Application of radix glehniae polysaccharide in prevention and treatment of respiratory syncytial virus

Abstract

The invention discloses application of radix glehniae polysaccharide in preventing and treating respiratory syncytial virus, and belongs to the technical field of biological medicine. The research finds that the radix glehniae polysaccharide in the radix glehniae can play a role in inhibiting the respiratory syncytial virus and can be used as a specific active ingredient for preventing and treating the respiratory syncytial virus for the first time. The research result of the invention overcomes the technical blind area that the overall efficacy of traditional Chinese medicine components cannot be attributed to specific components, accurately positions that the radix glehniae polysaccharide can play an anti-RSV role when being independently used, and provides a new target for designing a targeted RSV inhibitor and developing a combined medication strategy covering various respiratory viruses.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of radix adnate polysaccharide in prevention and treatment of respiratory syncytial virus. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the background information forms a part of the prior art already known to a person of ordinary skill in the art.

[0003] Respiratory syncytial virus (RSV) has been identified since 1957, and has become the most common pathogenic microorganism causing respiratory diseases in the world. Almost all adults have experienced RSV infection even repeatedly in childhood. Primary RSV infection usually leads to lower respiratory tract bronchiolitis and pneumonia. Severe RSV infection is characterized by cough, wheezing, shortness of breath, dyspnea, chest wall depression and cyanosis. More than 3 million people are hospitalized and about 60,000 people die worldwide due to RSV infection every year, which seriously threatens human health and safety. At present, there is no effective prevention and treatment method for RSV infection in China, and the main treatment is to support the treatment to relieve symptoms. The commonly used drugs are interferon, glucocorticoid and bronchodilator, but long-term use will cause drug resistance, hormone resistance or dependence and related complications. Therefore, it is of great significance to develop new drugs which are effective, safe and suitable for long-term use.

[0004] Radix adnate is the root of Adiantum capillus-veneris L. It has various components, rich in polysaccharides, volatile oil and coumarin compounds, amino acids and trace elements. It is recorded in Compendium of Materia Medica that radix adnate is sweet and cold, and its body is light and empty, so it can tonify lung qi, and benefit spleen and kidney. It is suitable for those who can be controlled by fire. It can clear lung fire and treat chronic cough and lung deficiency. Radix adnate is sweet, bitter and cold in nature, and belongs to lung and stomach channels. It has the effects of nourishing yin, moistening lung, benefiting stomach and generating saliva, and is the core drug for treating yin deficiency and deficiency of both qi and yin. It is often used in the treatment of lung heat and dry cough, labor cough with blood phlegm, stomach yin deficiency, heat disease with fluid loss, dry throat and thirst, etc. Radix adnate polysaccharide is a plant polysaccharide extracted from radix adnate, which has the functions of antioxidant and blood glucose reduction. At present, there is no report on the use of radix adnate polysaccharide in the prevention and treatment of RSV infection. SUMMARY

[0005] In view of the above prior art, the purpose of the present application is to provide the application of radix adnate polysaccharide in the prevention and treatment of respiratory syncytial virus, which solves the problems of lack of specific drugs in the existing treatment of respiratory syncytial virus, large side effects of western medicine and unclear active ingredients of traditional Chinese medicine, so as to provide reference for the research and development and clinical application of anti-RSV drugs.

[0006] To achieve the above object, the present application adopts the following technical solutions. In the first aspect of the present application, the use of polysaccharides in radix adnate glabrae in the preparation of a medicament for preventing and / or treating respiratory syncytial virus infection is provided.

[0007] The preparation method of the polysaccharides in radix adnate glabrae is as follows: (1) Lipid removal: dry radix adnate glabrae root decoction pieces are crushed and sieved, petroleum ether 60℃ is added and refluxed for 1-2h, the filtrate is collected by filtration, and the solvent is removed by drying at room temperature to obtain radix adnate glabrae powder; (2) Water extraction: distilled water is added to the radix adnate glabrae powder obtained in step (1), heated and boiled for 1-2h, the filtrate is collected by filtration, and concentrated at 60℃ to obtain a concentrated solution; (3) Protein removal: the protein impurity removal reagent and the concentrated solution obtained in step (2) are mixed in a volume ratio of 1:4, vortexed for 8-10min, ultrasonicated for 2-3min at a power of 200w, and the supernatant is collected by centrifugation to obtain a crude polysaccharide extract of radix adnate glabrae; (4) Alcohol precipitation: anhydrous ethanol is added to the crude polysaccharide extract of radix adnate glabrae obtained in step (3) until the final concentration of ethanol is 80% (v / v), and after sufficient reaction, the precipitate is collected by centrifugation and freeze-dried to obtain polysaccharides in radix adnate glabrae.

[0008] Preferably, in step (2), the solid-liquid ratio of the radix adnate glabrae powder and distilled water is 1:10.

[0009] Preferably, in step (3), the protein impurity removal reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1.

[0010] In the second aspect of the present application, a medicament for preventing and treating respiratory syncytial virus infection is provided, which uses the above-mentioned polysaccharides in radix adnate glabrae as the active ingredient.

[0011] The medicament also includes pharmaceutically acceptable solvent or auxiliary ingredients.

[0012] Preferably, the dosage form of the medicament is capsule, tablet, powder, pill, suppository, injection or granule.

[0013] The present application has the following advantages: The present application first discovers that the polysaccharides in radix adnate glabrae can inhibit respiratory syncytial virus and can be used as a specific active ingredient for preventing and treating respiratory syncytial virus. The research results of the present application overcome the technical blind spot that the overall efficacy of traditional Chinese medicine components cannot be attributed to specific components, and accurately locate the polysaccharides in radix adnate glabrae, which can exert anti-RSV effect when used alone, providing a new target for designing inhibitors targeting RSV and developing a combined drug strategy covering multiple respiratory viruses. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Fig. 1 is a picture of the appearance of the polysaccharide of Radix Adenophorae.

[0015] Figure 2 Fig. 2 is a picture of the content determination of the polysaccharide of Radix Adenophorae.

[0016] Figure 3 Fig. 3 is a picture of the infrared spectrum determination of the polysaccharide of Radix Adenophorae.

[0017] Figure 4 Fig. 4 is a picture of the monosaccharide composition determination of the polysaccharide of Radix Adenophorae and a standard substance control picture, wherein Figure 4 Fig. 4A is the standard substance control picture; Figure 4 Fig. 4B is the monosaccharide composition determination picture of the polysaccharide of Radix Adenophorae.

[0018] Figure 5 Fig. 5 is a picture of the surface morphology of the polysaccharide of Radix Adenophorae, wherein Figure 5 Fig. 5A is a picture of the surface morphology of the polysaccharide of Radix Adenophorae under a magnification of 500, Figure 5 Fig. 5B is a picture of the surface morphology of the polysaccharide of Radix Adenophorae under a magnification of 2000, Figure 5 Fig. 5C is a picture of the surface morphology of the polysaccharide of Radix Adenophorae under a magnification of 10000.

[0019] Figure 6 Fig. 6 is a picture of the CCK8 detection of cell activity.

[0020] Figure 7 Fig. 7 is a picture of the cytopathic effect.

[0021] Figure 8 Fig. 8 is a picture of the RSV virus titer.

[0022] Figure 9 Fig. 9 is a picture of the qRT-PCR detection of the RSV-F mRNA expression amount. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0024] The specific embodiments of the present application are described in further detail below with reference to the examples. The following detailed description is illustrative only and is intended to provide further description of the present application without limiting the scope of the present application.

[0025] The RSV virus used in the embodiments of the present application is provided by the Basic Medical Research Institute of Shandong Medical Science Academy, and is passed in the P2 laboratory of Shandong Academy of Chinese Medical Sciences, and is stored in a-80℃ refrigerator for use.

[0026] Wang X, Ren W, Wang P, et al. Investigating the active components and mechanistic effects of Forsythia suspensa Leaf against RSV via the PI3K / Akt-NLRP3 pathway. Heliyon . 2024;10(19):e38285. Published 2024 Sep 21. Example 1: Preparation of polysaccharides from Adenophora potaninii (1) Lipid removal: Take 100 g of dried Adenophora potaninii root decoction pieces (produced in Laizhang, Shandong, purchased from Shandong Baiweitang Chinese Herbal Decoction Pieces Co., Ltd.) and crush them through a sieve. Add the powder to 400 ml of petroleum ether 60°C and heat it to reflux for 2 hours. Collect the filtrate by suction filtration and dry it at room temperature to remove the solvent to obtain Adenophora potaninii powder.

[0027] (2) Water extraction: Add distilled water to the Adenophora potaninii powder at a solid-liquid ratio of 1:10. Heat it to boiling for 1 hour. Collect the filtrate by suction filtration and repeat the extraction three times. Combine the filtrates and concentrate them at 60°C to obtain about 200 ml of concentrated solution.

[0028] (3) Protein removal: Use the Sevage method to remove protein impurities. Mix chloroform and n-butanol at a volume ratio of 4:1 to prepare a protein impurity removal reagent. Mix the protein impurity removal reagent and the concentrated solution at a volume ratio of 1:4, vortex for 10 minutes. To improve the protein removal efficiency, ultrasonic at a power of 200 w for 2 minutes, centrifuge at 3000 g for 10 minutes, and take the supernatant to obtain the crude polysaccharide extract of Adenophora potaninii.

[0029] (4) Alcohol precipitation: Add anhydrous ethanol to the crude polysaccharide extract of Adenophora potaninii at a volume ratio of 1:4 until the final concentration of ethanol is 80% (v / v). After sufficient precipitation, centrifuge at 3000 g for 10 minutes, collect the precipitate, and freeze-dry it to obtain Adenophora potaninii polysaccharides Figure 1 ).

[0030] Example 2: Determination of the content of Adenophora potaninii polysaccharides and structural characterization (1) Determination of the content of Adenophora potaninii polysaccharides by phenol-sulfuric acid method: Dilute the glucose standard stock solution to prepare a standard curve. Measure the content of Adenophora potaninii polysaccharides by the phenol-sulfuric acid method, and determine the absorbance value at 490 nm on a full-wavelength microplate reader.

[0031] The glucose standard curve is as follows Figure 2With glucose content as the x-axis and absorbance as the y-axis, the glucose standard curve was obtained as Y = 0.008353*X + 0.09823, with R² = 0.9995. Substituting this into the calculation, the crude polysaccharide content of Adenophora adenophora was 82.32%.

[0032] Determination of functional group types of prepared Adenophora adenophora polysaccharides by spectrometry The results were analyzed by Nicolet iZ-10 Fourier transform infrared spectrometer. Figure 3 As shown, 3211.97cm -1 The significant signal is the OH stretching vibration absorption peak, which is the characteristic peak of sugars. -1 The absorption peak at 1012.9 cm is attributed to CH stretching vibration, which is consistent with the methylene and methine groups on the saturated alkyl chain or sugar ring. -1 There is an absorption peak at , which is attributed to the stretching vibration of CO, and is direct evidence of the ring structure and connecting bonds of sugars.

[0033] (3) Determination of monosaccharide composition of prepared Adenophora adenophora polysaccharide by mass spectrometry Depend on Figure 4 It can be seen that Adenophora adenophora polysaccharide is a heteropolysaccharide composed of glucose (Glc), galactose (Gal), arabinose (Ara), galacturonic acid (Gal-UA), and rhamnose (Rha), with molar percentages of 90.64%, 3.43%, 3.00%, 2.28%, and 0.65%, respectively. Among them, glucose is the main monosaccharide component.

[0034] (4) Scanning electron microscopy was used to examine the surface morphology of the prepared Adenophora adenophora polysaccharide Scanning electron microscopy was used to obtain the surface morphology of Adenophora adenophora polysaccharide. Figure 5 As shown in the figure, at a magnification of 500, the polysaccharide from Glehnia littoralis appears amorphous and flake-like. This is likely due to the high glucose content, which increases the number of hydrogen bonds between the chains, resulting in a highly folded, loose, flake-like structure that facilitates the dissolution of the polysaccharide. At higher magnifications, the polysaccharide from Glehnia littoralis exhibits a porous, reticular structure, and irregular granular structures are observed attached to the surface of the polysaccharide sample, indicating that it has good adsorption properties.

[0035] Example 3: CCK8 assay for cytotoxicity of Adenophora adenophora polysaccharide Human laryngeal epidermoid carcinoma cells (Hep-2 cells, provided by the Institute of Basic Medicine, Shandong Academy of Medical Sciences, passaged in the P2 laboratory of Shandong Academy of Traditional Chinese Medicine, and stored at -80°C) were seeded into 96-well cell culture plates. 100 μL / well was incubated in 5% CO2 at 37°C for 24 hours. The culture medium was discarded, and Adenophora australis polysaccharide was diluted in 2% FBS DMEM medium to concentrations of 12.5, 25, 50, 100, 200, 400, 800, and 1600 μg / mL. A control group containing only maintenance medium (2% FBS DMEM) was established. After 48 hours of culture, CCK-8 solution was added to each well at a ratio of 10:1. After incubation for 2 hours, absorbance was measured at 450 nm using a microplate reader.

[0036] Result: Passed Figure 6 It can be seen that different concentrations of Adenophora adenophora polysaccharides have no cytotoxicity to Hep-2 cells.

[0037] Example 4: Inhibitory effect of Adenophora dahurica polysaccharide on RSV virus (1) Observation of the degree of cytopathic effect Hep-2 cells were seeded into 96-well culture plates and cultured at 37°C with 5% CO2 for 24 h. The culture medium was discarded and 12.5, 50, 200, and 800 μg / mL of Adenophora adenophora polysaccharide were added and cultured for 2 h. The supernatant was discarded and washed with PBS. 100 μL of 100 TCID 50 RSV virus was incubated for 2 hours, and the supernatant was discarded and washed with PBS. Then, Glehnia littoralis polysaccharide at concentrations of 12.5, 50, 200, and 800 μg / mL was added, respectively. Cells not treated with polysaccharide and virus were used as the blank control group (mock). After culturing for 24 hours, the cell pathological changes were observed and recorded under a microscope.

[0038] Result: Passed Figure 7 It can be seen that compared with the blank control group, the cytopathic effect was alleviated after intervention with different concentrations of Adenophora adenophora polysaccharide. When the concentration was 200 and 800 μg / mL, it could exert a significant anti-RSV virus effect.

[0039] (2) Virus TCID 50 Determination Hep-2 cells were seeded into 6-well plates and cultured at 37°C with 5% CO2 for 24 h. The culture medium was discarded and 12.5, 50, 200, and 800 μg / mL of Adenophora adenophora polysaccharide were added and cultured for 2 h. After the supernatant was discarded and washed with PBS, 1 ml of 100 TCID was added to each well. 50RSV virus was incubated for 2h, the supernatant was discarded, and then PBS was used for washing. Then, 12.5, 50, 200, and 800 μg / ml of polysaccharides from Adenophora potaninii were added, respectively. Cells treated with no polysaccharides and virus were used as blank control group (mock). After 24h of culture, the supernatant was collected, diluted by 10 times in gradient, and then 10 -8 The virus TCID 50 was determined by using Karber method.

[0040] As shown in Figure 8 , compared with the blank control group, when the concentration of polysaccharides from Adenophora potaninii was 50, 200, and 800 μg / ml, the RSV virus titer of Hep-2 cells treated with polysaccharides from Adenophora potaninii was significantly reduced, indicating that polysaccharides from Adenophora potaninii had inhibitory effect on RSV, and the inhibitory effect increased with the increase of the concentration of polysaccharides from Adenophora potaninii.

[0041] (3) Detection of RSV-F mRNA expression The F gene (NC-001803.1) of respiratory syncytial virus is a key surface glycoprotein encoding virus, which plays a core role in the mechanism of RSV infection, especially in virus invasion of host cells, mediation of membrane fusion, and immune escape.

[0042] Hep-2 cells were inoculated in a 6-well plate, and cultured at 37℃ in 5% CO2 for 24h. Then, the culture solution was discarded, and 12.5, 50, 200, and 800 μg / ml of polysaccharides from Adenophora potaninii were added, respectively. After 2h of incubation, the supernatant was discarded, and then PBS was used for washing. Then, 1ml of 100 TCID 50 RSV virus was incubated for 2h, the supernatant was discarded, and then PBS was used for washing. Then, 12.5, 50, 200, and 800 μg / ml of polysaccharides from Adenophora potaninii were added, respectively. Cells treated with no polysaccharides and virus were used as blank control group (mock). After 24h of culture, the supernatant was collected, diluted by 10 times in gradient, and then 10

[0043] F (SEQ ID NO. 1): TTGGATCTGCAATCGCCA; R (SEQ ID NO. 2): CTTTTGATCTTGTTCACTTCTCCTTCT.

[0044] From Figure 9 As can be seen, compared with the blank control group, when the concentration of polysaccharide of radix adnatae is 50, 200 and 800 μg / mL, the expression of RSV virus F gene mRNA of Hep-2 cells after intervention of polysaccharide of radix adnatae is significantly reduced, which indicates that the polysaccharide of radix adnatae has inhibitory effect on RSV, and the inhibitory effect increases with the increase of the concentration of radix adnatae.

[0045] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and the present application can have various changes and variations for those skilled in the art. Any modification within the spirit and principle of the present application, equivalent replacement, improvement, etc. should be included in the protection scope of the present application.

Claims

1. Use of Adenophora scabra polysaccharide in the preparation of a medicament for preventing and / or treating respiratory syncytial virus infection.

2. The use according to claim 1, characterized in that The preparation method of the Adenophora adenophora polysaccharide is as follows: (1) Degreasing: Take dried Glehnia littoralis root slices, crush and sieve them, add petroleum ether, heat and reflux at 60°C for 1-2 hours, collect the residue by filtration, and dry at room temperature to remove the solvent to obtain Glehnia littoralis powder; (2) Water extraction: add distilled water to the Adenophora glauca powder obtained in step (1), heat and boil for 1-2 h, collect the filtrate by suction, and concentrate by rotary evaporation at 60°C to obtain a concentrate; (3) Protein removal: the protein impurity removal reagent and the concentrate obtained in step (2) were mixed in a volume ratio of 1:4, vortexed for 8-10 minutes, ultrasonicated at a power of 200W for 2-3 minutes, and centrifuged to obtain the supernatant to obtain the crude polysaccharide extract of Glehnia littoralis; (4) Alcohol precipitation: anhydrous ethanol is added to the crude polysaccharide extract of Glehnia littoralis obtained in step (3) until the final concentration of ethanol is 80%. After sufficient reaction, the precipitate is collected by centrifugation and freeze-dried in a vacuum to obtain Glehnia littoralis polysaccharide.

3. The use according to claim 2, characterized in that In step (2), the material-liquid ratio of the Glehnia littoralis powder and distilled water is 1:

10.

4. The use according to claim 2, characterized in that In step (3), the protein impurity removal reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:

1.

5. A drug for preventing and treating respiratory syncytial virus infection, characterized in that: The medicine uses the Adenophora adenophora polysaccharide described in claim 1 as an active ingredient.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable solvents or excipients.

7. The drug according to claim 6, characterized in that The dosage form of the medicine is capsule, tablet, powder, pill, suppository, injection or granule.

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