Machilus zhennan water-soluble extract as well as extraction method and application thereof

Abstract

The invention discloses a phoebe zhennan water-soluble extract as well as an extraction method and application thereof, and belongs to the technical field of medicine extraction. The method comprises the following steps: S1, crushing a phoebe zhennan raw material, adding 6 times of water, extracting twice, 1 hour each time, and combining filtrates; s2, concentrating the filtrate obtained in the step S1 under reduced pressure until the volume is 1: 1, standing at low temperature, and taking supernate; and S3, centrifuging the supernate obtained in S2, discarding the precipitate, concentrating under reduced pressure, and drying at low temperature to obtain the phoebe zhennan water-soluble extract. The invention discloses a phoebe zhennan water-soluble extract and an extraction method and application thereof. The phoebe zhennan water-soluble extract has efficient broad-spectrum antiviral activity and can effectively inhibit respiratory virus activity.

Description

Technical Field

[0001] The invention belongs to the technical field of drug extraction, and particularly relates to a water-soluble extract of Phoebe zhennan and an extraction method and application thereof. Background Art

[0002] Phoebe zhennan is a plant of the genus Phoebe in the Lauraceae family. Its application in traditional Chinese medicine has been documented. Ancient books such as Zhenglei Bencao and Taiping Shenghui Fang mention its use in infectious diseases, internal medicine, and dermatological diseases. Modern research mainly focuses on the extraction and separation of its volatile oil components and its antibacterial and antitumor biological activities. For example, volatile oil analysis has revealed that it contains terpenes, phenols and other compounds, and has verified its inhibitory effects on some bacteria and tumor cells. However, there are few studies on the water-soluble components of Phoebe zhennan, especially its application in the antiviral field has not been publicly reported, and there is no relevant literature on its treatment of respiratory diseases such as acute rhinitis.

[0003] In clinical practice, acute upper respiratory tract infections (such as viral rhinitis and asthma exacerbations) are mainly caused by influenza A virus (H1N1) and respiratory syncytial virus (RSV). Such viral infections can cause transient allergies in the nasal mucosa, triggering symptoms such as sneezing and runny nose. Currently, there is a lack of targeted antiviral treatment drugs in clinical practice, and symptomatic supportive treatment is mostly used, with limited effect. Although some plant extracts (such as Scutellaria baicalensis and Honeysuckle) have been reported to have antiviral effects, their inhibitory efficiency against H1N1 and RSV is insufficient (the killing logarithm is generally <3.0), and high concentrations are prone to cytotoxicity. Based on the existing technology's neglect of the antiviral activity of the water-soluble components of Phoebe zhennan, and the clinical demand for specific drugs against respiratory viruses, it is urgent to develop a high-efficiency, low-toxic antiviral component derived from Phoebe zhennan and a preparation method thereof. Summary of the Invention

[0004] The present invention aims to provide a water-soluble extract of Phoebe zhennan and an extraction method and application thereof. The water-soluble extract of Phoebe zhennan has high-efficiency and broad-spectrum antiviral activity and can effectively inhibit the activity of respiratory viruses.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A method for extracting a water-soluble extract of Phoebe zhennan, comprising the following steps:

[0007] S1. Crush the Phoebe zhennan raw material, add 6 times the amount of water to extract twice, each time for 1 hour, and combine the filtrates;

[0008] S2. The filtrate obtained in S1 was concentrated under reduced pressure to a volume ratio of 1:1, allowed to stand at low temperature, and the supernatant was collected;

[0009] S3. Centrifuge the supernatant obtained in S2, discard the precipitate, concentrate under reduced pressure, and dry at low temperature to obtain the water-soluble extract of Phoebe zhennan.

[0010] Preferably, in S2, the low-temperature standing temperature is 4-10°C, and the low-temperature standing time is 8-10 hours.

[0011] Preferably, in S3, the centrifugal speed is 8000-10000 rpm / min, and the centrifugal time is 20 min.

[0012] Preferably, in S3, the low-temperature drying temperature is not higher than 60°C.

[0013] The present invention also provides a water-soluble extract of Phoebe zhennan obtained by the extraction method.

[0014] The present invention also provides the use of the water-soluble extract of Phoebe zhennan in preparing anti-respiratory virus drugs.

[0015] Preferably, the respiratory virus is influenza A virus and / or respiratory syncytial virus.

[0016] Preferably, the drug contains 2% w / v of the water-soluble extract of Prunus zhennan.

[0017] Preferably, the drug is a preparation for treating viral acute rhinitis, upper respiratory tract infection or virus-induced asthma exacerbation.

[0018] The present invention also provides an anti-respiratory virus preparation comprising the water-soluble extract of Phoebe zhennan as an active ingredient and a pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] The present invention discloses a water-soluble extract of Phoebe chinensis, an extraction method, and applications thereof. The water-soluble extract prepared using the present invention exhibits a logarithmic killing effect greater than 4.50 against influenza A virus (H1N1, VR-1469) and greater than 4.47 against respiratory syncytial virus (RSV, VR-1803) at a concentration of 2%, both meeting the significant inhibition standards specified in the Technical Specifications for Disinfection. The extract can be directly used to prepare anti-respiratory viral drugs for the targeted treatment of viral acute rhinitis, upper respiratory tract infections, and exacerbated viral asthma, providing an effective means for addressing the clinical challenge of synergistic pathogenicity between viral infection and respiratory hypersensitivity reactions.

[0021] The technical solution of the present invention is further described in detail below through examples. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further illustrated by the following examples.

[0023] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0024] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0025] The virus strains were: influenza A virus (H1N1) (VR-1469), respiratory syncytial virus (RSV) (VR-1803).

[0026] The cell lines are: MDCK and HEP-2.

[0027] Example 1

[0028] A method for extracting a water-soluble extract of Phoebe zhennan, comprising the following steps:

[0029] S1. Grind 100 g of Phoebe zhennan raw material, add 6 times the amount of water and extract twice, each time for 1 hour, and combine the filtrates;

[0030] S2. The filtrate obtained in S1 was concentrated under reduced pressure to a volume ratio of 1:1, and allowed to stand at 4°C for 10 h. The supernatant was collected.

[0031] S3. Centrifuge the supernatant obtained in S2 at 10,000 rpm / min for 20 min, discard the precipitate, concentrate under reduced pressure, and dry at 60° C. to obtain a water-soluble extract of Phoebe zhennan, with an extract yield of 15%.

[0032] Take 2 g of the above-mentioned water-soluble extract of Phoebe zhennan and prepare it into a 4% solution with PBS. Filter it with a 0.22 μm microporous filter membrane, store it in a 1 mL EP tube as the original solution for the experiment, and store it in a -40°C refrigerator for use.

[0033] The effect was verified through the following tests:

[0034] The experimental methods are as follows:

[0035] Testing basis: "Disinfection Technical Specifications" 2002 Edition 2.1.1.10 Virus Inactivation Test

[0036] Action conditions: Disinfectant concentrations (12.5%, 25%, 50%, 100%) were applied for 2 minutes respectively; the original solution (4%) used in the experiment was used as 100% of the disinfectant concentration.

[0037] Methods for terminating the effect of residual disinfectants:

[0038] Physical disinfection method (preferential dilution method): The disinfectant (25%) is diluted in a gradient manner. After 10-fold dilution, 2.5% disinfectant has no effect on cells. Under this dilution condition, there is no obvious inhibitory effect on virus-infected cells, thereby obtaining the conditions for effectively terminating disinfection at different concentrations of disinfectant. Taking 25% disinfectant as an example, the following operations are performed:

[0039] Group 1: Pipette 0.9 mL of disinfectant into a test tube, place it in a 20℃±1℃ water bath for 5 minutes, then add 0.1 mL of virus suspension, mix well, and let it sit for 2 minutes, then measure the virus titer.

[0040] Group 2: Pipette 0.9 mL of disinfectant into a test tube, place it in a 20℃±1℃ water bath for 5 minutes, then add 0.1 mL of virus suspension, mix well, and let it sit for 2 minutes. Immediately dilute it 5-fold, mix well, and then measure the virus titer.

[0041] Group 3: Pipette 0.1 mL of virus suspension and add 0.9 mL of cell culture medium, dilute 5-fold, mix well, and then measure the virus titer.

[0042] Group 4: Pipette 0.1 mL of virus suspension and add 0.9 mL of cell maintenance medium. No disinfectant or other disinfection treatment is performed. Pipette the virus suspension according to the test requirements and perform the subsequent virus titer determination.

[0043] Group 5: Complete cell culture medium was added to the cell tube not inoculated with virus and cultured.

[0044] Inactivation test: Take 25% disinfectant as an example and perform the following operations:

[0045] (1) Pipette 0.1 mL of interfering substance (3% BSA) and add it to a 1.5 mL centrifuge tube.

[0046] (2) Add 0.1 mL of virus suspension to the centrifuge tube and mix. Then add 0.8 mL of product test solution and start timing after mixing.

[0047] (3) After the specified exposure time, immediately perform a 5-fold dilution. Then, prepare a 10-fold dilution series of mixed solutions (test mixture and cell maintenance solution) within 30 minutes. Calculate the virus kill rate based on the change in virus titer before and after the test.

[0048] The experiment was commissioned by Zhongke Testing Technology Service (Guangzhou) Co., Ltd., and antiviral testing was performed on a water-soluble extract of Phoebe chinensis, according to the Virus Inactivation Test Method, Section 2.1.1.10, of the "Technical Specifications for Disinfection," 2002 edition. The results of the water-soluble extract of Phoebe chinensis against influenza virus are shown in Tables 1 and 2.

[0049] Table 1 Results of the physical drug removal test for influenza virus (dilution method)

[0050]

[0051] Table 2 Killing effect of samples on influenza virus and inactivation logarithm

[0052]

[0053]

[0054] As shown in Tables 1 and 2, 2% of the water-soluble extract of Phoebe zhennan has a significant inhibitory effect on influenza A virus VR-1469, with a killing logarithm value greater than 4.50.

[0055] The results of the effect of the water-soluble extract of Phoebe zhennan on respiratory syncytial virus are shown in Tables 3 and 4.

[0056] Table 3 Respiratory syncytial virus physical removal identification test (dilution method)

[0057]

[0058] Table 4 Killing effect and inactivation logarithm of samples on respiratory syncytial virus

[0059]

[0060]

[0061] As shown in Tables 3 and 4, 2% of the water extract of Pseudostellaria zedoaria has a significant inhibitory effect on respiratory syncytial virus VR-1803, with a killing logarithm value greater than 4.47.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for extracting a water-soluble extract of Phoebe zhennan, characterized in that: The steps include: S1. Crush the Phoebe zhennan raw material, add 6 times the amount of water to extract twice, each time for 1 hour, and combine the filtrates; S2. The filtrate obtained in S1 was concentrated under reduced pressure to a volume ratio of 1:1, allowed to stand at low temperature, and the supernatant was collected; S3. Centrifuge the supernatant obtained in S2, discard the precipitate, concentrate under reduced pressure, and dry at low temperature to obtain the water-soluble extract of Phoebe zhennan.

2. The extraction method according to claim 1, wherein In S2, the low-temperature standing temperature is 4-10°C, and the low-temperature standing time is 8-10 hours.

3. The extraction method according to claim 1, wherein In S3, the centrifugal speed is 8000-10000 rpm / min, and the centrifugal time is 20 min.

4. The extraction method according to claim 1, wherein In S3, the low-temperature drying temperature is not higher than 60°C.

5. The water-soluble extract of Phoebe zhennan obtained by the extraction method according to any one of claims 1 to 4.

6. Use of the water-soluble extract of Phoebe zhennan as claimed in claim 5 in the preparation of anti-respiratory virus drugs.

7. The application according to claim 6, characterized in that The respiratory virus is influenza A virus and / or respiratory syncytial virus.

8. The use according to any one of claims 6-7, characterized in that: The medicine contains 2% w / v of the water-soluble extract of Prunus chinensis.

9. The application according to claim 8, characterized in that: The medicine is a preparation for treating viral acute rhinitis, upper respiratory tract infection or virus-induced asthma exacerbation.

10. An anti-respiratory virus preparation, characterized in that: The invention comprises the water-soluble extract of Phoebe zhennan as claimed in claim 5 as an active ingredient, and a pharmaceutically acceptable carrier.

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