributed in Yunnan, Guizhou, Sichuan and other places in China. Gynura japonica has very wide pharmacological effects, and modern pharmacological studies have proved that it has antibacterial, anti-inflammatory, antiviral, immune-enhancing and growth-promoting effects. Decanoyl acetaldehyde (i.e. Gynura japonica) is the main effective component of Gynura japonica. Due to its unstable properties, it is mostly in the form of Gynura japonica sodium salt in production and application.

[0003] Most of Gynura japonica exists in fresh leaves of Gynura japonica, and only a small amount exists in rhizomes. Gynura japonica belongs to natural plants, and its cell wall is relatively thick, which is composed of three parts of intercellular layer, primary wall and secondary wall, and contains various cellulose, hemicellulose, lignin, pectin and other substances, so that the cell wall has the characteristics of lignification, suberization, keratinization and mineralization. Conventional extraction methods mainly include steam distillation, alcohol extraction and / or ultrasonic assistance and supercritical extraction, etc. It is difficult to dissolve Gynura japonica in cells by using conventional extraction methods, and in addition, Gynura japonica is unstable, and is easily converted into methyl n-nonyl ketone during the long extraction process, which further leads to a decrease in yield.

[0004] In the patents such as CN1457810A, CN103719555A and CN111184794A, the raw material of Gynura japonica is subjected to conventional crushing treatment, and the particle size after crushing is 5-80 meshes, which is difficult to destroy the cell wall structure, and significantly increases the difficulty of subsequent extraction. CN202510543367 discloses a Gynura japonica extract and a preparation method thereof, wherein Gynura japonica is first crushed to 80-120 meshes, and then subjected to enzyme pretreatment. Although this method preliminarily crushes the raw material, the wall breaking is not sufficient, and then the enzyme treatment is used. Since the activity of the enzyme is easily affected by various factors such as temperature, PH, substrate concentration, enzyme concentration, activator and inhibitor, the enzyme is easily inactivated or has a reduced activity, and it is difficult to stably play a role, and multiple extractions are required, which is low in production efficiency.

[0005] In addition, Gynura japonica is sensitive to temperature and is easily degraded at high temperature, and the patents disclosed at present, such as CN201510471626, use steam distillation method with a distillation temperature of 100℃, and CN202310158936 uses subcritical extraction with a temperature as high as 130-170℃, which will lead to partial degradation and inactivation of Gynura japonica during the extraction process under the above high temperature conditions. Therefore, the present application proposes a Gynura japonica extract and a preparation method thereof to solve such problems. SUMMARY

[0006] Technical problems solved In view of the deficiencies of the prior art, the fishwort extract and the preparation method thereof are provided to solve the problems in the background art.

[0007] Technical scheme To achieve the above object, the fishwort extract is prepared by the following technical scheme: the fishwort extract takes fresh leaves of fishwort as the only raw material, the fresh leaves of fishwort are fresh leaves without mildew and pests, and the rhizome part is removed, and the processing is performed within 24 hours after picking.

[0008] The extract is prepared by a directional enrichment process, the core active ingredient is sodium houttuyniae extract, and the extract also contains water-soluble polysaccharide ingredients, wherein the purity of the sodium houttuyniae extract is greater than or equal to 99%, the extraction rate is greater than or equal to 98%, the polysaccharide content is greater than or equal to 3.0%, and the molecular weight of the polysaccharide ingredients ranges from 1000 to 5000 daltons.

[0009] The raw material is subjected to airflow ultrafine grinding treatment, and the particle size of the sodium houttuyniae extract after grinding ranges from 1 to 100 nanometers, preferably ranges from 1 to 50 nanometers, and the particle distribution uniformity is greater than or equal to 90%.

[0010] The content of the sodium houttuyniae extract in the extract decreases by less than or equal to 2% within 12 months of storage at room temperature in a sealed state, and the water content of the extract is less than or equal to 2%. The detection of the sodium houttuyniae extract is performed by high performance liquid chromatography, and the detection conditions are as follows: the chromatographic column is a C18 chromatographic column with a column length of 4.6 mm, a column diameter of 250 mm and a particle size of 5 microns, the mobile phase is a mixture of methanol and water with a volume ratio of 70 to 30, the detection wavelength is 254 nanometers, the flow rate is 1 milliliter per minute, the column temperature is 45 degrees Celsius, the injection amount is 20 microliters, and the theoretical plate number calculated according to the sodium houttuyniae extract peak is greater than or equal to 2500. The fresh leaves of fishwort and the removal of the rhizome as the only raw material are because modern pharmacological research has confirmed that the content of fishwort extract in fresh leaves is greater than or equal to 90%, the content in rhizome is less than or equal to 10% and rich in lignin and other impurities, which will increase the difficulty of extraction and purification and affect the purity and stability of the product. The processing within 24 hours after picking is because the oxidation rate of fishwort extract will increase by more than 3 times after the fresh leaves are dehydrated, and timely processing can maximize the retention of active ingredients.

[0011] The purity of the sodium houttuyniae extract is greater than or equal to 99%, the extraction rate is greater than or equal to 98%, the polysaccharide content is greater than or equal to 3.0%, and the molecular weight ranges from 1000 to 5000 daltons, because the sodium houttuyniae extract is the core active ingredient, high purity can ensure stable efficacy and reduce impurity interference; the extraction rate greater than or equal to 98% can fully utilize the raw material, reduce the production cost and improve the industrial economic efficiency; the polysaccharide with the molecular weight in the range has good water solubility, can coexist with the sodium houttuyniae extract, and does not affect the subsequent application.

[0012] The particle size of sodium houttuyniae extract is 1-100 nanometers, preferably 1-50 nanometers, and the uniformity is greater than or equal to 90%. The particle size interval has a large specific surface area, which is beneficial to absorption and utilization and can synergize with other components. The uniformity meets the standard to avoid performance fluctuations and ensure batch quality consistency. The content decreases by less than or equal to 2% and the water content is less than or equal to 2% after 12 months of storage at room temperature in a sealed container. The low water content can inhibit microbial growth, reduce degradation of active ingredients, and ensure stable quality during the shelf life.

[0013] The parameters of high-performance liquid chromatography are set because the C18 chromatographic column 4.6 mm x 250 mm x 5 microns has excellent separation effect on sodium houttuyniae extract, which can effectively distinguish target components from impurities. The volume ratio of methanol to water is 70 to 30, which can obtain suitable retention time for sodium houttuyniae extract, symmetrical peak shape, and separation degree meeting the standard. 254 nanometers is the characteristic absorption wavelength of sodium houttuyniae extract, with high detection sensitivity. The combination of 1 milliliter per minute flow rate, 45 degrees Celsius column temperature, and 20 microliter injection volume takes into account efficiency and accuracy. The theoretical plate number is greater than or equal to 2500 to ensure detection precision and avoid quantitative errors.

[0014] A preparation method of houttuynia cordata extract Step one, take the fresh leaves of the houttuynia cordata, wash and drain, and then dry them in a forced air drying machine. The drying temperature is 55 degrees Celsius, and the drying time is 2-4 hours. After drying, the water content of the raw material is less than or equal to 8%. Add the dried raw material to an air jet pulverizer, use nitrogen as an inert gas, control the nitrogen pressure at 0.5 megapascals, the pulverizer power is 150-500 kilowatts, preferably 200-400 kilowatts, the feeding speed is 150-260 meters per second, preferably 180-250 meters per second, the pulverizing time is 1-3 hours, and the continuous pulverizing time is 2-5 times, preferably 3-4 times. The particle size of the nanoscale houttuynia cordata powder is 1-100 nanometers, and the qualified rate is greater than or equal to 95%. In the raw material pretreatment, 55 degrees Celsius blast drying is a safe temperature, higher than which will cause the degradation of jinyingtaosu, and lower than which will result in insufficient drying efficiency, and 2-4 hours of drying time can reduce the moisture content to ≤8%, which can avoid caking during pulverization and also does not lose the effective components. Airflow pulverization uses nitrogen as the inert gas because nitrogen is chemically stable, can prevent the oxidation of jinyingtaosu and carry away the pulverization heat to avoid local overheating; the combination of 0.5 megapascal nitrogen pressure, 150-500 kilowatt power, 150-260 meters per second feeding speed, 1-3 hours of pulverization time, and 2-5 times of continuous pulverization can ensure that the raw material is pulverized to 1-100 nanometers, and the qualified rate of particle size is ≥95%; preferably 200-400 kilowatt power, 180-250 meters per second feeding speed, and 3-4 times of continuous pulverization, because this interval can reduce energy consumption and improve efficiency while ensuring effectiveness. In this step, the initial moisture content of fresh jinyingtao leaves is about 70%, jinyingtaosu is wrapped in the cell wall, and polysaccharides are dispersed in the cytoplasm, and the cell structure is complete; after 55 degrees Celsius drying for 2-4 hours, the moisture content is ≤8%, the cells shrink due to dehydration but the cell wall is intact, and the effective components have no chemical changes; after airflow pulverization, the cell wall is completely broken, jinyingtaosu and polysaccharides are exposed on the surface of the powder, the morphology changes from flaky to nanoscale particles, and the specific surface area increases by 100-1000 times, which lays the foundation for subsequent extraction.

[0015] Step two, mix the nanoscale jinyingtao powder with an ethanol solution with a concentration of 70%-95% at a solid-liquid ratio of 1:10 to 1:30, preferably with an ethanol concentration of 95% and a solid-liquid ratio of 1:15, soak and extract for 1-5 hours, preferably for 1-2 hours. After extraction is complete, filter using a plate and frame centrifuge, the centrifuge speed is 1200-2000 revolutions per minute, the filter cloth aperture is 10 microns, the flow rate is 60-100 cubic meters per hour, and collect the plate and frame filtrate. Add the filtered residue to a screw press to extract juice, the filter screen aperture of the screw press is 10 microns, the power is 5-10 kilowatts, and collect the pressed juice. Combine the plate and frame filtrate and the pressed juice to obtain the crude extract.

[0016] In the extraction step, 70% to 95% ethanol solution can take into account the fishy grassin liposolubility and polysaccharide water-solubility, and the concentration is too high to reduce the solubility of polysaccharide, and too low to reduce the extraction efficiency of fishy grassin, and 95% ethanol is preferred because the solubility of fishy grassin is the highest at this concentration. The solid-liquid ratio of 1 to 10 to 1 to 30 is calculated according to the specific surface area of nanoscale powder, which can ensure that ethanol fully wraps the particles to dissolve the effective components, and 1 to 15 is preferred to reduce the amount of ethanol and reduce the cost of recovery. The extraction time of 1 to 5 hours is because the nanoscale powder is broken after the wall, and the effective components are dissolved quickly, and there is no need for long soaking time, and 1 to 2 hours is preferred to improve the production efficiency and reduce the degradation of effective components. The plate and frame centrifuge 1200 to 2000 revolutions per minute, 10 microns filter cloth aperture, 60 to 100 cubic meters per hour flow, can quickly separate the extraction liquid and the filter residue, and intercept the coarse particles that are not completely crushed; the screw press 10 micron filter screen aperture, 5 to 10 kilowatt power, can squeeze out the extraction liquid adsorbed in the filter residue, and avoid waste. First, soak in ethanol to dissolve most of the effective components, then centrifugal filtration separation, and finally press the filter residue to recover the residual components, the three synergies can improve the extraction rate by more than 30%, and the combined filtration liquid and pressed juice can avoid the loss of effective components caused by batch processing, and ensure the continuous process. In this step, after the nanoscale powder is soaked, fishy grassin and polysaccharide are gradually dissolved to form a light yellow transparent mixed solution; after centrifugal filtration, coarse particles are removed to obtain a clear filtrate; the color of the pressed juice is slightly darker, and the effective component concentration of the combined crude extract is about 0.5 to 1.0 grams per liter, containing a small amount of chlorophyll, cellulose fragments and other molecules with a molecular weight of ≥10000 Dalton impurities.

[0017] Step three, the crude extract is filtered through ceramic membrane with pore size of 0.22 micron and filtration pressure of 0.3-0.5 MPa, and the microfiltrate is collected. An ion exchange column filled with XAD1600 macroporous resin with pore size of 150 micron is taken, the ion exchange column is first flushed with 2 column volumes of 50 mmol / L phosphate buffer with pH value of 6.5, then the microfiltrate is loaded into the ion exchange column for adsorption according to the treatment capacity of 10-15 g of active product per liter of resin, and the preferred loading amount is 10 g per liter of resin. The adsorption process is controlled at a flow rate of 4 column volumes per hour and pressure of 0.1-0.3 MPa. After the adsorption is completed, gradient elution is performed, and the gradient elution liquid is 2 column volumes of 50 mmol / L phosphate buffer with pH value of 6.5, 3 column volumes of 0.1 mol / L sodium chloride phosphate buffer with pH value of 6.5, 4 column volumes of 0.3 mol / L sodium chloride phosphate buffer with pH value of 6.5, 3 column volumes of 0.5 mol / L sodium chloride phosphate buffer with pH value of 6.5, and 2 column volumes of 0.7 mol / L sodium chloride phosphate buffer with pH value of 6.5, respectively. The elution flow rate is 4 column volumes per hour, the pressure is maintained at 0.1-0.3 MPa, and the eluate from the 0.3 mol / L sodium chloride phosphate buffer elution stage to the early stage of 0.5 mol / L sodium chloride phosphate buffer elution is collected, and the total collection amount is 6 column volumes, thereby obtaining the purified target component.

[0018] In the purification step, the 0.22-micron ceramic membrane can intercept macromolecular impurities with a molecular weight of ≥10,000 daltons in the crude extract, avoiding the blockage of the pore channels of the ion exchange column resin. The filtration pressure of 0.3-0.5 megapascals can ensure an appropriate flow rate, taking into account the efficiency and protection of the membrane module. The XAD1600 macroporous resin has a non-polar pore structure, which can preferentially adsorb non-polar agastachin and weakly adsorb polar impurities such as polysaccharides. The 150-micron pore size can intercept medium molecular weight impurities with a molecular weight of ≥1,000 daltons, allowing the fishy grass root extract with a molecular weight of 168 daltons and the target polysaccharides to pass through. First, the column is flushed with 2 column volumes of 50-millimolar phosphate buffer at pH 6.5 to balance the column environment and remove residual impurities from the resin. The loading amount of 10-15 grams per liter of resin, preferably 10 grams per liter, can ensure that the resin fully adsorbs the active ingredients and avoid adsorption saturation affecting the purification effect. Gradient elution sequentially increases the sodium chloride concentration to separate different components: 50-millimolar phosphate buffer elutes unadsorbed polysaccharides and small molecular polar impurities, 0.1-molar sodium chloride phosphate buffer elutes weakly adsorbed fat-soluble impurities, 0.3-0.5-molar sodium chloride phosphate buffer elutes the target component fishy grass root extract, and 0.7-molar sodium chloride phosphate buffer elutes residual strongly adsorbed impurities. The elution flow rate of 4 column volumes per hour and the pressure of 0.1-0.3 megapascals can ensure that the eluent and the resin are in full contact, avoid poor separation caused by excessive flow rate, and prevent the resin bed from loosening. Ceramic membrane filtration pretreatment removes macromolecular impurities, creating conditions for ion exchange chromatography. Resin adsorption and gradient elution achieve efficient separation of target components and impurities, and the combination of the two can increase the purity of fishy grass root extract from 80%-85% to 95%-98%. In this step, the crude extract is filtered by the ceramic membrane to obtain a colorless transparent microfiltrate with a significantly reduced impurity content. After the microfiltrate enters the ion exchange column, fishy grass root extract is adsorbed by the resin, and polar impurities such as polysaccharides are discharged with the effluent. The target component collected after gradient elution is colorless and transparent, the purity of fishy grass root extract is significantly improved, the polysaccharide content is reduced to 3%-5%, and the impurity content is ≤0.5%.

[0019] Step four, the purified target component is put into a crystallization tank, saturated sodium bisulfite solution is added for salt formation reaction, the reaction pH value is controlled at 8-9, preferably the pH value is 8.5, the reaction temperature is 40-60 degrees Celsius, preferably the reaction temperature is 55 degrees Celsius, the reaction time is 2-4 hours, preferably the reaction time is 3 hours. After the salt formation reaction is completed, 50% concentration ethanol solution is added to the crystallization tank at a ratio of 20 kg of 50% concentration ethanol solution per 1 kg of target component, and it is fully stirred and mixed. Hot water is introduced into the jacket of the crystallization tank to increase the temperature, until the temperature of the material liquid reaches 30 degrees Celsius, 10% concentration phosphoric acid is used to adjust the pH value of the material liquid to 6.5-6.8, preferably the pH value is adjusted to 6.8. Then ice salt water is introduced into the jacket, and the material liquid temperature is slowly reduced to 5 degrees Celsius at a uniform speed, and maintained for 18-24 hours, preferably the crystallization maintenance time is 20 hours. A centrifugal machine is used to centrifugal filtration at a speed of 1500 revolutions per minute, and the filter residue is collected. The filter residue is put back into the crystallization tank, 50% concentration ethanol solution is added at a ratio of 20 kg of 50% concentration ethanol solution per 1 kg of filter residue, which is pre-cooled to 5 degrees Celsius, and the 50% concentration ethanol solution is stirred and washed, and then centrifugal filtration is performed again at a speed of 1500 revolutions per minute, and the wet product is collected.

[0020] The saturated sodium bisulfite solution is added in the salt formation crystallization step because the houttuynia cordata extract is an aldehyde and is unstable and easily oxidized and degraded, and the reaction with sodium bisulfite generates sodium houttuynia cordata extract, and the oxidation rate is reduced by more than 90%; the reaction pH is 8-9, preferably 8.5, because the alkaline environment can promote the addition reaction and improve the salt formation conversion rate; the reaction temperature is 40-60 degrees Celsius, preferably 55 degrees Celsius, which can accelerate the reaction rate and avoid degradation of the active ingredients; the reaction time is 2-4 hours, preferably 3 hours, which can ensure that the salt formation conversion rate is ≥99%. After the salt formation reaction, 20 kg of 50% ethanol solution is added per kilogram of target component because the solubility of sodium houttuynia cordata extract in the ethanol of this concentration is moderate, meeting the crystallization requirements; the temperature is raised to 30 degrees Celsius and the pH is adjusted to 6.5-6.8, preferably 6.8, to optimize the crystallization environment, so that the crystals are easily precipitated and the particles are uniform; the ice-salt water is switched to uniform cooling to 5 degrees Celsius and maintained for 18-24 hours, preferably 20 hours, because the solubility of sodium houttuynia cordata extract decreases significantly with the decrease in temperature, and the solubility at 5 degrees Celsius is only 1 / 10 of that at 30 degrees Celsius; uniform cooling avoids crystal agglomeration, and long-term maintenance ensures complete precipitation; the filter residue is washed with pre-cooled 50% ethanol solution to remove impurities adsorbed on the surface of the crystals and avoid dissolving the crystals. The centrifuge speed of 1500 rpm can effectively separate the crystals from the mother liquor and avoid breaking the crystals, and the two centrifugation and filtration steps can ensure sufficient washing and improve the purity of the product. The unstable houttuynia cordata extract is first converted into stable sodium houttuynia cordata extract by salt formation reaction, and then further purified by ethanol crystallization. The temperature adjustment and pH adjustment create conditions for crystallization, and the cooling and incubation promote the precipitation of crystals. The whole process is closely linked, taking into account stability and purity. In this step, the target component before salt formation reaction is a colorless transparent liquid, and the solution becomes slightly turbid after reaction with sodium bisulfite; it returns to clarity after adding ethanol solution and adjusting the temperature and pH; white crystals gradually precipitate after the temperature is reduced to 5 degrees Celsius and maintained; the first centrifugation produces white crystal filter residue, which is washed with pre-cooled ethanol and subjected to secondary centrifugation. The wet product is a white crystal with a purity of ≥99%, a particle size of 1-50 nanometers, and a water content of 30%-40%.

[0021] Step five, the wet product is placed in a vacuum drying oven, the vacuum degree is controlled to 100-133 Pa, preferably 120 Pa, the temperature in the drying oven is 40-50 degrees Celsius, preferably 45 degrees Celsius, and the total drying time is 16-20 hours. After drying, the product is sieved and granulated with a 60-mesh shaking granulator, and then reloaded into the vacuum drying oven for further drying for 10-12 hours. After drying, the product is sieved again with a 60-mesh sieve to obtain the houttuynia cordata extract.

[0022] In the drying step, the 100-133 Pa vacuum degree, preferably 120 Pa, can reduce the boiling point of water, so that the water can be quickly evaporated at 40-50 degrees Celsius, preferably 45 degrees Celsius, to avoid high temperature leading to degradation of sodium houttuyniae; the two-stage drying, with a total time of 16-20 hours, is because the wet product contains surface moisture and internal moisture, the surface moisture is removed by drying for 6-8 hours first, the crystal agglomeration structure is destroyed after granulation, and then the internal moisture is removed by drying for 10-12 hours, so as to ensure that the water content is ≤2%; the 60-mesh swing granulator is used for sieving and granulation, so as to avoid caking during drying, make the finished product granules uniform, and facilitate subsequent application. The wet product is dried in the first stage to remove most of the moisture, and then granulated to ensure uniformity and fullness, and sieved twice through a 60-mesh sieve to ensure that the particle size meets the standard and improve the uniformity of the product. In this step, the wet product is a white crystal containing 30%-40% moisture, the water content is ≤5% after the first-stage drying, and it becomes a semi-dry product with agglomeration; after granulation, it becomes uniform granules; after the second drying, the water content is ≤2%, and the white powder-like extract is obtained after sieving again, the particle size passes through a 60-mesh sieve, the purity of sodium houttuyniae is ≥99%, and the polysaccharide content is ≥3.0%.

[0023] In the entire preparation method, the ultra-micro breaking of the raw material pretreatment is the premise, the broken cell wall exposes the effective components; the double-step juice extraction is the core of the extraction step, which fully recovers the effective components; the ceramic membrane filtration and ion exchange chromatography are the key of the purification step, which removes impurities and enriches target components; the salt formation and crystallization realize the stabilization and further purification of the effective components; and the drying step converts the wet product into the finished product.

[0024] Advantages The present application has the following advantages: (1) The houttuynia cordata extract and the preparation method thereof, by ultra-micro grinding of the raw material, the cells are completely broken, and the effective components can be fully released, which greatly shortens the extraction time; the effective component houttuyniae is subjected to salt formation reaction after ion exchange column adsorption and elution to generate sodium houttuyniae, which improves the stability and purity of the product in the post-processing process; the entire extraction process is carried out at low temperature, which further improves the stability and absorption rate of the product, and the purity of the houttuynia cordata extract of the present application is more than 99%, the extraction rate is more than 98%, the quality is stable, the safety is high, the process is simple, the production cost is low, and the product is safe and pollution-free.

[0025] Of course, implementing any product of the present application does not necessarily require achieving all the advantages described above at the same time. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure is a particle size diagram of the houttuynia cordata raw material of Example 1 of the present application. DETAILED DESCRIPTION

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1 A method for preparing houttuynia cordata extract, such as Figure 1 As shown, it includes the following steps: Preparation of Houttuynia cordata ultrafine powder: Fresh leaves of Houttuynia cordata were picked, washed with clean water to remove impurities such as mud and sand; dried in a blower dryer at 55 degrees Celsius for 3 hours. After drying, the powder was fed into an air jet mill at a pressure of 0.5 MPa, a power of 200 kW, and a feed speed of 180 m / s for 3 hours, followed by 4 consecutive grinding cycles, resulting in a particle size D50 of 12.80 nm.

[0029] Preparation of the alcohol extract: The ultrafine powder obtained in step 1 was mixed with a 95% ethanol solution at a solid-liquid ratio of 1:15 and extracted for 1 hour. The mixture was then filtered through a plate and frame filter at a speed of 1500 rpm, a filter cloth of 10 micrometers, and a flow rate of 70 cubic meters per hour. The alcohol extract was collected.

[0030] Treatment of filter residue: Press the juice using a twin-screw rotary press with a 10-micron filter screen and a power of 10 kilowatts, and collect the pressed juice.

[0031] Ceramic membrane filtration: The juice from the combined alcohol extract and filter residue is passed through a ceramic membrane for microfiltration with a pore size of 0.22 micrometers and a pressure of 0.3 MPa. The microfiltrate is then collected.

[0032] Ion exchange chromatography: The ion exchange column was washed with 2 column volumes of 50 mM phosphate buffer, and then loaded with ceramic membrane microfiltrate at a processing capacity of 10 g of active product per liter of resin to complete the sample loading and adsorption. The gradient elution sequence is as follows: 50 mM phosphate buffer solution, pH 6.5: 2 column volumes; 0.1 molar sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes; 0.3 mol sodium chloride-phosphate buffer solution, pH 6.5: 4 column volumes; 0.5 molar sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes; 0.7 mol sodium chloride-phosphate buffer solution (pH 6.5): collect the eluent at 2 column volumes, approximately 6 column volumes. During equilibration, adsorption, and elution, the flow rate was controlled at 4 column volumes per hour, and the pressure was maintained at 0.2 MPa.

[0033] Salt formation reaction: Add saturated sodium bisulfite solution to the eluent, control the pH at 8.5, the temperature at 55 degrees Celsius, and the reaction time at 3 hours.

[0034] Crystallization and Concentration: After the salt formation reaction is complete, add a 50% ethanol solution (1:20 ratio) to the reaction solution and stir thoroughly. Heat the hot water in the crystallizer jacket until the temperature of the solution reaches 30°C, then adjust the pH to 6.8 with 10% phosphoric acid. Switch the flow of ice-salt water into the jacket, stirring slowly to uniformly lower the temperature of the solution to 5°C and maintain this temperature for 20 hours. Centrifuge and filter at 1500 rpm. Return the filter residue to the crystallizer, add a pre-cooled 50% ethanol solution (1:20 ratio) to the residue, stir and wash, then centrifuge and filter again at 1500 rpm, collecting the wet product.

[0035] The wet product was vacuum dried at a vacuum level of 120 Pa and a drying oven temperature of 45 degrees Celsius for 6 hours. The semi-dried product was then released and granulated by sieving through a 60-mesh vibrating granulator, and then returned to the vacuum drying oven for another 10 hours. After sieving through a 60-mesh sieve again, the product was mixed and packaged to obtain the finished product.

[0036] Example 2 A method for preparing houttuynia cordata extract includes the following steps: Preparation of Houttuynia cordata ultrafine powder: Fresh leaves of Houttuynia cordata were picked, washed with clean water to remove impurities such as mud and sand; dried in a blower dryer at 55 degrees Celsius for 2 hours. After drying, the powder was fed into an air jet mill at a pressure of 0.5 MPa, a power of 300 kW, and a feed speed of 200 m / s for 2 hours, and continuously pulverized 3 times. The particle size D50 was 20.65 nm.

[0037] Preparation of the alcohol extract: The ultrafine powder obtained in step 1 was mixed with a 95% ethanol solution at a solid-liquid ratio of 1:15 and extracted for 1 hour. The mixture was then filtered through a plate and frame filter at a speed of 1500 rpm, a filter cloth of 10 micrometers, and a flow rate of 70 cubic meters per hour. The alcohol extract was collected.

[0038] Treatment of filter residue: Press the juice using a twin-screw rotary press with a 10-micron filter screen and a power of 10 kilowatts, and collect the pressed juice.

[0039] Ceramic membrane filtration: The juice from the combined alcohol extract and filter residue is passed through a ceramic membrane for microfiltration with a pore size of 0.22 micrometers and a pressure of 0.3 MPa. The microfiltrate is then collected.

[0040] Ion exchange chromatography: The ion exchange column was washed with 2 column volumes of 50 mM phosphate buffer, and then loaded with ceramic membrane microfiltrate at a processing capacity of 10 g of active product per liter of resin to complete the sample loading and adsorption. The gradient elution sequence is as follows: 50 mM phosphate buffer solution, pH 6.5: 2 column volumes; 0.1 molar sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes; 0.3 mol sodium chloride-phosphate buffer solution, pH 6.5: 4 column volumes; 0.5 molar sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes; 0.7 mol sodium chloride-phosphate buffer solution (pH 6.5): collect the eluent at 2 column volumes, approximately 6 column volumes. During equilibration, adsorption, and elution, the flow rate was controlled at 4 column volumes per hour, and the pressure was maintained at 0.2 MPa.

[0041] Salt formation reaction: Add saturated sodium bisulfite solution to the eluent, control the pH at 8.0, the temperature at 50 degrees Celsius, and the reaction time at 4 hours.

[0042] Crystallization and Concentration: After the salt formation reaction is complete, add a 50% ethanol solution (1:20 ratio) to the reaction solution and stir thoroughly. Heat the hot water in the crystallizer jacket until the temperature of the solution reaches 30°C, then adjust the pH to 6.8 with 10% phosphoric acid. Switch the flow of ice-salt water into the jacket, stirring slowly to uniformly lower the temperature of the solution to 5°C and maintain this temperature for 20 hours. Centrifuge and filter at 1500 rpm. Return the filter residue to the crystallizer, add a pre-cooled 50% ethanol solution (1:20 ratio) to the residue, stir and wash, then centrifuge and filter again at 1500 rpm, collecting the wet product.

[0043] The wet product was vacuum dried at a vacuum level of 130 Pa and a drying oven temperature of 50 degrees Celsius for 6 hours. The semi-dried product was then discharged and granulated by sieving through a 60-mesh vibrating granulator, and then returned to the vacuum drying process for another 11 hours. After sieving through a 60-mesh sieve again, the product was mixed and packaged to obtain the finished product.

[0044] Example 3 A method for preparing houttuynia cordata extract includes the following steps: Preparation of Houttuynia cordata ultrafine powder: Fresh leaves of Houttuynia cordata were picked, washed with clean water to remove impurities such as mud and sand; dried in a blower dryer at 55 degrees Celsius for 4 hours. After drying, the powder was fed into an air jet mill at a pressure of 0.5 MPa, a power of 400 kW, and a feed speed of 250 m / s for 1 hour, followed by two consecutive grinding cycles, resulting in a particle size D50 of 57.59 nm.

[0045] Preparation of the alcohol extract: The ultrafine powder obtained in step 1 was mixed with a 95% ethanol solution at a solid-liquid ratio of 1:25 and extracted for 2 hours. The mixture was then filtered through a plate and frame filter at a speed of 1500 rpm, a filter cloth of 10 micrometers, and a flow rate of 70 cubic meters per hour. The alcohol extract was collected.

[0046] Treatment of filter residue: Press the juice using a twin-screw rotary press with a 10-micron filter screen and a power of 10 kilowatts, and collect the pressed juice.

[0047] Ceramic membrane filtration: The juice from the combined alcohol extract and filter residue is passed through a ceramic membrane for microfiltration with a pore size of 0.22 micrometers and a pressure of 0.3 MPa. The microfiltrate is then collected.

[0048] Ion exchange chromatography: The ion exchange column was washed with 2 column volumes of 50 mM phosphate buffer, and then loaded with ceramic membrane microfiltrate at a processing capacity of 10 g of active product per liter of resin to complete the sample loading and adsorption. The gradient elution sequence is as follows: 50 mM phosphate buffer solution, pH 6.5: 2 column volumes 0.1 mol sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes 0.3 mol sodium chloride-phosphate buffer solution, pH 6.5: 4 column volumes 0.5 mol sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes 0.7 mol sodium chloride-phosphate buffer solution (pH 6.5): collect the eluent at 2 column volumes, approximately 6 column volumes. During equilibration, adsorption, and elution, the flow rate was controlled at 4 column volumes per hour, and the pressure was maintained at 0.2 MPa.

[0049] Salt formation reaction: Add saturated sodium bisulfite solution to the eluent, control the pH at 9.0, the temperature at 60 degrees Celsius, and the reaction time at 3 hours.

[0050] Crystallization and Concentration: After the salt formation reaction is complete, add a 50% ethanol solution (1:20 ratio) to the reaction solution and stir thoroughly. Heat the hot water in the crystallizer jacket until the temperature of the solution reaches 30°C, then adjust the pH to 6.8 with 10% phosphoric acid. Switch the flow of ice-salt water into the jacket, stirring slowly to uniformly lower the temperature of the solution to 5°C and maintain this temperature for 18 hours. Centrifuge and filter at 1500 rpm. Return the filter residue to the crystallizer, add a pre-cooled 50% ethanol solution (1:20 ratio) to the residue, stir and wash, then centrifuge and filter again at 1500 rpm, collecting the wet product.

[0051] The wet product was vacuum dried at a vacuum level of 100 Pa and a drying oven temperature of 50 degrees Celsius for 7 hours. The semi-dried product was then discharged and granulated by sieving through a 60-mesh vibrating granulator, and then returned to the vacuum drying process for another 12 hours. After sieving through a 60-mesh sieve again, the product was mixed and packaged to obtain the finished product.

[0052] Comparative Example 1 The purpose of this comparative study is to investigate the effect of insufficient cell wall disruption on the extraction rate of houttuynia cordata extract when using conventional pulverization treatment. A method for preparing houttuynia cordata extract includes the following steps: Simple crushing process for Houttuynia cordata raw materials: Pick fresh Houttuynia cordata leaves, wash them with clean water to remove mud and other impurities; place them in a blower dryer to dry at 55 degrees Celsius for 3 hours. After drying, crush them through an 80-mesh sieve to a particle size of 0.18 mm.

[0053] Preparation of the alcohol extract: The pulverized material obtained in step 1 was mixed with a 95% ethanol solution at a solid-liquid ratio of 1:15 and extracted for 1 hour. The mixture was then filtered through a plate and frame filter at 1500 rpm, using a 10-micron filter cloth and a flow rate of 70 cubic meters per hour. The alcohol extract was collected.

[0054] Treatment of filter residue: Press the juice using a twin-screw rotary press with a 10-micron filter screen and a power of 10 kilowatts, and collect the pressed juice.

[0055] Ceramic membrane filtration: The juice from the combined alcohol extract and filter residue is passed through a ceramic membrane for microfiltration with a pore size of 0.22 micrometers and a pressure of 0.3 MPa. The microfiltrate is then collected.

[0056] Ion exchange chromatography: The ion exchange column was washed with 2 column volumes of 50 mM phosphate buffer, and then loaded with ceramic membrane microfiltrate at a processing capacity of 10 g of active product per liter of resin to complete the sample loading and adsorption. The gradient elution sequence is as follows: 50 mM phosphate buffer solution, pH 6.5: 2 column volumes; 0.1 molar sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes; 0.3 mol sodium chloride-phosphate buffer solution, pH 6.5: 4 column volumes; 0.5 molar sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes; 0.7 mol sodium chloride-phosphate buffer solution (pH 6.5): collect the eluent at 2 column volumes, approximately 6 column volumes. During equilibration, adsorption, and elution, the flow rate was controlled at 4 column volumes per hour, and the pressure was maintained at 0.2 MPa.

[0057] Salt formation reaction: Add saturated sodium bisulfite solution to the eluent, control the pH at 8.5, the temperature at 55 degrees Celsius, and the reaction time at 3 hours.

[0058] Crystallization and Concentration: After the salt formation reaction is complete, add a 50% ethanol solution (1:20 ratio) to the reaction solution and stir thoroughly. Heat the hot water in the crystallizer jacket until the temperature of the solution reaches 30°C, then adjust the pH to 6.8 with 10% phosphoric acid. Switch the flow of ice-salt water into the jacket, stirring slowly to uniformly lower the temperature of the solution to 5°C and maintain this temperature for 20 hours. Centrifuge and filter at 1500 rpm. Return the filter residue to the crystallizer, add a pre-cooled 50% ethanol solution (1:20 ratio) to the residue, stir and wash, then centrifuge and filter again at 1500 rpm, collecting the wet product.

[0059] The wet product was vacuum dried at a vacuum level of 120 Pa and a drying oven temperature of 45 degrees Celsius for 6 hours. The semi-dried product was then released and granulated by sieving through a 60-mesh vibrating granulator, and then returned to the vacuum drying oven for another 10 hours. After sieving through a 60-mesh sieve again, the product was mixed and packaged to obtain the finished product.

[0060] Comparative Example 2 The purpose of this comparative study is to investigate the stability of houttuynia cordata extraction process after direct concentration and crystallization without salt formation reaction, and to analyze its impact on the extraction rate. A method for preparing houttuynia cordata extract includes the following steps: Preparation of Houttuynia cordata ultrafine powder: Fresh leaves of Houttuynia cordata were picked, washed with clean water to remove impurities such as mud and sand; dried in a blower dryer at 55 degrees Celsius for 3 hours. After drying, the powder was fed into an air jet mill at a pressure of 0.5 MPa, a power of 200 kW, and a feed speed of 180 m / s for 3 hours, followed by 4 consecutive grinding cycles, resulting in a particle size D50 of 12.80 nm.

[0061] Preparation of the alcohol extract: The ultrafine powder obtained in step 1 was mixed with a 95% ethanol solution at a solid-liquid ratio of 1:15 and extracted for 1 hour. The mixture was then filtered through a plate and frame filter at a speed of 1500 rpm, a filter cloth of 10 micrometers, and a flow rate of 70 cubic meters per hour. The alcohol extract was collected.

[0062] Treatment of filter residue: Press the juice using a twin-screw rotary press with a 10-micron filter screen and a power of 10 kilowatts, and collect the pressed juice.

[0063] Ceramic membrane filtration: The juice from the combined alcohol extract and filter residue is passed through a ceramic membrane for microfiltration with a pore size of 0.22 micrometers and a pressure of 0.3 MPa. The microfiltrate is then collected.

[0064] Ion exchange chromatography: The ion exchange column was washed with 2 column volumes of 50 mM phosphate buffer, and then loaded with ceramic membrane microfiltrate at a processing capacity of 10 g of active product per liter of resin to complete the sample loading and adsorption. The gradient elution sequence is as follows: 50 mM phosphate buffer solution, pH 6.5: 2 column volumes 0.1 mol sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes 0.3 mol sodium chloride-phosphate buffer solution, pH 6.5: 4 column volumes 0.5 mol sodium chloride-phosphate buffer solution, pH 6.5: 3 column volumes 0.7 mol sodium chloride-phosphate buffer solution (pH 6.5): collect the eluent at 2 column volumes, approximately 6 column volumes. During equilibration, adsorption, and elution, the flow rate was controlled at 4 column volumes per hour, and the pressure was maintained at 0.2 MPa.

[0065] Crystallization Concentration: Add a 50% ethanol solution (1:20 ratio) to the eluent and stir thoroughly. Heat the hot water in the crystallizer jacket until the feed solution reaches 30°C, then adjust the pH to 6.8 with 10% phosphoric acid. Switch the flow to ice-salt water in the jacket, stirring slowly to uniformly lower the feed solution temperature to 5°C and maintain this temperature for 20 hours. Centrifuge and filter at 1500 rpm. Return the filter residue to the crystallizer, add a pre-cooled 50% ethanol solution (1:20 ratio) to the residue, stir and wash, then centrifuge and filter again at 1500 rpm. Collect the wet product.

[0066] The wet product was vacuum dried at a vacuum level of 120 Pa and a drying oven temperature of 45 degrees Celsius for 6 hours. The semi-dried product was then released and granulated by sieving through a 60-mesh vibrating granulator, and then returned to the vacuum drying oven for another 10 hours. After sieving through a 60-mesh sieve again, the product was mixed and packaged to obtain the finished product.

[0067] Result detection Detection of the extraction rate of the active ingredient houttuynin Chromatographic conditions and system suitability test: The chromatographic column was a Waterssymmetry C84.6 mm × 250 mm, 5 μm; the mobile phase was methanol-water-10% tetrabutylammonium hydroxide solution (v / v) 55:45:0.3; the flow rate was 1 mL / min; the detection wavelength was 286 nm; the column temperature was 45 °C; and the injection volume was 20 μL. The theoretical plate number, calculated based on the houttuynia cordata peak, should be no less than 2500.

[0068] Solution preparation: Reference solution: Weigh 50 mg of sodium houttuynia cordata reference standard accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with the above mobile phase, shake well, accurately measure 2 mL, place it in a 100 mL volumetric flask, dilute to the mark with the mobile phase, shake well, and the solution is ready.

[0069] Sample solution: Accurately weigh an appropriate amount of Houttuynia cordata extract, approximately equivalent to 50 mg of sodium houttuynin, and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with the mobile phase, and shake well. Accurately measure 2 mL of the solution and place it in a 100 mL volumetric flask. Dilute to the mark with the mobile phase, and shake well. Calculate the houttuynin content in the Houttuynia cordata extract.

[0070] The formula for calculating the extraction rate of houttuynin is: actual extracted content / theoretical content in the medicinal material × 100%.

[0071] The test results are shown in the table below: Table 1 Extraction Rate Display Table Serial number Extraction rate Example 1 98.85 Example 2 98.29 Example 3 98.44 Comparative Example 1 26.17 Comparative Example 2 73.21 As shown in the table above, the Houttuynia cordata raw material in Examples 1, 2 and 3 was subjected to ultra-fine pulverization and combined with salt formation reaction crystallization concentration technology, resulting in the best extraction rate.

[0072] Stability Evaluation of Houttuynia cordata Thunb. Extract in Example 1 The stability test was carried out in accordance with the "Technical Guidelines for the Study of the Stability of Veterinary Chemical Drugs". The test conditions for the stress test were as follows: high temperature of 60±2 °C, high humidity of relative humidity 75±5%, and strong light of light intensity 4500±500 Lx. Samples were taken at 0, 5, and 10 days to determine the appearance, content, loss on drying, and identification, and compared with the results of the determination on day 0; the test conditions for the accelerated test were set as temperature 30±2 °C and relative humidity 65±5%. Samples were taken at the end of 0, 1, 2, 3, and 6 months to determine the appearance, loss on drying, and content, and compared with the results of the determination in month 0.

[0073] Stress test: The samples prepared in Example 1 were subjected to the stress test. The loss on drying of the samples should be ≤5%, and the content should be 90% - 110% of the labeled amount. The results are as follows in the table: Table 2 Results of the Stress Test of Houttuynia cordata Thunb. Extract Investigation factor Time (days) Character Loss on drying (%) Content (% of labeled amount) Light (4500 LX) 0 White powder 2.3 102.7 Light (4500 LX) 5 White powder 2.4 102.2 Light (4500 LX) 10 White powder 2.3 101.3 High temperature (60 degrees Celsius) 5 White powder 2.4 102.6 High temperature (60 degrees Celsius) 10 White powder 2.3 102.1 High humidity (RH 75%) 5 White powder 2.5 102.2 High humidity (RH 75%) 10 White powder 2.4 100.9 As can be seen from the above table, when the samples were placed for 10 days under the conditions of light, high temperature, and high humidity, their appearance did not change, and there was no obvious change in the loss on drying and content compared with day 0, indicating that the product quality of this Houttuynia cordata Thunb. extract is stable.

[0074] Accelerated test: The samples prepared in Example 1 were simulated in the commercial package and subjected to the accelerated test at 30±'2 °C and RH 65±5% for 6 months. Samples were detected at the end of 0, 1, 2, 3, and 6 months. The loss on drying of the samples should be ≤5%, and the content should be 90% - 110% of the labeled amount. The results are as follows in the table: Table 3 Results of the Accelerated Test of Houttuynia cordata Thunb. Extract Item Time (months) Character Loss on drying (%) Content (% of labeled amount) Example 1 0 White powder 2.3 102.7 Example 1 1 White powder 2.4 102.6 Example 1 2 White powder 2.4 102.2 Example 1 3 White powder 2.3 101.5 Example 1 6 White powder 2.3 100.8 As can be seen from the above table, when this product was accelerated for 6 months at 30±'2 °C and RH 65±5%, its appearance did not change, and there was no obvious change in the loss on drying and content compared with month 0, indicating that the product quality of this Houttuynia cordata Thunb. extract is stable.

[0075] Acute Toxicity Test of Houttuynia cordata Thunb. Extract in Example 1 on Mice Experimental animals: 40 clean-grade healthy ICR mice, weighing 18 - 22 g, with half males and half females, provided by the Comparative Medicine Center of Yangzhou University. The production license number of experimental animals is SCXK Su 2022 - 0009, and the license number for the use of experimental animals is SYXK Su 2022 - 0044. They were fed freely and given water, and the drinking water was tap water meeting the urban drinking water standard. They were adaptively raised in the experimental animal house environment for 3 days before the test, and were fasted for 12 hours before oral administration, and water intake was not restricted.

[0076] Tested drug: The Houttuynia cordata Thunb. extract in Example 1 was prepared into a solution with the required concentration using distilled water before use.

[0077] Administration of the test drug: The route of administration was oral, with mice receiving 0.2 ml of the test drug per 10 g of body weight. Mice were fasted for 12 hours prior to administration, but water intake was not restricted. Feeding was resumed 3 hours after administration.

[0078] Test procedure: The test operation shall be carried out in accordance with the "Limit test" method in GB15193.3-2014 "Acute Toxicity Test".

[0079] Preliminary Experiment: Four mice, half male and half female, were used in each group for the preliminary experiment to determine the 4 / 4 lethal dose b and the 0 / 4 lethal dose a. The preliminary experiment showed that the 0 / 4 lethal dose a of Houttuynia cordata extract in Example 1 was >5000 mg / kg body weight in mice. This dose is equivalent to administering the test substance at a concentration of 250 mg / mL and a maximum gavage volume of 0.2 mL / 10 g body weight to mice via gavage.

[0080] Formal Test: Based on the preliminary test results and the requirements of GB15193.3-2014 "Acute Toxicity Test", if the LD50 cannot be determined when the test substance is administered to test animals at the maximum permissible concentration and maximum permissible volume, the limit test can be used to determine the maximum tolerated dose. Therefore, the dosage for the formal test was determined to be 5000 mg / kg body weight. The maximum tolerated dose test in mice via oral administration was conducted in one dose group, with a test substance concentration of 250 mg / mL, administered via gavage at the maximum gavage volume of 0.2 mL per 10 g body weight, for a total dosage of 5000 mg / kg body weight.

[0081] Experimental animals were weighed, labeled, numbered, and grouped: 40 clean-grade healthy ICR mice, half male and half female, were used for formal experiments.

[0082] Preparation of test drug solution: The test drug was prepared into a solution of 250 mg / mL using distilled water and administered.

[0083] Administration method: The volume of test drug to be administered was calculated based on the body weight of each mouse in each group. The test drug was administered orally as described above, with a gavage volume of 0.2 ml per 10 g body weight.

[0084] Experimental observation: After the drug administration period, the general condition of the mice was observed twice daily, including weight, mental state, coat color, spontaneous activity, respiration, appetite, feces, oral and nasal secretions, symptoms of poisoning, and mortality. Gross anatomical examinations were performed on mice that died during the observation period, and all gross pathological changes were recorded. The observation was continued for 14 days. At the end of the experiment, surviving mice were euthanized, and necropsy findings were observed.

[0085] Data processing: Based on the experimental records, process and obtain various data.

[0086] Experimental results: Symptoms after administration: Mice administered orally had glossy fur, no discharge from the eyes, nose, or mouth; no abnormal behavior, activity, or mental state; normal feeding; and no abnormalities in urination or defecation.

[0087] Acute oral MTD: 14 ​​days after the end of administration, based on the administration concentration and gavage volume, it was found that the acute oral dose of Houttuynia cordata extract in Example 1 was greater than 5000 mg / kg body weight in mice.

[0088] Table 1-1 Results of acute oral MTD assay of Houttuynia cordata extract in mice in Example 1 Total dose (mg / kg) Quantity (units) Gavage volume and frequency Number of mice dead within 14 days (only) Acute Toxicity Reaction 5000 400.2 ml / 10 g body weight x 10 No acute toxicity reaction and death was found Table 1-2 Body weight change (x ± SD) of mice before and after oral administration of Houttuynia cordata extract of Example 1 Gender Number (only) 0-day weight (g) 14-day weight (g) Weight gain (g) Male 20 20.5±0.83 29.16±1.09 9.11±1.13 Female 20 19.42±0.71 27.09±1.42 7.67±1.32 Table 1-3 Compound oral acute toxicity grading standards Toxicity classification Mouse oral LD50 (mg / kg) Approximately lethal dose for a person weighing 70 kg Extremely toxic <1 a little, <7 drops Highly toxic 1-50 7 drops ~ 1 teaspoon Moderately toxic 51-500 1 teaspoon ~ 35 g Low toxicity 501-5000 35 ~ 350 g Practically non-toxic 5001-15000 350 ~ 1050 g Non-toxic >15000 > 1050 g Test Conclusion: The acute oral administration tolerance dose of Houttuynia cordata extract of Example 1 to ICR mice is greater than 5000 mg per kg of body weight, and the LD50 is greater than 5000 mg per kg of body weight. According to the compound oral acute toxicity grading standards, the acute toxicity grade of the test substance should be actually non-toxic.

[0089] It should be noted that the relational terms herein such as first and second and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0090] The preferred embodiments of the application disclosed above are only to help explain the application. The preferred embodiments do not describe all the details of the application, and the application is not limited to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the specification. The specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the application, so that those skilled in the art can well understand and utilize the application. The application is limited only by the claims and their full scope and equivalents.

Claims

1. A Houttuynia cordata extract, characterized in that, The fish-leaf extract is prepared by using the fresh leaves of the fish-leaf as the only raw material through a directional enrichment process, and the core effective component is sodium houttuynoid; The purity of the sodium houttuynoid is greater than or equal to 99%, and the extraction rate is greater than or equal to 98%. The raw material is subjected to airflow ultrafine pulverization treatment, and the particle size after pulverization is 1-100 nm. The sodium houttuynoid content of the extract decreases by less than or equal to 2% within 12 months of sealed storage at room temperature.

2. The extract of Houttuynia cordata according to claim 1, characterized in that, The particle size of the sodium houttuynoid is in the range of 1-50 nm, and the particle distribution uniformity is greater than or equal to 90%.

3. The extract of Houttuynia cordata according to claim 1, characterized in that, The fresh leaves of the fish-leaf are fresh leaves without mildew and disease and insect pests, and the rhizome part is removed, and the processing is performed within 24 hours after picking.

4. The extract of Houttuynia cordata according to claim 1, characterized in that, The detection of the sodium houttuynoid is performed by using high performance liquid chromatography, and the detection conditions are as follows: a C18 chromatographic column, a mobile phase of methanol-water, a detection wavelength of 254 nm, and a theoretical plate number calculated according to the sodium houttuynoid peak of not less than 2500.

5. The extract of Houttuynia cordata according to claim 1, characterized in that, The extract contains polysaccharide components, and the polysaccharide content is greater than or equal to 3.0%.

6. A method for preparing an extract of Houttuynia cordata, characterized by, The fish-leaf extract of any one of claims 1-5 is prepared by the following steps. Step one, raw material pretreatment: the fresh leaves of the fish-leaf are washed, drained, subjected to air drying, and then subjected to airflow ultrafine pulverization treatment to obtain nanoscale fish-leaf powder; Step two, extraction: the nanoscale fish-leaf powder is soaked and extracted in 70%-95% ethanol solution, the filter residue is subjected to screw press extraction after plate and frame filtration, and the two times of juice are combined to obtain a crude extract; Step three, purification: the crude extract is filtered through a 0.22 μm ceramic membrane, and then subjected to ion exchange chromatography by using XAD1600 macroporous resin, and the target component is collected by gradient elution; Step four, salt formation and crystallization: saturated sodium bisulfite solution is added to the purified target component for salt formation reaction, the pH is adjusted to 8-9, the reaction temperature is controlled to be 40-60 ℃, and crystallization is performed under low temperature conditions; Step five, drying: the crystallization product is subjected to vacuum drying treatment to obtain the high-purity fish-leaf extract.

7. The method for preparing a houttuynia cordata extract according to claim 6, characterized in that, In step one, the air drying temperature is 55 ℃, and the drying time is 2-4 h, and the water content of the raw material after drying is less than or equal to 8%.

8. The method of claim 6, wherein the Houttuynia cordata extract is prepared by the steps of: In step one, the process parameters of the airflow ultrafine pulverization are as follows: nitrogen pressure of 0.5 MPa, power of 150-500 kW, continuous pulverization for 2-5 times, and qualified rate of the powder particle size after pulverization of greater than or equal to 95%. ​ 9. The method of claim 6, wherein the Houttuynia cordata extract is prepared by the steps of: In step three, the gradient elution liquid is 50 mM phosphate buffer, 0.1 M sodium chloride phosphate buffer, 0.3 M sodium chloride phosphate buffer, 0.5 M sodium chloride phosphate buffer, and 0.7 M sodium chloride phosphate buffer in sequence, the elution flow rate is 4 times the column volume per hour, and the elution liquid collected is from the elution stage of 0.3 M sodium chloride phosphate buffer to the early stage of 0.5 M sodium chloride phosphate buffer, and the total collection amount is 6 times the column volume. ​ 10. The method of claim 6, wherein the Houttuynia cordata extract is prepared by the steps of: In step four, the low-temperature crystallization temperature is 5 ℃, and the crystallization time is 18-24 h; in step five, the vacuum drying pressure is 100-133 pa, the drying temperature is 40-50 ℃, and the total drying time is 16-20 h. ​

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