Extraction method and application of Liangzhisan active component capable of inhibiting neuraminidase

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an extraction method and application of active ingredients of Rhododendron simsii powder capable of inhibiting neuraminidase, and the extraction method comprises the following steps: (1) preparing a crude Rhododendron simsii powder extract: taking stiff silkworm, cicada slough, rheum officinale and turmeric, adding water, heating, refluxing, extracting, filtering, concentrating, cooling, adding absolute ethyl alcohol for suction filtration, and concentrating to obtain the crude Rhododendron simsii powder extract; concentrating to be dry to obtain a crude extract of the elevating and lowering powder; (2) extracting the Rhododendron simsii crude extract by adopting a three-phase extraction solvent, respectively collecting an upper phase, a middle phase and a lower phase, repeatedly extracting for 5 times, and concentrating and drying to obtain each part of extract; (3) carrying out high-speed counter-current chromatography separation on the middle-phase extract, carrying out main separation and secondary separation by using an eluent according to an elution procedure, and automatically receiving fractions; and (4) concentrating and drying. The extracted active ingredients are applied to preparation of drugs for inhibiting neuraminidase, and a basis is provided for development of the drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an extraction method of Shengjiang Powder, in particular to an extraction method and application of active components of Shengjiang Powder with neuraminidase inhibitory activity. Background Art

[0002] Neuraminidase (NA) is a key enzyme on the surface of the H1N1 influenza virus, responsible for hydrolyzing sialic acid residues on the surface of host cells and promoting the release of newly generated virus particles from host cells. Active components can bind to the active site of neuraminidase and block its enzyme activity through competitive or non-competitive inhibition.

[0003] Shengjiang Powder is composed of four medicinal materials: Rheum palmatum, Curcuma longa, Bombyx batryticatus, and Cryptotympana pustulata. It is mainly used to treat severe heat in the three energizers of the warm disease. There are only four medicinal materials in this prescription. Among them, Bombyx batryticatus and Cryptotympana pustulata dispel wind and relieve spasm, disperse wind-heat, and promote the lung qi, promoting the clear yang in yang; Rheum palmatum and Curcuma longa purge accumulation and promote blood circulation, clear pathogenic heat, and relieve warm toxin, descending the turbid yin in yin. However, the active components that inhibit neuraminidase are unclear, which limits the use and quality upgrade of products. Therefore, extracting and studying the active components that inhibit neuraminidase in Shengjiang Powder can provide theoretical and technical support for the clinical application and resource development of Shengjiang Powder. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an extraction method of active components of Shengjiang Powder with neuraminidase inhibitory activity.

[0005] Another object of the present invention is to provide the application of the product obtained by the above extraction method.

[0006] The present invention is achieved through the following technical solutions: The present invention provides an extraction method of active components of Shengjiang Powder with neuraminidase inhibitory activity, comprising the following steps: (1) Preparation of crude extract of Shengjiang Powder Take 80.92 g of Bombyx batryticatus, 40.62 g of Cryptotympana pustulata, 160.12 g of Rheum palmatum, and 120.98 g of Curcuma longa, add 10 times the total mass of water, heat under reflux for 2 - 3 h, filter, concentrate, cool the concentrated solution at room temperature, add anhydrous ethanol and mix evenly until the ethanol concentration is above 80%, refrigerate overnight at low temperature, filter by suction to obtain the medicinal liquid, and concentrate to dryness to obtain 38.32 g of crude extract of Shengjiang Powder.

[0007] (2) Three-phase solvent extraction of crude extract of Shengjiang Powder The three-phase extraction solvent is n-hexane / ethyl acetate / acetonitrile / water (HEAceWat) (8:2:6:4, v / v)(38.32 g of the crude extract of Shengjiang Powder was fully mixed with 200 mL of the three-phase extraction solvent and then transferred to a separatory funnel. After thorough shaking, it was allowed to stand for phase separation. After obvious three-phase separation, the upper phase, middle phase, and lower phase were collected separately. The middle phase was concentrated to dryness in a vacuum concentration device. The concentrated middle phase was re-extracted with 200 mL of fresh mixed solvent. According to the above steps, the extraction operation was repeated for the middle phase, a total of 5 times. After the extraction was completed, the upper and lower phases of the 5 extractions were combined. The three-phase solvent extraction parts were concentrated to dryness respectively.)

[0008] (3) HPLC analysis The template molecule of the Agilent 1260 HPLC system was used for HPLC analysis; the chromatographic column was Agilent ZORBAXSB - C 18 (5 μm, 4.6 × 250 mm) Mobile phase: water (A) - methanol (B), gradient elution: 0 - 45 min, 10% → 100% B; 45 - 55 min, 100% B. The flow rate was 1.0 mL·min -1 , the column temperature was 30 °C, and the injection volume was 10 μL. The peak area (A) of the target chromatographic peak was recorded.)

[0009] (4) HSCCC (High-speed countercurrent chromatography) separation The eluate of HSCCC was fractionated into test tubes, and 1 test tube (30 mL per tube) was collected every 6 min.)

[0010] The upper phase extraction part of Shengjiang Powder (183.23 mg) was separated using the n-hexane / methanol / water (HMWat) (10:7.7:2.3, v / v ) system for subsequent separation, and a total of 31 components were obtained for the inhibitory activity detection of NA. The middle phase extraction part (933.18 mg) was separated by HSCCC using the ethyl acetate / n-butyl alcohol / water (EBuWat) (8.2:1.8:10, v / v ) system to obtain 22 components for the inhibitory activity detection of NA, and n-hexane / ethyl acetate / methanol / water (HEMWat) (5:5:4:6, v / v ) to obtain 51 components for the subsequent inhibitory activity detection of NA.)

[0011] Beneficial effects Based on the three-phase solvent n-hexane / ethyl acetate / acetonitrile / water (HEAceWat) (8:2:6:4, v / vPerform three-phase extraction on Shengjiang Powder. Select the appropriate solvent system ratios for each part and perform HSCCC separation. Subsequently, measure the NA activity of the fractions obtained by HSCCC separation to determine the components with inhibitory activity. Through the correlation analysis of the inhibition rate of the active components and the HPLC chromatographic peak area, screen out potential NA inhibitors.

[0012] Apply the extracted active ingredients to the preparation of drugs for inhibiting neuraminidase, providing a basis for the development of such drugs. Description of the Drawings

[0013] Figure 1 It is the HPLC analysis chart of the crude extract of Shengjiang Powder and each extraction part, where A is the crude extract of Shengjiang Powder, B is the upper-phase extraction part, C is the middle-phase extraction part, and D is the lower-phase extraction part; Figure 2 It is the inhibitory activity of the crude extract of Shengjiang Powder against NA; Figure 3 It is the HSCCC-NA inhibitory activity correlation spectrum of the upper-phase extraction part; Figure 4 It is the HSCCC-NA inhibitory activity correlation spectrum of the middle-phase extraction part in the EBuWAt system; Figure 5 It is the HSCCC-NA inhibitory activity correlation spectrum of the extrusion part in the EBuWat system through the HEMWat system; Figure 6 It is the analysis of the active components of Shengjiang Powder; A is the HPLC analysis of the active components of Shengjiang Powder; B is the correlation analysis of the inhibition rate of the active components and the chromatographic peak area of the compounds; Figure 7 It is the correlation analysis of the peak area and inhibition rate of the potential active compounds of Shengjiang Powder; A is Compound 1, B is Compound 2, C is Compound 3, D is Compound 4, E is Compound 5, and F is Compound 6; Figure 8 It is the HPLC qualitative analysis of the potential inhibitors of Shengjiang Powder; A is the chromatogram of rhein standard, B is the chromatogram of the active components of rhubarb, C is the ultraviolet absorption curve of rhein standard, and D is the ultraviolet absorption curve of Compound 3; Figure 9 It is the inhibitory activity of rhein against NA; A is rhein, B is oseltamic acid, and C is diacerein; Figure 10 It is the structural formulas of rhein and diacerein; Figure 11 It is the schematic diagram of the key residues of the interaction between rhein and NA. Detailed Embodiments

[0014] The technical solution of the present invention will be further explained and illustrated below through specific embodiments. All experimental supplies used in the present invention are commercially available.

[0015] Example 1 I. Test method (1) Preparation of crude extract of Shengjiang Powder Take 80.92 g of Bombyx Batryticatus, 40.62 g of Cryptotympana pustulata Fabricius, 160.12 g of Rheum officinale Baill., and 120.98 g of Curcuma longa L., add 10 times the amount of water, heat under reflux for 2 - 3 h, filter, concentrate, and after the concentrated solution is cooled at room temperature, add anhydrous ethanol and mix evenly until the ethanol concentration is above 80%, refrigerate overnight at low temperature, filter by suction to obtain the medicinal liquid, and concentrate to dryness to obtain 38.32 g of the crude extract of Shengjiang Powder.

[0016] (2) Three - phase solvent extraction of the crude extract of Shengjiang Powder The three - phase extraction solvent is n - hexane / ethyl acetate / acetonitrile / water (HEAceWat) (8:2:6:4, v / v ). Mix 38.32 g of the crude extract of Shengjiang Powder with 200 mL of the three - phase extraction solvent, transfer it to a separating funnel, shake well and let it stand for stratification. After the three - phase stratification is obvious, collect the upper phase, middle phase, and lower phase respectively. Concentrate the middle phase to dryness in a vacuum concentration device. Use 200 mL of a new mixed solvent to perform secondary extraction on the concentrated middle phase. According to the above steps, repeat the extraction operation on the middle phase for a total of 5 times. After the extraction is completed, combine the upper and lower phases of the 5 extractions. Concentrate the three - phase solvent extraction parts to dryness respectively.

[0017] (3) HPLC analysis Use the Agilent 1260 HPLC system template molecule for HPLC analysis; the chromatographic column is Agilent ZORBAXSB - C 18 (5 μm, 4.6 × 250 mm). Mobile phase: water (A) - methanol (B), gradient elution: 0 - 45 min, 10%→100%B; 45 - 55 min, 100% B. The flow rate is 1.0 mL·min -1 , the column temperature is 30 °C, and the injection volume is 10 μL. Record the peak area (A) of the target chromatographic peak.

[0018] (4) HSCCC (High - speed counter - current chromatography) separation Pump the stationary phase into the high - speed counter - current chromatograph at a flow rate of 30 mL·min -1 , and after the stationary phase fills the chromatographic coiled tube, at a flow rate of 5 mL·min -1The mobile phase was pumped in at a flow rate and collected in a graduated cylinder, and the retention rate of the stationary phase was calculated. The sample solution was injected into the separation column, and the speed controller was turned on to make the separation column rotate clockwise at a speed of 800 rmp. The ultraviolet detector was turned on, and the detection wavelengths were 214 nm, 254 nm, and 280 nm. The eluate of HSCCC was fractionated into test tubes, and 1 test tube (30 mL per tube) was collected every 6 min.

[0019] The upper-phase extraction part (183.23 mg) of Shengjiang Powder was separated further using the n-hexane / methanol / water (HMWat) (10:7.7:2.3, v / v ) system, and a total of 31 components were obtained for the inhibitory activity detection of NA. The middle-phase extraction part (933.18 mg) was separated by HSCCC using the ethyl acetate / n-butanol / water (EBuWat) (8.2:1.8:10, v / v ) system to obtain 22 components for the inhibitory activity detection of NA, and the n-hexane / ethyl acetate / methanol / water (HEMWat) (5:5:4:6, v / v ) was used to obtain 51 components for the subsequent inhibitory activity detection of NA.

[0020] (5) Determination of the inhibitory activity of HSCCC components against NA 5.1 Preparation of sample solution 344.62 mg of the crude extract of Shengjiang Powder was accurately weighed, dissolved and mixed evenly in 2 mL of DMSO, and diluted by a 2-fold gradient to obtain the crude extract sample solution.

[0021] The components separated by HSCCC were concentrated to dryness and dissolved separately in 1 mL of DMSO to obtain the component sample solution.

[0022] 5.2 Enzyme activity experiment The inhibitory effects of each component of HSCCC on NA were determined using a neuraminidase inhibitor screening kit. The operation was carried out according to the kit instructions, and 322 nm and 450 nm were used as the excitation and emission wavelengths to measure the fluorescence intensity of the reaction system. Each sample had 3 parallel replicates, and the inhibition rate of each sample was calculated. The crude extract sample of Shengjiang Powder was used to plot the dose-effect curve according to the inhibition rate of each concentration, and its average 50% inhibitory concentration (IC 50 ) against NA was calculated.

[0023] (6) Target identification of potential NA inhibitors in Shengjiang Powder When conducting the NA inhibitory activity experiment on each component of HSCCC, due to the large differences in the concentrations of each component, it is impossible to judge the inhibitory effect on NA only based on the NA inhibition rate of different components. In order to more accurately identify the targets of potential NA inhibitors in Shengjiang Powder, this experiment established a correlation spectrum of HSCCC-NA inhibitory activity for different extraction parts. Combining the NA inhibition rates of different components with their response values in the HSCCC chromatogram can largely avoid the differences in NA inhibition rates caused by component concentration differences. Therefore, the components with higher NA inhibition rates at lower response values were selected as active components. Subsequently, HPLC analysis was performed on the active components to determine the main chromatographic peaks in the active components. The potential active compounds in the active components were determined by analyzing the correlation between the peak areas of the chromatographic peaks and the inhibition rates. Finally, based on the ultraviolet spectra of the compounds and the retention behavior of the chromatographic peaks, the structures of the potential active compounds were preliminarily judged, and standard substances were used for qualitative analysis to determine the structures of the potential NA inhibitors.

[0024] (7)NA inhibitory activity of potential inhibitors The NA inhibitory activity of potential inhibitors was measured, and the binding energy between the potential inhibitor molecules and the enzyme complex was studied by molecular docking technology to verify their NA inhibitory effects.

[0025] (II)Experimental results 1. HPLC analysis of Shengjiang Powder and each extraction part The crude extract of Shengjiang Powder and each extraction part were subjected to HPLC analysis according to the method under (3), and the results are shown in Figure 1 . The results showed that after extraction, the chemical components in the upper-phase extraction part mainly eluted after 40 min in the HPLC analysis, and the polarity range of the compounds was B%>90%. The compounds in this part were suitable for further separation using the HAWat system. The chemical components in the middle-phase extraction part mainly eluted within the time range of 15 - 40 min in the HPLC analysis, and their polarity range was 40%<B%<90%. The components in this part should be further separated using the EBuWat system combined with the HEAWat system. The HPLC analysis results of the lower-phase extraction part showed that its main components eluted in the first 10 min, which might be components with extremely high polarity and some polysaccharide components. Since this study mainly aimed to find small-molecule potential NA inhibitors in this compound prescription, this part was not further studied.

[0026] 2. Neuraminidase inhibitory activity of the crude extract of Shengjiang Powder First, the inhibitory activity of the crude extract of Shengjiang Powder on NA was detected, and its IC 50 ±SD was 913.3±17.85 (μg / mL) ( Figure 2 ). It indicated that the crude extract of Shengjiang Powder had a certain inhibitory effect on NA.

[0027] 3. HSCCC Separation of the Upper-Phase Extract Fraction and NA Inhibitory Activity of Each Component The crude extract of Shengjiang Powder was extracted, and finally 0.23 g of the upper-phase extract fraction, 1.43 g of the middle-phase extract fraction, and 32.49 g of the lower-phase extract fraction of Shengjiang Powder were obtained.

[0028] Under the conditions of a flow rate of 5.0 mL / min, a rotation speed of 800 rpm, and a detection wavelength of 214 nm, the retention rate of the stationary phase was 76.00%. 183.23 mg of the upper-phase extract fraction of Shengjiang Powder was separated by HSCCC. HMWat (10:7.7:2.3, v / v ) was used as the proportion of the HSCCC solvent system for this fraction. A total of 31 samples were detected for NA inhibitory activity, and the HSCCC-NA inhibitory activity correlation spectrum ( Figure 3 ) was obtained. The results showed that none of the components obtained by separating the upper-phase extract fraction by HSCCC had good NA inhibitory activity.

[0029] 4. HSCCC Separation of the Middle-Phase Extract Fraction and NA Inhibitory Activity of Each Component The EBuWat system is suitable for the separation of compounds in the range of 40% < B% < 55%.

[0030] For the middle-phase extract fraction, the EBuWat system was first used for separation. EBuWat (8.2:1.8:10, v / v ) was used as the proportion of the solvent system for HSCCC separation. Using EBuWat (8.2:1.8:10, v / v) as the proportion of the solvent system for HSCCC separation. Under the conditions of a flow rate of 5.0 mL / min, a rotation speed of 800 rpm, and a detection wavelength of 280 nm, the retention rate of the stationary phase was 58.30%. 933.18 mg of the middle-phase extract fraction sample was injected into the CCC for separation, and 1 test tube (30 mL per tube) was collected every 6 min. A total of 22 samples were collected within 132 min. Finally, the sample in the stationary phase was obtained by the extrusion-elution model to get the extrudate. The extrudate was concentrated to dryness and subjected to secondary separation using the HEMWat system.

[0031] The extruded fraction of the EBuWat system was separated by HSCCC. HEMWat (5:5:4:6, v / v)as the solvent system ratio for HSCCC separation of this part. Under the conditions of a flow rate of 5.0 mL / min, a rotation speed of 800 rpm, and a detection wavelength of 254 nm, the retention rate of the stationary phase was 68.30%. Inject 691.73 mg of the extruded part sample of the EBuWat system into CCC separation, collect 1 test tube every 6 min (30 mL per tube), and a total of 50 samples were collected within 300 min. Finally, the effluent of the sample in the stationary phase was obtained using the extrusion-elution model. Perform NA inhibitory activity detection on 22 component samples of the EBuWat system and a total of 51 component samples of the HEMWat system to obtain the HSCCC-NA inhibitory activity correlation spectrum of the EBuWat system ( Figure 4 )and the HSCCC-NA inhibitory activity correlation spectrum of the HEMWat system ( Figure 5 ). The results showed that none of the components obtained after HSCCC separation of the middle-phase extraction part by the EBuWat system had good NA inhibitory activity. However, among the components separated by the HEMWat system, the component samples within 204 - 234 min had high NA inhibitory activity at relatively low HSCCC response values, and their inhibition rates were all above 70%. Therefore, it was preliminarily determined that the component samples within 204 - 234 min were the active inhibitory components of NA.

[0032] To further determine the NA inhibitory active ingredients in the active components, perform HPLC analysis on the active components ( Figure 6 A). The results showed that there were 6 main chromatographic peaks in the component samples within 204 - 234 min, namely compounds 1 - 6. Perform combined analysis on the peak areas of the 6 main compounds and the sample inhibition rate ( Figure 6 B), and it was found that the peak areas of compounds 1, 3, and 6 had similar changing trends with the inhibition rate. Performing correlation analysis on the peak areas of compounds 1 - 6 and their component inhibition rates, it was found that the peak area of compound 3 had a strong correlation with the inhibition rate, and its r was 0.9390 ( Figure 7 ). Therefore, it was preliminarily determined that compound 3 was the potential NA inhibitor in Shengjiang Powder.

[0033] Figure 6 In, A is the HPLC analysis of the active components of Shengjiang Powder (perform HPLC analysis on the active components using an Agilent 1260 HPLC system; the chromatographic column is Agilent ZORBAX SB -C 18 (5 μm, 4.6 × 250 mm) mobile phase: 0.1% phosphoric acid water (A)-acetonitrile (B), gradient elution: 0 - 10 min, 10% B; 10 - 15 min, 10%→15% B; 15 - 16 min,15%→18% B; 16 - 30 min, 18%→20% B; 30 - 45 min, 20%→37% B; 45 - 60 min, 37%→42% B; 60 - 65 min, 42%→50% B; 65 - 75 min, 50% B; 75 - 78 min, 50%→80% B; 78 -85 min, 80%→90% B; 85 - 86 min, 90%→100% B; 86 - 92 min, 100% B. Flow rate: 1.0 mL min -1 , column temperature was 30 ℃, injection volume was 10 μL. ); B is the correlation analysis between the inhibition rate of active components and the chromatographic peak area of ​​the compounds.

[0034] 5. Identification of potential inhibitor structures In the present invention, in order to determine the source of compound 3 in Shengjiangsan, rhubarb and turmeric were extracted with methanol separately, and the retention time was compared and analyzed by HPLC. The results showed that compound 3 was consistent with the characteristic components in rhubarb extract, and it was determined that it originated from rhubarb. Further studies found that under the HPLC detection conditions described in (3), the retention time of compound 3 was about 61 min. However, when the phosphoric acid water in the mobile phase was replaced with pure water, the retention time of compound 3 was significantly advanced, indicating that its chromatographic behavior was affected by the composition of the mobile phase. In addition, through ultraviolet absorption spectroscopy analysis, compound 3 showed maximum absorption at 210 nm, 232 nm, 260 nm and 432 nm, which is consistent with the characteristic ultraviolet absorption curve of rhein. In order to further confirm the structure of compound 3, rhein standard was used for comparative analysis, and the results showed that the retention time and ultraviolet absorption characteristics of the two in HPLC were completely consistent ( Figure 8 ). Therefore, it can be determined that the potential inhibitor compound 3 in Shengjiangsan is rhein.

[0035] 6. Potential inhibitor NA inhibitory activity Using oseltamivir acid as the positive control drug, rhein was tested for its NA inhibitory activity. The results are shown in Figure 9 In the NA inhibitory activity assay, rhein showed moderate inhibitory activity with an IC 50 The value is 352.8±40.13 μM. Positive drug oseltamivir acid IC 50The value was 113.8 ± 22.04 μM. This indicates that the countercurrent chromatography-based neuraminidase inhibitory activity profiling is applicable and effective. To further analyze the key pharmacophore of the NA inhibitory effect in rhein, the NA inhibitory activity of its prodrug diacerein was determined. The results showed that diacerein, which was generated by the 1,8-dihydroxyacetylation of rhein, lost its NA inhibitory activity, and its IC 50 value was 593.5 ± 17.54 μM. Therefore, the 1,8-dihydroxy group in rhein may be the key pharmacophore for its NA inhibitory effect, and its structural formula is shown in Figure 10 .

[0036] Molecular docking is a computer simulation program used to predict the binding mode of receptor-ligand complexes and can also be used to predict the binding sites between receptors and ligands. The greater the negative value of the binding energy in molecular docking, the stronger the binding ability. A binding energy ≤ -5 kcal / mol is considered to indicate good binding activity between the ligand and the receptor protein. The binding energy of the potential inhibitor rhein was -8.5 kcal / mol, and the binding energy of the positive drug oseltamic acid was -6.5 kcal / mol. This indicates that the screened potential inhibitor rhein has a good inhibitory effect on NA, and its interaction active site model with NA is shown in Figure 11 .

Claims

1. A method for extracting an active ingredient of Shengjiangsan that inhibits neuraminidase, characterized in that: The following steps are involved: (1) Preparation of Shengjiangsan crude extract: Take silkworm pupa, cicada shell, rhubarb and turmeric, add water, heat and reflux to extract, filter, concentrate and cool, add anhydrous ethanol, filter and concentrate to dryness to obtain Shengjiangsan crude extract; (2) The crude extract of Shengjiangsan was extracted with a three-phase extraction solvent, and the upper phase, middle phase and lower phase were collected respectively. The extraction was repeated 5 times, and the extracts of each part were concentrated and dried; (3) subjecting the middle phase extract to high-speed countercurrent chromatography, performing primary separation and secondary separation using an eluent according to an elution procedure, and automatically collecting the fractions; (4) Concentrate and dry to obtain the product.

2. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 1, characterized in that: The specific method for preparing the crude extract of Shengjiang Powder in step (1) is as follows: take 80.92 g of Bombyx Batryticatus, 40.62 g of Periostracum Cicadae, 160.12 g of Rhubarb, and 120.98 g of Curcuma Longa, add 10 times the total mass of water, heat and reflux to extract for 2-3 hours, filter and concentrate, cool the concentrate at room temperature, add anhydrous ethanol and mix evenly until the ethanol concentration is above 80%, refrigerate overnight, filter to obtain the medicinal solution, and concentrate to dryness to obtain the crude extract of Shengjiang Powder.

3. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 1, characterized in that: In step (2), the three-phase extraction solvent is n-hexane, ethyl acetate, acetonitrile and water in a volume ratio of 8:2:6:

4.

4. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 1, characterized in that: The mass volume ratio of the crude extract of Shengjiangsan to the three-phase extraction solvent was 38.32 g:200 mL.

5. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 1, characterized in that: The eluent for the main separation in step (3) is composed of ethyl acetate, n-butanol and water in a volume ratio of 8.2:1.8:

10. In the main separation stage, the elution portion from 0 to 132 min is collected. The sample in the stationary phase is subjected to an extrusion-elution model to obtain an extrudate, which is concentrated to dryness for secondary separation.

6. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 1, characterized in that: The eluent for the secondary separation in step (3) is composed of n-hexane, ethyl acetate, methanol and water in a volume ratio of 5:5:4:

6. In the secondary separation stage, the elution portion from 0 to 300 min is collected.

7. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 6, characterized in that: The elution site in the second separation stage between 204 and 234 min was the active inhibitory component of NA.

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