Application of extracts from *Cephalotaxus fortunei* in antiviral infections

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of *Cephalotaxus fortunei* extract in antiviral infection control. The *Cephalotaxus fortunei* extract of this invention exhibits good antiviral activity and can be used to prepare antiherpesvirus and anti-influenza virus drugs or pharmaceutical compositions.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of extracts from *Cephalotaxus fortunei* in antiviral infection control. Background Technology

[0002] Viral infectious diseases are a major threat to human health; studies show that 60% of diseases are caused by viral infections. Currently, there is still a lack of highly specific drugs for treating viral infectious diseases. Clinically used drugs mainly fall into the following categories: antiviral drugs that inhibit viral replication; immunomodulators that enhance the body's immune function; antiseptics, analgesics, antipyretics, and anti-inflammatory drugs that target clinical symptoms; anti-infective drugs to prevent secondary infections; vaccines to prevent viral infection; and disinfectants to block viral transmission. Currently used antiviral drugs are actually only inhibitors of viral replication; they cannot directly kill or destroy the virus, otherwise they would damage host cells. Furthermore, these antiviral drugs have drawbacks such as significant toxic side effects, a tendency to cause viral gene mutations, and a high risk of rebound after discontinuation. Therefore, developing novel antiviral drugs is an urgent priority in the field of drug research and development.

[0003] *Cercis stellaria* is recorded in many Chinese medical books on medicinal plants. It possesses the effects of dispelling wind and dampness, dispersing blood stasis and relieving pain, and promoting bone healing. Clinically, it is often used for lower back and leg pain, rheumatic numbness, and externally for treating bruises, fractures, and traumatic bleeding. Years of clinical practice by Yao medicine practitioners in Guangxi have proven that *Cercis stellaria* has anti-tumor effects and can be used to treat various malignant tumors, hence its name "tumor vine" or "tumor-eliminating vine." Current research on *Cercis stellaria* mainly focuses on the extraction and separation of crude substances. To date, there are no reports on the use of *Cercis stellaria* extracts for treating herpes simplex virus and influenza virus infections, or on the preparation of antiviral drugs against these viruses. This invention aims to provide the effective antiviral components of *Cercis stellaria*, providing a basis for further development and utilization of *Cercis stellaria* resources. Summary of the Invention

[0004] Therefore, one object of the present invention is to provide the use of *Cephalotaxus fortunei* extract containing *Cephalotaxus fortunei* extract in inhibiting herpes simplex virus and influenza virus.

[0005] Another object of the present invention is to provide a pharmaceutical composition for the use of extracts of *Cephalotaxus fortunei* in inhibiting herpes simplex virus and influenza virus.

[0006] Pharmacodynamic experiments conducted by the inventors showed that the extract of *Cephalotaxus fortunei* has a certain inhibitory effect on influenza virus, and its IC50 against coronavirus and H1N1 influenza virus is [not specified]. 50The values ​​were 8.31 μg / ml and 18.52 μg / ml, respectively, and it showed good inhibitory effects on herpesviruses. It could significantly inhibit the replication of HSV-1 and HSV-2. The IC50 of the extract of *Cephalotaxus fortunei* against HSV-1 and HSV-2 viruses was [not specified]. 50 The values ​​were 3.74 μg / ml and 4.12 μg / ml, respectively, indicating that the extract of *Cephalotaxus fortunei* has a good inhibitory effect on a variety of viruses and can be used to prepare antiviral drugs or further to make antiviral drug compositions.

[0007] Therefore, the inventor provides the use of *Cercis stellaria* extract in antiviral drugs, the key being: the *Cercis stellaria* extract is prepared by the following method: *Cercis stellaria*, crushed or cut into small pieces, is extracted three times with 95% ethanol; the extracts are combined, and the solvent is recovered under reduced pressure to obtain a crude extract; the crude extract is extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively; the ethyl acetate phase is collected, and the solvent is recovered under reduced pressure to obtain a concentrated solution; the concentrated solution is subjected to column chromatography with gradient elution; the eluent is concentrated under reduced pressure until no alcohol odor remains, dried, and pulverized to obtain the product.

[0008] In the preparation method of the extract of *Cephalotaxus fortunei* of the present invention, the amount of extraction solvent used is 1-20 times (by weight) of the medicinal material, preferably 5-10 times, and the extraction time is 1-5 hours; the column chromatography medium can be silica gel, macroporous resin, polyamide, ion exchange resin, dextran gel, preferably macroporous resin or silica gel. The elution solvent for the first elution can be a chloroform-methane (1:0-80:1) mixed solvent system, and the elution solvent for the second elution can be a (6:1-1:1) chloroform-ethyl acetate system; the density of the concentrated solution to be spray-dried is within 1.0-1.3, preferably 1.05-1.10.

[0009] Furthermore, the extract of *Cephalotaxus fortunei* comprises compounds having formula I and / or compounds having formula II.

[0010]

[0011] Furthermore, the viruses include herpesviruses and influenza viruses.

[0012] Furthermore, the influenza virus is HCoV-OC. 43 Coronavirus, and H1N1 influenza virus.

[0013] Furthermore, the herpesvirus is HSV-1 virus or HSV-2 virus.

[0014] The purpose of this invention is to provide a pharmaceutical composition containing, as an active ingredient, a therapeutically effective amount of the extract of *Cephalotaxus fortunei* as described in claim 1 and pharmaceutically acceptable excipients.

[0015] Furthermore, the alkyl sesquiterpene acid includes compounds having formula I and / or compounds having formula II.

[0016]

[0017] The pharmaceutical compositions of the present invention may also include one or more commonly used pharmaceutical excipients, such as binders (e.g., microcrystalline cellulose), fillers (e.g., starch, glucose, anhydrous lactose and lactose beads), disintegrants (e.g., cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricants (e.g., magnesium stearate), as well as absorption promoters, adsorbents, flavoring agents, sweeteners, excipients, diluents, wetting agents, etc.

[0018] The pharmaceutical composition can be prepared according to conventional methods in the art and can be administered via the enteral, parenteral, or topical routes. Oral formulations include tablets, granules, suspensions, capsules, solutions, etc., while parenteral formulations include injections. Topical formulations include creams, patches, ointments, sprays, gels, etc.

[0019] The administration routes of the extract of *Cephalotaxus fortunei* and the pharmaceutical composition can be oral, sublingual, transdermal, intramuscular or subcutaneous, mucosal, intravenous, urethral, ​​vaginal, etc.

[0020] The dosage of the extract and pharmaceutical composition of the *Cephalotaxus fortunei* can vary depending on the route of administration, the patient's age and weight, and the severity of the disease. The daily dose can be 0.001-100 mg / kg and can be administered once or multiple times.

[0021] The present invention further proposes the use of extracts of *Cephalotaxus fortunei* or compositions thereof as basic active ingredients in the prevention and treatment of various viral diseases, including herpes labialis, herpetic keratitis, herpetic dermatitis, genital herpes, and viral influenza.

[0022] This invention discloses a novel use of Astrophytum asterias extract in antiviral therapy. The Astrophytum asterias extract extracted using the extraction method of this invention has a strong inhibitory effect on HSV-1, HSV-2, HCoV-OC43, and H1N1, indicating that the Astrophytum asterias extract of this invention has a good antiviral effect and can be used to prepare antiherpes virus and influenza virus drugs or drug compositions. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0024] Example 1: Extract of *Cephalotaxus fortunei*

[0025] 1.1 Ethanol crude extract: Take 20 kg of Crude Prunus cerasifera powder, add 5 times the amount of 95% ethanol to extract three times, each extraction for 2 hours, with an interval of 24 hours between extractions. Combine the extracts, and recover the solvent under reduced pressure to obtain the crude extract.

[0026] 1.2 Extraction concentrate: The crude extract was extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively. Each extract was concentrated under reduced pressure and centrifuged to remove impurities.

[0027] 1.3 Ethyl acetate extraction fraction: The concentrated ethyl acetate solution was loaded onto the pre-treated resin and eluted using a gradient elution with a chloroform-methane (1:0, 80:1, 40:1, 20:1, 10:1, 1:1) mixed solvent system as the mobile phase. The results were detected by TLC. The eluents with the same components were collected and combined. The eluents were concentrated and loaded again, and eluted with a (4:1) chloroform-ethyl acetate system. The solvent was recovered from the eluent under reduced pressure and the eluent was dried under vacuum to obtain the extract of *Cephalotaxus fortunei*.

[0028] Example 2: Inhibitory effect of *Cephalotaxus fortunei* extract on HSV-1 virus

[0029] 2.1 Experimental Materials:

[0030] Cells: Rectal cancer cells (HRT-18).

[0031] Virus: HCoV-OC43.

[0032] Test drug: The extract of *Cephalotaxus fortunei* was dissolved in DMSO to prepare a 60 mg / mL stock solution.

[0033] 2.2 Experimental Methods:

[0034] HRT-18 cells were cultured using standard methods. HRT-18 cells in the logarithmic growth phase were collected and diluted with culture medium to a concentration of 1×10⁶ cells / mL. 5Cells were seeded at a concentration of [value missing] mL in 96-well cell culture plates. After cell attachment, the cells were infected with HCoV-OC43 virus. The virus-containing culture medium was removed, and different concentrations of *Cephalotaxus fortunei* extract were added. After culturing for 48 hours, cytopathic effects were observed under an inverted microscope. Remdesivir treatment was used as a positive control. The experimental results are shown in Table 1.

[0035] Table 1. Inhibitory effect of *Trifolium repens* extract on HCoV-OC43 virus.

[0036]

[0037] Example 3: Inhibitory effect of *Cephalotaxus fortunei* extract on H1N1 virus.

[0038] 2.1 Experimental Materials:

[0039] Cells: Human embryonic kidney cells (293T).

[0040] Virus: H1N1.

[0041] Test drug: The extract of *Cephalotaxus fortunei* was dissolved in DMSO to prepare a 60 mg / mL stock solution.

[0042] 2.2 Experimental Methods:

[0043] HRT-18 cells were cultured using standard methods. Vero cells in the logarithmic growth phase were collected and diluted with culture medium to a concentration of 1×10⁶ cells / mL. 5 Cells were seeded at a concentration of [value missing] mL in 96-well cell culture plates. After cell attachment, the cells were infected with HCoV-OC43 virus. The virus-containing culture medium was removed, and different concentrations of *Cephalotaxus fortunei* extract were added. After 20 hours, cytopathic effects were observed under an inverted microscope. The experimental results are shown in Table 2.

[0044] Table 2. Inhibitory effect of *Trifolium repens* extract on H1N1 virus.

[0045]

[0046] Example 3: Inhibitory effect of *Cephalotaxus fortunei* extract on HSV-1 virus.

[0047] 3.1 Experimental Materials:

[0048] Cells: African green monkey kidney cells (Vero cells).

[0049] Virus: HSV-1.

[0050] Test drug: The extract of *Cephalotaxus fortunei* was dissolved in DMSO to prepare a 60 mg / mL stock solution.

[0051] 3.2 Experimental Methods:

[0052] Vero cells were cultured using standard methods. Vero cells in the logarithmic growth phase were collected and diluted with culture medium to a concentration of 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of [value missing] mL in 96-well cell culture plates. After cell attachment, the cells were infected with HSV-1 virus. The virus-containing culture medium was removed, and different concentrations of *Cephalotaxus fortunei* extract were added. After culturing for 22 hours, cytopathic effects were observed under an inverted microscope. Acyclovir treatment was used as a positive control. The experimental results are shown in Table 3.

[0053] Table 3. Inhibitory effect of *Trichoderma purpurea* extract on HSV-1 virus.

[0054]

[0055] Example 4: Inhibitory effect of *Cephalotaxus fortunei* extract on HSV-2 virus.

[0056] 4.1 Experimental Materials:

[0057] Cells: African green monkey kidney cells (Vero cells).

[0058] Virus: HSV-2.

[0059] Test drug: The extract of *Cephalotaxus fortunei* was dissolved in DMSO to prepare a 60 mg / mL stock solution.

[0060] 2.2 Experimental Methods:

[0061] Vero cells were cultured using standard methods. Vero cells in the logarithmic growth phase were collected and diluted with culture medium to a concentration of 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of / mL in 96-well cell culture plates. After cell attachment, the cells were infected with HSV-1 virus. The virus-containing culture medium was removed, and different concentrations of *Cephalotaxus fortunei* extract were added. The cells were incubated at 37°C, 5% CO2, and 100% relative humidity for 22 hours. Cell pathogenesis was then observed under an inverted microscope. Acyclovir treatment was used as a positive control. The experimental results are shown in Table 4.

[0062] Table 4. Inhibitory effect of *Trichoderma purpurea* extract on HSV-2 virus.

[0063]

[0064] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. Use of an extract of Ampelopsis acontifolia in the preparation of a drug against viral infections, characterized in that: The extract of the Adenosma glutinosum Wall. is prepared by the following method: the Adenosma glutinosum Wall. which is crushed or cut into small pieces is extracted with 95 % ethanol for three times, the extract is combined, and the solvent is recovered under reduced pressure to obtain a crude extract; the crude extract is extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively, and the ethyl acetate phase is recovered under reduced pressure to obtain a concentrated solution; the concentrated solution is subjected to column chromatography and gradient elution; the eluate is concentrated under reduced pressure until no alcohol smell is left, dried, and pulverized to obtain the product; the extract of the Adenosma glutinosum Wall. comprises a compound having formula I and / or a compound having formula II, Formula I Formula II; The virus is HCoV-OC 43 one or more of a coronavirus, an anti-H1N1 influenza A virus, an HSV-1 virus, an HSV-2 virus.

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