Extraction method and application of active ingredient of Shengjiangsan capable of inhibiting neuraminidase

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to an extraction method and application of an active ingredient of Shengjiang Powder that inhibits neuraminidase. The extraction method comprises the following steps: (1) preparing a crude extract of Shengjiang Powder: taking silkworm pupa, cicada shell, rhubarb, and turmeric, adding water, heating under reflux for extraction, filtering, concentrating, cooling, adding anhydrous ethanol, filtering, and concentrating to dryness to obtain a crude extract of Shengjiang Powder; (2) extracting the crude extract of Shengjiang Powder with a three-phase extraction solvent, collecting the upper phase, middle phase, and lower phase respectively, repeating the extraction 5 times, concentrating and drying to obtain extracts of each part; (3) subjecting the middle phase extract to high-speed countercurrent chromatography, using an eluent for primary separation and secondary separation according to the elution procedure, and automatically collecting the fractions; (4) concentrating and drying to obtain the active ingredient. The extracted active ingredient is applied to the preparation of a drug that inhibits neuraminidase, providing a basis for the development of such drugs.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for extracting Shengjiang Powder, and particularly relates to a method for extracting an active ingredient of Shengjiang Powder capable of inhibiting neuraminidase and an application thereof. 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 host cell surface, promoting the release of newly generated viral particles from the host cell. The active ingredient can bind to the active site of neuraminidase, blocking its enzymatic activity through competitive or non-competitive inhibition.

[0003] Shengjiang Powder (Shengjiang Powder) is composed of four herbs: rhubarb, turmeric, bombyx batryticatus, and cicada slough. It primarily treats severe fever in the triple burner (Sanjiao) caused by febrile diseases, both exterior and interior. This prescription contains only four herbs: bombyx batryticatus and cicada slough dispel wind and spasms, disperse wind-heat, and promote lung qi, promoting the clearing of yang within yang. Rhubarb and turmeric dissipate stasis, clear pathogenic heat, and resolve febrile toxicity, reducing the turbidity of yin within yin. However, the active components that inhibit neuraminidase are unknown, limiting the product's use and quality upgrades. Therefore, extracting and studying the active components in Shengjiang Powder that inhibit neuraminidase can provide theoretical and technical support for its clinical application and resource development. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for extracting the active ingredient of Shengjiangsan that can inhibit neuraminidase.

[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:

[0007] The present invention provides a method for extracting an active ingredient of Shengjiangsan capable of inhibiting neuraminidase, comprising the following steps:

[0008] (1) Preparation of crude extract of Shengjiangsan

[0009] Take 80.92 g of Bombyx batryticatus, 40.62 g of cicada slough, 160.12 g of rhubarb, and 120.98 g of turmeric, add 10 times the total mass of water, heat and reflux to extract for 2-3 h, filter, and concentrate. After cooling the concentrate at room temperature, add anhydrous ethanol and mix evenly until the ethanol concentration is above 80%, refrigerate overnight, filter and obtain the medicinal solution, and concentrate to dryness to obtain 38.32 g of Shengjiangsan crude extract.

[0010] (2) Three-phase solvent extraction of crude extract of Shengjiangsan

[0011] The three-phase extraction solvent was hexane / ethyl acetate / acetonitrile / water (HEAceWat) (8:2:6:4, v / v). Thoroughly mix 38.32 g of the crude extract of Shengjiang Powder with 200 mL of the three-phase extraction solvent and transfer it to a separatory funnel. Shake thoroughly and let it stand to separate the layers. Once the three-phase separation is obvious, collect the upper, middle, and lower phases separately. Place the middle phase in a vacuum concentrator and concentrate it to dryness. Extract the concentrated middle phase a second time using 200 mL of new mixed solvent. Repeat the extraction operation on the middle phase according to the above steps 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 separately to dryness.

[0012] (3) HPLC analysis

[0013] HPLC analysis was performed using an Agilent 1260 HPLC system and an Agilent ZORBAXSB-C column. 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. Flow rate: 1.0 mL min -1 , column temperature 30 ℃, injection volume 10 μL. Record the peak area (A) of the target chromatographic peak.

[0014] (4) HSCCC (High-Speed ​​Countercurrent Chromatography) Separation

[0015] The HSCCC eluate was fractionated into test tubes, with one test tube added every 6 minutes (30 mL per tube).

[0016] The upper phase extraction fraction of Shengjiang Powder (183.23 mg) was purified by using 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 NA inhibitory activity detection. The middle phase extraction part (933.18 mg) was extracted with ethyl acetate / n-butanol / water (EBuWat) (8.2:1.8:10, v / v ) system was used for HSCCC separation to obtain 22 components for NA inhibitory activity detection, n-hexane / ethyl acetate / methanol / water (HEMWat) (5:5:4:6, v / v ) 51 components were obtained for subsequent NA inhibitory activity detection.

[0017] Beneficial effects

[0018] The present invention is based on a three-phase solvent of n-hexane / ethyl acetate / acetonitrile / water (HEAceWat) (8:2:6:4, v / v) Shengjiang Powder was subjected to three-phase extraction. An appropriate solvent system ratio was selected for each fraction and HSCCC separation was performed. Subsequently, the fractions obtained from the HSCCC separation were assayed for NA activity to identify components with inhibitory activity. Potential NA inhibitors were screened by correlating the inhibition rate of the active components with HPLC peak area.

[0019] The extracted active ingredients are used to prepare drugs that inhibit neuraminidase, providing a basis for the development of this type of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 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;

[0021] Figure 2 The inhibitory activity of the crude extract of Shengjiangsan on NA;

[0022] Figure 3 is the correlation spectrum of HSCCC-NA inhibitory activity in the upper phase extraction site;

[0023] Figure 4 This is the HSCCC-NA inhibitory activity correlation spectrum of the EBuWAt system in the middle phase extraction site;

[0024] Figure 5 This is the correlation spectrum of HSCCC-NA inhibitory activity at the extrusion site of the EBuWat system via the HEMWat system;

[0025] Figure 6 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 between the inhibition rate of the active components and the chromatographic peak area of ​​the compounds;

[0026] Figure 7 Correlation analysis between the peak area and inhibition rate of the potential active compounds in 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;

[0027] Figure 8 HPLC qualitative analysis of the potential inhibitor of Shengjiangsan; A is the chromatogram of the rhein standard, B is the chromatogram of the active component of rhubarb, C is the UV absorption curve of the rhein standard, and D is the UV absorption curve of compound 3;

[0028] Figure 9 is the inhibitory activity of rhein on NA; A is rhein, B is oseltamivir acid, and C is diacerein;

[0029] Figure 10 It is the structural formula of rhein and diacerein;

[0030] Figure 11 Schematic diagram of the key residues in the interaction between rhein and NA. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further explained and illustrated by specific examples. The experimental supplies used in the present invention are all commercially available.

[0032] Example 1

[0033] 1. Test methods

[0034] (1) Preparation of crude extract of Shengjiangsan

[0035] Take 80.92 g of Bombyx batryticatus, 40.62 g of cicada slough, 160.12 g of rhubarb, and 120.98 g of turmeric, add 10 times the amount of water, heat and reflux to extract for 2-3 hours, filter, and concentrate. After cooling the concentrate at room temperature, add anhydrous ethanol and mix evenly until the ethanol concentration is above 80%, refrigerate overnight, filter and obtain the medicinal solution, and concentrate to dryness to obtain 38.32 g of Shengjiangsan crude extract.

[0036] (2) Three-phase solvent extraction of crude extract of Shengjiangsan

[0037] The three-phase extraction solvent was hexane / ethyl acetate / acetonitrile / water (HEAceWat) (8:2:6:4, v / v ). Thoroughly mix 38.32 g of the crude extract of Shengjiang Powder with 200 mL of the three-phase extraction solvent and transfer it to a separatory funnel. Shake thoroughly and let it stand to separate the layers. Once the three-phase separation is obvious, collect the upper, middle, and lower phases separately. Place the middle phase in a vacuum concentrator and concentrate it to dryness. Extract the concentrated middle phase a second time using 200 mL of new mixed solvent. Repeat the extraction operation on the middle phase according to the above steps 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 separately to dryness.

[0038] (3) HPLC analysis

[0039] HPLC analysis was performed using an Agilent 1260 HPLC system and an Agilent ZORBAXSB-C column. 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. Flow rate: 1.0 mL·min -1 , column temperature 30 ℃, injection volume 10 μL. Record the peak area (A) of the target chromatographic peak.

[0040] (4) HSCCC (High-Speed ​​Countercurrent Chromatography) Separation

[0041] At 30 mL·min -1 The stationary phase was pumped into the high-speed countercurrent chromatograph at a flow rate of 5 mL min after the stationary phase filled the chromatographic spiral tube. -1 Pump the mobile phase at a constant flow rate, collect it in a graduated cylinder, and calculate the retention rate of the stationary phase. Inject the sample solution into the separation column and activate the speed controller to rotate the separation column clockwise at 800 rpm. Use a UV detector at wavelengths of 214 nm, 254 nm, and 280 nm. Fractionate the HSCCC eluate into test tubes, adding one 30 mL test tube every 6 minutes.

[0042] The upper phase extraction fraction of Shengjiang Powder (183.23 mg) was extracted with 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 NA inhibitory activity detection. The middle phase extraction part (933.18 mg) was extracted with ethyl acetate / n-butanol / water (EBuWat) (8.2:1.8:10, v / v ) system for HSCCC separation to obtain 22 components for NA inhibitory activity detection, n-hexane / ethyl acetate / methanol / water (HEMWat) (5:5:4:6, v / v ) 51 components were obtained for subsequent NA inhibitory activity detection.

[0043] (5) Determination of the inhibitory activity of HSCCC components on NA

[0044] 5.1 Sample solution preparation

[0045] Accurately weigh 344.62 mg of the crude extract of Shengjiang Powder, dissolve it in 2 mL of DMSO, mix well, and then dilute it in a 2-fold gradient to obtain the crude extract sample solution.

[0046] The fractions separated from HSCCC were concentrated to dryness and dissolved in 1 mL of DMSO to obtain fraction sample solutions.

[0047] 5.2 Enzyme activity assay

[0048] The inhibitory effects of HSCCC components on NA were determined using a neuraminidase inhibitor screening kit. The procedure was performed according to the kit instructions, with excitation and emission wavelengths of 322 nm and 450 nm, respectively, and the fluorescence intensity of the reaction system was measured. Three replicates were run for each sample, and the inhibition rate was calculated for each sample. Dose-effect curves were drawn for the crude extract of Shengjiangsan based on the inhibition rate at each concentration, and the average 50% inhibitory concentration (IC) for NA was calculated. 50 ).

[0049] (6)Target Identification of Potential NA Inhibitors in Shengjiang Powder

[0050] When conducting the NA inhibitory activity experiments 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 rates 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 activities in 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 high NA inhibition rates at relatively small response values were selected as the active components. Subsequently, HPLC analysis was performed on the active components to determine the main chromatographic peaks in the active components. By analyzing the correlation between the chromatographic peak areas and the inhibition rates, the potential active compounds in the active components were determined. Finally, based on the ultraviolet spectra of the compounds and the retention behaviors 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.

[0051] (7)NA Inhibitory Activity of Potential Inhibitors

[0052] The NA inhibitory activity of the potential inhibitors was determined, 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.

[0053] (2)Experimental Results

[0054] 1. HPLC Analysis of Shengjiang Powder and Each Extraction Part

[0055] The crude extract of Shengjiang Powder and each extraction part were subjected to HPLC analysis according to the method under (3). 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 in combination 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.

[0056] 2. Neuraminidase Inhibitory Activity of the Crude Extract of Shengjiang Powder

[0057] First, the inhibitory activity of the crude extract of Shengjiang Powder against NA was detected, and its IC 50 ±SD was 913.3 ± 17.85 (μg / mL) ( Figure 2 ). It indicates that the crude extract of Shengjiang Powder has a certain inhibitory effect on NA.

[0058] 3. Separation of the upper - phase extraction part by HSCCC and the NA inhibitory activity of each component

[0059] The crude extract of Shengjiang Powder was extracted, and finally 0.23 g of the upper - phase extraction part, 1.43 g of the middle - phase extraction part, and 32.49 g of the lower - phase extraction part of Shengjiang Powder were obtained.

[0060] 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 extraction part of Shengjiang Powder was separated by HSCCC. HMWat (10:7.7:2.3, v / v ) was used as the HSCCC solvent system ratio for this part. The NA inhibitory activities of a total of 31 samples were detected, and a HSCCC - NA inhibitory activity correlation spectrum ( Figure 3 ) was obtained. The results showed that none of the components obtained by separating the upper - phase extraction part by HSCCC had good NA inhibitory activity.

[0061] 4. Separation of the middle - phase extraction part by HSCCC and the NA inhibitory activity of each component

[0062] The EBuWat system is suitable for the separation of compounds within the range of 40% < B% < 55%.

[0063] For the middle - phase extraction part, the EBuWat system was first used for separation. EBuWat (8.2:1.8:10, v / v ) was used as the HSCCC separation solvent system ratio. Using EBuWat (8.2:1.8:10, v / v) as the HSCCC separation solvent system ratio. 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 extraction part sample was injected into the CCC for separation, and 1 tube (30 mL per tube) was collected every 6 min. A total of 22 samples were collected within 132 min. Finally, the samples in the stationary phase were obtained using the extrusion - elution model to get the extruded solution. The extruded solution was concentrated to dryness and then separated twice using the HEMWat system.

[0064] The extruded part of the EBuWat system was separated by HSCCC. HEMWat (5:5:4:6, v / v) was used as the solvent system ratio for HSCCC separation at this site. 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%. 691.73 mg of the extruded site sample of the EBuWat system was injected into the CCC separation, and one test tube (30 mL per tube) was collected every 6 minutes. A total of 50 samples were collected within 300 minutes. Finally, the sample in the stationary phase was subjected to an extrusion-elution model to obtain its effluent. The NA inhibitory activity of 22 component samples of the EBuWat system and a total of 51 component samples of the HEMWat system were tested, and the HSCCC-NA inhibitory activity correlation spectrum of the EBuWat system was obtained ( Figure 4 ) and HSCCC-NA inhibitory activity correlation profiles of the HEMWat system ( Figure 5 The results showed that none of the fractions obtained from the mid-phase extraction site after HSCCC separation using the EBuWat system exhibited significant NA inhibitory activity. However, among the fractions separated using the HEMWat system, those separated between 204 and 234 min exhibited high NA inhibitory activity despite lower HSCCC response values, with inhibition rates exceeding 70%. Therefore, the fractions separated between 204 and 234 min were preliminarily identified as active NA inhibitors.

[0065] To further identify the active components that inhibit NA, the active components were analyzed by HPLC ( Figure 6 A), the results showed that there were 6 main chromatographic peaks in the component sample within 204 ~ 234 min, namely compounds 1 ~ 6. The peak areas of the 6 main compounds and the sample inhibition rate were combined for analysis ( Figure 6 B), it can be found that the peak areas and inhibition rates of compounds 1, 3 and 6 have similar changing trends. Correlation analysis of the peak areas of compounds 1 to 6 and their component inhibition rates shows that the peak area of ​​compound 3 has a strong correlation with the inhibition rate. r is 0.9390( Figure 7 Therefore, compound 3 was preliminarily identified as a potential NA inhibitor in Shengjiang Powder.

[0066] Figure 6 A is the HPLC analysis of the active components of Shengjiang Powder (HPLC analysis of the active components was performed using an Agilent 1260 HPLC system; the chromatographic column was an Agilent ZORBAX SB-C 18 (5 μm, 4.6 × 250 mm) mobile phase: 0.1% phosphoric acid in 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 ℃, and 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.

[0067] 5. Identification of Potential Inhibitor Structures

[0068] In the present invention, in order to determine the source of compound 3 in Shengjiang Powder, separate methanol extractions were performed on rhubarb and turmeric, and retention time comparison analysis was performed using HPLC. The results showed that compound 3 was consistent with the characteristic components in rhubarb extract, confirming 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, by 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. 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 Shengjiang Powder is rhein.

[0069] 6. Potential inhibitor NA inhibitory activity

[0070] 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 was 352.8±40.13 μM. Positive drug oseltamivir acid IC 50The value was 113.8±22.04 μM. This indicates that the neuraminidase inhibitory activity profile analysis based on countercurrent chromatography is applicable and effective. To further analyze the key pharmacophores of NA inhibition in rhein, its prodrug diacerein was tested for NA inhibitory activity. The results showed that when diacerein was generated after 1,8-dihydroxyacetylation of rhein, it lost its NA inhibitory activity and its IC 50 The value was 593.5±17.54 μM. Therefore, the 1,8-dihydroxy group in rhein may be the key pharmacophore for its NA inhibition effect. Its structural formula is shown in Figure 10 .

[0071] Molecular docking is a computer simulation program used to predict the binding mode of receptor-ligand complexes. It can also be used to predict the binding sites between receptors and ligands. The greater the negative value of the binding energy of molecular docking, the stronger the binding ability. A binding energy ≤-5kcal / mol indicates that the ligand and receptor protein have good binding activity. The binding energy of the potential inhibitor rhein is -8.5kcal / mol, and the binding energy of the positive drug oseltamivir acid is -6.5kcal / mol. This shows that the potential inhibitor rhein screened out has a good inhibitory effect on NA, and its active site model for interaction with NA is as follows: Figure 11 shown.

Claims

1. A method for extracting the 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 under reflux for extraction, filter, concentrate, cool, add anhydrous ethanol, filter, and concentrate to dryness to obtain the Shengjiangsan crude extract; (2) The crude extract of Shengjiang Powder was extracted with a three-phase extraction solvent, and the upper phase, middle phase and lower phase were collected separately. The extraction was repeated 5 times, and the extracts of each part were concentrated and dried. The three-phase extraction solvent was composed of n-hexane, ethyl acetate, acetonitrile and water in a volume ratio of 8:2:6:

4. (3) The middle phase extract was subjected to high-speed countercurrent chromatography, and the eluent was used for primary separation and secondary separation according to the elution procedure; the eluent for the primary separation was composed of ethyl acetate, n-butanol, and water in a volume ratio of 8.2:1.8:10; In the primary separation phase, the elution fraction from 0 to 132 min was collected; the sample in the stationary phase was subjected to an extrusion-elution model to obtain an extrudate, which was concentrated to dryness for secondary separation. The eluent for the secondary separation was composed of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:4:

6. During the secondary separation stage, the elution fraction from 0 to 300 min was collected. (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: 80.92 g of Bombyx Batryticatus, 40.62 g of Periostracum Cicadae, 160.12 g of Rhubarb, and 120.98 g of Curcuma Longa are taken, 10 times the total mass of water is added, and the mixture is heated under reflux for extraction for 2-3 hours. After filtration and concentration, the concentrated solution is cooled at room temperature, anhydrous ethanol is added and mixed evenly until the ethanol concentration is above 80%, and the solution is refrigerated overnight, and the medicinal solution is filtered and concentrated 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: The mass volume ratio of the crude extract of Shengjiang Powder to the three-phase extraction solvent was 38.32 g:200 mL.

4. The method for extracting the active ingredient of Shengjiangsan that inhibits neuraminidase according to claim 3, characterized in that: The elution site between 204 and 234 min in the secondary separation stage was the active inhibitory component of NA.

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